Chondrogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Induced By BMP-2under Hypoxic Microenvironment In Vitro

Objective：To investgate the role of bone morphogenetic protein-2(BMP-2)combined with hypoxic microenvironment in chondrogenic differentiation of rat bonemarrow mesenchymal stem cells (BM MSCs) in vitro.Methods：BMSCs were differentiated in the presence or absence of BMP-2under normoxic or hypoxic microenviroment for7-21days. Experiment is dividedinto four groups:A)Normoxia control group,B) Normoxia,BMP-2group,C)Hypoxiacontrol group,D)Hypoxia,BMP-2group. Then the cellular morphology was observedby microscope, the specific gene and protein expression were analysed by AlcianBlue immunohistochemical staining,reverse transcription-PCR,and Western blot.Results：After21days,BMP-2combined hypoxia-induced BMSCs(group D)became round,cell density was significantly reduced,and lacuna-like cells waswrapped in cell matrix,while this typical changes were not observed in groups A,Band C. Alcian blue staining in Group D was significantly more blue than that of othergroups, and with the induction time extension, the blue became darker, and to21da-ys, the cells were stained into pieces of deeply stained blue, the other experim-entalgroups at each time point were only observed a small amount of light stained bl-ue. Compareed with other experimental groups, the expression levels of collag-entype Ⅱ protein in group D were significantly higher (P<0.05). Compared with Gro-up A and B,HIF-1alpha protein expression levels of Group C and D were significan-tly higher (P <0.05).The cartilage differentiation-related mRNA indicators,such asCOL2α1,Agg,were the highest expression in group D,while the bone differentiation-related mRNA indicators,such as COL1α1,ALP, RUNX2,were the highestexpression levels in group B. Compared with the normoxic groups (A and B),thehypoxia-related mRNA indicators HIF-1α in group C and D significantly increased (P<0.05).Conclusion：Combined with hypoxia,BMP-2can be well induced differentiationof BMSCs to the cartilage phenotype,and inhibits osteoblast phenotype differentiation,HIF-1α is an important signaling molecule that is involved in the possible mechanism to promote Chondrogenic differentiation process;...