[Experiment of bone morphogenetic protein 2 induced chondrogenic differentiation of human Achilles tendon-derived stem cells in vitro].

OBJECTIVE: To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro.

METHODS: Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR.

RESULTS: Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P < 0.05).

CONCLUSION: BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.