| Objective The present study aimed at establishing a method to isolate, expand and identify mesenchymal stem cells (MSCs); describing their biological characteristics, and exploring the optimal combination of growth factors, including transforming growth factor-β1(TGF-β1), insulin-like growth factor-I (IGF-I) and bone morphogenetic protein(BMP-2), for inducing chondrogenesis of MSCs in vitro monolayer culture.Methods Density gradient centrifuge and anchoring screening was used to isolate MSCs.These MSCs were cultured in vitro.Cell morphology were investigated. At passage 4, MSCs were induced to differentiate into osteoblasts and adipocytes in different induction medium. Alizarin red stain and Oil red stain were used to identify osteoblasts and adipocytes, respectively. At passage 4 to 6, MSCs were induced to differentiate into chondrocytes in the medium containing the following growth factors, TGF-β1, BMP-2 and IGF-I, either alone or in combination. On day 21 of induction, we detected two cartilage specific markers in differentiated cells. Alcian blue stain was used for aggrecan and RT-PCR for collagen type II.Results MSCs isolated from bone marrow were of small size, high nucleus/cytoplasma ratio, spindle, with whirlpool growth pattern. Histochemistry showed that MSCs were differentiated into adipocytes, osteocytes and chondrocytes in special medium in vitro.The average level of Collagen type II mRNA measured by RT-PCR showed that TGF-β1 group was higher than IGF-I+BMP-2 group (0.48 vs 0.15, P<0.01). TGF-β1 group and TGF-β1+IGF-I group were similar (0.47 vs 0.48, P=0.104). TGF-β1 group was lower than TGF-β1+BMP-2 group (0.48 vs 0.65, P<0.01). TGF-β1+BMP-2+IGF-I group was the highest (0.80 compared with other groups, P<0.01).Conclusion MSCs could be isolated and purified with density gradient...
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