小口径生物型人工血管移植后的血管内皮再生

背景：经过生物化改造的人工血管特性更接近人体血管,移植后自体化程度也较高,但人工血管的内皮再生是解决血管长期通畅的关键。 目的：观察新型小口径生物型人造血管移植后不同时期移植材料的组织相容性及移植血管壁内膜再生的组织病理学变化。 方法：建立犬颈总动脉-人造血管端端连续缝合的动物模型。 结果与结论：①光镜：移植后12周于吻合口处见新内膜表面有不连续的内皮细胞生长;移植后6个月通畅的整段管腔内面均可见内皮细胞生长;移植后1.5年管腔通畅,部分内膜组织呈慢性炎症表现。②电镜：移植后12周新生血管内皮细胞排列规则,从吻合口向移植血管中段爬行;移植后6个月内皮细胞从吻合口向移植血管中段爬行,移植血管中段呈跳跃式片状生长的内皮细胞群落,细胞排列更致密,形态更接近成熟血管内皮细胞;移植后1.5年整段血管内壁均有致密内皮细胞覆盖,部分内膜组织呈慢性炎症表现。说明新型小口径生物型人造血管新生内皮形成早,血管内膜重构能力强,生物相容和稳定性好。     

体内环境培养的小口径组织工程血管

背景:人造血管是临床组织修复领域广泛应用的重要器官,但小口径人造血管易于栓塞、难以长期保持通畅,而组织工程血管虽有永久替代作用,培养时间长,不符合血管修复的临床实际。寻找既能及时修复血管、恢复血运,又能在体内形成生物性血管保持长期通畅,是最理想的血管修复重建方法。目的:观察以改性猪小肠黏膜下层组织为支架,在体内血流条件下培养小口径组织工程血管的可行性。方法:自犬隐动脉分离出血管内皮细胞、平滑肌细胞,与胶原蛋白凝胶均匀混合,分别种植于小肠黏膜下层膜表面,包绕3mm聚乙烯管制成30个3层管状支架,植入犬股动脉缺损处进行桥接吻合为实验组;植入犬皮下组织为对照组。术后进行彩超、组织学检测评价血管的形成过程和免疫组化鉴定。结果与结论:术后12周,实验组14个血管支架保持通畅,有明显的血管生物结构形成,种子细胞生长增殖良好,管腔有完整内皮细胞覆盖,平滑肌细胞形态、分布良好;对照组管腔结构不完整,种子细胞增殖弱,腔面无内皮细胞覆盖。结果表明通过体内组织工程技术可在血流条件下培养形成小口径组织工程血管。     

静脉组织碎片镶嵌法离体衬里人工血管的实验研究

目的 寻求一种理想的小口径血管代用品。方法 采用同体双侧对照法,以内径4mm的涤纶人工血管对16条犬进行双侧颈总动脉的间置移植。实验组人工血管用自体颈静脉组织碎片镶嵌后,体外培养7天,术后2、8周观察移植物的形态学和组织学变化。结果 实验组术后2周和8周的通畅率均为87.5％,而同期对照组仅为50％和12.5％,差异显著(P<0.01)。对照组人工血管仅在距吻合口6—8mm范围内发现有内皮细胞生长,而实验组内腔面完全被有序的内皮细胞覆盖。结论 实验说明自体静脉组织碎片离体衬里人工血管,可以有效地加速人工血管的内皮细胞化,有效地改善其近远期通畅率。     

初级组织工程小口径血管的制备

目的 探讨利用组织工程技术构建小口径组织工程化血管的可行性.方法 将犬骨髓细胞诱导为平滑肌细胞和内皮细胞后,用Brdu标记平滑肌细胞,DAPI标记内皮样细胞,观察细胞标记的成功率;血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面:将细胞和支架的复合体种植于犬皮下,并设立对照组,分别于植入后的第4、6、8周取出植入皮下的组织工程化血管,进行相关检查分析.结果 免疫荧光提示Brdu、DAPI标记种子细胞效果良好;核呈蓝色荧光的内皮样细胞均匀排列在支架表面:组织工程化血管材料植入动物皮下取出后观察,实验组管壁结构比较清晰,细胞数量也已减少、胶原成分增多,管壁分层明显,可见内膜、中膜和外膜结构,对照组无此特点;免疫荧光观察,8周实验组组织工程血管壁内有Brdu标记的种子细胞存在.结论 以动物的皮下为生物反应器,采用静态培养的方式可强化组织工程血管壁,该组织的结构与天然血管相似,种子细胞同时参与初级组织工程小口径血管的构建.     

