Abstract
ABSTRACTS (BY NUMBER)
Collagen scaffold with gallic acid-assisted crosslinking: exploring application in bone tissue engineering
Nattapak Darumas1, Panwa Promtep2, Tanapong Watchararot3, Atitaya Ruamwong3, Supansa Yodmuang3, Elizabeth Laird4, Lucy Bosworth4
1University of Liverpool, Liverpool - United Kingdom & Graduate Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, 2John Hopkins University, Baltimore (Maryland) - United States, 3Chulalongkorn University, Bangkok (Krung Thep) - Thailand, 4University of Liverpool, Liverpool - United Kingdom
Differentiating osteoblasts from Adipose-derived stem cells (ADSCs) is significantly more challenging than differentiating them from bone marrow-derived stem cells (BMSCs), primarily due to the higher adipogenic capacity of ADSCs. Enhancing osteoblast differentiation signals is one strategy for developing bone tissue from ADSCs. Gallic acid has been reported to clinically suppress adipocytes and to enhance osteoblastogenesis in mice BMSCs over the short term.
This study investigated the effect of incorporating gallic acid into collagen scaffolds during tissue development of ADSCs. Gallic acid, along with EDC/NHS coupling agents, was mixed with a collagen solution at 4°C and then lyophilized. The incorporation of gallic acid into the collagen scaffolds was confirmed using ssNMR, FTIR, and DSC techniques. The incorporated gallic acid showed a burst release in the first 24 hours, followed by continuous release up to 72 hours. The scaffold matrices degraded by 98.50±0.14% (w/w) during the 4-week biodegradation test.
The constituents of the osteogenic medium used in this study comprised dexamethasone, ascorbic acid, and β-glycerolphosphate. The cocktail stimuli were proven to cause the expression of an adipogenic regulator. ADSCs cultivated on the gallic acid-incorporated collagen scaffold showed significantly lower expression of PPARɣ but higher expression of COL1A1over 28 days. Histologically, glycosaminoglycans and oil droplets were absent in tissues developed on the gallic acid-incorporated scaffold, regardless of whether the tissue was treated with osteoblastic differentiation medium or general growth medium. Calcium particles were detected exclusively in the condition treated with osteoblastic differentiation medium.
In summary, gallic acid simultaneously limits adipogenic capacity via the PPARɣ -related pathway and enhances osteoblastogenesis signaling. Gallic acid plays an assistive role in bone tissue development; however, traditional osteoblastogenic stimuli remains mandatory. Gallic acid did not trigger differentiation into other ADSC lineages, neither adipogenesis nor chondrogenesis.
Innovative biomaterial-based strategy for systemic delivery of human mesangioblasts in an immunodeficient DMD mouse model
Somaya Amer1, Laricia Bragg1, Giulio Cossu1, Galli Francesco1
1Division of Cell Matrix Biology & Regenerative Medicine, FBMH, University of Manchester, UK, Manchester - United Kingdom
Systemic delivery of cell therapies in genetic disorders such as Duchenne Muscular Dystrophy (DMD) presents major challenges. we introduce a novel approach to systemic cell therapy utilizing a bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin fabricated via electrospinning can mimic ECM structure. This scaffold not only provides a substrate for cell-matrix attachment, supporting cell survival post-implantation, but also enhances long-term cell proliferation. Furthermore, our study demonstrated the general distribution, localization of cells, and dystrophin restoration in various muscles, including the diaphragm and heart, indicating sustained cell presence for 6-months in which cells slowly migrate from the scaffold to dystrophic muscle. Given that monotherapies have consistently demonstrated limited engraftment and functional improvement in clinical trials, we demonstrated that a combinatorial approach integrating both cell and gene therapy, via genetically corrected mesangioblasts expressing exon-skipping U7 snRNA, seeded onto the biomaterial scaffold had significant dystrophin restoration across dystrophic muscles. These findings underscore the potential of utilizing this bio-scaffold as a novel systemic delivery method for cell therapy in Duchenne muscular dystrophy.
Biomimetic and injectable aminated collagen-hyaluronic acid-glycosaminoglycan (aCol-HA-GAG) biomaterial scaffold for cell-based intervertebral disc (IVD) regeneration
Catherine Le Visage1, Chaaban Mansoor1, Yang Xingxing2, Cher Du-Hao1, Goel Tosmi1, Guicheux Jerome1, Fusellier Marion1, Chan Barbara P.2
1Nantes Universite, Nantes (Pays de la Loire) - France, 2The Chinese University of Hong Kong, Hong Kong
Background
Intervertebral disc (IVD) degeneration is a leading cause of chronic back pain, yet current treatments fail to restore its function. Here, we focused on a biomimetic scaffold that replicates the native disc matrix. This aminated collagen-hyaluronic acid (HA) -glycosaminoglycan (GAG) biomaterial was engineered to mimic the native ratio of native NP tissue. We investigated whether this biomaterial scaffold can support cell viability, proliferation, and differentiation. In a separate experiment, a method for evaluating scaffold localization within a disc was developed using an ex vivo bovine tissue.
Methods
Human adipose stromal stem cells (ADSCs) were mixed with the material and cultured for 28 days in hypoxic conditions. The projected surface area of the seeded scaffold was imaged every 3 days using a Leica Microscope over 28 days. The cells were cultured with transforming growth factor β-3 (TGF-β3) and growth differentiation factor-5 (GDF-5) to induce nucleopulpogenic differentiation. Cell viability, proliferation, metabolic activity, morphology, and differentiation were evaluated using CCK8, EdU, PicoGreen, and histological and imaging techniques at various time points, respectively. A micro-CT analysis was performed to visualize the material in ex vivo bovine discs. Samples were incubated with 3% phosphotungstic acid for 3 minutes before being injected into the IVD defect.
Results
Cell association with the collagen-HA-GAG biomaterial scaffold was successful. The CCK-8 assay showed increased metabolic activity, while EdU staining and Picogreen assay provided inconclusive results on proliferation due to interference from the biomaterial with the absorbance signal. The projected surface area initially presented a gradual decrease, indicating a contraction of the seeded material, as expected. Histological analysis with Alcian Blue staining revealed the presence of cells associated with the scaffold at all time points. The faint staining did not provide information regarding cell differentiation. Finally, Micro-CT imaging enabled a clear distinction between the scaffold and the surrounding bovine NP tissue.
Conclusion
The aminated collagen-HA-GAG scaffold supported cell viability during an in vitro differentiation protocol. Future work will focus on assessing specific markers for nucleopulpogenic differentiation and performing long-term studies in an ex vivo model.
The pomegranate theory: a systematic review on decellularization techniques for the development of tissue-engineered aortic scaffolds - SEMIT
Farida Alwakeel1, Rachel Duff2, Valerie Fiamavle3, Shakil Hussain4, Danni-Leigh Harrison5, Abdi Sahra6, Rachel Clough7
1HealthTech Research Centre in Cardiovascular and Respiratory Medicine. King's College London, London (London, City of) - United Kingdom, 2HealthTech Research Centre in Cardiovascular and Respiratory Medicine. King's College London, London (London, City of) - United Kingdom, 3Queen's University Belfast, Belfast - United Kingdom, 4City St. George's University of London, London (London, City of) - United Kingdom, 5Newcastle University, Newcastle upon Tyne - United Kingdom, 6King's College London, London (London, City of) - United Kingdom, 7Biomedical Engineering and Imaging Sciences. King's College London, London (London, City of) - United Kingdom
Aortic aneurysms affect approximately 35 million individuals worldwide, causing 153,927 deaths in 2021. Endovascular aortic repair (EVAR), the preferred approach for ruptured and unruptured aneurysms, involves inserting a synthetic stent graft into the aorta to reinforce weakened arterial walls and prevent rupture. However, metallic or polymeric graft materials cause biocompatibility complications such as heart failure, stent-migration, and endoleak. Tissue-engineered aortic grafts offer a promising alternative, replicating native aortic architecture to improve long-term outcomes through functional integration with the host tissue.
Currently, there is no standardized decellularization protocol for aortic tissue. This study systematically evaluates existing methods to advance biocompatible tissue-engineered grafts, comparing chemical, enzymatic, and physical approaches, including the emerging use of sonication, to identify the most effective strategy for maintaining extracellular matrix (ECM) integrity and preserving biomechanical properties.
A literature search was conducted using PubMed, Scopus, Embase, and IEEE Xplore databases from January 2000 to March 2025 following PRISMA guidelines. Eligible studies included original research on decellularization of healthy aortic tissue from large animal models. Study quality and risk of bias were assessed using an adapted QUIN tool.
Thirty-one studies met the inclusion criteria, most employing porcine models (n=26), with 6 performing in vivo implantation. Eighteen studies reported biomechanical assessments, including tensile (n=15), compression (n=3), and burst pressure (n=3) testing, and 21 quantified residual DNA content. Given the substantial heterogeneity in sample sizes, and intervention protocols, a comprehensive meta-analysis wasn’t feasible; however, sodium dodecyl sulphate (SDS, n=11), Triton X-100 (n=6), and sonication (n=10) were most frequently applied.
Multi-step detergent–enzymatic approaches incorporating sonication were most effective in removing cellular components while preserving ECM composition. However, no single protocol was identified as universally optimal. Conversely, chemical-only decellularization reduces tissue elasticity, compromising mechanical properties. Further validation of sonication-based decellularization is required, nonetheless it offers a promising foundation for functional aortic graft development.
Wireless nanobioelectronic systems for non-invasive modulation of cancer bioelectricity
Paola Sanjuan Alberte1, Catarina Jones1, Beatriz Simoes1, Diana Matias2, Luís Graça2, Frederico Castelo Ferreira1, Teresa Esteves1
1Institute for Bioengineering and Biosciences. Associação do Instituto Superior Técnico para a Investigação e Desenvolvimento (IST-ID), Lisboa - Portugal, 2Gulbenkian Institute for Molecular Medicine (GIMM), Lisboa - Portugal
Cancer's increasing prevalence highlights the pressing need for innovative treatment modalities. Recently, it has been established that cancer cells present abnormal bioelectrical properties, however, therapeutic strategies aimed at effectively interfering with cancer bioelectricity are still lacking. In this work, we explore novel nanobioelectronic systems consisting of a barium titanate core and a poly(3,4-ethylenedioxythiophene) shell (BTO@PEDOT NPs) (1). We hypothesise that the BTO@PEDOT NPs act as a nanoantenna, transducing a mechanical input provided by external ultrasound stimulation into an electrical output capable of interfering with the bioelectricity of two human breast cancer cell lines, MCF-7 and MDA-MB-231. Our results confirmed a reduction in cell viability in both cell lines, consisting of 31.05% and 24.03%, respectively, after US stimulation with the BTO@PEDOT NPs, compared to 94.45% of cell viability of of human mammary fibroblasts (HMF). Subsequently, evaluation was performed to elucidate the mechanisms involved behind this. ROS levels and intracellular Ca2+ concentrations were significantly increased in both cancer cell lines, highlighting the disruptive impact of BTO@PEDOT NPs on cancer cell bioelectricity. Furthermore, the analysis of the cell cycle revealed a marked <G1 population in the case of the MCF-7 and MDA-MB-231 indicating an increase in apoptotic and necrotic cells which was not evidenced in the case of the HMF cells. Preliminary in vivo evaluations also revealed a significant reduction in tumour size of mice treated with the BTO@PEDOT NPs. Overall, these results confirm the potential of nanobioelectronic systems as an emerging and promising strategy for cancer intervention.
1. Jones CF, Carvalho MS, Jain A, Rodriguez-Lejarraga P, Pires F, Morgado J, et al. Wireless Stimulation of Barium Titanate@ PEDOT Nanoparticles Toward Bioelectrical Modulation in Cancer. ACS applied materials & interfaces. 2025;17(6):8836-48.
Acknowledgements: This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., with dedicated funds from the project BIOMIMIC-CRC (2023.13896.PEX) and institutional funds from iBB (UIDB/04565/2020 and UIDP/04565/2020), and the Associate Laboratory i4HB (LA/P/0140/2020). This project also received financial support from “la Caixa” Foundation (ID 100010434) LCF/BQ/PI22/11910025.
Enhancing the therapeutic efficacy of freeze-dried stem cell extracellular vesicles in wound healing through hypoxia and 3D culture
Julia Monola1, Alisa Jokela1, Aleksi Kröger1, Elle Koivunotko1, Chris S. Pridgeon1, Riina Harjumäki1
1Faculty of Pharmacy. University of Helsinki, Helsinki (Southern Finland) - Finland
Introduction
Extracellular vesicles (EVs) have great potential for tissue regeneration that circumvent many of the drawbacks of stem cell therapies, including greater accessibility and decreased immunogenicity and cost. However, the development of standardised therapeutic EV production faces challenges related to consistent culture conditions, dosing efficacy, and compositional variability. To overcome these challenges, we generated stem cell-derived EVs under hypoxic conditions and stabilised them with freeze-drying in a biocompatible hydrogel, enabling controlled release for tissue regeneration applications.
Materials and methods
Human adipose-derived stem cells (hASCs) were cultured for 7 days under either normoxic (5% CO2) or hypoxic (5% CO2, 5% O2, 90% N2) conditions after one week hypoxic adaptation in 2D or 3D (ultra-low attachment (ULA) plates or nanofibrillated cellulose hydrogel). Conditioned media was purified by differential ultracentrifugation and EV yield was quantified by nanoparticle tracking analysis. The cells and EVs were characterised by total proteome analysis and qPCR. To improve stability and facilitate controlled release of EVs, samples were freeze-dried in hydrogel and evaluated with an in vitro scratch wound model.
Results
Hypoxic conditions and ULA culture improved MSC phenotype and increased EV yields compared to both normoxia and hydrogel culture. Surprisingly, EVs from 2D cultured hASCs outperformed those from 3D culture in the in vitro scratch wound assay. All systems showed clear differences in proteomic analyses highlighting the importance of optimised culturing conditions. By optimizing the hydrogel formulation, the release rate of EVs could be adjusted to the desired level while preserving their functionality.
Conclusions
We successfully developed a freeze-dried, biocompatible EV formulation with potential in wound healing applications, which will be studied further in vivo. The results of this study demonstrate the importance of culture conditions for EV production and are encouraging for future therapeutic applications.
Acknowledgements
Business Finland GeneCellNano flagship project, Cultural foundation, and MATRENA Doctoral school.
Engineering interstitial fluid pressure in a microfluidic device for in-vitro cell/tissue modelling
Pilar Alamán-Díez1, Silvia Ferrer-Royo1, Pablo Martín-Compaired1, Alejandra González-Loyola2, José Manuel García-Aznar1
1Multiscale in Mechanical and Biological Engineering (M2BE), Universidad de Zaragoza, Zaragoza - Spain, 2Aragon Health Research Institute (IISA), Zaragoza - Spain
Aim: The aim of this work was to establish a reliable method to apply controlled levels of interstitial fluid pressure (IFP) to 3D cell cultures. To achieve this, we designed and fabricated a syringe-holder assembly that enables the generation of defined pressure values through adjustable water columns using standard culture medium.
Methodology: The system consists of a 3D-printed modular structure designed to support syringes at adjustable heights, allowing control of hydrostatic pressure applied to microfluidic devices. The assembly includes a stable base for chip fixation and a set of column modules that can be combined to achieve different height configurations. To evaluate the system, pancreatic tumor spheroids embedded in collagen I hydrogels were cultured within the microfluidic device under increasing IFP levels. In addition, CAR-T and T cells were co-cultured with these spheroids under IFP.
Results: The 3D-printed structure enabled stable and reproducible generation of controlled IFP within culture chambers. Pressure was maintained over several days without observable fluid loss or structural deformation of the setup. The modular design allows parallelization of experiments and easy adjustment of pressure levels by varying column height. Validation experiments using pancreatic tumor spheroids demonstrated that sustained IFP exposure promoted enhanced spheroid growth compared to control conditions, consistent with pressure-driven tumor progression reported in vivo. Moreover, CAR-T cells efficiently eliminated tumor spheroids under atmospheric pressure, but their cytotoxic activity was reduced under IFP conditions.
Conclusions: We successfully developed and validated a system capable of applying and maintaining IFP in a microfluidic device. This platform provides a simple, reproducible, and low-cost approach to reproduce a key mechanical feature of the tumor microenvironment. Preliminary biological validation demonstrates its potential to study pressure-driven tumor behavior and immunotherapy treatment.
This work is part of a project that has received funding from the European Research Council (No:101018587 and No:101248308)
Retinoic acid-mediated differentiation and functional maturation of glandular organoids
Ya-Chuan Hsiao1, Jyue-Shi Yang2, Tsung-Lin Yang3
1Zhongxing Branch, Taipei City Hospital, Taipei (Mountain) - Taiwan, 2Massachusetts Institute of Technology, Cambridge (Massachusetts) - United States, 3Otolaryngology. National Taiwan University, Taipei (Mountain) - Taiwan
Tissue-specific organoids offer a robust model for investigating organogenesis, tissue regeneration, and disease. The salivary glands are vital for oral health, aiding digestion and hygiene through saliva production. However, their limited regenerative capacity poses a significant challenge in addressing dysfunction. This study systematically explores the regulatory impact of Retinoic Acid (RA), a critical derivative of vitamin A known for its role in embryonic development, on the differentiation potential of salivary gland organoids. We established foundational salivary gland organoids by isolating and culturing tissue-specific stem cells in a three-dimensional environment. Rigorous verification confirmed the organoids' capacity for self-organization and their morphological and functional resemblance to native glands. Our investigation then focused on the effects of RA treatment on their differentiation and functional specialization. We meticulously evaluated RA's influence on the formation of essential acinar and ductal structures, as well as their ability to synthesize saliva-related proteins. Furthermore, we delved into the molecular mechanisms underlying RA-mediated differentiation, conducting comprehensive gene expression analyses. These analyses aimed to unveil RA's effects on cell fate determination, proliferation, and morphogenesis within the organoids. These findings promise to yield valuable mechanistic insights into the regulatory mechanisms governing salivary gland organoid development, underscoring RA's potential as a powerful guide for tissue differentiation and maturation. Ultimately, the results obtained may pave the way for innovative regenerative therapies designed to restore function to dysfunctional salivary glands.
Application of biphasic dual-layer biomaterials for enhancing oral mucosal regeneration
Jyue-Shi Yang1, Ya-Chuan Hsiao2, Tsung-Lin Yang3
1Massachusetts Institute of Technology, Cambridge (Massachusetts) - United States, 2Ophthalmology. Zhongxing Branch, Taipei City Hospital, Taipei (Mountain) - Taiwan, 3Otolaryngology. National Taiwan University, Taipei (Mountain) - Taiwan
Oral mucosal wounds present significant healing challenges due to constant exposure to saliva, mechanical forces, and microbes, which conventional single-layer dressings cannot adequately address, often resulting in compromised healing efficiency. To overcome this, we developed a biphasic dual-layer biomaterial scaffold specifically engineered for simultaneous submucosal tissue integration and cavity-side moisture regulation. This asymmetric scaffold comprises a submucosal-facing layer designed to facilitate epithelial cell adhesion, proliferation, and migration into the biocompatible matrix, and a cavity-facing layer of hydrogel optimized to absorb excess salivary moisture, maintain optimal wound hydration, and provide mechanical protection. Physicochemical characterization confirmed that the system exhibited favorable porosity, swelling, high water absorption, and sustained integrity in simulated salivary conditions. In a mouse wound model, the dual-layer system significantly accelerated wound closure compared to individual layer or no-treatment controls. Histological analysis revealed enhanced re-epithelialization, increased granulation tissue formation, reduced inflammation, and improved collagen deposition, with immunohistochemical staining confirming the upregulated expression of regenerative markers. This biphasic design effectively meets the bilateral functional demands of oral mucosal wound healing by simultaneously promoting tissue regeneration at the interface and managing moisture dynamics at the surface, demonstrating significant therapeutic potential for managing various oral mucosal defects.
Computer-guided navigation to advance translational spine models - SEMIT
Bonilla Andres1, Nicole Erben2, Kristen Sack2, Howard Seim2, Jeremiah Easley1
1Clinical Sciences. Translational Medicine Institute, Fort Collins (Colorado) - United States, 2Translational Medicine Institute, Fort Collins (Colorado) - United States
Introduction:
Large animal spine models, such as sheep, goats, and pigs, are critical for advancing regenerative and translational spine research. Their anatomical and biomechanical similarity to the human spine makes them invaluable for developing and testing spinal fusion and intervertebral disc regeneration strategies. However, accurate pedicle screw placement in these models remains technically challenging due to anatomical variability and smaller pedicle dimensions, potentially compromising fixation stability and study reproducibility. Surgical navigation, widely validated in human spine surgery, offers an opportunity to enhance precision and safety in preclinical studies. This work evaluates the feasibility and accuracy of navigation-guided pedicle screw placement across three large animal species commonly used in spine research.
Materials and Methods:
Navigation-guided pedicle screw placement was performed on cadaveric lumbar spines from ovine, caprine, and porcine specimens. Two novice operators each placed five screws per specimen (N = 30) using an O-arm imaging system integrated with the StealthStation 8 platform (Medtronic, Minneapolis, MN). Polyaxial screws (3.5 × 30 mm) were inserted following preoperative planning. Planning and drilling times were recorded. Screw accuracy was assessed via postoperative CT using the Gertzbein–Robbins grading system.
Results:
Navigation enabled successful and accurate screw placement in all species. Planning and drilling times decreased progressively, indicating rapid adaptation to the technology. Mean accuracy scores were high across species (ovine 0.1 ± 0.3, goat 0.2 ± 0.4, pig 0.2 ± 0.4), with only minor cortical breaches observed. Anatomical differences influenced procedural efficiency, with porcine spines showing faster planning and drilling times.
Conclusion:
This work demonstrates that integrating surgical navigation into large animal spine models not only enhances procedural precision but also represents a critical step toward bridging preclinical and clinical spine research. By improving reproducibility, safety, and anatomical accuracy, navigation-guided techniques can accelerate the translation of regenerative therapies from bench to bedside.
Lactic acid as a therapeutic driver of cardiomyocyte reprogramming and cardiac regeneration
Elisabeth Engel1, Marina Cler2, Soledad Perez Amodio2, Elena Martinez Fraiz3, Ignasi Barba4, Freddy G Ganse5, Marta Consegal6, Cesare M Terracciano7, Antonio Rodriguez-Sinovas5
1Biomaterials for Regenerative Therapies. Universitat Politècnica de Catalunya, Barcelona - Spain, 2Biomaterials for regenerative therapies. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, 3Biomimetic Systems for Cell Engineering. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, 4Vall d’Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Barcelona - Spain, 5Cardiovascular Diseases Research Group. Vall d’Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Barcelona - Spain, 6Cardiovascular diseases research group. Vall d’Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Barcelona - Spain, 7National Heart & Lung Institute, Imperial College London, London (London, City of) - United Kingdom
Cardiac regeneration after an infarction has been for decades a major challenge for scientists and clinicians. Following injury, the heart exhibits a gradual regenerative capacity, potentially replacing damaged cardiac myocytes (CMs). This offers hope for treating myocardial infarction (MI) (1,2). Recent research suggests that manipulating cellular plasticity could be a promising strategy for cardiac regeneration (3). Studies in diverse organisms, from lower vertebrates to mammals, indicate that CM turnover often originates from existing CMs, rather than cardiac stem cells (4). Therefore, the focus has shifted to understanding the mechanisms regulating CM proliferation and how environmental factors can influence CM phenotype. In this work we demonstrate that a metabolic shift in the tissue can promote a phenotype change towards a proliferative stage.
In isolated mouse hearts subjected to ischemia-reperfusion, administering 20 mmol/L of L-lactic acid during the first 15 minutes, reduced infarct size by 23%. Inhibiting monocarboxylate transporter 1 (MCT1) reduced L-lactic acid’s protective effect to that of acidic Krebs. Metabolic analysis revealed significant alterations in pyruvate metabolism, fatty acid biosynthesis, and gluconeogenesis, indicating a metabolic shift with L-lactic acid treatment. Moreover, electrically stimulated human Living Myocardial Slices (LMS) treated with L-lactic acid for 48h, exhibited improved contractility, upregulation of structural and functional CMs components, stemness-related markers (Oct4, GATA4, LIN28A), introduction of the cell cycle (CDC7, CDC25A, CDC25C), and pro-angiogenic proteins.
The presented results support a cardioprotective role for L-lactic acid in both short- and long-term contexts, mediated in part by its uptake through the MCT1 transporter, induction of metabolic reprogramming, and gene expression modulation.
1. Bergmann O, et al. DOI: 10.1126/science.1164680; 2. Senyo SE, et al. DOI: 10.1038/nature11682; 3. Gong R, et al. DOI: 10.1038/s41392-020-00413-2; 4. Sadek H, Olson EN. DOI: 10.1016/j.stem.2019.12.004
Aknowledgements: Spanish National Research Agency (BIOCARDIO ref. RTI2018-096320-B-C21) and is part of the PDC2022-133755-I00 and by “ERDF A way of making Europe” and European Union Next Generation EU. The prize “ICREA Academia” for excellence in research. “La Caixa” Foundation fellowship LCF/BQ/DR19/11740025. EMBO Scientific Exchange Grant (ID 9684). LMS work by NC3Rs project grant NC/T001488/1.
Comparison of methods for human adipose-derived stem cells (hADSCs) differentiation into neural crest cells (NCCs)
Marć Małgorzata A.1, Marta Cadenas-Martin1, Ana I. Martin-Gonzalez1, Maria P De Miguel1
1Cellular Engineering Research Group. The Health Research Institute of La Paz University Hospital, IdiPAZ, Madrid - Spain
The aim of the studies was to compare the effectiveness of two independent methods of human adipose-derived stem cells (hADSCs) differentiation into neural crest cells (NCCs) for their subsequent use in ophthalmological transplants.
hADSCs were cultured in two culture media (Ali et al., PMID: 29847650 and Wagoner et al., PMID: 29685994) to induce their differentiation into NCCs. Furthermore, the culture plates were coated with laminin or vitronectin, to further enhance efficiency of differentiation. Cells were cultured in Ali medium for 11 days and 18 days in the Wagoner medium, following preliminary results. Specific NCCs gene expression analysis was performed by qRT-PCR (quantitative reverse transcription polymerase chain reaction) for genes SOX10, CXCR4, Noggin, PAX3, NGFR, Nestin, and immunofluorescence and confocal microscopy for NCCs markers Notch1, CXCR4 and Nestin. The experiments were repeated three times and analysed using one-way ANOVA with the Bonferroni’s post hoc test.
The obtained results allowed us to select an appropriate protocol for future research. Both protocols yielded NCCs, but with different levels of NCCs gene expression. Experiments conducted in Ali medium produced faster results and better cell growth rate. However, the level of gene expression characteristic for NCCs was significantly higher in the Wagoner medium. These results were confirmed by immunofluorescence experiments.
Obtained results support the strategy described by Wagoner. Although this procedure is more complex, longer, and demanding, it achieved higher levels of NCCs-characteristic gene expression.
Supported by: Biomimetic Cornea fabrication by 3D printing and adult Mesenchymal Stem Cell directed differentiation (MIMECOR), Marie Skłodowska-Curie Actions (MSCA),
HORIZON-MSCA-2024-PF-01-01 - MSCA Postdoctoral Fellowships 2024, Grant agreement ID: 101203685, DOI 10.3030/101203685, and the Ministry of Science and Innovation, Strategic Health Action, Instituto de Salud Carlos III (ISCIII), Proyectos de I+D+I en Salud (PI23/00207) and co-funded by the European Union.
Biofabrication based on combined 3D bioprinting and in-situ mechanical fiber spinning
Leonid Ionov
de University of Bayreuth, Bayreuth - Germany
Biofabrication is an emerging field of engineering aimed at creating tissues and tissue-like structures. A key technology in biofabrication is 3D bioprinting, which utilizes methods of precise layer-by-layer deposition of cell-containing bioingredients to form active 3D structures. While 3D bioprinting allows for the creation of some biologically relevant shapes and structures, its reliance on the use of isotropic hydrogels limits the mechanical properties of the formed structures, making them unsuitable for tissues such as cartilage, bone, and skin. The hydrogels themselves also lack the complex hierarchical ECM structure of native tissue. Combining 3D bioprinting with fiber fabrication can produce 3D hybrid structures with improved mechanical and biological properties that more closely mimic the architecture of native tissue at different scales. There are attempts to combine 3D bioprinting and electrospinning/electrospinning which, however, are limited in terms of how fibers are produced, how they are deposited, and what types of structures can be obtained.
We have developed a fundamentally new approach that combines mechanical fiber spinning and 3D bioprinting, offering significant advantages over methods such as electrospinning and electrospinning. Key advantages include (i) ultra-fast deposition of continuous fibers, (ii) deposition of freestanding fibers, and (iii) precise control of fiber direction. These fibers provide strong support for the structures created, eliminating the need for crosslinking hydrogels. This ensures cell motility, facilitating efficient cell migration and tissue formation.
Kitana V., Levario-Diaz V., Cavalcanti-Adam EA, Ionov L. Biofabrication of bioink-nanofiber composite structures: Influence of rheological properties of bioinks and on bioprinting and cell-cell interaction with aligned sensor nanofibers, Advanced Healthcare Materials 2024, 13, 2303343.
In vivo evaluation of ‘Skin in a Syringe’: evaluation of an injectable dermal construct in a porcine wound model
Rozalin Shamasha1, Sneha Kollenchery Ramanathan2, Nina Reustle2, Smilla Frenger3, Emilie Källåker3, Annika Starkenberg3, Jonathan Rakar1, Gunnar Kratz4, Daniel Aili2, Johan Junker1
1Center for Disaster Medicine and Traumatology; Experimental Plastic Surgery, Department of Biomedical and Clinical Sciences. Linköping University, LINKÖPING - Sweden, 2Laboratory of Molecular Materials, Division of Biophysics and Bioengineering, Department of Physics, Chemistry and Biology. Linköping University, LINKÖPING - Sweden, 3Experimental Plastic Surgery, Department of Biomedical and Clinical Sciences. Linköping University, LINKÖPING - Sweden, 4Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Hand and Plastic Surgery, Linköping University Hospital, LINKÖPING - Sweden
Severe burn injuries and full-thickness skin defects remain a major clinical challenge due to limited donor sites and the risk of scarring. This study aimed to evaluate the “Skin in a Syringe” concept toward clinical translation by evaluating an injectable, fibroblast-laden granular hydrogel, termed µInk, for dermal regeneration in a porcine wound model. The objective was to assess its biocompatibility, biodegradation, and ability to promote extracellular matrix (ECM) remodeling in vivo.
µInk was prepared by dispersing porous gelatin microcarriers (PGMs) within a hyaluronic acid–polyethylene glycol (HA-PEG) hydrogel network, forming a shear-thinning, self-healing construct suitable for extrusion or direct wound injection. Autologous dermal fibroblasts, isolated and expanded from two adult pigs, were seeded on PGMs for 72 h prior to transplantation. Each animal received twenty 2 cm full-thickness wounds, treated with µInk either with or without fibroblasts. Biopsies were collected after 2, 4, 8, and 12 weeks for histological, immunohistochemical, and gene expression analyses. Complementary in vitro experiments assessed fibroblast attachment and ECM production on PGMs.
In vitro, fibroblasts readily adhered to PGMs and synthesized key ECM components, including collagen I/III and elastin. In vivo, PGMs fully degraded within four weeks, demonstrating scaffold biocompatibility and remodeling. Wounds treated with fibroblast-laden µInk showed increased collagen deposition and formation of elastin clusters surrounding the former PGMs, suggesting active dermal regeneration rather than fibrotic repair.
The µInk platform supports fibroblast proliferation, ECM deposition, and in vivo tissue integration, confirming its potential as an injectable biofabricated treatment for dermal reconstruction. These findings strengthen the translational relevance of “Skin in a Syringe” and establish a foundation for large-scale preclinical evaluation.
An innovative insert for muscle tissue engineering – potential applications in drug discovery and safety assessment
Livia J Rocha Dos Santos1, Chris Gabbott1, Janelle Tarum2, Yang Wei1, John Hunt1, Hans Degens3
1Nottingham Trent University, Nottingham - United Kingdom, 2Loughborough University, Loughborough (Leicestershire) - United Kingdom, 3Manchester Metropolitan University, Manchester (Greater Manchester) - United Kingdom
Background: Cell culture inserts are small devices used in combination with carrier plates which instruct cells to organise in 3D, enabling the creation of physiologically relevant in vitro tissue models. Solutions to generate 3D muscle models at scale are vitally important for drug discoveries and toxicity testing, but are lacking. This study aims to assess an innovative insert patented by our lab (PCT/GB2023/053038) in generating muscle tissue in 3D in a higher-thoughput manner.
Methods: C2C12 myoblasts were suspended in a collagen/Matrigel™ mix, cast into the insert’s central channel, and cultured for two days in growth medium (DMEM, 10% FBS, 1% antibiotics). After two days, the tissue engineered muscle (TEM) either remained in the channel (control) or was transferred to pillars for maturation under static tension. At this stage, growth medium was replaced with differentiation medium (DMEM, 2% FBS, 1% antibiotics). Contractility was measured using a force transducer (403A AURORA) and morphology analysed by confocal microscopy on days 7, 14, and 21.
Results: TEM contractility was measured in response to electric stimulation. The tetanic forces were significantly higher in TEM cultured under tension compared to the control. Specifically, TEM under tension at day 7 exhibited a tetanic force of 63±8 µN vs 29±6 µN in the control (p=0.001), and at day 21, 130±20 µN vs 90±14 µN in the control (p<0.0001). Tetanic forces also increased significantly between day 7 and 21 in the TEM cultured under tension (p<0.0001). Morphological analysis revealed the presence of aligned cells in the TEM.
Conclusions: The proprietary cell culture inserts are effective in providing muscle cells morphogenic guidance and enanching maturation as evidenced by cellular alignment and improved functional contractility. This platform offers a robust, higher-throughput alternative to traditional models, with strong potential to accelerate drug discoveries, improve patients outcomes and reduce animals in pre-clincial trials.
Modeling duchenne muscular dystrophy: development of a 3D printed pathological muscle-on-chip
Léna Villerabel1, Maxime Mauviel2, Blaise Hebert1, Jean-Marie Chassot3, Olivier Thouvenin3, Mélina Motard4, Alba Marcellan4, Carole Aimé2, Christophe Hélary1
1Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Sorbonne Université, CNRS, Paris (Ile-de-France) - France, 2Chimie Physique et Chimie du Vivant (CPCV), Département de Chimie, Ecole Normale Supérieure, Université PSL, Sorbonne Université, CNRS, Paris (Ile-de-France) - France, 3Institut Langevin, ESPCI Paris, Université PSL, CNRS, Paris (Ile-de-France) - France, 4Sciences et Ingénierie de la Matière Molle (SIMM), ESPCI Paris, Université PSL, Sorbonne Université, CNRS, Paris (Ile-de-France) - France
Duchenne muscular dystrophy (DMD) is a genetic disease causing muscle degeneration due to absence of dystrophin. Consequently, the muscle extracellular matrix (ECM) becomes fibrotic, with excessive type I collagen replacing functional muscle tissue, leading to increased stiffness, reduced nutrient and oxygen perfusion, and impaired muscle contraction. Existing DMD models remain inadequate, as 2D cell cultures lack 3D structure and cell-ECM interactions, and animal models present species differences. To overcome these limitations, we developed a 3D printed collagen-based muscle ECM reproducing both healthy and fibrotic environments, integrated into a microfluidic chip. A multi-layered pattern was used, with intrinsic porosity controlled by adjusting the number of collagen filaments per layer. Porosity was quantified using optical coherence tomography (OCT). Increasing layer density reduced porosity from 6% to 2%, increasing the perfusion time from 10 min to 20 min, thus mimicking fibrotic muscle perfusion. Stiffness was increased by collagen chemical crosslinking using EDC/NHS reagents. By modulating chemicals concentrations, the mechanical properties were progressively increased from ∼20 kPa to ∼100 kPa as evidenced by tensile testing, replicating progressive stages of fibrosis and emphasizing disease progression. Tensile testing also showed that porosity did not significantly influence Young’s modulus, confirming that this parameter could be modulated independently of stiffness. The impact of reduced porosity was then evaluated after 3D cellularization into two 600 µm-diameter channels, combining immunofluorescence to observe cell morphology and dynamic OCT that provides label-free imaging of cell metabolism. Low porosity decreased C2C12 myoblasts metabolic activity (from 0.6-5 Hz to 0-0.3 Hz range), while cell morphology remained similar in both healthy and fibrotic conditions. These results highlight the significant impact of reduced perfusion on cell metabolism in 3D muscle ECM. This model accurately replicates the decreased porosity and increased stiffness of fibrotic muscle ECM, providing a tunable platform to study the impact of progressive fibrosis.
Biomimetic bone calcium phosphate-based scaffolds fabricated via ceramic vat polymerization: effect of porosity, sintering temperature, mineralogical phases and elements on the osteogenic potential
Antonia Ressler1, Roope Ohlsbom2, Virginia Alessandra Gobbo1, Markus Hannula3, Katharina Keck2, Toni-Karri Pakarinen4, Mehdi Mohammadi5, Jari Hyttinen3, Jonathan Massera3, Martin Schwentenwein5, Erkka J. Frankberg1, Erkki Levänen1, Arjen Gebraad2, Susanna Miettinen2
1Faculty of Engineering and Natural Sciences. Tampere University, Tampere (Southern Finland) - Finland, 2Faculty of Medicine and Health Technology, Tays Research Services. Tampere University, Tampere (Southern Finland) - Finland, 3Faculty of Medicine and Health Technology. Tampere University, Tampere (Southern Finland) - Finland, 4Tampere University Hospital. Tampere University, Tampere (Southern Finland) - Finland, 5Lithoz GmbH, Vienna (Wien) - Austria
In response to the growing demand for novel approaches in bone repair, scaffolds that mimic natural bone microstructure and mineralogical composition were developed using a cutting-edge ceramic vat photopolymerization method. Obtained scaffolds were complex and novel, pushing boundaries of possibilities in bone tissue engineering. Due to varying reported results regarding appropriate microstructural characteristics, this study aimed to clarify the optimal pore size distribution and porosity for an efficient osteogenic response. Scaffolds based on hydroxyapatite both support new bone formation by osteoblasts and can be resorbed by osteoclasts. An average pore size of ∼400 µm and porosity of 45.61% showed the best mechanical properties and osteogenic response, allowing cell penetration, and supporting cell-cell interactions and the differentiation process compared to scaffold with higher and lower pore size distribution and porosity. When Sr,Mg,Zn-substituted hydroxyapatite is used for scaffold fabrication, the required high sintering temperatures lead to the transformation of hydroxyapatite into β-tricalcium phosphate, a common calcium phosphate used in bone tissue engineering. However, the new mineralogical phase results in different surface properties that do not support appropriate cell attachment on scaffolds with higher negative surface charge. This work emphasizes the potential of ceramic vat photopolymerization in the development of biomimetic scaffolds that mimic natural bone tissue and provides guidelines on which microstructural characteristics are appropriate for efficient bone regeneration. This study also raises new questions regarding cell attachment on β-tricalcium phosphate-based scaffolds, which are currently under exploration. Study is a result from a HORIZON-MSCA- AffordBoneS project entitled “Development of personalized and affordable multi-substituted calcium phosphate based scaffolds for bone augmentation applications” that was done in collaboration with international companies Lithoz GmbH and Planmeca Oy, leaders in additive manufacturing and medical technology. Through joint efforts, project participants explored innovative solutions and new pathways for bone tissue regeneration, aiming to address unresolved challenges in the field.
3D-Printed scaffolds functionalized with extracellular matrix for bone regeneration
Thaís Amaral França1, Gabriela Morais Costa2, Janaina De Andrea Dernowsek2, Maria Carolina Coelho3, Sayuri Poli Suguimoto3, Karina Fittipaldi Bombonato-Prado3, Sônia Maria Malmonge1
1Postgraduate Program in Biomedical engineering. Federal University of ABC (UFABC), São Bernardo do Campo (Sao Paulo) - Brazil, 2Quantis Biotechnology, Sao Paulo - Brazil, 3Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto. University of São Paulo, Ribeirão Preto (Sao Paulo) - Brazil
Purpose/Objectives: Bone tissue engineering requires scaffolds with controlled architecture, surface bioactivity, and osteogenic performance. This study evaluated how solvent selection (chloroform or 2,2,2-trifluoroethanol (TFE)) affects the structural quality, cell adhesion, and biological activity of poly(ε-caprolactone) (PCL)/β-tricalcium phosphate (β-TCP) scaffolds fabricated by solvent-evaporation-based 3D printing. The study also assessed whether bioactivation with QMatrix, an extracellular matrix (ECM) derived from biofabricated dermal tissue, could enhance biological activity compared to rat tail collagen (RTC).
Methodology: PCL/β-TCP scaffolds were printed using chloroform or TFE. Morphology, filament fidelity, pore geometry, and qualitative cell adhesion were assessed by scanning electron microscopy (SEM). Chemical composition was evaluated by Fourier-transform infrared spectroscopy (FTIR) to confirm composite formation, solvent removal, and the absence of chemical bonding between PCL and β-TCP. Chloroform-printed scaffolds were bioactivated with RTC or QMatrix and seeded with MC3T3-E1 preosteoblasts (n=5). Cell viability (MTT, days 7–10), alkaline phosphatase (ALP, days 7–10), and mineralization (Alizarin Red, day 17) were quantified (ANOVA, p<0.05).
Results: SEM showed that chloroform produced regular filaments and consistent pores, whereas TFE caused sagging, fusion defects, and irregular porosity. Adhesion analysis revealed reduced cell spreading on chloroform scaffolds, while TFE structures supported more pronounced spreading and ECM deposition. FTIR confirmed characteristic PCL and β-TCP bands, no new peaks indicating chemical interaction, and full solvent removal. In biological assays, QMatrix increased viability at 7 and 10 days (p<0.05), and both RTC and QMatrix improved viability at day 10. ALP activity was highest for RTC at day 10 (p<0.0001), whereas mineralization was reduced for RTC (p<0.01). QMatrix showed mineral deposition comparable to control and higher than RTC.
Conclusion: Solvent choice strongly influenced scaffold architecture and adhesion: TFE produced more biologically favorable surfaces but lower geometric fidelity, while chloroform ensured structural precision. QMatrix enhanced adhesion, viability, and balanced mineralization more effectively than RTC, supporting its potential as a bioactivation strategy for synthetic bone scaffolds.
East meets West in maxillofacial surgery. Andrographolide boost immune response during the postoperative period
Lysann Michaela Kroschwald1, Heike Meissner1, Jan Bernhard Matschke1, Claudia Dietze2, Ina Prade2, Christian Rotsch3, Thomas Töppel3, Michael Werner3, Guenter Lauer1
1Department of Oral and Maxillofacial Surgery. University Hospital “Carl Gustav Carus” at Technische Universität Dresden, Dresden (Sachsen) - Germany, 2FILK Freiberg Insitute gGmbH, Freiberg (Sachsen) - Germany, 3Fraunhofer IWU, Dresden (Sachsen) - Germany
Objectives
Maxillofacial surgery is a complex field that requires innovative solutions to improve patient outcomes. Frequent post-op complications are partly caused by compromised innate immune responses. Combining traditional Eastern and modern Western medicine may create solutions. Andrographolide (AGP), a natural diterpenoid found in Andrographis paniculata, is used in traditional Asian medicine to treat fever, inflammation and infections. Its immunomodulatory, anti-inflammatory, antimicrobial, antiviral, anticancer and antioxidant properties may facilitate recovery after surgery.
Methods
Human osteoblast cells were cultivated and exposed to AGP, after which its biocompatibility was evaluated using a cytotoxicity assay. The release behavior and half-life of AGP were investigated using HPLC. ELISA and qPCR were employed to analyze the production and gene expression of antimicrobial peptides (AMPs), including human beta defensins 1–3 (hBD1–3) and LL-37. The antimicrobial effectiveness has been confirmed. Additionally, the expression of pro-inflammatory cytokines and differentiation markers for bone healing were investigated.
Results
The outcomes demonstrate that andrographolide is biocompatible at concentrations of up to 20 µM, confirming its potential application in biological systems. The release behavior and half-life period are influenced by several factors. Our in vitro results show an andrographolide half-life period of 24 hours, with the most potent effect occurring within the initial 48 hours after surgery. Stimulation with andrographolide in osteoblasts leads to AMP synthesis at a rate ten times higher than in untreated controls. Similar findings were observed for cytokines and markers of bone healing. This was confirmed by qPCR analyses and immunofluorescence staining, particularly for human beta defensins.
Conclusions and outlook
Integrating AGP into maxillofacial surgery could provide new treatment options that combine traditional Eastern medicine with Western technology, for example by using AGP as a coating for osteosynthesis plates or screws to stimulate the immune system, speed up healing and reduce the risk of infection after surgery.
Dual 3D printing of dense collagen and Laponite® to increase the cell-to-matrix fraction in muscle ECM models
Blaise Hebert1, Léna Villerabel1, Maxime Mauviel2, Carole Aimé2, Christophe Hélary1
1Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Sorbonne Université, CNRS, Paris (Ile-de-France) - France, 2Chimie Physique et Chimie du Vivant (CPCV), Département de Chimie, Ecole Normale Supérieure, PSL University, Sorbonne Université, CNRS, Paris (Ile-de-France) - France
Skeletal muscle consists of hierarchically organized fiber bundles embedded in a connective tissue network that ensures mechanical integrity and coordinated force transmission. This extracellular matrix (ECM), representing 10–20% of total muscle volume, combines anisotropic collagen organization, a stiffness of ∼15 kPa, and dense vascularization. Reproducing such structural and physico-chemical complexity in vitro remains a major challenge.
Three-dimensional (3D) printing of dense type I collagen provides a promising strategy to recreate the anisotropic and fibrillar architecture of native muscle ECM. However, forming internal channels dedicated to cell colonization without damaging the matrix remains challenging. Needle insertion has been used to generate channels but offers limited channel-to-matrix ratios and often disrupts scaffold integrity.
In this study, we aimed to increase the number and the size of channels dedicated to myoblast culture within biomimetic muscle matrices. For this purpose, dual-extrusion 3D printing of dense collagen and a sacrificial matrix was used. Several fugitive inks were tested such as Pluronic F-127, Laponite® and gelatin. Dual printing using gelatin by the FRESH method induced compaction of matrices. Pluronic-based inks were unstable under fibrillation conditions, leading to channel collapsing, while Laponite®/Pluronic composites improved stability but were difficult to handle. In contrast, pure Laponite® was easy to prepare, printable, stable in ionic media, flushable by distilled water, Alamar blue assay showed no cytotoxicity on C2C12 myoblasts after Laponite removal. The ultrastructure and fibrillary organization was not modified either.
Channels of different diameters (300 µm, 600 µm, and 1 mm) and layouts were printed to evaluate structural fidelity. Micro-CT imaging confirmed excellent printing fidelity and structural reproducibility. C2C12 cultured within matrices proliferated and differentiated in all conditions. Finally, dense collagen matrices of 8 mm diameter containing up to nine channels were synthesized, thereby mimicking the native-like muscle ECM architecture
Label-free in vitro modelling of nanoparticle and microorganism dynamics at cell surfaces
Genevieve Schleyer1, Eann A. Patterson2, Judith M. Curran1
1Department of Materials, Design and Manufacturing Engineering. University of Liverpool, Liverpool - United Kingdom, 2Department of Mechanical and Aerospace Engineering. University of Liverpool, Liverpool - United Kingdom
The development of biomaterials and antimicrobial technologies requires tools that can capture how nano- to microscale entities interact with complex cell environments. To design more effective materials at the cellular level, it is essential to investigate how particles move through cell-type-specific environments. Equally, understanding how microorganisms, such as bacteria, approach and colonise host surfaces is critical for advancing next-generation antimicrobial strategies. Progress has been limited by the lack of accessible techniques capable of visualising single entities in real time without fluorescent labels. There is a need for simple, label-free tracking methods that can directly quantify these interactions, advancing our understanding of the forces and mechanisms driving dynamics at the nano- and microscale.
We present a real-time optical tracking platform based on caustic phenomena, which converts a standard inverted microscope into a sensitive nanoparticle tracking system [1]. This approach amplifies optical signatures several orders of magnitude larger than the actual particles, enabling label-free detection and trajectory tracking. Positively and negatively charged gold nanoparticles (10–100 nm) were tracked up to 100 μm from human skin (HaCaT) and mesenchymal stem cell layers. Quantitative parameters, including diffusion coefficients and convex hull areas, were extracted from trajectories, and the method was extended to study bacterial motion within these cell models.
Nanoparticle diffusion was influenced by proximity to cell monolayers, revealing distinct zonal diffusion behaviour. Variations in diffusion coefficients and convex hull areas reflected the type of cell layer, indicating the influence of structural and biochemical cell-specific properties.
The caustics-based tracking technique supports development of detailed diffusion models describing how local cellular environments modulate nanoparticle and bacterial dynamics, providing a versatile, label-free platform for preclinical research in nanomedicine, drug delivery, and antimicrobial development.
1.Patterson, E. A., & Whelan, M. P. (2008). Tracking nanoparticles in an optical microscope using caustics. Nanotechnology, 19, 105502.
MSC-released extracellular NAD+ reprograms ischemic senescence in cardiac fibroblasts
Brent Bijonowski1, Nicholas Kurniawan2
1International Iberian Nanotechnology Laboratory, Braga - Portugal, 2Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
Following myocardial infarction, cell death and stress are abundant in and around the infarct injury. Cells found within this border region have been shown to undergo a transition to senescence following infarction. Adipose-derived mesenchymal stem cells (ASCs) exhibit a promising ability to regenerate various injury sites, particularly those affected by ischemic injury, due to their secretion of regenerative, angiogenic and anti-inflammatory cytokines. However, the role of ASCs in mitigating senescence induction in these injuries remains underexplored. Herein, we will show that nicotinamide adenine dinucleotide released by ASC aggregates is sufficient to reduce the induction of senescence in cardiac fibroblasts following ischemic injury. Specifically, co-culture with ASC aggregates resulted in reduced galactosidase activity and improved mitochondrial function and morphology following ischemic injury. These findings highlight both the importance of senescence post-ischemic injury and how adipose-derived mesenchymal stem cells can modify their environment through redox rebalancing.
Formation of cortical actin-based fence is critical for mesenchymal stem cell stemness in self-assembled aggregates
Brent Bijonowski1, Sarah Pragnere2, Nicholas Kurniawan3
1International Iberian Nanotechnology Laboratory, Braga - Portugal, 2Vascular Dysfunction and Hemostasis. École des mines de Saint-Etienne, SAINT-ETIENNE (Pays de la Loire) - France, 3Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
Self-assembly during aggregation induces multiple changes in the cells it comprises. This can be seen in changes to the metabolism, secretory profile and cytoskeletal structure. Many studies have observed a strong cortical actin ring, or actin fence, in such aggregations, but few have studied this event and its role in affecting the following aggregation. Therefore, we evaluated how self-assembled aggregation of adipose-derived mesenchymal stem cells (MSCs) utilizes the actin fence to manage stemness and how this actin fence allows the aggregate to transduce mechanical signaling. To this aim, we have explored the role of these cues by culturing MSC aggregates encapsulated in alginate gel. We observed an actin fence in the unencapsulated aggregates, which coincided with increased expression of Sox2 and Oct4 compared to aginate encapsulated aggregates. Treatment with promotors to enhance the integrated stress response rescued the actin ring in alginate encapsulated aggregates and improved Oct4 and Sox2 expression. Modulation of the membrane sensing pathway resulted in the re-establishment of the cortical actin ring and stemness, as well in algenate encapsulation. These results show that by altering the environmental cues, the overall stemness of the MSC aggregate can be changed in a non-adhesive system. By better understanding the role of these cues, we hope to also better understand how internal mechanical signals alter cellular and collective properties.This research project has received financial support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (FORTIFy, grant no. 101171765).
Age-and individual-dependent variability in MSC-derived angio-miRNA secretion and the potential of cell-free angiogenic therapy
Shoji Fukuda1, Tomomi Kusakabe2, Yoshiki Wada3, Akinari Iwahori2, Masato Obayashi2, Shinobu Akiyama2, Makoto Matsuda2, Ikki Kojima2, Shun Suzuki2, Masaki Kano2, Toshiki Fujiyoshi2, Toru Iwahashi2, Yusuke Shimahara2
1Department of cardiovascular Surgery. Tokyo Medical University, Shinjuku (Tokyo) - Japan, 2Department of Cardiovascular Surgery. Tokyo Medical University, Shinjuku (Tokyo) - Japan, 3Departmeny of Cardiovascular Surgery. Institute of Science Tokyo, Shinjuku (Tokyo) - Japan
Aim and Objective:
Although recent systematic reviews have failed to demonstrate consistent clinical efficacy of angiogenic therapy for critical limb-threatening ischemia (CLTI), extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have emerged as promising cell-free candidates. This study aimed to elucidate individual and age-related variability in angiogenic microRNA (miRNA) secretion from bone marrow–derived MSCs (BM-MSCs), to investigate how these differences may explain variable therapeutic responses, and to evaluate the potential of EV-based cell-free angiogenic therapy.
Material and Methodology:
Human BM-MSCs were cultured following MISEV2023 standards. Angio-miRNAs (miR-9, miR-105, miR-126, miR-135b, miR-210) were quantified in MSCs and their EVs by RT-qPCR. BM-MSCs were transduced with lentiviral vectors encoding miR-126, miR-135b, or miR-210, and EVs (EV126, EV135b, EV210) were isolated by ultracentrifugation. Their angiogenic capacity was tested by HUVEC tube formation and a murine hindlimb ischemia model following intramuscular injection (4 µg EVs). In a prospective clinical study, seven CLTI patients underwent autologous BM-MSC therapy and were followed for 12 months.
Results:
EVs were enriched in miR-126, miR-135b, and miR-210, but their expression decreased with donor age. Modified MSC-EVs significantly enhanced angiogenesis in vitro and in vivo (p<0.05). Clinically, five patients showed improved perfusion, while two experienced amputation or death within one year. These differences may reflect variability in endogenous miRNA secretion. Importantly, EVs reproduced the proangiogenic effects of MSCs, suggesting a viable, safe, and reproducible cell-free therapeutic option.
Conclusions:
Variability in angiogenic efficacy may arise from age- and donor-dependent differences in MSC-derived miRNA secretion. Enhancing angio-miRNA expression in MSC-EVs could overcome this limitation and provide an effective cell-free regenerative therapy for CLTI.
Multi-scale biomechanical analysis of mineralized spheroids derived from human mesenchymal stem cells
Jeonghyun Kim1, Takashi Inagaki2, Eijiro Maeda2, Takeo Matsumoto2
1Kyushu university, Fukuoka - Japan, 2Nagoya University, Nagoya (Aichi) - Japan
Three-dimensional (3D) culture systems that mimic bone formation in vitro are powerful tools for elucidating the mineralization process underlying osteogenesis. In this study, we established scaffold-free spheroids derived from human mesenchymal stem cells (hMSCs) and successfully induced mineralization through long-term culture. Moreover, we performed a comprehensive multiscale biomechanical analysis capturing both the morphological evolution of spheroids and the changes in their mechanical conditions, ranging from local stiffness at the microscale to overall viscoelastic and plastic behavior at the macroscale. Specifically, we investigated how overall mechanical properties (e.g., Young’s modulus), mechanical behaviors (e.g., elastic versus viscoelastic responses), and local mechanical environments (e.g., spatial heterogeneity in stiffness and morphology) evolved during the mineralization process. After 35 days of culture, the spheroids exhibited marked mineral deposition, accompanied by increased stiffness and plastic deformation at the macroscopic level as assessed by uniaxial compression tests. At the microscopic level, atomic force microscopy revealed heterogeneous distributions of stiffness, reflecting spatially uneven mineralization and extracellular matrix remodeling. These findings demonstrate that mineralization in scaffold-free hMSC spheroids is a dynamic and heterogeneous process that drives progressive mechanical maturation. This study provides novel insights into the biomechanical basis of bone formation in 3D culture models, offering a valuable platform for future research in bone tissue engineering and regenerative medicine.
Tissue engineering for the intervertebral disc
Benjamin Gantenbein
de Tissue Engineering for Orthopaedics & Mechanobiology, Bone & Joint Program, Department for BioMedical Research (DBMR). Medical Faculty, University of Bern, Bern - Switzerland
This keynote will address the key challenges in developing artificial intervertebral disc (IVD) implants using additive manufacturing, electrospinning, and embroidered or woven materials to mimic the IVD. The talk shall focus on selected biomaterials, such as silk and selected natural or synthetic hydrogels. Silk belongs to the traditional biomaterials. Biomimetic fiber-reinforced silk hydrogels could be promising materials for IVD regeneration. Silk has been investigated in biomedical applications for decades, especially in orthopedics. This talk will summarize the current status of silk in IVD regeneration applications. Another focus will be the application of fiber-reinforced hydrogels, possibly combined with other fibers for reinforcement, for disc repair. Here, some very promising formulations were introduced, such as fibrin genipin combinations (fib-gen) and methacrylated gellan gum. Also, highly appealing is hyaluronic acid cross-linked with collagen type 2.
I will also highlight some of our group’s research from the past 15 years. Among them, we highlight genetically engineered silk in B. mori expressing growth and differentiation factor 6 (GDF6), as well as studies in which GDF5 was covalently bound to silk fibers to facilitate “discogenic” or “pulpogenic” differentiation of mesenchymal stromal cells. Furthermore, upscaling scaffold-like or fully printed IVD, seeding these carriers, and then controlling the differentiation towards IVD-like cells is challenging. Finally, various silk-processing procedures have been established and can be further explored using electrospinning, biofabrication, or 3D printing methods.
Silk processed as foam, gel, or fibers in combination with hydrogels could be a promising solution for regenerating the intervertebral disc. Future research might even engineer microenvironments to enrich specific cell types, such as progenitor-like cells, and develop novel concepts for cell-delivery systems in combination with a scaffold.
ACKNOWLEDGEMENTS:
This Research was financed by a Weave Agency Grant of the Swiss National Science Foundation (# 320030E_224175) and the German Research Society (Deutsche Forschungsgemeinschaft, DFG, # 437213841), and by the Bridge Funding Program, which is co-financed by the Swiss National Science Foundation (SNF) and the Innosuisse, grant #211510.
Multimaterial-printed in vitro 3D skin model using a hybrid melt electrowriting and extrusion bioprinting technique
Sadjad Khosravimelal1, Rachael Moses2, Jessica Frith1, Neil Cameron1
1Materials Science and Engineering. Monash University, Melbourne (Victoria) - Australia, 2Dentistry and Health Sciences. University of Melbourne, Melbourne (Victoria) - Australia
The skin functions as a vital protective barrier, shielding the body from environmental insults and microbial invasion. Disruption of this structural integrity leads to wounds, underscoring the importance of understanding wound-healing mechanisms to advance therapeutic strategies. However, ethical constraints and interspecies anatomical differences limit the translational relevance of animal models. Consequently, in vitro three-dimensional (3D) skin models have gained increasing attention as physiologically relevant alternatives. Despite significant progress, many existing models exhibit limited mechanical robustness, poor structural fidelity, and low reproducibility, necessitating innovative approaches for next-generation skin model development. Here, we present a bioengineered skin equivalent composed of a polycaprolactone (PCL) and gelatin methacrylate (GelMA) composite scaffold incorporating human foreskin fibroblasts (HFFs) and keratinocytes (N/TERT-1). A melt electrowritten PCL mesh was fabricated to mimic the dermal architecture, providing mechanical stability and spatial separation between dermal and epidermal layers. GelMA hydrogel containing fibroblasts was bioprinted and photo-crosslinked using blue light to form the dermal compartment. The construct was subsequently rotated, and a thin GelMA layer laden with keratinocytes was printed atop to form the epidermal layer. Comprehensive characterization confirmed the model’s structural and functional resemblance to native skin. Rheological analysis revealed the shear-thinning and photo-reactive behavior of GelMA with a storage modulus of approximately 9 kPa. The PCL scaffold exhibited ∼70% porosity and a tensile modulus of 120 kPa. Live/Dead assays demonstrated high cytocompatibility up to seven days post-encapsulation. Furthermore, barrier function tests confirmed the model’s ability to restrict Lucifer Yellow permeation, and histological evaluation showed well-defined epidermal stratification after 21 days at the air–liquid interface (ALI). Overall, this engineered skin equivalent demonstrates excellent biomimicry and functional integrity, offering a robust platform for fundamental skin research and the development of advanced therapeutic and cosmetic strategies.
How cells sense geometry: computational insights into the role of surface curvature on mobility and alignment
Diogo A. S. Almas1, Andreas Roschger1, Thomas Antretter2, F. Dieter Fischer2, Peter Fratzl3, John W. C. Dunlop1
1Department of the Chemistry and Physics of Materials. Paris Lodron University of Salzburg, Salzburg - Austria, 2Chair of Mechanics. Montanuniversität Leoben, Leoben (Steiermark) - Austria, 3Department of Biomaterials. Max Planck Institute of Colloids and Interfaces, Postdam (Brandenburg) - Germany
Surface curvature is both a product and a driver of biological organization, shaping processes from the cellular to the tissue level [1]. Cells have been found to move, align and position themselves according to surface curvature. However, the mechanisms that cells use to read the geometrical cues of their surroundings is not yet understood. Some hints imply that mechanical signals may influence cell behaviour as determined through experiments where the inhibition of mechanosensitivity of cells influences curvature sensing [2].
This computational study uses a simplified model to explore how cells sense surface curvature mechanically, creating a link between the geometrical cues and possible internal mechanical response. A cell model positioned in different orientations on curved surface presents different strain and stress values. By calculating strain energy density and Von mises stress it was possible to create a link between surface curvature and mechanical signaling. These could be used as handles to describe the relationship between cell’s preference for alignment and mobility on different curved surfaces.
This in-silico study explores how cells might detect and react to surface curvature via mechanical signals. Our essay offers a starting point for computational models, which will then be validated by in vitro experiments. We plan to investigate how surfaces of revolution and constant mean curvature surfaces guide cell alignment and influence growth.
REFERENCES
[1] B. Schamberger et al., “Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales,” Adv. Mater., vol. 35, no. 13, p. 2206110, Mar. 2023, doi: 10.1002/adma.202206110Author A., Author B., Author C., Book’s title, Edition, Place, Publisher, Year.
[2] S. Ehrig et al., “Surface tension determines tissue shape and growth kinetics,” Sci. Adv., vol. 5, no. 9, p. eaav9394, Sep. 2019, doi: 10.1126/sciadv.aav9394.
Enhancing regenerative potential: effects of light exposure on adipose-derived mesenchymal stem/stromal cells in vitro
Sridharan Kaarthik1, Okikiola Tawakalitu Waheed1, Staehlke Susanne1, Riess Alexander2, Mand Mario2, Meyer Juliane3, Seitz Hermann4, Peters Kirsten1, Hahn Olga1
1Institute of Cell Biology, Rostock University Medical Center (Mecklenburg-Vorpommern) - Germany, 2Chair of Microfluidics, Faculty of Mechanical Engineering and Marine Technology, University of Rostock (Mecklenburg-Vorpommern) - Germany, 3Human Med AG, Schwerin (Mecklenburg-Vorpommern) - Germany, 4Department of Life, Light and Matter. Chair of Microfluidics, Faculty of Mechanical Engineering and Marine Technology, University of Rostock (Mecklenburg-Vorpommern) - Germany
Introduction: Light of different wavelengths can modulate cellular behavior and metabolic pathways, with promising applications in regenerative medicine. When applied therapeutically to modulate cellular responses, this process is called photobiomodulation (PBM). The modulation of cellular processes is harnessed through targeted illumination, yet wavelength-dependent cellular responses remain incompletely understood, especially in human adipose-derived mesenchymal stem/stromal cells (adMSCs). We therefore aimed to systematically investigate how different light wavelengths influence the viability, function, and differentiation of adMSCs to optimize their regenerative potential.
Methods: In the present study, adMSCs in suspension were exposed to light at 455 nm (blue), 660 nm (red), and 810 nm (near-infrared) under controlled dosage and duration conditions using an integrating sphere, both individually and in combinations. Key parameters analyzed included measurements of intracellular reactive oxygen species (ROS), cell viability, proliferation, cytokine secretion (IL-6, IL-8) and migration. In addition, adipogenic differentiation capacity was assessed after light exposure.
Results: Short-term exposure of adMSCs to these wavelengths showed clear cellular responses that are dependent on both wavelength and dosage. Blue light significantly increased ROS production in a dose-dependent manner, resulting in reduced proliferation, metabolic activity, and differentiation potential, accompanied by decreased IL-6/IL-8 secretion. Conversely, exposure to red and near-infrared light preserved viability and enhanced cell migration and mitochondrial activity, consistent with their known ability to stimulate both proliferation and mitochondrial function in mesenchymal stem cells. Overall, light exposure exerted wavelength-specific effects on adMSCs, with red and near-infrared light promoting favorable regenerative responses while blue light induced cellular stress.
Conclusion: These results underscore the critical role of wavelength and dosage in determining cellular outcomes under light exposure. The results provide mechanistic insight into how light modulates stem cell function and establish its potential as a complementary approach to improve MSC-based regenerative therapies.
Columnar scaffold design development and multiaxial bioreactor validation for the deep articular cartilage zone
Rachel Cordeiro1, Maja Schlittler2, Laura Mecchi2, Rui Alvites3, Ana C. Maurício4, Nuno Alves1, Carla Moura5, Martin J. Stoddart2
1Instituto de Ciências Biomédicas de Abel Salazar (ICBAS). Universidade do Porto, Porto - Portugal, 2AO Research Institute Davos. AO Foundation, Davos Platz (Graubunden) - Switzerland, 3Polytechnic and University Higher Education Cooperative (CESPU), Gandra (Porto) - Portugal, 4Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS). Universidade do Porto, Porto - Portugal, 5Research Centre for Natural Resources Environment and Society (CERNAS). Polytechnic University of Coimbra, Coimbra - Portugal
The deep zone (DZ) is the most challenging to mimic among the four zones comprising the articular cartilage due to its distinct architecture and mechanical properties. Adapted to sustain compressive loads, it unveils a fibrillar alignment perpendicular to the surface. This study investigates whether mimicking the DZ’s native structure and mechanical stimulation create a microenvironment that promotes cartilage differentiation/proliferation.
Columnar scaffolds with different distances between columns (1.5w, 2.0w, and 2.5w), along with a design control (well-known orthogonal scaffold), were manufactured using TPU. Scaffolds were morphologically and mechanically characterized, and latent TGF-β1 activation was quantified. After selecting the columnar scaffold with the best performance, a 21-day multiaxial mechanical study was performed using hMSCs. Scaffolds and medium were biochemically evaluated.
Morphologically, columnar scaffolds demonstrated porosities similar to native cartilage (between 83.50% and 88.86% while the orthogonal scaffolds showed a lower porosity (73.91 ± 0.96%). The TGF-β1 activation test showed that the 2.5w loaded scaffold activated more TGF-β1 under load (58.27 ± 15.56 pg/mL).
Results from the multiaxial study of 2.5w columnar and orthogonal scaffolds, comparing loaded versus non-loaded conditions, showed higher TGF-β1 and sGAG levels, as well as collagen type I expression in all loaded scaffolds. Additionally, columnar scaffold design expressed statistically higher RUNX2/SOX9 ratio when non-loaded, indicating chondrocyte hypertrophy/bone formation. TGF-β1 receptor analysis revealed lower TGFBRII and ACVRL-1 expression in all loaded scaffold designs compared with the non-loaded ones. Finally, the TGFBRI/TGFBRII ratio was evaluated, showing a higher ratio for loaded columnar scaffolds compared with non-loaded scaffolds, suggesting that they will follow the Smad2/3 signaling pathway, maintain the chondrocyte phenotype. Although there were no statistically significant differences between designs throughout the study, type II collagen was only detected in the loaded columnar scaffolds.
The results demonstrate the columnar scaffolds' chondrogenic differentiation potential, making them a promising design to guide DZ repair.
Bio-engineering of recombinant spider silk proteins for biomedical applications
Thomas Scheibel
de BIomaterials. University of Bayreuth, Bayreuth (Baden-Wberg Bayern) - Germany
Proteins reflect one fascinating class of natural polymers with huge potential for technical as well as biomedical applications. One well-known example is spider silk, a protein fiber with excellent mechanical properties such as strength and toughness. We have developed biotechnological methods using bacteria as production hosts, which produce structural proteins mimicking the natural ones. Further, we can specifically functionalize the recombinant silk proteins with cell-specific and bio-selective tags.
We employ silk proteins in different application forms such as hydrogels, particles or films with tailored properties, which can be employed especially for tissue engineering applications. In such applications, the performance of materials largely depends on their surfaces and is further strictly related to the materials biocompatibility. Spider silk hydrogels can be employed as new bioinks for biofabrication. Their elastic behavior dominates over the viscous behavior over the whole angular frequency range with a low viscosity flow behavior and good form stability. No structural changes occur during the printing process, and the hydrogels solidify immediately after printing by robotic dispensing. Due to the shape stability, it was possible to directly print multiple layers on top of each other without structural collapse. Cell-loaded spider silk constructs can be easily printed without the need of additional cross-linkers or thickeners for mechanical stabilization.
Our bio-inspired approach serves as a basis for new materials in a variety of tissue engineering applications such as heart muscle regeneration.
Self-assembling immuno-active peptides for spatiotemporal control of chronic inflammation
Jacek K. Wychowaniec
de Biomedical Materials. AO Research Institute Davos, Davos (Graubunden) - Switzerland
Numerous chronic, inflammation-driven conditions, such as tendinopathy, involve prolonged immune imbalance, with macrophages (MΦs) and their cross-talk with stromal cells playing a central role in dictating healing.[1] Biomaterial-based strategies can guide MΦ responses toward regeneration, particularly in severe injuries. Inspired by nature’s homochirality, self-assembling peptides (SAPs) provide a minimalistic platform for designing functional biomaterials. Here, we present an extended family of tyrosine-modified β-sheet-forming SAPs.[2] By varying hydrophobic/hydrophilic residues and tyrosine placement, as well as choosing the overall chirality state (L- versus D-), we were able to tune their physicochemical properties and interactions with MΦs. These SAPs were comprehensively characterized using physicochemical and molecular dynamics analyses, and their effects on MΦ polarization were evaluated in THP-1 and donor-derived PBMC-derived cells through molecular and cellular assays.
Among L-variants, we identified two negatively charged key modulators, EF8, which induced an anti-inflammatory M2 response (CD105, CD163, CD206 upregulation, IL-10 secretion), and YEF8, which favoured an M1-like phenotype (increased HLA-DR expression and TNF-α secretion).[2] Their cellular immunomodulatory responses were stronger as compared to existing hyaluronic acid-based therapies.[3] On the other hand, positively charged SAPs KYF8 and KYF8K revealed distinct immunomodulatory effects, with KYF8K exhibiting a unique Th1-promoting phenotype characterized by an increased IL-2, IP-10, MCP-1, TNF-α and IFN-γ, secretion. Homochiral D-variants of EF8 and YEF8 confirmed immunomodulatory induction of polarization, albeit with lower response as compared to L-variants. Our work highlights the pivotal role of SAP chemistry and chirality in MΦ modulation and introduces new strategies for spatiotemporal control of musculoskeletal inflammation, ultimately providing previously unseen therapeutic opportunities in regenerative medicine.
References
1. J. Lee, et al., Adv Healthc Mater, 2019, 8, e1801106.
2. J. K. Wychowaniec, et al., ACS Applied Materials & Interfaces, 2025, 17, 19, 27740–27758.
3. J. K. Wychowaniec, et al., Biomaterials Advances, 2025, 169, 214166.
Acknowledgements
This work was supported by the AO Foundation, EU H2020-MSCA-IF-2019 (no.893099-ImmunoBioInks), LEADING HOUSE MENA Research Partnership (RPG-2022-38) and Consolidation (COG-2023-35) grants.
Cell therapy in osteoarthritis: from concepts to clinical evaluation
Guicheux Jerome
de Inserm UMR 1229-RMeS, Regenerative Medicine and Skeleton. Nantes Universite, Nantes (Pays de la Loire) - France
Mesenchymal stem/stromal cell (MSC)-based therapies represent a promising and innovative approach for osteoarthritis (OA), offering disease-modifying potential beyond symptomatic management. Owing to their regenerative and immunomodulatory properties, MSCs can modulate the inflammatory microenvironment and promote joint tissue repair.
Preclinical and clinical studies have suggested that intra-articular (IA) MSC administration can alleviate OA symptoms and potentially slow disease progression through complementary mechanisms.
(i) Analgesic and functional effects: MSCs reduce pain intensity and improve joint mobility and quality of life.
(ii) Chondroprotective and reparative actions: MSCs enhance chondrocyte viability and metabolic activity, inhibit extracellular matrix degradation, and may contribute to cartilage regeneration.
(iii) Immunomodulatory effects: Through the secretion of cytokines, growth factors, and extracellular vesicles, MSCs attenuate pro-inflammatory signaling, stimulate anti-inflammatory pathways, and modulate immune cell phenotypes within the osteoarthritic joint.
Current evidence indicates that MSCs act predominantly via paracrine mechanisms, rather than by engraftment or differentiation, thereby functioning as “cellular biofactories” capable of dynamically restoring joint homeostasis. Ongoing research aims to optimize key parameters influencing therapeutic efficacy, including the cell source (bone marrow, adipose tissue, umbilical cord), dose, frequency of administration, and delivery systems such as biomaterial scaffolds or encapsulation technologies designed to improve cell persistence and bioactivity. In parallel, combinatorial strategies integrating MSCs with biomaterials or pharmacological agents are under investigation to enhance therapeutic synergy.
This presentation will summarize recent advances and translational progress in MSC-based interventions for OA, critically analyzing clinical outcomes, mechanistic insights, and current challenges to better define the clinical positioning and future potential of MSC therapy within evidence-based OA management.
Translational study of Osteogrow-C: A novel device for bone regeneration and spinal fusion containing rhBMP6 and synthetic ceramics
Nikola Stokovic1, Natalia Ivanjko1, Marko Pecin2, Drazen Maticic2, Slobodan Vukicevic1
1Laboratory for Mineralized Tissues. University of Zagreb School of Medicine, Zagreb (Grad Zagreb) - Croatia, 2Clinics for Surgery, Orthopedics and Ophthalmology. University of Zagreb Faculty of Veterinary Medicine, Zagreb (Grad Zagreb) - Croatia
Osteogrow-C is a novel therapeutic device for bone regeneration and spinal fusion, composed of recombinant human Bone Morphogenetic Protein 6 (rhBMP6) formulated in autologous blood coagulum (ABC) with ceramic particles. This innovative delivery system provides a natural environment for osteoinduction and controlled release of rhBMP6. The present study aimed to investigate the safety, osteoinductive potential, and efficacy of Osteogrow-C implants containing small (74–420 µm) ceramic particles in a rat subcutaneous assay and translate these findings to a rabbit posterolateral spinal fusion (PLF) model as preclinical validation of the therapeutic concept. Osteogrow-C osteoinductive implants were prepared by combining rhBMP6 with autologous blood and synthetic ceramic particles (TCP/HA 80/20, porosity 86%). In rats, implants containing 20 µg rhBMP6 were placed subcutaneously in the axillary region and analyzed by histology and microCT from 7 to 50 days post-implantation to assess ectopic bone formation and remodeling. In the rabbit PLF model, implants containing 125 µg rhBMP6 were similarly prepared and placed bilaterally between the L5–L6 transverse processes. After 52 weeks, spinal segments were evaluated using microCT, histology, and biomechanical testing to determine the extent, quality, and mechanical stability of the achieved fusion. In the rat subcutaneous assay, by day 7 post-implantation, zones of endochondral ossification appeared at the periphery, and by day 14, new bone was present throughout the implant. By day 21, bone formation was complete, and bone volume remained stable. In the rabbit PLF model, Osteogrow-C induced robust bone formation and achieved solid spinal fusion persisting through follow-up. Histology confirmed osteointegration between new and native bone, while three-point bending tests demonstrated biomechanical competence of the fusion. This translational study demonstrated rhBMP6-induced osteogenesis dynamics in rats and validated Osteogrow-C's safety and efficacy in rabbits, supporting its potential as a safe, effective therapeutic candidate for clinical trials.
Advancing dermal matrix with antimicrobial peptide therapeutics for the management of infection and inflammation in wound care
Van Vo1, Hanif Haidari2, Anteneh Amsalu1, Anna Antipov1, Allison J. Cowin1, Marcus J.d. Wagstaff3, Bronwyn Dearman3, Zlatko Kopecki1
1Future Industries Institute. Adelaide University, Adelaide (South Australia) - Australia, 2College of Medicine and Public Health. Flinders University, Adelaide (South Australia) - Australia, 3Adult Burn Service. Royal Adelaide Hospital, Adelaide (South Australia) - Australia
Recent technological advances have highlighted the potential of antimicrobial peptides (AMPs) as promising alternatives to conventional antibiotics in combating the overwhelming rise of antimicrobial resistance in wound care. This study harnesses the power of antimicrobial peptides for infection and biofilm management in wound care. For the first time, a dual antimicrobial peptide–coated dermal substitute (BTM-AMPs) was developed by integrating Nisin and LL-37 onto a commercially available dermal substitute, Novosorb® Biodegradable Temporising Matrix (BTM). BTM-AMPs demonstrated multiple synergistic effects beneficial for healing through the effects on polymicrobial biofilms and inflammation. In vitro results demonstrated strong antimicrobial activity of the BTM-AMPs, with an ability to eradicate mature polymicrobial biofilms consisting of common Gram-positive and Gram-negative pathogens. Direct tissue contact was identified as the primary mechanism of action, offering the advantage of higher efficacy and lower toxicity compared to biomaterials that rely on leaching. Using an ex vivo bioluminescent biofilm model, BTM-AMPs reduced the burden of metabolically active bacteria by >50% after 6 hours of treatment. Additionally, BTM-AMPs exhibited strong anti-inflammatory properties by significantly reducing the level of pro-inflammatory TNF-α cytokine and increased the phagocytotic activity of stimulated macrophages in vitro. Overall, the work provides a new strategy for the development of antimicrobial tissue implants to address the overwhelming issue of infection and inflammation in wound care and tissue regeneration.
Advancing intervertebral disc disease therapies: the role of biofabrication technologies
Catherine Le Visage
de Nantes Universite, Nantes (Pays de la Loire) - France
The intervertebral disc (IVD) is a fibro-cartilaginous structure that acts as a natural shock absorber for the spine. It consists of an outer ring of collagen fibers encasing a gelatinous core. Degeneration of IVD, for which there is currently no effective treatment, is a prevalent cause of chronic back pain and significantly impacts the quality of life for millions worldwide.
Preclinical animal models of the disease have been considered; however, alternatives to animal experimentation are needed due to ethical guidelines and concerns about animal welfare.
This talk will present various biofabrication techniques (extrusion, meltelectrowriting) to design an in vitro IVD model that replicates its intricate organization. Implementing bioprinted constructs that recapitulate the IVD’s complex architecture could be a crucial step toward assessing the effectiveness of cutting-edge therapeutic approaches that include cells, extracellular vesicles, mRNA, or miRNA, leading toward human and veterinary clinical trials.
Navigating the early seas: sharing the treasure map of a young PI in skeletal biofabrication
Gianluca Cidonio
de Department of Mechanical and Aerospace Engineering. Sapienza University of Rome, Rome (Lazio) - Italy
Becoming a Principal Investigator (PI) in tissue engineering and regenerative medicine (TERM) marks a significant transition, exchanging the focus of a post-doc for the multifaceted demands of leading a research group. This is not always straightforward, and often conceals struggles and a difficult journey ahead. There is a need to hold a solid ship, filled with the right tools and most importantly populated with fellows that would help you navigating the early seas.
This invited talk will delve into the challenges, as well as the success, experienced during the first few years as a young PI in the dynamic field of biofabrication and skeletal TERM. Drawing from my journey in the academic sea, from an AIRC Fellow and Bioprinting team Leader at the Italian Institute of Technology (IIT) to establishing the MoRe (Model and Regenerate) 3D Lab at Sapienza University of Rome, I will focus on key aspects of this career shift. I will provide insight on why the chance to secure initial independent funding (e.g., AIRC, ON Kick-Starter Grant) is key to manage and mentor a diverse research team (supervising multiple PhD and MSc students), and establish an international research profile in an always higher competitive domain.
To set sail in the academic sea, you will require a clear map of where you are headed, with specific aims and goals along the way. This is initially established by the finding of a research niche, and I will be using my interest on microfluidic-assisted 3D bioprinting as a case study to unravel a new pathway towards success. Highlighting the need for young PI in TERM research for a proportional interest shared between fundamental research innovation and translational application, I will be further delving on the requirements for a more carefree balance between academic work and personal life. Finally, I want to propose an interactive session to foster an environment for early career researchers to address their own fears and aspirations about leadership in TERM, to find together a treasure map that can be shared to navigate these early seas of our academic journey.
Interactive and anisometric colloidal building blocks for regenerative medicine and tissue engineering
Laura De Laporte
de DWI Leibniz Institute for Interactive Materials. DWI Leibniz Institute for Interactive Materials, Aachen (Rheinland-Pfalz) - Germany
We apply polymeric molecular and micron-scale building blocks to assemble into soft 3D biomaterials with anisotropic and dynamic properties. We focus on injectable materials that can be pipetted using automated systems, bioprinted or delivered in vivo in a low invasive manner. Spherical and rod-shaped microgels and fibers are produced by microfluidics, in-mold polymerization, and fiber spinning. To arrange the building blocks in a spatially controlled manner, self-assembly mechanisms and alignment by magnetic fields are employed. Reactive and/or bioactive spherical and rod-shaped microgels interlink and form macroporous constructs facilitating 3D cell growth and cell-cell interactions or cells are able to use microgels as bricks to build their own house. Chemically defined poly(ethylene glycol)-based microgels, produced via parallelized step-emulsification microfluidics, self-organize with induced pluripotent stem cells (iPSCs) into 3D constructs by robust cell-material interactions. The iPSCs expand and retain their pluripotency, after which they can be differentiated into the three germ layers, providing a suitable platform for organoid differentiation, which was exemplary demonstrated for cardiac organoids. This new organoid production technology enables iPSC expansion and differentiation in the same construct in a reproducible and scalable manner, compatible with high-throughput automation. On the other hand, magneto-responsive rod-shaped microgels form the core of the patented Anisogel technology, which offers a low-invasive therapy to regenerate sensitive tissues with an oriented architecture. It can be injected and structured in situ to guide cells in a linear manner. Finally, a thermoresponsive hydrogel system, encapsulated with plasmonic gold-nanorods, actuates by oscillating light and elucidates how rapid hydrogel beating affects cell migration, focal adhesions, extracellular matrix production, and nuclear translocation of mechanosensitive proteins, depending on the amplitude and frequency of actuation. The time spent in the in vitro gym seems to affect myoblast differentiation and fibrosis, while actuation seems to induce mesenchymal stem cell differentiation into bone cells.
In vitro and in vivo preclinical evaluation of a novel tissue-engineered vascular graft
David Durán-Rey1, Carlos Sánchez-Rumbo1, Ricardo Brito-Pereira2, Clarisse Ribeiro3, Silvie Ribeiro3, Juan Alberto Sánchez-Margallo1, Verónica Crisóstomo1, Unai Silván2, Senentxu Lanceros-Méndez2, Francisco Miguel Sánchez-Margallo1
1CCMIJU, Cáceres - Spain, 2BCMaterials, Leioa (Bizkaia) - Spain, 3University of Minho, Braga - Portugal
Tissue-engineered vascular grafts (TEVGs) are capable of replacing or repairing the biological functions of blood vessels. Poly(vinylidene fluoride) (PVDF) is a synthetic and biocompatible polymer that induces specific cellular responses and improve tissue regeneration due to its piezoelectric properties. Moreover, PVDF has been shown to promote endothelial cell formation. In this study, the performance of commercially available expanded polytetrafluoroethylene (ePTFE) grafts was compared with electrospun-PVDF grafts.
To simulate in vivo conditions, grafts were preincubated in blood serum before seeding with human umbilical vein endothelial cells (HUVECs). Scanning electron microscopy revealed that cells exhibited greater spreading and larger footprints on the electrospun PVDF scaffolds. Immunostaining against VE-cadherin (CD144) confirmed the presence of this adhesion molecule at intercellular junctions and within the nuclear compartment of endothelial cells. Quantification of nitric oxide (NO) production demonstrated the highest levels in HUVECs cultured on electrospun PVDF scaffolds, whereas considerably lower NO levels were observed on commercial ePTFE grafts.
Based on these favorable in vitro outcomes, twelve female Merino sheep were randomly assigned to either the electrospun-PVDF group (n = 6, test group) or the ePTFE vascular graft group (n = 6, control group). Both graft types had an inner diameter of 6 mm and were implanted into the right common carotid artery. After 6 weeks, thrombus and stenosis were confirmed in 2/6 of PVDF grafts and 4/6 in ePTFE grafts.
Although additional studies are necessary to confirm its translational potential, the results suggest that electrospun-PVDF vascular grafts constitute a promising alternative for applications in cardiovascular tissue engineering.
Precise chemically design in natural systems towards high-performance hydrogels
João Mano
de Department of Chemistry. CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal
Natural polymers and cell surfaces encode fascinating chemical cues finely tuned to their biological functions. Beyond their native chemical diversity, these systems can be further modified to generate biomaterials with enhanced structural and functional properties for tissue engineering.
Our group has explored diverse polysaccharides and proteins to create hydrogels that accommodate and transport cells for regenerative medicine. We have focused on introducing non-covalent chemistry routes to yield dynamically cross-linked hydrogels with injectability, printability, self-healing, and tunable viscoelasticity. For instance, coacervates of oppositely charged polysaccharides bearing thermo-responsive branches showed suitable rheology and gelation at body temperature, enabling the injection of micro-hybrid elements and formation of microtissues in situ. Similarly, coacervates with tannic acid displayed strong wet adhesion to biological tissues. Beyond electrostatic interactions, we also used host–guest chemistry to design dynamic, human protein–based hydrogels capable of supporting cells in 3D viscoelastic microenvironments.
In parallel, we engineered the surface of living cells to create cell-rich constructs reproducing tissue-like cell densities. These highly cell-dense, bioactive hydrogels—termed “cellgels”—employ glycoengineered cells as building blocks crosslinked via bioorthogonal click chemistry. Combining the mechanical adaptability and adhesiveness of conventional hydrogels with the intrinsic capabilities of living systems, these constructs exhibit environmental adaptability, biological responsiveness, and evolvability.
Organoid culture using organoid culture plates reduces variability and enhances screening efficiency
Douglas Kondro1, Michael Hiatt1, Selena Cen1, Aaron Ang1, Ryan Conder1, Sharon Louis1, Allen Eaves1
1STEMCELL Technologies Inc., Vancouver (British Columbia) - Canada
Organoids hold great promise for drug screening offering superior recapitulation of human biology compared to 2D cultures. However, widespread adoption of organoids in high-throughput workflows is limited by technical challenges including laborious manual operation and high culture-to-culture variability. To address these challenges, we developed high-throughput 96-well Organoid Culture Plates (OCP) designed to reduce evaporation, standardize growth conditions, and improve imaging. Intestinal and hepatic organoids were cultured in OCPs and compared to 10 µL Matrigel® domes in standard 96-well plates. Organoids cultured in OCPs exhibited superior growth (measured by mean cross-sectional area) and substantial reduction in the coefficient of variation (CV) by an average of 15% (intestinal) and 20% (hepatic) over five passages (n ≥ 85). Intestinal organoids derived from a patient with cystic fibrosis (ΔF508 mutation) demonstrated a 5% greater response to Orkambi® treatment in OCPs compared to standard plates (16 ± 9% vs 11 ± 10%) in forskolin-induced swelling assay. Drug screening with gefitinib produced a comparable IC50 as measured by CellTiter-Glo® 3D in OCPs (0.46 ± 0.08 µM) versus traditional domes (0.39 ± 0.09 µM; p ≤ 0.05, n = 3). A bile transport assay using a fluorescent bile acid analogue was used to determine the responses of hepatic organoids to treatment with common drug-induced liver injury (DILI)-causing drugs, including troglitazone, pioglitazone, nefazodone, buspirone, and cyclosporin A. In traditional domes, the response to increasing doses of DILI-causing drugs was variable due to irregularities in dome location and frequent presence of dead zones. In OCPs, imaging was standardized and clear dose-dependent responses were observed following treatment with DILI-causing drugs. OCPs enhance organoid workflows by reducing manual effort and decreasing experimental variability while enabling large-scale, high-throughput screening and functional assays.
High-throughput topographical screening identifies microfeatures that enhance nephrin localization and morphological differentiation of human podocytes
Marta Garcia Valverde1, Nikita Konshin2, Rosalinde Masereeuw1, Jan De Boer2, Silvia Maria Mihăilă1
1Division of Pharmacology, Utrecht Institute of Pharmacological Sciences, The Netherlands. Utrecht University, Utrecht - The Netherlands, 2Department of Biomedical Engineering and Institute for Complex Molecular Systems, The Netherlands. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
Podocytes are essential components of the glomerular filtration barrier (GFB), where their interdigitating foot processes and slit diaphragms ensure selective filtration of plasma. When cultured on flat substrates, podocytes lose their branched morphology and nephrin polarization, limiting the relevance of in vitro GFB models. To address this, we performed a systematic high-throughput screen of podocyte–surface interactions using the TopoChip platform containing 2,176 unique microtopographies generated from primitive geometrical combinations.
Human conditionally immortalized podocytes (ciPODs) were matured for 14 days on TopoChips and stained for nephrin, F-actin, and DNA. A CellProfiler-based image analysis pipeline processed over 1.2 million segmented objects, extracting >1,500 morphological and intensity descriptors per cell across 36,024 TopoUnits. Following multi-level quality control, supervised machine-learning classification in CellProfiler Analyst distinguished branched, nephrin-localized phenotypes from undifferentiated cells with an accuracy of 84%. The feature “Nephrin_Granularity_1”, defining the texture of the nephrin signal, emerged as a robust quantitative marker of nephrin clustering and foot process formation.
Integration of topographical design descriptors (TDDs) with phenotype data identified inter-pillar spacing (8–20 µm), feature eccentricity, and vertical spreading as dominant drivers of podocyte differentiation. Surfaces exhibiting these TDDs produced up to a six-fold reduction in granularity score compared with flat controls, reflecting enhanced nephrin polarization and cytoskeletal anisotropy.
This study provides the a quantitative link between defined microscale geometries and podocyte morphofunctional maturation. The identified “hit” topographies will be translated onto patterned, porous polyethersulfone (PES) membranes to generate perfusable GFB-on-chip systems. By coupling topographical design with membrane engineering, this approach establishes a blueprint for cell-instructive filtration interfaces with direct applications in nephrotoxicity testing, disease modeling, and regenerative nephrology.
This research was financially supported by the Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research (024.003.013).
An elastic journey: from the bench to the clinic
Anthony Weiss
de Charles Perkins Centre. University of Sydney, Sydney (Australian Capital Territory) - Australia
In every species of mammal, bird and reptile, and across almost the entire vertebrate world, the skin, lung, arteries and other tissues require elasticity to function. What bestows this elasticity is the protein elastin, which in turn is assembled from the structural protein building-block tropoelastin. We have found that tropoelastin can promote the repair many types of damaged tissues; and identified collaboratively that tropoelastin can allow stem cells to delay senescence, while retaining phenotype and function. Despite these critical abilities, and even though the tropoelastin gene is essential, paradoxically the production of tropoelastin drops precipitously with age.
This presentation will present advances in our mechanistic understanding in the use of tropoelastin to deliver organized elastin in vascular walls during replacement-repair in vivo, promote heart muscle survival and recovery in an in vivo model of ischemic injury, its ability to compress the inflammatory sequence displayed by macrophages, and their commercialization.
Collaboratively, we are seeing responses by mesenchymal stromal cells that extend to expression differences accompanied by delayed senescence that fundamentally reflect the role of the elastic extracellular matrix. In vivo and model studies will be presented that show the value of using tropoelastin to modify cell performance in these cases, in order to help heal surgical wounds, repair arteries and modulate immunity while delivering synthetic tissue-materials for building 3D constructs.
This bench-to-bedside commercialization has been facilitated by 174 granted international patents in 23 distinct families, comprising protein production, mutants, tropoelastin stability, elastin production, tissue regeneration, sealants for soft tissue, bone repair, injectables, triggered elastogenesis, scaled production of elastic materials, enhanced repair of wounds. Venture capital funding and 3 completed clinical trials led to Elastagen’s acquisition by Allergan, an AbbVie company, in one of the largest national transactions.
Ghovvati, M. et al. (2025) Science Translational Medicine 17,eadr6458
Hume, R.D. et al. (2023) Circulation Research 132,72
Lee, S. et al. (2024) Advanced Science 11,2402168
Wang, Z. et al. (2022) Advanced Materials 34,2205614
Wang, Z. et al. (2024) Acta Biomaterialia 184,56
Bioengineered collagen-coated scaffolds for advanced tendon regeneration
Francesca Romano1, Francesco Lopresti2, Chiara Di Marco2, Denise Murgia1, Vincenzo La Carrubba2, Roberto Di Gesù1
1Musculoskeletal Tissue Engineering lab. Fondazione Ri.MED, PALERMO (Sicilia) - Italy, 2Department of Engineering. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy
Tendons are dense connective tissues mainly composed of collagen I fibers, providing mechanical strength and stability to joints. However, their limited self-healing capacity, due to poor vascularity and cellularity, makes tendon ruptures a major clinical challenge. Surgical repair often leads to fibrotic tissue formation, compromising mechanical function. To overcome this, we propose a combined tissue engineering and drug delivery strategy aimed at mitigating post-surgical tendon fibrosis.
Our strategy exploits a 3D electrospun scaffold with tendon-like microarchitecture, cellularized with human tenocytes (hTCs), and loaded with Rolipram as antifibrotic agent to actively avoid the tendon fibrosis after surgical repair. In our study, we used PLA to produce aligned microfibers that closely mimic the natural arrangement of collagen fibers in native tendons, also promoting a native-like hTCs spatial arrangement. We provided microfibers with a type I collagen coating to modulate the drug delivery kinetics, and to induce a self-assembling of microfibers into fascicle-like substructures. These substructures were preserved in 3D scaffolds fabricated with a custom-made bundle-making device, which assembles microfibers into a helicoidal quaternary structure.
