Intra-articular injection of adipose-derived stem cells prevents cartilage destruction, new bone formation in the ligaments and lowers synovial activation

Abstract

Methods: Chondrocytes, MSC and a mixture of chondrocytes and MSC (1:1) were embedded in a 3D-fibringel and either kept in monoculture or were cocultured with cartilage or subchondral bone explants (obtained from arthroplastic surgery due to OA) for up to 28 days. Matrix production and chondrogenic characteristics of cells were determined immunohistologically (collagens I, II and III) and histologically (aggrecan) after 7, 14, 21 and 28 days. Gene expression of SOX9, COL1A1, COL2A1, COL3A1 and COL10A1 and of two miRNAs was analyzed after 7 and 28 days with quantitative real-time PCR. Protein expression in the cell-fibrin constructs was assessed with ELISA (collagen I / II), western blot (Sox9, phospho-Sox9 and collagen III) and glycosaminoglycan (GAGs) production was analyzed using a DMMB-assay. The culture supernatant was analyzed for collagen fragments by a hydroxyproline assay and for PTHrP with an ELISA. We separated the supernatants from MSC -monocultures versus cartilage cocultures with SDS-PAGE and identified differentially secreted proteins with MALDI-TOF. Results: MSC underwent chondrogenic differentiation with respect to collagen II expression in all culture conditions. In comparison to monocultures, collagen I, II and III gene expression and protein production was suppressed when MSC or chondrocytes were cocultured with cartilage or subchondral bone explants. Mixed cultures of chondrocytes and MSC showed a reduced collagen II production in all culture regimes. GAG production and total collagen content in the supernatant was altered in bone explant cocultures only. Phospho-Sox9 and PTHrP protein expression were not significantly affected by any culture condition. However, preliminary data indicate a correlation between miRNA expression and regulation of sox9 protein. MALDI-TOF analysis revealed several proteins/ factors present in coculture supernatants but not in the monocultured controls (e.g. stromelysin-1, chondroadherin, fibronectin and SPARC). Conclusions: Chondrogenic differentiation of MSC is affected by the microenvironmental conditions. Cartilage and subchondral bone explants from OA-patients suppress collagen I, II and III gene expression and production in MSC. This observation might be partly dependent on regulation of sox9 protein biosynthesis by miRNA. Future studies with ovine tissue will delineate if healthy cartilage/subchondral bone provokes different responses in MSC compared to OA derived tissues. Chondroadherin, fibronectin, SPARC and stromelysin-1 among other, yet unidentified proteins, were detected in coculture supernatants and might be associated with the reduced collagen content in OA-cartilage/subchondral bone cocultures of MSC. We will study a putative link between these factors and regulation of collagen expression and degradation in further analyses. T3 EFFECT THROUGH SCR2 ON CHONDROGENESIS IN VITROPurpose: There are evidences about the thyroid hormone role into chondrogenesis but its molecular implication is not clear. The current study treats to answer this question workin...