脱除猪主动脉瓣细胞的研究

组织工程心脏瓣膜是利用组织工程学技术 ,将受体活性细胞种植在生物可降解材料支架上 ,孵育出活性心脏瓣膜 ,无免疫原性 ,不需抗凝 ,耐久性强 ,有自身修复能力。本研究旨在探讨脱除猪主动脉瓣上细胞成分的方法 ,评价其效果 ,为组织工程心脏瓣膜的构建提供较理想的生物材料。一、材料与方法1.实验材料 :采集热缺血时间小于2h的成年猪心脏 5 0个 ,完整取下主动脉瓣 ,4℃Hanks液漂洗。随机分为两组 :A组 (2 5枚 ) :用去污剂 酶消化法[1] 进行脱细胞处理后分别行热皱缩温度、机械强度测定和组织学观察 ;B组 (2 5枚 )作为对照组。2 .脱细胞处…     

应用猪主动脉去细胞瓣膜支架体外构建组织工程心脏瓣膜

目的　探讨应用猪去细胞瓣膜支架体外构建组织工程心脏瓣膜的可行性。　方法　采用去垢剂、渗透压改变和核酸酶消化的方法制备猪主动脉瓣去细胞瓣膜支架 (实验组 ) ,用去内皮细胞主动脉瓣作对照 (对照组 ) ,并对两组含水量、可溶性蛋白含量、热皱缩温度、力学性能和组织相容性进行测定。培养犬主动脉壁间质细胞和内皮细胞 ,将其种植于实验组去细胞支架上 ,观察细胞生长情况 ,并测定内皮细胞合成前列环素的功能。　结果　实验组瓣膜细胞成分完全从瓣膜中去除 ,与对照组新鲜瓣膜相比 ,含水量增高 (P<0 .0 5 ) ,可溶性蛋白含量减少 (P<0 .0 5 ) ,热皱缩温度和抗张强度无明显变化。实验组瓣膜组织相容性试验显示 ,材料组织相容性好 ,体内降解时间为 10周 ;犬主动脉壁间质细胞和内皮细胞在瓣膜表面生长良好 ,内皮细胞具有合成分泌前列环素的功能。　结论　采用去垢剂、渗透压改变和核酸酶消化的方法制备猪主动脉瓣去细胞瓣膜支架 ,在去除细胞和可溶性蛋白质的同时保持了瓣叶的基本结构和力学性能 ;以其为支架体外构建组织工程心脏瓣膜的细胞不仅能在材料表面生长 ,还能合成、分泌血管活性物质 ,是具有生理功能的组织工程心脏瓣膜。     

去细胞猪主动脉瓣叶的获取和内皮细胞的种植

目的　探讨猪主动脉瓣叶去细胞后作为组织工程心脏瓣膜支架的可行性。　方法　经胰酶 - EDTA、表面活性剂和核酸酶处理 ,去除猪主动脉瓣叶的细胞成分 ,测定瓣叶去细胞前、后的生物力学特性 ,并在其表面种植新生牛主动脉内皮细胞 (BAECs) ;分别行大鼠皮下包埋实验。　结果　猪主动脉瓣叶中的细胞成分能完全去除 ,获得完整无细胞的纤维网状支架 ,断裂强度和断裂伸长率无明显变化 ;种植的 BAECs在去细胞瓣叶表面可形成一层连续的细胞层 ,其分泌前列环素 (PGI2 )的能力同直接种植在 2 4孔板中的比较 ,差异无统计学意义 (P>0 .0 5 )。　结论　猪主动脉瓣去细胞后获得的纤维支架可以用来构建组织工程瓣膜 ,适宜于血管内皮细胞的生长。     

组织工程心脏瓣膜内皮细胞的整合素β1表达

目的体外构建组织工程心脏瓣膜（TEHV）,初步探讨内皮细胞黏附生长的分子机制。方法猪主动脉瓣膜经胰酶-EDTA、表面活性剂、核酸酶处理,去除猪主动脉瓣叶的细胞成分,测定瓣叶脱细胞后的生物力学特性;将扩增的人脐静脉血管内皮细胞（HUVECs）种植在瓣叶上,体外静态构建TEHV,观察内皮细胞的生长状态。消化瓣膜内皮细胞,半定量RT-PCR检测内皮细胞整合素＆mRNA的表达,Western-Blot检测内皮细胞膜上整合素＆蛋白的表达。结果猪主动脉瓣膜中的细胞成分能完全去除,脱细胞瓣叶的生物力学特性同新鲜瓣叶相比无明显变化;种植的HUVECs在瓣叶表面生长状态良好,长成一层连续的细胞层。瓣膜内皮细胞可检测到整合素岛mRNA和蛋白的表达。结论脱细胞猪主动脉瓣膜作为支架,HUVECs做种子细胞可以成功构建TEHV,瓣膜内皮细胞可以表达整合素岛。     

