Biomaterials in myocardial tissue engineering

Cardiovascular disease is the leading cause of death in the developed world, and as such there is a pressing need for treatment options. Cardiac tissue engineering emerged from the need to develop alternative sources and methods of replacing tissue damaged by cardiovascular diseases, as the ultimate treatment option for many who suffer from end‐stage heart failure is a heart transplant. In this review we focus on biomaterial approaches to augmenting injured or impaired myocardium, with specific emphasis on: the design criteria for these biomaterials; the types of scaffolds – composed of natural or synthetic biomaterials or decellularized extracellular matrix – that have been used to develop cardiac patches and tissue models; methods to vascularize scaffolds and engineered tissue; and finally, injectable biomaterials (hydrogels) designed for endogenous repair, exogenous repair or as bulking agents to maintain ventricular geometry post‐infarct. The challenges facing the field and obstacles that must be overcome to develop truly clinically viable cardiac therapies are also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

心肌组织工程可降解支架材料的最新研究

健康细胞移植到心肌损伤部位是当前关注的热点.组织工程的三维支架材料可替代丢失和损伤的细胞外基质,对自我或移植细胞提供支持,避免或减少梗死区移植细胞被冲洗掉或死亡,并通过微环境线索获得组织的再生和修复,从而预防和改善心功能.就生物活性可降解材料的设计、修饰及目前常用的类型的研究现状做一综述,为可降解材料的应用研究提供理论依据.     

Myocardial tissue engineering: the quest for the ideal myocardial substitute

There has been an intense and competitive quest to manufacture bioartificial heart muscle in the last decade. Numerous biocompatible scaffolds and scaffold-free systems, enriched with various cell types, have been used to fabricate 3D grafts for myocardial repair. In spite of the impressive achievements in the myocardial tissue-engineering field, many issues remain to be addressed before clinical application of this strategy becomes feasible. This is largely due to the uniqueness of the heart’s structure and function. This review provides a survey upon the reported strategies, and indicates caveats and perspectives in the field of myocardial tissue engineering.     

心脏瓣膜组织工程中理想种植细胞的来源比较

背景:现有的人工瓣膜,无论是机械瓣膜还是物瓣膜,都存在与取材有关、无法从工艺制作方式改进解决的固有缺陷.组织工程心脏瓣膜概念的提出为解决该难题提供了新的方向.目的:通过比较成熟分化血管内皮细胞和骨髓间充质干细胞骨的获取手段、体外增殖能力以及在脱细胞基质上的生长情况,分析何者更适合作为制备组织工程人工心脏瓣膜的种植细胞.设计、时间及地点:单一样本观察,于2008 06/09在上海市胸科医院动物实验基地完成.材事斗:用胰蛋白酶(胰酶)EDTA、Triton X-100、DNase、RNase消化猪主动脉瓣膜获得脱细胞基质.从1只12 kg 2岁雄性杂交犬大隐静脉、股外侧动脉及肋骨骨髓获取种植细胞.方法:用胰酶消化并收集内皮细胞,收集骨髓细胞,分别进行培养.通过贴壁法获得内皮细胞及骨髓间充质干细胞.利用内皮细胞生长因子诱导骨髓间尤质干细胞向内皮细胞分化,并绘制细胞生长曲线.以抗Ⅷ因子抗体以及抗血管内皮生长因子抗体荧光染色,荧光显微镜观察.将细胞种植于脱细胞瓣膜上培养3 d后进行扫捕电镜观察.主要观察指标:①试验犬伤口愈合情况.②细胞培养、增殖情况.③骨髓来源骨髓间充质干细胞表型分析以及形态学观察.结果:成熟血管内皮细胞体外增殖能力低下,骨髓间充质干细胞则增殖旺盛,生长曲线显示骨髓间充质干细胞的倍增时间约30 h,而成熟内皮细胞生长曲线平直.对骨髓间充质干细胞进行抗Ⅷ因子抗体以及抗VEGF抗体荧光染色.可见荧光染色阳性.扫描电镜见种植骨髓间充质干细胞的脱细胞瓣膜上散在星状细胞贴附.结论:骨髓间充质下细胞体外增殖能力强,在内皮细胞生长因子诱导下可向内皮细胞转化,并可以生长贴附于脱细胞瓣膜,是作为组织上程人工心脏瓣膜制备的理想细胞来源.     

