| The principal means of atherosclerosis treatment is vascular transplantation, usually using autologous saphenous vein or internal mammary artery as material. But autologous vessel that can be available for transplantation is limited, and some patients not suitable for autologous vascular transplantation. Artificial blood vessels such as expanded polytetrafluoroethylene and Dacron(?) in large diameter (inner diameter>6mm) vascular transplantation has been successful, but in small diameter (inner diameter<6mm) vascular transplantation due to its low blood flow and high shear stress can lead to thrombosis and neointimal hyperplasia, the long-term patency rate is not ideal. At the same time, due to the lack of degradability and promoting tissue regeneration capacity, long-term transplantation in the presence of poor biocompatibility issues. In recent years, the development of tissue engineering for the study of biodegradable, biocompatible of small diameter vascular scaffolds provides new way.In our previous studies, the RGD spider silk protein (pNSR32) and polycaprolacton (PCL) with excellent mechanical properties and chitosan (CS) with good blood compatibility was blending. Then, pNSR32/PCL/CS small diameter vascular scaffold was prepared by electro spinning. The experimental results showed that with good blood compatibility and suitable mechanical properties, this vascular scaffold had the three-dimensional porous network structure which simulating the extracellular matrix structure. This study on the basis of that, biodegradation property will be discussed, and the vascular scaffold cotycompatibility should be do more in-depth study, such as the cell adhesion, proliferation, cell cycle, phenotypic expression and the development of cytoskeletal proteins. At the same time, in order to do further evaluation about small diameter vascular scaffold in vivo whether can keep stability and patency, SD rat abdominal aorta defect model will be bridged by this vascular scaffold.We got the results as following:1. The biodegradability of the small-diameter vascular scaffolds was investigated. With the degradation time, the loss rates of pNSR32/PCL/CS small diameter vascular scaffolds were increased, the degradation degree followed by in vivo degradation group> in vitro hydrolysis group> in vitro enzymatic group. pNSR32and CS and PCL were blended in a certain extent could promote the degradation of PCL, improved its surrounding microenvironment. It proved that the pNSR32/PCL/CS small diameter vascular scaffold was biodegradable.2. The cotycompatibility of the small-diameter vascular scaffolds were explored, such as cytotoxicity, adhesion, proliferation and physiological function.(1) The cytotoxicity of the small-diameter vascular scaffolds was studied. High purity SDRAECs were obtained by a tissue block method. MTT assay showed that extracts of different vascular scaffolds had not displayed cytotoxicity, in line with the requirements of the application of tissue engineering scaffolds. Cell relative growth rate was followed by pNSR32/PCL/CS group> pNSR32/PCL group> PCL group. This indicated that the cotycompatibility of PCL scaffold was improved by the addition of pNSR32and CS. Single cell gel electrophoresis showed that no DNA damage with cells cultured in extracts of pNSR32/PCL/CS scaffold, suggesting that pNSR32/PCL/CS was no significant carcinogenicity and genotoxicity. This laid the foundation for further test in vivo.(2) The impact of small-diameter vascular scaffolds on adhesion and proliferation of endothelial cells was analyzed. The experimental results indicated that the addition of pNSR32and CS can improve the cotycompatibility of scaffold. Among them, cell adhesion test showed the SDRAECs adhesion capacity of pNSR32/PCL/CS group was the strongest, the development of cytoskeletal proteins was the best. SEM test results displayed that the cells of pNSR32/PCL/CS group could maintain good growth form, and release matrix. Proliferation assay illustrated that the pNSR32/PCL/CS scaffold could promote the growth of SDRAECs, the cell number was more than pNSR32/PCL and PCL group. Flow cytometry showed that pNSR32/PCL/CS small diameter vascular scaffold promoted DNA synthesis and proliferation of cells by encouraging cells transforming from G0/G1to S phase. At the same time, the DI values in the range of1.0±0.1proved pNSR32/PCL/CS scaffold non-carcinogenic, and could be applied in vivo.(3) Try to study the cotycompatibility of small-diameter vascular scaffolds from the physiological function of cells. Nitrate reductase method for the determination of NO secretion tests showed that pNSR32/PCL/CS small diameter vascular scaffold could promote SDRAECs release of vasoactive substances NO, and the secretory volume was greater than pNSR32/PCL and PCL group. At the same time, compared with the control group, the difference was statistically significant (3,5,7d, P<0.05). Immunofluorescence studies showed that pNSR32/PCL/CS small diameter vascular scaffolds could support SDRAECs strong expression of endothelial cell markers---vWF, PECAM-1. It was visible that the cell biology characteristic was unaffected. pNSR32/PCL/CS small diameter vascular scaffold could be formed into a "function" vessel in tissue engineering.3. The feasibility of pNSR32/PCL/CS small-diameter vascular scaffolds used in the clini was explored. The SD rats abdominal aortic defect model was constructed. Then the pNSR32/PCL/CS small-diameter vascular scaffold was used to bridging defects by "double-cuff technique". During the transplant operation, pNSR32/PCL/CS small diameter vascular scaffold showed good operation. The postoperative animal recovered well, exercising and eating behavior maintained normal. The blood physiological and biochemical results showed the scaffold had not significantly affected on the liver and kidney function. The scaffold in vivo normal flow environment could support the growth of cells, and keep at least8weeks to maintain integrity and patency, but the long-term stability and patency was not clear. |
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