Tissue-engineered heart: electrophysiological properties and long-term safety

doi:10.3969/j.issn.2095-4344.0098

Abstract

Abstract:

BACKGROUND: Insufficiency of donor heart and ethics are the major obstacles to heart transplantation. Theoretically, a tissue-engineered heart is an important means to solve the donor heart insufficiency.
OBJECTIVE: To review the scaffold materials, seed cells and cell incubation methods in the construction of tissue-engineered heart, thus providing references for the future study on the tissue-engineered heart.
METHODS: A retrieval of PubMed and Web of Science databases was performed for the articles addressing the construction of tissue-engineered heart from 2004 to 2016. Totally 2 921 articles were searched, and finally 53 eligible articles were included in accordance with the inclusion and exclusion criteria.
RESULTS AND CONCLUSION: In vitro organ culture and the function of tissue-engineered heart are the difficulties in the construction of tissue-engineered heart. In vitro construction of tissue-engineered heart requires the supply of nutrients, gases, temperature and corresponding electrical stimulation. Myocardial cells, scaffold materials and organ culture system are indispensable for the tissue-engineered heart construction. Therefore, it is highly important to optimize the decellular process, select an ideal seed cell and improve its adhesion, proliferation and differentiation, improve the electrophysiological properties of the tissue-engineered heart by gene regulation, and confirm the long-term safety of the tissue-engineered heart.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Heart, Stents, Cells, Cultured, Tissue Engineering

CLC Number:

R318

Tong Cai-ling1, 2, 3, Li Ming-hui2, 3, Qi Zhong-quan2, 3. Tissue-engineered heart: electrophysiological properties and long-term safety[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(4): 606-611.

