Effect Of BMP9 On Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells Under Static Compression

Objective:To investigate the effect of BMP9 on osteogenic differentiation of human periodontal ligament stem cells under static compression,the expression of osteogenic factors ALP,RUNX2,OCN and mineralization of calcium salt nodules in hPDLSCs induced by BMP9 was detected after loading the suitable static compression for a certain period of time.Methods:1.hPDLSCs were cultured by the tissue adhering method and purified by limiting dilution method.Then the obtained hPDLSCs were identified and provided for subsequent experiments.2.The hPDLSCs suspensions(1×10~5 cells/ml.)were inoculated into six well plates.CCK-8 and flow cytometry method were used to detect the proliferation activity and cell cycle of hPDLSCs after loading different force levels(0,1,2,3,5g/cm~2)for different time(12h,24h,72h).Then,the appropriate loading force value and time that favored the proliferation of hPDLSCs were determined by the results of both.3.The Ad-BMP9 vector was constructed.Then the appropriate MOI value for Ad-BMP9 to transfect hPDLSCs was determined.Subsequently,the appropriate static compression was applied to the hPDLSCs transfected with Ad-BMP9.The virus-negative control group and the blank control group were set.After that,the expression of osteogenic factor ALP?RUNX2 and OCN was investigated though RT-qPCR and Western Blot analysis.The formation of mineral nodules was detected by alizarin red staining.Results:1.The hPDLSCs were successfully cultured and then confirmed after identification of cell cycle,cell proliferation and cloning,cell phenotype and differentiation ability.2.When appropriate force level(less than 5 g/cm~2)was loaded within a certain period of time(12h,24h),the OD value and PI value of hPDLSCs increased,meaning that the proliferation activity of hPDLSCs was promoted;but when the loading time was prolonged(72h),the cell OD value And the PI value decreased,meaning that the proliferation activity of hPDLSCs decreased.Meanwhile,the phenomenon of cell disintegration was observed in the 3 g/cm2force group.A excessive force level(5 g/cm~2)loading for a short period of time(12h)could increase the OD value and PI value of hPDLSCs,and promoted the cell proliferation activity.However,as the loading time was extended(24h,72h),a large number of cells would collapse.The OD value and PI value of hPDLSCs decreased significantly,indicating that the cell proliferation activity was significantly reduced.3.It showed that 3g/cm2 was the suitable force level,24h was the suitable loading time for hPDLSCs under static compression in vitro,which could promote the proliferation of hPDLSCs.4.The appropriate MOI value of Ad-BMP9 transfected hPDLSCs was 80,and the transfection time was 72h.And the comparison of the growth curves of each group showed that with a appropriate MOI value,Ad-BMP9 had little toxic effect on hPDLSCs,and the proliferation of hPDLSCs would not be inhibited.5.After loading the suitable static compression of 3g/cm~2 for 24h,The gene and protein exprssion of BMP9 increased greatly in the hPDLSCs with Ad-BMP9 transfected,indicating that the adenoviral vector overexpressing BMP9 was successfully constructed.Meanwhile,the expression of ALP,RUNX2,OCN and the production of calcium salt nodules of hPDLSCs transfected by Ad-BMP9 were significantly higher than those in the control group,indicating that BMP9 could enhance the osteogenic differentiation of hPDLSCs under the suitable static compression.Conclusion:The hPDLSCs were successfully cultured in this experiment.The weight loading model was used to determine the suitable static force value(3g/cm~2)and time(24 h)to promote the proliferation of hPDLSCs in vitro.Then the effect of Ad-BMP9 on hPDLSCs was verified,and it was confirmed that BMP9 can enhanced the expression of osteogenic differentiation factor ALP,RUNX2,OCN and mineralization of calcium salt nodules of hPDLSCs under the suitable static compression,which indicated that BMP9 can induced the osteogenic differentiation of hPDLSCs.