Experimental Study Of HGMSCs-MVs Enriched Cartilage Acellular Matrix Of Tracheal Patch To Promote Functional Reconstruction Of Tracheal Defect

BackgroundTracheal injury caused by various reasons seriously threatens the lives of patients.The artificial tracheal transplantation for surgical reconstruction in serious tracheal defect is needed,which is still a major challenge for clinicians.In recent years,the research on tracheal transplantation mainly focused on the application of tissue engineering technology,autologous tissue or allogeneic tissue for tracheal reconstruction.Due to the special anatomical structure and physiological characteristics of trachea,the tracheal reconstruction often ends in failure,which is caused by the insufficient blood supply after tracheal transplantation,the infection due to the continuous exposure of the trachea cavity in the external environment,the obstacle of airway epithelial regeneration or the mismatch of mechanical properties with the autologous trachea.An ideal tracheal transplantation and long-term stable recovery of airway function are still out of reach so far.The rationaltissue engineering trachea should have the mechanical strength matching with the autologous trachea,as well as the high histocompatibility to achieve rapid vascularization and airway epithelial regeneration after transplantation.Exploring a feasible method to construct a tissue-engineered trachea that can induce angiogenesis and airway epithelial regeneration is of great significance for the realization of functional reconstruction after tracheal injury.Construction of tissue engineering trachea has become an important research direction through the integration of scaffold,biomaterials and seeding cells.Acellular matrix(ACM),as one of the research hotspots in the field of biomaterials,retains the main components and structure of extracellular matrix(ECM)to a certain extent,and provides skeleton and material basis for wound healing and tissue regeneration in vivo.Although the tracheal grafts constructed by the ACM showed the good biological compatibility,there is still a high incidence of postoperative tracheal stenosis,and the reconstruction of airway epithelium is incomplete,which makes it difficult to restore the function of the airway and often leads to the failure of tracheal transplantation.Therefore,a large number of studies have applied bioactive components to modify tissue engineering materials and optimize the composition,microstructure and biological activity of the materials,so as to promote the rapid vascularization of tracheal graft and induce the regeneration of airway epithelium.In recent years,some studies have shown that there are matrix vesicles(MVs)integrated in ACM,acting as the carrier of biological signals between ECM and cells.These MVs play a regulator role in the differentiation of macrophages,the regulation of inflammation and cell proliferation,and finally promote functional tissue repair by providing signal molecules.In order to avoid the direct application of cells,more and more studies focused on loading the separation and extraction of the active components of stem cells in the microstructure of grafts to induce tissue repair.It has been proved that extracellular vesicle(EVs)derived from gingival mesenchymal stem cells(GMSCs)play an active role in anti-inflammatory and immunomodulatory in the repair of various tissue injuries.ObjectiveIn our previous study,segmental tissue engineered tracheal were constructed by rapid degradation elastomer polyglycerol sebacate(PGS)and polycaprolactone(PCL)ring,the first application of rapidly degraded biomaterials as the main materials for the tracheal construction,which showed rapid vascularization,cell recruitment and collagen deposition in vivo,without the regeneration and functional reconstruction of airway epithelium.We also confirmed the positive effects of ACM of chondrocyte sheets in promoting cartilage repair and regeneration in articular cartilage defects.Due to the complexity of tracheal reconstruction,more optimization and adjustment of the construction of grafts were needed to realize the tracheal regeneration.It is envisaged that an ACM tissue-engineered trachea with high biological activity can be constructed,endowed with biological efficacy of inducing angiogenesis and epithelial regeneration