Figures/Tables 2

SDS是心脏组织脱细胞最常用的灌注液。除此化学方法外，物理洗脱法和酶消化法也可达到去细胞的目的。前者如声波降解法、搅拌法等通常与化学洗脱法一起进行，达到去除破碎细胞成分的效果[22]。但由于心脏的脉管系统复杂且脆弱，故通常不会采用此法以免破坏支架的完整性。酶消化法主要是利用胰酶、胶原酶和核酸酶等的酶解作用破坏细胞膜蛋白进而脱去细胞。酶具有很强的特异性，所以不同的细胞和材料组成，在消化过程中酶的选择和作用时间也要做出相应改变，选择正确的酶处理方可体现其高效性。此外，酶的选择和处理不当还会导致胶原蛋白和纤维组成被破坏，得到不完整的心脏支架。 Rieder等[23]通过完全去除细胞的效率及再细胞化的潜力两方面比较了3种猪心脏瓣膜的脱细胞方法，发现使用0.25%叔辛基苯基聚氧乙烯/脱氧胆酸钠和核酸酶消化可得到一个再细胞化效果良好的支架。Hussein等[24]总结并分析了多种常用的物理、化学和生物洗脱液的作用原理及优缺点，可以为细胞洗脱液的初步选择提供参考。2015年，Papalamprou等[25]在其研究中通过抗原清除的方法构建细胞外基质支架，同时采用亲水性和亲脂性的异种抗原以分段的方式除去心脏组织中心肌细胞和非心肌细胞的抗原成分和细胞组成，也可成功保持原有心脏组织的结构、生化特性和力学特性，且后续实验结果表明，将小鼠或人的间充质干细胞接种至以抗原清除法构建的心脏支架进行再细胞化，细胞迁移较为深入，显示其具有良好的生物兼容性。而通过变性去污剂SDS洗涤得到的心脏支架再细胞化后较多细胞分布在外周区域，体内实验也显示移植后伴有长期的慢性炎症反应[26]。去细胞方法虽多，但还需要在实验中不断摸索，积累经验，找到最简单有效的去除方法和试剂作用的最佳浓度和时间，方可达到理想的洗脱效果。理想的心脏去细胞支架是无毒性、无致病性、无免疫原性的。去细胞后心脏支架内细胞残留情况和心脏支架的完整性及各项指标也是组织工程心脏成败的关键。去细胞支架材料的成分主要包括胶原蛋白、肌原蛋白、纤连蛋白和层粘连蛋白等[27]，可用染色分析其细胞残留情况，扫描电子显微镜观察其形态结构，用Western blot或质谱分析等技术对各成分做定量分析等，可通过各个方向的抗拉伸实验评估整个支架的弹性及强度，同时灌注有色染料可清晰观察到心脏支架内的大血管及脉管系统是否完整。Ott等[6]用苏木精-伊红染色和免疫荧光染色等评估脱细胞程度，发现经SDS洗脱12 h又经Triton-X100和洗涤液灌注后心脏支架内没有检测出细胞存在。通过组织学方法如苏木精-伊红染色来判断细胞是否清除完全是目前最常用的手段之一，还可以通过扫描电子显微镜观察脱细胞程度及残留组分。细胞未洗脱彻底会增强心脏支架的免疫原性，与后期种植的异种细胞不相容，移植到体内后会引起受体的免疫排斥反应，使组织工程心脏失去意义。同时也要注意，心脏组织脱细胞完全后需用洗涤液灌流冲洗，以防止洗脱液对支架结构的毒性作用。且避免洗脱液在支架内滞留时间过长，持续消化过度，破坏其纤维和胶原组成，降低支架机械强度，增大移植手术难度。近年来，人们在多种组织和器官中对去细胞支架材料也进行了系列研究，并评估了去细胞支架作为3维支架的可能性[28]。在软骨组织工程领域中，细胞外基质来源支架被已证明是很好的软骨组织工程仿生支架，被应用于临床实践中。虽然接种细胞后支架会出现萎缩，但可通过配合交联剂使用或其他物理化学方法来改进，克服缺陷[29]。杨立信等[30]将去细胞瓣叶植入SD大鼠背部皮下，按照不同的包埋时间取出瓣叶进行一系列组化实验，以此来评价去细胞支架的免疫原性及炎性反应。后期也可通过器官移植等体内实验来分析支架免疫原性的高低。 2.3 种子细胞的选取正常的心脏组织由多种细胞组成，心肌细胞是其主要的工作细胞。成熟的心肌细胞在体内增殖能力较低，心肌细胞的获取成为组织工程心脏构建的难题。近年来有人指出，心肌细胞要不断搏动以维持心脏的泵血功能，这也许是阻碍心肌细胞同时收缩与分裂的原因之一，并认为心肌细胞的增殖可以通过调控某些基因的表达来实现[31]。 Matsuyama等[32]发现敲除了新生鼠心肌细胞连接蛋白43的表达基因，p38 MAPK通路的抑制剂和表达上调的成纤维细胞生长因子能够刺激心肌细胞的增殖，也有研究结果表明抑制或过表达体内某些基因可以促进心肌细胞增殖[33]。另外有研究显示，心肌细胞干性的丧失是由于高氧环境中DNA损伤所致，低氧环境有利于心肌干细胞干性的维持[34]。但鲜有体外成功获得大量心肌细胞的报道。种子细胞作为组织工程心脏的关键，应同时满足易于获取、分化程度低、增殖能力强、免疫原性低等条件。所以，作为生命之源的干细胞成为组织工程器官构建中种子细胞的优秀候选者，主要因为干细胞具有以下特征： (1)具有自我更新能力。干细胞通过对称和非对称有丝分裂产生两种不同命运的细胞，一种细胞继续保持原有干细胞的功能，另一种为具有特定功能的细胞。子代干细胞可以不断的分裂下去并保持着干细胞的生物特性。这种分裂方式对维持机体器官的稳定性起到了非常重要的作用。 (2)具有多向分化性。胚胎干细胞具有全能分化性，能够分化为各谱系的细胞和特定组织干细胞，在特定环境中发育成完整的个体，也可用于临床细胞移植治疗疾病。但它存在着移植免疫排斥的限制和伦理学的困扰不能广泛获取和应用。成体干细胞虽只能在体外有限扩增，分化效率低，但这类细胞存在于宿主体内，可直接从患者自身获取，无移植排斥也无伦理道德的束缚。 (3)具有高度增殖能力和相对静止期。干细胞在体内可以连续分化增殖数代，也可以在较长时间内处于静止状态。当机体器官受损时，干细胞处于相对活跃的状态，可以增殖数代来修复受损的组织部位。 (4)干细胞的分化需要特定的微环境。在与不同的细胞接触或不同激素、试剂作用下，干细胞向着不同的方向进行特异性分化。间充质干细胞作为干细胞家族的重要成员之一因其来源广泛，提取和体外培养技术较成熟，易于扩增和纯化等优势，被认为是最有希望应用于临床治疗的一类种子细胞。间充质干细胞来源于发育早期的中胚层和外胚层，可取自全身结缔组织和器官间质中，脐血、肌肉、脂肪、骨骼等都有间充质干细胞存在，以成人骨髓组织中含量最多。除来源丰富外，间充质干细胞还具有低免疫原性、无MHC限制性、体外扩增迅速且稳定、损伤去化作用及归巢功能、免疫调节和组织修复能力强等特点[35]，是一类理想的种子细胞。 2011年Stubbs等[36]利用干细胞修复心脏。也有人将经过培养的间充质干细胞植入小鼠心肌梗死模型中，发现能有效减少心梗面积和心肌纤维化[37-38]。除骨髓来源的间充质干细胞外，脂肪组织中间充质干细胞的应用也日渐广泛。它在体内细胞基数大，且可通过微创等更安全的手段获得，具有比骨髓间充质干细胞更强的增殖能力和更稳定的增殖活性，比骨髓间充质干细胞有更大的优越性[39]。间充质干细胞可以沿着胶原纤维等胞外机制四处爬行[40]，这也是它作为去细胞支架种子细胞的重要原因之一。另有研究发现，用含有5-aza胞苷的培养基培养可成功在体外将间充质干细胞诱导成心肌细胞并检测到动作电位[41-43]，证实了在调节因子的作用下，间充质干细胞可以成功分化为心肌细胞。近年来，心肌干细胞的研究[44]，也为临床上终末期心脏病的治疗和受损心脏的修复带来了新的希望(图1)。"

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