| BACKGROUND: Cardiovascular disease is increasing year by year and has a high mortality rate,coronary artery disease and peripheral arterial disease are the main diseases leading to this high mortality rate,and reconstruction of blood flow in these regions by bypass surgery is the main treatment option.Although autologous vessels have been the gold standard for small-diameter vascular implantation,about 30% of patients cannot obtain appropriate autologous grafts due to trauma,infection,reoperation and other factors,therefore,there is a huge demand for small-diameter vascular grafts(< 6 mm).However,after small diameter vascular implantation,neointima formation and hyperplasia are easily caused by the lack of endothelial cell coverage,small caliber,hemodynamic changes and other factors,and the incidence of early thrombosis is high,resulting in vascular implantation failure.So far,there is still no suitable small diameter vascular graft for clinical application.Native vascular extracellular matrix(ECM)has complex three-dimensional structure and multiple biological components,which can regulate cell adhesion,migration and proliferation and rapid host cell infiltration in vascular grafts and promote vascular regeneration and remodeling.However,small diameter vascular xenografts will contact the blood system immediately after implantation,due to the lack of complete endothelial cell coverage,and trigger a series of biological response events,ultimately causing neointima formation and effective blood flow area reduction of the graft vessels resulting in hemodynamic disturbances and affecting the subsequent fate of the vascular graft.However,in the current study,there is no systematic study on the formation and evolution of neointima in small diameter vascular grafts,therefore,exploring the formation and regulation of neointima in vascular grafts is still a key issue that we need to solve.OBJECTIVE: The aim of this study was to construct decellularized small diameter vascular graft with good biocompatibility,and to investigate the evolution of neointima formation in acellular small-caliber vessels in vivo,as well as to regulate neointima formation and improve vascular patency by nanocoating modification of the vascular interfaceMETHODS: Construction a small diameter decellularized vascular graft with well-preserved ECM structure and low inflammatory response is the primary solution.In this study,we propose to construct decellularized small-diameter vascular grafts using bovine internal mammary artery(BIMA)and develop a new decellularization protocol that combines physical perfusion,sonication,chemical detergents,and biological enzyme treatment,and further improves the performance of decellularized BIMA using non-toxic,mild photooxidative cross-linking.To evaluate whether this decellularization protocol can sufficiently remove cellular components and antigenic material from tissues and preserve better ECM structure by in vitro and in vivo experiments,as well as the biocompatibility and in vivo performance of decellularized photooxidative cross-linked BIMA.Because decellularized small diameter vascular graft will form neointima on the luminal surface of vascular grafts early after implantation in vivo,the pathophysiological process of neointima formation is complex and variable and remains incompletely understood.Therefore,we established a rabbit abdominal aortic replacement model to observe the evolution of neointima within 4 weeks of implantation using decellularized photooxidized BIMA.Histological staining,immunohistochemistry,scanning electron microscopy and protein mass spectrometry analysis were performed on the neointima at days 1,4,7,14,21,and 28 to investigate the morphological characteristics of the neointima at each stage,changes in the main host cell types,changes in the main protein components and differential proteins,and related biological informationSubsequently,we focused on the biological processes such as protein adsorption,neutrophil infiltration,and platelet adhesion as important processes for early neointima formation when the decellularized vascular graft came into contact with the blood system according to the evolution of neointima in vivo implanted with decellularized small diameter vascular graft.We used a combination of chemical and biological methods to construct graphene oxide-heparin-polyethylene glycol(GHP)nanoparticles.Graphene oxide has a huge specific surface area and can be loaded with a large amount of heparin and polyethylene glycol to exert anticoagulant and anti-protein adsorption effects.In addition,based on the lamellar characteristics of graphene oxide,nanocoatings were formed at the basement membrane of acellular vessels by covalent binding to seal the exposed collagen fibers and pro-coagulant sites,and finally decellularized vascular graft modified with GHP nanoparticles were obtained,and the performance of vascular grafts modified with GHP nanoparticles was evaluated by in vitro and in vivo experiments.RESULTS: The BIMA were treated with physical perfusion,ultrasonic disruption,sodium dodecyl sulfate(SDS)and triatone(Triton X-100)combined with ribozyme to obtain decellularized small diameter vascular grafts with intact three-dimensional structure and almost complete removal of cellular components and α-Gal antigen.Then photocooxidative crosslinking was used to improve the enzymatic hydrolysis resistance of decellularized vascular grafts.Vascular implantation in a rat subcutaneous model demonstrated that decellularized BIMA treatment with photooxidative crosslinking reduced inflammatory responses and inhibited early and rapid degradation of ECM,but results from a rabbit abdominal aortic replacement model showed significant neointima formation in vascular grafts.For the first time,we investigated the evolution of neointima in decellularized small diameter vascular grafts at different time points from macroscopic,microscopic characteristics and proteomics in a rabbit abdominal aortic replacement model.From the perspective of gross specimens,histological characteristics,changes in cellular infiltration,and proteomic analysis,we initially divided neointima formation into six stages,stage I: protein adsorption and neutrophil accumulation;stage II:neutrophil chemotaxis and sustained protein adsorption;stage III:regression of neutrophils;stage IV: phagocytosis of macrophages and recruitment of fibroblasts;stage V: deposition phase of neo ECM;and stage VI: remodeling of ECM.Neointima involves many biological processes during its formation and progression,including protein adsorption,formation of mural thrombus,chemotaxis and regression of neutrophils,infiltration and differentiation of macrophages,migration and proliferation of fibroblasts,proliferation of smooth muscle-like cells,and remodeling of extracellular matrix.Based on the evolution of neointima formation in decellularized small diameter vascular grafts,we used graphene oxide as a carrier to load heparin and polyethylene glycol.The GHP nanoparticles containing a large amount of negative charge on its surface were constructed and has low cytotoxicity and good hemocompatibility.In addition,GHP nanoparticles were modified on decellularized photooxidation crosslinked BIMA.Functionalized small diameter vascular grafts with anti-platelet adhesion,anti-protein adsorption,good cytocompatibility and hydrophilicity were obtained.In a rabbit abdominal aortic replacement model,it was confirmed that the decellularized small diameter vascular grafts modified with GHP nanoparticles could reduce the thickness and area of neointima,reduce the adsorption of plasma proteins and the formation of mural thrombosis,and the infiltration of inflammatory cells,and significantly increase the effective blood flow area of vascular grafts,and improve the early patency of vascular grafts.CONCLUSION: In summary,decellularized small diameter vascular grafts with good three-dimensional ECM structure and resistance to rapid degradation were constructed by new decellularization protocols and photooxidation cross-linking.The evolution of early neointima formation in decellularized small diameter vascular grafts was studied for the first time in rabbit abdominal aortic replacement model.These results provide new ideas and innovations for the field of tissue engineering vascular grafts,and help to understand the pathophysiological process of neointima formation,and may provide promising theoretical basis and modification strategies for the design and development of the next generation of small diameter vascular grafts.Finally,we constructed GHP nanocoatings with anti-protein adsorption and platelet adhesion and inhibition of inflammatory cell infiltration on decellularized small diameter vascular grafts,which reduced the thickness and area of early neointima,increased the effective blood flow area of vascular grafts and maintained vascular patency.These results showed that the formation process of neointima could be regulated by modification of the interface of vascular grafts,providing a good basis for remodelling and functional regeneration of small diameter vascular grafts. |
