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。     

Fabrication of a Novel Hybrid Heart Valve Leaflet for Tissue Engineering: An In Vitro Study

The objective of this study was to fabricate biomatrix/polymer hybrid heart valve leaflet scaffolds using an electrospinning technique and seeded by mesenchymal stem cells. Mesenchymal stem cells were obtained from rats. Porcine aortic heart valve leaflets were decellularized, coated with basic fibroblast growth factor/chitosan/poly-4-hydroxybutyrate using an electrospinning technique, reseeded, and cultured over a time period of 14 days. Controls were reseeded and cultured over an equivalent time period. Specimens were examined biochemically, histologically, and mechanically. Recellularization of the hybrid heart valve leaflet scaffolds was significantly improved compared to controls. Biochemical and mechanical analysis revealed a significant increase of cell mass, 4-hydroxyproline, collagen, and strength in the hybrid heart valve leaflets compared to controls. This is the first attempt in tissue-engineered heart valves to fabricate hybrid heart valve leaflets using mesenchymal stem cells combined with a slow release technique and an electrospinning technique.     

组织工程心脏瓣膜构建现状与发展

为了克服目前临床应用的人工心脏瓣膜在抗凝和衰坏方面的缺点，随着组织工程学的发展、构建组织工程心脏瓣膜的研究已了得初步进展。综述近年来组织工程心脏瓣膜在种植材料、种子细胞、动物实验和基础研究方面取得的进展，并探讨组织工程心脏瓣膜在种植材料、种子细胞、动物实验和基础研究方面取得的进展，并探讨组织工程心脏瓣膜的优越性和可行性。     

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。     

Physiological performance of a detergent decellularized heart valve implanted for 15 months in Vietnamese pigs: surgical procedure, follow-up, and explant inspection

This study features the longest experimental follow‐up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X‐100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals (n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL‐treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham‐operated group (n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL‐treated valves during follow‐up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue‐engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self‐supporting substitute.     

In Vitro Hydrodynamic Evaluation of a Scaffold for Heart Valve Tissue Engineering

Although prosthetic heart valves have saved many lives, the search for a living substitute continues with the aid of tissue engineering. Much progress has been made so far, but the translation of this technology to clinical reality remains a challenge, especially due to the structural complexity of heart valves and the harsh environment they are in. In a joint effort, researchers from Federal University of ABC and Institute Dante Pazzanese of Cardiology have conceived a new bioresorbable scaffold for heart valve tissue engineering (HVTE), whose hydrodynamic performance was first assessed and described in this work. The scaffold was studied at the mitral position of a left heart simulator from Escola Politécnica of the University of São Paulo, under 60 bpm and with no cell seeding. In this condition, two‐dimensional particle image velocimetry was performed to investigate the flow during diastolic and systolic phases. The results indicate that the scaffold can withstand the required intraventricular pressures for a simulated normal physiologic condition in a bioreactor. Furthermore, the averaged (N = 150) velocity vector maps showed a smooth and well‐distributed flow during diastole and qualitatively demonstrated no‐significant regurgitation at systole.     

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.     

去细胞组织工程心脏瓣膜

组织工程心脏瓣膜的研究是心脏外科领域的研究热点之一 ,近年来这方面的研究正在不断拓展。在去细胞同种、异种瓣膜支架的基础上构建的组织工程瓣膜 ,具有低免疫原性、无细胞毒性、植入体内后宿主细胞再细胞化程度高、有较好的仿生性、耐久性和机械强度等多方面的优势 ,而成为未来组织工程心脏瓣膜研究发展的一个重要方向。现将去细胞组织工程瓣近年来的研究进展进行综述。     

Detergent Decellularization of Heart Valves for Tissue Engineering: Toxicological Effects of Residual Detergents on Human Endothelial Cells

