Effect Of Tensile Stress On Osteogenic Differentiation Of Bone Mesenchymal Stem Cells

Objective:To explore the effect of mechanical factors on osteoblasts differentiation by applying tensile stress to BMSCs,and to further investigate the mechanism of BMSCs osteogenic differentiation.Methods:Bone marrow mesenchymal stem cells were isolated from the femur and tibia of Kunming mouse and cultured in vitro.The cells of the third passage were seeded in specical 6-well tensile plates and identified by flow cytometry.A total of 3 groups were included in this experiment according to the various cell culture conditions,namely,the experimental group(A)which cultured in the ostegenesis inducing solution+stretch stress stimulation;the simple induction group(B)cultured in the ostegenesis inducing solution alone;and the blank control group in normal culture medium alone.Mechanical stimulation parameters is 6% tension amplitude,and the frequency is 0.5Hz,4h/d by the Flex-cell 5000 system.The cells in each group were cultured and medium was replaced every 2 days for 1 week.At the end of the experiment,the cells and culture medium were collected and expressions of BMP-2,Runx2,ALP m RNA in each group were determined by RT-PCR method and the content of Col1 was detected with Mouse Col1 chain ELISA Kit.In addition,alizarin red staining was employed to observe the formation of calcium nodules in each group.Results:1.Extracted primary cells were round or oval that contained more impurities,following changing medium firstly after 48 hours and wiping off floating dead cells,some cells were observed to adhere to the wall under the microscope,with an elongated or polygonal shape.Then,after a further culture for 48 hour,cells passage was performed in the ratio of 1: 2,the growth of passage cells was accelerated under microscope,which showed spindle-shaped and vortex-shaped growth.The results of flow cytometry analysis:CD29(96.4%)and CD90(97.2%)were strongly positive expression,CD34(1.7%)and CD45(1.3%)expression were negative expression.The P3 cells were highly purified and satisfied with experiment requirement.2.RT-PCR test:After 1 week of culture,mRNA expression levels of BMP-2,Runx2,ALP in group A and group B were significantly higher than those in group C(P<0.05).Furthermore,mRNA expression levels of BMP-2,Runx2,ALP in group A were higher than those in group B(P<0.05).3.The content of Col1 in group A and group B was higher than that in group C,and increased with the induction time.Meanwhile,the final concentration of group A was higher than that of group B,corresponding effect of inducing osteogenesis was the most obvious.4.Under the inverted microscope,group A and group B were found to be significantly stained with calcium nodules,suggesting that significant osteogenic differentiation was significant in both group,and the calcium nodules were more in group A than that in group B,without deep calcium nodules in group C.Conclusion:Tensile stress plays an important role in the differentiation of bone mesenchymal stem cells into osteoblasts,possible mechanism may be correlated with the promotion of bone mesenchymal stem cells differentiation into osteoblasts by activating the BMP-2and Runx2 expression.