Cells, scaffolds, and molecules for myocardial tissue engineering

Unlike heart valves or blood vessels, heart muscle has no replacement alternatives. The most challenging goal in the field of cardiovascular tissue engineering is the creation/ regeneration of an engineered heart muscle. Recent advances in methods of stem cell isolation, culture in bioreactors, and the synthesis of bioactive materials promise to create engineered cardiac tissue ex vivo. At the same time, new approaches are conceived that explore ways to induce tissue regeneration after injury. The purpose of our review is to describe the principles, status, and challenges of myocardial tissue engineering with emphasize on the concept of in situ cardiac tissue engineering and regeneration.     

Cell transplantation for heart failure,and tissue engineering of cardiovascular structures

The emergence of selective cell transplantation and tissue engineering has opened up new alternatives for the treatment of heart failure. These involve the use of stem and progenitor cells, as well as new biodegradable polymer scaffolds. The last two decades have seen a dramatic increase in our knowledge of how to fabricate a wide array of tissues, including cardiovascular structures. This article reviews current trends in selective cell transplantation and tissue engineering, and summarises recent achievements and remaining questions in constructing functional cardiac tissue.     

A new approach to heart valve tissue engineering: mimicking the heart ventricle with a ventricular assist device in a novel bioreactor

The ‘biomimetic’ approach to tissue engineering usually involves the use of a bioreactor mimicking physiological parameters whilst supplying nutrients to the developing tissue. Here we present a new heart valve bioreactor, having as its centrepiece a ventricular assist device (VAD), which exposes the cell–scaffold constructs to a wider array of mechanical forces. The pump of the VAD has two chambers: a blood and a pneumatic chamber, separated by an elastic membrane. Pulsatile air‐pressure is generated by a piston‐type actuator and delivered to the pneumatic chamber, ejecting the fluid in the blood chamber. Subsequently, applied vacuum to the pneumatic chamber causes the blood chamber to fill. A mechanical heart valve was placed in the VAD's inflow position. The tissue engineered (TE) valve was placed in the outflow position. The VAD was coupled in series with a Windkessel compliance chamber, variable throttle and reservoir, connected by silicone tubings. The reservoir sat on an elevated platform, allowing adjustment of ventricular preload between 0 and 11 mmHg. To allow for sterile gaseous exchange between the circuit interior and exterior, a 0.2 µm filter was placed at the reservoir. Pressure and flow were registered downstream of the TE valve. The circuit was filled with culture medium and fitted in a standard 5% CO₂ incubator set at 37 °C. Pressure and flow waveforms were similar to those obtained under physiological conditions for the pulmonary circulation. The ‘cardiomimetic’ approach presented here represents a new perspective to conventional biomimetic approaches in TE, with potential advantages. Copyright © 2010 John Wiley & Sons, Ltd.     

多普勒组织显像动态检测猪慢性心肌缺血心功能

目的：进一步了解心肌冬眠的发生机制和组织多普勒各指标识别心肌早期缺血的敏感性。方法：10头正常家猪，开胸通过Ameroid环套扎猪冠状动脉左回旋支（LCX），建立慢性心肌缺血模型，于术前及术后2周、5周进行超声检查，并与病理结果对照。结果：10头猪中5头猪成功建立冬眠心肌模型，病理结果显示心肌细胞变性为主，除心内膜下小灶性梗死外，无明显梗死灶。超声检查发现术后2周、5周心脏各腔室大小正常，回声无改变，有2头猪局部收缩明显异常，部位及范围与伊文思蓝显示的LCX供血区范围一致。放置Ameroid环2周后，局部心功能指标左心室后壁（心内膜、心外膜）的心肌运动速度（Vendo、Vepi)、收缩期及舒张期运动速度峰值跨壁梯度（MVGm 、MVGm^-)、乳头肌短轴观左心室后壁均厚率均明显下降，术后5周Vendo、Vepi和MVGm 较术前下降，但术后5周与术后2周比较差别无显著性意义。整体心功能指标左心室短轴缩短率（FS）、射血分数（EF）无明显变化。时间运动曲线的最大值无论是阻塞区还是对照区，放置Ameroid环2周和5周后均无明显变化。结论：通过对局部心功能指标的检测可以敏感地反映心肌血流灌注状态、侧支循环的形成情况。心电图T波时心内膜运动速度、心肌运动跨壁梯度可反映心肌内膜下缺血。     

