The Study On In Situ Endothelialization Of Small-caliber EPTFE Grafts Coated With Anti-CD133Antibodies Functionalized Heparin-Collagen Multilayer

PART I:Preparation of small-caliber ePTFE grafts coated with anti-CD133antibodies functionalized heparin-collagen layer-by-layer assembly multilayerObjective To investigate the preparation and stability of small-caliber expanded polytetrafluoroethylene (ePTFE) grafts coated with CD133antibodies functionalized heparin-collagen multilayer.Methods The composite small-caliber synthetic vessel was prepared by heparin-collagen layer-by-layer static electrical assemble onto ePTFE graft. Then, the CD133antibodies were coated on its surface. Scanning electron microscope(SEM), Infrared Spectrometer and Laser scanning confocal microscope (LSCM) were used to evaluate the whole procedures of preparation. Contact angle test was carried out to investigate the hydrophilicity changes of ePTFE grafts before and after surface modification with heparin-collagen layer-by-layer assemble coated with CD133antibodies. Surface modified ePTFE grafts were immerged into PBS and shaking with Decolourization Shaker for2h,6h,12h,24h,48h, LSCM was then used to observe the surface morphology and fluorescence intensity of CD133-PE antibodies.Results SEM revealed that ePTFE patchs were covered with obvious heparin-collagen films. The lines of Infrared Spectrometer changed in synchronization with the increase of heparin-collagen films and distinctive peaks of ePTFE, heparin and collagen appeared. LSCM indicated that CD133antibodies were successfully coated on the surface of heparin-collagen multilayer. Contact angle test shown that the hydrophilicity increased as the contact angle decreased from127.24to106.86before and after surface modification. After shaking with PBS for2h,6h,12h,24h,48h, modified ePTFE grafts were still observed with an obvious red fluorescence which indicated the stability of surface modification.Conclusions The preparation of composite small-caliber ePTFE grafts is feasible and achievable, and the surface modification is reliable. PART â…¡:Hemocompatibility and biocompatibility evaluation in vitro of small-caliber ePTFE vessel with heparin-collagen layer-by-layer assembly multilayerObjective To investigate the hemocompatibility and biocompatibility of small-caliber ePTFE grafts coated with CD133antibodies functionalized heparin-collagen layer-by-layer assembly multilayer.Methods The composite small-caliber synthetic graft was prepared by heparin-collagen layer-by-layer static electrical assemble onto ePTFE graft. Then, the CD133antibodies were coated onto its surface as stated above. According to European Standard of ISO10993and Chinese Standard of GB/T16886, blood platelet adhesion test, hemolysis test, plasma recalcification time (PRT) and APTT, PT, TT test were applied to evaluate the hemocompatibility of the composite blood vessel, while short-term subcutaneous implantation, cell adhesion test, CCK-8and LDH test were applied to evaluate the biocompatibility.Results Scanning electron microscopy (SEM) revealed obvious platelets adhesion on the bare ePTFE graft, while less occurred on the HEP/COL coated with CD133antibodies or without CD133antibodies groups (p<0.05). The hemolysis rates of HEP/COL coated with CD133antibodies or without CD133antibodies and bare ePTFE were1.56%,0.71%and2.41%, respectively. The fibrin was not seen as long as30min in HEP/COL coated with CD133antibodies or without CD133antibodies groups, significantly longer than that of the bare ePTFE graft (8.5min). Compared with bare ePTFE graft, the values of APTT, PT and TT were also significantly improved. Subcutaneous implantation shown that no significant difference was found of the inflammation between the modified grafts and the control group. Excellent cell adhesion of the modified ePTFE grafts was approved by coculture of ePTFE grafts with endothelial cells. CCK-8and LDH tests shown that heparin, collagen and CD133antibody had limited affection to the endothelial cells.Conclusions The composite small-caliber ePTFE graft has excellent antithrombotic acitivity, hemocompatibility and biocompatibility. PART â…¢:Carotid artery versus femoral artery small-caliber vascular graft transplantation model in pigsObjective To compare the superiority of carotid artery versus femoral artery small-caliber vascular graft transplantation model in pigs.Methods Twelve pigs were included in the present study. Carotid artery small-caliber vessel transplantation model was established in12pigs, while femoral artery small-caliber vascular graft transplantation model was established in4pigs. Two kind small-caliber vascular graft transplantation models were compared on aspects of vascular anatomy, surgical operation and post-operation observation.Results Carotid artery was deeper which made anastomosis difficult, but the vessel was larger and had less branches. Femoral artery was more superficial and easy to be found and dissected. It was easy to