Tissue Engineering Part A: Research Advances

Skin aging involves changes in extracellular matrix components, such as wrinkles and pigmentation. Caviar extract (CE) is a promising compound for skin rejuvenation, but effective topical delivery requires optimized carriers. This study evaluated polyvinyl alcohol/carboxymethyl chitosan (PVA/CMC) hydrogels loaded with CE at concentrations of 2%, 3.5%, and 5% as scaffolds to influence the epithelial differentiation of adipose-derived mesenchymal stem cells (ADMSCs). Hydrogels were synthesized using a freeze-thaw method and characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy, swelling and degradation tests, and mechanical analysis. Biocompatibility and cell migration were assessed using MTT and scratch assays; at the same time, expression of cytokeratin-18 (CK-18) and pan-cytokeratin (pan-CK) was measured via reverse transcription-quantitative polymerase chain reaction and immunocytochemistry (ICC), respectively. FTIR confirmed successful CE incorporation, and SEM revealed a porous structure. Hydrogels with 3.5% and 5% CE demonstrated a good balance between swelling and degradation over 336 h. The biocompatibility tests showed that 5% CE supported enhanced long-term cell growth. The scratch assay indicated improved cell migration, and transcriptional analysis revealed significantly higher CK-18 levels in ADMSCs treated with PVA/CMC/CE 5% (p < 0.001). ICC results showed significantly higher pan-CK expression at 3.5% CE (41.82%) and 5% CE (48.16%), suggesting that CE promotes repair processes. These findings suggest that 5% CE-loaded PVA/CMC hydrogel could be an effective option for skin regeneration and antiaging.

Graphical Abstract

This is a visual representation of the abstract.

While it is generally accepted that tendon healing following widespread extracellular matrix trauma is limited, tenocytes are thought to have the capacity to repair small amounts of microdamage generated through activities of daily living to maintain the mechanobiological function of the tendon. Despite this, few studies have directly studied the mechanisms governing this process. To address this, we developed a tunable ex vivo model of extracellular matrix microdamage in live tendon explants that enables us to track both clearance of denatured collagen and closure of a microsized defect in the tendon matrix. The purpose of this study was to controllably induce varying levels of localized microdamage to the tendon explants and identify (1) if thresholds for repair exist and (2) whether repair mechanisms are dependent on initial damage size. We found that within 3 weeks, all tendon explants were able to clear damaged matrix to some extent regardless of the damage size. Interestingly, larger injuries resulted in a more robust rate of damaged matrix clearance in the later weeks, while smaller injuries exhibited a more consistent rate that led to full clearance in two explants. While all groups demonstrated some closure of the hole over the course of culture, only a small subset of explants with smaller initial hole sizes demonstrated nearly complete closure. Greater than 50% clearance of denatured collagen was typically associated with an accompanying closure of the defect, suggesting a strong relationship between clearance and closure. Overall, our work characterizes a novel laser-induced microdamage model, which will be a powerful asset for investigating mechanisms of damage accumulation and/or repair important to maintaining the mechanobiological function of the tendon, as well as identifying local tendon-specific factors that can be leveraged for therapeutics.

Traditional approaches to tissue-engineered small intestine (TESI) require implanting a construct in the omentum of the abdominal cavity to allow for vascularization of the construct and tissue maturation. A second operation is then needed to implant the construct in continuity with the intestine. The timing of the second operation has historically been chosen based on predetermined biological endpoints. Currently, no well-validated method has been used to noninvasively monitor for vascular maturity which would better inform when the second operation should be performed. We hypothesized that photoacoustic imaging (PAI) could serve as a tool to noninvasively monitor regional and temporal changes in vascular maturity in scaffolds implanted in the omentum. For this pilot study, tubular scaffolds used for TESI were fabricated with electrospinning and implanted in the omentum of Sprague–Dawley rats for 1 and 2 months. Scaffolds were imaged with PAI and tissue oxygenation and hemoglobin concentration were quantified. PAI was then correlated with gross observations of vascularization at each time point. PAI was able to determine regional and temporal changes in tissue oxygenation and hemoglobin concentration. Specifically, the oxygenation and hemoglobin concentration of the top wall of the construct showed better vascular maturity compared with the bottom wall. In addition, vascular maturity seemed to improve in the top wall from 1 to 2 months. The bottom wall was not well covered by the omentum and thus did not vascularize as well as the top wall. The PAI findings were confirmed on gross examination of the scaffolds and upon quantification of the histological analysis of endothelial cell density. Thus, PAI may serve as a critical tool for monitoring vascular maturity in TE, specifically within the abdomen. This will be a critical tool in the preclinical development and clinical translation of TESI.

