Improved Hemocompatibility By Modifying Acellular Small Caliber Blood Vessels With Bivalirudin And Its Biocompatibility Evaluation

Background The incidence rate of cardiovascular diseases is increasing year by year,which has always threatened the health of people.In cardiac surgery,tissue-engineered blood vessels with a diameter larger than 6mm（large caliber blood vessels）have made a breakthrough and been used in clinic.However,substitutes for tissue-engineered blood vessels with a diameter less than 6mm（small caliber blood vessels）used in coronary artery bypass grafting have not been available.At present,such blood vessels are in great demand,but they are still mainly replaced by patients’ autologous blood vessels.Decellularized blood vessels have natural blood vessel foundation and have potential clinical application value,but their biocompatibility and anticoagulant ability need to be improved.Objective The acellular small caliber vascular scaffolds were prepared and modified with polydopamine grafted bivalirudin to enhance the mechanical properties and anticoagulant properties of the acellular vascular scaffolds,evaluated the mechanical properties,cytocompatibility,immune response and anticoagulant properties of the scaffolds in vitro,and further evaluated its subcutaneous entrapment in vivo degradability and in vivo remodeling after transplantation.In order to prepare a new small caliber vascular substitute with good blood compatibility,cytocompatibility and low immunogenicity.Methods1.Fresh abdominal aorta of SD rats（about 2mm in diameter）were taken and rinsed repeatedly,and then immersed in PBS containing penicillin,streptomycin and amphotericin B.The abdominal aorta of SD rats was cleaned and pruned on a super clean bench,and then randomly divided into three groups.Native group: the abdominal aorta of SD rats without experimental treatment.Decell group: the natural blood vessels were decellularized soon after pruning with Triton X-100.Then,the acellular vascular scaffolds were crosslinked in pre-prepared 2% gelatin solution and EDC/NHS crosslinking agent.Bivalirudin group: the vascular scaffolds on the basis of the decell group,were filled with dopamine solution,sealed at both ends,and then deposit in a sterile dark environment to form a polydopamine coating.After that,Bivalirudin solution was sealed in the vascular scaffolds and fully contacted with polydopamine to form a coating,and the bivalirudin modified acellular vascular scaffold was formed.2.The prepared acellular vascular scaffolds were stained histologically（HE staining and Masson staining）to observe the morphological changes.The bursting pressure,compliance and tensile strength were evaluated;Biological properties such as cytocompatibility and blood compatibility in vitro were evaluated by mixed culture with BMSCs,cytotoxicity assessment of materials,determination of anticoagulant related indexes,immunofluorescence staining,PCR and inflammatory reaction of scaffold.3.The vascular scaffolds were embedded under the skin of SD rats and its degradation was analyzed in vivo for 5 weeks.Three months after the scaffolds were transplanted into SD rats,the endothelialization of the grafts was observed by scanning electron microscope,the occlusion rate and patency rate of vascular grafts were analyzed by quantitative data,the changes of mechanical properties after scaffolds transplantation were detected,the grafts were subjected to HE staining,contraction test,relaxation test and calcification evaluation.the neovascularization was evaluated by anti-CD34 immunohistochemical staining.Immunofluorescence experiments were carried out on the grafts to observe the migration and value-added coverage of host ECs and VSMCs and the expression of inflammatory factors in vivo.Results1.We characterized the vascular scaffolds in detail.He staining showed that there were no cell residues in the decell group and bivalirudin group.Masson staining showed that the collagen fibers and elastic fibers in the decell group and bivalirudin group remained basically intact.The determination of collagen content in the two groups showed that collagen was retained.We also tested the mechanical properties of decell group and bivalirudin group.The results showed that there was no significant difference in vascular compliance or tensile strength between the two groups,but these mechanical properties were lower than those of natural blood vessels.Quantitative DNA analysis showed that the cells had been completely removed and showed low immunogenicity of the material.2.We evaluated the cytotoxicity of vascular scaffolds in decell group and bivalirudin group by MTT assay,and the results showed that it in decell group was lower than that in bivalirudin group.The two groups of vascular scaffolds were co-cultured with BMSCs respectively,and the growth of BMSCs in the two groups were observed by immunofluorescence staining.The results also confirmed that the cell viability in bivalirudin group was better than that in decell group.All these indicated that the hemocompatibility and biocompatibility of acellular vessels modified by bivalirudin were better.3.Through in vitro anti-inflammatory and pro-inflammatory related expression,we found that with the passage of time,both decell group and bivalirudin group vascular scaffolds promoted the differentiation of macrophages from pro-inflammatory macrophages to anti-inflammatory macrophages,and this trend was more obvious in bivalirudin group.We embedded the vascular scaffolds of two groups under rat skin to evaluate its histocompatibility.Through the determination of degradation rate for 5 weeks,no significant difference was found between the two groups.After 2 weeks of subcutaneous embedding,through pathological staining,we found that the immune response of vascular scaffolds in bivalirudin group was lower than that in decell group.4.Three months after transplantation,it was found that the grafts in decell group and bivalirudin group were covered with host cells by histological staining.It was further found that ECs and VSMCs migrated and infiltrated more in bivalirudin group by immunofluorescence staining.SEM showed endothelialization of the grafts in the bivalirudin group.Contraction and relaxation test of the grafts showed that the grafts had certain vascular function.Immunohistochemistry confirmed that the vascular density of bivalirudin group was higher,and the calcified area was relatively lower in bivalirudin group.Conclusion1.We prepared and investigated the biological characteristics and in vitro evaluation of acellular small caliber blood vessels modified by bivalirudin.It was found that the acellular vessels modified by bivalirudin had excellent hemocompatibility,low immunogenicity,biocompatibility and mechanical properties.It has more advantages than traditional acellular blood vessels.2.We found that the acellular small caliber vascular scaffold modified by bivalirudin has good host cell affinity,endothelialization and certain neovascularization ability.It may become a potential substitute for small caliber blood vessel transplantation,which provides a new idea for the construction of small caliber vascular grafts.