on the premise of guaranteeing mechanical strength,so as to initiate the key point of trachea regeneration in vivo and realize functional reconstruction.A tracheal patch was designed for enriched h GMSCs-MVs(g MVs)in the microstructure of the chondrocyte ACM(c ACM)which was combined with degradable PGS organically,and the biological activity of the patch,as well as its promoting effects and mechanism in the process of tracheal reconstruction in vivo were investigated.MethodsThis research includes four parts of experiments.1.Construction of a surgically suturable c ACM-PGS/PP tracheal patchChondrocytes(CHs)harvested from auricular cartilage of rabbits were seeded on the surface of PGS/polypropylene(PP)composite scaffolds with three different pores under dynamically culturing for 2 weeks to form cartilaginous ECM(c ECM);through the mechanical analysis,SEM,TEM,histological staining and quantitative analysis of ECM components,the most suitable pore size of PGS for the proliferation and ECM secretion of CHs was screened out according to the mechanical strength,microstructure,content of different components in ECM formed on different scaffolds;then three different methods of acellular precedure were performed on c ECM,and the optimal acellular procedure was selected according to the microstructure,the histological characteristics,the quantificationof ECM components of cartilaginous acellular matrix(c ACM).A surgically suturable c ACM PGS / PP tracheal patch was constructed.2.Construction of g MVs-c ACM-PGS/PP tracheal patch and its biological effect in vitrohGMSCs were isolated and cultured,then identified by flow cytometry and tripotential assessment;GMSCs and CHs were co-cultrued on PGS/PP scaffold at different ratio for dynamically culturing for 2 weeks in vitro,then the optimal ratio was screened out according to histological staining and gross analysis;the g MVs-c ACM-PGS/PP tracheal patch was constructed following the optimized acellular process in Experiment 1,then the differences of protein components and characteristics of microstructure between g MVs-c ACM PGS/PP and c ACM-PGS/PP were compared by mechanical analysis,SEM,TEM,histological staining,silver staining and western blot;the size,morphology and marker of g MVs were determined by NTA,TEM and western blot;proteomic analysis and ELISA were used to detect the active molecule of g MVs-c ACM-PGS/PP;Transwell,scratch test,immunofluorescence staining and western blot were used to trace the g MVs in vitro,and evaluate the effects of g MVs-c ACM-PGS/PP on the migration and proliferation of human bronchial epithelial cells(HBEs).3.Study on promoting the regeneration of airway ciliated epithelium by g MVs-c ACM PGS/PP tracheal patch in rabbit focal tracheal defect modelThe role of g MVs-c ACM-PGS/PP tracheal patch in the reconstruction of tracheal defect in vivo was evaluated using the rabbit focal tracheal defect model.The systemic and local toxicity of the patch was assessed through the histological staining at 1 and 3 weeks after being embedded under the rabbit subcutaneous;the in vivo imaging system was used to evaluate and trace the distribution and release of g MVs stained by Di O;PGS/PP,c ACM-PGS/PP and g MVs-c ACM-PGS /PP were used to reconstruct the focal tracheal defects in rabbits,and the effects of tracheal patches on the reconstruction of trachea,including the survival rate of the animals,stenosis rate of airway,cell typing and submucosal vascularization of new airway epithelial tissue were assessed according to the gross analysis,micro-CT histological staining,SEM and immunofluorescence staining.4.Study on the mechanism of gMVs-cACM-PGS/PP tracheal patch on promoting thefunction reconstruction of trachea defect in rabbitsThe regenerated airway epithelial tissues were analyzed by proteomics in the c ACM-PGS/PP and g MVs-c ACM-PGS/PP groups;according to the results of enrichment analysis,KEGG analysis and GO analysis of differentially expressed proteins,immunofluorescence staining and western blot were used to verify the different expression of proteins and the level of submucosal vascularization in the regenerated epithelial tissue between the two groups;the crosstalk of TGF-β1 and JAK2-STAT1 signaling pathway in the mechanism of the effect of g MVs-c ACM-PGS/PP tracheal patch on the behavior of HBEs was explored by