Preliminary Study Of Cyclic Tension Force BMP9 Regulates The Osteogenic Differentiation Of Human Periodontal Ligament Fibroblasts Via PI3K/AKT Signaling Pathway

ObjectiveA Flexcell-5000 cell mechanical loading system was used to construct a human periodontal ligament fibroblasts(h PDLFs)in vitro culture-periodic tension loading model,observe the changes of the cytoskeleton、proliferation activity、bone morphogenetic protein 9(BMP9)and related osteogenic marker proteins in h PDLFs expression under cyclic tension force,and study whether BMP9 activates the PI3 K / AKT signaling pathway to regulate osteogenic differentiation of h PDLFs under cyclic tension.MethodsIn this study,h PDLFs cultured in vitro were randomly divided into 3experimental groups and 1 control group,the experimental group used the Flexcell-5000 cell mechanical loading system to load a cyclic sine wave with a deformation rate of 10% and a frequency of 0.5 Hz.The action time was 6h,12 h and 12 h after the PI3 K signaling pathway inhibitor LY294002 was added,the control group was not active.The growth status of h PDLFs was observed under an inverted phase contrast microscope;Immunofluorescence experiments were used to detect the expression and distribution of cytoskeleton proteins;Flow cytometry was used to detect the proliferation of h PDLFs;The alkaline phosphatase(ALP)activity detection method was used to detect ALP activity of h PDLFs;The expressios of RUNX2,OCN,OPN,OSXm RNA were detected by real-time quantitative PCR;Western blot was used to detect the expressios of BMP9 and osteogenic marker proteins OCN and OPN,the expressios of AKT and P-AKT proteins and the expressions of osteogenic marker proteins OCN and OPN were changed after the PI3 K signaling pathway inhibitor LY294002 was added;Alizarin red staining was used to detect osteogenic differentiation of h PDLFs.ResultsThe h PDLFs inoculated into the Bioflex six-well culture plate showed good adherence to the cells,the h PDLFs grew well after periodic traction,and the arrangement order was consistent with the direction of the afterburner,and the expression of cytoskeletal protein was significantly increased and distributed along the direction of the force.Flow cytometry showed that the proportion of S-phase cells in the 6h group and 12 h group was significantly reduced compared with the control group,and the difference was statistically significant(P<0.05);The expression of ALP activity in the 6h group and 12 h groups was significantly increased compared with the control group,and the difference was statistically significant(P<0.05);The expressions of RUNX2,OCN,OPN,and OSX m RNA detected by real-time quantitative PCR were significantly increased in the 6h and 12 h groups compared with the control group,and the difference was statistically significant(P<0.05);The expressions of BMP9 and osteogenic marker proteins OCN and OPN detected by western blot in the 6h group and 12 h group were significantly higher than those in the control group,the difference was statistically significant(P <0.05).The level of ossification was decreased,and the expression levels of osteogenic marker proteins OCN and OPN were also significantly reduced,and the difference was statistically significant(P<0.05);Alizarin red staining showed that the bone calcium nodules formed in the cells in 6h and 12 h groups after having cultured in vitro for 21 days,and the amount of formation was significantly increased compared with the control group.Conclusions1.In this experiment,the Flexcell-5000 cell mechanical loading system was used to load a deformation rate of 10%,and a frequency of 0.5 Hz,and act on h PDLFs cultured in vitro.The h PDLFs grew well,and the h PDLFs in vitro culture-stress loading model was successfully constructed.2.Cyclic tensile force promotes osteogenic differentiation of h PDLFs.3.Cyclic tensile force can regulate osteogenic differentiation of h PDLFs through the BMP-PI3 K / AKT signaling pathway.