心肌组织工程研究进展

心肌组织工程的目的在于利用体外构建的组织修复、替换及再生受损心肌。我们综述了心肌组织工程中的最新方法及其在体内的初步应用,讨论了心肌组织工程的细胞来源和支架材料,提出了现存障碍。心肌组织工程的替代治疗具有极其诱人的前景,但是它还处于早期阶段,仍然需要对其真正的价值做出合适的评价。     

研究报告：MWNTs/PMMA薄膜对大鼠棕色脂肪来源的心脏干细胞生物学特性的影响

近年来,鉴于心肌组织独有的电生理特性,应用导电纳米材料作为细胞支架开展心肌组织工程研究取得明显进展．碳纳米管材料具有良好的力学性能和导电特性,前期研究表明,其具有促进新生鼠心肌细胞中未成熟心肌细胞增殖与成熟心肌细胞进一步发育的作用,但尚未见碳纳米管材料对棕色脂肪来源的心脏干细胞(CSCs)生物学特性影响的研究报道．为此,本研究首先采用凝胶-溶胶法制备了羧基化多壁碳纳米管和聚甲基丙烯酸甲酯 (MWNTs/PMMA)薄膜,并通过一系列细胞学、免疫细胞化学及电镜检测方法,系统评价了MWNTs/PMMA薄膜对大鼠棕色脂肪CSCs活力、增殖能力及向心肌分化效率的影响．研究发现,与明胶材料相比,MWNTs/PMMA薄膜对棕色脂肪CSCs活力、增殖能力有明显促进作用,并且明显增强其向心肌细胞分化的能力,分化的心肌细胞具有明显可见的肌管结构,并且表达介导细胞间电化学传导的缝隙连接蛋白connexin43．此外,超微结构观察发现碳纳米管与细胞膜间及细胞与细胞之间可直接形成紧密连接,调控细胞行为．本研究首次探讨了碳纳米管导电材料对棕色脂肪CSCs生物学特性影响的规律,证实碳纳米管导电材料具有良好的促心肌细胞分化作用．作为一种新型导电纳米材料,碳纳米管在心肌组织工程研究中具有良好的应用前景,未来有望在心肌组织体外构建及心肌梗死治疗性应用中发挥潜在的价值．     

心肌再生微环境构建策略

心肌细胞是一种高度分化的终末细胞,自我更新能力差,因而心梗发生后,坏死的心肌细胞不能得到有效的补充,梗死区域很快被纤维组织所取代,严重影响心功能。近年研究发现,利用组织工程手段构建的心肌补片能有效改善心梗区微环境,对心肌的再生能力有着重要的调控作用,能在一定程度上促进心肌再生,缓解心梗状态。该文综述了心肌微环境对心肌再生的调控机制,以及通过心肌补片的手段改善心肌微环境治疗心梗的相关研究,为心肌补片的设计和心梗的治疗提供参考。     

小鼠心室肌脱细胞化细胞外基质薄片的制备和评价

心肌细胞外基质(extracellular matrix,ECM)可由心肌经脱细胞处理制得,被广泛认为是一种理想的制备工程心肌的生物支架材料。然而目前的脱细胞方法尚存在不足,本研究拟联合使用经典去垢剂改良脱细胞方法,制备性能更为优良的心肌ECM薄片,以用于构建工程心肌片。用振荡切片机将包埋于低熔点琼脂糖中的成年昆明小白鼠心室肌组织沿心脏横轴切成300μm厚的薄片,随机分为正常对照组、SDS脱细胞组(0.1%SDS处理)和改良脱细胞组(0.1%SDS和0.5%Triton X-100联合处理)。通过总RNA和总蛋白质含量分析、HE染色和免疫荧光染色等方法评估各组的脱细胞程度和ECM成分保留状态;将改良脱细胞组ECM与小鼠胚胎干细胞源心肌细胞(murine embryonic stem cell-derived cardiomyocytes,m ES-CMs)和小鼠胚胎成纤维细胞(murine embryonic fibroblasts,MEFs)共培养以检测其生物相容性。结果显示:SDS脱细胞组和改良脱细胞组ECM中残留的总RNA及蛋白质含量均低于对照组。HE染色结果显示改良脱细胞组核质去除较SDS脱细胞组更彻底。改良脱细胞组可见胶原蛋白IV和层粘连蛋白两种ECM关键成分表达量和分布接近正常心肌组织,而SDS脱细胞组中这两种蛋白明显减少且分布紊乱。m ES-CMs和MEFs能存活于改良脱细胞组ECM表面12天以上并向内迁移。综上,SDS和Triton X-100联合脱细胞法制备ECM薄片效果明显,能更好地保留天然ECM成分和结构,具有良好的生物相容性。     