Detergents are powerful agents for tissue decellularization. Despite this, the high toxicity of detergent residua can be a major limitation. This study evaluated the efficacy of detergent removal from decellularized pulmonary valves (PVs) and the consequences of repopulation with human endothelial cells (HECs). Porcine PVs were treated with 1% sodium deoxycholate (SDC), group A; 1% sodium dodecyl sulfate (SDS), group B; and a mixture of 0.5% SDC/0.5% SDS, group C (n = 5 each). After each of 10 succeeding wash cycles (WCs), samples of the washing solution (WS) were analyzed by solid phase extraction and high performance liquid chromatography for the presence of detergents. Metabolic activity of HEC was also assessed in the WS samples (cytotoxicity and MTS assays). Decellularized and washed PVs were reseeded with HEC. Histological analysis demonstrated efficient tissue decellularization in all groups. Detergents' concentration in all WSs decreased exponentially and was below 50 mg/L after 6, 8, and 4 WCs in groups A, B, and C, respectively. This concentration resulted in no significant toxic influence on cell cultures, and scaffolds could be efficiently reseeded with HEC. In conclusion, intensive washing of detergent decellularized valvular scaffolds lowers the residual contamination below a hazardous threshold and allows their successful repopulation with HEC for tissue engineering purposes.     

In Vitro and In Vivo Validation Tests for Total Artificial Heart

Abstract: Properly planned in vitro and in vivo studies are mandatory to validate blood pump performance. However, standard procedures for in vitro and in vivo tests of total artificial heart (TAH) performance still do not exist. At Baylor College of Medicine, standard tests procedures for performance validation of the totally implantable TAH have been established. In this paper, these in vitro tests protocols (performance mapping tests, accelerated endurance test, hemolysis test, flow visualization tests, etc.) are described as well as in vivo test procedures (TAH implantation procedure, including surgical technique, postoperative management and tests, etc.). In addition, pathological protocols are presented. In our experience, these procedures are very simple, easy to perform, and inexpensive. These protocols are proposed as standard in vitro and in vivo tests for a TAH.     

In Vitro Evaluation of a Novel Autologous Aortic Valve (Biovalve) With a Pulsatile Circulation Circuit

We have used in‐body tissue architecture technology to develop an autologous valved conduit with intact sinuses of Valsalva (biovalve). In this study, we fabricated three different forms of biovalves and evaluated their function in vitro using a mock circulation model to determine the optimal biovalve form for aortic valve replacement. A cylindrical mold for biovalve organization was placed in a dorsal subcutaneous pouch of a goat, and the implant that was encapsulated with connective tissue was extracted 2 months later. The cylindrical mold was removed to obtain the biovalve (16 mm inside diameter) that consisted of pure connective tissue. The biovalve was connected to a pulsatile mock circulation system in the aortic valve position. The function of the three biovalves (biovalve A: normal leaflets with the sinuses of Valsalva; biovalve B: extended leaflets with the sinuses of Valsalva; biovalve C: extended leaflets without the sinuses of Valsalva) was examined under pulsatile flow conditions using saline. In addition, the mock circuit was operated continuously for 40 days to evaluate the durability of biovalve C. The regurgitation rate (expressed as a percent of the mean aortic flow rate during diastole) was 46% for biovalve A but only 3% for biovalves B and C. The durability test demonstrated that even after biovalve C pulsated more than four million times (heart rate, 70 bpm; mean flow rate, 5.0 L/min; mean aortic pressure, 92 mm Hg), stable continuous operation was possible without excessive reduction of the flow rate or bursting. The developed biovalve demonstrated good function and durability in this initial in vitro study.     

