| Objective: The aim of this is to explore the effect of BMP-2 and BMP-9 at different concentrations on osteogenetic differentiation of BMMSCs in vitro experiments and further research is explore the changes of osteogenic differentiation of BMMSCs mediated by different concentrations of BMP-2 and BMP-9 in the presence of inflammatory factor TNF-?.So as to get a preliminiary understanding the effect of BMP-2 and BMP-9 on osteogenic differentiation under inflammatory conditions.Methods: 1,cell extraction,culture and identification: The SD rat BMMSCs can be isolated an cultured by whole bone marrow culture,and the cell surface antifens were detected by the flow cytometer(FCM).Adipogenic and osteogenic differentiation to verify its multi-directional differentiation ability.2.Experimental group: The experiment was divided into 5groups:BMMSCs were cultured in osteogenic media and different concentrations of BMP-2,BMP-9 and TNF-? added in osteogenic media,which were respectively regarded as control group and experimental groups.Set the concentration:(1)BMP-2(50 ? 100 ? 150ng/ml)(2)BMP-9(50 ? 100 ?150ng/ml)(3)TNF-?(10ng/ml)(4)TNF-?(10ng/ml)with different concentration BMP-2(50?100?150ng/ml)(5)TNF-?(10ng/ml)with different concentration BMP-9(50?100?150ng/ml).3.ALP activity: ALP activity of cells was measured by AKP kit.4.Mineralization assay: Alizarin red staining was applied to observe the degree of mineralization;5.Real-Time PCR: RealTime PCR was used to detect the Osteogenesis-related indicators expression of Col??OCN.6.Statistical analysis: The statistical software SPSS 23.0 was used to analyze the data.The experimental results were expressed as mean ± standard deviation(x ± s).The LSD-t test was used to test the experimental data.P <0.05 was considered as the significant test standard.The difference was statistically significant Analytical test data were plotted using Graph Pad Prism 6.0.Results: 1.Cell extraction,culture and identification: FCM detected the positive expression rate of CD31,CD34,CD44,CD45 are 100%,2.9%,96.5%,0.8%.Alizarin red mineralized nodular staining and adipose red staining confirmed the multi-directional differentiation of cells.2.ALP activity tests.Compared with the control group,the ALP activity of BMMSCs was significantly decreased(P <0.05)at day3 day5 day7 after adding osteogenic media containing TNF-?.However after adding BMP-2 and BMP-9 into osteogenic media,the activities of ALP in BMMSCs were significantly increased(P <0.05),and all reached peak values at the concentration of BMP-2 and BMP-9 of 100 ng / ml.3.Alizarin red staining After culture cells in osteogenic media with different concentrations of TNF-?,BMP-2 and BMP-9 for 14 days,The content of calcium nodules in osteogenic induction group with TNF-? decreased(P <0.05),the content of calcium in osteogenic medium containing BMP-2 and BMP-9 increased significantly and the content of calcium nodules in BMP-9 group was higher than that in BMP-2 group(P <0.05).The content of calcium nodules in BMP-2,BMP-9 and TNF-? group((4),(5))was lower than that only in BMP-2 or BMP-9 group,but still higher than that in control group and TNF-? group(P <0.05).4.Real-Time PCR analyses Compared with the control group,the expression of OCN correlated with the level of osteogenic media containing TNF-?,BMP-2 and BMP-9 at different concentrations after 21 days of culture.The content of OCN in BMP-2 and BMP-9 groups increased with the increase of concentration,and reached the peak at the concentration of 100 ng / ml.And it did not have significant difference the concentration of 150 ng / ml expression(P > 0.05).The expression level of OCN in group(4)and group(5)at 21 days after induction was significantly lower than that in control group and only containing TNF-?(P <0.05)group.Col ? expression showed the same trend.Conclusion: BMP-2 and BMP-9 have a significant promoting effect on BMMSCs,and they tend to increase with increasing concentration,but both of them will reach the peak of bone-promoting effect when the concentration reaches 100 ng / ml,and the promotion function of BMP-9(ALP value,calcium nodule area,OCN and Col?expression)were higher than the same concentration of BMP-2.Osteogenic differentiation of BMMSCs was inhibited by the inflammatory cytokines TNF-?,which also inhibited the osteogenic differentiation induced by BMP-2 and BMP-9.However,BMP-9 still plays a role of osteogenesis better than that of BMP-2 at the same concentration of inflammatory cytokines.And its best concentration of osteogenesis is also due to the concentration of inflammatory cytokines. |
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