Collagen coating significantly increased the Young’s modulus of both drug-free (from 102.93 to 140.09 MPa) and drug-loaded (from 103.3 to 144.23 MPa) scaffolds, while Rolipram loading did not affect mechanical performance. Moreover, the coating modulated Rolipram release. Specifically, we didn’t notice any burst release neither from uncoated (PLA/Rol) nor from coated (PLA/Rol/Coll) scaffolds within first 2h. Excitingly, we obtained compelling result also from biological characterization of our tendon-like scaffolds. Histomorphological analysis performed via hematoxylin/eosin staining revealed hTCs in both inner and outer scaffold regions. This effect was enhanced in collagen-coated scaffolds that showed a more intense staining.
Overall our study shows encouraging results, demonstrating the potential high-impact that our methodology may have in the surgical repair of tendon ruptures, by inducing the regeneration of non-fibrotic tendons.
Multifunctional five-layer collagen laminates for integrated infection control and bone regeneration in contaminated fracture models - SEMIT
Tara Gaschik1, Claudia Eßbach2, Dirk Fischer2, Daniela Nickel2, Ulrike Ritz1
1Universitätsmedizin Mainz, Mainz (Rheinland-Pfalz) - Germany, 2Duale Hochschule Sachsen, Glauchau (Sachsen) - Germany
Open bone fractures, particularly those involving severe tissue damage and microbial contamination, pose major clinical challenges due to the dual need for infection control and bone regeneration. Conventional treatments, such as systemic antibiotics and bone grafting, often fail to achieve localized therapeutic concentrations or complete tissue healing.[1] To address these limitations, we developed a multifunctional, five-layer collagen laminate that combines structural support with the spatially organized delivery of bioactive molecules for antibacterial action, osteogenesis, and angiogenesis.
The laminates, composed of commercially available collagen sheets, were fabricated using a photochemical crosslinking method with rose bengal and green light, allowing rapid and biocompatible bonding of individual collagen layers. Each layer incorporated a specific therapeutic agent: vancomycin for localized infection control, bone morphogenetic protein-2 (BMP-2) for osteoinduction, and stromal cell-derived factor-1α (SDF-1α) for cell recruitment and vascularization. Material characterization included mechanical testing and drug release profiling. An in vitro osteomyelitis model was established to evaluate antibacterial, osteogenic, and pro-angiogenic properties.
The laminates exhibited strong mechanical integrity and layer-specific bioactive release. Vancomycin showed an initial burst, while BMP-2 and SDF-1α displayed sustained release, enabling temporal separation of antimicrobial and regenerative effects. In the in vitro osteomyelitis model, the laminates inhibited Staphylococcus aureus, promoted osteoblast differentiation, and enhanced endothelial cell migration, demonstrating synergistic regenerative potential.
This study introduces a multifunctional, five-layer collagen laminate that integrates antimicrobial defense, osteoinduction, and vascular recruitment to address the complex requirements of bone healing in contaminated or high-risk fractures. Its tunable, spatially organized therapeutic delivery offers a promising strategy for regenerative orthopedic and trauma applications.
[1] M. J. Flores et al., J. Clin. Med. 2022, 11, 7461 DOI: 10.3390/jcm11247461
Innovative RNA transfection kit based on hybrid polymer–lipid nanoparticles
Martina Coletto1, Camilla Paoletti1, Letizia Nicoletti2, Elena Marcello1, Giovanni Paolo Stola2, Francesco Schiavone1, Francesca Cossetta1, Ilaria Andreana3, Barbara Stella3, Silvia Arpicco3, Clara Mattu1, Valeria Chiono2
1DIMEAS. Politecnico di Torino, Turin (Italia) - Italy, 2PoliRNA Srl - DIMEAS. Politecnico di Torino, Turin (Italia) - Italy, 3University of Turin, Turin (Italia) - Italy
MicroRNAs (miRNAs) play crucial roles in several biological processes and therapeutic strategies based on their delivery appear extremely promising for tissue regeneration [1]. However, current delivery systems remain suboptimal: viral vectors are limited by safety issues and high costs, while commercial transfection agents exhibit poor stability, low loading efficiency, and uncontrolled release kinetics [2]. To overcome these challenges, novel hybrid polymer-lipid nanoparticles (H-NPs) were designed as innovative transfection agent for miRNAs delivery.
H-NPs were prepared using a patented nanoprecipitation method with a lipoplex core capable of encapsulating miRNAs and a polymeric shell to enhance stability [3]. H-NPs showed nanometric size, negative Z-potential, excellent miRNA encapsulation efficiency (99%), and good colloidal stability. Controlled miRNA release was observed for up to 9 days, compared with complete release within 72 h for commercial agent Lipofectamine RNAiMAX. As a proof of concept, two strategies were explored to promote cardiac regeneration following myocardial infarction: direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocytes using miRcombo (miR-1, 133, 208, 499) [4], and stimulation of cardiomyocyte proliferation in H9c2 cells with miR-199 [1]. AHCFs transfected with H-NPs had superior cell viability (100% vs. 70%) compared to RNAiMAX, while in H9c2 viability was comparable (100%). Both cell types efficiently internalized H-NPs. Transfection with miR-1-loaded H-NPs induced significant downregulation of TWF-1 after 48 h, while miRcombo/H-NPs increased cardiac troponin T expression in AHCFs at gene and protein levels after 15 days, indicating successful reprogramming. In H9c2, miR-199/H-NPs enhanced Ki67-positive cell counts after 72 h, confirming proliferative activity. Furthermore, H-NPs maintained physicochemical and biological properties after freeze-drying with trehalose and 14 days of storage.
These results highlight H-NPs versatility and potential for broader therapeutic and industrial applications, overcoming key limitations of existing transfection systems.
This work was supported by the European Research Council under the European Union’s Horizon research and innovation program (BIORECAR – 772168, POLIRNA - 101113522).
1. Gabisonia et al., Nature, 2019
2. Lee et al., J. Control. Release, 2019
3. Nicoletti, Paoletti et al. AHM, 2025
4. Paoletti et al., Front. Bioeng. Biotechnol., 2020
Comparative evaluation of mesoporous silica and polyplex-based nanocarriers for controlled RNAi release against post-traumatic stress disorder (PTSD)
Sofía Mares Bou1, Alessandra Morri2, Joaquín Ródenas Rochina3, Gloria Gallego Ferrer4, Joel Girón Hernández5, Piergiorgio Gentile6
1Center for Biomaterials and Tissue Engineering (CBIT), Valencia - Spain, 2Politecnico di Torino, Torino (Italia) - Italy, 3Center for Biomaterials and Tissue Engineering (CBIT), Valencia - Spain, 4Center for Biomaterials and Tissue Engineering (CBIT) & Center for Biomedical Research Network in Biomaterials, Bioengineering and Nanomedicine (CIBER-BBN), Valencia - Spain, 5Center for Biomaterials and Tissue Engineering (CBIT), Valencia - Spain, 6Center for Biomaterials and Tissue Engineering (CBIT) & Center for Biomedical Research Network in Biomaterials, Bioengineering and Nanomedicine (CIBER-BBN), Valencia - Spain
The development of nanocarrier systems capable of efficiently delivering RNA interference (RNAi) molecules, including small interfering RNA (siRNA) and microRNA (miRNA), represents a promising frontier in precision therapeutics. This study aimed to develop more effective therapies for post-traumatic stress disorder (PTSD) through the integration of nanotechnology with next-generation therapeutics. Specifically, we engineered two complementary nanoparticle systems: (i) amino mesoporous silica nanoparticles (SN) (size ∼20 nm), and (ii) chitosan/poly(lactic-co-glycolic acid) (Ch/PLGA) polyplexes nanoparticles (PN) (size ∼200 nm). Each nanocarrier was developed according to the physicochemical nature of the RNA cargo and the intended release kinetics, combining structural stability with biocompatibility. Indeed, silica nanoparticles were employed for RNAi surface loading, whereas Ch/PLGA polyplexes were produced via emulsion–solvent evaporation and electrostatic complexation for RNAi internal encapsulation. The RNAi agents included siRNA-PPP6C-mus-827, targeting the PPP6C gene involved in neuronal potassium channel modulation (Kv3.2), and miRNA-hsa-511-5p, a regulator of a PTSD-associated transcriptional gene (FKBP5), and also in the expression of inflammatory cytokines. Both systems were further modified using a Layer-by-Layer (LbL) assembly, testing different numbers of polyelectrolyte bilayers (pectin as polyanion and chitosan as polycation) to tune surface charge for enhancing their cell uptake, and RNAi retention and release behavior. Dynamic light scattering confirmed nanoscalesize distribution (SN/PN 20-90/200-300 nm), and tunable zeta potential (SN/PN +40/+25 mV) for polyplex-based nanoparticles, while fluorescence-based assays quantified encapsulation efficiency (SN/PN 95/97%). CryoSEM revealed well-defined morphologies and uniform surface coatings. RNAi release kinetics in PBS (37°C) demonstrated controlled and sustained release, strongly influenced by the number of LbL layers. In vitro cytocompatibility assays in SH-SY5Y neuroblastoma cells confirmed excellent biocompatibility and uptake demonstrated by flow-cytometry. qPCR has confirmed the successful modulation of FKBP5 expression, indicating that an in vitro PTSD model has been effectively established.
Cell-Dense tissue bioprinting
Y. Shrike Zhang
de Harvard Medical School, Boston (Massachusetts) - United States
Cell-dense tissues are essential in tissue engineering because they closely replicate the structural and functional characteristics of native organs. High cell density promotes extensive cell-cell and cell-matrix interactions, drives extracellular matrix production, and supports tissue-specific differentiation and maturation. These properties are crucial for achieving physiologically relevant mechanical strengths, metabolic activities, and long-term functions. However, current bioprinting approaches often fail to reproduce such dense yet sophisticated cellular architectures. Technical constraints in bioink formulation and printing methods typically result in constructs with insufficient cell content. This lack of cellular richness restricts the maturation and functional integration of bioprinted tissues. In this presentation, I will discuss our recent progress in developing technologies to fabricate functional, cell-dense engineered tissues. These approaches are likely to offer new opportunities in drug discovery, therapeutic screening, and regenerative medicine.
Miniature engineered heart tissues from human iPSC-cardiomyocytes on a hypoxia-on-chip platform
Lotta Kulmala1, Joona Valtonen1, Joose Kreutzer2, Mari Pekkanen-Mattila1, Katriina Aalto-Setälä3
1Heart Group, Faculty of Medicine and Health Technology. Tampere University, Tampere (Southern Finland) - Finland, 2Biogenium Microsystems Ltd. Tampere University, Tampere (Southern Finland) - Finland, 3Heart Hospital. Tampere University, Tampere (Southern Finland) - Finland
Ischemic heart disease (IHD) is the leading cause of death worldwide. It results from reduced myocardial blood flow, causing oxygen deprivation and scar tissue formation. While reperfusion therapy and antiarrhythmic drugs are standard treatments, reperfusion may aggravate tissue injury. Promising results from animal studies have not translated to clinical success, highlighting the need for human-based models. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a patient-specific platform for in vitro cardiac research. Our aim is to develop a more physiologically relevant model to study cardiac responses to hypoxia and support therapeutic development.
Here we present a novel 3D hypoxia-on-a-chip platform integrating miniature engineered heart tissues (mini-EHTs) with a system enabling precise oxygen control. The base of the platform is an OxyGenie mini-incubator (BioGenium Microsystems, Finland) combined with field stimulation electrodes and a custom EHT-insert. This setup enables electrical stimulation and real-time functional analysis of cardiac tissue under hypoxic conditions.
Mini-EHTs formed from iPSC-CMs begin spontaneously beating within the first week, with the beating rate stabilizing by week two. Fixed samples collected at weeks 2–4 show progressive cardiomyocyte alignment, indicating tissue development. Contractile force is quantified by measuring deflection of the EHT insert’s flexible poles. Functionality can be assessed real time during experiments, including beating rate, arrhythmia patterns, and contractile dynamics. During oxygen deprivation, hypoxic mini-EHTs are visualized using a live hypoxia reagent, supporting dynamic imaging and analysis. These preliminary results demonstrate strong potential for IHD modeling.
Our hypoxia-on-a-chip platform effectively combines iPSC-CMs and EHT technology to model cardiac hypoxia in vitro. This human-based system enables detailed functional analysis and may help bridge the translational gap between preclinical findings and clinical applications.
Cost-efficient iPSC-derived hepatocyte differentiation via solid-phase growth factor presentation
Estela Sanchez Gonzalez1, Oana Dobre2, Manuel Salmeron Sanchez3, Gloria Gallego Ferrer4, Laia Tolosa5
1Experimental Hepatology Unit. Health Research Institute of La Fe, Valencia - Spain, 2Centre for the Cellular Microenvironment. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, 3Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 4Centre for Biomaterials and Tissue Engineering (CBIT). Universitat Politècnica de València, Valencia - Spain, 5Health Research Institute of La Fe, Valencia - Spain
Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) represent a promising in vitro model thanks to their ability of self-renewal and in vitro expansion as well as their ability to recapitulate the effects observed in the individuals. However, conventional two-dimensional (2D) differentiation protocols often lead to immature HLCs and incur high costs due to the frequent supplementation with growth factors [1]. Three-dimensional (3D) models and solid-phase growth factor presentation have been shown to improve differentiation by providing biochemical cues similar to those of native extracellular matrices [2]. This study aimed to develop a 3D liver model based on collagen-fibronectin (COLFN) hydrogels with solid-phase presentation of hepatocyte growth factor (HGF), enhancing hepatic differentiation while reducing HGF and costs [3,4].
Hydrogels were characterised by rheology and Brillouin microscopy. Embryonic stem cells (ESCs) and iPSCs were differentiated into hepatoblasts using a multi-step 2D differentiation protocol. Hepatoblasts were aggregated and cultured in spheroids for 48h and encapsulated in hydrogels until day 28. The differentiation was monitored by qPCR, flow cytometry and immunofluorescence. Cell functionality was assessed by urea, albumin, and alpha-1 antitrypsin production.
Hydrogels showed liver-like mechanical properties and controlled HGF retention. The differentiation in 3D with HGF showed improved phenotype and functional maturation (higher expression of albumin, alpha 1-antitrypsin and cytokeratin 18) and gene analysis confirmed the upregulation of drug transporters and cytochromes. Cells in COLFN hydrogels with solid-phase HGF exhibited higher functionality compared to monolayer and spheroid controls.
This platform promotes efficient hepatic differentiation from iPSCs, enabling the generation of functional HLCs suitable for personalised models and drug testing.
REFERENCES
1. Tricot, T., et al. Cells 11(3), 442 (2022).
2. Zhang, S., et al., Communications Biology, 6(1), 62 (2023).
3. Dobre, O., et al. Advanced Functional Materials, 31(21), 2010225 (2021).
4. Sarrigiannidis, S., et al. Materials Today Bio, 20, 100641 (2023).
Establishing a 3D tendinopathy model in vitro by co-culture of tissue-engineered tendon constructs with pro- and anti-inflammatory macrophages
Ekaterina A. Oleinik1, Marguerite Meeremans2, Andreas H. Teuschl-Woller3, Sandra Van Vlierberghe4, Catharina De Schauwer2, Philipp J. Thurner1
1Institute of Lightweight Design and Structural Biomechanics. TU Wien, Vienna (Wien) - Austria, 2Veterinary Stem Cell Research Unit. Ghent University, Ghent (Oost-Vlaanderen) - Belgium, 3University of Applied Sciences Technikum Wien, Vienna (Wien) - Austria, 4Polymer Chemistry and Biomaterials Group. Ghent University, Ghent (Oost-Vlaanderen) - Belgium
Background: Tendinopathies are increasingly prevalent and pose significant healthcare burden, yet their pathogenesis remains poorly understood. Development of physiologically relevant in vitro models is essential to elucidate disease mechanisms and develop new therapeutic strategies. Currently, no standardized model reproduces complex inflammatory environment of tendinopathy. This study aims to establish in vitro tendinopathy model, incorporating key inflammatory aspects and allowing investigation of tissue adaptation and repair mechanisms.
Methods: Equine tendon-derived cells (TDCs) were encapsulated in fibrin scaffolds and cultured in Ebers TC-3 bioreactor. Tendon constructs underwent 7-day maturation under dynamic mechanical stimulation (10% nominal uniaxial tensile strain at 0.5 Hz for 6 h/day, followed by 3% strain at 0.5 Hz for 18 h/day). On day 8, either M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages, differentiated from peripheral blood monocytes using IFN-γ/LPS or IL-4 accordingly, were co-cultured with tendon constructs for up to 72h. In parallel, we incrementally increased nominal strain (up to 16%) to induce pathological overload. Cell viability, morphology and proliferation were assessed using Ca-AM/PI and Phalloidin/DAPI staining. Gene expression of tendon-specific, ECM-related, and inflammation-associated markers was analyzed by RT-qPCR, and transcriptomic profiles were examined by RNAseq (ONT).
Results: Both co-cultured groups (M1 and M2) showed markedly enhanced proliferation compared to CTRL (stimulation only). M1 group displayed upregulation of Il6, Cxcl8, and MMP1/9, indicating the acute inflammatory phase, whereas M2 group showed increased VEGF and TGFb1 expression, suggesting a shift toward the proliferative phase of the inflammatory cascade. RNAseq confirmed distinct gene clusters reflecting differential TDCs responses to macrophages depending on their subtype.
Conclusion: These results show successful co-culture of TDCs and macrophages within the bioreactor. Our approach offers a promising platform to investigate tenocytes-macrophages interactions, and holds promise to be used as a preclinical drug-testing platform.
Funding by Austrian Science Fund FWF (DFH-28), City of Vienna (SequenceTissue, MA23 #32-02), FWO funding (1S02822/24N, V418424N and 12E4523N), TENET COST Action (CA22170, STSM grant) and ESB Travel Award are gratefully acknowledged.
Effects of bovine milk-derived extracellular vesicles on a 3D intestinal stromal scaffold
Sharon Arcuri1, Georgia Pennarossa1, Madhusha Prasadani2, Fulvio Gandolfi3, Alireza Fazeli2, Tiziana Brevini1
1Department of Veterinary Medicine and Animal Sciences. UNIVERSITA' DEGLI STUDI DI MILANO, LODI (Italia) - Italy, 2Institute of Veterinary Medicine and Animal Sciences. Estonian University of Life Sciences, Tartu - Estonia, 3Department of Agricultural and Environmental Sciences-Production, Landscape, Agroenergy. UNIVERSITA' DEGLI STUDI DI MILANO, LODI (Italia) - Italy
Milk represents an important dietary component due to its rich nutritional profile and contains nanoscale extracellular vesicles (EVs), which are key mediators of intercellular communication. Milk-derived EVs (MEVs) have been shown to influence intestinal homeostasis by modulating inflammation, shaping gut microbiota composition, and supporting epithelial integrity. However, their effects on the intestinal connective tissue remain largely unexplored. Here, we investigated MEV effects on a a three-dimensional (3D) intestinal extracellular matrix (ECM)-based in vitro model.
Porcine primary intestinal stromal cells were cultured in ECM-derived bio-scaffolds and exposed to increasing concentrations of bovine MEVs (106, 108, 1010 particles/mL). Experimental groups included: (i) ECM pre-treated with MEVs before cell seeding, (ii) untreated ECM with MEVs added to stromal cells, (iii) untreated ECM with stromal cells and fetal bovine serum (CTR FBS), and (iv) untreated ECM with stromal cells without MEVs or serum (CTR w/o FBS).
MEVs induced a dose-dependent increase in stromal fibroblast proliferation. Treatment with 106 particles/mL did not significantly alter proliferation compared to CTR w/o FBS. In contrast, 108 and 1010 particles/mL applied directly to stromal cells restored proliferation to levels comparable with CTR FBS. Notably, pre-conditioning the ECM scaffolds with 108 or 1010 particles/mL MEVs further enhanced proliferation, yielding significantly higher values than CTR FBS.
These findings demonstrate the possibility to successfully generate functional intestinal biological scaffolds that recapitulate key features of the native tissue. The addition of bovine MEVs at concentrations higher than 106 particles/mL induced a statistically significant dose-dependent increase in the stromal cell density that was further boosted when MEVs were used to pre-condition the intestinal biological scaffolds.
Altogether these data suggest the possibility to use MEVs as in vitro enriching supplements, to reduce/replace FBS in intestinal cell cultures and, more in general, imply a potential trophic role on the intestinal stromal compartment.
Supramolecular injectable hydrogels with tunable microporous structures improve therapeutic efficacy of cell transplantation for tissue regeneration
Akihiro Nishiguchi1, Hana Yasue1, Tetsushi Taguchi1
1National Institute for Materials Science, Tsukuba (Ibaraki) - Japan
Injectable hydrogels hold promise as a cell delivery carrier to enable local administration of transplanted cells and improve therapeutic efficacy. However, hydrogels usually do not have micrometer-scale pores due to the densely cross-linked polymer networks, limiting the communication between transplanted cells and host tissues and resulting in low therapeutic efficacy.
Here, we aim to design cell-delivering, supramolecular injectable hydrogels with micropores by utilizing liquid-liquid phase separation (LLPS) [1-3]. We found that ureido-4[1H]-pyrimidinone (UPy) group-modified gelatin (GUPy) formed microcapillary network (μCN)-like LLPS structures in hydrogels. By mixing GUPy with thiolated gelatin (GTH), vinyl sulfonated G (GVS), and cells, phase-separated injectable hydrogels with tunable microporous structures were formed, and cells were encapsulated in hydrogels. The porous network structures help mass transport such as oxygen and nutrients and cell adhesion as a scaffold. Cells were encapsulated in GTH-GVS gel (non-porous) and GTH-GVS-GUPy gel (μCN), showing enhanced cell adhesion, extension, migration and proliferation in μCN. Furthermore, when MSC-encapsulated μCN hydrogels were administered to ischemic models, blood flow was recovered due to enhanced graft survival via material-host communication through the μCN structures. This approach is useful as a cell-delivering method to improve the therapeutic efficacy of cell transplantation therapy.
In this presentation, we will present the observation of LLPS hydrogels, encapsulation of mesenchymal stem cells (MSCs), evaluation of cellular functions (adhesion, spreading, migration, proliferation), and therapeutic efficacy in hindlimb ischemia models of mice.
References:
1. A. Nishiguchi et al, Biomaterials 305, 122451 (2024)
2. H. Yasue et al., Adv. Funct. Mater., in press.
Mimicking osteochondral architecture through integrated multi-scale 3D in vitro scaffolds
Aiste Pupiute1, Darius Ciuzas1, Odeta Baniukaitiene1, Dainius Martuzevicius1, Edvardas Bagdonas2, Eiva Bernotiene2, Tomas Ragauskas2, Edvinas Krugly1
1Faculty of Chemical Technology. Kaunas University of Technology, Kaunas (Kauno Apskritis) - Lithuania, 2Department of Regenerative Medicine. State Research Institute Centre for Innovative Medicine, Vilnius (Vilniaus Apskritis) - Lithuania
Introduction. Degenerative joint diseases such as osteoarthritis (OA) affect nearly one quarter of Europe’s population and remain a major therapeutic challenge. Cartilage and subchondral bone restoration requires precise tissue architecture and mechanical integrity. This study presents the development of a six-layer osteochondral model replicating the native cartilage-bone interface, aiming to provide an efficient tool for the in vitro modelling of cartilage regeneration
Methods. To mimic osteochondral tissue, six-layer 3D scaffolds were fabricated using combined melt and solution electrospinning. The cartilage region consisted of poly(ε-caprolactone) (PCL) and the bone region of PCL with 5% hydroxyapatite (HAp), separated by a nanofibrous tidemark-like interlayer. Scaffolds were treated with non-thermal plasma (NTP) to enhance hydrophilicity and cell adhesion, then characterized for mechanical and physicochemical properties. Human chondrocytes and osteoblasts were cultured on respective regions, and cell viability, proliferation, and morphology were evaluated by fluorescence staining and histology.
Results. The scaffolds exhibited interconnected porosity with gradient pore sizes varying from 112.1±28.0µm to 51.4±11.2µm, thus enabling homogeneous nutrient diffusion. The tidemark-like interlayer featured very small pores (0.87±0.18µm) and thin fibers (0.42±0.08 µm), preventing cell migration. The calcified cartilage layer was designed with the smallest pores (51.4±11.2µm), while the subchondral bone region with the largest (112.1±28.0µm). NTP treatment decreased the water contact angle from 108.0±1.7° to 49.6±1.5° and increased fiber surface roughness (Ra from 10.2±0.9nm to 36.0±2.1nm). The model seeded with both cell types supported high viability, robust proliferation, and homogeneous colonization with a gradual chondrocyte-osteoblast transition across the interface.
Conclusions. The biphasic PCL/PCL-HAp scaffolds support spatially organized chondrocyte and osteoblast growth, closely resembling native osteochondral architecture. This biomimetic in vitro model enables investigation of osteochondral interactions, disease progression, and therapeutic testing while advancing 3R principles and reducing animal use.
Spatially guiding collagen organization for functional tissue engineered cartilage
Jet Peters1, Sebastien Callens1, Keita Ito1
1Orthopaedic Biomechanics, Department of Biomedical Engineering and Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
Introduction
Articular cartilage is characterized by its unique extracellular matrix (ECM), where the highly organized alignment of collagen fibers plays a critical role in its mechanical function and durability. However, tissue-engineered cartilage grown in uncontrolled environments often lacks this organization, compromising long-term performance. Physical confinement can influence tissue architecture, which suggests a link between external physical cues and collagen fiber alignment [1]. This raises a fundamental question: can we deliberately direct microscale tissue organization, specifically collagen alignment, by guiding engineered cartilage growth through applied macroscale boundaries?
Methods
Mini-well arrays of varying geometries were fabricated by casting polydimethylsiloxane (PDMS) with stamps. To promote initial attachment and sustained cell interaction, PDMS walls were functionalized with type I collagen. Bovine chondrocyte-derived cartilage organoids [2] were seeded into the mini-wells forming a single layer. Tissues were cultured up to 4 weeks and tissue morphology and volume were monitored over time. The ECM organization and microstructural features were analyzed using histological staining, quantitative polarized light microscopy and scanning electron microscopy.
Results & Discussion
Preliminary results revealed that tissue morphology and collagen organization is clearly affected by the physical boundaries of the fabricated mini-wells. Using cylindrical wells, cartilage tissues adopt a cylindrical morphology with collagen predominantly aligning along the walls, while tissues grown on flat surfaces form a mushroom-like shape. In these tissues, peripheral type II collagen aligned along the curved surface, while internal collagen was predominantly oriented perpendicular to it, mimicking articular cartilage-specific collagen organization. Ongoing research explores the influence of physical boundaries and mechanisms behind growth-induced mechanics shaping tissue morphology and ECM organization, aiming to control collagen alignment and engineer cartilage with native functionality.
[1] Peters et al., Sci Rep. 2024
[2] Crispim JF, Ito K, Acta Biomater. 2021.
Financially supported by Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research (024.003.013).
Developing a fully synthetic, peptide-based animal-free model of pancreatic cancer
Emmanouela Mitta1, Luke Tappouni1, John Counsell1, Aline Miller2, Deepak Kalaskar3, Eirini Velliou1
1Department of Targeted Intervention. University College London (UCL), London (London, City of) - United Kingdom, 2Department of Materials, School of Natural Sciences, Faculty of Science and Engineering and The Henry Royce Institute, Royce Hub Building, The University of Manchester, Manchester - United Kingdom, 3Department of Orthopaedics and Musculoskeletal Science. University College London (UCL), London (London, City of) - United Kingdom
Pancreatic cancer (PC) is a lethal disease with high tumour tissue complexity, for the study of which human relevant models are needed. Similarly to many other human diseases, PC models are often built using animal-derived products to support cell attachment and growth. Despite offering good biocompatibility they lack accurate human tissue mimicry. In light of the recent Federal Drug Association (FDA) regulatory changes (1), efforts are made to reduce the use of animals and animal-derived products in drug screening as they fail to predict successful drug candidates. In this study we replace animal-derived protein coatings in our previously developed PC models(2–4), with synthetic peptides and assess their suitability (and potential differences) for developing fully synthetic models of PC. Polyurethane scaffolds were prepared (2) and coated with synthetic peptides derived from collagen, laminin and fibronectin using two methods; physisorption via centrifugation (3) or chemical modification using plasma and carboxylic acid grafting. Peptide topography within the scaffold was assessed by fluorescent imaging. Thereafter, the peptide coated scaffolds were seeded with cancer or stroma cells and cultured for 21 days. The effect of peptide type and concentration was assessed via monitoring of viability and metabolic activity. Coating via centrifugation and plasma modification yielded uniform dispersion of peptides within the scaffold. Plasma modification led to a longer lasting coating and more densely populated scaffolds compared to physisorption. When compared to historic data of animal protein-coated scaffolds, viability was similar to peptide-coated scaffolds. Synthetic peptides can therefore readily replace animal-derived proteins without compromising on biocompatibility.
1. FDA (2025) FDA Announces Plan to Phase Out Animal Testing Requirement for Monoclonal Antibodies and Other Drugs.
2. Kataki, AD et al. (2025) Mapping Tumor–Stroma–ECM Interactions in Spatially Advanced 3D Models of Pancreatic Cancer. ACS Applied Materials & Interfaces
3. Gupta, P et al. (2020) A Novel Scaffold-Based Hybrid Multicellular Model for Pancreatic Ductal Adenocarcinoma—Toward a Better Mimicry of the in vivo Tumor Microenvironment. Front Bioeng Biotechnol
4. Gupta, P et al (2024) Chemotherapy Assessment in Advanced Multicellular 3D Models of Pancreatic Cancer: Unravelling the Importance of Spatiotemporal Mimicry of the Tumor Microenvironment. Advanced Biology.
RENOVATE Project: Regenerative engineering innovation in early intervention of osteoarthritis
Zaida Ortega1, Chaozong Liu2, Miguel Oliveira3, Rubén Paz1, Renata Grzela4, Ilaria Caccioti5, Pablo Bordón1, Giulio Maccauro6, Dominika Gołubczyk7, Ricardo Donate1, Rocío Moriche8, Iulian Antoniac9, Mario Monzón1
1University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria (Las Palmas) - Spain, 2University College London, London (London, City of) - United Kingdom, 33IBs. University of Minho, Braga - Portugal, 4University of Warsaw, Warsaw (Mazowieckie) - Poland, 5University of Rome Niccolò Cusano, Rome (Lazio) - Italy, 6Policlinico Universitario Agostino Gemelli, Rome (Lazio) - Italy, 7Ticom, Olsztyn (Warminsko-Mazurskie) - Poland, 8Universidad de Sevilla, Sevilla - Spain, 9University Polytechnica of Bucharest, Bucharest - Romania
Recent advances in tissue engineering are helping to overcome key limitations in osteoarthritis (OA) treatment. While regenerative strategies for cartilage lesions offer promising alternatives to joint replacement, current approaches still face challenges in achieving durable and satisfactory outcomes throughout disease progression.
The biomedical engineering sector is rapidly evolving, demanding new knowledge, skills, and approaches to deliver robust therapeutic solutions. However, existing doctoral programmes only partially address these needs. This presentation introduces the RENOVATE project to the targeted and specialized attendants in the TERMIS community. The main scientific aims of the project are to:
- Define patient-specific functional and mechanical requirements for large osteochondral scaffolds and develop new therapeutic schemes
- Enhance the physicochemical, biological, and mechanical fixation properties of scaffolds
- Produce functionally graded large osteochondral scaffolds using advanced 3D printing technologies
RENOVATE brings together experts from clinical, academic, and industrial sectors to deliver a high-level, personalized transnational and interdisciplinary training programme. This initiative will provide future researchers with the skills to lead innovation in biomedical engineering and orthopaedics. The project’s core challenge is to develop a multinational, multisectoral, and multidisciplinary research and training framework focused on novel technologies and strategies for large osteochondral defect repair.
Acknowledgements
RENOVATE project has received funding from the European Union’s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101227121 (101227121 - RENOVATE - HORIZON-MSCA-2024-DN-01).
Injectable platelet rich fibrin-enhanced vascular spheroid units for regenerative applications
Xiaohe Liu1, Zhentao Xing1, Jiaxin Qin1, Laetitia De Kort1, Petra De Graaf1
1Department of Urology. University Medical Center Utrecht, Utrecht - The Netherlands
Background: Rapid vascularization is crucial for tissue healing and is particularly important in urethral reconstruction, where outcomes are often limited by poor microvascularization. Vascular spheroids (VS) provide pre-formed endothelial networks, while injectable platelet-rich fibrin (iPRF) supplies autologous growth factors and a supportive 3D matrix. Their combination may enhance angiogenesis and enable a personalized, minimally invasive option for urethral regeneration.
Methods: Green fluorescent protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) were co-cultured with human mesenchymal stem cells (MSCs; adipose- or bone marrow-derived) in 600 μm microwells to assemble VS. VS with HUVEC:MSC ratios of 1:1, 1:3, and 1:9 were compared. VS were cultured in endothelial growth medium-2 (EGM-2) or vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF)-depleted EGM-2. VS were mixed with 4 mg/mL type I collagen (static) or iPRF (injected via a 25G syringe), then either cultured in vitro or placed on chick chorioallantoic membranes (CAMs) for 4 days. Confocal microscopy, enzyme-linked immunosorbent assay (ELISA) for VEGF/bFGF, and alpha-smooth muscle actin (α-SMA) immunofluorescence were performed.
Results: Uniform VS formed within 24 h. The 1:9 condition exhibited the most regular morphology and strongest sprouting. VS sprouted in VEGF/bFGF-depleted medium; ELISA showed VEGF was higher in 1:9 VS than in lower-ratio VS (n=3, p<0.05), while bFGF did not increase, suggesting a VEGF-dominant paracrine mechanism from MSCs. In vitro, iPRF enhanced VS sprouting, resulting in greater sprout length than collagen-embedded VS on confocal imaging. On CAM, VS–iPRF constructs attracted host vessels growing towards and into the implant, whereas collagen alone did not. α-SMA staining showed MSCs reinforced spheroid structure and guided sprouting.
Conclusions: When combined with iPRF, VS forms injectable, prevascularized constructs in vitro. MSCs secrete VEGF, resulting in angiogenesis, whereas iPRF further enhances the angiogenic potential of VS. Together, they create an autologous matrix well-suited for personalized regenerative therapies.
Cell-Scale porosity: engineering immune-compatible biomaterials for tissue regeneration
Donald Griffin1, Colleen Roosa1, Jeremy Ortmann1, Ethan Nicklow1, Claudia Daboin1, Daniel Abebayehu1, Donald Griffin1
1Biomedical Engineering. University of Virginia, Charlottesville (Virginia) - United States
Traditional nanoporous hydrogels trigger foreign body responses that impede tissue regeneration, creating fibrous capsules that isolate implants from host tissue. This keynote starts with mechanistic evidence that microporous annealed particle (MAP) scaffolds with cell-scale porosity fundamentally alter how cells interpret biomaterial presence. Building on established work showing pore dimension effects on isolated macrophage behavior, our findings reveal how porosity orchestrates multi-cellular crosstalk networks that govern tissue development, including paracrine signaling between multiple cell lineages. Through integrated analysis using mass cytometry, single-cell RNA sequencing, and cytokine profiling, we reveal that MAP's pore architecture induces widespread downregulation of canonical foreign body recognition pathways at the transcriptional level. While nanoporous controls activate classical inflammatory cascades, cells responding to MAP scaffold implantation exhibit suppressed expression of key foreign body response genes including inflammatory cytokine signaling and fibrotic matrix deposition pathways. Critically, uncovering this mechanism required the complementary power of all three analytical approaches—mass cytometry for population-level cell phenotyping, single-cell RNAseq for discovering altered transcriptional programs, and cytokine analysis for validating functional outcomes.
This talk will also explore two of our emerging advances to extend MAP's capabilities for next-generation tissue engineering. First, we introduce time-controlled dynamic porosity through strategic incorporation of particle populations with differential in vivo stability profiles, enabling temporal evolution of scaffold architecture as regeneration progresses. Second, we present MAP-specific surface modifications utilizing selective extracellular matrix molecule conjugation to control initial cell-material interactions at the pore interface, directing early cellular responses. Together, these innovations establish design principles for immune-compatible biomaterials that actively promote healing through architectural control of cellular interpretation and multi-cellular coordination.
Tissue regeneration using adipose-derived stem cells (ADSCs) or micro-fragmented adipose tissue (MFAT): impact of cellular senescence
Beatrice Castiglioni1, Roberto Narcisi2, Carlo Tremolada3, Marica Imbimbo1, Michela Bosetti1
1Department of Pharmacological Science (DSF). University of Eastern Piedmont, Novara (Piemonte) - Italy, 2ERASMUS Medical Center Rotterdam, Rotterdam (Zuid-Holland) - The Netherlands, 3IMAGE Regenerative Clinic, Milano (Lombardia) - Italy
Despite the promising outcomes of stem cell–based therapies, clinical failure rates remain substantial. One of the main contributing factors is cellular senescence, which often arises during the in vitro expansion phase required for large-scale cell production. Senescent mesenchymal stem cells (MSCs) exhibit impaired proliferative capacity, altered secretory profiles, and reduced differentiation potential, ultimately compromising their therapeutic efficacy and regenerative performance.
In this study, we compared the senescence profiles of adipose-derived stem cells (ADSCs) isolated from enzymatically digested microfragmented adipose tissue (MFAT) obtained using the Lipogems technique with those obtained through the spontaneous outgrowth of cultured MFAT. By analyzing differences in senescence-associated markers between these two isolation strategies, our work aims to elucidate whether preserving the native stromal vascular niche can reduce cellular aging. These findings may contribute to optimizing ADSC preparation and improving the overall efficacy of MSC-based regenerative therapies.
Our results reveal distinct senescence patterns between ADSCs isolated by collagenase digestion of MFAT and those obtained through spontaneous migration from cultured MFAT fragments, suggesting that preservation of the native stromal niche in the spontaneous outgrowth model may mitigate the onset of senescence compared to enzymatic digestion.
These findings highlight the importance of preserving the stromal vascular niche, as in Lipogems-derived MFAT, to reduce senescence-related alterations in ADSCs and enhance their regenerative performance in clinical applications.
Acknowledgments: PRIN 2022HB7PNP
Interface engineering of polymer-ceramic core-shell nanofibers by alkaline hydrolysis enables controlled TGF-β3 delivery
Edvinas Krugly1, Aiste Pupiute1, Lauryna Bagdoniene1, Edvardas Bagdonas2, Eiva Bernotiene2, Darius Ciuzas1, Eidvyle Gasiulyte1, Dainius Martuzevicius1
1Faculty of Chemical Technology. Kaunas University of Technology, Kaunas (Kauno Apskritis) - Lithuania, 2Department of Regenerative Medicine. State Research Institute Centre for Innovative Medicine, Vilnius (Vilniaus Apskritis) - Lithuania
Aim. To engineer the surface chemistry of cryo-electrospun coaxial fibers composed of poly(ε-caprolactone) (PCL) - hydroxyapatite (HAp) core and cellulose acetate (CA) shell via alkaline hydrolysis, thereby enhancing wettability and modulating transforming growth factor-β3 (TGF-β3) release for cartilage tissue regeneration.
Materials and methods. Randomly oriented core-shell mats were fabricated by cryo-electrospinning and subjected to brief alkaline hydrolysis. Morphology (scanning electron microscopy, SEM; transmission electron microscopy, TEM), surface chemistry (Fourier transform infrared spectroscopy, FTIR; toluidine blue O assay, TBO), wettability (contact angle), thermal/crystalline states (thermogravimetric analysis, TGA; differential scanning calorimetry, DSC; X-ray diffraction, XRD), and fluid absorption (phosphate-buffered saline, PBS uptake) were assessed. TGF-β3 release was quantified over one week and fitted with a first-order model.
Results. Hydrolysis increased carboxylic functional groups (TBO: ∼2.4 × 104→∼4.2 × 104 μmol·g−1), reduced water contact angle (∼17%), and markedly increased PBS uptake (∼4-fold). Fiber diameter shifted from a median ∼0.6 to ∼0.9 μm, while pore size decreased from ∼7.5 to ∼4.5 μm; TEM revealed a core:shell ratio near 5:1. FTIR/TGA/DSC/XRD indicated partial regeneration of cellulose II from cellulose acetate. Treated scaffolds showed a lower day-1 burst release of TGF-β3 (from ∼39% to ∼31%), followed by sustained release through day 7 (cumulative ∼53% untreated vs ∼51% treated).
Conclusions. Alkaline hydrolysis provides a simple, scalable post-processing step that couples processing to surface chemistry and transport in PCL-HAp/CA coaxial fibers, yielding more hydrophilic, growth-factor-retentive scaffolds with suppressed burst and sustained TGF-β3 delivery. These materials show promise for cartilage tissue engineering; ongoing work will evaluate in vitro and in vivo performance.
Dual-crosslinked alginate hydrogel for sustained antioxidant nanoparticles delivery in colon-targeted therapy
Roberta Rovelli1, Eunhyung Kim2, Teresa Macchi3, Heungsoo Shin4, Serena Danti5
1Doctoral School in Life Sciences. University of Siena, SIENA (Toscana) - Italy, 2Department of Bioengineering. Hanyang University, Seoul (Seoul-tukpyolsi) - South Korea, 3Department of Translational Research and New Technologies in Medicine and Surgery. University of Pisa, Pisa (Toscana) - Italy, 4Department of Bioengineering. Hanyang University, Seoul (Seoul-tukpyolsi) - South Korea, 5Department of Civil and Industrial Engineering. University of Pisa, Pisa (Toscana) - Italy
Chronic psychological stress disrupts the gut-brain axis by activating the hypothalamic-pituitary-adrenal (HPA) pathway, promoting mucosal inflammation, oxidative stress, and microbial dysbiosis in the colon.
The oxidative burst damages epithelial integrity and perpetuates inflammatory signaling. Hence, localized modulation of oxidative stress represents a strategic target for restoring gut homeostasis 1.
To address this challenge, we designed a zinc-calcium crosslinked sodium alginate (SA) hydrogel for the sustained release of antioxidant mineral-tannic acid nanoparticles (mTNs) under colon oxidative-stress conditions.
Low molecular weight, mannuronic-rich SA (50-80 kDa, Mannuronic/Guluronic ratio = 1.56) was selected to form a flexible network suitable for homogeneous mTNs incorporation. A mild pre-gelation step with trace Ca2+/Zn2+ ions was introduced to improve ion diffusion and prevent surface-only crosslinking during subsequent dual crosslinking, thereby enhancing network density and mechanical stability. The inclusion of Zn2+, beyond its structural role, was motivated by its reported involvement in epithelial repair and redox regulation.
Composite hydrogels containing 2, 3, and 4% (w/v) SA and 1% (w/v) mTNs were prepared and characterized for encapsulation efficiency, mechanical integrity, swelling, degradation, and release behavior in simulated colon fluid, alongside cytocompatibility on Caco-2 cells.
Encapsulation efficiencies exceeded 80%, while dual ion crosslinking provided tunable mechanical properties comparable to native colon tissue. The composite hydrogels exhibited gradual matrix relaxation and sustained mTNs release over 72 hours, while preserving antioxidant functionality and excellent cytocompatibility.
Overall, this dual-crosslinked hydrogel platform demonstrates strong potential as a colon-targeted antioxidant delivery system to mitigate oxidative stress–driven mucosal injury associated with chronic stress–related gut disorders.
Keywords: Sodium alginate hydrogels; antioxidant nanoparticles; colon-targeted delivery
Reference
1. Algieri, F. et al. (2023). Lactobacillus paracasei CNCM I-5220-derived postbiotic protects from the leaky-gut. Frontiers in Microbiology, 14, 1157164. https://doi.org/10.3389/fmicb.2023.1157164.
Assessing the influence of macromolecular crowding in allogeneic and xenogeneic culture of human tenocytes
Dimitrios Zeugolis1, Andrea Rossoni1
1Regenerative, Modular & Developmental Engineering Laboratory (REMODEL). University College Dublin, Dublin - Ireland
Introduction: Macromolecular crowding (MMC), by inducing tissue specific extracellular matrix (ECM) deposition, and human platelet lysate (hPL), by inducing physiological cell function, are considered invaluable eukaryotic cell culture media supplements. Yet again, human tenocytes (hTCs) are still customarily extracted, expanded and grown in foetal bovine serum (FBS), despite the fact that FBS is responsible for their rapid phenotypic drift in vitro. Herein, we hypothesise that hTCs cultured in hPL, as opposed to FBS, and especially under MMC conditions will maintain physiological basic function, protein synthesis and gene expression.
Methods: hTCs were extracted in FBS or hPL. At passage 4, hTCs were cultured in their respective media without and with MMC for 4, 6 and 8 days and basic function, protein synthesis and gene expression analyses were conducted.
Results: hPL did not affect hTC morphology and viability and significantly increased hTC proliferation at all time points. In general, FBS did not affect hTC morphology, viability and proliferation at any time point. In a given media, MMC did not affect hTC morphology, viability and proliferation at any time point. At all time points, in FBS and hPL, MMC significantly increased collagen I deposition and hPL significantly increased collagen III deposition. In hPL under MMC conditions, fibrogenic markers were significantly downregulated, whilst in FBS under MMC conditions, ageing and senescence markers were significantly upregulated.
Discussion: We found that FBS is associated with early hTC ageing during ex vivo culture, whilst hPL not only increases hTC proliferation, but also delays hTC in vitro ageing. Further, our data show that MMC enhances and accelerates ECM deposition, without upregulating fibrogenic markers.
Conclusions: Overall, this work advocates the use of hPL and MMC in hTC culture.
Acknowledgements: This work has received funding from Research Ireland (grant number 19/FFP/6982) and the European Research Council (ERC) (grant number 866126).
Macromolecular crowding in the development of organotypic tissue engineered medicines
Dimitrios Zeugolis1, Laura Trujillo-Cubillo1
1Regenerative, Modular & Developmental Engineering Laboratory (REMODEL). University College Dublin, Dublin - Ireland
Introduction: Macromolecular crowding (MMC) by decreasing molecular diffusion increases molecular interactions. In eukaryotic cell culture, it has been proposed that MMC accelerates the enzymatic processing of collagen, which in turn results in enhanced extracellular matrix deposition. Despite its well-established history, the optimal MMC agent remains elusive and the precise mechanism of action has yet to demonstrated. Further, MMC has yet to be assessed in the development of tissue engineered medicines using 3D scaffolds.
Methods: Cell morphology, viability, proliferation, metabolic activity and protein deposition were assessed at 3 time points (4, 6 and 8 days) in hDF cultures as a function of 6 different MMC agents at 10 different concentrations each. ELISA, colorimetric and immunofluorescence techniques were utilised to assess the influence of MMC on collagen proteinases activity; lysyl oxidase and transglutaminase activity; and matrix metalloproteinases activity. For the development of tissue engineered medicines, 10 different scaffolds (tissue grafts, freeze dried sponges, films, fibrous mats) were assessed and the developed tissue engineered medicines were histologically assessed.
Results: None of the MMC agents assessed affected hDF morphology, viability, proliferation and metabolic activity at any time point. Among the different MMC agents, the λ-CR induced the highest collagen I deposition at all time points. MMC significantly increased procollagen I C-propeptide, lysyl oxidase, transglutaminase and matrix metalloproteinases levels at the cell layers. Among the different scaffolds assessed, the fibrous mats allowed for the development of organotypic tissue engineered medicines.
Discussion: Following the principles of excluded volume effect, we showed that MMC induces the accelerated conversion of procollagen into crosslinked collagen. When MMC is combined with an appropriate 3D culture environment, organotypic advanced therapy medicinal products can be developed within days, as opposed to months, that traditional approaches require.
Conclusions: This work further advocates the utilisation of MMC in the development of tissue engineered medicines.
Acknowledgements: This work has received funding from Research Ireland (grant number 19/FFP/6982) and the European Research Council (grant number 866126).
Tumor-induced cachexia: unraveling cellular and molecular drivers through preclinical modeling
Rita Lima-Sousa1, Lucília P. Silva1, Rui L. Reis1, David Caballero1
13B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics. University of Minho, Guimarães (Braga) - Portugal
Tumor-induced cachexia (TiC) is a severe metabolic syndrome marked by involuntary and progressive weight loss, muscle wasting, and systemic inflammation, often seen in 80% of advanced cancer patients. Despite extensive research, no FDA/EMA-approved drugs exist for TiC treatment. A critical factor in its progression is the interaction between tumor cells, skeletal muscle (SM), and adipocyte tissue (AT). Recent research highlights the role of extracellular vesicles (EVs) secreted by cancer cells, which carry molecular cargo (e.g., cytokines, proteins, nucleic acids, metabolites) that can influence both SM and AT, contributing to the onset and progression of TiC. Still, there is a lack of focus on how the tumor and EV origin impact TiC incidence. In this work, we hypothesize that cancer cells modulate cachexia remotely through EV and their molecular cargo, with tumor/EV origin influencing cachexia severity. We established a transwell co-culture system comprising SM or AT cells in the lower chamber, and cancer cells with varying cachexia incidences (high: pancreatic, moderate: lung, and low: breast) in the upper chamber. This configuration allowed for EV-mediated communication without direct contact. The results showed that the degree of muscle atrophy and lipolysis varied with tumor origin, and the molecular profiling of the EVs revealed distinct cargo signatures corresponding with different incidences. These findings lay the groundwork for developing targeted therapies and identifying new biomarkers for tumor-specific early detection.
R. L.-S. and D.C. acknowledge the support of the Portuguese Foundation for Science and Technology (FCT) through the RECOVER IC&DT project (https://doi.org/10.54499/2022.02260.PTDC) and the CEEC Institutional program (CEECINSTLA/00012/2022) (D.C.). ICVS/3Bs is supported by FCT through Funding “Base”: (https://doi.org/10.54499/UIDB/50026/2020); Funding “Programático” (https://doi.org/10.54499/UIDP/50026/2020); and Funding “Complementar - LA”: (https://doi.org/10.54499/LA/P/0050/2020).
Engineering vascularized 3D microenvironments to bridge regeneration and disease modelling
Cristina Barrias
de I3S – Instituto de Investigação e Inovação em Saúde, University of Porto. INEB – Instituto de Engenharia Biomédica, Oporto (Porto) - Portugal
Recreating functional vascularized stromal tissue represents a major frontier in tissue engineering, with broad implications for regenerative medicine and human-relevant in vitro models. The stroma – comprising the extracellular matrix (ECM), microvessels, and different cell types including fibroblasts, provides essential structural and biochemical cues that regulate tissue morphogenesis, repair, and pathological remodelling. Incorporating this level of complexity into 3D in vitro systems, particularly organoid-based models, is critical to capture native tissue dynamics and enhance the predictive power of experimental human cell-based platforms. This lecture will present recent advances from our group toward engineering vascularized stromal compartments capable of supporting diverse tissue models. We will discuss strategies spanning from modular vascularized microtissues to microfluidic organ-on-chip platforms that enable the bottom-up assembly of perfusable, multi-scale microvascular networks. Their integration with epithelial organoid cultures provides unique opportunities to increase the physiological relevance of these systems and to model complex cell–cell and cell–matrix interactions, with applications ranging from promoting large-scale organoid development to modelling disease. Collectively, these approaches demonstrate how integrating epithelial and vascular–stromal compartments within in vitro models can contribute to bridge the gap between engineered and native tissues, advancing both mechanistic understanding and translational potential.
References: Orge I et al. Bioactive Materials (2024) 38:499-511; Carvalho DTO et al. Biofabrication (2021) 13:035008; Feijão T et al. Biomaterials (2021) 279:121222; Torres AL et al. Biomaterials 228 (2020) 119554. Torres Al et al. Biomaterials 154 (2018) 34e47.
Acknowledgements: “MOBILIsE_Plus project, co-funded by the European Union through the NORTE 2030 Regional Program of the European Regional Development Fund (FEDER), under the operation with the code NORTE2030-FEDER-01801000”.
New commercial perspectives on human tissue-derived materials in tissue engineering
Michael Floren
de Innovation. AlloSource, Centennial (Colorado) - United States
Human tissue-derived medical products (TDMPs), including allogeneic grafts and implantable biologics, have been utilized for centuries in the treatment of a broad spectrum of clinical conditions. Traditionally employed in their native or minimally processed forms, these materials are now being reimagined through advances in tissue engineering and regenerative medicine. Such innovations enable the strategic manipulation of extracellular matrices (ECMs) and cellular components to create next-generation therapeutics with enhanced bioactivity, specificity, and clinical performance. This presentation will explore the historical and evolving role of TDMPs from the perspective of AlloSource, a world leader in tissue processing, highlighting their application across diverse medical indications. With a portfolio of over 100 issued patents, AlloSource has leveraged bioengineering principles to drive innovation in the design, formulation, and commercialization of regenerative products currently used in orthopedic, reconstructive, and other surgical repair procedures. We will further examine how the integration of advanced tissue engineering principals with allogeneic tissue, such as scaffold optimization, ECM signaling, and delivery systems, supports the development of novel TDMPs while reinforcing the mission of “honoring the gift of donation.” The session will conclude with a forward-looking discussion on the potential of human-derived materials to accelerate progress across multiple domains within tissue engineering and regenerative medicine, paving the way for the next generation of medical products.
Mechanically tuned hydrogels driving physiologically relevant intestinal cell differentiation for predictive in vitro drug absorption studies
Alessandra M. A. Rando1, Ziyuan Luo2, Gianfranco B. Fiore1, Marco Cantini2, Monica Soncini1
1Department of Electronics, Information and Bioengineering. Politecnico di Milano, Milano (Lombardia) - Italy, 2Centre for the Cellular Microenvironment. University of Glasgow, Glasgow (Glasgow City) - United Kingdom
Reliable in vitro models of intestinal drug absorption remain a major bottleneck in drug development, largely due to the oversimplified nature of standard Caco-2 monolayers. The small intestine, the primary site of oral drug absorption, exhibits a villus–crypt architecture with distinct cell populations and extracellular matrix mechanical properties. We hypothesized that mechanically tuned substrates mimicking crypt (0.5 kPa) and villus (3 kPa) stiffnesses could drive Caco-2 morphogenesis and sub-differentiation through passive mechanical cues in a bicompartmental environment provided by the TTOP insert(1).
Polyacrylamide hydrogels of 0.5 kPa, 3 kPa, and gradient hydrogels spanning 0.5–3 kPa were synthesized on TTOP microporous polycarbonate membranes. The integration into the TTOP insert enabled direct AFM stiffness measurements, validated against glass coverslips. Caco-2 growth and tight junction formation were monitored via trans epithelial electrical resistance (TEER) measurements, while occludin and lysozyme expression, cell density, and cell height were quantified as indicators of sub-differentiation.
Cells exhibited stiffness-dependent sub-differentiation and polarization: soft hydrogels induced Paneth-like phenotypes, a crypt-associated cell type, with elevated occludin and lysozyme expression, whereas stiffer hydrogels promoted enterocyte-like features, characteristic of the villus region.
Gradient hydrogels allowed the coexistence of multiple epithelial subtypes within a single sample.
Morphological analysis confirmed increased cell height and apical-basal polarization, consistent with native intestinal architecture. ROCK/RhoA pathway inhibition further highlighted the role of mechanotransduction in tight junction formation and differentiation.
The bicompartmental setup was essential for promoting polarization and sub-differentiation, while enabling proof-of-concept insulin permeability assays that yielded physiologically relevant transport values under regulatory-compliant conditions.
This scalable approach demonstrates that passive mechanical cues in a bicompartmental environment can drive physiologically relevant intestinal cell differentiation, enhance the predictive power and translational potential of in vitro drug absorption assays, while remaining compatible with standard protocols for large-scale pharmaceutical applications.
(1) Coppadoro et al., Adv Mater Technol, 2025, doi: 10.1002/admt.202500065
Hydrogel based models of muscle invasive bladder cancer for understanding radiotherapy response and resistance
Ellen Slay1, Eve Tipple2, Conrado Guerrero Quiles1, Luisa Vanessa Biolatti1, Julie Gough2, Olga Tsigkou2, Peter Hoskin3, Ananya Choudhury3
1Division of Cancer Sciences. University of Manchester, Manchester - United Kingdom, 2Department of Materials, School of Natural Sciences, Faculty of Science and Engineering and The Henry Royce Institute, Royce Hub Building, The University of Manchester, Manchester - United Kingdom, 3Division of Cancer Sciences. The Christie NHS Foundation Trust, Manchester - United Kingdom
Muscle-invasive bladder cancer (MIBC) presents a significant clinical challenge, characterised by poor five-year survival (∼21%) and high rates of radioresistance [1]. A driving cause of radioresistance is extracellular matrix (ECM) remodelling and tumour fibrosis. Current pre-clinical models fail to replicate the dynamic interactions of tumour cells, matrix and biomechanical cues.
To bridge this translational gap, biomimetic 3D self-assembling peptide hydrogel platforms (PeptiGel® Alpha1, Alpha2, Alpha4) spanning physiologically-relevant stiffness ranges (0.7–1.3 kPa, 3–5 kPa, 6–8 kPa) were used in this research. Using four MIBC cell lines (T24, J82, HT1376, UMUC3) cultured at 37°C and 5% CO2, viability was evaluated over 21 days with LIVE/DEAD™ assays (n=2) and radiation response was explored in the T24 line in Alpha 4 using a graded 0–10 Gy dose delivered via an Xstrahl CIX3 irradiator (n=1). Viability was quantified using ImageJ (Fiji).
T24 cells maintained robust viability across all stiffness conditions, 95.73% in Alpha 1, 93.75% in Alpha 2, and 81.35% in Alpha 4, while other lines exhibited increased cell death and detachment, emphasising inter‐cell-line heterogeneity within biomimetic matrices, also seen in 2D.
When exposed to increasing doses of radiotherapy, the proportion of viable T24 cells decreased (1±0.178 at 0Gy, 0.921±0.145 at 2Gy, 0.179±0.0.005 at 4Gy, 0.173±0.013 at 6Gy, 0.157±0.007 at 8Gy, and 0.086±0.002 at 10Gy) recapitulating a 2D dose response profile, but within a 3D context that better approximates native ECM architecture.
This work establishes a novel, clinically relevant, translational tool to explore radiotherapy response of MIBC cell lines. By integrating cell-matrix interactions and biomechanical cues, this 3D system enables the discovery of strategies that sensitise MIBC to radiotherapy and improve therapeutic outcomes. Ultimately, by anchoring pre-clinical testing in physiologically informed 3D microenvironments, we aim to shorten the path from bench to bedside.
[1] Catto, et al. (2023). BJU International
Fatigue resistant hydrogel interfaces for treating pediatric tracheomalacia
Alexander Nottegar1, Francois Gorostidi2, Kishore Sandu2, Dominique Pioletti1
1Laboratory of Biomechanical Orthopedics. EPFL, Lausanne (Vaud) - Switzerland, 2Department of Otorhinolaryngology. CHUV, Lausanne (Vaud) - Switzerland
Tracheomalacia (TM) is a life-threatening airway condition in infants, characterized by weakened and excessively collapsible tracheal cartilage, resulting in airway obstruction, respiratory failure, and increased risk of cardiopulmonary arrest. Severe TM currently requires invasive interventions, including tracheal reconstruction, pexy procedures, tracheostomies, or stenting, all of which carry significant risks, technical complexity, and complications. Hydrogel adhesives present a promising solution for the treatment of severe TM due to their biocompatibility, flexibility, and potential to provide structural support to the weakened trachea. Applied extraluminally to the malacic segment, hydrogel adhesives provide non-invasive mechanical support to prevent airway collapse. Hydrogels can be engineered to mimic the mechanical properties of the trachea, ensuring they conform to the dynamic movements of the trachea while maintaining airway patency. However, conventional hydrogel adhesives suffer from low interfacial fatigue resistance, making them vulnerable to debonding under cyclic respiratory stresses. To address this limitation, we developed fatigue-resistant hydrogel adhesives by leveraging polymer chain entanglements. Highly entangled hydrogels were synthesized via solvent-free polymerization with a low crosslinker-to-monomer ratio, producing long and entangled polymer chains. Mechanical properties, including hydrogel stiffness and interfacial fatigue threshold, were measured using a ZwickRoell uniaxial testing system. An ex vivo pediatric TM model was developed by removing a section of cartilage rings from recessed rabbit tracheas. Collapsing of the malacic trachea was observed using a bronchoscope under a negative pressure of 50 mbar. Solvent-free polymerization produced densely entangled hydrogel networks with enhanced mechanical properties. Stiffness values were over 33-fold greater than conventional hydrogels, and interfacial adhesion energies exceeded 300 J/m2, compared to ∼20 J/m2 for conventional hydrogels. When applied to malacic segments, the hydrogels completely prevented airway collapse, maintaining patency under repeated respiratory cycles. Highly entangled hydrogel adhesives provide strong, durable adhesion and mechanical support to prevent airway collapse in TM. This approach offers a biocompatible, non-invasive alternative to current surgical interventions, representing a promising strategy for the treatment of severe TM.
Prime-LS: a versatile cryoprotectant-free MSC-lyosecretome for injectable and oral treatment of ulcerative colitis
Edorta Santos-Vizcaino1, Maria Rossello-Gelabert1, Camino Garcia-Blasco1, Manoli Igartua1, Rosa Maria Hernandez1
1University of the Basque Country (UPV/EHU), Vitoria-Gasteiz (Álava) - Spain
Aim and objective
Ulcerative colitis, a major subtype of inflammatory bowel disease (IBD), remains an incurable disorder with limited therapeutic options and significant side effects [1]. Cell-free strategies based on the mesenchymal stromal cell (MSC) secretome have emerged as promising alternatives [2], but their clinical translation is hindered by loss of bioactivity during processing and storage [3]. This study aimed to develop Prime-LS, a next-generation lyophilized MSC-derived highly purified secretome designed as a versatile, stable, and clinically actionable platform for both subcutaneous and oral delivery.
Materials and methods
Prime-LS was generated from cytokine-licensed MSCs using a proprietary, cryoprotectant-free downstream process. Stability and immunomodulatory activity were assessed through Gal-9 and Il-1Ra content, and lymphocyte proliferation assays. Therapeutic efficacy was evaluated in a murine model of dextran sulfate sodium (DSS)-induced colitis after subcutaneous (SC) injection of Prime-LS in solution or oral administration of Prime-LS in pH-responsive polymer-coated capsules. Disease activity index, colon length, histology, myeloperoxidase activity, and colonic cytokine levels (IFN-γ, TNF-α, IL-1β, IL-6) were assessed.
Results
Prime-LS allowed flexible concentration while maintaining bioactivity and full immunosuppressive potency of the MSC-secretome. SC injection provided complete protection against DSS-induced colitis, maintaining normal disease activity index, colon length, basal cytokine levels and mucosal integrity. The oral Prime-LS formulation significantly reduced disease severity and normalized inflammatory cytokines, confirming effective colonic release and in vivo immunomodulation.
Conclusions
These results highlight the feasibility of translating MSC-secretome-based therapeutics into clinically relevant and patient-friendly formulations. By combining stability, potent immunomodulation, and versatile delivery routes, these advances position Prime-LS as a promising cell-free biologic for the treatment of IBD.
Acknowledgments
Supported by MCIN/AEI (PID2021-122577OB-I00) and the Basque Government (IT1448-22). M.R.-G. (PRE2022-102058), C.G.-B. (PRE_2024_2_0058).
References:
[1] Solitano V. et al., Nat. Rev. Gastroenterol. Hepatol. (2025)
[2] Rossello-Gelabert M. et al., Stem Cell Res Ther. (2025)
[3] Yang Y. et al., Biomed. Mater. (2023)
Organoids and light-driven cell patterning for advanced humanized engineered tissues
Riccardo Levato
de Utrecht University, Utrecht - The Netherlands
In the quest to capture the complex environment of living organs within lab-made tissues, light emerged as a uniquely powerful stimulus for enabling dynamic and spatio-temporal control over cell and biomaterial properties, opening new avenues in regenerative medicine and tissue engineering. Light-responsive materials permit to non-invasively trigger mechanical actuation and shape-changes in cell-laden constructs, to modulate stiffening or softening of the extracellular milieu, and to enable spatio-temporal control over cell behavior. Previously, we introduced volumetric bioprinting, an ultra-fast, layer-less visible light-based biofabrication approach, to resolve virtually any 3D geometrical patterns in less than 20 seconds by projecting tomographic patterns onto photosensitive hydrogels making it possible to sculpt cell-laden materials with unprecedented geometrical freedom into high resolution architectures. Using visible light volumetric bioprinting technologies complex mini-organ models, also termed organoids, can be safely assembled into centimeter scale living tissues in a matter of few seconds. Herein, the most recent advances in light-driven biofabrication will be presented, together with our efforts to engineer functional blood vessels, breast gland tissue, and pancreatic tissues as advanced biological models, using organoids as living building blocks. Vascularized environments can be built converging volumetric printing with microgel-based printable materials. Further advancing this technology, precise imaging strategies are leveraged to for enhanced metrology, quality control, and for introducing the concept of context-aware printing. In context-aware manufacturing, making printers that are able to detect objects, cells and features of interest within the printing vat, enables the creation of constructs that match the metabolic demands of the embedded cells, facilitates multi-material printing and overprinting, and permits to print across light-occluding elements, allowing for the creation of complex composite materials and living tissues. Introducing anisotropic organoid assemblies within volumetrically bioprinted constructs enables the biofabrication of freeform tissue models that more closely mimic the complex biochemical and structural composition of native tissues.
Modulation of extracellular matrix proteins in the glial and fibrotic scar
Siobhan Mcmahon1, Sorour Nemati1, Dimitrios Zeugolis2, James Phillips3
1Discipline of Anatomy. University of Galway, Galway - Ireland, 2Conway Institute of Biomolecular & Biomedical Research. University College Dublin, Dublin - Ireland, 3Department of Pharmacology. University College London (UCL), London (London, City of) - United Kingdom
Spinal cord injury (SCI) results in a cascade of cellular and molecular events that lead to permanent tissue damage and functional impairment. Following SCI, a glial and fibrotic scar is formed, and inhibitory extracellular matrix (ECM) molecules become upregulated. The aim of this project was to develop model systems that enable cells to produce ECM for testing therapeutic strategies for targeting inhibitory ECM molecules.
In this study, we employed a macromolecular crowding (MMC) technique to accelerate ECM deposition in vitro. This rapid and effective strategy created an environment rich in ECM in astrocytic cultures and leptomeningeal cultures to mimic the glial and fibrotic scar. Cells were cultured in media supplemented with the MMC Ficoll (FC). To mimic the injury environment in vivo, cells were exposed to physical and chemical injury. In addition we generated a 3D in vitro model of scarring whereby astrocytes were encapsulated in collagen hydrogels with FC.
The growth of astrocytes and leptomeningeal cells with FC in vitro did not affect cell morphology, metabolic activity, or cell viability. FC promoted a rapid upregulation of glial scar proteins and chondroitin sulphate proteoglycans. Cells supplemented with FC exhibited higher deposition of ECM proteins involved in fibrotic scar formation, including fibronectin, collagen IV, collagen I, and laminin. 3D hydrogels further promoted scar marker expression and ECM deposition, recapitulating key features of in vivo scar formation.
A key limitation of conventional cell culture is its clear difference from the naturally ‘crowded’ tissue environment, resulting in a slow rate of ECM protein deposition. Using the MMC approach, we successfully accelerated ECM protein deposition within 2D and 3D in vitro models of the glial and fibrotic scar, providing a valuable refinement in developing SCI in vitro models for drug screening and therapeutic applications.
Insights into improving male reproductive performance by integrating seminal plasma extracellular vesicle–based interventions
Monika Nõmm1, Anni Viljaste-Seera1, Fazeli Alireza2, Ülle Jaakma3
1Institute of Veterinary Medicine and Animal Sciences,Chair of Animal Breeding and Biotechnology. Estonian University of Life Sciences, Tartu - Estonia, 2Institute of Veterinary Medicine and Animal Sciences, Chair of Veterinary Biomedicine and Food Hygiene. Estonian University of Life Sciences, Tartu - Estonia, 3nstitute of Veterinary Medicine and Animal Sciences, Chair of Animal Breeding and Biotechnology. Estonian University of Life Sciences, Tartu - Estonia
Seminal plasma (SP) extracellular vesicles (EVs) play a conserved role in reproductive physiology by modulating sperm function. Understanding and harnessing these vesicle-mediated interactions offers new opportunities to develop regenerative and precision fertility strategies that bridge animal and human reproductive medicine.
SP–derived EVs from bulls with high (HF) or low (LF) in vitro fertility were isolated via size exclusion chromatography and characterized using nanoparticle tracking analysis. Firstly, EVs from HF bull SP were added to 100 µL IVF medium in three concentrations (A:200 ×106, B:400 ×106, C:600 ×106 EVs mL−1and D: control) and co-cultured with sperm and oocytes for 24 h under mineral oil. Secondly, EVs from LF bull SP were added to the IVF medium in a final concentration of 600 ×106 EVs mL−1. In both experiments, embryo development, morphology, and kinetics scores were assessed on Day 7 (D7) and Day 8 (D8). The statistical analysis was performed using GraphPad Prism 10 software. All data were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test with P ≤ 0.05.
On D7, the highest blastocyst morphological score was recorded in group C (3.0), which was 1.1 points higher than the control group morphological score (1.9). On D8, the difference in blastocyst rates (%) between group C and D was 19.8 ± 2.6 and 9.4 ± 0.9, respectively, but was insignificant with a P ≥ 0.05. The morphological score of D8 in group C was the highest (2.35), 0.55 points higher than group D (1.8). For LF bull SP treatment, there were no statistically significant differences between the treatment groups (P ≥ 0.05).
The preliminary results suggest improvement in embryo morphology and blastocyst rates when adding 600 × ×106 EVs mL−1 SP EVs from known HF bulls to the IVF media of known LF bulls. On the other hand, no differences were observed when adding 600 × 106 EVs mL−1 SP EVs from known LF bulls to the IVF media. More research is needed into the cargo components of SP EVs to understand the translational aspects of EV-mediated reproductive modulation across species. Supported by the Estonian Research Council (PRG1665).
Electrical stimulation promotes polarisation of primary human macrophages to pro-regenerative phenotypes
Aoife Mc Loughlin1, Meenakshi Suku2, Matteo Nosè2, Michael Monaghan2
1Discipline of Mechanical, Manufacturing and Biomedical Engineering. Trinity College Dublin, Dublin - Ireland, 2Discipline of Mechanical, Manufacturing and Biomedical Engineering. Trinity College Dublin, Dublin - Ireland
Macrophages follow a co-ordinated response to inflammation whereby pro-inflammatory (M1-like) macrophages initiate early resolution processes before switching to anti-inflammatory (M2-like) phenotype that completes tissue repair. In adverse cases, impaired macrophage modulation can lead to fibrosis, which in turn impedes normal organ behaviour and function. Macrophage function can be significantly altered by stimuli, and the potential of electrical stimulation (ES) holds great promise for therapeutic benefit; as all biological systems are electrically active. This study aims to investigate the immunomodulatory potential of ES on primary human macrophages, evaluating its role in shifting these cells towards an M2-like phenotype.
CD14+ monocytes were isolated from human blood and differentiated into macrophages with macrophage-colony stimulating factor. Macrophages were then further differentiated into an M1-like phenotype with lipopolysaccharide and interferon-gamma, or M2-like phenotype through interleukin-4 and interleukin-13 supplementation, before application of ES regimes over short (1 hr) and long-term (24 hr) periods. Cytokine secretion was performed through ELISA assays and phenotypic macrophage markers assessed through RT-PCR and flow cytometry. Immunometabolism was assessed using fluorescence lifetime imaging microscopy (FLIM), while calcium staining and RT-PCR was also undertaken to assess the fate of ion channels after ES.
Macrophages subjected to ES exhibited a more M2-like phenotype, with higher expression levels of surface markers CD163 and CD206 observed through flow cytometry and RT-PCR. Cytokine secretion assessment presented a suppression of M1 secretory cytokine release IL-6 after polarisation with pro-inflammatory stimuli. Analysis from FLIM unveiled higher levels of oxidative phosphorylation within ES macrophages while higher gene expression levels of metabolite markers associated with this pathway were observed in RT-PCR. Finally, macrophages demonstrated a signal-dependent response through calcium staining while ion channel gene markers showed altered expression under ES.
We demonstrate that ES can enhance a pro-regenerative, M2-like phenotype with a mechanistic link showing the potential ES can have in the immunomodulation of macrophages as a therapeutic.
This project is funded from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme Grant agreement No. 101125153.
Integrating articular cartilage progenitor cell–derived microtissues and extracellular matrix scaffolds to engineer zonally defined grafts for osteochondral defect repair
Aliaa Sherif Karam1, Gabriela Soares Kronemberger1, Kaoutar Chattahy1, Giovanni Gonnella1, Pieter Brama2, Daniel John Kelly1
1Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute. Trinity College Dublin, Dublin - Ireland, 2School of Veterinary Medicine. University College Dublin, Dublin - Ireland
Osteoarthritis is a debilitating joint disease characterised by progressive articular cartilage (AC) degeneration. Engineering functional cartilage grafts requires recapitulating the tissue’s arcade-like collagen architecture which is crucial for its biomechanical function. AC progenitors (ACPs) represent a promising cell source due to their capacity to generate phenotypically stable hyaline cartilage. Here, we combined the self-organizing potential of ACP-derived microtissues (µTs) with decellularized extracellular matrix (ECM) scaffolds to biofabricate scaled-up constructs for osteochondral repair. The regenerative capacity of these engineered implants was tested in a large animal (caprine) model of joint injury.
ACPs were isolated through differential adhesion to fibronectin. We optimized µT fusion by varying µT size (1000 or 2000 ACPs/µT), fusion number (2000–6000 µTs), and evaluated BMP-9 priming. The optimal configuration (4000 BMP-9–primed µTs of 1000 ACPs/µT) of µT were then fused under radial confinement onto AC or bone ECM scaffolds (Ø=6mm, h=5mm). Constructs were cultured for 6 weeks and evaluated in a goat osteochondral defect model (1- and 6-month endpoints; ongoing). Chondrogenesis and healing were evaluated with histology, immunohistochemistry, PCR, mechanical testing, biochemical assays, and micro-computed tomography.
ACP µTs demonstrated robust fusion capacity forming a white, smooth cartilage-like tissue. BMP-9 priming enhanced tissue expansion, matrix production and compressive strength. AC ECM scaffolds supported superior chondrogenesis of fusing µTs, resulting in a cartilage tissue with higher mechanical properties than that generated on bone ECM scaffolds. Polarized light microscopy revealed the engineered cartilage on both scaffolds possessed a biomimetic collagen architecture, with horizontal and vertical fibers in the superficial and deep zones, respectively. After one month in vivo, constructs demonstrated promising macroscopic healing. Further analysis is ongoing.
These findings highlight that radially confined ACP µTs, seeded onto the surface of ECM scaffolds, can fuse and self-organize into structurally organized, functional cartilage tissue, showing early potential for osteochondral defect repair.
Possibilities and limitations of 4D biofabrication
Leonid Ionov
de University of Bayreuth, Bayreuth (Baden-Wberg Bayern) - Germany
Most powerful biofabrication techniques, such as dispensing-based 3D bioprinting and stereolithography, still face major limitations that restrict their broader use for artificial tissue fabrication. These include insufficient resolution and cell density, challenges in creating multicomponent or hollow structures, limited cell orientation control, and poor oxygen diffusion.
We advanced biofabrication by developing a 4D biofabrication approach, which exploits shape transformation of printed structures to achieve complex geometries. This method uses shape-morphing polymers capable of controlled deformation under external stimuli. Unlike conventional techniques requiring molds or supports, 4D biofabrication enables fabrication of tubular or hollow structures without the need to remove a template. Cells can be uniformly seeded on a flat substrate, allowed to attach and proliferate, and subsequently enclosed as the construct transforms into a tube or scroll—resulting in homogeneous cell distribution and reduced shear stress compared to high-resolution printing.
A key challenge is enabling shape transformation under biocompatible conditions that maintain cell viability and function. We pioneered several polymeric systems—both solid and hydrogel-based—that change shape in response to mild stimuli such as temperature or calcium ions. These systems enable controlled cell encapsulation, scaffold porosity, and 3D cell patterning. Recent advances in applying 4D biofabrication for muscle and neural tissue regeneration will be discussed.
Evaluation of schwann and endothelial cell compatibility in relation to a biofabricated nerve guidance conduit
Lea Zausch1, Hassanbeiki Maryam1, Subburaj Sri Raam1, Simunovic Filip2, Zengerle Roland1, Kartmann Sabrina1, Zimmermann Stefan1
1Department of Microsystems Engineering. University of Freiburg, Freiburg (Baden-Wberg Bayern) - Germany, 2Aesthemedica, Freiburg (Baden-Wberg Bayern) - Germany
Nerve guidance conduits (NGCs) are used to treat peripheral nerve injuries (PNI), but are currently only reliable for short defect distances. Our work aims to overcome these size limitations by developing an enhanced NGC incorporating preformed human umbilical vein endothelial cell (HUVEC) strands and rat Schwann cells (RSC96) using a complex biofabrication process. The enhanced NGC should provide a defined microvascular structure accompanied by Schwann cells to guide ingrowing axons.
This study evaluates hydrogels suitable for 3D bioprinting with the two cell types by correlating cell compatibility and mechanical properties. Collagen I and alginate were tested in different concentrations and mixture ratios. In rheological measurements, collagen I (viscosity range: 6-100 mPas at 1-5 mg/ml) and alginate (30-215 mPas at 1-5%) show shear-thinning behavior. Composite hydrogels (1:1 ratio of 3 mg/ml collagen I and 1-5% alginate) show intermediate viscosities (25-95 mPas) increasing gradually with alginate content, allowing easy viscosity adjustment while preserving shear-thinning behavior. Reported values correspond to a shear rate of 100 s−1.
Alginate offers higher viscosity-based mechanical stability and reinforces collagen I, but reduces cell viability as shown by a live/dead assay. Collagen I (5 mg/ml) exhibited 85% viable RSC96 cells, whereas a 1:1 collagen I (5 mg/ml)/alginate (1%) blend reduced cell viability to 70%. A similar trend was observed for other mixing ratios. Consistent with these results, alginate had a negative impact on cell viability and self-assembly capacity of HUVECs too. Due to these results, we will use collagen I as hydrogel for our biofabrication approach. To compensate for the lower mechanical stability of collagen I, additional biofabricated layers should provide mechanical support. This study provides a comprehensive characterization of hydrogels suitable for 3D bioprinting with endothelial and Schwann cells, advancing nerve tissue engineering.
Acknowledgments
Funding by the Deutsche Forschungsgemeinschaft (FI 790/18-1, ZI 1326/6-1).
Advancing osteoarthritis therapy: in vitro and ex vivo modeling in bioreactor platforms
Zhen Li1, Kaihu Li1, Eda Ciftci1, Fatemeh Safari1, Huan Meng1, Sibylle Grad1
1AO Research Institute Davos, Davos (Graubunden) - Switzerland
Osteoarthritis (OA) is a progressive, chronic joint disease with rising global prevalence, largely driven by increased life expectancy and lifestyle changes. Current therapeutic approaches - primarily anti-inflammatory and analgesic medications - offer limited symptomatic relief and fail to halt cartilage degeneration. To support the preclinical evaluation of novel OA treatments, we have developed various in vitro and ex vivo OA models using joint tissues and 3D cell-scaffold cultures. These models incorporate a low-O2 workstation and a mechanical loading bioreactor to replicate the physiological conditions of articulating joints, thereby enabling mechanistic and efficacy studies in a more realistic microenvironment.
We established an ex vivo OA model using human osteochondral explants from the femoral head, which successfully recapitulates key pathological features of OA progression [1]. Considering the critical role of synovial tissue in modulating joint inflammation, we further developed a co-culture model combining osteochondral explants with synovial tissue [2]. These platforms were employed to evaluate the therapeutic potential of 5-Aminosalicylic acid in OA treatment [3].
Currently, we are investigating Glucagon-like Peptide-1 Receptor Agonists (GLP-1RAs) as potential disease-modifying agents for OA. While prior clinical studies have focused on systemic administration, we hypothesize that intra-articular delivery may more effectively target GLP1 receptors expressed across tissues within the articular joint. The efficacy of liraglutide, a GLP-1RA, was assessed in a 3D in vitro OA model composed of human chondrocytes embedded in scaffolds. Under mechanical loading, IL-1β stimulation induced robust inflammatory and catabolic responses, whereas liraglutide demonstrated significant anti-inflammatory activity and cartilage-protective effects.
References:
[1] K. Li, et.al. Frontiers in Bioengineering and Biotechnology 9 (2021) 787020.
[2] K. Li, et.al. Eur Cell Mater 47 (2024) 15-29.
[3] K. Li, et.al. J Orthop Translat 38 (2023) 106-116.
Funding: The present study was funded by AO Foundation, Eurostar project OA_BIO, and EU Horizon project SINPAIN (No. 101057778).
Engineering complex organoid-based implants as advanced therapeutics for regeneration of challenging skeletal defects
Ioannis Papantoniou
de Prometheus the Translational Division of Skeletal Tissue Engineering, Department of Development & Regeneration. KU Leuven, Leuven (Brabant) - Belgium
Tissue Engineered Products are classified by the European Medicines Agency (EMA) as advanced therapy medicinal products (ATMPs). To date only 4 products have obtained marketing authorization (first ATMP approved in 2009) as Tissue Engineered ATMPs (TE-ATMPs). Out of these four products, two have been withdrawn due to limitations in reimbursement and manufacturing capabilities which have been reported as major bottlenecks for all ATMPs. Concern and skepticism around manufacturing and scale-up challenges have also increased amongst the investment community for further investment. Therefore optimization and manufacturing of these ATMPs needs to be carried out in order to enhance robustness cost efficiency and sustainability. The rise of organoids is revolutionizing the field of regenerative medicine. Organoids can be seen as novel live building blocks which can be used for the manufacture of next generation TE-ATMPs. In order to address this manufacturing challenge and support translation of this new class of products automated manufacturing pipelines for scalable production. Skeletal organoids can be used in order to regenerate challenging skeletal defects such as critical size long bone defects as well as deep osteochondral defects of the knee joint. Organoids possess predetermined identity and function and hence constitute excellent building blocks for regenerating such challenging defects. In this presentation a variety of diverse organoid populations will be presented and their unique biologically features will be optimally employed through spatial arrangement within the engineered implant. This complexity will be more specifically focused on pre-endothelialisation as well as merger of bone forming and stable articular cartilage organoids. However clinical translation will required standardised and scalable production of these organoids. Hence, in this presentation we will provide a feasibility study for robotic-driven scalable biomanufacturing of organoid-based implants for regeneration of skeletal defects. This approach will pave the way for the development of optimised processes, automated and fully digitalised manufacturing of TE-ATMPs. Ultimately these technologies will further enable wide-spread adoption of “end to end” automated decentralised manufacturing as means to produce affordable, sustainable and accessible clinically-relevant TE-ATMPs.
Regulation of bone mechanosensation: the role of divalent cations and innovative peptides in modulating the immune-neural axis
Kelvin Yeung1, John Kubi1, Augustine Brah1, Wei Qiao2
1Orthopaedics and Traumatology. The University of Hong Kong, Hong Kong (Hong Kong (General)) - Hong Kong, 2Faculty of Dentistry. The University of Hong Kong, Hong Kong (Hong Kong (General)) - Hong Kong
Addressing the limitations of current bone regenerative agents necessitates innovative approaches that target fundamental physiological pathways. This presentation highlights our identification of divalent cations (Mg2+, Zn2+, Cu2+) and a novel plant-derived peptide, HKUOT-S2, as effective activators of the bone mechanosensory system. Our findings demonstrate that cations released from alginate hydrogels facilitate bone healing by specifically activating the mechanosensitive TRPM7 channel in macrophages, thereby modulating the immune-neural axis. In addition, HKUOT-S2, a peptide-based regulator with superior osteogenic potential compared to rhBMP-2, substantially upregulates mechanosensory ion channels (notably TRPM7) at both transcriptional and translational levels, serving as a mechanism essential for maintaining bone homeostasis, particularly in osteoporotic conditions. The research positions TRPM7 as a central therapeutic target and provides substantial evidence that both cation release systems and HKUOT-S2 peptides directly enhance mechanosensory channel function. By leveraging this mechanism, we propose a new paradigm for stimulating bone growth aimed at mitigating the adverse effects of reduced mechanical loading, such as those observed in microgravity or disuse osteoporosis. These discoveries represent a significant advancement in the understanding and therapeutic application of bone mechanobiology.
In vivo mechanical loading induces site-specific, partially reversible tissue adaptation in vertebrae
Cosima Erhard1, Tobias Thiele1, Agnes Ellinghaus1, Lukas Schönnagel2, Katharina Schmidt-Bleek1, Melih Ö. Celik3, Georg N. Duda1
1Julius Wolff Institute. Charite Universitatsmedizin, Berlin - Germany, 2Center for Musculoskeletal Surgery. Charite Universitatsmedizin, Berlin - Germany, 3Department of Anaesthesiology and Intensive Care Medicine, Charité Campus Benjamin Franklin. Charite Universitatsmedizin, Berlin - Germany
Introduction
Understanding how mechanical forces contribute to low back pain remains a clinical challenge [1]. Although a causal link between spinal loading, structural changes, inflammation, and pain has not been fully demonstrated, patients often associate their symptoms with mechanical strain, and surgery frequently targets morphological abnormalities as pain drivers [2]. Bone remodels site-specifically via osteoblast and osteoclast activity in response to mechanical load [3]. This study investigates whether similar load-induced remodeling also occurs in mouse caudal vertebrae, and whether these adaptations involve inflammatory and neurovascular responses, as well as their potential reversibility following load cessation.
Methods
Female C57BL/6 mice were assigned to loaded or sham groups. Titanium pins were inserted into caudal vertebrae 5 and 7 to apply cyclic axial compression (10 Hz, 5 min/session, three times weekly for four weeks), followed by four weeks of rest to assess reversibility. Bone structure was monitored longitudinally by in vivo micro-CT (μCT; MILabs U-CT xuhr). Image registration and post-processing were conducted using a semi-automated workflow (Xploraytion GmbH). For histology, vertebrae were embedded in a polyvinylpyrrolidone–gelatin matrix, and 50 μm cryosections were prepared for 3D visualization of collagen and angiogenesis.
Results
μCT imaging revealed load-dependent, site-specific bone adaptation. Loading increased bone formation and reduced resorption, with the proximal endplate showing notable vascular ingrowth and osteoprogenitor accumulation. During recovery, bone formation decreased but did not return to baseline. Whether neurovascular and inflammatory alterations persist and lead to long-term changes remains to be elucidated.
Discussion
Mechanical loading increases vertebral bone mass and induces spatially distinct remodeling that is partly reversible. Ongoing analyses compare endplate and trabecular compartments and explore parallels with human overload-related adaptations.
References
1. Von der Lippe et al, J Health Monit, 6(Suppl 3):2-14, 2021.
2. Vlaeyen et al, Nat Rev Dis Primers, 4(1):52, 2018.
3. Birkhold et al, Bone, 66:15-25, 2014.
Association of type I collagen with the marine bacterial exopolysaccharide HE800 enables the synthesis of dermal substitutes with enhanced mechanical and physical properties
Lena Villerabel1, Hemalatha Narassimprakash1, Maxime Mauviel1, Stéphane Cuenot2, Agata Zykwinska3, Christophe Helary4
1Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Sorbonne Université, CNRS, Paris (Ile-de-France) - France, 2Institut des Matériaux de Nantes Jean Rouxel, IMN, Nantes Université, CNRS, Nantes (Pays de la Loire) - France, 3Ifremer - MASAE - Microbiologie Aliment Santé Environnement, Nantes (Pays de la Loire) - France, 4Laboratoire de Chimie de la Matière Condensée. Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Sorbonne Université, CNRS, Paris (Ile-de-France) - France
The dermis is primarily composed of type I collagen, glycosaminoglycans (GAGs), and fibroblasts. Collagen provides tensile strength and elasticity to the skin, while GAGs contribute to its hydration. Collagen-based hydrogels have been widely developed as dermal substitutes; however, they present several limitations, including low stability and weak mechanical strength. Moreover, combining collagen with GAGs such as hyaluronic acid is challenging, as these polymers form polyionic complexes in solution.
In this study, type I collagen was combined with the bacterial exopolysaccharide HE800, produced by the deep-sea hydrothermal vent bacterium Vibrio diabolicus, to develop dermal substitutes with enhanced mechanical and physical properties. HE800 EPS was chosen because of its structural similarity to hyaluronic acid and its high hydrating capacity.
When mixed with native high molecular weight HE800 (600 kDa) at room temperature, collagen underwent complexation and precipitation with the polysaccharide. In contrast, at 4°C, the solutions remained stable for at least 10 minutes, enabling the formation of homogeneous composite hydrogels upon collagen neutralization. Scanning electron microscopy revealed that collagen/HE800 hydrogels exhibited large, elongated filaments within the collagen network. Compared to pure collagen hydrogels, the composites demonstrated higher mechanical properties and greater resistance to collagenase-mediated degradation. Conversely, hydrogels containing depolymerized HE800 EPS (300 kDa) showed destabilized structures.
Cell-laden collagen/native HE800 hydrogels containing human dermal fibroblasts remained stable for up to 7 days, with no visible contraction. In contrast, both pure collagen and collagen/hydrolyzed HE800 hydrogels contracted from day 1. Within the collagen/native HE800 composites, fibroblasts adhered and proliferated over 21 days. The cells displayed a dendritic morphology similar to that observed in pure collagen hydrogels.
Taken together, these results demonstrate that combining type I collagen with native HE800 EPS enables the fabrication of hydrogels with enhanced physical properties, making them promising candidates for novel dermal substitute materials.
Injectable hybrid hydrogen-releasing hydrogel for osteosarcoma treatment and bone regeneration
Vahid Shafiei1, Daniel Thuir2, Jihene Arfaoui3, Hajar Homa Maleki4
1Institute of Inorganic and Materials Chemistry, Department of Chemistry, University of Cologne, Greinstraße 6, 50939 and Center for Molecular Medicine Cologne, CMMC Research Center, Robert-Koch-Str. 21, 50931, KOLN (Nordrhein-Westfalen) - Germany, 2Institute of Inorganic and Materials Chemistry, Department of Chemistry, University of Cologne, Greinstraße 6, 50939 Cologne, Germany and, KOLN (Nordrhein-Westfalen) - Germany, 3Université Tunis El Manar, Laboratoire de Chimie des Matériaux et Catalyse, Département de Chimie, Faculté des Sciences de Tunis, Campus Universitaire Farhat Hached d'El Manar, 2092, Tunis - Tunisia, 4Institute of Inorganic and Materials Chemistry, Department of Chemistry, University of Cologne, Greinstraße 6, 50939 and Center for Molecular Medicine Cologne, CMMC Research Center, Robert-Koch-Str. 21, 50931, KOLN (Nordrhein-Westfalen) - Germany
Aim and objective: Osteosarcoma, common primary bone malignancy, often recurs after resection plus chemo/radiotherapy due to residual cells and lack of conformal, osteogenic fillers. To address these challenges, we designed a multifunctional injectable hydrogen (H2) and heat-releasing hybrid hydrogel. This hydrogel by mussel-inspired catechol chemistry assembly of oxidized silk fibroin (SFO), oxidized lignin (O-LN), polydopamine modified mesoporous magnesium silicate nanoparticles loaded with ammonia borane (AB) (MgSiO3-PDA@AB) as a H2 source for therapy and osteogenesis. The designed hydrogel combines mild photothermal (∼45°C) and H2 therapy for selective eradication of osteosarcoma while safeguarding healthy tissues from overheating followed by bone healing.
Material and methodology: SF from B. mori was extracted, tyrosinase-oxidized, and coordinated with Fe3+ to form catechol-Fe nodes. Alkaline lignin was demethylated and oxidized to yield O-LN. MgSiO3 nanoparticles were prepared via Stöber-derived SiO2 followed by hydrothermal Mg incorporation and coating with PDA, then loaded with AB. Self-assembly of SFO/Fe3+, OLN, and MgSiO3-PDA@AB afforded syringe-cast hydrogels. Formulations were optimized Fe3+, O-LN, nanoparticle content to achieve multifunctional hydrogel networks with controlled gelation, injectability, adhesiveness, photothermal response, and on-demand H2 release for osteosarcoma eradication and bone regeneration.
Results: Chemical analyses (1H-NMR, FT-IR, UV-Vis, XRD, SEM-EDX) verified oxidation/modification and nanoparticle integration. Gelation was tunable via Fe3+ and pH, yielding injectable constructs adhered to bone-mimetic and metallic surfaces. Under 808-nm irradiation, formulations reached mild photothermal with on/off cycling. MgSiO3-PDA@AB provided sustained, pH-responsive hydrogen release in tumor-relevant acidic conditions, supporting combined H2-thermal action. Overall, the platform offers injectability, adhesion, photothermal performance, and H2 delivery for osteosarcoma ablation and defect reconstruction.
Conclusions: We introduced hydrogen-thermal therapy as a novel approach for osteosarcoma via a multifunctional, stimuli-responsive hydrogel. Formulated from FDA-approved components, materials integrate photothermal conversion with pH-responsive H2 delivery, for an efficient tumor eradication. Next, we will assess cytocompatibility and in vitro and in vivo performance toward minimally invasive tumor control with bone repair.
Elucidating and modulating macrophage–fibroblasts crosstalk in response to nanoclay for chronic wounds
Zilian Fan1, Nicholas D. Evans1, Yang-Hee Kim1, Jon Dawson1
1Faculty of Medicine, School of Human Development and Health. University of Southampton, Southampton - United Kingdom
Impaired wound healing is a worldwide health problem with an estimated to cost the medical system of over US$25 billion per year, affecting around 6.5 million patients (Sen, 2019). Prolonged inflammation is one fundamental cause factor in the pathogenesis of chronic wounds, with a change of macrophage phenotype is a key mediator of this process. The failure of phenotype transition causes persistent inflammatory signalling, resulting in impeded repair (Falanga et al., 2022). With advances in understanding of macrophage phenotypes and their roles in tissue regeneration, there is growing interest in strategies to modulate the immune microenvironment surrounding implants. Gels formed from Laponite, a synthetic smectite clay, have shown potential to aid wound healing when applied to chronic wounds. The broad aim of this study was to elucidate the effect of laponite on macrophage response and the immune microenvironment on fibroblasts during chronic wound healing. We compared the migration patterns of mouse bone marrow-derived macrophages (BM-MØ) and mouse fibroblasts using in vitro time-lapse imaging and daily phase-contrast microscopy. We found that BM-MØ were particularly active on Laponite, especially along the edges of the hydrogel. They tended to infiltrate into the hydrogel and underwent marked morphological changes (time-lapse imaging), and phagocytosed the hydrogel (TEM). In addition, macrophages migrated along the hydrogel boundary or fused into multinucleated giant cells, suggesting an alteration of the immune microenvironment. Our previous data showed that the rate and quality of re-epithelialisation is significantly promoted in male db/db mice after Laponite treatment. The application of Laponite to appropriately polarize macrophages at the right time would be potential for promoting a new generation of therapies aimed at inflammation-resolution and regeneration, offering insights for designing better biomaterials for chronic wound healing.
