In Vivo Biocompatibility And Hemocompatibility Of A Polytetrafluoroethylene Small Diameter Vascular Graft Modified With Sulfonated Silk Fibroin

Background and objectivesCardiovascular disease (CVD) is the leading cause of death worldwide. The American Heart Association reported that CVD was responsible for 31.9% of all deaths in the United States in 2010, and the total number of in-patient cardiovascular operations or procedures was approximately 8 million in the same year in the USA. Expanded polytetrafluoroethylene (ePTFE) is a material with excellent biocompatibility that has been successfully used in replacing large diameter arteries. However, for arteries with a diameter less than 6 mm, ePTFE vascular grafts show very poor patency rates due to acute graft thrombogenesis and long-time intimal hyperplasia. Therefore, autologous vein and artery grafts remain the first choice for small diameter vessels in the cardiac bypass procedure. Not all patients have vessels suitable for use in the bypass approach though, because in some cases, the native vein and artery are of poor quality or were harvested in a previous bypass operation.Silk fibroin (SF) offers excellent biocompatibility along with acceptable degradation rates and minimally toxic degradation products, and importantly, SF elicits few adverse immune responses in host systems. Furthermore, when SF is sulfonated, it obtains anticoagulative properties, and these can be tuned by adjusting the amount of sulfonated groups introduced. Low-temperature plasma treatment is commonly used to modify the characteristics of a range of substrate surfaces without damaging surface morphologies. With the objective of creating a small diameter graft suitable for use in coronary artery bypass, in our previous research, we successfully fabricated a small-diameter ePTFE graft coated with SF sulfonated by low temperature plasma treatment. In our approach, a 4-mm diameter ePTFE graft is first treated with argon plasma, and then the graft is immediately coated with SF solution. After addition of the SF membrane to the surface of the graft, the composite graft is modified by lower temperature dioxide plasma treatment to obtain a film of sulfonated SF. Our in vitro studies demonstrated that the composite graft possesses excellent hemocompatibility. To further develop this graft for clinical use in coronary artery bypass, we herein investigated the in vivo biocompatibility and blood compatibility of the 4-mm sulfonated SF-coated ePTFE composite graft. We replaced the abdominal aorta of New Zealand rabbits with this graft and compared the resultant patency rate with that of the commercially available 4-mm ePTFE graft.Materials and methods2.1 Preparation and characterization of 4-mm sulfonated SF-modified ePTFE graftCommercially available 4-mm ePTFE grafts (MicroPort CO, Ltd, ShangHai, China) were used in this study. The ePTFE grafts were placed vertically in a plasma machine (DL-1, AoMiGe electromechanical Technology Co, Ltd, SuZhou, China) and modified by exposure to low-temperature argon plasma for 20 min with the electrical discharge voltage set at 70 Pa and the power at 60 W. After the procedure, the inner lumen of the modified grafts was coated in a 1.5 mg/ml solution of SF that had been purified from silkworm cocoons (SouthChina Agricultural University, GuangZhou, China) via the salting-out method. After coating with the SF film, the composite ePTFE grafts were exposed to sulfur dioxide plasma for 20 min with the electrical discharge voltage set at 20 Pa, the flow rate at 20 SCCM, and the power at 60 W. The prepared SF-coated ePTFE vessels were sterilized by ethylene oxide and packaged for artery replacement. The inner surface of the grafts was examined by scanning electron microscopy (SEM; S-3000N, Hitachi, Tokyo, Japan), and the amount of sulfonic groups on the inner surface was tested by X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Fisher Scientific, MA, USA).2.2 Establishing a rabbit model of small diameter vascular graft replacementTwenty-two New Zealand white rabbits (Research Animal Center of Southern Medical University, GuangZhou, China) were used for abdominal artery replacement experiments, including 12 females and 10 males. The animals were maintained according to the policies and guidelines of the People’s Republic of China on Research Animal Use (The State Council of the People’s Republic of China,1988) and the Guide for Care and Use of