Optimization Of Extracellular Matrix-based Tissue-engineered Bone Construction Strategy And Repair Of Femoral Defects

Background:Extracellular matrix(ECM)is rich in proteins and factors for tissue damage repair,which is one of the current research hotspots.Correspondingly,extracellular matrix-based tissue-engineered bone(ECM-TEB)is a newly developed and promising method for repairing bone defects.However,the dynamic balance of bone reconstruction is achieved by the cooperation of various cells in bone tissue.Therefore,if we can establish co-culture with different types of cells in the construction of ECM-TEB,it will more realistically simulate the microenvironment of osteogenesis in vivo.The two most important functional cells required to maintain bone homeostasis are osteoblasts(OBs)and osteoclasts(OCs).Among them,OC performs bone resorption,and OB dominates bone formation.The interaction and coupling of these two cells is the key to bone repair.Studies have found that platelet-derived growth factor-BB(PDGF-BB)secreted by preosteoclast(POC)can promote the mutual coupling of bone formation and vascularization,suggesting that osteoclast-related cells play a key role in bone repair and vascularization.Besides,endothelial progenitor cells(EPC)can differentiate into vascular endothelial cells(EC),generate blood vessels,and promote the homing and osteogenesis of mesenchymal stem cell(MSC)in specific microenvironments.Therefore,while MSC are used as osteogenic differentiation seed cells,introducing POC or EPC as multiple seed cells may be an effective strategy for repairing bone defects.Method:We constructed four matrix-dependent tissue engineering bones: MSC ECM-TEB,MSC+POC ECM-TEB,MSC+EPC ECM-TEB,MSC+POC+EPC ECM-TEB.First,the surface morphology was observed using a scanning electron microscope(SEM).Cell-dependent tissue engineering bone(Cell-TEB)was used as a control group,and ECM-TEB after lyophilization was used as an experimental group.Through mixed lymphocyte reaction(MLR)experiments,major histocompatibility complex-I and II(MHC-I and MHC-II),And deoxyribonucleic acid(DNA)immunofluorescence and DNA quantitative assay to detect the immunogenicity of the control group and the experimental group.In vivo,a 4 mm-diameter cylindrical femoral defect model was established in Sprague Dawley(SD)rats to evaluate osteogenesis,which was used as the important principles for evaluating bone defects repairation with ECM-TEB.In vitro,MSC ECM-TEB was used as a control group,ECM-TEB with the best repair effect was used as an experimental group.In addition to detecting the performance of ECM-TEB in terms of cell migration,adhesion,and osteogenic differentiation,iTRAQ-labeled mass spectrometry(MS)analysis was also performed to find out the key different components between different ECM-TEBs.Insulin like growth factor binding protein 5(IGFBP5)and C-X-C motif chemokine 12(CXCL12),the key different components analyzed by MS,were tested for their biological functions by the addition of neutralizing antibodies,real-time polymerase chain reaction(PCR),alizarin red,and alkaline phosphatase(ALP)staining,scratch experiments,and Transwell cell migration experiments.Result:The immunogenicity of ECM-TEB is significantly lower than that of Cell-TEB.The antigenic components(MHC-I,MHC-II,and DNA fragments)are not shown under a fluorescence microscope,and double-stranded DNA(dsDNA)is <50 ng / ml.In vivo experiments,micro-CT results and Masson section staining results show that compared with blank scaffolds and MSC ECM-TEB,most defects are repaired after the implantation of MSC+EPC ECM-TEB and MSC+POC+EPC ECM-TEB.After the implantation of MSC+POC ECM-TEB,the defect repair was completely completed,indicating that it has significant advantages in promoting osteogenesis.Subsequently,in vitro experiments using MSC ECM-TEB as the control group and MSC+POC ECM-TEB as the experimental group showed that approximately 3 mg/ml bioactive protein released from MSC+POC ECM-TEB also promoted MSC in vitro migration,adhesion and osteogenic differentiation.For the study of the mechanism,the results of MS analysis of isobaric labels for relative and absolute quantification(iTRAQ)labeling showed that 324 proteins were significantly up-regulated,of which CXCL12 and IGFBP5 were the most significant,which could promote the migration and osteogenic differentiation of MSC,respectively.The results were confirmed by neutralizing antibody experiments.Conclusion:In summary,this study optimized the construction of ECM-TEB by changing the type of seed cells,which could significantly enhance the effect of bone repair,which brought new technical strategies for bone tissue engineering.