gms | German Medical Science

Objectives: Multipotent mesenchymal stem cells (MSC) bear the potential to be used in regenerative medicine, e.g. for cartilage repair. But not much is known about the capacity of MSC for cartilage regeneration under osteoarthritic (OA) conditions. To improve the quality of MSC-derived cartilage-like tissue a better understanding of influences of the OA microenvironment like soluble factors secreted from neighboring cells and modulating effects of the OA cartilage tissue interface is necessary. Using an in vitro coculture model, the influence of OA versus "normal" cartilage explants on miRNA expression of cocultured chondrocytes and chondrogenically differentiating MSC is investigated and cell culture supernatants are analyzed for soluble factors like matrix metalloproteinases and interleukins.

Methods: Chondrocytes, isolated from OA cartilage and bone marrow-derived MSC are embedded in fibrin gels and cultured either on OA cartilage explants (coculture) or without explants (monoculture) as controls for 7 and 28 days in chondrogenic medium containing TGF-β. Expression of miR-124a, miR-675 and miR-29b is normalized to U6 snRNA expression. At day 28, supernatants of MSC in monocultures, cocultures with OA- and normal (derived from trauma patients) cartilage explants and of OA or normal cartilage explants covered with cell free fibrin gels are analyzed by a multiplex immunoassay including a multitude of analytes such as MMPs and interleukins. Data are validated by zymography and ELISAs.

Results and Conclusion: Coculture with OA cartilage leads to an increased expression of miR-124a, miR-675 and miR-29b in chondrocytes and MSC compared to monocultured cells. Expression of miR-675 is significantly increased in cocultured chondrocytes at day 7. Cocultured MSC (day 7 and 28) and chondrocytes (day 7) show significantly higher miR-124a expression while miR-29b expression is significantly increased only in chondrocytes at days 7 and 28. IL-8 concentration in cell culture supernatants from normal cartilage (w/o MSC) is higher than in corresponding supernatants from OA cartilage. Supernatants of cocultured MSC with normal or OA cartilage contain higher IL-8 level compared to MSC in monoculture but lower IL-8 level compared to normal or OA cartilage covered with cell free fibrin gels. MSC in coculture with normal or OA cartilage secrete less MMP-9 than MSC in monoculture whereas supernatants from normal or OA cartilage w/o MSC contain equal MMP-9 level.

Coculture of OA cartilage with chondrocytes/MSC results in an increased expression of miRNAs which may regulate essential genes for chondrogenesis however the targets of these miRNAs remain to be verified. Cocultured MSC reduce IL-8 release from cartilage indicating a regulatory effect on cartilage metabolism. Coculture with normal or OA cartilage has an inhibitory effect on MMP-9 secretion by MSC which might influence matrix composition and thus their chondrogenic differentiation.