Abstracts TERMIS-EU Palma de Mallorca April 20–24,2026

850. Adenosine-blended electrospun scaffolds for antioxidant and immune modulation in radiation-induced hypothyroidism

Maria Heim¹, Ella-Louise Handley², Daniel Grant³, Lizi Hegarty³, Elaine Emmerson³, Anthony Callanan²

¹Institute for Bioengineering & Centre for Regenerative Medicine. University of Edinburgh, Edinburgh (Edinburgh, City of) - United Kingdom, ²Institute for Bioengineering. University of Edinburgh, Edinburgh (Edinburgh, City of) - United Kingdom, ³Centre for Regenerative Medicine. University of Edinburgh, Edinburgh (Edinburgh, City of) - United Kingdom

Aim & Objective:

Up to 92% of patients suffer radiation-induced hypothyroidism (RIHT) following head and neck cancer radiotherapy (1). Current treatments address hormonal deficiency through thyroid hormone replacement but do not target the underlying tissue injury, leaving many patients with persistent symptoms and progressive gland dysfunction (2). This study aimed to develop an adenosine-blended scaffold to promote thyroid regeneration post radiation injury, informed by in vivo findings of oxidative and immune dysregulation, and validated through in vitro studies.

Materials & Methods:

Tissue dysfunction, including reactive oxygen species, fibrosis-associated deposition, and macrophage alterations, were evaluated through flow cytometry and immunofluorescent staining in a murine model of RIHT to define therapeutic targets. Subsequently, electrospun polycaprolactone scaffolds blended with 0.5–3% adenosine were fabricated, providing localized bioactivity. Scaffold morphology, mechanical integrity and release kinetics were assessed. In vitro assays were conducted using Nthy-ori 3-1 thyrocytes and THP-1–derived macrophages under irradiated and non-irradiated conditions, assessing protein and gene expression. Statistical analyses were performed on all data.

Results:

Irradiation of murine thyroid induced oxidative stress, increased fibrotic marker expression and macrophage remodelling. The 1% adenosine scaffolds supported enhanced thyrocyte proliferation, epithelial cohesion, expression of thyroid-specific and increased antioxidant enzyme expression, while reducing indicators of senescence and apoptosis. In macrophages, adenosine scaffolds promoted pro-reparative markers while suppressing pro-inflammatory markers.

Conclusion:

This study demonstrated that adenosine-blended polycaprolactone scaffolds provide a dual-action approach for addressing oxidative stress and immune dysregulation in RIHT. With further optimization, this approach could offer a single-intervention, biomaterial-based therapy to re-establishing thyroid function through targeted immune modulation, reducing the lifelong dependence on hormone replacement therapies.

Acknowledgements:

Funding: UKRI EPSRC (EP/T517884/1), UKRI MRC (MR/L012766/1, MR/X018733/1) and the Wellcome Trust Institutional Strategic Support Fund (ISSF).

References

1. Zhou et al., J Cancer, 12(2):451, 2021

2. Heim et al., Front Endo, 13, 2022

851. Deep learning enables detection of early chondrocyte degeneration beyond human annotation capability

Tonia Mack¹, Chengqiang Wang¹, Takeru Shiina², Kenjiro Tanaka², Ryuji Kato², Melanie L. Hart¹, Bernd Rolauffs¹

¹G.E.R.N. Research Center for Tissue Replacement, Regeneration & Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center. University of Freiburg, Medical Center, Freiburg (Baden-Wberg Bayern) - Germany, ²Laboratory of Cell- & Molecular Bioengineering, Division of Bioscience, Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences. Nagoya University, Nagoya (Aichi) - Japan

Objectives

Cartilage degeneration begins at the cellular level, but clinical imaging detects osteoarthritis only after structural damage appears. Early changes in chondrocyte clustering are difficult to quantify because segmentation models often fail in organizationally complex regions where human ground truth is impossible. We evaluated whether deep-learning models can segment chondrocytes across increasing organizational complexity, including cases where human annotations cannot be generated—critical for regenerative medicine.

Methodology

Isolated chondrocyte images (n=5 donors) were assigned to organizational grades: Grade-1 (single cells), Grade-2 (small clusters), Grade-3 (large non-overlapping clusters), and Grade-4 (overlapping clusters). Human ground truth masks were generated semi-automatically. Deep-learning CellPose Cyto3 models were trained on grade-specific subsets and evaluated using Intersection over Union (IoU; how predicted segmentation overlaps with ground truth), Average Precision at IoU ≥ 0.50 (AP@50), F1-score (harmonic mean of precision and recall), and Panoptic Quality (PQ), which jointly assess detection and segmentation accuracy.

Results

Performance varied with training-set complexity. The Grade-3 model achieved the highest accuracy (F1 = 0.95, IoU = 0.84, PQ = 0.84, AP@50 = 0.93). The Grade-1–3 model performed similarly (F1 = 0.92, IoU = 0.81, PQ = 0.82, AP@50 = 0.90). Including Grade-4 images—where human ground truth was not achievable but the model still produced plausible segmentations—reduced performance (F1 = 0.84, PQ = 0.76), despite high IoU (0.90) and AP@50 (0.91). Models trained only on low-complexity grades performed worst. Overall, intermediate-complexity training (Grade-3 or Grades 1–3) yielded the best generalization, while adding Grade-4 data was feasible but introduced a measurable cost.

Conclusion

Segmentation performance depended critically on training image complexity. Models trained on intermediate-complexity achieved the most accurate segmentation. Incorporating complex Grade-4 images—without human ground truth—was technically feasible but introduced a measurable performance cost. Nevertheless, this highlights AI’s potential to quantify early degenerative changes beyond human annotation capability.

852. Rheology-based laser printing of biomaterials and cells towards cartilage-mimetic architecture biofabrication

Stavroula Elezoglou¹, Antonis Hatziapostolou², Kyriakos Giannakopoulos², Chrysoula Chandrinou², Ioanna Zergioti²

¹Physics. National Technical University of Athens, Athens (Attiki) - Greece, ²Physics. National and Technical University of Athens, Athens (Attiki) - Greece

Laser-Assisted Bioprinting is an emerging promising technique for precise tissue fabrication due to its exceptional spatial resolution, non-contact operation, and compatibility with biomaterials. Laser-Induced Forward Transfer (LIFT) has gained attention for its ability to deposit living cells with remarkable accuracy (1,2). A pulsed laser is focused onto a thin sacrificial layer, which absorbs the energy and generates a rapidly expanding cavitation bubble, which propels the bioink onto a receiver substrate, enabling digitally controlled, high-precision deposition with spatial resolution of ∼10 μm, cell viability exceeding 95%, and jetting velocities approaching ∼30 m/s. Despite these capabilities, the complex interaction between laser-induced jet dynamics and bioink rheology is not fully understood, limiting predictive control over the printing process.

In this study, jet formation for both Newtonian and non-Newtonian bioinks is investigated, across a broad viscosity spectrum under nanosecond-pulsed LIFT. We introduce a depth-resolved cell-printing strategy for cartilage-mimetic constructs. Utilizing high-speed shadowgraphy combined with a dual LED flash lamp, we achieved 200 ns temporal resolution, allowing the visualization of jet evolution during the first microseconds and identification of two distinct jetting regimes. For low-viscosity bioinks, dual sequential jets are consistently observed, independent of rheology. At higher viscosities, Newtonian inks transition to a single-jet regime, whereas shear-thinning inks retain dual jets. This occurs because transient laser-induced shear reduces the apparent viscosity of non-Newtonian materials, permitting secondary jet formation even at elevated nominal viscosities.

Finally, we demonstrate tunable deposition of mesenchymal stromal cells (MSCs) within collagen-based and other extracellular matrix hydrogels by adjusting laser fluence. Cells are immobilized reproducibly at predefined depths, verified via confocal microscopy, enabling the fabrication of physiologically relevant 3D constructs. This approach paves the way for engineering cartilage-inspired grafts and ex vivo tissue phantoms for regeneration studies.

1) S. Elezoglou et al. https://doi.org/10.36922/IJB025100082

2) Chliara, M.A.; Elezoglou, S.; Zergioti https://doi.org/10.3390/bios12121135

853. Porous and printable: cell-remodelable granular PVA-based hydrogels for tissue engineering and biofabrication

Chrissie I. M. Baltzaki^{1 3}, Jonida Bushi^{1 3}, Julia Fernández-Pérez^{2 3}, Stefan Baudis^{1 3}

¹Christian Doppler Laboratory for Advanced Polymers for Biomaterials and 3D Printing, Technische Universität Wien, Austria; Institute of Applied Synthetic Chemistry, TU Wien, Austria, ²3D Printing and Biofabrication Group, Institute of Materials Science and Technology, Technische Universität Wien, Vienna, Austria, ³Austrian Cluster for Tissue Regeneration

Granular hydrogels combine increased surface area, modularity and porosity, making them attractive platforms for bioprinting and tissue engineering.1 In this work, a norbornene-functionalised polyvinyl alcohol2 (PVA-NB) was used as the hydrogel precursor to produce bulk and granular gels containing either cell-cleavable or non-cleavable crosslinkers. PVA-NB was rapidly (<60 s) crosslinked under UV-light in presence of Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and either a dithiolated MMP-sensitive peptide or a dithiolated polyethylene glycol (HS-PEG-SH). Initially, the reactivity, swelling, degradation and mechanical properties of the bulk hydrogels were investigated acellularly. Both matrices had similar reactivity, stability and swelling behaviour, with a storage modulus of ∼2 kPa. Enzymatic degradation showed complete degradation of the PVA-MMP hydrogels within 24 hours, whereas the non-cleavable PVA-PEG hydrogels only degraded 20% after 5 days, confirming tunable matrix degradability. Adult dermal fibroblasts were encapsulated in bulk gels to enable observation of the cell-matrix interactions. Viability after 2 weeks remained high, suggesting high biocompatibility. Increased cell protrusions and different morphologies were observed in PVA-MMP hydrogels, whereas cells remained round in PVA-PEG, underscoring the impact of degradability in cell spreading. Gels were fragmented into granular particles and, after packing, underwent a secondary crosslinking in different combinations of the above crosslinkers. Fibroblasts seeded on top migrated throughout the entire granular scaffold and adhered to the particle surfaces, showing high cellular accessibility. Moreover, volumetric strand dispensing of the granular hydrogels was optimised in terms of printing speed and extrusion speed to assess printability. Strand continuity and shape consistency suggested high printing fidelity. This versatile material is suited for clinical applications, providing a dynamic environment for tissue regeneration.

Acknowledgements

Christian Doppler Research Association, Austrian Federal Ministry of Economy, Energy & Tourism, National foundation for Research, Technology & Development.

References

1. A. C. Daly,Adv. Healthcare Mater. 2024, 13, 2301388.

2. X-H. Qin, Adv. Funct. Mater. 2015, 25, 6606–6617.

854. Reversible thermo-shrinkable hydrogels for dynamic stimulation of encapsulated cartilage progenitor cells in cartilage tissue engineering

Antonia G. Vasilopoulou¹, Sanne M. Van De Looij², Greta Di Marco², Ilenia Ciccarelli², Sylvia M. Mihăilă³, Jasmijn V. Korpershoek¹, Bas G.p. Van Ravensteijn², Jos Malda¹, Tina Vermonden²

¹Department of Orthopedics, University Medical Center Utrecht, Utrecht - The Netherlands, ²Division of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences. Utrecht University, Utrecht - The Netherlands, ³Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences. Utrecht University, Utrecht - The Netherlands

Chondrocytes depend on mechanical loading to activate pathways driving chondrogenesis and extracellular matrix (ECM) production. Dynamic mechanical stimulation of 3D cell cultures may therefore enhance in vitro tissue development. This study introduces a novel approach to dynamically stimulate encapsulated articular cartilage progenitor cells (ACPCs) using a thermo-shrinkable hydrogel with intrinsic, reversible shrinking-swelling properties.

The thermo-responsive polymer with methacrylate groups (Gel-NHMA) was synthesized by grafting chains of N-isopropylacrylamide (NIPAM) and 2-hydroxylethyl acrylate (HEA) onto a gelatin backbone. Hydrogels formed from this polymer exhibited temperature-dependent volume changes. ACPCs were encapsulated and assessed for viability across different polymer formulations using live/dead, alamarBlue, and lactate dehydrogenase assays. Swelling–shrinking cycle regimens (1x, 2x, or 5x weekly) were applied to assess how stimulation frequency influences hydrogel properties, cell metabolic activity, and chondrogenesis after 29 days in chondrogenic culture, compared to static controls.

The shrinking extent and gel stability were tuned by adjusting the NIPAM/HEA chain lengths and the degree of methacrylation, respectively, yielding a hydrogel that shrinks up to 65% in volume at 37°C. Encapsulated ACPCs showed high viability on days 1, 4, and 7, with no signs of cellular stress upon dynamic stimulation. No differences in glycosaminoglycan content were observed among the tested regimens or compared to static culture. Histological analysis confirmed proteoglycan and collagen deposition across all groups. Hydrogel shrinkage extent progressively decreased over time (from 30% on day 1 to 15% by day 29) and partially lost reversibility in the presence of cells.

Gel-NHMA hydrogels support cell encapsulation and reversible swelling–shrinking cycles, which were tolerated by the ACPCs and supported differentiation. Stimulation regimens did not significantly alter ECM deposition compared to static culture, although larger sample sizes and further characterization of ECM components are required for proper comparison.

Acknowledgments: Supported by LS-NeoCarE (NWA.1389.20.192) and the Dutch Research Council (NWO/VICI 18673).

855. Can spinal fusion for the elderly be improved? BMP2, L51P, and the EP4 agonist KMN159: synergistic osteogenesis in human annulus fibrosus cells

Benjamin Gantenbein¹, Shuimu Chen¹, Xinggui Tian², Laura González Blanco³, Oreste Gualillo⁴, Stefan Zwingenberger², Christoph E. Albers⁵, Sonja Häckel⁵

¹Tissue Engineering for Orthopeadics & Mechanobiology, Bone & Joint Program, Department for BioMedical Research (DBMR). Medical Faculty, University of Bern, Bern - Switzerland, ²Center for Orthopaedic, Trauma Surgery and Rehabilitation Medicine. University Medicine Greifswald, Greifswald (Mecklenburg-Vorpommern) - Germany, ³IDIS: Instituto de Investigación Sanitaria de Santiago C027 Group NEIRID. Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela (A Coruña) - Spain, ⁴SERGAS, Área Sanitaria de Santiago de Compostela e BarbanzaThe NEIRID (NeuroEndocrine Interactions in Rheumatic and Inflammatory Diseases,) Lab. Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela (A Coruña) - Spain, ⁵Department of Orthopaedic Surgery & Traumatology, Inselspital. Medical Faculty, University of Bern, Bern - Switzerland

Spinal fusion surgery is widely used to restore spinal alignment but remains challenging, particularly in older patients with comorbidities. Enhancing fusion through minimally invasive strategies could improve clinical outcomes. This study investigates factors that promote intervertebral disc fusion by inducing osteogenic differentiation in human annulus fibrosus cells (AFCs). We evaluated the effects of bone morphogenetic protein-2 (BMP2), its analog L51P, and KMN159, a prostaglandin E2 receptor 4 agonist known to modulate inflammation.

Primary human AFCs (n = 11) were cultured for 21 days and stimulated with BMP2, L51P, and KMN159 alone or in combination. Cell viability was monitored to detect potential cytotoxicity. Osteogenic differentiation was assessed at days 7, 14, and 21 by quantifying transcription levels of alkaline phosphatase (ALP), RUNX2, bone gamma-carboxyglutamate protein (BGLAP/osteocalcin), secreted phosphoprotein 1 (SPP1/osteopontin), osterix (SP7), and type I collagen (COL1). In addition, expression of BMP antagonists (Noggin, Gremlin-1, and Chordin) was measured by qPCR. ALP activity was evaluated at the protein level on day 14, and mineralization was examined on day 21 using alizarin red staining.

KMN159 showed no cytotoxic effects but induced weaker osteogenic responses compared with BMP2. On day 21, BMP2 produced higher fold changes in ALP, RUNX2, BGLAP, SPP1, SP7, and COL1 expression. However, combining KMN159 with BMP2 and L51P significantly enhanced ALP expression (P < 0.05), which was supported by increased ALP protein activity and greater calcium deposition at day 21. Although other osteogenic markers did not show statistically significant differences, their expression trended upward with the combined treatment. KMN159 had minimal influence on the expression of BMP antagonists.

These findings demonstrate that the combination of KMN159, BMP2, and L51P promotes osteogenic differentiation in human AFCs. This strategy may offer a promising minimally invasive approach to improve spinal fusion without removing the intervertebral disc, providing potential new treatment options for patients with low back pain.

Acknowledgements

S.C. was supported by the China Scholarship Council, assigned to S.C. no. 202208170027. The Spark programme of the Swiss National Science Foundation financially supported S.H., no. CSRK3_237950.

856. Accelerated chondrogenesis through modulation of actin regulating protein scinderin

Tess Uphof¹, Andrea Lolli¹, Ruud Das², Eric Farrell¹

¹Department of Oral and Maxillofacial Surgery. Erasmus MC University Medical Center Rotterdam, Rotterdam (Zuid-Holland) - The Netherlands, ²Scinus Cell Expansion Netherlands B.V., Zeist (Utrecht) - The Netherlands

Large bone defects pose a clinical challenge often addressed by autologous transplantation, with disadvantages like limited availability, donor site morbidity and the introduction of multiple surgical sites. Bone tissue engineering offers a promising alternative. Particularly through endochondral ossification, where mesenchymal stromal cells (MSCs) form a chondrogenic template that remodels into bone. However, the extensive culture time required for chondrogenesis of MSCs, typically 1-3 weeks, remains a major barrier to clinical translation.

To overcome this, we aimed to accelerate chondrogenesis by identifying and modulating early regulators. Bulk RNA sequencing of MSC pellets at day 3 (non-bone forming) and day 7 (bone forming) of chondrogenic differentiation revealed scinderin, an actin-regulating protein, as significantly upregulated during early chondrogenesis. Surprisingly, transient knockdown of scinderin in monolayer cultures led to early upregulation of chondrogenic markers (COL2A1, COL10A1, ALPL) and enhanced matrix deposition (GAGs, COL2A1, COL10A1) by day 7. To elucidate how the scinderin knockdown induced accelerated chondrogenesis, we performed bulk RNA sequencing at early timepoints comparing the knockdown to the scramble control (day 1 and 3 of differentiation). This revealed an upregulation of genes involved in ECM organisation, cell adhesion and cytoskeletal remodeling, with downregulation of proliferation, apoptosis, and immune signaling on day 1. Furthermore, gene set enrichment analysis showed upregulated pathways of homophilic cell-cell adhesion. By the 3rd day of chondrogenic differentiation, genes and pathways associated with cell adhesion remained upregulated in the knockdown samples, alongside pathways involved in cartilage condensation, cartilage development, and ossification. Additionally, key chondrogenic regulators, such as the SOX trio, showed consistent upregulation at both timepoints as well.

These findings demonstrate that transient scinderin knockdown enhances cell-cell adhesion and aggregation and ultimately accelerates early chondrogenesis in MSC pellets. This strategy could significantly reduce differentiation time, lowering culture costs and improving the feasibility of clinical bone tissue engineering via endochondral ossification.

857. A multiscale approach for the local release of therapeutics combining microfluidics and extrusion bioprinting

Eugenia Spessot¹, Xue Bai², Daniel Moranduzzo¹, Chen Zhao², Sam Butterworth², Devid Maniglio¹, Annalisa Tirella¹

¹BIOtech Research Centre - Dept. of Industrial Engineering. University of Trento, Trento (Italia) - Italy, ²Division 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

New approaches towards personalised drug delivery systems exploit composite injectable hydrogels and 3D printing. In this work, we selected alginate for its favourable shear-thinning properties and drug-loaded PLGA nanoparticles to 3D print new drug delivery technologies for cancer therapy.

PLGA-based nanoparticles were engineered for the sustained release of a small molecule (hydroxyl-FK866) and their surface modified to control the interaction with alginate hydrogels’ network. Composite hydroxyl-FK866-PLGA/alginate biomaterial inks were characterised and printability of the nanoparticles loaded-alginate ink was assessed with an extensive rheological characterization and shape fidelity evaluations.

The interaction of the hydroxyl-FK866-PLGA nanoparticles with blends of oxidized and pristine alginate was analyzed via AFM, highlighting the chitosan coating as an effective strategy for a better confinement of the nanoparticles in the hydrogel. An ionic crosslinking after printing was used to match the viscoelastic properties of human soft tissues and to guarantee stability of printed hydrogels. Finally, the efficacy of the hydroxyl-FK866 release from the alginate-based microsystem was evaluated in vitro both on human breast cancer cells (MDA-MB-231) and macrophages (THP-1).

Alginate-based biomaterial inks loaded with hydroxyl-FK866-PLGA nanoparticles were successfully 3D printed and designed to: 1) match the viscoelasticity of soft human tissues, hence reducing inflammatory effects after implant and 2) load and release a known concentration of cytotoxic compound, namely hydroxyl-FK866. Cytotoxic tests on both cancer cells and macrophages confirmed the efficacy of the composite 3D-printed systems vs free hydroxyl-FK866-PLGA nanoparticles, showing higher cytotoxicity values of approx. 20%.

The incorporation of nanoparticles into a 3D-printed hydrogel network is a promising strategy to ensure localization, increase retention time at the target site, hence control both the release kinetics and site of therapeutic agents.

Moreover, using minimally invasive procedures, discomfort to patients is reduced paving the way towards new delivery strategies for personalized medicine.

858. Multimaterial digital light processing for multiscaled and multimaterial vascular tissue constructs

Alba Fernandez Ferrer¹, Nadina Usseglio¹, Daniel Nieto¹

¹Advanced Biofabrication Laboratory - DNIETO LAB, A Coruña - Spain

Digital Light Processing (DLP) bioprinting is a powerful technology in tissue engineering and regenerative medicine, capable of fabricating high-resolution, geometrically complex, and biologically relevant three-dimensional (3D) constructs. In this work, we present a novel multimaterial DLP bioprinting platform designed to seamlessly print up to four distinct bioinks within a single construct, enabling the fabrication of heterogeneous tissue analogues with precise spatial control. The system’s performance was evaluated through extensive experimental testing, including quantitative assessments of printing accuracy and qualitative analyses of structural fidelity across multiple geometries. Viscosity-dependent behavior was first analyzed using polyethylene glycol diacrylate (PEGDA)-based photocrosslinkable solutions to determine its influence on meniscus formation and printing resolution. The platform successfully fabricated complex vascular-inspired geometries, such as bifurcated channels, multilayered tubular structures, and helical configurations, demonstrating high dimensional accuracy (≥ 90%) and structural integrity in both single and multimaterial configurations. Moreover, the influence of photoabsorber concentration on curing depth was characterized, establishing tunable light penetration profiles for accurate control of inner lumen formation. Finally, multimaterial bioprinting with cell-laden hydrogels confirmed high cytocompatibility, with fibroblast, cardiomyoblast, and endothelial cell viabilities. These results validate the system’s precision, reproducibility, and biocompatibility, highlighting its potential for fabricating vascularized and multi-tissue constructs for advanced tissue engineering applications

859. Enucleated adipose-derived MSCs as delivery vehicles for osteogenic proteins in bone fracture therapy

Amelie Frischer¹, Oona Jung¹, Johannes Oesterreicher¹, Magda Tyszkiewicz¹, Reema Jacob¹, Carina Kampleitner¹, Lydia Zopf¹, Patrick Heimel¹, Anna Mahringer¹, Kerstin Hauck¹, Maximilian Wagner¹, Madhusudhan Reddy Bobbili², Johannes Grillari¹

¹Ludwig Boltzmann Institute (LBI) for Traumatology, The Research Centre in Cooperation with AUVA, Vienna (Wien) - Austria, ²BOKU University, Vienna (Wien) - Austria

Several types of bone fractures require advanced therapeutic treatment and can be a substantial psychological and physiological burden for the patient. In non-union fractures, common surgical interventions such as autologous bone grafts and allografts are confronted with drawbacks such as restricted supply of donor tissue, donor site morbidity, and rejection of the implant by the host. Consequently, new strategies are necessary to complement conventional therapy.

Here, we aimed to establish enucleated mesenchymal stem/stromal cells (MSCs) – termed Cargocytes – for the treatment of bone fractures. Cargocytes are enucleated cells that are still capable of protein production and are viable for up to three days in vitro and in vivo. We established an enucleation protocol for immortalized human adipose-derived MSCs (ASC/TERT300) based on density gradient ultracentrifugation. The Cargocytes were then transfected with in vitro transcribed mRNA coding for Firefly Luciferase, and two therapeutic constructs. We characterized the resulting Cargocytes in terms of purity, viability, protein production and immunogenicity in vitro, and tested their therapeutic activity in a pilot in vivo study using a femoral drill hole model in rats. Preliminary data indicate a potential beneficial effect in defect healing.

The project presents a proof-of-principle study for the use of enucleated cells in bone fracture therapy.

860. Talin is associated with multiple diseases

Vesa Hytönen¹, Latifeh Azizi¹, Muktesh Athale¹, Alana R Cowell², Yasumi Otani², Lorena Varela², Neil Ball², Vasyl V Mykuliak¹, Paula Turkki¹, Benjamin T Goult²

¹Faculty of Medicine and Health Technology. Tampere University, Tampere (Southern Finland) - Finland, ²Department of Biochemistry. University of Liverpool, Liverpool - United Kingdom

Talin is a large cytoplasmic adaptor protein that links integrins to the actin cytoskeleton, thereby facilitating the transmission of mechanical forces between the cell and the extracellular matrix. This connection is critical for cellular processes such as spreading and polarization. Our research, along with studies from other groups, has focused on disease-associated talin mutations. Several missense mutations in talin have been linked to cardiovascular abnormalities, and alterations in blood cell composition have also been observed. In addition, patients harboring distinct talin variants frequently present with cataracts and dermatological symptoms. To elucidate the molecular mechanisms underlying these pathologies, we have investigated the physicochemical properties of the mutant talin proteins and assessed their functional impact in cellular models. Our findings indicate that these disease-associated mutations induce only subtle alterations in protein structure and function, and the resulting cellular phenotypes are relatively mild. This observation challenges bioinformatic predictions, as many computational algorithms fail to classify these variants as pathogenic. Collectively, our data support the notion that talin serves as a central hub for mechanosignaling within cells. Its mechanical integrity and the regulation of its force-dependent interactions are essential for maintaining proper cellular function. Notably, as these subtle mutations often result in complex and diverse phenotypic manifestations, we anticipate that there may be many, as of yet, unidentified disease caused by mutations in the talin genes, so we propose the name “talinopathies”.

References

Athale et al. (2025) De novo talin-1 variant L353F connects multifaceted clinical symptoms to alterations in talin-1 function. Biochem J. 482:1337

Azizi et al. (2024) Talin-1 variants associated with spontaneous coronary artery dissection (SCAD) highlight how even subtle changes in multi-functional scaffold proteins can manifest in disease. Hum Mol Genet. 33:1846.

Azizi et al. (2022) Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms. Hum. Mol. Genet. 31:4159.

861. Development of a 3D bioprinted tumour-on-chip platform for modeling gastrointestinal cancer migration and invasion

Adrian Garcia¹, Lia Jove², Maria Pereira², Angelica Figueroa², Daniel Nieto¹

¹Advanced Biofabrication Laboratory - DNIETO LAB. CICA - Centro interdisciplinar de Química e Bioloxía, A Coruña - Spain, ²Epithelial Plasticity and Metastasis Group. Instituto de Innvestigación Biomédica (INIBIC), A Coruña - Spain

Understanding how tumour cells migrate and invade surrounding tissues is fundamental to identify the early mechanisms of metastasis, a process responsible for the majority of cancer-related deaths. Gastrointestinal (GI) tumours, in particular, exhibit highly invasive behaviour influenced by both biochemical and mechanical factors within their environment. However, conventional two-dimensional (2D) cultures are unable to reproduce the spatial organisation and dynamic cell-matrix interactions that define native tissues. In contrast, while the use of animal models is informative, there are significant financial and time implications as well as ethical considerations. Consequently, there is a growing demand for versatile, rapid, and reproducible platforms capable of mimicking tissue architecture and cell behaviour under well-controlled experimental conditions.

Here, we present a customizable 3D bioprinted tumour-on-a-chip system designed to bridge the precision of bioprinting with the environmental control of microfluidics for GI cancer modelling. The device is fabricated using LCD-based 3D printing with a biocompatible resin, enabling fast redesign and adaptation to different experimental needs. Within its central chamber, digital light processing (DLP) bioprinting allows the in situ generation of complex, multicellular constructs that recapitulate the biochemical composition and structural organization of the tumour extracellular matrix. The bioink formulation, combining photocrosslinkable and biologically active hydrogel components, provides a tunable environment that maintains cell viability and supports interaction and migration, making it an attractive material for studying dynamic cancer processes.

This system enables the controlled reconstruction of tumour–endothelium interfaces and facilitates the study of cell migration, invasion, and vascular integration within physiologically relevant 3D contexts. Its modular design, accessibility and compatibility make it a flexible tool for observation of tumour progression, intravasation processes, and the early stages of the metastatic cascade under physiologically relevant flow conditions. Overall, this platform establishes a next-generation approach to tumour modelling, combining the precision of bioprinting with the versatility of microfluidics to accelerate the development of personalised and predictive cancer research models.

862. Enthesis engineering: understanding the pathophysiology and using molecular and mechanical cues to regenerate using scaffolds

Martijn Van Griensven¹, Elizabeth R. Balmayor², Carlos J. Peniche Silva¹

¹Cell Biology-Inspired Tissue Engineering. MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands, ²Experimental Orthopaedics and Trauma Surgery. RWTH Aachen University Hospital, Aachen (Nordrhein-Westfalen) - Germany

Tendons connect skeletal muscle to bone and are essential for joint movement and stability. The interfaces between muscle and tendon (myotendinous junction) and between tendon and bone (enthesis) are highly specialized tissues. Among these, the tendon-to-bone enthesis is particularly challenging to study and treat, owing to its complex transition between mechanically dissimilar tissues. This soft-to-hard gradient is characterized by opposing patterns of collagen alignment and mineralization, enabling efficient load transfer from tendon to bone. However, this architecture also renders the enthesis highly susceptible to injury. Healing typically results in fibrovascular scar tissue with inferior mechanical properties, predisposing patients to re-injury even after surgical repair.

Tissue-engineering strategies hold promises for improving enthesis regeneration by restoring native morphology and mechanical behavior. In a rodent model of patellar enthesis injury, we evaluated two biphasic silk fibroin scaffolds designed to mimic the tendon-to-bone transition. One scaffold exhibited a continuous, biomimetic phase transition, whereas the other displayed an abrupt interface. In vivo assessment demonstrated that the scaffold with an interconnected transition markedly enhanced enthesis regeneration compared to the abrupt variant, underscoring the critical role of morphological cues in interphase tissue repair.

Building on these findings, we next explored molecular regulators of early enthesis healing, focusing on fibrosis-related microRNAs (miRNAs). Analysis of injured patellar entheses revealed dysregulation of at least 13 miRNAs within the first 10 days post-injury. Target prediction indicated potential regulatory effects on key tendon and enthesis markers. Among these, miR-16-5p, one of the most strongly dysregulated, was selected for functional analysis.

Using a tendon-mimetic, magnetically responsive GelMA hydrogel loaded with human mesenchymal stem cells, we performed 3D transfections with miR-16-5p mimics or inhibitors. Mimic delivery led to sustained downregulation of SMAD3 and upregulation of tenogenic markers (tenomodulin, tenascin-C, decorin, etc.), alongside reduced collagen III expression. Opposite effects were observed with inhibitors, suggesting a tenogenic role for miR-16-5p.

Collectively, these results advance the understanding of enthesis healing mechanisms and support the development of biomimetic and molecularly informed regenerative strategies.

863. Bone defect therapy needs more than bone: microRNA modulation for bone-vessel-nerve triad regeneration

Virginie Joris¹, Noémie Tilquin¹, Martijn Van Griensven¹

¹Cell Biology-Inspired Tissue Engineering (cBITE). MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands

Large bone defects remain challenging to treat, as current therapies rarely achieve full recovery. The regeneration of bone first requires new innervation followed by angiogenesis and bone formation. While studies mainly investigate how to improve mineralization, a few are focusing on innervation and vascularization as adjuvants to bone regeneration. Recently, new components appeared as regulators of bone homeostasis: microRNAs (miRNA). In this work, we aimed to study the implication of the cluster miR-199a/214 on the bone-nerve-vessel triad.

To assess the effect of miR199a/214 on mineralization, hMSCs and osteoblasts (n=3) were transfected with inhibitor of miR-199a-5p or miR-214, or with a negative control (scramble) in calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA/mRNA expression by qPCR. To assess the effect of miR199a/214 on axon growth, iPSCs were differentiated into motorneurons or endothelial cells and transfected with the same inhibitors. Axon growth and tube formation were assessed by immunofluorescence and brightfield, respectively.

Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. Inhibition of miR-214 or miR-199a-5p in Ca++-treated cells, induced higher mineralization compared to scramble-transfected (p<0,01). Moreover, inhibition of miR-199a or miR-214 in iPSCs accelerated their specific differentiation into motorneurons compared to scramble-transfected with increases in axon length, junction number, and area coverage (p<0,05). Finally, inhibition of both miRNAs in iPSC-derived endothelial cells increases their tube formation potential when cultured in Matrigel.

To conclude, miR-199a-5p and miR-214 can modulate mineralization of hMSCs and osteoblasts. Moreover, these miRNAs also showed the ability to regulate axon growth and tube formation in transfected iPSC-derived motorneurons or endothelial cells. For future work, we aim to engineer a scaffold/mesh delivering nanoparticles carrying miR-199a/214 inhibitors to the three different cell types in a controlled, sequential manner, mimicking natural bone healing. This platform could help guide new strategies for treating bone diseases.

864. Injectable mineralized hydrogels for localized osteogenic and antibacterial bone regeneration

Anna Rubina¹, Artemijs Sceglovs², Ingus Skadins³, Anna Ramata-Stunda⁴, Kristine Salma-Ancane²

¹Baltic Biomaterials Centre of Excellence, Riga Technical University. Institute of Biomaterials and Bioengineering, Riga - Latvia, ²Baltic Biomaterials Centre of Excellence, Riga Technical University. Institute of Biomaterials and Bioengineering, Riga - Latvia, ³Department of Biology and Microbiology, Riga Stradins University, Riga - Latvia, ⁴University of Latvia, Department of Microbiology and Biotechnology, Riga - Latvia

Antimicrobial resistance (AMR) significantly contributes to the burden of bone infections, which remain major challenges in orthopaedic treatment due to their complexity and the limited efficacy of current therapeutic strategies. To address these limitations, non-antibiotic antibacterial injectable mineralized hydrogels were developed by incorporating hydroxyapatite nanoparticles (nHAp) into a covalently cross-linked ε-polylysine (ε-PL) and hyaluronic acid (HA) hydrogel network. These mineralized hydrogels were designed to combine the osteogenic and bioactive properties of nHAp with the inherent antibacterial activity of ε-PL, providing a dual-functional platform for localized bone reconstruction. The physicochemical and biological properties of the hydrogels were systematically evaluated. Rheological analysis confirmed their viscoelastic behavior, shear-thinning profile, and excellent injectability, supporting their suitability for minimally invasive clinical application. Adjusting the nHAp content enabled fine control of structural and mechanical performance. Enzymatic degradation studies demonstrated prolonged stability, with degradation times extending from 5 to 19 weeks. High nanoparticle loading did not compromise antibacterial performance. The hydrogels maintained rapid and sustained bactericidal activity against multidrug-resistant ESBL Escherichia coli and MRSA clinical isolates. In vitro biocompatibility assessments confirmed robust cell viability and proliferation, indicating a strong osteogenic potential. Overall, mineralized hydrogels represent a promising class of non-antibiotic injectable biomaterials that integrate antibacterial protection with osteogenic stimulation. These findings highlight their potential for the combined treatment of bone infections. The authors acknowledge financial support from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 857287 (BBCE).

865. Integration of functional nanomaterials with optical fiber sensors and microfluidics for the design of advanced biophotonic platforms in early cancer diagnosis

Giorgia Montalbano¹, Davide Luca Janner¹, Claudia Borri², Ambra Giannetti², Sonia Lucia Fiorilli¹, Chiara Vitale Brovarone¹, Francesco Chiavaioli²

¹DISAT. Politecnico di Torino, Torino (Italia) - Italy, ²Centro Nazionale delle Ricerche (CNR), Firenze (Italia) - Italy

Cancer’s impact on health, society, and the global economy makes early detection through biomarker identification in body fluids one of the greatest challenges of our time. Current gold-standard techniques remain labor-intensive and limited, highlighting the need for operando platforms with high sensitivity and selectivity. The FOCAL project (“Fiber Optic sensors as a platform for CAncer diagnosis and in vitro modeL testing”) addresses this gap by developing an advanced biophotonic platform that integrates optical fiber sensors, functional nanomaterials, and microfluidics for non-invasive cancer diagnosis and potential in vitro modeling.

To this end, D-shaped optical fibers combined with nanostructured metal oxides were employed to exploit lossy mode resonance (LMR), with analyte-induced peak variations analyzed via spectral transmission and data fitting. In parallel, electrospun nanofibrous substrates were fabricated upon optimization of material formulation and process parameters, comparing the use of Eudragit_L100 with a collagen/polycaprolactone (Coll/PCL) blend. Physico-chemical characterization and fluorescence assays confirmed the presence of functional groups suitable for biomolecule anchoring for both substrates, while osteosarcoma Saos-2 cells validated their biocompatibility.

Results demonstrated that D-shaped fibers with metal oxide nanolayers achieved ultra-high sensitivity, with LMR-based detection reaching sub-femtomolar limits in small biofluid volumes. Electrospun substrates exhibited homogeneous nanofibers (300–500 nm), structural stability in culture medium for up to seven days, and supported cell adhesion and proliferation, with superior performance observed in the Coll/PCL blend. Tailored deposition techniques enabled effective integration of polymeric substrates with optical fibers and their further incorporation into microfluidic systems. A preliminary prototype was validated using osteosarcoma biomarkers, achieving detection limits below 0.1 ng/mL.

This study introduces a novel approach for the design of optical fiber biosensor with exceptional sensitivity, paving the way for advanced diagnostic tools in oncology and tissue engineering.

866. Diels-Alder click chemistry as a dynamic-covalent crosslinking method in spheroid-encapsulating hydrogels for cartilage engineering

Antonia Vasilopoulou¹, Sanne Van De Looij², Lennard Spauwen¹, Antoinette Van Den Dikkenberg², Jasmijn Korpershoek¹, Mylene De Ruijter¹, Jos Malda¹, Bas G.p. Van Ravensteijn², Tina Vermonden²

¹Department of Orthopedics, University Medical Center Utrecht, Utrecht - The Netherlands, ²Division of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS). Utrecht University, Utrecht - The Netherlands

Aim: Multicellular spheroids are promising building blocks for engineering large and functional cartilage tissues. Dynamic hydrogels should facilitate spheroid fusion, creating a permissive environment for cartilaginous matrix deposition, while maintaining structural integrity to support maturation. Here, we present a hydrogel crosslinked by Diels-Alder (DA) click chemistry that was designed to promote chondrogenic differentiation and facilitate cartilage spheroid fusion under near-physiological conditions.

Methods: Hydrogels were formed via DA click chemistry using hyaluronic acid-furan, gelatin-furan, and 4-arm PEG-maleimide. The hydrogel formulation was optimized based on mechanical properties before cell encapsulation. Equine articular cartilage progenitor cell (ACPC) spheroids were pre-matured for 3 days, then encapsulated and cultured under chondrogenic conditions for 28 days. Cytocompatibility was assessed using Live/Dead, alamarBlue, and lactate dehydrogenase (LDH) assays. Histological analyses were performed to evaluate spheroid fusion behavior and extracellular matrix (ECM) deposition. Melt-electrowritten polycaprolactone scaffolds were fabricated following previously published methods1 and used to reinforce the hydrogels.

Results: Lowering the pH value during crosslinking (from pH 7.4 to 6.8) improved hydrogel properties, while maintaining high ACPC spheroid viability. Over 28 days, metabolic activity, spheroid size, and DNA content increased, and the spheroids deposited collagen type-II-rich matrix and progressively fused. Spheroid fusion became more evident at reduced inter-spheroid distances, which also enhanced cartilaginous ECM deposition. Reinforcement with a MEW PCL scaffold enhanced the compressive modulus 12-fold on day 1 and over 100-fold by day 28 compared to non-reinforced constructs, thereby enhancing its potential future applicability as a cartilage tissue implant.

Conclusion: In this study, we optimized the use of Diels-Alder click-chemistry as a crosslinking method for hydrogels for cartilage engineering.

Acknowledgements: Supported by the LS-NeoCarE (NWA.1389.20.192) and the Dutch Research Council (NWO/VICI 18673).

[1] Visser et al., Nature Communications, 2015.

867. Engineering the bone pre-metastatic niche with a millifluidic optically accessible platform for in vitro bone marrow culture

Leonardo Cherubin¹, Christina Capobianco², Xiaohua Gao², Christophe Merceron², Prakaimuk Saraithong², Chiara Martinelli¹, Claudio Conci¹, Manuela Teresa Raimondi¹, Annemarie Lang²

¹Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”. Politecnico di Milano, Milano (Lombardia) - Italy, ²Department of Orthopaedic Surgery. University of Michigan, Ann Arbor (Michigan) - United States

Bone metastases affect ∼75% of advanced breast cancer patients and are detected after extensive skeletal colonization, when treatments are limited. Intervention during pre-Metastatic Niche (pre-MN) formation, when tumor-secreted factors prime the bone marrow (BM), offers a crucial window for preventive therapy. Understanding the mechanistic crosstalk between tumor-secreted signals, BM remodeling and osteoclasts activation could improve therapeutic strategies for metastatic cancer. However, current in vitro models do not adequately preserve bone marrow viability and the complex niches architecture for accurate communication, while animal models lack the capacity for real-time monitoring and precise intervention1.

To overcome these limitations, we employed a miniaturized optically accessible bioreactor (MOAB)2 to study pre-MN dynamics via compartmentalized co-culture of breast cancer cells, BM explants, and osteoclasts. This system supports perfusion for up to 5 days, with medium flowing sequentially through three connected chambers: the first containing MDA-MB-231 breast cancer cells, the second accommodating ex vivo BM tissue, and the third housing osteoclasts on calcium-phosphate substrate simulating bone matrix. Sequential flow mimics physiological interactions, promoting the progressive accumulation of tumor-derived and marrow-processed factors.

Critically, perfusion yielded a 6-fold increase (6.18±0.79) in BM tissue viability by day 5 relative to static cultures, with functional architecture confirmed via confocal microscopy. This enhanced viability permits longitudinal assessment of pre-MN dynamics; preliminary molecular analysis currently investigates the time-dependent upregulation of pro-osteoclastic factors (RANKL, M-CSF) in tumor-primed marrow tissue. Osteoclast cultures are expected to depict and increased resorption activity and pit formation on calcium-phosphate substrates compared to controls, reproducing sequential pre-MN signaling.

This approach addresses critical gaps in pre-MN research by supporting quantitative analysis of sequential tumor-BM-osteoclast signaling and niche remodeling, providing a physiologically relevant platform for identifying molecular mediators and screening anti-metastatic therapies.

References

1. Jackett K.N. et al., DOI:10.1038/s43018-024-00854-6.

2. Laganà M., and Raimondi, M.T., DOI:10.1007/s10544-011-9600-0.

Acknowledgements

ERC, BEACONSANDEGG, G.A.101053122.

868. Different calcium chloride concentrations in the pre-crosslinking of a tuna skin collagen-based hydrogel for 3D bioprinting applications

Rebeca Mello Chaves¹, Rodrigo S. Vieira¹, Fábia K. Andrade¹, Francisco F. Pereira¹, Lidyane S. M. Marques¹, Érika P. C. Gomes¹, Thamyres F. Da Silva1¹, Pamela C. Bortoluzzi¹, Raimundo N. F. M. Filho1¹, Renata F. B. De Souza², Fernanda C. B. De Souza³

¹Department of Chemical Engineering. Federal University of Ceará, Fortaleza (Ceara) - Brazil, ²R-Crio Stem Cells, Campinas (Sao Paulo) - Brazil, ³R-Crio Stem Cells, Campinas (Sao Paulo) - Brazil

Three-dimensional (3D) bioprinting is a revolutionary technology to reproduce a 3D functional living tissue scaffold in vitro through controlled layer-by-layer deposition of biomaterials. Due to their biocompatibility, natural hydrogel polymers are commonly considered as scaffold materials and have been used in 3D bioprinting because it is suitable for the in vitro biological growth of human cells. However, the mechanical integrity of the hydrogel material, especially in 3D scaffold architecture, is an issue. In this study, a hydrogel composed exclusively of natural polymers was prepared using 0.25% (w/v) tuna skin collagen, 0.5% (w/v) agarose, and 1.5% (w/v) sodium alginate derived from marine bioproducts, and analyses of cell viability, rheological, and physicochemical properties were performed. In addition, the hydrogel was pre-crosslinked with 0.1%, 0.2%, and 0.3% calcium chloride, and systematic qualitative characterization tests were conducted to assess the impact on printability. The results show that the calcium chloride concentration in the pre-crosslinking process has a considerable impact on printing performance and that the tuna skin collagen–based hydrogel exhibits no cytotoxicity, making it suitable for use as a support material in 3D bioprinting applications.

869. In vivo evaluation of ACL injury–induced changes in bone remodeling in post-traumatic osteoarthritis

Michela Uberti¹, Bingbing Liang¹, Antonio Franchi², Tom Hodgkinson¹, Oran D Kennedy¹

¹Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine Royal College of Surgeons in Ireland (RCSI) 123 St. Stephen’s Green, Dublin 2, D02YN77, Ireland, Dublin - Ireland, ²Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland

Osteoarthritis(OA) is a leading cause of disability worldwide. Post-traumatic osteoarthritis (PTOA) accounts for 12% OA cases and develops following acute joint injury, enabling early intervention. Up to 80% of anterior cruciate ligament (ACL) injuries present subchondral bone marrow lesions (BMLs). BMLs are characterised by osteocyte apoptosis and recruitment of osteoclasts. Bisphosphonates inhibit osteoclast-mediated bone resorption and may decrease early pathological changes. This study aimed to characterise PTOA progression at multiple time points after injury and assess the effects of bisphosphonate treatment delivered intra-articularly or subcutaneously.

All procedures were approved by the HPRA (AE19127/P082). Forty-eight female Sprague Dawley rats were euthanised at 2-4-8 weeks post-injury (n=16/timepoint) and allocated into four groups: Control (no injury, no treatment), ACL (injury only), ACL+SC (injury + subcutaneous bisphosphonate), and ACL+IA (injury + intra-articular bisphosphonate). ACL rupture was induced bilaterally under anaesthesia using a single axial compressive load (1 mm/s, 10–20 N) following Buprenorphine analgesia. Zoledronic acid (0.1 mg/kg) was administered either subcutaneously or intra-articularly according to group allocation. Samples were fixed, stored in ethanol, analysed by microcomputed tomography (microCT), embedded in paraffin or polymethylmethacrylate (PMMA) for histology.

Overall, results obtained by microCT and immunohistochemistry demonstrated that the ACL group had a significant reduction of Bone Total Volume (BV/TV%) compared to the control groups, indicating early bone loss following joint mechanical trauma. In contrast, both bisphosphonate-treated groups preserved bone structure, showing no significant reduction in BV/TV. When comparing outcomes across time points, the ACL group at week 8 displayed the most pronounced decrease in bone volume and microarchitecture, consistent with progressive bone deterioration in the absence of therapeutic intervention.

These findings reveal that ACL injury triggers rapid and progressive osteoclasts-mediated subchondral bone loss, underscoring the critical importance of early therapeutic intervention: bisphosphonate administration mitigated this decline, suggesting a protective effect of the treatment.

870. Assessment of the effect of lipophosphonoxin DR-7072 in biodegradable nanofiber carrier systems for controlling Staphylococcus aureus infection in experimental murine wounds

Pavel Klein¹, Marek Kindermann¹, Elizabeth Tsibouki¹, Vera Jencova², Dana Kralova³, Vendula Peckova³, Eva Kuzelova - Kostakova², Maxim Lisenko², Kristyna Havlickova², Sarka Hauzerova², David Lukas², Katerina Chudejova³, Dominik Rejman⁴

¹Biomedical Center. Charles University, Faculty of Medicine in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic, ²Faculty of Science, Humanities and Education. Technical University of Liberec, Liberec - Czech Republic, ³Department of Microbiology. Charles University, Faculty of Medicine in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic, ⁴Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague (Hlavni Mesto Praha) - Czech Republic

Wound infections are a common cause of complications in the healing process, and in many cases, antimicrobial treatment is necessary. However, due to the development of pathogen resistance, it is desirable to expand the spectrum of antimicrobial agents that can be used to control bacterial infections of wounds and soft tissues. Lipophosphonoxins (LPPOs) appear to be a very promising group of substances suitable for such use. The aim of the study was to test the potential of LPPOs (molecule DR-7072) in biodegradable nanofiber carrier systems for the prevention of staphylococcal infections in skin wounds. A strain of CB6F1 mice immunosuppressed with cyclophosphamide and experimentally infected with Staphylococcus aureus wounds was used as an in vivo model. The result of the 7-day treatment was evaluated by scoring the appearance of the wounds and microbiological analysis. The results of the experiment confirmed the antimicrobial efficacy of DR-7072 and also showed that the incorporation of DR-7072 into a biodegradable nanofiber structure appears to be a promising application form.

Supported by the Ministry of Health of the Czech Republic, AZV project no. NW24-08-00073.

871. Reproducible 3D bioprinting and complex mechanical properties of alginate-gelatin hydrogel constructs with decreased concentrations

Anahita Ahmadi Soufivand¹, Monika Buss², Emine Deniz Bicer², Elmira Zanjani², Silvia Budday¹

¹Institute of Continuum Mechanics and Biomechanics (LKM). Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen (Baden-Wberg Bayern) - Germany, ²Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen (Baden-Wberg Bayern) - Germany

Extrusion-based 3D bioprinting has emerged as a promising technique for fabricating tissue replacements in tissue engineering. However, a main challenge is finding reproducible procedures that ensure good printability and yield final printed constructs with high shape fidelity, close similarity to the designed model, and controllable mechanical properties. In this study, we investigate the printability of multilayered 3D structures fabricated from various concentrations of alginate-gelatin (AG) hydrogels and quantify their complex mechanical properties. We used our recently developed procedure for the hydrogel with 2% and 5% (w/v) alginate and gelatin, respectively, which incorporates a pre-cooling step and optimized printing parameters informed by rheological and printability assessments. Through this procedure, we significantly improved the printability and flow stability of AG hydrogels, successfully fabricating well-defined constructs closely matching our design models. In this work, we decreased the bioink concentration by lowering the gelatin concentration to 4%, 3% and 2% (w/v) and kept the alginate concentration fixed to create a less dense microenvironment, which may reduce the shear stress during the extrusion and enhance biological properties when using cell-laden bioinks. Our printability results demonstrate that not all hydrogel concentrations are suitable for printing, and the hydrogel concentration significantly affects printability. Subsequent analyses reveal that the concentration markedly influences the rheological properties of the hydrogel. In addition, tuning the complex mechanical response of the printed constructs is possible by altering the hydrogel concentration. The presented approach and the corresponding results may inform future 3D bioprinting applications when aiming to produce replacements with tunable mechanical and biological properties, especially in soft tissue engineering.

Ref:

1. Ahmadi Soufivand, A., Faber, J., Hinrichsen, J. et al. Multilayer 3D bioprinting and complex mechanical properties of alginate-gelatin mesostructures. Sci Rep 13, 11253 (2023). https://doi.org/10.1038/s41598-023-38323-2

2. N. Murenu, J. Faber, A. A. Soufivand, M. Buss, N. Schaefer, and S. Budday, “Cell Behavior and Complex Mechanical Properties of 3D Printed Cell-Laden Alginate-Gelatin Macroporous Mesostructures.” Macromolecular Bioscience (2025): e00204. https://doi.org/10.1002/mabi.202500204

872. Nanoparticle delivery of STING immunotherapy to enhance anti-tumour immunity in metastatic Osteosarcoma

Aoise O'neill¹, Victor Burgueno¹, Jordan O'donoghue², Fiona Freeman¹

¹School of Mechanical and Material Engineering, Engineering and Materials Science Centre, UCD, Ireland. Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin - Ireland, ²School of Mechanical and Material Engineering, Engineering and Materials Science Centre, UCD, Ireland. Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin - Ireland

Osteosarcoma is the most common primary malignant bone tumour in children and adolescents and remains a therapeutic challenge due to the aggressive malignant phenotype and poor response to therapies [1]. Recent advances in cancer immunotherapy have highlighted the cGAS-STING pathway as a promising target to reprogram the tumour immune microenvironment (TIME). Systemic delivery of STING agonists has been limited by off-target toxicity and specificity. This study aims to compare nanoparticle (NP) formulations for the targeted delivery of a STING agonist, with the goal of selectively activating the STING pathway in osteosarcoma and reprogramming anti-tumour immune responses [2].

Two different NP formulations were synthesised: a polymer-based nanoparticle (pBAE) loaded with the STING agonist cyclic dinucleotide (CDN) with electrostatic interaction or direct conjugation. We generated a 3D Osteosarcoma spheroid model to mimic the Osteosarcoma TME, inclusive of cancer cells and bone marrow derived macrophages (BMDMs) or dendritic cells (DCs), to test NP delivery of a STING agonist. Subsequent activation of STING signalling in immune and cancer cells was assessed as type I interferon production by ELISA and measurement of STING mRNA expression by PCR. BMDM and BMDC activation was analysed by flow cytometry of MHC-II and CD80 expression.

Both cationic and conjugated STING NPs had efficient uptake by K7M2 Osteosarcoma cells and antigen-presenting cells in 2D and 3D in vitro studies, as seen by IFN-β secretion and an upregulation in STING expression. BMDM and BMDCs were activated with STING NP treatment as assessed by flow cytometric analysis of MHC-II and CD80/86 expression. In combination with doxorubicin chemotherapy, STING agonist delivery increased K7M2 cancer cell death and Osteosarcoma spheroid destruction.

Our findings highlight the immunotherapeutic potential of NP-mediated STING activation in Osteosarcoma. This novel therapeutic strategy may overcome current limitations in STING agonist delivery as immunotherapy and provides a foundation for combination therapy with chemotherapy to target resistant Osteosarcoma tumours.

1. Brar, G.S., et al., Osteosarcoma: current insights and advances. Explor Target Antitumor Ther, 2025. 6: p. 1002324.

2. O’Donoghue, J.C. and F.E. Freeman, Make it STING: nanotechnological approaches for activating cGAS/STING as an immunomodulatory node in osteosarcoma. Front Immunol, 2024. 15: p. 1403538.

873. Electrical conductivity in biomaterial design: from engineered in vitro cardiac tissues to in vivo myocardial repair

Kaveh Roshanbinfar¹, Felix B. Engel¹

¹Molecular Cardiology. Friedrich-Alexander University, Erlangen (Niedersachsen) - Germany

Myocardial infarction (MI) results in the formation of myocardial scar due to blockage of nutrients and oxygen to the myocardium. This scar tissue will comprise dense, acellular and electrically isolating tissue which does not beat with the remaining healthy myocardium. This results in the emergence of myocardial arrhythmia. In fact, nowadays, most people survive the MI itself, but they will die due to the subsequent complications of MI-related arrhythmia. Notably, cardiomyocytes function and communicate based on ionic movements across their cell membrane, causing membrane voltage changes. We hypothesized that conductive materials enhance ionic transportation across the cell membrane and between cells, thereby promoting cell contraction and intercellular communication. To test this hypothesis, we have evaluated multiple conductive materials with different conduction mechanisms (electronic or ionic conduction), such as polyaniline, carbon nanotubes, gold nanoparticles, and polythiophenes. We generated engineered cardiac tissues based on these conductive materials within extracellular matrix-mimicking biopolymeric matrices and eventually validated our hypothesis in vivo by enhancing the conductivity of the scar tissue. Our data showed that different conduction mechanisms affect cardiomyocytes differently, while improving cellular microstructure, contractile machinery, calcium handling, cellular connectivity, contractility, and force generation together with their maturation. Mixed ionic-electronic conductive microenvironments promote maturation of human pluripotent stem cell-derived cardiomyocytes (iCM) to adult level without any external stimulations. Moreover, such biohybrid conductive matrices are sufficient to electrify the myocardial scar tissue and protect the injured heart against myocardial arrhythmia. Eventually, these biocompatible conductive matrices allow simultaneous recellularization of the scar tissue by iCM delivery via the hydrogels. This partial remuscularization of the scar tissue improved cardiac output significantly. Collectively, our work demonstrates that mixed ionic–electronic conductive biomaterials enhance cardiac tissue maturation and provide great potential for translation to the treatment of electrically active tissues.

874. Multiscale heteromechanical and anisotropic fibrin assemblies enables vascularized muscle implants for volumetric muscle loss repair

Su Hyun Jung¹, Minjun Kim², Da-Yoon Kim¹, Min Kyu Kim¹, Yoonhee Jin², Joo H. Kang¹

¹Biomedical Engineering. Ulsan National Institute of Science and Technology, Ulsan (Ulsan-gwangyoksi) - South Korea, ²College of Medicine. Yonsei University, Seoul (Seoul-tukpyolsi) - South Korea

Volumetric muscle loss (VML), a severe injury involving irreversible loss of both muscle tissue and vasculature, presents a major obstacle to developing clinically viable muscle grafts. Functional restoration requires engineered constructs that not only integrate with host vasculature but also replicate the aligned architecture and region-specific mechanics needed to support simultaneous myogenesis and angiogenesis. Here, we introduce SPARC (Spatio-chimeric, Plasma-based, Anisotropic, and shear-Responsive Construct), a heteromechanical hydrogel system engineered via shear-guided fibrin assembly. By applying spatial gradients of microfluidic shear stress during polymerization, SPARC forms aligned fibrin bundles with localized stiffness, mimicking the structural and mechanical heterogeneity of native muscle. When co-cultured with myoblasts and endothelial cells, this anisotropic matrix enables spatially divergent lineage specification, supporting aligned myotube formation in the stiffer outer regions and perfusable vascular networks in the compliant core. Our theoretical model further corroborates shear-induced bundling and stiffness modulation by quantifying diffusion- versus shear-driven collision rates across relevant length scales. In vivo in a murine VML model, vascularized SPARC muscle grafts restore muscle architecture and function, promoting neovascularization, myofiber regeneration, and enhanced motor recovery. Through its spatially patterned architecture, SPARC enables integrated muscle and vasculature generation within a single construct. This work establishes a biofabrication strategy for guiding multicellular organization and offers a clinically scalable approach to repairing extensive muscle defects.

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by MSIT (Grant No. RS-2024-00344187) and the Nano & Material Technology Development Program through the NRF funded by MSIT (RS-2025-25424498).

875. Digital Light Processing(DLP) printed, bioactive chondroitin sulfate hydrogels for cartilage tissue engineering

Amanda A. Domingues¹, Monize C. Decarli², Prashant K. Sharma², Luiz H. Catalani¹

¹Institute of Chemistry. University of São Paulo (USP), Sao Paulo - Brazil, ²Biomaterials & Biomedical Technology (BBT). University Medical Center Groningen, Groningen - The Netherlands

Chondroitin sulfate (COS), a glycosaminoglycan (GAG) abundant in cartilage, contributes to its high compressive stiffness through effective water retention and is widely used in osteoarthritis oral therapies.[1] A COS-based bioink for DLP printing was designed to obtain high-resolution structures with minimum shear stress on cells.[2]

COS isolated from bovine trachea was methacrylated(COS-MA) to enable photopolymerizable hydrogels. Chondrocytes bind hyaluronic acid via CD44 and collagen via integrins, but not COS. Therefore, COS functionalized with histidine or bioactive peptides (COS-HIS, COS-GGGGHexyl, and future COS-GRGDS) was incorporated to confer bioactivity. The dual-function hydrogel forms a semi-interpenetrating network without additives, exhibiting favorable hydration, mechanics, biocompatibility, and bioactivity.

Functionalization was confirmed by ¹H NMR. COS-MA was synthesized using excess methacrylic anhydride, while COS-HIS and COS-GGGGHexyl were obtained via carbodiimide-mediated amide coupling. Photo-rheology and DLP working curves (Beer–Lambert model) were used to optimize printing. Inks containing COS-MA 24%(w/v) or COS-MA 23%+COS-HIS1%(w/v) with LAP 0.5%(w/v) in PBS (pH 7.0) were printed at 100 µm per layer with tartrazine/LAP ratios of 0–5%,and constructs were evaluated for printability, stiffness, and stress relaxation. The hMSC cell line will be encapsulated within the bioink.

COS-MA, COS-HIS, and COS-GGGGHexyl exhibited functionalization levels of 80%, 20%, and 40%, respectively. Optimal printing conditions (2% tartrazine/mol LAP, 15 s/layer) produced high-fidelity structures, including gyroid and logo-shaped scaffolds. COS films gelled in 5.7 ± 0.2 s, unaffected by tartrazine. The printed scaffolds reached 2.4 ± 0.2 MPa stiffness and 33 ± 3.4% stress relaxation, comparable to cartilage, ligaments, and tendons [3].Notably, this natural polymer achieved stiffness typical of synthetic systems.

COS-MA is a promising candidate for DLP-based cartilage tissue engineering, with ongoing studies evaluating post-printing cell viability and incorporating bioactive COS-GRGDS.

Acknowledgements:FAPESP

References

[1] D. Pal,S. Saha,RSC Adv. 2019,9,28061–28077.

[2] H. G. Hosseinabadi et al., ACS Biomater. Sci. Eng. 2022,8,1381–1395.

[3] C. F. Guimarães et al.,Nat. Rev. Mater. 2020,5,351–370.

