In Vitro Studies Of Small-caliber Vascular Grafts

Coronary artery and peripheral artery diseases caused by artherosclerosis harm human being's health to a large extent.Although saccule expanding and stent implanting can partially substitute artery transplantation,vascular bypass is still one of the main measures to treat severe vascular diseases.Vascular grafts,as the substitute for severe stenosis or occlusive blood vessel,have important application value and prospects in clinical study.In fact,after transplantation of small diameter vascular grafts,long-term patency rate is low due to lack of endothelial cells for lining and anti-thrombus characters.Purified human saphenous vein endothelial ceils(HSVECs) can be harvested enzymatically with a solution of collagenase from clamped cannulated vessels,which is a simple and easy-to-operate in vitro proliferation pathway of HSVECs.At the same time,expanded polytetrafluorethylene(ePTFE) artificial blood vessel possesses good biocompatibility and hematocompatibility,and can effectively inhibit thrombosis.This experiment is to investigate the feasibility of cultivation of HSVECs in vitro and seeding the HSVECs onto the surface of ePTFE small diameter artificial blood vessel,aiming at establishing a functional biological lining on the luminal surface.PartⅠCultivation of human saphenous vein endothelial cells in vitro and their identification.ObjectiveTo establish a better method to cultivate primary and subcultured human saphenous vein endothelial cells(HSVECs) in vitro and identify the cultured cells.Methods11 human saphenous veins were used in this study.HSVECs were isolated by means of collagenase perfusion,and then cultivated in M199 with 10%fetal bovine serum followed by immunofluorescence staining and transmission electron microscope examination.Finally,the factorⅧcontained in the supernatant of primary and subcultured passages was analyzed.ResultsExtremely high purified endothelial cells were isolated with the collagenase perfusion.The characterized Weibel-Palade("W-P") body was observed under the transmission electron microscope.Immunohistochemical analysis indicated that these cells were positively stained for factorⅧand CD31.Significant differences of the factor vWF contents were not detected between primary and subcultured passages in the supernatant(P＞0.05).ConclusionHighly pure endothelial cells can be isolated by infusing collagenase to the lumen of human saphenous veins.They may be an alterative choice of source cells for the endothelialization of the prosthetic grafts.PartⅡThe study of seeding HSVECs on small-caliber vascular grafts for the endothelializationObjectiveTo study the feasibility of seeding HSVECs onto luminal surface of prosthetic grafts for the endotheliazation of ePTFE small diameter artificial blood vessel.MethodsHSVECs were harvested and cultivated in vitro for 13-15 days.The proliferated HSVECs were seeded onto the surface of ePTFE small diameter prosthetic grafts precoated with fibrin glue.The ECs seeded graft were then placed in a rotative device (Endostrabilisator,Biegler,Austria) for 2 h to obtain homogeneous coverage.A total of 8 prostheses were used for this purpose.After another 9-12 days of cultivation, morphological change was observed under scanning electron microscope.ResultsAfter seeding the cells,the adhesion,crawling and spreading of the HSVECs were observed onto the surface of ePTFE prosthetic graft under the scanning electron microscope.On the 12th day,surface of ePTFE prosthetic graft was covered completely with endothelial monolayer.ConclusionEndothelial cells derived from human saphanous veins can be used as seed cells for the endothelialization of ePTFE prosthetic graft,which lays a theoretical foundation for the clinical application of endothelialized small-caliber prosthetic grafts.