The liver is a multifunctional organ essential for detoxification, protein synthesis, glucose regulation, bile secretion, and drug metabolism. However, persistent damage leads to chronic inflammation, excessive extracellular matrix deposition, and progressive fibrosis culminating in cirrhosis, for which liver transplantation remains the only curative option. Yet, the scarcity of donor organs and risks of immune rejection underscore the urgent need for physiologically relevant in vitro liver models to investigate pathogenesis and facilitate therapeutic discovery. Current two-dimensional cultures and animal models fail to recapitulate the multicellular interactions that govern liver homeostasis and disease progression. Hepatocytes (Heps) constitute the primary parenchymal population, while hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) coordinate fibrogenic, angiogenic, and regenerative responses. Dysregulation of this crosstalk drives fibrosis and architectural collapse, highlighting the necessity for multicellular systems that mimic native liver complexity. In this study, we established a three-dimensional (3D) microtissue platform that recapitulates both the structural and functional characteristics of the human liver. Human-induced pluripotent stem cells (hiPSCs) were differentiated into Heps, HSCs, and LSECs, which were subsequently cocultured within a self-organizing 3D microenvironment. We successfully reconstructed a miniaturized liver model that maintains hepatic functionality and exhibits steatogenic responses to alcohol exposure. This hiPSC-derived microtissue enables the modeling of chronic liver diseases, intercellular signaling, and fibrogenic pathways, thereby providing a translationally relevant system for mechanistic studies, drug toxicity testing, and personalized therapeutic development.

Normothermic machine perfusion (NMP) has emerged as a new technique for organ storage before liver transplantation, making viability testing of the organ feasible and possibly allowing for reconditioning of marginal liver grafts. Erythrocytes form a substantial component in the composition of NMP perfusates. The optimal hematocrit (HCT) for preserving organ function during NMP and the effects of the HCT on metabolic graft function and parameters commonly used to determine organ viability are still unclear. We retrospectively analyzed our rat NMP database of 123 perfusions and identified a possible link between HCT and liver damage parameters in 34 cases following a dual perfusion protocol. To further investigate this relationship, we perfused a total of 16 rat livers for 6 h each in our rat liver NMP setup with four different HCTs at 10%, 16%, 22%, and 28%. We performed blood gas analysis and measured common viability and damage parameters such as bile production, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, histological analysis, as well as enzyme-linked immunosorbent assay and colorimetric assay analysis for reactive oxygen species, glycogen content, and hypoxia-inducible factor (HIF), was performed. The perfusate HCT significantly impacted ALT and LDH levels during NMP of rat liver grafts, with lower HCT values corresponding to decreased damage parameters. We investigated differentiating oxygenation levels and identified a possible cause for our observations in HIF-1α stabilization in lower HCT groups. Furthermore, we demonstrate that HCT affects key viability parameters such as lactate, pH levels, and bile production. Our findings illustrate the importance of adequate HCT levels during NMP and propose mechanisms influencing the perfusion outcome.

Severe burn injuries pose significant clinical challenges, with high risks of infection, excessive inflammation, and impaired wound healing. Mesenchymal stem cells (MSCs) have shown regenerative and antimicrobial potential; however, their therapeutic efficacy is constrained by poor survival and engraftment. Here, we demonstrate that priming MSCs with insulin-secreting cells (ISCs) and encapsulating them in hydrogels (HEMI) enhances their regenerative function, leading to accelerated healing of full-thickness burns in a porcine model. By 5 weeks, 80% of HEMI-treated wounds achieved complete closure, compared with 50% of MSC-only wounds and 0% of standard-of-care control wounds. All groups exhibited partial wound closure over time, but no control wounds reached complete closure within the study period. Histological analysis revealed complete epidermal and dermal regeneration with minimal fibrosis in HEMI-treated wounds. Single-cell RNA sequencing and differentially abundant sequencing analysis identified distinct MSC subpopulations whose relative abundance and transcriptional profiles differed between insulin-primed and control MSCs, with insulin priming promoting pathways involved in extracellular matrix stabilization, immune modulation, and oxidative stress resistance. Our findings suggest that insulin priming enhances MSC-mediated tissue repair via paracrine mechanisms, providing a clinically translatable strategy for improving burn treatment and regenerative medicine applications.