876. Development and early validation of a 3D bioprinted skin-on-chip platform for drug testing applications

Sandra Ruiz-Alonso¹, Irene Bautista-López¹, Virginia Sáez-Martínez², Eider Sustatxa², Elfi Töpfer³, Claudia Gärtner³, Iker Ateca⁴, Jesús Izco⁴, Loli Gálvez⁴, Jose L. Pedraz¹, Denis Scaini¹

¹NanoBioCel Research Group. Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006, Vitoria-Gasteiz (Álava) - Spain, ²I+Med S. Coop., Alava Technology Park, Hermanos Elhuyar 6, 01510, Vitoria-Gasteiz (Álava) - Spain, ³microfluidic ChipShop GmbH, Jena (Thuringen) - Germany, ⁴Viscofan S.A., 31192 Tajonar, Aranguren (Navarra) - Spain

Skin models capable of recapitulating key aspects of the structural and functional complexity of human tissue are essential for drug screening, toxicity studies, and disease modeling. Traditional in vitro and animal models are limited by physiological relevance and ethical concerns, respectively. In this context, combining Organ-on-Chip (OoC) platforms with 3D bioprinting processes provides a reliable strategy for generating skin models with controlled architecture and dynamic microenvironments. Here, we report the development and early biological evaluation of a 3D bioprinted skin-on-chip platform within a multi-partner consortium.

Specifically, a customised hydrogel-based bioink co-developed with industrial partners was optimised for extrusion bioprinting and to support human dermal fibroblasts development in a genuine 3D environment. Constructs were bioprinted within a microfluidic chip designed to ensure structural confinement, nutrient exchange and optical access. Keratinocytes were added on top of the bioprinted dermal layer to promote epidermal formation and initiate stratified epithelium development. The chip supported high cell viability and progressive tissue organization. Fibroblasts showed elongated morphology and increasing extracellular matrix deposition, including collagen-rich fibrillar structures indicative of early dermal maturation. Keratinocytes adhered uniformly, proliferated across the surface and began to display features of early epidermal differentiation. Preliminary analyses revealed higher cell density in the suprabasal region and initial stratification, suggesting the onset of barrier-like architecture.

These findings represent an advance toward a functional 3D bioprinted skin-on-chip model for pharmaceutical and toxicological applications. Ongoing work focuses on improving epidermal stratification, enhancing barrier properties and assessing drug penetration and toxicity under dynamic flow to establish a reproducible platform for physiologically relevant skin assessment.

Acknowledgements: This work is part of the UNLOOC project funded by the Chips Joint Undertaking (Grant Agreement 101140192) and supported by the Joint International Programming Actions PCI2024-153423. Support was provided by the Basque Government (IT1448-22) and ICTS NANBIOSIS Drug Formulation Unit of CIBER-BBN UPV/EHU.

877. A multimaterial extrusion device with integrated optical crosslinking for minimally invasive cartilage repair

Marco Raffo¹, Amedeo Franco Bonatti², Theofanis Stampoultzis³, Carmelo De Maria¹, Marcy Zenobi-Wong³, Giovanni Vozzi¹

¹Department of Information Engineering. Research Centre “Enrico Piaggio”, Pisa (Toscana) - Italy, ²Research Centre “Enrico Piaggio”, Pisa (Toscana) - Italy, ³D-HEST. ETH Zurich, Zurich - Switzerland

Osteochondral defects represent a significant clinical challenge due to limited regenerative capacity and the anisotropic, zonal organization of articular cartilage, composed of hyaline, calcified cartilage, and subchondral bone. This heterogeneity hinders restoring mechanical stability and biological performance using conventional surgical approaches. In situ bioprinting offers a promising strategy by enabling layer-by-layer biomaterial deposition directly within the defect during surgery.

This work presents the design, development, and preliminary validation of an integrated toolhead for minimally invasive bioprinting that combines a motorized extrusion system with a fiber-optic-based Filamented Light (FLight) crosslinking module for real-time photopolymerization. This configuration enables the fabrication of anisotropic constructs that replicate the native OC unit, as the FLight system generates oriented filaments during crosslinking, leading to anisotropic architectures. Gelatin methacryloyl (GelMA) was selected as the base biomaterial for its photocrosslinking ability, tunable mechanical properties, and biological compatibility. Rheological testing on 5% and 10% GelMA at 20°C,25°C, and 37°C defined optimal extrusion windows, while preliminary deposition trials established correlations between flow rate, accuracy, and filament stability. The anisotropy of FLight-crosslinked constructs was then assessed through filament orientation analysis and mechanical testing under tensile and compressive loads, confirming directional reinforcement and reproducible organization. Finally, the performance of the combined system was validated through successive extrusion and FLight-crosslinking in PDMS molds and ex vivo bovine knees, showing effective material anchoring within OC defects. Live/Dead assays with embedded chondrocytes confirmed high post-extrusion viability, validating the system’s biocompatibility and fabrication efficiency.

This integrated platform advances intraoperative bioprinting by enabling precise fabrication of anisotropic, dual-material constructs, offering new potential for in situ OC tissue regeneration.

This project, granted under GA n° 101191804, is funded by the EU. The opinions expressed are those of the author(s) only and do not necessarily reflect those of the EU or the European Commission. Neither the EU nor the European Commission can be held responsible for them.

878. Bioengineering sclero-corneal limbal substitutes via cell-laden hydrogels functionalized with growth factors - SEMIT

Miguel Pérez-Garrastachu¹, Ainhoa Agirrebengoa-Arrieta², Ander Martin², Maddalen Rodriguez-Astigarraga¹, Cristina Romo-Valera¹, Noelia Andollo¹

¹Department of Cell Biology and Histology/BEGIKER Research Group. University of the Basque Country (UPV/EHU)/Biobizkaia Health Research Institute, Leioa/Barakaldo (Bizkaia) - Spain, ²Department of Cell Biology and Histology. University of the Basque Country (UPV/EHU), Leioa (Bizkaia) - Spain

The sclero-corneal limbus is a transitional region between the sclera and the cornea. It is precisely within this limbal zone where the niche of limbal epithelial stem cells (LESCs) resides. This niche is sustained by a highly specialized architecture (Vogt’s palisades), composed of a unique extracellular matrix as well as supportive stromal and epithelial cell populations and melanocytes. When this anatomical region is damaged, as occurs in limbal stem cell deficiency, the cornea loses its ability to regenerate; moreover, the histological boundary between the sclera and the cornea is lost, allowing the former to invade the latter, ultimately leading to pannus formation and progressive blindness.

Tissue engineering approaches offer potential solutions to restore corneoscleral limbal function, through the development of tissue substitutes capable of re-establishing the native histological organization of the cornea. Our recent work has focused on the use of hydrogels as the core component of such biosubstitutes.

In this study, we examine the behavior of LESCs cultured on methacrylated gelatin and collagen hydrogels in which primary corneal stromal cells have been embedded. Addditionally, these hydrogels have been functionalized with serum from plasma rich in growth factors (sPRGF), to harness its strong therapeutic potential. Our results demonstrate the capacity of these biomaterials to deliver bioactive compounds directly to the surrounding cellular environment. Furthermore, the stromal component embedded within the hydrogels, when stimulated by biochemical cues from sPRGF, enhances the adhesion and proliferation of corneal epithelial cells. Additionally, we investigate the cellular responses under inflammatory conditions, which are characteristic of corneal ulcerative processes and limbal stem cell deficiency.

Collectively, these results support the concept that hydrogels represent a promising strategy as regenerative scaffolds for repairing tissue defects in the cornea and the corneoscleral limbus.

Aknoledgements: This research study was supported by grants from the Department of Health of the Basque Government (2023111027, IT524-22) and the Spanish Ministry (PID2023-152436OB-100). Pérez-Garrastachu, M. was supported by a post-doctoral fellowship Margarita Salas.

879. Innovative multifunctional toolheads for the colonoscopic delivery of biomaterials in-situ

Edoardo Bilancia¹, Paolo Bertino¹, Gabriele Maria Fortunato¹, Carmelo De Maria¹, Giuliano Gorini², Ayca Bal-Ozturk³, Alberto Arezzo⁴, Federica Barontini⁴, Andreas Bernkop-Schnürch⁵, Sandra Van Vlierberghe⁶, Debby Laukens⁶, Benjamin Nottelet⁷, An Van Den Buckle⁸, Sonia Fiorilli⁹, Chiara Vitale-Brovarone⁹, Giovann Vozzi¹

¹University of Pisa, Pisa (Toscana) - Italy, ²ERA endoscopy, Pisa (Toscana) - Italy, ³AdBioInk Biosystem Technology, Kocaeli - Turkey, ⁴University of Turin, Torino (Italia) - Italy, ⁵ThioMatrix Forschungs- und Beratungs GmbH, Innsbruck (Tirol) - Austria, ⁶Ghent University, Ghent (Oost-Vlaanderen) - Belgium, ⁷IBMM, Montpellier (Ile-de-France) - France, ⁸4Tissue, Zwijnaarde, Belgium, Zwijnaarde (Oost-Vlaanderen) - Belgium, ⁹Politecnico di Torino, Torino (Italia) - Italy

Colorectal diseases (CRDs) encompass a broad spectrum of medical conditions that affect the mucosal and submucosal integrity of the colon walls, impacting over 2.2 million people within Europe annually. Current standard treatments involve the surgical removal of the lesions, alongside the removal of the entire rectum and part of the colon. These procedures frequently lead to prolonged healing times and to the risk of anastomotic leakage, underscoring the critical need for innovative and advanced strategies. In this scenario, the EU-funded Daedalus Project aims to pioneer a novel therapeutic paradigm based on advanced, minimally invasive in situ bioprinting and advanced biomaterials. In this context, its primary objective is the controlled intraluminal delivery and deposition of cell-laden functionalized gelatins and star-shaped polyesters [1], alongside with bioactive composites, directly onto the damaged colon site immediately after mucosa and submucosa resection, thus preventing the removal of the entire rectum.

To enable this vision, two specialized toolheads have been engineered to be integrated seamlessly with commercially available endoscopic devices. The two toolheads allow the controlled, minimally invasive deposition of the aforementioned cell-laden composite biomaterials through a multi-orifice extrusion system for precise material dispensing. These attachments are designed to handle materials of significantly different viscosities at zero-shear (mPa*s up to hundreds of thousands of Pa*s). Specifically, the first toolhead utilizes a high-pressure nozzle attachment optimized for spraying low-viscosity (1-5000 mPa*s)materials and hydrogels in situ; while the other is a soft, contact-based extruder developed for the precise deposition of higher-viscosity (up to 10 MPa*s) bioinks [2] by direct contact with the colon walls to conform to its morphology. Printability tests using commonly employed biomaterials (such as pluronic acid and methacrylated gelatin) and Daedalus composite biomaterials have been performed to optimize the design of the toolheads.

Eventually, this innovative approach not only sheds light on novel, less invasive treatments for CRDs but also establishes a technological foundation for next-generation, personalized regenerative medicine in gastroenterology.

[1] doi: 10.1002/mabi.202300016

[2] doi: 10.1039/D5TB00737B

This research has been supported by the EU's Horizon Europe research and innovation programme (GA:101178568 DAEDALUS).

880. Design of biocompatible GelMa/PVP/PA-MBGs hybrid scaffolds for bone regeneration

Sandra Sanchez-Salcedo¹, Patricia Álvarez Barrios², Jesus Lius Pablos¹, Daniel Arcos¹, Isabel Izquierdo-Barba³

¹Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, UCM; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN. Universidad Complutense de Madrid, Madrid - Spain, ²Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, UCM. Universidad Complutense de Madrid, Madrid - Spain, ³Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN. Universidad Complutense de Madrid, Madrid - Spain

The incorporation of MBGs into 3D scaffolds results in the combination of three aspects of bone regeneration: composition, hierarchical porosity and mechanical support. The 3D structure includes interconnected macro-mesopores, due to the combination of the properties of the MBGs and the design characteristics of the scaffolds, which provide nutrient transport, cellular metabolism, and the growth of blood vessels and tissues[1].

Three MBGs were prepared using a combination of sol-gel and EISA method a raw glass (MBG-BL) with a composition of 85SiO2–10CaO–5P2O5, doped with 2.5%SrO and 4%ZnO (MBG-4Zn), and doped with 4%CeO and 2.5%SrO (MBG-4Ce) (% mol). Scaffolds were obtained from previous MBGs and monomers of combined GelMa/Polyvinylpyrrolidone(PVP)/polyacrylamide(PA) copolymers using 3D printing Regemat3D and radical photopolymerization. A technique that allows precise control of morphology and porosity of scaffolds to ensure that they are suitable and allow for nutrient supply and proper cell adhesion [2]. Scaffolds were loaded with human lactoferrin (hLF), a protein with osteogenic properties, with the aim of establishing synergy with the ions loaded in the glasses and thus evaluating in vitro the regenerative capacity of these 3D systems [3,4].

Cytocompatibility tests shown that there was better cell proliferation in contact with SC-4Ce, especially if it incorporates hLF, and greater differentiation into osteoblasts in contact with SC-4Zn loaded with hLF. This suggests that the incorporation of hLF into scaffolds improves both viability and differentiation in combination with Ce/Sr and Zn/Sr respectively, as each of the ions contributes to different stages of cell development.

[1] Javier Jiménez-Holguín et al. Materials 2020,13,5526.

[2] Wang, Zhen, et al. Advanced Drug Delivery Reviews, 2021,504–534.

[3] Tian, Miao, et al. Frontiers in Endocrinology, 2023,1218148.

[4] Redwan, Elrashdy M., et al. Research in Microbiology, 2016,480–491.

Acknowledgements

Carlos III Health Institute through the FIS (PI20/01384) co-financed with European Regional Development Fund (ERDF) funds from the European Union. Spanish Ministerio de Ciencia e Innovación (PID2023-149093OB-I00, MAGEN4BONE) and Fundación Ramón Areces (FD5/22_01, Nano4Infection) for funding.

881. Translational advancement of hiPSC-derived cartilage combining dynamic bioreactor culture and human ex vivo models of osteoarthritis

Giorgia Mazzini¹, Sana Sayedipour¹, Helena Eka Suchiman¹, Marcella Van Hoolwerff¹, Ghazaleh Hajmousa¹, Yolande F.m. Ramos¹, Ingrid Meulenbelt¹

¹Biomedical Data Science. Leiden University Medical Center, Leiden (Zuid-Holland) - The Netherlands

Objectives

To optimize dynamic rotary suspension culture in the CERO bioreactor for hiPSC-derived cartilage generation and evaluate the translational potential of the resulting human induced chondrocyte-like (hiCHO) constructs using a human ex vivo osteochondral repair model.

Methods

hiPSCs were differentiated into chondroprogenitors (hiCPCs) using a stepwise protocol1. Manually selected hiCPC aggregates were cultured for 4 weeks under chondrogenic conditions either in static suspension or in rotary suspension in the CERO bioreactor. Construct size, extracellular matrix (ECM) composition, and gene expression were assessed by histology, immunohistochemistry, sGAG/DNA quantification, and RT-qPCR. Translational performance was tested by implanting hiCHO constructs into human osteochondral explants (RAAK study2,3,4), alongside human primary articular chondrocyte (hPAC) constructs as reference. Repair and matrix integration were evaluated after 4 weeks.

Results

Dynamic rotary culture markedly enhanced neo-cartilage formation compared with static conditions. hiCHO constructs showed larger volumes, intense proteoglycan staining, and homogeneous Collagen II and VI organization, indicating advanced chondrogenic maturation. Chondrogenic markers (COL2A1, ACAN, COMP, MGP) were strongly upregulated, while fibrotic and hypertrophic markers (COL1A1, COL10A1) were reduced, confirming a stable cartilage phenotype. In the ex vivo osteochondral model, hiCHO transplants demonstrated strong integration, progressive defect filling, and seamless ECM continuity with native cartilage. The regenerated tissue displayed rich glycosaminoglycan content and structural features comparable to hPAC reference constructs.

Conclusions

Dynamic bioreactor culture significantly improves the size, quality, and maturity of hiPSC-derived cartilage constructs, producing homogeneous, stable chondrogenic tissues. Their successful engraftment and integration in human osteochondral explants highlight the translational promise of this platform for developing scalable, off-the-shelf hiPSC-based cartilage grafts.

Acknowledgements

This work was supported by the NWA-ORC program project LS-NeoCarE (NWA.1389.20.192) financed (partly) by the Dutch Research Council (NWO).

References

1. Hajmousa G et al., Clin Epigenet. 2024

2. Ramos YFM et al., PLoS One. 2014

3. Houtman E et al. Human, Rheumatol Ther. 2021

4. Sayedipour SS et al., Comput Struct Biotechnol J. 2025

882. Influence of culture conditions on sweat gland regeneration in a tissue-engineered skin substitute

Henri De Koninck¹, Karel Ferland¹, Christian Martel², Danielle Larouche², Lucie Germain¹

¹Department of Surgery, Faculty of Medicine, Université Laval, QC, Canada. Centre de recherche en organogénèse expériementale de l'Université Laval/LOEX, CHU de Québec-Université Laval Research Centre, Québec, QC, Canada, Quebec - Canada, ²Centre de recherche en organogénèse expériementale de l'Université Laval/LOEX, CHU de Québec-Université Laval Research Centre, Québec, QC, Canada, Quebec - Canada

Severe burns represent complex injuries that are challenging to treat. To address this, the LOEX has developed a tissue-engineered skin substitute (TES) capable of covering large body surfaces. However, this model does not contain skin appendages. Among these, sweat glands play a central role in thermoregulation, but their regeneration after severe injury is very limited, and no effective treatment currently exists to restore their function. My project aims to integrate sweat gland cells (SGCs) into the TES in order to promote the formation of functional glands. The hypothesis is that SGCs can reform glandular structures when placed in an appropriate microenvironment.

To evaluate this hypothesis, SGCs were isolated from human skin and cultured either as monolayers (2D) or as spheroids (3D), and subsequently incorporated into TES constructs. They were then characterized by immunofluorescence and flow cytometry using specific markers. Our results showed that 2D culture modified the differentiation state of SGCs, inducing an expression profile similar to that of keratinocytes, and disrupted the distribution of SGC subpopulations. In contrast, 3D culture allowed SGCs to maintain their glandular phenotype, but reduced their proliferative capacity. Furthermore, after integration into the TES, SGCs cultured in 2D exhibited the ability to cluster into epithelial cell aggregates, though without regaining glandular differentiation. Conversely, SGCs derived from 3D cultures maintained the expression of markers characteristic of sweat glands up to 16 days after incorporation into the TES.

In summary, our results demonstrate that the culture method directly influences SGC differentiation and that the TES provides a favorable microenvironment for their clustering and maintenance of glandular identity. Additional analyses are underway to optimize the strategy for integrating SGCs into the TES. Ultimately, this work could lead to the production of the first bilayered skin substitute endowed with functional sweat glands for the treatment of severe burn patients.

883. Correlative spatiotemporal analyses of preclinically tested in situ tissue-engineered heart valves to illuminate mechano-inflammatory processes underlying tissue regeneration

Anthal Smits¹, Sofia Artamonova¹, Elmer Middendorp¹, Sylvia Dekker¹, Bente De Kort¹, Arturo Lichauco¹, Eva Brauchle², Jolanda Kluin³, Elena Aikawa⁴, Katja Schenke-Layland², Carlijn Bouten¹, Sandra Loerakker¹

¹Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven (Noord-Brabant) - The Netherlands, ²Department for Medical Technologies and Regenerative Medicine. Eberhard Karls University, Tübingen (Baden-Wberg Bayern) - Germany, ³Department of Cardiothoracic Surgery. Erasmus Medical Center, Rotterdam (Zuid-Holland) - The Netherlands, ⁴Division of Cardiovascular Medicine, Brigham and Women's Hospital. Harvard Medical School, Boston (Massachusetts) - United States

Introduction: There is a strong clinical need for living heart valve replacements that can grow and adapt with patients, especially for children with congenital heart defects. We previously developed resorbable microfibrous polymer-based valves, which are now in clinical trials. These valves promote in situ tissue regeneration while being immunologically resorbed by inflammatory cells. However, our preclinical studies revealed pronounced spatiotemporal variations in polymer replacement by regenerated tissue[1]. To better understand this, this study aimed to elucidate how local inflammation and mechanical loads influence the balance between tissue formation and scaffold resorption in a long-term in vivo model.

Methods: Electrospun supramolecular elastomer valves were implanted in the pulmonary position in sheep, as previously reported[2]. Explants at 6 and 12 months were analyzed using a 30-antibody panel to map the spatiotemporal distribution of inflammatory and tissue-producing cells and extracellular matrix composition. Raman microspectroscopy assessed local tissue and scaffold composition at the molecular level. Mechanical properties and thickness of implants and explants were measured and integrated into computational models for each individual valve to calculate strain distributions during remodeling.

Results and Conclusions: Overall, tissue gradually matured into a pseudo-layered structure partly resembling native valve composition at both tissue and molecular levels. Polymer resorption varied strongly across regions and was associated with local inflammatory and tissue-producing cell phenotypes, indicating that resorption is a cell-driven process rather than a passive time-dependent reaction, in line with previous findings[3]. Mechanical behavior transitioned from scaffold-dominated linear elasticity to collagen-dominated non-linear elasticity, reflecting polymer-to-tissue transition. Notably, the calculated local strains—but not stresses—correlated with local inflammatory markers (IL-10, CD163, TNF-α). These findings indicate that strain distribution influences inflammation and regeneration, providing design cues for optimizing valve microstructure and geometry to promote favorable in situ remodeling.

References: [1] De Kort, Acta Biomater 2021; [2] Kluin, Biomaterials 2017; [3] Wissing, Front Bioeng Biotechnol 2019.

884. Large-scale automated production of bone and cartilage organoids for osteochondral implants through closed robotics, bioprinting and noninvasive monitoring

Isaak Decoene¹, Dimopoulos Andreas¹, Krieger Judith², Nienhaus Florian², Nießing Bastian², Papantoniou Ioannis¹

¹Prometheus Division of Skeletal Tissue Engineering. KU Leuven, Leuven (Brabant) - Belgium, ²Department of Bio-adaptive Production. Fraunhofer Institute of Production Technology, Aachen (Nordrhein-Westfalen) - Germany

Replacement of functional tissues and organs by engineered alternatives is a challenge that has yet to become clinical and commercial reality. Current organoid-based approaches offer well controlled and high-quality building blocks that are compatible with modular biofabrication technologies such as 3D bioprinting. However, their production is labour intensive, not scalable and highly variable. Hence, during H2020 Jointpromise project, an end-to-end organoid factory was built converting those manual protocols into standardized automated-robotic workflows, providing precision, accuracy, and scalability from single-cell suspension to organoid production.

The current work showcases the evaluation of a fully automated workflow of the robotic platform compared to its manual process using human periosteum derived cells for bone-forming organoids and articular chondrocytes for cartilage organoids. First, cells were seeded via the robotic platform into nonadherent microwell plates. Both bone- and cartilage forming organoids were differentiated using the same chondrogenic medium for 14 and 7 days respectively. Medium changes were performed automatically every 3 days by the robotic platform, and after each action, the enclosed high-speed microscope was used as process monitoring tool. Finaly, organoids were harvested automatically, with the microscope providing analytics like organoid yield and homogeneity. Further analysis included histology and gene expression. The spheroids were subsequently mixed with collagen type I and bioprinted to form a cylindrical high-density bi-layered osteochondral construct.

Histological analysis supports high similarities in manual versus robotically produced organoids. While both populations were subjected to chondrogenic differentiation medium, articular cartilage organoids displayed a stable cartilage phenotype, while periosteal organoids display a fibrocartilaginous phenotype according to their function in endochondral fracture repair. While the robotically biofabricated spheroid-based osteochondral implant demonstrate homogeneity and distinct zonated hierarchical shape, composed by the two differentiated populations. To conclude, the future work will focus on the preclinical validation towards clinical translation, of human-sized-robotic-biofabricated osteochondral implants.

885. Magnesium-iron doped hydroxyapatite/collagen scaffolds for enhanced antimicrobial activity and bone regeneration

Diana Pacheco¹, Adriana Barroso¹, Rafaela Seabra¹, Lourenzo Apolloni², Belén Torrejón³, Gloria Ramírez-Rodríguez³, Joana Valente⁴, Liliana Gonçalves⁵, Carolina Armés⁶, Henrique Armés⁷, Lino Ferreira⁸, Artur Mateus⁹, Paula Faria¹, Nuno Alves¹, Abílio Sobral¹⁰, Telma Encarnação¹⁰, Andrea Ruffini², Silvia Panseri², Monica Montesi², Tatiana Patrício¹

¹CDRSP. Polytechnic of Leiria, Leiria - Portugal, ²CNR-ISSMC, Faenza (Italia) - Italy, ³Department of Inorganic Chemistry. University of Granada, Granada - Spain, ⁴Leiria. Polytechnic of Leiria, Leiria - Portugal, ⁵Hospital Veterinário de S. Bento, Rua de S. Bento (Lisboa) - Portugal, ⁶Hospital Veterinário de S. Bento, Rua de S. Bento (Lisboa) - Portugal, ⁷Hospital Veterinário de S. Bento, Rua de S. Bento (Lisboa) - Portugal, ⁸Faculty of Medicine at the University of Coimbra, Center of Neurosciences and Cell Biology (CNC), Coimbra - Portugal, ⁹ISISE - Institute for Sustainability and Innovation in Structural Engineering Department of Civil Engineering, Coimbra - Portugal, ¹⁰Coimbra Chemistry Centre-Institute of Molecular Sciences (CQC-IMS), Department of Chemistry, University of Coimbra, Coimbra - Portugal

Bone injuries represent a challenge in healthcare, increasing the interest in biomimetic and smart biomaterials [1]. Hence, hybrid magnetic materials were produced by heterogeneous nucleation of magnesium-iron-doped hydroxyapatite (HA) nanocrystals onto collagen type I, freeze-dried, dehydrothermally crosslinked (DHT) and functionalised with citrate. A comprehensive characterization showed chemical, thermal, morphological, mechanical, physical, magnetic, antimicrobial, and biological (in vitro and in vivo) favourable performance of these scaffolds. DHT treatment successfully improved the mechanical performance while preserving the scaffolds chemical composition, morphology and thermal characteristics. The results also showed that both the type of dopants had a substantial impact on the biological response. Antimicrobial assays demonstrated that iron and magnesium doping can significantly enhance the scaffolds’ ability to inhibit S. aureus, adding an additional advantage by reducing the risks of infection. The use of multi-ion doped hidroxiapatite/collagen scaffolds therefore represents a promising strategy to support mesenchymal stem cell differentiation and promoting new bone formation in vivo. Specifically, the magnesium-iron-doped HA/collagen scaffolds exhibited magnetic response, suitable mechanical performance and biological activity, suggesting its potential for future strategies involving magnetically stimulation for bone regeneration.

Keywords: Biomimicry; biomineralization; magnetic scaffolds; biological performance

Reference

[1] https://doi.org/10.3390/ijms252312766.

Acknowledgements

The authors acknowledge Fundação para a Ciência e a Tecnologia for its financial support via the CDRSP (DOI: 10.54499/UID/04044/2025) and ARISE (DOI: 10.54499/LA/P/0112/2020). The support of the project OP-SMARTherapy, 2022.04238.PTDC (FCT), BioRobotBeads (POCI-01–0247-FEDER-047081) (ANI) and PREDICTOS project, from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement No 101079372. To FCT by supporting this through PhD grant n° 2023.00333.BD (https://doi.org/10.54499/2023.00333.BD). We acknowledge funding from the Coimbra Chemistry Centre – Institute of Molecular Sciences (CQC-IMS) which is supported by the Fundação para a Ciência e a Tecnologia, Portuguese Agency for Scientific Research. through projects UID/PRR/00313/2025 (https://doi.org/10.54499/UID/PRR/00313/2025) and UID/00313/2025 (https://doi.org/10.54499/UID/00.

886. Detection of microbial DNA in gut and intervertebral discs of germ free and specific pathogen free mice

Ashish Kumar¹, Rosemary Abdoul Ahad², Nora Brigit Joseph², Junhua Wang², Ziad Al Nabhani², Alessandro Bertolo³, Benjamin Gantenbein⁴

¹Tissue Engineering for Orthopaedics and Mechanobiology, Bone and Joint Program, Department for BioMedical Research (DBMR). Medical Faculty, University of Bern, Bern - Switzerland, ²Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, Department for BioMedical Research (DBMR). Medical Faculty, University of Bern, Bern - Switzerland, ³Swiss Paraplegic Research, Nottwil (Luzern) - Switzerland, ⁴Department of Orthopaedic Surgery & Traumatology, Inselspital, Bern University Hospital. Medical Faculty, University of Bern, Bern - Switzerland

Introduction:

Recent evidence suggests that the microbiome may play a key role in intervertebral disc degeneration (IDD). Bacterial infiltration into the disc tissue could contribute to IDD and low back pain, and several studies have identified Cutibacterium acnes in pathological discs. This study evaluates whether mice are a suitable model for examining IVD microbial colonization by analyzing IVD and gut samples from specific pathogen-free (SPF) mice, which lack defined pathogens but retain normal microbiota, and germ-free (GF) mice, which are completely free of microorganisms.

Methods:

DNA was extracted using the QIAamp UCP Pathogen Mini Kit from IVD and gut tissues of female C57BL6J mice (n = 3) under SPF or GF conditions. DNA concentrations were measured with Qubit. A defined bacterial mixture (ZymoBIOMICS™ Spike-in Control I) was added before 16S rRNA amplification. Sequencing was performed using nanopore technology on a MinION device, followed by base calling with Dorado and filtering with Nanofilt (1400–1600 bp, q-score > 9).

Results:

No bacterial DNA was detected in any IVD samples from SPF or GF mice, indicating absence of microbial load. This finding matched the results from GF gut samples, confirming sterility. In contrast, bacterial DNA was detected in gut tissue from two of three SPF mice. Analysis of the ZymoBIOMICS™ Microbial Community Standard demonstrated a strong correlation between expected and measured relative abundances, supporting methodological accuracy.

Discussion:

These findings indicate that SPF mice may be unsuitable for studying a native IVD microbiome, as discs lacked detectable bacterial DNA. The study also confirmed the GF status of GF mice. Future work will include faecal samples to further understand the gut-disc axis and the potential relationship between gut microbes and IVD health.

Keywords: Microbiome, low back pain, intervertebral disc, degeneration

Acknowledgments:

Supported by SNSF Weave Grant (#320030E_224175) and DFG (#437213841).

887. A dual-purpose hydrogel platform for mechanical reinforcement and biological regeneration of the nucleus pulposus in dynamic bovine organ culture

Parisa Torabi¹, Ashish Kumar¹, Leon Schlagenhof¹, Mohammad Abdekhodaie², Michael Wöltje³, Benjamin Gantenbein⁴

¹Tissue Engineering for Orthopaedics and Mechanobiology, Bone and Joint Program, Department for BioMedical Research (DBMR) of the. Medical Faculty, University of Bern, Bern - Switzerland, ²Environmental and Applied Science Management, Yeates School of Graduate Studies. University of Toronto, Toronto (Ontario) - Canada, ³TUD University of Technology. Institute of Textile Machinery and High Performance Material Technology, Dresden (Sachsen) - Germany, ⁴Department of Orthopaedic Surgery and Traumatology, Inselspital, Bern University Hospital. Medical Faculty, University of Bern, Bern - Switzerland

Introduction: Low back pain, largely linked to degenerative disc disease (DDD), affects over 568 million people and arises from loss of water and proteoglycans in the nucleus pulposus (NP), leading to reduced intervertebral disc (IVD) height, altered biomechanics, and chronic pain. Existing treatments focus on symptomatic relief rather than restoring IVD function. To address both the mechanical and biochemical deficits of the IVD, this study introduces an injectable biomimetic hydrogel composed of hyaluronic acid (HA), collagen type II (COLII), and silk fibroin (SF) microfibers, engineered to replicate the native NP environment and support functional IVD repair.

Methods: HA and COLII were chemically modified with tyramine. Bone marrow–derived mesenchymal stromal cells (BMSCs) were encapsulated within HA/COLII and HA/COLII/SF hydrogels. Degenerated bovine IVD explants were generated by papain injection and subsequently treated with the hydrogels. The treated IVDs were then cultured in a custom bioreactor applying cyclic compression and torsion to simulate physiological loading.

Results: Live/dead staining (Calcein AM/ethidium homodimer-1) indicated high cell viability across all hydrogel formulations. Phalloidin/4′,6-diamidino-2-phenylindole staining revealed progressive actin filament organization and network formation over 15 days. After adding ZrO2 to the hydrogels as a contrast agent, micro-computed tomography confirmed homogeneous hydrogel filling within the NP region post-injection. Macroscopic examination revealed improved tissue integrity and mechanical stability. Magnetic resonance imaging (MRI) analysis further demonstrated that hydrogel-treated IVDs retained hydration and height under dynamic loading compared to papain-degenerated controls.

Discussion: This study demonstrated that injectable HA/COLII/SF hydrogels served as an effective carrier for stem cell delivery and promoted structural restoration in dynamically loaded IVD organ cultures. The system offers a promising platform for studying minimally invasive regenerative therapies targeting DDD.

Acknowledgments: This work was supported by the SNSF Weave Grant (#320030E_224175) and the DFG (#437213841). We thank Wolf-Dieter Zech, Julia Bruenig, and Andrina Auderset for MRI assistance.

888. Computational filamented-light: in-silico modelling for process optimization in the biofabrication of highly aligned tissue engineered constructs

Filippo Andreucci¹, Amedeo Franco Bonatti¹, Theofanis Stampoultzis², Carmelo De Maria¹, Marcy Zenobi-Wong², Giovanni Vozzi¹

¹Research Centre “Enrico Piaggio”, Pisa (Toscana) - Italy, ²ETH Zurich, Zurich - Switzerland

Filamented Light (FLight) Biofabrication represents one of the most promising approaches for the engineering of highly aligned and anisotropic tissues. It allows the fabrication of hydrogel networks characterized by unidirectional micro-filaments and interconnected micro-channels with diameters on the length scale of single cells, which provide the right topographical guidance for tissue growth and maturation.

Despite the understanding about the physical phenomena that lead to the formation of micro-filamented polymer networks during the FLight process, consisting in the projection of optically coherent light patterns in photo-crosslinkable resins, no attempts were made in designing computational models that could predict the outcome of the biofabrication process.

Such models could provide valuable insights into the optimization of the process, evaluating ideal light source parameters and resin properties to achieve the desired architecture for the engineering of different anisotropic tissues, such as muscle, cartilage and nervous tissue.

Here, a Matlab-based model of the FLight process is presented. The model takes light parameters such as power, wavelength, pattern and exposure time as inputs, along with the optical characteristics of the photo-resins, and produces three-dimensional models of the biofabricated constructs.

The computational model generates high fidelity three-dimensional representations of the biofabricated constructs for different light patterns and resin compositions, and could potentially be implemented to guide the fabrication of tissue-specific biologically relevant constructs. Validation of the model has been performed by quantitatively comparing the morphology of constructs obtained in-silico and experimentally under the same conditions.

Simulated and experimental results were compared in terms of morphological parameters such as porosity, micro-fiber and micro-channel diameters, and micro-fiber length.

Overall, the results suggest that the model can be a valuable tool for guiding the biofabrication of biologically relevant highly aligned constructs.

This project, granted under GA n° 101191804, is funded by the European Union. The opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them.

889. Ultrasound-modulated hydrogel for precision bone regeneration: on-demand release for enhanced healing

Ruth Gregg¹, David Hoey², Fiona Freeman³

¹CÚRAM Research Ireland Centre for Medical Devices, NUIG, School of Mechanical and Materials Engineering, UCD, Conway Institute, UCD,. University College Dublin, Dublin - Ireland, ²Trinity Centre for Biomedical Engineering, TCD, Department of Mechanical Manufacturing, and Biomedical Engineering, TCD, Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD. Trinity College Dublin, Dublin - Ireland, ³School of Mechanical and Materials Engineering, UCD, Conway Institute, UCD, CÚRAM Research Ireland Centre for Medical Devices, NUIG. University College Dublin, Dublin - Ireland

Bone is a dynamic tissue with strong regenerative capacity, but large-scale defects from traumatic injuries or tumour resection often result in non-union fractures. Tissue engineering offers promise for bone repair, yet clinical translation remains limited because therapeutic delivery typically requires supraphysiological dosages to elicit a therapeutic response, with little control over timing, which can cause off-target effects. Timing of healing stage progression remains an area of active research, regarding the role the immune system plays in bone healing. This is of interest in bone regeneration following tumour resection, where the patient’s immune system is compromised. Therefore, this project aims to develop an ultrasound-responsive hydrogel capable of drug release on-demand, offering spatiotemporal control of drug delivery to the defect site.

Sodium alginate (SA) (3.5% w/v) of at low, medium and high viscosity was dissolved in αMEM, then crosslinked using Calcium Chloride (CaCl₂) or Calcium Sulphate (CaSO₄) at a 25:9 ratio. 20kDa Blue Dextran allowed for assessment of drug release via spectrophotometric analysis, and degradation measured by gel mass. Ultrasound was applied at 20kHz for 30s with a 50% duty cycle for two weeks, incubating the gels in diH2O at 37.5°C between measurements.

We demonstrated SA is ultrasound-responsive where release/degradation is tailorable by viscosity and crosslinking agent, shown by accelerated degradation and increased drug release under ultrasound stimulation for all gels. CaSO4 slowed degradation and release compared to CaCl2, and as viscosity increased, degradation and drug release slowed. Future work will build upon these results by formulating microspheres of SA, characterised by size, encapsulation efficiency, and degradation. The gels will be validated as an on-demand delivery system via release of doxorubicin to osteosarcoma cell lines. Furthermore, we will explore the release profile for a range of model drugs +/-ultrasound application to harness the therapeutic effect of spatiotemporal control over the bone microenvironment.

890. Tissue-engineered oral mucosa epithelial grafts on fibrin scaffold for bilateral limbal stem cell deficiency: clinical outcomes with histological follow-up and complementary molecular characterization

Eustachio Attico¹, Elio Mignogna¹, Sybille Barvaux², Elena Enzo¹, Mattia Forcato³, Lorena Losi⁴, Silvio Bicciato³, Antonio Del Sol Mesa², Alessandro Lambiase⁵, Paolo Rama⁶, Graziella Pellegrini¹

¹Center for Regenerative Medicine “S. Ferrari”. University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ²Luxembourg Centre for Systems Biomedicine (LCSB). University of Luxembourg, Luxemburg (Luxembourg) - Luxemburg, ³Dept. of Molecular Medicine. University of Padova, Padova (Veneto) - Italy, ⁴Unit of Pathology, Dept. of Life Sciences. University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ⁵Dept. of Sense Organs. Sapienza University of Rome, Rome (Lazio) - Italy, ⁶SC Ophthalmology. IRCCS Policlinico San Matteo Foundation, Pavia (Lombardia) - Italy

Bilateral limbal stem cell deficiency (LSCD) poses a significant therapeutic challenge due to the lack of autologous limbal stem cells (LSCs) suitable for transplantation. While approaches such as Cultivated Limbal Epithelial Transplantation (CLET) are well established for unilateral disease, bilateral LSCD requires alternative autologous epithelial sources. In this context, we describe a tissue-engineering strategy that employs autologous oral mucosal epithelial cells expanded ex vivo on a fibrin scaffold.

Two patients with bilateral LSCD were treated using this fibrin-based epithelial graft. The production process adhered to stringent quality, safety, identity, and potency controls, including verification of epithelial lineage markers, assessment of stemness-associated profiles, sterility testing, and evaluation of graft integrity and functionality prior to release for clinical use.

Both patients experienced early functional improvement following transplantation. Each subsequently underwent penetrating keratoplasty (PK), offering the unique opportunity to perform an in-depth histological evaluation of the excised corneal buttons. These specimens were analysed for epithelial differentiation status, stemness markers, neovascularization, myofibroblast-associated fibrosis, and inflammatory cell infiltration, providing a comprehensive view of graft behaviour and host–tissue interaction.

Clinically, the patients were followed for over one year. Before the PK, one patient seemed to evolve towards progressive conjunctivalization, whereas the other developing a mixed corneal–conjunctival epithelial profile, whose long-term implications remain to be clarified.

To further elucidate the biological mechanisms underlying these divergent outcomes, we performed single-cell RNA sequencing (scRNA-seq) analyses of corneal, conjunctival, and oral mucosal cultured epithelial cells. This molecular profiling aims to identify the pathways governing epithelial identity, plasticity, and regenerative potential, ultimately supporting future innovations in tissue-engineered therapies and direct transdifferentiation strategies.

Overall, this work confirms the clinical feasibility of a fibrin-based, tissue-engineered autologous oral epithelial graft for bilateral LSCD, underscores the value of rigorous process controls and histological follow-up, and lays the groundwork for personalized regenerative strategies informed by high-resolution molecular data.

891. Evaluating intestinal organoids and primary monolayers as in vitro models for inflammation-driven changes in epithelial function

Tom Walraven¹, Jingxuan Wang¹, Mathias Busch¹, Nynke Kramer¹, Hans Bouwmeester¹

¹Department of Toxicology. Wageningen University & Research, Wageningen (Gelderland) - The Netherlands

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is characterized by chronic relapsing inflammation of the gastrointestinal tract. During active inflammation, disruption of the intestinal barrier increases susceptibility to foodborne contaminants and drugs, which is not fully addressed in risk assessments based on healthy individuals. In this project, we evaluated the suitability of adult tissue-derived intestinal organoids as a model for inflammation-related responses relevant to toxicological applications.

Intestinal organoids offer increased human relevance compared with Caco-2 cells, as they retain donor-specific genetic and phenotypic features. However, their 3D structure limits accessibility for apical exposure and transport studies. Therefore, we compared inflammatory responses in organoids with those of adult stem cell-derived monolayers, which provide improved experimental accessibility while maintaining key primary-cell characteristics. Cultures were exposed to bacterial TLR agonists as well as proinflammatory cytokines, and inflammatory activation was assessed through cytokine production, morphological changes, and transcriptional profiling. ADME-related endpoints, including barrier integrity, CYP3A4 activity and cytotoxicity were measured to evaluate how inflammation alters epithelial function across these complementary model systems.

Together, this work advances the development of in vitro intestinal models for studying IBD-related epithelial dysfunction and supports more tailored risk assessments for individuals with impaired intestinal barrier function.

892. Engineering of human bone marrow niches to investigate leukemic chemoresistance and metabolism

Marc Du¹, Carson C Cole², Victoria Lambrecht¹, Andrés García-García¹, Ivan Martin¹

¹DBM. University of Basel, Basel (Basel-Stadt) - Switzerland, ²DBSSE. ETH Zurich, Basel (Basel-Stadt) - Switzerland

Acute myeloid leukemia (AML) is a low-incidence, high-mortality cancer characterized by the clonal expansion of mutated hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). Bi-directional crosstalk between AML cells and BM niche environments is essential in regulating cancer attributes such as chemoresistance and metabolic adaptation. Current research primarily relies on mouse models, which present ethical, economic, and biological limitations, or 2D cultures and organoids that lack complexity, lumen accessibility, and have to be split regularly.

This project aims to utilize engineered 3D human BM niches as biomimetic in vitro models to study AML chemoresistance, metabolism, and cell-niche crosstalk. A scaffold was developed by crosslinking collagen I sponges using carbodiimide/N-hydroxysuccinimide (EDC/NHS), improving structural stability and preventing contraction during niche formation. The scaffold was populated with stromal vascular fraction (SVF) cells, subsequently seeded with healthy HSPC or AML cells, and treated with chemotherapy. Crosslinked scaffolds demonstrated enhanced HSPC seeding efficiency, viability, and stemness, while AML cells showed increased cancer marker expression and robust chemoresistance. Combining chemotherapeutics with fatty acid oxidation inhibitors showed synergistic effects, altering metabolism and cell proliferation. Confocal microscopy revealed a highly vascularized phenotype with vessel-adjacent CXCL12 expression and a gradient distribution of adipocytic and endothelial cells resembling red and yellow BM. Finally, the incorporation of biomimetic peptides into the scaffolds was explored to tailor the niche properties. Scaffold peptide modification altered niche composition, with differing effects on proportions of endothelial, pericytic, hematopoietic, and osteogenic cells within the niches. Spectral flow cytometry enabled quantitative readouts of characteristic populations found inside the niche and highlighted strong recapitulation of the physiological BM niches. Current work focuses on creating a composite niche model, taking advantage of scaffolds with different peptide modifications. First attempts showed promising results in recapitulating BM complexity in vitro. These engineered niches hold strong promise for studying AML pathophysiology.

893. Elastin-like recombinamer fibers as modular elements for the fabrication of tissue equivalents

Svenja Deichmann¹, Ikram El Maachi¹, Alexander Loewen¹, Sergio Acosta², Stephan Ruetten³, J. Carlos Rodríguez-Cabello², Stefan Jockenhoevel¹, Alicia Fernández Colino¹

¹Applied Medical Engineering, Department of Biohybrid & Medical Textiles (BioTex), Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen (Nordrhein-Westfalen) - Germany, ²Bioforge Lab, LaDIS. Universidad de Valladolid, Valladolid - Spain, ³Electron Microscopy Facility. RWTH Aachen University Hospital, Aachen (Nordrhein-Westfalen) - Germany

Introduction

Cardiovascular tissue mechanics rely on elastin and collagen to provide elasticity and long-term dynamic mechanical stability, yet most synthetic implants cannot replicate these properties. Elastin-like recombinamers (ELRs) offer a bioinspired approach for mimicking the native elastic behavior. In this work, we engineered ELR fibers with controlled diameters and porosities, paving the way for the design of hierarchical fiber assemblies to match the structural hierarchy and functionality of native tissues. This level of control supports more predictable mechanical behavior and advances the development of ELR-based vascular grafts and heart valves.

Materials and Methods

ELR fibers were produced via micro-molding combined with catalyst-free click chemistry, using chemically functionalized ELRs that crosslink efficiently under mild conditions. Microstructure was assessed with scanning electron microscopy and confocal microscopy, while tensile and cyclic tests under physiological conditions were performed to characterize stretchability, recoil, and fatigue resistance. Sterilization compatibility, cell response under dynamic conditions, and textile formation were also evaluated.

Results

The fabrication method enabled to manufacture fibers with precisely controlled diameter and porosity. The fibers showed exceptional elasticity (up to 500% stretchability) with excellent recoiling and mechanical stability under cyclic loading in physiological conditions. Human primary endothelial cells formed a confluent layer around the fibers and were able to align in response to cyclic stretching, making them a powerful tool for studying cellular behavior under physiological relevant dynamic conditions.

Conclusions

This study provides a versatile platform for producing elastin-like fibers with tunable mechanical and biological properties for cardiovascular implants. Their high stretchability and excellent recoil could improve implant durability and more closely replicate native tissue behavior than traditional materials. Tailorable ELR sequences allow control over cell adhesion and degradation, enhancing biocompatibility and integration. The elastic mechanical performance and reproducible fabrication position these fibers as promising candidates for the next generation of cardiovascular implants.

Reference

El Maachi I. et al, 2024, 10.1002/adfm.202313204

894. Dual-functional polydopamine spheres loaded with antimicrobial peptides to support bone regeneration in infected conditions

Laia Moliner-Carrillo¹, Giovanni Ferrandi¹, Carles Mas-Moruno¹, Maria-Pau Ginebra¹, Anna Diez-Escudero¹, Maria Godoy-Gallardo¹

¹Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering and Institute for Research and Innovation in Health (IRIS), Universitat Politècnica de Catalunya - BarcelonaTech, Barcelona - Spain

Introduction

Implant-related infections and impaired osseointegration remain major limitations in bone regenerative therapies. Although polydopamine (PDA) coatings are widely used, their application as multifunctional carriers have been scarcely explored. Here, we developed a multifunctional platform based on PDA spheres functionalized with antimicrobial peptides (AMPs; Lf1-11 and 4Dab-13) to simultaneously provide antibacterial activity and promote osteogenesis. To better mimic clinical scenarios, a cell–bacteria co-culture model was implemented, enabling evaluation interactions between hosts, pathogens and materials under infection conditions.

Methods

PDA spheres were synthesized via dopamine self-polymerization and covalently functionalized with AMPs. The platforms were immobilized onto biomaterial substrates to generate active surfaces. Surface morphology and chemistry were analysed using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Antibacterial activity against S. aureus and P. aeruginosa was evaluated through adhesion assays, metabolic activity measurements, colony-forming unit (CFU) counting, and live/dead staining. Human mesenchymal stem cells (hMSCs) were cultured on modified and control surfaces under standard conditions and in co-culture with bacteria to simulate early-stage implant infection. Cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and osteogenic gene expression (RUNX2, ALP, OCN) were assessed.

Results

PDA–AMP spheres were homogeneously distributed, increasing surface roughness and bioactivity. Functionalized surfaces significantly reduced bacterial adhesion and viability. Under co-culture conditions, the PDA–AMP platform protected hMSCs from bacterial induced damage, preserving viability and morphology. Additionally, PDA–AMP modification enhanced hMSC adhesion and proliferation and promoted osteogenic differentiation, evidenced by increased ALP activity and upregulation of osteogenic markers compared to unmodified surfaces.

Conclusion

PDA–AMP spheres create a dual-function interface capable of suppressing bacterial colonization while enhancing osteogenesis, even under infection mimicking conditions. This multifunctional strategy represents a promising approach for next-generation antibacterial and osteoinductive implants.

Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation (PID2023-148538OB-I00 and RYC2022-038428-I).

895. Engineering tuneable collagen microgels to control stem cell fate for enhanced bone regeneration

Zarina Issabekova¹, Lluís Oliver-Cervello¹, Jonathan Williams², Yecang Chen³, Marco Cantini¹, Cristina Gonzalez-Garcia¹

¹Centre for the Cellular Microenvironment. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, ²Centre for the Cellular Microenvironment. University of Strathclyde, Glasgow (Glasgow City) - United Kingdom, ³Biomedical Engineering Department. University of Glasgow, Glasgow (Glasgow City) - United Kingdom

Controlled delivery of therapeutic proteins, such as growth factors (GFs) [1, 2], combined with bioactive microcarriers can stimulate cell growth and differentiation, offering a promising strategy for bone tissue regeneration in cases of non-union defects. Injectable collagen (COL) microgels produced via flow-focusing droplet-based microfluidics enable precise control over biomechanical properties and protein release, making them suitable microcarriers for minimally invasive delivery and for promoting mesenchymal stem cell (MSC) differentiation toward the osteogenic lineage. This work investigates multiple strategies to enhance MSC differentiation when seeded on or encapsulated within COL microgels, including biochemical induction through GF delivery, polysaccharide-based stimulation using the Aloe Vera-derived acemannan to reduce or eliminate GF requirements, and mechanical stimulation via bioreactor culture. Building on these findings, we aim to develop an in vitro vascularised bone model composed of a COL microgel-hydrogel composite designed to better mimic the native bone extracellular matrix. In this model, “bone beads” generated via the aforementioned strategies are embedded within a bioactive bulk hydrogel populated with human umbilical cord endothelial cells, creating a supportive niche for cell migration, proliferation, and differentiation. Monodisperse COL microgels with tuneable mechanical properties and controlled degradation rates were successfully fabricated. In vitro studies revealed that COL microgels containing acemannan and GF support MSC viability and proliferation while promoting osteogenic differentiation, as demonstrated by increased expression of key markers such as alkaline phosphatase, osteocalcin, and osteopontin after 14 and 28 days of culture. Together, these findings establish tuneable COL microgels as a powerful injectable platform for controlled protein delivery and MSC osteogenic induction. Moreover, the integration of microgel-derived bone beads within a vascularised hydrogel bed provides a robust and physiological relevant in vitro model, advancing both regenerative therapies and the next generation of engineered bone tissue platforms.

(1) Oliveira et al. Int. J. Mol. Sci. 22(2), 903. 2021.

(2) Sarrigiannidis et al., 2021, 10.1016/j.mtbio.2021.100098.

896. Cryopreserved cartilage-mimetic implants for endochondral bone regeneration in maxillofacial defects

Flurina Staubli¹, Kenny Man¹, Leanne De Silva¹, Silke Nurmohamed¹, Nard Janssen¹, Eelco Bergsma¹, Alessia Longoni², Debby Gawlitta¹

¹Department of Maxillofacial Surgery and Special Dental Care. University Medical Center Utrecht, Utrecht - The Netherlands, ²Department of Orthopedics. University Medical Center Utrecht, Utrecht - The Netherlands

Inspired by developmental bone formation, endochondral bone regeneration (EBR) leverages a cartilage intermediate to orchestrate vascularized bone repair. We previously developed soft callus mimetics (CMs): small, devitalized cartilage units derived from allogeneic mesenchymal stromal cells (MSCs) that serve as modular, cartilage-mimetic scaffolds for EBR and previously supported healing of large bone defects in a goat orthopedic model[1]. While these findings establish CMs as a scalable, off-the-shelf scaffold platform, their performance in maxillofacial sites remains unknown, and the long-term functional stability of stored allogeneic constructs has not yet been evaluated. Therefore, this study assessed (i) the regenerative capacity of CMs in a clinically relevant goat alveolar cleft model and (ii) the impact of long-term cryostorage on CM bone-forming potency.

In a bilateral alveolar cleft model (n = 10), goats received a CM-based graft in surgically created maxillary defect and an autologous iliac crest graft contralaterally. After three months, microCT, histology, histomorphometry, and fluorochrome labeling showed that CM-treated defects achieved mineralization volumes (CM: 170.0±92.9mm³, autograft: 144.2±104.4mm³) and defect bone fractions (CM: 44.6±11.9%, autograft: 43.9±11.9%) equivalent to autograft controls. Bone architecture and temporal formation dynamics were likewise comparable, demonstrating that these devitalized cartilage-mimetic scaffolds support robust regeneration even in an intramembranous environment.

To evaluate scaffold stability during storage, CMs, either freshly prepared or cryopreserved for nine months at −80°C, were implanted into immunocompetent rats. After three months, microCT and histomorphometry showed comparable mineralization volumes (fresh: 2.6±1.6mm³, stored: 2.0±1.3mm³) and bone fractions (fresh: 26.3±8.4%, stored: 22.3±7.8%), indicating preserved regenerative potency following extended cryostorage.

Together, these findings show that modular, MSC-derived CMs function as a robust devitalized scaffold capable of effective bone regeneration in maxillofacial sites and maintain full bioactivity after long-term cryopreservation, supporting their translation as practical, off-the-shelf solution for scalable, donor-independent bone reconstruction.

[1] de Silva et al. Adv Healthc Mater. 2023

897. 3D in vitro microvascular model for non-penetrating traumatic injury research

Carla Verónica Fuenteslópez¹, Mark S. Thompson¹, Hua Ye¹

¹Institute of Biomedical Engineering. University of Oxford, Oxford (Oxfordshire) - United Kingdom

Microvascular injury critically influences the progression and severity of traumatic injuries, yet existing in vitro models often fail to replicate microvascular architecture and function accurately in 3D. This research presents the optimisation of a 3D hydrogel-based in vitro model that supports the formation and long-term stability of microvascular endothelial networks for trauma research.

Human dermal microvascular endothelial cells (MVECs) were embedded in fibrin-based hydrogels, and their performance was benchmarked against the widely used human umbilical vein endothelial cells (HUVECs). Systematic variations in hydrogel composition (fibrinogen source and concentration, crosslinking ratio, and medium) were examined to assess their effects on scaffold material properties and endothelial network formation, architecture, and longevity.

Network analysis showed that hydrogels formulated with high concentrations of human fibrinogen, a 200:10:1 fibrinogen:thrombin:CaCl2 crosslinking ratio, and either endothelial basal medium (EBM) or EBM supplemented with VEGF supported the most robust and durable microvascular networks, maintaining structural integrity for up to 14 days. In contrast, HUVEC-based models underwent rapid network degradation within 24 hours. Microrheometry revealed that increasing fibrinogen concentration significantly accelerated gelation kinetics, increased storage and loss moduli, and reduced creep compliance, thereby improving the constructs’ mechanical stability.

To investigate trauma mechanisms, controlled non-penetrating injuries (contusion, compression, and strain) were applied to optimised 3D microvascular constructs. This approach enabled the characterisation of immediate and short-term microvascular responses, establishing crucial links between trauma parameters and the resulting injury.

The optimised model offers a physiologically relevant platform for studying traumatic injury and evaluating therapeutic strategies to improve vascular repair and recovery.

898. Layer-by-Layer coatings of tannic acid and arginine for improved biological sealing of dental implant abutments

Inger T. M. Ernoe¹, Daria Zaytseva-Zotova¹, Catherine A. Heyward², Hanna Tiainen¹

¹Department of Biomaterials. University of Oslo, Oslo - Norway, ²Oral Research Laboratory. University of Oslo, Oslo - Norway

Successful integration of dental implants with the surrounding soft tissue is a requirement for long term clinical stability. Incomplete peri-implant sealing predisposes the site to bacterial colonisation, inflammation and tissue degradation. To date, an optimal implant abutment surface modification that promotes a tight biological seal has not been established. Tannic acid (TA) shows promise due to its anti-inflammatory properties and reduction of oxidative stress and pro-inflammatory cytokines. However, controlled release is necessary as high local concentrations of TA can hinder initial wound healing. In this study, we explored biological coatings for the functionalization of titanium dental implant abutments. The coatings consisted of tannic acid (TA) and arginine and were applied by a layer-by-layer (LbL) technique. The LbL coatings delivered a more controlled release of TA and exhibited moderate antioxidative effects compared to the TA coating. No acute cytotoxicity was observed, and metabolic activity remained high across all coating formulations. hGFs exhibited robust attachment and elongated fibroblast shape, although some cell aggregation occurred on TA coatings. The multilayer coated surfaces attenuated IL-6 secretion under inflammatory conditions while NF-κB activation was unaffected. These results demonstrate that TA-arginine multilayer coatings preserve hGF viability and exhibit selective anti-inflammatory activity. Such multi-component bioactive nanocoatings represent a promising strategy for engineering of anti-inflammatory dental implant abutments that can improve soft tissue sealing and thereby reduce peri-implant disease risk.

Acknowledgements: This project was financially supported by the Research Council of Norway (grant# 302590).

899. Bioactive nanoparticles combined with antibiotic and mucolytic: an immunomodulatory strategy against implant-associated infections

Isabel Izquierdo-Barba¹, Alberto Polo Montalvo², Monica Cicuendez², Daniel Arcos³

¹Química en Ciencias Farmaceuticas. UCM. CIBER de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Spain, Madrid - Spain, ²Química en Ciencias Farmaceuticas.UCM. Universidad Complutense de Madrid, Madrid - Spain, ³Química en Ciencias Farmaceuticas UCM. CIBER de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Spain, Madrid - Spain

Implant-associated infections represent a significant clinical challenge due to the severity of their consequences, which include persistent inflammation, bone loss, and potential implant failure, despite their relatively low incidence in most procedures. This problem has driven the development of therapeutic strategies capable of combating infection and, simultaneously, promoting bone regeneration. In this context, immunomodulation emerges as a promising approach, especially given the central role of macrophages in pathogen response, inflammation resolution, and tissue repair [1]. Currently, our research group has demonstrated that nanoparticles based on bioactive mesoporous glasses (MBGNs) display remarkable potential in the treatment of implant-associated infections when they combine the therapeutic action of an antibiotic (levofloxacin) with the biofilm-disrupting capacity of the mucolytic agent N-acetylcysteine. Previous studies have shown that N-acetylcysteine not only enhances biofilm dispersion but can also exert an immunomodulatory effect. Herein, we investigate the effects of these MBGNs—loaded with levofloxacin and covalently functionalised with N-acetylcysteine—on macrophages at different time points. Internalization assessed by transmission electron microscopy, cell viability, phenotype activation, and cytokine production were evaluated. The results show that these nanosystems are efficiently internalized by macrophages, preserve cell viability, and are capable of modulating macrophage activation toward phenotypes associated with both effective antimicrobial response and tissue repair, accompanied by a cytokine profile consistent with a balanced immunomodulatory effect. These findings highlight the promising potential of these nanosystems for the treatment of implant-associated infections.

1. Chen, Z., Wang, Y., Liu, X. y col. Harnessing osteoimmunity to treat peri-implant inflammatory osteolysis. Materials Advances. 2024; 5: 769-783. DOI:10.1039/D3MA00733B.

2. Polo-Montalvo, A.; Gómez-Cerezo, N.; Cicuéndez, M.; González, B.; Izquierdo-Barba, I.; Arcos, D. Osteogenic and Antibacterial Response of Levofloxacin-Loaded Mesoporous Nanoparticles Functionalized with N-Acetylcysteine. Pharmaceutics 2025, 17, 519. https://doi.org/10.3390/pharmaceutics17040519

Acknowledgements: Spanish Ministerio de Ciencia e Innovación (PID2023-149093OB-I00, MAGEN4BONE) and Fundación Ramón Areces (FD5/22_01, Nano4Infection) for funding

900. Vascularized tissue constructs designed for direct connection to the patient’s circulation

Michael Pflaum¹, Helms Florian¹, Klingenberg Melanie¹, Becker Imke¹, Kühne Hanna¹, Ruhparwar Arjang¹, Wilhelmi Mathias¹

¹HTTG/NIFE. Hannover Medical School, Hannover (Niedersachsen) - Germany

Introduction

The clinical translation of bioengineered tissues requires the integration of a patent, perfusable microvasculature capable of immediate blood flow post-implantation. To address this challenge, we developed a scalable platform technology enabling the formation of a pre-vascularized tissue construct with interconnected microchannels that can be directly anastomosed to the patient’s vasculature.

Methods

A custom flow chamber housed two 5 mm-diameter fibrin-based vascular prostheses, each connected to inlet and outlet ports for continuous perfusion with culture medium. The chamber was filled with a potentially autologous fibrinogen/thrombin hydrogel containing GFP-labeled endothelial cells (ECs) and adipose tissue-derived stromal cells (ASCs). After gel consolidation, removable cannulas were withdrawn to create microchannels between the prostheses, which were then luminally seeded with ECs. A removable top lid enabled air-liquid interface culture after keratinocyte seeding. Constructs were cultured under dynamic, physiological flow conditions for up to 14 days, followed by histological analysis and fluorescence microscopy.

Results

Within 14 days, ECs and ASCs formed a capillary-like network throughout the tissue construct. Keratinocytes formed a confluent epithelium covering the whole 3 × 3 cm area. Histological sections confirmed the vascular network integration. Notably, red blood cells introduced during the last 30 minutes of perfusion were detected within microvascular structures, indicating functional connectivity to the engineered microchannels and the pre-formed fibrin prostheses.

Conclusion

We demonstrate the feasibility of generating vascularized tissue constructs with immediate perfusion potential. This platform enables direct anastomosis of the fibrin prostheses to the host vasculature, offering a promising strategy for clinical applications in large-scale tissue reconstruction.

