Stem Cell-based Cartilage Regeneration Research Articles

Dual microparticles programmed delivery system regulating stem cell-based cartilage regeneration by cartilage-specific matrix hydrogels

Bone marrow mesenchymal stem cells (BMSCs)-based engineered cartilage usually faces the significant challenge of endochondral ossification tendency in an ectopic environment because of the inevitable vascular infiltration during chondrogenic differentiation and developmental stages. Additionally, current scaffold-free BMSC cartilage regeneration requires a long in vitro preinduction time for chondrogenic differentiation before in vivo transplantation, which limits its application in multiple cartilage defect repair. Therefore, ideal stem cell-based cartilage regeneration needs to meet time-dependent requirements of both early chondrogenic and late anti-angiogenic microenvironments in vivo. Here, we developed a cartilage-specific matrix hydrogel (CMH) with a dual microparticle-based programmed delivery system (dM-PDs) to dynamically regulate in vivo stem cell-based cartilage regeneration without in vitro preinduction. In this study, CMH scaffolds offer a three-dimensional matrix microenvironment for tissue regeneration, while dM-PDs has an early chondrogenic induction function to promote cartilage-specific differentiation and exerts a late anti-angiogenic effect to stabilize the cartilaginous phenotype. Stem cell-based cartilage regeneration was successfully achieved by sequentially dynamic regulation in vivo by dM-PDs, in which TGFβ3-loaded microparticles effectively activated the early-stage TGFβ/Smad signaling pathway and levatinib-loaded microparticles regulated the late-stage VEGF/TIMP signaling pathway. This study demonstrates a universal stem cell-based cartilage regeneration technique for clinical cartilage defects repair.

TNF-α activates RELA expression via TNFRSF1B to upregulate OPA1 expression and inhibit chondrogenic differentiation of human adipose stem cells

BackgroundTumor necrosis factor-alpha (TNF-α), one of the pro-inflammatory cytokines mediating the local inflammatory process in joints, inhibits cartilage formation and has a detrimental effect on stem cell-based cartilage regeneration for the treatment of osteoarthritis (OA). However, the mechanisms behind this inhibitory effect are still poorly understood. Mitochondrial morphological changes mediated by mitochondrial fusion and fission are highly plastic, are quite sensitive to environmental stimuli and play a crucial role in maintaining cell structure and function. In our study, chondrogenic differentiated human adipose stem cells (hADSCs) were exposed to TNF-α and the effect of TNF-α on the ability of hADSCs to chondrogenic differentiate and on mitochondrial fusion and fission was observed and analyzed. The aim was to investigate the role and mechanisms of mitochondrial fusion and fission regulation in the chondrogenic differentiation of hADSCs under normal conditions and under exposure to TNF-α.MethodsWe used flow cytometry to identify hADSCs immunophenotypes CD29, CD44, CD34, CD45, and HLA-DR. Alcian blue staining and Sirius red staining were used to observe the formation of proteoglycans and collagen during the chondrogenic differentiation of hADSCs, respectively. The mRNA and protein expression levels of the cartilage formation marker SOX9, type II collagen (COL2A1), and Aggrecan were measured by real-time fluorescent quantitative PCR (RT-qPCR) and western blot, respectively. The fluorescent probes MitoTracker® Red CMXRos and JC-1 were used to visualize mitochondria morphology and detect mitochondrial membrane electricity (MMP). Affymetrix PrimeView™ chips were used for gene expression profiling.ResultsThe results showed that the chondrogenic differentiation of hADSCs was inhibited in the presence of TNF-α that optic atrophy 1 (OPA1) expression was significantly upregulated and mitochondria were prolonged and interconnected during this process. Gene microarray and RT-qPCR data showed that the presence of TNF-α led to increased expression of TNFα receptor 2 (TNFRSF1B) and RELA during chondrogenic differentiation of hADSCs.ConclusionsTNF-α inhibits chondrogenic differentiation of human adipose stem cells by activating RELA expression through TNFRSF1B upregulating OPA1 expression thereby increasing mitochondrial fusion.