Electrical stimulation induces IL-6 secretion in N2a cells
Alberto Yúfera1, Daniel Martin1, Antonio Algarín1, Nuria Pastor2, Diego Ruano3, Luis Orta2, Akaitz Dorronsoro2, Paula Daza2
1Tecnología Electrónica. Universidad de Sevilla, Sevilla - Spain, 2Biología Celular. Universidad de Sevilla, Sevilla - Spain, 3Bioquímica y Biología Molecular. Universidad de Sevilla, Sevilla - Spain
N2a is a mouse neuroblastoma cell line commonly used to assess neuronal differentiation, neurite growth and nerve regeneration in tissue engineering studies. Under the right stimulus N2a cells can be directed to start the neuronal differentiation process, and one of those stimuli is electrical stimulation (ES). ES is a technique in which an electric field and its underlying current are applied through electrodes to a living tissue in order to induce a desired effect. We have previously reported that 500mV/mm-100Hz biphasic sqaure wave for 6 hours is the optimal protocol to start differentiation process in these cells, and we decided to investigate which molecules could be implicated in that mecahnism. We analyzed the cytokine secretion of N2a cells after 6 hours of 500mV/mm-100Hz biphasic pulsed ES using a commercial electrode platform (Applied Biophysics). ES cells upregulated IL-6 and downregulated IL-10 at the transcriptional level as of RT-qPCR assessment. ELISA analysis of cytokine levels in the stimulated medium showed an increase in IL-6 concentration compared to the control. Further experiments using recombinant IL-6 showed that the cytokine yielded effects similar to ES. Moreover, neutralization of IL-6 receptor with blocking antibodies reversed the effects induced by ES on N2a cells. Together, this results point to IL-6 as a molecule responsible for the effects of ES on these neuroblastoma cells differentiation.This publication is part of the project USECHIP (TSI-069100-2023-001), funded by the Secretary of State for Telecommunications and Digital Infrastructure, Ministry for Digital Transformation and Civil Service and by the European Union–NextGenerationEU/PRTR.
Two-photon polymerized 3D microscaffolds for guiding cell architecture and function
Emanuela Jacchetti
de Department of Chemistry, Materials and Chemical Engineering “G.Natta”. Politecnico di Milano, Milano (Lombardia) - Italy
Mechanobiology explores how physical cues within the cellular microenvironment influence cell behaviour, fate, and function. Our research aims to develop and apply advanced bioengineering tools to investigate how cells sense mechanical cues and to exploit mechanotransduction processes across scales—from molecular to tissue level.
We employ two-photon polymerization (2PP) to fabricate precisely controlled 3D microscaffolds recapitulating key physical features of the cell microenvironment with submicrometric precision. These engineered platforms allow us to design and vary scaffold architecture with high spatial precision, providing a versatile system to study how mechanical cues influence cell architecture and function.
By integrating high-resolution and time-resolved optical microscopy (confocal, two-photon, FLIM, Brillouin, and Raman imaging) with these custom-designed 3D scaffolds, we create defined microenvironments that allow real-time visualization of mechanical regulation in vitro and in ovo/in vivo. The combination with computational modelling and dynamic bioreactor systems further enables controlled modulation of both extracellular and intracellular forces, offering quantitative insight into how mechanical inputs drive stem cell differentiation, cancer progression, immune modulation, and regenerative processes.
Current applications include 3D scaffold for mesenchymal stem cell expansion, implantable devices for immunomodulation, perfused millifluidic chips for vascularized tissue models, and intravital imaging windows for monitoring tissue dynamics in real time. Overall, this integrative strategy strengthens our understanding of cell mechanobiology and fosters the rational design of next-generation regenerative and therapeutic systems.
Acknowledgement. This work would not have been possible without the valuable support and collaboration of G.Buccioli, C.Cortesi, C.Martinelli, A.Nardini, L.Cherubin, C.Conci, S.Carelli, R.Martinez, G.Cerullo, R.Osellame, R.Vanna, I.Barravecchia, D.Angeloni, G.Chirico. MT.Raimondi
Funding. MUR:project VISION, CUP D53D23007890001, EU, MSC-DN, project flIMAGING3D, G.A. 101073507, ERC, project BEACONSANDEGG, G.A. 101053122.
Bio-based self-healing pectin hydrogels for sustained delivery of antioxidant and anti-inflammatory curcumin-loaded zein nanoparticles in wound treatment
Francesca Tivano1, Elena Marcello1, Camilla Paoletti1, Alice Zoso1, Clara Mattu1, Irene Carmagnola1, Valeria Chiono1
1Department of Mechanical and Aerospace Engineering. Politecnico di Torino, Turin (Italia) - Italy
Oxidative stress at wound sites impairs cellular function and delays tissue regeneration, making antioxidant delivery a key therapeutic target in wound management1. Curcumin is an interesting natural antioxidant, but its poor water solubility limits its clinical effectiveness, requiring encapsulation strategies to enhance bioavailability2. A hybrid drug delivery system (DDS) combining nanoparticles and hydrogels enables controlled, localized release3. This work aimed to develop a bio-based hybrid DDS for enhanced wound healing composed of an injectable citrus-derived pectin hydrogel incorporating curcumin-loaded corn-derived zein nanoparticles (CurZNPs).
CurZNPs were produced by nanoprecipitation and showed high encapsulation efficiency (85%), sustained release, and strong in vitro antioxidant and anti-inflammatory activity. Oxidized pectin with different oxidation degrees (PDA, 2.5–5% oxidation) was synthesized to introduce aldehyde functionalities and blended with modified gelatin (mG) to obtain chemically crosslinked hydrogels. These hydrogels showed tunable rheological properties (50-350 Pa), low dissolution over 21 days (25-50% weight loss), injectability, self-healing behaviour, and high fibroblast adhesion after 7 days, thus enabling the identification of the optimal formulations. Incorporation of CurZNPs into PDA/mG hydrogels prolonged curcumin release for up to 28 days. In vitro studies using fibroblasts under oxidative stress and inflamed macrophages suggested that pre-treatment with CurZNP-loaded hydrogels exerts therapeutic effects, reducing oxidative damage and inflammatory signaling.
Overall, this study suggests the potential of a sustainable hybrid DDS based on bio-based materials for the controlled antioxidant and anti-inflammatory release. The injectable, self-healing PDA/mG hydrogels enable prolonged delivery of CurZNPs and effectively counteract oxidative stress and inflammation, highlighting their potential for wound healing and supporting future clinical applications.
FT acknowledges support from Research and Innovation NOP 2014-2020 for Doctoral Research programmes (Action IV.5), NODES project (MUR-M4C2 1.5 of PNRR-European Union-NextGenerationEU-ECS00000036). This study was carried out also within RECOVERY project (European Union-NextGenerationEU-PRIN 2022 PNRR program) and Horizon INJECTHEAL Project (101177924-2).
1 Zhang W et al., Front Bioeng Biotechnol 2021
2 Kumari A et al., Pharmaceutics 2022
3 Choi W et al., ACS Nano 2024
Fabrication and optimisation of a 3d printed polyhydroxybutyrate/MXene hybrid scaffold for bone regeneration
Mingzu Du1, Xuebin Yang1, Giuseppe Tronci2, David Wood3
1Biomaterials and Tissue Engineering Research Group, School of Dentistry. University of Leeds, Leeds - United Kingdom, 2Biomaterials and Tissue Engineering Research Group, School of Dentistry; Clothworkers’ Centre for Textile Materials Innovation for Healthcare, School of Design. University of Leeds, Leeds - United Kingdom, 3Biomaterials and Tissue Engineering Research Group, School of Dentistry; Bragg Centre for Materials Research. University of Leeds, Leeds - United Kingdom
Introduction: Owing to its intricate anatomy, complete recovery of an osteochondral defect becomes difficult when the lesion reaches the subchondral bone. Polyhydroxybutyrate (PHB) softens at elevated temperatures, allowing extrusion and reforming upon cooling to produce constructs with intricate geometry. However, it has limitations, such as high hydrophobicity. Titanium carbide (Ti3C2, MXene), a 2D material, has been shown to improve hydrophilicity. Titanium carbide (Ti3C2, MXene), a 2D material, has been shown to improve hydrophilicity of combined materials. Hence, we developed a novel hybrid ink of PHB and polycaprolactone (PCL) with MXene to support cell adhesion in bone-mimicking constructs.
Methods: PHB (BOC Sciences, USA) was mixed with MXene (NANOPLEXUS, UK) and PCL (Sigma-Aldrich, UK) at several ratios (80/5/15, 80/10/10, 80/15/5). Scaffolds were then printed using BIO X6 printer (Cellink, UK). Differential scanning calorimetry (DSC) and water contact angle measurement were employed to investigate the thermal denaturation of PHB and its hybrid samples, as well as the hydrophilicity. Cytotoxicity test was performed. CellTracker™ was used to track the attachment and adhesion behaviour of the human dental pulp stem cells (hDPSCs).
Results: MXene addition increased PHB melting temperature from 166.6°C to 175.7°C and reduced water contact angle from 88.2° to 71.3°, showing better wettability. CCK-8 and Live/Dead staining tests confirmed excellent biocompatibility. hDPSCs attached more efficiently to MXene-containing scaffolds, spreading well and forming higher cell densities. On PHB-only scaffolds, pre-treatment with 20% FBS greatly improved initial cell attachment, while plain medium led to minimal adhesion. After two days, cells were fully spread and displayed typical adhesion shapes, confirming that the scaffolds support both early attachment and sustained cell spreading.
Conclusions: PHB/PCL/MXene hybrid scaffolds are printable and cytocompatible. Serum pre-treatment further enhances early adhesion, and the scaffolds support proper cell spreading and morphology over time, highlighting their potential for osteochondral tissue regeneration.
Multilayered GBR membranes with mucoadhesive and osteoinductive properties
Claudia Dietze1, Benecke Lukas2, Klüver Enno3, Prade Ina3, Aibibu Dilbar2, Cherif Chokri2, Meyer Michael3
1Department Biopolymers and biological interactions. FILK Freiberg Institute gGmbH, Freiberg (Sachsen) - Germany, 2Institute of Textile Machinery and High Performance Material Technology. Dresden University of Technology, Dresden (Sachsen) - Germany, 3Department Biopolymers and biological interactions. FILK Freiberg Insitute gGmbH, Freiberg (Sachsen) - Germany
Introduction
Guided Bone Regeneration (GBR) is a cornerstone technique in oral and maxillofacial regenerative medicine to restore bone volume in areas with defects or deficiencies. The clinical success of GBR strongly depends on the performance of barrier membranes that prevent soft tissue ingrowth while supporting bone regeneration. However, currently available membranes often lack sufficient mechanical stability, bioactivity, and controlled degradation profiles. To overcome these limitations, we developed a biomimetic multilayer membrane that reproduces the natural transition between hard and soft tissues. The construct aims to simultaneously support bone regeneration and soft tissue integration while maintaining space stability during early healing.
Methods
A three-layer membrane system was fabricated using complementary processing techniques:
1. Chitosan layer with strong mucoadhesion and epithelial-shielding properties.
2. Electrospun PCL–silk fibroin layer (pore size ≈ 0.6 µm; fiber diameter ≈ 0.2 µm) providing a mechanical and cellular barrier.
3. 3D-printed calcium phosphate–enriched PCL layer designed with a defined pore geometry and mineral content for osteoinduction.
Plasma surface activation improved hydrophilicity and interlayer adhesion. Cell assays with human gingival epithelial cells and osteogenic cultures evaluated epithelial migration and osteogenic potential.
Results
The multilayer construct showed strong interfacial bonding and mechanical integrity. The chitosan and PCL–silk fibroin layers effectively inhibited epithelial cell migration, maintaining the protective barrier. The mineralized 3D-printed layer promoted osteogenic differentiation and mineral deposition. Structural analyses confirmed the desired pore architecture and compositional gradient resembling native bone–mucosa interfaces.
Conclusions
The developed biomimetic multilayer membrane combines mucoadhesive, barrier-forming, and osteoinductive functionalities in a single construct. Through the integration of electrospinning, 3D printing, and plasma activation, it offers a tunable platform for guided regeneration at complex hard–soft tissue interfaces. These results highlight its potential as a next-generation GBR membrane for translational applications in tissue engineering and regenerative dentistry.
Therapeutic action of FGF2-engaged stem cell spheroids in a critical limb Ischemia model: the role of tissue inhibitor of metalloproteinase 1and interleukin-8
Sangheon Kim1, Wonyoung Jang2
1Center for biomaterials. Korea institute of science and technolgy, Seoul (Seoul-tukpyolsi) - South Korea, 2center for biomaterials. Korea institute of science and technology, Seoul (Seoul-tukpyolsi) - South Korea
To enhance the survival and therapeutic efficacy of transplanted cell spheroids, we overexpressed tissue inhibitor metalloproteinase 1 (TIMP1) in human adipose-derived mesenchymal stem cells (hASCs) using an adenovirus-mediated gene delivery system and fabricated a spheroid (FECS-Ad) on an FGF2-tethered solid surface. TIMP1-overexpressing FECS-Ad showed significantly higher viability than normal FECS-Ad, whereas TIMP1-overexpressing hASCs did not demonstrate improved viability over their non-transduced counterparts. Doppler blood flow analysis confirmed that transplantation of TIMP1-overexpressing FECS-Ad into a critical limb ischemia (CLI) model enhanced therapeutic efficacy compared to normal FECS-Ad. Furthermore, HLA-A protein expression showed prolonged survival of TIMP1-overexpressing FECS-Ad up to 42 days post-transplantation, while normal FECS-Ad survived only until day 21. To further improve therapeutic efficacy while reducing the number of transplanted cells, we co-expressed interleukin-8 (IL-8), an angiogenic factor, with TIMP1 in FECS-Ad. Our findings demonstrate that IL-8/TIMP1 co-overexpressed FECS-Ad achieved comparable therapeutic effects (hemodynamic recovery and limb salvage) even when the number of transplanted cells was halved. Additionally, IL-8/TIMP1 co-overexpression significantly increased CD31 expression, indicating enhanced angiogenesis compared to IL-8 overexpression. In conclusion, the co-overexpression of TIMP1 and IL-8 enhances cell viability and angiogenesis, offering a promising strategy for stem cell-based therapy in CLI even with a reduced number of transplanted cell.
G-Force guided selfassembly of collagen hydrogels for transparent cornea equivalents
Luis Leo1, Sabina Eibichova2, Leon-Phillip Szepanowski1, Katharina Wiebe-Ben Zakour1, Joana Witt1, Gerd Geerling1, Florian Groeber-Becker1
1Department of Ophthalmology, Medical Faculty and University Hospital Düsseldorf. Heinrich Heine University Duesseldorf, Duesseldorf (Nordrhein-Westfalen) - Germany, 2Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany
The optical clarity and refractive index of the human cornea result from the highly organized architecture of its collagen type I fibrils. Collagen hydrogels have been extensively investigated for corneal tissue engineering; however, conventional fabrication methods often produce scaffolds with limited optical performance or inadequate mechanical strength. In this study, we present a scalable fabrication method for producing highly transparent, refractive collagen scaffolds with improved mechanical and biological properties. By precisely controlling the magnitude of G-force during gelation, collagen type I fibrils can be directed to self-assemble into planar ordered structures. The resulting scaffolds exhibit optical transparency comparable to that of the native cornea across the visible spectrum. The compact fibrillar organization enhances mechanical strength without compromising light transmission, closely mimicking the structure and function of the corneal stroma. The process is inherently cytocompatible and allows uniform stromal cell integration within the hydrogel matrix. In vitro culture studies further confirmed the retention of collagen’s native cyto- and biocompatibility. Overall, G-force–guided fibrillogenesis offers a robust and reproducible platform for fabricating advanced corneal equivalents, where optical transparency serves as a practical and quantifiable indicator of model health, supporting future applications in corneal tissue engineering and translational research.
A vascularized and contractile three-dimensional engineered heart ventricle can be fabricated using a functionalized fibrin–gelatin–reduced graphene oxide hydrogel
Kuckelkorn Christoph1, Baiker Marie1, Aksoy Ebru2, Jiang Yajuan2, Pfannkuche Kurt2, Fischer Horst1
1Department of Dental Materials and Biomaterials. RWTH Aachen University, Aachen (Rheinland-Pfalz) - Germany, 2Medical Faculty Center for Physiology and Pathophysiology. University of Cologne, Cologne (Nordrhein-Westfalen) - Germany
Objectives
Engineered cardiac tissues have emerged as a promising platform for cardiovascular research and therapeutic applications. The aim of this study was to develop a biomimetic three-dimensional engineered cardiac ventricle that replicates native cardiac functionality. This was achieved by embeddding human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a fibrin–gelatin–reduced graphene oxide (rGO) hydrogel.
Methodology
Composite hydrogels were prepared from fibrin or fibrin–gelatin matrices, each supplemented with different concentrations of rGO (0.004–0.4% w/v). The mechanical properties were assessed over a 28-day incubation period. In order to evaluate vascularization potential, human cardiac fibroblasts and human umbilical vein endothelial cells were incorporated into the hydrogels. After 21 days, vascular network characteristics, including total length, number of nodes, and vessel diameter, were quantified. To assess hiPSC-CMs contraction characteristics, 300 million cardiomyocytes per ml hydrogel were embedded in each formulation. A custom-designed stainless-steel mold enabled the reproducible fabrication of 3D ventricular constructs (12 mm diameter, 14 mm height, 2 mm wall thickness). They were then integrated into a flow bioreactor for dynamic culture and real-time flow monitoring.
Results
The storage modulus, lumen diameter of vascular structures, and contraction amplitude of cardiomyocytes all improved with increasing rGO concentration up to 0.04% (w/v) in fibrin–gelatin blends, after which higher concentrations led to a decline in these parameters. Furthermore, the engineered cardiac tissues showed expected changes in beating rate and contraction strength in response to cardioactive drugs (isoproterenol and carbachol).
Conclusions
We have developed a robust and reproducible approach to engineering vascularized, contractile 3D cardiac ventricles using fibrin-based reduced graphene oxide composite hydrogels. Including rGO significantly improved both mechanical integrity and biofunctional performance. Our approach has great potential for use in modeling cardiac disease and testing the effect of drugs, emphasizing the value of bioengineered cardiac tissue in translational research.
Polycaprolactone/bioactive glass 45S5 microparticles for bone regeneration: from in vitro characterization to in vivo validation in a cranial defect model - SEMIT
Mantas Liudvinaitis1, Dinas Tverijonas1, Ieva Šimoliūnė1, Milda Alksnė1, Povilas Daugėla1, Mindaugas Pranskūnas1, Egidijus Šimoliūnas1
1Department of Biological Models, Institute of Biochemistry, Life Sciences Center. Vilnius University, Vilnius (Vilniaus Apskritis) - Lithuania
Bone regeneration remains a significant challenge in clinical practice because it requires restoring both structural stability and biological function. Traditional grafting methods, such as autografts and allografts, are limited by donor-site morbidity, immune responses, and limited availability, which emphasizes the need for synthetic materials that are both bioactive and biocompatible.
We developed a composite free-packed microparticle system composed of polycaprolactone (PCL), a medically approved biodegradable polymer, and bioactive glass 45S5 (BG), recognized for its osteoconductive and osteoinductive properties. The microparticles were fabricated using a single-emulsion evaporation method, with polymer concentration, stirring speed and temperature adjusted to maximize the yield of spherical PCL/BG microparticles in the clinically relevant 100–500 µm range. Scanning electron microscopy revealed that the microparticles possess a rough, partially porous surface, well-suited for cell adhesion. Increasing agitation reduced particle size and surface roughness, while lower speeds produced larger and more irregular spheres.
Rat periosteum-derived mesenchymal stem cells readily adhered to the microparticles and maintained high viability. Although EdU incorporation method revealed a decrease in the proportion of proliferating cells over seven days, gene expression shifted under adipogenic, myogenic and osteogenic induction conditions, suggesting that the microparticle composition and surface influence mesenchymal stem cell differentiation.
In vivo testing in a rat critical-size cranial defect model has demonstrated that these microparticles support bone tissue ingrowth and integrate with the host bone over time. With their tunable architecture, inherent bioactivity and biocompatibility with mesenchymal stem cells, PCL/BG microparticles offer a promising alternative to conventional grafts and a foundation for future dynamic bone regeneration strategies.
Biodegradable scaffold design framework: guiding the future of oncological maxillofacial reconstruction
Milda Alksnė1, Ieva Šimoliūnė1, Mantas Liudvinaitis1, Dinas Tverijonas1, Ieva Gendvilienė2, Egidijus Šimoliūnas1
1Life Science Center, Institute of Biochemistry. Vilnius University, Vilnius (Vilniaus Apskritis) - Lithuania, 2Faculty of Medicine, Institute of Odontology. Vilnius University, Vilnius (Vilniaus Apskritis) - Lithuania
Large mandibular bone defects resulting from oncologic resections remain a major reconstructive challenge, as bone beyond a critical size loses its intrinsic regenerative capacity. Current graft-based approaches are limited by donor site morbidity, infection risk, and poor integration, while metallic implants lack bioactivity and often lead to long-term complications such as exposure or fibrous encapsulation.
To overcome these limitations, biodegradable scaffolds (BSs) are being developed. Their performance depends on achieving a delicate balance between mechanical stability, degradation kinetics, and vascularization. These interdependent parameters can be modulated through BS chemical composition and lattice design. However, the optimal relationship between degradation rate, mechanical behaviour, and vascular ingrowth remains largely unexplored.
This study aims to establish guidelines for the design and manufacture of BS tailored for large maxillofacial bone reconstruction. Well-characterized material combinations will be employed, integrating polymers with distinct degradation rates—polylactic acid for faster breakdown and polycaprolactone for slower—and ceramics with different dissolution speeds—calcium-deficient hydroxyapatite for slower resorption and Bioglass for rapid absorption. Using 3D printing technology and advanced lattice architectures (gyroid, diamond and YM), BSs will be fabricated to systematically evaluate how composition and structural design influence mechanical behaviour, degradation kinetics, and angiogenic potential during bone regeneration in vitro and in vivo.
The experimental findings will be complemented by finite element analysis to simulate mechanical evolution and bone regeneration dynamics. This integrated approach will generate comprehensive data on angiogenesis, bone formation, and long-term tissue compatibility during scaffold degradation. The combined experimental and computational results will provide insights into optimal micro- and macro-lattice configurations capable of maintaining mechanical stability while degrading in harmony with new tissue formation. Ultimately, this study aims to deliver a validated framework for the next generation of bioresorbable bone graft substitutes, offering precise design guidelines for large maxillofacial implants.
Harnessing electrical cues for tissue regeneration
Sahba Mobini
de Instituto de Micro y Nanotecnología, IMN-CNM, CSIC (CEI UAM+CSIC), Madrid - Spain
Electrical cues regulate cellular pathways, influencing key biological processes such as proliferation, migration, differentiation, and secretion.1 Since these cellular functions are main contributor in tissue repair, bioelectrical modulation offers a powerful therapeutic strategy in regenerative medicine; an emerging field known as electroceuticals.
Previously, we demonstrated that mimicking/amplifying endogenous bioelectrical signals through exogenous electrical stimulation (ES) accelerates bone tissue remodelling in vivo and in vitro.2 Ongoing studies in our laboratory using 2D neural cultures and 3D cerebral organoid systems have shown that specific ES protocols enhance neurite outgrowth and promotes neural maturation in healthy models,3 and support the reversal of apoptosis in pathological models.
Despite significant progress, challenges remain in defining optimal ES parameters and sustaining long-term functionality within complex tissue microenvironments. To address these challenges, we established a simple, standardized framework to improve experimental reproducibility and facilitate translation of ES parameters across various laboratory settings. Additionally, we explored indirect modulation strategy, “electrical cell priming”; results showed that controlled ES of secretory cells, e.g., mesenchymal stem/stromal cells (MSCs), enhances both the composition and potency of their secretome.4 Comparative analyses across MSC sources and stimulation paradigms reveal that this effect is reproducible and governed by specific electrical parameters.
Our results highlight the possible controllability of this process, reinforcing ES as a versatile, non-chemical and non-genetic tool for modulating cellular function and regeneration across both electroactive and non-electroactive tissues.
Acknowledgments
Grant PID2021-128611OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF/EU; Grant CNS2023-144736 funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR; Program of INVESTIGO 2023 of Madrid Community, co-financed by European Union.
References
[1] Thrivikraman G., et al (2018) Biomaterials 150, 60–86; [2] Leppik L. et al. (2018) Sci. Rep. 8, 6307; [3] Diego-Santiago M. et al. (2025) Sci. Rep. 15, 4772; [4] Mobini S., Schmidt C.E. (2020) US Patent US2133881 B2.
Investigating mechanosensitive alternative splicing in a 3D model of heart disease
Cristina Mazzotti1, Stefania Pagliari1, Giancarlo Forte1
1School of Cardiovascular and Metabolic Medicine & Sciences. King's College London, London (London, City of) - United Kingdom
Cardiac fibrosis is a hallmark of heart failure, driving extracellular matrix (ECM) remodelling. In turn, ECM remodelling alters myocardial anisotropy and architecture and disrupts cardiomyocyte function. Emerging evidence suggests that the mechanical stress caused by ECM remodelling rewires gene expression post-transcriptionally through alternative splicing (AS). The underlying mechanisms of mechanosensitive AS remain poorly understood due to a lack of physiologically relevant human models.
To address this, we developed a fully human, hiPSC-based three-dimensional engineered heart tissue (EHT) platform to investigate the interplay between fibrotic mechanical cues and RNA metabolism. Human iPSC-derived cardiac fibroblasts were activated with TGF-β1 to the myofibroblast phenotype depositing a fibrotic ECM with distinct mechanical and chemical properties. The fibrotic ECM was enzymatically fragmented into soluble bioactive fragments (matrikines), which were incorporated into EHTs together with hiPSC-derived cardiomyocytes.
EHTs incorporating fibrotic matrikines successfully developed functional contractility; however, they exhibited a progressive decline in force generation over prolonged culture, suggesting a sustained impact of fibrotic cues on their performance. Single-cell RNA sequencing revealed the transcriptional enrichment for cardiac pathology–associated pathways, including dilated and arrhythmogenic cardiomyopathy. Moreover, fibroblast clusters from fibrotic EHTs exhibited enhanced expression of focal adhesion genes, suggesting a shift toward an activated phenotype.
These findings demonstrate that fibrotic ECM-derived matrikines can trigger disease-relevant molecular programs in 3D cardiac tissues. This novel human-based model provides a foundation for dissecting the mechanosensitive regulation of alternative splicing in cardiac fibrosis and for identifying RNA-binding proteins and splice variants involved in heart failure progression.
Hydrogel-based cell bandage for human corneal epithelial cell transfer in the treatment of persistent corneal epithelial defects
Hugo Moreiras1, David E. Robinson1, Nardine Menassa2, Victoria R. Kearns1, Rachel L. Williams1, Hannah J. Levis1
1Eye and Vision Department. University of Liverpool, Liverpool - United Kingdom, 2Liverpool University Hospitals NHS Foundation Trust. University of Liverpool, Liverpool - United Kingdom
Introduction/Objectives: Persistent corneal epithelial defects (PCED) pose a significant therapeutic challenge in ophthalmology. Patients with PCED face an increased risk of corneal ulceration, infection, perforation, neovascularisation, and scarring, often leading to pain, discomfort, and vision loss. Standard treatments frequently prove ineffective, prolonging patient suffering and reducing visual acuity. To address this unmet need, we are developing a novel hydrogel-based cell bandage designed to deliver allogeneic human corneal epithelial cells (hCEpCs) directly to the ocular surface for the treatment of PCED.
Methods: We designed a methacrylated poly-epsilon-lysine (PeKMA) hydrogel, crosslinked with bis[sulfosuccinimidyl]suberate (BS3) before UV crosslinking and functionalised with an RGD peptide sequence, as a carrier for hCEpCs. The physical properties of the hydrogels were characterised by compressive modulus and handleability. The cytotoxicity of the hydrogels was assessed directly, by observing the attachment and morphology of hCEpCs on the hydrogels, and indirectly, by evaluating the viability of hCEpCs after exposure to media incubated with hydrogels for three days. Delivery of cells from hydrogels to the ocular surface was determined using decellularised human corneal discs and ex vivo porcine eyes.
Results: We demonstrated that our hydrogel supports attachment of hCEpCs, monolayer formation, and maintenance of cellular phenotype, including p63 expression — a key marker of epithelial progenitor cells. Viability data show that about 70% of cells retain normal metabolic activity. Over 60% of hCEpCs cultured on the hydrogels were successfully delivered onto the surface of the models within two days. Post-transfer, cells continued to proliferate for up to five days after removal of the bandage, contributing to epithelial regeneration.
Conclusions: This work introduces a promising platform for a cell-based therapy in PCED, enabling the delivery of a substantial number of hCEpCs to the corneal surface. Future studies will focus on in vivo safety tests in rabbits to assess the potential of our approach.
Calcitriol ameliorates myotonia in Myotonic Dystrophy type 1 muscle 3D models via an MBNL1-independent mechanism
Xiomara Fernández-Garibay1, Maria Sabater-Arcís1, Ainoa Tejedera-Villafranca1, Judit Núñez-Manchón2, Rubén Artero3, Mònica Suelves2, Gisela Nogales-Gadea2, Javier Ramón-Azcón1, Juan M. Fernández-Costa1
1Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, 2Germans Trias i Pujol Research Institute (IGTP), Badalona (Barcelona) - Spain, 3University Institute for Biotechnology and Biomedicine (BIOTECMED), University of Valencia, Valencia - Spain
Myotonic Dystrophy Type 1 (DM1) is a severe, multisystemic genetic disorder primarily characterized by progressive muscle weakness, atrophy, and myotonia. While traditional 2D culture systems and animal models have advanced our understanding of DM1 molecular pathways, they fall short in recapitulating the contractile and heterogeneous nature of the disease. To overcome these limitations, we engineered contractile 3D human skeletal muscle tissues using immortalized myoblasts from three DM1 patient-derived lines, representing juvenile, adult, and late-onset subtypes. These cells were embedded in biocompatible hydrogel scaffolds and anchored between flexible posts to support alignment, maturation, and spontaneous contraction. The resulting tissues faithfully reproduced key DM1 features, including MBNL1 sequestration in ribonuclear foci and widespread splicing defects. Importantly for the first time in vitro splicing alterations in CLCN1, the chloride channel associated with myotonia. Our model also successfully recapitulated patient-specific functional phenotypes such as transient and fixed muscle weakness and myotonia, previously only observed in vivo. Pharmacological treatment with small molecules that increase MBNL1 levels partially rescued molecular and functional defects. Intriguingly, treatment with Calcitriol significantly reduced myotonia without restoring MBNL1 distribution or CLCN1 splicing, indicating a novel MBNL1-independent mechanism of action. Transcriptomic analysis (RNA-seq) revealed that Calcitriol restores gene expression profiles altered in DM1: it upregulates genes involved in neuromuscular transmission, metabolism, and synaptic signaling, which are downregulated in our DM1 models, and downregulates genes associated with stress, inflammation, fibrosis, and dysregulated development, which are elevated in our DM1 models. This bidirectional gene expression correction aligns with the observed functional improvements. Altogether, our 3D DM1 muscle model represents a highly relevant, patient-specific platform for therapeutic testing. It enables the study of contractile phenotypes and uncovers Calcitriol as a promising modulator of DM1 pathology through an MBNL1-independent transcriptional mechanism.
Cardiomyocyte-derived extracellular vesicles production using 2D and 3D systems for cardiovascular tissue engineering
Xueqing Li1, Helder Almeida Santos1, Monize Caiado Decarli1
1Department of Biomaterials and Biomedical Technology. University Medical Center Groningen, Groningen - The Netherlands
Introduction: Cardiovascular diseases are the leading cause of global mortality and disability. Current pharmacological treatments are limited by low bioavailability and poor targeting. Extracellular vesicles (EVs), nanoparticles secreted by cells carrying bioactive molecules, hold cardiovascular clinical potential, but require controlled release and scalable production. Encapsulating EVs within hydrogels can provide favorable conditions to enhance EV-tissue retention, but the investigations of 3D bioprinted cardiovascular models loaded with EVs is rarely seen. Herein, our objective was to obtain cardiomyocyte spheroids and cardiomyocyte derived-EVs through 2D and 3D models. Methods: H9c2 cardiomyocytes were cultured in 2D and in a 3D-printed non-adhesive micromolded hydrogel. Spheroid morphology, cell viability, and metabolic activity were evaluated, and EVs were harvested over time. Nanoparticle Tracking Analysis assessed EV concentration and size, Bicinchoninic Acid Assay measured protein content, and Western Blot detected EV markers (CD63, CD81, TSG101). EV functionality testes via wound healing and tube formation assays are in progress. Results: H9c2 spheroids successfully formed within 7 days in the 3D system. Spheroids containing 3,000, 6,000, 9,000, and 12,000 cells had diameters of ∼100, 138, 165, and 231 µm, respectively, all highly spherical (sphericity≈0.93) and solid (solidity≈0.99). Live/Dead staining indicated high viability throughout all spheroids except the largest group (231 µm), which had a cell death core. ATP production increased with cell spheroid number, except in the largest spheroids, supporting the observed cell death. WB confirmed EV positive markers in both 2D and 3D-derived EVs and absence of Calnexin. While BCA showed no significant difference in total protein, NTA revealed a 10∼30 times higher EV yield in the 3D system when compared to 2D culture. Conclusion: The 3D micromold system generated viable cardiomyocyte spheroids and enhanced EV production (10∼30times), with lower medium consumption and simplified EV isolation, potential outcomes for scalable EV manufacturing and cardiovascular tissue engineering.
Rapid, autologous, and confluent endothelialization yields enhanced functionality and hemocompatibility of co-cultured vascular wall constructs
Kate D. Macquarrie1, Sahej Kaur Saini1, Jeremy A. Antonyshyn1, Stefan O. P. Hofer2, J. Paul Santerre1
1Ted Rogers Centre for Heart Research, Toronto (Ontario) - Canada, 2University Health Network, Toronto (Ontario) - Canada
Objectives
The patency of small-diameter vascular grafts is compromised by thrombosis resulting from poor endothelialization (1). Adipose tissue is a source of expandable human adipose-derived microvascular endothelial cells (HAMVECs) and adipose-derived stromal cells (ASCs). The indirect co-culture of these cells accelerates endothelialization from 14 days (HAMVEC monoculture) to <2 days (ASC/HAMVEC co-culture) (2). Here, we examined the phenotype and hemocompatibility of these endothelia, then generated endothelialized grafts.
Methodology
ASCs and HAMVECs were isolated from abdominal adipose tissue and seeded on opposite sides of electrospun polyurethane scaffolds (2). To assess partial vs. full endothelialization, HAMVECs were monocultured, or co-cultured with ASCs for 7 days (N=3, n=2). Whole blood was collected from healthy volunteers, and white blood cells, platelet-rich, or platelet-poor plasma were isolated and applied to scaffolds. Endothelial activation and hemocompatibility were assessed with scanning electron microscopy, immunostaining, and assays on media (3). Grafts were produced by rolling scaffolds into tubes, then endothelializing and conditioning in a perfusion bioreactor.
Results
Partially and fully endothelialized scaffolds had low hemolysis ratios (1.3% and 0.6%, respectively). HAMVECs in confluent endothelia expressed more endothelial nitric oxide synthase (p< 0.05), and less intercellular adhesion molecule-1 (p< 0.01), than those in partial endothelia. Half as many monocytes adhered to full vs. partial endothelia (p< 0.05), and preliminary results indicated that full endothelia reduced platelet activation.
Conclusions
Partial and full endothelia were non-hemolytic, but full endothelia adhered fewer blood components and had a more quiescent phenotype (3,4). Ongoing work is investigating clotting rates, complement cascade activation, and the effect of perfusion on endothelial phenotype and hemocompatibility (3).
References
1. Guo et al., Compos. B Eng, 2025, 301:112505.
2. Antonyshyn, et al., Acta Biomat, 2024, 175, 214–225.
3. Weber, et al., Front Bioeng Biotechnol, 2018, 6:99.
4. Allbritton-King & García-Cardeña, Front Cell Dev Biol, 2023 11:1278166.
Optimization of genipin crosslinking in electrospun blood protein scaffolds for autologous vascular implantation
Tom Bode1, Jan Drexler1, Kai Höltje1, Birgit Glasmacher1, Marc Müller1
1Institute for Multiphase Processes. Leibniz University Hannover, Hannover (Niedersachsen) - Germany
Objectives
Autologous vascular grafts, produced from a patient’s own blood proteins, offer a means of creating personalized, immunocompatible implants. The goal of this study was to develop a genipin-based crosslinking strategy for electrospun plasma protein scaffolds using porcine blood to create a preclinical model for autologous processing. The study optimized the ethanol-to-water solvent composition to achieve structural stability without fully denaturing the proteins.
Materials and Methods
Porcine plasma was purified by diafiltration and concentrated via rotary evaporation. Then, it was blended with polyethylene oxide (PEO) to achieve electrospinnability without the use of cytotoxic solvents. Nonwovens were fabricated using a vertical electrospinning setup and crosslinked with 5% w/w genipin in ethanol-water mixtures (30-96 vol%). Scanning electron microscopy (SEM) was used to assess morphology, while protein stability and secondary structure were evaluated using differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR).
Results
Scaffolds that were crosslinked in 70–90 vol% ethanol retained a stable fibrous morphology. Reduced amide band shifts in FTIR spectra indicated minimal denaturation and preserved secondary structure. Lower ethanol contents (<55 vol%) caused dissolution before crosslinking could occur. Very high ethanol concentrations (>90 vol%) promoted aggregation and reduced genipin reactivity due to limited amino group accessibility. Differential scanning calorimetry (DSC) revealed that scaffolds treated within the 70–90 vol % range maintained thermal stability comparable to that of native plasma fibers, confirming a balance between crosslinking and structural preservation. FTIR spectra confirmed reduced amide peak shifts under these conditions, indicating maintained protein conformation.
Conclusion/Outlook
This work presents a reproducible method of manufacturing protein-based vascular scaffolds from whole blood plasma. The process preserves the scaffolds' native biochemical features and structural porosity, paving the way for autologous graft fabrication using human plasma. Future studies will examine the mechanical performance and endothelialization of these scaffolds under physiological conditions, bringing us closer to developing clinically translatable, patient-specific vascular implants.
Engineering post-synaptic architectures with native biomembranes
Alice Hattar1, Janic Töx1, Nevena Stajkovic1, Mehdi Ravandeh2, Francesca Santoro1
1Institute of Biological Information Processing - IBI-3. Forschungszentrum Jülich, Jülich (Nordrhein-Westfalen) - Germany, 2Christian-Albrechts-University of Kiel (CAU), Kiel (Schleswig-Holstein) - Germany
Neuroelectronics aims for seamless integration at the interface between neuronal tissue and chip-based devices, which is crucial for effective cell-chip coupling1. To improve this coupling, many biomimetic approaches, such as Supported Lipid Bilayers (SLBs), have been developed as platforms that replicate the structure and composition of the biological plasma membrane2. These are formed through various techniques, with vesicle fusion (VF) being the simplest, relying on the spontaneous rupture and self-assembly of liposomes at a critical surface concentration. SLBs can be further functionalized by adding blebs. Blebs form as a result of actomyosin contractions in the cell's actin cortex, causing detachment from the plasma membrane. The cytosol then flows into the bleb, expanding it with the help of actin-membrane linker proteins like ezrin, until it detaches, carrying native plasma membrane components3. This combination of natural and artificial elements could enable manipulation of cell adhesion behavior by adjusting their ratio4. Building on existing strategies, we optimized the formation of hybrid membranes on both 2D conductive and non-conductive surfaces through VF. Blebs collected from neurons at different days in vitro (DIVs) were further analyzed using NanoSight Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) to determine their size, concentration, and surface charge. The hybrid SLBs, composed of neuronal blebs and positively charged artificial lipids, were then formed on glass and PEDOT-based surfaces and characterized via Fluorescence Recovery After Photobleaching (FRAP) and, for PEDOT, Electrochemical Impedance Spectroscopy (EIS). Additionally, mass spectrometry confirmed the presence of synaptic proteins, whose activity can be monitored using EIS, thereby enabling neuromorphic functionalities. In conclusion, this approach facilitates the formation of hybrid SLBs with native neuronal membranes on both conductive and non-conductive surfaces, with the potential to modulate device responses and advance the development of a functional neuro-hybrid synapse.
1. Organic Neuroelectronics: From Neural Interfaces to Neuroprosthetics.
2. Solid-supported lipid bilayers - A versatile tool for the structural and functional characterization of membrane proteins.
3. A short history of blebbing
4. Composite Lipid Bilayers from Cell Membrane Extracts and Artificial Mixes as a Cell Culture Platform.
Scaffold-mediated delivery of miRNA-29b mimic mitigates excessive collagen deposition and fibrotic marker expression in myofibroblasts
Juan Carlos Palomeque Chávez1, Marko Dobricic1, Amarachi Erugo1, Ahmed Almaini1, Jack Maughan2, James E. Dixon3, Cathal J. Kearney4, Shane Browne1, Fergal J. O'brien1
1Tissue Engineering Research Group. Royal College of Surgeons in Ireland, Dublin - Ireland, 2Tissue Engineereing Research Group. Royal College of Surgeons in Ireland, Dublin - Ireland, 3Nottingham Biomedical Research Centre. University of Nottingham, Nottingham - United Kingdom, 4Kearney Lab. University of Massachussetts Armhest, Armhest (Massachusetts) - United States
OBJECTIVES
During late wound healing, dermal fibroblasts remodel the extracellular matrix (ECM) to restore tissue integrity. However, fibroblast overactivation and excessive collagen-I (Col-I) deposition can lead to fibrosis.1 microRNA-29b (miRNA-29b) is known to regulate Col-I synthesis in fibroblasts. However, localised delivery of exogenous nucleic acids is still a challenge.2 Collagen-glycosaminoglycan (CG) scaffolds facilitate tissue repair and enable the controlled release of bioactive molecules.3 This study investigates the functionalisation of CG scaffolds with miRNA-29b nanoparticles to induce anti-fibrotic responses and minimise scar formation.
METHODS
miRNA-29b nanoparticles were generated via electrostatic complexation of miRNA-29b mimic with the non-viral GAG-binding enhanced transduction (GET)4 peptide. Human dermal fibroblasts (hDFs) were treated with TGF-B1 to induce a fibrotic phenotype before miRNA-29b administration. Col-I expression and deposition were assessed by PCR and histology, while a-SMA expression was evaluated by fluorescence microscopy. Functional outcomes were measured using a collagen gel contraction assay. CG scaffolds were then soak-loaded with miRNA-29b nanoparticles (CG-29b) and seeded with TGF-B1-treated hDFs. Gene expression, a-SMA levels, and cell viability were analysed to assess fibrotic attenuation.
RESULTS
miRNA-29b-treated hDFs exhibited significant reductions in Col-I expression and deposition without compromising viability. a-SMA expression and gel contraction were similarly decreased. hDFs cultured on CG-29b scaffolds demonstrated comparable reductions in Col-I and a-SMA expression, with no adverse effects on cell viability, as confirmed by gene and image analysis.
CONCLUSIONS
miRNA-29b nanoparticles effectively suppressed pro-fibrotic markers in hDFs. Incorporation within CG scaffolds generated biocompatible, anti-fibrotic platforms capable of modulating fibroblast activity during wound healing. This strategy offers a promising approach for preventing fibrosis-associated pathologies such as keloids and hypertrophic scarring.
REFERENCES
1 Wynn T.A., Jour. Pathol., 2013
2 Valatabar et al., Jour. of Nanobiotech., 2024
3 Palomeque Chavez et al., Biomat. Sci., 2025
4 Raftery et al., Biomaterials, 2019
ACKNOWLEDGEMENTS
Research Ireland Advanced Materials and BioEngineering Research (SFI/12/RC/2278_P2).
Bridging the gap between biological and synthetic scaffolds using Quantum Molecular Resonance (QMR) technology to develop next-generation scaffolds for tissue engineering
Martina Casarin1, Stefano Tedesco2, Niccolò Turetta2, Gianantonio Pozzato3, Maurizio Marzaro4
1Department of Surgery, Oncology and Gastroenterology. University of Padova, Padova (Veneto) - Italy, 2Telea Biotech Srl, Sandrigo (Veneto) - Italy, 3Telea Medical Srl, Sandrigo (Veneto) - Italy, 4AULSS2, Treviso (Veneto) - Italy
In tissue and organ regeneration, the concept of “organ memory” is crucial to promote cell adhesion, proliferation, and differentiation within a scaffold, ultimately enabling functional tissue restoration. Biological scaffolds provide natural biochemical cues but suffer from inter-sample variability and uncontrolled porosity. Conversely, synthetic materials allow tunable physicochemical properties but lack biological signaling, leading to limited biocompatibility.
To bridge this gap, we introduced Quantum Molecular Resonance (QMR) driven scaffold micro-perforation, a patented technique by Telea Biotech (EP2164536, EP2493522, EP20716109A) which exploits the QMR technology developed by Telea Medical to enhance the properties of decellularized tissues. This approach generates a dense network of microchannels, recreating a three-dimensional architecture comparable to that of synthetic scaffolds. QMR employs high-frequency energy and resonance phenomenon to selectively break atomic bonds without heat generation. Using a robotic system equipped with a QMR-connected needle, tissues can be micro-perforated with customizable patterns, enabling precise control of channel diameter and distribution.
The resulting increase in porosity enhances cell infiltration and repopulation, supporting faster and more homogeneous tissue regeneration. This approach has already demonstrated optimal in vivo outcomes in porcine models for both soft (esophagus, n=22 [10.3389/fbioe.2022.912617]) and hard (palate, n=6 [10.1038/s41598-021-93951-w]) tissues. Furthermore, QMR micro-perforation technology is being applied to MODA, a dermal matrix already approved for human clinical use [10.1007/s00266-017-1069-7 and 10.1177/1534734619884422], to assess whether micro-perforation can further accelerate healing and tissue integration. Recently, the method has been extended to thicker and more complex tissues such as the urinary bladder, showing promising in vitro results.
QMR driven micro-perforation represents an innovative strategy to modulate the microarchitecture of biological matrices, combining the advantages of natural scaffolds with enhanced porosity and improved regenerative potential. Its successful application to various tissue models highlights its versatility and paves the way for translational studies in regenerative medicine.
Toward fully autologous tissue engineering: human plasma as a functional scaffold in the PLATE method
Mahboobeh Amoushahi1, Elissa Elia2, Stéphane Chabaud3, Stéphane Bolduc4, Magdalena Fossum5
1Laboratory of Tissue Engineering, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Rigshospitalet, Copenhagen (Staden Kobenhavn) - Denmark, 2Centre de Recherche en Organogenèse Expérimentale de l’Université Laval/LOEX, Centre de Recherche du CHU de Québec‐Université Laval, Axe Médecine Régénératrice, Québec City, QC G1J 1Z4, Canada. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 3Centre de Recherche en Organogenèse Expérimentale de l’Université Laval/LOEX, Centre de Recherche du CHU de Québec‐Université Laval, Axe Médecine Régénératrice, Québec City, QC G1J 1Z4, Canada. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 4Centre de Recherche en Organogenèse Expérimentale de l’Université Laval/LOEX, Centre de Recherche du CHU de Québec‐Université Laval, Axe Médecine Régénératrice, Québec City, QC G1J 1Z4, Canada. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 5Laboratory of Tissue Engineering, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Rigshospitalet, Copenhagen (Staden Kobenhavn) - Denmark
Background and aim: The Perioperative Layered Autologous Tissue Expansion (PLATE) method enables intraoperative generation of three-dimensional tissue constructs using autologous micrografts embedded in collagen scaffolds. While collagen provides a supportive matrix, human plasma could allow a fully autologous graft with intrinsic growth-promoting properties. This in vitro study aimed to replace collagen with autologous human plasma, a xeno-free, growth factor–rich biomaterial, to enhance the biological and structural performance of PLATE constructs.
Material and methodology: Human foreskin tissue micrografts were embedded in three hydrogel formulations: (1) collagen-only (control), (2) plasma-collagen hybrid, and (3) plasma-only matrices. Constructs were fabricated and cultured in vitro for up to four weeks. Histological staining, scanning electron microscopy, and immunofluorescence (KI67, P63, K14, K10 and laminin-5) were performed to assess cell proliferation, migration, and differentiation.
Results: Plasma-only constructs demonstrated markedly enhanced cell proliferation and migration, early signs of epithelial differentiation, and higher epithelial layer thickness compared to collagen-based controls. The plasma-collagen hybrid exhibited intermediate results. Scanning electron microscopy revealed improved matrix integration and early tissue stratification in plasma-based constructs, consistent with enhanced biological activity of the autologous plasma matrix.
Conclusion: Autologous human plasma provides a bioactive, xeno-free scaffold that could replace collagen in the PLATE methodology. By taking advantage of the growth promoting factors in plasma, this approach may improve biological outcomes and support rapid, intraoperative tissue generation with strong translational potential for reconstructive surgery.
Effect of shear stress on vascular cell behavior with a 3D printed perfusion bioreactor system and topographically-modified electrospun scaffolds
Andrew P. Johnston1, Todd P. Burton1, Anthony Callanan1
1Institute for Bioengineering. University of Edinburgh, Edinburgh (Edinburgh, City of) - United Kingdom
Aim and Objective
Cardiovascular diseases (CVDs) are a significant cause of mortality. Bypass graft operations, used to treat CVDs, can be precluded by issues such as systemic diseases. Electrospun tissue-engineered vascular grafts are a promising alternative treatment method which consist of microfibers. Previous studies suggest that both modification of fiber topography and introduction of a dynamic fluidic environment can influence cell behavior [1, 2]. This study employed a combinatory approach, examining the influence of electrospun fiber surface topography on vascular cell behavior within perfusion bioreactors.
Material and Methodology
Both smooth and dimpled scaffolds were electrospun using 16%w/v polycaprolactone, with the former at ambient humidity in a chloroform/methanol solution, and the latter at increased humidity with the addition of dimethyl sulfoxide. Scaffold fiber morphology was assessed via Scanning Electron Microscopy (SEM). The computer-aided design model of the bioreactor was analyzed in Ansys Fluent Computational Fluid Dynamics (CFD). The bioreactors were 3D printed via stereolithography, and scaffolds seeded with two vascular cell types were maintained in static and dynamic culture over five days. Cell viability, proliferation, DAPI/Phalloidin and osmium-stained images were assessed. Statistical analyses were performed on all data.
Results
SEM images of the scaffolds confirmed smooth and dimpled fiber morphologies. CFD validated the bioreactor design, providing a physiologically relevant flow rate. Viability of both cell types was maintained under static and dynamic conditions and variations in behavior and morphology were noted between groups.
Conclusions
These results demonstrate that both the bioreactors and the fiber topography alter cellular response, warranting further investigation of these modalities. This 3D fluidic system offers enhanced physiological representation and therefore may contribute toward future treatment of CVDs.
Funding: EPSRC grant EP/W524384/1 and MRC grant MR/L012766/1.
References
1. Franzoni et al., Am J Ph Hea, 49-59, 2016.
2. Johnston et al., J Appl Polym Sci., 142: e57062, 2025.
Estimation of the scaffold storage stability using accelerated aging protocol
Boubacar Diallo1, Sylvie Changotade1, Didier Lutomski1, Philippe Djemia2, Rohman Geraldine1
1Unité de Recherche en Ingénierie Tissulaire, URIT, UR, F-93430. Université Sorbonne Paris Nord, Villetaneuse (Ile-de-France) - France, 2Laboratoire des Sciences des Procédés et de Matériaux (LSPM), CNRS. Université Sorbonne Paris Nord, Villetaneuse (Ile-de-France) - France
Introduction and objective
In tissue engineering applications, studies generally focus on the rate of polymeric scaffold degradation in an aqueous or cell-medium environment. Our Unit has developed a poly(ester-urethane-urea) (PEUU) elastomeric scaffod for tissue engineering applications [1-3] and recently observed that, after being stored at room temperature for 4 years, fibroblast cells still adhere and proliferate significantly on the aged scaffold, but stay round all over the pore surface. The aim of this study is to investigate how thermally accelerated aging of the scaffold affects cellular response.
Material and methodology
PEUU scaffolds were synthesized using the polyHIPE method [1] and thermally aged using the time-temperature principle from 2 to 50 days at 75°C and 90°C. Microscopy, FTIR, colorimetry, and Brillouin spectroscopy were used for characterization. For in vitro study, contact cytotoxicity as well as fibroblast adhesion, spreading and proliferation were evaluated.
Results
After aging, no modification of the porosity and the pore size were observed. FTIR analysis revealed no significant changes in the chemical structure. However, the scaffold yellowish aspect increased with the aging time. Brillouin scattering reveals that the frequency shift depends on aging time. A similar trend is observed for the full width at half-maximum. These variations intensify with higher aging temperatures.
After 3 days of contact cytotoxicity with aged scaffolds, cells are affected, adopt a rounded morphology at the well bottom. A similar observation was made regarding cell proliferation: cells stayed rounded and didn’t proliferate on aged scaffolds, unlike on fresh ones where they spread effectively and proliferate. These results allowed us to define the maximum storage time for these scaffolds.
[1] S. Changotade et al., Stem Cells Int., vol. 2015, p. 283796, 2015, doi: 10.1155/2015/283796.
[2] G. Rohman et al., Biomed. Opt. Express, vol. 10, no 4, p. 1649 1659, mars 2019, doi: 10.1364/BOE.10.001649.
[3] C. Langueh et al., Polym. Degrad. Stab., vol. 183, p. 109454, janv. 2021, doi: 10.1016/j.polymdegradstab.2020.109454.
Nanoparticle (PNP) uptake pathways and localization of mRNA when loaded on a PNP/lipid co-assembly
Suja Shrestha1, Emma A. Lindsay1, J. Paul Santerre1
1Faculty of Dentistry. University of Toronto, Toronto (Ontario) - Canada
Background: Messenger RNA (mRNA) therapies show promise for treating diseases such as Duchenne muscular dystrophy, and cardiovascular disease1. However, mRNA’s instability and negative charge require effective delivery systems. Oligomeric-urethane nanoparticles (PNPs) show high biocompatibility and immunomodulation2; and can co-assemble with PEGylated lipid (DSPE-PEG(2000)-amine) and mRNA to enhance transfection efficiency in mouse myoblasts (C2C12 cells)3. However, the mRNA/PNP/lipid uptake route in C2C12 cells remains unknown.
Objective: The objective of the study is to investigate the transfection of Cas9 mRNA using PNP/lipid co-assemblies in C2C12 cells and determine the cellular pathway responsible for uptake.
Materials and Methodology: Cas9 mRNA was purchased from Trilink Biotechnologies and PEGylated lipid was purchased from Avanti Polar Lipids. Fluorescein isothiocyanate (FITC)-labeled PNPs were synthesized based on a previously published protocol2 and co-assembled with PEGylated lipid and mRNA. C2C12 cells were treated with mRNA/PNP/lipid and localization was characterized using a fluorescence microscope and ImageJ. Cells were preincubated with endocytosis inhibitors, followed by lysosome and plasma membrane staining. Statistics were conducted using Prism 10.0 software (GraphPad).
Results: mRNA/PNP/lipid treatment yielded higher particle uptake/cell (10.8±2.1), compared to PNP alone (4.6±1.3, p<0.001), mRNA/PNP (8.5±2.4), and PNP/lipid (6.0±3.4, p<0.001) groups. Genistein treated groups showed the lowest % PNP uptake across all groups (PNP (26.2±8.6%, p<0.001), PNP/mRNA (26.4±10.9%, p<0.0001), PNP/lipid (37.1±14.4%, p<0.01), mRNA/PNP/lipid (26.4±14.4%, p<0.0001)) compared to other endocytosis inhibitors wortmannin, chlorpromazine and methyl-β-cyclodextrin indicating that the preferred particle uptake pathway is predominantly caveolae-mediated.
Conclusions: Unlike other endocytic pathways, caveolae-mediated uptake can bypass lysosomes4, organelles responsible for degrading biomolecules like mRNA. This characteristic makes PNP/lipid co-assembly an attractive route for delivering therapeutic agents, paving the way for potential innovative therapeutics against muscular dystrophies.
1. Nat Rev Drug Discov 2020, 19, 441-442.
2. Acs Appl Mater Inter 2021, 13, 58352-58368.
3. Acta Biomater 2025, 201, 457-470.
4. Cell Mol Life Sci 2009, 66, 2873-2896.
Repair of peripheral nerve injuries using a tissue-engineered nerve conduit in a rabbit model
Oumayma Hayouni1, Alexane Thibodeau2, Todd Galbraith3, Hélène Khuong2, François Berthod2
1Faculty of medecine, Department of Surgery. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 2Faculty of medecine, Department of surgery. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 3Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada
Peripheral nerve injuries are a significant concern in the medical field, due to their potential to cause long-term disability. Although autografts remain the gold standard for large-gap repair, they cause donor-site morbidity and have limited efficacy for extensive defects. The alternative is to use nerve conduits to guide axonal migration, but these have several clinical limitations.
This study aims to develop a prevascularized tissue-engineered nerve conduit in which a capillary network will be developed in vitro. This vascular network will be able to connect rapidly to that of the host, restoring blood flow and promoting long-distance axonal regeneration.
Methods: The prevascularized nerve conduit (PNC) is made of a living rolled fibroblast sheet seeded with endothelial cells to form a capillary-like network structure, compared to a non-prevascularized control (NC).
Conduits were grafted for 12 months into immunosuppressed New Zealand rabbits to bridge a 4 cm fibular nerve gap. Electrophysiological studies were performed to monitor muscle reinnervation. The Toe spread reflex (TSI) was used as an indicator of the onset of motor recovery in fibular nerve-dependent muscles. At the end of the experiment, the tibialis anterior muscles were analyzed for mass index, fiber area, and neuromuscular junction (NMJ) morphology.
Results: Electromyogram and TSI revealed motor recovery from the 6th month in autografts (p < 0.05) and from the 8th month in PNCs. Muscle analysis showed partial mass recovery, with higher muscle index and fiber area in autografts versus NC. At higher magnification, neuromuscular junctions appeared mature and branched in the autograft and PNC groups, while they were smaller and fragmented in the NC group.
Conclusion: Our approach, involving the graft of a pre-vascularized living nerve conduit able to significantly accelerate graft vascularization, could be a promising new clinical option for the repair of severe injuries (larger than 3 cm).
From rupture to regeneration: bioengineering anisotropic tendon replacements
Ane Llucia1, Sandra Camarero-Espinosa1
1BioSmarTE lab. POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain
Effectively treating full-thickness tendon ruptures is currently hindered by the lack of resilient support materials that can promote tissue formation while withstanding physiological loading conditions. The mechanical properties of tendon entheses are anisotropic, and the phenotype of cells within, heterotypic. To better mimic the physical and cellular characteristic of tendons, we developed 3D printed gradient poly(L-lactide-co- ε-caprolactone) (PLCL) scaffolds with diverse architectures, systematically varying pore geometry and dimensions. The resulting porosity was assessed by scanning electron microscopy (SEM). 3D constructs underwent tensile testing to determine their cyclic stress-strain responses and shape recovery capacity. Specific anisotropic scaffolds exhibiting mechanical properties optimal for replicating the native tissue were selected as candidates.
A novel tenogenesis protocol for bone marrow-derived MSCs was validated in 2D culture using qPCR and immunofluorescence (IF). This demonstrated the upregulation of tendon-specific markers (scleraxis, tenomodulin and tenascin-C) and the deposition of type I and type III collagen. Phenotypic stability was confirmed over 21 days of culture. MSCs were seeded on the chosen scaffolds and cultured in both basal and tenogenic media for three weeks. Fluorescence microscopy and DNA quantification revealed cell adhesion and proliferation, with cells exhibiting a spread and aligned morphology throughout the constructs. Differentiation studies further confirmed the ability of the scaffolds and media to drive MSCs toward a tenocyte-like phenotype, evidenced by qPCR upregulation of key markers. IF staining of ECM components, GAG quantification, and SEM imaging verified the de novo synthesis and deposition of tendon-characteristic proteins organized into well-aligned fibers, resulting in the formation of mature tendon-like tissue.
In summary, the selected gradient PLCL scaffolds successfully recreate the elastic and resilient mechanical properties of native tendon. These structures also provided a supportive microenvironment for MSC proliferation, differentiation, and subsequent matrix formation. These outcomes present a promising strategy for the effective regeneration of full-thickness tendon ruptures.
This work was supported by the grant AEI/10.13039/501100011033-CPP2021-008754, the University of the Basque Country (GIU21/033) and the Ramon y Cajal RYC2023-044860-I.
From microtissues to functional meniscal grafts: bioprinting of phenotypically defined fibrocartilage microtissues into melt electrowritten scaffolds to engineer regionally defined meniscal grafts
Gabriela Kronemberger1, Francesca Spagnuolo1, Tosca Roncada1, Kaoutar Chattahy1, Aliaa Karam1, Max Wirth1, Daniel J. Kelly1
1Trinity College Dublin, Dublin - Ireland
Lesions in the meniscus are common in sports injuries and significantly increase the risk of osteoarthritis, which leads to joint degeneration and disability. An estimated 1.5 million meniscal repairs are performed annually in the US and Europe, making it one of the most frequent orthopaedic procedures with substantial socioeconomic impacts. In tissue engineering, cellular microtissues are emerging as building blocks to biofabricate functional grafts. These microtissues can be incorporated into 3D-printed scaffolds to promote fusion, (re)modeling and enhanced mechanical properties, offering a promising strategy for meniscus regeneration. This study aimed to biofabricate zonally defined meniscus grafts by depositing fibrocartilaginous microtissues derived from meniscus progenitor cells (MPCs) from the inner and outer meniscus within melt-electrowritten (MEW) scaffolds. We investigated how scaffold architecture influences microtissue behavior using polycaprolactone (PCL) MEW scaffolds with different pore aspect ratios (0.8:0.8 mm and 0.4:1.6 mm). Following deposition, microtissues fused in the scaffolds within 24h, with tissue growth modulated by pore geometry. Scanning electron microscopy showed dense extracellular matrix formation and fiber alignment in anisotropic (0.4:1.6) scaffolds. Histology revealed intense sGAG deposition in the square (0.8:0.8) scaffolds seeded with inner MPC microtissues, while anisotropic scaffolds supported greater collagen deposition with inner and outer MPC microtissues. All groups were negative for calcium deposition, indicating the development of a stable fibrocartilaginous phenotype. Type I collagen expression and staining was strongest in the anisotropic (0.4:1.6) scaffolds. Outer MPC microtissue constructs were tested in a caprine meniscus explant model, demonstrating integration with native tissue, mimicking early healing responses. Finally, bioprinting inner and outer MPC derived microtissues (60,000 µT/mL) into either 0.8:0.8 and 1.6:0.4 scaffolds supported the development of a type I collagen rich fibrocartilage tissue resembling the native meniscus. These findings demonstrate the potential of combining regionally defined meniscal microtissues with MEW scaffolds to fabricate functional and zonally organized meniscus grafts.
Electroactive and adhesive hydrogel for myocardial infarction treatment
Vuong Pham Dac1, Seul-Gi Lee2, Arun Kumar Rajendran3, Hyewon Shin2, Yoonseo Kim2, Jaewoo Lee1, Jeong-Uk Kim1, Esther Jeong1, C-Yoon Kim2, Nathaniel Suk-Yeon Hwang1
1Chemical and Biological Engineering. Seoul National Univertsity, Seoul (Seoul-tukpyolsi) - South Korea, 2College of Veterinary Medicine. Konkuk University, Seoul (Seoul-tukpyolsi) - South Korea, 3School of Healthcare Science and Engineering. Vellore Institute of Technology, Vellore (Tamil Nadu) - India
Myocardial infarction (MI) is a common cause of morbidity and mortality worldwide, affecting up to 3 million people worldwide annually. MI is a critical cardiovascular disease caused by the occlusion of a coronary artery, resulting in an insufficient supply of oxygen to heart tissue and then the irreversible death of cardiomyocytes. This leads to the formation of poorly conductive fibrotic tissue, which is one of the main causes for the abnormal electrical activity of the cardiac tissue. In order to mitigate these pathological consequences, we have developed an electroactive and adhesive hydrogel to reconstruct the microenvironment for the infarcted cardiac tissue. The developed hydrogel demonstrated excellent adhesion strength to the epicardial surface with a rapid crosslinking time occurring in seconds. This property can help to minimize intraoperative risks by shortening the overall surgery procedure time. In addition, owing to its appropriate viscosity, desirable rheological properties, and minimal swelling ratio, the hydrogel can be applied to the epicardium with precise control over both the amount of hydrogel used and the painted area. Moreover, the incorporation of the electrically conductive and electrically stimulating materials into the hydrogel significantly improved the recovery of the myocardial tissue. In particular, it successfully established a microenvironment mimicking native cardiac ECM, thereby effectively restoring heart damage caused by MI. In another aspect, the hydrogel exhibited biocompatibility and degradability, offering an advantage over conventional conductive hydrogels based on conductive synthetic polymers, which highlights the strong potential of the hydrogel for future clinical applications in myocardial infarction therapy.
Effects of ceramic chemical composition on biomechanical outcomes of spinal fusion induced by Osteogrow-C implants
Natalia Ivanjko1, Nikola Stokovic1, Katarina Muzina2, Marina Milesevic1, Marko Pecin3, Drazen Maticic3, Slobodan Vukicevic1
1Laboratory for Mineralized Tissues. University of Zagreb School of Medicine, Zagreb (Grad Zagreb) - Croatia, 2 Department of Inorganic Chemical Technology and Non-metals. University of Zagreb Faculty of Chemical Engineering and Technology, Zagreb (Grad Zagreb) - Croatia, 3Clinics for Surgery, Orthopedics and Ophthalmology. University of Zagreb Faculty of Veterinary Medicine, Zagreb (Grad Zagreb) - Croatia
Degenerative spinal diseases are among the most prevalent medical conditions today and, in severe cases, may necessitate treatment via spinal fusion. Osteogrow-C is a novel therapeutic formulation designed to promote spinal fusion. It consists of rhBMP6 delivered in an autologous blood coagulum, with synthetic ceramics serving as a compression-resistant matrix. Given the wide variability in the chemical properties of synthetic ceramics, this study aimed to investigate how these characteristics affect the outcome and biomechanical properties of fused spinal segments. Osteogrow-C implants were prepared by adding rhBMP6 to autologous blood, which was then combined with ceramics of different chemical compositions (TCP, TCP/HA 80/20, TCP/HA 40/60, and HA) and left to coagulate. Implants were placed between the transverse processes of the lumbar vertebrae in rabbits. The porosity and surface area of the ceramics were measured by nitrogen adsorption analysis prior to the formation of implants. Spinal fusion success and biomechanical properties of the newly formed bone were evaluated using a three-point bending test after a one-year follow-up. TCP/HA 40/60 ceramics displayed significantly greater nitrogen adsorption, indicating the largest accessible surface area and highest porosity. Pore size distribution analysis confirmed these findings, showing the highest pore volume, especially for pores larger than 30 nm, reflecting a substantial population of larger mesopores. All Osteogrow-C implants induced successful spinal fusion that persisted one year post-surgery. While differences were not statistically significant, implants containing TCP or BCP with a high TCP proportion (TCP/HA 80/20) tended to outperform those with HA or BCP with high HA content (TCP/HA 40/60) in maximum force, elasticity, and work-to-break. These biomechanical advantages correlated with increased cortical thickness. Osteogrow-C implants represent a potential therapeutic solution for spinal fusion, reliably promoting durable fusion and biomechanically competent fusion masses across all tested ceramic compositions.
3D Bioprinting of perfusable renal tubular structures
Federico Sinnona1, Viola Sgarminato1, Giuseppe De Nisco1, Kristen Mariko Meiburger1, Hirofumi Hitomi2, Chiara Tonda-Turo1, Gianluca Ciardelli1
1Department of Mechanical and Aerospace Engineering. Politecnico di Torino, Torino (Italia) - Italy, 2Department of iPS Stem Cell Regenerative Medicine. Kansai Medical University, Osaka - Japan
The kidney maintains systemic homeostasis by filtering blood, reabsorbing solutes and water, and excreting metabolic waste. Its functional unit, the nephron, comprises specialized segments that work together to ensure selective filtration and reabsorption. Among these, the proximal tubule (PT) plays a central role, reclaiming up to 80% of solutes and water filtered in the glomerular region. Due to its high metabolic activity, the PT is particularly susceptible to drug-induced injury, making it a critical target for nephrotoxicity assessments and disease modeling [1].
Conventional preclinical models, however, fail to accurately replicate the renal microenvironment. Two-dimensional monolayers lack spatial organization and physiological flow, while animal models are limited by interspecies differences and ethical concerns. Recently, combining 3D bioprinting with perfusable systems has gained interest, as it enables the fabrication of geometrically accurate renal constructs that better mimic native tissue [2].
In this study, we optimized a coaxial 3D bioprinting approach to produce tubular structures resembling the microanatomy of the human PT. Printing parameters and bioink formulations were refined to enhance printability and reproducibility. Rheological analyses confirmed that the selected gelatine methacryloyl (GelMA)-based hydrogel exhibited suitable viscosity and crosslinking behavior for uniform extrusion. This hydrogel was tested with human endothelial and epithelial cells to evaluate biocompatibility and proliferation, demonstrating the feasibility of generating stable and perfusable tubular constructs.
Compared with existing approaches, coaxial bioprinting offers the advantage of directly depositing cells within the tubular lumen during fabrication, enabling a single-step seeding process. Moreover, this method can be integrated with embedded bioprinting strategies, expanding its versatility and compatibility with a wider range of cell types.
Acknowledgments
The PhD scholarship of Federico Sinnona runs under the joint agreement between Kansai Medical University and Politecnico di Torino.
References
1. Scott, R. P. et al. J Cell Biol, 2015.
2. Homan, K. et al. Sci Rep, 2016.
Macro-scale, scaffold-assisted model of the human bone marrow endosteal niche using hiPSC-vascularized osteoblastic organoids
Andres Garcia-Garcia1, Qing Li1, Marina T. Nikolova2, Gangyu Zhang3, Igor Cervenka4, Federica Valigi5, Dominik Burri4, Evelia Plantier1, Andrea Mazzoleni3, Anaïs Lamouline1, Juerg Schwaller5, Barbara Treutlein2, Ivan Martin3
1Department of Biomedicine. University of Basel, Basel (Basel-Stadt) - Switzerland, 2Department of Biosystems Science and Engineering. ETH Zürich, Basel (Basel-Stadt) - Switzerland, 3Departments of Biomedicine & Biomedical Engineering. University of Basel, Basel (Basel-Stadt) - Switzerland, 4Department of Biomedicine. Swiss Institute of Bioinformatics. University of Basel, Basel (Basel-Stadt) - Switzerland, 5Department of Biomedicine. University Children's Hospital. University of Basel, Basel (Basel-Stadt) - Switzerland
Endosteal bone marrow (BM) niches are crucial to sustain non-steady-state hematopoiesis but are challenging to be modelled in their cellular and molecular complexity in standardized, human settings. We report a developmentally-guided approach to generate a macro-scale organotypic model of BM endosteal niches (engineered vascularized osteoblastic niche, eVON) based on human induced pluripotent stem cells and porous hydroxyapatite scaffolds. The eVON contains long-lasting vascular networks covered by pericytes and neural fibers within an osteogenic matrix. Key niche signals (CXCL12, KITLG and VEGFA) are expressed in human-specific patterns. The system supports hematopoiesis in vitro and preserves HSPC multilineage repopulation capacity in vivo. eVON perturbations at cellular (removing vasculature) and molecular (deregulating VEGF and CXCL12 signaling) levels enabled to investigate the contribution of endosteal vasculature to myelopoiesis. The eVON faithfully captures phenotypic, structural and functional features of human endosteal BM, enabling the study of pathophysiological interactions with hematopoietic cells.
In vitro assessment of the biocompatibility and antimicrobial properties of silver-containing wound dressings
Milz Wieland1, Illner Sabine2, Kufahl Ann C.3, Lemken Anneke1, Foth Aenne1, Ficht Phillip K.1, Schultz Selina2, Eickner Thomas2, Grabow Niels2, Fiedler Tomas3, Emmert Steffen1, Boeckmann Lars1
1Clinic and Polyclinic of Dermatology, Venereology and Allergology, Rostock University Medical Center (Mecklenburg-Vorpommern) - Germany, 2Institute for Biomedical Engineering, Rostock University Medical Center (Mecklenburg-Vorpommern) - Germany, 3Institute for Medical Microbiology, Virology and Hygiene; University Medical Center Rostock, Rostock University Medical Center (Mecklenburg-Vorpommern) - Germany
Chronic wounds prone to infection pose significant clinical challenges for patients and the healthcare system. In order to prevent and treat microbial infections and hence to foster the healing process, silver-coated wound dressings are commonly used. However, besides the antimicrobial properties, silver may also exert detrimental effects on human cells. Against this background, the present study aims to evaluate the biocompatibility and antimicrobial efficacy of commercial silver-containing wound dressings, as well as novel electrospun poly-p-dioxanone (PPDO)-nanofiber nonwovens. Two-dimensional cell culture experiments were conducted on a human fibroblast cell line (GM637) and a human keratinocyte cell line (HaCaT). They were treated with extracts from different wound dressings and placed in direct contact with punches. A significant decrease in metabolic activity was observed, which indicates a cytotoxic effect of these silver-containing wound dressings. In order to investigate the role of reactive oxygen species (ROS) and the mutagenicity of the extracts, inhibitory concentrations were approximated using diluted extracts. The antimicrobial potential of the wound dressings was tested on four potential wound-associated bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. Ongoing investigations are focusing on the suitability of PPDO as a basic material for silver-containing wound dressings as well as on modifications of the silver-containing nanofiber nonwovens in order to reduce cytotoxicity while maintaining antimicrobial properties.
SPARC-deficient mice as a mechanistic model of tendinopathy
Andreas Traweger
de Inst. of Tendon and Bone Regeneration. Paracelsus Medical University, Salzburg - Austria
Recent advances have deepened our understanding of tendon biology, yet the precise mechanisms underlying tendinopathy remain incompletely understood. In addition to mechanical overload, local and systemic inflammation, and shifts in tendon-resident cell populations known to drive tendon degeneration, increasing evidence highlights the dynamic crosstalk between tendon cells and the extracellular matrix (ECM), where cellular responses to mechanical forces critically depend on ECM quality and maturation1. Our research focuses on the matricellular protein SPARC (Secreted Protein Acidic and Rich in Cysteine), which plays a central role in tendon tissue maturation and homeostasis2. Using a newly established SPARC conditional knockout mouse model, we observed the expected hallmarks of tendinopathy, including ECM degeneration, hypercellularity, and compromised biomechanical properties—interestingly, with varying severity across different tendons. Beyond these structural changes, the model also revealed profound alterations in cellular metabolism and mitochondrial function, providing new mechanistic insights into how SPARC deficiency compromises tendon resilience. Together, these findings underscore the role of SPARC as a key regulator linking ECM quality to cellular metabolism and tendon function. The model establishes a powerful experimental platform to deeply probe the mechanisms underlying tendinopathy.
(1) Gehwolf R, et al., Adv Sci (Weinh). 2025 Sep;12(36):e0644
(2) Wang T., Wagner A., Gehwolf R. et al., Sci Transl Med. 2021 Feb 24;13(582):eabe5738
Funded by Sparkling Science 2.0 (#SPSC_01_020), Land Salzburg/WISS2025 (#20102/F2400948-FPR), and Land Salzburg/WISS2030 (#20102/F2400949-FPR).
A biological and biomechanical description of the maturation process of fibroblast spheroids
Wolfgang Metzger1, Eugene Oh1, Fabian Krull2, Solenn Grolleau2, Sergiy Antonyuk2, Lilia Lemke3, Matthias Hannig3, Monika Bubel1, Franziska Lautenschläger4, Gagan Sharma4, Emmanouil Liodakis1
1Saarland University, Department of Trauma, Hand and Reconstructive Surgery, Homburg (Saarland) - Germany, 2Institute of Particle Process Engineering, RPTU University Kaiserslautern-Landau, Kaiserslautern, Germany, Kaiserslautern (Rheinland-Pfalz) - Germany, 3Department of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University, Homburg, Germany, Homburg (Saarland) - Germany, 4Center for Biophysics, Saarland University, Saarbrücken, Germany, Saarbrücken (Saarland) - Germany
Spheroids mimic the natural cellular environment more closely than traditional 2D cell cultures and hold promise for future tissue engineering approaches. In this study, we examined the biological and biomechanical maturation of normal human dermal fibroblast spheroids over time.
Spheroids were generated using liquid overlay technique (LOT) in 96-well plates containing 5,000, 10,000, or 50,000 cells per well. The spheroids were analyzed up to seven days after their generation. We assessed the size and morphology of both, vital and aldehyde-fixed spheroids via light and scanning electron microscopy (SEM). Cytoskeletal gene expression was analyzed using quantitative polymerase chain reaction (qPCR). We measured the Young’s modulus of single cells from 2D cultures and of single cells derived from dissociated spheroids using real-time deformability cytometry (RT-DC). The viscoelastic properties of vital spheroids were evaluated using compression and relaxation tests with a nanoindenter.
The LOT produced one spheroid per well consistently. The size of the spheroids decreased over time following an exponential decay. Depending on the spheroid's size and age, a central indentation could be seen; this indentation was more pronounced after aldehyde fixation. RT-DC and qPCR revealed an increased Young’s modulus in single cells and a downregulation of most analyzed cytoskeletal genes due to 3D organization. However, cytoskeletal gene expression increased within spheroids over time. As the spheroids matured, the viscous component of their viscoelastic properties decreased slightly, while the elastic modulus remained stable.
The reduction in spheroid size may result from single-cell shrinkage and internal reorganization. The central indentation may be due to high cell density settling on the concave agarose surface. Aldehyde fixation may have affected the initial results by cross-linking proteins. Although there was a reduced expression of cytoskeletal genes compared to 2D cultures, the increased stiffness of individual cells indicates cytoskeletal rearrangement or cross-linking.
Novel xeno-free hPLMA/dECM-MA biomaterial for 3D DLP bioprinting
Sara C. Silvério1, Sara C. Santos2, João F. Mano1, Catarina A. Custódio1
1CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal, 2Metatissue, Ílhavo (Aveiro) - Portugal
Over the years, Digital Light Processing(DLP)-based 3D bioprinting has emerged as a promising technology to develop platforms that accurately mimic native human tissues.[1,2] A fundamental factor in bioprinting techniques is the bioink’s composition and properties. Recently, researchers developed photopolymerizable biomaterials of human origin – human methacryloyl platelet lysates(hPLMA) and methacrylated decellularized extracellular matrix(dECM-MA) derived from placentas.[3-5] This work aimed to combine both, creating a novel xeno-free bioink for DLP bioprinting scaffolds for cardiac tissue engineering(TE). To achieve this, precursor solutions of hPLMA and dECM-MA were prepared at 15% and 1%(w/v), respectively, in a photoinitiator solution. The ink was prepared by combining the precursor solutions in equal parts and adding a photoabsorber. Simple structures were printed, demonstrating high shape fidelity, resolution, structural stability and reproducibility. Biocompatibility studies were conducted in hPLMA/dECM-MA hydrogels with mono- and co-cultures of human adipose stem cells, human cardiac fibroblasts and human umbilical vein endothelial cells. After 7 days, live/dead and nuclei/actin filaments staining of the hydrogels revealed good cell viability and proliferation across all cultures. Additionally, a bioink with nuclei-stained cells was bioprinted and the constructs demonstrated evenly distributed cells. Compression tests also revealed that by modulating the biomaterials’ concentration or functionalization degree, their mechanical properties can be finely tuned to meet specific TE requirements. Hence, the tunability of the herein developed bioink, combined with its human origin and inherent content of bioactive and structural proteins, positions it as a highly translational bioink for the fabrication of physiologically-relevant, stable scaffolds supporting long-term in vitro cell culture and potential clinical applications.
This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UID/50011/2025(DOI:10.54499/UID/50011/2025), LA/P/0006/2020(DOI:10.54499/LA/P/0006/2020) & COMPETE2030-FEDER-00836800, financed by national funds through the FCT/MCTES (PIDDAC).
[1] Yu Y. et al,2021,10.1016/j.engreg.2021.08.003
[2] Li H. et al,2023,10.1002/agt2.270
[3] Santos S. et al,2018,10.1002/adhm.201800849
[4] Martins E. et al,2024,10.1002/adhm.202401510
[5] Deus I. et al,2022,10.1016/j.msec.2021.112574
Modeling fibrosis and muscle function in Duchenne Muscular Dystrophy (DMD) using an engineered 3D co-culture system
Chiara Ninfali1, Xiomara Fernández-Garibay1, Ainoa Tejedera-Villafranca1, Elia López Serrano1, Jordi Díaz-Manera2, Javier Ramón-Azcón1, Juan M. Fernández Costa1
1Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, 2Institute of Genetic Medicine. John Walton Muscular Dystrophy Research Centre, Newcastle upon Tyne - United Kingdom
Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the DMD gene leading to loss of dystrophin, a cytoskeletal protein essential for muscle integrity. The absence of dystrophin results in progressive muscle degeneration and replacement of contractile tissue with fibrotic extracellular matrix, driven mainly by fibro-adipogenic progenitors (FAPs). Understanding how DMD-derived FAPs contribute to this pathological remodeling is critical for developing targeted antifibrotic therapies.
In this study, we developed a functional 3D in vitro model of human skeletal muscle using 3D-printed casting molds to co-culture primary myogenic precursors with FAPs embedded in a Matrigel–fibrin matrix. FAPs were isolated from three healthy donors and three DMD patients to capture inter-individual variability. The resulting engineered tissues exhibited organized myofiber formation and robust contractile responses to electrical stimulation. However, co-culture with DMD-derived FAPs led to a marked reduction in twitch and tetanic force, despite preserved myotube morphology and fiber-type distribution.
Bulk RNA sequencing revealed that DMD-FAP co-cultures displayed extensive transcriptional reprogramming, particularly in pathways related to extracellular matrix remodeling, fibrosis, and muscle structure and contraction. Consistent with these findings, DMD-FAP co-cultures showed elevated collagen I accumulation and increased secretion of fibrosis-associated proteins, as quantified by immunofluorescence and ELISA. Finally, antifibrotic drug testing demonstrated that selected compounds effectively reduced collagen deposition but failed to fully restore contractile function in 3D tissues.
Together, these results establish a robust human 3D muscle–FAP co-culture model that captures key aspects of DMD-associated fibrosis and functional decline. This model provides a physiologically relevant tool for dissecting cell–cell interactions in dystrophic muscle and for preclinical screening of antifibrotic therapeutics.
Accurate quantification of extracellular vesicles in the presence of hydrogels
Hannah Aris1, Yeyu Shen1, Xandra O. Breakefield2, Garry P. Duffy1, Meadhbh Á. Brennan1
1University of Galway, Galway - Ireland, 2Harvard Medical School, Boston (Massachusetts) - United States
Mesenchymal stromal cell (MSC)-EVs have regenerative effects in a variety of tissues. When EVs are delivered via hydrogels, they remain at delivery sites longer, improving therapeutic outcomes compared to EVs injected via saline. Researchers are developing materials with more controlled EV-release to optimize delivery to healing tissue. Numerous studies have evaluated EV release from hydrogel carriers in vitro, but no work has been conducted to investigate the impact of hydrogel presence on EV quantification. Measurements may be skewed by hydrogel particles, reducing understanding of EV release. We assessed the reliability of commercially available EV quantification techniques – nanoparticle tracking analysis (NTA), bicinchoninic acid (BCA) protein assay, CD9 ExoELISA, RNA quantification, and phospholipid assay (PLA) – alongside fluorescent and/or bioluminescent quantification of PalmtdTomato and PalmGRET (probes attached to the inner EV membrane following genetic modification of parent cells). EVs were derived from a murine bone-marrow MSC cell line, either naïve or genetically modified. Three commonly used hydrogels – methacrylated hyaluronic acid (Me-HA), gelatin methacryloyl (GelMA), and collagen (Col) – were crosslinked and allowed to incubate with phosphate buffered saline (PBS) for 3 h, 8 h, 24 h, 72 h, 2 wk, or 4 wk. This hydrogel-containing PBS was used to dilute EVs to known concentrations. EVs were quantified in the presence of Me-HA, GelMA, Col, or PBS alone, and the more measurements aligned between hydrogel-containing and PBS samples, the less interference. Ultimately, the quantification of fluorescent and/or bioluminescent EVs was not impacted by hydrogels. NTA, BCA, and CD9 ExoELISA proved unreliable, while RNA and PLA provided better but variable reliability when quantifying EVs with hydrogel. Therefore, fluorescent or bioluminescent EVs (like PalmtdTomato and PalmGRET) are recommended for use when measuring EV release from hydrogels. Ultimately, researchers must validate their EV quantification method with hydrogel present to ensure accurate EV release measurements and improve EV delivery.
SPARC deficiency impairs β-oxidation and mitochondrial function in tendons
Nevra Pelin Cesur1, Andrea Wagner1, Daniela Weber2, Christopher Gerner3, Ekaterina Oleinik4, Renate Gehwolf1, Andreas Traweger1
1Institute of Tendon and Bone Regeneration. Paracelsus Medical University, Salzburg - Austria, 2Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, University Hospital of the Paracelsus Medical University, Salzburg - Austria, 3Institute of Analytical Chemistry. University of Vienna, Vienna (Wien) - Austria, 4Institute of Lightweight Design and Structural Biomechanics. TU Vienna, Vienna (Wien) - Austria
The matricellular protein SPARC (Secreted Protein Acidic and Rich in Cysteine) is essential for extracellular matrix organization and tissue homeostasis in tendons. The loss of SPARC leads to impaired tendon function and tissue integrity. SPARC-null tendons are mechanically inferior, prone to spontaneous rupture and pathological remodelling, accompanied by extracellular matrix (ECM) disorganization, and lipid accumulation in tendon cells [1, 2]. Hence, we investigated metabolic and gene expression alterations in Achilles tendons of a tendon-specific SPARC conditional knockout (SPARCfl/fl:ScxCre+/-) model. Metabolomic profiling revealed a significant reduction in acylcarnitines and other intermediates of fatty acid β-oxidation. Further, phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylcholines, are markedly reduced in SPARC-cKO Achilles tendons, suggesting disrupted mitochondrial function, impaired membrane biogenesis, altered lipid metabolism, and enhanced lipid peroxidation. Consistently bulk RNA-Sequencing identified transcriptional downregulation of genes involved in mitochondrial fatty acid transport and β-oxidation (e.g. Ppargc1a or Cpt1/2), next to genes central in respiratory chain function and ATP production (e.g., NAHD dehydrogenase subunits, cytochrome c oxidase subunits or oxoglutarate dehydrogenase). Collectively, these findings suggest that SPARC deficiency may compromise mitochondrial metabolic homeostasis in tendon tissue, potentially reducing β-oxidation efficiency, impairing mitochondrial function, and enhancing oxidative stress. Whether this represents a direct consequence of SPARC loss or an indirect effect mediated by altered ECM quality causing tenocytes to perceive and respond to load differently—ultimately inducing mitochondrial stress—remains to be clarified in future studies.
(1) Gehwolf R. et al., Sci Rep. 2016 Sep 2;6:32635.
(2) Wang T., Wagner A., Gehwolf R. et al., Sci Transl Med. 2021 Feb 24;13(582):eabe5738.
Designing mesoporous silica nanoparticles for controlled ion delivery in tissue regeneration
Lei He1, Chloe Trayford1, Pichaporn Sutthavas1, Sabine Van Rijt1
1MERLN Institute for Technology-Inspired Regenerative Medicine. Maastricht University, Maastricht (Limburg) - The Netherlands
Bioactive ions are increasingly recognised as powerful regulators of cellular behaviour and tissue repair. Delivering these ions in a controlled and targeted way remains a major challenge in regenerative medicine. Here, we describe how mesoporous silica nanoparticles (MSNs) can be engineered as effective ion delivery systems and outline the design rules that determine their biological performance. Through a series of studies, we found that the position of the ion within the nanoparticle—whether incorporated in the silica framework, confined within the pores, or bound to the surface—strongly influences the release profile, degradation behaviour, and subsequent cellular response. Combining multiple ions proved consistently more potent than single-ion systems, as exemplified by calcium-strontium and calcium-strontium-zinc combinations, which enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and increased matrix mineralisation. We also observed that nanoparticle degradability is a key parameter: gradual dissolution supports sustained ion signalling without inducing cytotoxicity. Moreover, the way nanoparticles are presented to cells, either as a suspension or as a film coating, significantly affects uptake efficiency and osteogenic outcomes. Extending these principles beyond bone regeneration, MSN-based ion delivery has shown potential in cancer-related contexts, where combined regenerative and therapeutic effects may be achieved. Together, these studies highlight how rational control over ion composition, spatial localisation, and release dynamics can transform simple silica carriers into multifunctional bioactive platforms. The emerging design framework provides a foundation for developing next-generation nanomaterials that harness ionic signalling not only for tissue regeneration but also for broader applications such as antimicrobial and anti-aging therapies.
Matrix immaturity and osteoclast resorption promote vascular invasion in an in vitro model for endochondral ossification
Jonelle Meijer1, Rosa De Graaff2, Eelco Bergsma1, Moyo Kruyt3, Debby Gawlitta1
1Oral and Maxillofacial Surgery, Prosthodontics and Special Dental Care. University Medical Center Utrecht, Utrecht - The Netherlands, 2Bioengineering Technologies. University Twente, Enschede (Overijssel) - The Netherlands, 3Orthopedics. University Medical Center Utrecht, Utrecht - The Netherlands
Introduction:
Fracture healing is mediated through endochondral ossification (EO). Here, vascular invasion and matrix degradation are decisive events that enable the transition from cartilage to bone [1]. To understand and modulate this process, the work presented here aims to establish a comprehensive in vitro model for EO containing all essential cell types that permit vascular invasion and drive bone remodeling.
Materials and Methods:
Human bone marrow-derived mesenchymal stromal cells (MSCs) were differentiated in chondrogenic (21 and 28 days; 21d and 28d) and hypertrophic chondrogenic (28d) differentiation media to form spheroidal cartilaginous tissues. Next, these tissues were devitalized [1]. Peripheral blood mononuclear cells were isolated from blood, cultured on the devitalized cartilage tissues and stimulated for formation of osteoclasts. These cultures were placed in a fibrin gel together with a vascular organoid containing MSCs and endothelial colony forming cells (from cord blood). This tri-culture model was cultured for another 10 days.
Results:
Osteoclasts were formed on the devitalized cartilage tissues and penetrated deep into the core of hypertrophic spheroids via characteristic cutting cones but remained predominantly at the periphery of chondrogenic spheroids. Osteoclasts displayed distinct fusion dynamics, with more numerous and smaller cells in the 21d group compared to other groups. Strikingly, this group also exhibited osteoclast-driven vascular infiltration of the spheroids.
Conclusion:
This work presents an in vitro model that, given the achieved vascular infiltration, exceeds recent work on modeling EO and opens a unique avenue for pre-clinical testing of osteoinductive materials [2].
References:
[1] Longoni et al. Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material. Adv Sci. 2022 doi:10.1002/advs.202103284.
[2] Ji et al. Development of a Complex Human In Vitro Model of Endochondral Ossification. Tissue. Eng. Part C Methods. 2025 doi.org/10.1177/19373341251378152
Metabolic reprogramming of classically activated microglia by surface-functionalized enzymatic nanoreactors - SEMIT
Yanne Amassoka1, Aitor Larrañaga2, Ana Beloqui3
1Department of mining-metallurgy and materials engineering. Group of Science and Engineering of Polymeric Biomaterials (ZIBIO Group), POLYMAT, Bilbao (Bizkaia) - Spain, 2Department of mining-metallurgy and materials engineering. Group of Science and Engineering of Polymeric Biomaterials (ZIBIO Group), POLYMAT, Bilbao (Bizkaia) - Spain, 3Department of Applied Chemistry. POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain
Neuroinflammation, a vital immune response in the central nervous system (CNS), can become detrimental when dysregulated. Microglia represent the resident immune cells of the CNS and play a major role in this balance, rapidly switching between a pro-inflammatory (M1) and a neuro-protective (M2) phenotype with distinguishable cytokine profile release during brain assaults[1]. This phenotypic shift is tightly coupled with metabolic plasticity[2]. Therefore, regulating the metabolic pathways in microglia offers a promising approach to control neuroinflammation. The present study focuses on reprogramming microglial energy metabolism by depleting intracellular glucose to inhibit glycolysis and promote neuro-repair.
The nanoreactors were fabricated following the layer-by-layer approach with single enzyme nanogels (SENs) as enzymatically active building blocks. First, glucose oxidase (GOx) was encapsulated in a crosslinked polymeric network in which the functional monomer (3-Acrylamidopropyl) trimethylammonium chloride (APTAC) was included to achieve positively charged enzyme polymer hybrids. GOx@APTAC was then assembled within layers of poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) on a mesoporous silica template (∼200 nm). The surface of the nanoparticles was functionalized with a hydroxyl-terminated dendrimer to enhance colloidal stability and promote cellular uptake.
Upon treatment of lipopolysaccharide (LPS)-activated murine microglia (BV-2) with 10 µg/mL of nanoreactors, confocal images and flow cytometry indicated an optimal cellular uptake > 90%. The genetic profile of the treated group showed an approximate 70% decrease in pro-inflammatory cytokines (IL-1β and TNF-α) and a 5-fold increase in IL-4, a key cytokine associated with neuro-repair. Moreover, the undetectable IL-6 expression may further indicate a successful metabolic shift from M1 to M2 phenotypes [3].
Dendrimer-functionalized GOx@APTAC nanoreactors successfully decreased key inflammation markers in classically activated BV-2 cells, suggesting a modulation of their metabolism. This offers a biocompatible polymeric platform for the development of targeted therapies to reprogram altered metabolic pathways in neurodegenerative disorders.
We acknowledge grant PIBA_2025_1_0038 from the Basque Government.
References
[1] A. Mirarchi et al., 2024, Int. J. Mol. Sci., 25.
[2] J. Cheng et al., 2021, J. Neuroinflammation,18, 2021.
[3] T. Ageeva et al., 2024, Cells,13, 2024.
