Bone Marrow-derived Exosomes Regulate Osteoclast Precursor Migration And Fusion After Fatigue Loading

ObjectiveThe skeleton accumulates microdamage during fatigue loading in daily activities.This microdamage is repaired through bone remodeling,and osteoclasts are recruited first to the microdamage area for bone resorption.Osteoclasts renew themselves by fusing with mononuclear osteoclast precursors,whereas osteoclast precursor migration and fusion processes are regulated by several factors in the bone marrow microenvironment.From this microenvironment,bone marrow-derived exosomes(BM-exo)regulate adjacent and remote cells via micro RNAs(mi RNAs),but it is unclear if they regulate osteoclast precursors in localized bone remodeling due to fatigue loading.In this study,we investigated whether BM-exo regulated osteoclast precursor migration and fusion via mi RNAs after fatigue loading and examined associated mechanisms.Methods1.To simulate bone microdamage in the bone marrow microenvironment,in vivo fatigue loading tibia tests were performed on ovariectomized rats.Tibia microdamage and biomechanical properties were also examined.BM-exo were extracted and characterized by transmission electron microscopy,western blotting,and particle size analysis.Primary rat bone marrow-derived monocytes/macrophages(BMMs)were isolated and differentiated to identify the potential of osteoclast differentiation as osteoclast precursors using tartrate–resistant acid phosphatase(TRAP)staining and real-time quantitative polymerase chain reaction(RT-q PCR)assays on signature molecules.BM-exo were co-cultured with rat osteoclast precursors,and exosome uptake by BMMs was observed under laser confocal microscopy.Cell Counting Kit-8(CCK-8),TRAP,and RT-q PCR assays(on signature molecules)were performed to compare the effects of loading group BM-exo(LE)and non-loading group BM-exo(NLE)on osteoclast precursor proliferation and fusion.2.Due to the homogeneity and convenience of migration assays,Raw264.7 cells replaced BMMs for promoter studies.Rat BM-exo uptake by mouse Raw 264.7 cells was observed under laser confocal microscopy.Using the aforementioned assays(CCK-8,TRAP,and RT-q PCR),we compared LE effects on the osteoclast precursors between rat and mouse.Then,LE and their concentration gradient effects on Raw 264.7 cell migration were analyzed via scratch assay and transwell assay.3.Mi RNAs in LE and target genes were predicted on the basis of previous data,the literature,and mi RNAs target gene databases.Mi RNAs were detected by RT-q PCR.A mi RNA mimic was transfected into Raw264.7 cells,and a mi RNA inhibitor was transfected into LE before adding LE to Raw 264.7 cells.Raw 264.7 cell migration and fusion capacity were then determined via transwell and RT-q PCR assays.Molecules interacting with target genes were screened using the STRING protein interaction database,the previous literature,and RT-q PCR.Results1.BM-exo increased primary rat BMM activity at 10 μg/m L(low concentration)and decreased activity at 70 μg/m L(high concentration).Compared with NLE,LE(10 μg/m L)showed no statistical differences in affecting BMM proliferation but promoted osteoclast precursor fusion to generate more and larger osteoclasts and enhance differentiation [cathepsin K(Ctsk)and nuclear factor of activated T cells c1(Nfatc1)] and fusion marker genes expression [dendritic cell-specific transmembrane protein(Dcstamp)and ATPase H+ transporting V0 subunit d2(Atp6v0d2)].2.Raw 264.7 cell activity was enhanced by LE at 70 μg/m L(high concentration).Compared with NLE,LE(35 μg/m L)showed no statistical differences in affecting Raw 264.7 cell proliferation but promoted the formation of more and larger osteoclasts,with increased Ctsk,Nfatc1,Dcstamp,and Atp6v0d2 expression.Compared with NLE,LE and their concentration gradient increased the relative wound healing percentages and migration(transwell assays)in Raw 264.7 cells.3.Mi R-200b-3p was upregulated in LE.Mi R-200b-3p overexpression promoted Raw 264.7 cell migration,as reflected by an increased number of cells passing through transwell assay membranes.Raw 264.7 cells transfected with a mi R-200b-3p mimic exhibited enhanced fusion,possibly via targeted zinc finger E-box binding homeobox 1(Zeb1)downregulation and cadherin 1(Cdh1)upregulation.Increased Ctsk,Nfatc1,Dcstamp,Atp6v0d2,and Cdh1 expressions and decreased Zeb1 expression were also observed.Raw 264.7 cells cultured with mi R-200b-3p inhibitortransfected LEs showed reduced Ctsk,Nfatc1,and Dcstamp levels,implying weakened positive effects.Conclusions1.Bone marrow-derived exosomes and concentration gradient after fatigue loading accelerated osteoclast precursor migration.2.Bone marrow-derived exosomes after fatigue loading promoted osteoclast precursor fusion by upregulating mi R-200b-3p,with the mi R-200b-Zeb1-Cdh1 regulatory cascade as a possible pathway.