Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) do not have ideal specific surface markers, and there is no unified standard to identify BMSCs. Most identification depends on its morphological levels, functional characteristics and differentiational phenotype occurred during the culture.
OBJECTIVE: To culture, amplify and identify rabbit BMSCs in vitro, and observe the feasibility of differentiation into chondrocytes. 
METHODS: Rabbit BMSCs were extracted through the density gradient centrifugation. Cell morphology was observed using the inverted microscope. Cells were cultured in α-MEM medium containing 10% fetal bovine serum, and cells were counted daily to observe cell growth speed. Expression of CD45, CD29, CD44 on P0 and P3 BMSCs was detected using flow cytometry. Well-grew rabbit BMSCs at P3 were obtained and induced using special media supplemented with 10% fetal bovine serum, transforming growth factor (TGF)-1 10 μg/L, insulin-like growth factor (IGF)-Ⅰ 110 μg/L, transferring 6.25 mg/L, dexamethasone
10 mmol/L and vitamin C 0.05mmol/L. Morphological changes were observed using the inverted microscope. Specificity of cartilage collagen type Ⅱ was detected using immunohistochemical method.
RESULTS AND CONCLUSION: The cultured cells in the fusiform long, fibroblast growth of samples. The cells grow rapidly. Flow cytometry results showed that for the P0 cells about 62.4% of the cells expressed CD29, about 60.3% of the cells expressed CD44, 2.79% of the cells expressed CD45, and for the P3 cells, about 99.9% of the cells expressed CD29, about 99.6% of the cells expressed CD44, 2.62% of the cells expressed CD45. After the BMSCs in induction, the ability of expansion was decreased obviously, and the morphology changes after 21 days, and the collagen type Ⅱbecome dyeing. Results suggest that BMSCs can be successfully isolated and amplified by density gradient centrifugation. The third passage of BMSCs shows high purity. Under appropriate conditions, the BMSCs have the potential of differentiating itself into chondrocytes.