骨髓间充质干细胞-聚乙醇酸支架复合体种植动物皮下构建组织工程化小口径血管

背景：课题组的前期工作已证实骨髓间充质干细胞可以诱导分化为血管内皮细胞和血管平滑肌细胞,并证实所诱导的细胞和胶原包埋的聚乙醇酸支架具有良好的组织相容性。 目的：探讨利用动物皮下作为生物反应器构建小口径组织工程化血管的可行性。 方法：骨髓间充质干细胞诱导分化为血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸支架表面,然后将细胞-支架复合体种植于动物皮下,构建小口径组织工程化血管。 结果与结论：人工血管组织学观察见管壁结构清晰,其大体结构和天然血管相似,可承受26.6 kPa的血管腔内压力不破裂。皮下培养8周免疫荧光观察Brdu标记的部分细胞核呈现明亮的黄绿色荧光。结果说明利用动物的皮下作为生物反应器,采用静态培养的方式构建小口径组织工程化血管是可行的。     

小口径人工血管及制备方法的研究进展

血管移植是治疗心血管疾病的主要手段之一。小口径人工血管的研发也成为近十年来的研究热点。本文从制备小口径人工血管的材料和方法方面,首先简述了人工合成材料和天然生物材料,然后介绍了近些年来浸渍 沥滤法、混凝法、去细胞组织复合法、静电纺丝法、旋转曝光法、组织工程血管支架法及3 D快速成型技术应用等的研究进展。阐述了作为小口径人工血管的各种材料和制备方法的特点,为理想的小口径人造血管进一步的发展提供了一定的参考。最后对小口径人工血管发展前景做了展望。     

新型组织工程血管材料：静电纺复合纳米纤维小口径管状支架

背景：小口径人工血管对生物相容性和抗凝血的要求远远高于普通大口径人工血管,因此血管移植体内原位诱导组织再生成为了新的研究方向。 目的：总结近几年静电纺复合纳米纤维小口径管状支架的主要研究进展,并讨论其在体内原位诱导血管再生方面的重要应用。 方法：由第一作者检索中国期刊网CNKI全文数据库、万方数据库及ISI Web of Knowledge外文数据库,有关复合纳米纤维小口径管状支架的制备方法、血管支架仿生天然细胞外基质微环境的表面修饰以及种植体植入后生物相容性和安全性评价等方面的文献。 结果与结论：静电纺复合纳米纤维制备小口径管状支架,即将天然材料和合成材料共纺在一起,这样既能克服天然生物高分子材料力学性能的不足,又能避免合成材料在生物相容性和安全性的缺陷,成为制备小口径血管组织工程支架的必然趋势。同时制备多层血管,进行功能化修饰,模拟天然细胞外基质的结构和功能,将成为用于心血管组织修复及再生小口径血管组织工程研究的新方向。在获得上述新进展的同时,经动物实验检验的静电纺血管支架以聚合物为主。尽管这类支架采用了各种手段避免血栓、炎症等不良反应,其生物相容性仍旧无法与天然材料相比。由此可见,在天然材料与合成材料之间找到一个最佳比例,使复合材料的力学性能和血管相容性达到一个平衡,将会显著提高静电纺复合纳米纤维支架在小口径血管组织再生中的应用。     

组织工程化血管移植实验研究

目的 通过体外构建、体内培养的方法 构建组织工程化血管,并将其植入动物的股动脉,探讨利用小口径组织工程化血管进行血管移植的可能性.方法 首先用Brdu标记所有种子细胞,将血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面;然后将细胞和支架的复合体种植于动物皮下构建组织工程化血管,并移植到股动脉;最后行HE染色及免疫荧光等相关检查,了解移植后血管的特点及细胞来源.同时以移植物为单纯支架在皮下培养所形成的管状结构作为对照组.结果 移植后组织工程化血管通畅无血栓形成.HE染色见血管壁和正常生理性血管壁的结构相似.可分内膜、中膜和外膜三层.种植2周后Brdu标记细胞的免疫荧光观察证实血管壁细胞来源于所种植的种子细胞.结论 利用骨髓间充质干细胞分化而来的种子细胞和胶原包埋的PGA支架可以构建组织工程化小口径血管,而且可以移植入动物体内.     