901. Multiscale mechanical characterization of viscoelastic hydrogels for designing regenerative tissue scaffolds

Antonio Minopoli¹, Davide Evangelista¹, Matteo Marras¹, Giordano Perini¹, Valentina Palmieri², Marco De Spirito¹, Massimiliano Papi¹

¹Department of Neuroscience. Università Cattolica del Sacro Cuore, Rome (Lazio) - Italy, ²Consiglio Nazionale delle Ricerche, Rome (Lazio) - Italy

Mechanical cues play a fundamental role in guiding cell behavior, influencing processes such as adhesion, proliferation, differentiation, and matrix remodeling in regenerative medicine. Therefore, accurate assessment of scaffold mechanics across multiple length scales is essential for engineering biomaterials that closely replicate native tissue microenvironments. In this work, we integrate bulk rheological measurements with nanoscale mechanical mapping via atomic force microscopy (AFM) to characterize the viscoelastic response of hydrogels designed for use as regenerative scaffolds. While conventional Hertzian elastic models are commonly applied to AFM data, they neglect the intrinsic rate-dependent behavior of soft biological materials. Here, indentation curves were analyzed using established viscoelastic models capable of separating elastic and viscous contributions and accounting for the influence of loading rate.

Four commonly used hydrogel formulations (alginate, Cellink-RGD, GelMA, GelMA-A) were evaluated to map nanoscale stiffness variation and correlate it with macro-scale rheological behavior. All materials demonstrated non-linear stiffening with increasing indentation rate, reflecting polymer relaxation dynamics and crosslinking density. Additional tests on biological samples, including erythrocytes and zona pellucida, further highlighted how viscoelastic modeling enables discrimination of physiological and structural differences at the nanoscale.

These results demonstrate that multiscale viscoelastic characterization provides a more realistic picture of scaffold mechanics compared to purely elastic models. By predicting nanoscale stiffness as a function of deformation rate, our approach offers a practical design tool for developing hydrogels with mechanical properties tailored to specific tissue regeneration targets, ultimately supporting more reliable translation of biomaterial platforms into clinically relevant applications.

902. 3D-bioprinted breast tumour with embedded plasmonic biosensors for spatially resolved drug screening

Lara Troncoso Afonso¹, Paula Vázquez-Aristizábal¹, Gail A. Vinnacombe-Willson¹, Yolany M. Henríquez-Banegas², Patricia González-Callejo³, Pablo Valera-Sapena³, Malou Henriksen-Lacey³, Clara García-Astrain⁴, Luis Liz-Marzán³

¹Bionanoplasmonics. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ²Universidad del País Vasco, Donostia-San Sebastián (Gipuzkoa) - Spain, ³CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ⁴POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain

The development of antitumoral drugs is limited by the lack of in vitro platforms that simultaneously combine biological relevance, spatial complexity, and analytical sensitivity to effectively study cellular responses.[1] Despite many 3D in vitro models accurately mimic the tumour microenvironment, analysing drug responses within these complex environments remains challenging. To address this, we propose a new class of hybrid biomaterials that integrate hydrogel-forming polymers with gold nanorods, engineered for 3D bioprinting. These constructs support cell growth and organization while incorporating plasmonic functionality to allow in situ, label-free monitoring of molecular biomarkers via surface-enhanced Raman spectroscopy (SERS).

Gold nanorods (AuNRs) were embedded into a click cross-linkable mixture of thiolated alginate and methacrylated carboxymethylcellulose. These hydrogel forming polymers were synthesized and characterized by 1H-NMR and FTIR, while AuNRs were characterized by UV-VIS and TEM. The rheological properties and the kinetics of the crosslinking were determined. The microstructural features of the material were imaged by SEM and the SERS performance of the material was evaluated for different analytes. [2] Then, the material was used to 3D print pillar-shaped SERS-active structures within a complex 3D breast tumour model.[3]

The embedded SERS-active pillars enabled non-invasive, real-time monitoring of drug distribution and cellular responses within both tumor and stromal compartments. The treatment of the model with a chemotherapeutic drug (6-thioguanine) revealed differences in absorption between the core and the stroma, demonstrating the potential of the 3D breast cancer model to test chemotherapeutic efficacy within more realistic microenvironments. Overall, integrating plasmonic sensors into bioprinted tumor models can improve analytical sensitivity, potentially aiding in drug screening, biomarker identification, and personalized cancer research.

References

[1] L. Troncoso-Afonso, et al. Chem. Soc. Rev. 2024, 53, 5118-5148

[2] L. Troncoso-Afonso, et al. Biomater. Sci. 2025, 13, 2936-2950

[3] P. González-Callejo, et al. Mater. Today Bio 2023, 23, 100826

903. Influence of hyaluronan molecular weight on equine MSC phenotype and their responses to osteoarthritis-like inflammatory stimulation

Alice Ramesova¹, Claudia Bergow¹, Janina Burk¹

¹Physiology and Pathophysiology, Department of Biological Sciences and Pathobiology. University of Veterinary Medicine Vienna, Vienna (Wien) - Austria

Mesenchymal stem cells (MSCs) are a promising tool in regenerative therapy of osteoarthritis (OA). Due to similarities in joint size and cartilage properties, horses represent an excellent translational model for studying naturally occurring OA in humans. Sodium hyaluronate (HA) is widely used in OA treatment for its viscoelastic and anti-inflammatory properties; however, its exact molecular-weight–dependent effects remain unclear. High-molecular-weight HA (HMWHA) predominates in healthy joints and acts anti-inflammatory, while low-molecular-weight HA (LMWHA) increases in OA and is considered pro-inflammatory.

This study investigated whether HMWHA or LMWHA pretreatment modulates equine MSC responses to subsequent OA-like stimulation. Adipose-derived MSCs were pretreated for four days with 1 mg/ml HMWHA (2247 kDa) or LMWHA (29 kDa), or cultured without HA. To mimic an OA-like environment, cells were subsequently stimulated for two days with IL-1β (10 ng/ml) and TNF-α (50 ng/ml), or with cytokines together with LMWHA. Metabolic activity, proliferation, and protein/gene expression were assessed after pretreatment and inflammatory challenge.

Both HMWHA and LMWHA pretreatment significantly reduced MSC metabolic activity, while HMWHA increased the percentage of Ki-67⁺ MSCs, indicating higher proliferation. After inflammatory stimulation, Ki-67 increased, particularly in HMWHA-pretreated cells. Expression of adhesion markers CD44 and CD29 increased after HA pretreatment, especially in LMWHA-treated cells. Additional cytokine and LMWHA stimulation elevated CD29, suggesting an adhesion-related response specific to LMWHA. HA pretreatment also induced transcriptional changes. HMWHA shifted MSCs toward a matrix-stabilizing, anti-inflammatory phenotype (↓MMP9, ↓PTGS2, ↑MMP14, ↑TIMP2, ↑VCAM1, ↑HGF). Upon inflammatory challenge, VCAM1 markedly increased in HMWHA-pretreated MSCs, whereas PTGS2, MMP9, and MMP13 were similarly upregulated and HGF downregulated in all cytokine-stimulated groups, independent of pretreatment and concurrent LMWHA exposure.

In summary, HMWHA and LMWHA exert distinct effects on MSCs: HMWHA promotes a matrix-stabilizing, anti-inflammatory state, while LMWHA enhances adhesion-related signaling. These findings clarify how HA molecular weight modulates MSC behavior.

904. Copper-containing mesoporous bioactive glass nanoparticles in bone infection treatment

Mónica Cicuéndez¹, Marina Luna¹, Ana García¹, Blanca González¹, Carla Jiménez-Jiménez¹, Montserrat Colilla¹, Isabel Izquierdo-Barba¹

¹Universidad Complutense de Madrid, Madrid - Spain

In recent years, the use of nanomaterials for diagnosis and targeted therapy emerged as an alternative to conventional treatments. Among the different types of nanoparticles (NPs), mesoporous bioactive glass NPs present significant advantages [1, 2]. Staphylococcus aureus (S. aureus) is one of the most common pathogens in bone infections. Current treatment for this type of infections consists of a cocktail of antibiotics over a long period of time producing drug-resistant strains. In this scenario, the use of metal cations has emerged as a promising alternative to antibiotics. Regarding Cu2+ cations, is involved in cytokines action, expression of diverse matrix proteins or growth factors related to wound healing like vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF-2). Moreover, Cu2+ cations exhibit growth inhibition and bactericidal effects on both Gram-positive and Gram-negative bacteria. Despite these beneficial implications, Cu2+ cation is toxic at high concentrations because it induces a heavy rise in intracellular free radicals [1]. Herein, we developed a multifunctional nanoantibiotic based on radial mesoporous bioactive glass NPs in the SiO2-CaO system (RNPs), incorporating different antibiotics (levofloxacin or rifampicin) and Cu2+ cations in their surface, to combat wound healing, infection and promote bone regeneration. Cu2+-containing rifampicin-loaded radial mesoporous glass nanoparticles (in the SiO2-CaO) (RNPs) with dual antimicrobial and wound regenerative capacity were synthesized by a simple and versatile method. RNPs were externally functionalized with a third-generationpolypropylene dendrimer (PPI-G3), which through its tertiary amines allows the complexation of metal ions and that via its protonated primary amines favours bacterial internalization.The nanosystem promoted fast wound closure (98.1%), high biocompatibility (>70%) and a great capacity for migration and invasion. The versatility of the nanosystem obeys its capacity to load any antibiotic and its ability to complex any therapeutic metal ion according to clinical needs.

(1) Salvo, J. et al. 2022, 10.1093/burnst/tkab047.

(2) Vallet-Regí et al., 2021, 10.1016/j.mtbio.2021.100121.

(3) González, B. et al. 2018, 10.1016/j.actbio.2017.12.041

(4) Alvarez, E. et al. 2021 10.1016/j.actbio.2021.09.027

905. Life with little oxygen – attractive and worthwhile?

Yvonne Roger¹, Kirsten Elger¹, Miriam Jakoby¹, Mara Menzel¹, Mara Menzel², Heiko Fuchs³, Isabel Deppe⁴, Letizia Venturini⁴, Michael Stadler⁴, Andrea Hoffmann¹

¹Department of Orthopedic Surgery. Hannover Medical School, Hannover (Niedersachsen) - Germany, ²Department of Orthopedic Surgery. Department of Orthopedic Surgery. Hannover Medical School, Hannover (Niedersachsen) - Germany, ³Department of Ophthalmology. Hannover Medical School, Hannover (Niedersachsen) - Germany, ⁴Department of Hematology, Hemostaseology, Oncology and Stem Cell Transplantation. Hannover Medical School, Hannover (Niedersachsen) - Germany

The increasing life expectancy is leading to a growing number of patients with musculoskeletal disorders. For cell-mediated therapeutic approaches, human mesenchymal stromal cells (huMSCs) are a promising cell source. As cells with stem cell-features, the niche in which they reside in the body is of prime importance. One prominent feature of niches is their oxygen content, notably lower in all parts of bodies than in ambient air. This poses one urging challenge to conventional cell culture: upon isolation of the cells from bodies, subsequent culture manipulation, and eventually, upon transplantation back into bodies - steps associated with instantaneous dramatic changes in oxygen level. Recreating the natural oxygen environment can help to maintain the beneficial properties of huMSCs while understanding adaptations of cells to oxygen concentration will contribute to improving cell-mediated therapies based on optimized cultured cell procedures. To exclude as much air as possible during bone marrow harvest from the iliac crest of human healthy voluntary donors a three-way cock system was used, which was put into a hypoxic safety cabinet 24 h before use. After the bone marrow harvest the isolation of mononuclear cells was performed under hypoxic conditions and afterwards divided in normoxic and hypoxic conditions. Our studies reveal that specific features of huMSCs like surface antigens do not appear to be notably influenced by oxygen levels. On the contrary, low oxygen levels seem to have a positive effect on the gene expression of specific huMSC surface antigens like CD73, CD90 and CD105. Furthermore, genes like HIF1A, known to be upregulated under hypoxic conditions, as well as genes like GPNMB, FGF11, and PDG with less clear oxygen-related features are differentially expressed. Interestingly, our studies shed new light on HIF1A, downregulated in several donors under hypoxia.

906. Decoding bone-forming potential of engineered cartilage templates for endochondral bone regeneration

Debby Gawlitta¹, Flurina Staubli¹, Kenny Man¹, Eelco Bergsma¹

¹Department of Maxillofacial Surgery and Special Dental Care. University Medical Center Utrecht, Utrecht - The Netherlands

Endochondral bone regeneration (EBR) harnesses cartilage-mediated bone formation as a biomimetic strategy for bone repair. We previously developed soft callus mimetics (CMs): devitalized, allogeneic, cartilage constructs derived from mesenchymal stromal cells (MSCs) that are shelf-stable and support robust bone regeneration in small[1] and large[2] animal models. However, translation of cartilage matrix–based EBR remains limited by the lack of predictive quality attributes linking in vitro characteristics to in vivo bone formation. This study aimed to identify matrix constituents that predict bone-forming potential.

MSCs from six human donors were chondrogenically differentiated as spheroids (250 000 MSCs/spheroid, 28 days) using TGFβ (T) or TGFβ+BMP2 (TB). Following devitalization, CMs were characterized for matrix composition and differentiation markers, then implanted subcutaneously in immunocompromised rats (n = 8/group) and monitored by longitudinal microCT. After two months, samples were explanted and assessed by microCT to quantify mineralized tissue formation.

BMP2 increased spheroid size, GAG, VEGF and BMP2 content, ALP activity, and collagen II and X deposition. MicroCT revealed mineralization in 5.5± 5.1% of T and 45.3±20.1% of TB implants, with substantial donor variability. Five donors generated mineralizing TB CMs, whereas one donor failed to mineralize under either condition. One donor mineralized with T CMs, further enhanced under TB. These donor-dependent differences indicate that treatment conditions alone did not predict mineralizing capacity, despite overall improvement with TB. Neither, individual in vitro parameters nor marker combinations, correlated consistently with in vivo performance.

Ongoing endpoint histological analyses will determine whether the mineralized tissue represents mature bone. In parallel, proteomic profiling of the constructs will identify combinatorial molecular signatures predictive of mineralizing and bone-forming capacity. Establishing such multifactorial quality markers will support standardized manufacturing and quality control of EBR implants and facilitate broader clinical translation.

907. Magnetic control of neuronal alignment

Tasmin Nahar¹, Feng Xue¹, Monte Gates², Emilie Secret³, Neil Telling¹

¹School of Life Sciences. Keele University, Newcastle under Lyme (Staffordshire) - United Kingdom, ²School of Medicine. Keele University, Newcastle under Lyme (Staffordshire) - United Kingdom, ³PHENIX laboratory. Centre Interdisciplinaire de Recherche en Biologie (CIRB), Collège de France, CNRS, INSERM, Université PSL, Paris, France, Paris (Ile-de-France) - France

Aims and Objectives:

Magnetic nanoparticles (MNPs) enable the application of remote magnetic forces to guide axonal growth, offering a potential strategy for neural repair. While previous work has demonstrated magnetically induced neurite orientation, its impact on neuronal network formation and activity remains unclear. In parallel, it is not yet established whether distal axons can independently internalise magnetic material, which would be essential for applying forces directly to regenerating axons. This study combines functional electrophysiology with microfluidic free-axon analysis to examine how magnetic guidance influences both the structure and function of developing neuronal networks.

Materials and Methods:

Embryonic day 17 (E17) rat cortical neurons were cultured on microelectrode arrays (MEAs) and exposed to MNPs under a defined static magnetic field to induce oriented neurite outgrowth. Neurite orientation was assessed using two-dimensional Fourier transform analysis. Developing network activity was recorded longitudinally using the Axion Maestro system, examining firing behaviour, burst dynamics, and synchrony between magnetically aligned and control cultures.

In a complementary microfluidic platform, E17 neurons were seeded to allow axons to extend into isolated microchannels. Once axons had grown into the channels, magnetic nanoclusters (MNCs) were applied to determine whether distal axons could take up nanoparticles independently of their soma. Axonal uptake was assessed using fluorescence imaging.

Results:

Magnetically guided cultures showed neurite alignment along the magnetic force vector, the results assessing the functional trends will be presented. In the microfluidic system, free axons successfully internalised MNCs within the microchannels, confirming that distal axonal compartments can directly acquire magnetic material without soma involvement.

Conclusions:

These two complementary approaches demonstrate that magnetic nanoparticles influence both the structural organisation and functional maturation of neuronal networks, while free axons are capable of independently taking up magnetic material. Together, these findings support the feasibility of applying magnetic forces directly to regenerating axons and strengthen the translational rationale for magnetically guided strategies in neural repair.

908. Magneto-mechanotransduction: enhancing bone regeneration with magnetic nanoparticles

Manuel Estévez¹, Roberto Sánchez-Carrasco², Ana García¹, Blanca González¹, Mónica Cicuéndez¹, Daniel Arcos¹, Isabel Izquierdo-Barba¹

¹Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid - Spain, ²Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid - Spain

Bone fractures associated with osteoporosis remain a major global health challenge. Synthetic bone grafts are explored as alternatives to autografts, but the ideal material for bone repair has not been achieved. Magnetic nanomaterials enable active modulation of cellular behavior through remote mechanical stimulation [1]. Our research focuses on nanostructured scaffolds incorporating superparamagnetic iron oxide nanoparticles (SPIONs) to promote osteogenic differentiation via magneto-mechanotransduction.

SPIONs were synthesized via thermal decomposition and functionalized with the Arg-Gly-Asp (RGD) peptide to target cell integrins. Their osteogenic potential was assessed in vitro using human bone marrow mesenchymal stem cells (hBM-MSCs). SPIONs were integrated into two 3D scaffold systems: the first embedding SPIONs in type I collagen solutions to produce fibrous magnetic scaffolds via electrospinning; this design allows SPIONs to respond to external magnetic fields, generating subtle shifts in the fibre that could be perceived by adherent cells. The second approach employed polymeric scaffolds of poly(L-lactic acid), polycaprolactone, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), fabricated by fused deposition modeling and surface-functionalized with SPIONs via thermal inkjet printing, enhancing long-term nanoparticle–cell interactions and promoting osteogenic activity under magnetic stimulation.

In recent studies, our group has been investigating the osteogenic effects of SPIONs in the form of nanoflowers, with promising results. We are also exploring the incorporation of these SPIONs into hydrogels, which could improve cell-material interactions. Future studies will evaluate the integration of magnetic hydrogels in femoral heads from patients and analyze their osteogenic potential under magnetic fields.

These studies highlight SPION-based scaffolds and hydrogels as dynamic platforms for bone tissue engineering, advancing next-generation regenerative therapies through magneto-mechanotransduction.

[1] Wu C. et al., Adv. Mater. 2018, 30, 1705673.

Acknowledgements: Spanish Ministerio de Ciencia e Innovación (PID2023-149093OB-I00, MAGEN4BONE), (PID2023-150182OB-I00, BIOMAESTRO) and Fundación Ramón Areces (FD5/22_01, Nano4Infection) for funding.

909. Surgical navigation enhances precision and training in models for regenerative spine therapies

Andres Bonilla¹, Kristen Sack¹, Nicole Erben¹, Howard Seim¹, Jeremiah Easley¹

¹Translational Medicine Institute, Fort Collins (Colorado) - United States

Introduction:

Large animal spine models are essential for evaluating regenerative therapies, biomaterials, and implantable technologies due to their close anatomical similarity to humans. However, variability in implant placement remains a major limitation in preclinical tissue engineering studies, where the precision of delivery directly affects biological outcomes. Image-guided surgical navigation, widely used in clinical spine surgery, offers a powerful opportunity to refine these models by enabling accurate and reproducible placement of any implant, scaffold, biologic, or therapeutic device. Additionally, this technology provides an intuitive and safe platform for training new researchers, allowing students to learn advanced procedural skills relevant to regenerative medicine. This pilot study compares navigation-guided and freehand pedicle screw placement using novice operators as a proof-of-concept for broader application.

Materials and Methods:

Sixty pedicle screws were placed in lumbar vertebrae from six ovine cadavers. Thirty screws (n=30) were inserted with O-arm–based CT navigation, and thirty (n=30) were placed freehand. All procedures were performed by two veterinary students with no prior experience. Accuracy was evaluated using the Gertzbein–Robbins classification. Planning and drilling times were recorded to assess workflow and usability.

Results:

Navigation achieved high precision (80 percent Grade 0, 16.7 percent Grade 1) with no moderate breaches and only one severe violation (3.3 percent). Freehand placement resulted in lower accuracy with higher rates of moderate-to-severe breaches. Although navigation increased drilling time, planning was rapid, and novice operators demonstrated an immediate learning effect, successfully producing accurate placements on their first attempts.

Conclusion:

Navigation-guided instrumentation significantly improves precision, reproducibility, and safety in ovine spine procedures. Importantly, it also serves as an effective educational tool, enabling early-stage researchers to safely learn complex techniques that support consistent delivery of regenerative therapies. This technology holds strong potential to accelerate both the development and translation of tissue engineering strategies in large-animal models.

910. Personalized somatropin-loaded dressings for diabetic wound regeneration

Luis Diaz-Gomez¹, Maria Pita-Vilar¹, Diego Caicedo-Valdes², Angel Concheiro¹, Carmen Alvarez-Lorenzo¹

¹Departamento de Farmacología, Farmacia y Tecnología Farmacéutica. Santiago de Compostela University (USC), Santiago de Compostela (A Coruña) - Spain, ²Department of Vascular Surgery, Health Research Institute of Santiago de Compostela (IDIS), School of Medicine. Santiago de Compostela University (USC), Santiago de Compostela (A Coruña) - Spain

The development of advanced wound dressings is limited by traditional materials that lack tunable porosity and morphological control. In this work, 3D printing was used to fabricate personalized dressings based on carboxymethyl cellulose (CMC) and silk fibroin (SF), a protein valued for its anti-inflammatory activity yet limited by inherently low printability. Blending SF with CMC yielded inks with optimal rheology for high-fidelity 3D printing. The printed dressings were freeze-dried, crosslinked, sterilized, and subsequently loaded with somatropin (growth hormone, GH) to enhance regenerative activity. Structural analyses (SEM, micro-CT) confirmed a highly porous, interconnected architecture with strong fidelity to the digital design. SF-containing dressings exhibited improved exudate absorption, reduced swelling, and slower degradation compared to CMC-only controls. GH showed a rapid release profile, reaching 50% within 2 h and complete release within 24 h.

SF notably enhanced fibroblast proliferation, migration, and coagulation capacity, while in vitro cytocompatibility was maintained across all formulations. Chicken chorioallantoic membrane assays confirmed absence of tissue integration and demonstrated angiogenesis elicited by SF-containing dressings. In vivo testing in an ischemic diabetic wound model revealed accelerated wound closure and superior tissue regeneration in SF- and GH-loaded groups, with the CMC-SF-GH dressing achieving the most complete epithelialization and vascularization. Moreover, proteomic profiling showed early upregulation of proteins involved in fibroblast and endothelial proliferation, ECM remodeling, angiogenesis, and immune modulation, particularly in SF- and GH-treated wounds. By day 14, GH-treated groups maintained distinct regenerative signatures, including increased COL1A1/2, FIBG, FABP5, antioxidant enzymes, and annexins, alongside downregulation of inflammation- and degradation-associated proteins (e.g., MMP16, VIME, HSPB1).

In conclusion, the GH-loaded dressings developed in this study function as bioactive, patient-tailorable systems that enhance structural performance, modulate the wound microenvironment, and significantly accelerate healing, offering a promising platform for wound care.

911. Macrophage accumulation in deeper regions of equine exuberant granulation tissue suggests persistent fibroproliferative signaling

Lena Partusch¹, Stella Knüppel², Romanie Vervack¹, Jule K. Michler², Ward De Spiegelaere¹

¹Department of Morphology, Imaging, Orthopedics, Rehabilitation and Nutrition. Faculty of Veterinary Medicine, Ghent University, Ghent (Oost-Vlaanderen) - Belgium, ²Department of Biological Sciences and Pathobiology. University of Veterinary Medicine Vienna, Vienna (Wien) - Austria

In horses, being tight-skinned animals, second-intention wound healing is commonly observed in practice. Distal limb wounds — below the carpus and tarsus — are particularly prone to developing exuberant granulation tissue (EGT), a fibroproliferative disorder driven by dysregulated interactions between immune cells and hyperproliferating myofibroblasts. Previous studies have shown that EGT is histologically immature compared to control wounds, exhibiting altered collagen composition and abnormal myofibroblast activation. To further explore immune–fibroblast interactions in this pathology, we investigated the in situ distribution of macrophages within EGT.

Calcium-binding adapter molecule 1 (Iba1/AlF1), a widely used pan-macrophage marker in several species, has not previously been examined in EGT. In this study, 27 tissue samples from nine horses were analyzed using immunohistochemistry (IHC). For each sample, three 500 × 500 µm regions of interest were evaluated in both superficial and deep tissue layers. Deep regions contained significantly more Iba1-positive cells than superficially (Wilcoxon signed-rank test, p = 0.008).

To complement these findings, additional immunofluorescence (IF) staining was performed to qualitatively assess the spatial distribution and co-expression patterns of Iba1 with vimentin. IF confirmed the perivascular clustering of Iba1-positive cells observed in IHC. Many cells exhibited dual Iba1⁺/vimentin⁺ labeling, which may reflect vimentin expression in activated macrophages (macrophage to mesenchymal transition) and/or indicate close spatial interactions between macrophages and fibroblastic cells within the lesion.

In summary, this study provides the first in situ characterization of Iba1-positive cells in EGT and demonstrates their greater abundance in deeper tissue layers. This pattern is consistent with the deeper region’s more collagen type I–rich fibrotic matrix, suggesting that persistent macrophage–fibroblast interactions may contribute to maintaining the chronic fibroproliferative environment characteristic of EGT. Future work will involve an in vitro scratch assay to examine how macrophage-differentiated peripheral blood mononuclear cells influence migration, proliferation, and cytoskeletal dynamics of EGT-derived myofibroblasts.

912. HydroLIG: selective drug delivery via hydrogel/LIG smart actuators

Raquel Sanchez Diaz¹, Yawad Sbihi¹, Monsur Islam¹, De Yi Wang¹

¹IMDEA Materials Institute, Madrid - Spain

This work presents the development of an advanced drug delivery system combining poly(N-isopropylacrylamide) (PNIPAM) hydrogels with laser-induced graphene (LIG) electrodes, with the objective of enabling precise, on-demand, and localized drug release. PNIPAM hydrogels were synthesized with a lower critical solution temperature (LCST) around 37°C, ideal for biological applications. The hydrogels exhibited a swelling ratio of 13 times their dry weight and high porosity, ensuring excellent drug retention and release capabilities while maintaining stability at operational temperatures.

LIG electrodes, fabricated via laser engraving of flexible polyimide substrates, demonstrated high conductivity and rapid Joule-heating properties. These electrodes enabled precise thermal control of the hydrogel, reaching temperatures above its LCST in approximately 35 seconds. By varying the applied voltage, hydrogel temperatures could be modulated, even exceeding 55°C, allowing for faster or slower release rates depending on the therapeutic need.

Release functionality was validated using a dye solution as a drug model, with quantification via UV-Vis spectrophotometry confirming voltage-controlled, substance-specific release profiles. This system represents a significant advancement in the state-of-the-art by combining fast response times and precise thermal control. The hydrogel-LIG platform demonstrates significant potential for therapeutic applications, including advanced wound dressings, and addresses key technical and regulatory challenges for clinical translation.

913. Mechanically aligned Schwann cell constructs as biomimetic bands of Büngner for peripheral nerve regeneration

Carina Hromada¹, Dorota Szwarc-Hofbauer¹, Mai Quyen Nguyen², Janine Tomasch¹, David Hercher², Andreas Teuschl-Woller¹

¹Life Science Engineering. FH Technikum Wien, Vienna (Wien) - Austria, ²Ludwig Boltzmann Institute (LBI) for Traumatology, The Research Centre in Cooperation with AUVA, Vienna (Wien) - Austria

INTRODUCTION: Peripheral nerve injuries often result in poor functional recovery, highlighting the need for improved therapeutic strategies to effectively guide axonal regeneration. Schwann cells (SC) play a central role in this process by forming aligned cellular structures called bands of Büngner, which direct regenerating axons across nerve gaps. However, the formation of bands of Büngner is the major limiting factor during nerve regeneration.

AIM: This study aimed to engineer 3D bands of Büngner-like constructs by applying defined mechanical stimulation to SC–laden hydrogels. Specifically, the objectives were to: (i) induce longitudinal SC alignment via tensile strain, (ii) assess the repair SC phenotype, and (iii) evaluate whether mechanically aligned SC constructs promote axonal outgrowth.

METHODS: Primary rat SC were embedded in fibrin hydrogels and subjected to mechanical stimulation using a custom-made bioreactor. Incremental static strain was applied to induce SC elongation and alignment. Gene expression of repair-associated markers (BDNF, NGF, p75NTR) was quantified, and morphological alignment was assessed via immunofluorescence stainings. Functional evaluation was performed using co-culture with neonatal dorsal root ganglia, quantifying axonal extension and directionality.

RESULTS: Mechanical stimulation induced longitudinal SC alignment resembling native bands of Büngner and promoted a pronounced repair phenotype, demonstrated by significant upregulation of BDNF, NGF, and p75NTR. Functionally, aligned constructs supported significantly longer and more directed axonal outgrowth compared to unstimulated controls. Importantly, SC alignment remained stable after withdrawal of mechanical strain, highlighting structural robustness of these constructs for translational applications. Furthermore, first results will be presented showing the translatability from primary rat SC to human induced pluripotent stem cell (iPSC)-generated SC.

CONCLUSION: Mechanical stimulation presents a powerful strategy to engineer regeneration-promoting SC constructs capable of guiding axons in vitro. For potential future clinical applications as artificial nerve grafts, we are currently developing human SC constructs derived from iPSC.

914. Tissue-engineered reconstruction of a nasal pseudo-turbinate for empty nose syndrome

Giulia Galaverni¹, Davide Adamo¹, Anjana Chathanchirappattu Raj², Francesca De Carlo³, Simona Negoias⁴, Matteo Alicandri-Ciufelli⁵, Sandra Feliciano⁶, Andrea Barbero⁶, Ivan Martin⁶, Graziella Pellegrini²

¹Department of Life Sciences. Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ²Department of Surgical, Medical, Dental, and Morphological Sciences. Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ³Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ⁴Department of Otorhinolaryngology, Head and Neck Surgery. University Hospital of Basel, Basel (Basel-Stadt) - Switzerland, ⁵Department of Otolaryngology - Head and Neck Surgery, Azienda Ospedaliero-Universitaria Policlinico di Modena. University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ⁶Department of Biomedicine. University Hospital of Basel, Basel (Basel-Stadt) - Switzerland

Nasal turbinates are bony structures covered by submucosal tissue and respiratory epithelium that ensure proper warming and humidification of the inspired air. Surgical reduction or removal, commonly performed to treat turbinate hypertrophy or septal deviation, can inadvertently lead to Empty Nose Syndrome (ENS). ENS is an iatrogenic disorder marked by paradoxical nasal obstruction, dryness, crusting, and profound impairment of quality of life, often accompanied by severe psychological distress and an elevated risk of suicide.

While symptomatic treatments remain largely inadequate, experimental reconstructive approaches primarily aim to restore proper aerodynamics by reducing nasal cavity volume without targeting regeneration of the damaged nasal mucosa.

Here, we present a tissue-engineering strategy for nasal pseudo-turbinate reconstruction that integrates a bioengineered nasal cartilage graft, generated from a clinically validated collagen scaffold, with a functional human respiratory epithelium.

Our study focused on the in vitro co-culture of the two tissues: findings revealed the ability of nasal epithelial cells to stably adhere on the bioengineered cartilage while maintaining an adequate clonogenic, regenerative, and differentiation potential, and demonstrated sustained cartilage quality under extended culture in epithelial medium. Notably, we verified the maintenance of the epithelial stem cell population through clonal analysis, and conducted single-cell RNA-seq profiling to identify any subtle alterations in molecular pathways or differentiation processes.

Collectively, our study demonstrates the possible coexistence of multiple regenerated tissues in vitro, offering an innovative therapeutic perspective for patients suffering from ENS and tangible advancement for tissue-engineering strategies targeting complex organ reconstruction.

915. Native fibrillar-collagen bioink for cartilage tissue engineering

Teresa Zuñiga¹, Amaia Guembe², Iker Ateca², Tania Lopez-Martinez³, Froilan Granero-Molto³, Jesus Maria Izco⁴

¹Enabling technologies. Biomedical Engineering Program, CIMA, Pamplona (Navarra) - Spain, ²Viscofan SLU, Cáseda (Navarra) - Spain, ³Orthopedic Surgery and Traumatology. Clínica Universidad de Navarra Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona (Navarra) - Spain, ⁴Viscofan SA, Tajonar (Navarra) - Spain

In osteoarthritis (OA) progressive cartilage extracellular matrix (ECM) remodeling and degradation impair joint function. Biocompatible scaffolds offer promising strategies for cartilage repair and regeneration, and incorporating disease-relevant cell and matrix cues into 3D models is essential for developing physiologically accurate OA research platforms. In this study, we investigated the feasibility of a native fibrillar collagen type I 3D bioprinting bioink (Fibercoll-Flex-N^®) for cartilage tissue regeneration in comparison with alginate, a standard material with broad range of applications in tissue engineering. Half million of human nasal chondrocytes (hCN) were encapsulated in Fibercoll-Flex-N^® bioink (3% collagen concentration). A final volume of 80 μl per scaffold was extruded by hand and maintained three weeks in culture, or implanted ectopically into immunodeficient Rag2 mice (B6;129-Rag2tm1FwaIl2rgtm1Rsky/DwlHsd) for 12 weeks. In vitro, cultured scaffolds were mechanical characterized (elastic and viscous moduli, viscosity, and fiber length). Cell viability and proliferation were evaluated by AlamarBlue™ and Live/Dead™ assays respectively, as well as for the expression of chondrogenic markers. In vivo, ectopically implanted scaffolds were evaluated histologically through toluidine blue staining, and by immunofluorescence for the presence of chondrogenic maturation markers (Type II collagen, Type X collagen, MMP13). In vitro, collagen scaffolds showed higher viability, together with sustained expression of chondrogenic markers in comparison with the Alginate scaffolds. In vivo, after 12-week of implantation only Fibercoll-Flex-N^® bioink allowed sustained differentiation of chondrocytes and the production of and hyaline ECM as detected by Toluidine Blue metachromatic implants, and the presence of a rich type II collagen ECM, with reduced content of hypertrophic markers (Type X collagen, MMP13). In addition, no vascular invasion was detected in Fibercoll-Flex-N^® bioink based implants. In conclusion, Fibercoll-Flex-N^® scaffolds demonstrated suitability for supporting functional tissue models, and compatibility with human-derived chondrocytes, highlighting its significant potential for applications in cartilage tissue regeneration.

916. 3D bioprinting of hiPSC-derived neurons for brain-on-a-chip applications

Corinna Barella¹, Donatella Di Lisa¹, Alberto Lagazzo², Giulia Parodi¹, Sergio Martinoia¹, Laura Pastorino¹

¹Department of Informatics, Bioengineering, Robotics and Systems Engineering (DIBRIS). University of Genova, Genova (Italia) - Italy, ²Department of Civil, Chemical and Environmental Engineering (DICCA). University of Genova, Genova (Italia) - Italy

Modelling the functional and structural complexity of the central nervous system (CNS) in vitro remains one of the main challenges in neuroscience. To better reproduce cell-cell and cell-microenvironment interactions, advances in brain tissue engineering have driven the development of three-dimensional (3D) biofabricated brain models, exploiting biomaterials and emerging biofabrication technologies to replicate key features of brain tissue. In this context, the combination of human-induced pluripotent stem cells (hiPSCs) and 3D bioprinting represents a powerful strategy to engineer brain-like constructs with defined architecture and cellular heterogeneity.

This study presents the development of a novel thermosensitive chitosan-based bioink specifically designed for extrusion-based bioprinting of hiPSC-derived glutamatergic and GABAergic neurons co-cultured with astrocytes. Chitosan, a versatile and low-cost biopolymer, was selected for its ability to promote cell adhesion, neuronal growth, and network maturation. The bioink’s rheological and mechanical properties were characterized to evaluate shear-thinning behaviour, temperature-triggered gelation under physiological conditions, and stiffness values within the range of native brain tissue. A dedicated printing workflow was optimized to guarantee reproducible material deposition, high shape fidelity, and the fabrication of customizable patterned and compartmentalized 3D constructs.

The optimized formulation enabled the generation of stable neural constructs with well-preserved geometry, homogeneous cell distribution, and long-term cell viability. Morphological analysis confirmed the integration of neurons and astrocytes within the matrix. Functional characterization using high-density microelectrode arrays (HD-MEAs) revealed spontaneous electrophysiological activity and progressive network maturation under long-term culture conditions, validating the suitability of the bioink for functional neural modelling.

Overall, the developed chitosan-based bioink and optimized bioprinting protocol provide a scalable and reproducible platform for engineering functional 3D neural cultures. This approach offers significant potential for CNS modelling under both physiological and pathological conditions, paving the way for brain-on-a-chip models.

917. Environmental exposure as a driver of early pulmonary fibrosis onset

Annachiara Scalzone¹, Marco Lai², Claudia Mazio¹, Giorgia Imparato¹, Paolo A. Netti²

¹Istituto italiano di tecnologia, CABHC, Naples (Italia) - Italy, ²DICMAPI. University of Naples Federico II, Naples (Italia) - Italy

Pulmonary fibrosis (PF) is a progressive and irreversible interstitial lung disease whose early pathogenic mechanisms remain insufficiently understood. Although epidemiological evidence increasingly links chronic environmental exposure to PF development, the initial biological events that trigger the fibrotic cascade are still unclear. This gap is largely due to the absence of reliable human-relevant models capable of reproducing coordinated epithelial and stromal responses to inhaled irritants and the associated microenvironmental cues that shape connective tissue behaviour. As a result, the transition from early cellular injury to pre-fibrotic remodelling remains poorly characterised, despite its relevance for preventive therapeutic strategies.

To address this limitation, we developed a 3D human bronchial model that recapitulates key features of the airway microenvironment, including physiologic architecture, biomechanics and cell- extracellular matrix (ECM) interactions. The platform enables controlled exposure to environmental contaminants (silica nanoparticles) under realistic deposition conditions and allows us to fine-tune pollutant concentrations and exposure durations, reproducing patterns consistent with occupational or low-dose chronic exposure rather than acute, supraphysiological insults.

Under these optimised conditions, our data show that mitochondrial dysfunction arises as one of the earliest detectable responses, together with increased oxidative stress. In parallel, epithelial cells initiate a partial epithelial–mesenchymal transition (EMT), with reduced junctional integrity and cytoskeletal reorganisation. These epithelial alterations promote pathological crosstalk with the stromal compartment, where fibroblasts display early metabolic activation, enhanced contractile behaviour and initial ECM remodelling. Notably, this remodelling is accompanied by local matrix stiffening, a mechanical hallmark that amplifies fibroblast activation and accelerates progression toward a pre-fibrotic state. These events precede overt collagen accumulation, confirming the model’s ability to capture fibrogenesis at its onset.

Collectively, this 3D bronchial system provides a robust and human-relevant platform for dissecting pollutant-induced fibrotic pathways and identifying early biomarkers and preventive therapeutic targets.

Acknowledgments: BREATH project (CUP E53D23016840001)-Next Generation EU-PNRR-Mission 4, Component2-1.1, D.D. 14/09/2022 MUR).

918. Decoding tendinopathic TSPCs in a 3D ColMA tenogenic microenvironment

Giacomo Cortella¹, Erwin Pavel Lamparelli¹, Giovanna Della Porta¹

¹University of Salerno, Baronissi (Italia) - Italy

Tendon injuries remain difficult to treat due to their limited healing capacity and complex pathology. In this study, we developed a 3D bioprinted methacrylated type I collagen (ColMA) scaffold incorporating Growth Differentiation Factor-5 (GDF-5)–loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles to recreate a tenogenic microenvironment in vitro. Pathological human Tendon Stem/Progenitor Cells (hTSPCs), isolated from tendinopathic surgical explants, were encapsulated within this system to assess their extracellular matrix (ECM) remodeling deficits and associated inflammatory signaling.

GDF-5-loaded nanoparticles (140 ± 40 nm) were produced via microfluidic-assisted nanoprecipitation and uniformly integrated into the ColMA matrix, enabling controlled and sustained release of the growth factor. Dynamic perfusion culture (1 mL/min) ensured efficient mass transport and maintained cell viability above 70% over 21 days.

Pathological hTSPCs displayed marked ECM dysregulation, including the absence of type I collagen and a 2.56-fold increase in type III collagen by day 7, consistent with a fibrotic-like phenotype. Tenomodulin expression rose 5.31-fold at day 14, whereas ECM quantification revealed a pronounced type III/I collagen ratio of 4.5, further confirming impaired matrix organization. In parallel, the cells exhibited a strong pro-inflammatory profile, with sustained secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) from day 7 onward, reflecting their chronic inflammatory origin.

Altogether, this modular 3D ColMA-based construct effectively recapitulates critical aspects of tendinopathic microenvironments, offering a robust platform for mechanistic studies of tendon degeneration. By supporting long-term culture, sustained growth factor delivery, and quantitative readouts of ECM and inflammatory dynamics, this system provides a powerful in vitro model for advancing personalized regenerative strategies and for guiding the development of targeted therapeutics for chronic tendon disorders.

919. ALBUCOL: albumin-based biomaterials for skeletal tissue engineering

Emilien Lhospice¹, Jordan Beurton², Benoit Frisch², Eya Aloui³, Philippe Lavalle², Arnaud Scherberich¹

¹Department of Biomedicine. University of Basel, Basel (Basel-Stadt) - Switzerland, ²Biomaterials and Bioengineering. INSERM, Strasbourg (Alsace) - France, ³ALBUPAD, Biomaterials and Bioengineering. Université de Strasbourg, Strasbourg (Alsace) - France

Musculoskeletal disorders generally require tissue-engineered products. Commercially available collagen scaffolds often lack customization and are used for off-label applications. Albumin and collagen are promising natural materials, and their combination offers adjustable mechanics, adhesion sites and high biocompatibility. The aim of the ALBUCOL project is to build on this finding to develop customized collagen-doped albumin scaffolds for various clinical applications such as pediatric phalangeal reconstruction and biphasic tracheal graft.

Before combining biomaterials, different albumin sources have been tested for their intrinsic capacity to support chondrogenesis when using adipose stromal cells (ASC) as a cell source. Albumin scaffolds (here named ALBUCOL materials) originating from Egg-White, Ovalbumin and Bovine Serum Albumin were generated using an emulsion protocol with the use of various salts. ASC-seeded materials were cultured in vitro in chondrogenic conditions over 8 weeks and analyzed for cartilage differentiation in comparison to a commercial collagen scaffold.

After 4 and 8 weeks of in vitro culture, histological analysis showed a strong positive safranin-O staining with hypertrophic cartilage in all constructs. ALBUCOL-based cartilage tissues (CTs) were always larger but with comparable cartilage areas with collagen-derived CTs. This correlates with ALBUCOL materials not being remodeled in vitro, unlike collagen sponges. Glycosaminoglycans quantification normalized with DNA shows comparable CTs maturation but an increase in secreted cartilage matrix proteins in ALBUCOL-based CTs.

All ALBUCOL scaffolds supported ASCs chondrogenic differentiation and led to an increase in the size of the resulting construct. Their non-remodeling behavior in vitro could be further investigated for scaling up constructs for cartilage repair. Previous studies have shown that egg-white scaffolds (rich in ovalbumin) exhibit pro-angiogenic properties in vivo. Given that vascularization is necessary for the endochondral ossification of hypertrophic cartilage, ALBUCOL materials represent a promising innovation for potential bone regeneration applications.

Acknowledgement: This project is financed by the European INTERREG Upper-Rhine funding program.

920. Evaluation of the clinical usefulness of the NANOULCOR human bioengineered cornea in patients with severe corneal ulcers

Miguel Alaminos¹, Miguel Etayo-Escanilla², Carmen González-Gallardo³, Paula Ávila-Fernández², Jesús Chato-Astrain², Olimpia Ortiz-Arrabal², Fabiola Bermejo-Casares², Víctor Carriel², David Sánchez-Porras², Antonio Campos², Óscar Darío García-García²

¹Department of Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Department of Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ³Hospital Universitario Clínico San Cecilio and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Background: Cornea regeneration and repair is limited by the avascular nature of the human cornea, and severe ulcers affecting this organ are very difficult to heal.

Methods: Based on cultured human cornea epithelial and stromal cells combined with nanostructured fibrin-agarose biomaterials, we developed NANOULCOR as an anterior lamellar corneal substitute generated in a GMP facility as an ATMP. Histological evaluation of NANOULCOR revealed that this product reproduces the structure of the native organ, with a stratified epithelium on top and a subjacent bioengineered stroma containing abundant stromal cells immersed within the biomaterial. Analysis of relevant corneal markers showed positive epithelial expression of pancytokeratins PCK and AE1/AE3, cytokeratin KRT5 and crystallin alpha and lambda, along with several types of intercellular junctions. At the stromal level, these bioengineered tissues showed expression of collagen, proteoglycans, decorin, keratocan, and lumican. These results allowed us to implement a Phase-II clinical trial in patients with severe corneal ulcers, which was authorized by the Spanish Medicines Agency (EU CT number 2023-506856-25-00).

Results and conclusions: Preliminary results of the clinical trial demonstrate that the method is feasible, and NANOULCOR can be grafted at the eye surface of patients with severe corneal ulcers. In addition, no relevant side effects have been found to the date, suggesting that the procedure is safe for the patient. Although the results of this clinical trial should be confirmed at longer follow-up times, these findings suggest that NANOULCOR could be efficiently used for the clinical repair of corneal ulcers that are refractory to conventional treatments, using a novel regenerative approach based on a tissue engineered medicinal product.

Acknowledgements: Supported by grants ICI21/00010 (NANOULCOR) and FIS PI23/00335, Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, Spain. Cofinanced by the European Regional Development Fund, “Una manera de hacer Europa” program.

921. Continuous-fiber-reinforced 3D-printing: A novel manufacturing ap-proach for load-oriented 3D scaffolds in biohybrid implants

Alexander Loewen¹, Yasmin Kuhn¹, David Möllering¹, Lina Boughezala¹, Ingold Seidl², Mehdi Behbahani³, Stefan Jockenhoevel¹

¹Applied Medical Engineering, Department of Biohybrid & Medical Textiles (BioTex), Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen (Nordrhein-Westfalen) - Germany, ²Laboratory for Materials Engineering, FH Aachen - University of Applied Sciences, Aachen (Nordrhein-Westfalen) - Germany, ³Institute of Bioengineering, Biomaterials Laboratory, FH Aachen - University of Applied Sciences, Aachen (Nordrhein-Westfalen) - Germany

Introduction

Personalized medicine increasingly demands implant solutions tailored to individual mechanical and biological requirements. A major challenge lies in developing load-oriented biohybrid implants that combine structural integrity with biological compatibility. Existing textile-based and additively manufactured scaffolds offer valuable but distinct advantages — yet each has inherent limitations. This study introduces a novel fiber printing process designed to merge the tensile strength and flexibility of textile structures with the geometrical freedom of 3D printing, enabling the fabrication of load-oriented 3D scaffolds for biohybrid implants.

Methods

A fused deposition modeling (FDM) system was modified to enable continuous textile fiber feeding directly into the print head. Using this approach, composite scaffolds composed of a thermoplastic polyurethane (TPU) matrix reinforced with polyethylene terephthalate (PET) multifilament yarns were produced. The resulting structures were charac-terized in terms of mechanical performance, microstructural morphology, and biocompatibility.

Results

The fiber printing process successfully yielded thin-walled scaffold structures (≈150 µm) exhibiting the flexibility of TPU combined with the tensile strength and Young’s modulus typical of textile materials. The technique allowed the fabrication of single tracks, open-porous and closed surfaces, as well as complex 2D and 3D load-oriented geometries. Cell culture studies confirmed the absence of cytotoxic effects and demonstrated robust cell viability, adhesion, proliferation, and confluence on the printed scaffolds.

Conclusion

The developed fiber printing technique bridges the gap between textile engineering and additive manufacturing. By uniting mechanical advantages of continuous fiber reinforcement with the design versatility of additive manufacturing, it provides a new pathway for producing biomimetic, load-oriented scaffolds for biohybrid implants — offering a promising platform for personalized regenerative therapies.

922. A pathology-driven in vitro model of endothelial dysfunction revealing GEF-modulated oxidative and cytokine dysregulation

Saveria Batti¹, Arianna Mariacristina¹, Erwin Pavel Lamparelli¹, Myers Gina², Nicola Maffulli³, Giovanna Della Porta¹

¹Translational NanoMedicine Laboratory, Department of Medicine, Surgery and Dentistry. University of Salerno, Baronissi (Italia) - Italy, ²Incrediwear Holdings, INC., 3120 Thorntree DrChico, CA 95973, USA. University of Salerno, Baronissi (Italia) - Italy, ³Department of Trauma and Orthopaedics, Faculty of Medicine and Psychology, Sant’ Andrea Hospital, Sapienza University, 00189 Rome (RM), Italy. University of Salerno, Baronissi (Italia) - Italy

This study examines the biological activity of Germanium-Embedded Fabric (GEF) on inflammatory pathways using in vitro endothelial and monocytic models across single cultures, 2D co-cultures, and a 3D dynamic perfusion system. Initial optimization identified 10 ng/mL TNF-α and LPS as effective stimuli for inducing endothelial and monocyte activation, respectively, with a 3-day exposure providing consistent and reproducible inflammatory responses.

In isolated monolayer cultures, GEF did not alter cell viability but reduced reactive oxygen species (ROS) in HUVECs and modulated cytokine secretion in THP-1 cells, lowering IL-6 and increasing IL-2 levels. In 2D endothelial–monocyte co-cultures, GEF further decreased endothelial ROS and induced a biphasic ICAM-1 response, characterized by early upregulation followed by attenuation. Cytokine analysis revealed a transient rise in both pro-inflammatory (IL-8) and anti-inflammatory (IL-10, IL-4) mediators, suggesting a temporally balanced modulation of inflammatory signaling rather than a strictly suppressive or stimulatory effect.

Within the 3D bioprinted and perfused culture system, GEF maintained high cell viability and sustained its antioxidant activity, reducing ROS accumulation under inflammatory challenge. Cytokine profiles mirrored the 2D findings, with early peaks in IL-6 and IL-8 and a persistent but low release of IL-4, highlighting a controlled and nuanced immune response in a more physiologically relevant microenvironment.

Overall, these results indicate that GEF exerts a context-dependent, transient immunomodulatory effect characterized by an initial activation of inflammatory pathways followed by modulation and attenuation. The consistent antioxidant action across all models, coupled with the balanced cytokine response, suggests that GEF may help support controlled inflammation and promote tissue recovery. These findings position GEF as a promising bioactive material for regenerative medicine applications and for therapeutic strategies aimed at managing dysregulated inflammatory processes

923. The BioPacer – a tissue engineered biological pediatric pacemaker

Yasmin Kuhn¹, Alexander Loewen¹, Stefan Jockenhoevel¹

¹Applied Medical Engineering, Department of Biohybrid & Medical Textiles (BioTex), Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen (Nordrhein-Westfalen) - Germany

Introduction

Cardiac tissue engineering holds great potential for creating functional heart tissue in the lab. Here, we present an approach to design bioengineered cardiac tissue capable of reliably propagating electrical signals for the use as a biological pacemaker for children and newborns. This strategy addresses a key challenge in the field: enabling coordinated electrical conduction across engineered tissue, providing a solid foundation for future applications in regenerative medicine.

Methods

We designed a cylindrical bioengineered cardiac tissue construct using a tricellular culture that was cultured both statically and under uniaxial strain. Subsequent analysis by histology and immunostaining was performed to characterize tissue structure, composition, and the effects of dynamic cultivation on the construct. Furthermore, the tissue was analyzed in a specified experimental setup for controlled signal propagation after stimulation.

Results

The engineered cardiac tissue successfully propagated electrical signals from one end to the other. Conduction properties were influenced by physical parameters, for example, lower temperatures were associated with slower signal propagation. The constructs exhibited an intrinsic beating rhythm that also responded appropriately to pharmacological stimulation, such as epinephrine, with an increased frequency upon stimulation. Structural differences were observed between tissues cultured under static conditions and those subjected to uniaxial strain, highlighting the impact of mechanical stimuli on tissue organization.

Conclusion

These results demonstrate the potential of the engineered cardiac tissue in pediatric regenerative medicine, as it exhibits controlled electrical signal propagation and responds appropriately to physiological and pharmacological stimuli. Moreover, these properties highlight its promise as a potential building block for future biological pacemaker concepts.

924. Combining QCM-D with live cell imaging to reveal protein corona effects on fibroblast adhesion to tannic acid-coated surfaces

Enrique Oreja Remartinez¹, Hanna Tiainen², Daria Zaytseva-Zotova¹, Alejandro Barrantes Bautista¹, Josephine Franke¹

¹Department of Biomaterials. University of Oslo, Oslo - Norway, ²Department of Biomaterials. University of Oslo, Oslo - Norway

Interaction between proteins and surfaces is a determining factor that affects biomaterial integration. Proteins adsorption is the first step that occurs when a foreign body becomes in contact with physiological fluids and is the key to understand cell-biomaterial interactions. Our aim is to understand how proteins present in blood interact with tannic acid (TA) coated titanium surfaces and affect the interaction with human gingival fibroblasts (HGF)

Quartz crystal microbalance with dissipation (QCM-D) was used in combination with live-cell imaging to determine the dynamics of HGF adhesion onto TA-protein coated surfaces. The chemical/physical properties of the formed protein corona on TA-coated surfaces were investigated by FTIR, UV-VIS and surface energy and zeta potential measurements.

Fast individual adsorption of bovine serum albumin (BSA), fibrinogen (FNG) and fetal bovine serum proteins was observed in QCM-D on TA coated sensors. Surface saturation was only reached when working with higher protein concentrations, under the studied experimental conditions. Different phases of the protein corona formation were identified. QCM-D revealed that Vroman effect was observed when depositing BSA and subsequently FNG. Combined QCM-D and live cell imaging revealed the different phases of cell adhesion. Cell spreading was the highest when working with BSA-FNG. A correlation between cell area and changes in frequency and dissipation in QCM-D was made.

FTIR revealed conformational changes in secondary structure of proteins when deposited onto a TA layer. No evidence of new bands was observed in UV-VIS, although changes in the intensity of TA bands were observed, indicating changes in the molecular environment. Surface energy and zeta potential were reduced compared to TA-coated surfaces. Cell adhesion was delayed in presence of the protein corona compared to TA-coated surfaces.

925. 3D pathological hTSPC model for evaluating germanium-based anti-inflammatory intervention

Adamo Lancellotti¹, Claudia Orlanno¹, Erwin Pavel Lamparelli¹, Montella Federica¹, Myers Gina², Nicola Maffulli³, Giovanna Della Porta¹

¹Translational NanoMedicine Laboratory, Department of Medicine, Surgery and Dentistry. University of Salerno, Baronissi (Italia) - Italy, ²Incrediwear Holdings, INC., 3120 Thorntree Dr. Chico, CA 95973, USA. University of Salerno, Baronissi (Italia) - Italy, ³Department of Trauma and Orthopaedics, Faculty of Medicine and Psychology, Sant’Andrea Hospital, Sapienza University, 00189 Rome, Italy. University of Salerno, Baronissi (Italia) - Italy

This study investigates, for the first time, the biological effects of a semiconductor-based Germanium-Embedded Fabric (GEF) on human tendon-derived stem/progenitor cells (hTSPCs) from pathological tendon tissue. Morphological and elemental analyses confirmed that GEF retains its fibrous architecture and elemental stability after autoclave and UV sterilization, preserving its conductive and infrared-emitting properties. These features are thought to generate a bioactive microenvironment enriched in negative ions and mild infrared radiation capable of influencing cellular redox balance.

Comparative profiling of healthy versus pathological hTSPCs identified elevated IL-6, IL-4, and IL-8 in pathological cells, validating their use as an in vitro inflammation model to examine GEF-driven modulation. In 3D scaffold cultures, GEF exposure improved extracellular matrix (ECM) organization, with decreased collagen type III and enhanced collagen type I deposition, supported by gene-level changes (downregulated COL3A1, upregulated COL1A1). GEF also reduced reactive oxygen species (ROS) and improved cell viability, indicating antioxidant and cytoprotective effects. Cytokine analysis revealed a trend toward an attenuated inflammatory state, with lower IL-6 and IL-8 secretion and modest IL-4 release.

Overall, these findings demonstrate that GEF can beneficially modulate pathological hTSPCs within 3D scaffolds by reducing oxidative stress and supporting a more tendon-like ECM profile. Although preliminary, the results suggest that semiconductor-based fabrics may serve as supportive adjuncts for mitigating inflammatory events and promoting a more regenerative cellular response.

926. 3D-printed insert platform for controlled wound healing and tissue growth assessment in 3D skin models

Giulia Artemi¹, Caterina Perfili¹, Valeria Ferrara², Alberto Augello², Giordano Perini¹, Egidio Stigliano², Elena Benini², Vincenzo Arena², Francesca Sciandra³, Valentina Palmieri⁴, Marco De Spirito¹, Massimiliano Papi¹

¹Department of Neuroscience. Università Cattolica del Sacro Cuore, Rome (Lazio) - Italy, ²Policlinico Universitario Agostino Gemelli, Rome (Lazio) - Italy, ³Consiglio Nazionale delle Ricerche, Rome (Lazio) - Italy, ⁴Institute for Complex Systems. Consiglio Nazionale delle Ricerche, Rome (Lazio) - Italy

3D bioprinted skin models are engineered multilayer tissues mimicking human skin. This study introduces an innovative fully 3D-printed insert platform designed to enable growth and reproducible wound-healing assays in 3D bioprinted skin models. In line with the 3Rs (Replacement, Reduction, and Refinement) principles, the system advances in vitro skin research by reducing reliance on animal testing. Traditional biopsy-based wound creation often lacks precision, whereas this insert provides a stable, cost-effective, and standardized alternative.

The platform provides two main functions. It supports culture at the air–liquid interface and improves handling and stability of 3D skin constructs, with a grid-like base that ensures accurate tissue positioning. It also enables controlled wound induction through a conical pin that mimics a physiological injury. All parts were CAD-designed; the insert body was printed in polylactic acid, and the bridge and pin were produced via digital light processing with biocompatible resin. The structural features of the insert were characterized by scanning electron microscopy, whereas the 3D skin constructs were analyzed through confocal imaging, histological staining, and immunofluorescence assays. In addition, we evaluated morphometric and cellular parameters of the induced wounds to validate the reproducibility of the system.

Preliminary results show that the platform consistently maintains the air–liquid interface and enables reliable, adjustable, and reproducible wound formation across samples.

Overall, this versatile 3D-printed wound-healing insert enhances experimental control and reproducibility, providing an accessible and innovative tool for tissue-engineering applications and the evaluation of skin dressings.

927. Biofabrication of specialized dermal-epidermal structures in substitutes of the human skin, oral mucosa and palate using plastic compression nanostructuration

Miguel Alaminos¹, Jesús España-Guerrero², Paula Ávila-Fernández¹, Jesús Chato-Astrain¹, Miguel Etayo-Escanilla¹, Fernando Campos¹, Óscar Darío García-García¹, Carmen González-Gallardo¹, Ricardo Fernández-Valadés³, Ingrid Garzón¹, Antonio España-López⁴

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Histology. University of Granada, Granada - Spain, ³Pediatric Surgery. Hospital Universitario Virgen de las Nieves and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ⁴Stomatology. University of Granada, Granada - Spain

Background: Several models of bioengineered human skin, oral mucosa and palate have been developed to the date. However, none of these models is fully biomimetic to the native skin regarding the presence of the specialized dermal-epidermal structures, such as the rete ridges and dermal papillae. In the present work, we describe a biofabrication method to generate dermal-epidermal structures for use in tissue engineering.

Methods: 4 types of casts reproducing several dermal-epidermal patterns (irregular patterns, gross, medium and thin papillae) were fabricated using a 3D printer. Then, bioengineered stromal substitutes were generated using human fibroblasts immersed within fibrin and alginate biomaterials, and the casts were applied on the surface of each biomaterial, applying controlled pressure, to generate a surface pattern. Epithelial keratinocytes were cultured on top of each substitute to generate a bilayered substitute, and histological analyses were carried out after different follow-up times.

Results and conclusions: We found that plastic compression nanostructuration using prefabricated casts with specific surface patterns successfully generated structures resembling rete-ridges and papillae, especially in the case of the fibrin-based artificial tissues. Cells demonstrated to adapt to the environment of the pre-designed artificial stromas with good survival, especially in substitutes made with the larger patters of papillae. These results support the use of plastic compression nanostructuration to generate more biomimetic bilayered substitutes of the human skin, oral mucosa and palate.

Supported by FIS-PI23/00335, FIS-PI24/00006, FIS-PI25/00002, ICI19/00024-BIOCLEFT and ICI21/00010-NANOULCOR, by Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities (Plan Estatal de Investigación Científica, Técnica y de Innovación, and Plan de Recuperación, Transformación y Resiliencia). Co-financed by the European Regional Development Fund (ERDF-FEDER) through the “Una manera de hacer Europa” program. Supported by DGP_PIDI_2024_00361, grant DGP_PIDI_2024_01347 and grant C-CTS-032-UGR23, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía and University of Granada, Spain.

928. Endowing polypropylene meshes with advanced antibacterial drug-releasing coatings to prevent device-related infections

Bárbara Pérez-Köhler^{1 2 3}, Luis García-Fernández^{2 4}, Selma Benito-Martínez^{1 2 3}, Celia Rivas-Santos^{1 2 3}, Javier Molpeceres⁵, Cristina Abradelo⁵, María R. Aguilar^{2 4}, Gemma Pascual^{1 2 3}

¹Departamento de Medicina y Especialidades Médicas, Facultad de Medicina y Ciencias de la Salud, Universidad de Alcalá, 28805 Alcalá de Henares, Madrid, Spain, ²Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, 28029 Madrid, Spain, ³Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, ⁴Instituto de Ciencia y Tecnología de Polímeros (ICTP), CSIC, Madrid, Spain, ⁵Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Alcorcón, Madrid, Spain

Aim and objective. Postoperative infection remains a major complication in hernia repair, often requiring reintervention and prolonged antibiotic treatment. Functionalizing polypropylene meshes with drug-releasing coatings offers a cutting-edge strategy to deliver antibacterial agents locally while preserving the mechanical performance of the implant. This study evaluates two novel electrospun antibacterial coatings for polypropylene meshes, assessing their performance through in vitro and in vivo assays.