In vivo recellularization of plain decellularized xenografts with specific cell characterization in the systemic circulation: histological and immunohistochemical study

Decellularized biological scaffolds have been used for the tissue engineering of heart valves with good results in the pulmonary circulation. However, little information is available on the recellularization of plain decellularized biological scaffolds in the systemic circulation. The aim of this study was to determine whether plain decellularized xenografts (PDXs) can recellularize with specific cell characterization in the systemic circulation. The xenogenic aortic valved conduit grafts of rabbits were implanted in the abdominal aorta of dogs after decellularization. The grafts were explanted at 4, 12, or 24 weeks after implantation for histological, immunohistochemical examination, scanning electron microscope, and Western blot analysis. Although the valvular structures were completely lost after implantation, supravalvular conduits showed normal shapes and endothelialization. The PDXs were repopulated with basic vascular cell components in approximate natural proportions without immunological responses. The PDXs had been recellularized with specific cell characterization in the systemic circulation.     

组织工程化膀胱平滑肌结构的体外构建

目的 利用组织工程方法在体外初步构建膀胱平滑肌结构.方法 置备膀胱脱细胞基质移植物(BAMG)并以HE和Masson染色进行支架材料评价.酶消化法获得膀胱平滑肌原代细胞,分别以免疫荧光和RT-PCR进行细胞鉴定.经过体外培养和扩增后,将P3代膀胱平滑肌细胞接种在BAMG上.结果 支架上的膀胱平滑肌细胞经过体外培养4周后形成多层结构,同时免疫组化检测α-Smooth Muscle Actin表达阳性.结论 运用组织工程方法能够在体外进行膀胱平滑肌结构的初步构建,为进一步膀胱组织工程修复奠定技术基础.     

骨髓间质干细胞向软骨细胞表型定向诱导分化的实验研究

目的 研究体外培养的猪骨髓间质干细胞（Bone Marrow Stem Cells,MSCs)在特定培养液作用下向软骨细胞表型转化，探讨其作为组织工程化软骨的种子细胞的可行性。方法 取成年崇明长枫杂交猪髂骨骨髓5ml，在低糖DMEM完全培养液培养2周，传代后以高糖DMEM无血清特定培养液诱导（含胰岛素2mg/L、转铁蛋白3mg/L、丙酮酸100mg/L、地塞米松10^-7mol/L、TGF－β1 10ng/ml)，在相关显微镜和电镜下进行观察，免疫组化检测Ⅱ型胶原分泌，原位杂交检测Ⅱ型胶原mRNA表达。结果 细胞形态由成纤维样梭形向多角形、多边形转变，透视电镜观察见大量扩张粗面内质网、高尔基体、线粒体。诱导培养后第7，14dⅡ型胶原免疫组化阳性，同时原位杂交检测Ⅱ型胶原mRNA表达呈阳性。结论 MSCs在特定培养液诱导下能向软骨细胞表型转化，并能分泌软膏特异性基质，有可能成为软骨组织工程较理想的种子细胞来源的应用前景。     

Dynamic In Vitro and In Vivo Performance of a Permanent Total Artificial Heart

In vivo characterization studies were performed to compare the dynamic in vivo performance of the Penn State/3M Health Care electric total artificial heart to existing in vitro data. Fully implanted systems were utilized including the artificial heart, controller, backup batteries, compliance chamber, and transcutaneous energy transmission. Catheters were implanted to measure central venous pressure (CVP), left atrial pressure (LAP), right atrial pressure (RAP), pulmonary artery pressure (PAP), and aortic pressure (AoP). Cardiac output (CO) was determined from the implanted controller, and systemic vascular resistance (SVR) was calculated. Steady state data were collected for each animal along with data regarding the transient responses to changes in preload and afterload. Preload was manipulated through volume changes. Afterload changes were accomplished through vasoactive agents. Increased preload caused little change in cardiac output because the pump output was nearly maximum at baseline. LAP, AoP, and SVR increased with increasing RAP. Decreased preload caused a reduction in CO, LAP, and SVR. Afterload increase resulted in a slight decrease in flow and an increase in system power and SVR. Afterload reduction was accompanied by a decrease in preload and a concomitant reduction in flow. Overall, the system response was similar to the response observed in vitro.     