Evaluation of Autoregulated RUNX2 Suppression on Mesenchymal Stem Cell-based Cartilage Regeneration in Complex Pathological Environments

Evaluation of Autoregulated RUNX2 Suppression on Mesenchymal Stem Cell-based Cartilage Regeneration in Complex Pathological Environments

Inhibition of LDL receptor-related protein 3 suppresses chondrogenesis of stem cells, inhibits proliferation, and promotes apoptosis

Articular cartilage defects remain the most common and challenging joint disease. Cartilage lacks the self-healing capacity after injury due to its avascularity. Recently, stem cell-based therapy has been applied for cartilage regeneration. However, the critical target for stem cells during chondrogenesis remains unclear. We first reported that LDL receptor-related protein 3 (LRP3) expression was markedly increased during chondrogenesis in stem cells. Furthermore, LRP3 was an effective chondrogenic stimulator, as confirmed by knockdown and overexpression experiments and RNA sequencing. In addition, inhibition of LRP3 suppressed proliferation and induced apoptosis. Therefore, our study first defined a new chondrogenic stimulator, LRP3, with detailed clarification, which provided a novel target for stem cell-based cartilage regeneration.

Stem cell-based cartilage regeneration technology

Stem cell-based cartilage regeneration technology

Nanoparticle-Mediated TGF-β Release from Microribbon-Based Hydrogels Accelerates Stem Cell-Based Cartilage Formation In Vivo.

Conventional nanoporous hydrogels often lead to slow cartilage deposition by MSCs in 3D due to physical constraints and requirement for degradation. Our group has recently reported macroporous gelatin microribbon (μRB) hydrogels, which substantially accelerate MSC-based cartilage formation in vitro compared to conventional gelatin hydrogels. To facilitate translating the use of μRB-based scaffolds for supporting stem cell-based cartilage regeneration in vivo, there remains a need to develop a customize-designed drug delivery system that can be incorporated into μRB-based scaffolds. Towards this goal, here we report polydopamine-coated mesoporous silica nanoparticles (MSNs) that can be stably incorporated within the macroporous μRB scaffolds,and allow tunable release of transforming growth factor (TGF)-β3. We hypothesize that increasing concentration of polydopamine coating on MSNs will slow down TGF- β3 release, and TGF-β3 release from polydopamine-coated MSNs can enhance MSC-based cartilage formation in vitro and in vivo. We demonstrate that TGF-β3 released from MSNs enhance MSC-based cartilage regeneration in vitro to levels comparable to freshly added TGF-β3 in the medium, as shown bybiochemical assays, mechanical testing, and histology. Furthermore, when implanted in vivo in a mouse subcutaneous model, onlythe group containing MSN-mediated TGF-β3 release supported continuous cartilage formation, whereas control group without MSN showed loss of cartilage matrix and undesirable endochondral ossification. The modular design of MSN-mediated drug delivery can be customized for delivering multiple drugs with individually optimized release kinetics, and may be applicable to enhance regeneration of other tissue types.

An Injectable Bioorthogonal Dextran Hydrogel for Enhanced Chondrogenesis of Primary Stem Cells.

The purpose of this study is to corroborate the feasibility of an injectable dextran hydrogel, in situ formed through a catalyst-free bioorthogonal reaction, for cartilage regeneration with adult chondrocytes or adipose-derived stem cells (ADSCs). To this end, an injectable dextran (dextran-AN) hydrogel encapsulating the rabbit adult chondrocytes or rabbit ADSCs (rADSCs) or human ADSCs (hADSCs) was successfully prepared by simply incubating the cells with dibenzocyclooctyne-dextran conjugate and azide-dextran conjugate under physiological conditions through a copper-free bioorthogonal reaction without using additional reagents. The possibility of dextran-AN hydrogel/adult rabbit chondrocyte constructs for in vivo chondrogenesis was first assessed in a rabbit osteochondral defect model. Data indicated that the hydrogel was biocompatible against surrounding tissue and could promote adult chondrocyte survival and in vivo chondrogenesis within 8 weeks. Next, in vitro and in vivo chondrogenetic differentiation of rADSCs encapsulating in dextran-AN hydrogel was studied systemically. Compared to the agarose hydrogel as a positive control, the dextran-AN hydrogel elicited comparable ability to support in vitro chondrogenesis of rADSCs within 3 weeks but caused better cartilage regeneration within 6 weeks in a rabbit osteochondral defect model. Furthermore, miR29A downregulating of hADSCs was applied for in vivo chondrogenesis by subcutaneous in situ formation of dextran-AN hydrogel containing the hADSCs in a balb/c nude mouse model. Within 4-week regeneration, the hydrogel group illustrated upregulated mRNA expression level of chondrogenesis-related biomarkers. In addition, improved production of neocartilage extracellular matrices (type-II collagen and glycosaminoglycan) was found for miR29A-downregulatd hADSC group in comparison with miR29A-upregulated hADSC and untreated hADSC control groups. The results of this study show that bioorthogonal dextran-based hydrogel is capable of supporting in vitro and in vivo chondrogenesis of chondrocytes and human stem cells, thus highlighting high feasibility of bioorthogonal hydrogels for clinical translation in stem cell-based cartilage regeneration.

