| Background:Anterior cruciate ligament(ACL)injury is a common bony tendon interface injury in orthopedics and sports medicine.This ligament plays a vital role in maintaining the normal movement and stability of the knee joint.However,due to limited regenerative capacity,the ACL is often reconstructed with bone tunnel autogenous tendon transplantation to restore the integrity and stability of the knee joint.The ideal recovery state after ACL reconstruction surgery is not only that intraarticular grafts are quickly replaced by well-structured tendon tissues,but also that intraarticular grafts are firmly integrated into the bone wall through the new gradient gradient of tendon insertion to form an effective connection,so as to effectively maintain the stability of the knee joint.However,conventional ACL reconstruction grafts are designed solely to mimic the tendon structure.Such single-phase design can only meet the tendon tissue regeneration in the joint cavity,and the effective integration of the internal opening of the bone canal and the bony tendon interface in the bone canal is slow and difficult.Therefore,how to accelerate the generation of effective osseous tendon insertion in the bone canal and tendon tissue regeneration in the joint cavity after surgical reconstruction,and restore the biomechanical function of ACL reconstruction has become the focus of research in the field of sports medicine.This project breaks through the traditional single-phase structure design of ACL graft and intends to construct a bionic telescopic ACL reconstruction graft with optimized function of cartilage layer.The graft mimics the tendon-chondro-bone connection mode between the graft and the bone canal,allowing the scaffold to mimic mechanical properties and morphological structure.At the same time,exosomes that contribute to cartilage differentiation are innovatively combined with the graft through "linking peptides",so as to prolong the local action time of exosomes and improve the chondrogenic induction activity of the graft.This provides a new design idea for preparing ACL reconstruction tissue engineering grafts.Objectives:In this paper,we intend to construct a kind of double-similar telescopic ACL reconstruction graft with optimized function of cartilage layer,so as to provide a new tissue engineering graft for ACL reconstruction.Methods:(1)Material preparation and performance evaluation.By imitating the structural characteristics of the bone canal after ACL reconstruction,a new design concept of "biomimetic sleeve" was proposed to acquire "telescopic" cartilage tissue and "columnar" tendon tissue,and to prepare acellular cartilage sleeve scaffold and acellular tendon scaffold by using the self-designed "new negative pressure suction and decellular device",so as to achieve the biomimetic morphology,structure and mechanical properties of the above-mentioned scaffolds.Histology,synchrotron radiation Fourier infrared imaging(SR-FTIR),scanning electron microscopy(SEM),atomic force microscopy(AFM)and tensile tests were used to evaluate the physicochemical properties of the scaffolds from the aspects of morphology,structure and mechanical properties.Meanwhile,a co-culture system of scaffolds and mesenchymal stem cells was constructed to evaluate the effects of scaffolds on the viability,adhesion and proliferation of stem cells.(2)The subsets of chondroblast differentiated progenitor cells from adipose tissue were screened to extract exosomes and explore their functions.The subsets of chondroblast differentiated progenitor cells were separated from adipose tissue and identified by multi-channel flow separation technology.Secretory exosomes were extracted from chondroblast differentiated progenitor cell subsets and identified.Mesenchymal stem cells derived from bone marrow,fat and synovium were extracted and identified.The co-culture system of exosome-mesenchymal stem cells was established to determine the strength of the chondrogenic induction activity of subgroup exosomes on mesenchymal stem cells from different sources and to explore the optimal induction concentration.The effects of subgroup exosomes on migration of mesenchymal stem cells of different origin were investigated by scratch and longitudinal migration experiments.(3)Stable attachment and slow release of exosomes on acellular scaffolds are achieved by synthesis of "linking peptides".In view of the constant expression of CD9 antigen on exosomes,the molecular structure of CD9 antigen and CD9 antibody protein was obtained by homologous modeling in this study.The CD9 antigen and CD9 antibody were virtual interlinked with the computer to analyze the amino acid sequence of the binding site of CD9 antibody,and the polypeptide with CD9antigen-binding effect was calculated.The polypeptides with specific binding to exosome surface CD9 antigen(i.e.,CD9 binding peptide)were screened.Based on the recombinant protein expression technique,the collagen-binding peptide(CBP)with the collagen-binding property of the cell-free scaffold and the CD9 binding peptide were recombinant and expressed,and a "linking peptide" with the double binding property of collagen fiber and CD9 antigen was obtained by separation and purification.Exosomes with chondroblast inducing activity were stably attached to acellular cartilage sleeve scaffolds using "linking peptides",and the slow-release effect of exosomes and the optimization of scaffold stem cell inducing activity were evaluated.