Effects Of Periodic Hydrostatic Pressure On Osteogenic Differentiation And The Expression Of Vascular Endothelial Growth Factor Of Periosteal Cells And Its Mechanism

ObjectiveTo investigate the effects of periodic hydrostatic pressure on VEGF expression and osteogenic differentiation of cultured rats periosteal cells and its mechanism,.It may help us increase the understanding on the mechanisms of fracture healing and provide research basis for the application of periosteal cells in bone tissue engineering,and may provide a theoretical basis for clinically rational use of mechanical stimulation to accelerate fracture healing.MethodsRat periosteal cellsI were solated and cultured in vitro.Passaged 2-4 cells used in the experiments.After Periosteal cells cultured in osteogenic induction medium for 2 weeks,ALP(alkaline phosphatase)staining and alizarin red staining were used to determine the periosteal osteogenic potential.The rat periosteal cells were given periodic hydrostatic pressure(0-13 kpa,0.5Hz)stimulation,and the control group was not given pressure stimulation.Periosteal cells were pretreated by SP600125,SB203580 or PD98059 before given pressure stimulation to study the role of mitogen activated protein kinase(MAPK)signal pathway in the effects of periodic hydrostatic pressure on periosteal cells.After loaded for 6,12,24 h,the cell survival rate was detected by CCK-8(Cell Counting Kit-8)method.;ALP activity was detected by alkaline phosphatase assay kit;the osteogenesis-related genes(runx2,osteocalcin,type I collagen)and the expression of VEGF gene were detected by RT-PCR(real time quantitative polymerase chain reaction);P38、ERK1/2 and JNK protein expression and their phosphorylation levels were detected by Western blot.ResultsThe viability of cells under periodic hydrostatic pressure stimulation for 6h had no significant changes,but with increasing pressurization time,cell viability was inhibited.The alkaline phosphatase activity of periosteal cells increased under periodic hydrostatic pressure in time-dependent manner.The expression of osteogenesis-related genes(runx2,ALP,osteocalcin,type I collagen)and VEGF gene were also significantly increased.SP600125 and PD98059 could partly inhibit the the activity of ALP induced by periodic hydrostatic pressure.SP600125 could completely inhibit the expression of osteogenic related genes(runx2,ALP,osteocalcin,type I collagen)and VEGF gene of periosteal cells induced by the pressure expression,whereas SB203580 had no this effect.PD98059 could partly inhibit the expression of osteogenic related genes of periosteal cells induced by mechanical stimulation,but had no effect on the expression of VEGF of periosteal cells induced by this mechanical stimulation.ConclusionsThe appropriate intensity periodic hydrostatic pressure may promote the osteogenic differentiation of rat periosteal cells through the activation of JNK and ERK1/2 signaling pathway,and mediate VEGF expression through the activation of JNK signaling pathway,but this study indicated that p38 signaling pathway was not involved in the effects of this kind of pressure on the periosteal cells.