Experimental study on of Rhesus monkey MSCs cultivation and differentiation into osteoblasts in vitro

[Objective]To investigate the growth,expansion and differentiation into osteoblasts of non-human primate bone marrow mesenchymal stem cells(MSCs).The BMSCs were isolated from adult Rhesus monkeys,cultured and induced in vitro. [Methods]The Rhesus monkey bone marrow-derived MSCs were isolated and purified by density gradient centrifugation.We investigated the effect of cell culture condition,such as seeding density and interval of replacing medium.Osteoblasts were induced and differentiated by cultivation of confluent cells in the presence of osteogenic supplement(containing 10-7 mol/L dexamethasone,50 mg/L ascorbic acid,and 10 mmol/L beta-glycerophosphate) or 100 ng/ml recombinat human bone morphogenetic protein-2.Alkaline phosphatase activity,secretion of amino-terminal propeptide of type I procollagen,typeⅡprocollagen and osteocalcin were analyzed to characterize the osteoblasts and the effects of induction between two supplements were compared.[Results]Inverted microscope showed the splitting and proliferation of Rhesus monkey bone marrow-derived MSCs isolated and purified by density gradient centrifugation.It was found that MSCs expressed a small quantity alkaline phosphatase(ALP) activity and secrete less calcium.The condition of cell culture with 10×104 cells/cm2 to 30×104 cells/cm2 seeding density and semi-quantity medium replacement at 48 h and total medium replacement every 3 days thereafter showed good effects on cell proliferation.Cells induced and differentiated by osteogenic differentiation or recombinat human bone morphogenetic protein-2 from MSCs had the same morphologic feature with osteoblasts in vivo.Cells were alkaline phosphatase(ALP) positive with type I procollagen secretion,and no type Ⅱ procollagen secretion.There was an apparent increase in alkaline phosphatase activity and osteocalcin content at 3 days with a little increase at 14 days after cell fusion.There were no statistical differences in alkaline phosphatase activity and osteocalcin content at the same stage during MSCs differentiation into osteoblasts using osteogenic supplement(containing 10-7 mol/L dexamethasone,50 mg/L ascorbic acid,and 10 mmol/L beta-glycerophosphate) or 100 ng/mL recombinat human bone morphogenetic protein-2.[Conclusion]Rhesus monkey bone marrow-derived MSCs isolated and purified by density gradient centrifugation were able to split and proliferate in cell culture in vitro.MSCs can be induced and differentiated into osteoblasts by osteogenic differentiation or recombinat human bone morphogenetic protein-2.There were the same effects during the induction and differentiation between the osteogenic supplement and bone growth factors.