For addressing the challenges regarding muscle injuries, 3D printing has been a promising technique to fabricate patient-specific scaffolds and effectively guide myotube alignment. Although methacrylate-conjugated gelatin (GelMA) is widely used as an ink material for 3D printing because of its facile photo-crosslinking and cell-adhesive properties, its intrinsic low viscosity and weak mechanics require high concentrations of the polymer for 3D printing and matching with tissue-like modulus, while its limited tissue adhesion further restricts its applicability in on-muscle printing. In this study, we propose a printable and bioadhesive hydrogel ink (PBAink) with low polymer concentration of alginate tethered with phenylborate and methacrylate (AlMABA), which exhibits a storage modulus similar to that of muscle tissues and undergoes rapid crosslinking within 90 s under blue light irradiation, making it suitable for 3D printing. Additionally, it exhibits low swelling under physiological conditions and good biocompatibility, owing to its excellent hydrophilic properties imparted by phenylborate groups, making it a suitable material for direct on-muscle printing. Notably, because dynamic bonds between cis-diols and phenylborate groups are formed in phosphate-buffered saline environments, we optimized the salt concentration of the buffer solution mixed with AlMABA to enhance cohesion; this led to the development of PBAink, which enhanced fidelity of printing at low concentrations, while the methacrylate groups ensured structural stability via photo-crosslinking. Moreover, PBAink exhibits tissue-adhesive properties compared with methacrylate-conjugated alginate and GelMA, supporting the direct on-muscle printing and conformal integration of the printed hydrogel with the muscle tissue. The PBAink exhibited intriguing cell-adhesive properties, inducing C2C12 clustering, while also promoting cell spreading. Finally, these features contributed to increased cell density and enhanced F-actin coverage on 3D-printed PBAink scaffolds, thereby highlighting its potential as an effective alternative to conventional GelMA-based inks.

Stem cell-derived extracellular vesicles (EVs) play a crucial role in intercellular communication and reflect the functional characteristics of their parent cells. Despite the significant therapeutic potential of these EVs, the molecular features of EVs derived from different stem cells and their relationship with tissue specificity remain underexplored. In this study, we conducted integrated proteomic and transcriptomic analyses of EVs derived from six dental stem cells (DSCs) and three systemic stem cells (SSCs) to investigate the role of EVs in tissue-specific molecular transfer. This study found that although DSCs and SSCs share 92.1% of the core proteome, the EVs derived from each stem cell type displayed distinct molecular signatures. EVs from DSCs were enriched in signaling molecules, reflecting their parent cells’ roles in local tissue repair, whereas EVs from SSCs carried mitochondrial and metabolic proteins, indicating their preferential involvement in metabolic regulation. Furthermore, we uncovered that the parent cells transferred 83% of the core proteins to their EVs in a tissue-specific pattern, ensuring that dental EVs retain prominent signaling functions. Thus, understanding the relationship between EVs and their parent stem cells with respect to tissue origin would be helpful to harness EVs as targeted therapeutic agents, particularly in oral regenerative medicine.

Bone is a common site of breast cancer metastasis, which dramatically increases fracture risk. Recent in vivo bone metastasis studies show that mechanical loading is osteoprotective; however, little is known about how loading regulates breast cancer cell function in the unique bone mechanical environment, especially in combination with radiotherapy, one of the first-line treatments for advanced breast cancer patients. Here, we characterize the breast cancer cell response to a range of bone-mimicking fluid shears and determine how irradiation further modulates one candidate gene: SERPINE1. We found that irradiation, regardless of dosage, modulates SERPINE1 expression and is sensitive to the timing of administration. Additionally, protein expression of SERPINE1 accompanies a protumorigenic gene expression profile, which is elevated with higher-magnitude fluid shear stresses in a bone-mimicking 3D environment. Thus, we postulate that plasminogen activator inhibitor-1 (PAI-1) (encoded by SERPINE1) is a critical growth factor contributing to osteolytic lesion development in the bone metastatic vicious cycle of breast cancer.

To develop bone grafts with superior osteoconductivity, we have fabricated unidirectionally connected porous carbonate apatite (CAp) via gelatin gelation combined with freeze-drying. A composite of calcium carbonate (CaCO₃) and gelatin was first gelled at low temperature and then subjected to directional freeze-drying to create well-aligned macroporous channels. The samples were then sintered to remove the organic components and subsequently phosphatized to convert the composition into CAp. The resulting material exhibited a porosity exceeding 80% with fully interconnected unidirectional pores favorable for cell migration and vascular infiltration. Bone regeneration was evaluated by implanting the material into rabbit femoral bone defects; a comparison was also made with nonoriented porous controls. Histological and radiographical analyses demonstrated that the unidirectional porous structure significantly enhanced directional ingrowth of new bone tissue and accelerated early-stage bone formation relative to the control. This study demonstrates that unidirectional porous CAp fabricated via a gelatin-based freeze-casting method is promising for bone regeneration, particularly in clinical applications requiring guided bone formation.

