Mesenchymal stem cells (MSC) are considered suitable sources for cell-based therapies in cartilage engineering. However, their capacities to differentiate into chondrocytes and regenerate a fully functional and mature tissue need to be evaluated in a pre-clinical model. The objectives of this work was to establish a sheep model of cartilage repair and aimed at 1) characterizing the bone marrow-derived MSC and 2) evaluating their differentiation potential after implantation.
Methods. Bone marrow aspirates were seeded at the density of 1-3 x 105 mononuclear cells/cm2. Colonies of adherent cells formed after 10-15 days and were then passaged at the density of 103 cells/cm2. Chondrogenesis was induced by culturing the cells in micropellets in presence of TGF-β3 for 21 days. The expression of the differentiation markers (type II collagen, aggrecan) was determined by RT-PCR analysis and proteoglycan secretion was assessed by safranin O staining. Osteogenesis and adipogenesis were induced by culture in various media containing or not dexamethasone, NaH2PO4 or BMP-2. Expression of osteopontin and PPAR-γ was assessed by RT-PCR. Mineralization was detected by alizarin red S staining and lipid droplets were detected by oil red O staining.
Results. The CFU-F potential was determined to be 1-2 cells in 105 mononuclear cells. Cell proliferation was maintained for more than 10 passages with a doubling time of 12h during the first 3 months. The adherent cells were shown to be positive for CD44, CD105low, vimentin and, negative for CD34 and CD45, at least until passage 5. The cells were able to support hematopoiesis and the trilineage differentiation capacities of the cells were confirmed by the expression of specific differentiation markers and histological staining. Their immunosuppressive potential was evaluated in mixed lymphocyte reaction where the ovine cells were able to inhibit the proliferation of T cells induced by allogeneic cells.
In vivo, a preliminary experiment was performed to model a cartilage defect on 6 patellaes of 3 animals. Two partial-thickness lesions were created in the inner part of the patellaes and lesions were left empty or filled with autologous cells alone or, in presence of chitosan powder or TGFβ-3 embedded within a fibrin clot. Histological analysis confirmed the formation of cartilage after 3 months when MSC were combined with chitosan.
Conclusion. For the first time, the adherent cells of the bone marrow from sheep has been characterized in detail. This study provides evidence that the adherent cells isolated from bone marrow may be referred as MSC. The pre-clinical model of cartilage repair should prove useful to assess the regeneration potential of autologous MSC.