(4)The effect of biomimetic sleeve ACL reconstruction graft on cartilage layer was systematically evaluated.The reconstruction model of ACL injury in Beagle dogs was established to evaluate the regeneration ability of the graft using imaging,histology,biomechanics and molecular biology.In order to evaluate the immune microenvironment regulation mechanism of the grafts,a mouse ACL injury reconstruction model was established in this project.According to the characteristic markers of different types of immune cell groups,the type and number of immune cells in and around the grafts were detected by flow cytometry.The immunogenicity and rejection risk of the graft were evaluated using beagle inflammatory markers,blood routine,and H&E staining of visceral organs.(5)To explore the possible mechanism of chondrogenic differentiation of mesenchymal stem cells.Protein spectroscopy was used to compare and analyze the differences in protein components of exosomes.Based on bioinformatics analysis,key proteins leading to differences in chondrogenesis and induction characteristics were identified,and their mechanisms of action were explored.Results:(1)We prepared the "sleeve" cartilage and "column" tendon scaffolds with the new negative pressure decellularization device independently developed by us,and the experimental results proved that this method can thoroughly remove cells from tissues and retain the extracellular matrix components well.At the same time,the matrix composition and collagen composition of the scaffold were evaluated before and after the decellulation,which proved that the decellulated biphasic sleeve scaffold has the advantages of bionic morphology and matrix composition.The cartilage scaffolds after decellular treatment retained 89.26% collagen and 90.28% proteoglycan compared with those before treatment.The tendon scaffolds after decellular treatment retained89.37% collagen and 93.34% proteoglycan compared with those before treatment.The prepared acellular "sleeve" cartilage and "column" tendon scaffolds have the characteristics of good biocompatibility,no obvious cytotoxicity,good cell growth adhesion and low immunogenicity.(2)The subsets of chondroblast differentiated progenitor cells were successfully sorted and cultured,and the differentiation potential of the subsets was verified.The exosomes of adipose-derived mesenchymal stem cells and soft diaphylastic progenitor cells were extracted and identified.It was further confirmed that exosomes of chondroblast differentiated progenitor cell subset had the ability to promote chondrogenic differentiation and induce migration of adipose,synovial and bone marrow derived mesenchymal stem cells,and had the strongest effect on synovial mesenchymal stem cells.The optimal concentration of soft diaphysial progenitor cell subsets for mesenchymal stem cells was100μg/m L.(3)We obtained "linking peptides" that bind to exosome CD9 and bind to collagen,and the "link peptides" were synthesized by using the system expression of Escherichia coli.The bacteria with the highest expression of recombinant proteins were selected for mass amplification and culture,and 7 "link peptides" that could bind exosomes and scaffolds were obtained.By exploring the optimal binding ratio between linked peptide and exosome,we selected the best linked peptide and proved that it can be released continuously for 21 days in vitro and 15 days in vivo.The cartilage layer with optimized function of exosomes connected to the soft backbone progenitor cell subsets by linking peptides can effectively induce mesenchymal stem cells to differentiate toward chondrogenesis.(4)The results of the study proved that the function optimized cartilage bionic sleeve ACL graft has a good effect on promoting ACL injury repair,which is manifested as the characteristic bone-fibrochondro-tendon structure of regeneration between the graft and the bone canal.The formation of new bone and fibrocartilage can be seen,the arrangement of collagen fibers is regular and orderly,the deposition of proteoglycan matrix components is good,and the anti-tensile mechanics is strong.And the biphasic scaffolds with optimized cartilage layer had lower immunogenicity.(5)Through protein spectrum analysis,it was known that the key proteins AGRN and TNC related to chondroblast were differentially expressed in exosomes of chondroblast subsets.We successfully constructed lentiviruses that knockdown and up-regulate key protein expression,and obtained exosomes of corresponding chondroblast subsets.It is clear that AGRN in the exosomes of chondroblast subsets is the key factor leading to chondrogenic differentiation of synovial mesenchymal stem cells.It has been shown that exosomes of chondroblast subset induce chondrogenic differentiation of synovial mesenchymal stem cells by activating CREB and inhibiting classic Wnt/β-catenin.Conclusion:(1)We successfully constructed a double-similar telescopic ACL reconstruction graft with optimized cartilage layer function,which can promote the effective repair of bone tendon interface injury,and has the advantages of high new bone density,new fibrocartilage layer,good biomechanics,and low immunogenicity;(2)Exosomes can effectively promote the repair of bone tendon interface injury mainly by activating CREB and inhibiting classic Wnt/β-catenin to induce chondrogenic differentiation of synovium mesenchymal stem cells,while promoting stem cell migration to the injured site. |
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