The Animal Experiment Of Small Caliber Expended Polytetrafluoroethylene Artificial Blood Vessel Coated With Sulfonated Silk Fibroin

1 Research background and significanceWith the increasing morbidity and mortality of cardiovascular disease and peripheral vascular disease, human’s health and life expectancy were seriously affected. It is showed by the data that about 17 millions of people died from cardiovascular disease each year all over the world which accounted for 29% of all deaths. With the development of the world’s population aging and the increasing morbidity of chronic diseases such as diabetes, the number of deaths from cardiovascular disease will be rose to 23 millions in 2030. At present, there are about 290 millions of cardiovascular patients in our country, and cardiovascular cause death accounted for 45% of all deaths. When there is deterioration, atherosclerosis, vascular thromboembolism and lesions with the normal blood vessel, blood and oxygen supply to the normal tissues and organs will be affected. Artery bypass operation or vascular transplantation must be performed, when it’s seriously affected. The effect of the transplantation and long term patency rate of graft vessels are closely related with quality of artificial blood vessels. Autologous blood vessel is the golden standard material for its excellent anticoagulant capability, biocompatibility and mechanical properties. However, autologous blood vessel is not always available and the quality of the blood vessel is unequal, even more, it will done much harm to the body, so the application of the autologous blood vessel in clinical is limited. It’s difficult for the autologous blood vessels to meet the increasing demand of clinical use, so there is an urgent need for search a new substitute for blood vessels replacement. Artificial blood vessel, a kind of vascular prosthesis to repair and replace the lesion blood vessels, which is not come from body organs and tissues, is playing an more and more important role in the vascular transplantation. At present, the artificial blood vessels have achieved satisfactory clinical effect in replacement of large and medium sized artery. However, the replacement of small artery by artificial blood vessels remain the common problem for vascular surgeon, the researcher of material science and tissue engineering and other subjects. The main reason for this problem is that the poor blood compatibility and low blood velocity of small caliber artificial blood vessel make it easy to result in thrombosis and intimal hyperplasia which cause the machinery luminal stenosis. Therefore, improvement of hemocompatibility and biocompatibility of artificial blood vessels by surface modification is the key to solve this problem and research direction of small caliber artificial blood vessels in the future. Therefore, it’s an inevitable trend to develop a kind of safe small caliber artificial blood vessel to meet the clinical use for the clinical development.2 Objective2.1 To construct an animal model that can simulate the changes of the blood flow dynamics of the bridge vessel after coronary artery bypass grafting.2.2 To evaluate the anticoagulant effect and 8 weeks’ patency rate of small caliber expended polytetrafluoroethylene artificial blood vessels coated with sulfonated silk fibroin in vivo experiment.2.3 To explore the effectiveness of endothelialization of small caliber expended polytetrafluoroethylene artificial blood vessel coated with sulfonated silk fibroin in vivo experiment.3. Materials & Methods3.1 Artificial blood vessels for the substitutionOrdinary blood vessel:small caliber polytetrafluoroethylene artificial blood vessel (caliber =4mm, provided by Shanghai MicroPort medical equipment Co., Ltd); Composite blood vessel:small caliber expended polytetrafluoroethylene artificial blood vessel coated with sulfonated silk fibroin were prepared according to the previous research literature method.3.2 Experimental animalsTwenty two healthy New Zealand white rabbits, Weight 2.0-2.5kg, male or female unlimited, provided by the laboratory animal center of Southern Medical University and breeding by professionals. All the experimental animals got the approval of animal ethics committee of laboratory animal center of Southern Medical University. All the operations were performed in the clean operating room of old Zhujiang hospital of Southern Medical University according to the regulations of National Institutes of Health, where was disinfected by ultraviolet radiation. The surgical operating instruments were sterilized by high temperature and high pressure and the operation procedures were sterile.3.3 Experimental groupsHealthy New Zealand white rabbits were provided by the experimental animal center of Southern Medical University, conventionally breeding for one week before abdominal aorta artificial blood vessel transplantation surgery, and then dividing into A, B two groups randomly:Group A (n=12) was transplanted with small caliber expended polytetrafluoroethylene artificial blood vessels coated with sulfonated silk fibroin and Group B (n=10) was transplanted with ordinary expended polytetrafluoroethylene artificial blood vessel.3.4 Establishment of animal modelsThe rabbit was intravenous anesthesia with pentobarbital sodium through ear vein, and then the abdominal aorta was transplanted with a 3 cm long artificial blood vessel(caliber=4mm) to establish the animal model. Regular aspirin for anticoagulation in every rabbit after operation. Success rate of model building, anatomy of abdominal aorta, local hemodynamic changes and assessment methods of vascular patency condition were used to evaluate the model building.3.5 Doppler ultrasonography examinationDoppler ultrasonography examination was performed in every rabbit after operation immediately,4 weeks and 8 weeks to observe the flow velocity, vascular morphology and patency condition of grafted vessels. And then, compare the Doppler Ultrasound manifestations of obstructed and unobstructed artificial vascular grafts.3.6 Vessel patency condition and sample general observationThe patency rate of transplanted blood vessels in 