Materials and methodology. Carboxymethylcellulose/polyvinyl alcohol (CMC/PVA) microfibers (0.5% w/v), loaded with chlorhexidine (CHX) or rifampicin (RIF), were electrospun on both sides of the mesh, establishing four groups: uncoated (control), CMC/PVA, CMC/PVA+CHX, and CMC/PVA+RIF. In vitro assays (mesh size: 1 cm2) included drug release (high performance liquid chromatography, HPLC), antibacterial activity against Staphylococcus aureus (106 CFU/mL), and cytotoxicity on rabbit fibroblasts. Preclinical evaluation was conducted in a rabbit model of prosthetic hernia repair under S. aureus infection (n=6/group; mesh size: 5x2 cm). Antibacterial performance and biocompatibility were assessed 14 days postoperatively through macroscopic analysis, sonication, histology, light/scanning electron microscopy, and macrophage immunolabeling.

Results. RIF-functionalized meshes showed sustained release, complete bacterial clearance, and optimal tissue integration without exerting cytotoxicity or excessive inflammation. Contrary, CHX-based coatings exhibited mild cell toxicity (p<0.001) and limited antibacterial efficacy (p<0.05), which was corroborated preclinically by residual infectious niches found in areas of the implants adjacent to sutures. Regardless of the drug, electrospinning enabled uniform coating deposition and rapid biodegradation without compromising host response.

Conclusion. CMC/PVA+RIF coatings provide a robust strategy for infection prevention in hernia repair, outperforming CHX-based formulations and supporting their translational relevance. This electrospun microfiber platform represents a versatile approach to endow polypropylene meshes (and potentially other biomedical implants) with a local, effective antibacterial functionality.

Acknowledgements. Spanish Ministry of Science, Innovation and Universities, grant numbers PDC2021-121809-100, PID2023-152295OB-I00, PID2023-149301OB-I00. Support from CIBER-BBN (COATMESH Intramural Collaboration).

929. Engineering marine biopolymer-based hydrogels for advanced breast cancer tissue modeling in personalized medicine

Rizlène Bouhaya¹, Arnaud Petitpas¹, Virginie Pellerin¹, Irinka Séraudie², Richard Iggo², Laurent Rubatat¹, Susana Fernandes¹

¹IPREM. Universite de Pau et des Pays de l‘Adour, Pau (Midi-Pyrenees) - France, ²BRIC. Université de Bordeaux, Bordeaux (Midi-Pyrenees) - France

Breast cancer remains the most prevalent malignancy affecting women and a leading cause of cancer-related mortality worldwide [1]. In high human development index countries, one in eight women will develop the disease during their lifetime [2]. Despite multiples advances in the domain, current preclinical models often fail to fully reproduce the complexity of the mammary microenvironment, limiting their predictive value for drug response.

In this context, we aim to engineer biomimetic hydrogels using marine-biopolymers and bioactive molecules. These matrices with specific properties are designed to reproduce the extracellular matrix (ECM) in composition and structure of both healthy and tumoral breast tissues.

In this presentation, first, the hydrogels conception, and physicochemical, mechanical and morphological characterization, will be presented. After, the hydrogels serving as substrates for the development of breast tumoroid models, incorporating both established cell lines and also patient-derived xenografts (PDXs - human tumors that have proliferated in the mammary gland of an immunodeficient mouse) will be described in terms of cell viability, proliferation, and cellular behavior. These models aim to better capture tumor heterogeneity and, or cell–matrix interactions, thereby improving the relevance of preclinical drug screening. Overall, the results highlight the potential and interest of marine biopolymer-based hydrogels as platforms for 3D breast tumor modeling and drug screening in the context of personalized medicine.

1. Hu, C., Hart, S.N., Gnanaolivu, R., Huang, H., Lee, K.Y., et al. (2021) A population-based study of genes previ-ously implicated in breast cancer. New England Journal of Medicine., 384 (5), 440 − 451.

2. American Cancer Society (2025). Breast cancer Statistics: How common is breast cancer? https://www.cancer.org/cancer/types/breast-cancer/about/how-common-is-breast-cancer.html

930. From stem cells to functional airways: evaluating safety in respiratory tissue engineering

Davide Adamo¹, Giulia Galaverni¹, Anjana Chathanchirappattu Raj², Maria Macchia³, Francesca De Carlo³, Filippo Lococo⁴, Simona Negoias⁵, Matteo Alicandri-Ciufelli⁶, Graziella Pellegrini²

¹Department of Life Sciences. Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ²Department of Surgical, Medical, Dental, and Morphological Sciences. Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ³Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy, ⁴Thoracic Surgery Unit. Policlinico Universitario Agostino Gemelli, Rome (Lazio) - Italy, ⁵Department of Otorhinolaryngology, Head and Neck Surgery. Basel University Hospital, Basel (Basel-Stadt) - Switzerland, ⁶Department of Otolaryngology - Head and Neck Surgery, Azienda Ospedaliero-Universitaria Policlinico di Modena. University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy

Despite their life-saving potential, tissue engineering approaches for the treatment of extensive airway defects still face significant limitations. A major challenge is the inability to regenerate a functional airway epithelium containing the appropriate amount of stem cells required for long-term tissue renewal following transplantation of the bioengineered graft. We recently demonstrated that epithelial cells derived from different regions of the respiratory tract can be efficiently expanded in vitro using a clinical-grade culture system, while preserving the stem cell population and generating a fully differentiated respiratory epithelium. Building on this experience, the present study further investigated the safety profile of the expansion procedure through complementary assays including: (i) karyotype analysis, essential to rule out chromosomal abnormalities potentially accumulated during extensive epithelial cell manipulation; (ii) measurement of telomere length, to assess progressive physiological shortening; (iii) evaluation of cell growth dependence on adhesion via soft-agar culture, to exclude the possibility of invasion and metastasis; and (iv) assessment of the maintenance of proliferative potential in a tissue-specific growth factor–dependent manner. The results confirmed the safety of the expansion process, further supporting the use of this culture method for cell therapy and tissue engineering approaches aimed at reconstructing multiple segments of the respiratory system.

931. Fabrication and early characterisation of 47.5B and 1D bioactive glass scaffolds for perfusion-based studies

Elnaz Khorasani¹, Maria Erato Pianou², Bojana Obradović³, Enrica Verné², Francesco Baino²

¹Faculty of Technology and Metallurgy. University of Belgrade, Belgrade (Serbia) - Serbia, ²Department of Applied Science and Technology (DISAT). Politecnico di Torino, Turin (Italia) - Italy, ³Faculty of Technology and Metallurgy. University of Belgrade, Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia

Bioactive glasses (BAG) are osteinductive and osteoconductive biomaterials that are highly attractive for use in bone implants and scaffolds in bone tissue engineering. In the present study, we evaluated scaffolds obtained by a foam replication method based on two BAG types: 47.5B (47.5SiO2-2.5P2O5-20CaO-10MgO-10Na2O-10K2O mol.%) and 1D glass (46.1SiO2–6.2P2O5–28.7CaO–8.8MgO–4.5Na2O–5.7CaF2 wt.%). Polyurethane foams (45 ppi) were coated with an aqueous slurry containing fine BAG powder (<32 um 47.5B, <25 um 1D), water, and polyvinyl alcohol (PVA) as a binder. The coated templates were lightly compressed to remove excess slurry and dried gradually to avoid internal irregularities. The polymer template was removed through heat treatment, followed by sintering to stabilize the glass network while maintaining an interconnected pore structure. The resulting cylindrical scaffolds had average dimensions of approximately 8mm in diameter and 6 mm in height. Based on mass and volume measurements, the scaffolds displayed a high level of porosity (>50 vol.%) and retained an open architecture suitable for fluid transport (300 to 800 um pore diameters). Permeability of foam-replicated bioactive glass scaffolds typically lies in the range comparable to values reported for trabecular bone, which is also used as the reference for our newly prepared BAG scaffolds. Bioactivity and biological performance of the scaffolds were studied under perfusion conditions. The scaffolds were seeded with murine preosteoblasts (MC3T3-E1 Subclone 14; 15 million cells/cm³) and cultivated in a perfusion bioreactor (“3D Perfuse”) under continuous medium flow rate at a superficial velocity of 100 um/s. After 7 days of culture the cells remained viable and metabolically active, as confirmed by the MTT assay. Overall, both scaffold types have shown bioactivity and biocompatibility and potentials for further studies in bone tissue engineering.

Acknowledgement: This work was funded by the European Union’s Horizon Europe research and innovation programme (GA. 101119884).

932. Translating biomimetic design to oncology: decellularized ECM-Driven 3D platforms for breast, colorectal, and pancreatic cancer modeling

Julia López De Andrés¹, Laura De Lara-Peña², Carmen Griñán-Lisón³, Jesús Peña-Martín³, María Paz Zafra³, Juan Antonio Marchal³, Gema Jiménez³

¹Departamento de Anatomía y Embriología Humana. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ³Hospital Universitario Virgen de las Nieves and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Tumors are intrinsically heterogeneous structures composed of malignant, stromal, and immune cell populations embedded within an extracellular matrix (ECM) that shapes a dynamic tumor microenvironment (TME). The biochemical and biomechanical characteristics of the TME—continuously modulated by genetic, epigenetic, and environmental influences—critically regulate tumor progression, metastatic potential, and responses to therapy. Traditional two-dimensional (2D) culture systems fail to recapitulate these multifaceted interactions, limiting their translational relevance and contributing to the poor success rate of anticancer drug development.

Three-dimensional (3D) platforms derived from decellularized extracellular matrices (dECMs) have emerged as robust alternatives, as they retain native matrix proteins, growth factors, and mechanical cues, thereby supporting physiologically meaningful cell–ECM interactions. The integration of dECM-based bioinks enables the fabrication of 3D constructs that more closely mirror the in vivo tumor milieu, enhancing the evaluation of cancer cell behavior and treatment outcomes.

In this work, we developed dECMs generated from cell cultures, native tissues, and tumor-conditioned stromal sources to support the growth of patient-derived organoids. These 3D systems successfully reproduced essential TME features in models of breast (BC), colorectal (CRC), and pancreatic (PC) cancers. The platforms effectively influenced the phenotype and functional behavior of incorporated cell populations and recapitulated tumor-specific growth dynamics and chemoresistance patterns, underscoring the protective roles of ECM components. Collectively, our findings demonstrate that dECM-based 3D models provide powerful tools for dissecting tumor heterogeneity and advancing pharmacological testing and personalized therapeutic strategies in BC, CRC, and PC.

933. Investigating the role of epithelial-stromal interaction in bilayered human bioengineered tissues generated by tissue engineering

Miguel Alaminos¹, Jesús Chato-Astrain¹, Paula Ávila-Fernández¹, Fernando Campos¹, Víctor Carriel¹, Carmen González-Gallardo², Ricardo Fernández-Valadés³, Ingrid Garzón¹, Miguel Ángel Martín-Piedra¹, Olimpia Ortiz-Arrabal¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Ophthalmology. Hospital Universitario Clínico San Cecilio and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ³Pediatric Surgery. Hospital Universitario Virgen de las Nieves and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Background: The surgical treatment of patients affected by severe alterations of the human cornea, skin, oral mucosa and palate is very complex and requires the development of bioartificial tissues available for tissue reconstruction. Here, we fabricated a bilayered epithelial-stromal tissue substitute with or without cells at each layer, in order to determine which one is more biomimetic to the native tissues.

Methods: Complete bioartificial human epithelial-stromal substitutes were generated by tissue engineering using fibrin-agarose biomaterials with stromal cells within the biomaterial (as a stromal substitute), and epithelial cells cultured on top. Then, substitutes devoid of stromal cells and substitutes devoid of epithelial cells were generated to determine the role of each cell type. All substitutes were kept in culture and analyzed histologically to determine the presence of fibrillar and non-fibrillar components of the tissue extracellular matrix (ECM), such as proteoglycans, collagens, elastic and reticular fibers.

Results and conclusions: Artificial tissues without epithelial cells were devoid of an epithelial layer, whereas tissues with an acellular stroma were negative for most ECM components, although epithelial cells resulted in a uniform, bilayered epithelium. Complete models showed positive signal for non-fibrillar components, especially proteoglycans. These findings support the important role of epithelial-stromal crosstalk and interaction and suggest that both the epithelial and stromal cells should be used to develop human artificial cornea, skin, oral mucosa and palate.

Supported by grants FIS-PI23/00335, FIS-PI24/00006, FIS-PI25/00002, ICI19/00024-BIOCLEFT and ICI21/00010-NANOULCOR, funded by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Plan Estatal de Investigación Científica, Técnica y de Innovación, and Plan de Recuperación, Transformación y Resiliencia). Co-financed by European Regional Development Fund (ERDF-FEDER) through the “Una manera de hacer Europa” program. Supported by DGP_PIDI_2024_00361, grant DGP_PIDI_2024_01347 and C-CTS-032-UGR23, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía and University of Granada, Spain.

934. Wnt-coated radiopaque hydrogels as a novel injectable and trackable bone filler for osteoporosis

Michael Rotherham¹, Pietro Riccio¹, Richard Moakes¹, Alicia J. El Haj¹

¹Healthcare Technologies Institute. Institute of Translational Medicine, University of Birmingham, Birmingham - United Kingdom

Objectives

Engineering bone continues to be a major challenge. Success in this area is dependent on biomaterials that provide appropriate cues to cells, and development of materials that regulate cell behaviour and direct tissue formation is fundamentally important.

Wnt signalling plays a leading role in regulating the bone stem cell niche, so directing bone formation using stem cells and Wnt-functionalized materials is an attractive approach for bone tissue engineering. This project aims to develop an injectable and trackable Wnt-functionalised hydrogel to regulate the stem cell niche and augment bone formation for the treatment of osteoporosis.

Methods

Wnt3a was conjugated to Poly (ethylene glycol) diacrylate (PEGDA) using Michael addition and retention tested using Immuno-assays and X-ray fluorescence (XRF). Wnt-PEGDA signalling activity was tested using a Wnt pathway Green Fluorescent Protein (GFP) reporter cell line. The ability for Wnt-PEGDA to regulate osteogenesis was investigated in a human Mesenchymal Stem Cell (MSC) line using gene expression analysis, alkaline phosphatase (ALP) activity and histological staining. The Computed Tomography (CT) contrast agent Iohexol was incorporated into PEGDA and radiopacity tested using μCT.

Results

Wnt3a was successfully engrafted onto PEGDA, indicated by positive Wnt3a staining and the presence of Sulphur (cysteine residues) within Wnt3a (confirmed by XRF). Retention of Wnt3a activity after engraftment was confirmed by GFP+ expression in reporter cells. MSC osteogenesis was augmented by Wnt-PEGDA in osteogenic media, with increased bone gene expression, ALP activity and matrix staining. Iohexol increased the radiopacity of PEGDA, confirming its short-term trackable properties.

Conclusions

Wnt-functionalised hydrogels provide a novel way of presenting growth factors that spatially regulate stem cell behaviour in the bone niche. Wnt-PEGDA is injectable, trackable and compatible with cell therapies. Ultimately this approach can form a minimally invasive treatment, amenable with existing therapies, to augment bone repair and treat degenerative orthopaedic conditions.

935. A novel engineered plant-derived polysaccharide hydrogel for safer, enhanced bone regeneration

Cristina Gonzalez-Garcia¹, Xinyu Li¹, Aleixandre Rodrigo-Navarro², Lluís Oliver-Cervello¹, Oana Dobre¹, Manuel Salmeron-Sanchez²

¹Centre for the Cellular Microenvironment, Advanced Research Centre. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, ²Microenvironments for Medicine. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain

Critical-size bone defects remain a major clinical challenge due to the limited regenerative capacity of large skeletal injuries. While growth factor (GF) therapies – particularly bone morphogenetic protein-2 (BMP-2) – can enhance repair, clinically effective outcomes typically require supraphysiological doses that carry significant safety risks. This work presents a novel strategy that couples a bioactive, plant-derived polysaccharide, acemannan (ACE), with a customisable PEG-based hydrogel engineered for controlled GF delivery and synergistic osteogenic stimulation.

The osteoinductive potential of ACE was first assessed in 2D using human mesenchymal stem cells (hMSCs). ACE significantly improved hMSC viability and adhesion and markedly enhanced osteogenic differentiation, confirmed by Alizarin Red staining and OCN expression, establishing ACE as an osteogenic cue independent of 3D factors.

ACE was then incorporated into protease-degradable PEG-MAL hydrogels crosslinked with PEG-diSH and VPM peptides, creating 3D carriers with tuneable mechanical and degradation profiles. The fibronectin fragment (FNIII12-14) was integrated to introduce cell-adhesive sites and GF-binding motifs, enabling efficient BMP-2 loading and sustained release. In vitro, these PEG/ACE hydrogels supported hMSC viability and proliferation, and promoted differentiation, while the combination of ACE, FNIII12-14, and controlled BMP-2 release significantly amplified osteogenic differentiation, demonstrated by increased levels of osteogenic gene markers, highlighting the importance of both biochemical and biophysical cues.

The most promising formulations were evaluated in vivo using a mouse critical-size segmental bone defect model. Hydrogels containing ACE and low-dose BMP-2 promoted significantly greater new mineralised bone formation compared to BMP-2 alone at the same dose, demonstrating a clear synergistic effect and highlighting ACE’s capacity to potentiate osteogenesis while reducing GF requirements.

Together, these findings position ACE-loaded PEG hydrogels as a promising platform for safe, targeted, controlled GF delivery and efficient bone regeneration, potentially minimising reliance on high-dose BMP-2 in clinical applications.

936. Translating the lacuno-canalicular network into multi-scale bone scaffold design: a bio-inspired approach integrating nano-imaging, modeling, and advanced fabrication

Sara Sebastiani¹, Federica Buccino², Laura Maria Vergani²

¹Department of Mechanical Engineering (DMEC). Politecnico di Milano, Milano (Lombardia) - Italy, ²Department of Mechanical Engineering (DMEC) & IRCCS Galeazzi-Sant’Ambrogio. Politecnico di Milano, Milano (Lombardia) - Italy

Bone’s exceptional properties arise from its multi-scale hierarchical organization. The lacuno-canalicular network (LCN) provides micro- and nano-scale porosity, housing osteocytes in lacunae and enabling cell–cell interactions through canaliculi. Although LCN is recognized as critical for bone homeostasis and mechanosensing, its architecture is rarely incorporated into current bone tissue engineering (BTE) scaffolds. This study aims to translate nano-scale imaging of human LCN into design principles for BTE scaffolds, defining a bio-inspired approach supported by fluid-dynamics analyses and manufacturable by advanced fabrication.

High-resolution 3D overviews of the LCN are obtained via synchrotron-based nano-holotomography (pixel size 50 nm) and ptychography (down to 25 nm), creating a unique dataset of human femoral trabecular bone. Quantitative parameters, including porosity, interconnectivity, and branching patterns, are extracted. Computational fluid dynamics (CFD) simulations characterize native LCN fluid flow and shear stress distributions. Leveraging the optimized architecture and fluid-dynamics of LCN, key features are translated into generative design of bio-inspired scaffolds. After CFD-based screening, the most promising geometry is fabricated using biocompatible resin by two-photon polymerization (2PP), a high-resolution (down to 100 nm) 3D printing technique.

Results show that native LCN exhibits distinctive structural features and fluid-dynamic behaviors which can be effectively quantified and translated into a computational modeling workflow. CFD-guided screening highlights scaffold geometries capable of replicating flow pathways and shear-stress ranges characteristic of native bone. 2PP fabrication demonstrates manufacturability of bio-inspired scaffolds across scales, bridging micro-scale features with millimeter-scale architecture.

In conclusion, this work establishes a framework for integrating LCN-inspired microarchitecture into BTE scaffolds. By combining quantitative nano-imaging, CFD modeling, and high-resolution 2PP fabrication, the study provides a foundation for functional bio-inspired scaffold design. Future in-vitro studies will investigate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells on these constructs, evaluating their regenerative potential and the broader value of bio-inspired strategies in tissue engineering.

937. Usefulness of a bioengineered palate mucosa in children with cleft palate: the BIOCLEFT clinical trial

Jesús Chato-Astrain¹, Paula Ávila-Fernández¹, Miguel Etayo-Escanilla¹, Antonio España-López², Fernando Campos¹, Miguel Ángel Martín-Piedra¹, Ingrid Garzón¹, David Sánchez-Porras¹, Ricardo Fernández-Valadés¹, Antonio Campos¹, Miguel Alaminos¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Stomatology. University of Granada, Granada - Spain

Background: Cleft palate is a congenital disease in which palatal shelves fail to fuse together. The treatment consists in using healthy tissues to repair the central defect, thus reestablishing the physical separation between oral and nasal cavities. However, this treatment is associated to growth and development alterations. Here, we describe a pioneering clinical trial in which a palate mucosa substitute generated by tissue engineering is implanted in cleft palate children.

Methods: A tissue-engineered palate mucosa was generated using epithelial keratinocytes and stromal fibroblasts combined with fibrin and type-VII agarose biomaterials. This tissue was preclinically evaluated, demonstrating potential to prevent growth alterations in rabbits (1). With these results, we obtained preclinical assessment from the Spanish Medicines Agency, who required additional in vivo experiments. Then, we designed the BIOCLEFT clinical trial, the first in which an artificial palate mucosa is implanted in cleft palate children (ClinicalTrials.gov number NCT06408337). This trial was initiated in 2024, and 3 children have been grafted to the date.

Results and conclusions: First, we found that implant of the artificial tissue was feasible, and this tissue could be sutured at the patient’s palate with no complications. In terms of biosafety, all patients treated with the artificial palate mucosa demonstrated good biocompatibility and no side effects after 6-18 months of follow-up. These results support the feasibility and biosafety of BIOCLEFT in children with cleft palate.

Supported by Grant ICI19/00024 (BIOCLEFT), Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, and grant DGP_PIDI_2024_01347, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía, Spain. Cofinanced by the European Regional Development Fund (FEDER/ERDF) through the “Una manera de hacer Europa” program, European Union.

1. Fernández-Valadés-Gámez R, et al. Usefulness of a bioengineered oral mucosa model for preventing palate bone alterations in rabbits with a mucoperiostial defect. Biomed Mater. 2016;11(1):015015

938. Bioengineering miniaturized 3D bone-like tissue using human ECM-derived cryogels in a bone-on-a-chip model

Cátia F. Monteiro¹, Rita S. Ferreira¹, Elisa A. G. Martins¹, Catarina A. Custódio¹, João F. Mano¹

¹CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal

Non-union bone fractures remain a major clinical challenge, demanding physiologically relevant in vitro platforms to evaluate advanced therapeutic medicinal products (ATMPs) with higher predictive power. Organ-on-a-chips (OoCs) have emerged as cutting-edge microscale systems that mimic organ-level tissue architecture and fluid perfusion.

This work aims to create a novel biomimetic 3D bone-on-a-chip model integrating human extracellular matrix (ECM)-derived cryogels to investigate the effects of ATMPs on bone fracture regeneration. Cryogels composed of methacrylated (hECM-MA) and/or dopamine-modified (hECM-MA-DOPA) human ECM proteins were fabricated, exhibiting shape-memory behavior, interconnected macroporosity, and mechanical robustness for dynamic culture. A PDMS microfluidic chip comprising an open-top tissue chamber was designed to allow either the direct in-chip cryogel fabrication and future in situ mechanical fracture. These biomimetic scaffolds supported high infiltration and self-organization of human bone-marrow mesenchymal stem cells (hBM-MSCs), maintaining high viability and proliferation. Leveraging endogenous hECM trophic cues and dopamine-mediated hydroxyapatite nucleation, autonomous osteogenesis led to the formation of miniaturized bone-like tissue under long-term perfusion. The constructs exhibited mineralization and expression of osteogenic markers, confirming the intrinsic osteoinductive properties of hECM-MA-DOPA cryogels. Notably, when compared to static cultures, shear stress within the OoC device potentiated osteogenic differentiation through the upregulation of osteogenic markers. Medium collection for proteomic/metabolomic profiling is expected to further support comprehensive evaluation of tissue formation dynamics.

By simulating a non-union fracture into the matured cryogel-based bone tissue, the next step will assess the predictive capacity of this model to screen ATMPs as an ethically responsible alternative to animal models, opening a new avenue for the translation of biomaterials for bone repair.

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). This work was supported by the European Commission HORIZON-RIA grant ORTHO-ALLO-UNION (no.101137464).

939. Mapping biological strategies for bone toughness: high compressive residual strains compensate for the lack of osteocytes in fishbone

Andreia Sousa Da Silveira¹, Anton Davydok², Christina Krywka², Mario Scheel³, Timm Weitkamp³, Claudia Fleck⁴, Ron Shahar⁵, Paul Zaslansky¹

¹Department for Restorative and Preventive Dentistry. Charité - Universitaetsmedizin Berlin, Berlin - Germany, ²Helmholtz-Zentrum Hereon, Geesthacht (Schleswig-Holstein) - Germany, ³Synchrotron SOLEIL, Saint-Aubin (Ile-de-France) - France, ⁴Technical University of Berlin, Berlin - Germany, ⁵Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot (Israel) - Israel

Current bone tissue engineering often struggles to replicate the complex osteocyte lacuna-canalicular network (LCN) required for mechanosensation and tissue repair. However, the anosteocytic bone of most neoteleost fish functions without osteocytes, offering a “cell-free” model that relies on alternative mechanisms to withstand external loads [1, 2]. To understand these distinct design strategies, this study employed a multimodal imaging approach to compare osteocytic zebrafish and anosteocytic medaka bones. We combined microtomography (µCT) and Zernike phase-contrast nanotomography (nanoCT) for 3D structural analysis, complemented by X-ray fluorescence (XRF) for elemental mapping and X-ray diffraction (XRD) tomography for crystallographic characterization under loaded and unloaded conditions. NanoCT analysis quantified LCN porosity in zebrafish (0.83–2.25%), which contrasted with the dense, void-free architecture of medaka bone. Despite these structural differences, both species exhibited similar highly organized mineralized collagen fibril orientations and comparable elemental distributions of calcium, strontium, and zinc. However, XRD tomography revealed that medaka bone possesses significantly higher compressive residual strains (-0.100% ± 0.02) compared to osteocytic zebrafish bone (-0.071% ± 0.03). Findings suggest that anosteocytic bone compensates for the lack of osteocytes by structurally trapping higher compressive residual strains within its dense architecture. This “pre-strain” state likely enhances fracture toughness and resilience for microcrack initiation. By shifting the tissue model from active repair to passive damage resistance, this design offers a valuable blueprint for biomimetic engineering. Specifically, inducing compressive residual stresses in synthetic bone scaffolds could yield superior fracture toughness for load-bearing clinical applications.

940. Engineered biohybrid vasculature for colorectal organoids

Ria V. Vaishampayan¹, Gaurav Dave², Dana Levi², Jens Puschhof³, Federico Colombo², Christine Selhuber-Unkel²

¹Max Planck School Matter to Life, Heidelberg (Baden-Wberg Bayern) - Germany, ²Institute for Molecular Systems Engineering and Advanced Materials (IMSEAM), Heidelberg (Baden-Wberg Bayern) - Germany, ³Microbiome and Cancer Division. German Cancer Research Center, Heidelberg (Baden-Wberg Bayern) - Germany

Organoids are a widespread choice for studying tumors due to their structural and functional likeness to native tissue. However, the lack of a functional vasculature inside organoids remains a persistent limitation for their reproducibility and long-term use. In vivo, vasculature supports nutrient exchange in tissues while performing a paracrine function[1]. Our study aims to recapitulate some of these complexities by introducing a perfusable 3-dimensional vascular platform to endothelialize colorectal organoids. We have developed a hydrogel-based scaffold of micrometre-scale dimensions using 2-photon polymerization (2PP)[2]. The scaffolds, characterized by nanoindentations, have physiological values of stiffness (Young’s modulus) ranging from 1.5 to 5 kPa, making them suitable candidates for biomimetic vasculature[3]. Using confocal microscopy, we observed the attachment and growth of the endothelial cell line EA.hy926 for up to 12 days, both outside and inside the structures. The endothelialized vasculature will be integrated with healthy and tumor-derived colorectal organoids produced using the hanging drop method. The differential interactions of these two populations with endothelial cells on the scaffolds will help us gain insights into the interplay of colorectal cells, the microbiome and the dense gut vasculature involved in the progression of colorectal cancer. We aim to optimize this biohybrid platform to conduct uniform, controlled studies of tumor-vasculature dynamics, supporting downstream analysis of drug responses and immune interactions.

References

[1] S. Grebenyuk et al., “Large-scale perfused tissues via synthetic 3D soft microfluidics,” Nat. Commun., Jan. 2023.

[2] F. Colombo et al., “Two‐Photon Laser Printing to Mechanically Stimulate Multicellular Systems in 3D,” Adv. Funct. Mater., May 2024.

[3] T. R. Cox and J. T. Erler, “Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer,” Dis. Model. Mech., Mar. 2011.

941. Development and clinical translation of UGRSKIN as the first advanced therapies medicinal product authorized by the Spanish medicines agency for treatment of severely burnt patients

Paula Ávila-Fernández¹, Miguel Etayo-Escanilla¹, Óscar Darío García-García¹, Ingrid Garzón¹, David Sánchez-Porras¹, Víctor Carriel¹, Miguel Ángel Martín-Piedra¹, Fabiola Bermejo-Casares², Ricardo Fernández-Valadés³, Miguel Alaminos¹, Jesús Chato-Astrain¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Histology. University of Granada, Granada - Spain, ³Pediatric Surgery. Hospital Universitario Virgen de las Nieves and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Introduction: Management of severely burnt patients is very complex and reestablishing the protective skin barrier of the patient in order to prevent infections and to reduce the loss of key tissue component is challenging. In this work, we describe the clinical translation process that we carried out to generate a novel skin substitute medicinal product that has been authorized for consolidate use in the Spanish Health System.

Methods: UGRSKIN was generated using fibrin-agarose biomaterials and human epidermal and dermal cells isolated from patient biopsies and preclinically evaluated in immune-deficient mice (1). After a thorough histological, genetic and rheological characterization, UGRSKIN was grafted in severely burnt patients by compassionate use in the context of hospital exemption in a burn unit in Spain. The positive results obtained in the first cases, with 80% survival and good integration in the host tissue (2) allowed us to obtain authorization by the Spanish Medicines Agency for this product, as the first tissue engineering approved for use in the Spanish Health System, with a registry number 89618.

Results and conclusions: The clinical results obtained after more than 20 patients treated with this product were very positive and confirm the potential usefulness of UGRSKIN as a therapeutic tool. Although the translation process was complex, these results demonstrate the possibility to develop and register a skin substitute generated by tissue engineering for the clinical treatment of severely burnt patients.

Supported by grants C-CTS-032-UGR23 and DGP_PIDI_2024_00361, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía and University of Granada, Spain. Supported by Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, Grant FIS PI25/00002. Cofinanced by the European Regional Development Fund (FEDER/ERDF) through the “Una manera de hacer Europa” program, European Union.

1. Carriel V, et al. Cells Tissues Organs. 2012;196(1):1-12

2. Martin-Piedra MA, et al. Bioeng Transl Med. 2023 7;8(6):e10572

942. Challenges in testing the biocompatibility of electrospun scaffolds for wound dressing: insight from in vitro study

Aleksandra Zawadzka¹, Honorata Kraśkiewicz¹, Aleksandra Klimczak¹

¹Laboratory of Biology of Stem and Neoplastic Cells. Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Wroclaw - Poland

Electrospun polymeric scaffolds [Polycaprolactone/Polyethylene glycol (PLC/PEG blend)] have a promising potential in regenerative medicine. This is due to their fibrous structure that mimics the extracellular matrix (ECM). Their high surface area promotes cell attachment and supports biomolecule incorporation. Before their biomedical implementation, crucial parameters such as sterilization efficacy and biocompatibility must be optimized. The aim of this study was to develop and evaluate electrospun scaffolds sterilization method and to assess their in vitro biocompatibility. Scaffolds were subjected to UV exposure (2h) or UV exposure combined with incubation in 70% ethanol (20-minute) to determine how these sterilizations approaches influence on human fibroblasts (MSU-1.1 cell line) viability and adhesion. Cell metabolic activity and morphology were assessed using MTT assays and fluorescence microscopy following 24, 48 and 72 hours. Immunofluorescence analysis using DAPI and Phalloidin staining confirmed that fibroblast were able to attach to the membrane surface, however, with changed cytoskeleton morphology compared to cells seeded on glass discs. The LIVE/DEAD assay results revealed a high proportion of non-viable cells compared to positive control (live cells without scaffold), which resulted from the low hydrophilicity of scaffolds. Interestingly, cells that failed to attach to the membrane, adhered to the culture dish surface and retained viability and proliferative capacity at the higher level than cells attached to membrane surface [0.742 vs 0.220, respectively (absorbance measured at 570 nm)]. This indicates that the material itself was not cytotoxic for fibroblasts, however, their surface properties require modification to enhance hydrophilicity and support efficient cell colonization. Such optimization is essential to improve their functionality in regenerative medicine applications.

Acknowledgements: Study supported by National Science Centre, Poland (UMO-2023/05/Y/ST5/00251).

943. Biofabrication of a cell-laden intervertebral disc model using tissue-specific extracellular matrix bioinks

Valerie Kersten¹, Jijo Thomas¹, Tugdual Haffner¹, Conor Buckley¹

¹Trinity Centre for Biomedical Engineering. Trinity College Dublin, Dublin - Ireland

Lower back pain resulting from progressive intervertebral disc (IVD) degeneration represents a major epidemiological and socioeconomic burden 1. Current treatments focus primarily on symptom management while disease-modifying or regenerative therapies are yet to be clinically translated. To advance such therapies, physiologically relevant in vitro disc models are essential for studying pathology and testing regenerative strategies. However, replicating the distinct architecture and biochemical composition of the native IVD, particularly the gelatinous, proteoglycan-rich nucleus pulposus (NP) and fibrous, lamellar annulus fibrosus (AF), is challenging 2. Here we present a bioprinted disc model composed of ECM-based bioinks seeded with primary NP and AF cells, designed to mimic the structural and compositional complexity of native tissue.

Bovine caudal discs were characterised to define target dimensional, mechanical, and biochemical properties. NP and AF ECM was extracted through sequential decellularisation and solubilisation. ECM, elastin, and the glycosaminoglycan chondroitin sulphate (CS) were methacrylated (MA) and mixed in native tissue-derived concentrations to form NP, inner AF (iAF) and outer AF (oAF) bioinks. Primary NP and AF cells were encapsulated at native density in the bioinks and IVD constructs were bioprinted with a custom 3D-printer.

Tissue-specific NP, iAF, and oAF ECM-based bioinks were successfully formulated and 3D-printed with high G-code fidelity. All bioinks exhibited compressive strength comparable to native tissue. The bioprinting process and associated shear did not compromise NP or AF cell viability, showing no significant reduction after 7 days compared to cast hydrogels. Complete IVD constructs were successfully bioprinted with seamless integration between bioinks, demonstrating a physiologically relevant IVD model.

3D-bioprinting a full IVD construct with encapsulated cells represents an important step towards developing physiologically relevant in vitro research platforms. This study used well-established bovine caudal discs, however composition and dimensions can be readily adapted to healthy or degenerated human discs. Future work will focus on analysing matrix and cellular changes upon degeneration and developing a computational model for validation against human IVD data.

Acknowledgement

European Research Council (ERC-2019-CoG-864104: INTEGRATE).

Reference

1. Dieleman+ JAMA 2016 2 Zhu+ Mater Sci Eng C Biol Appl 2021

944. Evaluation of the nanostructured implant-tissue interface using a three-dimensional gingival tissue equivalent

Maria Antonia Llopis-Grimalt¹, Marta Munar-Bestard¹, Guillem Ramis-Munar², David Smith³, Tobias Starborg³, Karl Kadler³, Marta Monjo¹, Joana Maria Ramis¹

¹Cell Therapy and Tissue Engineering Group (TERCIT), Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears (UIB). Health Research Institute of the Balearic Islands (IdISBa). IUNICS-UIB, Palma de Mallorca (Illes Balears) - Spain, ²Cellomics Unit, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears (UIB). IUNICS-UIB, Palma de Mallorca (Illes Balears) - Spain, ³Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health. University of Manchester, Manchester - United Kingdom

Improved soft tissue integration around dental implants is essential to prevent peri-implantitis, a major cause of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicularly into the gingival tissue. In contrast, around dental implants collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Since nanostructured titanium surfaces have shown enhanced gingival cell responses in 2D cultures1, this study evaluated the implant- tissue interface using a 3D gingival tissue equivalent (GTE). GTEs were exposed to nanostructured (NN) and machined Ti surfaces and stimulated with Phorphyromonas gingivalis LPS to mimic peri-implantitis conditions. Viability (MTT assay), MMP1 and TIMP1 release (ELISA), and expression of extracellular matrix turnover genes (RT-PCR) were assessed. Implant- tissue interaction was further analysed by serial block-face SEM and collagen-1 orientation by immunofluorescence. Both surfaces showed similar GTE responses to LPS, but NN surfaces promoted a higher proportion of perpendicularly oriented collagen. These findings suggest that titanium nanostructuring can improve collagen fiber orientation and soft tissue sealing while maintaining comparable biocompatibility and LPS response.

ACKNOWLEDGMENTS

This research was funded by a grant from the Osteology Foundation (Switzerland; 13-069), by the Ministerio de Educación Cultura y Deporte (contract to M.A. L.G; FPU15/03412), by Santander Bank S.A. (short stay grant for PhD candidates to M.A.L.G.). Ministerio de Economía y Competividad (contract to M.M.; IEDI-2017-00941), and by the Instituto de Salud Carlos III, Ministerio de Economía y Competividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (contract to J.M.R.; MS16/00124 and to M.M.B; FI18/00104).

REFERENCE

Llopis-Grimalt MA, Amengual-Tugores AM, Monjo M, Ramis JM. Oriented Cell Alignment Induced by a Nanostructured Titanium Surface Enhances Expression of Cell Differentiation Markers. Nanomaterials. 2019; 9(12):1661. doi:10.3390/coatings10090907

945. Characterization of novel biomaterials derived from fish scales for corneal tissue engineering

Miguel Etayo-Escanilla¹, Juan Muñoz-Hurtado², Juan Pereira-Martínez², David Sánchez-Porras¹, Jesús España-Guerrero², Fabiola Bermejo-Casares³, Olimpia Ortiz-Arrabal¹, Carmen González-Gallardo⁴, Miguel Alaminos¹, Ingrid Garzón¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²University of Granada, Granada - Spain, ³Histology. University of Granada, Granada - Spain, ⁴Ophthalmology. Hospital Universitario Clínico San Cecilio and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Background: Biomaterials used in corneal repair and engineering must fulfill several requirements, including biocompatibility, mechanical resistance and elasticity and adequate optical properties (1). One of the biomaterials showing potential usefulness for corneal regeneration are fish scales, which are highly available and accessible at low cost. In this work, we evaluated several types of fish scales for use in cornea tissue engineering.

Methods: Fish scales were isolated from commercially available fish. These scales were preconditioned by acid treatment to eliminate the calcium deposits that are commonly found in these structures, and corneal epithelial cells were cultured on each scale. Then, histological and histochemical analyses were carried out to determine the ex vivo biocompatibility of each fish scale on corneal cells. Biomechanical analyses were also conducted on each type of material

Results and conclusions: We found that corneal epithelial cells were able to attach and differentiate on the surface of all fish scales, although morphological differences were detected among the different types of scales. Cells showed positive expression of limbal epithelial cell markers, and resistance was high in all the study groups, similar to native human corneas. These results suggest that fish scales can be used as efficient biomaterials for use in tissue engineering and open the door to the clinical evaluation of these products in patients with severe corneal damage.

Acknowledgements: Supported by grants ICI21/00010 (NANOULCOR) and FIS PI23/00335, Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, Spain. Cofinanced by the European Regional Development Fund, “Una manera de hacer Europa” program.

1. Manoochehrabadi T, Solouki A, Majidi J, Khosravimelal S, Lotfi E, Lin K, Daryabari SH, Gholipourmalekabadi M. Silk biomaterials for corneal tissue engineering: From research approaches to therapeutic potentials; A review. Int J Biol Macromol. 2025 May;305(Pt 1):141039

946. Mitochondrial targeting peptide restores chondrogenic capacity of osteoarthritic adipose human mesenchymal stem cells

Qiang Wang¹, Pieter J. Emans², Lorenzo Moroni³, Berta Cillero-Pastor⁴

¹Department of Cell Biology-Inspired Tissue Engineering and Department of Complex Tissue Regeneration. MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands, ²Laboratory for Experimental Orthopedics, Department of Orthopedic Surgery, CAPHRI Care and Public Health Research Institute Joint-Preserving clinic. Maastricht University Medical Center, Maastricht (Limburg) - The Netherlands, ³Department of Complex Tissue Regeneration. MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands, ⁴Department of Cell Biology-Inspired Tissue Engineering. MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands

Aim and Objective: Human Hoffa’s fat pad-derived mesenchymal stem cells (IFP-MSCs) represent a promising cell source for cartilage regeneration due to their accessibility and superior chondrogenic potential. However, the inflammatory milieu within injured or osteoarthritic joints severely compromises their ability to differentiate into functional chondrocytes, partly through mitochondrial dysfunction and oxidative stress. SS-31, a mitochondria-targeting peptide, is known to stabilize mitochondrial bioenergetics. This study aims to evaluate the efficacy of SS-31 in protecting IFP-MSCs against inflammation-induced impairment and rescuing their chondrogenic differentiation potential in vitro.

Material and Methodology: IFP-MSCs were isolated from donors with a history of cartilage damage, characterized (CD73⁺/CD90⁺/CD105⁺) and subjected to 3D spheroids culture for chondrogenic differentiation (28 days). Inflammation was mimicked by continuous exposure to IL-1β (10 ng/mL). The treatment group received SS-31 supplementation alongside inflammatory induction. Chondrogenic quality was assessed via Alcian Blue staining (for GAGs) and Immunohistochemistry for Collagen Type II and Collagen Type I. Quantification of sulfated glycosaminoglycan (s-GAG) content normalized to DNA. qPCR for chondrogenic markers (SOX9, ACAN, COL2A1) and hypertrophy/fibrosis markers (COL10A1, MMP13).

Results: Exposure to inflammation significantly suppressed the chondrogenic differentiation of IFP-MSCs, evidenced by reduced glycosaminoglycan (GAG) deposition and downregulated expression of chondrogenic markers (COL2A1, ACAN) compared to healthy controls. Conversely, SS-31 treatment significantly rescued chondrogenesis. SS-31 co-treated groups showed restored GAG synthesis and significantly higher expression of chondrogenic genes compared to the inflammation group.

Conclusion: These results demonstrate that SS-31 effectively counteracts IL-1β-induced mitochondrial dysfunction and thereby preserves chondrogenic differentiation of human IFP-MSC under inflammatory conditions. Mitochondria-targeted protection thus represents a promising strategy to enhance the regenerative potential of MSC-based cartilage repair therapies.

947. Sequentially loaded biofunctional demineralized bone matrix with therapeutic agents

Viorel Nacu¹, Vitalie Cobzac¹, Mariana Jian¹, Igor Casian², Ana Casian², Ludmila Motelica³, Iana Baranețchi⁴, Ovidiu Cristian Oprea³, Liliana Vereștiuc⁵, Roxana Trușca⁶, Anton Ficai³

¹Laboratory of tissue Engineering and Cells Cultures. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ²Phytochemistry and Bioanalysis Laboratory, Drug Development Center. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ³Faculty of Chemical Engineering and Biotechnologies. University Polytechnica of Bucharest, Bucharest - Romania, ⁴Laboratory of Tissue Engineering and Cell Cultures. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁵Department of Biomedical Sciences, Faculty of Medical Bioengineering. Grigore T Popa Faculty of Medicine and Pharmacy Iasi, Iasi - Romania, ⁶National Center of Micro and Nanomaterials. University Polytechnica of Bucharest, Bucharest - Romania

Bone grafting remains a key therapeutic strategy for repairing skeletal defects, yet the demineralization of bone matrix results in the loss of native osteoinductive factors. This study evaluates a protocol for dual sequential vacuum-loading, for incorporating into demineralized bone matrix (DBM) of vancomycin (VA) and human albumin (HA), to enhance bioactivity and modulate release behavior. DBM was prepared from bovine iliac bone using 1M HCl, processed according to Human Tissue Bank standards, lyophilized, and sequentially loaded with VA (100 or 200 mg/mL) followed by HA (10% or 20%). Material properties were assessed through swelling ratio, elastic modulus, enzymatic degradation, HPLC-based VA release, MTT assay and antibacterial activity.

Samples with high VA concentration had a reduced liquid absorption capacity (p<0.05), while 20% HA partially restored hydration properties, suggesting stabilizing protein–matrix interactions. VA incorporation significantly increased the elastic modulus (p<0.05), and all loaded samples exhibited reduced enzymatic degradation rates compared to native DBM (p<0.05). HA did not significantly modify VA release kinetics (p>0.05), while MTT assays demonstrated good cytocompatibility at 24 hours across all groups, with a moderate decrease at 48–72 hours due to high VA concentration. However, HA improved cell viability by 15–25%, particularly in high-VA samples, indicating a protective effect. Antibacterial testing confirmed strong VA mediated inhibition against Staphylococcus aureus and Enterococcus faecalis (30 mm), while Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa remained unaffected, consistent with intrinsic resistance profiles.

The sequential loading with VA and HA preserves cytocompatibility, maintains mechanical stability, ensures sustained antibiotic functionality, and does not impair VA release, making this strategy a promising approach for enhancing DBM-based grafts with combined structural and antibacterial benefits.

948. Genipin crosslinking enhances mechanical and biological properties of fibrin-agarose human tissue substitutes

Jesús Chato-Astrain¹, Miguel Etayo-Escanilla¹, Carmen González-Gallardo², David Sánchez-Porras¹, Miguel Ángel Martín-Piedra¹, Olimpia Ortiz-Arrabal¹, M. Carmen Sánchez-Quevedo¹, Antonio España-López³, Ingrid Garzón¹, Miguel Alaminos¹, Fernando Campos¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Ophthalmology. Hospital Universitario Clínico San Cecilio and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ³Stomatology. University of Granada, Granada - Spain

Background: Bioartificial tissue substitutes based on natural hydrogels have shown clinical potential in patients with diseases affecting the human skin, cornea, palate and oral mucosa (1). However, their mechanical strength and long-term stability remain limited. Natural crosslinkers like genipin (GP) offer a biocompatible strategy to improve scaffold performance while preserving safety.

Methods: Native and nanostructured fibrin-agarose hydrogels were fabricated and crosslinked with GP at 0.1%, 0.25%, 0.5%, and 0.75%. Biomechanical properties, ultrastructural features, and ex vivo biocompatibility were assessed to define optimal conditions for clinical-grade skin models.

Results and Discussion: GP significantly increased stiffness and elasticity, resembling the native dermis. Importantly, macrophages cultured on GP-treated scaffolds exhibited a shift towards a pro-regenerative phenotype (M2-like), suggesting an immunomodulatory effect that could enhance wound healing. High GP concentration (0.75%) compromised viability, while 0.1–0.5% maintained biocompatibility and promoted favorable macrophage polarization. These improvements address current limitations of fibrin-agarose and support its evolution toward more durable substitutes for burn patients.

Conclusions: Genipin-crosslinked fibrin-agarose scaffolds represent a new opportunity for skin, cornea, palate and oral mucosa tissue engineering, by combining enhanced mechanical properties with immunomodulatory potential. Further in vivo studies will confirm regeneration and biodegradability.

Acknowledgements: Supported by grants FIS-PI23/00335, FIS-PI24/00006, FIS-PI25/00002, ICI19/00024 (BIOCLEFT) and ICI21/00010 (NANOULCOR), funded by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Plan Estatal de Investigación Científica, Técnica y de Innovación, and Plan de Recuperación, Transformación y Resiliencia). Co-financed by the European Regional Development Fund (ERDF-FEDER) through the “Una manera de hacer Europa” program. Supported by DGP_PIDI_2024_00361, grant DGP_PIDI_2024_01347 and grant C-CTS-032-UGR23, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía and University of Granada, Spain.

1. Shafiee A and Atala A. Tissue Engineering: Toward a New Era of Medicine. Annu Rev Med. 2017;68:29-40

949. A modular coronary bifurcation bioreactor for controlled endothelial flow studies

Manuel Salinas

de Engineering. Nova Southeastern University, Fort Lauderdale (Florida) - United States

Coronary bifurcations are predilection sites for atherosclerotic plaque due to complex disturbed flow that is difficult to reproduce in vitro. We engineered a similitude-scaled distal-left-main bifurcation bioreactor that combines realistic hemodynamics with an enlarged working section, enabling endothelial tissue experiments and high-content imaging.

An idealized left main–LAD–LCx geometry was created in SolidWorks and scaled using similitude theory to a 13 mm inner-diameter straight segment while preserving physiologic Reynolds and Womersley numbers through appropriate flow-rate and viscosity adjustments. Daughter branch diameters and angles were selected to satisfy Murray’s law and reported coronary bifurcation angles. A transient CFD analysis was used to optimize vessel lengths, branch angles, and outlet boundary conditions so that time-averaged wall shear stress and oscillatory shear index reproduced hallmark coronary features, including low/oscillatory shear on lateral walls and elevated shear at the carina.

Human coronary artery endothelial cells (HCAECs, P3) were expanded in EGM-2, cryopreserved at low passage, and seeded onto substrates mounted in the 13 mm straight segment of the bifurcated platform. After reaching near confluence, monolayers were exposed in the bioreactor under controlled pulsatile flow for 24 h, without rocker-induced motion. Viability was assessed using a LIVE/DEAD assay (Calcein-AM/EthD-1), and junctional integrity was evaluated by VE-Cadherin immunofluorescence with DAPI nuclear counterstain.

Preliminary experiments showed that HCAEC monolayers remained highly viable after 24 h perfusion and that VE-Cadherin exhibited continuous junctional staining under baseline pulsatile flow, demonstrating robust attachment and barrier formation in the scaled bifurcation. This coronary-like bifurcation bioreactor enables controlled studies of endothelial responses to disturbed flow, providing a modular platform for future plaque-mimetic and multilayer tissue-engineered constructs relevant to atherosclerotic disease.

950. Establishment of a protocol to generate Kupffer cells from induced pluripotent stem cells

Gijs. J. J. Van Slobbe¹, Mathias Busch¹, Coen Govers², Hans Bouwmeester¹

¹Division of Toxicology. Wageningen University & Research, Wageningen (Gelderland) - The Netherlands, ²Cell Biology and Immunology Group. Wageningen University & Research, Wageningen (Gelderland) - The Netherlands

Kupffer cells (KCs) encompass the tissue-residing macrophage population of the liver. KCs play a vital role in tissue homeostasis of the liver by engulfing harmful particles and sensing danger signals like damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs). Human KCs are characterized by the expression of specific surface markers (folate receptor beta (FOLRβ), v-set and immunoglobulin domain containing 4 (VSIG4) and cluster of differentiation 163 (CD163)) and high phagocytic activity. Although KCs play a crucial role in tissue homeostasis, the biology of human KCs is poorly studied due to the lack of models that accurately resemble these cells. Therefore, this work focuses on the establishment of mature and functional KCs from induced pluripotent stem cells (iPSCs). For this, iPSCs were differentiated towards monocytes and subsequently subjected to macrophage colony-stimulating factor (M-CSF), dexamethasone, a liver x receptor (LXR) agonist and bone morphogenetic protein 9 (BMP9) to differentiate monocytes towards a KC-like phenotype, or towards a macrophage-like phenotype via exposure to M-CSF. Marker expression and phagocytic activity were measured to verify the establishment of a KC-like phenotype in iPSC-derived KCs (iKCs), and was subsequently compared to iPSC-derived macrophages (iMacs). The iMac and the iKC model both showed expression of the macrophage markers CD45 and CD14 as well as expression of the KC marker FOLRβ. Furthermore, KC markers VSIG4 and CD163 were upregulated in the iKC model, compared to the iMac model. Analysis of phagocytic activity showed that the iKC model had higher phagocytic activity compared to the iMac model. Collectively, the current iKC model showed elevated expression of KC markers and increased phagocytosis, which shows that the current set of growth factors successfully skews monocytes towards a KC-like phenotype. The established iKC model provides a promising tool to further study and understand the immune environment of the liver.

951. Evaluation of two xenogeneic-free culture conditions for human MSC used in tissue engineering. A comparative study

Paula Ávila-Fernández¹, Gloria Pérez-Ortiz², Andrea Rejón-Camacho², Carmen González-Gallardo³, Fernando Campos¹, Óscar Darío García-García¹, Miguel Alaminos¹, Miguel Ángel Martín-Piedra¹, M. Carmen Sánchez-Quevedo¹, Jesús Chato-Astrain¹, Ingrid Garzón¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²Histology. University of Granada, Granada - Spain, ³Ophthalmology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain

Background: Dental Pulp Stem Cells (DPSC) and Wharton's Jelly Stem Cells (WJSC) represent highly promising, cell sources in tissue engineering of the human oral mucosa, palate, cornea and skin, due to their high proliferative capacity, multipotency, and low immunogenicity. However, their clinical application is constrained by the use of xenogeneic material during cell expansion, especially, fetal bovine serum (FBS). Here, we compared the efficiency of human plasma (HP) and platelet lysate (PL) as xenogeneic-free supplements for the expansion and culture of DPSC and WJSC.

Methods: A comparative study was conducted to establish the optimal, xenogeneic-free culture conditions for DPSC and WJSC using DMEM-Complete medium supplemented with FBS (control), HP or PL. Cell behavior was functionally evaluated at each culture condition by using functional assays able to determine cell viability and metabolic activity.

Results and conclusions: Human-derived supplements significantly outperformed FBS in the expansion protocol of both DPSC and WJSC. Specifically, HP and LP increased up to 95% cell viability levels for both, DPSC and WJSC, in comparison with FBS. Both cell types showed optimal cell function and proliferation in the xenogeneic-free conditions. In general, these results confirm that HP and PL human blood derivatives are highly effective and safe for cell culture and support their use to generate heterotypical substitutes of the human oral mucosa, palate, cornea and skin.

Supported by grants FIS FIS-PI23/00335, FIS-PI24/00006, FIS-PI25/00002, ICI19/00024-BIOCLEFT and ICI21/00010-NANOULCOR, funded by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Plan Estatal de Investigación Científica, Técnica y de Innovación, and Plan de Recuperación, Transformación y Resiliencia). Co-financed by the European Regional Development Fund (ERDF-FEDER) through the “Una manera de hacer Europa” program. Supported by DGP_PIDI_2024_00361, grant DGP_PIDI_2024_01347 and C-CTS-032-UGR23, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía and University of Granada, Spain.

952. Two-photon-polymerization of polyacrylamide cell scaffolds with tunable ECM-tethering and stiffness

Annabelle Sonn¹, Målin Schmidt¹, Chantal Barwig¹, Christine Selhuber-Unkel¹

¹Institute for Molecular Systems Engineering and Advanced Materials (IMSEAM). Heidelberg University, Heidelberg (Baden-Wberg Bayern) - Germany

Polyacrylamide (pAm) hydrogels are commonly used for the fabrication of 2D and 3D cell scaffolds on the macroscale. Two-photon-polymerization (2PP) pAm can be used to print precise and complex structures on the micrometer length scale. In this study, we develop pAm microstructures printed by 2PP, that can be functionalized with extracellular matrix (ECM) proteins enabling cell-scaffold interactions. Depending on the formulation of the resin, it is possible to tune the ECM-tethering and therefore create cell-repellent and cell-adhesive structures. The stiffness of the scaffolds is tuned by adjusting the printing parameters, which is demostrated by nanoindentation. Finally, we show that multimaterial printing is possible to give rise to microstructures with locally regulated cell adhesion. In the future, these versatile resins will bear great potential for tissue engineering and biological studies with controlled chemical and mechanical signals on the micrometer length scale.

953. Forging an independent research path in tissue engineering in Spain: early lessons from a new PI

Pedro J. Diaz-Payno

de IMDEA Materials Institute, Madrid - Spain

Transitioning into an independent research role marks a decisive stage in the career of any scientist, especially within a rapidly evolving field such as tissue engineering. This presentation reflects on my path from early training to the establishment of a new research group at IMDEA Materials Institute, highlighting both opportunities and challenges encountered along the way. My scientific background spans biomimetic ECM scaffold development during my PhD in Ireland, 4D bioprinting in the Netherlands, and subsequent work on biodegradable metals for musculoskeletal repair. These experiences shaped a research vision focused on engineering functional tissues that respond not only to structural needs but also to biological and mechanical cues.

After several years abroad, I returned to Spain to build a family and be close to my own. This personal decision carried uncertainty, as funding opportunities and long-term career prospects are more limited compared to Northern Europe. Before joining IMDEA Materials, I worked in the marketing department of Pharmaceutical Laboratories ROVI. That period provided a valuable perspective on how industry, clinicians, and regulatory environments communicate, revealing gaps that often slow down translation. This understanding has influenced how I now plan research directions and evaluate potential clinical impact.

As a Ramón y Cajal researcher, I am leading a program dedicated to extracellular-matrix-inspired scaffolds reinforced with architected metamaterial designs, together with porous freeze-dried structures for bone, cartilage, ligament, meniscus, and other connective tissues. These strategies allow fine control over mechanics while keeping implants lightweight and permeable for cell infiltration and matrix deposition.

This contribution will discuss scientific, organizational, and personal aspects of becoming a PI in Spain. I will also reflect on the creation of SEMIT and how national scientific communities can help support early-career researchers. The goal is to share realistic lessons for those entering this stage of their careers.

954. Modified gellan gum nanoparticles as drug delivery vehicles

Ariana Gomes¹, Annalisa Perioli¹, Vânia I. B. Castro¹, Ana R. Araújo¹, Rui L. Reis¹, Ricardo A. Pires¹

¹3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on TERM; ICVS/3B’s–PT Government Associate Laboratory, Guimarães (Braga) - Portugal

Heart failure (HF), resulting from ischemic heart disease and myocardial infarction (MI), is the major cause of death globally, however, current treatment options remain limited, with heart transplantation being the most common strategy in advanced stages of HF (1). MI leads to the irreversible loss of cardiomyocytes and fibrotic tissue formation, impairing cardiac function (2). A strategy to induce the regeneration of myocardium is the use of paracrine modulators. However, their local delivery and retention at MI site is difficult, showcasing the necessity to develop sustained targeted delivery systems.

We developed cell-free delivery vehicles using gellan gum (GG), combined with GG conjugated with a cardiac homing peptide (i.e., CSTSMLKAC) and sulfated GG (to improve their capacity to capture and preserve growth factors in their bioactive conformation for extended periods of time – a known capacity of sulfated glycans. Nanoparticles of the combined systems (GGnps, 150nm average diameter) were produced using water-in-oil methodology previously published by our group (3). GGnps were characterized by their size, zeta potential and polydispersity index, monitored over 7 days in water, PBS and culture medium. Our results revealed that GGnps were stable under physiological conditions, confirmed by SEM. Biocompatibility and uptake studies using the cardiomyocyte cell line H9C2 showed that GGnps were non-toxic up to a concentration of 200µg/mL and were successfully internalized (microscopically confirmed using FITC labelling). We used BSA-FITC as a model loading protein and achieved an encapsulation efficiency of apx. 80% and a 10% release in 24h. Our results demonstrate that our system can act as a controlled delivery system capable of targeting cardiomyocytes and deliver its cargo in their intracellular space.

Acknowledgements: Founded by the European Commission, Portuguese Foundation for Science and Technology and CCDR-N: AmnioSMART project(CARDINNOV/0001/2023) and TERM RES Hub(Norte-01-0145-FEDER-022190, Norte2020/CCDR-N).

References: (1) Timmis et al.,European Heart Journal, 2020, 41(1), 12-85; (2) Bozkurt et al., Circulation: Cardiovascular Quality and Outcomes, 2021, 14(4); (3) Rodrigues et al., Biomaterials Research, 2022, 26(1)

955. Osteocytogenesis in-vitro for advanced microphysiological modelling of mature human bone tissue

Luke Madden¹, Rosario Milazzo¹, Daniel J. Kelly¹, David A. Hoey¹

¹Trinity Centre for Biomedical Engineering. Trinity College Dublin, Dublin - Ireland

Current bone models fail to replicate the dynamic environment of the mature tissue. This is due to a lack of efficacious methods to obtain human osteocytes, the most abundant bone cell type, which modulate bone physiology by regulating other bone cells [1]. Efficient osteocytic bone models for patient-specific organ-on-a-chip (OoC) systems would be a significant improvement on existing humanised bone OoC models, which cannot effectively model human osteocytes [2]. Therefore, we aim to develop methods to produce bulk and microtissue (µT) human bone containing functional osteocytes, readily incorporated into OoCs.

Collagen-nanoneedle (coll-nnHA) hydrogels were created by combining collagen type I (8 mg/mL), nanoneedle hydroxyapatite (0.27 mg/mL), and human mesenchymal stem cells (2 × 10⁶ cells/mL), forming cell-laden scaffolds. This mixture was cast into cylindrical moulds (⌀ = 6.25 mm, h = 2 mm) or directly into microfluidic devices and crosslinked for 60 minutes. µTs were formed by dispensing hydrogel volumes into ultra-low-attachment 384-well plates. Microfluidic chips were fabricated via PDMS replica moulding and plasma bonding to coverslips.

Coll-nnHA ECM induced osteogenesis and subsequent osteocytogenesis in hMSCs, evidenced by alizarin red staining at 21 days and upregulation of mature osteocyte markers SOST and MEPE at 42 days, coupled with sclerostin protein secretion. Manipulation of collagen gel contractility induced formation of µTs, which retain the potent osteogenic capacity of bulk coll-nnHA. Application of bulk coll-nnHA to OoC results in reduced mineralisation.

Current work involves implementation of pre-matured µTs to OoC to explore µT-on-chip models of mature human bone, addressing issues with bulk mineralisation on chip. Induction of diseased states and measurement of responses to therapeutics will enable assessment of coll-nnHA’s ability to form hMSC-derived models of bone disease.