间充质干细胞分化为心肌细胞的诱导方法研究进展

间充质干细胞作为一种取材方便、易于分离培养、体外扩增快、免疫原性低的成体干细胞,具有自我更新和多向分化潜能,可在体内外不同的诱导条件下分化为心肌细胞,是理想的心肌再生治疗的种子细胞。本文综述了间充质干细胞分化为心肌细胞的诱导方法,包括化学试剂、中药制剂、机械力和电磁刺激、心肌环境因子、损伤组织条件培养、组织工程方法等,为其在心肌损伤性疾病尤其是心肌梗死治疗中的应用提供基础。     

竹节参总皂苷抑制急性低压缺氧大鼠脑和心肌损伤的形态学观察

在模拟海拔8000米高空的急性低压缺氧条件下,研究了竹节参总皂苷对Wistar大鼠的脑、心肌组织和细胞损伤的预防效果.实验结果表明未给药的阴性对照组Wistar大鼠的脑和心肌组织以及细胞均遭到了严重损伤.而竹节参总皂苷和红景天提取物给药组的Wistar大鼠,其脑和心肌组织以及细胞的损伤程度明显减轻.其中竹节参总皂苷给药组的效果最明显.     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

CD4^＋T细胞在大鼠心肌缺血再灌注损伤中的作用

目的通过大鼠心肌缺血再灌注损伤的动物模型,分析CD4^＋T细胞在心肌组织损伤中的作用。方法结扎大鼠冠状动脉左前降支45min,随后恢复再灌的方法,制作缺血再灌损伤的动物模型,随机分为再灌注0、2、6、9、12h组及相应的对照组。II导联心电图及TTC确定模型,组织病理学观察心肌细胞的损伤情况,免疫荧光染色计数浸润的炎性细胞,半定量PCR进一步验证各型T细胞的表达。结果心肌的梗死面积与心肌缺血再灌时间成正相关,至观察结束未出现峰值;组织中浸润的中型粒细胞和T细胞分别在2h和12h有峰值出现,但CD4^＋T/CD3^＋T的比率几乎保持不变;观察所见CD4^＋T细胞是组织中存在最多的T细胞。结论大鼠缺血再灌注损伤中,心肌组织中浸润的CD4^＋T细胞作为主要的效应细胞,参与了持续稳定的心肌损伤过程。     

基质金属蛋白酶在心肌缺血再灌注损伤中的作用

肌缺血再灌注损伤是指缺血心肌组织在恢复血流供给后,其细胞代谢功能障碍及结构破坏反而加重的现象,主要表现在心肌收缩与舒张功能障碍、血管内皮功能障碍、微循环血流紊乱、细胞代谢失调、电解质平衡紊乱、细胞凋亡与坏死等,并伴随着氧自由基的大量产生和毒性损伤以及炎症反应的激活,是一个极其复杂的病理过程。基质金属蛋白酶(MMPs)及其组织抑制物(TIMPs)是心肌组织中多种细胞分泌的内源性细胞因子,其作用涵盖了细胞外基质降解、炎症反应激活、调节血管功能、影响细胞凋亡与存活等众多病理生理过程,而这些过程均在心肌缺血再灌注损伤中发挥着重要的作用。     

基质金属蛋白酶在心肌缺血再灌注损伤中的作用

肌缺血再灌注损伤是指缺血心肌组织在恢复血流供给后,其细胞代谢功能障碍及结构破坏反而加重的现象,主要表现在心肌收缩与舒张功能障碍、血管内皮功能障碍、微循环血流紊乱、细胞代谢失调、电解质平衡紊乱、细胞凋亡与坏死等,并伴随着氧自由基的大量产生和毒性损伤以及炎症反应的激活,是一个极其复杂的病理过程。基质金属蛋白酶（MMPs）及其组织抑制物（TIMPs）是心肌组织中多种细胞分泌的内源性细胞因子,其作用涵盖了细胞外基质降解、炎症反应激活、调节血管功能、影响细胞凋亡与存活等众多病理生理过程,而这些过程均在心肌缺血再灌注损伤中发挥着重要的作用。     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.     

Injectable cardiac tissue engineering for the treatment of myocardial infarction

Heart disease is a leading cause of morbidity and mortality worldwide. Myocardial infarction leads to permanent loss of cardiac tissue and ultimately heart failure. However, current therapies could only stall the progression of the disease. Thus, new therapies are needed to regenerate damaged hearts to overcome poor prognosis of patients with heart failure. The shortage of heart donors is also a factor for innovating new therapies. Although the cardiac performance by cell-based therapy has improved, unsatisfactory cell retention and transplant survival still plague this technique. Because biomaterials can improve the cell retention, survival and differentiation, cardiac tissue engineering is now being explored as an approach to support cell-based therapies and enhance their efficacy for cardiac disease. In the last decade, cardiac tissue engineering has made considerable progress. Among different kinds of approaches in the cardiac tissue engineering, the approach of injectable cardiac tissue engineering is more minimally invasive than that of in vitro engineered tissue or epicardial patch implantation. It is therefore clinically appealing. In this review, we strive to describe the major progress in the flied of injectable cardiac tissue engineering, including seeding cell sources, biomaterials and novel findings in preclinical studies and clinical applications. The remaining problems will also be discussed.     

