同种生物心脏组织工程瓣体外构建的初步研究

目的　在生物瓣支架上种植并静态培养自体内皮细胞 (ECs) ,形成完整的内皮细胞单层 ,为下一步脉动流培养并最终体外构建同种生物组织工程瓣 (TEHV)提供材料基础。方法　生物瓣支架选择经液氮保存的成人主动脉带瓣管道 ,用 0 1 %SDS脱去表面的ECs ;成人骨髓间充质干细胞 (MSCs)体外定向诱导分化的ECs作为种子细胞 ,高密度 (>1 0 5cell/cm2 )种植于瓣膜支架上静态培养 2 0d ,扫描电镜观察、摄片 ,以确定再内皮化程度。结果　同种生物瓣支架表面的ECs完全脱去 ,而细胞外基质成分保存良好 ;MSCs体外诱导分化的ECs与生物瓣支架复合体静态培养第 7、1 4和 2 0d ,再内皮化程度分别为 73%、85 %和 92 %。结论　静态培养条件下构建的TEHV基本上实现了体外再内皮化的预期目标。     

硝酸银染色法鉴定生物瓣支架材料体外再内皮化程度的实验研究

目的探讨生物瓣支架再内皮化程度的形态学观察方法,为体外构建心脏组织工程瓣提供手段。方法选择经液氮保存的同种主动脉带瓣管道作为生物瓣支架,0.1%十二烷基硫酸钠(sodiumdodecylsulphate,SDS)脱去表面的内皮细胞(endothelialcells,ECs),纤维连接蛋白(bironectin,FN;80μg/ml)预覆盖瓣膜;选择从正常成人骨髓间充质干细胞(mesenchymalstemcells,MSCs)经体外定向诱导分化的ECs作为种子细胞,以>1～2×105个/cm2密度种植于瓣膜支架上,采用含20%胎牛血清和血管内皮细胞生长因子等的DMEM-HG培养基静态培养,0.5%硝酸银染色,于培养的7、14和20d在立体显微镜下观察、摄片,确定其内皮化程度。结果同种生物瓣表面的ECs完全脱去,而细胞外基质成分保存良好;种植细胞与生物瓣支架复合体静态培养的第7、14和20天,其内皮化程度分别为73%、85%和92%。结论硝酸银染色鉴定生物瓣支架再内皮化的方法,简便、经济,是体外构建心脏组织工程瓣形态学观察的一种有效手段。     

应用四枝化状聚乙二醇-乙烯砜基交联去细胞带瓣管道的实验研究

目的利用四枝化状聚乙二醇-乙烯砜基（polyethylene glycol-VS,PEG-VS）交联去细胞主动脉带瓣管道,制备新型组织工程复合支架,研究其力学和生物学性能。方法采用胰酶法制备兔主动脉去细胞带瓣管道,利用四枝化状PEG-VS与兔去细胞带瓣管道交联构建复合支架,体外静态条件应用力学测试仪检测去细胞管道和复合支架力学性能。将纯种新西兰大白兔30只随机分为3组,每组10只,对照组：正常兔主动脉带瓣管道;去细胞组：去细胞带瓣管道;复合支架组：PEG复合支架。构建兔颈总动脉瓣膜移植模型,术后28 d分别用超声心动图检测3组移植管道通畅率,用HE染色、扫描电子显微镜观察微观形态和炎症细胞浸润,免疫荧光染色检测复合支架体内内皮化程度。结果体外力学测试结果示：PEG复合支架弹性模量及最大负荷力均较去细胞管道有明显提高（P弹性模量=3.1×10-9,P最大负荷力=1.1×10-6）。术后血管彩色多普勒超声心动图提示：复合支架组管腔通畅率高于对照组（P=0.054）和去细胞组（P=0.019）,腔内血栓形成率、管腔形变率明显降低;HE染色、扫描电子显微镜检测显示：复合支架组体内内皮化程度增高,内皮细胞于支架上均匀附着;免疫荧光检测显示：支架表面内皮细胞标记物CD34阳性率高于对照组和去细胞组。结论利用四枝化状功能化PEG-VS改性去细胞主动脉瓣带瓣管道,可明显改善组织工程支架生物力学性能和组织相容性。     