In tendons, ligaments, and menisci, collagen fibers running the length of the tissue are the primary source of strength and function. Cells assemble these fibers hierarchically from nanometer-wide fibrils into larger fibers and fascicles, increasing in size throughout development and with mechanical loading. These fibers largely do not regenerate after injury or with repair, limiting recovery options. Engineered replacements are a promising treatment option; however, it remains a challenge to produce the hierarchical collagen fibers essential to tissue strength, limiting their applications. To better repair, regenerate, and engineer these tissues, we must better understand how cells regulate hierarchical fiber formation and maintenance. It is well established that mechanical cues are critical for cell-driven hierarchical fiber formation, which cells sense through several mechanisms, such as integrin-mediated adhesions, cell-to-cell connections, mechanosensitive ion channels, primary cilia, and caveolae. These mechanisms of mechanosensation have been well studied at the fibril scale of collagen organization, but as tissues mature, the loading environment becomes more complex, with cells experiencing increasing secondary shear and compressive loads generated by the developing hierarchical structure. Mechanical cues in this environment are likely sensed through several pathways, each likely playing a role in tissue maturation and injury. There remains a clear gap in our understanding of the later stages of hierarchical fiber formation, which are crucial to better understand since large hierarchical fibers dominate the human musculoskeletal system. Here, we review the role of mechanobiology in hierarchical fiber development and maintenance, and highlight what still needs further research to better regenerate fibers in engineered replacements or in vivo after injury. A better understanding of the mechanisms by which cells form hierarchically organized collagen fibers could help to overcome the limitations of current tissue engineering techniques and help to create functional repairs and replacements.

Because articular cartilage (AC) lacks inherent repair capacity, research has focused on translating tissue-engineered cartilage to the clinic. Toward this, rapid and nondestructive methods would be useful for determining in-process and release characteristics during the manufacture of tissue-engineered products. The current work aims to introduce a Raman-based methodology for nondestructive qualitative and quantitative characterization of tissue development using AC. First, Raman shifts associated with critical biochemical components of AC, with particular emphasis on DNA, glycosaminoglycans (long chains of sugar molecules and a key component of cartilage), total collagen, as well as pyridinoline (a marker of collagen crosslinking and maturation), were collated. Next, verification of the molecular spectroscopic biomarkers was conducted by temporally tracking tissue maturation/development of nascent and mature AC, establishing a temporal reference dataset. Finally, validation was performed by correlating the spectroscopic biomarkers with traditional photometric biochemical assays and mass spectrometry. The results presented here include a Maturity Index for quantification of tissue development/maturation. Strong correlations were found between nondestructive spectroscopic-based measurements and destructive (photometric and mass spectrometric) measurements with high linearity for both nondestructive Raman (R² > 0.96) and destructive biochemical (R² > 0.97) assays, respectively. Uses of the proposed rapid and nondestructive method include in-line quality assessment (in which the sample is not removed from the process stream) to monitor the manufacturing of tissue-engineered medical products. This study shows that Raman spectroscopy has the capacity of being a powerful tool for nondestructive quality control and assurance in traditional biomanufacturing workflows, and the approach taken here may also be utilized as a template and research tool for studies on the development of other native and engineered tissues.

Volumetric muscle loss (VML) injuries result in an irrecoverable loss of muscle mass and function. VML injury causes loss of both contractile tissue and associated neuromuscular junctions (NMJs). Biosponge (BSG) scaffolds, composed of gelatin, collagen, and laminin-111, have improved recovery following VML. However, improvements in NMJ quantity were not observed. Glial cell line-derived neurotrophic factor (GDNF) is known to promote motor unit survival and stimulate neurite outgrowth. In this work, BSG scaffolds were electrostatically coupled with GDNF via gelatin nanoparticles (GNPs) to support myofiber regeneration and preserve NMJs post-VML in a rodent model. In vitro determination of release kinetics revealed an initial burst release of surface-bound GDNF with almost an equivalent amount of electrostatically bound GDNF retained within the BSG post 1 week of incubation at 37°C in phosphate-buffered saline. To create the VML injury in male Lewis rats (10–12 weeks old), ∼20% of the muscle mass was removed from the tibialis anterior (TA) muscle of both hindlimbs. Relative to BSG + GNP alone, treatment with BSG + GNP + GDNF showed a significant increase (∼25%) in peak isometric torque at 6 weeks postinjury. Qualitative and quantitative histological analysis of NMJs revealed an enhanced overlap between pre and postsynaptic structures in the BSG + GNP + GDNF group. Additionally, the incorporation of GDNF slowed BSG remodeling and degradation. Overall, these results suggest that BSG-mediated GDNF delivery is an effective strategy for mitigating NMJ loss and enhancing muscle recovery following VML.