层层静电自组装多聚电解质共混膜与组织工程心脏瓣膜细胞的黏附

背景:组织工程心脏瓣膜研究中的核心和难点是种子细胞在支架材料上的黏附.层层静电自组装技术能够将多聚阴阳离子组装成多层膜状结构增加材料表面的生物相容性.目的:拟对猪去细胞瓣膜在体外进行层层静电自组装成多聚电解质共混膜,并且检测共混膜列种子细胞黏附的效果.设计时间及地点:细胞学体外观察性实验,于2008-09/2009 02在解放军第二军医大学长海医院胸心外科实验室和复旦大学高分子科学系实验室完成.材料:新鲜猪心脏瓣膜取自上海五丰上食肉联厂,采用酶消化法将猪主动脉瓣叶行脱细胞处理.密度梯度离心法从骨髓中获取骨髓单个核细胞,体外扩增培养后鉴定.方法:通过层层静电自组装技术构建20,10,5,0层的壳聚糖,透明质酸共混膜履盖在猪去细胞瓣膜支架上.将骨髓基质干细胞在涂膜支架上种植培养并检测支架对种子细胞的黏附效果.主要观察指标:扫描电镜观察、细胞计数、MTT检测组织工程心脏瓣膜上种子细胞的形态和细胞黏附效果.结果:扫描电镜观察20层壳聚糖,透明质酸多层膜样品上细胞生长情况比较好,样品上可以铺满细胞.而在组装层数为5层和10层的样品上,细胞生长的比较少.构建层数为20层的瓣叶,其细胞计数及吸光值高于10层和5层构建的瓣叶(P<0.01).构建层数为10层和5层的瓣叶与0层比较差异无显著性意义(P>0.05).结论:通过层层静电自组装技术构建的共混膜能够促进种子细胞的黏附,而且有一定的量效关系.     

The future of heart valve replacement: recent developments and translational challenges for heart valve tissue engineering

Heart valve replacement is often the only solution for patients suffering from valvular heart disease. However, currently available valve replacements require either life‐long anticoagulation or are associated with valve degeneration and calcification. Moreover, they are suboptimal for young patients, because they do not adapt to the somatic growth. Tissue‐engineering has been proposed as a promising approach to fulfil the urgent need for heart valve replacements with regenerative and growth capacity. This review will start with an overview on the currently available valve substitutes and the techniques for heart valve replacement. The main focus will be on the evolution of and different approaches for heart valve tissue engineering, namely the in vitro, in vivo and in situ approaches. More specifically, several heart valve tissue‐engineering studies will be discussed with regard to their shortcomings or successes and their possible suitability for novel minimally invasive implantation techniques. As in situ heart valve tissue engineering based on cell‐free functionalized starter materials is considered to be a promising approach for clinical translation, this review will also analyse the techniques used to tune the inflammatory response and cell recruitment upon implantation in order to stir a favourable outcome: controlling the blood–material interface, regulating the cytokine release, and influencing cell adhesion and differentiation. In the last section, the authors provide their opinion about the future developments and the challenges towards clinical translation and adaptation of heart valve tissue engineering for valve replacement. Copyright © 2016 John Wiley & Sons, Ltd.     

四种组织工程瓣膜种子细胞功能的检测与分析

目的通过分析组织工程心脏瓣膜种子细胞的细胞功能指标,为种子细胞的临床筛选提供实验室依据。方法在体外分离、培养、扩增及传代hECs(人大隐静脉内皮细胞)、hMFbs(人大隐静脉壁肌成纤维细胞)、hMSCs(人骨髓间质干细胞)、hMSC-ECs(hMSCs体外向内皮细胞诱导分化细胞)四种种子细胞,按照试剂盒说明书分别测定其PGI2(前列环素2)、NO(一氧化氮)、ET-1(内皮素-1)及t-PA(组织纤溶酶原激活剂)等四个细胞功能指标的含量。结果hECs组的PGI2、ET-1指标明显高于其余三组(P<0.01);hECs组及hMSC-ECs组间的NO指标无差异(P>0.05),但均高于hMFbs组和hMSCs组(P<0.05);hECs组、hMSCs组及hMSC-ECs组的t-PA指标高于hMFbs组(P<0.05)。结论四种种子细胞各有其特点,但综合考虑,hMSC-ECs作为组织工程心脏瓣膜种子细胞更具优越性。     

