Intermittent Tension Promotes Bone Differentiation Of Mouse BMSCs Via P38MAPK-Osterix Signaling Pathway

Objective:The osteogenic of bone marrow mesenchymal cells caused by mechanical stretch is the key cellular basis of distraction osteogenesis, orthodontic tooth movement and new bone formation during fracture healing.BMSCs not only be a kind of multipotent stem cells, but also be one of the most sensitive cell of mechanical stress. Previous studies have demonstrated that stretch could induce BMSCs osteoblastic differentiation. P38MAPK signaling pathwayplay a crucial role in inflammatory reaction, cell cycle regulation and cellular differentiation, previous studies have found that p38MAPK pathwayhad a important rolein mesenchymal stem cells differentiate into osteoblasts.Osterix is a zinc finger transcription factor, specificly expression in osteoblast, is a key factor in osteoblast differentiation and bone formation. Researchs shows that mechanical stimulation can induce high expression of osterix. However, there has about the relationship between p38MAPK signaling pathway, osterix and osteoblastic differentiation of BMSCs by mechanical stretch.This paper intends to developing a mechanical stimulation model of BMSCs in vitro using a multichannel cell strain loading system and Osterix gene silencing of BMSCs model. To learn the relationship between p38MAPK signaling pathway, osterix and osteoblastic differentiation of BMSCs by mechanical stretch. Our study would contribute the understanding of the bone biomechanical stimulation mechanism, and for the further study, and provide a theoretical basis for clinical treatment.Methods:BMSCs primary culture:stripppingthe bilateral femur and tibia of the inbred strains of mice (C57BL/6J) in aseptic. Havesting BMSCs by whole marrow method of. observed the cell morphology under inverted microscope, multi-directional differentiation and cell surface molecular were identification. BMSCs served as the research object, and developing a mechanical stimulation model of BMSCs in vitrousing a multichannel cell strain loading system.The C57BL/6J BMSCs mice were divided into blank control group, strain group and inhibitor group (SB203580(a p38MAPK signal pathway inhibitor)+strain).Loading mechanical stimulation,0.5Hz,0.8%,2times a day,30min each time, by the multichannel cell strain loading system. Then harvest BMSCs cells on ld,3d and5d,respectively. Real time-PCR detect the expression changes of the ALP, COLI, OCN and osterix; Western blotting detect of osterix protein and P-p38MAPK protein after affecting by intermittent stretching force. Inhibitted p38MAPK signal pathway by SB203580,then Western blotting detection of osterix protein and P-p38MAPK protein;Real time-PCR detect the expression changes of the ALP, COLI, OCN and osterix. Knocking down the gene of osterix of mouse by siRNA, detecting the expression of the protein of osterix by Western blotting, Real time-PCR for the ALP, COLI, OCN mRNA. Results:1.Mechanical tension force can promote the expression of ALP, COLI, OCN and osterix mRNA. Compared with the control group, the differences of the corresponding gene of the strain group has statistics significance at the same time.(*P<0.05or**P<0.01) Western blotting showed the osterix protein and P-p38MAPK protein of the strain group increased significantly.Compared with the control group, the differences of the osterix protein and the P-p38MAPK protein of the strain group has statistics significance at the same time.(*P<0.05or**P<0.01)2.The expression of ALP, COLI, OCN and osterix mRNA decreased after using SB203580, and the results of the Western blotting also showed similar results.Compared with the strain group, the differences of the corresponding gene and protein of the inhibitor group has statistics significance at the same time.(*P<0.05or**P<0.01)3.Silencing of osterix could let ALP, COLI, OCN mRNA decrease. Compared with the strain control group, the differences of the corresponding gene of the strain-siRNA group has statistics significance at the same time.(*P<0.05or**P<0.01)Conclusion:1. Intermittent stretching force could promote the osteogenic differentiation of BMSCs2.The osteogenic differentiation of BMSCs caused by intermittent stretching force maythrough the p38MAPK-Osterix pathway.