In Vivo Morphological Studies Of Small-caliber Hybrid Vascular Graft Fabricated With Porcine Fibrin And Microprocessed Elastomeric Film

INTRODUCTIONSmall-caliber(＜6mm) arterial substitutes,which account for a majority of the demand,such as management of coronary heart disease,arteriosclerotic occlusive disease of the lower limb,chronic renal failure and congenital cardiovascular malformations in children,have generally possessed limited immediate or long-term patency largely because of acute thrombogenicity of the graft,anastomotic intimal hyperplasia.Currently,three major vascular grafts have been used in the clinical practice,such as:(1) autologous vessels,such as radial artery,internal mammary artery or saphenous vein.(2) allograft blood vessels.(3) synthetic grafts,such as expanded polytetrafluorethylene(ePTFE) and Polyethylene terephthalate(Dacron or PET) grafts.These vascular grafts always have various advantages and shortcoming respectively.Therefore,there is being urgent need to explore a novel constructing technology in an attempt to develop an successful small-caliber arterial graft,which must be able to be manufactured cheaply in a relatively short period of time,and in sufficient numbers with differing specifications(diameter,length,etc),must be biocompatible(noninflamatory,nontoxic,noncarcinogenic,nonimmunogenic) and biostable,and must have good long-term patency rate.Tissue engineering has emerged as a promising approach to address the shortcomings of current options.In our experiment vascular endothelial cells were harvested by the method of in situ cannulation from the peripheral vessel of adult mongrel dogs and followed by in vitro culture and proliferation.The endothelial cells were high-density seeded onto the inner surface of small-caliber tubular scaffold fabricated with porcine fibrin and polyurethane membrane(PU) and the cell-scaffold constructs were cultured in vitro continuously for 7-9 days.After the maturation,the endothelized small-caliber hybrid vascular grafts were implanted to replace the canine common carotid artery.Color Doppler ultrasound and CT angiography were performed to evaluate the patency and degree of anastomotic neointimal hyperplasia of hybrid vascular grafts postoperatively,and the remodeling process of the implanted hybrid vascular grafts in vivo were analyzed by scanning electron microscopy,histology and immunohistochemistry at different time interval. PARTⅠIn situ cannulation for endothelial cell harvest and its massculture and identification derived from adult canine superficial vesselObjectiveTo provide well functional and a sufficient number of autologous endothelial cells for the confluent in vitro lining of the small-caliber hybrid vascular grafts.MethodsEnzymatic endothelial cell detachment was achieved by the in situ application of typeⅠcollagenase to short segments of canine saphenous vein under anesthesia.The vascular endothelial cells were cultured and masscultured.The use of a microgrid technique enabled the daily follow up and quantification of endothelial cells in culture. These vascular endothelial cells were identified by the morphologic characteristics observed under phase-contrast microscope,transmission electron microscope(TEM) and immunohistochemical staining.The contents of both 6-keto-PGF_1α and vWF in the supernatant of primary and subcultured passages were analyzed to evaluate the endothelial function using the commercial ELISA kits.ResultsThe vascular endothelial cells were cultured and subcultured successfully.They presented typical "spindle-shaped",elongated,polygonal appearance and formed monolayer in "cobblestone-like" morphology under phase-contrast microscope.EC Weibel-Palade("W-P") body was sometimes observed under TEM. Immunohistochemical analysis indicated that these cells were positively stained for von Willebrand factor(vWF).In endothelial function assays,the content of both 6-keto-PGF_1α and vWF in the supernatant of primary and subcultured passages didn't have significant difference.ConclusionThe present study demonstrates that the ECs cultured may be used as cell source for the endothelialization of the hybrid vascular grafts. PARTⅡExperimental study on the biocompatibility of small-caliber hybrid vascular graft fabricated with porcine fibrin and the endothelialization of hybrid vascular graftObjectiveTo investigate the physico-mechanical property and biocompatibility of the porcine fibrin as the scaffold material and the possibility of vascular endothelial cells seeding onto the surface of