2w,4w and 8w were conducted by statistical analysis, and sample observation was conducted at the same time to evaluate its anticoagulant effect. The rabbit was killed immediately to get the grafted vessels sample for general observation if the transplanted blood vessel was obstructed. If not, the sample was got after 8 weeks for general observation and scanning election microscopy analysis to observe surface condition of surface thrombosis and endothelialization. General observation was conducted to evaluate the surface condition of surface thrombosis, endothelial cell growth, anastomotic stenosis and vascular patency condition.3.7 Scanning election microscopy analysisScanning election microscopy analysis was conducted to observe the surface condition of two group grafted vessels 8 weeks after operation, including surface micromorphology, thrombosis and endothelial cell growth, to preliminarily analyze the anticoagulation property and the effect of endothelialization of expended polytetrafluoroethylene artificial blood vessel coated with sulfonated silk fibroin.4 Results4.1 Results of animal modelAnimal models of small caliber artificial blood vessel transplantation for rabbit abdominal aorta were built successfully, success rate was 68%.Doppler ultrasonography examination data showed that grafted vessels were unobstructed. The anatomy of abdominal aorta showed that the abdominal aorta was closed to the inferior vena cava near the body surface and with less peripheral vascular branches. When the abdominal aorta was separated and exposed, there were almost no branching vessels, with good shallow location of the blood vessels, which is convenient for the vessel anastomosis. It was show that the graft was plump overall and pulsating well, with no bleeding and anastomotic stenosis after the artificial blood vessel anastomosis was completed. Rabbits showed paralysis of both lower extremities, hypermyotonia or muscle rigidity, incontinence and femoral arteries pulse disappeared. Doppler ultrasonography examination revealed that thrombus formation in the grafted vessels.lt’s proved that the patency condition of grafted vessels can be estimated through abnormal signs and Doppler ultrasonography examination.4.2 Doppler ultrasonography examinationDoppler ultrasonography examination was used to observe the flow velocity of proximal and distal region of anastomosis, vessels’ morphology and patency condition, after implantation of artificial blood vessels. It is showed by pictures of Doppler ultrasound of the obstructed grafts that the morphology of anterior wall, posterior wall and whole blood vessel were clearly showed, with no anastomotic stenosis, but high density echo shadow fill of the vessel lumen, proximal and distal region of anastomosis, which suggested that thrombogenesis in the vessel lumen. Blood flow signals were found in the proximal region of autogenous blood vessel by Doppler ultrasonography and the peak value was 52.0cm/s, while it was interrupted in the anastomotic stoma, which proved that there was almost no blood flow through the artificial blood vessels after the formation of thrombosis. Doppler ultrasonography examination data shown that the anterior and posterior wall of unobstructed grafted vessels 4 weeks after operation were displayed clearly, with no thrombogenesis and anastomotic stenosis. The uniform blood flow signals were found in the proximal and distal region of anastomosis through the all graft vessels, which the peak blood flow was 60.6cm/s.8 weeks after operation, the anterior and posterior wall of unobstructed grafted vessels were still can be detected by Doppler ultrasonography, while there was no thrombogenesis and anastomotic stenosis. And the peak blood flow was 61.7cm/s.4.3 Analysis of grafted vessels’patency condition and sample morphologyDifference of patency rates between composite blood vessel group and ordinary blood vessel group of 2 weeks,4 weeks and 8 weeks after implantation were analysis by Fisher’s Exact Test respectively, to evaluate their anticoagulation capability in vivo. Statistical analysis results showed that the patency rate and P value of composite blood vessel group and ordinary blood vessel group was:100% vs60%, P=0.029; 91.7% vs 40%, P=0.02; 91.7% vs 30%, P=0.006; respectively. It is showed by the sample morphology that the lumen of the obstructed blood vessels were filled with long striped thrombus from the proximal part to the distal part.8 weeks after implantation of artificial blood vessels, a layer of smooth, uniform and glossy intima was formed in the lumen of unobstructed composite blood vessel, without thrombus and anastomotic stenosis. While the lumen of the unobstructed ordinary blood vessels were filled with a layer of rough intima and local thrombogenesis.4.4 Results of scanning electron microscopy8 weeks later, results of scanning electron microscopy showed that the lumen of the ordinary blood vessel was covered with a mass of fibrous protein membrane material, with platelet adhesion, aggregation and erythrocyte sedimentation above it, but no endothelial cells covered. However, it was shown by results of scanning electron microscopy 8 weeks later that the lumen of the composite blood vessels were coated with a layer of uniform, dense.and smooth fibrous membranes, with vast regular and longitudinal fusiform cells covered which similar to the endothelial cells. The cell nucleus of fusiform cells were protuberate like island shape and the fusiform cells denser while closed to the anastomosis which connected to each other by the extracellular matrix.5 Conclusion5.1 Construction of small caliber blood vessel transplantation of rabbit model is feasible, which can better simulate the hemodynamic changes of bridge vessel after coronary artery bypass grafting, and observe more timely, convenient, high sensitivity, worthy of promotion.5.2 Expanded polytetrafluoroethylene artificial blood vessel coated with sulfonated silk fibroin have good anticoagulant capability and biocompatibility, which can improve the 2 weeks,4 weeks,8 weeks vascular patency rate and promote the rapid endothelialization of artificial blood vessels.