Hierarchical melt-electrowritten vascular grafts with spatially defined porosity and layer specific cell alignment
Jonelle Meijer1, Michael Bartolf-Kopp2, Eelco Bergsma1, Kruyt Moyo3, Jungst Tomasz2, Debby Gawlitta1
1Oral and Maxillofacial Surgery, Prosthodontics and Special Dental Care. University Medical Center Utrecht, Utrecht - The Netherlands, 2Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute. Julius-Maximilians-University Würzburg, Wurzburgo (Nordrhein-Westfalen) - Germany, 3Orthopedics. University Medical Center Utrecht, Utrecht - The Netherlands
Introduction:
There is a clinical need for large tissue substitutes that mimic the structure and function of native tissue, yet, engineering such constructs is limited by a lack of vascularization to the core [1]. This results in necrotic core formation. Vascularization strategies often target one scale of vasculature at a time while the interconnection of a vascular network at multiple levels is less studied [1]. Therefore, this study aims to achieve angiogenic sprouting of cells from the luminal side of small diameter vascular constructs into their surroundings for formation of an interconnected vascular network.
Materials and Methods:
An in-house developed melt electrowriting (MEW) printing set-up was used for printing of vascular grafts (⌀3mm) containing five membranes angled 0°, 45° and 70° for cell alignment of endothelial colony forming cells (ECFCs) seeded in the lumen, mechanical stability, and mesenchymal stromal cells (MSCs) seeded at the abluminal side, respectively. Each membrane contained a fiber shift in its design for regulated porosity.
Results:
Using MEW, we successfully fabricated controllable pores within the scaffolds at designed locations ranging from ⌀30 to ⌀50 μm, demonstrating microscale resolution. At the luminal side, ECFCs formed a monolayer and at the abluminal membrane, MSCs differentiated towards vascular smooth muscle cells. Both cell types at the luminal and abluminal side aligned with the filament direction. Ongoing research is looking into directed angiogenesis from the fabricated vessel into the surrounding hydrogel and interconnection with a surrounding vascular network.
Conclusion:
The findings presented here demonstrate the potential of introducing controlled porosity at a micro-scale level in MEW vascular grafts while supporting endothelialization.
Reference:
[1] de Silva L, Bernal PN, Rosenberg A, Malda J, Levato R, Gawlitta D. Biofabricating the vascular tree in engineered bone tissue. Acta Biomater. 2023 Jan 15;156:250-268. doi: 10.1016/j.actbio.2022.08.051. Epub 2022 Aug 28. PMID: 36041651.
Harnessing cancer cell-derived extracellular vesicles to improve the biomimetism of a 3D tissue-engineered bladder cancer model
Carignan Laurence1, Bollmann Enola1, Chabaud Stéphane2, Bolduc Stéphane1, Bordeleau François1
1Laval University, Quebec - Canada, 2LOEX Tissue Engineering Laboratory, Laval University, Quebec - Canada
Accurate cancer models are critical for understanding tumor biology and advancing therapeutic development. Yet, in vitro models fail to replicate the complexity of the tumor microenvironment, particularly the gradual stiffening of the extracellular matrix (ECM). This ECM stiffening, largely driven by cancer-associated fibroblasts (CAFs), is a key feature influencing cancer progression and treatment resistance. Our lab developed a tissue-engineered bladder cancer model that mimics the stiffness and architecture of tumors by activating fibroblasts with cancer cell-conditioned media. Further efforts are needed to recapitulate the tumor stroma across disease stages. This study investigates how specific cancer cell-secreted components modulate the activation of bladder fibroblasts, and how this activation affects the biophysical properties of our model.
Conditioned media from T24 bladder cancer cells was fractioned using sequential filtration to isolate microvesicles, exosomes, and soluble proteins. Bladder fibroblasts were treated with each fraction for 10 days, and CAF activation was assessed using immunofluorescence, western blotting and traction force microscopy. The stromal portion of our model was then produced using the self-assembly method where bladder fibroblasts were cultured in paper rings with ascorbic acid supplementation. Conditioned media fractions were introduced one week into culture and maintained for five weeks. Collagen deposition was quantified via picrosirius red staining, and ECM stiffness was measured using atomic force microscopy.
In cultured fibroblasts, microvesicles strongly induced αSMA expression (CAF marker) in a small subset of cells, whereas exosomes elicited weaker αSMA expression across a broader cell population. Complete conditioned media enhanced cellular contractility. In the engineered stromal tissues, histological analysis revealed that microvesicles promoted collagen deposition, while exosomes lead to thicker tissues, likely reflecting increased fibroblast proliferation.
These findings suggest that it is possible to modulate the biophysical properties of our 3D bladder cancer models. Ultimately, this model could serve as a better platform for validating therapeutic innovations.
Forces that shape the transcriptome: linking cellular mechanosensing to RNA processing
Pavel Šimara1 4, Helena Skálová1, Jan Vrbský1, Kristína Locker Kovačovicová2, Giancarlo Forte1 3 4
1International Clinical Research Center (ICRC), St. Anne's University Hospital, Czech Republic, 2PsychoGenics, Paramus, USA, 3School of Cardiovascular and Metabolic Medicine and Sciences, King’s College London, UK, 4Faculty of Medicine, Department of Biomedical Sciences, Masaryk University, Czech Republic
The role of mechanical forces in cellular function has become a widely recognized phenomenon. While the effects of mechanical cues on gene expression are well documented, their influence on RNA processing is only beginning to emerge. Our group has pioneered this field by providing evidence of mechanically induced alternative splicing (AS) in the human failing heart1. To build upon this work, we aim to elucidate the role of mechanically regulated AS in stem cells.
Our data show specific localization of RNA-binding proteins from the hnRNP family at the colony-edge regions of induced pluripotent stem cells (iPSCs) cultured on micropatterned 2D surfaces. The ring-like distribution of hnRNPs overlaps with areas of high mechanical tension2 and open chromatin regions. Similar edge localization was observed in iPSC spheroids grown in 3D. Co-immunoprecipitation confirmed that hnRNPs form complex assemblies with proteins involved in the spliceosome and Cajal bodies, suggesting the formation of a membraneless compartment controlling AS and potentially responsive to mechanical cues.
Treatment with the tension inhibitor Y-27632 resulted in reversible disruption of hnRNP patterning. RNA-seq analysis revealed upregulated pathways associated with RNA processing in colony edge–enriched cell samples. We are currently performing spatial visualization of selected transcript variants of hnRNP splicing targets using BaseScope (ACD) and MERFISH (Vizgen) technologies. Taken together, our data provide further evidence that mechanical forces regulate RNA processing.
[1] Martino F., et al., Sci Transl Med, 14(672), 2022. PMID: 36417487
[2] Muncie JM., et al., Dev Cell, 55(6), 2020. PMID: 33207224
This work was supported by the British Heart Foundation (BHF) Centre for Excellence Award (RE/18/2/34213) and BHF project grant PG/24/12045. This project has received funding from the European Union's Horizon Europe research and innovation programme under grant agreement No 101070546 (MUQUABIS). This work was funded by the EIC Pathfinder HopOn grant No 101115574 (CARDIOREPAIR).
Striation-free tomographic volumetric additive manufacturing using an LED light source
Felix Wechsler1, Riccardo Rizzo1, Christophe Moser1
1LAPD. EPFL, Lausanne (Vaud) - Switzerland
Tomographic Volumetric Additive Manufacturing (TVAM) rapidly prints centimeter-scale objects within seconds [1, 2] by projecting light patterns into a rotating, resin-filled container. Laser-based TVAM uses highly collimated beams to provide large depth of field but, despite being layerless, often produces layer-like striations. These artifacts arise from refractive index changes during polymerization that induce self-amplifying waveguiding [3, 4]. Striations can degrade appearance and function, yet they can be harnessed to guide anisotropic cell alignment, as in Filamented Light (FLight) bioprinting [5].
We show that an LED-based TVAM setup yields striation-free printing of cell-laden hydrogels. Here, the beam is deliberately defocused to introduce blur that suppresses waveguiding. High-resolution, striation-free results are obtained by explicitly modeling this blur within our computational framework, Dr. TVAM [6, 7]. The approach enables biofabrication of constructs with homogeneous density and isotropic cell growth, and supports bioprinting of perfusable architectures with smooth channel walls—improving adhesion of epithelial and endothelial cells compared with laser-based TVAM. The LED source also reduces cost and system complexity while maintaining biocompatible exposure conditions. Together, tailored optics and computation enable fast, gentle, and accurate volumetric bioprinting of tissues.
[1] Kelly BE et al. Volumetric additive manufacturing via tomographic reconstruction. Science 363, 1075–1079 (2019). doi:10.1126/science.aau7114
[2] Bernal PN et al. Volumetric bioprinting of complex living-tissue constructs within seconds. Adv. Mater. 31, 1904209 (2019). doi:10.1002/adma.201904209
[3] Kewitsch AS, Yariv A. Self-focusing and self-trapping upon photopolymerization. Opt. Lett. 21, 24–26 (1996). doi:10.1364/OL.21.000024
[4] Rackson CM et al. Latent image volumetric additive manufacturing. Opt. Lett. 47, 1279–1282 (2022). doi:10.1364/OL.449220
[5] Liu H et al. Filamented Light biofabrication of highly aligned tissue-engineered constructs. Adv. Mater. 34, 2204301 (2022).
[6] Nicolet B et al. Inverse rendering for tomographic volumetric additive manufacturing. ACM Trans. Graph. 43, 228:1–228:17 (2024). doi:10.1145/3687924
[7] Wechsler F et al. Overprinting with Tomographic Volumetric Additive Manufacturing (2025). arXiv:2507.13842
Beyond traditional methods: harnessing microfluidics for the synthesis of drug delivery systems
Sara Gimondi1, Daniel Mendanha1, Carlos Guimaraes1, Reis Rui L.1, Ferreira Helena1, Neves Nuno M.1
1I3B's Research Group. University of Minho, Braga - Portugal
Microfluidic-assisted nanoparticle (NP) synthesis offers unique advantages in precision, scalability, and reproducibility compared to conventional bulk methods, which often struggle to control physicochemical properties. Here, we demonstrate the versatility of a novel microfluidic platform for the synthesis of diverse NP types, including polymeric, polysaccharide-based, and lipidic NP such as large unilamellar liposomes (LUVs). We further highlight their impact on drug delivery for cancer and inflammation treatment.
The passive microfluidic device incorporates a herringbone-like structure that enhances mixing efficiency by modulating flow dynamics. This design enables the precise, uniform formation of NP by controlling the interactions between precursor streams.
Microfluidic-driven polymeric NP with diameters of 30, 50, and 70 nm were synthesized, characterized, and evaluated for their drug delivery performance. Notably, 30 nm NP achieved comparable efficacy to free dexamethasone in reducing macrophage PGE2 production under inflammatory conditions, highlighting their enhanced delivery efficiency.
Studies on internalization pathways revealed that NP size directs endocytic uptake and intracellular trafficking. For instance, 50 nm NP exhibited higher uptake and induced a 4x reduction in metabolic activity compared to 70 nm NP in doxorubicin-treated breast cancer cells.
Efforts were also made to adapt microfluidic synthesis for handling high-molecular-weight polysaccharides, such as chitosan–hyaluronan, resulting in smaller, biologically active nanoparticles with enhanced cytokine modulation under inflammatory conditions.
Finally, microfluidic synthesis of LUVs resulted to be 4x faster and required 60% fewer processing steps than the conventional thin-film hydration/extrusion method, providing a streamlined single-step strategy. This approach also facilitated efficient encapsulation of docosahexaenoic acid into LUVs, significantly enhancing its therapeutic efficacy in glioblastoma models compared to the free molecule.
In conclusion, by overcoming the limitations of conventional techniques, microfluidics allows precise tailoring of NP to meet specific biological needs. Its adaptability to diverse materials positions microfluidics as a highly versatile tool for nanomedicine applications.
Funding: SG grant (2023.06754.CEECIND/CP2841/CT0014) and TERM RES Hub (PINFRA/22190/2016; Norte-01-0145-FEDER-022190).
The impact of Adipose-Derived Stem Cells (ASCs) on Micro-RNAs of keratinocytes in Oral Lichen Planus (OLP) patients using 3D culture system: a pilot study - SEMIT
Claudio Catalano1, Ahmed Mohsen1, Gianluca Tenore1, Simona Ceccarelli2, Giulia Gerini2, Antonio Angeloni2, Umberto Romeo1
1Department of Odontostomatological and Maxillo-Facial Sciences. Sapienza University of Rome, Rome (Lazio) - Italy, 2Department of Experimental Medicine. Sapienza University of Rome, Rome (Lazio) - Italy
Background: Oral Lichen Planus (OLP) is a potentially malignant disorder with a neoplastic transformation risk of 0.07%-5.8%, lacking a definitive therapy. Adipose-derived stem cells (ASCs) exhibit immunomodulatory, pro-angiogenic, and oncosuppressive properties, potentially mediated by microRNAs (miRNAs), which may influence OLP progression.
Aim: This pilot study investigates the oncosuppressive effects of allogeneic ASCs on OLP by analyzing miRNA expression and cellular behavior in keratinocytes using 2D/3D culture systems (organoids).
Methods: Biopsies from 10 OLP patients and 10 healthy controls will be collected. One portion of the biopsy will be fixed for histopathological analysis, while a 1 mm fresh sample of the same biopsy, will be used for keratinocyte isolation and 2D/3D culture.
Keratinocytes will be co-cultured with allogeneic ASCs from healthy donors or with ASC secretome to assess changes in proliferation, migration, vitality, and miRNA expression (miR-375, miR-372, miR-10b) via qRT-PCR and Western Blot. A 3D microfluidic model mimicking oral mucosa will validate 2D findings, evaluating ASC effects on fibrosis, extracellular matrix, and neoplastic transformation markers.
Results: ASCs’ secretome significantly reduce oncogenic miRNA expression and inhibit proliferation and migration in OLP keratinocytes, potentially via paracrine mechanisms, with the organoid model confirming these effects in a near-in vivo setting.
Conclusion: This study aims to elucidate the therapeutic potential of ASCs in OLP and paving the way for cell-free therapeutic approaches, showing that cell therapy turns off chronic inflammation of the OLP at the molecular level, lowering the risk of carcinomatous evolution.
Sustainable processing of human amniotic membrane by supercritical CO2 for advanced ophthalmic implant development
Adam Lorinne1, Kerdjoudj Halima1, Rota Solène2, Bardonnet Raphaël2, Mauprivez Cédric1, Brenet Esteban1, Krawiec Elise1, Bouland Nicole1
1Université de Reims Champagne Ardenne, Reims (Champagne-Ardenne) - France, 2BIOBank, Lieusaint (Ile-de-France) - France
Transplant tissue banks provide various life-saving biological materials, including human amniotic membranes (hAM). For the successful clinical translation of hAM, adherence to good manufacturing practices, long-term storage stability and safe off-shelf availability are essential. Proper storage and sterilization of processed hAM remain critical issues and are expected to improve with the of advanced technologies. Identifying a sterilisation method that ensures sufficient bioburden reduction while minimizing mechanical and biological damage to hAM is essential. Over the past decade, efforts have been focused on using liquid and supercritical carbon-dioxide (scCO2) as an innovative technology for hard tissue processing (i.e. bone). The present study aims to evaluate the impact of scCO2 processing on hAM. Freeze-dried hAM samples were used as control. Compared to controls, scCO2-treated hAMs were sterile, as no bacterial colonies were detected on agar plates. Structural and physicochemical characterizations revealed no alteration in integrity, including porosity, thickness and swelling capacity. However, mechanical testing showed a significant increase in the elastic modulus of scCO2-treated hAMs, while residual water remained around 15%. Despite a measurable reduction in growth factor content, scCO2-treated hAMs were non-cytotoxic (ISO 10993-5), exhibited hemostatic and anti-inflammatory properties, and supported dermal fibroblast adhesion and proliferation. Moreover, scCO2-treated hAMs were successfully sutured onto porcine corneas, demonstrating their translational potential for corneal repair. These findings indicate that scCO2 treatment offers an efficient and sustainable process for the sterilization and long-term preservation of hAM. Ongoing in vivo studies aim to further evaluate the biocompatibility and antifibrotic activities of scCO2-treated hAMs.
Development of bismuth ferrite–reinforced bioactive glass scaffolds with improved osteogenic and biofunctional properties for bone regeneration
Sharun Khan1, Amitha Banu Shajahan1, Hasanur Rahaman2, Merlin Mamachan3, Swapan Kumar Maiti3, Amar Pal3, Subhadip Bodhak2, Vamsi Krishna Balla2, Abhijit Pawde3
1Department of Health Science and Technology. Aalborg University, Aalborg (Nordjylland) - Denmark, 2Biomaterials and Medical Devices Division. CSIR-Central Glass and Ceramic Research Institute, Kolkata (West Bengal) - India, 3Division of Surgery. ICAR-Indian Veterinary Research Institute, Bareilly (Uttar Pradesh) - India
Bone tissue engineering relies on the integration of cells, bioactive molecules, and scaffolds to restore or replace damaged bone. Among these components, the scaffold plays a crucial role by providing mechanical support and guiding cell attachment, proliferation, and differentiation. However, the development of an ideal scaffold that replicates the structural, mechanical, and electrical characteristics of native bone remains a major challenge. In this study, porous bioactive glass (BAG) scaffolds reinforced with varying concentrations of bismuth ferrite (BF) were fabricated to evaluate their potential for bone regeneration. Bismuth ferrite, a multiferroic material with inherent electrical activity, was incorporated to simulate the piezoelectric behavior of natural bone, thereby promoting osteogenic responses. The scaffolds were produced using the foam replication technique and subsequently characterized for their morphology, phase composition, porosity, mechanical performance, bioactivity, and antibacterial properties. In vitro studies using MC3T3-E1 pre-osteoblast cells revealed that the addition of BF significantly enhanced cell adhesion, viability, and proliferation, as confirmed by MTT and live/dead assays. The composite scaffolds exhibited improved compressive strength and superior apatite-forming ability in simulated body fluid, indicating enhanced bioactivity. Furthermore, the inclusion of BF imparted mild antibacterial activity without compromising cytocompatibility, although minimal haemolytic activity was observed at higher concentrations. Magnetic stimulation further amplified cellular proliferation, suggesting a synergistic effect between the scaffold’s electrical and magnetic characteristics. Overall, the BF-incorporated BAG scaffolds demonstrated a favorable combination of mechanical integrity, bioactivity, and biological performance, offering a promising strategy for electrically active bone substitute materials. This approach highlights the potential of integrating functional ceramics with conventional bioactive glass to develop next-generation scaffolds that can accelerate bone regeneration through electrical stimulation.
Biofabrication of polyhydroxyalkanoate-based dual drug-loaded mucoadhesive patches for localised therapy of oral lichen planus
Harshavardhan Budharaju1, Robyn Macartney2, Jonathan Knowles2, Ipsita Roy1
1School of Chemical, Materials and Biological Engineering. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom, 2Division of Biomaterials and Tissue Engineering. University College London (UCL), London (London, City of) - United Kingdom
This study aims to develop sustainable, patient-specific 3D-printed mucoadhesive patches for localised and prolonged dual-drug delivery in oral lichen planus (OLP) therapy. OLP is a chronic inflammatory mucosal disorder characterised by painful erosive lesions, epithelial degeneration, and frequent recurrences. Current clinical treatments mainly rely on topical corticosteroids and antifungal agents; however, their therapeutic efficacy is limited by poor mucosal adherence, short residence time, and inadequate drug retention, necessitating repeated applications and often resulting in inconsistent patient compliance. In this work, extrusion-based 3D printing was employed to fabricate mucoadhesive patches co-loaded with clobetasol propionate (an anti-inflammatory agent) and fluconazole (an antifungal agent) to achieve combined and sustained therapeutic effects. Medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biodegradable and biocompatible bacterial polyester, was selected as the base material due to its sustainable origin, mechanical flexibility, and excellent processability at low printing temperatures. The printed constructs exhibited smooth surface morphology, uniform drug distribution, and controlled release kinetics. Physicochemical characterisation confirmed successful drug integration and stability within the polymer matrix. Degradation and swelling studies demonstrated minimal expansion, indicating non-swelling behaviour that ensures comfort and prevents irritation during intraoral application. In vitro cytocompatibility assays using oral mucosal epithelial cells showed no cytotoxic effects, while wound-healing scratch assays confirmed enhanced cell migration and upregulation of genes associated with tissue regeneration. These findings establish mcl-PHA as a sustainable and printable polymer for fabricating dual-drug mucosal patches. Future work will focus on polydopamine surface functionalization to enhance mucoadhesion and in vivo evaluation for clinical translation.
Acknowledgments
This work was supported by the grants of the Engineering and Physical Sciences Research Council (EPSRC), UK [grant number EP/X026108/1].
Volumetric printing of porous constructs for enhanced transport and targeted ultrasound-mediated drug delivery in vascularized tissue models
Julia Siminska-Stanny1, Isaline Noirot2, Amir Abrishami2, Malavika Nair3, Dario Carugo3, Eleanor Stride3, Armin Shavandi2
1NDORMS & 3BIO-BioMatter. University of Oxford & Universite Libre de Bruxelles, Oxford (Oxfordshire) - United Kingdom, 23BIO-BioMatter. Universite Libre de Bruxelles, Brussels (Brussels Hoofdstedelijk Gewest) - Belgium, 3NDORMS. University of Oxford, Oxford (Oxfordshire) - United Kingdom
This work aims to establish a framework linking tissue architecture, acoustic stimulation, and controlled release to guide the design of more predictive and translational tissue models. Porous hydrogels that mimic native tissue transport are critical for tissue engineering and organ-on-chip applications, where nutrient exchange and waste removal sustain physiological function. Here, we employ volumetric 3D printing to fabricate GelMA–PEGDA hydrogels with tunable microarchitectures, enabling precise control over pore size, orientation, and distribution through digital design. The resulting constructs exhibit mechanical stability, biocompatibility, and enhanced mass transport.
Active perfusion studies demonstrated that solute diffusion depends on both pore arrangement (horizontal, vertical, or mixed) and molecular characteristics. Small, cationic molecules such as methylene blue diffused rapidly (∼1.8 mm/30 min), while larger or anionic species like Coomassie blue and bovine serum albumin (BSA) complexes showed slower transport (∼0.4 mm/30 min). Rhodamine–dextran (70 kDa), despite its higher molecular weight, achieved intermediate diffusion (∼1.0 mm/30 min) due to its compact hydrodynamic radius and low matrix affinity. Integration of microporosity with vascular-like channels (1mm diameter) further enhanced solute transport beyond the conventional ∼200 μm diffusion limit.
Bulk GelMA–PEGDA hydrogels exhibited a Young’s modulus of ∼15 kPa, decreasing to ∼12 kPa for porous constructs and ∼8–10 kPa under hydrated conditions, reflecting structural and environmental softening. As a proof of concept, we combine volumetric printing with ultrasound (US) and microbubble (MB)–mediated delivery to develop tissue models for targeted therapy studies. MBs (1–3 μm), formulated from dipalmitoylphosphatidylcholine (DPPC) and stabilized with PEG emulsifier, act as acoustic agents to enhance drug transport. By tuning acoustic parameters such as pressure (0.2–1 MPa), pulse repetition frequency (1-100 Hz) and duty cycle (5–20%), we aim to maximize permeation while probing cavitation and extravasation mechanisms that regulate molecular transport. There is evidence that US–MB exposure transiently increases vascular permeability; in this study we aim to evaluate whether this effect depends on scaffold porosity and acoustic input parameters.
COREX: scalable callus organoid-derived extracellular matrix granular bioink for bone regeneration
Jiarun Bai1, Hanna Svitina1, Isaak Decoene1, Ioannis Papantoniou1
1Prometheus Division of Skeletal Tissue Engineering. Skeletal Biology and Engineering Research Center, Department of Development and Regeneration. KU Leuven, Leuven (Brabant) - Belgium
Introduction
Bone fractures are common injuries, with 178 million cases worldwide in 2019 (GBD Fracture Collaborators 2021). Bone defects remain challenging and often result in non-unions. Callus organoids (COs) recapitulate early endochondral ossification, providing a blueprint for bone defect treatment (Hall et al. 2020). Nevertheless, such living implants face hurdles in preservation, transport and allogeneic use due to immunogenicity. One viable solution is to remove cells while retaining extracellular matrix (ECM) as ready-to-use biomaterials (Burdick et al. 2013). Cartilaginous ECM has been studied for bone regeneration (Longoni et al. 2021; Garcia et al. 2025), but using functionally preserved callus organoid ECM as micromaterials remains unexplored. Thus, we compared decellularization and devitalization methods for COs, evaluating morphology, biochemical composition and bioactivity. We assessed osteogenic potential subcutaneously in mice and further interpreted results using proteomics. Finally, dECM modules were crosslinked into an injectable granular bioink composed solely of dECM, providing a novel modular off-the-shelf biomaterial for treating bone defects.
Methods & Results
Human periosteum-derived cells were cultured in microwells with chondrogenic medium for 14 days to form COs. Various treatments (lyophilization, freeze-thaw, detergent, and enzyme) were applied to generate decellularized or devitalized ECMs (dECMs), which were compared to untreated COs. All methods preserved CO structure with slight shrinkage. Decellularization effectively removed > 99% of DNA (DAPI staining, DNA quantification). After recellularization, lyophilization-derived dECM best supported chondrogenic/hypertrophic differentiation (qRT-PCR, histology). Subcutaneous implantation in immunocompromised mice for 8 weeks led to formation of mineralized ossicles with cortex, trabeculae, and marrow in all groups (microCT, histology). Proteomics confirmed preservation of key proteins per Matrisome category especially in lyophilization and detergent groups. Finally, dECM modules were crosslinked via EDC/NHS into an injectable granular, modular bioink for scalable bone repair.
Conclusion
These findings demonstrate that decellularized and devitalized CO-derived ECM modules retain essential structural integrity, key Matrisome proteins and bone-forming capacity, serving as a potent scalable off-the-shelf granular bioink with promise for translation in bone defect regeneration.
Advancing collagen I-based tumor-on-chip models: from tunable mechanics to 3D mechanical sensing capability
Minye Jin1, Ali Al-Jaberi2, Séverine Le Gac2, Julieta Paez1
1Developmental BioEngineering. University Twente, Enschede (Overijssel) - The Netherlands, 2Applied Microfluidics for Bioengineering Research. University Twente, Enschede (Overijssel) - The Netherlands
Collagen (Col) is the most abundant structural protein in the body, playing an important role in tissue integrity and regeneration. It has been widely used as a hydrogel to model diverse tissue microenvironments. Yet, conventional physically crosslinked Col-I hydrogels are often too soft and unstable to accurately model stiffer tissues such as fibrotic or tumor environments.1 This project aims to advance in vitro tumor models by developing smart Col-I hydrogels that i) better replicate the mechanical and biological properties of healthy and tumoral breast tissues, ii) incorporate the capability to 3D-map scaffold mechanics, iii) are user-friendly and suitable for tumor-on-chip modelling.
First, we developed and compared two covalent crosslinking strategies (light-free vs. light-based) to complement the normal Col-fibrillogenesis process, which together enhanced hydrogel stiffness without prior chemical modification of the Col precursors. After process optimization, porous and fibrillar hydrogels exhibited a fivefold increase in stiffness (G′ = 400–3000 Pa), as measured by shear rheology, in relation to physically crosslinked controls. Such optimized elasticity range encompasses values from healthy to cancerous breast tissues, while maintaining high cell viability (MCF-7). Both chemical strategies are user-friendly, easy to implement and cytocompatible, while the light-based approach allows additional spatial-temporal control of gelation.
Secondly, we embedded thermo-responsive mechanical microsensors2 in the Col matrices to enable real-time monitoring of stiffness under physiological conditions. These sensors provided quantitative insight into local stiffness changes, which aligned with bulk rheology measurements. Exploration of these tailorable matrices for on-chip experimentation is ongoing to establish their suitability for tumor-on-chip modelling.
Our approach proposes Col-I hydrogels with tunable mechanical and sensing capabilities as a versatile platform for modeling both healthy and pathological tissues, studying disease progression, and improving preclinical testing of therapeutic strategies.
References
1. Levental et al., Cell 139, 891-906 (2009).
2. Moraes et al., Nat Commun 11, 4757 (2020).
Electrical pacing in a custom bioreactor accelerates maturation of MEW-based human engineered cardiac tissues
Maria Alejandra Rodriguez Pardo1, Peter Bennett2, Ilazki Anaut Lusar1, Eduardo Larequi1, Felipe Prosper3, Olalla Iglesias García1, Manuel Maria Mazo Vega4
1Biomedical Engineering Program. Cima Universidad de Navarra, and Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona (Navarra) - Spain, 2Boston Scientific, Clonmel (Tipperary) - Ireland, 3Hematology and Cell Therapy Area, Cancer Center, Clínica Universidad de Navarra (CCUN). Clínica Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdiSNA), and Hemato-Oncology Program, Cima Universidad de Navarra (CIMA), Pamplona (Navarra) - Spain, 4Biomedical Engineering Program, Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdiSNA), and Hematology and Cell Therapy Area, Cancer Center Clínica Universidad de Navarra (CCUN), Clínica Universidad de Navarra (CUN), Pamplona (Navarra) - Spain
Cardiovascular disease remains the leading cause of death worldwide, driving the development of engineered human cardiac tissue for regenerative therapies and drug discovery. Yet, most three-dimensional (3D) constructs exhibit a fetal-like phenotype that limits their translational value, making the delivery of appropriate maturation cues essential.
This study aims to optimize electrical stimulation to accelerate the maturation of hiPSC-derived 3D cardiac tissues. A custom bioreactor was designed in AutoCAD and fabricated by rapid prototyping. The chamber fits a standard Petri dish for incubator use and accommodates multiple tissues simultaneously, ensuring consistent and reproducible experimental conditions.
To emulate the native myocardial microenvironment, highly defined poly(ε-caprolactone) (PCL) scaffolds generated by melt-electrowriting (MEW) were embedded in a fibrin matrix containing hiPSC-cardiomyocytes. This composite provides both mechanical integrity and biological support for tissue formation. The fiber architecture reproduces native myocardial anisotropy, guiding cell alignment and synchronous contraction, while the fibrin hydrogel a 3D biocompatible environment and promotes robust cell–matrix coupling and compaction.
Four pacing parameters—field strength, frequency, pulse width, and waveform (mono- versus biphasic)—were systematically varied. Among the tested conditions, stimulation at 5V, 5ms, 2Hz with a biphasic pulse elicited the strongest up-regulation of cardiac maturation genes, while preserving cell viability (Live/Dead) and metabolic activity (Alamar Blue). Constructs maintained contractile behavior and structural integrity throughout the 21-day stimulation period.
These findings demonstrate that precisely tuned biphasic electrical conditioning enhances the transcriptional and functional maturation of engineered human cardiac tissues without compromising health. By combining MEW-fabricated scaffolds with optimized electrical pacing, this work provides a reproducible and scalable approach to improve the physiological relevance of human cardiac tissue models for regenerative medicine and drug testing.
REFERENCE
1. Sánchez-Bueno, A. et al. 3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells. J. Vis. Exp. 217, e67847 (2025).
GelMA-encapsulated of limbal mesenchymal stem cells and exosomes for corneal inflammation modulation in ocular surface chemical injuries
Merve Nur Soykan1, Burcugül Altug2, Didem Akincilar3, Gizem Yildirim4, Onur Sunnteci5, Nadiye Boncukcu4, Dilay Karacaoglu4, Eray Atalay6, Onur Ozalp6, Onur Uysal4, Ayla Eker Sariboyaci4
1Cellular Therapy and Stem Cell Production Application, Research Centre, ESTEM & Department of Stem Cell, Institute of Health Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, Eskisehir - Turkey, 2Cellular Therapy and Stem Cell Production Application, Research Centre, ESTEM, Eskisehir Osmangazi University, Eskisehir, Turkey & Department of Genetics, Faculty of Veterinary Medicine, Dokuz Eylül University, İzmir, Turkey, İzmir (Izmir) - Turkey, 3Cellular Therapy and Stem Cell Production Application, Research Centre, ESTEM, Eskisehir Osmangazi University, Eskisehir, Turkey & Department of Stem Cell, Institute of Health Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, Eskisehir - Turkey, 4Cellular Therapy and Stem Cell Production Application, Research Centre, ESTEM, Eskisehir Osmangazi University, Eskisehir, Turkey & Department of Stem Cell, Institute of Health Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, Eskisehir - Turkey, 5Cellular Therapy and Stem Cell Production Application, Research Centre, ESTEM, Eskisehir Osmangazi University, Eskisehir, Turkey & Department of Stem Cell, Institute of Health Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey, Eskisehir - Turkey, 6Department of Ophthalmology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey, Eskisehir - Turkey
Aims/Purpose:
The aim of this study is to investigate the effect of limbal mesenchymal stem cells (LMSCs) and their derived exosomes, when encapsulated with GelMA, on corneal regeneration and inflammation in ocular surface chemical injuries (OSCI).
Methods:
Human limbal mesenchymal stem cells (hLMSCs) and their exosomes were first isolated and characterized. A rabbit ocular surface chemical injury (OSCI) model was then created using NaOH. The hLMSCs and hLMSCs-exosomes were encapsulated in GelMA and crosslinked under visible light. Treatments were applied for 7, 21, and 60 days. At the end of these periods, samples were collected from the corneal surface via impression cytology. Expression changes in MUC1, CK19, IFN-ɣ, CCR2, IL-6, CCL2, CXCL12, CXCR4, IL-1β, CXCL-8, and MMP-9 genes were analyzed by Real-Time PCR, while HLA-DR, ICAM-1, CD8, CD31, and CD68 were examined using flow cytometry.
Results:
In the OSCI model, an increase in anti-inflammatory mediator gene expression levels (p<0.05) was observed in hLMSCs and hLMSC-exo treatment groups compared to control groups, while a decrease in inflammation marker gene expression levels was observed. Similarly, flow cytometry analysis revealed a decrease in inflammation severity and activation (p<0.05), and it was observed to regulate the cellular response.
Conclusions:
GelMA-encapsulated hLMSCs and their exosomes effectively suppressed inflammatory signaling targeted immunomodulation that decreases inflammatory activation while promoting corneal regeneration in OSCI. The authors acknowledge the support from Eskisehir Osmangazi University, Scientific Research Projects (ESOGU-BAP Grant ID: TDK-2024-3193 and 202046055) and Health Institutes of Türkiye (TÜSEB Grant ID: 37552), Scientific and Technological Research Council of Türkiye (TÜBİTAK Grant ID: 123S073).
Injectable thermosensitive hydrogel for localized delivery of amifostine to prevent radiation-induced side effects in head and neck cancer
Dario Castellana1, Oscar Castaño2, Elisabeth Engel3
1Biomaterials for Regenarative Therapies group. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 2Department of Materials science and chemical physical. University of Barcelona (UB), Barcelona - Spain, 3IMEM-BRT group, Department of Materials Science. Universitat Politècnica de Catalunya, Barcelona - Spain
Radiotherapy is the standard of care for head and neck carcinoma but causes debilitating side effects, including dry mouth, mucositis, and dysphagia that severely impact patients' quality of life. Amifostine is the only FDA-approved radioprotective drug capable of mitigating this effect; however, its clinical use is limited due to poor bioavailability and systemic toxicity following intravenous administration.
To address these limitations, we aim to develop an injectable, thermosensitive hydrogel system capable of delivering Amifostine locally and in a controlled manner to tissues subjected to X-ray irradiation. The system integrates Polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) encapsulating Amifostine within a Hyaluronic Acid (HA) and pNIPAM hydrogel matrix. The HA provides mechanical properties and tissue integration, while pNIPAM confers temperature-dependent phase transition, enabling in-situ implantation and sustained drug release.
Amifostine-loaded PLGA NPs were synthesized with 80% encapsulation efficacy, and different formulations were optimized to tune drug release kinetics. The HA-pNIPAM hydrogel was synthesized via polymer grafting, confirmed by 1H-NMR spectroscopy, and exhibited a sol-gel transition at approximately 35°C with mechanical properties suitable for injection and retention at the target anatomical site.
In vitro assays simulating clinical radiation doses demonstrated that Amifostine-loaded NPs significantly reduced reactive oxygen species (ROS) levels and improved cellular metabolic activity and maintained cell proliferation post-irradiation, confirming their radioprotective potential and lack of toxicity compared to the free drug.
In conclusion, this thermosensitive hydrogel-nanoparticle platform offers a promising strategy for localized Amifostine delivery, enhancing its therapeutic efficacy while minimizing systemic side effects. This approach could significantly reduce the severity and frequency of radiation-induced complications in head and neck cancer patients.
Induced mesenchymal stem/stromal cells (MSCs) produce less humoral response than bone marrow derived MSCs in the horse
Elvira Bernad Roche1, Ma Belén Serrano Pastor1, Marcos Del Cerro Brocal2, Arantza Vitoria Moraiz1, Sara Fuente Franco1, Francisco José Vázquez Bringas1, Antonio Romero Lasheras1, Pilar Zaragoza Fernández1, Alina Cequier Soler1, Clementina Rodellar Penella1, Laura Barrachina Porcar1
1LAGENBIO. University of Zaragoza, Zaragoza - Spain, 2University of Zaragoza, Zaragoza - Spain
Mesenchymal stem/stromal cells (MSCs) are promising for treating pathologies but undergo senescence, so deriving MSCs from induced pluripotent stem cells has been suggested as an alternative. Induced MSCs (iMSCs) can overcome certain limitations of primary MSCs, but their immunogenicity in vivo is unknown. The development of immune memory could limit clinical application and repeated administration. Allo-antibody production after administering allogeneic primary MSCs has been demonstrated in the horse, which is a highly relevant patient and translational model. However, the humoral response to equine iMSCs has not been investigated.
This study assessed the humoral immune response to equine iMSCs compared to bone marrow-derived MSCs (BM-MSCs) by evaluating the production of cytotoxic allo-antibodies directed against donor’s equine leukocyte antigen (ELA) after allogeneic major histocompatibility complex (MHC)-mismatched administration.
Either BM-MSCs or iMSCs were intradermally injected in one neck side of each recipient horse, divided into two groups (n=4 each group) and all of them MHC-mismatched with the donor. Peripheral blood from each horse was collected before and serially after (1, 3, 6 weeks). After two months, each animal was re-exposed to the same MSCs by repeating the procedure in the contralateral neck side.
The sera were used in microcytotoxicity assays, mixing it with the same MSCs administered to evaluate the presence of cytotoxic antibodies along the time.
The alloantibody production was moderate in all cases but higher at all time-points for BM-MSCs compared to iMSCs. For iMSCs, the cytotoxic scores barely increased after the first administration and remained stable until reaching a peak one week after the second administration.
This is the first time the humoral response against iMSCs has been described in the horse, showing that equine iMSCs are not more immunogenic than equine BM-MSCs. Further studies are needed to understand immune responses against cell therapies in order to develop safer therapies.
Developing a model of breast cancer dormancy in the bone marrow
Matthew Walker1, Massimo Cau2, Patrick Peschke2, Savvas Ioannou1, Molly Stevens2, Matthew Dalby1, Catherine Berry1
1University of Glasgow, Glasgow (Glasgow City) - United Kingdom, 2University of Oxford, Oxford (Oxfordshire) - United Kingdom
Aim and objective:
The bone marrow is the most common secondary site for breast cancer which often leads to recurrence after several years of dormancy. Within this niche, communication between mesenchymal stem cells (MSCs) and breast cancer cells via extracellular vesicles (EVs) is critical in regulating the activity state of secondary breast tumours. Previous work in our group has shown that specific bioactive metabolites secreted within MSC-EVs can stimulate a slow-growing phenotype from MCF-7 breast cancer cells (1). We aim to develop a dormant niche model using these metabolites, packaged in synthetic EVs, with MCF-7 spheroids and MSCs interacting with a bone/bone marrow-mimetic interface using a 2.5D/3D system. This model will be integrated into “on-chip” devices to allow therapeutic and diagnostic screening of breast cancer dormancy/recurrence.
Material & methodology:
MCF-7 cell culture, immunostaining, confocal microscopy, nanoparticle tracking analysis, qPCR, GraphPad Prism (statistical analysis performed by appropriate parametric/non-parametric ANOVA tests following outlier removal, descriptive statistics to assess the assumption of equal variance, and lognormality/normality tests).
Results:
Using previously established synthetic EVs (2-5), we have shown their capability to be engineered with similar size properties to natural EVs and have sustained storage stability at 4oC; these can also be uptaken by MCF-7 cells. Additionally, we have observed that bioactive metabolites can upregulate activity of dormancy markers p27 and COUP-TFI, via increased nuclear translocation, and can reduce expression of nuclear proliferation marker Ki-67. All experiments have been conducted so far in 2D.
Conclusions:
Synthetic EVs have suitable size characteristics as natural EV mimics, are suitable for long term storage, and can be actively uptaken by MCF-7 cells; this is promising for their use as metabolite delivery vehicles. Bioactive metabolites are capable of inducing a dormancy phenotype from MCF-7 cells which is encouraging for longer-term development of a dormancy model on-chip.
References:
1) Bartlome et al., BioRxiv (2022)
2) Nele et al., Advanced Materials (2020)
3) Ioannou et al., Stem Cell Research & Therapy (2025)
4) Lu et al., Int J Pharm (2018)
5) Nicolini et al., Biochimica et Biophysica Acta (2008)
Laser-microstructured Cu-doped bioresorbable glasses: tailoring cell response for regenerative medicine
Emanuela Peluso1, Marialaura Giannaccari1, Cristina Volpini1, Devanarayanan Meena Narayana Menon2, Eleonora Galleano3, Davide Jenner2, Paolo Minzioni3, Livia Visai1
1Molecular Medicine Department (DMM), Centre for Health Technologies (CHT), Unità di Ricerca (UdR) INSTM; Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R). University of Pavia, Pavia (Lombardia) - Italy, 2Department of Applied Science and Technology (DISAT) and RU INSTM. Politecnico di Torino, Torino (Italia) - Italy, 3Department of Electrical, Computer and Biomedical Engineering. University of Pavia, Pavia (Lombardia) - Italy
Introduction. Recent interest has grown in bioresorbable photonic components, capable of dissolving naturally within the body. Unlike bioresorbable electronics mainly used for sensing, photonic devices can also provide therapeutic effects, as light-based treatments show promise in cancer therapy, wound healing, and infection control. Despite the widespread use of optical tools in medicine, bioresorbable photonic materials have not yet reached clinical practice. This study employed an infrared (IR) pulsed laser (nanosecond, ns pulses) to microstructure Cu-doped calcium phosphate bioresorbable glass and evaluate the biological effects of surface-modified glasses with variable microstructure dimensions (height: 2–12 μm; width: 20–100 μm).
Materials and Methods. Cu-doped calcium phosphate glasses with composition (in mol%) ((50 P2O5–25 CaO–8 MgO–11.5 Na2O–2.5 B2O3–3 SiO2)x–(CuO)γ, where x = 1 when y = 0 and x = 0.99 when y = 1) were synthesized by melt-quenching, following established protocols (App. Sci. 2022, 12, 3516; Materials 2023, 16, 3899). Five sterilization methods were compared to preserve microstructures: ethylene oxide, autoclave, high temperature, UV exposure, and 70% ethanol. Surface morphology was analyzed by profilometry, while fibroblast (NIH-3T3) cultures were used to assess adhesion, viability via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and morphology by scanning electron microscopy (SEM). Results. Laser parameters (power, pulse duration, scan rate) were optimized to achieve precise micro structuring without cracks. UV sterilization best preserved surface features. Cell adhesion and morphology depended on structure dimensions: shorter spacing with low height or wider spacing with taller structures improved adhesion and proliferation. A linear correlation between adhered and remaining cells indicated ∼50% adhesion efficiency. In indirect tests, cell morphology improved with increasing medium dilution.
Conclusions. Cu-doped calcium phosphate bioresorbable glass proved biocompatible and resorbable, with surface microstructure significantly influencing cell response. These results demonstrate their potential for tissue engineering and resorbable photonic medical devices.
Acknowledgment. Part of the BioPhET project, funded by the EU “Next Generation EU” initiative through MUR (PRIN-2022-PNRR grant P2022ACLS2).
Plasma-assisted deposition of ultrathin collagen coatings enhances osteogenic cell response
Evelyn Knappe1, Fanny Mende1, Caroline Seidel1, Tom Schöbe1, Frauke Junghans1, Ina Prade1
1FILK Freiberg Institute gGmbH, Freiberg (Sachsen) - Germany
Aim and Objective
Successful osseointegration is essential for the long-term stability of orthopedic and dental implants. Collagen coatings can enhance biological integration; however, traditional wet-chemical methods often yield poor adhesion, limited mechanical stability, and complex manufacturing steps, restricting clinical translation. This study aims to develop a plasma-assisted technique for depositing ultrathin, mechanically stable collagen coatings that preserve biological activity and improve implant performance.
Material and Methodology
A non-thermal atmospheric pressure plasma system was combined with a commercial nebulization device to deposit collagen layers (<100 nm) directly onto titanium and polymer substrates relevant for tissue engineering, including polyethylene, polypropylene, polycaprolactone, and polyethylene terephthalate. Surface morphology, chemistry, and hydrophilicity were analyzed to assess coating uniformity and functionality. Coating stability was evaluated through mechanical testing and prolonged immersion in cell culture media. Human osteoblast-like cells were cultured on coated and uncoated substrates to evaluate cytocompatibility following ISO 10993-5 standards, adhesion, proliferation, and osteogenic differentiation.
Results
The plasma-assisted coatings exhibited strong adhesion and stability, maintaining integrity for at least 14 days under culture conditions. Enhanced surface hydrophilicity and topographical features promoted cell attachment and spreading without cytotoxic effects. Importantly, spectroscopic analyses confirmed that collagen retained its native conformation after plasma treatment. Quantitative assays showed significantly increased alkaline phosphatase activity and osteogenic marker expression on coated surfaces compared to controls.
Conclusions
This plasma-assisted, linker-free coating technique preserves collagen’s bioactivity while ensuring high mechanical stability, creating a favorable microenvironment for osteoblast growth and differentiation. The approach provides a scalable, cost-efficient platform for developing bioactive surfaces in tissue engineering applications and improving the integration of next-generation orthopedic and dental implants.
Engineered chitosan–lignosulfonate films as sustainable biomaterials for regenerative medicine
Marialaura Giannaccari1, Daniele Massari2, Nora Bloise1, Matteo Gigli2, Livia Visai1
1Molecular Medicine Department, Centre for Health Technologies (CHT), Research Unit (UdR) INSTM; Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R), University of Pavia Unit, Pavia (Lombardia) - Italy, 2Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Via Torino 155, 30172 Venezia Mestre, Italy, Venice (Veneto) - Italy
Introduction
The increasing demand for sustainable and multifunctional biomaterials is fostering innovation in regenerative medicine [doi.org/10.1007/s44347-025-00014-8]. This study aimed to develop and characterize eco-sustainable films based on deacetylated chitin nanocrystals (CsNCs) functionalized with varying concentrations of lignosulfonate (L), intended as potential supports for tissue regeneration.
Material and Methods
Comprehensive physicochemical analyses were performed using Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Nuclear Magnetic Resonance (NMR) spectroscopy, and Thermogravimetric Analysis (TGA) to assess morphology, chemical structure, degree of deacetylation, and thermal stability. Surface properties were evaluated through contact angle and water absorption measurements.
Biological performance was investigated using antibacterial assays against Staphylococcus aureus and Escherichia coli, antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and in vitro cytocompatibility and hemocompatibility studies with NIH-3T3 fibroblast cells and platelet assays, respectively.
Results
Analyses confirmed the needle-like morphology of CsNCs, their chemical integrity, and good thermal stability. The incorporation of lignosulfonate enhanced film wettability and long-term structural stability. The biomaterials exhibited selective antimicrobial activity against the tested bacteria. The presence of lignosulfonate conferred pronounced antioxidant activity, relevant for mitigating oxidative stress and supporting tissue healing. Biological assays demonstrated high cytocompatibility and hemocompatibility, confirming the suitability of CsNC/L films for biomedical applications.
Conclusion
The developed CsNC/L films represent promising, sustainable biomaterials for tissue regeneration. Further optimization is required to tailor biological responses for specific clinical applications, like wound healing or skin regeneration after surgery, and to enable scale-up from laboratory synthesis to industrial production. Future research will focus on in vivo validation and targeted surface functionalization to enhance tissue-specific regenerative outcomes and to ensure that these promising laboratory findings can evolve into the safe, practical, and impactful next-generation tissue regeneration biomedical solutions.
Essential (bio)signals and how to attain them in synthetic hydrogels for tissue engineering
Iva Pashkuleva
de I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics of University of Minho, Guimarães (Braga) - Portugal
Hydrogels are essential components of different tissue engineering (TE) approaches. Initially, advantages as minimally invasive delivery, high hydration ratio, and the ability to take the shape of the lesioned tissue area have been considered for their application but nowadays, additional assets that allow biospecificity and personalization must be also considered. Such assets include biofunctionalization, stimuli-responsiveness, self-healing, and printability.
In most TE approaches, hydrogels are used as a vessel for delivery of cells and growth factors. Therefore, the extracellular matrix (ECM) has been the main source of inspiration for designing synthetic biofunctional hydrogels. ECM is highly hydrated fibrous mesh composed from various amino acids and carbohydrate-based macromolecules specific for each tissue. Collagen, fibrin, and glycosaminoglycans (particularly hyaluronan) have been used to master ECM mimics employing different chemical modifications. Alternatively, short bioactive sequences and oligomers of these biopolymers can be also incorporated in synthetic polymers aiming better reproducibility, tunability, and biofunctional versatility. Among different approaches, supramolecular (non-covalent) assembly of bioinformation coding (oligo-) peptides and glycans is very attractive because allows formation of dynamic, adaptive and responsive hydrogels - just like the native ECM.[1,2] Our results demonstrated that bioactive peptides, glycans, and their amphiphiles can (co-) assemble into fibrous hydrogels that stabilize different growth factors (i.e. prolong their half-life) and can be used as cells vehicles.[3-5] Molecular design of these amphiphiles is not trivial and will be discussed. Particular attention will be given to sulfated glycans and their amphiphiles used as heparin mimics. The effect of the morphology and anisotropy at various scales (molecular to micron) will be also showcased.
Acknowledgements:
EU and the Portuguese FCT are acknowledged for the support through grants HORIZON-EIC-2022-PATHFINDEROPEN-01-101099063 (Aptadegrad) and 2023.17561.ICDT (SUPRAHELIX).
References:
1. A.Brito et al, Chem (2021) 7:2943.
2. J.Chen, X.Zou, Bioactive Materials (2019) 4:120.
3. A.Brito et al, Chem. Sci. (2019) 10:2385.
4. R.Novoa-Carballal et al, Chem. Eur. J. (2018) 24:14341.
5. R.R.Costa et al, Adv. Mat. Tech. (2023) 8: 2370102.
Improving bone regeneration potential via precision ultrasonic fragmentation of decellularized human bone
Alessio Bucciarelli1, Laura Gambari2, Leonardo Vivarelli3, Dante Dallari3, Francesco Grassi2, Devid Maniglio1
1Department of Industrial Engineering. Univeristy of Trento, Trento (Italia) - Italy, 2RAMSES Lab. IRCCS Istituto Ortopedico Rizzoli, Bologna (Italia) - Italy, 3Banca del Tessuto Muscoloscheletrico. IRCCS Istituto Ortopedico Rizzoli, Bologna (Italia) - Italy
Decellularized bone particles (dbPTs) provide structural and biochemical cues supporting bone regeneration; however, their osteogenic performance can be diminished during decellularization. Increasing evidence indicates that particle size is a key regulator of cellular response, yet reliable methods to finely control dbPTs size distribution remain limited (1,2,3). This study aimed to enhance the osteogenic capacity of dbPTs by developing a scalable ultrasonication-based microfragmentation process enabling precise size adjustment.
Human cortical bone was decellularized and pre-fragmented by ball milling. Ultrasonication parameters (power, duty cycle, duration) were systematically optimized to obtain controlled particle size profiles. Particle morphology and composition were characterized using SEM, confocal microscopy, FTIR, and thermogravimetric analysis. For biological assessment, dbPTs were embedded in methacrylated silk fibroin hydrogels seeded with adipose-derived stromal cells cultured under osteogenic conditions. Cell proliferation and differentiation were evaluated over 14 days using Alamar Blue and alkaline phosphatase (ALP) assays.
Ultrasonication reproducibly reduced particle size to ∼5 µm while preserving mineral content and increasing exposure of the underlying organic matrix. Although high-energy sonication led to partial degradation of organic components, smaller particles consistently promoted greater cell proliferation and osteogenic differentiation. The smallest particle fractions showed the highest ALP activity and osteogenic marker expression. Statistical analyses demonstrated a clear inverse correlation between particle size and osteogenic performance, likely due to increased surface area and enhanced accessibility of bioactive matrix domains.
This work demonstrates that controlled ultrasonic microfragmentation is an effective and scalable strategy to optimize dbPTs biological activity through precise particle size tuning. The method is simple, reproducible, and compatible with clinical workflows, offering a promising approach to improving the regenerative efficacy of bone graft materials.
References
(1) Pollock R. et al., 2008, 10.1007/s00586-008-0648-3
(2) Baldwin P. et al., 2019, 10.1097/BOT.0000000000001420
(3) Zhou X. et al., 2011, 10.1016/j.bjoms.2010.01.001
Acoustic patterning of vascularized engineered cardiac tissues
Niloofar Khoshdel Rad1, Oscar O`dwyer Lancaster-Jones1, Maja Bencun2, Mario Wisbar1, Russell Quinn1, Daniela Duarte Campos1
1Bioprinting & Tissue Engineering Group, Centrum for Molecular Biology (ZMBH). Heidelberg University, Heidelberg (Baden-Wberg Bayern) - Germany, 2Section of Bioinformatics and Systems Cardiology. Klaus Tschira Institute for Integrative Computational Cardiology. University Hospital Heidelberg, Heidelberg, Germany
Aim and Objective:
To build in vitro tissues for therapeutic applications, it is essential to replicate the spatial distribution of cells that occurs during morphogenesis in vivo. In this work, we present an acoustic bioassembly-induced morphogenesis strategy that harnesses acoustic radiation forces generated by standing waves to guide the arrangement of human iPSC-derived cardiomyocytes and supportive cells within fibrin-based matrices. This approach aims to recapitulate the native structures of the myocardium, utilizing a non-invasive patterning technique.
Materials and Methods:
Human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac fibroblasts, endothelial cells, and pericytes were encapsulated in soft fibrin hydrogels. Acoustic manipulation was applied during the polymerization process, resulting in parallel bands of cells. The constructs were cultured under cardiac-supportive conditions and characterized using immunostaining for cardiovascular markers, Live/Dead assays for viability, and image-based analyses to monitor spontaneous activity and tissue organization.
Results:
Preliminary data show that acoustic cues reproducibly organize mixed cardiac and vascular populations into well-defined multicellular lanes with high cell viability. Early constructs exhibited clear sarcomeric organization and visible contractile activity, alongside emerging microvascular structures within the same matrix. Molecular, electrophysiological, and calcium-imaging analyses confirmed acoustic patterning influences cellular maturation and tissue functionality.
Conclusions:
Acoustic patterning offers a rapid, non-invasive, and biocompatible method to orchestrate cell positioning and morphogenesis in engineered human cardiac tissues. The established multicellular fibrin model represents a versatile foundation for developing vascularized cardiac platforms with controlled microscale organization for regenerative and translational applications.
Silk fibroin methacrylate–keratin composite hydrogels for acetabular labrum regeneration - SEMIT
Alessio Bucciarelli1, Giorgia Borciani2, Michiela Battistelli3, Eugenia Spessot1, Devid Maniglio1, Keita Ito4, Eleonora Olivotto2
1Department of Industrial Engineering. Univeristy of Trento, Trento (Italia) - Italy, 2RAMSES Lab. IRCCS Istituto Ortopedico Rizzoli, Bologna (Italia) - Italy, 3Department of Biomolecular Sciences. Università degli Studi di Urbino Carlo Bo, Urbino (Italia) - Italy, 4Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
The acetabular labrum is a fibrocartilaginous structure essential for hip joint stability, fluid sealing, and load distribution (1). Degeneration of the labrum, commonly associated with femoroacetabular impingement, leads to pain, reduced mobility, and accelerated progression toward osteoarthritis (2). Current reconstruction options rely mainly on autografts or allografts, which present limitations in availability, geometry, and mechanical compatibility. Therefore, biomimetic scaffolds capable of supporting labral tissue regeneration are critically needed.
In this work, we developed a composite hydrogel based on silk fibroin methacrylate (SilMA) blended with wool-derived keratin (KI) at two concentrations (12.5 and 25% w/w). Keratin was selected to introduce fibrous reinforcement and bioactive cell-adhesion domains. Hydrogels were photocrosslinked under UV light, and their physico-mechanical properties and cytocompatibility were assessed.
Rheological measurements of the uncrosslinked precursors revealed near-Newtonian flow behavior with increasing viscosity proportional to keratin content, indicating tunability for injectable or extrusion-based fabrication. Mechanical testing of crosslinked hydrogels demonstrated that keratin significantly enhanced compressive modulus under both dry and hydrated conditions, without inducing brittle behavior. Transmission Electron Microscopy showed a uniform dispersion of keratin domains within the silk matrix.
Biological evaluation using C28/I2 human chondrocytes confirmed that all hydrogel formulations supported cell viability up to 14 days, with decreasing cytotoxicity after initial encapsulation stress. Histology and ultrastructural analyses revealed early extracellular matrix deposition, including proteoglycan presence, particularly in keratin-enriched hydrogels.
These results demonstrate that SilMA–keratin composite hydrogels provide a mechanically reinforced, biocompatible platform capable of supporting matrix-producing chondrocyte behavior. The materials show promising characteristics for further development into labrum-shaped scaffolds via 3D bioprinting, with potential translation toward minimally invasive hip preservation interventions.
References
(1) Bsat S. et al, 2026, 10.1302/0301-620X.98B6.37099
(2) Leunig M. et al, 2003, 10.1097/01.blo.0000073341.50837.91
Scalable cartilage tissue engineering using extracellular matrix-enriched organoids derived from human-induced chondrocytes
Daphne M.a. Menssen1, Giorgia Mazzini2, Marina Van Doeselaar1, Abinzano Florencia1, Sebastien J.p. Callens1, Yolande F.m. Ramos2, Ingrid Meulenbelt2, Keita Ito1
1Orthopaedic Biomechanics, Dept. of Biomedical Engineering, and Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands, 2Molecular Epidemiology, Dept. of Biomedical Data Sciences. Leiden University Medical Center, Leiden (Zuid-Holland) - The Netherlands
Objective: Current cartilage repair strategies using autologous chondrocytes require invasive two-step procedures and are hindered by expansion-induced dedifferentiation and the formation of inferior fibrocartilage in the defects. Therefore, we aim to improve the scalability and efficiency of cartilage tissue regeneration by using a prospective off-the-shelf cell source: human-induced chondroprogenitor cells (hiCPCs). We investigated the scalable maturation of hiCPCs into human-induced chondrocytes (hiCHOs) and explored the production of large volumes of cartilage by creating hiCHO-derived organoids in spinner flasks (SFs) using notochordal cell-derived matrix (NCM) as a culture additive.
Methodology: hiCPC aggregates were derived from hiPSCs (LUMC0099iCTRL04) and matured in a SF with chondrogenic differentiation medium (CDM) (1,2). Subsequently, some hiCHO aggregates were dissociated and cultured with NCM in SFs to produce cartilage organoids (3,4) and others were kept as aggregates to further mature in CDM. Afterward, these hiCHO organoids and aggregates were each fused in a scaffold-free manner to form larger cartilage constructs. The chondrogenicity and quality of the tissues were assessed with biochemical assays, immunohistochemistry, qPCR, and mechanical testing.
Results: hiCPCs successfully matured into hiCHOs as indicated by the SOX9 expression and the production of glycosaminoglycans and type II collagen. Going from single hiCHOs to organoids increased the tissue volume thirteenfold. When using the organoids as building blocks, coherent fusion occurred within seven days, forming a larger cartilage construct. Ongoing analysis aims to confirm the efficiency of the organoids over the continuously matured aggregates and to reveal the quality of the tissue at consecutive stages.
Conclusions: Intermediate results suggest that the hiCPCs can be efficiently matured towards hiCHOs using SFs and that these hiCHOs are capable of forming large volumes of cartilage organoids. The scalability and fusion potential of these building blocks highlight their potential for off-the-shelf, scalable cartilage tissue regeneration.
1. DOI:10.1007/s00441-021-03498-5
2. DOI:10.1186/s13148-024-01759-y
3. DOI:10.1016/j.actbio.2021.04.008
4. DOI:10.1177/19476035241313179
Biomimetic graphene oxide/PLGA scaffolds for Achilles regeneration: a platform to evaluate biocompatibility and function across species
Emine Berfu Ozmen1, Steven Newby1, Eli Christoph1, David Anderson1, Dustin Crouch2, Madhu Dhar1
1Tissue Engineering and Regenerative Medicine, Large Animal Clinical Sciences, College of Veterinary Medicine. The University of Tennessee, Knoxville (Tennessee) - United States, 2Department of Mechanical, Aerospace, and Biomedical Engineering, Tickle College of Engineering. The University of Tennessee, Knoxville (Tennessee) - United States
Achilles tendon (AT) injuries are common and challenging to treat due to their limited regenerative capacity. Existing interventions, like graft implantation, often lead to reinjury and incomplete functional recovery. As an interdisciplinary team of cell biologists, engineers, and surgeons, we aim to develop novel cytocompatible, biocompatible, and efficacious constructs to treat AT injuries. We hypothesized that biomimetic nanocomposite scaffolds can be adapted across species for Achilles tissue engineering and have the potential to be translated into clinical applications. We used extrusion-based 3D printing to develop graphene oxide (GO)-PLGA nanocomposites for evaluation in rat and rabbit models. Scaffolds were tested for their mechanical properties and combined with DiI-labeled mesenchymal stem cells (MSCs). Cytocompatibility was assessed in vitro, and groups of animals were treated with empty scaffolds and scaffolds with MSCs to assess biocompatibility and functionality in vivo. SEM images showed the characteristic surface topography with grooves suggestive of cell attachment sites. Tensile stress/strain showed the extension of the material, demonstrating elasticity with the addition of a plasticizer. Immunofluorescence and SEM revealed cell attachment, migration, and proliferation, confirming the cytocompatibility of the scaffolds. A rat model of AT defect confirmed the biocompatibility and demonstrated tissue repair. DiI-labeled cells were observed at the implantation site, and the nanocomposites were integrated into the host tissue, together with the expression of tenomodulin and Mohawk homeobox proteins. Subsequently, in rabbit scaffolds, the addition of a photo-initiator and aligned fiber design increased rigidity while maintaining elasticity. Cell alignment was observed, and significant morphological changes were quantified. Kinematics, biomechanics, and micro-CT scans of PTA-stained tissues confirmed functionality and tissue repair. Both species showed no adverse reactions with tissue integrity and biocompatibility. Altogether, we demonstrated that PLGA-GO-based scaffolds with modified composition and design, aligned with translational principles, can be advanced across species to support translational research in AT regeneration.
Development of nanoliposome-modified GelMA colloidal inks for 3D scaffold printing
Elaheh Omidvari1, Mohamadmahdi Samandari2, Delaram Ghanbariamin3, Evelyn Mollocana Lara3, Jacob Quint3, Farnoosh Saeedinejad3, Cyril Kahn1, Ali Tamayol3, Elmira Arab-Tehrany1
1University of Lorraine, Nancy (Lorraine) - France, 2Old Dominion University, nortfolk (Virginia) - United States, 3University of Connecticut, farmington (Connecticut) - United States
Bioprinting enables the fabrication of complex scaffolds that replicate the physical, chemical, and structural properties of native tissues. Porous scaffolds are particularly advantageous due to their enhanced diffusion of nutrients, oxygen, and waste. However, conventional methods for generating porosity require multiple processing steps, making them incompatible with bioprinting. To address this, our team developed a foaming technique for hydrogel precursors (Mostafavi et al., 2021) to create multi-scale porous structures, which were further functionalized with nanoliposomes (NL) to improve mechanical, physical, and biological properties before being processed into colloidal bioinks. We investigated the effects of varying NL concentrations on bioinks formulated with three different gelatin methacryloyl (GelMA) concentrations and characterized the resulting scaffolds.
Incorporation of NL allowed precise tuning of macropore diameters within the foam bioink—a highly desirable feature. At 10% GelMA, the addition of NL significantly enhanced mechanical performance. Notably, even at a 5% NL concentration, foam-embedded NL exhibited excellent compatibility with C2C12 cells, supporting their proliferation and differentiation without adverse effects. Moreover, the nanofunctionalized foam bioinks, composed entirely of natural materials, demonstrated substantial antioxidant activity and could be bioprinted in various geometries and dimensions.
The growing preference for natural compounds with antioxidant properties, driven by concerns over synthetic materials, highlights the relevance of this approach. Overall, our findings suggest that nanofunctionalized foam bioinks represent a promising new class of materials for 3D bioprinting, offering tunable structure, bioactivity, and oxidative balance to support tissue regeneration and biomedical applications.
Reference
Mostafavi, A., Samandari, M., Karvar, M., Ghovvati, M., Endo, Y., Sinha, I., Annabi, N., & Tamayol, A. (2021). Colloidal multiscale porous adhesive (bio)inks facilitate scaffold integration. Applied Physics Reviews, 8(4).
Acknowledgment: The authors thank the french ministry of higher education, research and innovation the LUE for funding Elaheh Omidvari.
Innovative decellularized extracellular matrix biomaterials for corneal regenerative medicine applications
Carolina Di Varsavia1, Grazia Maugeri2, Dalila Di Francesco3, Francesca Boccafoschi1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Biomedical and Biotechnological Sciences. University of Catania, Catania (Italia) - Italy, 3Tissuegraft Srl, Alessandria (Piemonte) - Italy
Corneal diseases represent a leading cause of visual impairment. Given the limitations of current therapeutic approaches, regenerative strategies based on decellularized extracellular matrix (dECM) have gained attention as a possible solution 1. dECM-derived matrix-bound nanovesicles (MBVs) were recently discovered showing a potent regenerative and immunomodulatory potential. MBVs are a subtype of extracellular vesicles, found entrapped in extracellular matrix, nanosized and presenting a tissue-specific bioactive cargo 2.
The present study aims to investigate the pro-regenerative effects of an hydrogel derived from decellularized bovine pericardium (dBP), a well-known regenerative biomaterial, and derived MBVs for corneal regeneration 3.
MBVs were isolated from dBP and characterized using nanoparticle tracking analysis and transmission electron microsocpy. Primary human corneal epithelial cells (HCECs) were useed. MBVs cytocompatibility was evaluated by cell viability assay, cellular uptake of MBVs and fluorescence staining. MBVs ability to maintain corneal epithelial barrier morphology was investigated in an air-liquid interface culture of HCECs. Moreover, its effect in cell migration was characterized. Corneal functionality was assessed using western blots cytokeratins 3 and 12 (CK3, CK12) expression. Metalloproteinases expression (MMPs) under basal and TNF-α conditions was evaluated by zymography.
dBP hydrogel and MBVs showed a good cytocompatibility and ability to enhance cell viability and preserve the morphology of corneal barrier. Scratch assays showed that MBVs promote wound closure. Moreover, CK3 and CK12 expression was significantly upregulated at lower MBV concentrations, supporting corneal epithelial differentiation. Under TNF-α challenge, MMP-9 expression remained stable across MBV treatments, suggesting an anti-inflammatory MBVs driven response.
In conclusion, dBP biomaterials are suitable for corneal regeneration. Their capacity to maintain epithelial integrity while modulating inflammation underscores their potential as next generation biotherapeutics for corneal regeneration.
1. Wu et al; 2024
2. Di Francesco et al; 2024
3. Di Francesco et al; 2025
Acknowledgements
Materials for this study were kindly provided by Tissuegraft.
Cardiac regenerative potential of decellularized bovine pericardium ECM hydrogel enriched with a novel small bioactive laminin-derived peptide
Simona Casarella1, Sasan Ejlalmaneshan1, Parnian Pezeshkpour1, Irene Bluma1, Geo Paul2, Dalila Di Francesco3, Leonardo Marchese2, Francesca Boccafoschi1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Science and Technological Innovation. University of Eastern Piedmont, Alessandria (Piemonte) - Italy, 3Tissuegraft s.r.l, Alessandria (Piemonte) - Italy
Acellular hydrogels have shown potential in cardiac repair by providing mechanical support. Our previous studies have characterized and demonstrated the regenerative potential of a decellularized bovine pericardium hydrogel (dBP), specifically in promoting angiogenesis and myogenesis. Furthermore, we have shown that a novel laminin-derived peptide KKGSYNNIVVHV (G2), which selectively binds adult cardiac-specific integrin α7β1, enhances the cardiac differentiation of human mesenchymal stem cells and modulates the contractile function of neonatal mouse cardiomyocytes. Therefore, this project aims to evaluate the cardiomyogenic regenerative potential of a dBP hydrogel (kindly provided by TissueGraft s.r.l.) enriched with G2 peptide (G2-dBP). Specifically, we characterized the G2-dBP in terms of degradation and chemical composition through ss-/ls-NMR to confirm the presence of the peptide, its stability and degradation rate, and its release kinetic. The in vitro potential of the G2-dBP has been evaluated using 3D spheroids with the H9C2 cardiomyoblasts. Cell viability, morphology and the differentiative potential of the G2-dBP have been tested, analyzing different cardiac markers such as GATA-4 and MEF2c through qRT-PCR. Results showed great cytocompatibility and, when in presence of G2, H9C2 presented cell clustering, spontaneous myotubes formation and enhanced cardiac markers expression in respect of the dBP alone (CTR), even in normal maintaining medium (DMEM 10% FBS) (p<0,05), confirming the differentiative potential. Moreover, we demonstrated the regenerative potential of G2-dBP by inducing inflammation of H9C2 spheroids with H2O2 (100 µM). Results showed a protective effect of G2-dBP by reducing the inflammatory cytokines and improving viability of cells (p<0,05). Taken together, these findings suggest that G2-dBP supports in vitro cardiac regeneration. Further experiments need to be done, such as testing in an in vivo myocardial infarction mouse model, in order to evaluate the in vivo potential. Combining natural hydrogels with active peptides may further improve cellular response by modulating cellular crosstalk and ECM remodeling.
Decellularized bovine pericardium-derived hydrogel (dBP) for traumatic brain injury (TBI) repair
Irene Regano1, Kiarah Leonard2, Dalila Di Francesco3, Federico Sesti2, Francesca Boccafoschi1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School. Rutgers University, Piscataway (New Jersey) - United States, 3Tissuegraft Srl, Alessandria (Piemonte) - Italy
Traumatic Brain Injury (TBI) is a major cause of long-term neurological disability, characterized by primary neuronal loss followed by inflammation, oxidative stress and glial scar formation, which reduce regeneration (1). Due to the limited repair capacity of the central nervous system, current therapies mainly attenuate symptoms without restoring tissue function. Hydrogels derived from decellularized extracellular matrix (dECM) provide biomimetic 3D environments that may support neural repair (2). In this study, a decellularized bovine pericardium-derived hydrogel (dBP) (kindly provided by Tissuegraft Srl, Italy) was evaluated as a potential regenerative approach for TBI.
A mouse model of TBI was established using Controlled Cortical Impact (CCI). After injury, dBP injection was performed at the lesion site. Post-surgery, animals underwent behavioral testing to assess cognitive recovery. After 7, 14 and 21 days, brain tissues were harvested for histological analyses. Neurodegeneration, astrocytes-related inflammation and apoptosis were evaluated by Western blot/densitometry and immunofluorescence. Finally, Blood-Brain Barrier (BBB) integrity was assessed by Evans Blue staining.
dBP-treated mice showed improved behavioral performance compared to sham TBI controls in the Rotarod and Barnes maze assays. Western blot and immunofluorescence analyses demonstrated a reduction in inflammatory markers (Iba1, COX-2) and astrocytic reactivity (GFAP) in the dBP group. Neurodegeneration and apoptosis were also reduced, as indicated by lower Fluoro-Jade C and cleaved Caspase-3 expression. Moreover, Evans Blue staining revealed better preservation of BBB integrity in dBP-treated mice, with reduced vascular permeability.
Overall, dBP hydrogel attenuated secondary injury mechanisms, reduced inflammation, and preserved BBB function, supporting its potential as a regenerative strategy for TBI treatment.
References
1. Gao Y. et al., 2024
2. Politrón-Zepeda G. A. et al., 2024
Immunomodulation and mechanical characterization of manuka honey incorporated near-field electrospun bioresorbable vascular grafts
Alexandra Snyder1, Evan Main1, Gary Bowlin1
1Department of Biomedical Engineering. University of Memphis, Memphis (Tennessee) - United States
Treatment of end-stage cardiovascular disease aims to restore blood flow to ischemic tissues via bypass grafting. Current synthetic grafts fail frequently due to anastomotic hyperplasia and thrombosis brought by mechanical mismatch and incomplete reendothelialization of the luminal surface. Polydioxanone near field electrospun (NFES) vascular templates feature programmable pore sizes to facilitate transmural ingrowth of endothelial cells and have demonstrated promise in mitigating mechanical mismatch but have yet to be investigated as an anti-inflammatory drug delivery system. It was hypothesized that incorporation of Manuka honey into vascular templates would reduce neutrophil extracellular trap (NET) release within a therapeutic window but would also cause a significant decrease in mechanical strength. NFES vascular templates were fabricated using 90 mg/mL of polydioxanone in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) with Manuka honey (ManukaGuard MGO 400) concentrations of 0%, 0.1%, 1%, and 10% v/v. Wall thickness, mechanical properties, Manuka honey elution, and in vitro NET release were quantified. The wall thickness of the templates ranged from 197 to 236 nm. Mechanical testing revealed that 1% and 0.1% Manuka honey templates demonstrated mechanical properties most closely mimicking native vessels than the other templates, outperforming the pure HFP control in ultimate tensile strength and burst pressure. The 10% templates exhibited significantly reduced tensile strength, Young’s modulus, suture retention, and burst pressure. However, none of the templates fully met the native vessel burst pressure. Elution study results demonstrated a burst of Manuka honey elution at 1 hour with very little increase in eluted Manuka honey at the following time points for all experimental groups. The 10% and pure HFP templates exhibited significantly more NET release than the unstimulated control but were not significantly different from the 1% and 0.1% templates. These results suggest low-dose Manuka honey incorporation maintains mechanical compatibility for grafting applications and prevents a significant increase in NET release.
Replicating skeletal muscle contraction dynamics in two-photon 3D-printed synthetic muscle fibers
Theresa Kühn1, André Tomalka2, Tobias Siebert2, Michael Heymann1
1Institute of Biomaterials and Biomolecular Systems. University of Stuttgart, Stuttgart (Baden-Wberg Bayern) - Germany, 2Institute of Motion and Exercise Science. University of Stuttgart, Stuttgart (Baden-Wberg Bayern) - Germany
Contractile force generation dynamics in native muscle tissues have been stringently optimized by evolution. Engineering muscles that exhibit such contractile dynamics are highly sought after for diverse applications, including actuators and in vitro modeling. Two-photon stereolithography can fabricate bio-scaffolds with micrometer resolution, ideally replicating micro-tissues in vitro.
Herein, we present a synthetic muscle made from bovine serum albumin that can be 3D-printed via two-photon stereolitography to realize 1 mm long contractile fibers. Using a custom microscale tensile strength device we show that pH-dependent contractions follow parabolic force-length relationships similar to biological muscles at a constant temperature of 20 ± 0.1°C. Achieved stress outputs of 0.78 ± 0.13 N/cm2 were comparable to smooth and cardiac muscle. Stretch-shortening work loops performed under different strain rates in turn revealed a viscoelastic behavior and significant velocity dependence of work and power output, more similar to skeletal muscle. Ultimately, two-photon cross-linked synthetic fibers can hence replicate contractile dynamics of skeletal muscle tissue.
Further, we seek to realize physiologically relevant muscle fibers in vitro, in which myocytes align and mature to exert contractile forces. For this, we induced topological defects in C2C12 precursor myocyte monolayers using two-photon 3D-printed confinements to identify minimal required geometries. We found a significant connection between confinement and tissue organization.
Our work offers avenues to further explore contractile synthetic materials. Further optimization should refine high-resolution 3D-printed fibers to incorporate functional geometries that promote myocyte maturation to achieve physiologically relevant synthetic muscle fibers in vitro.
A biofabricated human skin model for in vitro wound healing assays
Cintia D. S. Horinouchi1, Larissa C. M. Oliveira1, Samarah V. Harb1, Monielle S. A. Leal1, Julia C. M. Velho1, Ana Carolina M. Figueira1
1Brazilian Biosciences National Laboratory (LNBio). Brazilian Center for Research in Energy and Materials, Campinas (Sao Paulo) - Brazil
Chronic wounds of the skin represent a significant clinical problem, characterized by delayed repair and frequent complications such as infection. The underlying pathophysiology is multifactorial and still not completely elucidated, while available therapies remain largely ineffective. Progress in this field is further limited by the absence of advanced, human-relevant experimental models. Conventional in vitro systems fail to reproduce the complex architecture and dynamic repair processes of human skin, and animal models often show poor translational relevance. Therefore, developing reliable and physiologically representative models is essential to better investigate the mechanisms of wound healing under controlled conditions. The aim of this study was to establish a non-clinical in vitro human model of excisional skin wounds using a biofabricated full-thickness human skin equivalent with hypodermis (HSEH). The HSEH was engineered with three layers: a hypodermis composed of human mesenchymal stem cell–derived adipocytes embedded in type I collagen, a dermal layer formed by a collagen-based hydrogel containing human fibroblasts, and an epidermal layer consisting of stratified human keratinocytes. A standardized excisional lesion of 2 mm was induced using a punch biopsy needle. The wound gap was filled with a type I collagen–based hydrogel, which served as a scaffold to support the healing process. Healing responses were assessed by histology and gene expression analysis (qPCR) for markers including COL1A1, MMP2, INV, and TGM1. An excisional wound could be successfully performed and monitored over time, allowing the assessment of wound closure after maintenance in culture. The model exhibited cell infiltration, matrix remodeling, and gene expression profiles consistent with wound healing, demonstrating its biological responsiveness and regenerative potential. The HSEH constitutes a promising in vitro platform for modeling human skin wound healing, recapitulating key structural and functional attributes of native tissue and exhibiting dynamic responses to injury. It addresses a major gap in the field by providing a predictive, human-relevant system for regenerative medicine studies.
Metabolic reprograming via lipid priming improves the regenerative potential of human adipose mesenchymal stromal cells secretome in translational studies of spinal cord injury
Jonas Campos1, Bélem Sampaio-Marques1, João Afonso1, Ping Yip2, Marta Lima1, Alice Miranda1, Jorge Cibrão1, Sara Rito-Fernandes1, Filipa Antunes1, Susana Monteiro1, Andréia Monteiro1, Maria Moura1, Diogo Santos1, Alexandra Teixeira3, Sofia Baptista1, Sofia Serra1, Nuno Silva1, Adina Michael-Titus2, António Salgado1
1Life and Health Science Research Institute - ICVS. University of Minho, Braga - Portugal, 2Queen Mary University of London. Blizard Institute, London (London, City of) - United Kingdom, 3International Iberian Nanotechnology Laboratory, Braga - Portugal
Priming studies of mesenchymal stromal cells (MSCs) often evaluate cytokine exposure to modulate their disease-modifying effects. While these approaches have generated benefits in inflammatory settings, strategies that also account for the intrinsic biology of the MSC tissue source and the specific pathophysiology of the target disease remain limited. We hypothesized that priming human adipose-derived MSCs (hASCs) with docosahexaenoic acid (DHA) would enhance their functional properties and improve their regenerative potential in spinal cord injury (SCI), based on the expression of DHA receptors in hASCs and the established role of DHA in adipose tissue physiology. Using dose-escalation assays, metabolic viability readouts, temporally resolved morphological analysis, and free-fatty acid receptor-4 (FFAR4) engagement, we identified an optimal priming condition of 40 µM DHA for 72 h. Transcriptomic and metabolic profiling revealed enriched pathways linking metabolic sensing with increased biosynthetic capacity. These transcriptional responses were associated with enhanced glycolytic and mitochondrial activity, culminating in higher concentrations of proteins and extracellular vesicles in the secretome. Unbiased LC/MS proteomics further aligned transcriptional shifts with an increase in antioxidative and neurotrophic proteins. Functionally, DHA-primed secretome protected spinal cord cells and mitigated astrogliosis and microglial reactivity by reducing cellular proliferation after hyperosmotic stress in vitro. In a thoracic compression SCI model, DHA-primed secretome reduced astrocyte and microglial reactivity in grey matter and degenerating corticospinal tracts, indicating modulation of tissue injury responses. Although motor recovery was similar to standard secretome treatment, DHA-primed secretome differentially improved sensory and spasticity-related behavioral deficits, correlating with reduced aberrant peptidergic and non-peptidergic peripheral fiber innervation in the dorsal horn. Overall, this work introduces a biologically informed priming strategy that enhances hASC metabolic function and yields a secretome with improved therapeutic potential for SCI.
Immune profile of equine mesenchymal stem/stromal cells (MSCs): induced vs primary cells
Ma Belén Serrano Pastor1, Elvira Bernad Roche1, Arantza Vitoria Moraiz2, Antonio Romero Lasheras2, Francisco José Vázquez Bringas2, Sara Fuente Franco2, Pilar Zaragoza Fernández1, Clementina Rodellar Penella1, Laura Barrachina Porcar2, Alina Cequier Soler2
1Laboratorio de Genética Bioquímica LAGENBIO; Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-CITA); Instituto de Investigación Sanitaria de Aragón (IIS). University of Zaragoza, Zaragoza - Spain, 2Laboratorio de Genética Bioquímica LAGENBIO; Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-CITA); Instituto de Investigación Sanitaria de Aragón (IIS); Servicio de Cirugía y Medicina Equina, Hospital Veterinario. University of Zaragoza, Zaragoza - Spain
Primary mesenchymal stem/stromal cells (MSCs) are an interesting therapy in horses, but enter senescence during in vitro expansion, making it necessary to repeat invasive tissue harvestings, which also increases product heterogeneity. Deriving MSCs from induced pluripotent stem cells (iPSCs), referred to as iMSCs, could be a promising alternative. However, because iMSCs are derived from reprogrammed iPSCs, these cells may exhibit altered gene expression affecting their function and/or recognition by the immune system. However, little is known about the immunomodulatory profile of equine iMSCs. This study compared the immune profile of equine iMSCs and primary bone marrow-derived MSCs (BM-MSCs) by studying the gene expression and secretion of immunomodulatory molecules and immunogenic markers before and after exposing the iMSC/BM-MSC to activated allogeneic lymphocytes.
Equine iMSCs (n=3) and BM-MSCs (n=3) were co-cultured with mitogen-activated allogeneic lymphocytes (n=4). Before and after co-culture, the expression of immunomodulatory (VCAM1, IL6, COX2, iNOS, IDO) and immunogenic (CD40, CD80, MHC-I, MHC-II) genes was assessed by RT-qPCR in iMSCs/BM-MSCs, and the secretion of IL6 and PGE2 was measured by ELISA.
Lymphocyte exposure changed the immune gene expression of both equine iMSCs and BM-MSCs. BM-MSCs showed a higher basal immunomodulatory profile, while iMSCs showed a greater induction of immunomodulatory genes and secreted molecules after co-culture. Both MSC types presented low basal immunogenic profiles, but some immunogenic markers increased in iMSCs after the co-culture.
In conclusion, equine iMSCs poses a lower immunomodulatory profile in basal conditions but are more strongly activated upon interaction with reactive lymphocytes, which may have implications for their therapeutic effectiveness. Regarding the immunogenic profile, changes observed in iMSCs after their interaction with immune cells warrants further investigation to ensure that they could be safely administered. In short, further analysis and in vivo studies are necessary to determine whether iMSCs offer therapeutic advantages over primary MSCs in horses.
Harvesting immuno-mechanical interplay to boost regeneration
Duda Georg
de Julius Wolff Institute and BCRT, BIH at Charité. Charite Universitatsmedizin, Berlin - Germany
Tissue regeneration seems to be tightly intermingled with inflammation in the tissues undergoing healing. The patient’s immune competence defines the systemic environment within which the healing happens. Apparently, innate and adaptive immunity components are involved from the moment an injury occurs. Their interplay over time differentiate between success of regeneration or failure, eventually such as scarring. Using bone healing as a model system, we analyze the driving components determining the success of healing and scarfree regeneration. Bone is an organ positioned at the interface between immune system and mechanical constrains and thus regeneration of bones requires a tight interplay between mechanical conditions and local immune responses to injury. Hereby mechanical conditions affect the presense, activity and fate of stroma as well as immune cells and eventually differentiate the timely appearance and the niches in which healing may progress.
Comparing healing in both mechanically challenging vs less challenging conditions allows to identify distinct differences on their immuno-mechanical interplay and eventually carve out what determines an immune-mechanical niche that supports healing over one that drives towards scarring.
Optimising the mechanical conditions at a fracture side is one opportunity to enable healing in otherwise compromised settings. But sometimes this may not be possible. Biomaterial niches targeting to optimize this immuno-mechanical interplay are a novel option to enable healing also in cases where regeneration would otherwise not be possible or delayed.
Using pre-clinical model systems and clinical trial data examples of this immuno-mechanical interplay will be presented and opportunities of targeting a optimization by means of local immune modulation discussed.
Is knee joint distraction (KJD) effective in the setting of posttraumatic osteoarthritis?
Sabine Roth1, Max Praster1, Rald Groven1, Deniz Oezman1, Ulf Hofmann1, Nadav Goldstein1, Frank Hildebrand1
19Christian Weber1, 1Orthopaedics, Trauma and Reconstructive Surgery. RWTH Aachen University, Aachen (Nordrhine-Westphalia) - Germany.
Background: Posttraumatic knee osteoarthritis (PTOA) remains a challenging condition to treat, especially in younger and active individuals for whom joint replacement is undesirable. Knee joint distraction (KJD) has emerged as a joint-preserving technique aimed at promoting intrinsic cartilage repair and modifying disease progression.
Objective: This study reviews the biological and mechanical principles of KJD and evaluates its regenerative effects in posttraumatic knee osteoarthritis, supplemented by a clinical case illustrating its therapeutic potential.
Methods: Knee joint distraction involves temporary mechanical unloading of the tibiofemoral joint using an external fixation frame that maintains a controlled joint space (typically 5–7 mm) for 6–8 weeks. This controlled environment enables intermittent intra-articular fluid pressure changes during motion, stimulating chondrocyte metabolism, subchondral bone remodeling, and cartilage matrix synthesis. In addition to a literature review of recent clinical and imaging studies, we present the case of a 33-year-old female patient with posttraumatic knee osteoarthritis who underwent KJD following failure of conservative management.
Results: Published studies demonstrate that KJD provides significant pain reduction and functional improvement, comparable to or exceeding outcomes from high tibial osteotomy and microfracture at mid-term follow-up. Quantitative MRI and radiographic analyses show increased cartilage thickness and reduced subchondral bone sclerosis, reflecting genuine tissue regeneration. In our clinical case, the patient exhibited substantial symptomatic relief and functional recovery, accompanied by radiological signs of cartilage restoration at two-year follow-up.
Conclusion: Knee joint distraction represents a promising regenerative treatment option for posttraumatic knee osteoarthritis, capable of promoting structural cartilage repair and delaying arthroplasty in younger patients. The presented case supports the growing evidence for KJD as a viable biological joint-preserving intervention.
Keywords: knee joint distraction, posttraumatic osteoarthritis, cartilage regeneration, joint preservation, biomechanics, case report.
Jansen MP, Mastbergen SC. Joint distraction for osteoarthritis: clinical evidence and molecular mechanisms. Nat Rev Rheumatol. 2022
National Institute for Health and Care Excellence. Joint distraction for knee osteoarthritis without alignment correction (IPG529). 2024.
Decellularized plant scaffolds as 3R-aligned platforms for building a physiologically relevant and functional three-dimensional (3D) human intestine model
Didem P. Esmer1, Hilal Özdemir2, Merve Ünal3, Şükrü Güleç2, Serkan Dikici1
1Department of Bioengineering. İzmir Institute of Technology, İzmir (Izmir) - Turkey, 2Molecular Nutrition and Human Physiology Laboratory, Department of Food Engineering. İzmir Institute of Technology, İzmir (Izmir) - Turkey, 3Department of Biotechnology. İzmir Institute of Technology, İzmir (Izmir) - Turkey
The small intestine is a highly specialized organ responsible for nutrient absorption and secretion, featuring a polarized epithelial structure and complex cell-matrix interactions. Although in vivo animal models can closely mimic native tissue functions, ethical concerns and high costs highlight the need for reliable in vitro alternatives. However, conventional in vitro systems lack three-dimensional (3D) microenvironment required to recapitulate physiological relevance.
In this study, we aimed to develop a 3D human intestine model using decellularized plant scaffolds derived from parsley stems and spinach leaves, providing an ethical, sustainable, and 3R-aligned (Replacement, Reduction, and Refinement) alternative platform. After confirming the model's physiological relevance, the plant-derived gut model was further employed to investigate iron deficiency anemia.
Following the physical, chemical, and mechanical characterizations, cytocompatibility studies confirmed that the scaffolds were non-toxic and supported human colorectal adenocarcinoma cells (Caco-2) to attach, proliferate, and from a continuous epithelial monolayer. Our preliminary gene expression analyses under iron-deficient conditions further demonstrated functional cellular responses resembling native intestinal physiology.
Overall, this work presents a physiologically relevant, plant-derived intestine model that provides a promising alternative to animal-based research. The proposed 3R-aligned platform promotes the development of ethical, cost-effective, and sustainable in vitro models for investigating intestinal biology, nutrient absorption, and disease-related mechanisms, therefore advancing physiologically relevant in vitro systems for tissue engineering and drug discovery.
This project was financially supported by the Research Universities Support Program of Türkiye (ADEP) (2022IYTE-3-0002). The authors also acknowledge İzmir Institute of Technology, Integrated Research Centers (IzTech IRC) for their support. We thank to Betül Aldemir Dikici for her guidance on biological characterizations and are grateful to the DikiciLab and BaldemirLab group members for their valuable contributions.
Automated biofabrication of multicellular tendon in vitro models by embedded coaxial 3D bioprinting
Rosa F. Monteiro1 2 3, Simão P. B. Teixeira1, Meftune Ö. Öztürk-Öncel2 3 4, Syeda M. Bakht2 3, Manuel Gomez-Florit5, Manuela E. Gomes6, Rui M. A. Domingues1
1INL – International Iberian Nanotechnology Laboratory, Braga, Portugal, 23B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco – Guimarães, Portugal, 3ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal, 4Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, Turkey, 5IdISBa - Health Research Institute of the Balearic Islands, Palma, Spain, 6ICBAS - School of Medicine and Biomedical Sciences, University of Porto, Porto, Portugal
Representative multicellular in vitro models allowing the study of tendon (patho)physiology are highly needed to develop more effective treatments for tendinopathy, a complex and poorly understood disease for which current treatments fail in restoring native tissue functionality. Such models would not only accelerate further understanding on tendon homeostasis and degeneration mechanisms but also pave the way for the design of in vitro screening platforms for testing innovative treatment strategies. Here, we combine embedded bioprinting with coaxial printheads for the 3D writing of tendon-like fascicles within a cellulose nanocrystals (CNCs) fluid gel support. While CNCs support bath enables high resolution bioprinting and self-assembles into a tailor-made bioreactor for long-term in vitro culture[1], coaxial bioprinting allows the extrusion of single filaments with layer-specific bioinks to recreate tendon fascicle organization. Human derived tendon cells (hTDCs) and endothelial cells were used in core and shell bioinks, respectively, aiming to reconstruct the cellular patterns at the interface of the intrinsic (stroma) and extrinsic (e.g. vascular system) fascicle compartments. This approach allows the fast fabrication of multicellular models embedded within its own CNCs support for its in vitro maturation. hTDCs align and elongate along the fiber core over culture time, resembling tenocyte morphology, while, in the shell, endothelial cells reorganize into a vessel-like morphology, surrounding the tendon core compartment. Gene and protein expression analysis revealed that the long-term co-culture with vascular compartment suppresses the intrinsic pro-inflammatory and fibrotic signature of tendon core cell in monoculture. These results further indicate that hTDCs switch from a distressed state to a more quiescent and homeostatic state when co-cultured with endothelial cells, favoring the maintenance of a healthy tenogenic phenotype. Altogether, this strategy enables the rapid and reproducible 3D biomanufacturing of humanized tendon in vitro models that may be used for testing innovative tendinopathy therapies in the future.
Acknowledgements: EU Horizon 2020 and EU Horizon Europe for ERC grants No. 772817 and 101171765; FCT/MCTES for DOI:10.54499/2022.05526.PTDC, PD/BD/129403/2017, and 2023.01198.BD.
[1] R. F. Monteiro, et al. ACS Applied Materials & Interfaces 2023 15 (44), 50598-50611.
Beyond conventional repair: engineering personalized urethral substitutes
Elissa Elia1, David Brownell2, Stéphane Chabaud3, Julie Fradette2, Stéphane Bolduc2
1Department of surgery, Faculty of medicine, Université Laval, Québec, Qc, Canada. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 2Department of surgery, Faculty of medicine, Université Laval, Québec, Qc, Canada. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 3Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada
Aim & Objectives:
Urethral anomalies often require surgical reconstruction. When the foreskin is unavailable, buccal mucosa remains the gold standard, but its limited availability and donor-site morbidity highlight the need for new solutions. Despite extensive exploration of biomaterial-based tissue-engineered substitutes, most remain limited by poor tissue specificity and insufficient mechanical performance. Our team previously developed a self-assembled urethral substitute using bladder and dermal cells without exogenous biomaterials; however, this model required multiple biopsies and displayed suboptimal strength.
In this study, we aimed to design a fully autologous and organ-specific urethral substitute, hypothesizing that constructs made exclusively from urethral fibroblasts (UF) and urethral urothelial cells (UUC) could provide superior biological relevance, mechanical robustness, and simplified reconstruction.
Methods:
UF and UUC were isolated from human urethral biopsies (n=8). Using the self-assembly technique, UF were cultured in vitro in the presence of ascorbic acid to generate sheets that were subsequently stacked together to form a stromal layer, on which UUC were seeded. After air–liquid interface culture for epithelial maturation, the tissues underwent histology (Masson’s trichrome stain), immunofluorescence, scanning electron microscopy (SEM), permeability, and mechanical testing.