体外构建组织工程化小口径血管模型

目的体外构建组织工程化人工血管模型。方法将诱导细胞制成细胞ECMgel悬液培养,镜下观察细胞形态;将诱导的平滑肌细胞和内皮细胞悬液分层种植于支架内表面,以荧光显微镜、扫描电镜和HE染色光镜观察。结果在ECMgel凝胶中细胞生长良好;旋转培养的血管模型,质地均匀,内腔面光滑;HE染色可见血管壁三层结构:内皮细胞层、平滑肌细胞层、PGA 交联胶原层;扫描电镜可见内皮细胞融合成片;荧光显微镜观察可见均匀分布的内皮细胞。结论①ECMgel是携带细胞良好的基质;②旋转培养条件可以促进细胞的贴附和分化;③利用体外诱导的种子细胞分层种植可以构建成与天然血管结构相似的血管模型。     

细胞在小口径组织工程血管中的抗钙化作用

背景：小口径组织工程血管的远期结果研究极少,尚未见到研究组织工程血管分子水平、离子水平远期结果和平滑肌细胞与钙化关系的报道。 目的：利用脱细胞猪股动脉基质作为支架和犬血管壁细胞作为种子细胞体外构建小口径组织工程血管,植入种子细胞供体犬股动脉部位6个月,观察植入物中层平滑肌细胞与钙化的关系。 方法：12只实验犬被随机分为支架组(n=6)和再细胞化组(n=6),自体股动脉被作为对照组;支架组犬接受猪股动脉经脱细胞后的基质支架植入双侧股动脉,再细胞化组犬接受受体血管壁细胞共同培养、联合种植于脱细胞的猪股动脉基质并体外预适应后植入血管壁细胞供体双侧股动脉位置;6个月后测定植入物和对照组股动脉组织钙含量、平滑肌密度和病理学变化。 结果与结论：小口径组织工程血管植入后6个月检查见2组植入物无明显狭窄和扩张,扫描电镜示内表面均已完全内皮化,有管壁僵硬和局部钙化斑块形成,以上改变以支架组植入物更明显。支架组管道组织钙含量显著高于再细胞化组和自体股动脉(P < 0.01),再细胞化组植入物组织钙含量亦显著高于自体股动脉(P < 0.01);病理学检查示再细胞化组植入物平滑肌密度高于支架组(P < 0.01),再细胞化组和支架组植入物平滑肌密度均低于对照组(P < 0.01);超声检查见2组管道植入术后即刻与6个月后舒缩幅度较邻近自体股动脉舒缩幅度小,有部分管道无舒缩功能。结果提示,猪股动脉常规脱细胞方去获得的基质作为支架体外构建组织工程血管时,平滑肌细胞难于迁移至支架中层,中层平滑肌密度低,植入体内6个月后中层平滑肌细胞密度仍低,平滑肌细胞有抗血管钙化作用。     

Study of a Novel Small Caliber Vascular Graft in a Canine Model with Optical and Scanning Electron Microscopy

A novel biological small-diameter vascular graft was evaluated in a canine model. 3 cm long segments with 4 mm I. D. were implanted end-to-end in the carotid position of 12 dogs for 6 months. Color Doppler sonography was performed at the first week post-operation, and angiography was then administered to 9 grafts at 4th week, 12th week and 24th week respectively to monitor the graft pantency and blood flow characteristics. Vascular samples containing the grafts were collected at 1st week, 8th week, 12th week and 24th week after implantation. Morphological changes of the grafts were observed by optical and scanning electron microscopic （SEM） studies and compared with that of the original prosthesis and the normal host vessel. All grafts were patent throughout the experiment except one graft. Histopathology and SEM demonstrated both a nearly complete inner capsule of varied thickness lining the graft luminal surface and connective tissue adventitia formation at one-week post-operation. The neointima became confluent at 8 weeks and then compact but had no signs of hyperplasia up to 12 weeks; meanwhile on the neointimal surface newly grown endothelial-like cells were migrating from the stoma to the middle portion. The grafts also illustrated endothelialization in many ＂islands＂ in the mid-segment luminal surface of the grafts. In addition, the closer distance the cells towards the stoma were, the more morphological similarity the cells with the normal endothelial were. Taken together, the biological vascular graft remained patent for 24 weeks as a carotid prosthesis, characterized by the early and complete neointima formation plus endothelialization starting before 12 weeks post grafting. Therefore, the graft seems suitable for reconstruction of vascular lesions in dogs. Further studies may be carried out to extend the graft application for the clinical use.     

