Signaling Pathways In The Early Responses To Compressive Stress In Osteoblasts

Background and ObjectiveForce is very important in the process of bone remodeling.For example,bone density will increase in the long-term and weight-bearing condition.However,bone density will decrease which leading to osteoporosis in the weightless condition.In the clinical treatment of oral orthodontics,vertical compressive stress can intrude teeth into sockets,and horizontal compressive stress leads to teeth move by inducing bone resorption around the roots.The progressive loading can prompt bone formation in the implant treatment.There are many treatments to reconstrust bone by compressive stress.In vitro,osteoblasts secrete a variety of cytokines to regulate bone remodeling by the mechanical force.The changes of cytokines are mediated by intracellular cellular signaling systems.Osteoblasts are stimulated by mechanical stress to produce osteogenic and osteoclastic effects,which are associated with complex biomechanical signaling pathways.Mitogen-activated protein kinase and protein kinase B signal transduction are important during the remodeling of bone.There have been a large number of experiments confirmed that mechanical stress can activate these two signal pathways and regulate functions of osteoblasts.However,how do these signaling pathways regulate bone remodeling when the cells are exposed to compressive stress.MAPK signaling pathway involves a large number of proteins which are related to complex reactions.There are no further study to confirme this mechanism.In the present experiment,using the phosphorylation protein array technique to detect expression of related proteins and explore the early responses of signal pathways to compressive stress.Screening out the key signal factors to different compressive stress.Methods1.Making the calcium alginate gel culture well to culture cells.The rat type I collagen was used as the three-dimensional culture medium of osteoblasts.The osteoblasts were exposed to compressive stress of 2 g/cm2 and 5 g/cm2 and the control group without force.The cultured cells were exposed for 6 h under different compressive stresses.Then the cell microscopy was observed by fluorescence confocal microscopy after staining the rhodamine phalloidin.Verified the viability of MC3T3-E1 cells by CCK8.2.MC3T3 cells were exposed to Og/cm2,2g/cm2,5 g/cm2 of compressive stress.The operation of applying compressive stress was same as the first step.The cells were exerted for 45 min,3 h or 6 h at 37 ?.Then detect the expression of the phosphorylated proteins by using the phosphorylation protein array technique.Results1.The results of immunofluorescence microscopy shown that the cell attachment points still distributed three-dimensional after compressive stress.The results of CCK8 shown that there was no significant difference in OD among this three groups.2.When the cells were exposed for 6 h,the phosphorylated protein expression of CREB,AKT,HSP27,GSK3a,GSK3P,JNK,MSK2,p38,p53,p70s6k,ERK1/2 in the group of 2 g/cm2 were significantly higher than the control group(P<0.05).The phosphorylated protein expression of CREB,MSK2,p38,p53,p70s6k and ERK1/2 in the group of 5 g/cm2 were significantly higher than the control group(P<0.05).The expression of AKT,GSK3? and HSP27 in the group of 5 g/cm2 were significantly lower than the group of 2 g/cm2(P<0.05).There was no significant difference between the group of 2 g/cm2 and 0 g/cm2 after exerted for 45 min or 3 h(P>0.05).In the group of 5 g/cm2 after exerted for 45 min,the phosphorylated protein expression of p70s6k was significantly higher than the control group(P=0.038<0.05).The expression level of RSK1 was significantly lower than the control group(P=0.019<0.05).In the group of 5 g/cm2,the phosphorylated protein expression of p70s6k,HSP27 and RSK2 were significantly higher than the control group after exerted for 3 h(P<0.05).There were no significant differences in the expression of other phosphorylated proteins between the group of 5 g/cm2 and 0 g/cm2(P>0.05).Conclusions:1.MC3T3 cells could maintain three-dimensional growth pattern under the compressive stress of 2 g/cm2 or 5 g/cm2 lThe cell viability was not inhibited by the compressive stress of 2 g/cm2 or 5 g/cm2.A compressive stress loading model was successfully established and could be used as an experimental platform for compressive stress relevant studies.2.The expression of phosphorylated proteins had significantly changed under different compressive stresses exerted for 6 h,which was therefore regarded as the critical point of time.3.The activation of the relevant signaling pathways in the early response of osteoblasts to compressive stress was related to the magnitude of compressive stress.The heavier compressive stress of 5 g/cm2 was able to induce signaling pathways activated earlier than 2 g/cm2.The group of 2 g/cm2 could active the signal pathways more wildly than 5 g/cm2 groups after the cells were exerted for 6 h.4.p70s6k may be the key protein kinase in the present study,which participated in the early response of osteoblasts.But its specific mechanism needs to be further explored.