人骨髓基质干细胞构建组织工程心脏瓣膜的研究

目的探讨人骨髓基质干细胞种植在去细胞猪主动脉瓣叶上体外构建组织工程心脏瓣膜的可行性。方法经1%TritonX100、0.01%胰酶0.02%EDTA、DNaseI及RNaseI处理制备去细胞猪主动脉瓣叶支架,测定瓣叶去细胞前、后的生物力学特性;人骨髓基质干细胞体外分离、培养、扩增后种植在去细胞瓣叶表面,观察细胞生长情况。结果猪主动脉瓣叶去细胞后获得完整无细胞的纤维网状支架,瓣叶去细胞前、后的断裂强度和断裂伸长率差异无统计学意义(P>0.05);体外培养的人骨髓基质干细胞(MSCs)在去细胞瓣叶表面形成一层基本连续的细胞层。结论去除细胞成分的猪主动脉瓣叶组织是一种良好的纤维支架,可以用于构建组织工程心脏瓣膜;人骨髓基质干细胞种植在去细胞猪主动脉瓣叶上构建组织工程心脏瓣膜是可行的。     

活性带支架绵羊主动脉瓣的制作及组织学和细胞活性研究

目的　制作活性带支架绵羊主动脉瓣 ,观察液氮深低温保存 3个月后的组织结构 ,证实细胞活性 ,为同种二尖瓣置换实验做准备。方法　无菌条件下取 4～ 12个月 ,体重 (4 0～ 60 )kg的绵羊主动脉瓣 8个 ,经修剪、抗菌素灭菌后缝制于弹性支架上、制成绵羊带支架主动脉瓣 ,1℃ /min的速度程控降温至 -80℃后投入液氮冻存。 3个月后解冻 ,取其中 6个 ,剪取瓣叶 ,流式细胞技术评价细胞活性 ,同期冻存的 6个无支架绵羊主动脉瓣对照。另外 2个带支架绵羊主动脉瓣解冻后用光镜及电镜研究其组织结构 ,新鲜主动脉瓣 2个对照。结果　带支架组和无支架组细胞活性分别为 (90 .92± 2 .90 ) %和 (91.69± 3 .5 0 ) %,两组比较差异无显著性 (P >0 .0 5 )。带支架组细胞外基质、内皮细胞及大多数成纤维细胞的超微结构与新鲜瓣膜差异无显著性 ,仅部分成纤维细胞的超微结构有轻微的可逆性改变。结论　带支架设计不影响液氮深低温保存的同种瓣的细胞活性及超微组织结构 ,可用于制作新型的人同种主动脉瓣。     

应用聚乳酸聚乙醇酸膜构建组织工程心脏瓣膜的实验研究

目的;探讨应用聚乳酸聚乙醇酸（PLGA）构建组织工程心脏瓣膜的可行性。方法：扫描电子显微镜观察PLGA结构特点,将PLGA在兔皮下包埋,分别于2周、4周、6周、8周和12周观察材料的生物相容性和降解率,培养犬主动脉瓣间质细胞、主动脉壁间质细胞和皮肤成纤维细胞,对照其生长曲线、平滑肌α肌动蛋白表达和扫描电子显微镜特点。将犬主动脉壁间质细胞和内皮细胞种植于PLGA上,观察其形态并测定细胞合成胶原和前列环素的功能。结果：PLGA呈网孔状结构,孔径179μm。皮下包埋显示PLGA生物相容性好,体内降解时间为12周。犬主动脉瓣间质细胞和主动脉壁间质细胞平滑肌α肌动蛋白均为部分阳性表达,细胞内有大量粗面内质网,生长曲线相似。细胞种植显示细胞在材料表面生长良好,并具有合成胶原和前列环素的功能（P＜0．05）。结论：以PLGA为支架体外构建组织工程心脏瓣膜细胞不仅能在PLGA表面生长,还能合成细胞间质和血管活性物质,初步提示应用本组材料和方法构建组织工程心脏瓣膜是可行的。     

