The Effect Of PTP1B Tyr152 And Laminin α4 On Bone Regeneration And Angiogenesis In Different Types Of Tissue Engineering Bone

Background:The treatment of bone defect is a difficult problem in orthopedics.Autologous bone graft is the most effective treatment.However,there are some complications in the bone graft donor site,and some people have insufficient bone mass or even no.To solve this problem,many researchers are involved in the research and development of bone graft alternatives.A large number of new bioactive materials have achieved exciting results in early animal experiments.However,further improvement is needed in clinical practice.In the traditional tissue engineering bone,autologous stem cells were cultured and amplified in vitro,and then transplanted into scaffold materials.However,it is difficult to be widely used due to its complex production process,difficult transportation and high pollution risk.With the rapid development of bone tissue engineering,tissue engineering bone can be divided into self-recruited tissue engineering bone and stem cell enrichment tissue engineering bone.Our group has researched many different types of tissue engineering bone for years.We found that osteogenesis and vascularization were still not enough in the bone defect.Therefore,it is very important to consider both osteogenesis and angiogenesis for different types of bone defect repair.For critical bone defects,self-recruited tissue engineering bone has certain advantages because it does not require additional cells to be planted on the scaffold.Currently,many studies have reported that tissue engineering bone is modified with various cytokines or chemokines could recruit bone marrow mesenchymal stem cells(MSCs)and endothelial cells to the bone defect.Despite the good effect of bone defect repair in animal experiments,these cytokines all exceed the physiological dose and often have biosafety problems.MSCs and endothelial cells play a key role in bone defect repair.Most MSCs are distributed around blood vessels in vivo.Endothelial cells can affect the micro-environment which they live in.The first step of bone defect repair is to recruit enough MSCs and endothelial cells.This process involves initiating cell-cell adhesion from cadherin-mediated to integrin-dependent cell-extracellular matrix adhesion and migration.Protein tyrosine phosphatase 1B(PTP1B)is a non-receptor tyrosine phosphatase.Phosphorylation of tyrosine-152(Tyr152)in PTP1 B plays an important role in stabilizing cadherin and β-catenin adhesion complexes,which is key to enhancing cell-to-cell adhesion and reducing cell migration.In our previous study,we designed the PTP1 B Tyr152 region mimetic peptide(152RM),and verified that it could effectively inhibit the phosphorylation of tyrosine at site 152 and enhance integrin-related signaling pathways.However,does phosphorylation inhibition of PTP1 B Tyr152 cause migration of MSCs or endothelial cells? Would loading it on scaffolds be effective in recruiting cells? So far,there is no relevant report.For segmental bone defects,it is difficult to collect sufficient cells in large bone defect.The advantages of stem cell-enriched tissue engineering bone are more obvious.Stem cell enriched tissue engineering bone utilizes selective cell retention technology(SCR)to effectively enrich MSCs and other cells into the scaffold.The previous study of our group successfully increased the number of MSCs in scaffolds and promoted bone defect repair.However,due to a lack of attention to improve the angiogenesis of the scaffold,their ability to repair segmental bone defect is still limited in vivo.Laminin(LN)is a major component of the basement membrane and extracellular matrix.Laminin,as the binding motif of adhesion ligand,mediates stem cell differentiation through integrin.Laminin-α4(LN-α4)is very important in vascular basement membrane,which plays a key role not only in maintaining endothelial cell proliferation,but also promoting osteoblast proliferation and osteogenic differentiation.Therefore,we attempted to use the core functional sequence of LN-α4 to modify DBM scaffold to increase the enrichment of MSCs and endothelial progenitor cells in SCR technology through integrin-mediated cell adhesion and differentiation.Methods:Part I: Firstly,the DBM-MSN/152 RM scaffold was constructed.The characterization,hydrophilicity,biomechanical strength and biosafety of the scaffold were evaluated.Secondly,transwell migration assay was used to observe the effect of scaffolds on the migration of MSCs and endothelial cells.In addition,the possible molecular mechanism of the migration effect of PTP1 B Tyr152 region mimicking peptide(152RM)on MSCs and endothelial cells in vitro were evaluated by cell scratch assay,RNA high-throughput sequencing(RNA-seq),q RT-PCR,and western blotting.Subsequently,the critical-size femoral bone defect of rat was established,and the DBM-MSN/152 RM scaffold was implanted.The healing of bone defect and angiogenesis were observed by X-ray,Micro-CT,calcein-xylenol orange double fluorescence labeling,routine pathological sections,immunofluorescence staining,Von Kossa staining and angiography