Biophysical regulation during cardiac development and application to tissue engineering

Tissue engineering combines the principles of biology, engineering and medicine to create biological substitutes of native tissues, with an overall objective to restore normal tissue function. It is thought that the factors regulating tissue development in vivo (genetic, molecular and physical) can also direct cell fate and tissue assembly in vitro. In light of this paradigm, tissue engineering can be viewed as an effort of "imitating nature". We first discuss biophysical regulation during cardiac development and the factors of interest for application in tissue engineering of the myocardium. Then we focus on the biomimetic approach to cardiac tissue engineering which involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic cell signaling in native myocardium, subpopulations of neonatal rat heart cells were cultured at a physiologically high cell density in three-dimensional polymer scaffolds. To mimic the capillary network, highly porous elastomer scaffolds with arrays of parallel channels were perfused with culture medium. To mimic oxygen supply by hemoglobin, culture medium was supplemented with an oxygen carrier. To enhance electromechanical coupling, tissue constructs were induced to contract by applying electrical signals mimicking those in native heart. Over only eight days of cultivation, the biomimetic approach resulted in tissue constructs which contained electromechanically coupled cells expressing cardiac differentiation markers and cardiac-like ultrastructure and contracting synchronously in response to electrical stimulation. Ongoing studies are aimed at extending this approach to tissue engineering of functional cardiac grafts based on human cells.     

Fibrin as a scaffold for cardiac tissue engineering

Fibrin is a natural biopolymer with many interesting properties, such as biocompatibility, bioresorbability, ease of processing, ability to be tailored to modify the conditions of polymerization, and potential for incorporation of both cells and cell mediators. Moreover, the fibrin network has a nanometric fibrous structure, mimicking extracellular matrix, and it can also be used in autologous applications. Therefore, fibrin has found many applications in tissue engineering, combined with cells, growth factors, or drugs. Because a major limitation of cardiac cell therapy is low cell engraftment, the use of biodegradable scaffolds for specific homing and in situ cell retention is desirable. Thus, fibrin-based injectable cardiac tissue engineering may enhance cell therapy efficacy. Fibrin-based biomaterials can also be used for engineering heart valves or cardiac patches. The aim of this review is to show cardiac bioengineering uses of fibrin, both as a cell delivery vehicle and as an implantable biomaterial.     

Gene expression studies on bio-electrosprayed primary cardiac myocytes

Cardiovascular pathology accounts for the greatest number of mortalities in the western world and thus the development of ex vivo cardiac tissue has vast potential in cardiac therapy. Bio-electrosprays (BES), a recently discovered direct cell engineering protocol, has demonstrated tremendous applicability for regenerative and therapeutic medicine. For bio-electrospraying to be carried forward as a novel method of cardiac tissue engineering, it is important that the process does not adversely affect cellular physiology. Our previous work has shown that bio-electrospraying does not induce cell death, activate intracellular stress pathways or induce DNA damage in primary cardiac myocytes. Here we show for the first time using genome-wide microarray analysis, that bio-electrospraying has no negative effects on global gene expression in cardiac myocytes. Moreover, we show that bio-electrospraying does not lead to endothelial cell activation. These data suggest that BES has minimal effect upon the physiology of cardiac myocytes and endothelial cells and thus paves the way for the development of BES in cardiac tissue engineering.     

Engineered cardiac tissues

Cardiac tissue engineering offers the promise of creating functional tissue replacements for use in the failing heart or for in vitro drug screening. The last decade has seen a great deal of progress in this field with new advances in interdisciplinary areas such as developmental biology, genetic engineering, biomaterials, polymer science, bioreactor engineering, and stem cell biology. We review here a selection of the most recent advances in cardiac tissue engineering, including the classical cell-scaffold approaches, advanced bioreactor designs, cell sheet engineering, whole organ decellularization, stem cell-based approaches, and topographical control of tissue organization and function. We also discuss current challenges in the field, such as maturation of stem cell-derived cardiac patches and vascularization.     

Immune response to stem cells and strategies to induce tolerance

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.     

Cardiac tissue engineering using perfusion bioreactor systems

This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is 'biomimetic' in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2-4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research.