1. Bonewald, L.F., The amazing osteocyte. J Bone Miner Res, 2011.

2. Fois, M.G., et al., Mini-bones: miniaturized bone in vitro models. Trends in Biotechnology, 2024.

956. Next-generation therapies for spinal fusion and segmental bone defects in clinical trials

Reinhard Windhager

de Department of Orthopedics and Trauma Surgery. Medical University of Vienna, Vienna (Wien) - Austria

Bone regeneration remains a major clinical challenge in the treatment of fractures, spinal fusion, and nonunions, as the gold standard—autologous bone grafting—has significant limitations, including limited availability and donor site morbidity. To overcome these drawbacks, recombinant human Bone Morphogenetic Protein 6 (rhBMP6) delivered within autologous blood coagulum (ABC) has been developed as a novel, biologically compatible bone graft substitute, known as Osteogrow. This approach utilizes the body’s own coagulum as a natural carrier, allowing controlled release of rhBMP6 and promoting osteoinduction in various clinical settings. These clinical studies aimed to evaluate the safety, tolerability, and efficacy of rhBMP6/ABC in multiple orthopedic indications, including high tibial osteotomy, distal radius fracture, lumbar interbody fusion for degenerative disc disease, and posttraumatic tibial nonunion. In a randomized, placebo-controlled trial of high tibial osteotomy, rhBMP6/ABC was applied at the osteotomy site, and bone healing was monitored by quantitative CT analysis. In a phase 1 randomized study of distal radius fractures, callus formation, cortical rebridgement, and trabecular remodeling were evaluated radiographically at 5, 9, and 13 weeks. The OSTEOproSPINE non-inferiority trial examined a single dose of rhBMP6/ABC in patients undergoing posterior lumbar interbody fusion, with imaging used to assess fusion stability and micromotion. A dose-ranging study using rhBMP6/ABC supplemented with synthetic ceramics (Osteogrow-C) was initiated for posttraumatic tibial nonunion, with radiological follow-up at 3–12 months. Patients treated with rhBMP6 in high tibial osteotomy showed accelerated bone healing at 9 and 14 weeks, without serious adverse events or anti-BMP antibody formation. In distal radius fractures, rhBMP6-treated patients demonstrated faster early bone regeneration, though differences decreased by week 13. Overall, rhBMP6 delivered in autologous blood coagulum demonstrates strong potential as a safe and effective therapy for enhancing bone healing and regeneration.

957. Biodegradable nanofibrous dressing incorporating GOAg for enhanced antimicrobial performance in chronic wound treatment

Thamyres F. Da Silva¹, Francisco F. Pereira¹, Ana L. De B. Soares¹, Rebeca M. Chaves¹, Rodrigo S. Vieira¹, Fábia K. Andrade¹

¹Department of Chemical Engineering. Federal University of Ceará, Fortaleza (Ceara) - Brazil

Chronic wounds represent a major global health challenge, affecting an estimated 1–2% of the world population over their lifetime. Such wounds often present persistent microbial colonization. Current commercial dressings commonly rely on relatively high concentrations of silver, which may induce cytotoxic effects while offering limited control over release kinetics. Electrospinning has emerged as a promising technology to fabricate nanofibrous membranes with high surface area, porosity, and improved contact with the wound bed. The incorporation of graphene oxide–silver nanocomposites (GOAg) into polymeric matrices has demonstrated enhanced antimicrobial and antibiofilm activities, as well as improved mechanical performance through stable hydrogen-bond interactions. The objective of this study was to develop and characterize a wound dressing composed of PVA-sodium alginate-GOAg. The work was structured into three stages: production of GOAg nanocomposite; preparation and characterization of electrospun membrane incorporating the optimized GOAg. The nanocomposite was characterized by AFM and Raman spectroscopy. Antibacterial and antibiofilm activities were assessed against Staphylococcus aureus and Pseudomonas aeruginosa. After, nanofibrous membrane was produced by electrospinning PVA-SA-GOAg. The membrane underwent AFM imaging, antimicrobial tests and cytotoxicity assays. The PVA-SA-GOAg nanofibers demonstrated uniform morphology and high surface area conducive to sustained antimicrobial release. The membrane showed biocompatibility to fibroblasts cells in vitro, presents a promising alternative for the management of chronic wounds, antimicrobial performance with low silver content, and favorable biological compatibility.

958. Composite biomaterials on collagen with antimicrobial activity

Doina Fosa¹, Mariana Jian², Vitalie Cobzac³, Andrei Mostovei⁴, Valeriana Pantea⁵, Ianos Coretchi⁶, Ludmila Motelica⁷, Ovidiu Cristian Oprea⁷, Denisa Ficai⁷, Anton Ficai⁷

¹surgery. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ²Bioology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ³traumatologi. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁴stomatology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁵farmacy. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁶Biochimist. State Medical and Pharmaceutical University ”Nicolae Testemitanu”, Chisinau - Moldova, ⁷nanomateriale. University Polytechnica of Bucharest, Bucharest - Romania

Bone tissue engineering is a complex field of research involving pure regeneration, diagnosis and treatment of specific bone-related diseases such as osteoporosis, osteomyelitis, osteosarcoma. Considering the composite nature of bone tissue, the morphology induces the properties of bone tissue, the most biomimetic solution is based on composite materials based on collagen polymer and hydroxyapatite (COLL/HA) and their properties can be further corrected by loading with different an improvement of biological activity – from terostructures, drugs, drugs.

The purpose of this study was to evaluate antimicrobial activity of composite biomaterials based on collagen, hydroxyapatite, mesoporous silica (MCM 41) loaded with coriander oil (Col/HA/MCM-41/OIL composite materials).

Col/HA/MCM-41/OIL composite materials were obtained and used in hard tissue engineering as drug delivery system.

The antimicrobial activity was analyzed by agar diffusion test. The bacterial strain Staphyloccocus aureus (t.209) was cultured on the medium, and the composite samples were placed on the agar surface. After the incubation period, the inhibition zones were visually examined.

The results of the diffusimetric test of Col/HA/MCM-41 composite materials loaded with coriander oil completely inhibited the growth of the Stafilococus aureus (t.209) strain, demonstrating a pronounced antibacterial efficacy.

959. Carboxymethyl tunicate cellulose bioink for adipose tissue reconstruction

Kristin Oskarsdotter¹, Peter Apelgren², Rikard Agrenius³, Edwin Eliasson³, Karin Säljö², Stina Simonsson³, Paul Gatenholm⁴, Lars Kölby¹

¹Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg (Vastra Gotaland) - Sweden, ²Region Västra Götaland, Sahlgrenska University Hospital, Department of Plastic Surgery, Gothenburg (Vastra Gotaland) - Sweden, ³Department of Medicinal Chemistry & Cell Biology, Institution of Biomedicine, Sahlgrenska University Hospital, Gothenburg (Vastra Gotaland) - Sweden, ⁴Advanced Polymer Technology AB, Gothenburg, Sweden, Gothenburg (Vastra Gotaland) - Sweden

3D bioprinting of adipose tissue (AT) holds significant promise for reconstructive surgery, as reliable soft-tissue substitutes could reduce the need for complicated and invasive surgery. Realizing this potential requires the development of bioinks that provide appropriate biological, mechanical, and physiochemical properties to produce viable tissues. Cellulose nanofibrils (CNF) isolated from tunicates have been recently evaluated as a biocompatible hydrogel for AT engineering (Säljö et al. 2022), which through combination with the polysaccharide alginate (ALG) can be dimensionally stabilized through ionic crosslinking. Through further surface modification by carboxymethylation, CNF can acquire the ability to crosslink through a similar mechanism, eliminating the need for additional components to ensure dimensional stability.

Here, we evaluated carboxymethyl tunicate cellulose (CTC) as a one-component bioink for reconstruction of AT through implantation of solid grafts produced through 3D bioprinting.

Bioink formulations composed of pure CTC, CTC:AT and CTC:AT:ALG were prepared, and square scaffolds were 3D printed and crosslinked through ionic gelation (Ca2+) and implanted in Balb/c mice. After 30 days, the scaffolds were explanted, studied macroscopically and harvested for evaluation. Rheological evaluations were performed on scaffolds on the day of implantation and explantation, and the elastic modulus was measured through unconfined compression and nanoindentation.

In vivo, 3D printed CTC or CTC:AT scaffolds did not retain its 3D shape. However, through the addition of ALG, the 3D graft remained dimensionally stable for 30 days in vivo, with high tissue retention. Unconfined compression confirmed that although initial stiffness dropped significantly over the time in vivo, the resulting stiffness after 30 days remained within the same order of magnitude as native AT.

From our preliminary results, we conclude that CTC is a promising biomaterial as a bioink for 3D printing AT for soft tissue reconstruction that warrants further study and optimization.

960. Get a grip: finding the best combination of tissue engineering fabrication practices for the creation of pillar pulling, artificial skeletal muscles

Vasileios D. Trikalitis¹, Cintia Rivares Benitez¹, Niek Klein Hofmeijer¹, Jose M. Rivera Arbelaez², Alessandro Iuliano³, Pim Pijnappel³, Robert Passier², Jeroen Rouwkema¹, Massimo Sartori¹

¹ Biomechanical Engineering. University Twente, Enschede (Overijssel) - The Netherlands, ²Applied Stem Cell Technologies, Department of Bioengineering Technologies, Cardiovascular Health Technology Centre, TechMed Centre, University of Twente, Enschede, The Netherlands, ³Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, 3015GE, The Netherlands. Department of Pediatrics, Erasmus MC University Medical Center, Rotterdam, 3015GE, The Netherlands. Center for Lysosomal and Metabolic Diseases, Erasmus MC University Medical Center, Rotterdam, 3015GE, The Netherlands

Skeletal muscles comprise, on average, 30-40% of a human’s total body mass. Their function in everyday activities, their role in metabolic processes, as well as the variety of diseases and injuries they are involved, make it one of the most important target tissue categories for tissue engineering. There is a plethora of approaches in skeletal muscle engineering, including the use of fibrin, Matrigel, collagen, hyaluronic acid, or a combination of those as scaffolds. Moreover when fibrin is used as the scaffold, aprotinin and/or 6-Aminocaproic acid (EACA) are used to delay the fibrin matrix remodeling. The cell type, ranging from immortalized mouse myoblasts (C2C12) to Induced Pluripotent Stem Cell (iPSC) derived myoblasts, as well as the cell number used for skeletal muscle engineering is also not standardized. In all cases, the skeletal muscle tissue must grip around pillars to enable functional contractions. Thus, in this work we present a systematic comparison where self-assembled, cell-only skeletal muscle tissues wrapped around inert pillars are used as a control, and are compared with tissue made using fibrin scaffolds with and without the addition of EACA and/or aprotinin. We demonstrate the effect of those parameters in the resulting formation of skeletal muscle tissue in terms of morphology over time and ability to grip around pillars, and present the best combination practice for C2C12 and iPSC-derived myoblasts.

961. Bioadhesive hydrogel for intra-articular delivery of lipid and mesoporous silica nanoparticles for osteoarthritis treatment

José Carlos García Perdiguero¹, María Natividad Gómez Cerezo¹, Miguel Manzano García², María Vallet Regí³

¹Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid - Spain, ²Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid - Spain, ³Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Instituto de Investigación Sanitaria, Hospital 12 de Octubre i+12, Madrid - Spain

Osteoarthritis (OA) is a major global health and economic burden. OA arises from a multifactorial factors resulting in the disruption of cartilage homeostasis and a pro-inflammatory environment. OA treatment represents a challenge due to cartilage’s limited regenerative capacity, its avascular nature, and the rapid clearance of intra-articular therapeutics.

In this context, materials science and tissue engineering combined with gene therapy offer promising strategies to treat this multifactorial disease. The use of gene therapy has shown substantial potential in regenerative medicine. Even though its translation is still limited, nanoparticles can address these limitations. This work focuses on the development of a hydrogel designed for intra-articular delivery of nanoparticles with the aim of improving gene transfer to chondrocytes and mesenchymal stem cells.

The hydrogel was based on hyaluronic acid (HA) and fibrin. HA was oxidized and functionalized with dopamine. Dopamine, inspired by the adhesive chemistry of mussels in aqueous environments, confers strong tissue adhesion. The combination of modified HA and fibrin resulted in a hybrid network with enhanced structural properties. We investigated how this new formulation affects the release profile of therapeutic molecules and nanoparticles. In addition, the hydrogel was evaluated on chondrocytes and immune system cells. The capacity of the hydrogel to induce macrophage polarization was examined using human macrophages, showing pro-regenerative M2 phenotype, which is of particular interest in an OA context.

The hydrogel was loaded with two classes of nanoparticles: lipid nanoparticles (LNPs) and mesoporous silica nanoparticles (MSNs). Nanoparticles were synthesized with an average size of approximately 50 nm, a dimension to enhance penetration to deeper regions of the tissue. The cellular internalization of LNPs and MSNs released from the hydrogel was evaluated, along with the expression of GFP messenger RNA delivered by these systems in chondrocytes and human mesenchymal stem cells.

962. Automated assembly of liquid capsules to establish self-guided bone-forming environments

Maria Clara Gomes¹, Ana Margarida Almeida¹, Ana Rita Pinho¹, Magda Carvalho Henriques¹, Mano João¹

¹CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal

Engineering bone tissue in vitro that captures the behavior of all bone resident cells requires microenvironments that support viability, guide cell organization, and allow for dynamic remodeling. Here, we introduce a modular strategy based on liquefied capsules that act as supportive osteogenic units and can be positioned with high precision using a robotic arm. Each capsule contains a liquid core that improves nutrient and oxygen transport and promotes natural three-dimensional remodeling, while the surrounding protein-based shell provides stability, controlled permeability, and compatibility with external handling requirements.

The robotic assembly step enables the predictable placement of individual capsules to create organized architectures and spatial patterns that are difficult to achieve with traditional biomaterials. This combination of a fluid internal environment with automated positioning supports the emergence of bone like features in a controlled and self-directed manner. The capsules integrate well within larger engineered constructs and create discrete osteogenic niches that can sustain long term tissue formation.

Overall, this capsule-based and automation-driven approach offers a simple but powerful platform for introducing stable and functional bone-forming units inside complex bioengineered tissues, advancing the development of more biomimetic bone models.

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). We also acknowledge the European Research Council Advanced Grant ‘‘REBORN’’ (grant agreement no. ERC-2019-ADG-883370), and the METABONE project (COMPETE2030-FEDER-00784100).

963. Native fibrillar-collagen scaffolds for cardiac tissue engineering

Teresa Zúñiga Arrarás¹, Amaia Guembe Lapuente¹, Iker Ateca Blanco¹, Pilar Montero-Calle², Ilazki Anaut-Lusar², Edaurdo Larequi³, Felipe Prósper⁴, Manuel Maria Mazo⁵, Jesús Maria Izco⁶

¹Viscofan SLU, Cáseda (Navarra) - Spain, ²Biomedical Engineering Program, Enabling Technologies Division, CIMA Universidad de Navarra, Foundation for Applied Medical Research, and IdiSNA, Navarra Institute for Health Research, Pamplona (Navarra) - Spain, ³Hematology and Cell Therapy, Clinica Universidad de Navarra, Pamplona (Navarra) - Spain, ⁴Hematology and Cell Therapy, Clínica Universidad de Navarra, and Hemato-Oncology Program, Cancer Division, CIMA Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona (Navarra) - Spain, ⁵Biomedical Engineering Program, Technological Innovation Division, Cima Universidad de Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), and Hematology and Cell Therapy, Clínica Universidad de Navarra, Pamplona (Navarra) - Spain, ⁶Viscofan SA, Tajonar (Navarra) - Spain

Ischemic heart disease (IHD) is the leading single cause of mortality in Europe1. Cardiac tissue engineering (cTE) is focused on creating functional heart tissue using cells, scaffolds, and bioactive signals. Fibrillary collagen I is the main component of the cardiac extracellular matrix, but its use in cTE is limited to its soluble and less biomimetic form. The objective of this work is to develop a fibrillar collagen-based scaffold for cTE to be used as a 3D regenerative option for IHD. Freeze-dried collagen scaffolds were developed and crosslinked with EDC/NHS or with a dehydrotermal treatment (DHT) and compared with a non-crosslinked one. For this, the two main types of the cells found in the cardiac tissue (fibroblasts and cardiomyocytes) were differentiated from hiPSCs and seeded in a 1:9 ratio. Cell viability was measured via Alamar Blue^®, whilst cell morphology and distribution were observed with a Live/Dead^® Assay, Immunofluorescence and histology. Beating rate was also recorded and gene expression analysis with RT-qPCR, after a 3-week culture period. Cell metabolism, measured by Alamar blue, was strongly influenced by scaffold type, showing the non-crosslinked scaffolds a much lower viability than the crosslinked ones (EDC and DHT) at day 21 (0.11, 0.5, 0.84 Alamar Blue units respectively). Cell internalization was also distinct between the formed structures although cell organization appeared to be similar between the three types. The beating rate of the crosslinked scaffolds was higher than the non-crosslinked ones (20 beats/min and 12 beats min respectively), in line with the higher cell viability. RT-qPCR however showed little variation, probably due to the highly similar mechanical properties. In conclusion, the lyophilized collagen scaffolds seem to be an optimal solution to generate 3D cardiac functional tissues.

1. Wilkins E, Wilson L, Wickramasinghe K, Bhatnagar P, Leal J, Luengo-Fernandez R, Burns R, Rayner M, Townsend N (2017). European Cardiovascular Disease Statistics 2017. European Heart Network, Brussels.

964. From micro to macro-tubular constructs: exploring biofabrication methodologies to achieve higher anatomical mimicry using blood-derived polymers

Rita Sobreiro-Almeida¹, Andreia Malafaia¹, Leila Pimentel¹, João M. M. Rodrigues¹, Marcelo Costa¹, João F. Mano¹

¹CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal

Tubular tissue engineering is at the forefront of regenerative medicine, aiming to replicate the structure and function of natural tubular organs. These organs, including vessels, trachea, esophagus, ureter, urethra, etc., play critical roles in human physiology. The complexity of their anatomical and physiological characteristics presents unique challenges in creating bioartificial counterparts.

Two biofabrication strategies were developed to generate tubular constructs using blood derived polymers: 3D extrusion printing and layer-by-layer 4D assembly. In the first, low-viscosity photoclickable inks composed of hyaluronic acid-norbornene (HA-Nor) and human platelet lysate (hPL) were formulated and rheologically characterized. Structures were printed using embedded 3D extrusion within a xanthan gum support bath, followed by rapid thiol-ene photo-gelation. Mechanical properties and cytocompatibility with endothelial cells were assessed. For the second, methacrylated bovine serum albumin (BSA-MA) was assembled into 200-layer membranes using a layer-by-layer dipping robot with alternating BSA-MA/PEI layers. Crosslinking gradients were introduced via patterned photomasks. Membranes were characterized structurally, mechanically, and by swelling-driven curvature analysis.

HA-Nor/hPL inks exhibited shear-thinning behavior, ultrafast gelation (<20 s), and produced constructs with soft-tissue-relevant stiffness (20–30 kPa). Embedded printing enabled high-fidelity fabrication of straight and branched tubular geometries. Stem cells remained viable and metabolically active, confirming the biocompatibility of the hPL-enriched matrices. Additionally, patterned BSA-MA membranes showed rapid, programmable self-rolling upon hydration, forming hollow tubes within seconds. Curvature direction and magnitude correlated with photopattern-induced crosslinking gradients, enabling controlled shape morphing with good mechanical integrity.

Together, these methods demonstrate two complementary routes for creating bioactive tubular architectures of micro to macro sizes, relevant to diverse tissue engineering approaches.

Acknowledgements: This work was supported by micro2Macro Horizon EU project (101191729) and the FCT-funded project LEGO (COMPETE2030-FEDER-00827000). Authors wish to acknowledge the PhD grant of A.M and the CEEC grants of R.S-A (10.54499/2022.04605.CEECIND/CP1720/CT0021) and J.M.M.R (10.54499/2023.07239.CEECIND/CP2840/CT0004).

965. Tailoring material properties through network topology control: mid-chain versus end-group crosslinking in poly(globalide) photo-crosslinkable resins

Noemi S. P. Kimura¹, Gabriel S. Michelini¹, Isabela L. A. Dourado¹, Flávia Gonçalves², Luiz H. Catalani¹

¹Institute of Chemistry. University of São Paulo (USP), Sao Paulo - Brazil, ²Department of Odontology. University Santo Amaro, Sao Paulo - Brazil

In tissue engineering, the ability to modulate the chemical, physical, and biological responses of a polymer-based scaffold through control of its chemical composition is crucial for designing materials tailored to different target tissues.1 In this work, four novel photocurable resins based on linear and star-shaped poly(globalide) (PGl) derivatives were developed to study the impact of pre-polymer architecture (linear or star-shaped), crosslinking mechanism (chain or step growth polymerization), and crosslinking position (from mid-chain or terminal double bonds) on material properties. PGl polymers were synthesized via enzymatic ring-opening polymerization and subsequently acrylated to yield photo-curable resins.2 The position of the reactive double bonds proved to strongly influence network topology, crosslink density, and their thermal and mechanical performance. Resins crosslinked from terminal double bonds via chain polymerization reaction showed crystallinity values 90% higher and an elongation at break at least sevenfold greater than systems crosslinked via the globalide-native mid-chain unsaturations crosslinked via thiol-ene reactions. Photo-rheology indicated rapid gelation times (5–10 s), while accelerated hydrolysis experiments revealed tunable degradation rates. Cytocompatibility assays showed non-cytotoxicity behavior towards NIH-3T3 and HEK-293 cell lines. The materials developed addresses the growing demand for new photocurable resins for 3D printing that are both cytocompatible and hydrolytically degradable. Finally, 3D-printed tests validated their printability and processability. Overall, these results highlight the versatile potential of globalide-based resins, notably due the presence of mid-chain double bonds that can be fully or partially preserved for post-crosslinking functionalization,2,3 thereby broadening their applicability.

References

1. Yu, C. et al. Chem. Rev. 120 (19), 10695–10743 (2020).

2. Rebouças, L. O. et al. Polym. Chem. 16, 2962-2977 (2025).

3. Guindani, C. et al. Mater. Sci. Eng. C, 94, 477-483 (2019).

966. A human neuromuscular platform for ALS modelling and drug testing

Afonso Malheiro¹, Katharina Hennig², David Barata², Inês Martins¹

¹Accelbio, Lisboa - Portugal, ²Gulbenkian Institute for Molecular Medicine (GIMM), Lisboa - Portugal

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by progressive motor neuron loss. Approximately 90% of ALS cases are sporadic, limiting the relevance of animal models and creating a critical need for human-based systems that can capture patient heterogeneity and accelerate drug discovery. Human in vitro platforms therefore represent a promising strategy to model sporadic ALS in a rapid, biologically relevant, and cost-effective manner.

We developed a microfluidic platform containing a functional human neuromuscular tissue with a mature, biomimetic architecture. The tissue is formed by optogenetically modified iPSC-derived motor neurospheres, which exhibit robust electrical activity and express key maturation markers (ChAT, ISL1/2), co-cultured with iPSC-derived skeletal muscle fibers showing aligned organization and characteristic mature features, including striation, peripheral nuclei, and myosin heavy chain expression. Blue-light stimulation enables remote activation of neuromuscular junctions (NMJs), allowing quantification of muscle contractions as a direct readout of functional NMJ connectivity.

To model sporadic ALS, two patient-derived iPSC lines were incorporated into the system. Both ALS models displayed markedly reduced and smaller NMJs (synapsin/BTX co-localization), fewer and slower muscle contractions, decreased cell viability, and hallmark ALS pathological features such as pTDP-43 cytoplasmic inclusions and stress-granule formation.

As a proof-of-concept for drug screening, we tested ropinirole, previously reported to confer therapeutic benefit in motor-neuron-only cultures1. Ropinirole treatment enhanced neurite outgrowth, improved cell viability, and reduced pathological markers in our system. Ongoing work is evaluating its ability to rescue NMJ functionality within the platform, providing insight into its potential as a candidate ALS therapy.

Overall, this microfluidic human neuromuscular model offers a powerful tool for studying sporadic ALS mechanisms and holds broad applicability for preclinical testing across neuromuscular diseases.

1. Fujimori K. et al., “Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent”, Nat Med, 2018

967. Restoration of type XVII collagen in skin cells from junctional epidermolysis bullosa patients to improve dermal-epidermal junction adhesion strength

Anne-Julie Bernier¹, Martin Barbier¹, Mbarka Bchetnia¹, Danielle Larouche¹, Manuel Caruso², Mélissa Saber³, Lucie Germain¹

¹Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX, Department of Surgery, Faculty of Medicine, Université Laval and Regenerative Medicine Division, Centre de recherche du CHU de Québec-Université Laval, Quebec - Canada, ²Department of Molecular Biology, Faculty of Medicine, Université Laval and Centre de Recherche sur le cancer de l'Université Laval, Quebec - Canada, ³Department of Medicine, Université de Montréal and Centre de recherche du CHUM, Université de Montréal, Montreal (Quebec) - Canada

Junctional epidermolysis bullosa (JEB) is a rare genetic skin disease with no curative treatment. It is characterised by skin fragility at the dermal-epidermal junction. JEB can be caused by a deficit in type XVII collagen (COLXVII), a protein involved in dermal-epidermal junction adhesion strength and expressed only by keratinocytes. Tissue-engineered skin substitute(s) (TES) produced with the self-assembly approach represent a useful model to study epidermolysis bullosa. In combination with ex vivo gene therapy, TES could be developed to treat patients’ chronic wounds. We obtained a biopsy from a JEB patient carrying a mutation in the gene COL17A1 leading to a complete loss of COLXVII expression. We successfully extracted and amplified JEB primary keratinocytes and fibroblasts in culture to establish a cell bank. The objective of this study is to investigate the therapeutic potential of ex vivo gene therapy of primary keratinocytes combined with tissue engineering to produce corrected skin substitutes. To permanently restore the expression of COLXVII, we used a self-inactivating retroviral vector, highly effective at transducing primary keratinocytes and regularly used in clinical trials for its safety profile. A viral titer of 2.16E5 infectious units/mL was obtained with the first production of viral particles containing COL17A1. With these particles, primary JEB keratinocytes were transduced and recombinant COLXVII was detected in up to 21% of the keratinocytes. Transduced keratinocytes will be used with the patient’s fibroblasts to produce TES. Recombinant COLXVII deposition at the dermal-epidermal junction and strength of the dermal-epidermal junction will be assessed through immunostainings and mechanical peeling tests, providing information about the functionality of the recombinant COLXVII. This strategy has already been used by our team to design a gene therapy currently in clinical trial for recessive dystrophic epidermolysis bullosa. Hopefully, this work will also lead to the beginning of a clinical trial for JEB.

968. In vivo experimental study of composite biomaterials based on collagen

Mariana Jian¹, Doina Fosa², Vitalie Cobzac³, Andrei Mostovei⁴, Iana Baranetchi⁵, Ludmila Motelica⁶, Ovidiu Cristian Oprea⁶, Denisa Ficai⁷, Anton Ficai⁷

¹Biology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ²surgery. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ³traumatology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁴stomatology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁵epidemiology. State Medical and Pharmaceutical University “Nicolae Testemitanu”, Chisinau - Moldova, ⁶nanomateriale. University Polytechnica of Bucharest, Bucharest - Romania, ⁷nanomaterials. University Polytechnica of Bucharest, Bucharest - Romania

Composite biomaterials based on collagen are used in oral-maxillo-facial surgery for restoring bone defects. The most widely used are collagen-based grafts of xenogeneic origin, which still have certain shortcomings in use, due to the possibility of transmitting zoonoses. Thus grafts based on human collagen, extracted from the umbilical-placental complex, would be a solution for maxillofacial bone reconstruction.

The purpose of this study was to evaluate biocompatibility of composite biomaterials based on collagen extracted from the umbilical-placental complex.

Collagen/hydroxyapatite composite materials were obtained by mineralizing the collagen structure using soluble calcium salt precursors and soluble phosphates. Crosslinking of the biomaterial was performed with 1% glutaraldehyde. The composites obtained was washed for several days in distilled water and then lyophilized to obtain a potentially reliable graft for cellular population.

The control group consisted of a composite based on collagen extracted from the bovine Achilles tendon, whereas the experimental group consisted of a composite based on collagen isolated from the umbilical-placental complex. The obtained grafts were transplanted into Wistar rats in a 5mm calvarial defect. At one and three months, the rats were euthanized and blood samples were collected for determination of acid phosphatase analysis, antioxidant system indicators and oxidative stress markers.

Comparative analysis at one and three months shows that the human graft presents a more efficient bone integration and is less traumatic for the body, evidenced by a progressive decrease in the level of acid phosphatase. The xenogeneic graft induces a prolonged inflammatory reaction and an accentuated resorption, reflecting a lower biocompatibility.

969. Nanofiber scaffolds functionalized with mesenchymal stem cells conditioned media for enhanced human tendon regeneration

Lucie Wolfová¹, Karolína Vocetková¹, Veronika Hefka Blahnová¹, Tomáš Janoušek²

¹Institute of Experimental Medicine, Czech Academy of Sciences, Prague (Hlavni Mesto Praha) - Czech Republic, ²Nanoprogress, z.s., Pardubice - Czech Republic

Human tendon injuries represent a significant clinical challenge due to the limited natural regenerative capacity of tendons. Tissue engineering approaches using nanofiber scaffolds and mesenchymal stem cell (MSCs)-derived factors offer promising strategies to enhance tendon healing.

For this purpose, we developed a novel oriented nanofiber scaffold composed of a mixture of polylactide acid, polycaprolactone, hyaluronic acid, and collagen. To enhance tendon healing efficiency, the scaffolds were further functionalized using conditioned media obtained from human MSCs isolated from Wharton's jelly (hWJ-MSCs).

In this study we present biological properties of these scaffolds tested in vitro in the presence of human tenocytes and hWJ-MSCs cultured in tenogenic cell culture medium. Cell viability, morphology, proliferation and expression of specific tenogenic markers including Tenomodulin, Collagen I, Scleraxis, and Actin was evaluated. RT-PCR, immunochemistry staining and confocal microscopy were employed for detailed cellular and molecular assessment.

Our interim results showed that the newly designed functionalized nanofiber scaffold effectively supported cell viability, proper cell morphology and proliferation and promoted expression of tendon-specific extracellular matrix proteins, essential for effective tendon regeneration.

These findings suggest that this innovative scaffold combining oriented nanofiber architecture and biochemical functionalization by MSCs conditioned media, provides necessary cues to enhance tendon regeneration, making it promising biomaterial for effective tendon injury treatment and potential improvement of tendon regenerative therapies.

Acknowledgements: Supported by the Operational Program Technologies and Application for Competitiveness - OP TAC (CZ.01.01.01/01/22_002/0000798).

970.  

971. Aerosol Jet^® printing of gold nanoparticle ink for bio-conductive micro-patterns guiding Schwann cell migration in nerve conduits

Yuexi Zhuang¹, Miriam Seiti¹, Karen Libberecht², Tim Vangansewinke², Ivo Lambrichts², Eleonora Ferraris¹

¹KU Leuven, Leuven (Brabant) - Belgium, ²UHasselt, Hasselt (Brussels Hoofdstedelijk Gewest) - Belgium

Nerve guidance conduits (NGCs) require strong biocompatibility and electrical conductivity to mimic the native bioelectrical microenvironment of neural tissue[1], thereby supporting Schwann cell (SC) adhesion, migration, and myelination. Yet the confined internal geometry of NGCs necessitates microscale, high-resolution conductive patterning with well-defined boundaries and structural continuity[2]. Aerosol Jet^® Printing (AJ^®P), an additive manufacturing technology capable of depositing functional inks onto complex surfaces, offers a promising solution for integrating bioelectronic functionality into NGCs. This study systematically optimises AJ^®-printing of gold nanoparticle (AuNP) ink to fabricate high-resolution, bio-conductive micro-patterns[3]. We examined the effects of nozzle diameter, carrier gas flow rate (CG), and focus ratio (Rf) on printed linewidth (Lw), identifying CG as the dominant parameter with strong CG–Rf coupling. Using optimised settings, we achieved a minimum Lw of 30 μm and established practical guidelines for selecting parameters for metal-based inks across varying printing requirements. The biological efficacy of the Aerosol Jet^® Printed (AJ^®P) conductive micro-patterns was demonstrated using Dental Pulp Stem Cell-derived Schwann Cells (DPSC-SCs). The AuNP exhibited excellent biocompatibility, actively supporting DPSC-SC adhesion, proliferation, and differentiation. Approximately 73% of DPSC-SCs adhered to gold tracks, and 93% displayed aligned, directional growth when cultured on the 40/160 μm structures. These findings highlight the potential of AJ^®P-printed conductive micro-patterns for developing next-generation neural interfaces and conductive NGCs.

[1] Rahman M, Mahady Dip T, Padhye R, Houshyar S. Review on electrically conductive smart nerve guide conduit for peripheral nerve regeneration. J Biomed Mater Res A. 2023;111(12):1916-1950.

[2] Zhuang YZ; Seiti M; Ferraris E. Design, biomaterials, and 3D bioprinting technologies in nerve guidance conduits for injured peripheral nerves: A review. Int. J. Biop. 2025, 11(4), 32–65.

[3] Wilkinson NJ., Wilkinson N.J., Smith MAA., et al. A review of aerosol jet printing-a non-traditional hybrid process for micro-manufacturing. Int. J. Adv. Manuf. Technol. 2020, 105(11), 4599-4619.

972. The evolution of in vitro skin models: bridging epidermis, full-thickness models, and hiPSC-derived skinorganoids

Reigl Amelie¹, Köder Nina², Zöphel Saskia³, Klose Pauline⁴, Scherer Johanna⁴, Flütter Lynn⁴, Groeber-Becker Florian⁵, Groneberg Dieter⁴

¹TigerShark Science, Würzburg (Baden-Wberg Bayern) - Germany, ²in vitro skin test systems. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany, ³IZKF. University Hospital Würzburg, Würzburg (Baden-Wberg Bayern) - Germany, ⁴in vitro skin test systems. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany, ⁵TLZ-RT. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany

Mimicking the complex structure of human skin in vitro is a major challenge due to its multi layered composition of epidermis, dermis, and hypodermis, which houses various cell types and structures such as hair follicles. Traditional 2D cell cultures, while enabling high-throughput rapid testing, are limited in their representation of this complexity.

Our laboratory utilizes a range of in vitro skin models, from Reconstructed Human Epidermis (RHE) to more complex Full-Thickness Skin Equivalents (FTSE) and hiPSC-derived skinorganoids (SO).

The RHE models are suitable for the testing of skin barrier functions by conducting impedance measurements, which is a non-invasive measurement technique. These models are particularly effective for high throughput screening and rapid testing of epidermal functions. RHE also facilitates the examination of pathological conditions like epidermolysis bullosa simplex by using patient-derived keratinocytes. Furthermore, RHE can also be employed in researching epidermal burn wounds. Whereas both RHE and FTSE are utilized in wound healing studies. FTSE models are more complex than RHE, incorporating a Type I collagen-based dermal component. They allow for study of the skin's dermal layer. In parallel, the development of hiPSC-derived SO mimicking the skin can recapitulate the architecture of the human skin, including all skin layers and the formation of hair-like structures. They can be used for a wide range of applications, e.g. melanoma cancer models. In the SO we identified several skin-specific cell types such as keratinocytes (CK5), dermal cells (vimentin), adipocytes (nile red) and complex hairpeg formation (indicated by the expression of CK5, CK17). By integrating immunocompetent into the different in vitro skin test-systems, more comprehensive examinations of skin functions, skin diseases and drug tests can be carried out.

Our research shows that there is no “one-skin-model-fits-all” solution. Combining these skin systems allows precise, question-driven investigation of human skin biology.

973. Capsule-based bioinks to overcome diffusion limitations in 3D bioprinted constructs

João F. Mazeda¹, Magda C. Henriques¹, Bruna R. Ferreira¹, Maria C. Gomes¹, João F. Mano¹

¹COMPASS RG. CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal

3D bioprinting is a powerful application in tissue engineering, however it remains facing limitations with nutrients, oxygen and growth factors diffusion, especially in macroscopic constructs. These transport barriers leads to uneven tissue maturation and hinders cell viability across the final constructs. In this work, we introduce a capsule-based bioink that overcomes these limitations by combining liquefied capsules containing microparticles designed to serve as bioactive cues to guide stem cells differentiation. Meanwhile, the surrounding hydrogel matrix provides structural stability to the construct. The liquid-core of the capsules provide localized instructive microenvironments for cell proliferation and tissue maturation, while aiding in overall diffusion throughout the whole construct. The addition of instructive cues given by the microparticles requires minimum use of exogeneous biochemical factors, helping to mitigate the typical limitations with dense hydrogels.

By integrating microparticle-laden capsules within a stable matrix, this bioink approach offers enhanced versatility for tissue engineering applications. It allows for the fine-tuning of the cellular microenvironment in discrete regions without compromising the mechanical properties required for handling and implantation. Such a system can potentially improve the efficiency and precision of stem cell differentiation in complex tissue constructs, facilitating the development of biomimetic tissues with heterogeneous cellular compositions and functions.

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). We also acknowledge the European Research Council Advanced Grant ‘‘REBORN’’ (grant agreement no. ERC-2019-ADG-883370) and the Horizon Europe project PRISM-LT (GA: 101070913).

974. Mechanoregulation of cartilage intermediate layer protein (CILP) in cardiac in vitro models

Minerva Corrales Terrón¹, Amber Evens¹, Chiara Rossi², Julia Krause², Kate Herum², Blanche Schroen¹, Frans A. Van Nieuwenhoven¹

¹Physiology. Maastricht University, Maastricht (Limburg) - The Netherlands, ²Mechanistic biology. Novo Nordisk, Måløv (Kobenhavn) - Denmark

Heart failure results from maladaptive remodeling in which cardiac fibroblasts produce excessive extracellular matrix, causing fibrosis, stiffening, and impaired heart function. The matricellular protein cartilage intermediate layer protein (CILP) has emerged as both a biomarker and a possible driver of cardiac fibrosis. In vitro studies show that, similar to cartilage, transforming growth factor (TGF-β) strongly induces CILP expression in cardiac fibroblasts (CF), the main cellular source of CILP in the heart. Although CILP is known to be mechano-responsive and to regulate ECM production in cartilage, its precise regulation, role, and mechanism of action within cardiac tissue are still not understood.

Three-dimensional engineered heart matrix (3D EHM) and self-assembling beating cardiac organoids are used in this project to elucidate how mechanical cues and fibrotic stimuli affect CILP expression. 3D EHM was subjected to physiological cyclic stretch (10%, 1Hz) using the Flexcell^® strain system. Cardiac organoids were derived from human induced pluripotent stem cells (hiPSC). TGF-β was used to recapitulate a fibrotic environment in both 3D EHM and cardiac organoids. Moreover, to mimic mechanical stimulation in standard culture plates, hiPSC-CF were stimulated with Yoda1, a selective activator of the mechanosensitive channel Piezo1, known for its importance in cellular mechanotransduction.

Our preliminary data show that CILP expression is upregulated in our 3D EHM following TGF-β stimulation. Notably, both Piezo1 activation via Yoda1 and cyclic stretch reduced CILP expression. These findings suggest that the regulation of CILP is also mechanosensitive in cardiac tissue, highlighting its possible role as a modulator of fibrosis in the heart. The self-assembling beating cardiac organoid protocol was successfully validated. These organoids contain multiple cardiac cell types, including fibroblasts, cardiomyocytes, and endothelial-like cells, and seem to respond to fibrotic stimulation, making them a promising platform for studying CILP in a more physiologically relevant environment.

975. Radiosensitizing and mucoadhesive dual-layer electrospun gastric patch: 2D and 3D in vitro evaluation

Sara Guerreiro¹, Tiago Ribeiro², Tiago Dos Santos², Daniel Ferreira², Anabela Dias³, Pedro Granja², Juliana Dias¹

¹CDRSP. Polytechnic of Leiria, Leiria - Portugal, ²i3S-Instituto de Investigação e Inovação em Saúde, Porto, Portugal, Oporto (Porto) - Portugal, ³Medical Physics, Radiobiology and Radiation Protection Group, IPO Porto Research Center (CI-IPO), Portuguese Oncology Institute of Porto (IPO/Porto), Oporto (Porto) - Portugal

Introduction: Gastric cancer is the fourth leading cause of cancer-related death, with over 700,000 deaths annually and complication rates of 3–23% [1]. Expanding treatment options to include radiotherapy is crucial, although its use remains limited by collateral toxicity and suboptimal dose distribution [2]. Gold nanoparticles (AuNPs) are promising radiosensitizers to enhancing local dose through X-ray–induced ionization [4].

Aim: Herein, a dual-layer electrospun patch with mucoadhesive and radiosensitizing properties was designed to provide localized AuNPs-mediated radiosensitization in gastric tumours without systemic exposure, preventing rapid clearance and off-target accumulation of AuNPs.

Materials & Methods: The dual-layer patch is composed by a (i) blend of polycaprolactone and gelatin which offers mucoadhesion and biocompatibility, and (ii) a layer of thermoplastic polyurethane nanofibers that can sequester the AuNPs at the target site while exhibiting resistance to degradation. The patch was subjected to morphological, physiochemically, mechanical and biological characterization. The biological response was evaluated using 2D and 3D gastric adenocarcinoma (AGS) models.

Results: The patch showed suitable tensile strength (2.1±0.6MPa), mucoadhesion (0.6±0.2MPa), and long-term structural stability over 60 days in simulated gastric environment. In 2D AGS models, the preserved radiosensitizing effect compared to patch absence was demonstrated by increased oxidative stress (1.53-fold), reducing post-irradiation viability (0.49-fold), and reduced clonogenic survival. Additionally, 3D AGS spheroids were established to evaluate spatial treatment responses, with the patch significantly reducing metabolic activity (0.20-fold) and spheroid volume (0.50-fold) following irradiation.

Conclusions: These findings support the patch as a localized platform for AuNP-mediated radiosensitization, highlighting its potential to improve the therapeutic efficacy of radiotherapy in gastric cancer.

References

1. Yanagimoto Y, et al. Ann Gastroenterol Surg. 2023;7(5):698–708.

2. Lordick F, et al. 2021;32(3):287–9.

4. Guerreiro SFC, et al. Acta Biomater. 2025.

Acknowledgments - This work was supported by FCT doctoral scholarship (2021.05893.BD) and ANI project Nanofilm (CENTRO2030-FEDER-01469100).

976. Extrusion–inkjet bioprinting of a vascularised human skin equivalent for patient-specific wound modelling

Adrian Perez Barreto¹, Carlo Tremolada², Marco Domingos³, Adam Reid⁴, Jason Wong⁴

¹Department 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, ²IMAGE Regenerative Clinic, Milano (Lombardia) - Italy, ³Department of Materials & Henry Royce Institute, The University of Manchester, UK. NIHR Biomedical Research Centre, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, UK., Manchester - United Kingdom, ⁴Division of Cell Matrix Biology & Regenerative Medicine, FBMH, University of Manchester, UK, Manchester - United Kingdom

Complex and chronic wounds pose a major clinical challenge due to their impaired healing, persistent inflammation, and disrupted vascularisation. Conventional treatments often fail to restore normal skin architecture, highlighting the need for advanced in vitro models that replicate the structural and cellular complexity of human skin. Recent advances in 3D bioprinting are promising for overcoming these limitations by providing precise spatial organisation and enabling the creation of guided structures such as blood vessels. The development of such models is also essential to reduce reliance on animal testing following the 3Rs (Replacement, Reduction, Refinement). Despite notable progress, most existing constructs lack integrated vascular networks, which are fundamental for maintaining tissue homeostasis and replicating the wound microenvironment.

Here, we present a human skin equivalent produced by extrusion and inkjet bioprinting with in situ photo-crosslinking on the BIO X6. The constructs reproduced key structural and functional hallmarks of native skin. Immunofluorescence and immunohistochemistry for vimentin, keratin-14, involucrin, and pan-cytokeratin demonstrated stratification and marker expression across layers compared with skin. Moreover, basement membrane protein deposition, such as collagen IV, was found, demonstrating dermal-epidermal binding and skin integrity. Functional assessment of the epidermal barrier using Lucifer Yellow diffusion and ZO-1 staining confirmed barrier formation. Gene expression analysed by quantitative PCR showed collagen I, collagen III, and fibronectin expression, indicative of extracellular matrix deposition, fibroblast activity, and epithelial maturation. Furthermore, the incorporation of endothelial cells and their confirmation via CD31 staining demonstrate the potential for vascular integration within the construct.

This approach integrates extrusion, inkjet bioprinting, and in situ photo-crosslinking to achieve high spatial precision while preserving cell viability, functionality, improving bioprinting standarisation, and providing an ethically alternative to animal testing. This work aims to develop a platform for advancing personalised wound therapies.

977. Multifunctional cardiac patch for the regeneration of the infarcted heart tissue

Chiara Vitale-Brovarone¹, Sonia Fiorilli¹, Jacopo Barberi², Giorgia Montalbano¹, Alice Benedetto-Mas², Ornella Ieropoli³, Wing Tai Tung⁴, Priscila Melo⁴, Kenneth Dalgarno⁴, Lino Ferreira⁵, Miguel Lino⁵, Carolina Mendes⁵, Rita Gomes⁶, Diana Nascimento⁶, Rui Cerqueira⁷, Pedro Mendes-Ferreira⁷, David Galan⁸

¹Department of Applied Science and Technology, INSTM UdR POLITO. Politecnico di Torino, Torino (Piemonte) - Italy, ²Department of Applied Science and Technology. Politecnico di Torino, Torino (Piemonte) - Italy, ³Corcym srl, Corcym srl (Italia) - Italy, ⁴University of New Castle, Newcastle upon Tyne - United Kingdom, ⁵Center for Neurosciences and Cell Biology (CNC), Coimbra - Portugal, ⁶Institute for Research and Innovation in Health, University of Porto. i3S, Oporto (Porto) - Portugal, ⁷Department of Cardiothoracic Surgery, Hospital Universitario de Sao Joao. Department of Surgery and Physiology, Faculty of Medicine, University of Porto, Oporto (Porto) - Portugal, ⁸Bioinicia, Paterna (Valencia) - Spain

Heart failure post-myocardial infarction (MI) is among the leading causes of mortality worldwide, primarily due to the limited regenerative capacity of myocardial tissue. The REBORN project aims to electrospinning a piezoelectric, multifunctional fibrous patch able to promote the regeneration of ischemic tissue and restore the physiological cardiac functionality. The patch is designed to promote cardiomyocyte activity thanks to the aligned fibres, electromechanical coupling, the release of anti-inflammatory and anti-fibrotic agents, as well as cardiomyocyte proliferative agents to control biological events after MI.

Polyvinylidene fluoride (PVDF) formulations for ESP were prepared using a solvent mixture combining acetone and dimethyl sulfoxide. Aligned nanofibers were produced using a rotating collector, and electrospinning parameters optimized up to a pilot scale system. The beneficial effect of aligned fibre morphology was confirmed by culturing cardiomyocytes on both random and aligned scaffolds, with clear cellular alignment observed on the anisotropic patch. Low-pressure oxygen plasma was used to modify the hydrophobicity of PVDF to a super hydrophilic-like behavior, improving biocompatibility and reducing inflammatory response, as observed on infarcted mice seven days after implantation. Drug loading in the fibrous patch was achieved by electrospinning hybrid formulations incorporating mesoporous silica loaded with anti-inflammatory drugs, as well as by solubilizing ibuprofen and pirfenidone directly into the formulations. The patch also incorporates extracellular vesicles engineered with miRNA clusters to induce cardiomyocyte proliferation. The electrospun patch was characterized in terms of piezoelectrical and mechanical properties, obtaining good piezoelectric response due to the high amount of ß phase (d33= -6.3 pm/V) and suitable mechanical properties (E=65±12 MPa; σR=11±2 MPa). Cytotoxicity assays were performed using human cardiomyocytes and fibroblasts. The patch delivery to the epicardium was proven using different approaches including an ad hoc designed nitinol frame on which the patch was sutured. In vivo test on mice and Landrace pigs are ongoing.

This project has received funding from Horizon Europe research and innovation programme (GA 101091852 -REBORN).Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or HaDEA.

978. Endothelialization of perfused vascular models using tunable gelatin-based biomaterials for advanced drug screening

Cesare Gabriele Gaglio^{1 2}, Lucia Napione¹, Francesca Frascella¹, Lorenzo Moroni², Candido Fabrizio Pirri^{1 3}

¹Dipartimento di Scienza Applicata e Tecnologia, PolitoBIOMed Lab, Politecnico di Torino, ²Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health Medicine and Life Sciences, Maastricht University, Maastricht, Netherlands, ³Center for Sustainable Futures Technologies, Italian Institute of Technology, Turin, Italy

Three-dimensional (3D) in-vitro models provide a more physiologically relevant representation of in-vivo conditions than traditional 2D cultures. Among the key features for recapitulating native tissue environments, vascularization is central, enabling nutrient and oxygen transport, waste removal, and improved biomimicry by reproducing endothelial barrier function.

In this study, a PDMS-based millifluidic platform was developed to perfuse gelatin-derived tissue constructs containing a vascular compartment. These hydrogels were selected for their biocompatibility, tunable mechanics, and established use in tissue engineering. This system is designed for integration into diseased models for angiogenesis, tumor-vasculature interactions, and drug-response studies.

To fabricate smooth, patent channels compatible with endothelialization, different manufacturing strategies—extrusion-based bioprinting, light-based techniques, and sacrificial templating—were evaluated. Ultimately, a ∼700 µm diameter channel was fabricated via sacrificial templating. A parallel auxiliary channel was added to host different 3D structures such as spheroids or organoids at a controlled distance.

Endothelialization was optimized using human umbilical vein endothelial cells (HUVECs), comparing different bulk materials, channel coatings, photocrosslinking methods, seeding densities, and seeding techniques.

To support HUVEC attachment and monolayer stability, two approaches were evaluated. Firstly, HUVECs were subjected to pathophysiologically relevant shear stress using a peristaltic pump and a custom platform. Secondly, human mesenchymal stem cells (hMSCs) were introduced in a direct co-culture to provide support and paracrine signaling, enhancing endothelial adhesion and promoting prolonged culture viability. Specific cell markers were used to distinguish both cell types and assess hMSC differentiation towards a mural cell-like phenotype.

In conclusion, this work establishes dynamic culture conditions in gelatin-derived vascular models, supporting endothelialization under peristaltic flow. Co-culture provided improved vessel stability. Ongoing barrier function characterization and the introduction of diseased compartments exploiting the auxiliary channel will further validate this platform’s robustness and flexibility, integrating vascular compartments into diverse 3D tissue models, enabling advanced disease modelling and drug screening applications.

979. Context-dependent challenges in short peptide self-assembly

Ena Dražić¹, Helena S. Azevedo², Daniela Kalafatović¹

¹University of Rijeka, Faculty of Engineering, Rijeka (Primorsko-Goranska) - Croatia, ²i3S-Instituto de Investigação e Inovação em Saúde, Porto, Portugal, Oporto (Porto) - Portugal

Peptide-based supramolecular assemblies are attractive in regenerative medicine and drug delivery applications as they offer responsive and tunable behavior. However, the sequence-to-assembly relationship has still not been fully understood. The ability of peptides to assemble into ordered supramolecular structures is critically influenced by sequence composition and experimental context, making it challenging to identify the optimal conditions for their application [1]. For this purpose, we set out to determine the influence of sequence composition and pH-dependent ionization on two hexapeptides, IMGIIA and FMGIIF, obtained by generative AI [2]. We aim to understand how the substitution of aliphatic isoleucine and alanine side chains with the aromatic phenylalanine ones affects the peptide assembly behavior. Moreover, we assess how the ionization of free N- and C- termini modulates self-assembly under acidic and neutral conditions. Initial results revealed that FMGIIF has a higher self-assembly propensity, as evidenced by the lower critical aggregation concentration using thioflavinT assay and a more prominent β-sheet content compared to IMGIIA, shown by circular dichroism. Transmission electron microscopy confirmed the presence of nanostructures for both peptides, with morphology modulated by pH. Furthermore, Fourier-transform infrared spectroscopy is expected to elucidate supramolecular arrangement of both peptides while titration will provide insight into pH-dependent charge states. These findings will underscore the self-assembly mechanisms of IMGIIA and FMGIIF, providing a better understanding of how inherent side-chain features and context-dependent charge states govern peptide assembly.

References

[1] E. Dražić, et al. ACS Nano 2025, 19, 20295–20320

[2] M. Njirjak, et al. Nat. Mach. Intell. 2024, 6, 1487–1500

980. 3D lung tumor modeling using hydrocolloid bioinks with enhanced angiogenic potential

Özüm Yildirim-Semerci¹, Ahu Arslan-Yildiz¹

¹Department of Bioengineering, Faculty of Engineering, Izmir Institute of Technology, Izmir, Türkiye, İzmir (Izmir) - Turkey

Lung cancer remains the leading cause of cancer-related mortality worldwide, responsible for 18.7% of all deaths, primarily due to absence of detectable symptoms in early clinical stages. To overcome the limitations of conventional 2D cell culture and animal models—which fail to recapitulate intricate tumor microenvironment—advanced in vitro 3D systems have emerged as transformative tools in drug screening. In this context, present study introduces plant-derived hydrocolloid bioinks engineered to recapitulate lung tumor models (LTM) with enhanced angiogenic capacity and to serve as a drug-screening platform. Hydrocolloid bioinks were extracted from plant sources and characterized using FTIR-ATR and SEM, confirming their chemical content and favorable morphological features. Rheological profiling and printability assays verified their optimal viscosity and fluid behavior for extrusion-based bioprinting. Using optimized bioprinting parameters, 3D LTMs were fabricated via co-culture of A549 human lung cancer cells and healthy WI-38 human lung fibroblasts. SEM imaging and immunostaining revealed highly organized cellular distribution and progressive ECM deposition throughout long-term culture. Angiogenic performance was examined using in ovo chorioallantoic membrane assay, validating the pro-angiogenic character of the bioink. Drug-screening experiments with Doxorubicin and Cisplatin further highlighted the physiological relevance of platform, as 3D models exhibited significantly increased IC50 values compared to 2D controls, reflecting enhanced chemoresistance consistent with in vivo tumors. Overall, this study shows the performance of plant-based hydrocolloids as proper bioinks for bioprinting of 3D LTMs. Their excellent print fidelity and biocompatibility position these bioinks as environmental-friendly alternative to conventional bioinks. Moreover, angiogenic potential and reliable drug-screening performance of resultant 3D LTMs effectively bridge the gap between traditional in vitro assays and preclinical research, enabling more predictive and sustainable therapeutic development.

This study was supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK; 120C155, 125M167) and the IZTECH Scientific Research Project (IYTE-BAP; 2024IYTE-1-0051).

981. Injectable antibacterial hydrogels from ɛ-polylysine and hyaluronic acid as a vehicle toward antibiotic-free infection treatment

Artemijs Sceglovs¹, Anna Rubina¹, Ingus Skadins², Anna Ramata-Stunda³, Claudia Siverino⁴, Jacek K. Wychowaniec⁴, Fintan T. Moriarty⁴, Juta Kroica², Kristine Salma-Ancane¹

¹Institute of Biomaterials and Bioengineering. Riga Technical University, Riga - Latvia, ²Department of Biology and Microbiology, Riga Stradins University, Riga - Latvia, ³University of Latvia, Department of Microbiology and Biotechnology, Riga - Latvia, ⁴AO Research Institute Davos, Davos (Graubunden) - Switzerland

The growing threat of antimicrobial resistance has created an urgent demand for non-antibiotic biomaterials capable of preventing infections without promoting bacterial resistance. Therefore, the objective for this research was to develop resistance-preventing antibacterial hydrogels based on a biopolymers – ε-PL (ε-polylysine) and HA (hyaluronic acid).

Synthesis of hydrogels was performed by alternate mixing of reagents in interconnected syringes. ε-PL and HA were chemically cross-linked together in different mass ratios. Prepared hydrogel compositions were compared by comprehensive characterisation, e.g., molecular structure, surface morphology and topology, gel content, swelling behaviour, autoclavability, enzymatical degradation and rheological properties. In vitro evaluation included advanced antibacterial studies against both, reference and clinically isolated, including multidrug-resistant bacterial strains, and biocompatibility studies via direct/indirect assays using human dermal (HDFs) and mouse fibroblast (Balb/c 3T3) cell lines.

Chemical cross-linking approach resulted in formation of stable biopolymer-matrix with gelation time up to 50 min. Hydrogels showed autoclavability with minimal impact on its gel fraction (∼55%), morphology (60-200 μm), stiffness (10-15 kPa) and antibacterial activity. Rheological studies confirmed hydrogels are injectable with recovery capability up to 98%. Swelling capacity (up to 450%), zeta-potential, bactericidal effectiveness, as well as biocompatibility on different fibroblast cell lines were found as a function of ε-PL mass ratio in hydrogel composition.

To sum up, here we present perspective hydrogels based on naturally-derived components. While HA provides injectability, mechanical stability and ECM-mimicking environment for cells, ε-PL showed resistance-preventing properties and bactericidal activity against wide range of bacteria, including biofilms. Presented biomaterials show perspective potential in development of new non-antibiotic antibacterial strategies.

The authors acknowledge financial support from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 857287 (BBCE).

The authors acknowledge support and contribution from AO Research Institute (ARI), Davos, Switzerland in different development stage of this research.

982. Engineering and characterization of a retinal-mimetic bioink for optimized bioprinting of precursor and cancer cell models

Alvaro Plaza Reyes¹, Berta De La Cerda², Lourdes Valdés², Francesco Ganzerli³, Elin Pernevik⁴, Marta Torres⁵, Volodymyr Kuzmenko⁴, Elena Veronesi³, Massimo Dominici⁶, Francisco Diaz Corrales²

¹CABIMER. Fundación Progreso y Salud, Sevilla - Spain, ²CABIMER. Fundación Progreso y Salud, Sevilla - Spain, ³Democenter. Tecnopolo Mario Veronesi, Mirandola (Emilia-Romagna) - Italy, ⁴CELLINK Bioprinting AB, Gothenburg (Vastra Gotaland) - Sweden, ⁵Fundación Progreso y Salud, Sevilla - Spain, ⁶University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy

The development of physiologically relevant retinal models requires bioinks with mechanical, biochemical, and printing properties tailored to the unique architecture of the human retina.

Here, we present the engineering and characterization of a biomimetic bioink optimized for the extrusion bioprinting of single-cell suspensions, including dissociated induced pluripotent stem cell (iPSC)-derived retinal precursors and retinal cancer cells. Fluorescent reporter iPSC lines generated using CRISPR-Cas9 enabled the enrichment of photoreceptor and bipolar progenitors, ensuring defined cell populations for downstream bioprinting applications.

Bioink optimization was conducted primarily using the Y79 retinoblastoma cell line printed onto mature ARPE19 retinal pigment epithelium (RPE) cultured on transwell membranes. This allowed systematic evaluation of formulation performance under biologically relevant conditions. Rheological characterization, including shear thinning behavior, viscosity profiles, and storage/loss moduli, guided the selection of a composition supporting controlled extrusion and high shape fidelity. Photocrosslinking kinetics were refined through assessment of photoinitiator concentration and exposure time, balancing rapid gelation with minimal phototoxic effects.

Biocompatibility was confirmed through cell viability assays, which identified formulations supporting >70% post-print survival. Optimization of extrusion pressure, nozzle diameter, and printing speed produced constructs with consistent filament stability and uniform cell distribution. Y79 bioprinting demonstrated robust adhesion to ARPE19 monolayers and maintained proliferative behavior, highlighting the bioink’s suitability for establishing retinal cancer models.

Subsequent printing of enriched iPSC-derived retinal precursor cell suspensions on RPE validated the applicability of the optimized conditions for engineering retinal tissue constructs with relevance for disease modeling and regenerative medicine.

This work provides a comprehensively characterized retinal-mimetic bioink and a refined bioprinting workflow with demonstrated utility in both retinal tissue engineering and the fabrication of in-vitro retinal cancer models, expanding opportunities for therapeutic development and mechanistic research.

983. Large-area atomic force microscopy (AFM) mapping for mechanobiology studies in cells and tissues

Alexander Dulebo¹, André Körnig¹, Joan-Carles Escolano¹, Thomas Henze¹

¹BioAFM. Bruker Nano Surfaces Division, Berlin - Germany

Mechanical properties regulate mechanotransduction and transport processes, shaping signaling, molecular trafficking, and tissue organization. Atomic force microscopy (AFM) enables nanoscale characterization of stiffness, adhesion, and viscoelasticity, and is also widely used for high-resolution structural analysis – critical for understanding force-driven transport in complex biological systems. However, conventional AFM is limited by sample roughness and restricted lateral range, constraining large-scale analysis of heterogeneous specimens.

We present an advanced AFM imaging approach using SmartMapping technology, which synchronizes AFM head motors with XYZ-piezo stage movement. This innovation enables automated, high-resolution mapping across extended areas without user intervention, improving reproducibility and throughput for multi-region mechanobiological studies.

Using this technology, we analyzed Cytochalasin D-treated 3T3 fibroblasts versus controls, revealing time-dependent changes in mechanics and cytoskeletal architecture that influence intracellular transport. Highly corrugated 3D SKOV-3 spheroids (>100 µm) and zebrafish tumors (>300 µm) exhibited structural and mechanical heterogeneity relevant to diffusion and signaling. Mapping mouse brain tissue along the anterior-posterior axis uncovered stiffness gradients plausibly linked to neuronal transport. Finally, 600 µm-thick neuroblastoma tumors embedded in agarose validated the performance on rough and complex tissues.

This integrated approach establishes a new standard for large-area mechanobiological imaging, enabling spatially resolved analysis of mechanical cues that govern cellular and tissue-level transport.

984. Translational strategies to accelerate bone regeneration in osteogenesis imperfecta

Josephine T. Tauer¹, Bertrand David², Umebayashi Mayumi³, Kavaseri Kyle⁴, Rauch Frank⁵, Hamdy Reggy⁶, Willie Bettine M.⁷

¹Department of Pediatrics and Adolescent Medicine. Johannes Kepler University, Linz (Upper West) - Austria, ²McGill University, Department Experimental Surgery. Shriners Hospital for Children, Montreal (Quebec) - Canada, ³Shriners Hospital for Children, Montreal (Quebec) - Canada, ⁴McGill University, BBME Department. Shriners Hospital for Children, Montreal (Quebec) - Canada, ⁵McGill University, Department of Pediatrics. Shriners Hospital for Children, Montreal (Quebec) - Canada, ⁶McGill University, Department of Orthopaedics. Shriners Hospital for Children, Montreal (Quebec) - Canada, ⁷McGill University, Faculty of Dental Medicine and Oral Health Sciences. Shriners Hospital for Children, Montreal (Quebec) - Canada

Osteogenesis imperfecta (OI) is a complex bone disorder characterized by altered extracellular matrix organization, bone remodeling, and mechanoadaptive capacity, all of which impair bone regeneration. These deficits strongly affect clinical outcomes, particularly delayed healing and non-union following corrective osteotomies in children with severe OI. To address this, we evaluated cell-based therapies, biomaterial scaffolds, and mechanobiological responses to identify translational strategies for improving bone repair in OI.

Our first approach assessed the adipose stromal vascular fraction (SVF) as an autologous stem-cell source for pediatric OI patients. OI-derived SVF maintained cell viability, population composition, and tri-lineage differentiation capacity comparable to healthy controls, supporting the feasibility of adipose tissue–based regenerative therapies. We then tested SVF-seeded collagen and collagen–β-TCP scaffolds in critical-sized femoral defects in wild-type and OI mice and found similar bone healing in SVF–collagen and saline–collagen groups. Regenerative outcomes were also comparable between SVF–collagen and SVF–collagen–β-TCP scaffolds.