the hybrid vascular grafts.MethodsA coagulation film and tubular scaffold were constructed with porcine fibrin gel using the technique of lyophilization.The fibrin tubular scaffold was wrapped with a laser-processed micropored segmented polyurethane(SPU) film and SEM was used to observe the 3-dimensional structure of small-caliber hybrid vascular grafts. Biocompatibilities of porcine fibrin were evaluated in vivo and in vitro by the means of cell adhesion test and cytotoxicity test,short-term test of subcutanous implantation, acute hemolysis test according to the requirements of ISO 10993.The compliance value,burst and suture-holding strength were measured in vitro by insuffiation and pull-through techniques in order to evaluate the biomechanical properties of the hybrid vascular grafts.Endothelial cells which were seeded onto the tubular scaffold were harvested from canine saphenous vein by collagenase digestion and subcultured. After another 7-9 days culture that enabled the maturation of cytoskeleton of ECs,the morphological characteristics of vascular EC on the inner surface of the hybrid vascular grafts were studied with light and electron microscopy.ResultsThe porcine fibrin tubular scaffold were made successfully using the technique of lyophilization and then wrapped with a laser-processed micropored SPU film.These small-caliber hybrid vascular grafts had elasticity and tenacity,and could maintain their shapes in culture medium for 2 weeks.Their grid structure and almost uniform pores were observed under electron microscope.Biocompatibility tests of the porcine fibrin provided evidences for good cell adhesion,growth viability and morphology of human umbilical vein endothelial cells and no cytotoxicity.Hemolysis test and subcutanous implantation approved the porcine fibrin as a biocompatible biomaterial and complete degradation was observed in about 8 weeks.Compared with fresh jugular vein,the hybrid vascular grafts had similar in vitro burst (2040.83±239.17mmHg vs 2248.33±300.89mmHg,P＞0.05) and suture-holding (190.75±41.47g vs 169.15±41.84g,P＞0.05) strength.Confluent lining of vascular EC on the inner surface of the hybrid vascular grafts was observed by light and electron microscopy.ConclusionsThe techniques of lyophilization and wrapping to fabricate the small-caliber hybrid vascular grafts are feasible.The hybrid vascular graft has good biocompatibility and provides structural basis for vascular ECs seeding.PARTⅢMorphological study of small-caliber hybrid vascular grafts upon replacement of canine common carotid arteriesObjectiveTo inveatigate the in vivo morphological change of hybrid vascular graft made of porcine fibrin and microprocessed SPU film under the hemodynamic condition for predetermined period of time.MethodsAutologous vascular ECs derived from the saphenous veins of 6 adult mongrel dogs were isolated by collagenase digestion.The cells were cultured and proliferated in vitro in M199 culture medium,prelined on the luminal surface of lyophilized porcine fibrin tube,and wrapped with a SPU thin film with multiple micropores as a compliant scaffold.After canine common carotid arteries were bilaterally replaced with these vascular grafts for 4 weeks,light and electron microscopy,radiological imaging examination and immunohistochemistry were performed to evaluate the patency and remodeling in vivo.ResultsEight of 10 engineered vascular grafts were patent(patency rate 80%),one graft was broken 5 days postoperatively,and another one was occluded as a result of thromboembolism comfirmed by color Doppler ultrasound 2 weeks postoperatively. The patent grafts possessed thin,smooth and glistening luminal surfaces at the predetermined observation period up to 4 weeks.The intimal layer was covered with confluent,cobblestone-like monolayered cells that were positively stained with factorⅧ-related antigen.The thickness of the neoarterial walls was approximally 900μm at 4^th week after implantation.Vascular medial tissue rich in smooth muscle cells in the hierarchic structures of arterial wall was observed,and more interestingly,mass of elastic fiber was found in the medial tissue as early as 4 weeks postoperatively.ConclusionsThe small-caliber hybrid vascular graft made of porcine fibrin tube wrapped with microprocessed SPU which was endothelialized can be used to replace the canine common carotid artery.The endothelialized hybrid vascular grafts can be transformed to similar hierarchic structures of arterial walls under the hemodynamic condition for as early as 4 weeks.