Tissue Responses to Endovascular Stent Grafts for Saccular Abdominal Aortic Aneurysms in a Canine Model

We investigated tissue responses to endoskeleton stent grafts for saccular abdominal aortic aneurysms (AAAs) in canines. Saccular AAAs were made with Dacron patch in 8 dogs, and were excluded by endoskeleton stent grafts composed of nitinol stent and expanded polytetrafluoroethylene graft. Animals were sacrificed at 2 months (Group 1; n = 3) or 6 months (Group 2; n = 5) after the placement, respectively. The aortas embedding stent grafts were excised en bloc for gross inspection and sliced at 5 to 8 mm intervals for histopathologic evaluation. Stent grafts were patent in all except a dog showing a thrombotic occlusion in Group 2. In the 7 dogs with patent lumen, the graft overhanging the saccular aneurysm was covered by thick or thin thrombi with no endothelial layer, and the graft over the aortic wall was completely covered by neointima with an endothelial layer. Transgraft cell migration was less active at an aneurysm than at adjacent normal aorta. In conclusion, endoskeleton stent grafts over saccular aneurysms show no endothelial coverage and poor transgraft cell migration in a canine model.     

Evaluation of urokinase-type plasminogen activator and its receptor in neointima of polyester vascular grafts

The aim of the study was to assess the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in neointima of polyester vascular grafts. Anastomotic areas were examined at 1, 4 and 12 months after prosthesis implantation in dogs, as well as in human vascular grafts. Immunohistochemistry was performed for uPA and uPAR. Graft neointima in dogs was positively stained for uPA with increased intensity at 4 and 12 months, whereas uPAR expression appeared at 4 and its intensity was increased at 12 months. Intensive uPA and positive uPAR labeling was shown in human grafts. The results demonstrated that in the early period of the healing process of polyester vascular grafts only uPA is present in the neointima in the region of the graft to adjacent artery anastomosis, whereas in healed grafts in dogs and humans uPAR is found as well.     

Explant pathology study of decellularized carotid artery vascular grafts

The purpose of this study was to evaluate the morphologic findings in small-diameter freeze-dried decellularized carotid artery grafts implanted in goats as carotid artery interposition grafts for 6-7 months. Unimplanted decellularized carotid artery grafts did not contain intact cells; however, remnants of smooth muscle cells were present in the media. The extracellular matrix was well preserved. All decellularized grafts were patent at explant, without significant dimensional changes or aneurysm formation. Their luminal surfaces were lined by a thin neointima, consisting of myofibroblasts, collagen, and a discontinuous layer of endothelial cells. Histologic evidence of calcification within the explants was not observed; however, electron microscopy showed calcification of minute remnants of cell membranes. Inflammatory cells were not present in the graft wall. Host cell migration was greatest in the adventitia along the length of the graft. Migration of host cells into the media was more apparent close to the anastomoses, forming cellular nests rich in extracellular proteoglycans, whereas cell migration into areas subjacent to the lumen was minimal. Ingrowth of host blood vessels was not observed. These results demonstrate satisfactory structural and morphologic features of a decellularized carotid artery small-diameter graft implanted for up to 7 months.     

Expression of fibrinolysis activators and their inhibitor in neointima of polyester vascular grafts

The aim of the study was to evaluate dynamic changes in the expression of fibrinolytic system components in neointima forming in polyester vascular grafts.