同种带瓣心外管道的临床应用

目的　探讨同种带瓣主动脉和肺动脉心外管道治疗复杂先天性心脏病的临床效果。　方法　应用深低温保存同种带瓣主动脉或肺动脉心外管道重建右心室流出道治疗复杂先天性心脏病 31例 ,其中 ,右心室双出口 6例 ,矫正型大动脉转位 2 1例 ,完全型大动脉转位 2例 ,三尖瓣闭锁 1例 ,感染性心内膜炎合并主动脉瓣关闭不全 1例。应用同种主动脉 2 7例 ,同种肺动脉 4例。　结果　术后同种管道吻合口通畅 ,无压差、无扭曲和受压 ,同种瓣膜活动良好。　结论　同种带瓣主动脉和肺动脉心外管道具有生物活性和完整的瓣膜功能 ,可从解剖学和血流动力学上矫正心脏畸形     

复杂性先天性心脏病应用同种带瓣管道的术中护理体会

许多复杂先天性心脏病 ,尤其是动脉发育不良的病人 ,同种带瓣管道的应用 ,为其治疗带来了广阔的前景 ,它对于解决心外科肺动脉、升主动脉的重建 ,发挥了重大的作用 ,使得一些肺动脉闭锁 ,大动脉转位伴肺动脉发育不全 ,重症法乐氏四联症等危重病人有了治愈的机会。同种带瓣管道以其优良的血流动力学性质 ,高度符合人体生理结构 ,并且无需抗凝的特性 ,是一良好的血管替代产品。临床资料本院自 2 0 0 1年 2月～ 2 0 0 3年 2月 ,应用同种带瓣管道(VH ) ,包括同种带瓣主动脉 ,同种带瓣肺动脉 ,为 8例复杂性先天性心脏病人进行了手术治疗 ,其中男…     

同种带瓣管道应用的远期效果

目的　评价同种带瓣管道应用后的远期效果。方法　1988年1月至1997年12月的10年间应用同种带管道瓣治疗165例多种复杂先天性心脏病(先心病)，其中主动脉带瓣管道136例、肺动脉带瓣管道29例。病人年龄1.3～22.0岁，平均7.6岁。体重9.5～58.0kg，平均22.0kg。管道直径17～22mm，平均19mm。结果　术后早期（30d内）死亡40例，死亡率为24%。术后随访10个月～12.1年，平均(37.0±8.6)个月。病人术后5、10年生存率分别为94.6%、83.8%，带瓣管道完好率分别为83%、58%。结论　应用同种带瓣管道治疗复杂先心病，远期疗效比较满意。     

三种不同手术方式行同种异体血管主动脉根部置换的临床效果

目的　比较 3种应用低温保存的同种异体带瓣管道行主动脉根部重建术式的近期和远期效果。　方法　85例主动脉瓣膜疾病患者采用同种异体带瓣管道行主动脉根部重建 ,其中 Freehand手术 16例 ,Miniroot手术 4 4例 ,Total aortic root手术 2 5例。　结果　 3种术式患者灌注时间、主动脉阻断时间、呼吸机辅助时间和住 ICU时间差别均无统计学意义 (P>0 .0 5 ) ;随访 38.9± 13.2个月 ,Miniroot手术患者主动脉瓣膜中度反流发生率低于其他 2种术式 (P<0 .0 5 )。　结论　用同种异体带瓣管道行主动脉根部重建术治疗主动脉瓣膜疾病 ,3种手术方式均有效 ,其中Miniroot手术术后瓣膜反流的发生率低于其它 2种术式。     

