Mechanism Of SDF-1/CXCR4-mediated Host Mesenchymal Stem Cells Migration In Bone Repair

Background and objectives:Large defects of bone caused by trauma,severe bone infections,and tumors are common.Autologous bone grafting is the common effective treatment methods.However,bone resources are limited.And the bone removal surgery will cause new trauma at resecting sites.Tissue-engineered bone(TEBs)constructed with mesenchymal stem cells(MSCs)which were used as seeded cells have been widely studied and applied,and are considered as one of the most promising methods for treating large bone defects.Large numbers of basic research and clinical applications have proven that tissue engineering bones inoculated with mesenchymal stem cells had better bone repair effect than pure scaffold material.This revealed that the inoculated seed cells played an important role.With subsequent studies,most of the mesenchymal stem cells had apoptosis or died after tissue engineering bone transplantation.Within newly-formed bone tissues,the proportion of cells originated from inoculated MSCs was very low.At the same time,many host-derived mesenchymal stem cells would be recruited to participate in bone repair.However,it is unclear how these host stem cells were recruited to the bone defect area by tissue engineering bone.Our previous research found that after tissue engineering bone transplantation,a certain SDF-1 concentration gradient was formed in the bone defect transplantation area,peripheral blood and bone marrow.SDF-1 recruits host mesenchymal stem cells to the injured area through the CXCR4 receptor,but the specific mechanism is not clear.This article will focus on the transcriptomics analysis of host mesenchymal stem cells recruited to the bone defect area by TEB or DBM,and the mechanism of the SDF-1 / CXCR4 pathway in promoting host mesenchymal stem cell migration.Methods:(1)Through non-invasive bioluminescent imaging and flow cytometry,we detected the ability of tissue engineering bone and demineralized bone matrix(DBM)in recruiting exogenous and host MSCs in peripheral blood to the injured area.After TEB or DBM transplanted,host mesenchymal stem cells that had migrated to bone defect were sorted by flow sorting technology.After mRNA extraction,cDNA library construction and sequencing,the transcriptomic databases of these cells were established.After the library screening and comparison of the reference genome,the differential gene expression profiling was obtained.Then GO enrichment were performed based on the differentially expressed genes.At last GSEA enrichment were performed.(2)Cell migration experiments and QRT-PCR technology were used to verify the role of Rap1 and Rac1 in CXCR4-mediated mesenchymal stem cell migration.(3)Cell migration experiments,QRT-PCR,immunofluorescence,and tissue staining technology were used to verify the role of Arpin in CXCR4-mediated mesenchymal stem cells migration.Results:(1)Compared with DBM,more mesenchymal stem cells were recruited to the bone defect area by TEB.The quality of extraction RNA,library construction and sequencing depth was good,and the base distribution was uniform,so the sequencing quality met the analysis requirements.All percentages of the total mapped read were higher than 94%.Between the TEB and DBM groups,there were significant differences in the mRNA expression levels of 859 genes,and 284 genes were up-regulated while 575 genes were down-regulated.The different expression levels of genes function analyses including GO.GO enrichment results showed that they were related to the regulation of bone resorption,myeloid cell apoptotic process,positive regulation of osteoclast differentiation,chemokine receptor activity and chemokine binding.GSEA enrichment results showed that cytokine-cytokine receptor interaction signaling pathway was enriched,and the CXCR4 gene expression increased in TEB groups.(2)After blocking Rap1 and Rac1,the number of migrated MSCs to SDF-1 was reduced.And the mRNA expression levels of Rap1 and Rac1 were reduced after CXCR4 was blocked by AMD3100.(3)Silencing Arpin protein expression will enhance BMSCs migration and accelerate "wound healing”.2 days after the operation,immunofluorescence results showed that there was no significant difference between the two groups and almost no labeled cells migrated.At 7 days after surgery,relative to the empty vector control group,more cells migrated to the transplanted area in the Arpin expression inhibition group.4 weeks after the operation,Micro-CT results showed that compared with the normal group,the Arpin expression inhibition group had better bone formation quality.Pathological staining showed that there were more new bone tissues in the Arpin expression inhibition group.Conclusion:(1)RNA-seq analysis reveals that TEB has significant effects on the transcriptomic profile of BMSCs which were recruited to bone defect areas,and the functions of the different expression levels genes are related to the regulation of bone resorption,positive regulation of osteoclast differentiation,chemokine receptor activity and chemokine binding.Cytokine-cytokine receptor interaction signaling pathway was enriched,and the CXCR4 gene expression increased in TEB groups.Which may be related to the mechanism of TEB recruiting MSCs.(2)Rap1/Rac1 and Arpin are downstream molecules of SDF-1/CXCR4,and play a role in the migration of mesenchymal stem cells to the bone defect area.