干细胞--心脏组织工程瓣种植细胞的新来源

心脏组织工程瓣(tissue-engineering heart valve，TEHV)的研究是组织工程和“再生医学(regeneration of medical science)”的一个分支领域。TEHV的先决条件是种植类似于天然瓣膜的内皮细胞(endothelialceus，ECs)和成纤维细胞，由于ECs具有强烈的同种异体免疫原性，因此种植细胞来源应是宿主的自体细胞。寻找一种经济、便捷、有效地获取种植细胞的方法，是TEHV体外构建应首先解决的核心问题。     

组织工程心脏瓣叶的体外培养

目的 探讨瓣叶支架种植细胞后,不同体外培养时间瓣叶的生成情况. 方法杂种猪9只,取主动脉,分离内皮细胞、成纤维细胞和平滑肌细胞,培养扩增.将成纤维细胞、平滑肌细胞和内皮细胞按顺序种植在预湿处理的支架材料上,以首次种植成纤维细胞、平滑肌细胞为起点,取7天、14天和28天样本使用戊二醛固定,用扫描电子显微镜(SEM)检测,对培养28天的样本进行组织学检查. 结果 SEM检测显示:随着培养时间的延长,细胞数量增加,细胞间连接出现,并有基质生成;至28天,瓣膜支架上可见大量细胞黏附,细胞融合成片,表面可见基质形成.支架的细胞覆盖率也随时间的延长明显增加.组织学检测见大量细胞黏附材料,细胞已长入材料中部. 结论体外培养的瓣叶存在生长活性,使用组织工程方法有可能生成组织工程心脏瓣膜.     

脱细胞猪主动脉瓣膜与可降解聚合材料共同构建复合组织工程瓣膜

目的对新型复合组织工程瓣膜进行体外生物力学和动物体内移植试验，为临床应用过渡提供依据。方法以脱细胞猪主动脉瓣作为支架，用可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯（3一hydroxybutyrate—co-3-hydroxyhexanoate，PHBHHx）涂层，构建新型复合组织工程瓣膜。（1）复合组织工程瓣膜、新鲜猪主动脉瓣和脱细胞猪主动脉瓣各12枚，用单轴生物拉伸机进行体外生物力学测试；（2）小尾寒羊10只，其中5只在全身麻醉非体外循环下接受复合组织工程瓣膜，移植到羊的肺动脉瓣位；其余接受脱细胞猪主动脉瓣作为对照。术后18周处死动物，取出移植瓣膜，进行组织学、免疫荧光染色、扫描电子显微镜检查和钙含量测定。结果复合组织工程瓣膜保持了自然瓣形态，抗拉强度显著提高（P〈0．05）；瓣膜柔软，表面光滑无血栓；免疫荧光染色检测，瓣膜新生内膜中内皮细胞呈CD31阳性反应，沿瓣表面连续排列，间质细胞呈现单克隆鼠抗人平滑肌actin（sMA）阳性反应；复合组织工程瓣膜钙含量明显低于脱细胞猪主动脉瓣（P〈0．05）。结论复合组织工程瓣膜具有自然瓣膜的三维形态结构，良好的生物力学特性、生物相容性和细胞引导性，初步具备组织工程瓣膜雏形。     

Tissue engineering of heart valves in vivo using bone marrow-derived cells

In this study, we tissue-engineered heart valves in vivo using autologous bone marrow-derived cells (BMCs). Canine BMCs were differentiated into endothelial cell (EC)-like cells and myofibroblast (MF)-like cells. Decellularized porcine pulmonary valves were seeded with BMCs and implanted to abdominal aorta and pulmonary valve of bone marrow donor dogs. Histological examination of the explants identified the regeneration of valvular structures expressing CD31 and smooth muscle alpha-actin, indicating the presence of EC-like and MF-like cells in the grafts at 3 and 1 week, respectively, after implantation. Fluorescent microscopic examinations identified the presence of fluorescently labeled cells in the explants, indicating that the implanted BMCs survived and participated in the heart valve reconstitution. This study reports, for the first time, on tissue engineering of heart valve in vivo using BMCs.     

Tissue-Engineered Heart Valve: Future of Cardiac Surgery

Background

Heart valve disease is currently a growing problem, and demand for heart valve replacement is predicted to increase significantly in the future. Existing “gold standard” mechanical and biological prosthesis offers survival at a cost of significantly increased risks of complications. Mechanical valves may cause hemorrhage and thromboembolism, whereas biologic valves are prone to fibrosis, calcification, degeneration, and immunogenic complications.

Methods

A literature search was performed to identify all relevant studies relating to tissue-engineered heart valve in life sciences using the PubMed and ISI Web of Knowledge databases.