perfusion.Finally,the effect of 152 RM peptides on the osteogenic differentiation of MSCs was detected by osteogenic specific staining,and the possible mechanism was analyzed.Meanwhile,the effect of 152 RM peptides on angiogenesis of endothelial cells and their possible molecular mechanism were investigated by vascular tube formation experiment and molecular biological methods.Part II: Firstly,the DBM/CBD-LN scaffold was constructed.The characterization and biosafety of the scaffold were evaluated.Secondly,the effect of DBM/CBD-LN scaffold on cell adhesion and enrichment in SCR were identified by flow cytometry,conventional pathological sections and immunofluorescence staining.Thirdly,the segmental bone defect model was established,and the DBM/CBD-LN scaffold was implanted to the bone defect.The healing and angiogenesis of bone defects were observed by micro-CT,calcein-xylenol orange double fluorescence labeling,pathological section,immunofluorescence staining and perfusion angiography.Finally,the effect of DBM/CBD-LN scaffold on osteogenic differentiation of MSCs was detected by osteogenic staining,and the possible mechanism was analyzed by molecular biological methods.And the vascular tube formation assay was used to detect the effect of the DBM/CBD-LN scaffold on vascular formation of endothelial progenitor cells,and the possible mechanism was analyzed by molecular biological methods.Results:(1)The DBM-MSN/152 RM scaffold was successfully constructed.The increase of BET specific surface area could increase the loading amount of 152 RM peptides and achieve the purpose of stable and sustained release.The hydrophilicity and biomechanical strength of the DBM-MSN/152 RM scaffold did not decrease,and the biocompatibility was good.It did not affect the viability,proliferation and spreading ability of cells,and could meet the strength requirements of bone regeneration without weight bearing.(2)The DBM-MSN/152 RM scaffold had a strong ability to recruit MSCs and endothelial cells in bone defect repair.152 RM peptides could enhance the migration ability of MSCs,which might be mediated by CXCR4 and integrin αvβ3,and activated the downstream FAK/STAT3 signaling pathway.152 RM peptides also enhanced endothelial cell migration,possibly by enhancing the crosstalk between integrin αvβ3 and VEGFR2 signaling,inducing FAK-ERK1/2-dependent endothelial cells migration.(3)The DBM-MSN/152 RM scaffold could promote the mineralization and remodeling of new bone in the bone defect,and promote the rapid growth of H-type blood vessels,realizing the organic coupling of bone formation and angiogenesis.(4)152RM peptides could induce osteogenic differentiation and matrix mineralization of MSCs,might partly through down-regulating the level of endoplasmic reticulum stress,promoting β-catenin into the nucleus,thereby activating the classical wnt pathway.152 RM peptides significantly enhanced the vascular tube formation of endothelial cells,possibly by activating the Notch signaling pathway.(5)The DBM/CBD-LN scaffold was constructed,and CBD-LN could stably bind to DBM surface through collagen binding domain to form a stable scaffold.The BET specific surface area of the DBM/CBD-LN scaffold increased,which could increase cell adhesion and spread space.The DBM/CBD-LN scaffold showed good biocompatibility and had no inhibition on cell viability and spreading ability.(6)The DBM/CBD-LN scaffold showed high adhesion ability to MSCs and endothelial progenitor cells.Integrin α5β1 may be the main molecule to enhance MSCs adhesion to the DBM/CBD-LN scaffold.Integrin αvβ3 may be the main molecule to enhance endothelial progenitor cells adhesion in the DBM/CBD-LN scaffold.(7)The DBM/CBD-LN scaffold not only had strong ability of new bone formation and remodeling,but also could promote the angiogenesis in the bone defect,especially for the rapid growth of H-type blood vessels.The DBM/CBD-LN scaffold realized organic coupling of bone formation and angiogenesis.(8)The DBM/CBD-LN scaffold could promote osteogenic differentiation of MSCs,possibly through inducing activation of FAK-ERK1/2 signaling pathway.The DBM/CBD-LN scaffold might activated the HIF-1α/VEGF pathway in endothelial progenitor cells,thereby speeding up angiogenesis.Conclusions:Through this research subject,different types of tissue engineering bone were developed based on integrin-mediated events of cell recruitment,migration and adhesion.For critical bone defects,we revealed that the DBM-MSN/152 RM scaffold effectively recruited MSCs and endothelial cells,coupled bone formation and angiogenesis in vivo.These results suggested that phosphorylation inhibition of PTP1 B Tyr152 could enhance the integrin-mediated motility of MSCs and endothelial cells,promote osteogenic differentiation and angiogenesis,which providing a new idea for critical bone defect repair.For segmental bone defects,the DBM/CBD-LN scaffold was constructed,which could enhance the integrin-mediated adhesion of MSCs and endothelial progenitor cells in SCR technology.And the DBM/CBD-LN scaffold could couple bone formation and angiogenesis,which providing a new perspective for segmental bone defect repair.