四种组织工程瓣膜种子细胞功能的检测与分析

目的 通过分析组织工程心脏瓣膜种子细胞的细胞功能指标,为种子细胞的临床筛选提供实验室依据.方法 在体外分离、培养、扩增及传代hECs(人大隐静脉内皮细胞)、hMFhs(人大隐静脉壁肌成纤维细胞)、hMSCs(人骨髓间质干细胞)、hMSC-ECs(hMSCs体外向内皮细胞诱导分化细胞)四种种子细胞,按照试剂盒说明书分别测定其PGI2(前列环素2)、NO(一氧化氮)、ET-1(内皮素-1)及t-PA(组织纤溶酶原激活剂)等四个细胞功能指标的含量.结果 hECs组的PGI2、ET-1指标明显高于其余三组(P＜0.01);hECs组及hMSC-ECs组间的NO指标无差异(P＞0.05),但均高于hMFbs组和hMSCs组(P＜0.05);hECs组、hMSCs组及hMSC-ECs组的t-PA指标高于hMFbs组(P＜0.05).结论 四种种子细胞各有其特点,但综合考虑,hMSC-ECs作为组织工程心脏瓣膜种子细胞更具优越性.     

MRI of myocardial infarction with tissue tagging

Myocardial tagging with MRI has been available for three decades as a method for direct noninvasive quantification of regional myocardial motion for assessing the impact of ischemia, electrical asynchrony, and heart failure, among other conditions. In recent years, new developments in imaging sequences, hybrid techniques, and automated postprocessing have brought tagging closer to clinical application. Improvements in acquisition strategies have increased tag-tissue contrast and persistence, making automated tag detection easier and more robust. Imaging techniques such as harmonic phase MRI and displacement encoding with stimulated echoes that quantify pixel-by-pixel myocardial motion have simplified postprocessing. Parallel imaging and the increased signal-to-noise ratio available at the higher field strengths of new clinical scanners have moved the assessment of cardiac function into the real-time imaging domain. All of these developments have made myocardial motion mapping with tagged MRI a gold standard for the quantification of regional function within the research world, with potential to become an extremely valuable clinical technique.     

组织工程研究新进展

自 198 7年提出“组织工程学”概念以来 ,由于其重要的科学意义、潜在的经济效益 ,组织工程受到世界各国科学家、企业家及政府的高度重视。在政府基金、企业资金支持下 ,组织工程学基础研究已取得显著的进展 ,在某些领域已进入临床前研究或产业化阶段 ,组织工程皮肤已被美国食品及药物管理局 (ＦＤＡ)批准上市。预计在不久的将来还会有新的产品应用于临床治疗。1种子细胞的研究进展为支架材料提供生命源泉、并能形成组织的功能细胞称为种子细胞。依据其来源可分为自体、同种异体和异种细胞。自体细胞可由活检或穿刺所得到的组织进行分离培养…     