Discussion

Tissue engineering is a new, emerging alternative, which is reviewed in this paper. To produce a fully functional heart valve using tissue engineering, an appropriate scaffold needs to be seeded using carefully selected cells and proliferated under conditions that resemble the environment of a natural human heart valve. Bioscaffold, synthetic materials, and preseeded composites are three common approaches of scaffold formation. All available evidence suggests that synthetic scaffolds are the most suitable material for valve scaffold formation. Different cell sources of stem cells were used with variable results. Mesenchymal stem cells, fibroblasts, myofibroblasts, and umbilical blood stem cells are used in vitro tissue engineering of heart valve. Alternatively scaffold may be implanted and then autoseeded in vivo by circulating endothelial progenitor cells or primitive circulating cells from patient’s blood. For that purpose, synthetic heart valves were developed.

Conclusions

Tissue engineering is currently the only technology in the field with the potential for the creation of tissues analogous to a native human heart valve, with longer sustainability, and fever side effects. Although there is still a long way to go, tissue-engineered heart valves have the capability to revolutionize cardiac surgery of the future.     

改性聚乙二醇水凝胶在组织工程心脏瓣膜中的应用研究

目的 探索改性聚乙二醇(PEG)水凝胶在改善种子细胞和去细胞生物材料支架的复合中的效果.方法 猪主动脉瓣进行去细胞处理后分两组(n=8),A组:山羊自体骨髓间充质干细胞(BMSCs)作为种子细胞包裹于改性PEG水凝胶中贴附于去细胞猪主动脉瓣;B组:单纯种植BMSCs于去细胞猪主动脉瓣;并随机取两组中的8只山羊自身主动脉瓣为对照组(C组).A、B组静态培养7 d后,植入细胞供体羊的腹主动脉内;16周后取材进行形态学、组织学、B超、扫描和透射电镜观察以及生物力学检测.结果 在张力强度[(12.9±1.3)MPa对(8.8±0.4)MPa]、内皮细胞覆盖率(84.6%对14.8%)、附壁血栓形成率(0/8对8/8)等方面A组明显优于B组(P<0.05).生物力学强度A组和C组差异无统计学意义.BMSCs于体内微环境下向内皮细胞和肌成纤维细胞分化.结论 利用改性PEG水凝胶复合去细胞生物支架材料以及自体间充质干细胞构建组织工程瓣膜具有可行性.它可进一步改善种子细胞和支架材料之间的复合关系,并保护种子细胞在动脉流环境下的生长和分化.     

内皮祖细胞和去细胞猪主动脉瓣构建组织工程瓣膜的研究

目的将内皮祖细胞(EPCs)诱导分化为内皮细胞后种植在去细胞猪主动脉瓣膜(APV)上,以期为组织工程瓣膜(TEHV)的研制提供一个新的思路。方法采用去垢剂联合胰蛋白酶法去除猪主动脉瓣膜细胞。采用密度梯度离心法从羊骨髓中分离出EPCs,并诱导分化后,免疫组织化学染色鉴定。将诱导分化得到内皮细胞种植到APV上。采用光镜和电镜检测猪主动脉瓣膜的去细胞效果。对内皮细胞在APV上的生长效果进行观察。结果猪主动脉瓣内的细胞完全去除。EPCs经定向诱导后,具有表达CD-31,CD-34,von Willebrand因子等内皮细胞特征。诱导分化后的EPCs可以在APV表面黏附生长,并且表达CD-31和yon Willebrand因子。结论EPCs可以定向诱导分化为内皮细胞并可以在APV上种植生长。     

