【作者】 施德兵；

【导师】 符伟国； 何红兵； 陈斌；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 冠心病、下肢动脉硬化性闭塞症、慢性肾功能衰竭行血液透析以及小儿心血管先天性畸形等,这些疾病较多需要行小口径（直径＜6mm）血管移植手术。目前在临床上使用血管移植物主要有如下三类:1)自体血管移植物如内乳动脉、桡动脉和大隐静脉;2)同种异体血管移植物;3)人工合成材料血管,由高分子有机材料,如膨体聚四氟乙烯（ePTFE）,涤纶（Dacron）,聚氨酯（PU）等制备。以上三种血管各有缺陷,如来源有限、免疫排斥或移植术后因血栓形成、吻合口内膜增生而导致近、远期通畅率不理想。因此,人们迫切需要研制新的小口径动脉替代品,使得不仅具有丰富的原料来源,宿主对移植物不产生免疫排斥反应,安全可靠,并且远期通畅率高。组织工程（tissue engineering,TE）技术的发展,为这一目标的实现开辟崭新的途径。本实验通过原位获取犬浅静脉内皮细胞（enthothelial cell,ECs）,体外培养和扩增后高密度种植于猪纤维蛋白/微孔聚氨酯弹性膜（PU）制备的小口径管形支架内表面,继续体外静态培养7～9d,后将这种完全内皮化的小口径血管移植物置换实验犬颈总动脉,术后行彩色多普勒超声和CT血管造影（CT angiography,CTA）随访移植血管血流通畅度和吻合口内膜增生程度,并于术后分节点取出移植的血管行扫描电镜、组织学和免疫组织化学检测以了解置换的小口径血管移植物在体内重塑情况。第一部分实验犬浅静脉内皮细胞原位获取、培养、鉴定和功能检测目的为小口径血管支架体外内皮化提供足够数量和功能良好的ECs。方法实验犬麻醉后前肢隐静脉原位插管,采用Ⅰ型胶原酶消化法获取ECs,培养、传代并大规模扩增。采用微格法每日计数培养的细胞总数。倒置显微镜观察细胞生长情况。细胞免疫化学和透射电镜进行细胞鉴定。同时用试剂盒检测原代和传代细胞条件培养液中6—酮—前列腺素F_1α(6-keto-PGF_1α)和von Willebrand因子（vWF）的含量。结果成功培养、传代并大规模扩增静脉ECs,倒置显微镜下观察ECs呈典型的“纺锤样”梭形细胞,单层细胞贴壁生长至融合时呈铺路石样排列。透射电镜下见ECs胞浆中特征性Weibel-Palade小体。细胞免疫化学显示Ⅷ因子相关抗原抗体染色（+）。原代及传代细胞培养上清液中6—酮—前列腺素F_1α和vWF的含量差异无显著意义。结论所培养的静脉ECs为小口径血管支架体外内皮化提供足够数量和功能良好的细胞来源。第二部分猪血纤维蛋白材料的生物相容性及体外小口径血管支架内皮化的实验研究目的研究猪血纤维蛋白材料的生物相容性和静脉ECs种植于小口径血管支架腔内表面的生长情况。方法采用模具成形和冷冻干燥技术制备猪血纤维蛋白胶凝固膜和管形支架,于纤维蛋白管形支架外包裹微孔聚氨酯膜,观察制备的小口径血管支架的大体形态并用扫描电镜观测其三维结构。利用纤维蛋白胶凝固膜及其浸提液进行生物相容性检测:细胞粘附实验、MTS细胞毒性实验、急性溶血性实验和短期皮下植入实验。对制备的小口径血管支架进行生物力学性能检测。采用酶消化法从犬浅静脉中分离、培养和体外扩增血管ECs,并接种于制备的小口径血管支架腔内表面,体外静态培养7～9d,再进行组织学和扫描电镜观察。结果应用模具成形和冷冻干燥技术成功制备猪血纤维蛋白胶凝固膜和管形支架,并于纤维蛋白管形支架外包裹微孔聚氨酯膜。这种小口径管形支架具有一定弹性,在培养液中2周可保持管腔形态不变形。扫描电镜显示其具有网格状结构,孔径较均匀。细胞粘附实验提示人脐静脉ECs株ECV304在猪血纤维蛋白凝固膜上粘附生长较好;MTS细胞毒性实验示纤维蛋白凝固膜组吸光度较阴性对照组无明显差异（P＞0.05）;急性溶血性实验示纤维蛋白凝固膜浸提液溶血率为3.43±0.003%;短期皮下植入实验示纤维蛋白凝固膜周围组织炎症反应轻微,在体内约8周完全降解。体外制备小口径血管支架具有类似于自体颈静脉的爆破强度（2040.83±239.17mmHg vs 2248.33±300.89mmHg;P＞0.05）和最大缝持张力（190.75±41.47g vs 169.15±41.84g;P＞0.05）。组织学和扫描电镜观察表明体外培养扩增的ECs在血管支架腔内表面形成较完整的ECs单层,生长状况良好。结论猪血纤维蛋白具有良好的生物相容性;应用模具成形、冷冻干燥和外包裹缝合等技术制备小口径血管支架的方法切实可行,这种管形支架可作为血管组织工程机理研究较理想的支架选择。第三部分小口径血管移植物置换实验犬颈总动脉的实验研究目的探讨以猪血纤维蛋白/微孔聚氨酯弹性膜为管形支架内皮化构建的小口径血管移植物在体内血流动力条件下血管壁组织结构重塑过程,为进一步研究奠定基础。方法采用酶消化法分离、培养和体外扩增实验犬浅静脉ECs,并将这些细胞高密度种植于以猪血纤维蛋白/微孔聚氨酯弹性膜为管形支架腔内表面,体外培养待形成完整的ECs单层,然后用这种小口径血管移植物置换6只实验犬自体双侧颈总动脉,于术后1d、1w、2w、4w行影像学检查、组织学和免疫组织化学检测,以及扫描电镜检查以评价移植物血管在体内重塑情况。结果10根小口径血管移植物中有8根仍保持通畅（通畅率80%）;1根于术后5d破裂后取出,另一根于术后2w时彩超显示闭塞。8根通畅的血管在术后至4w的不同时点取出发现内表面菲薄、光滑。血管壁内表面覆盖一连续、鹅卵石样单层细胞,该细胞Ⅷ因子相关抗原抗体染色阳性。这种移植于体内的小口径血管移植物于术后4w时新生动脉壁厚度约为900μm,并于管壁中层可见较多血管SMC和外膜层主要为成纤维细胞,而且最早于术后4w时在血管壁中层就可见弹力纤维。结论猪血纤维蛋白/微孔聚氨酯弹性膜管形支架内皮化体外构建的小口径血管移植物在体内血流动力条件下经过重塑后,可形成具有类似自体动脉壁结构。更多还原

【Abstract】 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.更多还原