The Regulation Of NPNT By Mechanical Tension On Osteoblast

Objective:Bone is a highly vascularized tissue containing an extensive vascular network of large vessels and capillaries,which provide oxygen and nutrients for bone formation and development through regulation of different signaling pathways between bone cells and endothelial cells.Local vascular changes are also closely related to the development of many bone diseases,such as osteoporosis,osteonecrosis,osteoarthritis,rickets,ischemic necrosis,and the occurrence and metastasis of bone tumors.Bone vascularity is associated with bone formation.Bone cells can secrete angiogenic factors to trigger signals,causing different cells?including vascular endothelial cells,chondrocytes,osteoblasts,osteoclasts,etc.?to release cytokines and then promote angiogenesis.At the same time,the factors released by the bone vascular endothelial cells also play a role in the chondrocytes and osteoblasts.In recent years,studies have reported that NPNT is an important angiogenic factor secreted by osteoblasts and plays an important role in the regulation of bone metabolism.Therefore,this study investigated the role of mechanical tension on osteoblasts in the regulation of NPNT,providing a theoretical basis for further exploring whether exercise can regulate the angiogenesis of bone microenvironment through osteoblast-expressing cytokines,and also providing new ideas for the treatment of osteoporosis and other related bone diseases.Methods:1.The cultured MC3T3-E1 osteoblast-like cells were divided into 4 groups?group C,group S1,group S2,and group S3?and were seed on BioFlex 6-well plates with1×10⁵/ml.The cells were grown to 80%and Flexcell-5000 tension systerm were used to perfrom mechanical tension.The group S1 were treated with 6%elongation,0.5Hz,2h for 5days;the group S2 were treated with 6%elongation,0.5Hz,4h for 5days;the group S3 were treated with 4%elongation,0.5Hz,4h for 5 days,while group C was static culture.The cells were changed medium every 48 hours.On the 5^thh day,the cells were collected after the mechanical tension.Real-time PCR was used to detect the expression of ALP,RUNX2,Osterix and NPNT mRNA and western blot was used to detect the expression of NPNT protein.2.The third generation of primary osteoblasts in C57BL/6 mice were seed on BioFlex6-well plates with 1×10⁵/ml.The cells were grown to 80%and Flexcell-5000 tension systerm were used to perfrom mechanical tension.The strain group was treated with3%elongation,0.5Hz,2h and 4h for 7days,and stimulated by mechanical tension on the 1^st,3^st,5^stt and 7^stt day while control group was static culture.The cells were changed medium every 48 hours.The medium were collected to culture the SVEC cells,and scratch wound healing assays were performed to observe the effect of medium on the proliferation and migration of SVEC cells.On the 7^stt day,the cells were collected after the mechanical tension.Detection of ALP activity of osteoblasts by ALP staining;Real-time PCR was used to detect the expression of ALP,RUNX2,Osterix and NPNT mRNA;western blot and immunohistochemical staining was used to detect the expression of NPNT protein.Results:1.Effect of MC3T3-E1 Osteoblasts on the differention and the expression of NPNT after mechanical tension?1?The results of Real-time PCR:Mechanical tension can promote the expression of ALPmRNA in MC3T3-E1 osteoblasts.The best result were achieved with 4%elongation,0.5Hz,4h for 5days?P<0.05?,but significantly decreased with the treat with 6%elongation,for 5 consecutive days?P<0.01?.Compared with the group C,The expression of RUNX2 mRNA was significantly up-regulated under mechanical tension?P<0.05?with the treat of 6%elongation,0.5Hz,2h for 5days and4%elongation,0.5Hz,4h for 5days.The expression of Osterix mRNA was significantly up-regulated under mechanical tension with the treat of 4%elongation,0.5Hz,4h for 5days?P<0.01?.At the same time,the mechanical tension with the treat of 4%elongation,0.5Hz,4h for 5days promoted the expression of NPNT mRNA obviously?P<0.01?.?2?The results of western blot showed that the mechanical tension with the treat of6%elongation,0.5Hz,4h for 5days made NPNT protein significantly up-regulated?P<0.05?.2.Effect of primary osteoblasts on the differention and the expression of NPNT after mechanical tension?1?The results of Real-time PCR:The expression of ALP mRNA and RUNX2 mRNA was significantly up-regulated under mechanical tension with the treat of 3%elongation,0.5Hz,2h for 7days?P<0.05?;The expression of ALP mRNA and RUNX2mRNA was significantly up-regulated under mechanical tension with the treat of 3%elongation,0.5Hz,4h for 7days?P<0.05?;in addition,The expression of Osterix mRNA and NPNT mRNA was significantly up-regulated under mechanical tension with the treat of 3%elongation,0.5Hz,4h for 7 days only?P<0.05??2?The results of ALP staining showed that the mechanical tension with the treat of3%elongation,0.5Hz,4h for 7 days promoted the secretion of ALP in osteoblasts.?3?The results of western blot showed that the mechanical tensionwith the treat of 3%elongation,0.5Hz,4h for 7 days made NPNT protein up-regulated,but there was no significant difference.?4?The results of immunohistochemical staining showed that mechanical tensionwith the treat of 3%elongation,0.5Hz,4h for 7days promoted the secretion of NPNT by osteoblast.?5?The result of scratch wound healing assays showed that the mechanical tension on osteoblast could promote the proliferation and migration of SVEC cells.Conclusion:Mechanical tension can promote the differentiation of osteoblast and upregulate the expression of NPNT;Mechanical tension on osteoblasts can promote the proliferation and migration of SVEC cells.It may be that mechanical tensionon osteoblasts promote the expression of NPNT,while NPNT was one of the pro-angiogenic factors that promoting the proliferation and migration of SVEC cells,but the specific mechanism needs further verification.