Functional Protein-based Tissue-engineered Bones Promote Endothelial Cell Migration,Vascularization And Bone Repair Via Activating CXCR2

Background and objectivesLarge segmental bone defects due to severe trauma,bone tumor resection,and bone infection are very common and difficult to treat.In order to solve the dilemma of limited autologous bone sources,the osteogenic repair ability of tissue-engineered bone(TEB),which is constructed by inoculating seeded mesenchymal stem cells(MSC)with osteogenic differentiation potential into scaffold material using bone tissue engineering technology,is close to that of autologous bone,the "gold standard" of clinical treatment.Despite the efficacy of TEB in the treatment of bone defects,the clinical application of TEB is limited by technology and cost because the construction of TEB requires the application of live cells.After the deep-freeze-drying treatment of TEB,no live cells existed on the scaffold material but most of the cell-secreted functional proteins were retained,and the new scaffold was named functional protein-based tissue-engineered bones(FP-TEB).Unlike other organs or tissues transplanted,FP-TEB does not have existing blood vessels and living cells inside,and its internal vascularization level is poor after transplantation,which cannot effectively improve the unfavorable environment of local ischemia and hypoxia and cannot provide sufficient blood supply in time,resulting in limited material exchange inside the transplanted material and affecting the targeted migration of host osteogenic and vascularization-related cells to the bone defect area.Therefore,one of the key issues for further research,development and application of FP-TEB is to clarify how to rapidly recruit host osteoblastassociated cells to accelerate the bone formation of scaffold materials.Our group has shown that the protein retention rate of FP-TEB after freeze-drying is more than 85% compared with that of TEB,and the protein microarray assay has confirmed that it contains a large number of cytokines and chemokines.Therefore,bioactive factors within FP-TEB may be important factors in host cell recruitment and osteogenesis and vascularization,but the mechanism is unknown.In this paper,we initially explored the regulatory mechanism of FP-TEB induced migration of host endothelial cells(EC)into bone defects to participate in the vascularization process and observed the regulatory effects of EC on MSC and related signaling pathways after migration into bone defects.Methods:Part one: The mice bone marrow mesenchymal stem cells(m BMSC)were first extracted,and the cell phenotype was identified by flow cytometry.m MSC were purified and inoculated on DBM to construct TEB,and further freeze-dried to construct FP-TEB.TEB and FP-TEB were observed by scanning electron microscopy to observe the cell and extracellular matrix on the scaffold.The osteogenic and angiogenic abilities of FP-TEB were evaluated by histological staining,RT-PCR,Western blot,and vascular perfusion assays.Part two: The recruitment of FP-TEB to EC and MSC was observed in vitro by Transwell assays and wound healing assays.The recruitment ability of DBM,TEB,and FPTEB to host EC and MSC was observed by immunofluorescence staining.Part three: First,we used lentivirus to interfere with the expression of potential chemokine receptors of FP-TEB that regulate EC migration,and screened the major chemokine receptors of FP-TEB that regulate EC migration in vitro by Transwell assay and wound healing assay.The role of chemokine receptors in the pro-angiogenesis and bone reconstruction of FP-TEB was further evaluated by histological staining and immunofluorescence staining after using inhibitors and constructing gene edited mouse.Finally,the downstream signaling molecules were screened and their effects on FP-TEB angiogenesis and osteogenesis were verified by combining a series of inhibitors with Transwell migration,cell scorting and tube formation assays and in vivo histological staining,immunofluorescence staining and vascular perfusion assays.Part four: By simulating the inflammatory microenvironment of bone defects in vitro,we observed the ability of EC to recruit MSC in the inflammatory environment,and screened the signaling pathway of EC recruitment to MSC and its downstream signaling molecules by a series of inhibitors and lentivirus combined with in vitro Transwell assay and wound healing assay.Inhibitors and tail vein transfusion of gene-edited MSCs were used to validate the signaling pathways screened in in-vitro experiments.Results:Part one: The extracted cells were identified as positive for CD73,CD90,CD105,and negative for CD31,CD45.Scanning electron microscopy showed a large amount of m BMSC on TEB and uniformly crinkled extracellular matrix on FP-TEB.H&E and Masson staining showed that FP-TEB had similar bone formation ability as TEB.Further RT-PCR and WB showed that the expression of osteogenesis-related genes Run X2,ALP,and OCN were higher in FP-TEB and TEB,with Run X2 being the most highly expressed in FP-TEB,and vascular perfusion experiments showed that FP-TEB had the similar angiogenic capacity to that of TEB.Part two: Transwell assays and wound healing assays showed that FP-TEB had similar recruitment effect on EC and MSC.Immunofluorescence results showed that both FP-TEB and TEB could recruit large amounts of host EC and MSC to the bone defect area,but FPTEB had a stronger recruitment effect on H-type vessels compared with TEB.Part three: In vitro experiments revealed that the ability of FP-TEB to promote EC migration was significantly reduced when CXCR2 expression was disrupted by lentivirus.In vivo experiments showed that the ability of FP-TEB to recruit host EC and to promote angiogenesis and bone repair in bone defect areas was significantly reduced when CXCR2 expression on mouse EC was knocked down by inhibitors or gene editing.In vitro cell migration assays identified two potential signaling molecules downstream of CXCR2--Src and p38 MAPK,and in vivo experiments revealed that FP-TEB recruitment to host EC and the ability to promote angiogenesis and bone repair in bone defect areas were significantly reduced when the expression of Src or p38 MAPK was inhibited by inhibitors.Part four: In the mimicked inflammatory microenvironment,MSC migration towards EC was significantly promoted.In vivo experiments confirmed that EC is the major PDGF-BBsecreting cell in the bone defect area,and immunofluorescence staining showed that the scaffolds were unable to recruit MSC after knockdown of PDGFRβ,Src,or Akt.Together,these findings suggest that in the inflammatory microenvironment,MSCs migrate towards ECs via PDGF-BB/PDGFRβ and the downstream Src-Akt signal pathway.Conclusion: We demonstrated for the first time that FP-TEB promotes EC migration through CXCR2 and its downstream Src and p38 MAPK signaling pathways.When EC migrated to the bone defect area,it will activate PDGFRβ receptor and its downstream SrcAkt in MSC by secreting PDGF-BB to promote MSC migration to the bone defect.These results reveal the molecular mechanism of FP-TEB vascularization and provide a new target for improving the bone repair ability of FP-TEB.