gms | German Medical Science

Introduction: Damaged articular cartilage has limited regeneration capacity mainly due to lack of chondroprogenitor cells. Pluripotent mesenchymal stem cells (MSCs) are the only known adult chondrocyte progenitor cells. MSCs may be used for therapeutic purposes e.g. for the repair of focal defects in articular cartilage. However, information about mutual paracrine effects of surrounding tissue and newly transplanted implant is not sufficient. In-vitro studies on chondrogenesis in MSCs cocultured with cartilage may not only give information regarding molecular control but it may also be directly relevant to the clinical application. We have determined paracrine effects of cartilage on chondrogenic differentiation of MSCs. Here, we present identity and temporal secretion pattern of signalling factors derived from cartilage during coculture with MSCs.

Methods: A coculture system is set up in culture plates holding inserts of 1 µm pore size. Cartilage chips are kept in the bottom of the wells and rat MSCs suspended in alginate beads are kept in inserts. Culture is carried out in serum-free chondrogenic medium with and without TGFß-3 for three weeks. Gene expression rates are established with quantitative RT-PCR. Protein expression level is measured with immunoblotting, immunofluorescence and C14-proline assays. Soluble signaling factors were detected using an antibody array from RayBiotech™.

Results: Increased gene expression for collagen II, aggrecan and COMP concurrent with upregulation of transcription factor sox9, indicates cartilage specific extracellular matrix formation during the time course of culture. Monocultured MSCs express and synthesize collagen X at day 21 while cartilage suppresses collagen X production. Array analysis shows a time dependent decrease in VEGF secretion and increase in TIMP-1 secretion by cartilage accompanied by TIMP-2 production from day 14 onwards. Increasing amounts of pro-MMP-2 are detected in coculture supernatants from day 7 on, however, relative activation status remains unchanged compared to day 1. In absence of cartilage active MMP-2 is not detectable after day 7. Neither cartilage nor MSCs secrete pro-inflammatory cytokines except for monocyte chemoattractant protein, MCP-1 which is restricted to the first week of culture. Discussion: Early elevated gene expression level of sox9 indicates onset of chondrogenesis in cocultured MSCs. Culture time dependent progressive elevation of TIMP-1 and TIMP-2 secretion was reciprocated by less active MMP-2 in the culture supernatant. Suppression of collagen X in presence of cartilage depicts delayed hypertrophy in our coculture system. Elevated concentration of TIMPs in the late stage of coculture may account for delayed hypertrophy. We suggest that secretion of VEGF, MMP-2, TIMP1 and -2 from differentiated chondrocytes may play a regulatory role in early induction of chondrogenesis and in late hypertrophy, important for the fate of differentiating MSCs.