Laboratory Animals (Guangdong Province,2010). The study protocol was approved by the Institutional Animal Care and Use Committee of Southern Medical University. Prior to surgery, the rabbits were weighed, and general anesthesia was induced by intravenous injection of 1% sodium pentobarbital through the auricular vein at the dose of 30 mg/kg (Yike Guang Zhou Biological Technology Co, Ltd, GuangZhou, China). The rabbits were then fixed upon the experimental platform, the abdominal hair was cut off, and the skin was sterilized using iodine and alcohol. An incision was made along approximately 1/3 of the midline of the abdomen, extending from the symphysis pubis to the xiphoid. Local anesthesia with 1% lidocaine was applied at the incision site. After a 4-cm incision was made through the skin and linea alba, a mastoid distraction was used to expose the abdominal viscera. The intestinal tract and bladder were pushed away by wet warm gauzes. Then the lower abdominal aorta was exposed, and its branches were ligated. During surgery, systemic heparinization was achieved by administering 375 IU/kg heparin (Chengdu Hepatunn Pharmaceutical Co, Ltd, ChengDu, China). The abdominal aorta was separated from the abdominal aortic vein carefully to prevent the vessel from bursting, and the diameter of the aorta was measured using a Vernier caliper (500-712-10, Mitutoyo, Kawasaki, Japan) in the middle section of the divided abdominal aortic arteries. The abdominal aorta was occluded at both posterior and anterior positions in the divided part and then transected at the median section. A proximal longitudinal arteriotomy was made at a length of 3 mm from each end. Next, 3-cm ePTFE grafts were anastomosed to the abdominal artery end-to-end with continuous 6-0 Prolene sutures (Prolene, Johnson & Johnson, Piscataway NJ). The distal anastomosis was performed similarly.The distal anastomosis was not tied at the last suture. The clamp at the proximal end was loosened gently to push the air out of the graft and to detect any leakage. Then the proximal clamp was closed, and the clamp at the distal end was released to release the air in the vessel. Then the distal suture was tied, and a small block of abdominal muscle was excised as a shim for a mattress-suture to stop the bleeding from the anastomosis. Finally, all the clamps were released. The linea alba was closed with interrupted suturing with No.4 silk line, and the skin was closed with No.1 silk line sutures after hemostasis. The incision was cleaned with alcohol and covered with gauze fixed with tape. The operative time and aortic clamping time were recorded.Rabbits were housed in separated cages after they awoke from anesthesia. Penicillin at a dose of 50,000 IU/kg was injected intravenously twice per day to prevent infection. Aspirin at a dose of 3 mg/kg was given once per day as antiplatelet therapy. Ranitidine at a dose of 5 mg/kg was given twice per day to inhibit gastric acid production and prevent gastric ulcer formation or hemorrhage of the gastrointestinal tract. In the postoperative observation period, all rabbits were fed as normal.2.3 Postoperative observationThe primary aim of observation was to confirm whether the abdominal artificial graft became occluded or not. The rabbits were observed two times each day, and the lower limbs in particular were examined carefully to identify any physical findings of paralysis, increased limb muscle strength, or gatism, which would indicate the possibility of occlusion. In addition, femoral arteries on both sides were palpated inside the lower limbs. A good pulse at the arteries was considered indicative of the patency of the artificial grafts. Ultrasonic examination was performed by a sonographer using an ultrasound machine for vessels (iE33, Philips, Eindhoven, The Netherlands) if any of the physical findings listed above were detected. The secondary objective during observation was to identify infection or dehiscence of the incision.The ePTFE grafts and abdominal aortic arteries were examined by ultrasound along the transversal and longitudinal axes every 3 days from implantation to the end of the study period to confirm the patency of the grafts, independent of the presence of any related physical findings. The presence of thrombosis, stenosis, dilation, or anastomotic pseudoaneurysm formation were also noted.Rabbits in which graft occlusion was diagnosed were euthanized by intravenous injection of 1% sodium pentobarbital (120 mg/kg, a lethal dose) through the auricular vein, and the grafts were harvested. In all other rabbits, the grafts were harvested using the same methods at 3 months after operation. After gentle washing with saline, the harvested grafts were cut longitudinally at both points of anastomosis and transected at the middle. Then the grafts were fixed and processed for SEM observation as described in our previous report. The inner surface of the grafts was examined to determine the patency rate for each graft type. Patency rates based on the absence of occlusion at 3 days after operation and from 4 days to 3 months after operation as well as the rates at the end of the 3-month observation period based on imaging examinations were calculated and compared between the experimental and control groups. In the analysis of data from 4 days to 3 months after the operation, data for grafts found to be occluded within the first 3 days were eliminated for the purpose of excluding the impact of previous embolization in this period. In addition, using Metamorph software (Molecular Devices, Sunnyvale, CA, USA), the percentage of the lumen covered with endothelium was determined in SEM images.2.4. Statistical analysisSPSS 13.0 software (SPSS, Inc, Chicago, IL, USA) was used for statistical analysis. Continuous data are expressed as means ±standard deviation (SD) and were analyzed by Student’s t-test followed by a post-hoc test when appropriate. Patency rates are presented as percentages, and Fisher’s exact probabilities were used to assess differences in patency rates. Statistical significance was assumed at a value of P<0.05.Results3.1. Material, animal, and operative characteristicsSEM imaging confirmed that the inner surface of the control graft consisted of ePTFE fiber under SEM, whereas the inner surface of the modified graft was covered by SF film. The rabbits’weight and aortic artery diameter as well as operative details including operation time and aortic clamping time did not differ between the two groups (all P>0.05).3.2. Graft patencyThe patency rates for the experimental and control ePTFE grafts 3 days after abdominal aortic artery replacement,4 days to 3 months postoperatively, and at 3 months postoperatively are presented in tables. At 3 days after artery replacement, the patency rate in the experimental group was significantly higher than that in the control group (100%VS60% P<0.05), and none of the SF-coated ePTFE grafts were occluded. Color Doppler ultrasonography confirmed the grafts were unobstructed, and color Doppler flow imaging revealed blood flow through the SF-coated grafts. In contrast, blockage was readily observed in the unmodified ePTFE grafts, and thrombosis as well as a lack of blood flow were observed in the interior of the control grafts.From 4 days to 3 months and at 3 months after abdominal aortic artery replacement, the patency rates in the experimental group remained significantly higher than those in the control group (91.7%VS33.3% P<0.05). SEM images of grafts harvested 3 months after graft placement revealed that endothelial cell (EC) adhesion to and proliferation on the experimental grafts were much better than on the control grafts. Proliferating ECs covered approximately 84% of the whole lumen of the SF-modified grafts, and the ECs were growing longitudinally and showed extensive cell-cell connections.In comparison, ECs covered only approximately 11% of the inner lumen of the unmodified ePTFE grafts, and the inner surface of these grafts was covered with activated platelets, erythrocytes, and newly formed extracellular matrix.ConclusionIn summary, we fabricates a 4-mm ePTFE graft coated with a SF film sulfonated by low-temperature plasma treatment with advanced parameters, which is much better than our previous study. And we developed a small diameter vascular graft replacement animal model, in which the patency of grafts can easily by confirmed according to clinical physical findings and ultrasound examination.Our study demonstrates that a 4-mm ePTFE graft coated with a SF film sulfonated by low-temperature plasma treatment had a higher patency rate than the 4-mm unmodified ePTFE graft with our animal model, indicating the SF-modified grafts offer much better hemocompatibility and biocompatibility than the unmodified grafts. SEM revealed that the inner surface of the SF-modified grafts were almost completely covered by ECs, which is a predictor of long-term patency.