be exposed for vascular graft anastomosis in operation; however, femoral artery had more perforating branches. In both carotid artery and femoral artery small-caliber vascular graft transplantation models, no abnormal clinical manifestations were observed when thrombosis happened. Clinical observation could not help detecting whether the synthetic vascular grafts thrombosed or not.Conclusion The establishment of carotid artery and femoral artery small-caliber vascular graft transplantation model was possible, both of them could be used as models for the research of small-caliber synthetic grafts. However, both of the two models have their own advantages and disadvantages. PART IV:The animal experiment in vivo of6mm-caliber ePTFE graft coated with anti-CD133antibodies functionalized heparin-collagen layer-by-layer assembly multilayerObjective Early rapid endothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve the early and late patency for synthetic vascular grafts. In the present study, anti-CD133antibodies functionalized multilayer of heparin-collagen onto ePTFE graft is developed for acceleration of early endothelialization via layer-by-layer assemble.Methods Both femoral artery and carotid artery small-caliber vascular graft transplantation models were included in the present study. In4pigs, bare ePTFE (n=2), HEP/COL-CD133antibody-coated ePTFE (n=3) and HEP/COL-CD34antibody-coated ePTFE (n=3) grafts were implanted into the carotid artery or femoral artery (anyone side). Ultrasonography or CTA was used to test the proximal and distal anastomotic stoma morphologies, diameters and blood velocities. Computer-based local hemodynamic situation was simulated to analyze the local hemodynamic disturbance. After7days, the pigs were terminated, and the implanted ePTFE grafts were excised for general and histopathological analysis. SEM was used to observe the luminal morphology of the grafts.Results All implanted ePTFE grafts were keeping patency. Anastomotic stoma were tested without any obvious abnormalities. Computer-based local hemodynamic simulation shown that hemodynamic changes around both proximal and distal anastomotic stoma were superior to that of4mm samll-caliber ePTFE grafts. The HE staining and IHC results indicated that anti-CD133antibody could accelerate the attachment of endothelial cells onto the ePTFE grafts. SEM shown that more endothelial cell coverage was observed on the CD133antibody-coated ePTFE and CD34antibody-coated ePTFE grafts than the bare ePTFE grafts.Conclusions Anti-CD133antibodies functionalized heparin-collagen multilayer onto ePTFE grafts could achieve early endothelialization. However, the anti-thrombotic ability needed further evaluation. PART V:The reasons of thrombosis for4mm small-caliber ePTFE graft coated with anti-CD133antibodies functionalized heparin-collagen layer-by-layer assembly multilayerObjective Small-caliber (4mm) ePTFE grafts can not be widely used in clinical because of early thrombosis and occlusion. However, this problem is still not be effectively resolved and appears to be underestimated and is worth being investigated further.Methods Both femoral artery and carotid artery small-caliber vessel transplantation models were included in the present study. In12pigs, HEP/COL-CD133antibody-coated ePTFE (n=6), HEP/COL modified ePTFE (n=4), HEP/COL-CD34antibody-coated ePTFE (n=3) and bare ePTFE (n=3) grafts were implanted into the carotid artery or femoral artery (anyone side). Ultrasonography was used to test the proximal and distal anastomotic stoma morphologies, diameters and blood velocities. CTA was used to reconstruct the aortic arch and supra-arch vessels. Thrombosis instrument (CAST-I) was applied to examine the blood coagulation state. Computer-based local hemodynamic situation was also simulated to analyze the hemodynamic disturbance. After7days, the pigs were terminated, and the implanted ePTFE grafts were excised for general and histological analysis.Results87.5%(14/16) implanted ePTFE grafts happened thrombosis. Anastomotic stoma and blood coagulation state were tested without any obvious abnormalities. Computer-based local hemodynamic simulation shown that low flexibility of synthetic ePTFE grafts indeed cause the hemodynamic disturbance around both proximal and distal anastomotic stoma. General observation found that thrombus located on both proximal and distal anastomotic stoma for those thrombosed grafts. Imcomplete endothelial cell coverage was observed in the three groups-bare ePTFE, HEP/COL-CD133-coated ePTFE and HEP/COL modified ePTFE grafts.Conclusions The patency of small-caliber ePTFE grafts for clinical use is impaired by early thrombosis and results from mixed causes. Local hemodynamic disturbance was the most powerful predictor of early thrombosis of small-caliber ePTFE grafts. Improving the quality of anastomotic stoma, strengthening anticoagulation and promoting rapid endothelialization may combat local hemodynamic disturbance and increase the patency.