Differentiation of bone marrow mesenchymal stem cells into chondrocytes in transforming growth factor beta-1 culture medium in two-dimensional culture in vitro

Abstract：BACKGROUND: Bone marrow mesenchymal stem cells may directionally differentiate into chondrocytes.OBJECTIVE: To establish an inducing system for differentiation of bone marrow mesenchymal stem cells into chondrecytes in two-dimensional culture in vivo, to analyze tbe best concentration of transforming growth favor beta-1 (TGF-β1) to induce tbe differentiation and the correlated influence factors for the directional differentiation, and to observe the changes of cell form and phenotype. DESIGN: Randomized controlled animal study.SETTING: Animal Experimental Center, Guiyang Medical College.MATERIALS: This study was performed at Animal Experimental Center, Gulyang Medical College from September 2005 to November 2006. Twenty-four 4-week-old male SD rats weighing (98.23±7.97) g were provided by Animal Center of Guiyang Medical College. The animal experiment received informed consent from the local ethic committee.METHODS: Bone marrow mesenchymal stem cells were separated and purified from femur and tibia using attachment culture method. Surface antigen of the fourth-generated bone marrow mesenchymal stem cells was determined by using flow cytometry. Subsequendy, the fourth-generated bone marrow mesenchymal stem cells were induced with TGF-β1 culture medium (1, 5, 10, 15, and 20 μ g/L) in two-dimensional culture. On the other hand, bone marrow mesenchymal stem cells in the control group were induced with negative culture medium. Collagen type Ⅱ was qualitatively determined by using immunohistochemical method after two weeks, and glycosaminoglycan expression of extracellular matrix was quantitatively detected by using dimethyl-methylene-blue colorimetric method.MAIN OUTCOME MEASURES: Surface antigen, collagen type Ⅱ, and glycosaminoglycan expression of extracellular matrix.RESULTS: Bone marrow mesenchymal stem cells were separated and purified by using attachment culture method. The fourth-generated bone marrow mesenchymal stem cells were positive for surface antigen CD44, but negative for surface antigen CD34 and CD45. Cell form was irregular two weeks after induction. Immunohistocbemical staining on collagen type Ⅱ indicated that positive cells were observed in TGF- β 1 (1, 5, 10, 15, and 20 μg/L) groups. On the other hand, dimethyl-methylene-blue colorimetric method demonstrated that glycosaminoglycan expression of extracellular matrix in the TGF-β1 groups were significantly higher than in the control group (P < 0.01). In particular, glycosaminoglycan expression in the 10 μ g/L TOF- β 1 group was significantly higher than in other concentration groups (P < 0.01), and the capability of excreting glycosaminoglycan of extracellular matrix was positively correlated with cell densities (r=0.822, P < 0.01). CONCLUSION: A high cell density is beneficial to differentiation of bone marrow mesenchymal stem cells into chondrocytes induced by TGF-β1 (10 μg/L) in two-dimensional culture.