Recognizing that matrix brittleness and reduced periosteal expansion contribute to bone fragility, we further examined mechanoadaptive responses using in vivo tibial loading. Wild-type mice exhibited robust periosteal bone formation, whereas OI mice showed a markedly blunted response despite experiencing similar mechanical strain. This indicates that impaired mechanosensation—potentially via altered osteocyte activity—or downstream signaling limits cross-sectional growth and structural reinforcement in OI bone.

To explore whether additional mechanosensitive bone cell types might compensate for deficient osteocyte function, we used anosteocytic teleost fish as a comparative model. We found that these species can still mount transcriptional responses to mechanical load, indicating that non-osteocytic cell populations can contribute to mechanosensation. These insights raise the possibility that alternative mechanosensitive pathways could be leveraged therapeutically in OI.

Overall, our findings demonstrate how integrating cell biology, biomaterials engineering, and mechanobiology can advance translational treatments for traumatic and genetic bone defects in OI.

985. Evaluation of apoptotic-induced cell death after cryopreservation of an artificial oral mucosa model

Paula Ávila-Fernández¹, S Aguilar-Pérez², Miguel Etayo-Escanilla¹, Óscar Darío García-García¹, Olimpia Ortiz-Arrabal¹, Miguel Alaminos¹, Ismael Ángel Rodríguez³, Fabiola Bermejo-Casares⁴, Antonio España-López⁵, Ingrid Garzón¹, Miguel Ángel Martín-Piedra¹

¹Histology. University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²University of Granada, Granada - Spain, ³Histología B. Universidad Nacional de Córdoba, Cordoba - Argentina, ⁴Histology. University of Granada, Granada - Spain, ⁵Stomatology. University of Granada, Granada - Spain

Introduction: Diverse disorders can cause severe oral mucosa lesions requiring surgical treatment using tissue grafts. However, large oral mucosa defects are limited by the scarcity of available tissue. Biofabrication of artificial oral mucosa by tissue engineering may offer an alternative solution, but the ideal oral mucosa substitutes should support cryopreservation for long-term storage and immediate availability (1). The aim of this study is to evaluate the effect of different cryopreservation protocols on the viability of human oral mucosa stroma substitutes (HOMS).

Methods: HOMS models based on a fibrin-agarose matrices with human gingival fibroblasts were developed and subjected to four cryoprotective solutions (10% DMSO; 10% glycerol; 0.35M trehalose; CryoStor^®) at different temperatures (-20°C; -80°C) for diverse times. As controls, non-cryopreserved HOMS (CTR) and HOMS cryopreserved in PBS (CTR-) were used. Immunohistochemical analyses were carried out for Caspase-7 and TUNEL assays to evaluate the percentage of cells undergoing apoptosis.

Results and conclusions: Caspase-7 immunohistochemistry was not able to find any differences among groups. However, TUNEL analysis found that the percentage of apoptotic cells was low in HOMS cryopreserved in DMSO and CryoStor^®. Other cryoprotective agents showed similar values to CTR-. These findings imply that using cryoprotective agents based in DMSO, as CryoStor^®, could be useful for a future optimization of the protocols related to long-term storage of HOMS for clinical use.

Acknowledgements: Supported by by Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, Grant FIS PI24/00006. Cofinanced by the European Regional Development Fund (FEDER/ERDF) through the “Una manera de hacer Europa” program, European Union.

1. Ziani K, Saenz-Del-Burgo L, Pedraz JL, Ciriza J. Advances in Cryopreservation Strategies for 3D Biofabricated Constructs: From Hydrogels to Bioprinted Tissues. Int J Mol Sci. 2025 Jul 18;26(14):6908

986. 2D histology vs. 3D micro-CT: a comparative study of bone-implant contact (BIC) assessment in biodegradable metal-based orthopaedic implants

Marek Kindermann¹, Lucie Vistejnova¹, Pavel Klein¹, Filip Hanak², Dominik Durica², Robert Kral³, Peter Minarik³, Milos Svoboda⁴, Jan Benes⁴, Jitka Lunackova⁵, Martin Bartos⁵, Patrik Mik⁶, Martina Dolejsova⁷, Yaroslav Kolinko⁶, Vojtech Havlas²

¹Biomedical Center. Charles University, Faculty of Medicine in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic, ²Second Faculty of Medicine, Charles University, Prague (Hlavni Mesto Praha) - Czech Republic, ³Faculty of Mathematics and Physics, Charles University, Prague (Hlavni Mesto Praha) - Czech Republic, ⁴New Technologies – Research Centre, University of West Bohemia in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic, ⁵Institute of Dental Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague (Hlavni Mesto Praha) - Czech Republic, ⁶Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Pilsen (Plzensky Kraj) - Czech Republic, ⁷Charles University, Faculty of Medicine in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic

Successful orthopaedic implants require intimate bone–implant contact (BIC), a critical parameter influencing stability and long-term success. Traditionally, 2D histological methods (2D-BIC) assess this by measuring the bone-implant interface length along selected section planes. In contrast, micro-CT analysis (3D-BIC) enables a more comprehensive, three-dimensional evaluation across the entire implant surface. This in vivo study compared a commercial Mg-Y-RE-Zr alloy (MAGNEZIX^®) with three experimental biodegradable Mg-Y-(Li)-based alloys, implanted as screws/pins into rat femurs for six months. The interface was evaluated using high-resolution micro-CT (∼6μm voxel size) for 3D-BIC, and corresponding 2D-BIC was assessed from histological sections. The primary objective is to critically evaluate the accuracy and limitations of 2D methods. The results will quantify the variability of 2D-BIC across different planes and determine if single-plane 2D-BIC accurately represents the overall 3D-BIC. This research will clarify methodological considerations for BIC evaluation, guiding the development of more effective metal-based bone implants.

987. Dissecting epithelial cell-cell communication in cystic fibrosis using single-cell RNA-seq data and in vitro stem cell-based disease models

Marta Vilà González¹, Míriam Salvà Barceló¹, Josep Muncunill Farreny², Marta Monjo Cabrer¹, Joana Maria Ramis Morey¹

¹Cell Therapy and Tissue Engineering Group (TERCIT), Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears (UIB), Health Research Institute of the Balearic Islands (IdISBa). IUNICS-UIB, Palma de Mallorca (Illes Balears) - Spain, ²Genomics and Bioinformatics Platform. IDISBA - Health Reseach Institute of the Balearic Islands, Palma de Mallorca (Illes Balears) - Spain

Cystic fibrosis (CF), caused by biallelic mutations in the CFTR gene, is characterised by impaired ion and fluid homeostasis in the airway epithelium, yet the contribution of individual epithelial cell types and the intercellular networks that coordinate them remain incompletely understood. Recent single-cell transcriptomic studies have revealed a diverse epithelial landscape that includes rare but functionally critical populations such as ionocytes[1,2]. However, how epithelial intercellular signalling is rewired in CF, and how these changes shape epithelial dysfunction, has not been systematically explored.

Here, we re-analysed publicly available single-cell RNA-sequencing data[3] of cells from healthy donors and people with CF using CellPhoneDB[4], a computational tool that enables a high-resolution interrogation of ligand-receptor interactions across airway epithelial cell types based on RNA expression data. This analysis uncovered distinct communication modules involving ionocytes, secretory cells, and basal progenitors, and revealed CF-specific alterations in pathways associated with adhesion and barrier organisation, repair signalling and differentiation. These findings suggest that CF pathophysiology arises not only from cell-intrinsic CFTR dysfunction but also from disruptions in the multicellular signalling networks that maintain epithelial homeostasis.

To validate these predictions, we established a human induced pluripotent stem cell (hiPSC)–derived airway epithelial model using isogenic wild-type and CF cells, enabling controlled assessment of how CFTR mutations alter epithelial crosstalk. By integrating computational cell-cell interaction analysis with a stem cell-derived disease model that recapitulates the epithelial diversity of the airway, this approach provides a framework for dissecting multicellular network dysfunction in CF and identifying signalling axes that could guide regenerative therapeutic strategies.

We thank the Vallier lab (BIH Berlin), the Hart lab (UCL London) and the Cystic Fibrosis Foundation for the gift of cell lines. This work was funded by the Fundación Respiralia, MICIU (BG23/00054) and ISCIII co-funded with ESF+ European Social Fund Plus (FI24/00159).

1. doi:10.1038/s41586-018-0394-6

2. doi:10.1038/s41586-018-0393-7

3. doi:10.1038/s41591-021-01332-7

4. doi:10.1038/s41596-025-01140-0

988. Enhanced fibrosis modeling in human skin organoids through hiPSC-derived macrophage integration

Dieter Groneberg¹, Anna-Sophie Hauser Hauser¹, Amelie Reigl¹, Nina Köder¹, Saskia Zöphel¹, Pauline Klose¹, Johanna Scherer¹, Maximilian Schinke², Florian Groeber-Becker³, Nico Lachmann²

¹Skin Research. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany, ²Fraunhofer Institute for Toxicology and Experimental Medicine. Hannover Medical School, Hannover (Niedersachsen) - Germany, ³Translational Center Regenerative Therapies. Fraunhofer Institute for Silicate Research, Würzburg (Baden-Wberg Bayern) - Germany

Animal models are limited in translational value due to fundamental differences between murine and human skin. To address these issues, we generated a complex three-dimensional skin organoids (SO) from human induced pluripotent stem cells (hiPSCs), based on a modified protocol by Lee et al. The SO replicate all three skin layers, including hair formation. We characterized the SO using molecular markers for the epidermal layer (CK5, CK17), dermal layer (vimentin), and hypodermal layer with adipocytes using lipid staining (Nile Red).

To model fibrotic remodeling, we stimulated mature SO with TGF-β for seven days and analyzed tissue responses via immunofluorescence for α-SMA, FAP, collagen I, and Ki67. To capture immune tissue interactions, we integrated RFP-labeled hiPSC-derived macrophages (provided by the group of Prof. Nico Lachmann) into the SO and maintain long-term co-culture. During the incubation we monitored the effects of macrophages on the composition of the extracellular matrix, tissue architecture, and cell proliferation. Significant transcriptional differences in fibrosis-associated markers (e.g. ACTA2, FAP) were observed by qPCR with and without the presence of macrophages indicated by both conditions formation of myofibroblast. In the condition with macrophages altered Ki67 and reduced expression of Col1A1 were observed suggesting a modulating role of immune cells during fibrotic remodeling. We treated the organoids with the antifibrotic drug nintedanib, which attenuated the fibrotic phenotype.

Our model enables the functional testing of fibrosis-inducing stimuli and immune interactions in a translational human test system. This provides an alternative to animal models for the study of fibrotic skin conditions and integration of macrophages led to a more translational in vitro system.

989. Bioengineering surfaces to preserve mesenchymal stromal cells’ growth in vitro

Yusuf Ayten¹, Monica P Tsimbouri¹, Vineetha Jayawarna², O.c. Oreffo Richard³, Manuel Salmeron-Sanchez², C Catherine Berry¹, Matthew J Dalby¹

¹Centre for the Cellular Microenvironment, School of Molecular Biosciences. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, ²Centre of Cellular Microenvironment, James Watt School of Engineering. University of Glasgow, Glasgow (Glasgow City) - United Kingdom, ³Bone and Joint Research Group, Centre for Human Development, Institute of Developmental Sciences. University of Southampton, Southampton - United Kingdom

Mesenchymal stromal cells (MSCs) have been characterised for their immunomodulatory, self-renewal, and multipotent differentiation capacities, making them important candidates for cellular therapies [1,2]. However, their low numbers in native tissues, such as the bone marrow, require extensive in vitro expansion to achieve sufficient numbers [1,2]. Long-term culture creates mechanical and biochemical stress for MSCs, leading to senescence, decreased proliferation, and loss of phenotypic and functional identity [3]. Here, we hypothesise that bioengineered surfaces functionalized with laminin could support MSCs in reversing these detrimental effects through controlled mechanotransduction. Poly(ethyl acrylate) (PEA) promotes the self-assembly of laminin into a biomimetic nanonetwork, which has been shown to promote adhesion and cell growth while reducing intracellular tension [4,5,6]. Therefore, laminin-containing PEA surfaces may create a physiological niche that preserves MSC functionality and restores their maintenance during long-term in vitro expansion.

Materials and methods

To optimise cell culture conditions, the culture surfaces are coated with PEA, which was applied by plasma polymerisation of the monomer onto polystyrene culture plates. Stro-1-selected MSCs were cultured on the surfaces. Following culture, in-cell western, flow cytometry, and immunostaining analysis were carried out for phenotypical analysis.

Results and Conclusion

According to the results, PEA-laminin surfaces preserved MSC proliferation and morphology in the early stages of culture and supported the recovery of functional properties that were diminished by long-term expansion on conventional plastics. This has implications in MSC therapy manufacturing.

References

1. Ding DC et al., Cell Transplant. 2011.

2. Jovic D et al., Stem Cell Rev Rep. 2022.

3. McMurray RJ et al., Nat Mater. 2011.

4. Kechagia Z et al., Nat Mater. 2023.

5. Dalby MJ et al., Nat Rev Mater. 2018.

6. Llopis-Hernández V et al., Sci Adv. 2016.

Acknowledgements

This PhD is funded by the Ministry of National Education in Turkey.

990. Biodegradable injectable polymer/calcium phosphate bone paste with antibacterial properties

Lucie Vistejnova¹, Marek Kindermann¹, Pavel Klein¹, Iveta Paurova¹, Lucy Vojtova², Lenka Michlovska², Dana Kralova¹, Vendula Peckova¹, Katerina Chudejova¹

¹Charles University, Faculty of Medicine in Pilsen, Pilsen (Plzensky Kraj) - Czech Republic, ²CEITEC - Central European Institute of Technology, Brno University of Technology, Brno (Jihomoravsky Kraj) - Czech Republic

INTRODUCTION AND CLINICAL MOTIVATION: Infections of bone and bone marrow (osteomyelitis) still represent a significant risk in orthopedics. The pathogens most often causing infections of bone tissues are gram-positive staphylococci, with Staphylococcus aureus being the most common. Other participants include, for example, MR Staphylococcus aureus, Staphylococcus epidermidis, or gram-negative Escherichia coli Pseudomonas aeruginosa or Klebsiella pneumoniae. Further, clinicians in orthopedics seek resorbable bone defect fillers that stimulate new bone formation, prevent infections and accomplish bone mechanical load. The aim of the study is to verify the antibacterial effects of newly developed biodegradable polymer/calcium phosphate bone paste with prolonged injectability enriched with selected antibiotics (vancomycin, gentamicin, tigecycline, colistin) (ATB) effective against the above-mentioned pathogens at the in vitro level, and to prepare a functional animal model of bone infection for studying the antibacterial effects of bone paste under physiological conditions.

IN VITRO ANTIBACTERIAL EFFICACY: Diffusion microbiological tests showed that all 4 tested antibiotics were effectively released from the bone paste and formed concentration-dependent growth inhibition zones for all tested bacterial strains.

IN VIVO SIMULATION OF BONE INFECTION: Four concentrations of Staphylococcus aureus (SA) (102, 104, 106, 108 CFU/ml) were tested in the form of a 1% agar block when applied to a defect in rat femur measuring 2 x 5 mm. The defects were sealed by bone paste without antibiotics. After 4, 11 and 28 days, the defects were analyzed for the presence of bacteria (dilution test) and for the damage of bone tissue due to bacteria (µCT and histology). The highest SA concentration caused painful movements of animals and was excluded from analysis. All lower concentrations caused stable SA positivity without painful movements and showing the base for selection of SA concentration for ATB efficacy evaluation.This work was supported by the Ministry of Health of the Czech Republic under project no. NW24-05-00202

991. Chicken or egg: exploring the role of the malignant bone marrow microenvironment in acute myeloid leukemia via 3D in vitro models

Louise Roolfs¹, Annamarija Raic², Nadine Schadzek¹, Peter Schertl¹, Mark Ringhoffer³, Michael Heuser⁴, Felicitas R. Thol⁵, Vincent Felde⁶, Jörg U. Hammel⁷, Constantin Von Kaisenberg⁵, Karen Bieback⁸, Frank Schaarschmidt¹, Cornelia Lee-Thedieck¹

¹Institute of Cell Biology and Biophysics. Leibniz University Hannover, Hannover (Niedersachsen) - Germany, ²Karlsruhe Institute of Technology (KIT), Karlsruhe (Baden-Wberg Bayern) - Germany, ³Medizinische Klinik III. Städtisches Klinikum Karlsruhe, Karlsruhe (Baden-Wberg Bayern) - Germany, ⁴University Hospital Halle (Saale), Halle (Saale) (Sachsen-Anhalt) - Germany, ⁵Hannover Medical School, Hannover (Niedersachsen) - Germany, ⁶Institute of Earth System Sciences. Leibniz University Hannover, Hannover (Niedersachsen) - Germany, ⁷Institute of Materials Physics. Helmholtz-Zentrum Hereon, Geesthacht (Schleswig-Holstein) - Germany, ⁸Universitätsmedizin Mannheim, Universität Heidelberg, Mannheim (Baden-Wberg Bayern) - Germany

Introduction

Acute myeloid leukemia (AML) is an aggressive malignancy leading to rapid growth of leukemic cells, adversely affecting the blood-forming, hematopoietic system in the bone marrow. AML is characterized by high relapse rates and chemoresistance, particularly among elderly patients, leading to poor prognoses. The interaction between hematopoietic stem cells (HSCs), leukemic cells and the bone marrow microenvironment plays an important role in these processes.

Materials and Methods

To investigate the potential impact of hypothesized malignant niches in AML, we developed a 3D in vitro model to simulate the bone marrow microenvironment in different stages of AML onset and progression and in the healthy state. For this purpose, protein-based macroporous hydrogels were used, seeded with healthy and leukemic hematopoietic and stromal cells. The cellular compositions include primary human hematopoietic stem and progenitor cells (HSPCs), AML blasts, healthy mesenchymal stromal cells (MSCs) and leukemic MSCs (LMSCs).

Results

Our findings reveal the differential effects exerted by MSCs and LMSCs on HSPCs and leukemic blasts, dependent on the cell type and the dimensional culture context. The ability of LMSCs to support HSPCs in a manner similar to healthy MSCs was highly donor dependent. The similarity in LMSC behavior to healthy MSCs correlated with positive patient responses to clinical induction therapy. Drug testing experiments with cytarabine (AraC) and daunorubicin (DNR) affirmed the model's effectiveness in evaluating therapeutic interventions and studying chemoresistance.

Conclusions

The developed in vitro AML model demonstrates clinical relevance, as the obtained in vitro data correlate with patient outcomes. The study supports the hypothesis that a malignant bone marrow niche critically contributes to AML progression and suggests that the niche's malignancy could be a parameter in predicting patient outcomes. The application of the developed in vitro model in drug testing underscores its potential for use in preclinical and pharmaceutical studies aimed at enhancing and personalizing AML patient treatments.

992. Porcine myocardial extracellular matrix-derived hydrogel crosslinked with genipin for cardiac tissue engineering

Angélica Raquel Rivera Contreras¹, José Luis Hidalgo Vicelis¹, Andrés Eliú Castell Rodríguez¹, Beatriz Hernández Téllez¹, Gabriela Piñón Zárate¹, Katia Jarquín Yáñez¹, Gertrudis Hortensia González Gómez², María Alicia Falcón Neri²

¹Laboratorio de Inmunoterapia e Ingeniería de Tejidos, Departamento de Biología Celular y Tisular. Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacán (Distrito Federal) - Mexico, ²Laboratorio de Biofísica Funcional, Departamento de Física. Facultad de Ciencias, Universidad Nacional Autónoma de México, Coyoacán (Distrito Federal) - Mexico

Myocardial infarction (MI) is one of the leading death causes. Cell therapy has been used for post-MI repair; however, this approach is limited by the lack of a suitable extracellular environment in the infarct region. In this regard, injectable hydrogels derived from decellularized tissues are an attractive therapeutic option because the resulting extracellular matrix (ECM) is biomimetic and its administration is minimally invasive. Decellularization affects the degradation rate and mechanical properties of the ECM, so the addition of a crosslinking agent such as genipin would improve them (1). Thus, a porcine myocardial ECM-derived hydrogel crosslinked with genipin was developed in this study. To prepare the hydrogel, the porcine myocardial ECM obtained by decelullarization was lyophilized and solubilized (2). Then, genipin was added at different concentrations, and the hydrogels were incubated at 37°C for self-assembly. The concentration of genipin played an important role on the hydrogels properties. For example, the porosity and degradability of the hydrogels decreased with a higher concentration of genipin. Furthermore, a low concentration of genipin did not compromise the viability of cardiomyocytes and improved their contractile activity. Therefore, this study demonstrates that crosslinking with genipin allowed altering the properties of the porcine myocardial ECM-derived hydrogel to achieve the potential tissue engineering requirements for the tissue regeneration post-MI.

Acknowledgments: To PAPIIT-DGAPA-UNAM IT200525, IN216723 and IA203025 projects for their financial support.

References

1. Jeffords ME, Wu J, Shah M, Hong Y, Zhang G. Tailoring material properties of cardiac matrix hydrogels to induce endothelial differentiation of human mesenchymal stem cells. ACS Appl Mater Interfaces. 2015;7(20):11053–61. http://dx.doi.org/10.1021/acsami.5b03195

2. Hidalgo-Vicelis JL, Rivera-Contreras AR, Hernández-Téllez B, Piñón-Zárate G, Fiordelisio-Coll T, et al. Thermosensitive porcine myocardial extracellular matrix hydrogel coupled with proanthocyanidins for cardiac tissue engineering. Gels. 2025;11(1). http://dx.doi.org/10.3390/gels11010053

993. Personalised 3D-bioprinted urethral grafts using autologous cells: a proof-of-concept in a porcine model

Teresa Olsen Ekerhult¹, Johanna Odbratt², Matilda Öjmertz², Patrik Stenlund³, Mattias Berglin⁴, Stina Simonsson⁵, Anthony Atala⁶, Joakim Håkansson⁷

¹Urology Department. Sahlgrenska University Hospital, Urology department, Institute of Clinical Sciences, University of Gothenburg, Gothenburg (Vastra Gotaland) - Sweden, ²Chalmers University of Technology, Gothenburg (Vastra Gotaland) - Sweden, ³Research Institute of Sweden. Reaserch Institute of Sweden, Gothenburg (Vastra Gotaland) - Sweden, ⁴Reaserch Institute of Sweden, Gothenburg (Vastra Gotaland) - Sweden, ⁵Department of Laboratory Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg (Vastra Gotaland) - Sweden, ⁶Wake Forest Institute for Regenerative Medicine, Winston-Salem (North Carolina) - United States, ⁷RISE. Reaserch Institute of Sweden, Gothenburg (Vastra Gotaland) - Sweden

Background: Urethral strictures and congenital or iatrogenic urethral defects remain major clinical challenges, with current reconstructive options relying largely on autologous buccal mucosa grafts associated with donor-site morbidity. Tissue-engineered alternatives could enable personalized, cell-based reconstruction while avoiding intraoral complications.

Methods: We developed a personalized, multilayer 3D-bioprinted urethral graft integrating autologous porcine urothelial and smooth muscle cells within a GelMA-fibrinogen hydrogel and reinforced by polycaprolactone spirals. Biopsies of the urinary bladder were harvested to isolate and expand autologous cells. The grafts were fabricated on a rotating rod to mimic native architecture and cultured for 14 days prior to implantation. In four pigs, a 4-cm urethral segment was surgically excised and replaced with a personalized graft, followed by anastomosis and soft-tissue closure. At four weeks post-implantation, graft patency was assessed using contrast radiography and macroscopic dissection.

Results: The bioprinted constructs reproduced key structural components of the urethra with distinct epithelial and smooth muscle layers. Surgical implantation was feasible and uneventful in all animals. At four weeks, all grafts demonstrated full patency without signs of leakage on contrast imaging. Macroscopic examination revealed intact luminal continuity and integration with surrounding tissues.

Conclusions: This study provides the first in vivo proof-of-concept in a large animal model demonstrating that a personalized, cell-laden 3D-bioprinted urethral graft can be surgically implanted, maintain luminal patency, and support structural integrity at short-term follow-up. These findings highlight the potential of advanced biofabrication as a future alternative to autologous tissue harvesting in urethral reconstruction.

994. Role of LGR5 and LGR6 in the regulation of human epithelial stem cell niche in the interfollicular epidermis

Joelle Giroud¹, Katarzyna Michalak-Micka¹, Ueli Moehrlen³, Thomas Biedermann¹, Agnes S. Klar¹

¹Tissue Biology Research Unit, University Children’s Hospital. University of Zurich, Zurich - Switzerland, ²Tissue Biology Research Unit, University Children’s Hospital. University of Zurich, Zurich - Switzerland, ³University Children’s Hospital. University of Zurich, Zurich - Switzerland L. Gugler¹, A. Aebischer¹, J. Giroud¹, K. Michalak-Micka¹, T. Biedermann¹, U. Moehrlen², A.S. Klar¹

(1) Tissue Biology Research Unit, University Children's Hospital Zurich, Zurich, Switzerland & Children's Research Center (CRC), University Children's Hospital Zurich, Zurich, Switzerland & University of Zurich, Zurich, Switzerland, (2)Tissue Biology Research Unit, University Children's Hospital Zurich, Zurich, Switzerland & Children's Research Center (CRC), University Children's Hospital Zurich, Zurich, Switzerland & University of Zurich, Zurich, Switzerland & University Children's Hospital Zurich, Zurich, Switzerland

The human interfollicular epidermis (IFE) is sustained by a tightly regulated balance between slow-cycling epidermal stem cells (ESCs) in the basal layer and more proliferative transient amplifying cells (TACs). Defining the spatial organization and functional properties of these keratinocyte (KC) subsets is essential for understanding epidermal homeostasis and regeneration. Here, we identified the leucine-rich repeat-containing G protein-coupled receptors LGR5 and LGR6 as robust and spatially distinct markers of basal versus suprabasal KC populations in the human IFE.

Immunofluorescence revealed LGR5 expression confined to CK15⁺/integrin β4⁺ (Iβ4⁺) basal KC, which also co-express melanoma-associated chondroitin sulfate proteoglycan (MCSP), further supporting their identity as a specialized basal stem-like subset. In contrast, LGR6 localized exclusively to CK10⁺ suprabasal layers. Flow cytometry confirmed this organization, showing that LGR5⁺ cells represent a small fraction of Iβ4⁺ basal KC (5 ± 1%), while most Iβ4^- suprabasal KC were LGR6⁺ (57 ± 19%). Functional assays demonstrated that LGR5⁺Iβ4⁺ basal KC possess markedly superior clonogenic capacity compared with LGR6⁺Iβ4^- cells. Consistent with their proliferative potential, the transcriptional regulator YAP was localized exclusively to the nuclei of LGR5⁺Iβ4⁺ KC but absent from LGR6⁺ cells.

To evaluate regenerative capacity, dermo-epidermal skin substitutes (DESS) generated from unsorted KC, purified LGR5⁺Iβ4⁺ basal KC, or LGR6⁺Iβ4^- suprabasal KC were transplanted onto immunodeficient rats. After four weeks, DESS derived from LGR5⁺Iβ4⁺ cells exhibited superior basal layer organization and enhanced epidermal regeneration.

Collectively, these findings identify LGR5⁺Iβ4⁺MCSP⁺ KC as a stem-cell–enriched basal population with enhanced proliferative, clonogenic, and regenerative properties. Unlike murine skin, where LGR5 expression is restricted to hair follicles, the human IFE contains a distinct LGR5⁺ basal subset that significantly contributes to epidermal maintenance and repair.

995. Metamaterial based microstructures as mechanically adaptive environments for cells

Gaurav Dave¹, Barbara Schamberger¹, Krishna Ramesh¹, Tolga Meydanacar¹, Federico Colombo¹, Fereydoon Taheri¹, Målin Schmidt¹, Christine Selhuber-Unkel¹

¹Institute for Molecular Systems Engineering and Advanced Materials (IMSEAM). Heidelberg University, Heidelberg (Baden-Wberg Bayern) - Germany

The influence of matrix stiffness has been extensively studied for its effect on cell behaviour such as migration speed, traction forces and morphology.[1] Additionally, surrounding factors such as geometry, macro and micro topographies, and curvature have been shown to influence the cell behaviour.[2] One major gap in understanding the cell-material interaction has been the scale at which the engineered environment is designed. With the development of Direct Laser writing (DLW)-based 3D printers, one can go to biologically relevant sizes of a few micrometres. By utilizing design-based approaches like metamaterials, mechanical properties can be tuned via design/geometry changes rather than material chemistry, we can develop a better understanding of cell-material interactions.

In this project, we combine the fabrication capabilities of 2PP-DLW with mechanical metamaterial designs to create engineered “soft” environments for cells with geometric features on a biologically relevant scale. To achieve this, we use a snakeskin-inspired metamaterial design. Mechanical characterization of macro (mm) and microscale(µm) structures show a drop of effective Young’s Modulus by 80-90% for polymeric materials compared to the bulk properties. In vitro experiments highlight the capacity of cells to actively integrate into the mesh of the structure and deform it via traction forces during migration. Such in vitro model systems offer a unique insight into mechanobiology and offer a passive yet dynamic environment for cells. Additionally, such design-based approaches combined with the fabrication capabilities of 2PP-DLW have potential for a new class of engineered materials for tissue engineering and tissue regeneration.

1) Discher DE. Tissue Cells Feel and Respond to the Stiffness of Their Substrate. Science. 2005 Nov 18;310(5751):1139–43.

2) Schamberger B, Roschger A, Ziege R, Anselme K, Martine Ben Amar, Bykowski M, et al. Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales. Advanced Materials. 2023 Feb 15;35(13).

996. Electrospun PCL–Pluronic F127 scaffolds to enhance hydrophilicity and mechanical performance for AV-graft applications - SEMIT

Inmaculada De Dios Pérez¹, Inmaculada De Dios Pérez², Amy Morgan³, Ílida Ortega Asencio³, Patricia Pérez Esteban⁴

¹Department of Chemical Engineering. University of Salamanca, Salamanca - Spain, ²Chemical Engineering. Institute of Translational Medicine, University of Birmingham, Birmingham - United Kingdom, ³School of Clinical Dentistry. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom, ⁴Chemical Engineering. Institute of Translational Medicine, University of Birmingham, Birmingham - United Kingdom

Arteriovenous (AV) grafts remain essential for long-term haemodialysis, yet up to 70% fail within the first year due to thrombosis, infection, leakage and insufficient tissue integration (1). Enhancing graft biocompatibility through surface modification is therefore critical. This study reports the development of electrospun fibrous scaffolds intended to coat the outer surface of AV grafts to improve surface properties and support better integration.

Poly(ε-caprolactone) (PCL) is widely used in vascular engineering due to its mechanical robustness and biodegradability; however, its intrinsic hydrophobicity restricts early cell adhesion and tissue integration (2,3). To address these limitations, we fabricated electrospun scaffolds composed of PCL blended with Pluronic^® F127 (PF), an amphiphilic triblock copolymer that enhances wettability (4). The aim was to optimise the scaffold by studying different PCL–PF ratios.

Electrospinning solutions were prepared by dissolving PCL/PF in dichloromethane/dimethylformamide. Rheological characterisation was performed prior to electrospinning to examine the influence of PF on solution viscoelasticity and jet stability. Resulting scaffolds were assessed by optical and scanning electron microscopy; tensile testing was used to evaluate mechanical performance, and static contact angle measurements quantified hydrophilicity.

Increasing PF content altered fibre diameter and improved wettability. Nevertheless, scaffolds containing a 50:50 PCL–PF ratio displayed the most balanced overall behaviour, whereas higher PF proportions led to loss of mechanical integrity, with elasticity decreasing when PF exceeded 25% relative to PCL. A bilayer configuration, combining a mechanically robust PCL base layer with a 50:50 PCL–PF surface layer, achieved optimal strength and is proposed as the most promising architecture for graft coating, supporting its further evaluation for future vascular tissue-engineering applications.

References

(1) Sagmeister MS. et al., Front Endocrinol, 2022

(2) Ntrivala S. et al., Materials Today, 2025

(3) Woodley D. et al., Biomaterials Science, 2023

(4) de Dios-Pérez I. et al., Eur J Pharm Sci, 2023

997. Development of novel bioengineered tissue constructs to model idiopathic pulmonary fibrosis pathophysiology in vitro

Jessica Simpson¹, Stefan Przyborski²

¹Biosciences. Durham University, Newcastle upon Tyne - United Kingdom, ²Biosciences. Durham University, Newcastle upon Tyne - United Kingdom

Idiopathic pulmonary fibrosis (IPF) is a degenerative interstitial lung disease, characterised by epithelial dysregulation, aberrant activation of interstitial fibroblasts, and excessive extracellular matrix (ECM) deposition within the alveolar interstitium (1,2). The continued reliance on in vivo animal models of pulmonary fibrosis has impeded the discovery of effective therapeutic compounds, highlighting the need for more physiologically representative, humanised models of IPF (3).

Utilising Alvetex^® Scaffold technology to support fibroblast infiltration, proliferation, and de novo endogenous ECM deposition, we have bioengineered a novel in vitro construct of the alveolar epithelium, comprised of distinct epithelial, macrophage, and fibroblast tissue compartments. Within this model system, A549 cells form an integral epithelial barrier that recapitulates the alveolar type II pneumocyte (ATII) phenotype, characterised by apical microvilli, mature surfactant protein synthesis, and multilamellar body biogenesis.

Alveolar models constructed using fibroblasts isolated from non-fibrotic lung or IPF tissue display innate differences in ECM deposition and composition, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) homeostasis, inflammatory cytokine secretome and fibroblast activation. Models were further validated for use as an in vitro testing platform following application of disease-appropriate stimuli, to mimic low grade chronic injury to the alveolar epithelium, and further induce fibrotic remodelling. Finally, the efficacy of Nintedanib, Pirfenidone and Saracatinib were assessed in IPF models, in isolation, or in combination with pro-fibrotic stimuli; Saracatinib demonstrated notable anti-fibrotic capacity, reducing ECM deposition and turnover, myofibroblast activation, and IL-6, IL-8 and IL-1B expression.

These results highlight Alvetex^® alveolar constructs as a robust and reproducible platform for modelling the complex cellular mechanisms that drive IPF pathophysiology, thereby offering improved translational relevance for drug target discovery and screening.

References

(1) Booth, A et al., Am J Respir Crit Care Med. (2012).

(2) Adams et al., Sci Adv. (2020).

(3) Cruwys et al., Drug Discov Today (2024).

998. A highly porous dermal matrix featuring rete ridge topography as an artificial stem cell niche towards improved full-thickness wound healing

Amy Morgan¹, Frederik Claeyssens¹, Joey Shepherd¹, Ílida Ortega Asencio¹

¹University of Sheffield, Sheffield (South Yorkshire) - United Kingdom

Acellular dermal matrices for full-thickness wounds offer promising alternatives to autologous skin grafts. However, products like Integra^® and Novasorb^® do not fully replicate the architecture of the dermis. The dermis of healthy skin contains peg-like dermal papillae that interlock with downward projections of the epidermis called rete ridges. Rete ridges are a putative stem cell niche (SCN) for cells involved in epidermal regeneration. To recapitulate this SCN in a wound healing construct, a highly porous methacrylated poly-glycerol sebacate (PGS-M) dermal matrix is fabricated by high internal phase emulsion polymerisation (polyHIPE) then cast and cured into ridged silicone moulds.

PGS-M polyHIPE provides a cytocompatible matrix for human dermal fibroblasts (HDFs) shown by increasing proliferation (PicoGreen™) and metabolism (resazurin assay) across days 7, 14 and 21. Defined pseudo-dermal papillae are formed on the polyHIPE surface with a near-native width and height of 300-400 µm and 115-150 µm, respectively. Pore morphology analysis via scanning electron microscopy (SEM) imaging reveals a mean pore and interconnect size of 29.0 ± 11.0 µm and 5.8 ± 2.1 µm, respectively. Through these highly interconnected pores HDFs infiltrate 410 ± 237 µm into the bulk over 21 days. Unlike motile fibroblasts, N/TERT-2G keratinocytes remain nearer the seeded surface (62.7 ± 23.9 µm at day 21). DAPI/Phalloidin-TRITC fluorescence imaging shows N/TERT-2G are guided by the pseudo-papillae to congregate in inter-papillae spaces (IPS). N/TERT-2G stratification on the surface, visualised by H&E staining, is achieved on ridged and flat matrices, with thicker areas of stratification forming from cells located in the IPS.

Ongoing studies involve immunofluorescence imaging of key markers collagen IV and integrin β-1, focussing on differences in expression between ridged and flat matrices.

In conclusion, highly porous emulsion templated dermal scaffolds featuring pseudo-dermal papillae have the potential to support full-thickness wound healing by offering a skin-equivalent SCN.

999. Injectable alginate-based dynamic hydrogels formed by reversible acylhydrazone bonds using oxidized polysaccharides for tissue engineering scaffold - SEMIT

Fereshteh Kazemi Aghdam¹, Zuzana Varchulová², Luboš Danišovič³, Igor Lacík¹, Abolfazl Heydari¹

¹Polymer Institute of the Slovak Academy of Sciences, Dúbravská cesta 9, 845 41. Slovak Academy of Sciences, Bratislava - Slovakia, ²Comenius University Bratislava, Faculty of Medicine, Špitálska 24, 813 72 Bratislava, Slovakia, Bratislava - Slovakia, ³Comenius University Bratislava, Faculty of Medicine, Špitálska 24, 813 72 Bratislava, Slovakia, Bratislava - Slovakia

Injectable hydrogels are widely employed in tissue engineering as scaffolds that provide hydrated, cell-supportive environments while enabling minimally invasive delivery into the body. Among them, shear-thinning dynamic hydrogels formed through reversible covalent bonds are particularly attractive, as they combine shear-responsive flow with rapid self-healing during and after injection [1]. In addition, the stress-relaxation behavior of these materials more closely mimics the cell-adaptive mechanical environment of the native extracellular matrix (ECM) [2]. However, limitations in controlling crosslinking kinetics and simultaneously balancing injectability, network reversibility, and long-term stability under physiological conditions remain challenges for systems relying on reversible covalent bonds.

In this study, modified sodium alginate was crosslinked with two oxidized polysaccharides, enabling the formation of a dynamic network through reversible acylhydrazone linkages. Modulation of the oxidized polysaccharide crosslinkers allowed systematic and on-demand tuning of hydrogel properties, most notably the crosslinking kinetics. Hydrogels also exhibited shear-thinning and rapid self-healing behavior, allowing smooth injection through a needle and rapid recovery of the storage modulus following injection via syringe needle. Owing to the reversible nature of these linkages, post-injected hydrogels displayed time-dependent mechanical responses, including stress relaxation. Cell-laden hydrogels showed high viability and uniform distribution of encapsulated MSCs, confirming the cytocompatibility of the gelation conditions. The hydrogels further retained their mechanical stability under physiological conditions for several weeks. Collectively, these results underscore the potential of injectable dynamic alginate hydrogels as an advanced scaffold providing adaptive mechanics of native ECM.

Acknowledgment

This work was supported by APVV-22-0568, and FLAG-ERA grant GRAPH-OCD, by the Slovak Academy of Sciences under the grant number FLAG ERA III/2023/808/GRAPH-OCD.

1. Guvendiren, M., H.D. Lu, and J.A. Burdick, Shear-thinning hydrogels for biomedical applications. Soft matter, 2012. 8(2): p. 260-272.

2. Webber, M.J. and M.W. Tibbitt, Dynamic and reconfigurable materials from reversible network interactions. Nature Reviews Materials, 2022. 7(7): p. 541-556.

1000. The nature of the dermal component of self-assembled skin substitutes influences the differentiation of epithelial stem cells of preformed hair

Cattier Bettina¹, Caillaud Alizée¹, Larouche Danielle¹, Germain Lucie¹

¹Centre de recherche de l'Université Laval/LOEX, CHU de Québec-Université Laval Research Centre, Québec, QC, Canada, Quebec - Canada

Hair follicle integration into engineered skin remains a major challenge in regenerative medicine. We hypothesized that the dermal microenvironment, modulated by fibroblast origin and age, influences the capacity of epithelial stem cells to differentiate into hair lineage. To test this, self-assembled dermal substitutes were produced using fibroblasts from four populations: neonatal and adult mouse (FVBN), and neonatal and adult human donors. Pre-formed hair follicle buds (HFB) from neonatal C57/Bl6 mice (<12 h) were seeded onto those dermal constructs and grafted on athymic mice for six weeks. HFB represent an excellent model for hair regeneration because they are sufficient to induce hair growth and contain all essential components for hair formation, including the dermal papilla.

Our results show that dermis reconstructed from neonatal mouse fibroblasts supports mature hair follicle development after grafting. In contrast, even if dermis generated from adult mouse or human fibroblasts also allowed mature hair shafts development, a reduced density has been observed.

To identify dermal factors that promote appendage sustainability, proteomic analysis was performed on murine dermal constructs prior to HFB addition. We identified 199 differentially expressed proteins: 126 upregulated in neonatal fibroblast-derived dermis and 73 in adult-derived dermis. Over-representation analysis revealed enrichment of developmental processes in neonatal dermis, whereas adult dermis showed biological processes related to extracellular matrix. These findings suggest that neonatal fibroblast-derived dermis retains inductive signals essential for appendage morphogenesis, while adult dermis exhibits a more mature phenotype.

Future work will focus on matrisome characterization, comparison between human dermis, and identification of candidate proteins to enhance hair follicle integration in engineered skin.

1001. Development of a smart antifungal biomaterial device with artificial microenvironments for enhanced corneal healing

Mehmet Gunen¹, Ilida Ortega Asencio¹, Frederik Claeyssens², Joey Shepherd¹

¹School of Clinical Dentistry. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom, ²School of Chemical, Materials and Biological Engineering. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom

Introduction

Corneal blindness caused by limbal stem cell deficiency remains a major global health challenge. Simple limbal epithelial transplantation (SLET) offers a promising solution by transferring limbal tissue explants onto an amniotic membrane (AM) using fibrin glue. However, AM presents several drawbacks, including infection risk, ethical and sourcing issues, and batch variability, while fibrin glue can impede epithelial proliferation. This study aims to develop a smart, ring-shaped corneal device fabricated from antifungal polymeric materials to replace both AM and fibrin glue, enhancing the efficacy and accessibility of SLET.

Materials and Methods

The device integrates a polycaprolactone (PCL) polyHIPE ring featuring an interconnected porous structure that mimics limbal microenvironments, and an electrospun poly(lactic-co-glycolic acid) (PLGA)–polyethylene glycol (PEG) membrane offering transparency and rapid degradation. PLGA-PEG solutions containing natamycin were electrospun under controlled voltage and flow rates. Structural and physicochemical properties were evaluated via SEM, EDS, profilometry, tensile testing, and contact angle analysis. Antifungal efficacy was examined using UV–Vis spectroscopy and disc diffusion assays. Biocompatibility was tested using indirect cell culture assays and porcine air–liquid interface (ALI) corneal cultures.

Results

Electrospun membranes exhibited uniform nanofibers with successful natamycin encapsulation and minimal bead formation. PEG incorporation enhanced flexibility and hydrophilicity. The PCL polyHIPE demonstrated high internal porosity, supporting cell infiltration and nutrient exchange. Strong antifungal activity against Candida albicans was confirmed, and indirect biocompatibility assays indicated excellent cytocompatibility. Preliminary ALI cornea experiments showed effective tissue responses under wounded, infected, and combined wounded+infected conditions.

Conclusion

The developed device combines biocompatibility, mechanical integrity and handability whilst achieving sustained antifungal function. These findings demonstrate its strong potential as a fully synthetic alternative to the amniotic membrane in SLET, offering a scalable and clinically translatable corneal medical device.

1002. Tuning biomimetic bioreactor operating conditions for osteochondral tissue engineering and research

Jovana Zvicer¹, Fatemeh Safari², Mia Milosevic³, Jasmina Stojkovska¹, Sibylle Grad², Martin J. Stoddart², Zhen Li², Bojana Obradovic⁴

¹University of Belgrade, Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia, ²AO Research Institute Davos, Davos Platz (Graubunden) - Switzerland, ³University of Belgrade, Innovation center of the Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia, ⁴University of Belgrade, Faculty of Technology and Metallurgy, Belgrade (Serbia) - Serbia

Designing effective experimental setups particularly for osteochondral research is challenging. Cartilage and bone tissues are distinctively different regarding composition, structure and biophysical signals: cartilage is gelatinous, avascular, and exposed primarily to dynamic compression, whereas bone is porous, vascularized, and responsive to hydrodynamic shear stresses, which are known osteogenic stimuli in vitro. Therefore, we designed a biomimetic bioreactor capable of recapitulating these conditions. The bioreactor was first used for developing osteochondral constructs using bilayer scaffolds consisting of an upper gellan gum hydrogel layer mimicking cartilage that was well integrated to the bottom macroporous layer of gellan gum with bioactive glass (BAG) particles representing bone. The scaffolds were evaluated in a bioreactor exhibiting dynamic compression to the upper layer (frequency 0.68 Hz) and continuous perfusion of simulated body fluid through the bottom layer (400 µm/s superficial velocity) over 14 days, resulting in enhanced BAG transformation to hydroxyapatite. In the next step the bioreactor was used to culture osteochondral explants and synovium tissue. In these ex vivo experiments, we observed reduced inflammation, as explants cultivated in the bioreactor showed a trend toward decreased expression of IL-6 and ADAMTS-4 compared to the static culture. However, histological analyses revealed reduced cell viability within cartilage, suggesting that nutrient and oxygen transport may not have been fully optimized under the applied conditions. To investigate these findings, we performed mathematical modeling of mass transport incorporating diffusion, advection, and tissue-specific glucose consumption rates. The model successfully predicted glucose and oxygen profiles within the explants offering insights into spatial limitations of mass transport. These results emphasize biomimetic bioreactors as powerful platforms for advancing scaffold development and ex vivo joint models; however, they are not inherently pre-optimized systems. Integrating mathematical modeling enables rational refinement of culture parameters to better meet scaffold and tissue specific requirements.

1003. Localized immunomodulation via engineered subcutaneous niche for cancer, autoimmune tolerance and in situ cell therapy

Nikitha Kota¹, Danilo Settis¹, Alessandro Grattoni¹, Corrine Ying Xuan Chua¹

¹Houston Methodist Research Institute, Houston (Texas) - United States

Conventional cell therapies are limited by laborious ex vivo processing, systemic dispersion, and poor cell survival. To address these challenges, we developed the NanoLymph, a subcutaneous platform designed for the in situ enrichment and reprogramming of immune cells. We aimed to demonstrate its versatility in two opposing therapeutic contexts: eliciting antigen-specific anti-tumor immunity and inducing immune tolerance for autoimmune diseases or allogeneic cell transplantation.

The NanoLymph device comprises interconnected drug and cell reservoirs separated by a nanoporous membrane. This architecture allows for the sustained, localized elution of bioactive cues into the cell reservoir, creating a gradient to continuously recruit and reprogram immune cells in situ. We evaluated the platform's efficacy in murine models by loading it with specific combinations of immune-modulating agents and antigens to drive either immunogenic or tolerogenic immune cells.

For cancer immunotherapy, localized elution of dendritic cell (DCs) stimulatory cues and immune adjuvants recruited and activated DCs. These cells engaged with co-delivered antigens, eliciting a systemic antigen-directed CD8+ T cell response that inhibited B16F10 melanoma growth. Conversely, to create an immunosuppressive microenvironment, localized elution of CCL22 and IL2 recruited and expanded regulatory T cells (Tregs) in situ and promoted induction and maintenance of tolerance towards allogeneic cell grafts. Separately, NanoLymph released tolerogenic agents (e.g., retinoic acid) can reprogram recruited DCs into a tolerogenic phenotype.

The NanoLymph serves as a versatile, tunable platform for in situ cell therapy. By enabling the localized enrichment and functional reprogramming of specific immune cells, it overcomes the limitations of systemic toxicity and cell dispersion associated with conventional approaches. This platform offers a flexible solution for disease-specific immune modulation, ranging from cancer vaccination to the protection of transplanted cells in autoimmune settings.

1004. A scaffold-based platform for nanoparticle-mediated mRNA delivery in breast cancer immunotherapy

Na Zhao

de Tissue Engineering Research Group, Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin - Ireland

Breast cancer remains a major cause of cancer-related mortality among women worldwide, and the development of effective postoperative immunotherapeutic strategies is crucial for preventing tumour recurrence and metastatic progression[1]. Although mRNA holds considerable potential for the local production of therapeutic proteins, its clinical use is restricted by rapid degradation and limited intracellular delivery[2]. This project therefore aims to establish a scaffold-based platform capable of supporting nanoparticle-mediated mRNA delivery, using three-dimensional collagen–hyaluronic acid (Col/HyA) scaffolds to provide localised and sustained release following tumour resection[3].

Three mRNA delivery systems—MessengerMAX lipid nanoparticles, the peptide-based carrier mGET, and extracellular vesicles (EVs)—were prepared and evaluated according to established laboratory protocols. Their transfection efficiency and cytocompatibility were systematically assessed in MDA-MB-231 breast cancer cells under two-dimensional culture conditions and within Col/HyA scaffolds. Transfection performance was quantified using firefly luciferase mRNA assays and fluorescence microscopy of EGFP mRNA expression, while biological safety was assessed through AlamarBlue metabolic activity measurements, PicoGreen DNA quantification, and Live/Dead staining. MessengerMAX exhibited the highest transfection efficiency overall, whereas mGET enabled effective mRNA expression when the N/P ratio was appropriately optimised.

Building on these findings, the most effective nanocarrier will be combined with TRAIL mRNA and incorporated into Col/HyA scaffolds. Following tumour removal, these scaffolds will be positioned at the resection site to enable sustained local mRNA release and in situ TRAIL protein production, with the aim of inducing apoptosis in residual tumour cells.

In conclusion, this study establishes a scaffold-based platform for nanoparticle-mediated mRNA delivery, demonstrating strong potential for enhancing the performance of localised postoperative immunotherapy in breast cancer.

Acknowledgements

Funding: the RCSI-Soochow PhD Program.

References

[1] Rebecca L Siegel (et al.), CA Cancer J Clin. Jan 16;75(1): 10-45, 2025.

[2] Xiang Li (et al.), Theranostics. Jan 14(2): 738-760, 2024.

[3] Yue Zhang (et al.), Adv Mater. Dec 33(48): e2106768, 2021.

1005. Volumetric tissue monitoring of structure and function based on optical coherence tomography

Jonas Golde¹, Antonia Starcke¹, Bernhard Schwarze¹, Stephan Becker¹, Frank Sonntag¹

¹Fraunhofer Institute for Material and Beam Technology, Dresden (Sachsen) - Germany

Extended, volumetric tissues, such as spheroids, organoids and printed compounds of cell-laden biomaterials attract an increasing interest in tissue engineering, pharmacology and in-vitro drug testing but challenge the established light microscopy techniques due to their increased thickness. While modern techniques such as light-sheet fluorescence and confocal multiphoton microscopy are typically limited to penetration depths of up to 100 µm, optical coherence tomography (OCT) is an interferometric imaging modality, which measures the depth-resolved backscattering of near-infrared light at more than 1 mm in dense scattering tissue. We have developed OCTbiolab, a novel imaging system based on OCT that enables rapid, label-free exams of live tissues in different sizes and conditions. Based on time-resolved speckle dynamics and polarized light, the OCT contrast is further enhanced to track not only volumetric growth but also necrotic, vascular or extra-cellular matrix (ECM) changes over time, thus providing quantitative metrics on the same sample for improved assay throughput and reduced variability in tissue engineering.

Supported by the Free State of Saxony and the European Union EFRE within the SAB projects “OGD-LOC” and “LOC-OGD-Sys”.

1006. Human chondrocytes show higher viability and maturity after 3D-bioprinting and 30 days in vitro when cultured in microcarriers (3D) compared to monolayer (2D)

Karin Säljö¹, Kristin Oskarsdotter¹, Susann Li², Peter Apelgren¹, Lars Kölby¹

¹Department of Plastic Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg (Vastra Gotaland) - Sweden, ²Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicin, Sahlgrenska University Hospital, Gothenburg (Vastra Gotaland) - Sweden

The overall goal is to develop a new minimal invasive surgical treatment for children born without an external ear, i.e. microtia. We will use chondrocytes isolated from the patient’s contralateral ear and 3D-bioprint a replica of the same. The conventional surgical method is an invasive surgical approach associated with high donor site morbidity, as well as risk for severe complications and variable esthetic outcomes.

We have previously shown long-term survival of 3D-bioprinted constructs with human chondrocytes in animal models. However, culturing chondrocytes in monolayer is associated with dedifferentiation. This study aims to compare the viability and functionality of chondrocytes cultured in porous gelatine microcarriers (PGMs) and monolayer, as well as how they survive in 3D-bioprinted constructs in vitro.

We isolated human chondrocytes from auricular cartilage. The chondrocytes were expanded in monolayer with DMEM/F12 medium (group 1) and cultured in a stirring flask with PGMs (group 2) for 72h. The bioink consisting of 5% enzymatically pretreated nanocellulose hydrogel and 3% alginate solution at an 80:20 ratio (v/v) was prepared. Single cell suspension (group 1) and PGM containing chondrocytes (group 2) was 3D-bioprinted into circular scaffolds (5x1mm) using a 22 G conical nozzle at 5 mm/s and 40 respectively 22kPa. All scaffolds were crosslinked with 100 mM CaCl2 and cultured in vitro for 30 days. The viability was assessed after 7, 14 and 30 days by calcein-AM/propidium iodide staining. Samples (n=6) were taken at each time point and fixated, paraffin embedded, sectioned (5μm) and stained with Alcian blue-van Gieson for histology.

The PCR analysis of the chondrocytes showed significantly increased expression of Col2A, COL2B and ACAN in the cells cultured in PGMs compared to monolayer indicating a more mature and healthy state. The cell viability was 98% in both groups prior to 3D bioprinting. However, after 30 days in vitro the live/dead staining showed a considerably higher cell viability in group 2 compared to group 1. The histology confirms the presence of functional chondrocytes inside the PGMs producing extracellular matrix at day 30.

In conclusion, chondrocytes cultured in PGMs show less signs of dedifferentiation than in monolayer and higher survival in vitro following 3D-bioprinting. Hence, 3D-bioprinted constructs with chondrocyte containing PGMs can be of interest for future clinical applications such as ear reconstructions.

1007. Single-cell-derived breast cancer spheroids for real-time growth and multi-Omics analysis

Margarida Esteves¹, Francisca Oliveira¹, Alexandra Teixeira¹, Nuria Estévez-Gómez², João M. Alves², Andrea Garcia-Lizarribar³, Iratxe Madarieta³, Beatriz Olalde³, David Posada², Miguel Xavier¹, Sara Abalde-Cela¹

¹International Iberian Nanotechnology Laboratory, Braga - Portugal, ²CINBIO Universidade de Vigo, Vigo (Pontevedra) - Spain, ³Tecnalia, Donostia-San Sebastián (Gipuzkoa) - Spain

Introduction and Aim: Metastatic breast cancer(mBC) remains a leading cause of cancer mortality, driven by tumour heterogeneity and metabolic adaptation. Understanding these processes at the single-cell level is essential for identifying metastatic drivers and therapeutic targets. This work aims to establish a human-relevant 3D platform that enables clonal expansion of single BC cells into spheroids for real-time metabolic monitoring, and to integrate genomic and transcriptomic analysis to trace clonal evolution and phenotypic diversity within tumour cell populations. After validating the system in BC cell lines, we will apply it to patient-derived circulating tumour cells(CTCs) to uncover traits associated with metastatic progression.

Materials and Methodology:Single MCF-7 cells were cultured within human and porcine decellularised adipose extracellular matrix(adECM) hydrogels to develop spheroids over 28 days. Gold nanostars(GNSs;0.5-4mM) were incorporated to enable surface-enhanced Raman scattering(SERS) analysis. Spheroid growth, proliferation, viability, metabolic activity, and nucleic acid content were evaluated. A protocol for simultaneous DNA and RNA extraction from few-cell spheroids was established using the AllPrep^®DNA/mRNA Nano Kit, followed by whole-exome and whole-transcriptome sequencing.

Results:Both adECM supported ∼30% spheroid formation and long-term viability. GNSs concentrations ≤2mM preserved cell morphology and viability, whereas 4mM significantly impaired growth(p<0.05). Genomic analysis revealed consistent mutation patterns across spheroids, with most variants already reported in the Sanger COSMIC Cell Lines Project. Copy number variants matched publicly available MCF-7 data. Transcriptomic analysis showed highly consistent expression profiles, including expected MCF-7 markers (GFRA1,HIPK1,TRIM37). SERS enabled robust metabolite diffusion and in situ detection.

Conclusions:This integrated platform enables reproducible clonal spheroid development, non-destructive metabolic monitoring, and combined genomic-transcriptomic profiling. These advances support future application to patient-derived CTCs, with the overarching goal of identifying cellular features associated with metastatic potential and advancing preclinical systems for studying tumour development.

Acknowledgements:This work was funded by the EU(HORIZON-EIC-2022-PATHFINDEROPEN-01-01,grant101099066) and UKRI under the UK government Horizon Europe guarantee(grant10063360).

1008. Investigating the effects of mechanotherapy on macrophages in vitro

Parand Shokrani¹, Hannah Prendeville¹, Rachael Dillon¹, Eimear Dolan¹

¹Biomedical Engineering. University of Galway, Galway - Ireland

Aim and Objectives: We previously showed that actuation of a mechanotherapeutic implant disrupts the host foreign body response (FBR), reducing fibrous capsule formation and improving device performance1,2. Actuation generates local fluid flow at the biotic–abiotic interface, modulating neutrophil, macrophage, and myofibroblast infiltration. However, the cellular mechanisms underlying these effects remain unclear. Recent work from our group suggests that mechanical loading above a defined threshold can alter fibrotic behaviour in human myofibroblast-like cells by decreasing collagen and IL-1β while increasing anti-inflammatory IL-103. The present study investigates how fluid flow influences macrophages, which are key regulators of FBR progression.

Methods: Bone marrow was isolated from C57BL/6J mouse tibias and fibulas to generate bone marrow–derived macrophages (BMDMs) using DMEM supplemented with 20% L929-conditioned media. Naïve BMDMs were seeded on Superfrost slides and exposed to pulsatile laminar flow at 0.5 or 0.8 Pa at 0.5 Hz for 10 minutes per day over two days using a parallel plate flow chamber. Wall shear stress was calculated using the equation for steady, fully developed laminar flow.

τ_w=6Qμ/(bh^2)

Flow cytometry quantified viability (Live/Dead Aqua), macrophage activation (CD11b, F4/80), classically (CD86) and alternatively (CD206) activation markers, and total cell numbers.

Results: CD11b and F4/80 expression increased in flow-stimulated groups, indicating enhanced macrophage activation relative to static controls. Viability and CD86/CD206 expression were unchanged, suggesting that this fluid-flow regimen did not alter the polarisation state of naïve BMDMs. Both flow conditions resulted in reduced total cell numbers, likely due to detachment of loosely adhered cells during shear exposure. Ongoing study incorporate LPS pre-treatment to mimic the early inflammatory environment following implantation.

Conclusions: Macrophages are mechanosensitive cells4 that strongly influence the FBR. Our results show that pulsatile fluid flow enhances macrophage activation without shifting polarisation. Future cytokine profiling of TNF, IL-6, and IL-10 will help elucidate how mechanical forces modulate macrophage behaviour in the context of mechanotherapeutic implant design.

Ref:

1. Whyte et al, Nature Communications, 2022

2. Dolan et al, Science Robotics, 2019

3. Ward et al, Acta Biomaterialia, 2024

4. Tang et al, Frontiers in Immunology, 2023

1009. DLP bioprinting of hydroxyapatite scaffolds for bone regeneration: effects of HA concentration on light penetration, printability, and cellular response

Nicolás E. Ruiz San Jose¹, Alberto Otero¹, Nadina Usseglio¹, Daniel Nieto¹

¹Advanced Biofabrication Laboratory - DNIETO LAB, A Coruña - Spain

Digital light processing (DLP) bioprinting has emerged as a powerful tool for fabricating scaffolds with precise architecture for bone tissue engineering. Incorporating hydroxyapatite (HA), a bioactive ceramic, into photopolymerizable resins can enhance osteoconductivity, but it also significantly affects light absorption and curing behavior, impacting print fidelity and resolution.

In this study, we systematically investigated the effects of HA concentration on the DLP printing process and scaffold performance. By varying HA content, we analyzed the resulting changes in light penetration depth and their influence on layer curing, structural integrity, and achievable resolution. Scaffolds were designed with a 400 µm interconnected pore network to mimic trabecular bone architecture, and printability was assessed through resolution fidelity and dimensional accuracy. Our results demonstrate a clear correlation between HA content and light scattering/absorption: higher HA concentrations reduced penetration depth, which challenged curing uniformity but enhanced scaffold opacity, influencing final geometry.

Optimizing the balance between HA content and printing parameters allowed the production of well-defined, reproducible scaffolds with controlled porosity. The biocompatibility of printed scaffolds was evaluated using bone-derived cells, revealing excellent cell adhesion, proliferation, and viability across all HA concentrations tested. These findings indicate that careful tuning of HA incorporation in DLP-resins can achieve scaffolds that combine structural fidelity, controlled porosity, and biological functionality. This study provides key insights into the interplay between material composition and DLP printing parameters, offering practical guidelines for the fabrication of HA-based scaffolds tailored for bone regeneration applications.

Overall, this work advances the design of bioactive ceramic scaffolds and highlights the potential of DLP bioprinting for producing customizable, osteoconductive constructs suitable for clinical translation.

1010. Versatile multiplexing device for the in vivo testing of bone organ inducing cells and microenvironments

Matteo Monticelli¹, Bianca Maria Carrara¹, Martin Ehrbar¹

¹Laboratory for Cell and Tissue Engineering, University Hospital Zurich. University of Zurich, Zurich - Switzerland

Introduction:

Over the past years the ability of human bone marrow mesenchymal stromal cells (hBM-MSC) to regenerate bone replicas when transplanted in vivo has been reported. However, the identity and function of hBM-MSCs remain unknown. Due to the vast number of cells required for the experiment and the restricted number of samples that can be implanted in one animal, with currently available methods in vivo testing has limited screening capacity. Here, we provide a high throughput platform to study and test low-abundant hBM-MSC in vivo behavior.