Results:
The UF-UUC constructs closely replicated the native urethral structure, displaying a well-differentiated pseudostratified columnar urothelium. Immunofluorescence confirmed urothelial marker expression, and SEM revealed superficial umbrella cells with uroplakin-positive apical coverage. The low permeability demonstrated functional epithelial integrity, while mechanical testing showed clinically relevant mechanical properties and confirmed surgical-grade strength and handling.
Conclusion & Perspectives:
We report, for the first time, the successful reconstruction of a fully autologous, organ-specific human urethral substitute generated without any exogenous biomaterial. This novel and unique model represents a major advancement in urethral tissue engineering, with direct translational potential for clinical urethroplasty. Preclinical in vivo evaluation in animal models is currently underway to pave the way toward future clinical application.
Engineered skeletal muscles for robotic and biomedical applications
Leonardo Ricotti
de Scuola Superiore Sant'Anna, Pisa (Toscana) - Italy
Skeletal muscle tissue engineering is a fascinating and interdisciplinary field that integrates bioinstructive materials, biofabrication technologies, stem cell biology, and biophysical stimulation to promote tissue maturation.
This talk will focus on the research conducted at the Regenerative Technologies Lab of Scuola Superiore Sant’Anna, in which skeletal muscle tissue engineering is pursued with a twofold goal.
On one hand, efforts focus on developing biohybrid soft machines that integrate artificial components with living tissues to exploit the unique properties of biological systems, particularly muscles, refined through millions of years of evolution [1]. Biohybrid technologies hold great promise for soft robotics, enabling systems that are efficient, silent, scalable, biodegradable, and potentially self-healing. In this context, the talk will describe different approaches, including ultra-thin films [2] and flexural mechanisms for biohybrid intravascular devices [3].
On the other hand, efforts are directed toward tissue-engineered muscle constructs for biomedical applications, particularly for creating regenerative peripheral nerve interfaces. By promoting the integration of engineered muscle grafts with peripheral nerves, this strategy supports advanced neural control of robotic prostheses. Preclinical studies will be presented in which bioinstructive hydrogels are combined with ultrasound stimulation and piezoelectric nanomaterials. Acting as intracellular nanotransducers, piezoelectric nanoparticles deform under ultrasound exposure, producing localized stimuli that activate specific intracellular pathways and modulate cell behavior [4,5]. This approach enhances muscle maturation and neural integration, demonstrating its potential in vivo.
[1] Ricotti L. et al. Science Robotics. 2(12): eaaq0495 (2017)
[2] Hasebe A. et al. ACS Biomater. Sci. Eng. 5(11): 5734-5743 (2019)
[3] Bartolucci A. et al. Adv. Intell. Syst. 2400989 (2025)
[4] Cafarelli A. et al. ACS Nano. 15(7), 11066-11086 (2021)
[5] Vannozzi L. et al. Small Science. 5(4): 2400439 (2025)
Biofabrication of the osteochondral unit through biphasic layering of wild-type and RUNX2 KO primary human MSCs
Simone Ponta1, Anna Puiggali-Jou1, Siyi Chen1, Martin J. Stoddart2, Goncalo Barreto3, Marcy Zenobi-Wong1
1ETH Zurich, Zurich - Switzerland, 2AO Research Institute Davos, Davos (Graubunden) - Switzerland, 3University of Helsinki, Helsinki (Southern Finland) - Finland
The osteochondral unit, composed of hyaline cartilage and subchondral bone, provides structural and load-bearing support. Osteochondral defects cause joint instability and lead to osteoarthritic degenerative changes, yet current surgical treatments cannot fully restore the native tissue. Tissue engineering of the osteochondral unit using mesenchymal stem cells (MSCs) represents a promising tool for regenerative approaches, however, MSCs committed to the cartilaginous phenotype undergo instead endochondral ossification, hindering the formation of distinct cartilage and bone layers. Furthermore, the in vitro maturation of biphasic osteochondral constructs is achieved using transwell systems and multiple culturing media, or separate maturation processes followed by suturing. Here we propose a novel strategy for the engineering the osteochondral unit by using wild-type (WT) and runt-related transcription factor 2 (RUNX2) knockout (KO) primary human MSCs to create bi-layered constructs that can be fully cultured in the same media and ultimately lead to functional tissue maturation.
In this study, we used clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) to KO RUNX2 with a 95% efficiency in multiple primary human MSCs donors. We subsequently generated WT, KO and bilayered grafts using cartilage decellularized extracellular matrix (dECM) and Filamented Light (FLight) biofabrication and evaluated chondrogenesis and mineralization in vitro. Strikingly, KO MSCs grafts displayed significantly higher glycosaminoglycans and collagen type II deposition and mechanical properties matching the levels of the native tissue, while simultaneously showing complete resistance to calcification and full retention of chondrogenic properties under hypertrophic stimulation. Bilayered grafts resulted in a precisely localized WT MSCs calcified region, and a specific KO MSCs hyaline cartilage area, as confirmed by histology and scanning electron microscopy (SEM).
Ultimately, we envision our strategy to be suitable not only for future clinical treatment of osteochondral defects but additionally for the creation of a novel in vitro model of the osteochondral unit.
Magnetically responsive nanoplatforms for otoprotection: liposomal corticosteroid delivery and protocatechuic acid–engineered iron oxide nanoparticles
Loredana Iftode1, Adeline Josephine Cumpata1, Camelia Mihaela Zara Danceanu2, Anca Niculina Cadinoiu3, Luminita Labusca4, Luminita Radulescu5
1Grigore T Popa Faculty of Medicine and Pharmacy Iasi, Iasi - Romania, 2National Institute Of Research and Development In Technical Physics Iasi Romania, Iasi - Romania, 3.“Ioan Haulica” Institute, Faculty of Medicine, “Apollonia” University of Iasi, Iasi - Romania, 4Magnetic materials and nanosensors. National Institute of Research and Development in Technical Physics Iasi Romania, Iasi - Romania, 5Grigore T Popa Faculty of Medicine and Pharmacy Iasi, Iasi - Romania
Hearing loss remains a major clinical burden, with platinum compounds and aminoglycosides causing dose-limiting ototoxicity. To address the poor access of therapeutics to inner-ear targets, we developed two complementary magnetic nanoplatforms and evaluated their otoprotective potential in an in-vitro model. First, we fabricated cationic liposomes coated with carboxymethyl chitosan and loaded their aqueous core with magnetite nanoparticles (Fe3O4) and dexamethasone phosphate. Formulations were optimized for hydrodynamic size, ζ-potential, and temporal drug retention, and the lead candidate was advanced to biological testing. Second, we synthesized Fe3O4 nanoparticles whose surface was engineered with protocatechuic acid (3,4-dihydroxybenzoic acid) with or without sodium citrate (Fe3O4@3,4-DHAB[±citrate]) aiming to induce antioxidant/anti-inflammatory functionality and colloidal stability. HEI-OC1 auditory cells exposed to cisplatin or gentamicin were co-treated with the nanomaterials. Across both platforms, we assessed cell viability, mitochondrial membrane potential, and senescence-associated β-galactosidase activity. All magnetic nanoformulations showed good biocompatibility (viability typically ≥85% at high doses) and significantly mitigated drug-induced injury. Magnetically addressable, polymer-coated liposomes preserved mitochondrial polarization and reduced senescence while enabling corticosteroid delivery; PCA-functionalized Fe3O4 likewise sustained viability, stabilized mitochondrial function, and dampened senescence, consistent with the redox-modulatory properties of the ligand. Together, these results provide convergent proof-of-concept that magnetic, multifunctional nanomaterials—combining physical guidance, drug carriage, and intrinsic cytoprotective chemistry—can counteract ototoxic stress in auditory cells. These findings motivate in-vivo studies to explore magnetically assisted inner-ear targeting and guide the design of translational strategies for hearing preservation.
Magnetic nano-emulsions carrying dexamethasone and ascorbic acid are effective in treating collagenase induced osteoarthritis in rats
Luminita Labusca1, Camelia Mihaela Zara Danceanu1, Eusebiu Viorel Sindilar2, Daniel Dumitru Herea1, Nastasa Valentin2, Mihai Mares2, Aurelian Sorin Pasca3
1National Institute of Research and Development in Technical Physics Iasi Romania, Iasi - Romania, 2“Ion Ionescu de la Brad” University of Life Sciences (IULS), Faculty of Veterinary Medicine, Iasi - Romania, 3“Ion Ionescu de la Brad” University of Life Sciences (IULS), Faculty of Veterinary Medicine, Iasi - Romania
The global burden of osteoarthritis (OA) continues to increase, eroding quality of life and escalating healthcare costs. Advanced, ‘smart’ drug delivery systems may improve targeting and boost the effectiveness of current and emerging OA interventions. We present a dual-mode intra-articular platform that combines magnetic nanoparticles (MNPs) with nano-emulsions (NEs) engineered to carry dexamethasone (Dex) and ascorbic acid (AA) as a local modular therapy for OA.
Physicochemical characterization of NE–MNP formulations included dynamic light scattering (DLS), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), and Fourier-transform infrared spectroscopy (FTIR) to assess size, morphology, magnetic response, composition, and stability. Cytocompatibility was evaluated in vitro by MTT and LIVE/DEAD assays. Senescence modulation, wound repair, and chondrogenic potential were examined via senescence-associated β-galactosidase (SA-β-gal) activity, protein-level analyses by Western blotting and immunocytochemistry (ICC), scratch-wound closure, and chondrogenic readouts in adipose-derived stem cells (ADSCs) exposed to or loaded with NE–MNP–Dex/AA. Systemic tolerance and biodistribution were explored following intraperitoneal administration in mice, whereas therapeutic efficacy was tested after a single intra-articular injection in a collagenase-induced OA model in rats. Follow-up included hematology and C-reactive protein (CRP), histology of target organs (lung, liver, spleen, heart, skeletal muscle, kidney, cerebellum, brain), and joint evaluation by magnetic resonance imaging (MRI) and histology at 1-, 3- and 5-months post-injection. NE–MNP carriers loaded with Dex and AA were nanoscale (20–60 nm), non-aggregating, and highly stable (maintaining properties for up to two years at RT). In vitro, ADSCs and fibroblasts exhibited a marked reduction in SA-β-gal activity and enhanced ADSC chondrogenesis after loading with NE–MNP formulations. Systemic administration did not provoke hematologic or inflammatory disturbances and produced no histologic injury in examined organs; transient activation of Kupffer cells in the liver was noted. In the rat OA model, a single intra-articular dose led to attenuation of synovial inflammation at 1 month and sustained effects at 2 and 4 months, accompanied by improved cartilage thickness.
These data support the feasibility, safety, and therapeutic activity of Dex and AA-loaded magnetic nano-emulsions for OA.
Advancing in vitro 3D corneal models for toxicity, regeneration, and graft preservation
Fabiola Walz1, Knetzger Nicola2, Schwebler Julian1, Eibichova Sabina1, Christian Lotz2
1Functional Materials in Medicine and Dentistry. University Hospital Würzburg, Würzburg (Baden-Wberg Bayern) - Germany, 2Translational Center Regenerative Therapies TLC-RT. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany
The cornea, the transparent front surface of the eye, is constantly exposed to external influences that can compromise its function and lead to vision loss, requiring a transplant. Advanced human 3D corneal models provide new approach methodologies (NAMs) to investigate epithelial wound healing, predict substance induced irritation and recovery, and improve storage conditions for corneal grafts.
Multilayered in vitro corneal constructs were generated comprising a differentiated epithelium and stromal compartment. These systems formed a versatile platform for comparative assessment across wound-healing, toxicological testing, and preservation studies.
For wound healing assessments, standardized mechanical and excimer laser injuries were applied. Epithelial closure was monitored non-invasively using optical coherence tomography (OCT) and confirmed through histological analysis. Reproducible re-epithelialization was demonstrated by the platform, with progressive wound recovery and initial closure being observed by day 7 and full surface coverage by day 14.
Further in vitro corneal models were used to replace the Draize eye test by conducting chemical irritation testing of substances with defined hazard classifications. Non-invasive transepithelial electrical resistance (TEER) measurements were combined with MTT viability and histology, allowing reversible and irreversible epithelial damage to be distinguished. The established ImAi-test was able to discriminate category 1 (90.9%) from category 2 (83.3%) chemicals over 14 days after exposure. This enables a quantitative prediction of tissue recovery rather than endpoint-based MTT classification.
To evaluate graft preservation, porcine corneas were maintained under controlled flow in a custom dynamic bioreactor designed to enhance nutrient distribution and provide physiological mechanical cues. Corneal tissues preserved in dynamic culture retained epithelial integrity, metabolic activity, and transparency over 14 days, outperforming static storage conditions.
Together, these integrated 3D ocular surface models provide a predictive, NAM-aligned platform to study tissue damage, repair, and preservation in controlled culture conditions.
Cell-source dependent phenotype preservation and plasticity in hydrogel-bioceramic osteochondral engineering
Marcin Kotlarz1, Philipp Fisch1, Anna Puiggalí-Jou1, Simone Ponta1, Haobo Guo2, Zufu Lu2, Hala Zreiqat2, Marcy Zenobi-Wong1
1ETH Zurich, Zurich - Switzerland, 2University of Sydney, Sydney (Australian Capital Territory) - Australia
Introduction: Achieving stable hyaline cartilage layer and hypertrophic cartilage layer integrating with subchondral bone requires balancing phenotype preservation in mature chondrocytes with the plasticity of progenitor cells such as mesenchymal stromal cells (MSCs). Hydrogel-bioceramic constructs provide a platform in which different cell sources can establish and interact within osteochondral-like microenvironments.
This study aimed to assess the influence of initial cell source on in vitro engineered cartilage formed on a bioceramic scaffold designed to support the layered organisation of an osteochondral unit.
Methods: Articular chondrocytes (ACs), nasal chondrocytes (NCs) or MSCs were encapsulated in a 0.5% hyaluronan-transglutaminase (HATG) and 0.25% alginate (Alg) hydrogels. Viability (Live/Dead), mechanical properties, and matrix deposition (glycosaminoglycan and collagen content/DNA), were assessed to evaluate cell-source-dependent phenotypic outcomes. Collagen fibre orientation within the engineered cartilage atop the 3D-printed strontium-hardystonite-gahnite scaffolds was examined.
Results: NC and AC groups exhibited stronger ECM matrix formation and maintained cartilage-specific markers, indicating robust phenotype preservation. Despite exposure to pro-hypertrophic cues (BMP2), AC constructs resisted mineralisation, reinforcing their intrinsic stability within the osteochondral-like environment. In contrast, MSCs showed greater expression variability, higher collagen type I deposition, and responsiveness to hypertrophic cues, leading to partial mineralisation and illustrating their adaptive plasticity.
Conclusion: The cell-laden hydrogel-bioceramic bilayers support osteochondral construct fabrication and reveal distinct, cell-source-dependent trajectories: chondrocytes preserve phenotype, whereas MSCs display adaptive plasticity. Ongoing work investigates how ionic release from the bioceramic modulates this balance and whether it can be leveraged to generate tri-layered osteochondral grafts.
Modulation of the neural cell response through piezoelectric stimulation
Teresa Marques-Almeida1, Clarisse Ribeiro1, Igor Irastorza2, Unai Silvan3, Senentxu Lanceros-Mendez3
1Physics. University of Minho, Braga - Portugal, 2Faculty of Medicine, University of the Basque Country. University of the Basque Country, Leioa (Bizkaia) - Spain, 3BCMaterials, Basque Center for Materials, Applications and Nanostructures. Ikerbasque, Basque Foundation for Science, Bilbao (Bizkaia) - Spain
Electric cues are determinant in different physiological functions of cells and tissues. Thus, the ability to control and direct electric signals within tissue-engineered constructs is critical for proper tissue engineering strategies, as they guide tissue development towards more functional and biomimetic outcomes. Smart piezoelectric materials convert mechanical solicitations into electrical potential variations and vice versa and, therefore, allow to induce specific cell behaviors through electric cues, improving biomimicry of the cell microenvironment.
In fact, the presence of piezoelectric properties in different tissues has been proven and the effect of surface charge and piezoelectric stimulation has been positively evaluated in different cells, including mesenchymal stem cells, myoblast and osteoblasts.
Here we report on the response of primary neurons when cultured on piezoelectric polymer surfaces with different surface electric charge and electric charge type. The used poly(vinylidene fluoride), PVDF, electroactive films promote attachment, viability and maturation of primary neurons, which are particularly influenced by the presence of negative surface charge.
Further, the effect of surface charged PVDF films on adhesion, proliferation and differentiation of neural-derived cell line, under static and dynamic mechanoelectrical stimulation was assessed.
Upon mechanoelectrical stimulation, PVDF with average positive surface charge allows to significantly improve cellular adhesion. Independently of the PVDF surface charge type, the applied electrical stimulus improved neurite extension and differentiation.
The obtained results highlight the suitability of the PVDF based surface-charged substrates, opening the way to novel therapeutic strategies for the regeneration of neural tissues based on dynamically surface charge variations.
Acknowledgements
Fundação para a Ciência e Tecnologia (FCT): Strategic Programs UID/FIS/04650/2025 and contract 2020.04163.CEECIND (CR). Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD): PID2019-106099RB-C43/AEI/10.13039/501100011033. Advanced Materials program supported by MCIN, European Union NextGenerationEU (PRTR-C17.I1) and the Basque Government under the IKUR and Elkartek programs.
Structural polymers as suspending media for lightsheet fluorescent microscopy
Joseph Weightman1, Paige-Louise White2, Thomas E Robinson1, Richard J A Moakes1, Mueller Ferenc2, Liam Grover1
1Healthcare Technologies Institute. University of Birmingham, Birmingham - United Kingdom, 2Department of Cancer and Genomic Sciences. University of Birmingham, Birmingham - United Kingdom
Long-term live imaging of three-dimensional (3D) cell and tissue constructs remains technically challenging, as maintaining biological viability and optical stability simultaneously is difficult. In this study, we developed an optically clear, rheologically tunable hydrogel system based on low-acyl gellan gum to function as a structural suspension medium for light sheet fluorescence microscopy (LSFM). Our aim was to create a culture-compatible material that provides sufficient mechanical support for temporal imaging while maintaining long-term cell viability.
Fluid gels were produced by dissolving gellan gum (1-2% w/v) in deionised water supplemented with phosphate-buffered saline and NaCl, followed by controlled cooling under shear to form a soft particulate network. Rheological analysis confirmed an elastically dominant response over the physiological temperature range (25-40°C), indicating mechanical stability during imaging. FUCCI-expressing colorectal cancer organoids were encapsulated within the gels and imaged for up to 14 days using a Zeiss Z1 LSFM system. The organoids demonstrated sustained proliferation and preserved morphology throughout the observation period, allowing spatial and temporal tracking of cell cycle dynamics without positional drift or structural collapse.
This study reveals that gellan-based fluid gels can serve as transparent, cytocompatible suspension matrices for extended LSFM imaging. Their tunable viscoelasticity and thermal resilience provide a controllable environment for maintaining 3D biological models under live-cell conditions. Beyond imaging, this approach offers a framework for designing bio-optical scaffolds capable of supporting tissue morphogenesis and regenerative model systems, bridging the interface between biomaterials engineering and advanced microscopy.
A higher throughput heart tissue model for predictive drug screening
Sílvia Major1, Kelvin Chung2, Chris Denning2, Yang Wei1, Karah Dring1, Lívia Santos1
1Nottingham Trent University, Nottingham - United Kingdom, 2University of Nottingham, Nottingham - United Kingdom
Background: Cardiovascular diseases (CVDs) are the leading global cause of death, yet only 2% of newly developed drugs target CVDs compared to 45% for cancer. This disparity is partly due to suboptimal translational relevance and scalability of current preclinical models. To address this, the present study aims to develop and characterise a higher-throughput platform to generate engineered heart tissue (EHT).
Methodology: EHT was generated using H9c2(2-1) cardiomyoblasts and a patented culture insert (PCT/GB2023/053038) which allow to scale EHT manufacturing by 40x in relation to commercial platforms. Cells were embedded in collagen I/Matrigel™, cast into the platform, and cultured in growth media. Maturation occurred under uniaxial tension in differentiation media. Tissue integrity and remodelling were monitored via bright-field microscopy for up to 21 days. Viability was assessed at day 14 using LIVE/DEAD™ assay. Cardiomyogenic marker expression for sarcomeric-α-actinin (SAA) and cardiac troponin T (cTnT), was evaluated by confocal microscopy, and contractile force measured via video-optical analysis (EHT Technologies and CellOPTIQ®).
Results: Optimal cell seeding density for EHT generation was determined at 3.9x105 cells/construct, with a proliferation of 4 days prior to initiating differentiation. Over a 21-day culture period, EHT thickness decreased by 18% (p < 0.001), indicating substantial tissue remodelling, while cell viability was approximately 83% (p < 0.001). Despite remodelling and viability, expression of cardiomyogenic markers declined across the first week, and no contractile activity was observed. Contractility was also absent in organoids derived from the same cell line. EHT derived from human induced pluripotent stem cells (hiPSC)-cardiomyocytes (CMs) is currently under development.
Conclusions: We developed and characterised a higher-throughput platform to generate EHT at scale. However, expression of cardiomyogenic markers was not sustained and contractility was lacking. EHT derived from hiPSC-CMs is currently being developed to enhance the physiological and biological relevance of this newly developed approach.
Phosphate-specific regulation of osteoblast mineralisation informs design of regenerative bone scaffolds
Joseph Weightman1, Ellie Northall2, Simon W Jones2, Amy Naylor2, Liam Grover1
1Healthcare Technologies Institute. University of Birmingham, Birmingham - United Kingdom, 2Department of Inflammation and Ageing. University of Birmingham, Birmingham - United Kingdom
The chemical form of phosphate available to osteoblasts is a critical determinant of mineral deposition, matrix organisation, and mineral phase development. Understanding how cells process different phosphate sources is essential for replicating physiological bone formation and designing biomaterials that emulate native mineralisation pathways. This study investigated how distinct phosphate donors influence osteoblast differentiation, mineral formation, and mineral composition. Murine pre-osteoblastic MC3T3-E1 cells and primary human osteoblasts were cultured under osteogenic conditions supplemented with defined phosphate sources, including orthophosphate, β-glycerophosphate, ATP, and phosphocholine. Mineralisation was assessed using Alizarin Red staining and phase-contrast imaging to evaluate extracellular matrix deposition. Raman spectroscopy was utilised to characterise the chemical composition and mineral phase through phosphate and carbonate vibrational band analysis, providing insight into mineral maturity and crystallinity. To determine transcriptional responses to phosphate environment, quantitative PCR was conducted to evaluate the expression of key osteogenic genes, including ALPL, RUNX2, COL1A1, and BGLAP. Phosphate source strongly influenced the progression and morphology of mineral deposition, suggesting differences in mineral growth dynamics and matrix organisation across treatment groups. Phosphocholine supplementation produced extensive, well-organised matrix mineralisation, whereas orthophosphate led to more diffuse deposits. Gene expression analysis revealed a phosphate-dependent regulatory pattern, with reduced ALPL and BGLAP expression under orthophosphate supplementation, while phosphocholine significantly increased BGLAP expression, indicating enhanced osteoblast maturation and mineral-associated protein synthesis. Raman spectroscopy showed compositional variation between phosphate sources, showing subtle shifts in phosphate ν1 and ν3 bands and carbonate-associated peaks, consistent with differences in mineral phase development and biological substitution. Collectively, these findings identify phosphate chemistry as a fundamental regulator of osteoblast-driven mineralisation and mineral phase evolution. This work provides mechanistic insight into phosphate utilisation during bone formation and supports the rational design of regenerative biomaterials that reflect physiological mineralisation pathways.
4D piezoelectric scaffolds with tunable bioelectric cues for skeletal muscle repair
Noemi Ravaglia1, Arianna Rossi1, Maurizio Vignolo2, Pietro Galizia1, Diana Pacheco3, Federica Arienti1, Carlo Baldisserri1, Monica Montesi1, Rosa Mancinelli4, Massimiliano Labardi5, Tatiana Patricio3, Julia Glaum6, Carla Cunha7, Elisa Mercadelli1, Giorgio Luciano2, Silvia Panseri1
1National Research Council of Italy (ISSMC-CNR), Faenza (Italia) - Italy, 2Consiglio Nazionale delle Ricerche (CNR), Genova (Italia) - Italy, 3Polytechnic of Leiria, Leiria - Portugal, 4University of Studies “G. D’Annunzio, Chieti (Italia) - Italy, 5National Research Council-Institute for Chemical and Physical Process, Pisa (Toscana) - Italy, 6Norwegian University of Science and Technology, Trondheim (Nord-Trondelag) - Norway, 7i3S-Instituto de Investigação e Inovação em Saúde, Porto, Portugal, Oporto (Porto) - Portugal
Volumetric muscle loss (VML) remains a major clinical challenge due to the inherently limited regenerative capacity of skeletal muscle and the lack of bioactive scaffolds capable of providing both structural support and dynamic stimulation. In this study, we introduce a novel class of piezoelectric scaffolds composed of egg white proteins (EWP) integrated with barium titanate (BTO) nanoparticles, fabricated through a rapid, sustainable, and versatile microwave-assisted method. EWP:BTO scaffolds exhibited a favourable balance of porosity, mechanical compliance, and degradation stability, effectively mimicking the physicochemical environment of native muscle tissue. Permanent polarization of the BTO phase was achieved through corona poling, enabling the scaffolds to generate localized electrical signals in response to externally applied ultrasound.
In vitro experiments using C2C12 myoblasts demonstrated that the polarized EWP:BTO scaffolds support robust cell adhesion, proliferation, infiltration, and early organization within the 3D matrix. Importantly, ultrasound-driven activation synergized with scaffold piezoelectricity to enhance mechanosensitive and electroactive gene expression, highlighting the capacity of this platform to integrate structural and dynamic stimuli essential for myogenic commitment. These findings suggest that electromechanical transduction can be remotely triggered in a controlled, non-invasive manner, providing a powerful modality to guide early muscle regeneration.
A pilot in vivo study further confirmed the biocompatibility, stability, and absence of local or systemic toxicity of the scaffolds over 28 days, supporting their suitability for future preclinical development. Overall, EWP:BTO piezoelectric scaffolds represent a sustainable, biocompatible, and functionally active system capable of converting mechanical energy into targeted bioelectric cues. By coupling biomimetic architecture with ultrasound-driven electromechanical activation, this platform advances a promising 4D regenerative approach for VML repair. These results lay the groundwork for further investigations in orthotopic muscle injury models, where controlled ultrasound stimulation may enable dynamic, on-demand modulation of muscle regeneration.
Engineering the tumor microenvironment to generate next-generation in vitro prostate cancer models
Stephane Bolduc1, Pellerin Felix-Antoine2, Chabaud Stephane3, Carignan Laurence4, Alavi Reza5, Germain Lucas6, Pouliot Frederic7, Audet-Walsh Etienne8, Bordeleau François9
1Regenerative Medicine Division, CHU de Quebec-Université Laval research center. Department of Surgery, Faculty of Medicine, Université Laval, Québec City, QC G1V 0A6, Canada, Quebec - Canada, 2Regenerative Medicine Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 3Regenerative Medicine Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 4Oncology Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 5Oncology Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 6Endocrinology and Nephrology Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 7Oncology Division, CHU de Quebec-Université Laval research center. Department of Surgery, Faculty of Medicine, Université Laval, Québec City, QC G1V 0A6, Canada, Quebec - Canada, 8Endocrinology and Nephrology Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 9Oncology Division. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada
Background:
Prostate cancer (PCa) remains a leading cause of cancer-related morbidity and mortality worldwide, yet treatment for advanced disease is largely palliative. A major barrier to therapeutic innovation is the lack of physiologically relevant models that replicate tumor-stroma interactions and microenvironmental stiffness—key drivers of progression and therapy resistance. Tissue engineering offers a transformative approach to bridge this gap. Our goal was to develop a human-derived 3D prostate model, integrating cell line spheroids or patient-derived organoids, to study PCa initiation, invasion, and microenvironment remodeling for precision medicine applications.
Methods:
Primary human prostate fibroblasts were assembled into stromal sheets using the self-assembly method and seeded with epithelial cells to form a testosterone-responsive glandular epithelium. PCa spheroids (LNCaP (non-invasive), DU145 (invasive)) or patient-derived organoids (2 patients Gleason grade 7, 2 patients Gleason grade 8-9) were incorporated to model tumor invasion. Stromal stiffness was modulated by inducing cancer-associated fibroblasts (CAFs) via DU145-conditioned media. Atomic force microscopy (AFM) and immunofluorescence characterized tissue architecture, mechanical properties, and tumor-stroma interactions.
Results:
The engineered model reproduced native prostate architecture characterized by the presence of acini and adequate cell polarity. Prostate-specific metabolism was confirmed by measuring citrate secretion. Following CAF induction, we observed increase extracellular matrix remodeling and stiffening of the stroma compared to those produced using normal prostatic fibroblasts. Incorporation of cell line spheroids or patient-derived organoids enabled clinically relevant modeling of tumor heterogeneity, including the assessment of tumor cell invasion.
Conclusion:
This human-derived 3D prostate platform closely mimics native tissue and dynamic tumor-stroma interactions, offering a powerful tool for mechanistic studies and precision oncology. By integrating patient-derived organoids, this model paves the way for personalized therapeutic strategies and accelerates translational research in PCa.
An engineered iPSC-based model of the human thymus supports multi-lineage epithelial development and in vitro T cell generation
Yann Pretemer1, Yuxian Gao1, Kaho Kanai1, Takuya Yamamoto1, Kohei Kometani1, Manami Ozaki1, Karin Nishigishi1, Huaigeng Xu2, Akitsu Hotta1, Yoko Hamazaki1
1Center for iPS Cell Research and Application. Kyoto University, Kyoto - Japan, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research. University of California, San Francisco, San Francisco (California) - United States
Aim and objective: In the thymus, the organ responsible for T cell generation and selection, congenital disorders and age-related involution can lead to immunodeficiency and autoimmunity. Due to the limited availability of human samples and species differences in murine models, thymic dysfunction remains difficult to study and treat. Therefore, we sought to establish a faithful model of the human thymus in vitro.
Material and methodology: Here, we engineered a human iPSC-based model by inducing multi-lineage thymic epithelial cells (TECs), the major stromal component in the thymus supporting T cell development. We devised a stepwise induction through each developmental stage followed by long-term self-directed differentiation, after which we benchmarked induced TECs (iTECs) against human pediatric thymus samples through single cell profiling. We then co-cultured iTECs in artificial thymic organoids (ATOs) with CD4/CD8 double-positive T cell precursors to evaluate their functionality.
Results: Our system allowed the differentiation of diverse TEC-like cells essential for thymic function, including cortical and medullary lineages with clear spatial separation mimicking the architecture of the human thymus. We discovered that precisely controlled retinoid signaling was necessary and sufficient for the specification of FOXN1+ progenitors, after which we observed the autonomous differentiation of mature HLA-DR+ populations with transcriptomic profiles similar to their human primary counterparts. Within ATOs, iTECs supported the generation of naïve CD4+ and CD8+ T cells with diverse TCR repertoires, as well as FOXP3+ cells reminiscent of regulatory T cells, and further matured into AIRE+ and mimetic lineages crucial for the establishment of self-tolerance.
Conclusions: Together, these results demonstrate our model’s faithful reconstitution of human thymus development and function in vitro, presenting a significant advantage over current systems. By providing a renewable source of iTECs for T cell production, drug discovery, and regenerative applications, our model is expected to widely contribute to new treatments for thymic dysfunction.
Engineering the renal tubule epithelium-endothelial interface using organ-on-chip technology
Marta G Valverde1, Lydia Tsocha2, Andreas Hierlemann2, Daniela Gabriel3, Ralph Gruber3, Mario M. Modena2
1Biosystems Science and Engineering. ETH Zürich & BioMedical Research. Novartis Pharma AG, Basel (Basel-Stadt) - Switzerland, 2Biosystems Science and Engineering. ETH Zürich & BioMedical Research, Basel (Basel-Stadt) - Switzerland, 3Novartis Pharma AG, Basel (Basel-Stadt) - Switzerland
Chronic kidney disease (CKD) is a major global health burden with limited therapeutic options and a high unmet medical need. At the nephron level, dysregulation of the proximal tubule (PT) membrane-bound transporters alters solute reabsorption, secretion, and xenobiotic clearance.
Conventional in vitro systems based on primary cells and cell lines fail to maintain transporter expression or replicate the physiological environment necessary for functional assessment of PT epithelial cells (PTECs). Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have emerged as promising model systems to recapitulate renal physiology, displaying superior epithelial maturity and transporter expression compared to traditional systems. However, the lack of vascularization and limited access to the apical surface of PTECs remain major barriers to the quantitative analysis of solute transport and drug disposition.
To overcome these limitations, we developed a PT-on-chip (PToC) platform integrating PTECs magnetically sorted from kidney organoids using the PT brush-border marker lotus tetragonolobus lectin (iPTECs-LTL+). Early geno- and phenotypic characterization of the iPTECs-LTL+ show improved maturity and transporter expression compared to conventional cell culture methods. To further drive iPTECs-LTL+ polarization and maintain a stable culture, different coatings with extracellular matrix (ECM) proteins were evaluated. We sought to investigate whether combining iPTECs-LTL+, ECM proteins, and fluid shear stress improves the maturation and transport functionality of iPTEC, so as to recapitulate renal physiology and disease conditions. We designed a PToC device featuring two adjacent channels separated by a porous membrane, which enables controlled perfusion conditions to induce shear stress on both the epithelial and endothelial layers, and direct epithelial-endothelial interaction. This design supports the long-term culture of polarized iPTEC-LTL+ monolayers and provides direct access to both apical and basal compartments for transport assays. This system establishes a physiologically relevant foundation for studying kidney function, drug disposition, and transporter-related mechanisms in CKD, ultimately advancing the development of targeted therapies and precision medicine approaches for kidney disease.
Mechanical vibration drives intervertebral disc degeneration via inflammatory and mechanotransduction pathways
Luisa De Roy1, Claudia Goerlich1, Astrid Schoppa1, Melanie Haffner-Luntzer1, Anita Ignatius1, Cornelia Neidlinger-Wilke1, Graciosa Q. Teixeira1
1Institute of Orthopaedic Research and Biomechanics, Ulm University Hospital, Ulm (Baden-Wberg Bayern) - Germany
Introduction/Objectives
Intervertebral disc (IVD) degeneration is a key contributor to chronic low back pain. Mechanical stimuli such as low-magnitude high-frequency vibration (LMHFV) are widely applied in rehabilitation and exercise, yet their biological effects on human IVD tissue remain controversial. While mechanosensitive ion channels such as Piezo1 have been implicated in disc mechanotransduction, the molecular pathways involved in LMHFV are not fully understood. This study investigated the effects of LMHFV and Piezo1 activation on human IVD cells and tissues, integrating RNA sequencing (RNA-seq) to identify underlying mechanobiological signaling pathways.
Methods
Human IVD cells (n=5 donors; mean age 43.3±6.2) and tissues (n=7 donors; mean age 26.0±4.5) obtained from disc degeneration patients were exposed to LMHFV, Yoda1 (Piezo1 agonist), or combined treatment for 2 days in vitro and 21 days ex vivo. RNA-seq was performed on IVD cells exposed to LMHFV versus untreated controls. Gene expression of senescence (CDKN1A, CDKN2A), inflammatory (PTGS2, IL6, IL8), and matrix remodeling (MMP3, MMP13, COL1A1, COL2A1) markers was quantified by qPCR. Tissue mechanics (annulus fibrosus [AF] tensile modulus, endplate [EP] stiffness) were assessed by indentation testing, and matrix composition (collagen, glycosaminoglycan [GAG]) by histology and biochemical assays. Statistical analysis: Kruskal–Wallis test (p<0.05).
Results
KEGG and Reactome analyses highlighted Toll-like receptor (TLR2–6) and MyD88/NF-κB cascades, apoptosis, and IL-4/13 signaling. LMHFV increased CDKN1A and IL6 expression and reduced EP stiffness (p<0.05), whereas Yoda1 downregulated matrix-related genes and enhanced AF stiffness (p<0.05). LMHFV+Yoda1 reduced EP stiffness relative to Yoda1 alone. AF tensile modulus and matrix composition were unchanged across groups.
Conclusions
LMHFV induced pro-inflammatory, catabolic, and structural changes characteristic of IVD degeneration, partly independent of Piezo1 activation. RNA-seq revealed stress-responsive transcriptional signatures consistent with previous findings (Widmayer et al., 2023). These data identify novel mechanotransduction networks that may guide development of targeted mechanical and molecular therapies for disc regeneration.
Physiological induction and culture of pluripotent stem cell-derived mesenchymal stem cells
Alisa Jokela1, Julia Monola1, Kerttu Airavaara1, Chris S. Pridgeon1, Riina Harjumäki1
1Drug Research Program, Faculty of Pharmacy. University of Helsinki, Helsinki (Southern Finland) - Finland
Introduction
To advance the clinical use of mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs), challenges such as cell heterogeneity, early senescence, and variability in quality must be addressed. To achieve stable and scalable production, we aim to identify an optimal induction and culture protocol for induced pluripotent stem cell -derived MSCs (iMSC). We hypothesize that mimicking in vivo MSC conditions during induction and culture will yield more tissue-like cells and EVs with improved regenerative potential and scalability.
Methods
Potential iMSC induction protocols from literature were tested, and MSC marker expression, proliferation, and cell yield were analysed. Based on integrin expression in adipose-derived MSCs, different protein coatings were evaluated to promote iMSC induction. The most promising protocol was further optimised with a novel approach by inducing the cells under physiological 5% oxygen. Cells were analysed using qPCR, proteomics, and immunocytochemistry. EVs were isolated and analysed according to the Minimal information for studies of EVs (2023) guidelines, and their efficacy was tested in an in vitro wound model and compared with platelet lysate.
Results
Induction via a neural crest-like intermediate cells produced typical MSC gene and protein expression, strong proliferation, and high cell yield. Compared to tissue-derived MSCs, iMSCs had reduced batch variation and increased proliferation. Fibronectin coating enhanced the induction, and 5% oxygen altered the cell phenotype. Preliminary results from in vitro wound assay showed promise in wound closure when used iMSC-EVs prior to optimisation.
Conclusion
Our findings highlight a promising method for generating consistent, high-yield iMSCs suitable for cell- and EV-based regenerative therapies. Optimised culture conditions can further enhance the therapeutic potential of iMSCs, and upcoming wound healing assays will help define their regenerative efficacy and translational potential in clinical applications.
Funding
Research Council of Finland: GeneCellNano flagship project [grant number #337430].
Development of a hydrogel-based dermal equivalent for a 3D-bioprinted melanoma skin model
Shakti Mara Schröder1, Nicole Teusch2, Doris Heinrich3, Sebastian Schröder1
13D-SCT. Institute for Bioprocessing and Analytical Measurement Techniques e.V., Heilbad Heiligenstadt (Thuringen) - Germany, 2Institute of Pharmaceutical Biology and Biotechnology. Heinrich Heine University Duesseldorf, Duesseldorf (Nordrhein-Westfalen) - Germany, 3Analytical Measurement Techniques. Institute for Bioprocessing and Analytical Measurement Techniques e.V., Heilbad Heiligenstadt (Thuringen) - Germany
The human skin comprises a dermal layer of fibroblasts producing extracellular matrix (ECM) components and an epidermal layer mainly consisting of keratinocytes interacting with melanocytes. While keratinocytes proliferate and differentiate during migration to the surface, melanocytes remain at the epidermal–dermal junction, regulating pigmentation in response to UV radiation. Melanocytes are the precursor cells of melanoma, one of the most aggressive and metastatic cancers, and play a crucial role in tumor progression. Due to the rising incidence of malignant melanoma, physiologically relevant 3D skin models are essential for studying tumor biology, drug development, and reducing animal experiments.
The proposed melanoma model is based on a functional dermis supporting keratinocyte adhesion. The dermal hydrogel is optimized to promote spindle-shaped fibroblast morphology, ECM production, and physiological dermal stability and elasticity.
Primary human dermal fibroblasts, neonatal (HDFn), human epidermal keratinocytes (HEK), and melanocytes (HEM) were incorporated into a 3D bioprinted construct using an optimized hydrogel blend of gelatin-methacryloyl (GelMA), elastin-methacryloyl, and hyaluronic acid. A design-of-experiments approach guided the hydrogel optimization to enhance HDFn viability and printability. Rheological and bioprinting properties were analyzed using a plate-to-plate rheometer and a droplet based bioprinting method (RegenHU). HEKs and HEMs were printed onto the dermis equivalent in a physiological ratio, and the newly formed skin equivalent is characterized using immunohistochemistry.
The optimized hydrogel supports HDFn proliferation and ECM production (such as fibronectin, collagen IV), while providing a physiological dermal stability. It shows shear-thinning properties that enable a bioprinting process with droplets <500 µm in diameter and <100 nl in volume. The dermis equivalent allows HEKs and HEMs to adhere forming a basal layer.
The dermis equivalent mimics native architecture and physiology, providing a promising platform for introducing melanoma cells to study tumor invasion, stromal remodelling, and melanoma–tumor microenvironment interactions under controlled 3D conditions.
Advancing alginate-based bioinks through collagen incorporation and biomimetic mineralisation to study osteoblast-to-osteocyte differentiation
Anne Bernhardt1, Dolphee Khurana1, Johannes Windisch1, Michael Gelinsky1
1Centre for Translational Bone Joint and Soft Tissue Research. University Hospital “Carl Gustav Carus” at Technische Universität Dresden, Dresden (Sachsen) - Germany
In vitro bone models mimicking structure, function and cell-cell communication of bone tissue are advantageous to study the interaction of bone with biomaterials, drugs and bioactive substances. Bioprinted in vitro models offer the possibility to generate spatially defined tissue constructs. Aim of the present study was to develop hydrogel inks, containing the main components of bone extracellular matrix - collagen and hydroxyapatite - as basis for bioprinted in vitro bone models. Furthermore, the differentiation of primary human osteoblasts to osteocytes in the bioprinted constructs was examined.
Based on an already established hydrogel ink, composed of 3% alginate, 9% methylcellulose and fresh frozen human blood plasma [1], collagen from bovine skin was incorporated with concentrations up to 9 mg/ml. Collagen solutions were sterilized by supercritical CO2 [2], maintaining the native collagen structure. Collagen-containing inks showed excellent rheological performance and printability. Printed constructs were subjected to biomimetic mineralization for three days using a previously published protocol [3] with slight changes. Precipitation of calcium phosphate increased during mineralization from day 1 to 3, verified by von Kossa staining and quantified by colorimetric calcium assay after dissolving the samples. Primary human osteoblasts were bioprinted in collagen-containing inks, constructs were mineralized and cultivated for up to two weeks for osteoblast-to-osteocyte differentiation. Osteocyte differentiation was verified by gene expression analysis of osteocyte markers E11, sclerostin, matrix extracellular phosphoglycoprotein and dentin matrix protein 1 as well as dendritic morphology, which was evident after fluorescence staining.
In conclusion, inks with a more bone-like composition compared to pure polysaccharide inks were generated showing excellent printability and supporting the differentiation of osteoblasts to osteocytes.
References
1. Ahlfeld, T. et al. ACS Appl. Mater. Interfaces 12, 12557–12572 (2020).
2. Bernhardt, A. et al. PloS One 10, e0129205 (2015).
3. Thrivikraman, G. et al. Nat. Commun. 10, 3520 (2019).
Multispheroid bioassembly through meltelectrowriting for thyroid in-vitro models
Camilla Mussoni1, Indong Jun2, Taufiq Ahmad1
1FMZ. University Hospital Würzburg, Würzburg (Baden-Wberg Bayern) - Germany, 2Korean Institute of Science and Technology Europe, Saarbrücke (Saarland) - Germany
Extensive research has been conducted on the fabrication of human tissues as models to test the effects of a wide range of chemicals on human health and the environment (1). Thus, the need for the development of scalable and modular tools that provide reliable results in identifying and regulating the risks of potentially harmful substances. This study presents a precisely arranged fibrous architecture mesh, fabricated via melt electrowriting (MEW) of Polycaprolactone as spheroid culture support for 3D in vitro models. This platform facilitates multi-spheroid bioassembly, overcoming single spheroid limitations such as hypoxia and promoting both cell-cell communication. The technique has been employed to recapitulate different human tissues, for this study the targeted tissue was the thyroid.
Fibers are precisely deposited in the shape of squared boxes, in a variety of sizes between 100 µm and 1 mm (2). The scaffolds perimeter was reinforced with a 3D printed Poly Lactic Acid ring for better handling and coated in polydopamine for improved cell compatibility (3). Human thyroid epithelial cells (huThyrEC) spheroids are formed and after 3 days of culture-supplied with thyroid-stimulating hormone- are transferred in the scaffold. The assemblies are further cultured for 7 more days followed by evaluating thyroid hormones, thyroid-related protein, metabolomic and transcriptomic changes. Results are compared between monoculture, traditional spheroid culture, the proposed PAFA method and compared to zebra fish embryo model, proving our method as reliable and comparable, if not better in some cases than more traditional models.
1) La Merrill MA, Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification, DOI: 10.1038/s41574-019-0273-8
2) McMaster R, Tailored Melt Electrowritten Scaffolds for the Generation of Sheet-Like Tissue Constructs from Multicellular Spheroids, DOI: 10.1002/adhm.201801326
3) Lamberger Z, Streamlining the Highly Reproducible Fabrication of Fibrous Biomedical Specimens toward Standardization and High Throughput, DOI:/10.1002/adhm.202402527
Urethral organoids' response to dense collagen matrices
Isabelle Martinier1, Nijhuis Jules1, De Kort Laetitia1, De Graaf Petra1
1Urology. Regenerative Medicine Center Utrecht, Utrecht - The Netherlands
Understanding urethral epithelium biology and interactions with the surrounding tissue is important for improving the treatment of urethral diseases, but is limited by the lack of accurate models. Several urogenital and male reproductive organs have already been miniaturized using organoids, including the prostate, the testis, and the bladder. However, because the urethral epithelium differs from the bladder epithelium, the bladder organoid model cannot be transposed to the urethra—limiting our comprehension of physiological phenomena and their relevance for urethral treatment. Besides the lack of urethral organoids, the current method of culturing epithelial organoids relies on tumorigenic and chemically undefined hydrogels such as the basement membrane extract (BME), which cannot be translated into clinic. Considering this, there is an urge to replace BME with more physiologically relevant support1.
To address those limitations, we introduce for the first time urethral organoids with epithelium that is different from the bladder, derived from patient tissue. First, epithelial cells were grown in BME or type I collagen hydrogels to establish urethral organoids. We further developed new materials for organoid growth composed solely of type I collagen, at physiological concentrations of 100-200 mg.mL-1 and up to pathological concentrations of 400-900 mg.mL-1. For this, we fabricated non-porous collagen matrices via evaporation and porous matrices via ice-templating, fibrillated without the use of cross-linker2. Results reveal how the concentration and the porosity of biomimetic collagen impact the behaviour and morphology of organoids. Our materials represent a new alternative for organoid culture, which is compatible with clinical translation and closely mimic biological tissues.
1. Kretzschmar, K. & Clevers, H. Dev Cell 38, 590-600 (2016).
2. Martinier, I. et al. Biomater Sci 12, 3124-3140 (2024).
Dynamic loading enhances meniscus-like matrix formation on non-woven PET scaffolds seeded with human mesenchymal stromal cells
Graciosa Quelhas Teixeira1, Luisa De Roy1, Anna-Lotta Feldmeier1, Mubashir Ahmad1, Sofia Pilão1, Maria Ahrens1, Anita Ignatius1, Carsten Linti2, Andreas Martin Seitz1
1Institute of Orthopaedic Research and Biomechanics, Ulm University Hospital, Ulm (Baden-Wberg Bayern) - Germany, 2German Institutes of Textile and Fiber Research, Denkendorf (Baden-Wberg Bayern) - Germany
Introduction/Objectives
Meniscal injury is a major risk factor for early-onset osteoarthritis, yet therapeutic alternatives to meniscectomy remain limited. This study investigated the potential of non-woven polyethylene terephthalate (PET) scaffolds to support human mesenchymal stromal cell (MSC) proliferation and differentiation, and evaluated whether mechanical stimulation could enhance meniscus-like matrix formation.
Methods
MSCs were seeded onto PET scaffolds (400–420 g/m2, 85% porosity) and cultured for 7, 14 and 21 days under three conditions: basal medium (Ctr), chondrogenic medium (ChD), or ChD with dynamic loading (ChD + Dyn; 12% strain, 1 Hz, 1 h/day, 5 days/week). Metabolic activity and DNA content were assessed at each time point using resazurin and PicoGreen assays, respectively. Glucose consumption and lactate production were analysed in culture supernatants using biochemical assays. Quantification of DNA, glycosaminoglycan (GAG), and collagen was performed on digested constructs. Viscoelastic properties were evaluated through confined compression testing at 10% strain. RNA sequencing was performed to compare ChD versus Ctr and ChD+Dyn versus ChD groups. Statistical analysis: Kruskal-Wallis or one-way ANOVA (significance, p<0.05).
Results
PET scaffolds supported uniform MSC adhesion, proliferation, and differentiation. Dynamic loading promoted cell proliferation and collagen production, transiently upregulating chondrogenic markers (SOX9, ACAN, COL1A1 and COL2A1) at day 14, and suppressing hypertrophic COL10A1 expression, while reducing GAG accumulation relative to ChD alone. Despite increased ECM synthesis, particularly collagen, no significant changes in mechanical properties were observed over 21 days. RNA sequencing revealed distinct transcriptional profiles between ChD and ChD+Dyn groups, highlighting enrichment of pathways associated with ECM organization, actin cytoskeleton regulation, growth factor responsiveness, and cell adhesion and migration.
Conclusions
Overall, non-woven PET scaffolds provide a robust platform that supports MSC proliferation and differentiation. Importantly, mechanical stimulation directed MSC fate and enhanced meniscus-like tissue formation by targeting several pathways related to matrix metabolism, highlighting its potential for improving meniscus repair strategies.
Abiotic cell-derived constructs and secretome hydrogels for immunomodulation and tissue regeneration
Mariana Oliveira
de CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal
Cells generate diverse products with therapeutic potential, including soluble factors, vesicles, and extracellular matrix. In the pursuit of safer and more predictable therapies, cell-free approaches have expanded significantly over the past decade. Despite advances in the field, using the full capabilities of cells and their products to build safe and predictable materials with complex functions is still at an early stage.
Abiotic cellular materials derived from fixed mesenchymal stromal cells (MSCs) capture key biological cues while avoiding risks (e.g., migration, undersired differentiation) associated with viable cells. Fixed MSC-based biomaterials exhibit robust immunomodulatory activity in vitro and in vivo, influencing both innate and adaptive immune populations. They attenuated monocyte inflammatory responses through NF-κB signaling, and promoted regenerative macrophage phenotypes. In T lymphocytes, these materials increased FOXP3 expression in CD4+ cells while reducing cytotoxic CD8+ markers [1]. Pre-conditioning and processing strategies further enable precise modulation of immune and regenerative functions.
MSCs also generate secretomes with immunomodulatory and regenerative properties. Their mixture with exogenous biomaterials has enabled their localized delivery, despite the difficulty to predict effects of those materials in the overall performance of therapies. To address this, hydrogels were generated directly from purified secretomes obtained from pristine or IFN-γ-primed MSCs, yielding materials with tunable viscoelasticity, composition, and biological activity [2]. A 100-fold scale-up in secretome-derived raw material production demonstrates the emerging translation potential of the technology.
Collectively, abiotic and secretome-based platforms establish complementary, modular routes towards more predictable regenerative therapies through the preservation of cellular cues without relying on living cells.
References
[1] Sousa et al., Advanced Materials, 2024; doi: 10.1002/adma.202405367.
[2] Santos-Coquillat et al., Matter, 2025; doi: 10.1016/j.matt.2025.102431.
Acknowledgements. This work was developed within the scope of the project LF-OC-23-001396 (Leo Foundation) and CICECO Aveiro Institute of Materials, UID/50011/2025 (DOI 10.54499/UID/50011/2025) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC).
Linking cytoplasmic fluidity to extracellular stiffness during wound repair
Eloisa Torchia1, Melissa Pezzotti1, Moises Di Sante1, Francesco Silvio Pasqualini1
1Synthetic Physiology Laboratory. University of Pavia, Pavia (Lombardia) - Italy
Tissue regeneration relies on coordinated cell migration across mechanically diverse environments [1,2]. While extracellular stiffness and topography are established regulators [3], the cytoplasm’s mechanical state, its viscosity and fluidity, is rarely considered in regenerative design [4]. Yet this internal rheology governs how forces and biochemical cues propagate inside cells, shaping how they sense and respond to their surroundings [5,6].
Here, we combine genetically encoded multimeric nanoparticles (GEMs) with engineered microenvironments to investigate how intracellular viscosity can be measured alongside collective migration during wound closure. GEMs self-assemble as fluorescent nanoparticles within the cytoplasm and act as passive microrheological probes. We integrate live-cell fluorescence imaging with photopatterned wound-healing assays on glass and polyacrylamide substrates featuring pathophysiological stiffness and extracellular matrix cues.
By tracking GEM trajectories in migrating cells, we extract diffusion coefficients that can be translated into estimates of cytoplasmic viscosity. These measurements demonstrate the feasibility of quantifying intracellular rheology in parallel with wound closure dynamics across different mechanical contexts. This integrated platform enables controlled comparisons between extracellular mechanics and the mechanical state of the cytoplasm.
Altogether, this work establishes a methodological framework that brings intracellular microrheology into regenerative biomaterial design, paving the way for future studies that aim to link extracellular cues with the physical state of the cytoplasm during tissue repair.
References
[1] Discher et al., Science (2005).
[2] Yi et al., Bioact. Mater. (2022).
[3] Dalby et al., Nat. Mater. (2014).
[4] Eskandari et al., J. Cell Sci. (2025).
[5] Wirtz et al., Annu. Rev. Biophys. (2009).
[6] Ladoux et al., Nat. Rev. Mol. Cell Biol. (2017).
Topography affects macrophage-driven fibroblast physiology
Streggi Vandersteene1, Els Alsema2, Nicholas A. Kurniawan1, Jan De Boer1
1Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands, 2Center for Health Protection. RIVM National Institute for Public Health and the Environment, Bilthoven (Utrecht) - The Netherlands
The foreign body response (FBR) is a complex inflammatory and fibrotic process that can lead to implant failure. Macrophages, as primary mediators, polarize into pro- or anti-inflammatory phenotypes and secrete cytokines that recruit fibroblasts. These fibroblasts proliferate, differentiate, and contribute to fibrous encapsulation through extracellular matrix deposition. We hypothesized that surface topography modulates macrophage behavior in ways that indirectly shape fibroblast physiology, thereby influencing the fibrotic outcome. Surface topographies offer a powerful tool to study how mechanical cues influence macrophage and fibroblast behavior in the FBR. Building on TopoChip screenings that revealed topography-induced effects on macrophage and fibroblast attachment and phenotype1, we investigated how microscale topographies modulate macrophage morphology, YAP localization, and cytokine secretion. Macrophages were cultured on poly(styrene-block-isobutylene-block-styrene) surfaces with distinct topographies, and conditioned media were transferred to fibroblasts to assess morphological changes and α-smooth muscle actin (α-SMA) expression. This approach allowed us to isolate the paracrine effects of macrophage-secreted factors, shaped by topography, on fibroblast physiology. Our results show that topography markedly influences macrophage behavior, driving distinct cytokine profiles, including TNF-α, IL-6, IL-1β, IL-10, and IL-1RA, compared to flat controls. These changes were accompanied by altered cell morphology and YAP localization, suggesting that physical cues from the substrate play a key role in shaping macrophage function. Fibroblasts exposed to conditioned medium exhibited distinct morphological changes compared to those treated with TGF-β1, highlighting the influence of macrophage-derived signals. While TGF-β1 induced expected stress fiber formation and high α-SMA expression, the conditioned medium elicited a broader spectrum of responses, suggesting a complex and physiologically relevant interplay. By linking macrophage responses to fibroblast outcomes, we identify novel mechanisms in the FBR and provide insights to guide biomaterial design for improved implant integration.
[1] Sudarsanam, P. K. Engineering Topography Mediated Tissue Response in Foreign Body Reaction. (Eindhoven University of Technology, Eindhoven, 2024).
From fixation to function: an adhesive cell carrier enabling enhanced delivery and reproducible cartilage matrix formation
Peyman Karami1, Robin Martin1, Alexis Laurent2, Virginie Philippe1, Lee Ann Applegate3, Dominique P. Pioletti4
1Department of Orthopedic Surgery and Traumatology. Lausanne University Hospital, Lausanne (Vaud) - Switzerland, 2Manufacturing Department. LAM Biotechnologies SA, EPALINGES (Vaud) - Switzerland, 3Regenerative Therapy Unit, Reconstructive and Hand Surgery Service. Lausanne University Hospital, EPALINGES (Vaud) - Switzerland, 4Laboratory of Biomechanical Orthopaedics. EPFL, Lausanne (Vaud) - Switzerland
Despite advances in autologous chondrocyte implantation (ACI), durable cartilage repair remains limited by inefficient cell delivery and retention, inadequate fixation, and variable clinical outcomes. Existing ACI cell carriers rely on non-adhesive scaffolds or secondary fixation methods that lack sufficient interfacial strength to native tissue. A next-generation carrier must therefore encapsulate and protect chondrocytes while anchoring them securely to cartilage and subchondral bone and maintaining viability and reproducible biological function.
We developed an injectable, intrinsically adhesive hydrogel as a 3D carrier for human autologous chondrocytes (HACs). After demonstrating successful tissue integration of acellular hydrogel in large animal models, this study evaluates its potential as an advanced ACI carrier and identifies an optimal formulation that supports viability, hyaline-like matrix formation and donor-independent performance.
HACs from five donors were encapsulated at 15 × 106 cells/mL in precursors at 10-20 wt% polymer contents and cultured for 21 days under chondrogenic conditions. Viability, biochemical, histological, immunohistochemical and mechanical analyses were performed. The hydrogel maintained initial viability above 85% and more than 70% at 21 days, with no significant donor-related variability. GAG/DNA increased significantly with a formulation-time interaction, with greater accumulation in lower polymer-contents. Collagen II/aggrecan staining confirmed hyaline-like matrix synthesis, and construct stiffness increased in parallel with ECM deposition. Donor variability affected matrix and mechanical outcomes but not the ability to maintain viable and functional cells, indicating that while the hydrogel maintains reproducible viability and phenotype retention, biochemical and mechanical performance are further influenced by patient-specific cellular capacity. Tensile adhesion testing on human cartilage and subchondral bone was about fivefold higher than fibrin glue after sterilization. We identified the 15 wt% formulation as the translational optimum, balancing initial stiffness/adhesive stability with progressive matrix development for in situ ACI delivery.
By combining strong adhesion with chondrocyte encapsulation in one reproducible material, this technology can overcome the delivery limitations in ACI and support early fixation and hyaline cartilage formation using a suture-free, membrane-free implantation approach.
Nanovibrational stimulation for mesenchymal stromal cell osteogenesis: investigating the relationship between osteogenesis and inflammation
Udipt R. Das1, Peter G. Childs2, Peter S. Young3, Dominic Meek4, Stuart Reid2, Penelope M. Tsimbouri1, Matthew J. Dalby1
1Centre for the Cellular Microenvironment, School of Molecular Biosciences, Advanced Research Centre. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, 2SUPA Department of Biomedical Engineering. University of Strathclyde, Glasgow (Glasgow City) - United Kingdom, 3Department of Orthopaedics. University Hospital Ayr, Ayr (South Ayrshire) - United Kingdom, 4Department of Orthopaedics. Queen Elizabeth II University Hospital, Glasgow (Glasgow City) - United Kingdom
Bone anomalies pose a significant global burden, underscoring the need for deeper insights into bone biomechanics, pathophysiology, and regenerative strategies [1]. Conventional bone tissue engineering relies on exogenous agents, such as growth factors (e.g., BMP2) and corticosteroids (e.g., dexamethasone), to promote osteogenesis, however, limitations exist, including a lack of differentiation specificity and side effects [2]. We developed a nanovibrational bioreactor capable of inducing mesenchymal stem cell (MSC) osteogenesis using 30 nm amplitude, 1 kHz stimulation (NK30), without chemical supplementation. We examined whether increasing amplitude to 100 nm would enhance osteogenesis or drive reactive oxygen species (ROS) and inflammation, as suggested in [3]. Therefore, high-amplitude nanovibration (100 nm, NK100, 1 kHz) was investigated to assess effects on MSC osteogenesis, redox balance, and inflammation. Primary bone marrow MSCs were cultured at NK30, NK100, osteogenic media (OGM), and basal control for 6 weeks. Gene expression of osteopontin (OPN), osteocalcin (OCN), IL-6, and NF-κB was assessed via RT-qPCR. Phospho-p38/p38 ratio, OPN, and OCN protein expression were quantified by in-cell western. Matrix metalloproteinase (MMP)-mediated remodelling was evaluated by zymography, and mineralization by Alizarin Red S and Von Kossa staining. IL-6 and TNF-α were measured using ELISA, and ROS production by flow cytometry. Immunostaining assessed activating transcription factor 4 (ATF4) and OPN localisation. NK100 significantly upregulated OPN and IL-6, with increased phospho-p38/p38, OPN, and OCN protein expression. Both nanovibrational stimulations increased MMP-2 remodelling activity and late-stage mineralization, with elevated nuclear ATF4 and cytoplasmic OPN localisation. Despite greater osteogenic effects, IL-6 and TNF-α secretion remained consistent, and ROS levels were reduced. These findings demonstrate that high-amplitude nanovibration enhances osteogenesis while maintaining redox and inflammatory homeostasis.
Acknowledgment
We acknowledge funding from EPSRC (EP/X013057/1).
References
[1] Shen Y et al. Front. Endocrinol., 2022.
[2] Childs PG et al. Biochem. J., 2020.
[3] Orapiriyakul W et al. A.C.S. Nano., 2020.
Ageing causes structural and cellular alterations in the mouse muscle–tendon junction
Nodoka Iwasaki1, Chavaunne Thorpe1
1Comparative Biomedical Sciences. Royal Veterinary College, London (London, City of) - United Kingdom
Introduction: The muscle-tendon junction (MTJ) is a specialised interface that transmits contractile force from muscle to tendon. Compared with either tissue alone, the MTJ is more prone to injury, and its vulnerability increases with age. Current treatments remain inadequate, often resulting in scar tissue formation and high re-injury rates. Despite its clinical importance, the mechanisms underlying MTJ ageing and associated functional decline are poorly understood. This study aimed to characterise age-related structural and cellular changes in the murine MTJ using high-resolution imaging and molecular techniques.
Methods: Achilles MTJs were isolated from young (3-month-old) and old (23-month-old) C57BL/6 mice. The samples were stained with 1% phosphotungstic acid and imaged in 3D using micro-computed tomography (µCT) to assess structural parameters (n=4). Whole-tissue immunostaining was performed using endothelial (von Willebrand factor, VWF) and muscle (laminin-α2) markers (n=4). Two-dimensional immunolabelling and in situ hybridisation were performed to assess expression of senescence markers, p16 and p21, the MTJ marker collagen-22, and VWF (n=4).
Results: µCT analysis revealed a 27% reduction in muscle fibre diameter with age (p=0.029), accompanied by a trend towards increased MTJ surface area (209%; p=0.057) and a 19% reduction in pennation angle (p=0.0289), indicating reduced force transmission efficiency. Whole-tissue immunostaining showed a 49% decrease in VWF-positive endothelial cell volume (p=0.029) with age, suggesting reduced vascularity across the MTJ. Senescence markers p16 and p21 were markedly upregulated in endothelial and MTJ-specific cells in aged tissues, particularly in MTJ-specific cells, showing 270% and 310% increases, respectively (p<0.0001; p=0.0038).
Discussion/conclusions: These findings suggested that vascular and MTJ-specific cells are particularly susceptible to ageing and may collectively contribute to functional decline of the MTJ in aged individuals. Understanding these mechanisms may help with developing targeted therapeutic strategies to preserve or restore MTJ integrity and function in ageing populations.
Funder: Gill Malone Award (Royal Veterinary College).
Induced cellular senescence disrupts vascular organisation in an in vitro vascularised tendon model
Nodoka Iwasaki1, Elizabeth Finding1, Caroline Wheeler-Jones1, Chavaunne Thorpe1
1Comparative Biomedical Sciences. Royal Veterinary College, London (London, City of) - United Kingdom
Introduction: Tendon degeneration is common, and its risk increases with age. Tendon regeneration and healing are limited due to inherent low cell density and vascularisation, and current treatments are insufficient. We have shown that tendon vascularisation decreases, and tendon vascular cells become senescent, with ageing; therefore, targeting the vascular network represents a promising therapeutic avenue for age-related tendon injuries. This study developed an in vitro 3D co-culture model to establish the effects of cellular senescence on tendon vasculature. Horse superficial digital flexor tendons (SDFTs) were used as they are functionally and clinically similar to human Achilles tendons.
Methods: Tenocytes (TCs) and endothelial cells (ECs) were isolated from equine forelimb SDFTs (age: 2-15 years, n=3). Hydrogen peroxide (600µM; 2 hours; two consecutive days) was used to promote stress-induced senescence in ECs prior to co-culture. TCs were cultured in 12-well plates until confluent, and untreated or senescent ECs were seeded onto this TC layer. Co-culture was performed for 7 days with fibroblast growth factor (10 ng/ml) and vascular endothelial growth factor (0.5 ng/ml) to promote vessel formation. Senescence induction was confirmed through immunostaining for p16. Vessel length and diameter were measured in co-cultures immunostained for the EC marker CD146.
Results: Hydrogen peroxide treatment of ECs significantly upregulated the senescence marker p16, with a 145% increase in mean fluorescence intensity (p=0.018). Vessel-like structures were formed in TC:EC co-cultures. In senescent cultures, vessel length decreased by 38% (p=0.0096), whereas vessel diameter increased by 149% (p=0.026).
Discussion/conclusion: Hydrogen peroxide treatment successfully induced senescence in ECs. Co-cultures of TCs and ECs supported vascular network formation, with senescence leading to shorter, wider and disorganised vessel-like structures. This 3D TC:EC co-culture model provides a valuable platform for investigating the impact of senescence on the tendon vasculature and for screening therapeutic compounds.
Funder: Horserace Betting Levy Board (prj/804).
A versatile 3D bioprinting platform for engineering physiologically relevant and high throughput human blood-brain barrier models
Gal·la Vinyes I Bassols1, Anna Vilche1, Oscar Castaño1, Anna Lagunas1, Josep Samitier1
1Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain
The blood–brain barrier (BBB) is a highly selective interface preserving central nervous system (CNS) homeostasis by regulating molecular exchange and blocking toxins, pathogens, and inflammatory mediators. While essential for protection, the BBB’s restrictive permeability hampers therapeutic delivery for neurodegenerative diseases, highlighting the need for physiologically relevant in vitro models that replicate human neurovascular architecture and cellular dynamics.
We present a versatile three-dimensional (3D) bioprinting platform to generate physiologically relevant, high-throughput human BBB models. This platform employs a novel bioink optimized for rheological properties and biocompatibility, compatible with microvalve-based embedded 3D bioprinting—a technique not previously applied to BBB modeling. The bioink allows precise, low-shear deposition while maintaining excellent cell viability. Using this approach, 48 uniform 3D ring-shaped hydrogel scaffolds are fabricated in nine minutes, demonstrating scalability and reproducibility.
Human brain microvascular endothelial cells (BMECs) bioprinted within fibrin-rich constructs remain viable and proliferative over seven days, organizing into microvascular-like structures. The scaffolds sustain structural integrity and biological functionality, validating the material and printing parameters.
While this study focuses on the endothelial component, the platform’s modular and adaptable design offers future potential for integration with co-cultures of astrocytes and pericytes, as well as incorporation into microfluidic systems and brain organoids. These advancements could further enhance physiological relevance and translational capacity by combining the strengths of multiple contemporary BBB models.
This work represents the first successful application of microvalve-based embedded 3D bioprinting for BBB modeling, establishing a rapid, reproducible, and adaptable platform with broad utility for drug screening, disease modeling, and neuroengineering applications.
A stratified 3D in vitro model of HPV-positive and HPV-negative head and neck squamous cell carcinoma for precision drug testing
Agnieszka Nikitiuk1, Yuan Zhou2, Anna Vilche3, Oscar Castaño4, Elisabeth Engel5, Nuno Coelho1
1Biomaterials for Regenerative Therapies. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 2Biomaterials for Regenrative Therapies. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 3Biomaterials for Neural Regeneration. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 4Biomaterials for Regenrative Therapies. University of Barcelona (UB), Barcelona - Spain, 5Materials Science and Engineering (EEBE). Universitat Politècnica de Catalunya, Barcelona - Spain
Head and neck squamous cell carcinoma (HNSCC) is one of the seventh most common malignancies worldwide and remains a therapeutic challenge due to its high heterogeneity, complex tumor microenvironment, and frequent metastasis. Furthermore, human papilloma virus (HPV) status significantly influences chemotherapy response and drug resistance. Current preclinical models fail to recapitulate the histopathological and microenvironmental complexity of HNSCC, limiting new therapeutic development. To address these limitations, we developed a physiologically relevant HPV-stratified 3D in vitro HNSCC model that uniquely recapitulates tumor-stroma interactions and heterogeneity. Multicellular spheroids were generated from HPV-positive (SCC152) or HPV-negative (Cal27) HNSCC cell lines, co-cultured with human microvascular endothelial cells (HMEC-1) and human dermal fibroblasts (hdF). These spheroids were embedded in biologically relevant matrices (collagen, fibrin or cell derived matrix (CDM) produced by human adipose-derived mesenchymal stem cells (hAMSCs).
Morphological analysis revealed distinct behaviors between HPV+ and HPV-spheroids. HPV+ spheroids remained compact and cohesive, while HPV-exhibited extensive invasion of the matrix and branching, with threefold increase in size. Models containing fibroblasts showed evidence of matrix remodeling, highlighting stromal influence. Drug testing with doxorubicin or cisplatin demonstrated matrix- and cell composition-dependent chemoresistance: complex fibroblast-containing models were more resistant than simpler co-cultures, and HPV- ones consistently showed higher resistance than HPV+ models, reflecting clinical observations. Immunohistochemistry confirmed formation of distinct epithelial, mesenchymal and endothelial compartments within models maintained for up to 21 days.
This physiologically relevant, HPV-stratified 3D HNSCC platform faithfully reproduces tumor heterogeneity and drug response, offering a powerful tool for precision medicine applications and preclinical screening of novel therapies.
Optimisation of synthesis and study on biological activity of silver-doped calcium hydroxyapatites for tissue engineering
Edita Vernickaite1, Judita Zeideryte1, Gabriele Karvelyte2, Gabija Stachneviciute2, Darius Stukas2, Zilvinas Dambrauskas2, Daiva Tomkute-Luksiene1
1Research and development department. TRATE, UAB, Kaunas (Kauno Apskritis) - Lithuania, 2Institute for digestive research, Laboratory of surgical gastroenterology. Lithuanian University of Health Sciences, Kaunas (Kauno Apskritis) - Lithuania
Silver-doped calcium hydroxyapatites (Ag-CHA) were synthesized and optimized for tissue engineering applications, focusing on controlled antimicrobial and cellular response. Ag-CHA compounds with varying silver concentrations were prepared via wet precipitation methods, including microwave (MW) and ultrasound (US) assisted synthesis. The influence of subsequent thermal annealing on crystallinity and crystallite size was also investigated. Structural characterization was performed using X-ray diffraction, scanning electron microscopy/energy dispersive X-ray spectroscopy, inductively coupled plasma-optical emission spectrometry, Fourier-transform infrared spectroscopy, simultaneous thermal analysis, Brunauer–Emmett–Teller method and mercury intrusion porosimetry. Biological activity was investigated by subjecting MG-63 human osteosarcoma cells, S. Oralis, E. Coli and S. Salivarius bacteria to Ag-CHA in 96-well plates and measuring the metabolic activity by resazurin reduction assay.
Reaction temperature was found to be an important factor, with a higher silver incorporation rate into target compounds observed at 80°C compared to 60°C. Furthermore, a rapid addition of diammonium hydrogen phosphate into the solution of silver nitrate and calcium nitrate and subsequent MW irradiation resulted in a lower doped-silver concentration compared to slow precursor addition using conventional wet precipitation. The biological activity assays showed an ambiguous, Ag concentration dependent effect both for cells and bacteria. Annealing significantly increased the antibacterial activity of the compounds compared to non-thermally treated analogues. However, this enhanced activity was coupled with a higher cytotoxicity toward the human osteosarcoma cells.
While the synthesis process shows promise, Ag-CHA shows ambiguous biological activity results suggesting that optimal synthesis conditions are necessary to balance antimicrobial efficacy and cytocompatibility.
Acknowledgment: The authors are grateful to the European Regional Development Fund (Project No. 02-048-K-0011) for the financial support for this research.
Key words: silver-doped hydroxyapatite, antibacterial activity, biomaterials, tissue engineering.
Effect of zinc content on physicochemical characteristics and biological activity of synthesized Zn-doped hydroxyapatite
Judita Zeideryte1, Edita Vernickaite1, Gabriele Karvelyte2, Gabija Stachneviciute2, Darius Stukas2, Zilvinas Dambrauskas2, Daiva Tomkute-Luksiene1
1Research and development department. TRATE, UAB, Kaunas (Kauno Apskritis) - Lithuania, 2Institute for digestive research, Laboratory of surgical gastroenterology. Lithuanian University of Health Sciences, Kaunas (Kauno Apskritis) - Lithuania
Introduction
Hydroxyapatite (HAp) is a bioceramic material widely used for bone regeneration due to its excellent biocompatibility and chemical similarity to natural bone mineral. However, limited osteoinductivity, antibacterial activity and relatively low mechanical strength motivate the incorporation of dopant ions to improve functional properties. In the present work Zn-doped HAp was synthesized to investigate the influence of dopant content on structural incorporation and its potential advantages in biomedical applications.
Methods
HAp was synthesized by a wet chemical precipitation method, including microwave-assisted synthesis. Zinc was introduced at varying concentrations. The obtained materials were characterized using X-ray diffraction, scanning electron microscopy-energy dispersive x-ray spectroscopy, inductively coupled plasma-optical emission spectrometry, Fourier-transform infrared spectroscopy, and simultaneous thermal analysis. The biological activity of the synthesized compounds was assessed by subjecting MG-63 human osteosarcoma cells and bacterial strains (S. Oralis, E. Coli, and S. Salivarius) to the Zn-doped HAp in 96-well plates, and the resulting metabolic activity was subsequently measured using the resazurin reduction assay.
Results
The results confirmed the formation of a single-phase apatite structure. The Ca/P ratios exceeded the stoichiometric value of 1.67. HAp characterization indicated successful Zn2+ incorporation, however the zinc content was slightly lower than the nominal precursor ratio, suggesting partial retention in solution. Biological activity studies on Zn-doped hydroxyapatite demonstrated enhanced antibacterial effects against S. Oralis, E. Coli, and S. Salivarius, and favourable, dose-dependant effect to viability of human osteosarcoma cell line compared to hydroxyapatite.
Conclusion
The chosen synthesis parameters provide a biologically active, Zn2+ incorporated HAp which demonstrates an enhanced antibacterial effect and a favourable effect to viability of human cells, showing promising results as a bioactive material.
Acknowledgement
Research is partially financed by European Regional Development Fund (Project No. 02-048-K-0011).
Keywords: zinc-doped hydroxyapatite, antibacterial activity, bone graft.
Patient-specific intestinal models of IBD via multimodal imaging-guided 3D bioprinting
Florencia Diaz1, Zhe Wang1, Matthias Schewe2, Christine Selhuber-Unkel1, Federico Colombo1
1Institute for Molecular Systems Engineering and Advanced Materials. Heidelberg University, Heidelberg (Baden-Wberg Bayern) - Germany, 2Department of Medicine II. Universitätsmedizin Mannheim, Universität Heidelberg, Mannheim (Baden-Wberg Bayern) - Germany
Inflammatory Bowel Diseases (IBD), characterized by the localized inflammation of different regions of the intestine and colon, have been estimated to affect between 10 and 40 per 100.000 people in Europe, with an increasing burden over the last few decades [1].
Despite extensive clinical research, in vitro models that accurately replicate the structural and mechanical complexity of inflamed intestinal tissue remain limited. Current organoid [2] systems fail to capture patient-specific variations in fibrosis, tissue stiffness, and collagen organization, hindering progress in personalized drug testing and tissue engineering approaches.
In this work, we’ve analyzed the structural differences between inflamed and healthy pairs of intestinal biopsies obtained from the same patient. The collagen architecture of the snap-frozen samples was analyzed using second harmonic imaging microscopy (SHIM), a non-destructive technique that enables the formation of three-dimensional images of the collagen content encompassing the entire thickness of the sample. Simultaneously, the samples were scanned using a Brillouin microscope to investigate the gradients of viscoelastic properties resulting from the fibrotic and healthy regions of the intestine.
3D reconstructions were obtained from this data, and subsequently 3D printed using 2-photon polymerization with biocompatible hydrogel inks that match the stiffness and architecture of the native tissue. These patient-specific models support long term cell attachment and proliferation, and could provide a mechano-physiologically relevant platform for advanced 3D cell culture and drug testing, with broad applicability in tissue engineering and regenerative medicine.
References
[1] - Caron B, et al. Epidemiology of Inflammatory Bowel Disease across the Ages in the Era of Advanced Therapies. J Crohns Colitis. 2024 Oct 30;18.
[2] - Ray, K. Next-generation intestinal organoids. Nat Rev Gastroenterol Hepatol 17, 649 (2020).
A vertically integrated system for tracking and assessing cell-cycle-aware phenotypes under confinement
Melissa Pezzotti1, Eloisa Torchia1, Julius Zimmermann1, Sara Rigolli1, Alessandro Enrico1, Moises Di Sante2, Francesco Pasqualini1
1Department of Civil Engineering and Architecture. University of Pavia, Pavia (Lombardia) - Italy, 2Department of Internal Medicine and Medical Therapy. University of Pavia, Pavia (Lombardia) - Italy
Decoding how cells coordinate migration, cytoskeletal remodeling, and cell-cycle (CC) progression at engineered interfaces requires high-resolution, time-resolved imaging and analytical frameworks that resolve mechanically driven transitions. Foundational work showed that adhesion geometry and cytoskeletal tension dictate proliferative commitment1, and spatial confinement has since emerged as a potent regulator of CC fidelity. Prolonged geometric restriction increases aberrant CC states, constrains proliferative expansion, and promotes slow-cycling, drug-tolerant phenotypes2. This positions CC–migration coupling at the center of the “go-or-grow’’ balance and frames confinement as both a mechanistic vulnerability and a potential driver of therapy resistance.
Here we present a vertically integrated system unifying multiplexed CC reporters, photopatterned ECM islands, fabrication-to-microscopy registration, and high-content longitudinal imaging. A four-reporter HT1080 line (LifeAct-EGFP, RFP-tubulin, CFP-G1, iRFP-S/G2/M) enables simultaneous structural and CC readouts³. Using adhesive islands of 10000, 2500, and 625 µm2, we quantify how confinement reshapes CC fidelity, cytoskeletal organization, and motility at single-cell resolution. Our Python-based Fab2Mic pipeline converts fabrication layouts into imaging coordinates, ensuring reproducible multi-position acquisitions across engineered interfaces.
Dynamic tracking revealed a strong confinement dependence: CC abnormalities increased from 17% in free space to 78% at 625 µm2, driven by prolonged G1 and confinement-specific S/G2/M-G1 slippage. These abnormal states were invisible to static snapshots and correlated with reduced motility and altered cytoskeletal remodeling. Together, this framework advances real-time, high-speed, AI-augmented phenotyping of cells at material interfaces and provides a scalable route to interrogate how spatial constraints regulate the coupling of proliferation and migration.
1. Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M. & Ingber, D. E. Geometric control of cell life and death. Science 276, 1425–1428 (1997).
2. Hunter, M. V. et al. Mechanical confinement governs phenotypic plasticity in melanoma. Nature 1–11 (2025)
3. Di Sante, M. et al. CALIPERS: Cell cycle-aware live imaging for phenotyping experiments and regeneration studies.
Coupling a bioartificial tendon/liver on chip to evaluate drug toxicity: a feasibility study
Vigneron Pascale1, Ceron Rozenn1, Jellali Rachid1, Legallais Cécile1
1BMBI. Université de Technologie de Compiègne, Compiègne (Picardie) - France
Some medications, such as corticosteroids, statins, or quinolones, are known to induce tendinopathies as adverse side effects, notably due to the alteration of collagen I, the main protein in the tendon extracellular matrix, and to tenocyte dysfunction [1]. These effects may arise from the parent drug or from metabolites produced after hepatic biotransformation [2]. To date, no test is available to evaluate the iatrogenic effects of drugs on tendon in vitro. We thus aimed to develop a new in vitro model to evaluate the effects of xenobiotics on tendon by adapting the parallelized platform allowing co-culture developed at University of Compiègne [3]. Coupling two compartments in a closed-loop microfluidic circuit—one containing an in vitro engineered tendon and the other a biochip seeded with HepG2C3A hepatic cells—enables the analysis of drug effects after liver metabolization. Bioartificial tendons were obtained from human mesenchymal stem cells (MSCs) differentiated into tenocytes on polycaprolactone (PCL) electrospun scaffolds under controlled mechanical stimulation using CellScale mechanical stimulation system. Cells aligned along the PCL fibers and adopted a tendon-like morphology with elongated nuclei. Tendon markers tenomodulin, scleraxis, and collagen I, were confirmed by immunostaining. Dexamethasone and atorvastatin were used as test compounds in the co-culture model. First analyses showed an increase in cell mortality measured via LDH activity, a decrease in collagen I expression in tendon cells, and a destabilization of the cell layer. Although preliminary, our results are encouraging and support continued development of a non-animal alternative method integrating inter-organ crosstalk to evaluate drugs that could induce tendinopathy.
[1] Bolon, Toxicologic Pathology, 2017
[2] Shaoyi Sun et al Bioorganic & Medicinal Chemistry Letters, 2021
[3] Madiedo-Podvrsan et al., Toxicology in Vitro, 2023
Ultrasound-activated 4D scaffolds for selective cell differentiation
Maite Garcia-Hermosa1, Hector Lafuente1, Sandra Camarero-Espinosa1
1BioSmarTE. POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain
Tissue engineering is a promising solution to treat osteochondral defects. Yet, it fails to promote the development of a well-structured and functional tissue. This is mainly due to the “static” character of traditional scaffolds while tissue development is a dynamic process. Moreover, the application of physical stimuli to cell cultures has been shown to modulate cell differentiation.
Ultrasound are pressure waves that can be used to make scaffolds deflect, transmitting this deformation as a load to surrounding cells. Exploiting this concept, we have developed dynamic scaffolds based on PLA:PCL blends that can be activated on demand through ultrasound stimulation. A computational model was developed to optimize these stimuli by analyzing material deflection across different ultrasound frequencies. The computational model confirmed that scaffolds achieved the maximum deflection under low-frequency conditions.
Mechanical loading of human mesenchymal stem cells (hMSC) cultured on scaffolds activated for 30 minutes over 1, 7, and 14 days was demonstrated qualitatively and quantitatively through analysis of Lamin A/C localization, chromatin expression, the formation of stress-fibers and nuclear morphology. Remote scaffold stimulation through ultrasounds at varying frequencies and duration times, promoted overall cell proliferation and matrix deposition in seeded hMSCs. Variations on scaffold printing pattern enabled control over deflection amplitude and, thus, the selective activation of key mechanoreceptors that lead, downstream, to chondrogenic and osteogenic differentiation.
This work was supported by the grants (Project No. AEI/10.13039/501100011033-46 114901RA-100 and AEI/10.13039/501100011033-47 153333OB-I00) of the Ministry of Science and Innovation (MICINN), State Investigation Agency (AEI) (Project No. PID2020-114901RA-I00 and PID2023-153333OB-I00), the University of the Basque Country (GIU21/033), the Ministry of Science and Innovation of the Government of Spain (“María de Maeztu” Programme for Center of Excellence in R&D, grant CEX2023-001303-M funded by 55 MICIU/AEI/10.13039/501100011033) and the Ramon y Cajal RYC2023-044860-I.
[1] T. Vinikoor et al., “Injectable and biodegradable piezoelectric hydrogel for osteoarthritis treatment,” Nature Communications 2023 14:1, vol. 14, no. 1, pp. 1–18, Oct. 2023, doi: 10.1038/s41467-023-41594-y.
[2] A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, “Matrix Elasticity Directs Stem Cell Lineage.”
3D-printed skin dressings manufactured with fish skin collagen enriched with unsaturated fatty acids for enhanced wound healing
Flavia De Oliveira1, Mariana C. Simões1, Danielle S. Reis1, Pietra M. S. Costa1, Marcelo Assis1, Amanda De Souza1, Lais C. S. Silva1, Mirian Bonifacio1, Karolyne S. J. Sousa1, Giovanna A. Grasser2, Elson Longo2, Guadalupe Rivero3, Ana C. M. Rennó1
1Departamento de Biociências. Universidade Federal de São Paulo, Santos (Sao Paulo) - Brazil, 2Departamento de Química. Universidade Federal de São Carlos, São Carlos (Sao Paulo) - Brazil, 3Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA, CONICET-UNMdP), Mar del Plata (Buenos Aires) - Argentina
Objective: To develop and characterize 3D-printed collagen-based skin dressings combined with unsaturated fatty acids (FA) and to evaluate the in vivo effects of these components on wound healing morphology in rats. Material and Methodology: Marine collagen was extracted from Syacium spp. and mixed with sodium alginate and a blend of FA, resulting in a bioink used to manufacture dressings through 3D printing. The material was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Cytocompatibility was assessed in vitro using the Alamar Blue assay with L929 murine fibroblast cells and HDFn human dermal fibroblasts. In vivo evaluation was conducted in Wistar rats subjected to skin incision. The animals were distributed into three groups: control group (CG); treated group one (TG1), receiving fish-skin collagen dressings; and treated group two (TG2), receiving fish-skin collagen dressings combined with FA. Morphological assessment of wound healing was performed 14 days post-injury using Hematoxylin and Eosin staining for histopathological analysis and Sirius Red staining for collagen evaluation. Results: FTIR confirmed the presence of collagen, alginate, and lipid components, each showing characteristic peaks. SEM analysis revealed that the collagen–alginate matrix exhibited a porous structure, which became less porous with the addition of FA. The Alamar Blue assay demonstrated enhanced cell adhesion and proliferation, confirming the biocompatibility of the material. Histopathological analysis revealed a re-epithelialized hyperplastic epidermis in all groups. The dermis of TG1 and TG2 showed abundant collagen fibers undergoing organization, whereas the CG displayed inflammatory infiltrate. In the papillary dermis of TG2, the presence of hair follicles indicated a more advanced stage of skin regeneration. Conclusions: The 3D-printed collagen-based dressings incorporating FA exhibited a favorable porous structure, biocompatibility, and stimulatory effects on cellular activity. The in vivo morphological findings demonstrated enhanced dermal collagen deposition and accelerated skin regeneration.
Acknowledgments. FAPESP (2024/18524-0);CNPq (310139/2025-2).
Piezoelectric PLA/BCZT composite scaffolds with optimized gyroid architecture for bone regeneration
Ricardo Donate1, Álvaro Quintana1, Rocío Moriche2, Rubén Paz1, María Jesús Sayagués3, Joaquim Miguel Oliveira4, Mario Monzón1
1Departamento de Ingeniería Mecánica. University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria (Las Palmas) - Spain, 2Departamento de Física de la Materia Condensada. Universidad de Sevilla, Sevilla - Spain, 3CSIC-US. Instituto de Ciencia de Materiales de Sevilla, Sevilla - Spain, 4ICVS/3B's - PT Government Associate Laboratory. I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics of University of Minho, Guimarães (Braga) - Portugal
Recent advances in Tissue Engineering and stem cell-based therapies have considerably improved clinical strategies for bone repair. Nevertheless, currently available biomaterials still lack the multifunctionality required to achieve effective regeneration of complex osseous defects. One promising approach to overcome this limitation is the development of advanced piezoelectric scaffolds capable of stimulating bone tissue growth through electromechanical cues.
In this work, innovative piezoelectric composite scaffolds were fabricated by combining a polylactic acid (PLA) matrix with a lead-free ceramic, xBaZr0.2Ti0.8O3–(1-x)Ba0.7Ca0.3TiO3 (BCZT), recognized for its relatively high piezoelectric response. BCZT powders were synthesized via mechanochemical processing, confirming the formation of a pseudocubic perovskite structure and identifying the composition with x = 0.4 (Ba0.82Ca0.18Zr0.08Ti0.92O3) as optimal. The BCZT powder obtained from the milling process was subjected to heat treatment at 1450°C for 4 hours. A PLA/BCZT 90/10 (%v/v) composite was extruded into filaments and processed by material extrusion (MEX) to obtain scaffolds with gyroid-type architectures and pore sizes ranging from 0.3 to 0.7 mm. Data from morphological and mechanical characterization were fed into a MATLAB-based genetic algorithm, which determined the 0.7 mm pore size as optimal for maximizing surface area and interconnectivity.
Enzymatic degradation assays using Proteinase K enzymes, together with SEM analysis and mechanical testing, revealed controlled degradation behavior and adequate structural integrity of the manufactured composites. Furthermore, cell viability assays performed in a perfusion bioreactor confirmed the viability of human osteoblastic cells seeded on the PLA/BCZT 90/10 scaffolds. Overall, the combined structural, mechanical, and biological results demonstrate the potential of these piezoelectric scaffolds for advanced bone tissue engineering applications.
Acknowledgements
This contribution is part of the RENOVATE project funded by the European Union’s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101227121 (101227121 - RENOVATE - HORIZON-MSCA-2024-DN-01) and the PIZAM project (PID2020-117648RB-I00) funded by MCIN/AEI/10.13039/501100011033.
An individual-based modeling framework for magnetic biofabrication using tissue spheroids
Maya Guilliams1, Konstantinos Ioannidis2, Dimitrios Lefas2, Dimitrios Sakellariou3, Ioannis Papantoniou2, Bart Smeets1
1MeBioS. KU Leuven, Leuven (Brabant) - Belgium, 2Skeletal Biology and Engineering Research Center. KU Leuven, Leuven (Brabant) - Belgium, 3cMACS. KU Leuven, Leuven (Brabant) - Belgium
Large bone defects remain a significant clinical challenge. Bottom-up tissue engineering approaches that use spheroids derived from human periosteum-derived stem cells (hPDCs) are a promising way to regenerate bone tissue by assembling cellular building blocks. Among biofabrication strategies, magnetic biofabrication is a rapid, scalable, and controllable method of assembling magnetized spheroids while delivering mechanical cues that promote tissue maturation.
To better understand and control this process, we developed an individual-based model that explicitly represents individual spheroids and predicts the structure and evolution of hPDC-derived tissue assembloids under magnetic influence based on a mechanical force balance. The model captures early-stage aggregation and long-term fusion dynamics, revealing how magnetic forces shape tissue architecture. Based on parameters such as magnetic nanoparticle loading, spheroid size, spheroid number, magnet geometry, and the differentiation stage of the spheroids, the model can predict both the final assembled area and the rate of tissue formation. Moreover, the computational model provides predictions for the distribution of mechanical stress in the assembloid. The predicted stress consists of a vertical component due to the direct magnetic force on individual spheroids but also a concentrated radial component that scales with the size of the assembloid. Since hPDCs are highly mechanosensitive, these mechanical cues are expected to enhance endochondral ossification by mimicking the biomechanical environment that guides native bone development.
This modeling framework provides new insights into the coupling between magnetic forces, cell behavior, and tissue-level mechanical properties in magnetic biofabricated constructs. Looking ahead, this framework will serve as a foundation for inverse modeling approaches that identify optimal magnetic field parameters for fabricating tissue assembloids with predefined structures and mechanical characteristics.
Real-time insights into biomaterial-host interactions: intravital imaging of the cellular and infectious response to wettability-tuned PDMS coatings
Elles Boonstra1, Lisa Tromp2, Paul Jutte1, Henny Van Der Mei2, Theo Van Kooten2, Patrick Van Rijn2, Jelmer Sjollema2
1Orthopedics. University Medical Center Groningen, Groningen - The Netherlands, 2Biomaterials & Biomedical Technology. University Medical Center Groningen, Groningen - The Netherlands
The success of implanted biomaterials is influenced by the foreign body response (FBR), a complex multifaced immune response that governs fibrotic encapsulation. Moreover, implant success is also hindered by difficult-to-eradicate biomaterial-associated infections (BAI). To optimize implant design and increase their success, a deeper understanding of the interactions between biomaterial surfaces and host cells is crucial.
This study investigated the impact of surface wettability on the biocompatibility og polydimethylsiloxane (PDMS) coatings, modified via plasma treatment to yield 3 distinct wettability profiles. Both antimicrobial properties and cellular responses of fibroblasts and macrophages were evaluated in vitro. The coatings significantly modulated myofibroblast differentiation and bacterial cell adhesion, whereas macrophage polarization exhibited less variation across the different coatings.
To translate these findings to in vivo, a novel “FBR-through-a-window” model was applied, enabling longitudinal two-photon imaging of immune cell dynamics and fibrotic encapsulation using an imaging window as the foreign body itself. This approach revealed distinct temporal and spatial patterns of neutrophils, macrophages, fibroblasts and collagen for the different coatings. The induction of a biomaterial-associated infection influenced the spatiotemporal response, but differences in biofilm formation were less pronounced.
These findings of this study underscore the pivotal role of material design in modulating the FBR and infection outcomes. Moreover, they highlight the value of intravital imaging for elucidating dynamic cell-material interactions, offering a powerful platform to inform the rational design of next-generation biomaterials with enhanced biocompatibility and infection resistance.
Acknowledgements: This publication is part of the DARTBAC project (with project number NWA. 1292.19.354 of the research programme NWA-ORC which is (partly) financed by the Dutch Research Council (NWO).
Hydrogels derived from cell culture extracellular matrix for tissue engineering vascularization purposes
Rogério P. Pirraco
de 3B’s Research Group, I3Bs, University of Minho, Guimarães (Braga) - Portugal
Vascularization is widely recognized as a critical step to ensure the adequate performance of Tissue Engineering (TE) constructs. Several vascularization strategies have been proposed by researchers with the most common involving the use of endothelial and other ancillary cells combined with vasculogenic/angiogenic growth factors, to induce the in vitro formation of a prevascular network in the construct. These approaches have achieved varying degrees of success, with researchers exploring different combinations of cell types and growth factor cocktails. The role of the extracellular matrix (ECM) – or ECM surrogate - on the formation of such prevascular networks is often underemphasized. This is because, typically, the ECM surrogate or scaffold used in a TE strategy primarily recapitulates the bulk properties of the target tissue, which may not be ideal for vascularization. Therefore, given the importance of this process, ensuring the compatibility of scaffold properties with vascularization is essential.
