The Mechanism Of Matrix-remodeling Associated 7(Mxra7) In Mediating The Differentiation Of Bone Marrow Mesenchymal Stem Cells Toward Osteoblast

Objective:Bone marrow mesenchymal stem cells(BMSCs)are a subpopulation of adult stem cells with the ability of self-renewal and multipotent differentiation.BMSCs are critical for healthy hematopoiesis and osteogenesis,thus alterations of the BMSCs differentiation might result in diseases,or verse versa.In this study,we tried to identify the mechanism of matrix-remodeling associated 7(Mxra7)in mediating the differentiation of bone marrow mesenchymal stem cell toward osteoblast.Methods:(1)Micro-computerized Tomography(MicroCT)analysis of Wild Type(WT)and Mxra7 deficiency(Mxra7-/-)mice(8-weeks and 9-months old,male).(2)The femurs of WT and Mxra7-/-mice were embedded,sectioned and stained with H&E.The growth plate and the trabecular bone were analyzed.(3)The paraffin sections of femurs were stained with TRAP and the number of osteoclasts was analyzed.(4)Analyze the number of osteoblasts by immunoflurescence staining of osteocalcin,the specific marker of osteoblast,in femur sections of WT and Mxra7-/-mice.(5)Bone mesenchymal stem cells(BMSCs),the progenitor of osteoblast plays critical role in the regulation of osteogenesis.The BMSCs derived from WT and Mxra7-/-mice were isolated and cultured to study the mechanism of Mxra7 in the regulation of osteogenesis.(6)BMSCs derived from WT mice were subjected to osteoblast differentiation and adipocyte differentiation and then examined the mRNA and protein expression of Mxra7 during differentiation.(7)WT and Mxra7-/-BMSCs were induced to differentiation toward osteoblast or adipocyte,and the potency of the differentiation was compared.(8)The signaling pathway which was involved in mediating the effect of MXRA7 on BMSCs differentiation was examined by Western blot.Results:(1)MicroCT analysis and three-dimensional reconstructions of the femurs of WT and Mxra7-/-mice showed that Mxra7-/-mice had shorter femur length and lower Bone Volume per Tissue Volume(BV/TV)in the metaphyses of the distal femur than WT mice.Moreover,the osteopenic phenotype was more profound with aging.(2)HE staining of femur revealed thinner growth plate thickness and lower trabecular bone amount in Mxra7-/-mice than WT mice.(3)The TRAP staining of femur sections showed that the number of osteoclasts in Mxra7-/-mice femur was similar with WT mice.(4)The immunoflurescence staining of osteocalcin in femur bone sections showed less osteoblasts number in Mxra7-/-mice than WT mice.(5)We measured the expression of Mxr7-during WT BMSCs differentiation toward osteoblast or adipocyte.We found that the mRNA level and protein expression of Mxra7 were significantly increased during the differentiation of BMSCs toward osteoblast and decreased when BMSCs were induced to differentiation toward adipocyte as assessed by qRT-PCR and western blot.(6)We compared differentiation potency or gene expression in BMSCs from WT and Mxra7-/-mice.Mxra7-/-BMSCs showed significantly decreased osteogenic potential and increased adipogenic potential compared with WT BMSCs.Treatment of BMSCs from Mxra7-/-mice with MXRA7 protein could increase osteogenic potential.(7)Furthermore,we found that Mxra7 deficiency suppressed the activation of extracellular signal-regulated kinase(ERK1/2)phosphorylation and active?-catenin in differentiation of BMSCs toward osteoblast.Conclusion:Our study demonstrated Mxra7 as a novel factor that influenced bone formation via regulating the balance between osteoblast differentiation and adipocyte differentiation of BMSCs.It not only advanced our understanding of molecular mechanism of BMSCs differentiations,but also suggested a new therapeutic target for enhancing bone formation or regeneration.