Methods:

We designed PDMS multiplexing devices featuring multiple inlets of varying sizes to allow encapsulation of multiple small tissue constructs of 3.7, 1.2, and 0.4 μl per device, to increase the in vivo screening power taking into account different number of hBM-MSC and environmental conditions. For in vitro evaluations transglutaminase cross-linkable poly(ethylene glycol) (PEG) hydrogel which contained hBM-MSCs were loaded in multiplexing devices and cultured for 2 weeks in presence of 0 or 50ng of BMP2 or TGFβ3. For in vivo evaluations miniaturazed PEG hydrogels containing 20 or 10x106 ml-1 of hBM-MSCs and 0, 5 or 12.5 μg ml-1 of BMP2 or TGFβ3. After 8 weeks of subcutaneous implantation in Fox1nu mice samples devices were harvested and analyzed through micro-CT and histological characterization.

Results:

hBM-MSC cultured in miniaturized TG-PEG hydrogels showed comparable cell survival, osteogenic potential, ECM protein deposition as large tissue constructs. MicroCT and histological analysis revealed hBM-MSC function in ectopic bone and bone marrow formation with different degree of remodiling for different environmental conditions. Interstingly hBM-MSC supplemneted with TGFβ3 show comparable bone and bone marrow tissue formation to BMP2 samples.

Conclusions:

We report an innovative, versatile, and scalable PDMS-based platform for high-throughput in vitro and in vivo screening of hBM-MSCs, which also enables a significant reduction in the number of animals required.

1011. Bioengineered immunosuppressive tumor microenvironment models for screening immunotherapies

Maria Monteiro¹, Margarida Henriques Pereira², Vitor M. Gaspar², João F. Mano²

¹Cellularis Biomodels, Ílhavo (Aveiro) - Portugal, ²CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Aveiro - Portugal

Human tumors exhibits pronounced resistance to current treatment options, largely due to its highly immunosuppressive tumor microenvironment (TME). Tumor microenvironemnt directly impacts the efficacy of cell therapies and prompts disease progression. Modelling the complexity and recreating the immunosupressive hallmarks of human neoplasias in vitro remains highly challenging. Herein, we bioengineered spherical, miniaturized tumor-stroma platforms using superhydrophobic surfaces, that not only emulate the native tumor composition but key tummor signatures such as the recognized immunessupression.

The immunosuppressive potential of well-established 3D tumor-stroma models, that closely recapitulates tumor composition and bioarchitecture, was evaluated. The ability to resemble the complex tumor-immune system interplay and the inherent immunosuppressive TME was examined, through co-culture with different immune cells surrogates, namely Jurkat T cells, dendritic cells and monocytes, providing insights into tumor-driven immune modulation and compartmentalized tumor-stroma models potential for immunotherapy screening.

1012. Modular engineering of miniaturized bone-like tissues with Janus microgels

Ke Song¹, Esra Güben Kaçmaz¹, Francesca Giacomini¹, Filippo Zanforlin¹, Wen Chen¹, Hans Duimel², Lorenzo Moroni¹, Roman Truckenmüller¹, Pamela Habibović¹, Zeinab Niloofar Tahmasebi Birgani¹

¹MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht (Limburg) - The Netherlands, ²Microscopy CORE Lab, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht (Limburg) - The Netherlands

Microgels made from naturally derived proteins, such as collagen and gelatin, have emerged as promising biomaterial building blocks in modular tissue engineering, mimicking the extracellular matrix (ECM) of native tissues and supporting cell differentiation. However, in many tissues, including vascularized bone, the ECM presents sharply contrasting regions with different compositions and mechanical properties that conventional homogeneous microgels cannot replicate. Janus microparticles, which contain two compositionally and mechanically distinct regions, offer a powerful platform for engineering matrices that integrate both soft and stiff ECM regions and recreate such heterogeneity. In this study, we fabricated Janus microgels containing gelatin and gelatin-nanohydroxyapatite compartments using a solvent-free film-based microparticle patterning technique for modular engineering of vascularized bone-like tissues. This method enabled precise control over microscaffold structure, and the resulting microgels could be fabricated with well-defined geometries as well as two distinct soft and stiff regions. The Janus microgels self-assembled with human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in non-adherent microwells and formed hybrid microtissues. Our results indicated that Janus microgels supported osteogenic differentiation of hMSCs while enabling the sprouting of HUVECs. Furthermore, the microgels allowed spatially controlled loading of vascular endothelial growth factor and bone morphogenetic protein-2 into their soft and stiff compartments, respectively. The subsequent release of the growth factors from the microgels further enhanced their osteogenic and angiogenic effects. Integration of magnetic nanoparticles into Janus microgels enabled the controlled bottom-up assembly of the hybrid microtissues into larger tissues. This work presents a novel method for fabrication of Janus microgels and underscores their potential as versatile microscaffolds for modular engineering of complex tissues with spatial definition.

1013. From cells to living materials: dynamic mechanical and morphological changes of chondrogenic spheroids in 3D culture

Karolina Z. Dąbrowska^{1 2 3}, Marta Tosini⁴, Hanna Svitina^{2 3}, Ioannis Papantoniou^{2 3}, Bart Smeets^{1 3}

¹KU Leuven, Department of Biosystems, Division of MeBioS, Leuven, Belgium, ²KU Leuven, Skeletal Biology and Engineering Research Center, Department of Development and Regeneration, Leuven, Belgium, ³KU Leuven, Prometheus, Division of Skeletal Tissue Engineering, Leuven, Belgium, ⁴Politecnico di Torino, Department of Mechanical and Aerospace Engineering, PolitoBIOMed Lab, Turin, Italy

Multipotent human periosteum-derived cells (hPDCs) are promising for bone tissueengineering, especially when formed into 3D aggregates that mimic living tissue buildingblocks. These 3D spheroids enhance cell-cell interactions and extracellular matrix (ECM)secretion, crucial for driving cell behaviour and mechanical properties duringdiNerentiation. Understanding how spheroid morphology and mechanics evolve cansupport their use as tailored living materials in regenerative medicine.In this study, hPDCs were cultured in 2D monolayers and 3D spheroids underchondrogenic conditions for 21 days. Mechanical properties were measured using nondestructiveindentation techniques, as Atomic Force Microscopy (AFM) andNanoindentation. Cytoskeletal organization and chondrogenic marker expression wereassessed by immunostaining imaging, while spheroid structure was evaluated by AFMand Scanning Electron Microscopy.Results revealed that hPDCs in both 2D and 3D cultures initially softened (day 3), likelydue to cytoskeletal reorganization. As chondrogenesis begins, vimentin networkresponsible for mediating the cell’s mechanical resilience undergoes remodelling and nolonger surrounds the nucleus in a cage-like structure. By day 21, 3D spheroids produceda dense ECM and increased stiNness, forming a tissue-like microenvironment. Peripheralcells in spheroids softened after two weeks, reflecting further cytoskeletal changes. Incontrast, 2D-cultured hPDCs showed no significant stiNness increase and expressedchondrogenic markers, as COL2A1 and SOX9, primarily in the cytoplasm, indicating aninactive transcriptional state and limited diNerentiation.These findings highlight the essential role of 3D culture environments in promoting ECMdeposition and functional maturation during chondrogenic diNerentiation. This workrepresents a first step towards understanding the dynamic interplay between cellular,structural, and material properties within 3D chondrogenic spheroids, necessary tofurther advancing their use as living building blocks in regenerative medicine.Authors thank Magdalena Kobielarz (Wrocław University of Science and Technology,Poland) for guidance and help during SEM images acquisition.

1014. Hybrid 3D-printed hybrid nerve guides show promising structural stability and regenerative performance in a rat sciatic nerve injury model

Miguel Etayo Escanilla¹, David Sanchez Porras², Paula Avila Fernandez², Oscar Dario Garcia Garcia², Jesus Chato Astrain², Fernando Campos², Sandra Vieira³, Marta Pegueroles⁴, Victor Carriel²

¹University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ²University of Granada and Instituto de Investigación Biosanitaria ibs.GRANADA, Granada - Spain, ³University of Aveiro, Aveiro - Portugal, ⁴1Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering and Institute for Research and Innovation in Health (IRIS), Universitat Politècnica de Catalunya - BarcelonaTech, Barcelona - Spain

Peripheral nerve injuries often result in long-term disability, and although 3D-printed nerve guides have emerged as promising alternatives to conventional fabrication techniques, they still lack sufficient biological activity to support optimal regeneration. To address this limitation, this study proposes combining 3D-printed PLLA tubular meshes with nanostructured fibrin-agarose based hydrogels, aiming to integrate mechanical consistency with biomimetic properties.

PLLA conduits were 3D-printed and subsequently coated with fibrin-agarose (PLLA-FA) or genipin-crosslinked fibrin-agarose (PLLA-FAGP). Structural and mechanical properties were assessed through FE-SEM and uniaxial compression testing. Then, a 10-mm defect was created in the sciatic nerve of Lewis rats to evaluate the therapeutic efficacy of these nerve guides, comparing the results with the NeuraGen^® nerve guides. After 14 weeks, muscle atrophy was evaluated and the general nerve morphology, regeneration and myelinization were analyzed histologically.

Both hydrogel coatings formed continuous layers around PLLA filaments, with PLLA-FAGP showing superior compactness and structural integrity. Regarding the in vivo evaluation, muscle morphometry revealed similar atrophy in both groups. Histology demonstrated that genipin-crosslinked coatings enhanced biomaterial preservation and reduced fibrotic encapsulation. Moreover, PLLA-FAGP supported the formation of numerous newly regenerated micro fascicles and strong axonal regeneration, although NeuraGen^® showed slightly higher myelin staining intensity.

These results demonstrate that PLLA-FAGP nerve guides achieve structural stability and regenerative outcomes comparable to NeuraGen^®. However, future enhancements such as the use of intraluminal bioactive hydrogels or cells could further optimize their therapeutic performance.

This work was financed by the “Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica” of Instituto de Salud Carlos III and co-financed by FEDER funds (Grant FIS PI23/00337); (MCIN/AEI/10.13039/501100011033 and European Union-NextGenerationEU/PRTR) (Grant CPP2021-009070). MEE by FPU Fellowship Grant FPU21/06183 of the Spanish Ministry of Universities.

1015. Human microcirculation-on-a-chip: a microphysiological platform to model lung cancer progression

D. Caballero^{1 2}, C.M. Abreu^{1 2}, A.C. Lima^{1 2}, N.M. Neves^{1 2}, S.C. Kundu^{1 2}, R.L. Reis^{1 2}

¹3B’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, ²ICVS/3B’s-PT Government Associate Laboratory, Braga, Guimarães, Portugal

The microcirculatory system is pivotal in cancer progression, but its multicellular and dynamic complexity is difficult to replicate in vitro. Most microfluidic models focus solely on endothelialized blood vessels, limiting physiological relevance. Herein, we developed a human microcirculation-on-chip integrating self-organized blood and lymphatic microvessels co-cultured with 3D tumors. Using lung cancer as model, we studied how tumor-derived mediators and cell interactions drive vascular remodeling and invasion, uncovering key molecular and biophysical regulators.

The PDMS chip, fabricated by UV/soft lithography, comprised 5 channels mimicking native architecture. Human pulmonary microvascular (blood/lymphatic) and lung cancer cells with distinct lymphangiogenic profiles were compartmentalized. Tumor spheroids embedded in Coll I gel occupied the central channel. Adjacent channels were seeded with blood and lymphatic cells forming 3D perfusable networks. Immunostaining (Podoplanin/CD31/VE-Cad/F-actin/DAPI) characterized vessel morphology, and Luminex assays quantified angiogenic, lymphangiogenic and inflammatory cytokines.

The model reconstituted lung-specific blood and lymphatic microvessels, exhibiting native-like biomarker expression and organization. Upon tumor introduction, distinct remodeling patterns emerged: lymphangiogenic tumors induced stronger lymphatic sprouting, vessel dilation, and tumor invasion compared to non-lymphangiogenic tumors. Lymphatic networks displayed larger diameters and sprouting densities correlating with tumor lymphangiogenic potential. Cytokine profiling identified elevated G-CSF, IL6, IL8 and HGF levels in lymphangiogenic tumors suggesting that inflammatory signaling promoted vessel remodeling and tumor invasion.

Overall, our model reproduced the structural and molecular complexity of human lung microvasculature enabling mechanistic dissection of tumor-driven vascular remodeling and invasion.

Acknowledgements: This work is funded by national funds through the Portuguese Foundation for Science and Technology (FCT) under the CEEC Institutional (CEECINSTLA/00012/2022) (D.C) and Individual (2022.07521.CEECIND/CP1718/CT0010) (A.C.L.) programs, and the IC&DT projects 2MATCH (PTDC/BTM-ORG/28070/2017) and RECOVER (2022.02260.PTDC). 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). Authors thank the “TERM RES Hub–Scientific Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190).

1016. Engineering the cell-matrix interface to probe and direct tissue regeneration

Loebel, C.^{1 2}, Ahmed, D.³, Liu, J.^{1 2}

¹Department of Bioengineering, University of Pennsylvania, ² Center for Precision Engineering for Health (CPE4H), University of Pennsylvania, ³ Department of Biomedical Engineering, University of Michigan

Cells do not simply reside within the extracellular matrix (ECM); they actively build, remodel, and interpret it. Across development, regeneration, and disease, this dynamic reciprocity at the cell - matrix interface governs tissue function. Yet, much of our understanding of ECM biology has overlooked the newly synthesized, nascent ECM that cells assemble at their immediate interface and experience first. In this talk, I will discuss engineering approaches to reveal the origins, composition, and function of nascent ECM and why this transient matrix is a critical regulator of cell fate and tissue outcomes.

I will first highlight recent in vivo work on epithelial cells as active and early contributors to fibrotic matrix remodeling following lung injury. Using lineage-specific visualization of newly secreted ECM, we show that epithelial transitional cells deposit collagen- and laminin-rich matrices within days of injury, well before fibrotic remodeling. These findings challenge the traditional view that fibroblasts are the primary contributors to fibrotic ECM and suggest that early epithelial-derived matrices may prime fibrotic remodeling.

I will then transition to engineered in vitro systems that allow us to systematically dissect how biomaterial properties shape nascent ECM deposition and how this process feeds back to regulate cell fate. Using chemically defined hydrogels, we demonstrate that subtle changes in polymer chemistry - independent of bulk mechanics - alter nascent ECM composition and organization, which directs lineage-specific cell fate. These results suggest that nascent ECM acts as an essential intermediary between engineered materials and biological responses.

I will conclude by discussing our ongoing studies on how nascent ECM may be engineered to probe tissue regeneration and to design more predictive biomaterials for translational applications.

Disclosure of Conflicts of Interest: The authors have no conflicts of interest.

1017. In silico modelling of engineered cells

S.L.Waters¹

¹Mathematical Institute, Radcliffe Observatory Quarter, University of Oxford, Oxford

Cell therapies in regenerative medicine hold enormous potential for restoring, replacing, or regenerating damaged tissues and organs by delivering living cells that directly target the biological origins of disease or injury. The safe delivery of these therapies, minimisation of immunogenicity, and optimisation of cell engraftment and function at the target site, are critical to their clinical success. Next generation cell therapies that encapsulate cells with biocompatible and biodegradable immunoprotective micromatrices, enabling modulation of cell behaviour, genotype and phenotype, offer exciting opportunities to tailor therapies to specific clinical challenges. However, the optimsation of such advanced therapies requires a detailed understanding of the complex interplay between the delivered cells and their surrounding biomechanical and biochemical environment, as well as the influence of encapsulation on these interactions. Mechanistic mathematical modelling, integrated with in vitro and in vivo experimental approaches, provides a powerful framework for gaining fundamental insights into these complex biological systems. In this talk, we will showcase how mathematical modelling can be used to elucidate the biomechanical cues experienced by engineered cells as they transit through the vasculature to sites of injury. We will further discuss how insights from such in silico approaches can generate experimentally testable hypotheses leading to new treatment strategies, optimise cell therapy protocols, and ultimately support the successful translation of novel cell therapies from bench to bedside.

1018. Biomaterials advances and engineering approaches for osteochondral tissue engineering scaffolding

Oliveira J.M.^{1 2}

¹ 3B'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, ² ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal.

In this invited lecture, the fundamentals and latest developments in field of natural-based biomaterials for osteochondral (OC) tissue engineering and regenerative medicine will be presented and discussed in depth. The biofabrication and biodesign approaches, traditional and advanced, used in the processing of multiscale and hiearchical scaffolds will be overviewed. It will follow a disucssion of the main key concepts, principles and challenges involving the interplay between biomaterials and cells in OC tissue regeneration. Several examples of the significant contributions arising from Dr. Oliveira team (I3Bs-UMINHO) will be highlighted and serve as references. Lastly, the progress on in vitro 3D OC tissue models that have been used in the preclinical stage as reliable alternatives to 2D in vitro cell culture methods and in vivo models will be also presented. Some new avenues of research in osteochondral tissue engineering will be also proposed.

The author acknowledges the funding obtained through the projects EngVIPO and RENOVATE funded by the European Union under Grant Agreements 101183041 and 101134024, respectively.

1019. Combining tissue bioengineering and extracellular vesicles in a one health perspective

Tiziana Brevini

de Department of Veterinary Medicine and Animal Science. UNIVERSITA' DEGLI STUDI DI MILANO, LODI (Italia) - Italy

Tissue bioengineering and extracellular vesicles (EVs) represent complementary strategies in regenerative medicine, with broad implications for a One Health framework encompassing human, animal, and environmental health. Over the past two decades, advances in tissue bioengineering have enabled the development of three-dimensional (3D) scaffolds that, not only recapitulate key structural and functional aspects of native tissues, but also provide controlled microenvironments to study cell behavior, tissue function, and disease progression. Such platforms are increasingly used to model organ-specific physiology, investigate pathological mechanisms, and screen therapeutic interventions in a more predictive and ethically sustainable manner, reducing reliance on in vivo models. In parallel, EVs—lipid-bound nanoparticles carrying proteins, lipids, and nucleic acids—have been recognized as critical mediators of intercellular communication, capable of modulating immune responses, tissue repair, and regenerative pathways. EVs exhibit remarkable stability, biocompatibility, and the ability to cross biological barriers, positioning them as promising bioactive agents for both human and veterinary applications. This presentation will discuss the integration of EVs into 3D tissue culture systems, aiming at improved regenerative outcomes, through the generation of platforms that investigate EV-mediated mechanisms in physiologically relevant contexts, to optimize therapeutic strategies for tissue repair. Overall, the synergistic application of EVs and bioengineered tissues provides a robust translational framework, bridging fundamental biology with clinical and veterinary applications, and exemplifies the potential of One Health-oriented strategies in regenerative medicine.

1020. 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.

1021. From heat to insight: tracking bacterial behavior on scaffolds with isothermal microcalorimetry

Carmen Alvarez-Lorenzo

de Dept. Pharmacology, Pharmacy and Pharmaceutical Tecnology. Santiago de Compostela University (USC), Santiago de Compostela (A Coruña) - Spain

In the field of scaffolds for regenerative medicine, two pressing challenges remain unresolved: (a) how to prevent bacterial colonization on a scaffold immediately after implantation, and (b) how to effectively intervene when infection is already established, as in chronic wounds that frequently harbor persistent biofilms. In the era of increasing antimicrobial resistance, the development of strategies to both prevent and eradicate biofilm formation is essential. However, designing materials capable of resisting or counteracting bacterial colonization requires advanced analytical tools that can sensitively and reliably characterize scaffold–bacteria interactions.

Isothermal microcalorimetry (IMC) emerges as a powerful technique within this context. By enabling continuous, real-time monitoring of metabolic heat flow at constant temperature, IMC offers significant advantages over conventional end-point microbiological assays. The method is non-destructive, highly sensitive, and suitable for studying a wide variety of biomaterials. Moreover, IMC can capture the dynamic evolution of material properties in biorelevant environments and provide quantitative insights into interactions with both human and microbial cells [1].

This presentation will highlight recent advances in applying IMC to the development and evaluation of scaffolds engineered with antimicrobial or antibiofilm functionalities [2,3]. Several representative case studies will illustrate how IMC can guide material optimization, assess antimicrobial performance, and support the design of next-generation scaffolds for infection-resilient regenerative therapies.

Acknowledgments: Work supported by Spain Ministerio de Ciencia, Innovación y Universidades MICIU/AEI/10.13039/501100011033 [PID2023-150422OB-I00], ERDF A way of making Europe, cofunded by the European Union, and Xunta de Galicia [ED431C 2024/09].

References

[1] C. Alvarez-Lorenzo, A. Concheiro, Adv. Colloid Interf. Sci. 346, 103681 (2025).

[2] X. Farto-Vaamonde, L. Diaz-Gomez, A. Parga, A. Otero, A. Concheiro, C. Alvarez-Lorenzo, J. Control. Release 352, 776-792 (2022).

[3] N.F. Virzi, P. Diaz-Rodriguez, A. Concheiro, A. Otero, A. Mazzaglia, V. Pittala, C. Alvarez-Lorenzo, Carbohydr. Polym. 351, 123069 (2025).

1022. Disclosing inflammation in intervertebral disc: lessons from novel in vitro models

Raquel Goncalves

de i3S, Institute of Research and Innovation in Health. University of Porto, Oporto (Porto) - Portugal

Intervertebral disc (IVD) degeneration is a major contributor to chronic low back pain, a leading cause of disability worldwide. Inflammation plays a pivotal role in the onset and progression of disc degeneration by disrupting the homeostasis of disc cells and extracellular matrix (ECM) components. Pro-inflammatory cytokines, namely interleukin(IL)-1β mediate IVD catabolic cascades that enhance metalloproteinases (MMPs) activity, leading to ECM deregulation including proteoglycans loss and collagen degradation, which then weaken the structural and mechanical integrity of the disc, accelerating degeneration [1].

In vitro models have become essential tools for investigating the molecular mechanisms underlying disc inflammation and for evaluating potential therapeutic interventions. Organ culture models can preserve disc architecture and ECM organization, essential to enable the study of tissue-level responses to inflammatory stimuli under controlled conditions [2]. Three-dimensional (3D) humanized IVD models can be specifically designed to better mimic the physiological matrix and cell–matrix interactions, allowing for more representative inflammatory responses [3].

Co-culture models incorporating immune cells, models that recreate native ECM of the IVD, or thigh control of inflammatory conditions, enable dynamic monitoring of cell behavior and intercellular communication in the IVD. These models may contribute to a deeper understanding of the complex interplay between inflammation and IVD degeneration, supporting the development of targeted therapies aimed at restoring disc homeostasis, or constituting a platform for preclinical screening of anti-inflammatory and regenerative therapies. Overall these models can offer high reproducibility, ethical advantages, and contribute to the progress of managing IVD diseases.

References: [1] Molinos M et al. doi: 10.1098/rsif.2014.1191; [2] Teixeira GQ et al. doi:10.1089/ten.tec.2015.0195; [3] Castro AL et al. doi: 10.1016/j.actbio.2025.01.060.

Acknowledgements: Alexander Humboldt Foundation and Portuguese Foundation for Science and Technology (Project IMMUNOSECRET).

1023. Rationally tuned hyaluronic acid-based hydrogels as extracellular matrix mimics for central nervous system organoid culture

Anna Vilche¹, Francisco M. Saez¹, J. Alberto Ortega², Oscar Castaño³, Zaida Alvarez¹

¹Biomaterials for Neural Regeneration. 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 Electronic and biomedical engineering. University of Barcelona (UB), Barcelona - Spain

The complex organization of the central nervous system (CNS) makes it challenging to reproduce its extracellular matrix (ECM) and cellular interactions in vitro, limiting the generation of physiologically relevant neural models. To address this, we developed a rationally tuned library of ECM-mimetic bioinks that reproduce the biochemical and mechanical features of the native CNS matrix. This approach provides defined and customizable materials to support brain organoid growth and maturation, eliminating dependence on commercial matrices such as Matrigel.

A library of hyaluronic acid (HA)-based hydrogels with variable molecular weights was synthesized and functionalized at hydroxyl groups with methacrylic or carbic anhydride (norbornene). These reactive groups enable covalent crosslinking and precise control over porosity, stiffness, and degradation kinetics. The incorporation of PEG chains of different molecular weights further tuned the morphology and rheological properties of the hydrogels. In addition, specific ECM proteins were integrated into selected hydrogel formulations to provide biochemical cues that guide neural cell adhesion, differentiation, and maturation within the 3D environment. Bioinks were then tested with human brain organoids to evaluate cell integration and maturation over time.

FTIR and NMR confirmed successful HA functionalization with methacrylic and norbornene groups. Rheological analysis showed mechanical moduli within the physiological range of CNS tissue. SEM imaging revealed distinct and tunable microarchitectures, while swelling assays demonstrated strong hydrophilicity and diffusion capacity. When combined with human brain organoids, the bioinks promoted robust neural cell integration, sustained viability, and progressive maturation during long-term culture, maintaining structural integrity throughout. The rational tuning of composition, mechanics, and biochemical cues enables reproducible, ECM-like microenvironments optimized for neural development and study.

PEG-tuned, HA-based bioinks with controlled crosslinking, architecture, and ECM protein incorporation effectively replicate CNS-like mechanical and biochemical properties, supporting brain organoid growth and maturation in vitro. These results highlight the potential of rationally tuned ECM-mimetic bioinks as a versatile platform.

We acknowledge EIC-2021-PATHFINDER-OPEN-01-01-101047099 4DBR), Grant P199, RTI2018-097038-B-C22, PID2021-124575OB-I00, R01AG086270.

1024.  

1025. Engineering a novel in vitro polyhydroxyalkanoate (PHA) based lung cancer model with dynamic perfusion

Samruddhi Mujumdar¹, Gwendolen Reily², Ipsita Roy²

¹Department of Chemical Biological and Material Engineering. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom, ²Department of Chemical, Biological and Material Engineering. University of Sheffield, Sheffield (South Yorkshire) - United Kingdom

Lung cancer is a leading cause of global mortality due to the highly heterogeneous and dynamic tumour microenvironment (TME) across patients. In vitro models have been instrumental in both the disease pathophysiology and treatment landscapes. However traditional models are unable to replicate the complexities of the TME limiting their translatability. Newer generation models need to better replicate physiological and biochemical properties in vivo to improve disease relevant phenotypes.

This work involves the use of the highly biocompatible and sustainable medium chain length polyhydroxyalkanoate (mcl PHA)-based ink for 3D printing to generate highly reproducible 3D models that better align to native tissue characteristics. Combined with the use of the A549 human lung adenocarcinoma cell line cultured in a dynamic air to liquid perfusion system this novel platform aims to provide more control over spatial organisations, thereby improving both cell-cell interactions and cell phenotypes.

3D mcl-PHA scaffolds were analysed through SEM and optical microscopy as well as tensile testing to establish compliance to lung tissue. Cytocompatibility using 2D static condition was assessed using the A549 cell line and solvent cast mcl-PHA films. Viability, functionality, and metabolic activity were assessed using LDH cytotoxicity, ELISA and Alamar Blue assays respectively. Further assessments of functionality was assessed using lung specific antibodies and immunofluorescence.

The results obtained show that at a 2D static level, using solvent cast films, cell viability measured using Alamar Blue assays and LDH cytotoxicity assays confirm that mcl PHA performs comparable to TCP and collagen coated samples. Additionally, functionality was measured using an E-cadherin ELISA and results indicated good epithelial barrier formation and immunofluorescence using the MUC5B, CK18 and P63 markers show evidence of mucus formation and metastatic potential respectively. Further the 3D printed models show improved spatial and mechanical properties that better align with the lung tissue and can be made to a range of dimensions with high reproducibility for high throughput applications. All results show a marked improvement through the induction of dynamic perfusion yielding patterns more consistent with in vivo behaviour.

This platform thus provides a versatile, reproducible, and human-relevant system for mechanistic studies of lung cancer progression, microenvironmental interactions.

1026. Dual-functional polydopamine spheres loaded with antimicrobial peptides to support bone regeneration in infected conditions

Laia Moliner-Carrillo¹, Giovanni Ferrandi¹, Carles Mas-Moruno¹, Maria-Pau Ginebra¹, Anna Diez-Escudero¹, Maria Godoy-Gallardo¹

¹Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering and Institute for Research and Innovation in Health (IRIS), Universitat Politècnica de Catalunya - BarcelonaTech, Barcelona - Spain

Introduction

Implant-related infections and impaired osseointegration remain major limitations in bone regenerative therapies. Although polydopamine (PDA) coatings are widely used, their application as multifunctional carriers have been scarcely explored. Here, we developed a multifunctional platform based on PDA spheres functionalized with antimicrobial peptides (AMPs; Lf1-11 and 4Dab-13) to simultaneously provide antibacterial activity and promote osteogenesis. To better mimic clinical scenarios, a cell–bacteria co-culture model was implemented, enabling evaluation interactions between hosts, pathogens and materials under infection conditions.

Methods

PDA spheres were synthesized via dopamine self-polymerization and covalently functionalized with AMPs. The platforms were immobilized onto biomaterial substrates to generate active surfaces. Surface morphology and chemistry were analysed using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Antibacterial activity against S. aureus and P. aeruginosa was evaluated through adhesion assays, metabolic activity measurements, colony-forming unit (CFU) counting, and live/dead staining. Human mesenchymal stem cells (hMSCs) were cultured on modified and control surfaces under standard conditions and in co-culture with bacteria to simulate early-stage implant infection. Cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and osteogenic gene expression (RUNX2, ALP, OCN) were assessed.

Results

PDA–AMP spheres were homogeneously distributed, increasing surface roughness and bioactivity. Functionalized surfaces significantly reduced bacterial adhesion and viability. Under co-culture conditions, the PDA–AMP platform protected hMSCs from bacterial induced damage, preserving viability and morphology. Additionally, PDA–AMP modification enhanced hMSC adhesion and proliferation and promoted osteogenic differentiation, evidenced by increased ALP activity and upregulation of osteogenic markers compared to unmodified surfaces.

Conclusion

PDA–AMP spheres create a dual-function interface capable of suppressing bacterial colonization while enhancing osteogenesis, even under infection mimicking conditions. This multifunctional strategy represents a promising approach for next-generation antibacterial and osteoinductive implants.

Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation (PID2023-148538OB-I00 and RYC2022-038428-I).

1027. A 3D-printed human spinal cord organoid-on-a-chip for modelling neural injury and repair

Palash Chandravanshi¹, Dakota Coloroso¹, Marta Cuenca¹, Marco Fusco², Susana Rodriguez Gonzalez¹, Eloi Montañez³, J. Alberto Ortega⁴, Zaida Álvarez¹

¹Biomaterials for Neural Regeneration. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, ²Biomaterials for Neural Regeneration. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, ³Institut d'Investigació Biomèdica de Bellvitge, Barcelona - Spain, ⁴Department of Physiological Sciences, Faculty of Medicine and Health Sciences. University of Barcelona, Barcelona - Spain

Objectives

Spinal cord injury (SCI) results in severe and often permanent neurological deficits, yet translation of preclinical research into effective human therapies remains limited.1 Current in vitro models lack the structural and functional complexity needed to study human-specific injury responses.1 This study aims to develop a human-relevant, 3D-printed spinal cord organoid-on-a-chip (SCO-on-a-chip) platform to model SCI with high reproducibility, scalability, and compatibility for multimodal analyses.

Methods

Human-induced pluripotent stem cell (iPSC)-derived spinal cord organoids (SCOs) were grown in an optimized 3D-printed organ-on-a-chip device that allows for geometric confinement. The platform's design was optimized using COMSOL Multiphysics simulations to refine fluid dynamics and minimize shear stress on the organoids. Multiple biocompatible resins were assessed for printability, mechanical stability, and neural cytocompatibility. The optimal device, fabricated using stereolithography (SLA), incorporated a glass-bottom base for live imaging and removable chambers for electrophysiological studies and imaging. Furthermore, a precisely engineered lid allowed controlled mechanical impact using automated impactor to induce reproducible SCI.

Results

The optimized SCO-on-a-chip supported stable long-term culture of up to 48 organoids per six-well plate, maintaining neuronal viability and cytoarchitecture. Controlled mechanical impact produced localized and reproducible injury, enabling quantitative assessment of neuroinflammatory responses, glial scar formation, and early regenerative activity. Imaging and electrophysiological readouts showed injury-associated signatures consistent with patterns described in established in vivo SCI models.

Conclusions

The 3D-printed SCO-on-a-chip is a scalable, physiologically relevant platform for modelling human spinal cord injury. Its modular design allows semi–high-throughput culture and simultaneous analysis of multiple organoids. By combining human iPSC-derived SCOs with a precision mechanical impactor for controlled contusion in the device, it bridges the gap between traditional in vitro systems and the structural and functional complexity of the human spinal cord. Continued optimization will help establish its use in therapeutic screening, including testing biomaterials designed to enhance repair and regeneration after SCI.

Reference

[1] Omelchenko A, 10.1016/j.cobme.2020.05.002

1028. Ensuring safe clinical translation of decellularized sural nerve grafts through structured risk mitigation

Cristina Castells-Sala¹, Laia Ruiz-Ponsell¹, Núria Nieto-Nicolau¹, Patricia Lopez-Chicon¹, Esther Udina², Xavier Navarro², Raquel Bermudo¹, Paula Muñoz Tomas¹, Diego Fernandez Alvarez¹, Enric Tarragona Carretero¹, Roger Oliva Palau¹, Andrés Savio¹, Jaime Tabera¹, Oscar Fariñas¹, Anna Vilarrodona¹

¹Banc de Teixits. Banc de Sang i Teixits, Barcelona - Spain, ²Institute of Neurosciences, DeptCell Biology, Physiology and Immunology. Universitat Autonoma de Barcelona, Barcelona - Spain

Background

Peripheral nerve injuries represent a significant cause of permanent disability in adults. Autografts remain the clinical gold standard for repairing large gaps, but their use is limited by donor site morbidity and restricted availability. Decellularized nerve scaffolds represent a promising alternative for the development of clinically applicable nerve substitutes. However, to enable their safe and reproducible use, systematic characterization and validation are essential.

Methods

A novel preparation method for decellularizing small-calibre peripheral nerves was evaluated through a structured validation pipeline following EuroGTP-II quality and safety principles. The process included an initial risk identification phase followed by a set of targeted in vitro studies designed to assess scaffold quality, efficacy of decellularization, and biosafety. To support translational feasibility, the resulting grafts were subsequently tested in a large-animal model.

Results

The EuroGTP-II risk assessment indicated an initial medium risk, which was subsequently lowered through in vitro characterization demonstrating preservation of extracellular matrix architecture, as confirmed by mechanical testing, histology, and protein analysis. Residual DNA showed a >99% reduction, supporting the high efficiency of the decellularization process. Microbiological and cytotoxicity assays verified sterility and the absence of adverse cellular responses, leading to a substantial reduction in overall risk.

To complement these findings, and further reduce the risk, decellularized grafts were implanted in an ovine model bridging a 5-cm nerve defect. After 9 months, the grafts supported robust axonal regeneration, with no safety concerns detected. The combined results markedly reduced the initial uncertainty associated with the new preparation method and supported its suitability for controlled clinical evaluation.

Conclusions

This work establishes a comprehensive validation framework for small-calibre decellularized nerve grafts, demonstrating their structural integrity, biosafety, and regenerative potential. The robust preclinical evidence provides a solid foundation for advancing these scaffolds to a randomized controlled clinical trial in patients with monofascicular peripheral nerve injuries.

1029. Autologous ECFC and MSC combinations from chronic spinal cord injury patients enable robust vascular regeneration

Angela Santos De La Mata¹, Mario Martínez Torija¹, Francisco J. Espino Rodríguez², María A. Ruiz De Infante¹, Matilde Castillo Hermoso¹, Pedro F. Esteban Ruiz³, Eduardo Molina Holgado³, Rafael Moreno Luna¹

¹Pathophysiology and Regenerative Medicine Group. Hospital Nacional de Parapléjicos, IDISCAM, SESCAM, Toledo - Spain, ²Plastic and Reconstructive Surgery Service. Hospital Nacional de Parapléjicos, IDISCAM, SESCAM, Toledo - Spain, ³Neuroinflammation Group. Hospital Nacional de Parapléjicos, IDISCAM, SESCAM, Toledo - Spain

Chronic spinal cord injury (SCI) is linked to adiposopathy and vascular dysfunction. Adipose tissue harbors mesenchymal stem cells (MSCs) and endothelial colony forming cells (ECFCs) that are being explored for tissue regeneration. To determine whether these cells remain functional despite chronic injury, we compared the angiogenic and vasculogenic potential of ECFCs and MSCs isolated from SCI patients and healthy donors.

We collected subcutaneous adipose tissue from 15 SCI patients with pressure injuries and 15 age and sex matched controls. After enzymatic digestion, ECFCs (CD31⁺CD90^-CD45^-) and MSCs (CD90⁺CD31^-CD45^-) were purified by magnetic separation and expanded separately. We assessed ECFC clonogenicity, migration and TNF α responsiveness, and evaluated MSC trilineage differentiation. To test vasculogenic potential in vivo, ECFCs and MSCs were mixed in a 40:60 ratio, embedded in Matrigel and implanted subcutaneously into immunodeficient mice; perfused microvessels were quantified after seven days.

Both cell types were successfully isolated from all samples. Expanded cell cultures exceeded 98 % purity and cryopreservation viability was above 95 %. ECFCs from SCI patients and controls showed comparable morphology and expression of CD31 and von Willebrand factor, maintained clonogenic growth and migrated in response to TNF α. MSCs retained CD90/CD73 expression and differentiated into osteogenic, chondrogenic and adipogenic lineages across groups. Although SCI derived cells showed slight reductions in some functional assays, these differences were not statistically significant. In vivo, ECFC/MSC co implantation produced networks of perfused microvessels in every implant, with no differences between patient and control groups.

These results demonstrate that ECFCs and MSCs from chronic SCI patients maintain their phenotype, proliferative capacity, and vasculogenic function. Thus, subcutaneous adipose tissue from SCI patients provides a viable autologous source for angiogenic and vasculogenic therapies targeting chronic wound repair and tissue regeneration. This study was supported by grant SBPLY/23/180225/000083, funded by ERDF/EU and the Regional Government of Castilla-La Mancha (JCCM) through the INNOCAM program.

1030. 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.

1031. Development of a 3D bioprinted tumour-on-chip platform for modeling gastrointestinal cancer migration and invasion

Adrian Garcia¹, Lia Jove², Maria Pereira², Angelica Figueroa², Daniel Nieto¹

¹Advanced Biofabrication Laboratory - DNIETO LAB. CICA - Centro interdisciplinar de Química e Bioloxía, A Coruña - Spain, ²Epithelial Plasticity and Metastasis Group. Instituto de Innvestigación Biomédica (INIBIC), A Coruña - Spain

Understanding how tumour cells migrate and invade surrounding tissues is fundamental to identify the early mechanisms of metastasis, a process responsible for the majority of cancer-related deaths. Gastrointestinal (GI) tumours, in particular, exhibit highly invasive behaviour influenced by both biochemical and mechanical factors within their environment. However, conventional two-dimensional (2D) cultures are unable to reproduce the spatial organisation and dynamic cell-matrix interactions that define native tissues. In contrast, while the use of animal models is informative, there are significant financial and time implications as well as ethical considerations. Consequently, there is a growing demand for versatile, rapid, and reproducible platforms capable of mimicking tissue architecture and cell behaviour under well-controlled experimental conditions.

Here, we present a customizable 3D bioprinted tumour-on-a-chip system designed to bridge the precision of bioprinting with the environmental control of microfluidics for GI cancer modelling. The device is fabricated using LCD-based 3D printing with a biocompatible resin, enabling fast redesign and adaptation to different experimental needs. Within its central chamber, digital light processing (DLP) bioprinting allows the in situ generation of complex, multicellular constructs that recapitulate the biochemical composition and structural organization of the tumour extracellular matrix. The bioink formulation, combining photocrosslinkable and biologically active hydrogel components, provides a tunable environment that maintains cell viability and supports interaction and migration, making it an attractive material for studying dynamic cancer processes.

This system enables the controlled reconstruction of tumour–endothelium interfaces and facilitates the study of cell migration, invasion, and vascular integration within physiologically relevant 3D contexts. Its modular design, accessibility and compatibility make it a flexible tool for observation of tumour progression, intravasation processes, and the early stages of the metastatic cascade under physiologically relevant flow conditions. Overall, this platform establishes a next-generation approach to tumour modelling, combining the precision of bioprinting with the versatility of microfluidics to accelerate the development of personalised and predictive cancer research models.

1032. 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.

1033. 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.

1034. 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.

1035. 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.

1036. Engineering regenerative niches for intestinal therapy via encapsulation of iPSC-derived mesenchymal stromal cells in synthetic degradable microgels

Pablo Rodríguez-Alonso¹, Ana Mora-Boza², Junqiao Lyu¹, Zaki Ahmedin³, Alan Y. Liu⁴, Andrés J. García³, Abhay Pandit¹

¹CÚRAM, Research Ireland Centre for Medical Devices, Biomedical Sciences. University of Galway, Galway - Ireland, ²School of Life Sciences. Technical University Munich, Freising (Baden-Wberg Bayern) - Germany, ³George W. Woodruff School of Mechanical Engineering. Georgia Institute of Technology, Atlanta (Georgia) - United States, ⁴Optics11 Inc., Boston (Massachusetts) - United States

Aims and objectives

Intestinal Mesenchymal Stromal Cells (IMSCs) are structural stem cells that promote epithelial regeneration by modulating Wnt/BMP signalling and immune tolerance, making them attractive for intestinal therapy[1]. We assessed whether encapsulating IMSCs derived from human induced pluripotent stromal cell-derived human intestinal organoids (HIOs) in four-arm polyethylene glycol norbornene (PEG-4aNB) microgels modulate their secretome through microcarrier mechanics to enhance epithelial regeneration.

Materials and Methods

Induced Pluripotent Stem Cell-derived HIOs were developed using Matrigel^® then, dissociated into single cells after day 21 and sorted by Magnetic-Activated Cell Sorting into EpCAM+ (Epithelial) and IMSCs followed by immunostaining characterization. IMSCs were encapsulated in PEG-4aNB microgels within a flow-focusing microfluidic device. PEG-4aNB microgels were fabricated at different concentrations bearing a protease-sensitive linker and RGD cell adhesion peptide[2]. Secretome from naïve and IFN-γ-stimulated IMSCs was quantified by flow cytometry using LEGENDplex™. Conditioned media were then applied to HIOs for RNA-seq analysis.

Results

EpCAM⁺ HIOs displayed heterogeneous epithelial lineages (EpCAM+/MUC2+/LYZ+/CHGA+/MUC17+/FBP2+), whereas IMSCs expressed a Col-I/Vimentin/α-SMA. PEG-4aNB microgels (6, 8, and 10 wt%) were fabricated, showing effective Young’s moduli of 78.3±21.0, 158.3±68.1, and 301.8±53.5Pa, respectively with a size of ∼350µm and supporting IMSCs viability over 21 days for all formulations.

IFN-γ increased inflammatory cytokine secretion, mitigated by higher stiffness. RNAseq of HIOs treated with IMSC-conditioned media revealed a pro-regenerative signature associated with epithelial proliferation and stemness, while modulating immune-related processes. Data deconvolution using XCell2, indicated a higher enrichment of Transit-Amplifying cells in HIOs exposed to secretomes from IFN-γ-conditioned microgels, positively correlated with increasing stiffness.

Conclusion

PEG-4NB microgels stand as an effective system for stem cell-based intestinal therapies. IMSCs remained highly viable in microgels, with stiffness mitigating proinflammatory responses. Their secretome induced a stiffness-dependent enhancement of epithelial regeneration, demonstrating the potential of HIOs for regenerative medicine.

[1] J.Chen et al.,Stem CellResTher,2024

[2] A.Mora-Boza et al.,AdvHealthcMater,2023

1037. Cardiac organoids: novel regenerative therapy for myocardial infarction

Maria Kalil¹, Sarkawt Hamad², Daina Martínez-Falguera¹, Ebru Aksoy², Albert Teis³, Júlia Aranyó³, Esther Jorge¹, Georgina Iraola-Picornell¹, Felipe Bisbal³, Antoni Bayés-Genís³, Kurt Pfannkuche², Carolina Gálvez-Montón¹

¹ICREC. Germans Trias i Pujol Research Institute (IGTP), Badalona (Barcelona) - Spain, ²Marga-and-Walter-Boll-Laboratory for Cardiac Tissue Engineering, köln (Nordrhein-Westfalen) - Germany, ³iCor. Hospital Universitari Germans Trias i Pujol, Badalona (Barcelona) - Spain

Introduction

Cell therapy for acute myocardial infarction (AMI) faces two key challenges: cellular integration and post-transplantation arrhythmias. This study aimed to develop a scalable platform for producing human cardiac organoids (COs) and to evaluate their safety and efficacy in a translational swine AMI model.

Methods

Human induced pluripotent stem cell–derived COs were generated using a single, scalable bioreactor workflow. Twelve immunosuppressed pigs underwent AMI induction and were randomized into Short-term CO (8-day follow-up, n=2), Long-term Control (30-day follow-up, n=4), and Long-term CO (30-day follow-up, n=6) groups. Animals received 3500 COs (CO groups) or Plasmalyte (control) via 5–6 intramyocardial injections 30 minutes post-AMI. CO engraftment in CO groups was assessed by immunohistofluorescence (IF) and spatial transcriptomics. Additionally, in long-term groups, heart rhythm was monitored for 15 days using ECG Holter recording; cardiac function and scar size were evaluated by magnetic resonance (MRI) at days 2 and 29; and electrophysiologic properties and arrhythmia inducibility at day 30 by high-density mapping (HDM) and programmed electrical stimulation, respectively.

Results

IF confirmed human nuclear antigen–positive cells within infarct and border zones at days 8 and 30, demonstrating CO survival. Spatial transcriptomics showed presence of human cells with reduced profibrotic gene expression at day 30 versus day 8. All animals maintained sinus rhythm without major ventricular arrhythmias. In the Long-term CO group, significant improvements in left ventricular (LV) stroke volume, LV ejection fraction, cardiac output, and cardiac index, along with smaller scar size were noticed while these changes were not observed in controls. HDM did not reveal electrophysiologic differences between CO or Control animals.

Conclusions

Our bioreactor platform enables highly scalable and reproducible CO production. Following implantation, COs integrated robustly into the host myocardium, maintained long-term viability, and did not elicit severe arrhythmias. Early findings pointed toward meaningful improvements in cardiac function and scar reduction, and larger ongoing studies are expected to further substantiate their therapeutic potential.

1038. 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.

1039. Bioactive nanoparticles combined with antibiotic and mucolytic: an immunomodulatory strategy against implant-associated infections

Isabel Izquierdo-Barba¹, Alberto Polo Montalvo², Monica Cicuendez², Daniel Arcos³

¹Química en Ciencias Farmaceuticas. UCM. CIBER de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Spain, Madrid - Spain, ²Química en Ciencias Farmaceuticas.UCM. Universidad Complutense de Madrid, Madrid - Spain, ³Química en Ciencias Farmaceuticas UCM. CIBER de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Spain, Madrid - Spain

Implant-associated infections represent a significant clinical challenge due to the severity of their consequences, which include persistent inflammation, bone loss, and potential implant failure, despite their relatively low incidence in most procedures. This problem has driven the development of therapeutic strategies capable of combating infection and, simultaneously, promoting bone regeneration. In this context, immunomodulation emerges as a promising approach, especially given the central role of macrophages in pathogen response, inflammation resolution, and tissue repair [1]. Currently, our research group has demonstrated that nanoparticles based on bioactive mesoporous glasses (MBGNs) display remarkable potential in the treatment of implant-associated infections when they combine the therapeutic action of an antibiotic (levofloxacin) with the biofilm-disrupting capacity of the mucolytic agent N-acetylcysteine. Previous studies have shown that N-acetylcysteine not only enhances biofilm dispersion but can also exert an immunomodulatory effect. Herein, we investigate the effects of these MBGNs—loaded with levofloxacin and covalently functionalised with N-acetylcysteine—on macrophages at different time points. Internalization assessed by transmission electron microscopy, cell viability, phenotype activation, and cytokine production were evaluated. The results show that these nanosystems are efficiently internalized by macrophages, preserve cell viability, and are capable of modulating macrophage activation toward phenotypes associated with both effective antimicrobial response and tissue repair, accompanied by a cytokine profile consistent with a balanced immunomodulatory effect. These findings highlight the promising potential of these nanosystems for the treatment of implant-associated infections.

1. Chen, Z., Wang, Y., Liu, X. y col. Harnessing osteoimmunity to treat peri-implant inflammatory osteolysis. Materials Advances. 2024; 5: 769-783. DOI:10.1039/D3MA00733B.

2. Polo-Montalvo, A.; Gómez-Cerezo, N.; Cicuéndez, M.; González, B.; Izquierdo-Barba, I.; Arcos, D. Osteogenic and Antibacterial Response of Levofloxacin-Loaded Mesoporous Nanoparticles Functionalized with N-Acetylcysteine. Pharmaceutics 2025, 17, 519. https://doi.org/10.3390/pharmaceutics17040519

Acknowledgements: Spanish Ministerio de Ciencia e Innovación (PID2023-149093OB-I00, MAGEN4BONE) and Fundación Ramón Areces (FD5/22_01, Nano4Infection) for funding

1040. Exploring cellular interactions in inflammatory bowel disease using an immunocompetent 3D hydrogel model

María García-Díaz¹, Anna Vila¹, Núria Torras¹, David Bartolomé¹, Aitor Otero¹, Elena Martínez¹

¹Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain

Inflammatory bowel disease is a complex, multifactorial condition characterized by chronic inflammation of the gastrointestinal tract, which significantly affects patients’ quality of life. During inflammation, the intestinal epithelial barrier is compromised and the crosstalk between the stromal cells, immune cells, and epithelial cells is crucial for responding to inflammatory triggers. However, robust immunocompetent in vitro models of the intestinal mucosa that incorporate all these compartments are still scarce. To address this gap, we have developed a hydrogel-based 3D cell culture model of the intestinal mucosa that integrates the epithelial, stromal, and immune compartments, facilitating the study of this cellular crosstalk.

In our model, THP-1 monocytes and CCD-18Co myofibroblasts were encapsulated within a hydrogel co-network of polyethylene glycol diacrylate (PEGDA) and gelatin methacryloyl (GelMA), while Caco-2 cells were seeded on top to form a differentiated epithelial barrier. Inside the hydrogel, the CCD-18Co elongated and interacted with the epithelial cells, promoting the development of the monolayer. Importantly, the encapsulated THP-1 cells spontaneously differentiated into a M2 phenotype in response to the biomaterial, reproducing the main phenotype of intestinal-resident macrophages. This differentiation was significantly enhanced in the presence of the myofibroblasts and/or the epithelial cells, indicating effective paracrine signaling.

Upon induction with DSS and LPS, our model reproduced key features of bowel inflammation, including compromised epithelial barrier integrity and increased secretion of pro-inflammatory cytokines such as TNF-a, IL-8, IL-6 and IL-1b. Notably, treatment with the first-line IBD drug 5-ASA partially reversed these inflammatory markers, demonstrating the potential of this immunocompetent 3D model for IBD drug development.

1041. 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.

1042. Fast transport of soluble antimicrobials by porous meshes enables bacterial eradication in distal locations

Sajad Mohammadi¹, Ana Laura Pereira¹, Alessia Maranesi¹, Chiara Spasiano¹, Aldo Ferrari¹

¹Hylomorph AG, Zurich - Switzerland

Implantable polymeric meshes provide structural support, reinforce soft tissue, and promote regeneration in patients with abdominal wall damage, pelvic prolapse or undergoing breast reconstruction after mastectomy. In addition, meshes are used in combination with soluble drugs to ensure therapeutic protection and mechanical reinforcement.

Cardiac implantable electronic devices (CIEDs) are susceptible to potential complications, including post-implantation infections (1.7-5.6%) and device migration. Absorbable protective envelopes made from polymeric meshes and including a drug eluting component, sustain the local elution of antimicrobial agents and significantly reduce the risk of CIED infection and biofilm formation. In this context, uniform antimicrobial distribution and homogenous elution in the CIED surgical pocket are vital for infection prevention. Herein, we highlight the importance of understanding the interaction mechanisms between soluble molecules and polymeric meshes.

We propose a Multiphysics model that capture the interplay between material microstructure, local tissue environment, and drug transport phenomena. In silico experiments provide mechanistic insights into how polymeric mesh distributes antimicrobial activity. The results are validated experimentally by demonstrating the functional capacity to inhibit and kill bacteria in vitro in distal locations. By connecting computational modeling with biological validation, this work underscores how predictive simulation can accelerate biomaterials innovation and ensure reproducibility in translational applications.

1043. 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.

1044. 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.

1045. Hybrid silk fibroin–GelMA scaffolds as next-generation platforms for vascular tissue engineering

Carlos Sánchez Rumbo¹, Sylvie Ribeiro², David Durán Rey¹, Ricardo Silva Carvalho³, Ricardo Brito Pereira², Senentxu Lanceros Méndez⁴, Francisco Miguel Sánchez Margallo⁵

¹Laparoscopy. CCMIJU, Cáceres - Spain, ²Laboratory of Physics. CF-UM-UP, Physics Centre of Minho and Porto Universities, University of Minho-Campus de Gualtar, Braga, Portugal, Braga - Portugal, ³CEB - Centre of Biological Engineering, University of Minho, Braga - Portugal, ⁴Applications and Nanostructures. BCMaterials, Basque Center for Materials, Applications and Nanostructures, Leioa (Bizkaia) - Spain, ⁵Scientific Director. CCMIJU, Cáceres - Spain

Despite significant progress in cardiovascular tissue engineering, recreating scaffolds that simultaneously mimic native vessels mechanics and support endothelialisation remains a major challenge. In this work, it was developed and comprehensively evaluated hybrid electrospun silk fibroin (SF) membranes functionalized with a GelMA hydrogel layer, combining the mechanical stability of SF with the bioactivity and tunable degradation of GelMA to create a promising platform for vascular graft applications.

Silk fibroin was electrospun into aligned and randomly fibrous membranes, which were subsequently coated with 3% (w/v) photocrosslinkable GelMA to generate hybrid scaffolds. The constructs were characterized by extensive physicochemical analysis, wettability, mechanical behaviour under hydrated and dry conditions, and long-term degradation assays in physiological media. Additionally, cytocompatibility was assessed using indirect cell viability and proliferation assays with human cardiac fibroblasts (HCFs).

Morphological analysis confirmed a uniform and stable GelMA coating, resulting in a three-fold increase in mean fiber diameter. The hydrogel layer significantly enhanced surface hydrophilicity, significantly reducing the water contact angle. Mechanically, GelMA functionalization modulated scaffold stiffness, achieving an additional 11% reduction under hydrated conditions, thus enhancing flexibility toward native vessel compliance. The hybrid scaffolds constructs exhibited a controlled degradation profile, retaining 89% of their mass after 5 weeks. Crucially, the constructs supported robust cytocompatibility, maintained high viability, exceeding 110% at 72 hours, alongside enhanced HCF proliferation, confirming a non-cytotoxic and pro-endothelialisation microenvironment.

In conclusion, SF/GelMA hybrid scaffolds successfully integrate mechanical stability, bioactivity, hydrophilicity and controlled degradation, establishing a multifunctional and clinically relevant platform for vascular tissue engineering. These findings position the system as a strong candidate for future bioactive molecule delivery and next-generation vascular-grafts.

1046. An innovative culture platform enabling decellularization and recellularization of small vessels under complex stimuli

Lucrezia Moro¹, Elia Pederzani¹, Alessia Bolandrina¹, Gianluca Perrucci², Sara Rega², Giorgia Liotti¹, Lucrezia Gilli¹, Gianfranco B. Fiore¹, Monica Soncini¹

¹Department of Electronics, Information and Bioengineering. Politecnico di Milano, Milano (Lombardia) - Italy, ²Centro Cardiologico Monzino, Milano (Lombardia) - Italy

Cardiovascular diseases remain the leading cause of death worldwide. For coronary and peripheral revascularization, autologous conduits are the gold standard but suffer from limited availability and variable quality. Extracellular matrix (ECM)-based grafts offer a promising alternative thanks to their native architecture and remodelling potential, yet challenges related to immunogenicity and mechanical instability still hinder clinical translation. Moreover, preclinical assessment of ECM-based tissue-engineered vascular grafts (TEVGs) relies mainly on animal models, which poorly replicate the complex human hemodynamic [1].

To address these concerns, we developed an innovative integrated platform enabling automated decellularization, recellularization, and fluid-dynamic conditioning of multiple small-caliber grafts under controlled, physiologically relevant conditions. The platform integrates a control unit that manages different actuators to allow perfusion and pulsatility in the samples and a multi-chamber architecture housing the samples themselves. Accessory modules include in-line chemical sensors (pH, O₂) and feedback-based medium/solution exchange protocols, reducing manual intervention and improving process reproducibility.

Bench validation demonstrated stable communication among the main platform actuators and accessory modules, ensuring precise control of automated perfusion and flow-induced stimuli and allowing the system to reliably reproduce the desired hemodynamic conditions. Moreover, preliminary biological tests using rat aortic arch samples confirmed the platform’s suitability for both decellularization and recellularization protocols, resulting in optimal decellularization performance and encouraging early recellularization outcomes. Overall, these results highlight the system’s potential for scalable and standardized TEVG maturation.

The proposed platform represents a versatile and automated preclinical tool for the standardized evaluation of ECM-based tissue-engineered vascular grafts in in vivo-like physiological conditions, bridging the gap between in vitro and in vivo animal testing and enabling reproducible development and validation of vascular grafts.

[1] M. Kimicata, P. Swamykumar, and J. P. Fisher, “Extracellular Matrix for Small-Diameter Vascular Grafts”, Tissue Eng Part A, vol. 26, no. 23–24, pp. 1388–1401, Dec. 2020, doi: 10.1089/ten.tea.2020.0201.

1047. A modular coronary bifurcation bioreactor for controlled endothelial flow studies

Manuel Salinas

de Engineering, Nova Southeastern University, Fort Lauderdale (Florida) - United States

Coronary bifurcations are predilection sites for atherosclerotic plaque due to complex disturbed flow that is difficult to reproduce in vitro. We engineered a similitude-scaled distal-left-main bifurcation bioreactor that combines realistic hemodynamics with an enlarged working section, enabling endothelial tissue experiments and high-content imaging.

An idealized left main–LAD–LCx geometry was created in SolidWorks and scaled using similitude theory to a 13 mm inner-diameter straight segment while preserving physiologic Reynolds and Womersley numbers through appropriate flow-rate and viscosity adjustments. Daughter branch diameters and angles were selected to satisfy Murray’s law and reported coronary bifurcation angles. A transient CFD analysis was used to optimize vessel lengths, branch angles, and outlet boundary conditions so that time-averaged wall shear stress and oscillatory shear index reproduced hallmark coronary features, including low/oscillatory shear on lateral walls and elevated shear at the carina.

Human coronary artery endothelial cells (HCAECs, P3) were expanded in EGM-2, cryopreserved at low passage, and seeded onto substrates mounted in the 13 mm straight segment of the bifurcated platform. After reaching near confluence, monolayers were exposed in the bioreactor under controlled pulsatile flow for 24 h, without rocker-induced motion. Viability was assessed using a LIVE/DEAD assay (Calcein-AM/EthD-1), and junctional integrity was evaluated by VE-Cadherin immunofluorescence with DAPI nuclear counterstain.

Preliminary experiments showed that HCAEC monolayers remained highly viable after 24 h perfusion and that VE-Cadherin exhibited continuous junctional staining under baseline pulsatile flow, demonstrating robust attachment and barrier formation in the scaled bifurcation. This coronary-like bifurcation bioreactor enables controlled studies of endothelial responses to disturbed flow, providing a modular platform for future plaque-mimetic and multilayer tissue-engineered constructs relevant to atherosclerotic disease.

1048. 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.”

1049. Effect of mechanical properties in mimetic hydrogels on cartilage formation

Pablo Martin¹, Dorleta Jiménez De Aberasturi², Ander Abarrategi³

¹Hybrid biofunctional materials. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ²HYBRID BIOFUNCTIONAL MATERIALS. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ³Dpto. Biología Celular e Histología Facultad Medicina y Enfermería. University of the Basque Country (UPV/EHU), Bilbao (Bizkaia) - Spain

Articular cartilage regeneration remains a major clinical and scientific challenge due to the tissue’s avascular nature, low cellularity, and limited intrinsic repair capacity. Current approaches often fail to replicate the biochemical and mechanical properties needed for stable chondrogenic differentiation. This study aimed to design and evaluate a biomimetic hydrogel system capable of supporting cell viability, promoting chondrogenesis, and enhancing the formation of functional cartilage-like tissue. The main objective was to create a hydrogel platform combining methacrylated polymers with decellularized cartilage extracellular matrix (dECM) and cell lines to achieve mechanical and biochemical properties of native cartilage.

A library of varying stiffness hydrogels was fabricated by adjusting polymer concentration of hyaluronic acid methacrylate (HAMA) and gelatin methacrylate (GelMA). Selected hydrogels were combined with porcine decellularized cartilage ECM to enrich biochemical complexity. In vitro studies were performed using cells seeded within the hydrogels. Viability, proliferation, and morphology were assessed to evaluate cytocompatibility and early responses to stiffness and ECM content. Long-term chondrogenic differentiation was then compared between hydrogel-encapsulated cells and conventional pellet cultures. Gene expression analysis focused on collagen type II (COL2A1) and collagen type X (COL10A1), with complementary histological staining after four weeks to examine tissue organization and matrix deposition.

Softer hydrogels supported significantly higher cell viability and proliferation than stiffer formulations, indicating that mechanical compliance strongly influences early cell behavior. Cells within hydrogels showed marked upregulation of COL2A1 and COL10A1, exceeding levels observed in pellet cultures under identical conditions. Histology confirmed chondrocyte-like morphology and new extracellular matrix deposition.

Overall, this work demonstrates that HAMA–GelMA hydrogels, with or without dECM, can support key processes underlying cartilage regeneration. Hydrogel stiffness shows a stronger impact on cellular responses than compositional differences in this concentration range. These findings highlight the potential of customizable biomimetic hydrogels as advanced platforms for next-generation cartilage repair strategies.

1050. 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.

1051. 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.