Hydrogels are increasingly used in TE due to their biophysical and biochemical similarity to native ECM. This bio-similarity is even more evident when the hydrogel is derived from solubilized ECM, offering tissue-specific biochemical cues that can significantly impact cells’ phenotype and function. We propose the use of ECM derived from cultured cell sheets to produce hydrogels especially supportive of the vascularization process. Furthermore, we explore different protocols that target the main caveats related with ECM-derived hydrogels relative to synthetic counterparts, namely their limited mechanical properties and susceptibility to enzymatic degradation, demonstrating that these disadvantages can be mitigated while preserving the bioactivity edge.
Acknowledgements
EU Horizon 2020 research and innovation programme under the ERC StG CapBed (805411). UID/PRR/50026/2025: https://doi.org/10.54499/UID/PRR/50026/2025.
Towards GMP-compatible biofabrication and MRI-based quality monitoring of implantable adipose tissue constructs
Yilbert Gimenez1, Gilmus Valernst2, Cowles Elliott3, Dumoulin Chloe4, Essayan Lucie3, Semet Vincent2, Cabrera Michel2, Magalon Jérémy5, Lambert Simon2, Petiot Emma3, Christophe Marquette3
1Univ Lyon, Université Claude Bernard Lyon 1, INSA Lyon, Ecole Centrale de Lyon, CNRS, Ampère, UMR5005, Villeurbanne (Auvergne-Rhone-Alpes) - France, 2Univ Lyon, Université Claude Bernard Lyon 1, INSA Lyon, Ecole Centrale de Lyon, CNRS, Ampère, UMR5005, Villeurbanne (Auvergne-Rhone-Alpes) - France, 33d.FAB, CNRS, INSA, CPE-Lyon, UMR5246, ICBMS, Université Claude Bernard Lyon 1, Villeurbanne (Auvergne-Rhone-Alpes) - France, 4Cell Culture and Therapy Laborator. Assistance Publique Hôpitaux de Marseille, Marseille (Provence-Alpes-Cote d Azur) - France, 5Cell Culture and Therapy Laboratory. Assistance Publique Hôpitaux de Marseille, Marseille (Provence-Alpes-Cote d Azur) - France
Relevance to Theme Biofabrication, Bioprinting & Advanced Manufacturing: This study presents an integrated platform for GMP-compatible bioproduction and non-invasive monitoring of living tissues through confined bioprinting and low-field MRI.
Impact/Novelty: We introduce a closed and MRI-compatible bioreactor enabling large-scale, sterile, and monitorable biofabrication of implantable microvascularized adipose tissue. A proof of concept demonstrates the generation of functional adipose tissue directly from the stromal vascular fraction (SVF) as the sole biological input.
Introduction: The clinical translation of 3D bioprinting requires production environments that ensure sterility, scalability, and quality control compliant with Good Manufacturing Practice (GMP). Simultaneously, the maturation of complex tissues calls for non-invasive tools to track structure, perfusion, and differentiation. To address these dual constraints, we developed a confined bioprinting and culture platform coupled with low-field (0.3 T) magnetic resonance imaging (MRI), enabling longitudinal assessment of tissue formation within a sealed and sterile environment.
Methods/Approach: The proposed soft, MRI-transparent bioreactor integrates microextrusion bioprinting, controlled perfusion, and culture fluid management. Its flexible polymeric envelope maintains sterility while minimizing magnetic susceptibility artifacts, allowing direct MRI monitoring without removing the construct. Constructs of at least 25 mL were fabricated using isolated human SVF or adipose derived mesenchymal stem cells, cultured under perfusion for several weeks, and monitored with customized 0.3 T MRI sequences to evaluate flow distribution and tissue organization.
Results: The closed bioreactor maintained sterility and tissue perfusion with culture media. MRI specific sequences enabled the monitoring of perfusion path and tissue morphology changes. Clear histological and biochemical evidences of adipogenesis and microvascular formation, confirmed the generation of adipose tissue from SVF within the MRI-compatible bioreactor.
Conclusion: This approach establishes the feasibility of large-scale, GMP-compatible adipose tissue production with integrated MRI-based quality monitoring, paving the way toward automated and traceable manufacturing of implantable soft tissues.
Magnetic torque-based stretching platform for 3D human functional engineered skeletal muscle tissues
Carolina Rodríguez-Gallo1, Sabater Arcis Maria2, Fernandez-Garibay Xiomara1, Fernandez-Costa Juan M.3, Ramón Javier1
1Biosensors for bioengineering. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 2Biosensors for bioengineerinf. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 3Biosensors for bioenginnering. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain
Three-dimensional (3D) tissue bioengineering has emerged as a powerful strategy for constructing physiologically relevant in vitro models. By controlling the cellular microenvironment, these systems promote morphogenesis, differentiation, and functional maturation [1]. Among them, 3D human skeletal muscle models—composed of myoblasts encapsulated in biomaterials compacted around two flexible, biocompatible pillars—are widely used to study muscle physiology. Matrix contraction aligns cells along the pillar axis, leading to the formation of multinucleated myofibers capable of generating measurable contractile forces. The deflection of the pillars upon electrical stimulation enables quantitative assessment of tissue functionality. Such systems have successfully recapitulated markers of myogenic differentiation and sarcomere organization, modeled neuromuscular pathologies [2], and identified therapeutic targets.
However, the role of mechanotransduction—how cells sense and respond to mechanical stimuli—remains insufficiently explored in these platforms. To address this gap, we developed a novel magneto-responsive system that enables dynamic and tunable mechanical stretching of 3D muscle microtissues. Ferromagnetic nanoparticles embedded within PDMS pillars generate controlled bending (up to 400 µm) under an external magnetic field through magnetic torque [3]. By modulating the field strength, we precisely regulate the applied strain and define distinct mechanical loading regimes. This approach allows systematic investigation of how dynamic mechanical cues influence muscle differentiation, gene expression, and fiber-type specialization via mechanotransductive pathways. Ultimately, this platform provides a versatile tool for dissecting the interplay between mechanical forces and muscle function, advancing both fundamental mechanobiology and the development of biomimetic muscle models.
[1] Nature Reviews Bioengineering 1, 545–559 (2023)
[2] Biofabrication 15 045024 (2023).
[3] Microsystems & Nanoengineering 9, 153 (2023)
Fabrication of tungsten-doped manganese nanoflowers and evaluation of their photothermal therapeutic efficacy
Jeong Man An1, Yoo-Wook Kwon2, In-Kyu Park3, Yong-Kyu Lee1
1Korea National University of Transportation; Chungju-si(Chungcheongbuk-do); Republic of Korea, 2Biomedical Research Institute, Seoul National University Hospital, Department of Internal Medicine, Seoul National University College of Medicine Seoul National University Hospital, Jongno-gu, Seoul, Republic of Korea, 3Chonnam national university hwasun hospital; Hwasun-gun(Jeonnam-Do); Republic of Korea
Pristine manganese nanoflowers (MNF) have potential applications in photothermal therapy. The objective was to fine-tune the morphology to reach the desired temperature while avoiding permanent damage, a goal successfully accomplished through tungsten doping. In this study, we effectively incorporated tungsten metal into the flower morphology, resulting in defects and the formation of secondary mesopores within the channel, which subsequently led to a temperature reduction of nearly 20°C. The flawed surface clearly exhibited a peak in the mesopore-micropore region, which can be linked to the introduction of foreign metal doping. This approach to doping can be extensively employed to adjust the molecular structure during the application of photothermal therapy treatment.
An optimal construct for trans-affirmative vaginoplasty
David Brownell1, Matisse Duval1, Elissa Elia1, Félix-Antoine Pellerin1, Stéphane Chabaud2, Alexis Laungani3, Eric Philippe4, Stéphane Bolduc1
1Regenerative medicine division (LOEX), CHU de Québec-Université Laval research centre, Québec City, QC Canada. Department of Surgery, Faculty of Medicine, Université Laval, Québec City, QC G1V 0A6, Canada, Quebec - Canada, 2Regenerative medicine division (LOEX). Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada, 3GrS Montréal. Division science et snseignement, Département de chirurgie plastique, Université de Montréal, Montréal, QC Canada, Montreal (Quebec) - Canada, 4Laboratoire d'anatomie. Department of Surgery, Faculty of Medicine, Université Laval, Québec City, QC G1V 0A6, Canada, Quebec - Canada
Aim & objectives:
Gender-affirming surgery (GAS) significantly improves psychological well-being in transgender individuals. In transfeminine (TF) patients, vaginoplasty usually employs penile skin inversion, but the keratinized, dry, and heterotopic nature of this tissue often leads to complications. To address these limitations, we developed a novel tissue-engineered vaginal substitute using cells from the fossa navicularis (FN), a region of the distal male urethra sharing embryological origins and key features with vaginal epithelium, including glycogenation, lubrication, and pathogen resistance. We further explored the use of allogenic vaginal fibroblasts to enhance construct functionality.
Methods:
FN epithelial cells and fibroblasts were isolated from biopsies of 14 TF patients (ages 19-71) undergoing vaginoplasty. Enzymatic digestion protocols using dispase or thermolysin and collagenase ± elastase were compared to optimize cell yield. FN fibroblasts cultured with 2-phospho-L-ascorbate produced cell-assembled matrix sheets that were stacked to form stroma. FN epithelial cells were seeded, cultured submerged for one week, and at the air-liquid interface for three weeks to promote differentiation. Constructs using vaginal fibroblasts and/or estradiol supplementation were compared to vaginal and urethral controls. All constructs were grafted on silicone stents and implanted subcutaneously in nude mice. Histological and immunophenotyping analyses included Masson’s Trichrome, PAS, and epithelial markers.
Results:
Dispase and collagenase + elastase yielded optimal cell recovery. FN constructs formed stratified squamous epithelia with glycogenation and estrogen receptor expression, resembling vaginal mucosa. Vaginal fibroblast stromata enhanced vaginal marker expression (keratin 10, mucin 1), while estradiol increased stromal thickness and improved epithelial differentiation. In vivo, constructs maintained morphology and p63+ basal stem cell populations.
Conclusions:
This is the first biologically relevant substitute for transfeminine vaginoplasty. This autologous, fossa navicularis-derived vaginal-like construct has the potential to improve long-term surgical outcomes, particularly in patients with limited penile tissue due to puberty blocker use.
Photobiomodulation enhances antioxidant defense, mitochondrial recovery, and myogenic differentiation in a 2D skeletal muscle injury model
Haohua Liu1, Sandy Macrobert1, Umber Cheema1, Darren J Player1
1Division of Surgery and Interventional Science. University College London (UCL), London (London, City of) - United Kingdom
Aim and Objective:
Acute skeletal muscle injury induces oxidative stress, mitochondrial dysfunction, and impaired regeneration. Photobiomodulation therapy (PBMT) is a promising biophysical approach to enhance cellular recovery. This study compares preconditioning and post-injury PBMT regimens to assess their effects on antioxidant capacity, mitochondrial function, and myogenic differentiation in C2C12 myoblasts.
Material and methodology：
A 2D C2C12 myoblast injury model was established using 12% BaCl2 for 6 hrs. Groups included: (1) injury-only control, (2) PBMT pre-conditioning (18 mW/cm2, 5J/cm2, 280 s, once daily × 3 days), and (3) PBMT post-injury (12 mW/cm2, 3J/cm2, 250 s, once daily × 3 days). Irradiation used an LED at 810 nm continuous mode. AlamarBlue, T-AOC, MitoTracker, Phalloidin/DAPI, and MHC8 staining were used to evaluate metabolism, antioxidant capacity, mitochondria, cytoskeleton, and differentiation at day 2 and 5 post-injury.
Results:
PBMT significantly enhanced metabolic activity and antioxidant capacity in both preconditioning and post-treatment conditions compared with injury-only controls (AlamarBlue: 1.42 ± 0.08 vs 1.00 ± 0.05; T-AOC: 1.58 ± 0.11 vs 1.00 ± 0.07; p < 0.01). The preconditioning group demonstrated the highest T-AOC values and earlier mitochondrial recovery with stronger MitoTracker fluorescence intensity (1.6-fold increase, p < 0.01). Post-injury PBMT promoted sustained mitochondrial network integrity through day 10. Phalloidin/DAPI imaging showed improved cytoskeletal alignment, with a 1.7 ± 0.2-fold increase in myotube width compared to injury-only controls (p < 0.01). MHC8 staining confirmed enhanced myotube maturation and fusion in PBMT-treated groups, with the post-treatment protocol showing the highest fusion index (74 ± 5% vs. 28 ± 3%, p < 0.01).
Conclusion:
PBMT at 810 nm enhances antioxidant capacity, restores mitochondrial, and promotes myogenic differentiation in a 2D myoblast injury model. These novel findings support PBMT as a potent biophysical modulator of oxidative stress and muscle regeneration following injury, providing a foundation for translation to 3D tissue-engineered systems and pre-clinical models.
Bioactive gelatin based hydrogels with extracellular vesicles for corpus spongiosum regeneration
Jiaxin Qin1, Isabelle Martinier1, Zhentao Xing1, Xiaohe Liu1, Laetitia De Kort2, Petra De Graaf1
1Urology. Regenerative Medicine Center Utrecht, Utrecht - The Netherlands, 2Urology. University Medical Center Utrecht, Utrecht - The Netherlands
Aim and Objective: Urethral stricture disease causes fibrotic narrowing and urinary obstruction. The most effective treatment is a urethroplasty, which in some cases requires autologous grafts. Current grafts restore the epithelium but fail to regenerate the corpus spongiosum (CS), a highly vascularized structure whose loss promotes fibrosis and stricture recurrence. Cell-free extracellular vesicle (EV) therapy delivers paracrine factors without cell-transplantation risks. This study aims to develop a growth factor functionalized gelatin methacryloyl (GelMA) hydrogel incorporating adipose-derived stem cell EVs (ADSC-EVs) to promote CS regeneration and enable personalized reconstruction via 3D bioprinting.
Materials and Methodology: GelMA (50% degree of functionality) was synthesized and functionalized with basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) through N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry. Growth-factor release was quantified by ELISA. Patient-derived ADSCs were isolated, characterized, and co-cultured with human umbilical vein endothelial cells (HUVECs) in unmodified or functionalized GelMA. ADSC-EVs were obtained by ultracentrifugation, characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and multiplex flow cytometry. Effects on HUVEC proliferation, migration, and tube formation by the ADSC-EVs were analysed.
Results: ADSCs expressed mesenchymal stem cell (MSC) markers and showed multipotent differentiation. In unmodified GelMA, ADSCs enhanced HUVEC tube formation, which typically occurred after 5-7 days, whereas in bFGF-GelMA, HUVECs formed 3D networks within 3 days. ELISA showed controlled release of bFGF/VEGF. ADSC-EVs (∼150 nm) exhibited cup-shaped morphology with CD9/CD63/CD81 and adhesion markers (CD29, CD44, CD49e). Functionally, they enhanced HUVEC proliferation, migration, and tube formation (p < 0.01).
Conclusions: ADSCs are an effective EV source, and growth-factor-functionalized GelMA provides a promising bioactive platform. The GelMA-EV hydrogel offers a novel, cell-free strategy integrating growth-factor release with ADSC-EV signaling to induce vascularized CS-like tissue. Future work will include 3D bioprinting to fabricate CS-like scaffolds, extending urethral reconstruction beyond epithelial repair towards corpus spongiosum regeneration.
Glucocorticoid responses in a degenerative human meniscus ex vivo culture model
Sofia Pilão1, Neila Ouldali1, Luisa De Roy1, Anita Ignatius1, Jan Tuckermann2, Andreas Seitz1, Graciosa Quelhas Teixeira1
1Institute of Orthopedic Research and Biomechanics, Ulm University Hospital, Ulm (Baden-Wberg Bayern) - Germany, 2Institute of Molecular Endocrinology and Physiology, University of Ulm, Ulm (Baden-Wberg Bayern) - Germany
Introduction/Objectives
Glucocorticoids (GC) are widely used anti-inflammatory and analgesic drugs for the treatment of meniscal injuries associated with inflammation and degeneration (Reichardt et al., 2021). However, their direct effects on meniscus tissue remain poorly understood. This study investigated the impact of GC treatment on degenerative human meniscus tissue cultures.
Methods
Degenerated human menisci (n=17, 5 male/12 female, age=73±7, Pauli score 2-3) were obtained from patients undergoing knee arthroplasty with ethical approval and informed consent. Explants were cultured up to 21 days under basal conditions, with IL-1β (10 ng/mL), or with IL-1β followed by methylprednisolone (MP, 1 µM). Time-dependent responses to IL-1β and MP were assessed up to 72 hours. Expression of pro-inflammatory (IL1B, IL6, IL8, PTGS2), catabolic (MMP1, MMP3), and GC-regulated (DUSP1, FKBP5, TSC22D3, NR3C1α) genes was analyzed by qPCR. DNA, glycosaminoglycans (GAGs), and collagen were quantified biochemically. IL-6 and MMP-3 production was examined via immunohistochemistry, and biomechanical properties were assessed by indentation testing. Statistics: Kruskal-Wallis test (significance, *p<0.05).
Results
IL-1β significantly upregulated pro-inflammatory and catabolic genes (p<0.05) as early as 6 hours, with effects persisting up to 21 days. MP treatment significantly upregulated GC target genes (p<0.05) and downregulated pro-inflammatory and catabolic markers (p<0.05) compared with IL-1β treatment alone, with effects sustained for up to 72 hours after administration. At protein level, MP reversed the IL-1β–induced increase in IL-6 and MMP-3 production (p<0.05). DNA, GAGs, and collagen content, as well as overall biomechanical properties, were unaffected.
Conclusions
IL-1β induced a pro-inflammatory and catabolic phenotype in human meniscus explants, which was effectively suppressed by MP, in line with previous studies in osteoarthritis (Kydd et al. 2007; Tian et al. 2018). Overall, this human meniscus ex vivo model was validated as a platform for investigating inflammatory responses associated with meniscus degeneration (Conaghan et al., 2018; Tammachote et al., 2016).
Synthetic mRNA-guided cell engineering to modulate the implant-tissue interface
Max Gerlein1, Martin Santocildes-Romero2, Santiago Castillo3, Hanin Alkhamis2, Katarzyna Polak-Kraśna2, Manfred Gossen2, Hanieh Moradian2
1Department of Biology, Chemistry, Pharmacy. Freie Universität Berlin, Berlin - Germany, 2Institute of Active Polymers. Helmholtz Zentrum Hereon, Teltow (Brandenburg) - Germany, 3Faculty of Engineering Science. University of Bayreuth, Bayreuth (Baden-Wberg Bayern) - Germany
The demand for effective and robust biomedical implants is steadily increasing in our aging society due to the multitude of life-threatening degenerative indications. The immune system plays a central role in orchestrating events at the biomaterial-tissue interface, where uncontrolled inflammation can trigger fibrotic encapsulation and ultimately lead to implant failure. Thus, strategies to modulate immune responses, shifting them from chronic inflammation toward constructive tissue integration, are essential to advance implant-based treatments. To promote constructive integration of implant, we aim to engineer immune cells at biomaterial interface to produce immunomodulatory factors using synthetic messenger RNA (mRNA). Given their central role in the foreign body reaction, macrophages were selected as a primary target for immunomodulation. Here, we systematically screened mRNA chemistries to identify formulations that are non-immunogenic yet support robust transgene expression. We evaluated immunogenicity by measuring the co-stimulatory surface markers and secretion of pro-inflammatory and antiviral cytokines. Our findings indicate uridine modifications as key to reducing innate immune activation, with 5-methoxyuridine (5moU) entirely abrogating these responses. This exploration of mRNA chemistry-dependent immune responses provides a reference for selecting optimal mRNA formulations for biomaterial-related applications. Future work will focus on integrating chemically modified mRNA–lipid nanoparticle formulations into degradable implant coatings to achieve localized and time-specific delivery. Collectively, this study lays the foundation for next-generation implants that can modulate immune responses and promote effective host integration.
2D and 3D modeling of Parkinson’s disease: lysosomal and pH dysregulation in 3K-SNCA neuron-like cells
Valentina Pavanati1, Donatella Di Lisa1, Valentina Onesto2, Helena Iuele2, Loretta Del Mercato2, Laura Pastorino1
1DIBRIS. Università degli studi di Genova, Genova (Italia) - Italy, 2CNR NANOTEC - Institute of Nanotechnology, Lecce (Puglia) - Italy
Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by neuronal degeneration and α-synuclein accumulation. Physiologically relevant in vitro models are essential for investigating the molecular mechanisms of PD, which remain unclear. In this study, we characterized an innovative PD model, the 3K-SNCA cell line that overexpresses α-synuclein, whose aggregation is a hallmark of PD. We compared it with the SH-SY5Y control line, commonly used in PD research, focusing on lysosomal dysfunction and intracellular pH alterations in two-dimensional (2D) and three-dimensional (3D) culture systems. Both SH-SY5Y and 3K-SNCA cells were differentiated into neuron-like cells and validated by immunofluorescence for MAP2 and α-synuclein, while lysosomal function was assessed using LysoTracker Red. The 3D model was developed by encapsulating cells in a thermogelling chitosan-based hydrogel, characterized by rheological testing. Intracellular pH was measured using ratiometric microsensors (FITC/Cy3), calibrated in both 2D and 3D systems within the pH range 4.5–7.5. Time-lapse (2D) and z-stack (3D) confocal imaging monitored sensor internalization and intracellular acidification, with quantitative analyses performed using ImageJ and GraphPad software. In 2D cultures, 3K-SNCA cells exhibited impaired lysosomal function, as shown by a significant reduction in LysoTracker fluorescence compared to SH-SY5Y cells. Calibration of the ratiometric microsensors confirmed their stability and linear responsiveness to pH variations within the range 4.5–7.5 in 2D and 5.5–7.5 in 3D systems, with the latter remaining stable for up to seven days. Rheological analysis demonstrated the compatibility between the chitosan-based hydrogel and the embedded sensors. In 2D, real-time tracking of intracellular pH revealed normal acidification in SH-SY5Y cells (pH 5.5–6.2) but markedly elevated pH values in 3K-SNCA cells (7.4–8.3). The 3D model reflected this trend, showing similarly increased intracellular pH levels (7–8) in 3K-SNCA cells. These results indicate that the 2D and 3D 3K-SNCA models successfully recreate lysosomal dysfunction and provide a more comprehensive model for studying PD, such as in the study of intracellular alkalization. The integration of ratiometric microsensors within chitosan-based constructs offers a multimodal and physiologically advanced approach for investigating Parkinson’s disease.
Early hypertrophic chondrocyte-like pellets trigger vascular network formation and invasion in a 3D in vitro endochondral callus model
Anita Jose1, Esther Wehrle1, Martin Stoddart1, Eric Farrell2, Sophie Verrier1
1AO Research Institute Davos, Davos (Graubunden) - Switzerland, 2ERASMUS Medical Center Rotterdam, Rotterdam (Zuid-Holland) - The Netherlands
Bone is a highly vascularised tissue with remarkable regenerative capacity. However, approximately 10% of fractures develop non-unions [1], often because of insufficient or excessive vascularisation [2]. Understanding how blood vessels develop and interact with the endochondral bone healing process is crucial for advancing non-union therapies.
To address this, we developed a 3D in vitro microvascular invasion model using PKH26-labeled human mesenchymal stem cell (hMSC) pellets primed with Transforming Growth Factor-β for 3, 7, or 14 days. These timepoints were chosen based upon existing open-source bulk sequencing data of pellets cultured under identical conditions [3] which showed early hypertrophy as shown by increased COL10A1 expression over time, while MMP13, VEGFA, and ALPL peaked at day 3 as compared to day 7 or 14. At each time point, pellets and their conditioned media (CM) were collected for analysis. To create the model, primed pellets were embedded in LunaGel hydrogel containing Green Fluorescent Protein-Human Umbilical Vein Endothelial Cells (GFP-HUVECs) and hMSCs, in a 90:10 ratio, and cultured in Endothelial Basal Medium for 21 days.
Histology and immunohistochemistry analysis showed rising proteoglycan and collagen X, II deposition between days 7 and 14. Cytokine analysis of pellet CM showed elevated angiogenic factors (VEGF, SDF-1, MCP-1, HGF, FGFs) at day 3, with high angiogenin levels, alongside increased matrix-remodelling factors (TIMP-1, TIMP-2, osteopontin, IL-6). The CM from all pellets effectively promoted endothelial network formation in 2D HUVEC assays on Matrigel. Confocal microscopy showed that day 3 primed pellets induced robust vascular networks and showed signs of pellet invasion, as compared to day 7 or 14 pellets.
Overall, our data shows that 3 days primed pellets present early hypertrophic-chondrocyte characteristics, support vascular network formation and show signs of invasion.
References
1. Holmes.D, 2017, Nature
2. Menger et.al 2022, Angiogenesis
3. GSE128554, Vail et.al, 2020
Reconstituting the mammary ductal network from human milk–derived mammary epithelial cells: tissue-specific ECM guides branching and lactogenic output
Amelia Hasenauer1, Valerie Pascetta1, Maxwell Mccabe2, Anthony Saviola2, Simone Ponta1, Stefan Prekovic3, Kirk Hansen2, Marcy Zenobi-Wong1
1Department of Health Science and Technology. ETH Zurich, Zurich - Switzerland, 2Department of Biochemistry and Molecular Genetics. University of Colorado, Colorado Springs (Colorado) - United States, 3Center for Molecular Medicine. University Medical Center Utrecht, Utrecht - The Netherlands
Introduction
Human milk-derived mammary epithelial cells (milk MECs) are an accessible cell source for studying human mammary biology. The ability of these cells to reconstruct breast tissue architecture remains unclear. Here we studied the role of extracellular matrix (ECM) cues on MEC organoid morphogenesis. We hypothesized that tissue-specific matrices would bias organization toward ductal versus alveolar states and establish milk MECs as a cell source for breast tissue engineering and human-relevant models.
Methods
Self-gelling decellularized ECM (dECM) hydrogels were prepared from human breast and lactating bovine udders. Proteomics confirmed compositional differences, with bovine dECM enriched in basement membrane components. Mechanical properties of the hydrogels were tuned to ∼800 Pa (Matrigel-like). Milk MECs from breastfeeding donors were encapsulated in dECM, Matrigel, or collagen-I and benchmarked against MCF10A (normal breast cell line). Branching, polarity, and cytoskeletal organization were quantified by imaging, epithelial phenotype by flow cytometry, milk expression (β-casein and milk-fat globules ± prolactin (PRL) stimulation) by Western Blot, complemented with proteomics.
Results
dECM supported self-organization of milk MECs into polarized, branched networks that maintained epithelial identity (Cytokeratin 8, Cytokeratin 14, E-cadherin), whereas Matrigel supported formation of more spherical organoids. Luminal progenitors were the most dominant cell type (∼80%) across conditions, but dECM/collagen-I better supported differentiated lactocytes than Matrigel (∼18% vs ∼8%). Branch extension in dECM/collagen-I coincided with higher proliferation (Ki67+ ∼36% in dECM vs ∼10% in Matrigel), actin alignment, and protrusive growth along collagen fibrils, features reduced in basement-membrane-rich Matrigel. Expression of milk proteins was robust: β-casein and milk-fat globules were detected ± PRL stimulation, indicating lactogenic memory and reduced PRL dependence. Findings reproduced in MCF10A support generalizability across mammary cell sources.
Conclusions
Milk MECs with tissue-specific dECM constitute a noninvasive, scalable platform that reconstitutes mammary branching and function in vitro. Coupling collagen-guided growth with preserved lineage programs and secretion establishes milk MECs as a powerful tool for breast tissue engineering, longitudinal in vitro lactation studies, matrix-guided morphogenesis, and drug screening.
Effects of the biomaterial carrier on macrophage cell therapy in regenerative medicine
Samuel Sung1, Phoebe Chua1, Eva Kraus1, Ricardo Whitaker1, Kara Spiller1
1Drexel University, Philadelphia (Pennsylvania) - United States
Macrophage cell therapy holds great potential in regenerative medicine because of its central role in coordinating tissue repair but is limited by two main challenges: 1) retention of transplanted macrophages within the site of injury, and 2) sustained expression of the desired macrophage phenotype. Biomaterial carriers may be able to overcome these challenges, but their impact on transplanted macrophages is poorly understood. We compared the effects of hydrogels or porous scaffolds on the retention and phenotype of encapsulated macrophages in vitro and in vivo in a volumetric muscle loss model, and analyzed the host response to the biomaterials with or without encapsulated macrophages. We hypothesized that the hydrogel would increase retention of macrophages within the site of injury, whereas the scaffolds would better support host cell interactions.
Primary murine macrophages were pre-polarized to a regenerative phenotype with interleukin-4 and then encapsulated within gelatin methacryloyl (GelMA) hydrogels or macroporous gelatin scaffolds. In vitro, GelMA hydrogels caused macrophages to dramatically increase secretion of several proteins related to immune cell recruitment, including CCL2, CCL5, CXCL1, and CXCL2. Flow cytometry showed that the hydrogel caused encapsulated macrophages to switch from pro-inflammatory at day 1 to a reparative phenotype by day 7, while porous scaffolds had minimal effects on macrophage phenotype. In vivo, macrophage retention was 7 times higher when using scaffolds compared to the hydrogels at day 1, which was linked to low viability of macrophages in hydrogels. Compared to their carrier controls, macrophages in the porous scaffold initially decreased neutrophil recruitment at day 1 and increased recruitment of dendritic cells by day 7. Overall, recruitment was driven by the biomaterial; however, when imaging tissue cross-sections stained for endothelial cell marker CD31, the macrophage-encapsulated hydrogel had increased presence along the interface between the injury and transplanted biomaterial supporting angiogenesis and repair by day 3.
Tailoring a chitosan-ECM hydrogel platform to mimic the tumor microenvironment of equine sarcoid for comparative oncology
Alessia Zanacchi1, Floriana Fruscione2, Elisabetta Razzuoli2, Laura Pastorino1, Donatella Di Lisa1
1Department of Informatics, Bioengineering, Robotics and Systems Engineering (DIBRIS). Università degli studi di Genova, Genova (Italia) - Italy, 2National Reference Center of Veterinary and Comparative Oncology (CEROVEC). Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Genova (Italia) - Italy
Equine sarcoid is the most common cutaneous neoplasm in horses, characterized by the lack of effective treatments. Furthermore, it’s associated with bovine papillomaviruses (BPVs), making it a valuable model for comparative oncology investigations of papillomavirus (PV)-associated neoplasms [1]. In line with the growing applications of three-dimensional (3D) systems, this study aimed to develop a biomaterial that more faithfully recapitulates the tumor microenvironment (TME) of equine sarcoid.
A protocol was optimized for the decellularization of tumor spheroids to obtain a decellularized extracellular matrix (dECM) [2] derived from a primary cell line established from a BPV-1 induced equine sarcoid tumor. The resulting dECM was incorporated into previously developed and validated chitosan (CHITO) and chitosan-collagen (CHITO-COL) hydrogels [3] to enhance the biological and structural fidelity of the model.
The formulations were subjected to rheological, morphological via scanning electron microscopy (SEM), and mechanical analyses to assess the influence of dECM incorporation on material properties. Biological characterization, including confocal microscopy and real-time qPCR, was performed to evaluate changes in cellular organization and gene expression. Data were analyzed by one-way ANOVA with Tukey’s test (p < 0.05).
Rheological analyses indicated that the inclusion of dECM did not significantly alter gelation kinetics, ensuring suitability for cell viability. Mechanical testing revealed decreased stiffness in CHITO-COL + dECM hydrogel, consistent with microstructural differences observed by SEM. Confocal microscopy highlighted better cellular morphology in CHITO + dECM hydrogel, and gene expression analyses suggested that dECM incorporation may contribute to the maintenance of mesenchymal features characteristic of the tumor phenotype.
Overall, these findings demonstrate that the developed dECM-enriched hydrogels represent a promising 3D model for mimicking the equine sarcoid TME, providing a relevant tool for future applications in comparative oncology.
[1] Munday, J. S., 2014, 1063-1075.
[2] Zhang, Y., et al., 2023, 378-388.
[3] Zanacchi A., et al., Manuscript under review.
3D microstructures for modeling physiological tissue microenvironments and immune responses
Chiara Martinelli1, Alberto Bocconi1, Srijan Chakraborty1, Giovanni Buccioli1, Matteo Vicini1, Leonardo Cherubin1, Claudio Conci1, Giulio Cerullo2, Roberto Osellame2, Giuseppe Chirico3, Emanuela Jacchetti1, Manuela Teresa Raimondi1
1Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”. Politecnico di Milano, Milan (Lombardia) - Italy, 2Institute for Photonics and Nanotechnologies (IFN), CNR and Department of Physics. Politecnico di Milano, Milan (Lombardia) - Italy, 3Department of Physics. University of Milano-Bicocca, Milan (Lombardia) - Italy
Introduction: Subcutaneous implants elicit immune responses driven by macrophage recruitment and require efficient tissue repair to restore the physiological dermal microenvironment. Three-dimensional (3D) in vitro microstructures offer valuable tools for modeling these processes and can be used to recreate microvascular networks under perfusion. Upon proper engineering of their physicochemical properties, they can efficiently modulate macrophage activity.
Methods: 3D microstructures with large (50 × 50×20 μm³)1 and small pores (15 × 15×15 μm3)2 were fabricated by two-photon polymerization of the biocompatible photoresist SZ2080. Human dermal fibroblasts and endothelial cells were cultured on large pore scaffolds integrated into a Miniaturized Optically Accessible Bioreactor (MOAB) to enable perfusion (5μl/min), in the presence and absence of VEGF (50ng/mL) and TGF-β1 (5 ng/mL). Macrophages, cultured on both substrates, were polarized with lipopolysaccharide (M1) or interleukin-4 (M2). Confocal laser scanning microscopy was performed upon staining collagen I and CD31 (co-cultures) at day 7, inducible nitric oxide synthase (iNOS, M1), and arginase 1 (Arg1, M2). Morphological changes and metabolic activity of macrophages were analyzed.
Results: The perfused platform revealed a reliable model of dermal regeneration, demonstrating the formation of primordial tubules (128.1±8.6 μm) and enhanced collagen I deposition in the presence of microstructures and VEGF. Both substrates significantly altered macrophage morphology and metabolism. When combined with chemical stimulation, large pores slightly enhanced Arg1 expression (+5%), favoring an anti-inflammatory activity, whereas small pores strongly upregulated iNOS (+121%), promoting a pro-inflammatory profile.
Conclusions: The 3D scaffolds integrated into the MOAB successfully recapitulated a model of physiological dermal perivascular microenvironment and, owing to their tunable architecture, provided fine regulation of macrophage behavior. Future experiments will focus on characterizing macrophage responses in the perfused bioreactor upon chemical stimulation.
References
1. Martinelli et al., Lab Chip, 2025, 25, 423-439
2. Martinelli et al., Mater Today Bio, 2025, 35, 102328
Acknowledgements: ERC, BEACONSANDEGG, G.A.101053122; EU, MSCA-DN, flIMAGIN3D, G.A.101073507; EU, FET-OPEN, IN2SIGHT, G.A.964481; MUR, VISION, CUP D53D23007890001.
4D-printed thermoresponsive plasmonic scaffolds recapitulating alveolar dynamics for in vitro lung modeling
Ane Urigoitia-Asua1, Irune Villaluenga2, Dorleta Jimenez De Aberasturi1
1Hybrid Biofunctional Materials. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, 2Inorganic-polymer hybrid and nanostructured materials. POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain
Advances in three-dimensional (3D) in vitro alveolar models are essential to improve the physiological relevance of respiratory tissue systems for disease modeling or drug screening. Replicating native lung microarchitecture and breathing dynamics is critical for functional tissue development [1]. Building upon previous work in our group [2], we developed a stimuli-responsive, biocompatible hybrid resin for vat photopolymerization printing (VPP) to fabricate four-dimensional (4D) scaffolds that mimic alveolar geometry and support dynamic expansion-contraction simulating breathing motions [3]. The resin contained the thermoresponsive monomer vinyl caprolactam (VCL) and near-infrared (NIR, 800 nm) plasmonic gold nanorods (AuNRs) functionalized with thiolated polyarginine and polyethylene glycol (PA/PEG-SH). This surface modification was essential for biocompatibility, while polyarginine introduced positive surface charges enhancing cell adhesion. Upon NIR irradiation, the photothermal effect of AuNRs induced localized heating, triggering a phase transition of VCL across its Lower Critical Solution Temperature (LCST) at physiological temperature. Cyclic NIR exposure generated reversible contraction-expansion of 6% on the material, consistent with physiological lung strain (5-12%). We 4D-printed and characterized honeycomb-patterned scaffolds (400 µm cavity diameter) resembling native alveoli. These scaffolds supported lung epithelial cell proliferation, forming monolayers in 21 days. Incorporation of a porcine-derived decellularized lung extracellular matrix (lung dECM) layer significantly accelerated epithelial coverage (complete by day 14) and improved uniformity. Under cyclic NIR stimulation, cells exhibited upregulated expression of mechanotransduction-associated genes (SNAI1, CTGF, ANKRD1), while heat shock proteins (HSPD1) remained unaffected, demonstrating their potential to reproduce physiological cues. Furthermore, the model was engineered to support endothelial cell integration and microvascularization. This dynamic 4D-printed system represents a promising platform for respiratory disease modeling and high-throughput drug testing, bridging material actuation with functional tissue responses.
[1] Fallert et al. Nanoscale 2024, 16, 10880
[2] Aizarna-Lopetegui et al. J. Mater. Chem. B 2023, 11, 9431
[3] Urigoitia-Asua et al. Adv. Optical Mater. 2025, e01142
tUnabLe smarT biomateRiAl for CARTilage REGENERAtion (ultracart regenera)
Denise Murgia1, Gaetano Burriesci2, Francesco Lopresti3, Francesca Romano1, Sofia Di Leonardo2, Vincenzo La Carrubba4, Roberto Di Gesù1
1Musculoskeletal Tissue Engineering. Fondazione Ri.MED, PALERMO (Sicilia) - Italy, 2Bioengineering lab. Fondazione Ri.MED, PALERMO (Sicilia) - Italy, 3Engineering dept. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy, 4Engineering dept. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy
Introduction
Rheumatoid Arthritis (RA) and Osteoarthritis (OA) are among the most prevalent musculoskeletal disorders, leading to progressive degradation of articular cartilage and joint dysfunction1. Matrix-Assisted Autologous Chondrocyte Implantation (MACI) represents a promising approach for cartilage repair; however, post-surgical rehabilitation often fails to reproduce the optimal mechanical environment necessary for effective regeneration2. To address this limitation, we developed a smart scaffold capable of delivering localized, tunable mechanical stimuli to human articular chondrocytes (hACs), integrating mechanobiological and biomaterial engineering principles.
Methods
Magneto-responsive, piezoelectric scaffolds were fabricated from aligned electrospun poly(vinylidene fluoride–trifluoroethylene) (PVDF-TrFE) fibers alone or incorporating Fe3O4 magnetic nanoparticles (MNPs). The aligned structure conferred mechanical anisotropy, while MNPs enabled remote activation via external magnetic fields. After hAC seeding, daily magnetic stimulation for up to 14 days mimicked physiological loading, activating the piezoelectric response and generating localized electromechanical cues.
Results and Discussion
MNPs were successfully integrated within the polymer fibers, enabling the fabrication of PVDF and PVDF–Fe3O4 matrices. The scaffolds showed reproducible deformation under magnetic actuation and high biocompatibility. Stimulated constructs exhibited enhanced deposition of type II collagen and a higher type II/type X collagen ratio, indicating a stable chondrogenic phenotype and reduced hypertrophy. These outcomes align with previous evidence that electromagnetic and electrical cues can modulate cartilage metabolism and promote regenerative signaling3. Our results suggest that combined mechanical and piezoelectric stimulation supports hyaline cartilage formation and improves chondrocyte function.
Conclusions
This multifunctional scaffold provides controlled electromechanical cues to embedded chondrocytes, effectively reproducing joint-like stimuli and promoting cartilage regeneration. The approach offers a promising advancement toward personalized and functionally effective treatments for chondral defects.
References
1. Ostrowska et al., 2018, Reumatologia.
2. Seok-Jung et al., 2009, J. Med. Case Rep.
3. Ongaro et al., 2011, Bioelectromagnetics
Acknowledgment
This work was supported by the ON Foundation (CH), starting grant n° 22-006/2022.
Coaxial 3D printing of core-shell inks: engineering 3D printed strands to produce meniscus implants with robust mechanical strength and enhanced biological performance - SEMIT
Francisco A. P. Rodrigues1, Pedro L. Granja2, Sang Jin Lee3, Ana L. Oliveira1, João B. Costa1
1CBQF - Centre for Biotechnology and Fine Chemistry Faculty of Biotecnology Universidade Católica Portuguesa, Porto, Oporto (Porto) - Portugal, 2Instituto de Investigação e Inovação em Saúde. University of Porto, Oporto (Porto) - Portugal, 3Wake Forest Institute for Regenerative Medicine. Wake Forest University, Winston-Salem (North Carolina) - United States
Knee meniscus lesions are a common injury that significantly impact an individual's quality of life. Meniscus tissue lack of intrinsic regenerative capacity requests efficient treatments that simultaneously offer robust mechanical integrity and promote rapid tissue integration. Current treatments fail in long-term, accelerating osteoarthritic changes in the knee [1]. To address the inherent trade-off between strength and bioactivity, this study employs a coaxial printing technique to fabricate core–shell constructs, where the core ink provides robust mechanical strength, and the shell ink enhances biological function.
The coaxial technique enabled the simultaneous extrusion of two distinct inks through a single nozzle, creating a 3D strand composed by a core and a shell. This strategy allowed the combination of two materials: the core, composed of poly(ε-caprolactone) (PCL), a synthetic polymer that provides mechanical stability; and the shell, composed of silk fibroin (SF), a natural biopolymer that enhances cell attachment and long-term viability. Rheology tests were performed, and 3D printed implants were produced and analyzed in terms of morphology and mechanical performance. The in vitro biological properties were assessed through cell metabolic activity, proliferation, and microscopic imaging.
The developed coaxial inks showed adequate rheological properties for printability and structural fidelity. Scanning electron microscopy (SEM) analysis confirmed the morphological characteristics and confirmed the distinct core–shell architecture of the printed constructs. Uniaxial tensile and compressive tests revealed robust mechanical properties, with obtained elastic modulus values within the physiological range. In vitro biological assays, performed with human primary fibrochondrocytes, showed that the SF shell significantly enhanced initial cell adhesion and long-term viability.
Overall, this novel coaxial ink presented both enhanced mechanically and biologically performance, making it a promising candidate for 3D printing of personalized meniscus implants.
Acknowledgements: FCT through F.R. Doctoral Research Grant (2024.00742.BD) and Project IBEROS+(0072_IBEROS_MAIS_1_E - (POCTEP)2021-2027).
[1] J.Twomey-Kozak et al., Clin Sports Med, 2020, doi: https://doi.org/10.1016/j.csm.2019.08.003.
Non-invasive characterization of 3D conjunctival models using OCT
Fabiola Walz1, Julian Schwebler1, Geraldine Beer1, Sebastian Häusner1, Sarah Nietzer1, Oliver Pullig1, Christian Lotz2
1Department for Functional Materials in Medicine and Dentistry. University Hospital Würzburg, Würzburg (Baden-Wberg Bayern) - Germany, 2Translational Center Regenerative Therapies. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany
Physiologically relevant conjunctival in vitro models are essential for studying the ocular surface and related diseases. However, achieving key characteristics—such as a non-keratinized, stratified epithelium with mucus-secreting goblet cells—requires careful optimization of culture conditions. In this study, we systematically investigated how serum concentration, addition of a collagen matrix, and fibroblast co-culture affect epithelial development and differentiation in 3D conjunctival tissue models.
To enable repeated, non-destructive measurements, we used optical coherence tomography (OCT), which provides cross-sectional and volumetric imaging at micrometer-scale. This approach enabled continuous, real-time tracking of model development under standardized conditions while minimizing the need for invasive sampling. As a result, we obtained a more comprehensive and longitudinal view of the maturation process through predominantly non-invasive observations. Alongside qualitative imaging, we performed OCT-based quantification of epithelial thickness, keratinization, and goblet cell density, and compared the results to histology, qPCR, and ELISA for validation.
OCT enabled clear visualization of all major layers of the conjunctival models, including the epithelium, keratinized areas, and the collagen scaffold. Using this setup, we were able to detect distinct effects of the different culture conditions. Lower serum concentrations promoted epithelial differentiation, while higher levels hindered development. Adding a collagen matrix supported epithelial organization and conjunctival-specific features. Fibroblasts did not directly influence differentiation, but increased epithelial stratification, improving overall tissue architecture.
In summary, we demonstrate how serum levels, a collagen matrix, and fibroblasts influence the development of 3D conjunctival models. OCT proved to be a reliable, non-invasive tool for tracking tissue maturation over time and adds important complementary information to conventional endpoint analyses. Overall, this approach establishes a robust foundation for further optimization of conjunctival tissue engineering and can be readily adapted to other complex 3D tissue models such as bone and cartilage.
Bioadhesive hydrogels based on collagen and combined with gallium-bioglass nanoparticles for bone regeneration
Margarida Fernandes1, Cátia Correia1, Albina R. Franco1, Rui L. Reis1, Natália M. Alves1, Daniela Peixoto1
13B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics; ICVS/3B’s–PT Government Associate Laboratory, University of Minho, Guimarães (Braga) - Portugal
Bone can self-regenerate after fractures; however, when fractures are extensive, bone grafts may be necessary. Conventional orthopaedic implants lack antibacterial activity, are non-biodegradable, and exhibit limited biocompatibility, thereby increasing the risk of inflammation and infection. Since this option presents several limitations and risks for the patient, hydrogels represent a promising alternative for bone regeneration, as they possess a structure similar to the extracellular matrix, are biocompatible, and can incorporate molecules that stimulate osteogenesis. Collagen (Coll) is a primary component of bone, making it an ideal material for developing new scaffolds for bone applications. Here, we develop hydrogels based on Coll modified with catechol groups. This biomimetic approach is inspired by the exceptional adhesion ability of marine mussels to wet, irregular surfaces. These hydrogels were combined with Gallium-doped bioglass nanoparticles (Ga-BGNPs) to improve osteoblast cell viability and proliferation, thus promoting osteoconductivity and biomineralization. The physical and chemical properties of the hydrogels and Ga-BGNPs were analyzed, along with their cytocompatibility with human osteoblast cells, and their antibacterial and antibiofilm properties. The modification of collagen with catechol groups significantly enhanced the hydrogel’s adhesive properties, as demonstrated in adhesion tests on rat femur and porcine skin. The incorporated Ga-bioglass nanoparticles exhibited antibacterial activity against clinically relevant microorganisms. The hydrogel/Ga-BGNPs system showed the greatest effectiveness against P. aeruginosa, a pathogen known for its antibiotic resistance and clinical challenges in bone infections. Additionally, the Ga-BGNPs promoted the formation of hydroxyapatite and supported osteoblast adhesion and proliferation.
Overall, the produced hydrogels represent a promising system for bone regeneration, combining enhanced adhesive capacity, bioactivity, antibacterial properties, cytocompatibility, and stimulation of cell proliferation.
We acknowledge the FCT for funding UID/PRR/50026/2025, PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), and projects SeaJellySpine – Operação n°15520 (COMPETE2030-FEDER-00653700), and Jelly4Bone – Operação n°15969 (COMPETE2030-FEDER-00710400 co-financed by ERDF, through the Innovation and Digital Transition Thematic Programme (COMPETE 2030), under Portugal 2030.
Distinct matrix viscoelasticity guides macrophage polarization in the fracture hematoma
Raphael S. Knecht1, Duncan M. Morgan2, Matthias R. Kollert1, Christian H. Bucher1, David J. Mooney2, Katharina Schmidt-Bleek1, Georg N. Duda1
1Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration. Charite Universitatsmedizin, Berlin - Germany, 2John A. Paulson School of Engineering and Applied Sciences. Harvard University, Cambridge (Massachusetts) - United States
Physical cues from the extracellular matrix (ECM) are key regulators of cell function and tissue regeneration. After fracture, the hematoma forms an initial transient niche where immune cells and repair processes are coordinated. However, how viscoelastic properties of the ECM modulate immune cell behavior remains poorly understood. Here, we identify distinct hematoma stress relaxation states as mechanical cues guiding macrophage polarization from pro-inflammatory to pro-regenerative phenotypes. Analysis of human fracture hematomas revealed progressive changes in ECM mechanics over time, with a significant increase in the late-phase stress relaxation time constant (τ2) with days post-injury. To study the influence of τ2 on macrophage polarization, we used low- and high-molecular-weight alginate to develop hydrogels with tunable τ2 but constant stiffness. Low-molecular-weight alginate produced low τ2 values (fast late-phase stress relaxation), whereas high-molecular-weight alginate resulted in high τ2 values (slow relaxation), mimicking early and late hematoma viscoelastic states, respectively. Macrophages cultured in high-τ2 matrices exhibited pro-inflammatory phenotypes, whereas low-τ2 environments promoted anti-inflammatory, pro-regenerative phenotypes, as assessed by surface marker expression and cytokine secretion. These effects persisted under toll-like receptor co-stimulation, underscoring the robustness of viscoelastic control over inflammatory signaling. Single-cell RNA sequencing revealed distinct transcriptional programs associated with different ECM τ2 values, with many of the differentially expressed genes related to metabolic processes. Importantly, in vitro macrophage profiles induced by low-τ2 hydrogels mirrored in vivo macrophage gene expression patterns in the early fracture hematoma, suggesting that late-phase stress relaxation of the fracture hematoma guides macrophage polarization in vivo. This work establishes hematoma viscoelasticity as a regulator of macrophage polarization. Since hematoma formation is an early step after injury in many tissues, the identified τ2-dependent mechanism likely operates across multiple contexts. Thus, ECM stress relaxation provides a tunable parameter to guide immune responses that can be harnessed for biomaterial design and regenerative therapies.
Hybrid 3D-printed PLA and ionic exchange calcium alginate scaffold for articular cartilage repair
Liliana Lizarazo-Fonseca1, Adriana Lara-Bertrand1, Leonardo Prieto-Abello1, Gustavo Salguero-Lopéz1, Ingrid Silva-Cote1
1Unidad de Ingeniería Tisular. Instituto Distrital de Ciencia Biotecnología e Innovación en Salud-IDCBIS, Bogotá D.C. (Cundinamarca) - Colombia
Articular cartilage repair remains a major challenge in regenerative medicine due to the tissue’s avascular nature and limited intrinsic regenerative capacity. Following injury, hyaline cartilage is typically replaced by fibrocartilaginous tissue with inferior biomechanical properties, leading to progressive joint degeneration, pain, and functional impairment. Osteoarthritis (OA) is a leading consequence of this process and currently affects an estimated 528 million individuals worldwide according to the World Health Organization, with prevalence driven by aging, obesity, sedentary behavior, and joint injury. The knee is most commonly affected, contributing to substantial disability and socioeconomic burden globally.
To address this challenge, we developed a hybrid osteochondral scaffold combining a 3D-printed polylactic acid (PLA) framework for subchondral bone anchorage with a calcium alginate hydrogel mimicking the cartilage layer. 3D-design incorporates an internal cavity that can be filled with autologous bone obtained during surgical debridement to enhance osseointegration and mechanical support. Mechanical testing confirmed that the scaffold exhibits compressive strength and elastic modulus comparable to native trabecular bone, supporting its capacity for physiological load-bearing. SEM analysis revealed a uniformly interconnected porous microstructure that promotes cell adhesion, proliferation, and nutrient diffusion. Moreover, the scaffold can be functionalized with human Wharton’s jelly mesenchymal stromal cells (hWJ-MSCs), which offer significant advantages for osteochondral repair due to their high proliferative capacity, low immunogenicity, and potential differentiation. The combination of the scaffold’s bioactive architecture with the regenerative properties of hWJ-MSCs may enhance tissue integration, accelerate matrix formation, and improve long-term functional recovery, while implantation in anatomical femoral condyle and patellar defect models demonstrated that the scaffold precisely conforms to defect geometry, maintains structural stability, and is easily handled during placement. Together, these findings indicate that the scaffold exhibits appropriate mechanical behavior, biological responsiveness, and strong potential for clinical application in articular cartilage repair.
Developing a humanised model of traumatic brain injury compatible with clinical imaging methods
Zoe Dombros-Ryan1, Christopher Adams2, Mahon Maguire3, Harish Poptani4, Divya M. Chari1
1School of Medicine. Keele University, Newcastle under Lyme (Staffordshire) - United Kingdom, 2School of Life Sciences. Keele University, Newcastle under Lyme (Staffordshire) - United Kingdom, 3Centre for Preclinical Imaging. University of Liverpool, Liverpool - United Kingdom, 4Centre for Preclinical imaging, Department of Molecular and Cellular Cancer medicine. University of Liverpool, Liverpool - United Kingdom
Traumatic brain injury (TBI) is the leading cause of adult disability, associated with wide-ranging motor/cognitive deficits, with no regenerative therapies available. Neuromimetic and accessible in vitro models of TBI are needed to screen novel therapies and reduce reliance on live animal experiments. We previously reported a 3D-neurotrauma model (mouse-derived multicellular neural network, encapsulated in a 3D polymer matrix) with induction of a reproducible penetrating injury. Key neuropathological responses were observed including glial scarring and microglial activation, highlighting benefits of the model for therapeutic screening. [1]. However, a humanised version of the model is needed to enhance translational relevance. This study demonstrates that human neural cells derived from neuroblastoma (SH-SY5Y) and glioblastoma (T98G) cell lines respectively can be reliably encapsulated in a 3D polymer (collagen) matrix. Robust 3D cell growth was achieved, with successful induction of a focal penetrating injury in the matrix. To assess if the injury foci could be imaged using clinical imaging tools, samples were subject to MRI/MRS imaging (9.4T Bruker MR imaging scanner). Preliminary data show it is possible to locate focal injury using MRI, with cellular metabolites identifiable on MRS spectra, in constructs encapsulating both neuronal and glial cell lines. These data suggest that it will be possible to develop a humanised 3D in vitro neurotrauma model compatible with clinical imaging tools, to reduce reliance on live animal experiments/animal-derived reagents (e.g. neural antibodies) to study neural injury/repair. Future work will focus on co-encapsulation of neuronal and glial cells with inclusion of human immune cells (microglia), to simulate cardinal neuropathological events.
[1] Wiseman JP, Dombros-Ryan Z, Griffiths J, Adams C, Chari DM. Development of a Three-Dimensional Pathology-Simulating Model of Neurotrauma Using a Polymer-Encapsulated Neural Cell Network. Gels. 2025 Mar 27;11(4):247.
3D-printing of electroconductive MXene-based micro-meshes in a biomimetic hyaluronic acid-based scaffold directs and enhances electrical stimulation for neural repair applications
Ian Woods1, Dahnan Spurling2, Sandra Sunil3, O'callaghan Anne3, Jack Maughan4, Gutierrez-Gonzalez Javier4, Mcguire Tara3, Liam Leahy3, Adrian Dervan3, Valeria Nicolosi5, Fergal O'brien4
1Research Ireland FutureNeuro Centre. Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland, 2Advanced Materials and BioEngineering Research (AMBER) Centre. School of Chemistry, Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, Dublin - Ireland, 3Tissue Engineering Research Group, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland, 4Advanced Materials and BioEngineering Research (AMBER) Centre. Tissue Engineering Research Group, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland, 5Advanced Materials and BioEngineering Research (AMBER) Centre. School of Chemistry, Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, Dublin - Ireland
Traumatic brain injury and neurotrauma effect approximately 1 million people per year in Europe and no effective treatments are currently available. However, recent advances in electrical stimulation provide promising neural tissue repair applications but much is still unknown about the role of electric fields in activating neuronal repair. Furthermore, the conductive biomaterials necessary to harness this potential for clinical applications are lacking. To address this challenge, it was hypothesized that structured integration of an electroconductive biomaterial into a tissue engineering scaffold could enhance electroactive signaling for neural regeneration in a manner dependent on its spatial distribution. To accomplish this, electroconductive 2D Ti3C2Tx MXene nanosheets were synthesized from MAX-phase powder, demonstrating excellent biocompatibility with neurons, astrocytes and microglia. To achieve spatially-controlled distribution of these MXenes, melt-electrowriting was used to 3D-print highly-organized PCL micro-meshes with varying fiber spacings (low-, medium-, and high-density), which were functionalized with MXenes to provide highly-tunable electroconductive properties (0.081 ± 0.053 - 18.87 ± 2.94 S/m). Embedding these electroconductive micro-meshes within a neurotrophic, immunomodulatory hyaluronic acid-based extracellular matrix (ECM) produced a soft, growth-supportive MXene-ECM composite scaffold. Electrical stimulation of neurons and olfactory bulb-derived neurospheres seeded on these scaffolds promoted axonal outgrowth, influenced by fiber spacing in the micro-mesh. In a multicellular model of neural cell behavior, neurospheres stimulated for 7 days on high-density MXene-ECM scaffolds exhibited significantly increased axonal extension and neuronal differentiation, compared to low-density scaffolds and MXene-free controls. The results demonstrate that spatial-organization of electroconductive materials in a neurotrophic scaffold can enhance repair-critical responses to electrical stimulation and that these biomimetic MXene-ECM scaffolds offer a promising new approach to neurotrauma repair.
Acknowledgements:
This study was funded by a joint funding initiative of the Irish Rugby Football Union Charitable Trust (IRFU-CT) and the Research Ireland Advanced Materials and Bioengineering Research (AMBER) Centre (SFI/12/RC/2278) and an Irish Research Council Postdoctoral Fellowship (Government of Ireland), Grant Number: GOIPD/2021/262.
Development of electrospun hollow fibres for muscle tissue engineering
Marie Beatrix Kruth1, Cathy Hua Ye2, Philippa Hulley1, Pierre-Alexis Mouthuy1
1Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences. University of Oxford, Oxford (Oxfordshire) - United Kingdom, 2Department of Engineering Science. University of Oxford, Oxford (Oxfordshire) - United Kingdom
Muscle tissue engineering holds great promise for developing human in vitro models that could replace animals in research. A major hurdle, however, is the diffusion limit which restricts gas and nutrient exchange in thicker engineered tissues. Specifically, muscle’s high metabolic demands require efficient oxygen and nutrient supply, yet passive diffusion only reaches depths of 100–200µm [1].
This work aims to develop tissue-engineered muscle with high physiological relevance for drug testing applications. Human muscle cells will be grown on electrospun hollow fibres (eHFs) integrated into a soft flexible bioreactor chamber. eHFs offer promise for perfused three-dimensional tissue culture, as they can mimic vasculature by providing nutrient and oxygen transport via a semi-permeable membrane [2]. Additionally, eHFs can serve as a scaffold for growing muscle tissue by mimicking the extracellular matrix and providing anchorage points for cells [3].
Polycaprolactone-based eHFs were produced through electrospinning using a dissolvable conductive collector. The collector was synthesized by wetspinning a PVA-Pedot:PSS solution. It was found that a PVA concentration of ∼41% correlated to the highest continuous collector length, and a Pedot:PSS-to-PVA ratio of 16:84 provided sufficient electrical conductivity for electrospinning.
The produced eHFs were stretched for matrix alignment, and their dimensions were determined using SEM. eHFs exhibited an average wall thickness of ∼103µm pre-stretch (StD: 12) and ∼41µm post-stretch (StD: 11), the latter being more favourable for diffusion. eHF stretching also resulted in the rearrangement of microfibres, with ∼80% of fibres exhibiting alignment post-stretch.
Future work will investigate fibre porosity and integrate dynamic perfusion for intratissue diffusion. Additionally, cellular responses to stretched and non-stretched eHFs under growth and differentiation conditions will be investigated.
Acknowledgements: NC3Rs, St. Peter’s College Oxford.
[1] T. Rademakers et al., 2019, J.TissueEng.Regen.Med., 13(10), pp. 1815–1829.
[2] H. Eghbali et al, 2016, Int.J.Artif.Organs, 39(1), pp.1–15.
[3] I. Jun et al, 2018, Int.J.Mol.Sci., 19(3), p.745.
Magnetic liquefied-core microcapsules as remotely actuated bioreactors for bone tissue regeneration
Mariana Carreira1, Manauel Pires-Santos1, Sara Nadine1, Syeda M. Bakht1, João F. Mano1
1CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal
Mechanical cues are fundamental regulators of bone regeneration; however, most current biomaterial systems are static and fail to recapitulate the dynamic mechanical environment of native bone tissue. To overcome this limitation, we developed self-regulated liquefied-core microcapsules capable of delivering localized and tunable mechanical stimulation to human umbilical cord-derived mesenchymal stem cells (hUCMSCs) via external magnetic actuation. We hypothesized that co-encapsulating hUCMSCs with magnetically responsive poly(ε-caprolactone) microparticles in a remotely actuated bioreactor would establish a dynamic mechanobiological niche that mechanically triggers osteogenic commitment.
Microcapsules were fabricated through electrohydrodynamic atomization, resulting in constructs with: (i) a semi-permeable membrane enabling nutrient and waste exchange, (ii) surface-functionalized magnetic microparticles serving as internal adhesion substrates, and (iii) hUCMSCs. Cultures were subjected to controlled magnetic stimulation for 21 days, and osteogenic differentiation was evaluated using confocal microscopy, alkaline phosphatase assays, and RT-qPCR.
Magnetic actuation modulated internal fluid flow, generating shear stresses that improved mass transport and provided dynamic mechanical cues. This led to enhanced mechanotransduction signaling and earlier upregulation of osteogenic markers, indicating accelerated lineage commitment. Building on this approach, the magnetic methodology could be adapted for in vivo regenerative applications through minimally invasive implantation, offering a versatile and biomimetic platform for studying mechanoregulation and advancing next-generation dynamic biomaterials for bone tissue engineering.
Acknowledgments:
The authors greatly acknowledge the Portuguese Foundation for Science and Technology (FCT)/MCTES in the scope of the project “O2Cells” (2022.04237.PTDC), and the European Research Council for the project “REBORN” (ERC-2019-AdG-883370). M. Carreira and M. Santos acknowledge financial support from FCT through doctoral grants 2021.04542.BD and 2024.01120.BD, respectively. This work was developed within the scope of the project CICECO Aveiro Institute of Materials, UID/50011/2025 (DOI 10.54499/UID/50011/2025) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC).
Polarized BCZT-polymer composites as piezoelectric platforms for ultrasound-assisted cell stimulation
Mario Monzón1, Ricardo Donate1, Pablo Bordón1, Rocío Moriche2, María J. Sayagués3, Rubén Paz1
1Mechanical Engineering Department. University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria (Las Palmas) - Spain, 2Department of Condensed Matter Physics, Faculty of Physics. University of Sevilla, Sevilla - Spain, 3Institute of Materials Science of Sevilla (CSIC-US), Sevilla - Spain
The incorporation of lead-free piezoelectric ceramics into biocompatible polymers represents a promising route to enhance cell stimulation and bone tissue regeneration through electromechanical coupling. In this study, disc-shaped piezoelectric composites were manufactured by compression molding using either polylactic acid (PLA) or poly(vinylidene fluoride) (PVDF) as polymeric matrices. Each polymer was combined separately with one of two ceramic phases: barium titanate or xBaZr0.2Ti0.8O3–(1–x)Ba0.7Ca0.3TiO3 (BCZT), the latter being a lead-free material with a relatively high piezoelectric coefficient.
BCZT powders were prepared by mechanosynthesis, and X-ray diffraction confirmed the formation of a pseudocubic perovskite structure, with the composition with x = 0.4 (Ba0.82Ca0.18Zr0.08Ti0.92O3) selected as optimal. The BCZT powder obtained from the milling process is subjected to a heat treatment at 1450°C for 4 hours. The samples later produced by compression moulding were evaluated through in vitro assays using human osteoblastic cells, aiming to assess the influence on cell viability of polymer matrix, ceramic filler, polarization, and external mechanical stimulation. Polarization was performed at 4 kV/mm and 100°C for 30 min, maintaining the field during cooling to align dipoles and enhance piezoelectric activity. Ultrasound stimulation was applied through a custom-designed system comprising a sonicator, a water bath, and a holder for the cell-seeded samples.
Results showed that BCZT-containing composites, particularly polarized PLA/BCZT 90/10 (%v/v) samples exposed to ultrasound, exhibited the most favorable biological response. No significant differences were observed among non-polarized or non-stimulated groups. These findings highlight the potential of lead-free BCZT–polymer composites as multifunctional piezoelectric biomaterials for advanced biomedical applications.
Acknowledgements
This contribution is part of the RENOVATE project funded by the European Union’s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101227121 (101227121 - RENOVATE - HORIZON-MSCA-2024-DN-01) and the PIZAM project (PID2020-117648RB-I00) funded by MCIN/AEI/10.13039/501100011033.
In vitro evaluation of PCL-based biodegradable scaffolds incorporated with gold nanoparticles
Vasileios Tzirtziganis1, Chudobová Ema Chudobová2, Kuželová Košťáková Eva2, Lukáš David3, Filová Eva1
1Department of Tissue Engineering. Institute of Experimental Medicine, Czech Academy of Sciences, Prague (Hlavni Mesto Praha) - Czech Republic, 2Department of Bioengineering. Technical University of Liberec, Liberec - Czech Republic, 3Department of Chemistry. Technical University of Liberec, Liberec - Czech Republic
Tissue engineering focuses on creating functional biomaterials that facilitate bone regeneration by replicating the extracellular matrix and stimulating osteogenic differentiation. Polycaprolactone (PCL)-based scaffolds have attracted significant interest for their biocompatibility, biodegradability, and capacity to promote cell adhesion and proliferation. This study evaluates the viability of Bone Marrow-derived Mesenchymal Stem Cells (bmMSCs) and the temporal expression of key osteogenic markers when seeded on PCL-based scaffolds with Gold Nanoparticles (AuNP) of three different concentrations under osteogenic conditions, with an emphasis on their potential for bone regeneration.
BmMSCs were seeded in scaffolds and cultured in osteogenic induction medium. Cell proliferation and viability analysis was performed with MTS test. Alkaline phosphatase activity (ALP) was measured spectrophotometrically. The expression levels of osteogenic markers, including alkaline phosphatase (ALP), osteopontin (SPP1), and collagen type I (COL1) were analyzed using quantitative real-time PCR (qRT-PCR). Fluorescence imaging, DiOC, ColI and OCN stainings, was used to visualize cell morphology over time.
The ALP results show that the combination of PCL-based scaffolds with AuNPs appear to promote osteogenic differentiation. The temporal expression of osteogenic markers aligns with established osteogenesis stages, with ALP promoting early mineralization, SPP1 peaking in the matrix maturation stage, and collagen I expression remaining consistently high throughout differentiation, emphasizing its crucial role in extracellular matrix formation and mineralization. The microscopic analysis for COL1A1, DiOC and OCN revealed morphological changes consistent with osteoblast differentiation.
This study demonstrates the progressive expression of osteogenic markers in bmMSCs that were seeded in PCL-based scaffolds with AuNP nanoparticles, confirming their differentiation potential. The integration of molecular, biochemical, and microscopic analyses strengthens our understanding of osteogenesis. These results contribute to the advancement of stem cell-based bone regeneration strategies and the development of optimized protocols for osteogenic induction.
The project was supported by the Ministry of Education, Youth, and Sports of the Czech Republic, project OPJAK CZ.02.01.01/00/22_008/0004562.
Biopolymeric scaffold enriched with tannic acid and ferulic acid as a potential material for periodontal regeneration - SEMIT
Anna Vakuliuk1, Beata Kaczmarek-Szczepańska2, Katarzyna Cholewa-Kowalska3, Anna Maria Osyczka4
1School of Medicine, Medical Sciences and Nutrition. University of Aberdeen, Aberdeen (Grampian) - United Kingdom, 2Laboratory for Functional Polymeric Materials, Faculty of Chemistry. Nicolaus Copernicus University in Toruń, Toruń (Kujawsko-Pomorskie) - Poland, 3Department of Glass Technology and Amorphous Coatings. University of Krakow, Krakow - Poland, 4Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research. Jagiellonian University, Krakow - Poland
Periodontitis is a chronic inflammatory condition that destroys the supporting bone around teeth and, if left unchecked, leads to tooth loss. In tissue engineering there is a clear need for biomaterials that not only support regeneration but also counter oxidative stress and persistent inflammation. In this work we prepared multifunctional chitosan–collagen composite hydrogels modified with tannic acid (TA) and ferulic acid (FA), chosen for their antioxidant potential. The materials were formed by ionic gelation. Incorporation of the polyphenols into the polymer network was verified by ATR-FTIR spectroscopy. We then assessed compressive performance, monitored degradation in phosphate-buffered saline (PBS), measured antioxidant activity using the DPPH radical-scavenging assay, and evaluated the release kinetics of the polyphenols. Adding TA increased scaffold strength and slowed degradation relative to FA, while both formulations retained high antioxidant activity throughout testing; TA-containing composites were the most effective in quenching free radicals. Release of the bioactive components was gradual and controllable over time. Biological evaluation—specifically cytotoxicity and hemocompatibility—will follow in the next phase in 2026. Taken together, these results indicate that polyphenol-enriched chitosan–collagen composites are promising candidates for periodontal tissue regeneration, combining mechanical stability with bioactivity and the capacity to modulate inflammatory and oxidative conditions in the defect site.
The authors would like to thank the National Science Center (Grant no: UMO-2023/51/B/NZ5/01403) for providing financial support to this project.
Bioactive bilayer osteochondral scaffold integrating 3D PCL architecture and platelet lysate–enhanced hydrogel
Luz Stella Correa Araujo1, William Antonio Cárdenas Aguazaco1, Adriana Lorena Lara Bertrand1, Ingrid Zulay Silva Cote1, Gustavo Andrés Salguero Lopez1
1Unidad de Ingeniería Tisular. Instituto Distrital de Ciencia Biotecnología e Innovación en Salud-IDCBIS, Bogotá D.C. (Cundinamarca) - Colombia
Articular cartilage defects represent a major clinical challenge due to the tissue’s limited intrinsic regenerative capacity. Current treatments, such as microfracture and mosaicplasty, offer inconsistent and often suboptimal outcomes, underscoring the need for advanced regenerative strategies.
At the IDCBIS Tissue Engineering Unit, we have developed a biomimetic bilayer osteochondral scaffold using a hybrid fabrication approach that integrates 3D extrusion printing with physical freeze–thaw crosslinking. The lower layer, designed to support subchondral bone regeneration, consists of a porous polycaprolactone (PCL) scaffold fabricated by fused deposition modeling. The upper layer, intended for cartilage repair, is composed of a polyvinyl alcohol (PVA) hydrogel crosslinked via freeze–thaw cycles and enriched with lyophilized human platelet lysate (LPL), which provides bioactive growth factors to promote cell recruitment and chondrogenic differentiation.
Scanning electron microscopy confirmed the structural integration of the two layers and the desired pore architecture. Mechanical testing demonstrated a biphasic profile resembling native osteochondral tissue, with high stiffness in the PCL layer and viscoelastic behavior in the PVA hydrogel. In vitro assays using Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) showed that the scaffold is non-cytotoxic and that LPL enrichment enhances cell proliferation. Additionally, implantation in anatomical osteochondral defect models demonstrated excellent geometric adaptability and ease of surgical handling.
In summary, this bilayer osteochondral scaffold is non-cytotoxic, promotes cell growth, exhibits adequate mechanical properties and shows potential to improve clinical outcomes in the repair of osteochondral defects.
Development of an in-vitro system for studying the chronic exposureof liver microenvironment to the pro-metastatic effect of extracellular vesicles released by colorectal carcinoma cells
Antonio Filippone1, Francesco Lopresti2, Simona Fontana1
1Biomedicine, Neuroscience and Advanced Diagnostic. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy, 2Engineering. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy
Traditional 2D cell systems fail to replicate the complex tissue microenvironment and organ-specific cell interactions. In contrast, Organ-on-Chips offer dynamic microfluidic control, enabling co-culture of multiple cell types and precise regulation of their microenvironments. This allows more realistic modelling of tissue-specific functions and cellular communication, providing deeper insight into organ-level responses. To develop a suitable system for studying how the extracellular vesicles from colorectal cancer cells (CRC_EVs) may affect the hepatocyte–macrophages cross-talk during the pre-metastatic niche formation, we have developed a Liver-on-Chip (LoC) model in which healthy human hepatocytes (THLE2) and human monocytes (THP-1) are seeded into two interconnected chambers. This setup allows for a continuous flow from the CRC_EVs-conditioned THLE2 to the THP-1-derived M0 macrophages.
We selected alginate hydrogels as the most suitable scaffold to ensure the biocompatibility and biomimetic conditions required for the 3D culture of THP-1 monocyte cells and their subsequent differentiation. This scaffold successfully promoted spontaneous THP-1 differentiation into M0 macrophages, as assessed by confocal imaging of macrophage marker CD68 expression. Subsequently, we evaluated the effect of the conditioned medium of THLE2 cells treated with CRC_EVs on the THP-1-derived M0 macrophages, to investigate if and how the CRC_EVs-conditioned hepatocytes may activate the M0 macrophage pro-tumor phenotype, supporting pre-metastatic niche formation, observing via confocal imaging and ELISA the expression of the M2 TAM markers (eg. CD206) and the release of cytokines such as TGF-β and IL-10.
At the same time, we designed the LoC setup previously mentioned, enabling continuous flow from the CRC_EVs-conditioned THLE2 chamber to THP-1-derived M0 macrophages. This setup supports chronic CRC_EVs exposure, creating a more biomimetic environment. Results showed that this system improves understanding of hepatocyte–immune cell, such as macrophages, cross-talk during pre-metastatic niche formation, overcoming limitations of acute CRC_EVs treatment.
Designing animal free 3D in vitro models for drug screening and disease progression
Aline Miller1, Julie Gough2
1Manchester Institute of Biotechnology, School of Engineering. University of Manchester, Manchester - United Kingdom, 2Department of Materials, School of Natural Sciences, Faculty of Science and Engineering and The Henry Royce Institute, Royce Hub Building, The University of Manchester, Manchester, UK, Manchester - United Kingdom
Despite decades of reliance on animal models – including rodents (mice, rats), rabbits, and larger mammals such as dogs and pigs – translation to human treatments needs to be more efficient. Over 100 million animals are used annually in research worldwide, with rodents comprising 85–90% of these cases. In the UK alone, 3.06 million procedures involving live animals were conducted in 2021, with 51% dedicated to basic research.
Conventional cell culture in 2D does not recapitulate the complexities of tissues, but revolutions in three-dimensional (3D) cell culture systems are leading to increased attention in drug discovery, tissue engineering and regenerative medicine due to their clear advantages in providing more physiologically relevant information and more predictive data for in vivo tests. This can facilitate understanding of mechanisms of disease progression with potential identification of therapeutic targets. Importantly, with the right cell and molecular methodologies, these models can be completely animal reagent free.
Here, we will outline recent progress in the use of synthetic, self-assembling peptide hydrogels that recapitulate the in vivo microarchitecture and chemical functionality to generate 3D tissue and disease models including for example 3D organoids and tumour models. We will demonstrate how appropriate models can mimic the tumour microenvironment, in terms of ECM architecture, stiffness, incorporate multiple cell types, spheroids and organoids, include immune cells and cancer associated fibroblasts. These can be used to study disease progression, effects of a hypoxic environment and test therapeutics. For drug toxicity testing, we will show how hepatocytes can be incorporated along with other relevant cell types to provide a physiologically relevant environment for investigating drug toxicity mechanisms, ultimately showing how we can drive the 3R’s ultimately reducing the use of animals in research, and critically how these can be developed to be completely animal reagent free.
Engineering material-mediated bioelectric cues for stem cell response
Amy Gelmi
de RMIT University, Melbourne (Victoria) - Australia
Electrically conductive biomaterials will control stem cell fate; engineering these cues for a specific response is the current challenge in the field.[1,2] The charge transport between a biomaterial and a stem cell is inherently complex due to the heterogenous nature of the cell-material interface but is critical to understand in relevance to electrically sensitive components within stem cells. Knowing how these components are modulated is key as they are initiators for downstream signalling, leading to fate decisions.
How the stem cells transduce an electrical signal into a biological response is explored via different classes of conductive biomaterials, targeting proliferation and osteogenesic responses. Immediate changes in the stem cells during and post-stimulation is characterised, using live cell bio-AFM for morphological and biomechanical changes, complemented with biological characterisation. The advanced bioAFM technique delivered unprecedented intracellular biomechanical information of live cells undergoing simultaneous electrical stimulation.[3,4]
Here I will present our research platform which explores the impact of electrical stimulation on stem cell response; from biomedical engineering approaches using custom electrical stimulation culture devices, through to advanced bio atomic force microscopy (AFM) to capture real-time changes in stimulated cells.
[1] A. Gelmi, C. E. Schutt, Stimuli-Responsive Biomaterials: Scaffolds for Stem Cell Control. Adv. Healthcare Mater. 2020, 10, 2001125.
[2] K. Zhang, D. De Maria, M. Jebakumar, J. Collins, K.E. Fox, and A. Gelmi. The Bionic Interface: Considering the Material Mediated Electrical Stimulation of Stem Cells. Adv. Mater. 2025: e12399.
[3] K. Zhang, C. L. Reeves, M. Moghaddar, K. E. Fox, A. Elbourne, and A. Gelmi, At the Pointy End of Mechanobiology: AFM for Transient Biomechanical Analysis. Adv. Healthcare Mater. 2025: e02026
[4] K. Zhang, C. L. Reeves, J. D. Berry, K. E. Fox, A. Elbourne, and A. Gelmi, Live Quantitative Characterization for Stem Cell Biomechanics. Adv. Mater. Interfaces 2025: e00403
Modulating macrophage phenotype under hypoxia for bone regeneration using hemoglobin-based microparticles
Michelle Jansman1, Fan Yang2
1Department of Health Technology, Technical University of Denmark. Denmark & Department of Orthopaedic surgery, Stanford University, Stanford (California) - United States, 2Department of Orthopaedic surgery & Department of Bioengineering, Stanford University, Stanford (California) - United States
Introduction: Critical-sized bone defects are difficulty to heal, and are associated with persistent hypoxia and inflammation. Following injury, macrophages play a pivotal role in orchestrating the immune responses, where timely transition from acute inflammation (M1-like) to regenerative (M2-like) phenotype is key to effective healing. However, sustained hypoxia within the defect can hinder this transition, thereby delaying healing. We hypothesize that hypoxia changes macrophage polarization, and modulating oxygen can promote macrophage transition to regenerative phenotype and subsequently enhance stem cell osteogenesis. To test this hypothesis, we are investigating oxygen tension as a key regulator of macrophage phenotype and are exploring oxygen delivery as a strategy to promote regenerative immune response using hemoglobin-based microparticles (Hb-MPs).
Methods: RAW264.7 macrophages were first primed to M1 to mimic acute inflammation, and then cultured under hypoxia (5.0% or 2.5% O2) in both 2D and in 3D gelatin microribbon scaffolds. Hypoxic response and macrophage polarization were assessed at various time points using PrestoBlue, qPCR, and ELISA. Hb-MPs were synthesized via the desolvation process, optimized to obtain colloidally stable MPs (1.6 µm), with 80% yield and confirmed O2-releasing capacity. Particles were added to the macrophages for an additional 24 h, after which cellular responses were reassessed.
Results: Under severe hypoxia (2.5% O2), macrophages exhibited upregulation of HIF-1α, VEGFA, and Glut-1, confirming hypoxic stress. Hb-MPs furthermore showed excellent cell viability across a wide range of dosage (0-5 mg/mL). Treatment with Hb-MPs increased oxygen levels, and induced a change in hypoxic- and phenotypic expression of macrophages. Ongoing work focuses on characterizing cytokine secretion under oxygen modulation and evaluating macrophage-MSC crosstalk to link oxygen-mediated immune modulation with osteogenic outcomes. Together, these results will establish oxygen-modulation as a promising immunomodulating strategy to enhance bone regeneration.
Facial nerve regeneration promoted by scaffold-free Bio 3D conduits derived from human dental pulp stem cells
Yoko Kawase-Koga1, Yuri Matsui-Chujo2, Ayano Hatori2, Monika Nakano1, Yudai Miyazaki3, Yoko Torii3, Daichi Chikazu2, Shizuka Akieda3
1Division of Maxillofacial Surgery and Stomatology, Department of Oral and Maxillofacial Surgery. Tokyo Women's Medical University, School of Medicine, Tokyo - Japan, 2Department of Oral and Maxillofacial Surgery. Tokyo Medical University, Tokyo - Japan, 3Cyfuse Biomedical K.K., Tokyo - Japan
Background: Facial nerve injury resulting from trauma or tumor resection causes severe functional and aesthetic impairments, including difficulties in speech and facial expression. Although autologous nerve grafting remains the standard treatment, it has limitations such as donor site morbidity and restricted graft length. To overcome these issues, we developed a novel strategy to promote nerve regeneration by transplanting three-dimensional (3D) nerve conduits composed solely of human dental pulp stem cells (hDPSCs) fabricated using a Bio-3D printer, taking advantage of their high proliferative capacity and neurogenic potential. This study aimed to investigate whether these Bio 3D conduits could also promote regeneration of the facial nerve.
Materials and Methods: hDPSCs were isolated and cultured from a patient’s extracted tooth to form spheroids, which were then layered using a Bio-3D printer to create scaffold-free Bio 3D conduits. These conduits were transplanted into immunodeficient rats with facial nerve defects. Functional recovery was evaluated by whisker movement observation. Histological and immunohistochemical analyses were performed 12 weeks after transplantation.
Results: Twelve weeks after transplantation, whisker movement was observed in both groups. The number of myelinated axons was greater in the Bio 3D group than in the silicone group. Moreover, the myelinated axon diameter and myelin thickness of regenerated axons in the Bio 3D group were significantly larger (p < 0.01, p < 0.05), indicating enhanced axonal maturation and remyelination.
Conclusion: This study confirmed the nerve regeneration potential of Bio 3D structures fabricated from hDPSCs transplanted into a rat model of facial nerve injury, suggesting their efficacy for facial nerve regeneration and supporting future validation studies in the maxillofacial region.
Tailoring type I collagen properties through functionalization with natural compounds and biomolecules
Anna Rossanese1, Jovana Curcic2, Silvia Spriano1, Milka Malesevic2
1Material Science and Technology. Politecnico di Torino, Torino (Italia) - Italy, 2Institute of Molecular Genetics and Genetic Engineering, Belgrade (Serbia) - Serbia
Type I collagen is one of the main components of the extracellular matrix and plays a crucial role in providing structural support and regulating cellular processes. In this work, a system based on type I collagen functionalized with bioactive molecules was developed to enhance the biological properties and application potential of the material. The functionalization strategy was designed to preserve the structural integrity of collagen while ensuring the stable immobilization of the selected bioactive molecules. Morphological, chemical, and physicochemical analyses confirmed the success of the surface modification and the presence of the desired functional groups. Preliminary tests demonstrated good biocompatibility, suggesting an improvement in biological performance compared to the untreated material. The proposed approach, owing to its versatility and the possibility of tuning the nature of the bioactive molecules, offers a promising platform for the development of advanced biomimetic materials intended for various biomedical applications, including tissue engineering, regeneration, and controlled release of therapeutic agents.
Application of a 3-level full factorial experimental design for the synthesis of chlorogenic acid-loaded transethosomes
Ance Bārzdiņa1, Dace Bandere1, Agnese Brangule1
1Riga Stradins University; Baltic Biomaterials Centre of Excellence, Riga - Latvia
Transethosomes are ultra-flexible lipid nanoparticles designed to efficiently penetrate the stratum corneum and deliver various therapeutics to the deeper layers of skin. This study aimed to apply a design of experiment (DoE) approach to optimize the synthesis of chlorogenic acid (CGA)-loaded transethosomes to achieve particles with parameters suitable for transdermal delivery.
The DoE was performed on the MODDE software. Impact of 3 factors at 3 levels – concentration of phosphatidylcholine (1%; 2%; 3%), Tween 80 (0.2%, 0.3%, 0.4%), and CGA (1 mg/mL; 3 mg/mL; 5 mg/mL) – on the vesicle size, polydispersity index (PDI), encapsulation efficiency (EE%) and pH of the formulation were investigated. The target formulation parameters were defined as a size under 250 nm, PDI under 0.3, EE% higher than 70% and a pH range of 4.5 to 7. The particles were prepared using the cold method. The most promising formulation was further incorporated in a Carbopol-based hydrogel. Additionally, the zeta potential, FTIR spectra, drug release, antioxidative properties (DPPH method), and stability of formulations were investigated.
A full-factorial design (30 formulations) was chosen (model power – 91%). Only formulations with 1% and 2% phosphatidylcholine had a size under 250 nm (141 to 171 nm and 172 to 233 nm, respectively). EE% ranged from 81.2% to 95.6% with a positive correlation between EE% and phosphatidylcholine concentration. Formulations with 0.4% Tween 80 had the lowest PDI (0.10 to 0.17) and showed no formation of agglomerates over 3 months. Only the 3 mg/mL CGA formulations had a pH suitable for transdermal delivery (pH 4.85 to 4.89). In 3 hours, 96.6 ± 1.3% of CGA was released from transethosomes, while from hydrogel, only 65.9 ± 3.8% was released in 8 hours.
CGA-loaded transethosomes with the most suitable parameters for transdermal delivery can be synthesized using 2% phosphatidylcholine, 0.4% Tween 80, and 3 mg/mL CGA.
PIC hydrogels: synthetic, tailorable and easy-to-use Matrigel replacement
Paul Kouwer
de Radboud University, Nijmegen (Gelderland) - The Netherlands
Researchers, industry, society, governments and regulating bodies increasingly realize that animal experiments have severe limitations. Phasing out animal tests, however, requires good in vitro alternatives. The most promising alternatives include organoids, either patient-derived or from pluripotent stem cells. Besides complete organ models, organoids have the important advantage that they are human and provide human-relevant data.
Establishment, expansion and application of these organoids often use murine sarcoma basement membrane extract (BME), the best-known is Matrigel. The disadvantages of Matrigel are well known, the animal and cancerous origin and the variability and poorly defined composition hampers progress in the field and adaptation by industry. Yet, it is the only matrix material that supports the necessary growth rates and maturation profile.
In this talk, I will introduce PIC hydrogels as an alternative for Matrigel in organoid research. PIC gels are completely synthetic and have a well-defined composition. They are tailorable to match desired applications. In addition, PIC gels are extremely easy to use. Gels will form by heating to 37°C and dissolve within seconds after cooling to 5°C, facilitating very easy organoid extraction without the risk of damaging the cells. [1]
Most importantly, in PIC gels we observe organoid expansion and passaging at rates comparable to or faster than Matrigel cultures. In our first studies, some together with the Clevers group at the Hubrecht Institute, we cultured patient-derived organoids from lung, colon, small intestine [2] and head and neck cancer.
References:
[1] Yuan, …, Kouwer “Fibrous polyisocyanide hydrogels for 3D cell culture applications” Nat. Protoc. 20, 3339-3360 (2025).
[2] Wijnakker, …, Kouwer & Clevers, “Invasin-functionalized PIC hydrogels enable long-term 3D culture of epithelial organoids” Proc. Natl. Acad. Sci. U. S. A. 122, e2507500122 (2025).
Electrospun gelatin–carbon black scaffolds for anisotropic cardiac tissue models
Luca Di Nunno1, Arman Mirzaei1, Federica D’erchia1, Diego Mantovani2, Francesca Boccafoschi1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Mineral, Metallurgical, and Materials Engineering. Centre de Recherche du CHU de Québec‐Université Laval, Quebec - Canada
Cardiac cells phenotype and functionality are lost when cultured in vitro, thus, predicting cardiac behaviour in current experimental models is challenging. A significant advance in improving tissue functionality is represented by biomaterials combining biochemical, topographical and electrical cues1. To replicate the complexity of tissue microenvironments, electrospinning offers a versatile platform for creating nanofibrous scaffolds with controlled size and orientation. Gelatin is a promising candidate for electrospinning, that promotes biological responses, however, its electrically insulating properties severely restrict cellular electromechanical coupling2. In this context, electroconductive nanoparticles could compensate this limitation, however, they present concentration-dependent cytotoxicity3. This study aims to develop a physiologically relevant anisotropic cardiac tissue model. To do so, scaffolds made of aligned electrospun gelatin-carbon black (Gel-CB) were characterized.
Highly electroconductive Carbon black Super P® was added to gelatin (15% w/v) at different concentrations (0.1%, 0.5%, 1%, and 2% w/v). Scanning electron microscopy was used for fibers size and orientation analysis. Swelling, degradation, and energy-dispersive X-ray spectroscopy were performed. Nanoindentation was used to evaluate the micro-mechanical properties and electroconductive properties of the cardiac model were characterized. H9C2 cardiomyoblasts were used for cytocompatibility testing.
Carbon black nanoparticles, averaging 63nm, were dispersed with minimal aggregation in the scaffolds. Results showed uniformly aligned nanofibers ∼180 nm in diameter with ∼80% alignment efficiency. Scaffolds were stable over one month in PBS with approximately 400% swelling rate. Young's modulus recorded were ∼50 kPa on average. MTT assay confirmed non-cytotoxicity of the carbon black concentrations tested, additionally cardiac cells demonstrated alignment with the scaffold structure. Electrospun Gel-CB nanofibers demonstrated adequate anisotropic properties, mechanical stability, and cytocompatibility, hence holding great potential to advance biomaterials for cardiac tissue applications.
1) Tadevosyan K. et al., Int. J. Mol. Sci. 2021
2) El-Seedi, H.R. et al., Heliyon. 2023
3) Ou L. et al., Part Fibre Toxicol. 2016
New Bench-Top incubator for continuous microscopic observation of microfluidic devices
Pedro Moreo1, Marta Mainar1, Víctor Alastrué1, Javier Conte1
1R&D. EBERS Medical Technology S.L., Zaragoza - Spain
Introduction.
Microscopic monitoring of cell culture behavior in microfluidic devices presents significant challenges due to the need to maintain the system inside an incubator. It is often necessary to transfer the microfluidic circuit from the incubator to the microscope when observation is required. This process entails considerable risks, as removing the circuit from the controlled environment of the incubator leads to loss of regulated gas concentrations and temperature, as well as increased risks of contamination or leakage due to handling.
Materials & methods
The core of the system is a gas mixing chamber that receives controlled flows of the gases required to achieve the desired concentrations. These flows are independently regulated by mass flow controllers. The mixed gases are injected into a microscopy chamber equipped with a heating system that regulates its temperature. Both, temperature and gas concentrations can be precisely controlled directly within the microscopy chamber, enabling continuous observation without manipulating the microfluidic device.
The system also allows monitoring of the gas mixture to ensure proper composition and can operate in different control modes depending on the gases available to the user, including CO2, N2, and/or O2.
Results
The device presented here eliminates these risks by maintaining, within the microscopy chamber itself, the same controlled conditions normally provided by an incubator, allowing continuous observation without the need for an external incubator.
The gas flow rates can be regulated within a range from 0.5 mL/min to 0.5 L/min, minimizing gas consumption and allowing for an almost instantaneous change in incubation parameters, without the need for the typical stabilization time required in a conventional incubator.
The use of micropumps enables the configuration of complex flow patterns as well as the integration of various inline sensors (pH, glucose, temperature, biosensors, …).
Early therapeutic approaches to treat neurogenic lower urinary tract dysfunction after spinal cord injury
Elena Keller1, Karin Roider1, Michael Kleindorfer2, Evelyn Beyerer1, Georgina Brandtner3, Lukas Lusuardi2, Ludwig Aigner1, Sophina Bauer2
1Institute of Molecular Regenerative Medicine. Paracelsus Medical University, Salzburg - Austria, 2Department of Urology. Paracelsus Medical University, Salzburg - Austria, 3Department of Pediatric Surgery. Paracelsus Medical University, Salzburg - Austria
Neurogenic lower urinary tract dysfunction (NLUTD) is one of the most debilitating consequences of spinal cord injury (SCI), often leading to detrusor-sphincter-dyssynergia (DSD), bladder fibrosis, and long-term renal complications. Despite major advances in neuro-urology, no causal therapies are currently available. This presentation highlights translational approaches focusing on early neuro- and pharmacomodulatory interventions to prevent the onset of NLUTD.
Sacral neuromodulation (SNM) has been shown to be effective and safe for treating NLUTD in selected neuro-urological patients. However, its therapeutic potential in complete SCI remains under investigation. In a preclinical minipig model, early initiation of SNM preserved bladder compliance, prevented DSD, and reduced fibrotic remodeling, underscoring the pivotal role of timing and supporting SNM as a promising preventive therapy. Complementing these findings, a cross-sectional study involving 86 patients with chronic SCI revealed that over 60% would have consented to early intervention, independent of quality of life, emphasizing the need for patient education and engagement to facilitate clinical translation.
Parallel rodent studies investigated early pharmacomodulation. Early montelukast, a cysteinyl-leukotriene receptor-1 antagonist, treatment after complete SCI improved bladder function in rats over 4 weeks post SCI. However, an ongoing long-term study over 2 and 6 months indicates complex immunomodulatory effects, with preliminary results suggesting potential adverse impacts on bladder function and structure, underscoring the need for a more tailored post-SCI anti-inflammatory and pharmacomodulatory strategy.
Together, these translational findings demonstrate that early interventions can preserve bladder structure and function after SCI. They provide a robust methodological foundation for clinical implementation, including preparations for a multicenter clinical trial on early SNM in acute SCI. Ultimately, these efforts aim to shift NLUTD management from symptomatic control toward mechanism-based, preventive treatment strategies.