Link Protein N-Terminal Peptide as a Potential Stimulating Factor for Stem Cell-Based Cartilage Regeneration.

Background Link protein N-terminal peptide (LPP) in extracellular matrix (ECM) of cartilage could induce synthesis of proteoglycans and collagen type II in cartilaginous cells. Cartilage stem/progenitor cells (CSPCs), the endogenous stem cells in cartilage, are important in cartilage degeneration and regeneration. We hypothesized that LPP could be a stimulator for stem cell-based cartilage regeneration by affecting biological behaviors of CSPC. Methods CSPCs were isolated from rat knee cartilage. We evaluated the promoting effect of LPP on proliferation, migration, and chondrogenic differentiation of CSPCs. The chondrogenic differentiation-related genes and proteins were quantitated. Three-dimensional culture of CSPC was conducted in the presence of TGF-β3 or LPP, and the harvested pellets were analyzed to assess the function of LPP on cartilage regeneration. Results LPP stimulated the proliferation of CSPC and accelerated the site-directional migration. Higher expression of SOX9, collagen II, and aggrecan were demonstrated in CSPCs treated with LPP. The pellets treated with LPP showed more distinct characteristics of chondroid differentiation than those with TGF-β3. Conclusion LPP showed application prospect in cartilage regeneration medicine by stimulating proliferation, migration, and chondrogenic differentiation of cartilage stem/progenitor cells.

Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation

BackgroundMesenchymal stem cells (MSCs) hold promising translational potential in cartilage regeneration. However, the efficacy of MSC-based tissue engineering is not satisfactory in the treatment of cartilage defect because of the inevitable cellular functional changes during ex vivo cell expansion. How to maintain the chondrogenic capacity of MSCs to improve their therapeutic outcomes remains an outstanding question.MethodsBone marrow-derived MSCs were firstly primed in chondrogenic induction medium which was then replaced with normal growth medium to attain the manipulated cells (M-MSCs). Methacrylated hyaluronic acid (MeHA) was synthesized as a scaffold to encapsulate the cells. The MSC- or M-MSC-laden constructs were treated with dynamic compressive loading (DL) in a bioreactor or with free loading (FL) for 14 days. Afterwards, the constructs were implanted in nude mice or rat models of osteochondral defects to test their efficiency in cartilage regeneration or repair.ResultsData showed that the resulting M-MSCs exhibited superior chondrogenic differentiation potential and survivability compared with untreated MSCs. More importantly, we found that DL significantly promoted neocartilage formation in the MeHA hydrogel encapsulated with M-MSCs after 30 days of implantation in nude mice. Furthermore, the constructs laden with M-MSCs after DL for 14 days significantly enhanced cartilage healing in a rat model of osteochondral defect.ConclusionsFindings from this study highlight the importance of maintaining chondrogenic potential of MSCs by in-vitro chondrogenic preconditioning and a synergistic effect of mechanical stimulation in cartilage engineering, which may shed light on the stem cell-based tissue engineering for cartilage repair.

Stepwise preconditioning enhances mesenchymal stem cell-based cartilage regeneration through epigenetic modification.

This study is to investigate the functions and underlying mechanisms of mesenchymal stem cells (MSCs) underwent stepwise preconditioning in chondrogenic medium before expansion, then further explore their therapeutic effects in a surgically induced osteoarthritis (OA) model. MSCs isolated from the adult rats expressing Green Fluorescence Protein (GFP) were incubated in basal medium or primed in chondrogenic medium before expansion. The multipotency including cell proliferation, differentiation, and survivability was compared between chondrogenic manipulated MSCs (M-MSCs) and untreated MSCs. Methylation modification of Nanog and Oct4 were detected by bisulfite genomic sequencing. Loss-of-function phenotype in M-MSCs induced by shNanog was also observed. Then the therapeutic effect of the cells was evaluated in a surgically induced OA rat model by single intraarticular injection. The injected GFP-labeled cells in the joints were monitored invivo. These rats were sacrificed and subjected to histological examinations and microstructural analysis after 4 weeks. We found that cell clonogenicity, proliferation, survivability, and chondrogenic property were enhanced after stepwise preconditioning. We then further found that the expression level of Nanog and Oct4 was temporarily increased in the M-MSCs. Results of epigenetic analysis revealed that demethylation happened in Nanog and Oct4 after the stepwise preconditioning. Results of invivo imaging showed more GFP-labeled cells in the M-MSCs-injected group. And results of histology and micro-CT analysis also indicated a superior therapeutic effect of M-MSCs on the surgically induced-OA. These findings indicated a feasible method to obtain a cell population with high survivability and chondrogenic commitment for the treatment of OA.