冷冻保存同种带瓣管道的活性研究

目的　探讨液氮冷冻保存对带瓣管道的细胞活性的影响。方法　将 40份同种主、肺动脉管道 ,经修剪、抗生素液处理 ,液氮冷冻保存 4～ 12个月后取出 ,留取主、肺动脉壁片、二尖瓣作细胞培养、光镜和电镜检查 ,并取新鲜主、肺动脉壁片、二尖瓣 4份作对照。结果　冷冻保存 4～ 7个月的管道 ,于 4周后成纤维细胞长满视野 ,细胞收获数为 :二尖瓣 (67.64± 2 5 .60 )万～ (74.5 8±12 .3 4)万 ,主肺动脉壁片 (3 2 .45± 4.75 )万～ (3 8.67± 18.3 2 )万 ,冷冻 1年的管道极少量细胞生长 ,为 (3 0 .61± 17.3 3 )万和 (15 .64± 8.73 )万。保存 4～ 7个月组和新鲜组相比 ,差异无显著性 (P >0 .0 5 ) ,与保存 12组相比 ,差异有非常显著性 (P <0 .0 1)。病理改变见内皮细胞层消失 ,电镜下见成纤维细胞及平滑肌细胞形态正常 ,胶原纤维排列整齐。结论　液氮冷冻保存 7月内 ,带瓣管道仍具有较强的细胞活性。     

Rastelli术中应用同种带瓣管道的中远期结果分析

目的总结Rastelli手术中应用冷冻保存同种带瓣管道的中远期结果, 探索影响管道衰败和再次干预手术的危险因素。方法 2002年4月至2013年12月, 应用冷冻保存的同种带瓣管道完成52例Rastelli手术并长期随访, 其中男36例, 女16例, 手术中位年龄4岁(3～14岁), 中位体质量14 kg(10～36 kg)。术中应用同种主动脉瓣管道30例, 肺动脉瓣管道22例, 根据同种带瓣管道直径的大小分为3组:组Ⅰ(16 mm)22例, 组Ⅱ(16～20 mm)22例, 组Ⅲ(≥20 mm)8例。术后随访同种带瓣管道衰败的发生时间和病理改变。结果术后随访中位时间12年(7～18年), 所有患儿随访中都出现不同程度的同种带瓣血管衰败, 衰败的主要病理改变是管道狭窄。37例患儿因为同种带瓣管道严重衰败接受右心室流出道再次重建手术, 两次手术中位间隔时间12年(9～18年)。单因素统计学分析结果显示, 影响同种带瓣血管术后发生衰败时间的危险因素是手术年龄<5岁(P<0.001), 同种带瓣管道的直径大小(P<0.001), Rastelli手术后右心室/主动脉压比值&...     

经液氮保存有活性同种带支架瓣膜的实验研究

目的 检测经液氮保存的人同种带支架瓣膜的细胞活性和流体力学性能.方法 制作同种带支架瓣膜经液氮保存3个月后,利用流式细胞仪技术定量检测瓣叶细胞活性(实验组,n=6),采集新鲜同种瓣膜作为对照(对照组,n=6).利用国产脉动流实验装置,分别测试实验组21#、23#、25#同种带支架瓣膜的流体力学性能,测试各流量下的跨瓣压差、有效瓣口面积和回流百分比;并使用相应型号的国产Perfect牛心包生物瓣膜进行对比研究.结果 实验组内皮细胞死亡率与对照组比较差异无统计学意义(10.24%±1.71% vs.9.09%±2.72%,P=0.441);实验组平滑肌细胞死亡率与对照组比较差异无统计学意义(8.76%±1.82% vs.7.84%±0.59%,P=0.178);实验组总细胞死亡率与对照组比较差异无统计学意义(8.79%±1.44% vs.7.40%±0.49%,P=0.072).两种21#、23#、25#人工生物瓣膜的跨瓣压差对流量具有非常大的依赖性,随着流量的增大而增大.同种带支架瓣膜的跨瓣压差较Perfect牛心包生物瓣膜的大.两种各型号瓣膜的回流百分比均随测试流量的增大而略有减小,同样,同种带支架瓣膜的回流百分比较Perfect牛心包生物瓣膜的大.两种各型号瓣膜的有效瓣口面积均有随流量增加而增大的趋势,同种带支架瓣膜的有效瓣口面积较Perfect牛心包生物瓣膜的略小.结论 经液氮保存同种带支架瓣膜的细胞活性保存良好,流体力学性能较满意.     