Delivery of cells using innovative nanoclay gels for cartilage regeneration
Zahra Hosseinzadeh1, Jonathan I Dawson2, Agnieszka Janeczek3, Richard O.c. Oreffo2, Roxanna S. Ramnarine-Sanchez4
1Bone and Joint Research Group, Human Development and Health. University of Southampton, Southampton - United Kingdom, 2Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton, SO16 6YD, UK. University of Southampton, Southampton - United Kingdom, 3Renovos Biologics Ltd, Science Park, 2 Venture Rd, Chilworth, Southampton, SO16 7NP, UK. University of Southampton, Southampton - United Kingdom, 4Faculty of Medicine, Department of Human Development and Health, University of Southampton, Southampton, SO16 6YD UK. University of Southampton, Southampton - United Kingdom
Osteoarthritis affects approximately 8% of the global population, leading to an urgent need to establish a low-cost method for cartilage repair without invasive surgeries. Cartilage regeneration remains a major clinical challenge due to the tissue’s limited self-repair capacity. Although cell therapies are now a viable alternative to joint replacements in some cases, the lack of an effective cell carrier system has, to date, resulted in poor cell retention and high cell death, highlighting the need for biomaterials that offer both structural support and biochemical signals for improved cartilage repair. Synthetic nanoclays form biocompatible colloidal gels capable of supporting cell growth within the body. The high surface-area-to-volume ratio and dual-charged surface of nanoclay, which increase cell adhesion and protein adsorption, respectively, make the nanoclay a promising biomaterial for biomedical applications. The aim of this study is to evaluate nanoclay hydrogel–cell constructs for cartilage tissue engineering applications. To evaluate the cytocompatibility of the nanoclay and determine its ability to support cell viability during encapsulation, a live/dead assay was performed. The viability of the human Bone Marrow Stromal Cells (hBMSCs) was evaluated in different concentrations of the nanoclay hydrogel (2.5%, 3%, and 3.5%) to determine whether increasing hydrogel stiffness adversely affects cell viability; no significant differences were observed across the tested concentrations. To compare the cell viability of hBMSCs and human articular chondrocytes (HACs) in the optimised condition, a 2-week live/dead study was performed. On Day 14, both cells maintained high viability. Histological evaluation was performed using Alcian Blue/Sirius Red and Haematoxylin and Eosin staining to assess glycosaminoglycan production, collagen deposition, and chondrocyte morphology. In 3% nanoclay hydrogel containing HACs, lacuna-like structures with rounded chondrocytes were observed, alongside positive Sirius Red staining indicating collagen deposition. Together, these methods provided a robust platform for the systematic assessment of nanoclay hydrogel for cartilage regeneration.
Enhancement of the bioactivity of titanium surfaces by combining laser texturing and calcium phosphate deposition
Jaroslav Fojt1, Tomas Kovarik2, Ema Volkova1, Denys Moskal2, Jiri Martan2, Vojtech Hybasek1
1Department of metals and corrosion engineering. University of Chemistry and Technology in Prague, Prague (Hlavni Mesto Praha) - Czech Republic, 2New Technologies Research Centre. University of West Bohemia in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic
Titanium and its alloys are extensively used in biomedical applications, particularly for orthopedic and dental implants, owing to their excellent mechanical strength, corrosion resistance, and biocompatibility. However, their intrinsic bioinertness limits direct bone bonding and may delay osseointegration after implantation. Therefore, surface modification is essential to improve the biological response of titanium and accelerate the integration of implants with surrounding bone tissue.
Laser surface texturing represents an effective approach to modify titanium surfaces by generating well-defined micro- and nanoscale topographies. Such hierarchical structures can enhance surface roughness and wettability, promote protein adsorption, and facilitate osteoblast adhesion and proliferation. In addition, the application of bioactive coatings such as calcium phosphates further enhances bioactivity by providing nucleation sites for hydroxyapatite and promoting chemical bonding with bone.
In this study, titanium substrates were modified by laser texturing to create micro- and nanoscale surface features. Subsequently, calcium phosphate layers were deposited from simulated body fluid (SBF) using a potentiostatic two-step polarization technique. The bioactivity of the modified surfaces was evaluated by immersion tests in SBF, while the kinetics of mineralization were monitored using electrochemical impedance spectroscopy.
The obtained coatings exhibited Ca:P ratios between 1.6 and 2.0, confirming the successful formation of calcium phosphate phases. Immersion experiments revealed accelerated hydroxyapatite precipitation on laser-textured titanium compared to smooth surfaces, with a further enhancement observed for samples combining laser texturing and calcium phosphate deposition.
Overall, the synergistic effect of laser surface structuring and electrochemically induced calcium phosphate coating provides a promising strategy to improve the bioactivity and osseointegration potential of titanium-based implants.
This study was supported by the project “Mechanical Engineering of Biological and Bio-inspired Systems” (No. CZ.02.01.01/00/22_008/0004634), funded by the Johannes Amos Comenius Program.
High-resolution imaging platform to unravel pathophysiological mechanisms of Pseudomonas aeruginosa infections of the human upper airway epithelium
Amanzhol Kurmashev1, Julia A. Boos1, Benoît-Joseph Laventie2, A. Leoni Swart2, Rosmarie Sütterlin2, Tina Junne2, Urs Jenal2, Andreas Hierlemann1
1Biosystems Science and Engineering. ETH Zürich, Basel (Basel-Stadt) - Switzerland, Basel (Basel-Stadt) - Switzerland, 2Biozentrum. University of Basel, Basel (Basel-Stadt) - Switzerland
Pseudomonas aeruginosa poses a significant threat as a leading cause of hospital-acquired pneumonia and has been designated a priority pathogen by the World Health Organization (WHO) due to its alarming antibiotic resistance [1]. Critical aspects of P. aeruginosa infections are effective colonization and breaching of the lung mucosal surface. Yet, understanding the precise mechanisms governing the host tissue responses remain elusive. Although biomimetic in-vitro models of the human airway have enabled significant insights into the infection process, current methodologies feature limited capability of studying the underlying pathophysiological mechanisms at single-cell resolution in real time.
Here, we introduce a novel microphysiological platform designed for live-cell high-resolution imaging of transwell-based lung organoids [2]. The platform allows for seamless alternation between air-liquid and liquid-liquid interface conditions. While the air-liquid interface is used to culture the lung tissue under natural conditions, the liquid-liquid interface permits imaging of the infected tissue at high spatiotemporal resolution. This plastic-based microfluidic platform facilitates straightforward insertion and retrieval of transwell inserts, providing standard well plate conditions for tissue differentiation and analysis.
We continuously monitored Pseudomonas aeruginosa infections in human stem-cell-derived bronchial epithelial tissue in real time. We gained valuable insights into bacterial invasion of the apical surface of the lung epithelium, as well as the evolving dynamics of tissue breaching and destruction over time. The developed airway tissue culture system is a powerful tool for visualizing and understanding critical aspects of pathophysiological mechanisms underlying P. aeruginosa infections and antimicrobial resistance and may contribute to developing novel antibiotic treatment strategies.
[1] Langendonk R.F., Neill D.R., Fothergill J.L., (2021) Frontiers in Cellular and Infection Microbiology 11, doi: 10.3389/fcimb.2021.665759
[2] Kurmashev A., Boos A. J., Laventie B.-J., et al., (2023) Advanced Materials Technologies, 9, 2400326, doi: 10.1002/admt.202400326
Development of an engineered periosteum based on a melt electrowritten PCL mesh, combined with bioprinted hydrogel layers
Yakui Liu1, Xinggui Tian2, Max Von Witzleben1, Anne Bernhardt1, Anja Lode1, Stefan Zwingenberger2, Michael Gelinsky1
1University Hospital Carl Gustav Carus and Faculty of Medicine, Centre for Translational Bone, Joint and Soft Tissue Research. Dresden University of Technology, Dresden (Sachsen) - Germany, 2Center for Orthopaedics, Trauma Surgery and Rehabilitation Medicine. University Medicine Greifswald, Greifswald (Mecklenburg-Vorpommern) - Germany
Background: The periosteum is essential for bone remodeling and repair but autologous periosteal grafts are limited by tissue availability and patient condition [1]. Engineered periosteum scaffolds based on melt electrowriting (MEW) offer a solvent-free, highly organized 3D architecture, which further can be integrated with bioactive hydrogels to recapitulate the native osteogenic and angiogenic microenvironments.
Method: A tri-layer periosteum-mimetic construct was fabricated combining a MEW polycaprolactone mid-layer with two hydrogel compartments. An alginate/methylcellulose bioink containing human osteoblasts (hOB) formed the osteogenic layer, and a fibrin/gelatin bioink containing human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) formed the angiogenic layer. After separate optimization of scaffold architecture and single-layer performance, tri-layer constructs were assembled and cultured in the same co-culture medium. Three configurations were compared: osteogenic-only (3O, only hOB), angiogenic-only (3A, NHDF+HUVEC) and tri-layer (3T, all cell types). Alkaline phosphatase (ALP) activity, CD31 immunofluorescence staining and osteogenic/angiogenic gene expression were assessed.
Results: In 3O, ALP activity remained low without significant change over 28 days (pNp < 0.1 µmol/mL at all time points), and osteogenic genes (ALPL, BGLAP, BMP-2) showed no significant differences over time. By contrast, 3T exhibited a time-dependent increase in ALP, reaching ∼1 µmol/mL pNp by day 42, accompanied by significant upregulation of osteogenic genes. CD31 immunostaining revealed dense, interconnected vascular-like networks in 3A at days 14 and 21, whereas 3T displayed sparse structures at early stages but formed comparable networks after day 28. Consistently, angiogenic genes (CD31, vWF, CDH5) increased significantly in 3A by day 14, while 3T showed significant elevations at days 28–35.
Conclusion: The tri-layer MEW/hydrogel periosteum supports sustained osteogenesis while preserving angiogenic capacity. This platform provides a controllable model for studying osteo–angiogenic coupling and a promising new strategy for vascularized bone regeneration.
Reference
[1] Yang, Yuhe, et al., J. Orthop. Transl. 36 (2022): 18-32.
Cobalt modified calcium phosphate cement for promotion of angiogenesis during bone repair
Chuandong Ye1, Richard Frank Richter1, Anne Bernhardt1, Michael Gelinsky1
1Centre for Translational Bone, Joint and Soft Tissue Research. University Hospital “Carl Gustav Carus” at Technische Universität Dresden, Dresden (Sachsen) - Germany
Introduction:
Calcium phosphate cements (CPC) are widely investigated bone replacement materials used clinically due to their biocompatibility, osteoconductivity, and injectability. Self-setting CPC typically include a liquid phase and hydraulic reactive calcium phosphate precursors like α-tricalcium phosphate (α-TCP), enabling high modifiability [1]. Angiogenesis is essential for bone repair, delivering oxygen and nutrients. Cobalt ions are known to stimulate angiogenesis, promoting vascularization and bone regeneration [2]. This study modifies CPC with cobalt(II) (Co-CPC) to enhance angiogenic properties and improve its use as bone replacement material.
Methods:
Co-CPC samples were prepared by partially substituting CaCO3 with CoCO3 in the traditional CPC formulation (60 wt% α-TCP, 26 wt% CaHPO4, 10 wt% CaCO3, 4 wt% HAp) [3]. Samples were characterized for setting behavior, mechanical strength, ion release, degradation behavior, and physicochemical properties. In vitro studies analyzed osteogenic differentiation of hMSC (human mesenchymal stem cells) and hOB (human osteoblasts), and angiogenesis of HUVEC (human umbilical vein endothelial cells) in co-culture with hOB.
Results:
CoCO3 substitution enabled controlled Co2+ ion release over 28 days in HPLM (Human Plasma-Like Medium) and HEPES media. Under a specific ratio, the cements showed strong compressive strength, which can easily meet the strength requirements for clinical application. Osteogenically differentiated hMSC and hOB proliferated and expressed alkaline phosphatase (ALP) when cultivated with Co-CPC extracts. Furthermore, HUVEC in co-culture with hOB showed significantly increased tube-formation in response to Co-CPC.
Conclusion:
Co(II)-substitution in CPC maintains essential mechanical properties while significantly enhancing angiogenesis through controlled Co2+ release. Improved vascularization supports better bone regeneration, positioning Co-CPC as a promising bone replacement material.
References:
1. R. F. Richter et al., Bioact. Mater. 2023, 28, 402.
2. C Wu et al., Biomaterials 2012, 33, 2076-2085.
3. F. C. M. Driessens et al., J. Biomed. Mater. Res. A 1997, 38, 356-360.
Human iPSC-based blood–brain barrier models as predictive tools for CNS drug development
Catarina Gomes1, Elisabetta Traggiai2, Andreas Hierlemann3, Agostino Cirillo2, Mario M. Modena3
1Biosystems Science and Engineering. ETH Zürich, Basel (Basel-Stadt) - Switzerland & BioMedical Research. Novartis Pharma AG, Basel (Basel-Stadt) - Switzerland, Basel (Basel-Stadt) - Switzerland, 2BioMedical Research, Novartis Pharma AG, Basel (Basel-Stadt) - Switzerland, 3Biosystems Science and Engineering. ETH Zürich, Basel (Basel-Stadt) - Switzerland, Basel (Basel-Stadt) - Switzerland
The human blood–brain barrier (BBB) is a highly specialized and dynamic multicellular interface that strictly regulates molecular and cellular exchange between the systemic circulation and the central nervous system (CNS). Modeling the BBB still relies greatly on animal models, yet these do not fully recapitulate human-specific neurovascular interactions and CNS drug permeability, which limits their predictive value for translational research. Despite progress in developing in vitro human BBB models, faithfully recapitulating the full physiological and pathophysiological complexity of the native barrier remains a major challenge, limiting the translational relevance of current systems for CNS drug development and disease modeling.
In this study, we present a robust, human induced pluripotent stem cell (hiPSC)-derived in vitro BBB platform that recapitulates key structural and functional features of the native barrier. Using optimized differentiation and co-culture protocols, we generated brain endothelial cells (iECs) with enhanced molecular identity and barrier functionality. When co-cultured with iPSC-derived neural cells and pericytes, the resulting multicellular BBB model exhibited a distinct brain endothelial signature, receptor polarization (e.g., luminal: P-gp, ABCG2; abluminal: SLC6A13), and expression of known transcytosis receptors, such as the transferrin receptor (TfR). Exchanging a conventional PET membrane with an extracellular matrix (ECM)-based scaffold further improved intercellular contact and promoted a more physiologically relevant BBB phenotype. Permeability assays using small compounds validated the tight junction integrity of the hiPSC-derived BBB, showing selective diffusion patterns that recapitulated in vivo barrier behavior, underscoring the translational potential of our model for CNS drug testing.
This engineered hiPSC-derived BBB system provides a scalable, human-relevant platform for investigating drug transport, neurovascular interactions, and potentially BBB dysfunction in neurodegenerative diseases. It is ultimately aimed at accelerating preclinical development of CNS-targeted therapeutics.
Directing signaling profiles and mechanotransduction in tendon bioengineered systems using magnetic-instructive strategies
Ana Gonçalves1, Bárbara Adão1, Manuela Gomes1
1ICBAS - School of Medicine and Biomedical Sciences, University of Porto, Porto - Portugal
Tendon pathologies represent a diverse spectrum of conditions that affect the functional integrity and biomechanics of tendons. The regeneration of this tissue requires the coordinated activation of complex signaling networks that regulate cell fate, matrix synthesis, and tissue organization. Mechanical cues play a central role in these processes, yet their controlled modulation in bioengineered systems remains a major challenge.
Integrating temporally tunable magnetic mechano-actuation with magnetic nanoparticles to remotely deliver functional mechanical stimuli to therapeutic or diseased cells provides a tool for regulating tenogenic regenerative mechanisms and/or in the modulation of signaling profiles in both homeostasis and disease.
Signaling cascades are the main routes of communication between the membrane and intracellular regulatory targets involved in mechanotransduction. In this study, we aim at targeting key mechanosensitive membrane receptors with functionalized magnetic nanoparticles and magnetic stimulation to direct cellular mechanotransduction processes, influencing signaling cascades and investigating its role in tendon homeostasis, regeneration, and tendon tumor progression. Particularly, the Transforming growth factor (TGF)-β signaling pathway which mediates adaptive responses towards tendon homeostasis and regeneration, and the Hippo–YAP/TAZ pathway, a central regulator of mechanosensitive transcriptional responses also associated to the tenosynovial giant cell tumor progression. The activation of ligand-receptor complexes drives a cascade pf phosphorylation events inducing structural changes in the cytoskeleton and promoting regulated transcriptional responses.
Moreover, 3D cultures represent more natural cell-cell and cell-matrix interactions and closer mimic the physiological 3D microenvironment of tissues. As such, the construction of 3D magnetic spheroids offers the possibility to work with complex cell cultures while providing local stimulation and better engineered microenvironments, maximizing health/disease models and facilitating targeted therapy.
Overall, mechanotransduction-driven insights are expected to advance the understanding of how mechano-magnetic environments shape cellular signaling in musculoskeletal repair and open new avenues for bioinstructive regenerative therapies.
Acknowledgements: FCT grant 2024.09198.CEECIND, FCT Project COMPETE2030-FEDER-00656100, and TENET COST Action CA22170.
The role of titanium surface properties in directing macrophage polarization and promoting osseointegration
Biagio Matera1, Ottavia Cannatella1, Francesca Rossi2, Ludovica Parisi3, Edoardo Manfredi1, Simone Lumetti1, Maddalena Manfredi1, Benedetta Ghezzi1
1Center of Dental Medicine, Medicine and Surgery. University of Parma, Parma (Emilia-Romagna) - Italy, 2IMEM-CNR, Institute of Materials for Electronics and Magnetism-National Research Council, Parma (Emilia-Romagna) - Italy, 3Department of Orthodontics and Dentofacial Orthopedics. Medical Faculty, University of Bern, Bern - Switzerland
Endosseous implants are the gold standard for the replacement of missing tooth and their long-term osseointegration is influenced by several factors (e.g. age, general health, bone quality).At a molecular level, impaired bone healing might be due to a combination of i) inflamed micro-environment, ii) presence of pro-inflammatory M1-like instead of anti-inflammatory alternatively activated macrophages (M2), and iii) impaired osteoprogenitors functionality. Therefore, the aim of this study is to ascertain if titanium implant surfaces characterized by different material composition, surface micro-topography and hydrophilicity (SLA, SLA+, R and R+), can affect the polarization of human macrophages (THP-1) and the consequent response of osteoprogenitors (MG-63).
The polarization of THP-1 through M1/M2 phenotype has been induced on the selected surfaces and gene expression (e.g. IL-6, IL-8, TNF-α, IL-10, etc.), as well as cytokine release (e.g. BMP-2, IL-4, IL-6, IL-10, OPG, etc.) have been observed through qRT-PCR and Luminex assay. Simultaneously, the culturing medium resulting by THP-1 has been used to stimulate the mineralization of MG-63 to corroborate the effectiveness of the interaction between the lineages.
Gene expression analysis showed a clear effect of SLA+ as a substrate capable of inducing a more pronounced anti-inflammatory effect, as demonstrated by most of the considered markers. Luminex analysis highlighted that cytokine/chemokine release was often greater in the presence of hydrophilic surfaces (SLA+/R+) without major differences between M1/M2. The induced mineralization on MG-63, even if not statistically significant, was in agreement with the results of protein quantification, underlining a higher mineralization on SLA+.
All the obtained results showed that SLA+ surface apparently reversed the effects of M1-induced polarization toward the M2 pathway, inhibiting the inflammatory response and confirming that the optimization of surface characteristics plays a pivotal role in the development of innovative and more effective titanium implants able to control immune response and stimulate bone matrix deposition.
Mechanical stimulation driven cellular responses in a 3D corneal model cultured in a custom made bioreactor system
Matthia Bonizzi1, Mark Ahearne1
1Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute. Trinity College Dublin, Dublin - Ireland
Introduction
Mechanical stimuli play a key role in regulating cellular behavior. Anterior cornea is subject to shear and stretch forces. While the effect of stretching on corneal epithelial cells (CEpiCs) and corneal stromal cells (CSCs) has been studied, little is known about how corneal cells respond to different magnitudes of shear stress. Therefore, this study aims to design and validate a bioreactor system that mimics the tear movement during the eyeblink to understand corneal cells behavior in a physiological-like environment.
Methods
CSCs are embedded in a collagen-type-I:PEG-SG (polyethylene glycol-succinimidyl glutarate) hydrogel (stromal layer) and inserted into a custom-made chip. An electrospun PCL (poly-ε-caprolactone) membrane with CEpiCs (epithelial layer) is placed atop. The chip is connected to a fluidic pump that controls fluid flow and varies shear stress (0, 0.25, and 1Pa) provided to the corneal model during up to 3 days stimulation.
Results
The bioreactor system has been validated through computational, gene expression, and immunocytochemistry analyses. Differences in CEpiCs response to stimulation were detected when CSCs are embedded or not in the underneath hydrogel.
qPCR and immunocytochemistry analyses of the epithelial and stromal layers showed that the co-culture model (0Pa) reduced epithelial functions (MUC1 downregulated) and drove CSCs towards a fibroblastic phenotype (α-SMA upregulated). However, following the mechanical stimulation, particularly 1Pa shear stress, the epithelial function was improved (ZO-1 and MUC1 upregulated), and CSCs showed healthy keratocyte phenotype (ALDH3A1 upregulated). Moreover, collagen quantification assay showed that the stimulation also increases the production of extracellular matrix (ECM).
Conclusions
This study demonstrated that a co-culture model is necessary but not sufficient as a cornea model. Indeed, a physiological-like stimulation is required to maintain cellular phenotypes and improve cellular activity and ECM production.
Effect of solvent composition on the viscosity and stability of emulsions during the synthesis of PCLU scaffolds in tissue engineering through poly(HIPE) method
Massil Mourah1, Philippe Djemia2, Géraldine Rohman1
1Unité de Recherche en Ingénierie Tissulaire, Bobigny (Ile-de-France) - France, 2Laboratoire des Sciences des Procédés et de Matériaux (LSPM), CNRS, Villetaneuse (Ile-de-France) - France
Introduction:
Porous polymeric scaffolds are essential in tissue engineering due to their ability to support cell adhesion, proliferation, and nutrient transport. Various fabrication techniques - such as gas foaming, particle leaching, and thermally induced phase separation - have been explored to obtain such materials. In this study, an emulsion-based approach was employed to synthesize poly(caprolactone-urethane) (PCLU) scaffolds. Using the poly(high internal phase emulsion) (PolyHIPE) method, highly porous PCLU structures can be obtained. The objective of this work was to assess how solvent composition affects emulsion viscosity and stability, and consequently, the pore size and morphology of the resulting scaffolds.
Materials and Methods:
Scaffolds were synthesized following a protocol as previously described [1]. Toluene and dichloromethane were used as solvents for the organic phase in different volume ratios (100:0, 80:20, 50:50, 20:80, 0:100). Dynamic viscosity was determined using a ROTAVISC IKA® rotary rheometer, while static viscosity was measured by Brillouin spectroscopy through analysis of frequency shift and full width at half maximum (FWHM) of the spectral peaks. Stability and pore size were characterized using Numerical 3D and SEM microscopies.
Results:
When the amount of dichloromethane increases in the organic phase, the viscosities of emulsion increase from 49,7 to 76,4 and 1,22E-03 to 1,63E-03 Pa.s for dynamic and static viscosities, respectively. With pure toluene, the emulsion was fluid, stable, and homogeneous, producing scaffolds with variable pore sizes and well-defined polymer walls. Adding 20% dichloromethane led to smaller pores and thicker walls, while maintaining good stability. At a 50:50 solvent ratio, the emulsion became more viscous but remained stable, generating even smaller pores. When dichloromethane exceeded 50% of the total solvent volume, the emulsion became highly viscous and did not produce any scaffold.
Conclusion:
Solvent composition strongly influences emulsion behavior and scaffold morphology. Low-viscosity emulsions are unstable and fail to polymerize, whereas overly viscous emulsions hinder polymerization. An intermediate viscosity, achieved with a balanced toluene/dichloromethane ratio, ensures optimal stability and scaffold formation.
[1] S. Changotade, G. Radu-Bostan, A. Consalus, F. Poirier, J. Peltzer, J.-J. Lataillade, D. Lutomski, G. Rohman. Stem Cells International 2015, vol.2015, 283796
A fully integrated hiPSC-derived 3D blood-brain barrier-on-chip model
Beatriz Amanda Barbosa Rangel dos Passos123, Marie Celine Lefevre1, Maria Cristina Ceccarelli1, Katarzyna Krukiewicz4, Aitor Larrañaga3, Matteo Battaglini1, Gianni Ciofani1
1Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Pontedera, Italy, 2Scuola Superiore Sant’Anna, The Biorobotics Institute, Pontedera, Italy, 3Department of Mining-Metallurgy Engineering and Materials Science, POLYMAT, Bilbao School of Engineering, University of the Basque Country (UPV/EHU), Bilbao, Spain, 4Silesian University of Technology, Department of Physical Chemistry and Technology of Polymers, Gliwice, Poland
The human blood–brain barrier (BBB) remains a significant challenge for central nervous system (CNS) drug delivery studies due to its complex multicellular structure and selective permeability. Here, we present a fully integrated BBB-on-chip platform combining human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (hiPSC-BECs) and astrocytes (iAstrocytes), co-cultured within a microfluidic device designed for real-time electrophysiological monitoring. Utilizing defined protocols, hiPSCs were sequentially differentiated into brain endothelial-like cells, neural progenitor cells (NPCs), and astrocytes, achieving cell populations that recapitulate the molecular and functional features of the human BBB. The hiPSC-BECs expressed tight junction proteins ZO-1 and occludin in a continuous membrane pattern and exhibited high impedance and low permeability to FITC-dextran 70 kDa, confirming barrier maturation. Neural differentiation resulted in SOX-1+/Nestin+ NPCs and S100β+ astrocytes, validated by immunocytochemistry and RT-qPCR.
Within the microfluidic chip, iAstrocytes were embedded in Matrigel to form a 3D parenchymal matrix that interacted with hiPSC-BECs cultured in the vascular channel. The co-culture exhibited high cell viability, continuous ZO-1 localization on the endothelial compartment, and TEER values up to 250 ± 50 Ω·cm2, indicative of robust barrier integrity. Solute permeability assay revealed low permeability (Papp: 10−6 cm/s), surpassing the performance of conventional models. Drug crossing studies using temozolomide (TMZ) and doxorubicin (DOX) confirmed selective endothelial permeability: TMZ crossed the barrier in minimal quantities under both static and dynamic conditions, whereas DOX was more restricted, consistent with active efflux mechanisms observed in vivo.
This work establishes a reproducible, hiPSC-derived 3D BBB-on-chip model that integrates physiological relevance, functional barrier properties, and the potential for personalization. The platform offers a versatile tool for further patient-specific studies of BBB dysfunction, disease modeling, and CNS drug screening under physiologically relevant conditions.
Acknowledgements
This work was supported by the Marie Sklodowska-Curie Actions program (TheraTools, 101073404).
Development of a novel wound-on-a-chip platform to study extracellular matrix-mediated regulation of normal and pathological healing
Lucía García-Villagrasa1, José Manuel García-Aznar1, Elena García-Gareta1
1Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon 50018, Spain, Zaragoza - Spain
Introduction
Normal wound healing is a tightly coordinated process encompassing inflammation, proliferation, and remodeling to restore tissue structure and function. This regulation fails in pathological healing such as keloids, resulting in persistent fibroblast activation and excessive, stiffened and disorganized extracellular matrix (ECM) deposition. The role of ECMs appearing during wound healing in regulating fibroblast behavior in pathological wounds has been overlooked. Understanding how ECM influences fibroblast behavior is crucial to uncovering mechanisms underlying aberrant healing. Our aim was to develop wound healing-on-a-chip models to study how ECM composition and organization influence fibroblast behavior leading to pathological wounds.
Material & Methods
A three-dimensional microfluidic platform was developed to recreate key aspects of the wound microenvironment under controlled ECM conditions. Normal and keloid-derived fibroblasts were embedded in fibrin- and collagen-based hydrogels mimicking different tissue repair phases. The device enables the creation of well-defined hydrogel interfaces that reproduce ECM gradients and discontinuities observed in vivo. Fibroblast migration, invasion, and ECM deposition were monitored over 21 days using live imaging, allowing real-time visualization of fibroblast-matrix interactions.
Results
The platform successfully generated reproducible ECM interfaces and supported sustained cell viability and invasion, allowing real-time visualization of fibroblast dynamics and matrix remodeling. Fibroblasts exhibited distinct invasion patterns and matrix organization depending on ECM composition, demonstrating the model’s ability to capture differential cellular behaviors between normal and pathological wound conditions.
Conclusion
This microfluidic 3D model provides a physiologically relevant, versatile platform to investigate ECM-driven mechanisms of wound closure. By reproducing normal and pathological matrix environments, it offers a promising approach to study fibroblast-ECM interactions, wound healing dysregulation and explore ECM-targeted therapeutic strategies aimed at restoring controlled tissue repair.
Acknowledgements
Work funded by CNS2024-154389, MICIU/AEI/10.13039/501100011033. E.G-G funded by RYC2021–033490-I, MCIN/AEI/10.13039/501100011033 and “NextGenerationEU/PRTR”. L.G-V funded by the Government of Aragón (Spain) Grant No. 2025-2029.
Engineering self-assembled 3D human bladder models using FN-silk scaffolds and micrografts: structural and transcriptomic characterization
Clara Chamorro Jimenez1, Dennis Rootsi2, Savvina Gkouma3, Nageswara R. Boggavarapu4, My Hedhammar3, Magdalena Fossum5
1Laboratory of Tissue Engineering, Department of Women’s and Children’s Health Karolinska Institutet and, Faculty of Health and Medical Sciences, University of Copenhagen Copenhagen, Denmark. Karolinska Institutet, Stockholm (Stockholms Lan) - Sweden, 2Department of Women's and Children's Health. Karolinska Institutet, Stockholm (Stockholms Lan) - Sweden, 3Division of Protein Technology, School of Biotechnology. KTH Royal Institute of Technology, AlbaNova University Center, Stockholm (Stockholms Lan) - Sweden, 4Division of Obstetrics and Gynecology, Department of Women's and Children's Health, Karolinska Institutet, and Karolinska University Hospital, Stockholm (Stockholms Lan) - Sweden, 5Division of Pediatric Surgery, Department of Surgery and Transplantation, Copenhagen University Hospital Rigshospitalet and Laboratory of Tissue Engineering, Department of Women’s and Children’s Health Karolinska Institutet, Copenhagen (Staden Kobenhavn) - Denmark
Introduction and aims: Bladder reconstruction is clinically challenging due to the organ’s multilayered architecture, dynamic mechanical demands, and limited availability. We created biologically relevant, self-assembled 3D bladder tissue models using an extracellular matrix (ECM) mimicking scaffold made of a recombinant functionalized spider silk protein, FN-silk, combined with tissue-derived micrografts. Our objective was to evaluate the structural organization and gene expression compared to native bladder tissue.
Materials and methods: Porcine bladder mucosa and submucosa was harvested and minced mechanically into tissue micrografts. Three composite constructs were created with FN-silk and:(1) epithelial-only (mucosa micrografts),(2) stromal-only (submucosa micrografts),(3) full-thickness (mucosa + submucosa).
The micrografts were embedded into freshly made FN-silk scaffolds and cultured at air–liquid interface for 2–3 weeks. Morphologic characterization included histology (H&E), immunostaining (pancytokeratin, Ki67, αSMA), and western blotting. RNA-seq was performed on all constructs, with native bladder tissue for comparative transcriptomics analysis.
Results: The recombinantly produced FN-silk provided a highly porous, and biocompatible matrix that allowed mechanical handling and supported tissue integration. The micrografts offered an accessible, autologous source of viable cells with native extracellular matrix cues. Histological analysis revealed early epithelial stratification, proliferative activity, (Ki67), and keratin expression in epithelial constructs The stromal-only models showed matrix remodeling, cell migration, and expression of fibroblast/myofibroblast markers. The combined mucosa + submucosa full-thickness constructs demonstrated clear epithelial–stromal layering and tissue organization, similar to the native bladder wall.All 3D models exhibited consistent activation of early wound healing pathways including: inflammation (IL6, CXCL8), ECM remodeling (MMP1, MMP9), hypoxia response (NDUFA4L2, EGLN3), and metabolic adaptation. The full-thickness construct showed the strongest regeneration and had a gene expression profile most similar to Conclusions FN-silk scaffolds combined with bladder-derived micrografts supported the formation of biologically active 3D bladder models. These constructs replicate key aspects of early tissue repair and demonstrate a strong potential as translational platform for regenerative native bladder tissue.
Tissue-engineered 3D human model of peritoneal endometriosis using self-assembling peptide hydrogel composites
Raul Silva1, Debbie Fischer2, Kay Marshall2, Julie Gough3, Aline Miller4, Alberto Saiani5, Sarah Herrick1
1Blond McIndoe Laboratories. Division of Cell Matrix Biology & Regenerative Medicine, FBMH, University of Manchester, UK, Manchester - United Kingdom, 2Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, M13 9PT, UK, Manchester - United Kingdom, 3Department of Materials, School of Natural Sciences, Faculty of Science and Engineering and The Henry Royce Institute, Royce Hub Building, The University of Manchester, Manchester - United Kingdom, 4Manchester Institute of Biotechnology, The University of Manchester, UK, Manchester - United Kingdom, 5Division of Pharmacy and Optometry & Manchester Institute of Biotechnology, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, M13 9PL, UK, Manchester - United Kingdom
Endometriosis is a chronic oestrogen-dependent disorder in which endometrial-like tissue establishes on the peritoneal surface as lesions. Despite its high prevalence, therapeutic options remain limited, in part due to incomplete understanding of disease mechanisms. Cellular interactions driving lesion development are poorly represented in existing models, constraining efforts to elucidate pathogenic pathways, identify therapeutic targets, and evaluate potential treatments. To address this gap, we have developed a three-dimensional (3D) human peritoneal construct that models key structural and cellular organisation of the disease microenvironment using defined self-assembling peptide hydrogel composites as a supporting scaffold.
To recapitulate the interface between the mesothelial surface and underlying stroma, primary human fibroblasts were embedded within a peptide hydrogel, then mesothelial cells seeded on the surface. A comparative analysis of distinct peptide hydrogel formulations of varying stiffness, charge, and pH demonstrated that composite peptide hydrogel–collagen I matrices provide superior mechanical stability, cell distribution, and viability compared with single-component scaffolds. Histological and immunohistochemical analyses confirmed the formation of a cohesive mesothelial monolayer with a basement membrane, a uniformly distributed fibroblast network, and cell marker expression consistent with native peritoneal tissue.
To mimic menstrual-shed tissue fragments, patient-derived endometrial epithelial organoids co-cultured with stromal cells were established in collagen–Matrigel, and exhibited gland-like morphology and cell marker expression characteristic of endometrial epithelium and stroma. Integrating endometrial organoids with the peritoneal construct enables investigation of early cell-cell interactions in developing lesions.
Together, a reproducible and versatile hybrid 3D human model of endometriosis is established, offering improved physiological relevance over conventional 2D assays and animal systems. This platform provides a foundation for studying peritoneal–endometrial cell interactions and advancing mechanistic and translational research to further understanding of endometriosis.
Towards personalized prevention of cranial osteonecrosis: cellular composition of outgrown cells from surgery derived bone fragments as elements of tissue engineering approaches
Jonas Kubat1, Franziska Mitrach1, Benita M. Burghardt2, Sigrid Uxa3, Stefan Simm4, Johannes Wach5, Michael Cross3, Erdem Güresir5, Stefan Kalkhof2, Michaela Schulz-Siegmund1
1Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine. Leipzig University, Leipzig (Sachsen) - Germany, 2Department of Preclinical Development and Validation. Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig (Sachsen) - Germany, 3Clinic and Polyclinic for Hematology. University of Leipzig Medical Center, Leipzig (Sachsen) - Germany, 4Institute for Bioanalysis, Faculty of Applied Natural Sciences. University of Applied Science Coburg, Coburg (Baden-Wberg Bayern) - Germany, 5Clinic and Polyclinic for Neurosurgery. University of Leipzig Medical Center, Leipzig (Sachsen) - Germany
Hemicraniectomy is a surgical intervention that involves the removal of a portion of the skull to relieve increased intracranial pressure. It is performed in medical emergencies such as traumatic brain injury or stroke when swelling compresses brain tissue and impairs vital functions. As a standard procedure, the removed part of the skull is stored at -80°C for weeks to months and reinserted once the brain swelling has diminished. In about 22% of hemicraniectomy patients an aseptic bone flap resorption (ABFR) develops. In order to prevent ABFR, autologous cell-based tissue engineering may provide promising prevention in enhancing the integration between the existing cranial bone and the reimplanted bone flap, reducing the need for additional surgeries.
To address this therapeutic potential, we isolated cells from bone pieces of patients undergoing hemicraniectomies or craniotomies. These bone pieces arise during the surgeries and cannot be reinserted. For isolation, the cells were allowed to migrate from the bone fragments to tissue culture flasks and proliferate. In order to analyze their composition, various methods were employed and different cell passages and donors compared. These methods included bulk RNA sequencing and deconvolution. Additionally, the cells were analyzed by FACS to confirm the results of NGS and deconvolution and to separate different populations of cells for further tissue engineering experiments.
We could demonstrate proficient osteogenic potential of the isolated cells. Further characterization revealed a heterogeneous population comprising mesenchymal stem cells, osteoblasts, endothelial cells, and hematopoietic cells. Such cellular diversity makes them highly promising for bone tissue engineering applications, as they encompass the major cell types involved in bone regeneration. This opens opportunities for coculture and crosstalk studies to unravel the mechanism behind ABFR and possible measures for personalized prophylactic therapies.
acknowledgement: this project is supported by funding of the Studienstiftung des deutschen Volkes e.V.
Optimization of adipose-derived mesenchymal stem cells spheroids for migration studies in space-time warped environments
Wendy Balestri1, Adele Magi1, Paolo Signorello1, Francesco Fontana1, Ludovica Cacopardo1
1Department of information Engineering. University of Pisa, Pisa (Toscana) - Italy
Cell migration is a process characterizing immune response, tissue regeneration, and embryogenesis, while also serving as a hallmark of metastatic progression1. Migration is regulated by chemical cues, and physical factors including stiffness, topography, and external forces like gravity2. On soft viscoelastic substrates, force typically change over time and cells adhesion is less stable, displaying nuclear and cytoskeletal alterations and forming clusters that deform the substrate proportionally to cell number3.
This preliminary work, in the context of the INTERCELLAR project (FIS-2023-00416), aims to investigate the influence of cluster mass (related to cell number) and surface coating (i.e. gelatine, type I collagen, and poly lysin) on the migration of adipose-derived mesenchymal stem cells (ADSCs) spheroids.
Different cell seeding densities, from 2.000 to 200.000 cells/spheroids, will be seeded in ultra-low cell adhesion plates for 48 hours. The spheroids will be then transferred to 96-well plates on agarose gels with gelatine. At day 1 and 5 brightfield images will be acquired and analysed for spheroids size, integrity and intensity. At day 5 Live/Dead assay will be performed too to assess cell viability. The cell seeding numbers allowing for cell survival and shape stability will be used to assess cell response on other coatings. Live imaging will be performed with ZenCell for 5 days to study migration and extrapolate cell binging and unbinding rate (kon and koff).
We will use this data to predict cell migration taking into account gravitational effects. In further study, we will combine different cluster to investigate cell migration in a complex warped environment.
1. Guan, J.-L. 294, (Springer Science & Business Media, 2008)
2. Wu, D. et al. Comprehensive Biotechnology 521–528 (2011)
3. Califano, J. P. et al. Cell Mol Bioeng 3, 68–75 (2010)
Programmable hydrogel-nanocapsule protein delivery system targeting dense cartilage regeneration for osteoarthritis therapy
Qiuwen Zhu1, Wei Sun1, Hang Su1
1School of medicine. Zhejiang University, Hangzhou (Zhejiang) - China
The clinical translation of protein therapeutics for osteoarthritis has been hampered by rapid synovial clearance, poor penetration into cartilage, and insufficient retention under joint loading. We developed hydro/nano-bFGF, a programmable system that integrates a FGF-loaded nanocapsule (nano-bFGF) with an injectable, cartilage-adhesive hydrogel. Nano-bFGF was generated through interfacial polymerization of monomers with enzyme-cleavable crosslinkers directly on the protein surface, forming a nanoscale shell that preserves bioactivity while enabling pathology-specific disassembly. The hydrogel anchors nano-bFGF to the cartilage surface and undergoes gradual degradation under disease-associated enzymatic activity, allowing spatiotemporally programmed release aligned with the joint microenvironment. This spatiotemporally controlled design enhanced intra-cartilage transport, prolonged local retention, and maintained growth factor functionality. In human osteoarthritic cartilage explants, hydro/nano-bFGF doubled glycosaminoglycan deposition, tripled type II collagen expression. In a rat model, it restored cartilage integrity, preserved subchondral bone architecture, and achieved a full cartilage thickness reversing. Owing to its modular design, this strategy is broadly adaptable to diverse protein therapeutics and degenerative joint diseases, offering a generalizable platform for spatiotemporally precise biologic delivery.
Stretching immunity: cyclic strain drives pro-inflammatory polarization in human macrophages
Sofia Artamonova1, Hasnae El Showk1, Carlijn V. C. Bouten1, Anthal I. P. M. Smits1
1Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands
Mechanical cues play a pivotal role in shaping immune cell behavior within tissue-engineered constructs [1,2]. For example, we recently found that local inflammation in in situ tissue-engineered heart valves correlates with local strains in preclinical studies. As key regulators of inflammation and regeneration, macrophages exhibit diverse phenotypes in response to environmental cues [3]. Although increasingly recognized as mechanosensitive, the effect of cyclic strain on human macrophage polarization and its interaction with biochemical stimuli remains unclear. This study investigates the combined effects of cyclic stretch and biochemical cues on human macrophage behavior.
Human monocyte-derived macrophages were cultured on flexible PDMS membranes (uncoated or collagen I coated) and exposed to cyclic uniaxial strain (12%, 0.8 Hz, 48 h) using a Flexcell stretching bioreactor. Prior to stimulation, cells were polarized with IL-4/IL-13 or IFN-γ/LPS to induce M2 or M1 phenotypes, respectively; unstrained cultures served as static controls. Cytoskeletal organization and podosome formation were assessed by immunofluorescence, and cytokine secretion (IL-6, IL-10, TNF-α) quantified by ELISA.
Cyclic strain markedly increased IL-6 secretion versus static controls, particularly in M1-polarized cells. Actin organization showed limited alignment along the strain axis, suggesting cytoskeletal remodeling is not essential for mechanical activation. IL-6 upregulation occurred independently of coating type, indicating a robust mechano-inflammatory response, while IL-10 and TNF-α remained minimally affected. These findings show that cyclic strain primes macrophages toward a pro-inflammatory phenotype, potentially influencing tissue remodeling in mechanically active environments and informing the design of next-generation immunomodulatory scaffolds.
References
1. Du, Huixun, et al. “Tuning immunity through tissue mechanotransduction.” Nature Reviews Immunology 23.3 (2023): 174-188.
2. Petrousek, S. R., et al. “Mechano-immunomodulation of macrophages influences the regenerative environment of fracture healing through the regulation of angiogenesis and osteogenesis.” Acta Biomaterialia (2025).
3. Brown, Bryan N., et al. “Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine.” Biomaterials 33.15 (2012): 3792-3802.
Mechanobiological engineering of organoids for tissue engineering and regeneration
Jose Manuel García-Aznar2, Isabel Villaoslada1, Soraya Hernández-Hatibi2, Paula Guerrero-López2, Pilar Alaman2, Patricia Balsas2, Nieves Movilla2
1Aragon Health Research Institute (IISA), Zaragoza - Spain, 2Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon 50018, Spain, Zaragoza - Spain
Introduction
Although organoid technology has been successfully applied to disease modelling, drug discovery and regenerative medicine, challenges remain regarding its real-world application to personalized medicine. In particular, the variability of organoids in terms of growth and self-organization, as well as limited access to and analysis of them, hinder their applicability. This work aimed to present strategies for improving organoid development by engineering the mechano-microenvironment, including the matrix, cell co-culture and nutrients.
Methodology
Different types of organoids are seeded in microfluidic devices that are embedded in various matrices, either culture alone or together with immune cells. To evaluate the dynamics of organoid initiation and progression, a microscopy-based image quantification approach is developed to assess different variables such as organoid size and structural organization [1]. Additionally, a physics-based model with a deep learning algorithm is presented to simulate morphogenetic patterns of 3D organoids [2].
Results
Our results indicate that alterations in matrix properties, such as stiffness, pore size and permeability, significantly influence the growth patterns and organization of organoids [1]. Furthermore, the phase in which each individual cell is found can also affect the size of the organoid [2]. These findings highlight the strong interplay between biophysical cues and cellular dynamics, underscoring how fine-tuning the microenvironment can lead to more reproducible and physiologically relevant organoid models.
Conclusions
The heterogeneity associated with cell and matrix characteristics is crucial in determining how organoids grow and organize. However, the availability of oxygen and nutrients is also relevant in regulating the evolution of organoids, including tumor organoids. Our overall results involve a better understanding of the mechanobiological conditions governing organoid development and open new perspectives for enhancing their reliability and translational potential in personalized medicine.
Ackowlegdements
This work is part of the project (ICoMICS ERC grant agreement No 101018587).
References
[1] Hernández-Hatibi (2025). DOI: 10.1063/5.0242490
[2] Camacho-Gómez (2023). DOI: 10.1016/j.isci.2023.107164
Bioprinting shear stress exposure effects on cell viability
Pablo Martín Compaired1, Mario Mora2, Hippolyte Amaveda2, María Ángeles Pérez1, Elena García Gareta1
1Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon 50018, Spain, Zaragoza - Spain, 2Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC and University of Zaragoza, Zaragoza, Aragon, 50018, Spain., Zaragoza - Spain
Aim and objectives.
Bioprinting is advancing across different fields such as tissue engineering, regenerative medicine and drug-screening applications. Despite progress in implementing this technology, the mechanical stresses experienced by cells, particularly shear stress during extrusion, remain overlooked. This study aims to investigate the relationship between shear stress exposure and post-printing cell viability during extrusion-based bioprinting.
Methods.
Cell viability was assessed through complementary experimental and computational approaches. Experimentally, PANC-1, U251-MG, MG-63 and YT cell lines were bioprinted with a custom bioink formulation at two initial cell densities using a BIO X extrusion-based bioprinter. Viability was quantified on days 1, 4, 7 and 10 post-printing. Computational fluid-dynamics (CFD) simulations were performed in OpenFOAM using realistic three-dimensional geometries of commercial bioprinting tips. The bioink's non-Newtonian rheological behaviour was incorporated, and cells were modeled as Lagrangian tracer particles to evaluate their trajectories and local shear stress exposure during extrusion.
Results.
Experimental findings indicated that post-printing cell viability is influenced by both the cell line and the cell density. CFD simulations proved a shear stress increase associated with higher extrusion pressures, with tracer particles experiencing variable stress levels along the printing pathway. These simulated shear stress patterns were subsequently aligned with the experimentally observed viability responses across all printing conditions.
Conclusions.
Cell viability after bioprinting is influenced by both cell type and seeding density, and computational analysis demonstrates that shear stress exposure is a key determinant of post-printing viability. Integrating CFD based shear stress characterisation with experimental results provides a quantitative framework to optimise bioprinting parameters and improve cell preservation during extrusion processes.
Acknowledgements.
This work is supported by the project PID2023-146072OB-I00 funded by MCIU/AEI/10.13039/501100011033 and FSE+. E.G.G is funded by a Ramon and Cajal Fellowship (RYC2021-033490-I, funded by MCIN/AEI/10.13039/501100011033 and the EU “NextGenerationEU/PRTR”).
Biocompatible polymeric nanoparticles for potential application in antimicrobial skin regeneration therapies
María Isabel Quiñones Vico1, Inmaculada Mulero Valle2, Ana Ubago Rodríguez1, Ana Fernández González1, Olga Espinosa Ibáñez1, Antonio Lizana Moreno1, Jorge Guerrero Calvo1, Natividad Fernández Porcel1, Pablo Martín Urdangaray1, Fátima Fernández Álvarez2, José L. Arias2, José Gutiérrez Fernández3, Salvador Arias Santiago4
1Cell Production and Tissue Engineering Unit. Virgen de las Nieves University Hospital, Granada - Spain, 2Department of Pharmacy and Pharmaceutical Technology. University of Granada, Granada - Spain, 3Microbiology Department. Virgen de las Nieves University Hospital, Granada - Spain, 4Cell Production and Tissue Engineering Unit. Hospital Universitario Virgen de las Nieves, Granada - Spain
Severe skin injuries often compromise the healing process and predispose patients to infections and other complications. Bioengineered Artificial Skin Substitutes (BASS) are used in the management of extensive burns, but bacterial infections, particularly by Pseudomonas aeruginosa (P. aeruginosa), remain a critical challenge. This study proposes an innovative wound care strategy by integrating antibiotic-loaded polymeric nanoparticles (NPs) within BASS to enhance their antimicrobial performance while maintaining biocompatibility. Chitosan (CS) and poly(lactic-co-glycolic acid) (PLGA) NPs were synthesized by ion gelation and double emulsion methods, respectively, and characterized in terms of size, zeta potential, and colloidal stability. Their effects on human fibroblasts and BASS were evaluated through metabolic, proliferative, histological, and immunohistochemical assays. Colistin (COL), selected for its efficacy against multidrug-resistant P. aeruginosa, was encapsulated within PLGA NPs, achieving high encapsulation efficiency and sustained release under acidic conditions. In contrast, CS NPs showed limited loading capacity. When incorporated into the BASS matrix, COL-PLGA NPs were homogeneously distributed and preserved the microstructure of the dermal scaffold. Antimicrobial tests confirmed a slight inhibition of P. aeruginosa growth, supporting the potential of this system as a localized antibiotic delivery platform. In conclusion, BASS incorporating antibiotic-loaded NPs offers a promising and biocompatible strategy for clinical translation. Their potential is particularly relevant in reconstructive surgery, where the physiological skin pH favors a controlled and sustained antibiotic release. Moreover, in chronic or severe wounds, the progressive acidification that accompanies tissue repair could further enhance the localized delivery of the drug. Future in vivo studies are required to confirm the therapeutic efficacy, stability, and safety of this system in complex wound environments.
Allogeneic mesenchymal stem cells in trilayered artificial skin: incorporated or injected? A GMP preclinical study
Ana Ubago-Rodríguez1, Magdalena Zafra-Castellano1, María I. Quiñones-Vico1, Ana Fernández-González1, Olga Espinosa-Ibáñez1, Antonio Lizana-Moreno1, Jorge Guerrero-Calvo1, Natividad Fernández-Porcel1, Pablo Martín-Urdangaray1, Salvador Arias-Santiago1
1Cell Production and Tissue Engineering Unit. Virgen de las Nieves University Hospital, Granada - Spain
(1) Background: The treatment of skin ulcers, such as the chronic ones diabetic patients suffer, are a medical challenge that still needs to be addressed and skin substitutes represent a promising alternative treatment for this. Most of the skin substitutes are single-layered or bilayered, thus there is a lack of full thickness trilayered artificial skin substitutes, which would emulate skin function better.
(2) Methods: In this study we present a novel Bioengineered Artificial Skin Substitute (BASS) with three layers, containing human keratinocytes (HKTs), fibroblasts (HDFs), and adipose tissue derived stromal mesenchymal cells (hAT-MSCs) in a hyaluronic acid scaffold. It was implanted into nude athymic mice, some of which were also injected with hAT-MSCs intradermally. Several parameters including transepidermal water loss (TEWL), erythema and pigmentation were measured. After 4 or 6 weeks they were euthanized, and biopsies of the engraftments were taken for its subsequent histological analysis.
(3) Results: TriBASS showed a suitable clinical integration and epithelization after six weeks. Homeostasis analysis indicated similar values to Healthy Skin of temperature, pH, TEWL and elasticity. Histological results showed that TriBASS presented better skin structuration and higher expression of cytokeratins.
(4) Conclusions: This new TriBASS is a feasible treatment for skin ulcers and once its characteristics are fully stablished, a clinical trial should follow these findings in order to confirm the preclinical results.
Dual regulation of spheroid bioenergetics by ECM composition and glucose availability in tissue-engineered cancer models
Paula Guerrero López1, Gorana Drobac2, Eduardo A. Silva3, Hanne R. Hagland4, José Manuel García Aznar1
1Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon 50018, Spain, Zaragoza - Spain, 2Department of Process Technology,Nofima-Norwegian Institute of Food,Fisheries and Aquaculture Research, Stavanger (Rogaland) - Norway, 3School of Chemical, Materials & Biomedical Engineering, University of Georgia, Atlanta (Georgia) - United States, 4Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Stavanger (Rogaland) - Norway
Introduction
Recent studies show that extracellular matrix (ECM) stiffness strongly influences cancer metabolism. Tumors typically exhibit increased stiffness and residual stress, making this an important feature of the tumor microenvironment (TME). To investigate how stiffness shapes tumor metabolism, we employed 3D cell culture models that model physiological conditions. Hydrogels with different mechanical properties were used to mimic the ECM, combined with varying glucose levels. This study examines how these factors modulate the metabolic phenotype of A549 lung and Panc1 pancreatic cancer spheroids.
Methodology
Collagen and fibrin hydrogels with varying concentrations were used. AlamarBlue® Cell Viability Reagent was used to study cell proliferation. The analysis of the metabolic flux of tumor cells in each matrix was performed using SeaHorse technology. Finally, metabolites at end point in the culture medium were determined by colorimetric assays.
Results
In this study, we observed that while glucose availability predominantly dictates metabolic profiles in collagen-based matrices, particularly influencing A549 cell behavior, metabolic adaptation in fibrin hydrogels was co-regulated by matrix properties and glucose levels. Notably, lung cancer cells shifted towards glycolysis under high glucose in collagen, whereas pancreatic cancer cells, inherently more glycolytic, exhibited metabolic rigidity, especially under low glucose, irrespective of collagen stiffness. Conversely, fibrin matrices generally induced a less noticeable, more quiescent metabolic state in both cancer cells, particularly under glucose deprivation. Specifically, higher collagen concentrations tended to support anaerobic metabolism, especially under glucose scarcity.
Conclusion
This study reveal an interplay where ECM composition tunes the sensitivity of cancer cells to nutrient availability, underscoring the necessity of integrating both matrix-specific mechanical cues and nutrient gradients in advanced 3D tumor models for identifying context-dependent metabolic vulnerabilities.
Acknowledgements
Funding provided by the DGA (Grant No. 2021-25 and MVT_10_24), the Spanish Ministry of Economy and Competitiveness (PID2021-122409OB-C21), the ERC (ICoMICS - 101018587) and the project ASPIRE-AECC.
Early development of a soft bioreactor for ex-vivo study of carpal tunnel syndrome
Grzegorz Koc1, Jinrong Lin1, Akira Wiberg1, Dominic Furniss1, Pierre-Alexis Mouthuy1
1NDORMS. University of Oxford, Oxford (Oxfordshire) - United Kingdom
Carpal tunnel syndrome (CTS) is characterised by fibrotic remodelling of sub-synovial connective tissue (SSCT) in response to repetitive mechanical stress, ultimately leading to median nerve compression and neuropathy. The pathways transducing mechanical stress to fibrotic changes remain poorly understood, largely due to the lack of physiologically relevant ex-vivo models. Here, we present the design and initial testing of a soft bioreactor chamber engineered to simulate the biomechanical environment of the carpal tunnel for ex-vivo culture of human SSCT explants.
The bioreactor chamber was designed and manufactured using soft biomaterials, building on previous work by our team [1]. The chamber holds SSCT between two artificial tendons that undergo reciprocating shear motion, mimicking in-vivo tendon gliding dynamics [2]. Key functional requirements included endurance of >60,000 movement cycles (estimated one week of daily hand use [3]) and controlled maintenance of 30-150 mmHg hydrostatic pressure, representative of elevated carpal tunnel pressure in CTS. Human SSCT explants were cultured in the bioreactor for one week under static conditions, with viability quantified daily via PrestoBlue, followed by histological analysis.
Mechanical testing confirmed the chamber’s integrity over >60,000 shear cycles. Hydrostatic pressure was found to decrease exponentially with a half-life of ∼24h, due to compliance of the chamber’s flexible walls. Explant culture showed no significant decline in metabolic viability compared to controls, indicating the chamber has suitable biocompatibility. Picro Sirus Red staining revealed preserved tissue architecture, with pathological features indistinguishable from controls.
These preliminary results demonstrate the bioreactor enables ex-vivo culture of SSCT and can withstand relevant mechanical stresses. The novel platform provides a foundation for studying mechanobiology-driven fibrosis in CTS, enabling future studies involving mechanical stimulation, transcriptomic profiling, and preclinical drug testing.
1. Mouthuy et al. 2022, Communications Engineering 1:2.
2. Amadio et al. 2018, J Electromyogr Kinesiol 38:232-239.
3. Dollar 2014, Springer STAR 95:201-216.
From injectable gels to macroporous scaffolds: polyisocyanide-based materials for soft tissue repair
Paul Kouwer
de Radboud University, Nijmegen (Gelderland) - The Netherlands
Repairing soft tissues requires biomaterials that actively guide regeneration. Current hydrogel systems often fall short because they lack the architecture and instructive nature of native extracellular matrix (ECM).
Polyisocyanide (PIC) hydrogels address this gap through a unique combination of synthetic tailorability and biomimetic mechanics. Upon warming to physiological temperature, PIC polymers self-assemble into a fibrous network with mechanics resembling collagenous ECM. This property allows the gel to dynamically respond to cellular forces, providing an instructive mechanical environment that supports spreading, migration, and matrix remodeling.[1-3]
As fully synthetic materials, PIC hydrogels offer reproducible control over stiffness and biofunctionalization, while remaining cytocompatible and easily processable. Their reversible sol–gel transition enables injectable or surgical delivery and immediate in situ formation—features particularly attractive for minimally invasive tissue repair.
To further enhance the scope of PIC gels, we recently developed PIC cryogels [4]: macroporous scaffolds fabricated via controlled cryopolymerization. These cryogels retain the thermoresponsiveness and ECM-like mechanics of PIC hydrogels while introducing large, interconnected pores that enhance cellular and nutrient transport, and potentially, vascularization, and tissue infiltration.
In summary, PIC hydrogels represent a new generation of adaptable, cell-instructive matrices for soft tissue repair and regeneration. In this presentation, I will provide an overview of the structure and properties of the gel platform and discuss the current status in soft tissue applications.
References
[1] Liu, …, Kouwer “Cell-matrix reciprocity in 3D culture models with nonlinear elasticity” Bioact. Mater. 9, 316-331 (2022).
[2] Yuan, …, Kouwer “Fibrous polyisocyanide hydrogels for 3D cell culture applications” Nat. Protoc. 20, 3339-3360 (2025).
[3] Gudde, …, Kouwer, Roovers & Guler “Injectable polyisocyanide hydrogel as healing supplement for connective tissue regeneration in an abdominal wound model” Biomaterials 302, 122337 (2023).
[4] Gerrits, …, Kouwer “Tailoring of Physical Properties in Macroporous Poly(isocyanopeptide) Cryogels” Biomacromolecules 25, 3464-3474 (2024).
Alveoleye – an alveolus-on-a-chip model
Federico Farina1, Chiara Tonda-Turo2, Gianluca Ciardelli2, Michela Licciardello2, Lorenzo Moroni3
1Department of Mechanical and Aerospace Engineering. Politecnico di Torino, Turin (Italia) - Italy, 2Department of Mechanical and Aerospace Engineering. Politecnico di Torino, Torino (Italia) - Italy, 3MERLN Institute - Department of Complex Tissue Engineering. Maastricht University, Maastricht (Limburg) - The Netherlands
Alveolar tissue displays a highly complex multiscale morphology, composed by a thin epithelial layer organized in a 3D architecture surrounded by a branched capillary network, which nowadays remains difficult to reproduce in vitro[1].
Most platforms still rely on flat membranes that poorly recapitulate lung anatomy and physiology, limiting biomimicry of alveolar structure and vascular topology[2].
Here we introduce an alveolus-on-a-chip that integrates an alveolus-like digital light processing (DLP) printed scaffold (3Dresyn, Bioflex A10 MF), placed directly on a cellularized gelatin methacryloyl (GelMA) hydrogel The alveolus-like scaffold provides a Voronoi patterned substrate that recapitulates the alveolar packing inside the lung tissue with physiological pores dimension, generating an array of units for epithelial cell seeding. The GelMA hydrogel is co-laden with human umbilical cord endothelial cells (HUVECs) and lung fibroblasts (MRC-5) to promote capillary morphogenesis and it is laterally confined by micropillars while culture medium flows in adjacent channels The bottomless chip is DLP-fabricated in Liqcreate BioMed Clear resin and bonded to a cover glass using polydimethylsiloxane (PDMS), enabling easy optical evaluation during cell culture. A removable lid integrates tubing anchors and allows air flow to perform an air-liquid interface cell culture.
This study was carried out within the BREATH project (a Bioprinted alveolaR lung-on-chip devicE to assess The role of inHaled pollutants towards pulmonary fibrosis onset) – funded by European Union – Next Generation EU within the PRIN 2022-PNRR program (D.D. 104 - 02/02/2022 Ministero dell’Università e della Ricerca).
1. Knudse L. et Ochs M. Histochemistry and Cell Biology (2018), 10.1007/s00418-018-1747-9
2. Baptista D. et al, ACS Biomater Sci Eng (2022), 10.1021/acsbiomaterials.1c01463
Advanced 3D bioprinted hydrogels as in vitro platforms for breast cancer microenvironment modeling - SEMIT
Giovanni Lo Bello1, Amirhossein Elahi2, Mariella Rosalia3, Nora Bloise4, Livia Visai4, Domenica Scumaci1
1Department of Experimental and Clinical Medicine. University Magna Graecia of Catanzaro, Catanzaro (Italia) - Italy, 2Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R), University of Pavia Unit, 27100 Pavia, Italy; Pavia (Lombardia) - Italy, 3Department of Drug Sciences, University of Pavia. University of Pavia, Pavia (Lombardia) - Italy, 4Molecular Medicine Department, Centre for Health Technologies (CHT), Research Unit (UdR) INSTM; Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R), University of Pavia Unit, Pavia (Lombardia) - Italy
Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide, highlighting the urgent need for physiologically relevant in vitro models [1]. Conventional 2D cultures and animal models often fail to reproduce the complex mechanical and biochemical features of the tumor microenvironment. Three-dimensional (3D) bioprinting provides an effective strategy to fabricate tissue-mimetic constructs with tunable architecture and stiffness [2, 3]. In this work, alginate-gelatin (ALG-GEL) hydrogels were designed and optimized to reproduce the viscoelastic properties of breast tumor tissue. The formulations exhibited high swelling capacity and gradual degradation with sustained gelatin release over 21 days, ensuring structural stability and bioactivity. Fourier-transform infrared spectroscopy confirmed the presence of gelatin within the alginate network, while rheological analysis demonstrated a storage modulus close to 10 kPa, comparable to tumor stiffness. The hydrogels showed excellent printability and were used to fabricate 3D constructs embedded with MDA-MB-231 breast cancer cells. Confocal microscopy revealed homogeneous cell distribution and high viability up to 14 days of incubation, confirming the suitability of the printed constructs for long-term culture. RNA extraction from the bioprinted scaffolds confirmed cellular activity and the potential of these systems for subsequent gene expression studies. The combination of appropriate mechanical performance, controlled degradation, and cellular compatibility demonstrates that ALG-GEL hydrogels provide a reliable bioink for fabricating breast tumor-mimetic 3D models. These results support the use of bioprinted hydrogels as advanced in vitro tumor platforms for studying cancer progression and therapeutic response, ultimately contributing to the reduction of animal testing and the advancement of precision oncology research.
Acknowledgments:
The authors acknowledge the support of the Interuniversity Center for the Promotion of the 3Rs Principles, the Nanotechnology Lab at Istituti Clinici Scientifici Maugeri IRCCS, and the PNRR program.
References
[1] M. Arnold, https://doi.org/10.1016/j.breast.2022.08.010.
[2] M. Kapałczyńska, https://doi.org/10.5114/aoms.2016.63743.
[3] A. Guller, https://doi.org/10.3390/bioengineering10010017.
Low-protein 3D bioprinted hydrogel bioscaffolds as a research platform for studying the role of adipose-derived stem cells in the extracellular matrix deposition towards full-thickness in-vitro skin
Aleksandar Atanasov1, Liam Grover1, Anthony Metcalfe1
1School of Chemical Engineering. University of Birmingham, Birmingham - United Kingdom
The Collagen I-rich composition of native skin’s ECM makes protein-based bioscaffolds the default choice for mimicking native skin’s microenvironment. In the context of understanding the inter-skin cell type interactions resulting in the formation of tissues, the abundance of readily available ECM protein in-vitro impedes studying ECM deposition in 3D. To evaluate the potential of low-protein bioscaffolds to address this, the suspended layer additive manufacture (SLAM) 3D bioprinting technology was implemented with bioinks of pectin-Collagen I ratios, varying from 100:0 to 0:100. Cell metabolic activity testing over 21 days was employed to determine the minimal amount of Collagen I, capable of maintaining for both human dermal fibroblasts (HDF) and adipose-derived stem cells (ADSC). To validate the relevance of low-protein bioscaffolds for the field of in-vitro 3D skin modelling, the optimised formulation was then used to 3D bioprint full-thickness dermal-epidermal 3D skin models - with or without a hypodermis-like ADSC-laden layer. Histological and transcriptomic analyses were performed to study the bioscaffolds’ ECM deposition in the two configurations. The data indicated that while Collagen I elevates the cells’ metabolic activity, functionalisation beyond the minimal tested 1.25 mg/ml does not yield statistically significant changes, while live/dead data indicated the percentage of live cells increasing up from 73% at day 4 to 98% by day 21. Qualitative histological assessment revealed that the deposition of ECM at the dermal-epidermal junction depends on the introduction of the hypodermal ADSC layer. Transcriptomic analysis revealed possible protein targets, through which this effect may be mediated; namely, statistically significant increases in Collagens IV, VII and XV; as well as growth factor signalling. Together, the obtained results indicate that SLAM 3D bioprinted low-protein pectin bioscaffolds provide an accessible in-vitro 3D research platform for studying the mechanisms of ADSC-mediated ECM deposition.
Macrophage plasticity and fusion dynamics at the biomaterial interface: insights into foreign body giant cell biology
Thijs S. Conner1, Sanne D.h. Ten Damme1, Carlijn P.m.e. Metz1, Frank P.t. Baaijens1, Livia Angeloni2, Carlijn V.c. Bouten1, Anthal I.p.m. Smits1
1Department of Biomedical Engineering and the Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands, 2Department of Basic and Applied Sciences for Engineering. Sapienza University of Rome, Rome (Lazio) - Italy
Given the increasing clinical use of biomaterials, a comprehensive understanding of the host immune response to these materials is critical. Fused macrophages, e.g. foreign body giant cells (FBGCs), are known key mediators of this response, in cross-talk with other cells, such as macrophages, T-cells and fibroblasts. FBGCs are linked to fibrous encapsulation, biomaterial degradation, and oxidative stress cracking, potentially leading to clinical device failure. However, the exact formation and functions of FBGCs remains elusive. Therefore our research focuses on systematically unraveling the influence of biochemical and biomaterial cues on macrophage fusion and FBGC function. Specifically, we studied the influence of (1) polarizing cytokines on macrophage fusion and FBGC plasticity in monoculture, as well as in co-culture with T-cells and fibroblasts, and (2) biomaterial topographical features on macrophage fusion.
To develop physiologically relevant models of FBGC formation, human peripheral blood mononuclear cell derived monocytes were used, differentiated to macrophages using colony stimulating factors, polarized into M1/M2a/M2c macrophages, and induced to fuse using interleukin-4. Interestingly, even though M1 macrophages showed increased IL-4Ra on protein level and increased fusion related gene expression when compared to M2a macrophages, they were not found to show increased fusion potential. Furthermore, providing Th cell-associated polarization cues to FBGCs showed that FBGCs are able to plastically respond to cytokines in a similar way as macrophages. When in direct co-culture, T-cells were able to induce fusion, depending on the type of co-culture (juxtacrine vs paracrine). Furthermore, we showed that fibroblasts were activated to myofibroblasts in response to FBGCs in co-culture. Finally, when cultured on 2.5D biomaterial substrates with well-controlled topographical features, FBGCs were determined to sense and respond to the physical cues exerted by biomaterials.
Taken together, these results show that human FBGC behavior is plastic and dependent on the local biochemical and biophysical cues in the biomaterial environment.
Towards “live” biocomposites: immobilization and release of probiotics from activated charcoal pads
Tanja Krunic1, Andrea Osmokrovic2
1University of Belgrade, Innovation center of the Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia, 2University of Belgrade, Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia
The aim of this study was to develop and evaluate biocomposites composed of probiotic bacteria immobilized onto activated charcoal (AC) fabric pads, specifically designed for wound dressing applications.
Lactobacillus plantarum (Lp299v), Lactobacillus rhamnosus (LGG), and Lactobacillus gasseri (LG) were incubated twice for 18 h, washed with normal saline (NS; 0.9% w/v NaCl), centrifuged, and diluted in NS. The bacterial suspensions were then aseptically applied onto AC pads, frozen, and freeze-dried for 24 h. The obtained pads were incubated under normoglycaemic (5 mM glucose; N) and hyperglycaemic (25 mM glucose; H) conditions at 37°C in 2 mL of simulated wound fluid (50% NS and 50% fetal bovine serum). At various time points (10 min, 3, 6, 24, and 48 h), viable cell counts and pH were determined.
The AC pads were approximately 12 mm in diameter and weighed 0.017 ± 0.001 g. Probiotic immobilization efficiency was 76.25% for LGG, significantly higher than for Lp299v (72.85%) and LG (73.48%). During the first 6 h, all probiotics exhibited better release under normoglycaemic conditions, whereas at later time points no statistically significant differences were observed. However, after 48 h, metabolic activity was significantly higher under hyperglycaemic conditions: the pH of the medium decreased from the initial 8.15 to 4.04 (LGG and LG) and 4.20 (Lp299v), while under normoglycaemic conditions the pH reached 4.99 in all probiotic cultures.
In summary, probiotic release was slightly higher during the initial 6 h under normoglycaemic conditions, whereas after 24 h and 48 h the hyperglycaemic environment supported comparable probiotic viability but significantly enhanced metabolic activity.
Acknowledgment: This research was supported by the Science Fund of the Republic of Serbia, Grant No 9802, Activated Charcoal as a Carrier of Probiotics: A New Approach for Pathogen Elimination in Wounds-ProHealingAC.
3D-printed pyrolytic carbon microlattices: a novel scaffold platform for musculoskeletal tissue engineering
Monsur Islam
de Mechanical Engineering Department. Universidad Politécnica de Madrid, Madrid - Spain
Carbon-based materials have recently attracted growing attention in tissue engineering due to their unique combination of biocompatibility, electrical conductivity, and mechanical robustness, properties that directly address key shortcomings of polymeric scaffolds such as mechanical mismatch, degradation instability, and poor long-term functionality. Among the many carbon allotropes explored, including graphene oxide, carbon nanotubes, and glassy carbon, most approaches still rely on complex synthesis routes and offer limited control over scaffold geometry. To overcome these challenges, our work leverages high-resolution 3D printing followed by pyrolytic conversion to create architected pyrolytic carbon (PyC) microlattices with tunable geometry, stiffness, and surface functionality.
These 3D-printed PyC scaffolds exhibit exceptional structural fidelity, mimicking the hierarchical mechanics of native musculoskeletal tissues while providing conductive interfaces that promote cell communication and mechanotransduction. We have systematically investigated their biocompatibility with osteoblasts and myoblasts, demonstrating excellent cell adhesion, proliferation, and preservation of morphology across both soft and hard tissue environments. Furthermore, advanced imaging and quantitative analysis were employed to correlate lattice design parameters, such as strut size, porosity, and surface roughness, with cellular organization and tissue-specific responses.
Our findings highlight the potential of PyC as a new class of multifunctional scaffold material that integrates mechanical resilience, biointerface conductivity, and architectural precision. The ongoing in vitro and in vivo studies suggest that these microengineered carbon lattices can serve as universal platforms for musculoskeletal repair, providing new insights into how carbon architecture and microstructure can be tailored to regulate biological performance. This work establishes a pathway toward next-generation bioactive carbon scaffolds that can bridge the gap between structural mechanics and functional tissue regeneration.
Screening synthetic dynamic biomaterials for cancer spheroid culture
Rita Correia1, Cristina Romo Valera1, Hongning Sun1, Nicola Contessi Negrini1, Adam D. Celiz1
1Department of Bioengineering, Imperial College London, London (London, City of) - United Kingdom
Cancer spheroids and organoids provide powerful platforms for investigating cancer biology and therapeutic responses. Culturing these 3D structures requires a matrix that provides mechanical support and mimics the tumour microenvironment. However, the most used matrix, Matrigel, presents several drawbacks, including batch-to-batch variability, xenogeneic origin, and limited mechanical tuneability. Synthetic alternatives offer improved reproducibility but often rely on irreversible covalent bonds hinder spheroid growth restricting dynamic remodelling of the matrix. Here, we designed and synthesised polyethylene glycol (PEG)-based hydrogels incorporating both dynamic (boronate-ester) and static (Michael addition) crosslinks to achieve stable yet viscoelastic materials.
A library of PEG precursors was synthesised by functionalising PEG end groups with static and/or dynamic moieties, confirmed by 1H NMR spectroscopy. Hydrogels were prepared by varying polymer concentration (5–10%) and crosslinking type, yielding static, dynamic, and dual formulations. Mechanical properties were characterised by rheology and compression testing, while cytocompatibility was evaluated using live/dead staining. Epithelial ovarian cancer spheroid invasion was quantified by changes in area and circularity and by immunostaining for invasion markers to determine which matrices best supported invasive behaviour.
All PEG precursors were successfully synthesised with >90% modification. Hydrogel stiffness (1.5–5 kPa) and relaxation times (101–105 s) were tuneable through polymer concentration and crosslinking chemistry, respectively. Matrigel exhibited lower stiffness (50–300 Pa) but comparable relaxation to dual and dynamic gels. Purely dynamic hydrogels, while mimicking Matrigel’s viscoelasticity, dissolved after three days, highlighting the need for static crosslinks. Both static and dual gels supported cell viability (>95%) after 14 days. Spheroids in dual gels displayed reduced circularity, increased area, and elevated invasion marker expression compared with static gels and Matrigel, highlighting the combined importance of stiffness and viscoelasticity.
We successfully developed and tuned PEG-based hydrogels as synthetic matrices cancer spheroid invasion assays. We are currently investigating these matrices for organoid culture.
Humanized models for studying spinal cord injury and developing therapeutic strategies
Francisco M. Sáez1, Marta Cuenca2, Eloi Montañez3, J. Alberto Ortega2, Zaida Álvarez1
1Biomaterials for Neural Regeneration Group. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, 2Department of Pathology and Experimental Therapeutics, Institute of Neurosciences. Universitat de Barcelona (UB), L’Hospitalet de Llobregat (Barcelona) - Spain, 3Department of Physiological Sciences, Faculty of Medicine and Health Sciences. Universitat de Barcelona (UB), L’Hospitalet de Llobregat (Barcelona) - Spain
Background: Spinal cord injury (SCI) is a devastating condition causing irreversible motor and sensory deficits. Despite extensive research, no regenerative therapy has reached the clinic, largely due to the poor translational relevance of preclinical models [1]. Animal systems fail to reproduce the human spinal cord’s cellular diversity and injury responses, limiting therapeutic success [2]. This study establishes humanized SCI models by integrating neural and vascular systems from human induced pluripotent stem cells (hiPSCs) into physiologically relevant platforms for translational research.
Methodology: Human spinal cord and vascular organoids (hSCOs, hVOs) were derived from hiPSCs by directed differentiation into neuroectodermal and mesodermal lineages. Fusion at defined stages produced vascularized spinal cord organoids (hVSCOs) which were mechanically injured using the Infinite Horizon impactor to model SCI in vitro. Tissue morphology and neurovascular organization were analyzed by immunohistochemistry, and neuronal activity recorded in injured and control organoids with multielectrode arrays (MEAs). In parallel, an in vivo humanized model was generated by transplanting GFP-expressing hSCOs into the spinal cord of immunodeficient juvenile mice. Surgeries resulted in 100% survival of the transplanted hSCOs within the mouse spinal cord, with no macroscopic disruption of host tissue and no detectable motor deficits.
Results: Fusion of hSCOs with vascular organoids resulted in robust, stage-dependent vascularization with CD31+/VE-Cadherin+ vessels and parenchymal Iba1+ microglia infiltration. Upon injury, hVSCOs reproduced canonical SCI responses—GFAP-driven glial scar formation, caspase-3-mediated cell death, Ki67+ progenitor expansion, and loss of vascular integrity—alongside a marked reduction in electrophysiological activity.
Conclusions: Our humanized models recreate key SCI phenotypes and offer a translational framework for mechanistic discovery and therapy development.
References
1. Hutson TH et al., Nat Rev Neurol. 2019;15:732–745.
2. Hanna A et al., Neural Regen Res. 2019;14:7.
Acknowledgments: Funding from “la Caixa” Foundation (LCF/PR/HR25/52450044), MICIU/AEI PID2024-162574OB-I000 (10.13039/501100011033, ERDF/EU), and NIH NIA R01AG086270. The content solely reflects the author’s views.
In vivo live imaging of human iPSC-derived endothelial cells seeded on decellularized small-diameter vascular grafts
Ryo Kawabata1, Michiyo Koyanagi-Aoi2, Hiroaki Takahashi1, Kenji Okada1, Takashi Aoi2
1Department of Cardiovascular Surgery. Graduate School of Medicine, Kobe University, Kobe (Hyogo) - Japan, 2Division of Stem Cell Medicine. Graduate School of Medicine, Kobe University, Kobe (Hyogo) - Japan
＊ Aim and Objective:
Small diameter vascular grafts pre-seeded with endothelial cells (ECs) have shown better patency compared with acellular grafts, and seeded ECs are gradually replaced by host-derived cells after implantation. However, no quantitative approach has been available to assess the survival duration of the seeded ECs and the timing of replacement by host ECs in vivo. This study aimed to establish a novel longitudinal imaging platform that enables in vivo visualization of seeded cell dynamics within the same animal over time.
＊ Methodology:
Rat abdominal aortas were decellularized using 0.5% sodium dodecyl sulfate (SDS) and seeded with human induced pluripotent stem cell–derived endothelial cells (hiPSC-ECs) on the luminal surface. The grafts were transplanted into the abdominal aortas of the nude rats. The hiPSC-ECs were transduced with the Venus-Akaluc reporter gene, and cell dynamics were monitored using an in vivo imaging system (IVIS). Immunofluorescence staining was performed to confirm re-endothelialization, and macrophage infiltration was compared with that of the acellular control grafts.
＊ Results:
The bioluminescence signal of hiPSC-ECs transiently increased during the early postoperative phase and gradually declined, nearly disappearing within two weeks. Immunofluorescence analysis confirmed that the seeded cells initially covered the luminal surface and were subsequently replaced by host ECs. Macrophage infiltration was milder in EC-seeded grafts than in controls, indicating that re-endothelialization of the grafts attenuated early inflammatory responses.
＊ Conclusions:
This study demonstrates a novel in vivo imaging platform that enables real-time tracking of seeded ECs from engraftment to replacement by host cells within the same individual. This quantitative approach provides insight into the transitional phase of endothelial replacement and offers a foundation for designing rational endothelialization strategies for small-diameter vascular grafts.
Biofabrication of an in situ hypoxia-delivery scaffold for hyaline cartilage regeneration
Roberto Di Gesù1, Antonio Palumbo Piccionello2, Giampiero Vitale3, Silvia Panzavolta4, Maria Francesca Di Filippo4, Angelo Leonarda5, Monica Cuccia5, Riccardo Gottardi6
1Musculoskeletal Tissue Engineering (MsTE) lab. Fondazione Ri.MED, PALERMO (Sicilia) - Italy, 2Organic chemistry dept. Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy, 3MusculoSkeletal Tissue Engineering (MsTE) lab. Fondazione Ri.MED, PALERMO (Sicilia) - Italy, 4Organic chemistry dept. University of Bologna, Bologna (Italia) - Italy, 5Orthopaedic and traumatologic surgery division. Buccheri La Ferla Hospital, PALERMO (Sicilia) - Italy, 6Pulmunology division. The Children's Hospital of Philadelphia, Philadelphia (Pennsylvania) - United States
Osteoarthritis (OA) is a debilitating joint condition affecting millions of people worldwide, triggering painful chondral defects (CDs) that ultimately compromise the overarching patients’ quality of life. Currently, several reconstructive cartilage techniques (RCTs) (i.e.: matrix-assisted autologous chondrocytes implantation has been developed to overcome the total joint replacement limitations in the treatment of CDs. However, there is no consensus on the effectiveness of RCTs in the long term, as they do not provide adequate pro-regenerative stimuli to ensure complete CDs healing. In this study, we describe the biofabrication of an innovative scaffold capable to promote the CDs healing by delivering pro-regenerative hypoxic cues at the cellular/tissue level, to be used during RCTs. The scaffold is composed of a gelatin methacrylate (GelMA) matrix doped with hypoxic seeds of GelMA functionalized with a fluorinated oxadiazole (GelOXA), which ensures the delivery of hypoxic cues to human articular chondrocytes (hACs) embedded within the scaffold. We found that the GelMA/GelOXA scaffold preserved hACs viability, maintained their native phenotype, and significantly improved the production of type II collagen. Besides, we observed a reduction in type I and type X collagen, characteristic of unhealthy cartilage. These findings pave the way for the regeneration of healthy, hyaline-like cartilage, by delivering hypoxic cues even under normoxic conditions. Furthermore, the GelMA/GelOXA scaffold’s ability to deliver healing signals directly to the injury site holds great potential for treating OA and related CDs, and has the potential to revolutionize the field of cartilage repair and regenerative medicine.
Mass production of uniform spheroids using acoustic standing waves
Mira Ritter1, Johannes Hahn1, Ebru Aksoy2, Sarkawt Hamad2, Kurt Pfannkuche2, Horst Fischer1
1ZWBF. RWTH Aachen University, Aachen (Rheinland-Pfalz) - Germany, 2Center of Physiology and Pathophysiology. University of Cologne, Cologne (Nordrhein-Westfalen) - Germany
Introduction
Spheroids are three-dimensional structures derived from human induced pluripotent stem cells (hiPSCs) or mature cell types, capable of mimicking organ-specific functions. Conventional spheroid production is labor-intensive, slow, and yields variable sizes. We hypothesized that acoustic standing waves could enable rapid, large-scale generation of uniformly sized spheroids.
Materials and Methods
We developed a custom acoustic device featuring a square PMMA chamber (30 × 30 mm2 or 60 × 60 mm2) with piezoceramic transducers (2 MHz) on all four sides. Each transducer was driven independently by a multi-channel radio frequency (RF) generator. The chamber temperature was maintained at 36.5°C. hiPS cell or cardiac fibroblasts, endothelial cells and cardiomyocytes suspensions were introduced into the chamber, and acoustic fields were applied immediately. After 24 hours of continuous acoustic exposure, the resulting spheroids were collected for further differentiation or characterization.
Results
The system generated a stable acoustic pressure field that reproducibly organized cells into uniform aggregates. Spheroid diameters ranged from 70–320 µm depending on initial cell density. Up to 30,000 spheroids could be produced in a single run. Acoustic exposure did not impair cell viability, and hiPSC-derived spheroids retained pluripotency [1]. Additionally, sequential introduction of mature cardiac cell types enabled formation of multi-layered spheroids with defined spatial organization, highlighting the platform’s capacity to produce complex tissue structures.
Conclusions
This acoustic patterning system offers a scalable and cost-effective alternative to traditional spheroid fabrication. It enables rapid, reproducible production of uniformly sized spheroids while maintaining cell viability and differentiation potential. The ability to generate structured spheroids with controlled architecture positions this technology as a powerful tool for disease modeling and regenerative medicine applications.
1. Hahn J, Aksoy E, Hamad S, Kuckelkorn C, Gómez Montoya A, Ritter M, Pfannkuche K, Fischer H. Mass production of uniform embryoid bodies by acoustic standing waves. Small Methods, https://doi.org/10.1002/smtd.202501283 (2025)
Cell-free regeneration in Parkinson’s disease: the therapeutic signature of iMSC secretome
Filipa Ferreira-Antunes1, Ana Marote1, Jorge H. Fernandes1, Jonas Campos1, Carla Teixeira-Pereira1, Sofia C. Serra1, Sandra Barata-Antunes1, Bárbara Mendes-Pinheiro1, Daniela Monteiro-Fernandes1, Carina Soares-Cunha1, Ana V. Domingues1, Stephanie Oliveira1, Sandra I. Anjo2, Patrícia Patrício1, Bruno Manadas2, Andreia Teixeira-Castro1, Luísa Pinto1, Belém Sampaio-Marques1, António J. Salgado1
1Life and Health Science Research Institute - ICVS, Braga - Portugal, 2Universidade de Coimbra, Coimbra - Portugal
The multifactorial etiology of Parkinson’s disease (PD) demands therapeutic strategies capable of targeting multiple dysregulated pathways beyond dopaminergic replacement. Mesenchymal stem cell (MSC)-based approaches show promise in promoting neuroregeneration and neuroprotection. However, their clinical translation is limited by donor variability and lack of standardization. Induced MSCs (iMSCs) overcome these challenges, yet their therapeutic potential in PD remains largely unexplored.
We compared the efficacy of iMSC-derived secretome with bone marrow MSC (BM-MSC)-derived secretome in a 6-hydroxydopamine rat model. Both secretomes significantly improved motor function, while the iMSC secretome uniquely alleviated anhedonia-like behavior and enhanced preservation of dopaminergic neurons in the striatum, substantia nigra pars compacta (SNc), and ventral tegmental area. Proteomic profiling of SNc revealed that BM-MSC secretome primarily engaged proteostasis and redox pathways, whereas iMSC secretome activated a broader network of neuroprotective mechanisms, including AMPK-dependent signaling, NRF2-mediated antioxidant defense, synaptogenesis, and endocannabinoid signaling. Enrichment in immunomodulatory factors further contributed to an anti-inflammatory, neuroprotective environment.
To enhance translational relevance, we developed a non-invasive intranasal delivery method for secretome administration, bridging the gap toward clinical application. Complementary studies in Caenorhabditis elegans confirmed preventive and protective effects of iMSC secretome against α-synuclein–induced dopaminergic neurodegeneration. Using transgenic reporter strains, we demonstrated that treatment enhanced glutathione-dependent antioxidant defenses, mitigating oxidative stress under neurotoxic conditions. Moreover, we demonstrated that the iMSC secretome modulates autophagy, promoting the formation of autophagosomes and activating key autophagy-related pathways.
Overall, iMSC secretome reproduces and expands the neuroprotective actions of BM-MSC secretome through multi-pathway modulation. Its efficacy and suitability for non-invasive administration establish iMSC secretome as a potent, cell-free, and clinically translatable therapeutic candidate for Parkinson’s disease.
Mechanotransduction of soft matrix viscoelasticity: a molecular clutch perspective
Mariana Azevedo Gonzalez Oliva1, Manuel Salmeron Sanchez2
1Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, 2Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain
The extracellular matrix (ECM) is increasingly recognized to mechanically behave as a viscoelastic solid, exhibiting fluid-like energy loss (viscosity) and solid-like energy storage (elasticity) in response to applied deformations. ECM viscoelasticity has been shown to profoundly influence cell behavior, including proliferation, differentiation as well as disease progression. Notably, the brain is among the most viscoelastic tissues in the body, and alterations in its mechanical properties are hallmarks of neurodegeneration [1]. However, most in vitro brain models rely on unphysiologically stiff, purely elastic substrates that fail to capture the dynamic mechanics of native tissue [2,3].
Our work has previously demonstrated the importance of the concerted action of Piezo1 and the molecular clutch complex in transducing viscoelasticity [4]. For this, we employed polyacrylamide (PAAm) hydrogels with tuneable elastic and viscous moduli as well as a modified viscoelastic molecular clutch model that incorporated the crosstalk of Piezo1 and integrin complexes. With our experimental set-up we demonstrated integrin–Piezo1 concerted action is a key regulator of the mechanotransduction of viscoelasticity in soft matrices. Building on these findings, we are now extending this approach to neuronal systems to decode how viscoelasticity shapes neuronal mechanosensing, activity, and communication. Using induced pluripotent stem cells (iPSCs) derived neurons (iNeurons), we investigate cell response to physiological and pathological viscoelasticity and assess how and whether these changes influence neuronal communication, investigating the key regulators of circuitry development. By integrating advanced biomaterials, live-cell imaging, and computational modeling, we aim to reveal how the dynamic mechanical properties of the brain microenvironment modulate synaptic and cytoskeletal dynamics.
References
1. Chaudhuri et al., 2020. “Effects of Extracellular Matrix Viscoelasticity on Cellular Behaviour.” Nature.
2. Lantoine et al., 2016. “Matrix Stiffness Modulates Formation and Activity of Neuronal Networks of Controlled Architectures”. Biomaterials.
3. Rabadan et al., 2022. “An in Vitro Model of Neuronal Ensembles.” Nature Communications.
4. A. G. Oliva et al., 2025. “Piezo1 Regulates the Mechanotransduction of Soft Matrix Viscoelasticity.” Nature Communications.
Development and validation of clinically-relevant large animal model of meniscal degeneration
Daniele D'arrigo1, Pietro Conte1, Giuseppe Anzillotti1, Barbara Canciani2, Valentina Rafaela Herrera Millar2, Alessia Di Giancamillo3, Laura De Girolamo2, Giuseppe Peretti2, Alberto Maria Crovace4, Elizaveta Kon5
1Humanitas University, Pieve Emanuele (Lombardia) - Italy, 2IRCCS Galeazzi-Sant'Ambrogio Orthopaedic Institute, Milan (Lombardia) - Italy, 3University of Milano, Milan (Lombardia) - Italy, 4University of Sassari, Sassari (Sardegna) - Italy, 5IRCCS Humanitas Research Hospital, Rozzano (Lombardia) - Italy
Degenerative meniscal tears represent a major clinical and socio-economic burden, as they are also strongly associated with the onset and progression of knee osteoarthritis (OA), underscoring the urgent need for effective preventive and regenerative strategies. Several tissue-engineering and cell-based approaches have been proposed to restore the meniscal structure and function, but their translation toward the clinics is still limited, partly because of the lack of reliable and pathophysiologically relevant preclinical models that mimic human meniscal degeneration process.
This study aimed to develop and validate a large animal model that reproduces the degenerative changes occurring in meniscus and in the other articular tissues, as cartilage, after meniscal injury. Three surgical procedures were performed and compared in an ovine model (n=32 stifle joints): 1) direct arthroscopic mechanical injury in medial meniscus; 2) peripheral devascularization and denervation; 3) full-thickness cartilage lesion in medial femoral condyle. Contralateral joints served as controls. After three months, morphological and histological evaluations were conducted to quantitatively assess the degenerative process.
Distinct lesion patterns emerged across the experimental groups already after macroscopic inspection. Interestingly, these lesions involved both meniscal and cartilaginous tissues, indicating the onset of OA-related cartilage degeneration, and were characterized by a different severity and localization. In particular, arthroscopic meniscal injury induced milder and gradual degeneration, better replicating the relatively slow and progressive degeneration typical of human meniscal pathology and meniscal-related OA.
This validated large animal model provides a robust, reproducible platform to test and compare regenerative and tissue-engineering approaches aimed at preventing or restoring meniscal integrity and function. By offering a controlled and physiologically relevant environment for evaluating biomaterials, scaffolds, and cell-based treatments, it will allow to bridge the translational gap between bench and bedside in musculoskeletal regeneration.
Funded by the European Union – Next Generation EU – PNRR M6C2 – Project PNRR-MAD-2022-12375978, CUP: E23C22000770006.
3D-printed composite scaffolds for bone tissue engineering based on Chondrosia reniformis collagen and Sr-doped codfish bones
Miguel Rocha1, Catarina Marques1, Sandra Pina1, Miguel Oliveira1, Rui Reis1, Tiago Silva1
13B's Research Group. University of Minho, Guimarães (Braga) - Portugal
Bone defects are highly prevalent worldwide, and despite extensive research current therapies remain inefficient. To tackle this issue, marine-origin materials have emerged as promising alternatives for advanced biomaterials, offering sustainable sourcing from industrial by-products while avoiding zoonosis or religious restrictions associated with mammalian materials.
In this work, collagen was isolated from Chondrosia reniformis, a sustainably maricultured marine sponge. Its high glycosylation enhances cell attachment and proliferation, improving regenerative potential. Calcium phosphates were obtained from codfish bones (FB), an abundant by-product from fish processing industry, through calcination. To enhance osteogenic performance, FB were doped with strontium [FB(Sr)]. The main objective was to develop bioinspired and bioresorbable inks for additive manufacturing, combining C. reniformis collagen (MColl) and FB, never explored before, to produce scaffolds towards bone regeneration. Because collagen-based materials often require rheological adjustment for 3D printing, alginate (Alg) was incorporated. Therefore, three formulations were prepared: Alg+FB (control), MColl+Alg+FB, and MColl+Alg+FB(Sr).
The success of the FB Sr-doping procedure was confirmed by XRD and FTIR-ATR, which demonstrated the incorporation of Sr into the calcium phosphate structure while maintaining its crystallinity. The inks exhibited shear-thinning behaviour, stable at 37°C, thus enabling 3D printing of structures considered for application at physiological temperature. SEM and μCT analyses confirmed accurate scaffold printing, with pore size and interconnectivity conducive to cell migration. Among the MColl-containing inks, MColl+Alg+FB(Sr) showed the highest compressive modulus (∼12 MPa). Biological assays using Saos-2 osteoblast-like cells demonstrated that MColl+Alg+FB(Sr) increased more significantly the cell metabolic activity, proliferation and alkaline phosphatase expression, which was further confirmed by live/dead and SEM analyses.
Overall, the incorporation of marine collagen and FB-derived CaPs, especially Sr-doped, enabled the fabrication of 3D-printed scaffolds with improved biological performance, highlighting their strong potential for application in bone regeneration therapies.
Integrating population genetics into tissue engineering applications: characterization of the “village-in-a-dish” as an innovative cellular model for investigating variability in DNA damage response
Anna Bertocci1, Sara Costa2, Pierangela Chiafele1, Mehrnaz Ghazvini1, De Pinho Gonçalves Joana2, Miao-Ping Chien1, Joyce Van Meurs1, Roberto Narcisi1
1Erasmus Medical Center, Rotterdam (Zuid-Holland) - The Netherlands, 2TU Delft, Delft (Zuid-Holland) - The Netherlands
The use of induced pluripotent stem cells (iPSCs) for in vitro population genetics has gained popularity, enabling the modelling of complex (non-monogenetic) diseases. This approach also has a major impact on regenerative medicine and tissue engineering, which are increasingly focused on integrating genetic factors into regenerative processes. However, traditional iPSC-based approaches are often time-consuming, expensive, and subject to major technical variability. To overcome these limitations, the “village-in-a-dish” strategy has emerged.