Glioblastoma (GBM) tumors are characterized by an excess of extracellular glutamate, one important source of which is the tumor cells themselves. This abundance of glutamate promotes GBM proliferation, migration, and therapeutic resistance, and causes excitotoxicity in nearby neurons. However, despite glutamate’s clear role in promoting GBM aggression, the exact mechanisms through which excess glutamate drives these phenotypes, particularly three-dimensional (3D) invasion, remain incompletely understood. To address this gap, we used a 3D brain-mimetic hyaluronic acid (HA) hydrogel to investigate the role of glutamate signaling in GBM 3D invasion. We demonstrate that inhibiting the glutamate N-methyl-d-aspartate receptor (NMDAR) reduces invasion from 3D tumorspheres, a result that is reproducible across multiple continuous culture models and a patient-derived xenograft cell line. We then conducted glutamate-driven invasion studies in 3D HA-based devices that can be microdissected to isolate and differentially analyze invasive and noninvasive cells. Transcriptomic analysis of invasive, noninvasive, and drug-treated populations of cells reveals that NMDAR inhibition suppresses several pathways associated with the mechanobiology of invasion, including matrix remodeling and collagen deposition. Correspondingly, supplementation with exogenous collagen VI partially rescued 3D invasion. Our work speaks to the potential value of biomaterial platforms for identifying the autocrine and paracrine mechanisms through which neurotransmitters fuel GBM invasion.

Maternal and fetal health during pregnancy is closely tied to contractile modulation of the uterine myometrium, as dysfunctional contractions underlie pregnancy complications such as preterm labor and postpartum hemorrhaging. Structural abnormalities increase the risk for pregnancy complications, yet the complex interplay between biochemical and biomechanical cues in the myometrial microenvironment is not well defined. Significant gaps in our understanding of myometrial physiology and the lack of physiologically relevant research tools to enable systematic investigation have resulted in a largely ineffective and nonspecific therapeutic landscape for myometrial contractile modulation during pregnancy. We address this unmet need by developing a widely accessible approach for in vitro hydrogel platform fabrication to advance the study of agonist-mediated calcium responses in myometrial cells. We directly fabricated polyacrylamide hydrogels in polypropylene multiwell plates and Petri dishes and validated their mechanical properties and use as a mechanically tunable cell culture substrate. Using “physiological” matrix conditions during pregnancy, a fluorescent calcium mobilization assay was implemented in a plate-reader-based workflow to determine the dose sensitivity of myometrial cells to the endogenous agonist oxytocin. Using hydrogels with elastic moduli spanning physiological, pathological, and supraphysiological conditions in the myometrium, we assessed stiffness-mediated effects in myometrial cell behavior, including agonist-mediated calcium responses. We observed the mechanosensitive modulation of calcium response curve amplitude in oxytocin-stimulated myometrial cells. Myometrial cell behavior on mechanically tunable substrates was compared against widely used tissue culture plastic, and the observed changes to myometrial cell morphology and calcium responses highlight the significant influence of supraphysiological substrates. To explore complex pathological microenvironments, myometrial cells were exposed to the proinflammatory trigger lipopolysaccharide (LPS) before agonist stimulation. In our studies, LPS exposure altered calcium responses during oxytocin stimulation in cells cultured on supraphysiological substrates. Altogether, we present an easily adoptable in vitro hydrogel platform with scalable fabrication and versatile application use, including new directions in myometrial mechanobiology.