去细胞猪主动脉瓣移植于犬腹主动脉内构建组织工程瓣

目的 探讨在犬腹主动脉内构建组织工程心脏瓣膜的实验方法。方法 将预种犬血管间质细胞和内皮细胞的去细胞猪主动脉瓣叶(猪瓣),移植于6条犬的腹主动脉内,于术后4,6,8和10周对移植瓣叶进行形态、组织结构及免疫组化染色观察。结果 (1)移植术后4周时瓣叶周边有多层细胞长入,新细胞外基质形成,原支架组织部分吸收。(2)10周末猪瓣组织完全吸收,为宿主细胞及新合成的细胞外基质取代。基质中间质细胞主要为成纤维细胞和肌纤维母细胞。细胞外基质成分主要为Ⅰ、Ⅲ型胶原和少量弹力纤维,并含有中性和酸性黏多糖。(3)内皮细胞覆盖于瓣叶表面。结论 (1)移植于犬腹主动脉内的去细胞猪瓣于移植术后10周末基本构成组织工程瓣叶;(2)腹主动脉内异位移植是一种可供选择的实验研究方法。     

Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches

Tissue‐engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long‐term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)‐based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose‐derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM‐cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.     

Improvement of Decellularization Efficiency of Porcine Aorta Using Dimethyl Sulfoxide as a Penetration Enhancer

Decellularization of tissues and organs enables researchers to obtain extracellular matrix (ECM) with the natural conformation and chemical composition of specific tissues. However, drawbacks exist such as the structural alteration of ECM or loss of some important components in ECM due to overexposure to chemicals during the decellularization process. In this study, porcine aorta was decellularized by sodium dodecyl sulfate (SDS). Dimethyl sulfoxide (DMSO) was used as a penetration enhancer in the decellularization process to enhance the penetration of SDS, consequently reducing the exposure time of SDS to treated tissues. It is revealed that by addition of DMSO to the decellularization process 64.4% more DNA was removed when compared with just SDS exposure within a 3 h reaction. Cross‐validation by DAPI staining showed that, in the presence of DMSO, the penetration of SDS was improved and almost all cells were removed from the aorta within the 3 h exposure time. Collagen staining revealed that just SDS treatment showed less polarized collagen fibers, while the DMSO addition groups revealed denser and organized collagen fibers. Moreover 77% glycosaminoglycan content was preserved by addition of DMSO in resultant tissues. Scanning electron microscopy analysis of decellularized aortic matrix showed that ECM components remained in the adventitia layer with the addition of DMSO treatment, while the layer was removed with just SDS treatment. Biocompatibility assays proved that after washing the decellularized samples with media supplemented with 3% antibiotic and antimycotic solution for 2 days there was no cytotoxic effect related to the SDS + DMSO decellularization protocol. This study demonstrates that the new decellularization protocol not only improves the removal efficiency of cellular components but also protects the crucial ECM components.     

组织工程角膜生物材料载体制备的比较性研究

目的 比较用不同的方法脱细胞处理异种（猪）角膜基质制备组织工程角膜支架材料，并对其生物相容性进行研究。方法 成年York猪角膜基质材料，以Triton联合0．25％胰酶，处理30min 3h为材料1；Triton联合DNA—RNA酶，处理8～14h为材料2；Triton联合0．25％胰酶、DNA—RNA酶，处理3～5h为材料3。3种材料用无水氯化钙脱水干燥保存。在材料上接种兔角膜基质成纤维细胞，观察细胞生长情况。新西兰大白兔24只，随机分为3组，每组8只。右眼为实验眼，左眼为空白对照眼。将材料植入兔眼角膜基质层中，术后每日裂隙灯检查术眼；1、4、8和12周分别随机取2只兔眼角膜作HE染色，观察材料的生物相容性。结果 材料1、2细胞脱出不完全，处理最佳时间分别是2hN10～12h，角膜基质网状间隙无明显增大；接种兔角膜基质成纤维细胞后，3～4d细胞死亡；植入兔角膜基质中有明显的免疫排斥反应。材料3脱出细胞完全，其脱细胞处理的最佳时间为4h，角膜基质纤维结构无破坏，呈三维网状结构，网状间隙明显增大；接种兔角膜基质成纤维细胞后，细胞在材料上贴附生长良好；将其植入兔角膜基质中无炎性及排斥反应，可逐渐降解吸收，有良好的生物相容性。结论 Triton联合0．25％胰酶、DNA—RNA酶法处理的异种（猪）角膜基质具有良好的生物相容性，可作为一种组织工程角膜的支架材料。     