The study was carried out on 18 mongrel dogs divided into three groups, that underwent replacement of abdominal aorta with a polyester double velour prosthesis. Grafts were removed at 1, 4 and 12 months. The specimens were fixed according to AMeX method. Immunohistochemical labeling for von Willebrand factor (vWf), tissue plasminogen activator (t-PA), urokinase (u-PA), its receptor (u-PAR), plasminogen activator inhibitor type 1 (PAI-1) and D-dimer (DD) was performed.

Increasing intensity of vWf expression on the graft luminal surface was found in successive periods of the study. A light positive t-PA and u-PA staining was shown in neointima at 1 month and its intensity was significantly increased at 4 and 12 months. Expression of u-PAR appeared at 4 months. A light positive PAI-1 and DD staining was demonstrated in neointima in all periods of the study.

The results demonstrated increasing expression of fibrinolysis activators in neointima of polyester vascular grafts. Intensive expression of plasminogen activators, when compared to their inhibitor may reduce thrombotic properties of graft neointima particularly in the late period after prosthesis implantation.     

小口径组织工程血管支架：如何产生一种具有生理重塑活性的材料

文题释义：小口径血管支架材料：①机械强度：包括生理的顺应性,即可承受长期的血流动力学压力,不会导致动脉瘤形成;②良好的生物相容性;③愈合时不会发生炎症、增生及纤维囊形成;④易于手术的操作和缝合;⑤低免疫原性。 丝素蛋白：存在于产丝节肢动物的腺体中,通过蜕变过程纺成纤维,是一种天然的生物衍生材料。其具有良好的机械性能、生物相容性、稳定化学性能等优点,因此丝素蛋白在血管组织工程领域中应用比较广泛。 背景：心血管疾病目前临床上常用的治疗方法是血管重建术,包括经支架介入治疗、冠状动脉旁路移植术和血管成形术。 目的：总结天然衍生支架材料、人工合成高分子材料和复合材料等组织工程血管支架材料的最新研究进展,为小口径血管置换提供理论依据。 方法：检索PubMed 数据库、万方数据库和中国全文期刊数据库2008年1月至2019年7月相关文献,以检索词为“组织工程;生物材料;支架材料;血管”“Tissue engineering,biological material,scaffold material,blood vessel”进行中英文检索。排除内容较陈旧及结论重复性文献,对最终入选的52篇文献进行探讨。 结果与结论：自体血管移植物,如隐静脉和胸内动脉,是小口径血管的最佳替代物,然而在移植后可能诱发血管腔再次狭窄,以及导致血栓形成、感染和移植失败发生率增加,因而严重阻碍在临床上的应用。考虑到存在的这些局限性,研究者将组织工程血管移植物嵌入细胞,从而产生一种具有生理重塑活材料,这一潜在的解决方案可为血管移植物的未来带来希望。 ORCID: 0000-0002-0841-0464(杨磊) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Mouse Model of Venous Bypass Graft Arteriosclerosis

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18⁺) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (α-actin⁺) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases.     

砷涂层支架置入后的血管组织相容性

背景：研究认为,三氧化二砷可以抑制血管平滑肌细胞的增殖,促进其凋亡,那么砷对血管平滑肌细胞的增生是否也有同样的抑制作用,砷涂层血管支架能否与血管组织相容,早期较好地被血管内膜覆盖或达到减少内膜过度增生的作用？ 目的：观察砷涂层血管支架的血管组织相容性。 方法：取大耳白家兔14只,随机分为2组,分别在腹主动脉处植入砷涂层316 L不锈钢支架和316 L不锈钢裸支架,植入28 d后结扎支架部位血管的远端和近端,取下支架部位的血管行苏木精-伊红染色,光镜检查。 结果与结论：①大体观察：支架处的血管外径稍大于相邻处血管的外径,呈扩张状态,无肉眼可见的血栓,切开支架,支架表面可见光滑的新生内膜形成,新生内膜表面光滑。②光镜观察：支架丝位于血管的中层,中层平滑肌被压,支架丝周边,血管内膜平滑肌增生,使血管内膜增厚。支架丝的血管腔面可见新生的血管内膜形成并覆盖支架丝,支架丝与血管组织之间可见一薄层黑色物质,为涂层药物砷及其化合物,证明砷涂层支架可以被血管组织覆盖,具有良好的血管组织相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：