In Vitro Evaluation of Linear Motor-Driven Total Artificial Heart

Abstract: This paper deals with the in vitro evaluation of a newly developed linear motor-driven total artificial heart (linear TAH). The linear TAHs have been developed and evaluated in mock testing and acute animal experiments. The new linear TAH was made based on experience with acute animal experiments. The maximum static thrust of the new linear pulse motor is 146 N with exciting current of 1.69 A. The weight and the volume of this linear TAH are 1.9 kg and 560 ml, respectively. The linear TAH has a kinetic thrust of 85 to 43 N and provides the maximum flow rate of 6.8 L/min in mock circulatory testing.     

反应器内构建组织工程血管平滑肌层的实验研究

目的 探讨生物反应器内构建组织工程血管的可行性.方法 于6个月龄犬颈总动脉血管获取平滑肌细胞,经体外培养扩增后接种于聚羟基乙酸(PGA)上,形成细胞-材料复合物,并将其置于生物反应器内培养.实验组模拟成年哺乳动物循环系统的参数予以动态力学刺激培养(搏动频率:75次/分;扩张量＜5%);对照组为静态培养,其余与实验组相同,分别于3与6周后取材检测.结果 生物反应器内培养的血管大体观察具有良好的弹性,管腔圆,色泽光亮;HE染色显示平滑肌纤维成分排列较规则,有层次感;弹力纤维染色显示弹力纤维成分较多而密;免疫组织化学检测显示为棕黄色层状排列的平滑肌纤维.对照组弹性欠佳,管腔塌陷,色泽暗淡;平滑肌纤维与弹力纤维成分较少且排列紊乱,层次感差.结论 应用生物反应器可构建具有良好结构的血管样结构的平滑肌层组织.     

房室连接不一致右心室双出口根治手术的选择与适应证优化

目的　回顾性分析房室连接不一致右心室双出口(doubleoutlet right ventricle,DORV)的病理解剖特点、手术方法选择和治疗结果,探讨手术适应证和手术方法的优化。　方法　1990年9月至2 0 0 4年8月对7例房室连接不一致的DORV患者施行根治手术治疗,年龄3～7岁,其中形态学左心室与肺动脉心外管道连接和室间隔缺损修补术3例,double- switch(双调转)手术2例,改良Fontan手术2例。　结果　手术早期死亡1例(14 .3% ,1/ 7) ,为手术后2个月并发Hom ograft感染。 度房室传导阻滞1例,安装永久心外膜起搏器;1例改良Fontan手术后并发上腔静脉回流梗阻和心包积液,症状改善后出院。Double- switch手术后平均监护时间最短为术后6 d。　结论　房室连接不一致DORV根治手术应首选double- switch手术,尤其对形态学右心室功能不全和三尖瓣关闭不全者,如果合并多种复杂畸形解剖无法矫治,可选择单个心室的修补方案。     

去细胞猪主动脉瓣支架及兔骨髓干细胞构建组织工程心脏瓣膜的初步研究

目的探讨应用去细胞猪主动脉瓣支架(ace llu larized porc ine aortic va lve scaffo ld,APAV S)与兔骨髓干细胞(bone m arrow strom a l ce lls,BM SC s)体外构建组织工程瓣膜的可行性。方法采用去垢剂+核酸酶消化法处理,去除猪主动脉瓣细胞成分,并作去细胞前后的形态学检查和生物力学测定;在去细胞支架上种植兔BM SC s,行形态学检查和免疫组织化学染色观察。结果光学显微镜、扫描及透射电子显微镜下可见猪主动脉瓣膜中的细胞成分可完全被去除,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(642±102g/mm2vs.636±127g/mm2)和断裂伸长率(62.2%±18.1%vs.54.4%±16.0%)差别无统计学意义(P>0.05)。种植的兔BM SC s在APAV S表面形成一层连续的细胞层。免疫组织化学检查α-平滑肌动蛋白抗体(+),CD 31(-)。结论种植兔BM SC s于APAV S上,可在体外构建组织工程心脏瓣膜。