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.     

脱细胞天然支架生物力学性能变化及预处理改善组织相容性的实验研究

目的观察脱细胞猪主动脉瓣的生物力学性能变化,探讨不同预处理改善天然支架组织相容性的效果。方法新鲜猪主动脉瓣经酶加去污剂法去除细胞,力学测试仪检测其最大负荷、最大应力、最大应变和弹性模量的变化,苏木精-伊红（HE）染色、Ⅰ型胶原免疫组化染色和扫描电镜观察其病理形态学变化;将脱细胞瓣膜分别予磷酸缓冲液、多聚赖氨酸和未灭活胎牛血清包被处理,然后种植大鼠主动脉肌成纤维细胞,甲基噻唑基四唑试验检测细胞黏附率,HE染色和扫描电镜观察形态学变化。结果酶加去污剂法能完全脱去瓣膜细胞,基本维持胶原纤维的空间结构,但其最大负荷、最大应力及弹性模量下降,最大应变上升（P〈0．05）;胎牛血清预处理去细胞瓣能显著提高肌成纤维细胞的黏附率,促进细胞生长、分化和增殖,并在瓣膜表面形成连续的细胞层（F值=129．26,P=0．000）。结论酶加去污剂法可较完全去除猪主动脉瓣膜细胞并保持细胞外基质的三维结构,但其生物力学性能有所下降;胎牛血清预处理能改善脱细胞瓣天然支架的细胞黏附、生长和繁殖。     

液氮保存同种带支架人工瓣膜流体力学测试

目的：利用脉动流模拟实验装置测试液氮保存同种带支架瓣膜流体力学性能，同时与国产perfect牛心包人工瓣膜对比研究。方法：采集同种瓣膜，缝制成21^#、23^#、25^#同种带支架主动脉和同种带支架肺动脉人工瓣膜，经液氮保存，使用国产脉动流实验装置测试瓣膜流体力学性能，采用ISO／FDA浮估标准，分别测量各流量下的跨瓣压差、有效瓣口面积(EOA)和回流比。并与相应型号国产perfect牛心包人工瓣膜对比研究。结果：21^#、23^#、25^#同种带支架主动脉和肺动脉瓣膜的跨瓣压差和回流百分比差异无显著性，但较perfect瓣大。同种带支架主、肺动脉瓣膜的EOA差异无显著性，但较perfect牛心包生物瓣膜的略小些。同种带支架瓣膜实际开口面积(AOA)小于perfect牛心包瓣，但有效瓣口面积，实际开口面积比值无差别。结论：同种带支架主动脉和肺动脉人工瓣膜流体力学性能满意，同种带支架主动脉瓣膜与同种带支架肺动脉瓣膜流体力学性能无差别。     

Decellularized vein as a potential scaffold for vascular tissue engineering

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.     

The failure modes of biological prosthetic heart valves

Bioprosthetic heart valves have been used since the 1960s, starting with the use of homograft aortic valves obtained from human cadavers. Today prosthetic heart valves are used widely, and bioprostheses account for close to 40% of all heart-valve replacements. Although most bioprosthesis are still stented porcine aortic valves, the introduction of stentless valves and the increasing use of cryopreserved homograft valves has led to an upsurge of interest in bioprosthesis. There have been significant changes in the handling and fixation of porcine aortic valves; however, their modes of failure remain virtually unchanged, although many bioprosthetic valves now last for considerably longer periods. This article reviews the modes of failure of bioprosthetic heart valves.