This strategy implies the culturing and of iPSCs from multiple donors at the same time in a single dish. This method increases throughput, reduces technical variability, and lowers costs, making it particularly useful for population genetic studies. Our goal has been to characterize the village-in-a-dish model and implement its use in investigating diversity in the DNA damage response, one of the most common mechanisms underlying tissue degeneration.
We generated or purchased 85 fibroblast-derived iPSC lines representing the “normal” Caucasian population (ages 22–76), and we adapt all of them to the same culturing conditions. We then characterized the lines by proliferation rate and assembled mini-villages by pooling five lines with comparable proliferation rates, otherwise, highly proliferating lines may overgrow the low-proliferating ones in the long term. Moreover, using 5 lines cultured independently or as a mini-village, we confirmed that proliferation rate was not significantly influenced by the different culture conditions. As additional characterization step, we examined the qualitative composition of iPSC colonies within a village, demonstrating that cluster composition arises largely by random chance. After, a Functional Single Cell sequencing analysis confirmed that transcription profile of cell lines that grow as mono or multi cluster is comparable, supporting the validity of the model.
To investigate the DNA damage response, we induced DNA damage using X-ray irradiation, in 3 rounds of experiments consisting in 3 villages of 25-30 iPSC lines each. Single-cell RNA sequencing was then performed, and through eQTL analysis, we associated the specific transcriptional responses of individual cells with the genotypes of their respective donors.
The ability to capture inter-individual variation in cellular responses is a powerful tool for advancing tissue engineering and regenerative strategies.
Human nasal olfactory stem cell-derived extracellular vesicles enhance functional and structural repair of peripheral nerve gaps in rats
Maxime Bonnet1, Seblani Mostapha2, Witters Marie3, Marqueste Tanguy4, Jaloux Charlotte3, Morando Philippe5, Decherchi Patrick4, Feron François2, Guiraudie-Capraz Gaëlle2
1Department of Biomedical and Clinical Sciences. University of Milan, Milan (Lombardia) - Italy, 2Nasal Olfactory Stemness and Epigenesis (NOSE). Institut de Neuropathophysiologie, Marseille (Provence-Alpes-Cote d Azur) - France, 3Department of Hand Surgery and Reconstructive Surgery of the Limbs. Assistance Publique Hôpitaux de Marseille, Marseille (Provence-Alpes-Cote d Azur) - France, 4Plasticité des Systèmes Nerveux et Musculaire (PSNM). Institut des Sciences du Mouvement: Etienne-Jules MAREY, Marseille (Provence-Alpes-Cote d Azur) - France, 5Gliomagenesis and MicroEnvironment (GlioMe). Institut de Neuropathophysiologie, Marseille (Provence-Alpes-Cote d Azur) - France
Introduction: Peripheral nerve injuries cause long-term functional impairment and remain a major clinical challenge, particularly when loss of substance prevents spontaneous regeneration. Human olfactory ectomesenchymal stem cells (OEMSCs) have demonstrated regenerative potential through their paracrine activity, notably via extracellular vesicles (EVs), carrying proteins, cytokines, and signaling molecules. This study evaluated the therapeutic potential of OEMSC-derived EVs for repairing segmental loss of peripheral nerves.
Methods: A 7 mm gap in the peroneal nerve in rats was bridged using an autologous venous conduit filled with either freshly purified or cryopreserved OEMSC-derived EVs. Control groups included unoperated rats and autografted animals (nerve sutured in inverted position, Gold Standard). Three months post-surgery, sensorimotor and locomotor recovery, muscle contractile properties, muscle mass, axon number, and myelination were assessed.
Results: Treatment with OEMSC-derived EVs significantly improves locomotor recovery, partially preserves the contractile phenotype and mass of target muscles, and increases the number of regenerating axons. Freshly purified EVs induce superior recovery compared to autologous grafting which is the Gold Standard in this field.
Conclusions: OEMSC-derived EVs exert potent pro-regenerative effects on peripheral nerve repair, closely mimicking the benefits of their parent stem cells. These findings position EVs as a promising, cell-free alternative to stem cell therapy for peripheral nerve reconstruction and as a scalable approach for regenerative medicine applications.
Acknowledgments: Aicha AOUANE (IBDM, AMU-Marseille), member of ANR-10-INBS-04 and member of the Marseille Imaging Institute, Aix Marseille University A*MIDEX, French “Investissements d’Avenir” programme (AMX 19 IET 002), NeuroSchool Marseille, Département des Bouches-du-Rhône.
Transcriptomic profiling of pulsed electromagnetic field (PEMF)-stimulated biomimetic bone tissue model cultured in a direct perfusion bioreactor - SEMIT
Farah Daou1, Beatrice Masante2, Stefano Gabetti2, Federico Mochi3, Giovanni Putame2, Eleonora Zenobi3, Elisa Scatena3, Federica Dell’atti1, Francesco Favero1, Massimiliano Leigheb1, Costantino Del Gaudio3, Cristina Bignardi2, Diana Massai2, Andrea Cochis1, Lia Rimondini1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Mechanical and Aerospace Engineering and PolitoBIOMed Lab. Politecnico di Torino, Turin (Italia) - Italy, 3Hypatia Research Consortium, Rome (Lazio) - Italy
Pulsed electromagnetic field (PEMF) stimulation is extensively used in clinical practice for promoting bone repair [1]. While several signaling pathways associated with its osteogenic and anti-inflammatory properties have been identified, the majority are still unknown, hindering the standardization and optimization of therapeutic procedures [2]. To identify PEMF-induced signaling pathways, this study used a research platform, based on a previously developed direct perfusion bioreactor [3], to culture human mesenchymal stem cells (hMSCs) onto 3D-printed polylactic acid (PLA) scaffolds resembling trabecular bone. For 21 days, these constructs were cultivated in a perfusion bioreactor (0.3 mL/min) with either basal or osteogenic media, and with or without PEMF stimulation (1.5 mT, 75 Hz, 4 h/day). Static cultures were used as a reference. Real-time qPCR and RNA sequencing (RNA-Seq) were carried out. The findings showed that even in the absence of biochemical cues, PEMF has a noticeable impact on cells. The four stages of bone healing—inflammatory, fibrovascular, bone creation, and bone remodeling—are specifically targeted by PEMF stimulation in basal media, even in the absence of a pathological condition, according to RNA-Seq. As a result, the adopted in vitro research platform enabled the discovery of the signaling pathways triggered by PEMF, and represents a powerful tool for studying bone biology.
REFERENCES
1. Massari, L. et al. Int. Orthop., 43(3), 539–551, 2019.
2. Cadossi, R. et al. JAAOS Glob. Res. Rev., 4(5), e1900155, 2020.
3. Gabetti S. et al. Sci. Rep, 12(1):13859, 2022.
“Study carried out within the BIGMECH project – funded by European Union – Next Generation EU within the PRIN 2022 program (D.D. 104 - 02/02/2022 Ministero dell’Università e della Ricerca).”
Native mechanobiological regulation of human mesenchymal stem cells on 3D-printed PLA trabecular scaffolds via dual mechanical stimulation - SEMIT
Shahd Alahmad1, Farah Daou1, Stefano Gabetti2, Beatrice Masante2, Eleonora Zenobi3, Carlotta Achille4, Elisa Scatena3, Simone Israel2, Cristina Bignardi2, Diana Massai2, Andrea Cochis1, Lia Rimondini1
1Department of Health Sciences. University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Mechanical and Aerospace Engineering and PolitoBIOMed Lab. Politecnico di Torino, Turin (Italia) - Italy, 3Hypatia Research Consortium, Rome (Lazio) - Italy, 4E. Amaldi Foundation, Rome (Lazio) - Italy
In this study, we aimed to develop an in vitro platform for bone biology research to investigate how tissue-specific geometry and mechanical cues influence bone healing, thereby clarifying the impact of the local environment on cellular responses. The platform utilizes 3D bone tissue models constructed using human bone marrow-derived mesenchymal stem cells (hBM-MSCs) cultured within polylactic acid (PLA) scaffolds. The latter were rationally designed to mimic the distinct native architectures of the trabecular bone of the iliac crest (PLA600) and bones, such as the ulna, femur, and tibia (P3S3). The 3D models were cultured for a period of 14 days within a custom direct perfusion bioreactor. This system enabled the simultaneous application of two physiological mechanical stimuli crucial for bone remodeling: continuous shear stress (0.3 mL/min) and intermittent hydrostatic pressure (15 kPa, 2 h/day). Using our platform, we were able to identify the genes upregulated under hydrostatic pressure that are directly related to the osteogenic differentiation of hBM-MSCs. Our investigation demonstrates the value of recapitulating the complex environment of bone tissue in vitro for better understanding the effect of physiological loading on bone regeneration.
“Study carried out within the BIGMECH project – funded by European Union – Next Generation EU within the PRIN 2022 program (D.D. 104 - 02/02/2022 Ministero dell’Università e della Ricerca).”
Hyperglycemia and IL-1β synergistically derail osteoblast maturation in a human bone-on-a-chip model
Verónica Sosa-Castellano1, Valeria Valez-Medina2, María Ángeles Pérez1, Elena García-Gareta1
1Multiscale in Mechanical & Biological Engineering Research Group, Aragon Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragon 50018, Spain, Zaragoza - Spain, 2CEINBIO: Center for Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo - Uruguay
Diabetes mellitus (DM) is associated with impaired bone regeneration due to dysregulated angiogenesis, deficient osteoblastic activity, and a persistent pro-inflammatory environment. As a consequence, a state of oxidative stress is established, resulting from redox imbalance and further limiting the effectiveness of current regenerative strategies. However, conventional in vitro models do not accurately reproduce these pathological conditions, hindering the evaluation of targeted therapeutic approaches.
In this study, we validated a human bone-on-a-chip platform to model diabetic and inflammatory microenvironments. Primary human osteoblasts were cultured under four conditions representing physiological control, inflammatory stimulation (IL-1β), high glucose (HG), and the combined diabetic-inflammatory microenvironment (HG + IL-1β). Cell morphology was quantified through morphometric parameters (area, perimeter, solidity, form factor), while osteogenic differentiation was assessed by nuclear translocation of Runx2 (early marker), alkaline phosphatase (ALP) activity (intermediate marker), and cytoplasmic expression of osteocalcin (late marker), using immunofluorescence and spectrophotometry.
Control cultures progressively expanded, developed cytoplasmic extensions, and formed interconnected osteoblastic networks by day 21, accompanied by dynamic Runx2 localization. In contrast, HG and IL-1β induced compact, rounded morphologies with reduced cytoskeletal organization and altered differentiation; the combined HG + IL-1β condition exhibited the most pronounced effect.
These results demonstrate a synergistic negative effect of metabolic and inflammatory stress on osteoblastic function and establish the bone-on-a-chip platform as a physiologically relevant and scalable model for simulating diabetic bone pathophysiology, enabling the preclinical screening of biomaterials and therapeutic strategies aimed at restoring bone regeneration.
Acknowledgements
This work is supported by the project PID2023-146072OB-I00 funded by MCIU/AEI/10.13039/501100011033 and FSE+. E.G.G. is funded by a Ramón and Cajal Fellowship (RYC2021-033490-I, funded by MCIN/AEI/10.13039/501100011033 and the EU “NextGenerationEU/PRTR”). V.S.C. is supported by a Banco Santander–Universidad de Zaragoza Predoctoral Fellowship for Ibero-American and Equatorial Guinean students.
Soft by nature: human protein-based hydrogels for next-generation liver models
Maria C. Lobo1, Ana T. Rufino1, João F. Mano2, Catarina A. Custódio1
1Metatissue, Ílhavo (Aveiro) - Portugal, 2Department of Chemistry. CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal
The pharmaceutical industry faces challenges in the process of development of new drugs, with approval processes exceeding a decade and the success rates from phase I to launch below 10%. Drug-induced liver injury (DILI) causes 18-30% of withdrawals.1 Animal-based models poorly mimic human liver physiology and metabolism, highlighting the urgent need for human-relevant 3D preclinical models to improve hepatotoxicity prediction while reducing animal use.
This work aims to engineer a human liver model using protein-based biomaterials that mimic the ECM’s structure, signals, and softness for hepatocyte function. A fully xeno-free culture system for HepG2 cells was established by comparing media supplemented with FBS and human platelet lysates (hPL). Comparable cell proliferation, metabolic activity, and hepatic functionality (urea, albumin, CYP450 activity) were observed, confirming hPL as a viable human-derived alternative for maintaining hepatocyte-like cell performance. To further recreate the liver microenvironment, HepG2 cells were encapsulated in photocrosslinkable human protein-based hydrogels composed of methacryloyl platelet lysates (hPLMA) and methacryloyl-modified decellularized ECM (dECM-MA). Systematic optimization revealed that hydrogels containing 10% hPLMA and 0.7% dECM-MA (1:1) with 10 × 106cells/mL provide a soft, ECM-rich matrix that closely mimics the liver’s natural mechanical properties, supporting high cell viability and metabolic activity. Overall, integrating hPLMA and dECM-MA establishes a human-derived, xeno-free platform that sustains hepatic function and leverages the natural ECM’s biochemical and mechanical cues.2 This biomimetic approach bridges traditional in vitro systems and physiologically relevant human tissues, enabling more predictive drug safety assessment while adhering to ethical principles.
Acknowledgements
This work was developed within the scope of the project CICECO Aveiro Institute of Materials, UID/50011/2025 (DOI 10.54499/UID/50011/2025) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC). MCL thanks FCT through PDQI for her PhD grant ref. 2024.03688.BDANA.
References
1. Dowden H. & Munro J. Nat. Rev. Drug Discov. 18, 495–496 (2019).
2. Xia T. et al. Polymers 12, 1903 (2020).
Bioink development and characterisation for 3D bioprinting of muscle spindle models - SEMIT
Yuannan Kang1, Deepak Kalaskar2, Darren J. Player1
1Centre for 3D Models of Health and Disease, Division of Surgery and Interventional Science, Faculty of Medical Sciences. University College London (UCL), London (London, City of) - United Kingdom, 2Department for Surgical Biotechnology, Division of Surgery and Interventional Science, Faculty of Medical Sciences. University College London (UCL), London (London, City of) - United Kingdom
Muscle spindles are principle proprioceptive mechanoreceptors, yet once damaged, there is currently no defined way to reconstruct the sensory circuit or spindle itself. Current two-dimensional (2D) models poorly replicate the complex ECM required for spindle maturation, leading to incomplete phenotypes. Our work employs a three-dimensional (3D) bioprinting approach to engineer controlled scaffold with tailored bioinks that guide spindle fibre specification through defined geometries, mechanics, cell distribution and bioactive cues. We hypothesised that recreating a native-like ECM within a spatially defined scaffold will promote spindle fibre maturation.
Candidate biomaterials, including alginate (3-5% w/v), guar gum (2, 4% w/v), collagen type I (0.5-3.68 mg/ml) and GelMA (7, 10% w/v)-based formulations, were screened and optimised for their cytocompatibility, printability, rheological and mechanical performance. Optimised formulations and cell suspensions were printed via extrusion bioprinting (CELLINK BioX3) to fabricate spatially defined constructs alongside acellular and 2D controls. All constructs were cultured under identical differentiation conditions, with analyses performed at 4, 7, 14 and 21 days. Outcome measures included morphological (fluorescence and immunofluorescence for myotube formation) and cellular (Live/Dead assay) analysis. Myotube fusion and orientation were quantified by fusion index analysis and F-actin staining.
Among four candidate biomaterials tested, collagen/GelMA blends exhibited improved viscoelastic properties while maintaining shear-thinning behaviour suitable for extrusion bioprinting, as characterised by an extended linear viscoelastic region (LVR) and decline in viscosity with increasing shear rate. This led to smooth extrusion, high structural fidelity, and excellent cytocompatibility (>90%) over 14 days. F-actin staining confirmed myogenic differentiation, while hybrid bioinks promoted enhanced cell-matrix interactions and tuneable stiffness relative to single-component controls. These findings demonstrate that collagen-based hybrid bioinks support robust myogenic differentiation within 3D printed constructs, providing a promising platform for generating aligned, physiologically relevant muscle spindle models for future regenerative and neuromuscular studies.
Towards PDAC-on-chip mimicking tumor–stroma interactions for drug testing
Matteo Bortolameazzi1, Marialucia Rubicondo1, Viola Sgarminato1, Gianluca Ciardelli1, Clara Mattu1, Chiara Tonda-Turo1
1DIMEAS. Politecnico di Torino, Torino (Italia) - Italy
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to late diagnosis and limited therapeutic options, mainly ascribed to the presence of a dense desmoplastic stroma that impairs drug delivery.
To overcome these challenges, a microfluidic PDAC-on-chip model was developed to reproduce tumor-stroma crosstalk and to evaluate nanotherapeutic strategies under physiologically relevant conditions. Designed in Fusion 360 and fabricated by PDMS replica molding, the PDAC-on-chip platform integrates stromal and tumor compartments within a single architecture comprising an external channel for medium perfusion, a central fibroblast-laden collagen gel that mimics the stromal barrier, and a dedicated chamber for PDAC cell culture. Micropillar arrays separate the compartments, whereas a 3D-printed holder supports an electrospun polycaprolactone/gelatin (PCL/Gel) membrane acting as a biomimetic substrate for PDAC cells adhesion. Plasma bonding with a glass coverslip provides direct optical access for real-time monitoring. Optimization of micropillar geometry ensured hydrogel confinement while preserving nutrient and drug diffusion. Static co-cultures revealed homogeneous cell distribution and physiological morphology throughout the device, as well as the permeability of the stromal barrier to hNPs loaded with a novel colchicine-derived compound. Future studies will introduce dynamic perfusion to investigate the therapeutic response of both cell populations following treatment.
In parallel, a complementary transwell system incorporating the same electrospun PCL/Gel membrane confirmed higher hNP internalization in PDAC cells compared to fibroblasts, validating their selective targeting capability.
Collectively, the PDAC-on-chip platform offers a promising, biomimetic and ethically sustainable approach for studying tumor–stroma interactions and assessing nanotherapeutic efficacy, bridging the gap between conventional in vitro assays and in vivo models while adhering to the 3R principles.
Acknowledgment: Supported by Fondazione Compagnia di San Paolo – Trapezio Call for Proposals, Target 1 project “Exocrine Glandular Tissue Models Through Precisely Designed Biomimetic Environments (ERATOSTHENES).”
Exploring extracellular matrix as biomimetic material for regenerative medicine and tissue engineering applications
Katja Schenke-Layland
de Eberhard Karls University, Tübingen (Baden-Wberg Bayern) - Germany
The human body is a complex compilation of myriads of gear wheels, where malfunctioning, misplaced, or missing cogs negatively affect the whole system. Apart from the DNA, the blueprint that defines and controls the body’s functions, internal and external forces in the nano-, micro-, and macroscale are major contributors to the maintenance of the machinery. These biomechanical cues control, guide, and impact cellular processes and cell fate decisions on the subcellular level, resulting in functional homeostasis, just like a well-lubricated gearbox. Defective mechanosensing, mechanotransduction, or biomechanical force creation, the equivalent of a sabulous gearbox, contributes to disease development and therefore results in malfunctioning in the long run.
With our scientific work, we aim to make contributions to a better understanding of early human development and translate our findings into applications for regenerative medicine and tissue engineering. Our research has a strong focus on extracellular matrix (ECM) proteins and how ECM developmentally guides cell fate decisions and impacts human cell physiology and pathophysiology. We have previously identified the basement membrane glycoprotein Nidogen-1 (NID1) and the small leucine-rich proteoglycan decorin (DCN) as crucial pancreatic niche molecules and important ECM proteins that drive early human development, promote cell/tissue survival and regeneration under hypoxic/ischemic conditions, making them ideal biomaterials for different applications in tissue engineering and regenerative medicine. In my presentation, I will provide insight into how NID1 and DCN-functionalization can rescue the insulin production of human β-cell-composed pseudo-islets cultured in a pancreas-on-a-chip in vitro model that mimics the ischemic environment post β-cell transplantation. To non-invasively analyze and quantify the cell response and monitor artifact-free phenotypic and functional changes within the in vitro model, marker-independent Raman microspectroscopy, Raman imaging, and fluorescence lifetime imaging microscopy (FLIM)-based metabolic mapping were employed. The data presented suggest that NID1 and DCN are powerful therapeutic candidates that may have multiple clinical applications in the future.
Human iPSC-derived hepatic models for the study and therapeutic targeting of liver disorders
Estela Villanueva Bádenas1, María Teresa Donato Martín2, Laia Tolosa Pardo3
1Experimental Hepatology. Health Research Institute of La Fe, Valencia - Spain, 2Department of Biochemistry and Molecular Biology, Faculty of Medicine and Odontology. Universitat de València, Valencia - Spain, 3Experimental Hepatology Research Group. Health Research Institute of La Fe, Valencia - Spain
Liver diseases are a growing global health problem, creating the need for in vitro models that better reproduce the complexity of the human liver for disease research and therapeutic development. An important factor in developing realistic liver models is the presence of multiple interacting cell types. The liver contains hepatocytes along with non-parenchymal cells, such as hepatic stellate cells and Kupffer cells, and these heterotypic interactions are essential to maintain hepatocyte function. Induced pluripotent stem cells (iPSCs) offer a promising approach because they can be differentiated into various liver cell types that closely resemble their in vivo counterparts. From a single iPSC line, we can obtain hepatocyte-like cells (HLCs) and hepatic stellate-like cells (HSCs), allowing the creation of co-culture systems that better mimic the hepatic microenvironment. The aim of this study was to generate HLCs and HSCs from iPSCs, to characterize their phenotype and function, and to optimize their co-culture conditions. The HLCs produced albumin, exhibited ureogenic capacity, and showed cytochrome P450 enzymes activity, confirming hepatocyte-like functionality. The HSCs expressed typical stellate cell markers and responded to TGF-β stimulation by increasing the expression of fibrosis-related genes. When co-cultured as 3D spheroids, both cell types maintained their key characteristics. Overall, these results show that iPSC-derived liver cells reproduce essential aspects of liver physiology and pathology, supporting their use as a relevant platform for studying liver disease mechanisms and developing new therapeutic strategies.
Gap junction-mediated intercellular communication between CD27+ expressing γδT cells and EO771 breast cancer cells alters cytotoxic activity and immune cell recognition
Brian Harkin1, Robert Wiesheu2, Nadia Iqbal3, Charlotte Turner1, Meadhbh Brennan1, Seth Coffelt3, Laoise Mc Namara1, Eoin Mc Evoy1
1Discipline of Biomedical Engineering. University of Galway, Galway - Ireland, 2Dana-Farber Cancer Institute. Harvard Medical School, Boston (Massachusetts) - United States, 3Cancer Research UK Scotland Institute. University of Glasgow, Glasgow (Glasgow City) - United Kingdom
Direct tumour-immune cell interactions are crucial for effective anti-tumour responses, yet the mechanisms regulating these interactions remain poorly understood. Gap junction channels are key mediators of cell-cell communication, allowing the transfer of ions and small molecules [1]. To investigate their role, we modulated intercellular communication between cancer and T-cells in co-culture killing assays using pharmacological agents to assess innate cytotoxic potential. We focus on the lesser-studied γδT cells, which can kill cancer cells through MHC-independent recognition, enabling them to bypass common tumour immune-evasion stategies.
Various concentrations of GJ-mediators, aCT1-peptide, retinoic acid, dipyridamole, and resveratrol were used to treat EO771 mammary carcinoma cells for 24-hours. These drugs collectively promote gap junction formation, preserve existing communication, or boost connexin expression. Cancer cells were then plated in a flat-bottom 96-well plate. After 3-hours, C57BL/6J-derived γδT cells were added to the wells. Co-cultures were incubated for 24-hours. Cells were collected and blocked with TruStain-FcX, then stained with CellTraceViolet, anti-CD107a, and CD3. Zombie Green was added as a live/dead marker (n=4).
After 24-hours, cytotoxicity decreased in response to all drugs compared with the vehicle and positive control. Drug treatments significantly reduced IFN-γ and perforin secretion in co-culture supernatants (ELISA), consistent with decreased γδT activation and degranulation. This was confirmed by reduced surface CD107a expression. We also confirmed expression and localisation of GJ-protein Cx-43 were altered using each drug.
Overall, we show that modulation of gap junctions in E0771 cells altered susceptibility to γδT cell-mediated cytotoxicity. Increased Cx-43 expression weakened perforin and granzyme-dependent killing, demonstrating that enhanced intercellular communication disrupts successful immune recognition and cytotoxic signalling. This supports recent literature focusing on the pro-tumorigenic implications of altered Cx-43 levels, with increasing evidence highlighting tumour-promoting roles [1,2].
1. Busby et al. (2018). Molecular Sciences Vol. 19
2. Zhang et al. (2024). Cellular and Molecular Medicine Vol. 28
3D printable hydrogels for regenerative medicine
Samarah Vargas Harb1, Cintia Delai Da Silva Horinouchi1, Ana Carolina De Aguiar1, Mariane Aparecida Risso2, Helga Caputo Nunes Holzhausen2, Caroline Nascimento Barquilha2, Mônica Alves2, Ana Carolina Migliorini Figueira1
1Brazilian Biosciences National Laboratory. Brazilian Center for Research in Energy and Materials, Campinas (Sao Paulo) - Brazil, 2Department of Ophthalmology. State University of Campinas (UNICAMP), Campinas (Sao Paulo) - Brazil
The development of effective strategies for tissue regeneration remains a major challenge in medicine. This study focuses on the formulation and characterization of 3D printable hydrogels based on methacrylated gelatin (GelMA) enriched with bioactive components, including decellularized extracellular matrix (ECM), ECM proteins (fibronectin, elastin, laminin), platelet-rich plasma (PRP), and plasma rich in growth factors (PRGF). The hydrogels were loaded with mesenchymal stem cells (MSCs) to further enhance their regenerative potential through the paracrine release of trophic factors. Rheological analyses demonstrated that all formulations maintained suitable shear-thinning behavior for extrusion-based 3D printing, while scanning electron microscopy (SEM) revealed interconnected microporous structures favorable for cell infiltration and nutrient diffusion. Mechanical testing confirmed that the elastic moduli of the composites could be tuned to approximate those of native soft tissues. Biological assays indicated that the enriched GelMA formulations supported high MSC viability and accelerated wound closure. ELISA and mass spectrometry analyses confirmed the release of growth factors and cytokines that promote angiogenesis, modulate inflammation, and stimulate endogenous tissue repair, demonstrating their adaptability for different soft tissue regeneration applications.
Differential extracellular matrix compartmentalization in 3D bioartificial muscle engineering promotes neuromuscular junction formation
Yagmur Filiz1, Ludo Van Den Bosch2, Lieven Thorrez1
1KU Leuven Campus Kulak, Kortrijk (West-Vlaanderen) - Belgium, 2KU Leuven, Leuven (Brabant) - Belgium
Introduction: Neuromuscular junctions (NMJs) are critical for motor control, yet replicating their structural and functional complexity in vitro remains challenging. This study compares 3D bioartificial muscle (BAM) models differing in cellular ratio’s, matrix composition (fibrin vs. collagen/Matrigel), compartment engineering strategy (one-compartment vs. two-compartments), and culture duration (2 vs. 4 weeks) to identify conditions that best support NMJ formation.
Methods: BAMs were engineered by embedding C2C12 myoblasts and NSC-34 motor neuron-like cells within fibrin or collagen/Matrigel hydrogels at motor neuron-to-myoblast ratios of 1:100 or 1:200. In one-compartment BAMs, C2C12s and NSC-34s were co-cultured from day 0. In two-compartment BAMs, first C2C12s were cultured for 7 days to form myotubes, then NSC-34s were added as a second compartment as previously described [1]. One-compartment and two-compartment BAMs were maintained for 2 or 4 weeks with agrin supplementation. Immunofluorescence staining was performed to assess myotube alignment (myosin heavy chain), motor neuron integration (βIII-tubulin), and NMJ formation (α-bungarotoxin). Images were quantified for fusion index, fiber area, axonal outgrowth and acetylcholine receptor (AChR) cluster counts.
Results: Two-compartment BAMs better reflected in vivo organization, showing spatial organization of myotubes and neurons at 1:200 motor neuron-to-myoblast ratio. Fibrin supported myoblast fusion, whereas collagen/Matrigel promoted axon outgrowth. Extended culture duration did not improve myotube or NMJ formation in one-compartment BAMs; however, two-compartment BAMs exhibited aligned myotubes and NMJ-like structures. The NMJs displayed diffuse and elongated morphologies, with AChR clusters distributed along the myotubes, consistent with early stages of NMJ maturation.
Conclusion: Matrix composition, compartmenting strategy, and culture duration synergistically influence NMJ development. The two-compartment 3D BAM model provides a promising platform for investigating NMJ formation. Future studies will focus on improving functional readouts and integrating human-derived cells to increase translational relevance.
Reference
[1] Gholobova, D., Terrie, L., Mackova, K. et al. (2020). Biofabrication, 12(3), 035021. doi:10.1088/1758-5090/ab8f36
Extracellular vesicles derived from microfragmented adipose tissue modulate osteoclast differentiation and may enhance fat tissue transplant efficacy in osteoarthritis
Marica Imbimbo1, Sara Nembrini2, Beatrice Castiglioni1, Adrian Perez Barreto3, Carlo Tremolada4, Annalisa Chiocchetti2, Michela Bosetti1
1Department Pharmacological Sciences (DSF). University of Eastern Piedmont, Novara (Piemonte) - Italy, 2Department of Health Sciences, Center for Translational Research on Autoimmune and Allergic Disease (CAAD). University of Eastern Piedmont, Novara (Piemonte) - Italy, 3Blond-McIndoe Laboratory, Division of Cell Matrix Biology and Regenerative Medicine, School of Medical Sciences, Faculty of Biology, Medicine, and Health. Division of Cell Matrix Biology & Regenerative Medicine, FBMH, University of Manchester, UK, Manchester - United Kingdom, 4IMAGE Regenerative Clinic, Milano (Lombardia) - Italy
Extracellular vesicles (EVs) are emerging as key mediators of cell–cell communication, with increasing evidence supporting their anti-inflammatory and pro-regenerative roles in musculoskeletal disorders. Adipose tissue, as microfragmented fat (MFAT) transplantation, is largely used in intra-articular regenerative therapies and it naturally releases EVs that may contribute to the therapeutic outcome.
This study, performed in collaboration with Exogems, aimed to investigate the biological effect of EVs isolated from MFAT on osteoclast differentiation, a process strongly implicated in subchondral bone remodeling and cartilage degeneration in osteoarthritis (OA).
Adipose tissue was processed using the Lipogems device and EVs were isolated through differential ultracentrifugation followed by size-exclusion chromatography (SEC) for enrichment and purification. RAW 264.7 murine macrophages were stimulated with RANKL to induce osteoclastogenesis and subsequently treated with MFAT-derived EVs.
Quantitative and morphological analyses revealed a reduction in multinucleated osteoclast formation in the presence of EVs, suggesting an inhibition of osteoclast differentiation and a potential role of adipose-derived EVs in maintaining a balanced bone–cartilage interface by attenuating excessive bone resorption.
In conclusion, EVs derived from microfragmented adipose tissue appear to modulate osteoclast activity, potentially enhancing the regenerative microenvironment following adipose tissue transplantation. These results open new perspectives for optimizing MFAT-based therapies in OA and other cartilage-degenerative conditions.
Injectable elastin-like hydrogels with tunable degradation profile promote osteochondral repair in long-term rabbit models
Desiré Venegas-Bustos1, Gonzalo Martínez2, Sonia Martínez-Páramo3, Israel González De Torre1, Francisco Lamus3, María Isabel Alonso3, Ángel Gato3, Ángel José Álvarez-Barcia4, Aurelio Vega-Castrillo2, Mercedes Alberca5, José Carlos Rodríguez-Cabello1
1Bioforge Lab, LaDIS, CIBER-BBN, Edificio LUCIA. Universidad de Valladolid, Valladolid - Spain, 2Hospital Clínico Universitario de Valladolid. Universidad de Valladolid, Valladolid - Spain, 3Departamento de Anatomía y Radiología, Facultad de Medicina. Universidad de Valladolid, Valladolid - Spain, 4SIBA-UVA: Servicio de Investigación y Bienestar Animal. Universidad de Valladolid, Valladolid - Spain, 5IBGM (Institute of Molecular Biology and Genetics) and University Scientific Park. Universidad de Valladolid, Valladolid - Spain
Osteochondral defects compromise both articular cartilage and subchondral bone, demanding biomaterials that provide time-coordinated cues. We developed an injectable elastin-like recombinamer (ELR) hydrogel with tuneable degradation, including a layered design to address cartilage and bone requirements. ELRs incorporated an RGD adhesion motif and protease-sensitive domains with differential kinetics: a slow-degrading DRIR sequence and a fast-degrading GTAR sequence. The formulation is delivered in liquid form and gels in situ, conforming to irregular defect geometry.
Critical-size osteochondral defects were created in a rabbit model. Experimental groups included homogeneous and heterogeneous fast- or slow-degrading hydrogels, a layered degradation profile, and cell-augmented conditions with mesenchymal stem cells (MSCs) and/or chondrocytes. At 6 months, repair was assessed by biochemical glycosaminoglycan (GAG) content, histology, and immunohistochemistry for type II collagen. Quantitative evaluation used a modified O’Driscoll score with significance at p < 0.05.
Even cell-free ELR hydrogels supported bone and cartilage-like matrix formation, yielding GAG-rich tissue and type II collagen deposition. Homogeneous hydrogels (fast and slow) achieved significantly higher modified O’Driscoll scores than untreated defects and chondrocyte-only controls (p < 0.05). Adding MSCs further improved structural organization and matrix quality, consistent with paracrine support of chondrogenesis. The layered degradation design was feasible and adaptable in situ; trends toward improved osteochondral integration were observed, motivating powered comparisons across profiles.
In summary, injectable ELR hydrogels with tunable (including layered) degradation provide a versatile, minimally invasive platform for long-term osteochondral repair. Material alone promotes durable cartilage-like regeneration, while MSC loading enhances outcomes, supporting tailored ELR systems as promising candidates for clinically relevant osteochondral therapies.
Acknowledgements: the authors acknowledge funding from the Spanish Government (MCIN/AEI/10.13039/501100011033 and ERDF, EU), the Junta de Castilla y León (Department of Education, ERDF cofunded grants), and the Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León.
Extracellular matrix protein-decorated hyaluronic acid hydrogels enhance growth factor retention and vascularisation
Yu-Yin Joanne Chang1, Louise Hosty1, Noémie Petit1, Tom Hodgkinson1, Shane Browne1
1Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland
Introduction
The formation of functional vasculature remains a critical barrier in tissue engineering [1]. Extracellular matrix (ECM) composition and properties play a central role in regulating cell behaviours, including vascular network formation. Hyaluronic acid (HyA)-based hydrogels offer a tuneable, biocompatible platform for synthetic ECMs (sECMs) [2]. We hypothesise decorating HyA-based sECMs with vascular niche proteins enhances network formation of encapsulated human vascular cells.
Materials and Methods
HyA was functionalised with acrylate groups (AcHyA), and the degree of modification confirmed by nuclear magnetic resonance (NMR) [3]. Fibronectin (FN) or laminin (LM) was conjugated to AcHyA by Michael-type addition and crosslinked with dithiol crosslinkers. Biophysical properties, including sECM swelling, degradation, and storage modulus were characterised. Growth factor sequestration, modelled using vascular endothelial growth factor (VEGF), was assessed using enzyme-linked immunosorbent assay (ELISA) and fluorescence recovery after photobleaching (FRAP). Human vascular cells were encapsulated within sECMs and visualised using F-actin staining.
Results
NMR confirmed ∼22% acrylation of HyA and decoration with FN/LM. Protein decoration did not affect sECM swelling, degradation, or stiffness (600–800Pa). ELISA demonstrated enhanced VEGF sequestration in FN- and LM-decorated sECMs (∼55% release after 5 days) versus HyA-only (∼65%), corresponding to reduced VEGF diffusivity by FRAP. Vascular cells encapsulated in decorated sECMs showed increased elongation and cell area, indicating enhanced vascular network formation.
Conclusions
Decoration of HyA-based sECMs with FN or LM enhances growth factor retention and vascular cell elongation without altering hydrogel mechanics. This tuneable sECM platform supports the development of 3D vascularised tissue constructs, and will be integrated into a microfluidic system to introduce physiological flow as a dynamic, pro-vasculogenic cue.
Acknowledgments
CÚRAM (13/RC/2073_P2), EPSRC-Research Ireland funded LifETIME-CDT (18/EPSRC-CDT/3583).
References
[1] Bae H et al. Sci. Transl. Med., 2012.
[2] Petit N. et al. Mater. Today Bio, 2025.
[3] Browne S et al. ACS Biomater. Sci. Eng., 2019.
Integration of liver tumor organoids and in silico methods to address drug resistance (LIVERtera)
Marc Terrones1, Bilge Sen2, Marta Duran-Güell1, María J. Lozano1, Daniel Rodrigo-Torres1, Estephan Arredondo1, Aline Roch2, Eric Herrero3, Mireia Casulleras1, Natalia Sánchez1
1BeCYTES Biotechologies S.L. - BioIVT, Barcelona - Spain, 2DOPPL S.L., Lausanne (Vaud) - Switzerland, 3Hepatobiliopancreatic Surgery Unit. Hospital Universitari Mútua de Terrassa, Terrassa (Barcelona) - Spain
Introduction
Approaching drug resistance is essential to meet current clinical needs, particularly in hepatocellular carcinoma (HCC) and liver-metastatic colorectal cancer (mCRC). In the case of mCRC, resistance-conferring mutations affecting the extracellular domain of EGFR determine its interaction with cetuximab, as it has been described with S468R. However, other clinically reported variants like G465R/E or S464L remain to be functionally characterized. LIVERtera integrates computational modeling to predict the functional impact of EGFR mutations, evaluate the ADME properties of sorafenib and identify novel active compounds against both malignancies.
Methods
Patient-derived organoids (PDOs) were established from HCC and mCRC tumor resections as well as matched normal adjacent tissue (NAT), which were histologically characterized. In parallel, the in silico study started by generating EGFR mutated residues by means of chemoinformatic tools to establish the systems. Next, potential steric impediments between cetuximab and the interface surface area of EGFR were evaluated by quantifying changes in hydrogen bonds, hydrophobic interactions and Van Der Waals contacts.
Results
We observed that S468R abolished hydrogen bond formation, generating steric impediments with T47 and Y104 residues of the cetuximab light chain (chain C). G465R induced intramolecular steric impediments with K443 and intramolecular clashes with residues W52 and D58 of the cetuximab light chain. G465E generated an intramolecular clash with S418 and an additional clash with residue W52 of cetuximab light chain. Additional interactions such as cationic and π-stacking were mapped and, interestingly, an unexpected increase in events was observed in the cetuximab light chain, possibly representing artifacts of residue mutations.
Conclusion
Our study identifies novel atomic-level interactions between EGFR and cetuximab that underlie resistance mechanisms, consistent with similar findings in literature. Future work will focus on screening candidate compounds to overcome these clashes and evaluating sorafenib’s ADME profile in functionally characterized PDOs.
Immune–tumour modelling in osteosarcoma using a dynamic microfluidic platform
Amanda Guitián-Caamaño1, Finola Cliffe2, Mark Lyons3, Fiona Freeman1
1School of Mechanical and Materials Engineering, Engineering and Materials Science Centre. University College Dublin, Dublin - Ireland, 2Hooke Bio Ltd, Limerick - Ireland, 3Hooke Bio Ltd, Limerick - Ireland
Osteosarcoma (OS) remains the most common primary bone malignancy in children and adolescents, yet patient outcomes have stagnated since the introduction of multi-agent chemotherapy in the 1970s. A key limitation in current preclinical research is the poor physiological relevance of traditional in vitro systems, which fail to reproduce the complex physical and cellular cues of the tumour microenvironment (TME). Three-dimensional (3D) culture models have begun to address this gap, but most still lack a critical component—physiological fluid flow—that profoundly influences immune cell trafficking, activation, and tumour–immune interactions.
In collaboration with Hooke Bio, we aim to develop a high-throughput system using the Mera platform—a microfluidic device with automated imaging and real-time monitoring—to analyse immunotherapy function and toxicity in OS. To test the impact of physiological flow, OS spheroids (MG63) were co-cultured with human peripheral blood mononuclear cells (PBMCs) at a 1:10 ratio. PBMCs were either activated or inactivated before co-culture and exposed to fluid shear using a rocker to simulate flow for seven days. Tumour destruction (spheroid size), immune activation (flow cytometry), cytokine release (ELISA) and immune infiltration (confocal imaging) were measured.
Our results demonstrate that PBMC activation is required to elicit anti-tumour responses in the absence of flow. Under flow conditions, activated PBMCs exhibited enhanced tumour cell killing and increased IL-6, IL-10 and IL-2 secretion by days 3–4. Furthermore, we observed increased T cell and macrophage activation, a higher CD4+/CD8+ ratio and upregulation of exhaustion markers, irrespective of PBMC activation status. Together, these findings demonstrate that incorporating physiological flow improves the realism and predictive power of preclinical immunotherapy models by better replicating immune cell infiltration and response within the TME.
From wood to regenerative medicine: softwood lignin-derived carbon dot characterization, in vitro stem cell response, and in vivo wound healing - SEMIT
Eli Christoph1, Isaac Cutshall2, Emine Berfu Ozmen3, Steven Newby1, David Harper4, Madhu Dhar1
1Tissue Engineering and Regenerative Medicine, Large Animal Clinical Sciences, College of Veterinary Medicine, Knoxville (Tennessee) - United States, 2Department of Mechanical, Aerospace, and Biomedical Engineering, Tickle College of Engineering, Knoxville (Tennessee) - United States, 3Genome Science and Technology, The Bredesen Center for Interdisciplinary Research and Graduate Education, Knoxville (Tennessee) - United States, 4Center for Renewable Carbon, Knoxville (Tennessee) - United States
Carbon dots (CDs) hold promise in a variety of biomedical applications, including regenerative medicine and wound healing. A prominent focus is synthesizing CDs from biobased precursors, such as lignin- an inexpensive byproduct of wood and pulp industries found in the tissues of woody plants. Herein, we build on our earlier studies that demonstrate cytocompatibility of softwood lignin-derived CDs (L-CDs) and explore the following hypothesis: based on material properties, doped and undoped L-CDs will invoke variable responses of human mesenchymal stem cells (hMSCs), including extracellular matrix (ECM) expression, and, hence, provide grounds for variable application. We synthesized L-CDs, undoped and doped with nitrogen (N@L-CDs), and deposited onto poly (lactic-co-glycolic acid)-based electrospun scaffolds. L-CD has an amorphous structure and sizes between 5-10 nm. Carboxyl, hydroxyl, and amine functional groups were identified. Both L-CD types exhibited negative zeta potential, with N@L-CD being slightly higher. Both L-CD types supported hMSC attachment and extensive filopodia networking. Immunofluorescence confirmed ECM expression on all scaffold types. CD31 was expressed by hMSCs seeded on both iterations of CDs, suggesting an important role in angiogenesis. Quantitative analysis showed significant increases in collagen type I expression of hMSCs on L-CD scaffolds, while fibronectin and collagen type III expressions were comparable across all samples. A mass-dependent cytotoxic effect was observed for N@L-CDs at higher coating densities, while undoped L-CDs remained cytocompatible. An in vivo pilot study of full thickness skin defect mouse model showed skin regeneration and healing in mice treated with both L-CDs after 14 days. Results indicate that L-CDs influence in vitro ECM expression and organization and may promote in vivo wound healing, providing promise for further translational applications.
CRISPR-functionalized 3D printed biomaterials for spatiotemporal gene editing
Joshua Graham1, Chiebuka Okpara1, Abolfazl Moghaddam1, Lesley Chow2, Tomas Gonzalez-Fernandez1
1Bioengineering. Lehigh University, Bethlehem (Pennsylvania) - United States, 2Bioengineering & Materials Science and Engineering. Lehigh University, Bethlehem (Pennsylvania) - United States
Non-viral CRISPR gene editing in vivo is limited by transient expression and rapid nanoparticle diffusion or accumulation, which reduce editing efficiencies, induce local inflammation, and increase off-target risks. In situ biomaterial-mediated CRISPR delivery can localize editing, but current strategies rely on non-specific substrate adsorption, offering poor control over CRISPR loading, release kinetics, and spatial patterning. To address these limitations, our objective is to engineer a CRISPR-functionalized 3D-printed platform for precise spatiotemporal control of gene editing.
We previously optimized the RALA cell-penetrating peptide system for delivering CRISPR RNAs and ribonucleoproteins for knock-out, knock-in, and activation. To control scaffold-mediated release of RALA-CRISPR nanoparticles, we used a solvent-casting technique to functionalize 3D-printed poly(caprolactone) (PCL) scaffolds with short chemical linkers. We employed the SnoopTag-SnoopCatcher bioconjugation system for rapid covalent binding, allowing RALA nanoparticles to be tethered to scaffolds via a Snoop-RALA construct. Circular dichroism confirmed alpha helicity, and dynamic light scattering was used to optimize nanoparticle size and charge.
PCL scaffolds were printed with SpyTag peptides which were tethered to Snoop-RALA nanoparticles via a SnoopCatcher-SpyCatcher fusion protein. Cy5-labeled nanoparticles were bound and characterized over one week: 60.7 ± 12.9% were released over 72 h before plateauing, and 37.6 ± 11.5% remained on the scaffold after one week. Mesenchymal stem cells seeded onto scaffolds loaded with nanoluciferase nanoparticles showed time-dependent transfection, with a maximal bioluminescent signal of 79.9 ± 15.8% compared to monolayer controls, though expression was not sustained beyond seven days.
Next, we will assess nanoparticle release kinetics and CRISPR editing efficiencies in the 3D printed system via transwell co-culture, and cellular infiltration. We hypothesize that sustained release of RALA nanoparticles through covalent tethering will prolong CRISPR activity, enhance editing efficiencies, and localize therapeutic outcomes.
References
1. Graham et al., Adv. Funct. Mater. (2025).
2. Veggiani et al., PNAS (2015).
3. Camacho et al., Biomater. Sci. (2019).
Hyaluronic acid-based hydrogel loaded with phloretin and triamcinolone acetonide-encapsulated PLGA nanoparticles for intraarticular osteoarthritis therapy
Natalia Izquierdo1, Marina Frutos2, Manuel Arruebo3, Gracia Mendoza4
1Departamento Ingeniería Química y Tecnologías del Medio Ambiente (Unizar). Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza - Spain, 2Departamento de Ingeniería Química y Tecnologías del Medio Ambiente (Unizar). Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza - Spain, 3Departamento de Ingeniería Química y Tecnologías del Medio Ambiente (Unizar). Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC and University of Zaragoza, Zaragoza, Aragon, 50018, Spain., Zaragoza - Spain, 4Aragon Institute for Health Research (IIS Aragon), Miguel Servet University Hospital, Zaragoza - Spain
Osteoarthritis (OA) remains difficult to treat due to persistent inflammation and progressive cartilage degradation, while current intraarticular (IA) treatments show short-lived efficacy [1], [2]. This study aims to develop and evaluate an injectable hyaluronic acid (HA) hydrogel incorporating PLGA nanoparticles (NPs) loaded with triamcinolone acetonide (TA), a synthetic corticosteroid, and phloretin (PHL), a natural compound, hypothesizing that a dual synergistic drug delivery would provide superior anti-inflammatory effects.
The HA-based hydrogel was synthesized by photopolymerization. NPs were prepared via single emulsion and characterized for size, ζ-potential and encapsulation efficiency. TA and PHL release was quantified by absorbance measurement. NPs were incorporated into the gel by dispersing them in the water used to dissolve HA. Rheology assessed IA injectability and stability. In vitro, IL-1β-stimulated chondrocytes were analyzed for inflammatory (iNOS, IL8, IL6) and cartilage catabolic markers (MMP-3, MMP-13) via qPCR. Statistical analysis used one-way ANOVA (p < 0.05).
Co-loaded TA and PHL NPs produced a significant reduction in NO levels produced by iNOS compared to single-drug controls confirming a synergistic effect. Embedding NPs in the HA hydrogel enabled sustained release and preserved hydrogel viscoelastic properties suitable for IA injection.
This smart nanosystem provides dual-acting, controlled drug delivery targeting both inflammation and cartilage matrix degradation. The approach here proposed shows strong potential to locally prolong therapeutic residence time and improve clinical outcomes in OA.
[1] Yao, Q., Wu, X., Tao, C., Gong, W., Chen, M., Qu, M., Zhong, Y., He, T., Chen, S., & Xiao, G. (2023). Osteoarthritis: Pathogenic signaling pathways and therapeutic targets. Signal Transduction and Targeted Therapy, 8(56). https://doi.org/10.1038/s41392-023-01330-w
[2] Han, Z., Wang, K., Ding, S., & Zhang, M. (2024). Cross-talk of inflammation and cellular senescence: A new insight into the occurrence and progression of osteoarthritis. Bone Research, 12, Article 69. https://doi.org/10.1038/s41413-024-00375-z
Cyclic mechanical stimulation of TDPCs improves tendon tissue maturation in vitro by enhanced collagen fibril formation
Philipp J. Thurner1, Ekaterina A. Oleinik1, Felix Gross1, Magdalena Fuchs1, Lune Teplitchi-Menou1, George Gourgi2, Büsra Kulekci2, Andreas H. Teuschl-Woller2
1Institute of Lightweight Design and Structural Biomechanics. TU Wien, Vienna (Wien) - Austria, 2University of Applied Sciences Technikum Wien, Vienna (Wien) - Austria
Background: Mechanical loading is a key regulator of tendon development, maintenance, and repair, and thus an essential parameter in tendon tissue engineering (TTE). Despite progress in in vitro TTE, a standardized tendon model is still lacking. Current strategies primarily employ either static or cyclic stimulation; static loading is technically simple but poorly mimics physiological strain, while cyclic loading better reproduces native mechanical cues yet requires specialized equipment. This study compared the effects of static (SL) and cyclic loading (CL) on tendon-derived progenitor cells (TDPCs) to identify optimal conditions for maintaining tenocyte function in TTE.
Methods: Rat TDPCs (4x106 cells/mL) were embedded in fibrin scaffolds (10 mg/mL fibrinogen, 0.625 KIU thrombin) and subjected to mechanical stimulation in a custom-made bioreactor (MagneTissue). We applied 10% uniaxial tensile strain for 6 hours daily, followed by 18-hour resting period (at zero position), over 7 consecutive days. Static (SL) and cyclic loading (CL) modes were compared. CL was applied with 0.5 Hz frequency throughout the stimulation period to replicate physiological conditions. Cell proliferation (EdU), survival (TUNEL), and morphology (Actin/DAPI) were evaluated. Gene expression was analysed by RNAseq (ONT) and RT-qPCR for extracellular matrix (ECM) and tendon-specific markers. Construct functionality was assessed by uniaxial tensile testing (Zwick), collagen deposition by MSB staining, and fibril formation by atomic force microscopy (AFM).
Results: CL induced pronounced cell alignment, along with higher cell proliferation and survival compared to SL. RNAseq identified two distinct transcriptomic clusters: CL upregulated tendon-specific and ECM-related genes (Scx, Tnc, Tnmd, Sparc, Col1a1, MMP3), indicating enhanced ECM remodelling and mature tendon phenotype, while SL upregulated inflammatory markers (Il6, Ccl2, Cxcl1/3). Histological and AFM analyses confirmed enhanced collagen deposition and fibril formation upon CL. Mechanical testing revealed a threefold higher tensile modulus in CL constructs, indicating superior structural integrity.
Conclusion: CL constructs significantly outperformed SL ones in functionality, ECM organization, and mechanics.
Funding by Austrian Science Fund FWF (DFH-28) and City of Vienna (SequenceTissue, MA23 #32-02) are gratefully acknowledged.
Pro-regenerative and antimicrobial degradable metallo-elastomer
Cole Latvis1, Mark Garren2, Nathaniel Wright1, Katelyn Ge1, Zhenyu Li1, Hanshuang Shao3, Hitesh Handa2, Elizabth Brisbois2, Simon Van Herck1, Alan Wells3, Yadong Wang1
1Meinig School of Biomedical Engineering. Cornell University, Ithaca (New York) - United States, 2School of Chemical, Materials and Biomedical Engineering. University of Georgia, Athens (Georgia) - United States, 3Department of Pathology. University of Pittsburgh, Pittsburgh (Pennsylvania) - United States
Copper is an essential micronutrient that is involved in various aspects of tissue regeneration, including angiogenesis, protein crosslinking, and reactive oxygen species (ROS) suppression. These properties are mediated by metalloenzymes, such as lysyl oxidase and superoxide dismutase, which contain copper ions coordinated by several imidazole rings. Cu-PIAS, a novel coordination-crosslinked degradable elastomer, mimics these active sites and, in turn, inherits some of the same catalytic bioactivity. Cu-PIAS exhibits broad spectrum anti-ROS activity against hydrogen peroxide, superoxide anions, and hydroxyl radicals, and frees nitric oxide from its endogenous reservoirs, both contributing to a reduction of inflammation and scarring in a wound or implant environment. The copper ions are tightly bound within and on the surface of the material, thus limiting the free copper ion concentration far below its toxicity limits and well within the micronutrient level. In vitro assessments reveal no cytotoxicity against mammalian cells, while mouse subcutaneous implantation studies reveal minimal inflammation and scarring induced by the material and potentially enhanced angiogenesis. In addition to anti-inflammatory activity, the copper ions also make Cu-PIAS antimicrobial, further enhancing its utility in healing of compromised wounds. Catalysis is a powerful tool in biological systems but is often overlooked in biomaterial design. By integrating bioinspired motifs here, we have created an actively pro-regenerative material by several mechanisms with broad potential in tissue regeneration.
Understanding fabrication variability in core-shell biomaterials using stochastic AI for in vitro BBB models
Maria Alexaki1, Lília M. S. Dias2, Marie Celine Lefevre1, Raquel Gonçalves3, Dinis O. Abranches3, Albano N. C. Neto2, Rute A. S. Ferreira2, Paulo S. B. André4, Attilio Marino1, Gianni Ciofani1, João F. Mano3, Mariana B. Oliveira3
1Institute of BioRobotics - SSSA. Istituto Italiano di Tecnologia, Pontedera (Toscana) - Italy, 2Department of Physics. CICECO-Aveiro Institute of Materials, Aveiro - Portugal, 3CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal, 4Department of Electrical and Computer Engineering. Instituto de Telecomunicações, Instituto Superior Técnico, Lisboa - Portugal
The development of physiologically relevant in vitro blood-brain barrier (BBB) models is essential for drug permeability and neurotherapeutic testing, requiring biomaterials that replicate the geometry, flexibility, and selective permeability of cerebral microvessels. In this work, soft tubular membranes were fabricated via polyelectrolyte complexation of alginate and ε-poly-L-lysine, providing compliant and self-standing constructs that better reflect the architecture of small brain vessels and support brain endothelial adhesion for barrier modeling. Comprehensive material characterization showed that key properties such as yield of production, permeability, porosity, thickness, opacity, and swelling ratio remained stable despite variations in fabrication and post-fabrication parameters. This consistency highlights the robustness and reproducibility of the method, supporting scalable and controlled tube fabrication. Endothelialization was observed along the luminal surface, and endothelialized constructs showed reduced permeability compared to non-cellular tubes, indicating early barrier-like behavior. An artificial intelligence (AI) framework based on Gaussian process modeling was used to uncover correlations between processing parameters and material performance. This data-driven analysis provides valuable insights for process optimization and prediction of material behavior. Overall, this study introduces a robust and tunable tubular biomaterial platform that better reproduces the geometry and mechanical compliance of brain microvessels for BBB modeling, and demonstrates how AI tools can enhance understanding of biomaterial fabrication.
Development and characterisation of a novel antibacterial drug delivery system for the management of bacterial vaginosis
Elzarie De Wet1, Shohreh Jafarinejed2, Adam Ward3, Antony Scimone2, Stephen Sikkink4, Michael J Raxworthy5, Anant Paradkar3, Mojgan Najafzadeh2, Farshid Sefat1
1Department of Biomedical Engineering. University of Bradford, Bradford (West Yorkshire) - United Kingdom, 2Faculty of Life Sciences. University of Bradford, Bradford (West Yorkshire) - United Kingdom, 3Centre for Pharmaceutical Engineering Sciences. University of Bradford, Bradford (West Yorkshire) - United Kingdom, 4Centre for Skin Sciences. University of Bradford, Bradford (West Yorkshire) - United Kingdom, 5Neotherix Ltd, York - United Kingdom
Bacterial vaginosis (BV) is a recurrent vaginal infection affecting 23 – 29% of women globally. A key species in the development of BV is Gardnerella vaginalis, which is associated with biofilm formation and antibiotic resistance. Standard treatments such as metronidazole and clindamycin demonstrate recurrence rates of 50 - 80% within six-twelve months. To address these limitations, this study explores the development of a novel antimicrobial agent and its encapsulation within β-cyclodextrin complexes as a basis for a targeted intravaginal delivery system.
Two β-cyclodextrin inclusion complexes (ICs) were synthesised by encapsulating two small-molecule bioactive compounds and characterised using nuclear magnetic resonance, Fourier-transform infrared spectroscopy and mass spectrometry to confirm complex formation. Physicochemical stability was evaluated using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Drug-release studies were conducted using reverse-phase high performance liquid chromatography at 282 and 322 nm, and release profiles of the raw compounds and ICs were compared. Antibacterial screening against Gardnerella vaginalis biofilms is underway. Standard curves were generated (n=3, r2 > 0.99); comparative release studies will be analysed by two-way ANOVA.
Spectroscopic and mass spectrometry analyses confirmed successful encapsulation. DSC demonstrated shifts in the parent drug thermal transition after complexation (IC 1: + 62°C; IC2: -105°C), and consistent with reduced crystallinity and encapsulation. TGA revealed β-cyclodextrin-associated moisture loss and enhanced high-temperature stability of the included agents. Initial release studies indicated a gradual, time-dependent release profile, and the small molecules showed strong antibacterial activity at non-cytotoxic concentrations. ICs are expected to enhance stability and potentially modulate antibacterial performance.
These findings demonstrate successful fabrication of β-cyclodextrin inclusive complexes with modified thermal behaviour and gradual release characteristics, forming the foundation for incorporation into electrospun or pessary-based intravaginal delivery systems, to evaluate local antibacterial performance and clinical potential in bacterial vaginosis management.
Acknowledgements: NBIC/BBSRC for funding and Neotherix for industrial support
Biofunctional supramolecular polymeric biomaterials for advanced regenerative therapies
João Borges
de Department of Chemistry. CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal
Nature provides us with a unique source of fascinating structures formed via non-covalent intermolecular interactions. Those include the molecular motor proteins, the cell membrane, the DNA double-helical structure or the native extracellular matrix (ECM) of tissues and organs which are formed by the supramolecular self-assembly of the fundamental building blocks of life, including saccharides, nucleobases, peptides, and their derivatives. Such complex and dynamic supramolecular landscapes have been boosting scientists to the supramolecular design and development of self-assembled biofunctional materials to emulate the structural complexity and functional dynamic nature of living systems and develop advanced regenerative therapies.
In this lecture emphasis will be given to the synergistic use of polysaccharides, nucleosides, peptides and/or proteins, and bottom-up approaches towards the supramolecular engineering of a library of multifunctional self-assembling nanobiomaterials for addressing a plethora of biomedical applications and fulfilling healthcare needs. In particular, the molecular design, synthesis and development, and physicochemical, mechanical and biological characterization of chemically programmable, dynamic and responsive soft supramolecular polymeric hydrogels, hydrogel-based bioinks and layer-by-layer nanobiomaterials will be presented. Moreover, their potential to be used as advanced vehicles for controlled drug/therapeutics/cell delivery, as 2D/3D cell culture platforms, and as bioinstructive matrices to elucidate cell-biomaterial interactions and stimulate cell-signaling pathways that are pivotal in tissue engineering and regenerative medicine will be discussed.
Acknowledgments: This work was funded by the European Union’s Horizon Europe research and innovation programme under grant agreement No. 101079482 (“SUPRALIFE”), and by FEDER - Fundo Europeu de Desenvolvimento Regional through the COMPETE 2030 in the framework of the project COMPETE2030-FEDER-00874400 (“SUPRANEURO”). This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UID/50011/2025 (DOI 10.54499/UID/50011/2025) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC). J. Borges gratefully acknowledges Fundação para a Ciência e a Tecnologia (FCT) for the individual Assistant Researcher contract (DOI 10.54499/2020.00758.CEECIND/CP1589/CT0007).
Smart theragenerative hydrogels: engineered living materials for bone regeneration
Maria Grazia Raucci1, Anna Mariano2, Federica Zuppardi3, Alessandra Soriente3, Ashkan Bigham3, Gomez D'ayala Giovanna3, Luigi Ambrosio3
1Institute of Polymers, Composites and Biomaterials. National Research Council (IPCB-CNR), Naples (Italia) - Italy, 2Institute of Polymers, Composites and Biomaterials. National Research Council (IPCB-CNR), Naples (Italia) - Italy, 3Institute for Polymers, Composites and Biomaterials. National Research Council (IPCB-CNR), Naples (Italia) - Italy
Within the framework of the Engineered Living Materials (ELM) and BIOACTION EU projects, this study aims to develop bioactive and responsive materials capable of guiding cellular behavior for enhanced bone regeneration. Engineered Living Materials represent an emerging class of biomaterials that integrate biological functionality into synthetic matrices, enabling dynamic and adaptive responses to the cellular environment. In this context, we focused on designing hybrid hydrogel-based systems with intrinsic regenerative and immunomodulatory properties.
Bone defects resulting from trauma, tumor resection, congenital malformations, or degenerative diseases remain a major clinical and socioeconomic challenge. Conventional grafting methods, such as autografts and allografts, are effective but constrained by donor site morbidity, immune rejection, and limited availability. Hydrogels have emerged as promising candidates for bone regeneration due to their biocompatibility, tunable mechanics, and extracellular matrix (ECM)-like porosity that supports cell survival and nutrient exchange. Their injectability and in situ gelation further enhance their suitability for irregular bone defects.
Here, hydrogels composed of Pluronic F127 and alginate were developed and bioactivated by incorporating hydroxyapatite (5–10 wt%) or bioactive glass (2.5–5 wt%), yielding nanocomposite formulations. Physicochemical properties were characterized by Raman spectroscopy and scanning electron microscopy. Biocompatibility was assessed using human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs). Osteogenic differentiation was evaluated through collagen I and osteocalcin expression and cytoskeletal organization in hMSCs. Anti-inflammatory and angiogenic effects were studied using RAW 264.7 macrophages and HUVECs, respectively.
The results demonstrate that bioactivated hydrogels are highly biocompatible and multifunctional, promoting bone regeneration, angiogenesis, and inflammation modulation—key objectives of the ELM and BIOACTION initiatives.
Acknowledgements: Funded by the European Union - BOOST EIC Booster Grant Programme - ELM portfolio. GA – BOOS_03_04 and HORIZON-EIC-2022-PATHFINDEROPEN-01 (GA 101098972): Bacteria Biofilm as bio-factory for tissue regeneration – BIOACTION.
Microfluidic synthesis of collagenase-encapsulated chitosan microparticles for breast cancer treatment
Martina La Rosa1, Gabriele Lo Buglio1, Gaia Pascolo2, Simona Campora1, Alessandra Lo Cicero1, Giulio Ghersi1, Francesco Lopresti1, Annalisa Tirella2, Vincenzo La Carrubba1
1Università Degli Studi di Palermo, PALERMO (Sicilia) - Italy, 2Univeristy of Trento, Trento (Italia) - Italy
Aim and objective
As a solid tumor, breast cancer is embedded in a complex extracellular matrix where type I collagen is overexpressed and associated with treatment failure and tumor aggression. Previous studies demonstrated that the proteolytic activity of collagenase could improve the permeability of tumors to therapeutic agents. However, its short serum half-life makes collagenase delivery extremely challenging in vivo, necessitating innovative delivery systems that can prolong its bioactivity. To overcome the rapid enzymatic degradation of collagenase, we developed a novel microfluidic platform for the fabrication of chitosan microparticles that enable controlled collagenase release.
Methodology
Collagenase-loaded chitosan microparticles were fabricated using a multidisciplinary approach that relied on the use of an innovative rapid prototyping technique for the fabrication of the microfluidic chip. Autodesk Fusion 360™ software was chosen to structure and slice the microfluidic chip into three layers, which were fabricated using polymethyl methacrylate sheets. The system employed a flow focusing junction, where the chitosan phase and oil-surfactant mixture were introduced through separate inlets, creating the conditions for droplet formation. A CO2 laser cutter enabled high-precision process of channel geometries, and the final product was obtained by hot pressing the layers. The chip's architecture and transparency also facilitated real-time monitoring of microparticle formation.
Results
The results confirmed that the chip geometry enabled the formation of monodisperse chitosan microparticles and allowed improved size uniformity compared to conventional methods. Rheological characterization determined the operating window for stable droplet formation, with shear-thinning behavior enabling predictable control over droplet size and formation rate. Extensive validation tests were conducted by modulating viscosity through flow velocity to evaluate the microparticle size. Tests conducted on the collagenase-embedded microparticles demonstrated preservation of enzyme activity and release profile.
Conclusions
These findings highlight an interesting microfluidic method for the synthesis of monodisperse particles with controllable size in the micrometre range.
Polypropylene nanoplastic exposure on a human airway barrier-on-chip and protein corona formation insights through multiscale analysis
Omur Sert1, Eyup Bilgi2, Reyhan Coban1, Pelin Saglam-Metiner1, Basar Dogan1, Zeynep Imir-Tekneci3, Suleyman Selim Cinaroglu1, Ozlem Goksel4, Ceyda Oksel Karakus2, Ozlem Yesil-Celiktas1
1Department of Bioengineering, Faculty of Engineering, Ege University, İzmir (Izmir) - Turkey, 2Department of Bioengineering, Faculty of Engineering, Izmir Institute of Technology, İzmir (Izmir) - Turkey, 3Department of Chemical Engineering, Faculty of Engineering, Ege University, Izmir, Turkey, İzmir (Izmir) - Turkey, 4Translational Pulmonary Research Center (EgeSAM), Ege University, İzmir (Izmir) - Turkey
Introduction
Inhaled micro- and nanoplastics have become a growing concern for human health. Among them, polypropylene (PP) nanoplastics stand out because of their extensive use and strong hydrophobicity, yet their effects on respiratory barriers remain unclear. To improve our understanding, we established a microfluidic airway barrier-on-chip model that mimics the physiological environment of the human lung and investigated how PP nanoplastics interact with the epithelial layer and blood proteins.
Methods
PP nanoplastics were synthesized and characterized by physicochemical methods. The particles were introduced into the epithelial barrier-on-chip under peristaltic perfusion to simulate physiological conditions. Barrier integrity and cellular responses were evaluated through electrical resistance, cytotoxicity, and immunostaining analyses. In parallel, protein corona formation was examined both experimentally and via molecular dynamics (MD) simulations to understand the molecular mechanisms.
Results
Preliminary data indicate that when introduced into the airway-on-chip, exposure caused a reduction in barrier integrity and tight-junction organization. As such, PP nanoplastics immediately interact with serum proteins, forming a stable corona that modifies their surface properties and biological behavior. Simulation results further supported the hydrophobicity-driven binding of human serum albumin onto the PP surface.
Conclusion
By combining experimental and computational approaches, this study investigates how hydrophobic nanoplastics interact with epithelial barriers and plasma proteins. These findings contribute to a better understanding of the mechanisms behind inhaled nanoplastic exposure and its potential health implications.
Keywords: polypropylene nanoplastics; organ-on-chip; protein corona; epithelial barrier; molecular dynamics
Acknowledgement: The funding provided by TUBITAK through 123M406 project is highly appreciated.
Harnessing machine learning to advance scaffold-guided neural tissue regeneration
Sing Yian Chew
de School of Chemistry, Chemical Engineering and Biotechnology. Nanyang Technological University, Singapore (Singapore (General)) - Singapore
Regeneration of the central nervous system (CNS) remains a formidable challenge due to the intricate interplay of biophysical and biochemical cues that govern cellular behavior and tissue repair. Our work focuses on understanding the role of the extracellular matrix (ECM) in this process and how host cells respond to engineered neural tissue constructs.
Building on the principles of ECM-mediated regulation, we have designed biofunctional scaffolds that recapitulate key structural and signaling features of the native microenvironment. These scaffolds are tailored for the delivery of nucleic acid therapeutics or human spinal cord progenitor cells, with the goal of enhancing neural regeneration and remyelination following CNS injury.
In this presentation, we shall share our latest advances in scaffold design and therapeutic integration, and demonstrate how machine learning approaches are being employed to analyze tissue regeneration outcomes in greater depth. By integrating machine learning-based analysis with biomaterial and cell therapy, we aim to develop more predictive and adaptable strategies for next-generation neuroregenerative therapies.
Understanding the forces that control cell fate and disease progression
Yongsung Hwang
de Department of Pathology, School of Medicine. Soonchunhyang University, Seoul (Seoul-tukpyolsi) - South Korea
Although the biomechanical properties of native extracellular matrix (ECM) and its contribution to the cell-cell and cell-matrix interactions play a critical role in maintaining tissue homeostasis and disease progression, an efficient method to understand these biological events during various fibrosis-associated disease progression is still a daunting task. Thus, in this talk, I will describe the dynamic role of cell-cell/cell-matrix interactions of various cells, which are cultured on biomaterials with varying matrix stiffnesses, in regulating disease progression by employing traction force microscopy, intracellular microscopy, and monolayer stress microscopy to understand the cell-generating forces and their subsequent contribution to the disease progression. Such a cell culture platform can offer novel strategies to understand the pathophysiology of various diseases.
Extended ex vivo preservation of human osteochondral tissue at ambient conditions through enhanced culture medium formulation
Leonardo Prieto1, William Cárdenas1, Adriana Lara1, Luz-Stella Correa1, Andrea Lizarazo1, Leidi Mendez2, Cristian Garcia2, Marco-Tulio Caicedo2, Gabriel Fletscher2, Jhon Bello2, Gustavo Salguero3, Ingrid Silva1
1Unidad de Ingeniería Tisular. Instituto Distrital de Ciencia Biotecnología e Innovación en Salud-IDCBIS, Bogotá D.C. (Cundinamarca) - Colombia, 2Banco Distrital de Tejidos. Instituto Distrital de Ciencia Biotecnología e Innovación en Salud-IDCBIS, Bogotá D.C. (Cundinamarca) - Colombia, 3Instituto Distrital de Ciencia Biotecnología e Innovación en Salud-IDCBIS, Bogotá D.C. (Cundinamarca) - Colombia
Preserving the structural integrity and cellular viability of human osteochondral tissues is essential for successful transplantation and functional cartilage repair. However, current preservation strategies rely heavily on refrigeration and cold-chain infrastructure, increasing logistical complexity and limiting widespread clinical access. Prolonging osteochondral allograft viability without refrigeration could simplify tissue banking processes and increase the availability of functional grafts. For this study, we used human femoral condyle sections from donors aged ≤45 years who were free of articular pathology and serologically non-reactive for HIV, HBV, HCV, syphilis, CMV, Chagas, and HTLV.
Tissue sections were preserved at room temperature for up to 42 days in two preservation media. Medium-1 contained amino acids, vitamins, minerals, plasma-derived proteins, and human growth factors. Medium-2 consisted of high-glucose DMEM supplemented with human growth factors, sodium pyruvate, and ascorbic acid. Chondrocyte viability measured via LIVE/DEAD fluorescence remained >98% at day 14, ∼97% at day 28, and >89% at day 42 in Medium-1. Medium-2 showed a marked decline (75%, 41%, and ∼40% at the respective time points). Histological evaluation (H&E, Masson’s trichrome, and Alcian Blue) demonstrated preservation of the osteochondral unit architecture in both media, including clear layer organization of the articular cartilage (superficial, mid, deep, and calcified zones), an intact tidemark, and underlying subchondral bone. However, samples preserved in Medium-1 showed higher lacunar occupancy, viable isogenous chondrocytes, and strong proteoglycan retention, evidenced by uniform Alcian Blue staining, compared to those preserved in Medium-2. Viable chondrocytes were still recoverable from Medium-1 at day 42, indicating sustained cell survival during extended storage.
These findings demonstrate that Medium-1 enables effective long-term room-temperature preservation of osteochondral tissue, outperforming DMEM-based conditions. This refrigeration-free strategy reduces reliance on cold-chain logistics. It improves tissue bank efficiency, facilitating broader clinical distribution of high-viability allografts for the treatment of focal cartilage defects and osteochondral injuries.
Urinary bladder matrix promotes tissue-engineered vascular graft remodeling through macrophage-mediated immunomodulation
Min Leon1, Oishi Mayumi1, Maestas David1, Wagner William1, Vande Geest Jonathan1
1Bioengineering. University of Pittsburgh, Pittsburgh (Pennsylvania) - United States
Coronary artery disease affects over 18 million people in the US, with nearly 400,000 bypass procedures performed annually. While saphenous vein remains standard for small-diameter bypass, its limited availability, invasive harvesting, and 40% failure rates have driven the development of tissue-engineered vascular grafts (TEVGs). Urinary bladder matrix (UBM), a decellularized extracellular matrix from porcine bladder, supports cell migration, proliferation, and promotes macrophage polarization toward an anti-inflammatory (M2) phenotype. Despite its clinical success in soft tissue repair, UBM’s application in TEVGs remains underexplored.
This study investigates how UBM influences TEVG remodeling and early host response. TEVGs were fabricated with an electrospun inner layer of PESBUU, an antithrombogenic polymer, with or without an outer UBM wrap. In vitro 3D culture system was used, culturing grafts in direct contact with explanted rat aortas (male Sprague Dawley rats, n=3) to assess UBM’s effect on cell migration and phenotype. Two-photon intravital microscopy evaluated metabolic activity weekly, and immunofluorescence (IF) assessed contractile (calponin) and synthetic (ICAM-1) vascular smooth muscle cell (VSMC) phenotype at 6-week endpoint. For in vivo evaluation, TEVGs were implanted as interpositional abdominal grafts in 6-week-old male rats for 8 days. Explants were analyzed by qRT-PCR for contractile VSMC (Acta2, Cnn1), macrophage (Cd68, Arg1), and endothelial (Cd31) gene expressions, using native aorta as baseline.
In vitro, cells migrated into UBM-wrapped TEVGs, showing increased metabolic activity by 2 weeks, earlier than in non-UBM grafts. IF revealed a mixed phenotype expressing both synthetic and contractile markers. Preliminary in vivo data suggest that Cd68 was similarly upregulated in both groups compared to the aorta, while Arg1, an M2-associated gene was markedly higher in UBM-wrapped than in non-UBM grafts. Contractile VSMC genes were strongly downregulated in both groups.
These findings highlight UBM’s potential to guide TEVG remodeling through immunomodulation. Ongoing studies include additional timepoints, replicates, and IF validation.
A 3D bioprinted neural tissue model using a novel photoinitiator-free, visible light-crosslinking hydrogel
Samaneh Eftekhari1, John S. Forsythe1, Vinh X. Truong2, Timothy F. Scott3, Helena C. Parkington4, Jessica E. Frith5
1Materials Science and Engineering. Monash University, Melbourne (Victoria) - Australia, 2Institute of Sustainability for Chemicals. Energy and Environment (ISCE2), Agency for Science, Technology and Research (A*STAR), Singapore (Singapore (General)) - Singapore, 3Chemical and Biological Engineering. Monash University, Melbourne (Victoria) - Australia, 4Department of Physiology, Biomedicine Discovery Institute. Monash University, Melbourne (Victoria) - Australia, 5Materials Science and Engineering. Monash University, Melbourne (Victoria) - Australia
Hydrogels that replicate the 3-dimensional (3D) microenvironment and mechanical features of native tissues play a key role in tissue engineering. Conventional photocurable hydrogels typically depend on small-molecule photoinitiators that generate free radicals under ultraviolet or visible light to trigger polymerization. However, such radical-based reactions can introduce oxidative stress and potential genetic damage to cells within the gel. To address these challenges, we developed a photoinitiator-free gelatin-based hydrogel (Gel-Pyr) functionalized with acrylamidylpyrene moieties, which undergoes visible-light-induced [2 + 2] cycloaddition to form crosslinked networks. The material was subsequently used to generate a 3D platform that mimics the sophisticated architecture of the brain cortex for neural network studies. Gel-Pyr demonstrated on-demand polymer network formation and tuneable mechanical properties by adjusting light exposure time, polymer concentration, and/or light intensity. It maintained structural integrity for over 30 days under cell culture conditions, confirming its suitability for long-term in vitro tissue modelling. Due to its strong shear-thinning properties, Gel-Pyr allowed smooth extrusion and accurate layer stacking during 3D bioprinting, producing well-defined, multilayered structures that promote nutrient diffusion. Gel-Pyr’s light-controlled step-growth polymerization allows its mechanical properties to be tuned to mimic different tissues. Embryonic (E18) rat cortical neurons and astrocytes were bioprinted in Gel-Pyr to form layered constructs and irradiated for 20 seconds, resulting in brain-like mechanical properties. These constructs supported neuronal cell viability and extended axonal connections, demonstrating its potential as a platform for neurodevelopmental and brain disease studies. Overall, this visible-light-activated hydrogel offers a gentle, cell compatible and printable platform for tissue engineering and advanced in vitro modelling.
Fiber-reinforced spider silk hydrogels
Christina Heinritz1, Thomas Scheibel1
1Chair of Biomaterials, Faculty of Engineering Science. University of Bayreuth, Bayreuth (Baden-Wberg Bayern) - Germany
We investigated how embedding fibers into soft hydrogels composed of recombinant spider silk proteins can improve their structural performance [1]. Hydrogels based on recombinant spider silk proteins represent a sustainable and scalable biomaterial [2] and are known for their biocompatibility and biodegradability [3]. However, if too high concentrated, low proliferative activity has been observed for cells inside these stable physically entangled networks. The use of low-concentrated silk hydrogels led to improved cell proliferation inside the scaffolds, but their mechanical weakness limits their application. Therefore, electrospun fiber meshes made of recombinant spider silk were introduced into the hydrogels using a layer-by-layer approach and analysed using scanning electron microscopy and rheological tests, resulting in significantly increased bulk stiffness and structural integrity without comprising their softness or biocompatibility. The improved mechanical properties make these materials promising candidates for applications in tissue engineering and regenerative medicine. This work contributes to the development of advanced biomaterial designs by combining the unique properties of spider silk with structural reinforcement strategies to overcome existing limitations.
References
[1] C. Heinritz and T. Scheibel, Macromolecular Rapid Communications 2025, e00475.
[2] D. Huemmerich, C. W. Helsen, S. Quedzuweit, J. Oschmann, R. Rudolph, T. Scheibel, Biochemistry 2004, 43.
[3] D. Steiner, S. Winkler, S. Heltmann-Meyer, V. T. Trossmann, T. Fey, T. Scheibel, R. E. Horch, A. Arkudas, Biofabrication 2021, 13.
Optimizing non-viral CRISPR delivery in MSCs by minimizing unintended transcriptomic and inflammatory effects
Josh Graham1, Alexander Arteaga1, Tomas Gonzalez-Fernandez1
1Bioengineering Department. Lehigh University, Bethlehem (Pennsylvania) - United States
CRISPR gene editing has revolutionized biomedical research by enabling precise control of gene expression for knock-out, transgene insertion, and epigenetic regulation. However, clinical translation is limited by the challenge of safely and efficiently delivering CRISPR components to target cells. Viral vectors provide strong expression but risk immune responses and insertional mutagenesis. Non-viral approaches—including electroporation, lipid, polymeric, or peptide-based nanoparticles—offer safer alternatives but vary in efficiency, toxicity, and impact on cell phenotypes.
To optimize CRISPR delivery for mesenchymal stem cell (MSC) therapies, we systematically characterized the transcriptomic and phenotypic effects of different delivery systems and molecular formats. MSCs were transfected with eGFP or Cas9 mRNA using lipofectamine, polyethyleneimine (PEI), electroporation, or the RALA cell-penetrating peptide. RNA sequencing via the kallisto-sleuth pipeline revealed that RALA and electroporation induced significantly fewer differentially expressed genes (DEGs, 1,243 and 2,485) compared to lipofectamine and PEI (8,570 and 5,973). To assess inflammatory impact, transfected MSCs were co-cultured with THP-1 macrophages. RALA and electroporation minimized pro-inflammatory M1 markers (CD86, TNF-α), while no groups altered pro-regenerative M2 markers (CD206, IL-10).
To determine optimal CRISPR molecular format, MSCs were transfected with Cas9 and guide RNA targeting the AAVS1 safe-harbor locus in plasmid DNA (pDNA), RNA, or ribonucleic protein (RNP) formats. pDNA caused the most transcriptomic changes, upregulating 352 genes common to RALA and electroporation, primarily involved in cytokine and interferon signaling. Combined with inferior editing efficiencies of pDNA, we conclude that Cas9 delivery in RNA or RNP formats must be considered for MSC gene editing strategies.
These findings demonstrate that CRISPR delivery reagents and molecular format critically influence MSC transcriptomic profiles and immunomodulatory function. Minimizing off-target dysregulation is essential to preserve self-renewal and multipotency and maximize therapeutic potential. Strategic selection of non-viral delivery systems and molecular formats will be crucial for advancing CRISPR-based therapies in regenerative medicine.
Heat-regulated human hepatoma culture system with enhanced hepatic function for drug response evaluation
Nana Shirakigawa1, Masato Shimoyama1, Silas Habimana1, Yoshinori Kawabe1, Masamichi Kamihira1
1Department of Chemical Engineering. Kyushu university, Fukuoka - Japan
[Introduction]
In drug discovery, the establishment of an in vitro toxicity evaluation platform capable of predicting human hepatotoxicity is highly desirable. Although animal experiments are widely used, they have inherent limitations due to interspecies differences and ethical concerns, thus, the development of human cell-based liver models is needed. We have been developing hi-Hep cells—a genetically engineered human hepatoma cell line whose liver function can be induced by heat treatment. In this study, we investigated whether embedding hi-Hep cells in type I collagen gels enhances hepatic function. Furthermore, we encapsulated hi-Hep cells in collagen–alginate mixed gel beads and evaluated their liver functions.
[Methods]
hi-Hep cells were embedded in type I collagen gels, followed by heat treatment the next day. After 3 days, the ammonia metabolism and albumin secretion rates were measured. For comparison, cells cultured on collagen-coated plates and cells embedded in collagen gels after heat treatment were similarly evaluated. In addition, hi-Hep cells were encapsulated in collagen–alginate mixed gel beads at various mixing ratios. After heat treatment followed by 3 day-culture, hepatic functions were assessed.
[Results and Discussion]
Hepatic functions were higher in cells heat-treated after embedding in collagen gels compared with those embedded after heat treatment, suggesting that heat-treated cells became more sensitive to physical stress. hi-Hep cells cultured in collagen gels showed higher albumin secretion than monolayer cultures. Moreover, hi-Hep cells encapsulated in collagen–alginate beads exhibited increased albumin synthesis and ammonia removal rates with increasing collagen content, indicating the beneficial role of collagen. These results indicate that collagen-based three-dimensional culture enhances hepatic function in hi-Hep cells. Optimization of alginate concentration and cell density will allow the development a simple and scalable platform for drug testing and bioartificial liver applications.
Acknowledgements:
This work was supported by JSPS KAKENHI Grant Number JP24K08174.
Critical geometric attributes of titanium lattices for enhanced osteointegration in mandibular reconstruction
Reza Sanaei1, Ayda Farhoudi1, Babatunde Ayodele1, Helen Davies1, George Dimitroulis2, Alastair Sloan3, Charles Pagel1
1Melbourne Veterinary School, Faculty of Science. The University of Melbourne, Parkville (Victoria) - Australia, 2MAXONIQ Pty Ltd, Melbourne (Victoria) - Australia, 3Melbourne Dental School, Faculty of Medicine, Dentistry and Health Sciences. The University of Melbourne, Parkville (Victoria) - Australia
Endoprosthetic reconstruction following jaw resection surgery is essential in avoiding autografts. Whilst still not fully adopted in practice, the recommended pore size to enhance osteoblastic penetration into metallic implants is said to be 400–600 μm. However, such pore sizes result in similarly-sized ingrown bony spicules, rendering osteointegration insufficient for large implants. Pore sizes of millimetres are more suitable, effectively converting the implant into a lattice. This work aimed to identify the critical geometric attributes of such lattices to achieve maximum osteointegration.
A fit-for-purpose surgical approach to the mandibular ramus in sheep was developed through cadaver experiments. Five Merino sheep were used. Each animal received four titanium implants (MAXONIQ, Australia), two per side, each bearing a unique design. Animals were intravitally labelled using three different fluorochromes at two-week intervals. Implants were retrieved 24 weeks postoperatively. MicroCT and histomorphometry were used for evaluation. An in vitro cell culture model was subsequently developed to reproduce results and evaluate novel lattice designs.
Repeated measures ANOVA using microCT data indicated that larger lattice units with larger pores promoted better osteointegration (Partial η2 = 0.84, F = 20.42, p < 0.001). Dynamic histomorphometry of intravital labels revealed that, once formed, the ingrown bone in all of the lattices had similar ‘mineralising surfaces’, ‘mineral apposition rate’ and ‘bone formation rate’. In vitro, various experimental variables were adjusted to reproduce in vivo results without success. Notably, one experimental setup using 3D cocultures of endothelial-mesenchymal cells ranked lattice structures in reverse order – poorer performing lattices in vitro performed better in vivo. Our in vivo results indicate that lattice shape and porosity are important predictors of operative success. Critically, our work suggests that utilising in vitro models to replace or expand in vivo assessment of biomaterials requires extensive experimental refinement and alternative analytical methods for effective interpretation.
Stepwise subcutaneous injection as an in vivo bioreactor strategy for time-sequenced vascularized skeletal muscle regneration
Pin-Hsun Chiu1, Jun-Zhi Dai1, Shih-Yen Wei2, Ying-Chieh Chen1
1Department of Materials Science and Engineering. National Tsing Hua University, Hsinchu - Taiwan, 2Department of cardiac surgery, boston children’s hospital. Harvard Medical School, Boston (Massachusetts) - United States
While previous studies have advanced vascularized or functional skeletal muscle constructs, most rely on perfusion bioreactors, pre-formed scaffolds, pedicled flaps, or long implantation periods, and seldom achieve millimeter-scale thickness with uniform vascularization and mature myofibers under simple subcutaneous conditions.
Here, we introduce a biomimetic, stepwise subcutaneous injection strategy that alternately delivers blood-vessel-forming (human umbilical vein endothelial cells (HUVECs) and white adipose tissue–derived mesenchymal stem cells (MSCs)) and induced pluripotent stem cell (iPSC)-derived myoblast–laden collagen hydrogels, using the host as an in vivo bioreactor to emulate sequential vascular–muscular development. The subcutaneous niche provides a perfused and controllable environment for prevascularization and monitoring prior to transplantation, thereby reducing ischemic failure risks of direct in situ treatments. In vitro, multiple injections at two-day intervals enhanced cell spreading (∼25%) and maintained high viability (∼90%) compared with single bulk formation. In vivo, sequential subcutaneous layering (200 µL every four days, total ≈ 800 µL) generated mature and stable microvascular networks (∼50 vessels mm−2, ∼60% smooth-muscle coverage, and ∼95% host vessel replacement) throughout 3.5-mm-thick, 10-mm-diameter hydrogels within 12 days. Subsequent myoblast delivery yielded uniformly distributed, mature myofibers (myosin heavy chain positive (MF20)+ encircled by laminin) with enhanced differentiation.
This study establishes a novel in vivo bioreactor–based approach that mimics the natural sequence of vascular and myogenic development through temporally controlled subcutaneous injections. Although ectopic assembly in subcutaneous space is challenging due to its relatively low vascular density and limited perfusion, its immune-privileged and minimally invasive nature provides a controllable niche for early cell survival, vascular maturation, and host-vessel integration; matured constructs can then be reimplanted orthotopically to reduce immune mismatch and accelerate repair. Unlike previous bioreactor- or scaffold-dependent systems, this stepwise platform enables thick, uniformly vascularized muscle formation under simple physiological conditions, providing a translatable route toward large-volume muscle regeneration.
Biofabrication and preclinical testing of mechanically functional meniscal grafts engineered using meniscus progenitor cell-derived microtissue
Kaoutar Chattahy1, Gabriela S. Kronemberger1, Aliaa S. Karam1, Pieter Brama2, Daniel J. Kelly1
1Mechanical, Manufacturing and Biomedical Engineering. Trinity College Dublin, Dublin - Ireland, 2School of Veterinary Medicine. University College Dublin, Dublin - Ireland
Meniscal injuries remain challenging to repair, as current treatments rarely restore the native tissue’s load-bearing capacity. Functional regeneration requires grafts that recapitulate the meniscus’s mechanical and structural integrity. This study aimed to engineer mechanically functional scaled-up meniscal grafts using meniscus progenitor cell (MPC) derived microtissues (μTs) and test their regenerative capacity in a large animal model of meniscal repair.
Meniscus (mECM) and tendon (tECM) were decellularized to generate solubilized ECM. Alginate microgels (∼200 µm) were produced by electrospraying. MPC μTs were preconditioned with BMP9 for 4 days and mixed with each of the following bulking agents (1:1 v/v): microgels, mECM, tECM, type I collagen, or a composite system (μTs: μgels: type I collagen at 2:1:1 ratio). Scaled-up constructs were engineered in 3 × 5 mm cylindrical moulds, evaluated by live/dead staining, compression testing, histology, and biochemical assays. The composite constructs were sutured into cylindrical defects (3 mm diameter) in the outer region of caprine menisci.
mECM, tECM, and type I collagen formed stable gels within 30 min. In the composite system, μTs contracted by day 3 and subsequently expanded to fill the mould, unlike the other groups that contracted. Live/dead staining showed high cell viability across groups. Biochemical quantification revealed higher collagen content and collagen/sGAG ratio in tECM and mECM groups. Macroscopically, all constructs retained their cylindrical shape, with only the composite and type I collagen constructs expanding to ∼5 mm height. The compressive modulus of these constructs was higher, approaching 600 kPa. Based on these findings, the composite constructs were implanted into cylindrical meniscal defects (analysis of this in vivo study is ongoing).
The composite system supported μT fusion, high cell viability, fibrocartilaginous matrix deposition, and the development of a geometrically defined, mechanically functional meniscal graft, demonstrating its suitability for translational meniscus repair.
Advanced Sr-HT-Gahnite bioceramic scaffolds: Integrating 3D printing, microstructural modelling, and machine learning toward functional bone regeneration
Haobo Guo1, Marcin Kotlarz2, Philipp Fisch2, Anna Puiggali-Jou2, Zufu Lu1, Marcy Zenobi-Wong2, Hala Zreiqat1
1Biomedical Engineering School. University of Sydney, Sydney (Australian Capital Territory) - Australia, 2Department of Health Sciences & Technology. ETH Zurich, Zurich - Switzerland
The creation of bioceramic scaffolds that combine mechanical robustness, interconnected porosity, and biological functionality remains central to bone tissue engineering. Our developed Sr-HT-Gahnite; an emerging bioactive ceramic, offers superior strength, controlled ionic release, and high osteoconductivity compared with conventional clinical bioceramics. This study presents an integrated digital–experimental approach combining 3D printing, microstructural analysis, and machine learning to design Sr-HT-Gahnite scaffolds for optimal bone regeneration.
We developed an integrated workflow that converts micro-CT scans of bone into 3D models that accurately reproduce trabecular architecture and porosity. The pipeline first preprocesses the raw images to minimize artifacts, segments bone and pore regions, and aligns the results along a normalized depth axis from the cartilage surface to the trabecular interior. From these processed datasets, depth-dependent structural features such as local porosity and curvature are extracted. Machine-learning models trained on these structural descriptors capture geometric and connective trends across depth, which are then used to guide the design of new scaffolds and vascular networks. The optimized scaffold was fabricated using an Sr-HT-Gahnite ceramic slurry through LCD-based 3D printing. After sintering, the constructs exhibited dense microstructures with well-interconnected pores. This workflow establishes a data-driven bridge between imaging, machine learning, and fabrication, enabling reproducible design of biomimetic scaffolds for bone regeneration.
Direct and indirect osteogenesis by bone-resident MSCs during ageing is epigenetically regulated by the pioneering transcription factor FOXO1 - SEMIT
George Soultoukis1, Marina Leer2, Mareen Storbeck3, Katharina Schmidt-Bleek3, Tim Schulz2
1German Institute of Human Nutrition, Nuthetal (Brandenburg) - Germany, 2Adipocyte Development and Nutrition. German Institute of Human Nutrition, Nuthetal (Brandenburg) - Germany, 3Julius Wolff Institute, Berlin Institute of Health at Charité. Charite Universitatsmedizin, Berlin - Germany
Introduction: Direct and indirect ossification are two distinct but critical aspects of bone homeostasis during tissue growth and regeneration following injury. Osteogenesis declines as a result of ageing, which is associated with osteoporosis, increased fracture risk, marrow fat accumulation, impaired haematopoiesis and compromised immunoregulation. Despite progress in this field of research, the conditions of lineage specialization of mesenchymal stromal cells (MSCs) towards an osteochondrogenic vs. an adipogenic fate remain elusive. This study aims to characterize the age-related alterations in discrete cell states and the regulatory mechanisms modulating these events and contribute to impaired bone maintenance.
Material & Methods: Single cell-based transcriptomic and epigenetic approaches are used to characterize MSC subset heterogeneity as a function of aging. To map MSC subset distribution throughout the bone, spatially resolved bulk transcriptomic analyses are used in combination with computational methods for cell type deconvolution and histology approaches.
Results: We observe an age-related decline in specialized MSCs with osteo-chondrogenic differentiation potential (ocMSCs) while MSC subsets with an adipogenic differentiation potential expand. Using the surface protein CD200 as marker of this cellular subset, this depletion is confirmed to localize to the epiosteal areas of mineralized bone and the growth plates. Epigenetic analyses of chromatin structure reveal a pro-osteochondrogenic molecular signalling network that depends on the molecular interaction of MSCs with native, non-mineralized type-1 collagen which is mediated by the (pioneer) transcription factors FOXO1 and JUN. Specially, in vitro exposure of multipotent MSCs to type-1 collagen promotes osteogenic differentiation and inhibits adipogenic differentiation.
Conclusions: We report that the age-dependent disruption of collagen-I homeostasis depletes microanatomical regions conducive to an ERK-dependent FOXO1-mediated osteogenic commitment that in young bones occurs through pro-osteogenic transcription regulation of Runx2, Col1a1, and chromatin remodeling.