The clinical success of bone regeneration relies on developing a functional, vascularized bone. Insufficient vascularization of tissue constructs remains a challenge in stem cell-based approaches, leading to poor graft integration and necrosis of newly formed bone. To overcome current challenges, using pre-committed stem cells for endothelial lineages is a novel approach to developing a vascularized tissue construct. Human gingiva-derived mesenchymal stem cells (GMSCs) are a unique cell population that is readily accessible, has a high proliferation rate, and exhibits multipotent differentiation. This study aimed to investigate the in vitro differentiation potential of GMSCs into the endothelial lineage. GMSCs were induced with 0, 10, 50, or 100 ng/mL recombinant vascular endothelial growth factor (VEGF) for 1 week. The relative mRNA expressions of vascular cell adhesion molecule 1, protocadherin 12, VEGF receptor 1 (fms-like tyrosine kinase 1), VEGF receptor 2 (kinase insert domain receptor), and platelet endothelial cell adhesion molecule-1 were measured by quantitative reverse transcriptase polymerase chain reaction. The expression of all endothelial marker mRNAs was significantly upregulated in a dose-dependent manner in GMSCs induced by VEGF, and maximum expression was observed at 50 ng/mL VEGF induction. A Matrigel assay demonstrated the tube-forming ability of pre-differentiated GMSCs. Our findings demonstrated that GMSCs have the potential to differentiate into endothelial progenitor-like cells. Thus, our study identifies potential options for using GMSCs as an autologous or allogeneic stem cell source for craniofacial bone regeneration.

Vascularization and perfusion are essential for tissue engineering of large-scale bioartificial tissues and organs. Both fluid flow and hypoxia have been shown to influence capillary tube formation in bioartificial matrices. However, the ideal timing and combination of these two factors for optimizing capillary tube formation in tissue engineering approaches still remains unclear to date. Capillary tube formation was evaluated in 3D flow chamber wells, in which human microvascular endothelial cells (HMVEC) and adipogenous stem cells (ASCs) suspended in fibrin-based tissue matrices were exposed to fluid flow and hypoxia under defined conditions. Here, both factors were either applied alone or in combination, either from the beginning of the incubation period or after initial capillary tube formation on day 3. Capillary tube formation was analyzed by fluorescence microscopy and quantified two times on days 3 and 6. Furthermore, large-scale fibrin matrices surrounded a perfused microchannel under selected conditions. Exposure of HMVEC in fibrin-based matrices to flow and hypoxia alone or in combination immediately from the beginning of the in vitro culture resulted in impaired capillary tube formation compared with static or normoxic culture conditions in all settings. When initiated after initial tube formation under static conditions for 3 days, exposure to physiological fluid shear stress with or without parallel exposure to hypoxia was associated with significantly increased network complexity. The inhibition of capillary tube formation by continued fluid flow could be compensated for by adding exposure to hypoxia after 3 days of initial tube formation. The impact of both fluid flow and hypoxia was highly dependent on the timing of onset as well as their application alone or combined. Both fluid flow and hypoxia were identified as potent tools for regulating capillarization in vascular tissue engineering, but timing and combination are crucial for optimized in vitro tube formation.

Chondrogenesis by chondrogenic progenitor cells (CPCs) has emerged as a promising avenue for articular cartilage regeneration and early-stage treatment of posttraumatic osteoarthritis. The mechanism of chondrogenesis involves the differentiation of CPCs residing in the cartilage extracellular matrix (ECM). CPC chemotaxis to damaged or degenerated cartilage, followed by in situ proliferation, is an essential first step in restoring tissue integrity. Extracellular vesicles (EVs) have gained significant attention in regenerative medicine due to their several advantages, such as intercellular communication, minimally invasive administration, and reduced immune rejection. In particular, chondrocyte-derived small extracellular vesicles (CC-sEVs) contain highly and differentially expressed microRNAs that enhanced chondrogenesis in our previous studies. In this study, we aimed to evaluate the effects of CC-sEV on CPC homing and chondrogenic differentiation in both monolayer and three-dimensional (3D) hydrogel culture systems. Treatment with 1 × 10¹⁰/mL CC-sEV (CC-sEV-H) significantly enhanced CPC chemotaxis and proliferation compared with untreated control. Moreover, reactive oxygen species production involved in cartilage degradation was reduced in CC-sEV. Gene expression analysis revealed that CC-sEV upregulated chondrogenic markers, including SRY-box transcription factor 9, collagen type 2 alpha 1, and aggrecan and downregulated catabolic markers, including a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), matrix metalloproteinases 3 (MMP3), and MMP13. In a fibrin/hyaluronate hydrogel culture system, CC-sEV-H was effective in promoting ECM formation via glycosaminoglycan and collagen depositions. Thus, our findings from in vitro experiments revealed that CC-sEVs could significantly attract CPCs and induce chondrogenesis.