人-猪异种生物瓣瓣膜材料的实验构建

目的　探讨用组织工程的方法实现异种生物瓣瓣膜材料的内皮化 ,制备新一代的生物瓣瓣膜材料。方法　取自肉联厂宰杀后 2小时内 4℃冰水保存的 8只猪心 ,在无菌条件下取出主动脉瓣分成 4组 :1组戊二醛 (GA) ,2组环氧氯丙烷 (EC) ,3组环氧氯丙烷 +左旋谷氨酸 (EC+L - GA) ,4组环氧氯丙烷 +左旋谷氨酸 +细胞提取液 (EC+L -GA +CE)。再将新生儿脐带 (离体后 6小时内 )用胰蛋白酶消化制备种子细胞 ,接种于加入 M199(10 %的胎牛血清 +2 0 %的混合血清 +黏附蛋白 +内皮细胞生长因子 )的培养液中进行种植 ,每日用倒置相差显微镜观察细胞生长情况 ,并进行内皮细胞 因子的定性检测、细胞生长覆盖密度的检测、光学和电子显微镜观察。　结果　倒置相差显微镜观察 :1组未见细胞生长 ,2组可见有细胞呈散在性生长覆盖 ,但较 3、4组的细胞数及覆盖率明显降低 (P<0 .0 1)。光镜和电镜观察 :1组未见有内皮细胞生长 ;2组可见有散在的内皮细胞生长 ;3组内皮细胞局部生长并可见细胞脱落 ;4组内皮细胞生长于原瓣叶的无细胞纤维支架上 ,并呈单层紧密排列 ,少见有细胞脱落或丢失 ,细胞沿纤维嵌合排列 ,细胞下组织呈间隙稀疏构成 ,有少量的基质成分和散在的胶原纤维 ,细胞沿纤维间隙排列、嵌合生长 ,形成空间三维排列生长的内皮     

Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde-fixed porcine aortic valves

OBJECTIVE: This study represents the development of a treatment and seeding procedure to improve endothelial cellular adhesion on glutaraldehyde-fixed valves. METHODS: Porcine aortic valves were fixed with 0.2% glutaraldehyde. Wall pieces of these valves had either no additional treatment (n = 4), incubation in M199 Earle (1x), with sodium carbonate at 2.2 g/L without l-glutamine for 24 hours (n = 4), or additional pretreatment with 5%, 10%, or 15% citric acid (three groups, n = 4 each). Thereafter the pieces were washed and buffered to a physiologic pH. This was followed by seeding of human endothelial cells (5 x 10(6) cells). On the basis of the results of these pilot tests, complete glutaraldehyde-fixed aortic roots treated with 10% citric acid were subjected to cell seeding. The valves were seeded with endothelial cells (4.3 x 10(6) cells) either alone (n = 4) or in combination with preseeding of autologous fibroblasts (2.4 x 10(7) cells, n = 4). After each seeding procedure specimens of the free wall of the grafts were taken. In addition, one leaflet was taken for histologic examination after endothelial cell seeding, after 7 days, and after 21 days. Finally, two commercially available stentless aortic valve prostheses (Freestyle; Medtronic, Inc, Minneapolis, Minn) were treated with 10% citric acid and seeded with human fibroblasts and endothelial cells. Specimen were taken according to the glutaraldehyde-fixed aortic roots. Specimen of all experiments were examined with scanning electron microscopy. Frozen sections were stained immunohistochemically for collagen IV, factor VIII, and CD31. RESULTS: On untreated glutaraldehyde-fixed aortic wall pieces, only poor adhesion (24%) was seen. No viable cells were found after 1 week. Cellular adhesion was best on aortic wall pieces pretreated with 10% citric acid. After 7 days, the cells formed a confluent layer. Endothelial cell seeding on citric acid-treated complete aortic valves showed 45% adhesion, but no confluent layer was found after 1 week. Preseeding of these valves with autologous fibroblasts resulted in an endothelial cellular adhesion of 76% and a confluent endothelial cell layer after 7 days. The layer remained stable for at least 21 days. Results of staining for collagen IV, factor VIII, and CD31 were positive on the luminal side of these valves, indicating the synthesis of matrix proteins and viability of the cells. Pretreatment of commercially available porcine valves with 10% citric acid and preseeding with autologous fibroblasts followed by endothelial cell seeding resulted in an adhesion of 78%. The cells formed a confluent cell layer after 7 days. CONCLUSIONS: Pretreatment of glutaraldehyde-fixed porcine aortic valves with citric acid established a surface more suitable for cellular attachment. Preseeding these valves with autologous fibroblasts resulted in a confluent endothelial cell layer on the luminal surface. Flow tests and animal experiments are necessary for further assessment of durability and shear stress resistance.     

Tissue engineered heart valves: autologous cell seeding on biodegradable polymer scaffold

We previously reported on the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts then subsequently coated with endothelial cells. Using these methods, autologous cell/polymer constructs were implanted in 6 animals. In 2 additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, using cardiopulmonary bypass, the right-posterior leaflet of the pulmonary valve was resected completely and replaced with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. After 6 h and 1, 6, 7, 9, and 11 weeks, the animals were sacrificed and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. Animals receiving leaflets made from polymer without cell seeding were sacrificed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were degraded completely leaving no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group; 4-hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. In the tissue-engineered heart valve leaflet, transplanted autologous cells generated proper matrix on the polymer scaffold in a physiologic environment at a period of 8 weeks after implantation.