1052. 4D-printed thermoresponsive plasmonic scaffolds recapitulating alveolar dynamics for in vitro lung modeling

Uxue Aizarna-Lopetegui¹, Adrian Lluveras¹, Malou Henriksen-Lacey², Jesús Ruiz-Cabello³, Dorleta Jimenez De Aberasturi¹

¹Hybrid Biofunctional Materials. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ²Bionanoplasmonics. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ³Molecular and Functional Biomarkers. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain

Mechanical forces are a critical factor to consider when designing physiologically relevant three-dimensional (3D) in vitro models, as they play fundamental roles in various human organs by inducing physical deformation and activating mechanotransduction pathways that regulate cellular behavior. However, these forces are often neglected in model design, despite their particular relevance in the cardiovascular system, where the cyclic mechanical stresses generated by the rhythmic contractions of the heart are continuously present and exert significant influence on tissue function, cellular alignment and crosstalk, and phenotype. In this line, thanks to the incorporation of plasmonic nanoparticles (pNPs) in different hybrid extracellular matrixes we have achieved highly controllable cyclic expansion-contraction changes that have demonstrated to induce cellular mechanotransduction related gen expressions [1-3]. Considering this, we have explored the use of volumetric printing [4] and embedded bioprinting [5] to obtain dynamic 3D printed multilayered vasculature models that recreate the physical forces to which cells are exposed during arterial pulsation. For that we have developed two different inks, an elastic one containing the pNPs for the external tunica adventitia layer and a cell-laden bioink consisting of human pulmonary artery smooth muscle cells embedded in a dECM-based hydrogel, which is biocompatible and fosters optimal cell growth and proliferation, allowing the reproduction of tunica media layer. Moreover, we have demonstrated how these kinds of in vitro models are ideal platforms for real-time 4D flow magnetic resonance imaging (MRI) to investigate the effect of flow dynamics in vascular diseases.

1053. Osteomimetic assembly of vascularized human-based mineralized tissue via microfluidic-assisted 3D bioprinting approach

Lucia Iafrate¹, Edoardo Brandi², Caterina Sanchini², Biagio Palmisano³, Mara Riminucci³, Giancarlo Ruocco⁴, Karl H. Schneider⁵, Yang-Hee Kim⁶, Gianluca Cidonio¹

¹Department of Mechanical and Aerospace Engineering. Sapienza University of Rome, Rome (Lazio) - Italy, ²Department of Biology and Biotechnology “C. Darwin”. Sapienza University of Rome, Rome (Lazio) - Italy, ³Department of Molecular Medicine. Sapienza University of Rome, Rome (Lazio) - Italy, ⁴Department of Physics. Sapienza University of Rome, Rome (Lazio) - Italy, ⁵Center for Biomedical Research and Translational Surgery. Medical University of Vienna, Vienna (Wien) - Austria, ⁶Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences. University of Southampton, Southampton - United Kingdom

Objectives:

Current strategies to repair critical-size bone defects, such as autologous grafts or metal implants, often fail to achieve long-term regeneration due to limited availability, lack of osteoinductive cues, or insufficient physiological remodeling. To address these challenges, new biomimetic strategies are required. Here, we developed a human 3D OSTEOMimetic MICrofluidic bioprinted model (OSTEOMIMIC) combining decellularized human bone (hbECM) and placenta (hpECM) extracellular matrix-based bioinks, encapsulating human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) respectively. This platform enables spatially controlled deposition of osteogenic and angiogenic compartments to promote mineralization and vascularization, offering a new framework for personalized skeletal repair.

Methods:

hBMSCs and HUVECs were encapsulated in methacrylated hyaluronic acid (HAMA)–fibrinogen–hbECM and HAMA–fibrinogen–hpECM hydrogels, respectively. Single and gradient bioprinted constructs were cultured up to 21 days. Alkaline phosphatase (ALP) activity, Real-Time quantitative PCR, immunostaining of bone and vascular compartments were used to evaluate osteogenic differentiation, mineralization, and vascular functionality. A stability assay under cell encapsulation assessed ECM production, while the chorioallantoic membrane (CAM) assay evaluated angiogenic potential.

Results:

Spatially controlled deposition of bone–vascular bioinks enhanced integration and direct hBMSC–HUVEC crosstalk. Tissue-specific cells showed functional viability over time. ALP staining indicated a steady increase in osteogenic activity without supplements, confirming hbECM bioactivity. Gene expression and immunostaining revealed late osteogenic markers and adherents junctions. The stability assay showed reduced mass loss compared to acellular controls, suggesting ECM deposition reinforced scaffold integrity. The CAM assay further confirmed the angiogenic functionality of the human-derived bioinks.

Conclusions:

Human decellularized matrices retain essential cues regulating cell behavior and mineralisation. Incorporating hbECM and hpECM into bioprinted constructs recreated a coupled osteogenic–angiogenic niche supporting coordinated cellular activity. Altogether this mineralisation model offers a promising route toward an advanced 3D bioprinted construct for personalized regenerative medicine and drug testing.

1054. Monitoring approaches for 3D bioprinted cell models: integrating biosensors to track pathology

Clara García-Astrain¹, Ana María Muñoz-Mateo¹, Francesca Perin¹, Lara Troncoso-Afonso²

¹POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain, ²CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain

Bioprinting is transforming the fabrication of 3D scaffolds enabling the recreation of physiologically relevant microenvironments.1 This progress, however, requires sensing technologies adapted to complex 3D structures.2 Integrating sensors directly into bioinks provides a promising strategy to embed real-time sensing into bioprinted constructs. Here, we present several 3D models as smart tissue models for studying disease mechanisms and therapeutic responses. Different scaffolds configurations and cell combinations were explored by combining different printing techniques. Sensors were incorporated either as gold nanorods (AuNRs) for Surface-Enhanced Raman Spectroscopy (SERS) detection or as water-soluble conjugated polymers (WSCPs) for colorimetric and fluorescent sensing. First, 3D breast cancer models incorporating AuNRs were used for SERS-based monitoring of chemotherapy. Grid-like scaffolds supported cancer cell growth and the SERS signal of paclitaxel at 1002 cm⁻¹ enabled assessment of drug resistance. Decellularized extracellular matrix-based core–stroma models containing cancer cells and fibroblasts were also fabricated, where plasmonic hydrogel pillars allowed monitoring of 6-thioguanine consumption both at the core and the stroma. Additionally, polydiacetylene vesicles, which undergo optical changes upon exposure to bacteria, were used to monitor infection in 3D. Their incorporation into methacrylated gelatin-based hydrogels showed color and fluorescence response to pH changes and enzymatic activity, supporting their use in infection-responsive models. Taken together, embedding polymers and sensors within bioinks enables smart 3D tissue models capable of real-time monitoring, offering valuable insights into disease progression and treatment response.

References

(1) C. Mota et al. Chem. Rev. 2020, 120, 19, 10547–10607

(2) Hansel C. S. et al., Biomaterials 2020, 226, 119406

(3) Troncoso-Afonso L. et al., Chem. Soc. Rev., 2024,53, 5118-5148

Acknowledgements

Financial support was provided by MICIU PID2024-162567OA-I00 and a 2025 Leonardo Grant for Scientific Research and Cultural Creation from the BBVA Foundation.

1055. From barrier to breach: a 3D microvascular model to assess barrier disruption and recovery

Leanne De Silva¹, Patrick Kuntschke¹, Katinka Theis¹, Alexander Radüchel¹, Jürgen Groll¹, Matthias Ryma¹

¹Department for Functional Materials in Medicine and Dentistry. University Hospital Würzburg, Würzburg (Baden-Wberg Bayern) - Germany

Evaluating compounds that disrupt or restore the endothelial barrier function requires in vitro systems that can model both barrier breakdown and subsequent recovery. Traditional static assays often produce unstable barriers, and organ-on-chip systems, although more physiologically relevant, are typically constructed with small channels and limited 3D complexity. This work aims to recreate pathological permeability using disease-associated factors and to establish an improved recovery model suitable for screening compounds that target leaky vasculature. To meet these needs, the BasicVasc platform was used. BasicVasc is a perfused 3D microvascular system that uses a removable sacrificial scaffold to cast a defined channel within a hydrogel matrix. Once endothelialized, the lumen provides a consistent vessel for assessing baseline barrier function, induced leak and responses to recovery treatments. Its 3D architecture and larger vessel geometry create a more realistic microvascular environment for evaluating barrier function and leak.

Human umbilical vein endothelial cells (HUVECs) were seeded into fibrin-casted channels and cultured for seven days to establish a stable, confluent endothelium. Pathological permeability was then induced using tumour necrosis factor-alpha (TNFα), lipopolysaccharide (LPS) and thrombin. Permeability was quantified using 70 kDa fluorescein isothiocyanate (FITC) dextran and transendothelial electrical resistance (TEER). Thrombin produced an immediate rise in dextran leakage, defining the upper limit of the assay window, while TNFα and LPS generated a moderate increase in permeability after 24 hours.

For the recovery model, stabilising agents were added to test the platform’s ability to detect partial repair, measured by reduced dextran flux and improved TEER compared with controls. Junctional markers (ZO-1 and VE-cadherin) served as qualitative checks of endothelial organisation before and after a treatment. Initial studies indicate that this platform provides a practical and adaptable route for modelling pathological permeability and for evaluating compounds that disrupt or restore the microvascular barrier.

1056. Reconstructing the tumor immune microenvironment on a chip: a primary cell-derived model for studying T cell migration in colorectal cancer

David Bartolomé-Català¹, Pau Canaleta Vicente¹, Jordi Comelles¹, María García-Díaz¹, Elena Martínez¹

¹Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain

The immune system plays a pivotal role in cancer progression and response to therapy, with T cells acting as central mediators of anti-tumor immunity. Despite significant progress, the mechanisms governing T cell migration within the tumor microenvironment (TME) remain poorly understood. The inherent complexity and limited accessibility of in vivo systems make these processes difficult to study, driving the development of advanced in vitro models. In this context, the use of primary cells is particularly critical, as it preserves the physiological heterogeneity and authenticity of native tissues, ensuring that experimental outcomes closely reflect in vivo conditions. Here, we present a three-dimensional in vitro approach to investigate T lymphocyte migration in colorectal cancer. Using organ-on-a-chip technology, we established a microphysiological system consisting of three interconnected channels: an epithelial channel lined with a 3D tubular monolayer derived from healthy or tumor mouse colon organoids, a central stromal channel containing primary intestinal fibroblasts embedded in a collagen–Matrigel hydrogel, and an immune channel where primary T cells are introduced. This architecture mimics the spatial organization of the colonic wall and enables the study of T cell migration through the stromal compartment toward the epithelial tumor region. We demonstrate that tumor-derived organoids retain key pathological hallmarks within the chip, including enhanced proliferative capacity, reduced differentiation, and the formation of aberrant crypt-like structures. In parallel, we developed an automated imaging and analysis workflow for real-time tracking and quantitative assessment of immune cell migration dynamics. Our findings reveal that tumor-associated factors modulate T cell motility and trajectories, underscoring the critical influence of TME on immune cell behavior. This tumor-on-a-chip model provides a versatile platform for dissecting immune–tumor interactions and offers a physiologically relevant tool for advancing the understanding of T cell migration in cancer.

1057. 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.

1058. Next-generation neurovascular stents: design strategies linked to modern manufacturing techniques

Daniel Frape¹, Fernanda Zamboni²

¹School of Engineering. University of Limerick, Limerick - Ireland, ²School of Engineering, Health Research Institute, Bernal Institute. University of Limerick, Limerick - Ireland

Introduction:

Neurovascular stents are designed to treat complex cerebrovascular conditions such as aneurysms. Continued development of novel stent designs is essential to improve deployment precision in the brain’s delicate vasculature and to accelerate endothelialization, which supports vessel healing while lowering the risks of clot formation and stroke. These devices represent a major advancement in minimally invasive therapy and vascular reconstruction, offering safer and more effective alternatives to open surgery.

Objectives and Aims:

This work aims to generate multiple neurovascular stent designs tailored for manufacture using braiding, laser cutting, and 3D printing.

Methods:

Neurovascular anatomy involves small vessel diameters (2–5 mm), tortuous intracranial pathways, and thin arterial walls; thus, stents must provide high flexibility, low deployment profiles, precise placement, strong fatigue resistance, and reduced thrombogenicity. Geometries are created in CAD software (SolidWorks) to model the essential structural features. Material selection is informed by a comparative evaluation of melting behaviour, mechanical strength, biocompatibility, radiopacity, and suitability for each manufacturing process. Finalised concepts undergo flow-dynamic assessment using CFD tools to evaluate hemodynamic performance.

Results:

Process-specific minimum feature sizes were defined, and stent models were developed for laser cutting, additive manufacturing, and braiding, including crimped and expanded states. Designs incorporated metal strut thicknesses of 60–150 µm with filleted junctions to minimise stress. Mesh pore-size was 0.5–1.5 mm for scaffold stents and <0.5 mm for flow diverter stents. Additional refinements such as tapered struts, atraumatic flared ends, variable braid angles, and selective wire diameters were also developed. Enhancements included microgrooved or nano-aligned surfaces to accelerate endothelialization, radiopaque markers for accurate placement, and hybrid cell configurations to balance scaffolding and flexibility. Mechanical and flow simulations identified areas prone to strain or adverse flow.

Conclusion:

This project outlines emerging neurovascular stent designs optimised to promote faster endothelialization and reducing clot risk while defining key geometric, material, and manufacturing considerations.

1059. Electrochemical considerations for the design of electrical stimulation protocols

María Ujué González¹, Andrés Sánchez-Pérez¹, Gaurav Kulkarni¹, Miriam Isasi-Campillo¹, Sahba Mobini¹

¹Instituto de Micro y Nanotecnología, IMN-CNM. Consejo Superior de Investigaciones Científicas, CSIC, Tres Cantos (Madrid) - Spain

Direct electrical stimulation (ES) of cells and tissues has attracted significant attention from researchers in biology and biomedicine over the past few decades. Low-voltage ES (<300 mV/mm) with diverse signal shapes and periodicities has been investigated as a physical tool to modulate cellular responses, primarily aiming to trigger regenerative processes such as proliferation, differentiation, and migration. Although applying electrical signals to cell cultures may appear straightforward, the selection of appropriate ES protocols and the establishment of reproducible procedures remain complex challenges.

The design of an effective ES protocol should foremost be guided by the specific biological objective. The biological goal is critical for establishing meaningful ES conditions in terms of the magnitude and orientation of the electric field delivered inside the culture area, the dynamics of the delivered charge and the temporal characteristics of stimulation events. These parameters determine the specific ES protocol, i.e. the applied electric signal amplitude, waveform and frequency, which are calculated in accordance with the electrical properties of the stimulation platform. Given that biological environments typically involve electrolytic media, the electrical behaviour of the system is governed by electrochemical interactions at the electrode–electrolyte interface. Therefore, it is essential to incorporate electrochemical design principles to ensure safe, effective, and reproducible ES protocol.

Here, we will present a description of the electrochemical basis of direct ES, including the basic characterization needed to describe our systems and devices. We will discuss the use of equivalent circuits to describe the platforms, and the obtention of the voltage window that reduces harmful electrochemical reactions. All these considerations will be aimed to the obtention of standardized ES protocols that facilitate the comparison of ES conditions between experiments and laboratories.

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 INVESTIGO 2023 of Madrid Community, co-financed by EU.

1060. Modulation of LPS-induced inflammation by white and black garlic extracts in an intestinal epithelial co-culture

Joel Girón-Hernández¹, Sofía Mares Bou², Andrea Martelli³, Gloria Gallego-Ferrer², Piergiorgio Gentile²

¹Centre for Biomaterials and Tissue Engineering (CBIT). Universitat Politècnica de València, Valencia - Spain, ²Center for Biomaterials and Tissue Engineering (CBIT). Universitat Politècnica de València, Valencia - Spain, ³Department of Engineering. University of Modena and Reggio Emilia, Modena (Emilia-Romagna) - Italy

Intestinal inflammation and increased epithelial permeability are hallmarks of several functional gastrointestinal disorders. Phenolic compounds with antioxidant and anti-inflammatory properties, together with probiotics, have been proposed as complementary strategies to modulate these alterations. We investigated the individual and combined effects of probiotics and white or black garlic extracts on inflammation and barrier function in an intestinal epithelial model generated from a Caco-2/HT29 (9:1) coculture differentiated for 21 days, stimulated for 72 h with LPS and subsequently exposed to probiotics and in vitro–digested garlic extracts.

Barrier integrity was assessed by transepithelial electrical resistance (TEER), ZO-1 distribution and Lucifer Yellow (LY) permeability, and cytokine expression (TNF-α, IL-1β, IL-6) was quantified using a ELISA kit assays. Inflamed tissues showed a significant reduction in TEER after 72 h (924 ± 126 vs 1129 ± 74 Ω·cm² in non-LPS controls), while LY permeability remained <3% for all conditions, indicating overall preserved barrier function; the highest permeability (2.1%) was observed in tissues exposed to LPS plus probiotic bacteria. ZO-1 imaging revealed more pronounced disruption and widening of tight junctions in tissues treated with white garlic, whereas black garlic–treated tissues more closely resembled non-LPS controls.

IL-6 displayed the greatest increase after 72 h of LPS stimulation, and treatments containing garlic (white or black) produced the largest reductions in IL-6, with white garlic lowering levels to below 100% of the baseline reference. No significant changes were detected in TNF-α or IL-1β expression. In summary, white and black garlic exerted an attenuating effect on IL-6 expression, while probiotics did not demonstrate a clear modulatory effect on LPS-induced inflammation and appeared to increase epithelial stress and permeability, except when combined with white garlic, which may confer additional antimicrobial protection.

1061. Human dental pulp stem cells (hDPSCs) secretome inhibits inflammation and cartilage degeneration in a rat model of osteoarthritis

Lucía Bravo-Baranda¹, Àlvar Soler-García², Lara Milián¹, Mauro Llop-Miguel¹, María Sancho-Tello¹, Cristina Martínez-Ramos¹, José Marcelo Galbis-Caravajal³, Manuel Mata¹

¹Department of Pathology. Universitat de València, Valencia - Spain, ²Servicio de Cirugía Ortopédica y Traumatología. Hospital Clínico Universitario de Valencia, Valencia - Spain, ³Servicio de Cirugía Torácica. Hospital de La RIbera, Alzira (Valencia) - Spain

Osteoarthritis (OA) is a chronic degenerative joint disease and one of the leading causes of disability worldwide, placing a significant socioeconomic burden on healthcare systems. Current treatments are primarily palliative, focusing on pain and inflammation relief rather than restoring cartilage integrity. Therefore, innovative regenerative strategies are urgently needed.

Our aim was to evaluate the anti-inflammatory and regenerative effects of secretome obtained from high-density cultured mesenchymal stem cells (MSCs) under chondrogenic stimuli, as a potential cell-free therapy for OA in an in vivo rat model. The knee joint was destabilized by surgical resection of the medial meniscus and anterior and posterior cruciate ligaments. Four weeks later, animals received weekly intra-articular injections of hDPSC secretome for an additional four weeks, after which they were sacrificed. Control animals underwent the same surgery but did not receive secretome injections. Inflammation and tissue degeneration were assessed by in vivo imaging analysis and histological studies.

Imaging analysis revealed a significant anti-inflammatory effect in the treated animals compared to the controls. Histological studies showed a trend toward improved cartilage organization and matrix regeneration in the secretome-treated group.

These findings suggest that the therapeutic benefits traditionally attributed to MSC transplantation may be largely mediated by their paracrine activity. The use of secretome as a cell-free regenerative strategy could avoid the risks associated with cell implantation and represents a promising alternative for the treatment of osteoarthritis. Moreover, hDPSCs constitute a realistic and accessible alternative to other MSC sources, including bone marrow-derived MSCs (BM-MSCs).

1062. 3D-bioprinted breast tumour with embedded plasmonic biosensors for spatially resolved drug screening

Lara Troncoso Afonso¹, Paula Vázquez-Aristizábal¹, Gail A. Vinnacombe-Willson¹, Yolany M. Henríquez-Banegas², Patricia González-Callejo³, Pablo Valera-Sapena³, Malou Henriksen-Lacey³, Clara García-Astrain⁴, Luis Liz-Marzán³

¹Bionanoplasmonics. CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ²Universidad del País Vasco, Donostia-San Sebastián (Gipuzkoa) - Spain, ³CIC biomaGUNE, Donostia-San Sebastián (Gipuzkoa) - Spain, ⁴POLYMAT, Basque Center For Macromolecular Design And Engineering - UPV/EHU, Donostia-San Sebastián (Gipuzkoa) - Spain

The development of antitumoral drugs is limited by the lack of in vitro platforms that simultaneously combine biological relevance, spatial complexity, and analytical sensitivity to effectively study cellular responses.[1] Despite many 3D in vitro models accurately mimic the tumour microenvironment, analysing drug responses within these complex environments remains challenging. To address this, we propose a new class of hybrid biomaterials that integrate hydrogel-forming polymers with gold nanorods, engineered for 3D bioprinting. These constructs support cell growth and organization while incorporating plasmonic functionality to allow in situ, label-free monitoring of molecular biomarkers via surface-enhanced Raman spectroscopy (SERS).

Gold nanorods (AuNRs) were embedded into a click cross-linkable mixture of thiolated alginate and methacrylated carboxymethylcellulose. These hydrogel forming polymers were synthesized and characterized by 1H-NMR and FTIR, while AuNRs were characterized by UV-VIS and TEM. The rheological properties and the kinetics of the crosslinking were determined. The microstructural features of the material were imaged by SEM and the SERS performance of the material was evaluated for different analytes. [2] Then, the material was used to 3D print pillar-shaped SERS-active structures within a complex 3D breast tumour model.[3]

The embedded SERS-active pillars enabled non-invasive, real-time monitoring of drug distribution and cellular responses within both tumor and stromal compartments. The treatment of the model with a chemotherapeutic drug (6-thioguanine) revealed differences in absorption between the core and the stroma, demonstrating the potential of the 3D breast cancer model to test chemotherapeutic efficacy within more realistic microenvironments. Overall, integrating plasmonic sensors into bioprinted tumor models can improve analytical sensitivity, potentially aiding in drug screening, biomarker identification, and personalized cancer research.

References

[1] L. Troncoso-Afonso, et al. Chem. Soc. Rev. 2024, 53, 5118-5148

[2] L. Troncoso-Afonso, et al. Biomater. Sci. 2025, 13, 2936-2950

[3] P. González-Callejo, et al. Mater. Today Bio 2023, 23, 100826

1063. 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.

1064. Three-dimensional nichoid cubic scaffold enhances hADMSCs expansion, stemness, and functional recovery in a rat model of spinal cord injury

Stephana Carelli¹, Bonnet Maxime², Maresca Federico³, Conci Claudio³, Cherubin Leonardo³, Dardi Marko², Berardo Clarissa¹, Messa Letizia¹, Zuccotti Gianvincenzo², Cereda Cristina¹, Raimondi Manuela³

¹Department of pediatrics. Functional Genomics and Rare Diseases Unit, Milan (Lombardia) - Italy, ²Department of Biomedical and Clinical Sciences. University of Milan, Milan (Lombardia) - Italy, ³Department of Chemistry. Politecnico di Milano, Milan (Lombardia) - Italy

Introduction: Spinal cord injury (SCI) leads to irreversible sensorimotor and autonomic deficits, and current therapies fail to restore function. Human adipose-derived mesenchymal stem cells (hADMSCs) provide neuroprotective, immunomodulatory, and trophic effects. Yet, achieving therapeutic doses remains challenging, as conventional 2D culture induces senescence and loss of potency. To overcome these limitations, we developed the Nichoid Cubic Scaffold (NCS), a 3D-microstructured scaffold fabricated by two-photon polymerization, designed to mimic a native stem cell niche. This study aimed to biologically validate the 3D-microstructured NCS and to evaluate the efficacy of NCS-expanded hADMSCs following SCI in rats.

Methods: Fabrication parameters were optimized to yield a mechanically stable 30 × 30×30 µm³ lattice unit arranged in two stacked planes, upscaled into an 8 mm-diameter biocompatible scaffold. Finite element analysis (FEA) was used to design a structurally stable structure having the desired pore stiffness distribution. Biological validation was performed with hADMSC donors to evaluate viability, colonization, stemness-related gene expression, and multilineage differentiation potential. Finally, NCS-expanded hADMSCs (patent WO2020183343) were transplanted into a rat model of SCI to assess locomotor and electrophysiological recovery after 6 weeks.

Results: FEA demonstrated high mechanical stability and homogeneous pore stiffness, confirming the robustness of the designed structure. The NCS supported high cell viability and proliferation while maintaining the controlled expression of pluripotency-related genes. hADMSCs exhibited enhanced plasticity, retaining mesodermal commitment and neurogenic transdifferentiation potential. In the SCI rat model, NCS-expanded hADMSCs significantly improved locomotor and electrophysiological recovery.

Conclusions: The NCS provides a biomimetic and mechanically stable microenvironment that enables efficient hADMSC expansion and reprogramming. The combination of 3D culture and cell transplantation promoted functional recovery after SCI in rats, highlighting the NCS as a promising platform for scalable and clinically translatable regenerative therapies.

Acknowledgements: Funded by the Ministry of Business and Made in Italy under NRRP, supported by the European Union – NextGenerationEU.

1065. Metastasis-on-a-chip: Engineering a hydrogel-based 3D model to study patient-derived CTCs extravasation in colorectal cancer

Melika Parchehbaf Kashani¹, Nadia Saoudi González², Jordi Comelles¹, Elena Élez², Lenie Van Den Broek³, Karla Queiroz³, María García-Díaz¹, Elena Martínez¹

¹Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona - Spain, ²VHIO Vall d'Hebron Institute of Oncology, Barcelona - Spain, ³MIMETAS B.V., Oegstgeest (Zuid-Holland) - The Netherlands

Colorectal cancer (CRC) metastasis remains a major cause of cancer-related mortality worldwide. Extravasation, the step in which tumor cells exit the vasculature to establish secondary tumors, is critical for metastatic progression. However, studying this process in vivo is challenging due to complex tumor–endothelial interactions and patient-specific variability. Circulating tumor cells (CTCs), which exhibit diverse molecular and phenotypic profiles depending on disease stage and treatment, further influence metastatic behavior. Therefore, physiologically relevant in vitro models that replicate endothelial barrier function and stromal cues are essential for studying CTC dynamics and therapy response under controlled conditions.

In this work, we developed a hydrogel-based 3D endothelial model that recreates the vascular and stromal interface of metastatic tissue, allowing investigation of CRC cell extravasation. The bioink, composed of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and fibrin, was bioprinted using digital light processing (DLP) and assembled into Transwell^® inserts. Human intestinal fibroblasts were embedded within the hydrogel, while primary human umbilical vein endothelial cells (HUVECs) formed a confluent monolayer with robust barrier properties. To mimic the premetastatic inflammatory niche, the endothelial layer was stimulated with TNF-α and TGF-β, thereby increasing permeability. To account for dynamic endothelial conditions, the model was integrated into the OrganoPlate^® microfluidic platform.

Using this platform, we evaluated the extravasation potential of CTCs isolated from fresh blood samples of metastatic CRC patients with distinct clinicopathological profiles, alongside three different CRC cell lines (SW620, SW480, and HT29). Immunofluorescence analysis confirmed that patient-derived CTCs remained viable and actively transmigrated across the endothelial barrier, only upon inflammatory conditions. Quantitative assessment revealed clear inter-patient variability but with significant trends. The patient tumor burden correlated with the number of extravasated CTCs, whereas the shorter chemotherapy-to-CTC isolation interval, the higher tissue penetration was observed. Overall, this bioprinted metastasis-on-chip model provides a robust platform for studying patient-specific CTC extravasation and identifying potential therapeutic strategies to prevent CRC metastasis.

1066. Immune profile of equine mesenchymal stem/stromal cells (MSCs): induced vs primary cells

M^a 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.

1067. Surgical navigation enhances precision and training in models for regenerative spine therapies

Andres Bonilla¹, Kristen Sack¹, Nicole Erben¹, Howard Seim¹, Jeremiah Easley¹

¹Translational Medicine Institute, Fort Collins (Colorado) - United States

Introduction:

Large animal spine models are essential for evaluating regenerative therapies, biomaterials, and implantable technologies due to their close anatomical similarity to humans. However, variability in implant placement remains a major limitation in preclinical tissue engineering studies, where the precision of delivery directly affects biological outcomes. Image-guided surgical navigation, widely used in clinical spine surgery, offers a powerful opportunity to refine these models by enabling accurate and reproducible placement of any implant, scaffold, biologic, or therapeutic device. Additionally, this technology provides an intuitive and safe platform for training new researchers, allowing students to learn advanced procedural skills relevant to regenerative medicine. This pilot study compares navigation-guided and freehand pedicle screw placement using novice operators as a proof-of-concept for broader application.

Materials and Methods:

Sixty pedicle screws were placed in lumbar vertebrae from six ovine cadavers. Thirty screws (n=30) were inserted with O-arm–based CT navigation, and thirty (n=30) were placed freehand. All procedures were performed by two veterinary students with no prior experience. Accuracy was evaluated using the Gertzbein–Robbins classification. Planning and drilling times were recorded to assess workflow and usability.

Results:

Navigation achieved high precision (80 percent Grade 0, 16.7 percent Grade 1) with no moderate breaches and only one severe violation (3.3 percent). Freehand placement resulted in lower accuracy with higher rates of moderate-to-severe breaches. Although navigation increased drilling time, planning was rapid, and novice operators demonstrated an immediate learning effect, successfully producing accurate placements on their first attempts.

Conclusion:

Navigation-guided instrumentation significantly improves precision, reproducibility, and safety in ovine spine procedures. Importantly, it also serves as an effective educational tool, enabling early-stage researchers to safely learn complex techniques that support consistent delivery of regenerative therapies. This technology holds strong potential to accelerate both the development and translation of tissue engineering strategies in large-animal models.

1068. Soft and deformable bioreactor enables long-term perfusion and actuation of engineered muscle for biohybrid robotics

Patricia Zoio¹, Jordi Comelles¹, Florencia Lezcano², Usama Mahmood³, Stefano Lai³, Samuel Sánchez², Elena Martínez¹

¹Biomimetic Systems for cell Engineering. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, ²Smart Nano-bio-devices Laboratory. Institute for Bioengineering of Catalonia (IBEC), Barcelona - Spain, ³Department of Electrical and Electronic Engineering. University of Cagliari, Cagliari (Sardegna) - Italy

Biohybrid robotics merges engineered muscle tissues with synthetic structures to achieve lifelike, adaptive motion. Proof-of-concept systems generate contractile force, yet most lack integrated platforms for continuous perfusion, humidity control, and protection from contamination, critical for stable function beyond submerged laboratory setups. Organ-on-chip technologies offer precise microfluidic environments, but their rigid construction and reliance on external sealing components hinder integration with soft, deformable robotic systems, limiting their utility for autonomous operation and real-world biohybrid applications.

To overcome these limitations, we developed a soft, deformable perfusion bioreactor fabricated by extrusion-based 3D printing of ultra-soft silicone and a self-sealing encapsulation method. Leveraging silicone’s curing and conformal sealing properties, the device forms a fully enclosed, adhesive-free chamber that deforms synchronously with muscle contraction both in liquid and in air.

3D skeletal muscle tissues were biofabricated by mold-casting a hydrogel laden with C2C12 myoblasts into ring-shaped molds, followed by differentiation and transfer into the soft bioreactor chamber. Custom-designed PDMS anchoring structures ensured stable attachment and efficient force transmission. Constructs comprising two or four muscle bundles were electrically stimulated (0.2–2.0 V/mm) and cultured under continuous perfusion for up to six days. Functional performance was assessed by bending angle analysis, force output, viability assays (LDH release, AlamarBlue, Live/Dead) and immunostaining for myosin heavy chain and sarcomeric α-actinin.

Encapsulated constructs remained viable and contractile, achieving voltage-dependent deformation of the soft chamber. Multi-bundle constructs reached bending angles of 16 ± 2° at 2 V/mm and preserved motion and structural integrity under both submerged and air-exposed conditions. This platform provides a robust foundation for long-term muscle culture and soft actuation, advancing biohybrid robotics toward more autonomous, durable, and physiologically relevant systems.

1069. 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.

1070. 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.

1071. 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.

1072. 3D in vitro microvascular model for non-penetrating traumatic injury research

Carla Verónica Fuenteslópez¹, Mark S. Thompson¹, Hua Ye¹

¹Institute of Biomedical Engineering. University of Oxford, Oxford (Oxfordshire) - United Kingdom

Microvascular injury critically influences the progression and severity of traumatic injuries, yet existing in vitro models often fail to replicate microvascular architecture and function accurately in 3D. This research presents the optimisation of a 3D hydrogel-based in vitro model that supports the formation and long-term stability of microvascular endothelial networks for trauma research.

Human dermal microvascular endothelial cells (MVECs) were embedded in fibrin-based hydrogels, and their performance was benchmarked against the widely used human umbilical vein endothelial cells (HUVECs). Systematic variations in hydrogel composition (fibrinogen source and concentration, crosslinking ratio, and medium) were examined to assess their effects on scaffold material properties and endothelial network formation, architecture, and longevity.

Network analysis showed that hydrogels formulated with high concentrations of human fibrinogen, a 200:10:1 fibrinogen:thrombin:CaCl2 crosslinking ratio, and either endothelial basal medium (EBM) or EBM supplemented with VEGF supported the most robust and durable microvascular networks, maintaining structural integrity for up to 14 days. In contrast, HUVEC-based models underwent rapid network degradation within 24 hours. Microrheometry revealed that increasing fibrinogen concentration significantly accelerated gelation kinetics, increased storage and loss moduli, and reduced creep compliance, thereby improving the constructs’ mechanical stability.

To investigate trauma mechanisms, controlled non-penetrating injuries (contusion, compression, and strain) were applied to optimised 3D microvascular constructs. This approach enabled the characterisation of immediate and short-term microvascular responses, establishing crucial links between trauma parameters and the resulting injury.

The optimised model offers a physiologically relevant platform for studying traumatic injury and evaluating therapeutic strategies to improve vascular repair and recovery.

1073. Biocompatibility and mechanical characterization of silica-enriched PLA composites for 3D-printed bone scaffolds

Carolina Centeno-Cerdas¹, Sergio Paniagua², Jorge Oviedo-Quirós³, Alfonso García-Piñares⁴, Leonardo Lesser-Rojas⁵

¹Centro de Investigación en Biotecnología. Tecnológico de Costa Rica, Cartago - Costa Rica, ²National Nanotechnology Laboratory (LANOTEC), National Center for High Technology (CENAT), San José (San Jose) - Costa Rica, ³Faculty of Dentistry, Universidad de Costa Rica. Craniomaxillofacial Cleft Palate Unit, National Children’s Hospital “Dr. Carlos Sáenz Herrera”, San José (San Jose) - Costa Rica, ⁴Universidad de Costa Rica. Centro de Investigación en Biología Celular y Molecular, San José (San Jose) - Costa Rica, ⁵Universidad de Costa Rica. Laboratory of Nano Bio Systems, San José (San Jose) - Costa Rica

Introduction/Objectives:

Autologous bone grafting remains the standard for cleft palate repair and other bone defects, yet it entails donor site morbidity and surgical complexity. This study aims to develop and characterize biocompatible, biodegradable scaffolds based on polylactic acid (PLA), calcium phosphate (CP), and diatomaceous earth (DE) (a silica-rich additive) to mimic bone architecture and promote osteoblast proliferation. Emphasis was placed on evaluating biocompatibility, a critical parameter for clinical translation of new biomaterials.

Methods:

Composites were prepared with varying ratios of PLA, CP, and DE, cast and extruded into filaments, and 3D-printed using fused deposition modeling (FDM). Sterilization was performed via gamma irradiation, moist heat, and oxygen plasma to identify the most appropriate method. Biocompatibility was assessed using MC3T3-E1 cells following ISO 10993 guidelines, with MTT and LDH assays. Mechanical properties were evaluated via rheometry, thermal behavior via DSC, and microstructure via SEM and EDX. Scaffold degradation was monitored under physiological conditions (pH 7.4, 37 °C). Cell adhesion and proliferation were analyzed over 30 days using Alamar Blue and fluorescence microscopy on scaffolds printed at three porosity scales (130%, 180%, 230%).

Results:

Gamma irradiation preserved scaffold integrity and supported high cell viability, while plasma sterilization significantly reduced viability due to surface oxidation, and moist heat caused deformation due to PLA’s low Tg (∼60 °C). Composites showed mechanical stability over 13 weeks, with Young’s moduli exceeding that of cancellous bone (∼0.442 GPa). DE increased scaffold porosity and enhanced cell adhesion. The 130% porosity scale yielded the highest metabolic activity at day 30, suggesting optimal pore morphology for osteoconduction from the evaluated scales. Silica from DE promoted osteoblast proliferation and differentiation, though excessive DE slightly reduced viability.

Conclusions:

The 20PLA/1CP/1DE composite demonstrated suitable biocompatibility, mechanical performance, and osteoconductive potential, making it a promising candidate for bone tissue engineering. Characterizing biocompatibility across sterilization methods and scaffold architecture is essential for the safe and effective clinical application of novel biomaterials. Future work will focus on gene expression profiling and in vivo validation of these 3D-printed scaffolds.

1074. Toward a greener approach for next-generation functional pancreatic ECM in diabetes treatment - SEMIT

Simone C. Sá¹, Joana Sá¹, Carlos Pazmino¹, Sara Amorim¹, João B. Costa¹, Raquel Soares², Ana L. Oliveira¹

¹Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal, Oporto (Porto) - Portugal, ²FMUP-Faculdade de Medicina da Universidade do Porto, Porto, Portugal, Oporto (Porto) - Portugal

Current decellularization methods rely on harsh chemicals compromising the extracellular matrix(ECM) biochemical structure, particularly in soft tissues like pancreas. Supercritical carbon dioxide(scCO₂) offers a sustainable, non-toxic alternative, preserving structural and biochemical integrity, while improving immune tolerance1. This study establishes an integrated scCO₂-based workflow for porcine pancreas decellularization and sterilization, to develop an immunocompetent decellularized ECM(dECM) platform for diabetes cell-based therapies.

Pancreas decellularization was achieved using a scCO₂-assisted method(sc-dECM) and compared with a conventional detergent-based protocol. Efficiency and ECM preservation were assessed through DNA quantification, histology, SEM/TEM, biochemical assays(glycosaminoglycans(GAGs),collagens) and proteomics. ScCO2-assisted sterilization was validated through turbidity assay. Pyrogenic safety was studied through endotoxin quantification. Cytotoxicity(ISO10993-5:2009) was evaluated through metabolic activity, proliferation assays, and immunofluorescence imaging.

Both methods achieved DNA levels below 50ng/mg of dry tissue2. However, sc-dECM preserved higher GAGs content, with enhanced collagen-network preservation. Proteomics revealed that sc-dECM better preserves functional, pancreas and insulin pathway-related proteins, along with elastic fibers, indicating enhanced retention of tissue-specific biomolecules. ScCO₂-assisted decellularization was more effective in reducing endotoxin content(0.027EU/mg in scCO₂vs.0.413EU/mg in detergent-treated samples). ScCO₂ sterilization ensured Bacillus subtilis inactivation. Non-cytotoxicity was confirmed by metabolic activity and cell proliferation. DAPI/Phalloidin staining confirmed normal morphology and cytoskeleton organization. Ongoing study investigates whether Damage-Associated Molecular Patterns trigger macrophage inflammation.

Overall, scCO₂ enables fast, efficient decellularization, preserving dECM integrity and cytocompatibility. This greener approach delivers functional pancreatic dECM as potential platform towards pancreatic tissue engineering for diabetes.

Acknowledgements:FCT(ERC-PT LESSisMORE;UID/50016/2025;2024.00955.BDANA; 2025.01322.BDANA;CEECINSTLA/00040/2022/CP2993/CT0005);be@t–Textile Bioeconomy(TC-C12-i01,No.02/C12-i01.01/2022);IBEROS+(0072_IBEROS_MAIS_1_E,Interreg-POCTEP2021-2027).

1. de Wit et al.,Acta Biomater.,2023

2. Crapo et al.,Biomaterials,2011

1075. Fractal architected pyrolytic carbon scaffolds: hierarchical design strategies for geometry-governed mechanics in musculoskeletal regeneration - SEMIT

Adrían Martinez Cendrero¹, Andrés Díaz Lantada¹, Monsur Islam¹

¹Mechanical Engineering Department. Universidad Politécnica de Madrid, Madrid - Spain

Musculoskeletal tissues rely on finely tuned mechanical environments and multiscale structural cues to regulate cell behavior, matrix organization, and the transmission of forces. However, current scaffold materials often lack mechanical robustness, long-term stability, and control over the architectural interface at the micro- and nano-scales. Pyrolytic carbon (PyC) offers a compelling alternative, combining exceptional mechanical resilience, chemical stability, and electrical conductivity, properties attractive for guiding mechanosensitive cell populations and supporting electrically active tissues such as bone and muscle. To this promise, our group has demonstrated that DLP-printed PyC microlattices enable strong cell attachment, migration, and osteogenic differentiation, underscoring their biological potential. Yet, translating PyC toward clinically relevant constructs remains challenging, as carbonization induces ∼90% shrinkage and can produce deformation modes such as wrinkling, hollow bulges, and partial collapse. These defects hinder geometry retention and limit reproducibility across physiologically meaningful sizes.

To overcome these limitations, we investigate fractal-inspired, multiscale lattice designs engineered to redistribute stress during pyrolysis and preserve geometric fidelity. Using computational modeling and parametric design, we create lattices that embed microscale patterns, such as gyroid, FCC, BCC, and other TPMS geometries, within larger structural frameworks. For example, circular disks composed of gyroid units incorporate secondary micro-architectures to provide additional stiffness control and deformation resistance. This hierarchical approach enables tuning of the mechanical properties of PyC scaffolds from the microstructural level and is expected to enhance biological responses by providing tissue-relevant mechanical and topographical cues.

Our ongoing efforts include evaluating the mechanical performance of these fractal scaffolds, assessing their correlations with the architectural features, and characterizing the biological performances of these scaffolds. This work establishes foundational design and fabrication principles for next-generation architected carbon scaffolds tailored for musculoskeletal regeneration, paving the way for future biological studies and clinical translation.

1076. Decoupling elastic and viscous components in an ELR hydrogel system

Sheila Lorenzo Sanchez¹, Sergio Acosta¹, Luis Quintanilla¹, Matilde Alonso¹, José Carlos Rodríguez-Cabello¹

¹Bioforge Lab, LADIS, CIBER-BBN. Universidad de Valladolid, Valladolid - Spain

The mechanical properties of the extracellular matrix (ECM) are fundamental regulators of cell behavior. Beyond stiffness, the viscoelastic nature of the ECM—its ability to dissipate stresses and resist flow—critically influences processes such as migration, proliferation, and differentiation. However, since stiffness and viscoelasticity are often coupled, new materials that enable their independent modulation are required to study mechanobiological responses.

This work introduces a hydrogel platform composed of three elastin-like recombinamers (ELRs) designed to decouple elastic and viscous components through orthogonal chemistry and liquid–liquid phase separation. Two ELRs forming the continuous solid network were crosslinked by strain-promoted azide–alkyne cycloaddition (SPAAC) [1], while a third coacervating ELR generated a secondary liquid phase at 37°C, enhancing viscous behavior without affecting the elastic modulus.

Rheological analyses showed that incorporating the coacervate increased the loss modulus (G’’) by up to one order of magnitude while maintaining constant stiffness, demonstrating independent control of viscosity and elasticity. Hydrogels containing coacervates exhibited slower stress relaxation than purely elastic matrices, indicating persistent internal stress. Human mesenchymal stem cells (hMSCs) cultured on these slow-relaxing hydrogels maintained high viability (>95%) but displayed reduced spreading and a rounded morphology. YAP/TAZ localization remained largely cytoplasmic, and osteogenic gene expression decreased, suggesting limited mechanotransduction and a shift toward multipotent or adipogenic states, consistent with a mechanically restrictive microenvironment.

This ELR-based hydrogel system provides precise control over viscoelastic parameters, enabling systematic studies on how time-dependent mechanical cues regulate stem cell fate. Its tunability makes it a promising platform for engineering biomaterials and designing viscoelastic scaffolds optimized for regenerative medicine applications.

Reference

1. González de Torre, I., et al. Acta Biomaterialia 10, 2495–2505 (2014).

Acknowledgments:

Supported by MCIN/AEI/10.13039/501100011033, FEDER (PID2021-122444OB-100, PID2022-137484OB-I00), Junta de Castilla y León (VA188P23), and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León.

1077. Digital Light Processing(DLP) printed, bioactive chondroitin sulfate hydrogels for cartilage tissue engineering - SEMIT

Amanda A. Domingues¹, Monize C. Decarli², Prashant K. Sharma², Luiz H. Catalani¹

¹Institute of Chemistry. University of São Paulo (USP), Sao Paulo - Brazil, ²Biomaterials & Biomedical Technology (BBT). University Medical Center Groningen, Groningen - The Netherlands

Chondroitin sulfate (COS), a glycosaminoglycan (GAG) abundant in cartilage, contributes to its high compressive stiffness through effective water retention and is widely used in osteoarthritis oral therapies.[1] A COS-based bioink for DLP printing was designed to obtain high-resolution structures with minimum shear stress on cells.[2]

COS isolated from bovine trachea was methacrylated(COS-MA) to enable photopolymerizable hydrogels. Chondrocytes bind hyaluronic acid via CD44 and collagen via integrins, but not COS. Therefore, COS functionalized with histidine or bioactive peptides (COS-HIS, COS-GGGGHexyl, and future COS-GRGDS) was incorporated to confer bioactivity. The dual-function hydrogel forms a semi-interpenetrating network without additives, exhibiting favorable hydration, mechanics, biocompatibility, and bioactivity.

Functionalization was confirmed by ¹H NMR. COS-MA was synthesized using excess methacrylic anhydride, while COS-HIS and COS-GGGGHexyl were obtained via carbodiimide-mediated amide coupling. Photo-rheology and DLP working curves (Beer–Lambert model) were used to optimize printing. Inks containing COS-MA 24%(w/v) or COS-MA 23%+COS-HIS1%(w/v) with LAP 0.5%(w/v) in PBS (pH 7.0) were printed at 100 µm per layer with tartrazine/LAP ratios of 0–5%,and constructs were evaluated for printability, stiffness, and stress relaxation. The hMSC cell line will be encapsulated within the bioink.

COS-MA, COS-HIS, and COS-GGGGHexyl exhibited functionalization levels of 80%, 20%, and 40%, respectively. Optimal printing conditions (2% tartrazine/mol LAP, 15 s/layer) produced high-fidelity structures, including gyroid and logo-shaped scaffolds. COS films gelled in 5.7 ± 0.2 s, unaffected by tartrazine. The printed scaffolds reached 2.4 ± 0.2 MPa stiffness and 33 ± 3.4% stress relaxation, comparable to cartilage, ligaments, and tendons [3].Notably, this natural polymer achieved stiffness typical of synthetic systems.

COS-MA is a promising candidate for DLP-based cartilage tissue engineering, with ongoing studies evaluating post-printing cell viability and incorporating bioactive COS-GRGDS.

Acknowledgements: FAPESP.

References

[1] D. Pal,S. Saha,RSC Adv. 2019,9,28061–28077.

[2] H. G. Hosseinabadi et al., ACS Biomater. Sci. Eng. 2022,8,1381–1395.

[3] C. F. Guimarães et al.,Nat. Rev. Mater. 2020,5,351–370.

1078. Integration of liver tumor organoids and in silico methods to address drug resistance (LIVERtera) - SEMIT

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.

1079. Engineering functional microvasculature: defined ECFC/MSC combinations outperform SVF, ASC and MSC preparations from human adipose tissue

Rafael Moreno Luna¹, Ángela Santos-De-La-Mata¹, Mario Martínez-Torija¹, Francisco J. Espino-Rodríguez², Matilde Castillo-Hermoso¹, María A. Ruiz De Infante¹, Pedro F Esteban³, Eduardo Molina-Holgado³

¹Pathophysiology and Regenerative Medicine Group. Hospital Nacional de Parapléjicos, IDISCAM, SESCAM, Toledo - Spain, ²Plastic and Reconstructive Surgery Service. Hospital Nacional de Parapléjicos, SESCAM, Toledo - Spain, ³Grupo de Neuroinflamación. Hospital Nacional de Parapléjicos, IDISCAM, SESCAM, Toledo - Spain

Despite extensive research on adipose-derived cell therapies, it remains unclear which approach most consistently supports vascular repair or the formation of functional microvascular networks. To identify the most effective strategy, we systematically compared stromal vascular fraction (SVF), adipose-derived stem cells (ASCs), mesenchymal stem cells (MSCs) and defined endothelial colony-forming cell/MSC (ECFC/MSC) combinations. All populations were isolated from the same subcutaneous adipose tissue sample in each donor, enabling rigorous and direct inter-population comparison.

Paired (n = 5) subcutaneous adipose samples from adult donors were processed in parallel. SVF was obtained by enzymatic digestion; ASCs derived from adherent culture; MSCs and ECFCs were purified and expanded separately. Angiogenic and vasculogenic potential was assessed in vivo using subcutaneous Matrigel implants, evaluating host-derived sprouting and the formation of new human vessels. Perfusion of newly formed vessels was confirmed by host erythrocytes and human endothelial markers.

SVF exhibited pronounced heterogeneity, low post-thaw survival and variability, which prevented consistent angiogenesis or vasculogenesis. ASCs showed angiogenic and vasculogenic activity, but outcomes were inconsistent across donors, with perfused human vessels forming only in a subset of implants. MSCs promoted robust sprouting angiogenesis but not vasculogenesis, indicating they cannot assemble functional microvasculature alone. The defined ECFC/MSC 40:60 combination consistently formed interconnected microvessels in vitro and highly organized, perfused vascular networks in vivo, outperforming all other adipose-derived preparations. ECFC/MSC constructs also consistently demonstrated superior cell expansion, enhanced survival following cryopreservation and remarkably uniform behaviour across donors.

These findings indicate that the regenerative potential of adipose tissue does not reside in any single cell population, but emerges from cooperative interactions among specific subsets. By enabling reproducible generation of functional vasculature, defined ECFC/MSC combinations provide a robust platform for vascular tissue engineering. This study was supported by grant PID2022-137080OB-I00 funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU.

1080. Conjunctiva mimetic hydrogel for the delivery of extracellular vesicles to the ocular surface

Yolanda Diebold¹, Yolanda Diebold², Ismael Romero-Castillo¹, Antonio López-García¹, Laura García-Posadas¹

¹Instituto de Oftalmobiología Aplicada (IOBA). Universidad de Valladolid, Valladolid - Spain, ²Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN). Instituto de Salud Carlos III, Madrid - Spain

Purpose: Hydrogels derived from natural extracellular matrix (ECM) components have gained attention in ophthalmology for their suitable properties for ocular surface (OS) applications. We developed a hydrogel based on the conjunctival ECM composition (Conj-ECMh) and assessed its potential for delivering conjunctival mesenchymal stromal cells (Conj-MSC)-derived extracellular vesicles (EVs) to the OS. Final purpose is to achieve a novel regenerative therapy for OS pathologies.

Methods: The Conj-ECMh was prepared using collagen type I, hyaluronan, fibronectin, and heparan sulphate. To facilitate hydrogel visualization in ocular tissues, two dyes (Coomassie blue or Syrian red) were added into the Conj-ECMh. In another set of experiments Conj-MSC-derived EVs, previously isolated by precipitation and labeled with IRDye^® to enable macroscopic identification, were incorporated into the Conj-ECMh prior to gelation. 25-30 μl of Conj-ECMh either stained, blank or loaded with labelled Conj-MSCs EVs (200 μg/ml) were subconjunctivally injected into ex vivo rabbit eyes obtained from a local slaughterhouse. Then, eyeballs were processed for histopathological evaluation.

Results: Stained Conj-ECMh effectively gelled in situ after subconjunctival injection. Sirius red demonstrated hydrogel successful retention at the injection site up to 2 hours. Evaluation of hematoxylin/eosin-stained tissue sections confirmed the presence of the subconjunctival cavity corresponding to the site of Conj-ECMh administration, while in control eyes the conjunctival structure appeared intact. IRDye^® labelling confirmed EV retention within the hydrogel matrix. Conj-ECMh either blank or loaded with labelled EVs also gelled in situ after subconjunctival injection. A fluorescent signal corresponding to labelled EVs was detected in tissue sections at the injection site up to 48 hours; however, no fluorescence was detected in sections from eyes injected with blank hydrogel. These results demonstrate adequate EV delivery.

Conclusions: Conj-ECMh shows promising potential as a delivery platform for mesenchymal EVs in OS therapies, offering efficient in situ gelation and targeted localization.

Funding: RTI2018-094071-B-C21 and PID2023-148252OB-C21, MICIU/AEI/10.13039/501100011033 and FEDER, UE.

1081. Algorithmic design and laser powder bed fusion of woven NiTi bio-metamaterials for cardiovascular repair

Andrés Díaz Lantada¹, Carlos Aguilar Vega¹, Óscar Contreras-Almengor², Mónica Echeverry-Rendón², Jon Molina-Aldareguia²

¹Mechanical Engineering. Universidad Politécnica de Madrid, Madrid - Spain, ²IMDEA Materials Institute, Getafe (Madrid) - Spain

Cardiovascular diseases remain the leading cause of mortality worldwide and cardiovascular devices constitute a dominant segment of the medical device market, presenting a steady growth driven by a rising global prevalence of chronic diseases, an aging population, an increasing demand for minimally invasive procedures and several transformative technological advances, which also enable treatment of neonatal and pediatric patients with congenital defects. In recent decades, minimally invasive transcatheter devices have revolutionized treatment of cardiovascular issues, thanks to incorporation of superelastic alloy structures based on Nitinol or NiTi as biomaterials facilitating percutaneous interventions. Despite remarkable technological advancements in transcatheter device design, currently available solutions, either surgical or transcatheter, still face significant limitations: mass-produced devices lack personalization, existing options restore biological structures mechanically but lack healing ability to prevent fibrosis in surrounding tissues, and employed biodevices do not evolve with patients.

To address these problems, our team presents an algorithmic design strategy for woven NiTi bio-metamaterials, personalized to cardiovascular structures requiring repair, leading to biomedical devices whose mechanical properties mimic compliance and functional gradients in cardiovascular tissues. Personalized designs can be manufactured by laser powder bed fusion, whose fine-tuning for additive processing of NiTi and related postprocesses enables patient-specific solutions based on the mentioned alloy and design strategy. Following this approach, patient-specific minimally invasive surgeries may be fostered for several transcatheter devices, including stents, valve replacements, customized clot retrievers, tapered flow diverters, braided bifurcations, and fenestrated stents. Current capabilities and challenges are discussed and illustrated through innovative prototypes, whose biocompatibility is analyzed by indirect and direct cell cultures employing endothelial and smooth muscle cells, representatives of cell types usually interacting with these devices.

1082. 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.

1083. Bioengineering sclero-corneal limbal substitutes via cell-laden hydrogels functionalized with growth factors

Miguel Pérez-Garrastachu¹, Ainhoa Agirrebengoa-Arrieta², Ander Martin², Maddalen Rodriguez-Astigarraga¹, Cristina Romo-Valera¹, Noelia Andollo¹

¹Department of Cell Biology and Histology/BEGIKER Research Group. University of the Basque Country (UPV/EHU)/Biobizkaia Health Research Institute, Leioa/Barakaldo (Bizkaia) - Spain, ²Department of Cell Biology and Histology. University of the Basque Country (UPV/EHU), Leioa (Bizkaia) - Spain

The sclero-corneal limbus is a transitional region between the sclera and the cornea. It is precisely within this limbal zone where the niche of limbal epithelial stem cells (LESCs) resides. This niche is sustained by a highly specialized architecture (Vogt’s palisades), composed of a unique extracellular matrix as well as supportive stromal and epithelial cell populations and melanocytes. When this anatomical region is damaged, as occurs in limbal stem cell deficiency, the cornea loses its ability to regenerate; moreover, the histological boundary between the sclera and the cornea is lost, allowing the former to invade the latter, ultimately leading to pannus formation and progressive blindness.

Tissue engineering approaches offer potential solutions to restore corneoscleral limbal function, through the development of tissue substitutes capable of re-establishing the native histological organization of the cornea. Our recent work has focused on the use of hydrogels as the core component of such biosubstitutes.

In this study, we examine the behavior of LESCs cultured on methacrylated gelatin and collagen hydrogels in which primary corneal stromal cells have been embedded. Addditionally, these hydrogels have been functionalized with serum from plasma rich in growth factors (sPRGF), to harness its strong therapeutic potential. Our results demonstrate the capacity of these biomaterials to deliver bioactive compounds directly to the surrounding cellular environment. Furthermore, the stromal component embedded within the hydrogels, when stimulated by biochemical cues from sPRGF, enhances the adhesion and proliferation of corneal epithelial cells. Additionally, we investigate the cellular responses under inflammatory conditions, which are characteristic of corneal ulcerative processes and limbal stem cell deficiency.

Collectively, these results support the concept that hydrogels represent a promising strategy as regenerative scaffolds for repairing tissue defects in the cornea and the corneoscleral limbus.

Aknoledgements: This research study was supported by grants from the Department of Health of the Basque Government (2023111027, IT524-22) and the Spanish Ministry (PID2023-152436OB-100). Pérez-Garrastachu, M. was supported by a post-doctoral fellowship Margarita Salas.

1084. Multiscale nonlinear mechanics of collagen I hydrogels under stretch

Elena Montero-Bragado¹, Marc Rico-Pastó², Héctor Sanz-Fraile³, Jordi Alcaraz⁴, Ramon Farré⁵, Jorge Otero⁶, Ignasi Jorba¹

¹Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut. University of Barcelona (UB), Barcelona - Spain, ²Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut. Institute for Bioengineering of Catalonia (IBEC). University of Barcelona (UB), Barcelona - Spain, ³Escola Politècnia Superior d’Enginyeria de Manresa. Universitat Politècnica de Catalunya, Barcelona - Spain, ⁴Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut. Institute for Bioengineering of Catalonia (IBEC). 4Thoracic Oncology Unit, Hospital Clinic Barcelona. University of Barcelona (UB), Barcelona - Spain, ⁵Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut. CIBER de Enfermedades Respiratorias. Institut Investigacions Biomediques August Pi Sunyer (IDIBAPS). University of Barcelona (UB), Barcelona - Spain, ⁶Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut. CIBER de Enfermedades Respiratorias. University of Barcelona (UB), Barcelona - Spain

Successful tissue engineering regenerative strategies require of biomaterials with an appropriate biochemical and biophysical properties, in particular, their multiscale mechanical properties. Natural-derived biomaterials remain the most promising option but demand precise control and characterization of their multiscale mechanical behavior. The objective of the present work was to characterize the multiscale mechanical properties of collagen hydrogels with different crosslinking degrees and applied stretches, and to correlate their microstructure with the resulting mechanical response. Atomic force microscopy (AFM) was used to characterize the mechanical properties at the microscale. A stretching device compatible with simultaneous AFM measurements was first designed and built to explore hydrogel micromechanics under applied stretch. Collagen I pre-gels (9 mg/mL) were mixed with Ru/SPS photocrosslinker. Neutralized mixture was pipeted on top of the stretching device and thermally crosslinked. Afterwards, gels were illuminated with blue light (455 nm) for 0, 5 or 30 minutes. The stretching device was placed on the AFM stage and the hydrogels were measured at strains up to 30% to extract its viscoelastic properties. Macromechanical properties of (un)crosslinked hydrogels were assessed by tensile testing. An analytical model modeling collagen I fibers as springs connected through nodes was developed to correlate macromechanical properties with the microstructure (crosslinking degree) of the hydrogels. Collagen I hydrogels exhibited a baseline stiffness of ∼100 Pa (uncrosslinked), ∼900 Pa (crosslinked, 5 min), and ∼1200 Pa (crosslinked, 30 min). Notably, we report for the first time, AFM measurements of collagen I hydrogels under stretch. The stiffness displayed significant nonlinearity under strain, increasing ∼5 fold at 30% strain, independently of the crosslinking degree. Moreover, the analytical model well predicted the macromechanical properties measured by tensile test, consistent with immunofluorescent image fiber analysis. This study presents the first report of nonlinear micromechanical behavior in collagen hydrogels, offering insights for developing mechanically relevant tissue engineered approaches.

1085. A 3D-bioprinted mechanobiology platform for high-fidelity fibrosis modelling and mechanopharmacology

Kevin Coffey¹, Ailbhe Coughlan², Robert Texidó¹, Nuria Oliva¹

¹Bioengineering. Instituto Quimico de Sarria (IQS), Barcelona - Spain, ²Royal College of Surgeons in Ireland, Dublin - Ireland

Background: Dysregulated mechanosensing, driven by increased tissue stiffness, is a central mechanism of pathological fibrosis. The high failure rate of antifibrotic drugs in clinical trials is linked to the inadequacy of conventional 2D cultures and natural ECM hydrogels, which lack the precise, tuneable mechanical control necessary to accurately model the fibrotic microenvironment [1]. To address this translational bottleneck, a highly reproducible, mechanically tuneable 3D-bioprinted platform based on silk methacrylate (SilMA) and gelatin methacrylate (GelMA) was developed. This system enables the study of stiffness-induced myofibroblast activation by modulating mechanics without altering material composition or cell-adhesion ligand density.

Methods: Bioprinted SilMA scaffolds, formulated with 0–4% GelMA, were fabricated, allowing the storage modulus (G') to be tuned reliably across a pathologically relevant range, from 2 kPa (soft tissue analog) to 50 kPa (stiff fibrotic tissue analog). Human dermal fibroblast viability was confirmed using Presto Blue and fluorescence microscopy after staining with CellTracker, and myofibroblast phenotype transition was evaluated via ACTA2 gene expression and alpha-Smooth Muscle Actin (alpha-SMA) immunofluorescence.

Results: Scaffold stiffness was reliably and quantitatively modulated by controlling SilMA molecular weight and degree of methacrylation. Cells maintained robust metabolic activity across all stiffnesses. Importantly, biological results showed a direct, dose-dependent correlation between matrix stiffness and myofibroblast activation. ACTA2 gene expression increased by over 5-fold in the stiffest scaffolds. Preliminary analysis confirmed that increased stiffness correlated positively with enhanced collagen type I deposition.

Discussion & Conclusion: The 3D-bioprinted SilMA/GelMA platform provides a high-fidelity model that effectively decouples mechanical and biochemical cues, enabling rigorous investigation of stiffness-dependent mechanotransduction. Ultimately, these scaffolds serve as a critical component in the emerging field of mechanopharmacology [2], offering unprecedented capabilities for screening antifibrotic therapies and investigating how the mechanical microenvironment influences drug efficacy.

References: (1) Krishnan, R., et al., Trends Pharmacol Sci 37, 87–100 (2016). (2) Nizamoglu, M. et al., European Respiratory Review 32, 230042 (2023).

1086. 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.