Our study indicates that synovial activation rapidly drives anti-inflammatory and protective effects of intra-articularly deposited ASCs in experimental OA which is reflected by decreased S100A8/A9 levels.

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Backgound and Objectives Synovitis is evident in a substantial subpopulation of patients with early osteoarthritis (OA) and has been associated with development of pathophysiology. Recently we have shown that adipose-derived stem cells (ASC) inhibit joint destruction after local application to knee joints with experimental OA. In the current study we explored the effect of synovitis on the immunomodulatory capacity of ASCs after local administration in two experimental OA models differing in synovitis. Material and Methods ASCs were isolated from fat surrounding the popliteal lymph nodes. ASCs were injected into knee joints after induction of collagenase-induced OA (CiOA) characterised by synovitis and surgically induced DMM model in which synovitis is scant. Synovial activation, chondrogenesis in ligaments and osteophytes were measured using histology. Cytokines in synovial washouts and serum were determined using Luminex. Active TGFβ was measured using the CAGAluc assay. Results ASCs injected into knee joints at different time-points after induction of DMM (day 7, day 14 or day 7 and day 14) had no effect on development of ligament damage or osteophyte formation. In contrast, ASC treatment of collagenase-induced OA knee joints, rapidly inhibited synovitis and ligament damage when administered at day 7 after induction. Washouts of synovium taken at different time points after injection of ASCs (6 hrs, day 2, day 14 and day 42) showed significantly decreased levels of IL-1β and S100A8/A9 already at 48 hrs after ASC treatment. No effect was found on levels of active TGFβ. Serum levels of S100A8/A9 were significantly decreased (85% lower) at day 14 whereas IL-1 levels were not detectable at that time-point. Next, we explored the effect in CiOA in a condition with less synovial inflammation. Synovial thickness at day 42 was 62% lower when compared to the former study. Injection of the same dose of ASCs at day 7 after induction of CiOA, only marginally inhibited synovial thickening when measured at day 42. Serum levels of S100A8/A9 were low at day 14 (around 50 ng/ml compared to 800 ng/ml in the first experiment) and were not altered by the ASC treatment. Chondrogenesis in collateral and cruciate ligaments but also osteophyte formation at the bone margins was also not diminished. Conclusion Our study indicates that synovial activation rapidly drives anti-inflammatory effects of ASCs after local administration in murine OA knee joints with synovitis protecting against development of ligament damage and osteophyte formation.

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...