A Pre-Clinical Animal Study for Zonal Articular Cartilage Regeneration Using Stratified Implantation of Microcarrier Expanded Zonal Chondrocytes.

OBJECTIVE: The zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. In this study, the efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a porcine chondral defect model. DESIGN: Autologous chondrocytes extracted from articular cartilage in the non-weight bearing trochlea region of the knee were subjected to an expansion-sorting strategy, integrating dynamic microcarrier (dMC) culture, and spiral microchannel size-based zonal chondrocyte separation. Zonal chondrocytes were then implanted as bilayered fibrin hydrogel construct in a porcine knee chondral defect model. Repair efficacy was compared with implantation with cell-free fibrin hydrogel and full thickness (FT) cartilage-derived heterogenous chondrocytes. Cartilage repair was evaluated 6 months after implantation. RESULTS: Sufficient numbers of zonal chondrocytes for implantation were generated from the non-weight bearing cartilage. Six-month repair outcomes showed that bilayered implantation of dMC-expanded zonal chondrocytes resulted in substantial recapitulation of zonal architecture, including chondrocyte arrangement, specific Proteoglycan 4 distribution, and collagen alignment, that was accompanied by healthier underlying subchondral bone. CONCLUSION: These results demonstrate that with appropriate expansion and isolation of zonal chondrocytes, the strategy of stratified zonal chondrocyte implantation represents a significant advancement to Autologous Chondrocyte Implantation-based cartilage regeneration, with the potential to improve the long-term integrity of the regenerated tissues.

Electrospun fibers enhanced the paracrine signaling of mesenchymal stem cells for cartilage regeneration.

BACKGROUND: Secretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage. METHODS: Conditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated. RESULTS: We showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production. CONCLUSIONS: This study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

Human mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

An analysis of soft tissue allograft anterior cruciate ligament reconstruction in a rabbit model: a short-term study of the use of mesenchymal stem cells to enhance tendon osteointegration.

BACKGROUND: Soft tissue allografts are essential for revision and multiple ligament surgeries in the knee, where donor-site morbidity is an issue. However, the use of allografts is associated with a higher failure rate of osteointegration. Mesenchymal stem cells (MSCs) are investigated as potential agents to enhance bone tunnel and tendon healing. PURPOSE: This study was conducted to analyze the effect of coating allografts with MSCs on the quality and rate of osteointegration at the allograft tendon and bone interface, and the biomechanical properties of these enhanced anterior cruciate ligament (ACL) grafts compared with controls. STUDY DESIGN: Descriptive laboratory study. METHODS: Bilateral ACL reconstructions using Achilles tendon allografts were performed in 36 rabbits. On 1 limb, the graft was coated with autogenous MSCs in a fibrin glue carrier, while the contralateral limb served as a control with no MSCs. The reconstructions were assessed histologically and biomechanically at 2, 4, and 8 weeks. RESULTS: At 8 weeks, histologic analysis of the controls revealed the development of mature scar tissue resembling Sharpey fibers spanning the tendon-bone interface. In contrast, the MSC-enhanced reconstructions showed a mature zone of fibrocartilage blending from bone to the allograft, strongly resembling a normal ACL insertion. On biomechanical testing, the MSC-enhanced grafts had significantly higher load-to-failure rates than controls. However, the stiffness and Young's modulus were lower in the treatment group. CONCLUSIONS: The application of MSCs at the allograft tendon-bone interface during ACL reconstruction results in the development of an intervening zone of fibrocartilage. The use of MSCs to enhance allograft osteointegration is a novel method offering the potential of more physiologic and earlier healing, although further investigation must be conducted to improve the biomechanical strength. CLINICAL RELEVANCE: Mesenchymal stem cells can improve the biologic properties of soft tissue allograft healing. Combined with the decrease in donor-site morbidity, allografts are a viable choice for the sports medicine surgeon.