Mesenchymal Stem Cell-Based Cartilage Regeneration Approach and Cell Senescence: Can We Manipulate Cell Aging and Function?

Aging is the most prominent risk factor triggering several degenerative diseases, such as osteoarthritis (OA). Due to its poor self-healing capacity, once injured cartilage needs to be reestablished. This process might be approached through resorting to cell-based therapies and/or tissue engineering. Human mesenchymal stem cells (MSCs) represent a promising approach due to their chondrogenic differentiation potential. Presently, in vitro chondrogenic differentiation of MSCs is limited by two main reasons as follows: aging of MSCs, which determines the loss of cell proliferative and differentiation capacity and MSC-derived chondrocyte hypertrophic differentiation, which limits the use of these cells in cartilage tissue regeneration approach. The effect of aging on MSCs is fundamental for stem cell-based therapy development, especially in older subjects. In the present review we focus on homeostasis alterations occurring in MSC-derived chondrocytes during in vitro aging. Moreover, we deal with potential cell aging regulation approaches, such as cell stimulation through telomerase activators, mechanical strain, and epigenetic regulation. Future investigations in this field might provide new insights into innovative strategies for cartilage regeneration and potentially inspire novel therapeutic approaches for OA treatment.

Sustained Release of Bone Morphogenetic Protein 2 via Coacervate

Category: Basic Sciences/Biologics Introduction/Purpose: Currently, there is no cure for osteoarthritis (OA) with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and can undergo chondrogenic differentiation when cultivated in chondrogenic medium in vitro and can differentiate into chondrocytes and repair injured articular cartilage (AC) in vivo. MDSCs retrovirally transduce to express chondrogenic proteins (BMPs) to differentiate into chondrocytes and enhance cartilage repair in vivo. Gene therapy is a promising approach to promote the chondrogenic potentials of MDSCs for AC repair. We have developed a unique sustained growth factor delivery platform comprised of native heparin and a synthetic polycation incorporated with BMP2 (BMP2 coacervate) which can sustain delivery of BMPs to stimulate the chondrogenesis of MDSCs for AC repair. Methods: MDSC were stimulated in vitro with single dose of free BMP2, multi-dose BMP2, BMP2 coacervate, coacervate alone (vehicle only), or in the absence of BMP2 and coacervate (control). BMP2 effects on MDSCs were evaluated by RT-PCR. 20μl of a MIA solution was injected into the knee joint of C57B6 mice to induce osteoarthritic lesions. Two weeks after MIA injection, 20μl of PBS (PBS control), 1×106 MDSCs with 1μg of free BMP2 (MDSC-free BMP), or MDSCs with 1μg BMP2-coacervate (MDSC- BMP coacervate) were injected into the knee joints of the OA injured mice. After 4 weeks, macroscopic and histologic evaluations of cartilage regeneration were conducted. Results: mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (P < 0.05). Multi-dosage free BMP2 demonstrated significantly higher Aggrecan expression compared to single dose free BMP2 (p < 0.05). Col2A and aggrecan expression in the BMP coacervate group was superior to both single and mult-dose free BMP2 delivery (p < 0.05) (Fig 1A). Histologic examination demonstrated superior cartilage repair and integration in the BMP2 coacervate group (Fig. 1B). Conclusion: This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle- derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Prolonged delivery of BMP2 via coacervate led to enhanced MDSC chondrogenesis in vivo and improved cartilage regeneration in vivo. Our results demonstrate an effective method for prolonged exposure to BMP2 and is more clinically translatable.

Sustained Release of Bone Morphogenetic Protein 2 via Coacervate improves Muscle Derived Stem Cell Mediated Cartilage Regeneration in MIA-induced Osteoarthritis

Objectives:Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model.Methods:mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies.Results:Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (P<0.05). Multi-dosage free BMP2 demonstrated significantly higher Aggrecan expression compared to single dose free BMP2 (p<0.05). Col2A and aggrecan expression in the BMP coacervate group was superior to both single and mult-dose free BMP2 delivery (p<0.05) (Fig 1A). Histologic examination demonstrated superior cartilage repair and integration in the BMP2 coacervate group (Fig. 1B).Conclusion:This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

Supramolecular GAG-like Self-Assembled Glycopeptide Nanofibers Induce Chondrogenesis and Cartilage Regeneration.