transwell,scratch test,immunofluorescence staining and western blot in vitro.Results1.Through the screening of the pore characteristics of PGS and the optimization of acellular procedure,the pore size of PGS was determined to be 30-38 μm which was more conducive to the adhesion and proliferation of CHs,as well as the secretion and deposition of c ECM;at the same time,it was determined that elution with 1% SDS for 6 hours could achieve complete decellularization of c ECM with minimal damage and impact on ECM components.A suturable c ACM-PGS/PP tracheal patch was successfully constructed.2.It is proved that GMSCs highly express the marker proteins of MSCs,CD105,CD44 and CD29,and are tripotential to differentiate into osteogenic cells,adipogenic cells,and chondrogenic cells suggesting the stemness of cultured GMSCs;GMSCs and CHs were co-cultured in vitro at a ratio of 1:4 to construct g MVs-c ACM-PGS/PP tracheal patch on the basis of Experiment 1;the protein composition of g MVs-c ACM was significantly different from that of c ACM,and the expression levels of CD9,CD81 and TSG101,which were the marker proteins of EVs,contained in g MVs-c ACM were significantly higher than those of c ACM;the size and morphology of g MVs showed the characteristics of g MVs accord with those of EVs;the results of proteomics showed that the differentially expressed proteins between g MVs-c ACM and c ACM were mainly enriched in the extracellular domain and related to the signal pathways of cell proliferation and angiogenesis;ELISA showed that g MVs were rich in TGF-β1,VEGF and KGF;in vitroexperiments confirmed that g MVs can be released from the g MVs-c ACM and taken up by HBEs;the migration and proliferation of HBEs was promoted by g MVs,accompanied by high expression of CD44,MMP9,Cyclin D1 and Axin2 in HBEs.3.Histological results confirmed that there was no obvious systemic and local tissue toxicity in rabbits induced by g MVs-c ACM;Di O labeled g MVs can be released and maintained at a certain concentration in vivo;in the model of rabbit focal tracheal defect,the airway keep patent and the survival rate was significantly improved in the g MVs-c ACM group,but the degree of airway stenosis and mortality in the c ACM group were significantly higher than those in the g MVs-c ACM group;the histological results showed that g MVs-c ACM could significantly increase the level of submucosal vascularization and promote the regeneration of airway ciliated columnar epithelial cells,marked by CK5+8 and β-Tubulin Ⅳ,while Mucin 5AC,a marker of goblet cells,was highly expressed in the structurally disordered regenerated epithelium of c ACM group.4.According to proteomic analysis and immunofluorescence staining,it was confirmed that the expression of p STAT1 and vascular endothelial marker protein CD31 in regenerated airway epithelial tissue was significantly increased in g MVs-c ACM group,which were significantly higher than that in c ACM group;TGF-β1 in g MVs was confirmed to have a crosstalk with JAK2-STAT1 signaling pathway in HBEs for promoting the migration and proliferation of HBEs in vitro,which indicated the molecular mechanism of g MVs-c ACM to realize airway epithelial regeneration and functional reconstruction in tracheal defect.ConclusionIn this study,GMSCs-MVs with high biological activity were successfully enriched into the microstructure of c ACM.And g MVs-c ACM-PGS/PP tracheal patch was constructed using the elastic porous degradable PGS as the carrier by artificial tissue engineering methods,avoiding the resources shortage of ACM and enhancing the c ACM regeneration efficiency.The ability of g MVs-c ACM to promote airway epithelial migration and proliferation were confirmed in vivo and in vitro,breaking down the barriers of airway epithelial regeneration and functional reconstruction in trachealreconstruction for a long time and providing a new strategy for the construction of segmental tracheal grafts.At the same time,it also clarified the molecular mechanism of g MVs in promoting airway epithelial regeneration,which provided a theoretical basis and drug target for clinical transformation in the future.In summary,as a cell-free tissue engineered tracheal graft with high biological activity,g MVs-c ACM-PGS/PP tracheal patch can meet the needs of clinical customization and has a high value of clinical application.