Mechanical Stimulus Promotes Bone Remodeling Through Stem Cell Recruitment By Activating ERK/CCL2 In Osteoblast

Objective:Osteoporosis,which is characterized by bone mass loss and increased bone fragility,has threaten over 200 million people's health because of high morbidity and even high mortality.As life style changing and population ageing,situation can become worse.Therefore,studies have focused on prevention and therapy of osteoporosis,looking for promising solution for the disease.Exercises have been demonstrated to be accessible and efficient in preventing osteoporosis and improving bone metabolism,as well as other chronic diseases.However,the intrinsic mechanism have not been elucidated clearly.On this basis,we designed and performed the present study in order to get a better understanding of how exercises influence bone structure and bone metabolism.Chapter 1 Moderate running increases long bone bone volume and osteoprogenitor number in miceMethods and Resullts:C57BL/6 mice were grouped randomly,running on treadmill for moderate intensity or move freely for 5 weeks.Micro-CT scanning of tibia showed increased bone volume/tissue volume(BV/TV)and trabecular number(Tb.N),along with decreased trabecular spacing(Th.Sp)in running mice.Histochemistry staining revealed increased OCN positive cell on the trabecular bone surface in running mice while no significant difference was detected in TRAP positive cell number between groups.Significant increased number of endosteal osteoprogenitors(EOP)was found by flow cytometry while no significant change in bone marrow stromal cells(BMSCs)number.CFU-F and alirizan red staining of EOP suggested higher EOP number and osteoblastogenic potential in running than control mice.Statistical significant increase in Osterix positive osteoprogenitor number on trabecular bone surface was detected by immunofluorescent staining.Conclusions:Moderate running increase cell number and osteoblastogenic potential of osteoprogenitors on trabecular bone surface.Chapter 2 Mechanical stimulus promote bone marrow stromal cells migration through ERK-CCL2 in osteoblastsMethods and Resullts:Module analysis of protein-protein interaction network,heatmap and time-expression line chart were performed to analysis dataset GSE50707 and GSE22286,indicating that chemokines such as CCL2,CCL7,CXCL1,CXCL13,CXCL16,et al.were up-regulated after mechanical stimulus.Taking together the results of qRT-PCR,WB and immunohistochemistry staining of running mice sample,CCL2 was screened out as potential key genes in mechanical regulation of BMSCs migration.Furthermore,MC3T3-E1 preosteoblast cell line was treated by U0126 and subjected to mechanical stimulus by cyclic strain for 12 h.Results of qRT-PCR and WB suggested that ERK was activated after mechanical stimulus and ERK blockade by U0126 can suppress CCL2 expression up-regulated by mechanical stimulus.Conditioned medium of stimulated MC3T3-E1 could promote BMSCs migration and this effect was abrogated by U0126.Surprisingly,administration of CCL2 rescued the effect of U0126.Conclusions:As a result of mechanical stimulus,CCL2,which is up-regulated by ERK activation in osteoblast,promotes bone marrow stromal cells migration.