Glycosaminoglycans (GAGs) and glycoproteins are vital components of the extracellular matrix, directing cell proliferation, differentiation, and migration and tissue homeostasis. Here, we demonstrate supramolecular GAG-like glycopeptide nanofibers mimicking bioactive functions of natural hyaluronic acid molecules. Self-assembly of the glycopeptide amphiphile molecules enable organization of glucose residues in close proximity on a nanoscale structure forming a supramolecular GAG-like system. Our in vitro culture results indicated that the glycopeptide nanofibers are recognized through CD44 receptors, and promote chondrogenic differentiation of mesenchymal stem cells. We analyzed the bioactivity of GAG-like glycopeptide nanofibers in chondrogenic differentiation and injury models because hyaluronic acid is a major component of articular cartilage. Capacity of glycopeptide nanofibers on in vivo cartilage regeneration was demonstrated in microfracture treated osteochondral defect healing. The glycopeptide nanofibers act as a cell-instructive synthetic counterpart of hyaluronic acid, and they can be used in stem cell-based cartilage regeneration therapies.

Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

As a tissue-specific stem cell for chondrogenesis, synovium-derived stem cells (SDSCs) are a promising cell source for cartilage repair. However, a small biopsy can only provide a limited number of cells. Cell senescence from both in vitro expansion and donor age presents a big challenge for stem cell based cartilage regeneration. Here we found that expansion on decellularized extracellular matrix (dECM) full of three-dimensional nanostructured fibers provided SDSCs with unique surface profiles, low elasticity but large volume as well as a fibroblast-like shape. dECM expanded SDSCs yielded larger pellets with intensive staining of type II collagen and sulfated glycosaminoglycans compared to those grown on plastic flasks while SDSCs grown in ECM yielded 28-day pellets with minimal matrix as evidenced by pellet size and chondrogenic marker staining, which was confirmed by both biochemical data and real-time PCR data. Our results also found lower levels of inflammatory genes in dECM expanded SDSCs that might be responsible for enhanced chondrogenic differentiation. Despite an increase in type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Wnt and MAPK signals were actively involved in both expansion and chondrogenic induction of dECM expanded cells. Since young and healthy people can be potential donors for this matrix expansion system and decellularization can minimize immune concerns, human SDSCs expanded on this future commercially available dECM could be a potential cell source for autologous cartilage repair.

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell-material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell-cell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin-specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration.

Hyaluronan initiates chondrogenesis mainly via CD44 in human adipose-derived stem cells

Cell-matrix adhesion is one of the important interactions that regulates stem cell survival, self-renewal, and differentiation. Our previous report (Wu SC, Chang JK, Wang CK, Wang GJ, Ho ML. Biomaterials 31: 631-640, 2010) indicated that a microenvironment enriched with hyaluronan (HA) initiated and enhanced chondrogenesis in human adipose-derived stem cells (hADSCs). We further hypothesize that HA-induced chondrogenesis in hADSCs is mainly due to the interaction of HA and CD44 (HA-CD44), a cell surface receptor of HA. The HA-CD44 interaction was tested by examining the mRNA expression of hyaluronidase-1 (Hyal-1) and chondrogenic marker genes (SOX-9, collagen type II, and aggrecan) in hADSCs cultured on HA-coated wells. Cartilaginous matrix formation, sulfated glycosaminoglycan, and collagen productions by hADSCs affected by HA-CD44 interaction were tested in a three-dimensional fibrin hydrogel. About 99.9% of hADSCs possess CD44. The mRNA expressions of Hyal-1 and chondrogenic marker genes were upregulated by HA in hADSCs on HA-coated wells. Blocking HA-CD44 interaction by anti-CD44 antibody completely inhibited Hyal-1 expression and reduced chondrogenic marker gene expression, which indicates that HA-induced chondrogenesis in hADSCs mainly acts through HA-CD44 interaction. A 2-h preincubation and coculture of cells with HA in hydrogel (HA/fibrin hydrogel) not only assisted in hADSC survival, but also enhanced expression of Hyal-1 and chondrogenic marker genes. Higher levels of sulfated glycosaminoglycan and total collagen were also found in HA/fibrin hydrogel group. Immunocytochemistry showed more collagen type II, but less collagen type X, in HA/fibrin than in fibrin hydrogels. Our results indicate that signaling triggered by HA-CD44 interaction significantly contributes to HA-induced chondrogenesis and may be applied to adipose-derived stem cell-based cartilage regeneration.