心脏磁共振组织追踪技术评估冠心病心肌梗死的诊断价值

目的探讨心脏磁共振组织追踪技术(cardiovascular magnetic resonance tissue tracking technology,CMR-TT)与冠心病患者左心功能相关性及定量化评价心肌梗死的诊断价值。材料与方法对23例冠心病患者和16名健康志愿者进行3.0 T SSFP电影序列以及对比延迟增强扫描,用CVI42软件测量心功能,并使用组织追踪技术对左心室整体和节段的应变进行分析,得到心肌径向、周向、纵向三个方向CMR-TT 3D应变参数值。对上述参数值进行组间均值比较、组内相关系数(ICC)分析、Pearson相关性分析、Logistic回归模型、受试者工作特性(receiver operating characteristic,ROC)曲线分析。结果左心室整体径向应变(radial strain,RS)(ICC=0.944)、周向应变(circumferential strain,CS)(ICC=0.988)、纵向应变(longitudinal strain,LS)(ICC=0.987)均表现较好的可重复性。冠心病组左心室整体的径向应变、周向应变、纵向应变低于健康人组(30.35%±17.26%和45.46%±8.90%、-13.92%±5.7 7%和-1 9.3 4%±2.3 0%、-11.3 0%±4.7 5%和-1 6.5 4%±2.4 0%,P值均0.01)。左心室射血分数与径向应变呈强相关(r=0.774,P0.001),与周向应变呈强相关(r=0.778,P0.001),与纵向应变呈极强相关(r=0.802,P0.001)。冠心病组延迟强化(late gadolinium enhancement,LGE)阳性心肌节段径向应变、周向应变、纵向应变峰值低于LGE阴性心肌节段(9.95%和41.42%,-7.67%和-17.2%,-6.68%和-13.83%,P值均0.01)。心肌径向应变(AUC=0.914)、周向应变(AUC=0.911)在诊断心肌节段梗死中具有较高的诊断价值,纵向应变(AUC=0.819)具有一定的诊断价值;当心肌径向应变截断值为16.83%时,诊断准确度较高(Youden指数=0.7399);当周向应变截断值为-11.44%时,诊断准确度较高(Youden指数=0.7511);纵向应变在截断值为-9.41%时,诊断准确度较低(Youden指数=0.5552)。当使用径向应变和周向应变联合诊断冠心病心肌节段梗死时,AUC和特异度较各指标独立诊断时提高,在联合诊断时纵向应变无预测意义。结论心脏磁共振组织追踪技术在临床应用中具有较好的可行性及可重复性;径向应变、周向应变、纵向应变与冠心病左心室射血分数具有较强的相关性;径向应变、周向应变在诊断冠心病心肌节段梗死的诊断价值较高,纵向应变诊断价值有限。当使用径向应变和周向应变两项指标联合诊断时可提高诊断效能。这提示CMR-TT的应变分析在无需对比剂的情况下识别冠心病心肌梗死节段具有潜在的临床诊断价值。     

Airway tissue engineering

Introduction: Prosthetic replacements, autologous tissue transfer and allografts have so far failed to offer functional solutions for the treatment of long circumferential tracheal defects and loss of a functioning larynx. Interest has therefore turned increasingly to the field of tissue-engineering which applies the principles and methods of bioengineering, material science, cell transplantation and life sciences in an effort to develop in vitro biological substitutes able to restore, maintain or improve tissue and organ function.

Areas covered: This article gives an overview of the tissue-engineering approach to airway replacement and will describe the encouraging results obtained so far in tracheal regeneration. The recent advances in the field of tissue-engineering have provided a new attractive approach towards the concept of functional substitutes and may represent an alternative to the shortage of suitable grafts for reconstructive airway surgery. We summarize fundamental questions, as well as future directions in airway tissue engineering.

Expert opinion: The replacement of active movement, as would be necessary to replace an entire larynx introduces another order of magnitude of complexity, although progress in this area is starting to bear fruit. In addition, the stem cell field is advancing rapidly, opening new avenues for this type of therapy.     

Airway tissue engineering

INTRODUCTION: Prosthetic replacements, autologous tissue transfer and allografts have so far failed to offer functional solutions for the treatment of long circumferential tracheal defects and loss of a functioning larynx. Interest has therefore turned increasingly to the field of tissue-engineering which applies the principles and methods of bioengineering, material science, cell transplantation and life sciences in an effort to develop in vitro biological substitutes able to restore, maintain or improve tissue and organ function. AREAS COVERED: This article gives an overview of the tissue-engineering approach to airway replacement and will describe the encouraging results obtained so far in tracheal regeneration. The recent advances in the field of tissue-engineering have provided a new attractive approach towards the concept of functional substitutes and may represent an alternative to the shortage of suitable grafts for reconstructive airway surgery. We summarize fundamental questions, as well as future directions in airway tissue engineering. EXPERT OPINION: The replacement of active movement, as would be necessary to replace an entire larynx introduces another order of magnitude of complexity, although progress in this area is starting to bear fruit. In addition, the stem cell field is advancing rapidly, opening new avenues for this type of therapy.     

三种可降解聚合物构建组织工程心脏瓣膜的可行性对照

目的:评价作为组织工程心脏瓣膜支架的聚乙醇酸/聚乳酸共聚物,聚羟基丁酸/聚羟基戊酸共聚物,聚羟基丁酯3种生物可吸收材料的组织相容性和皮下吸收情况。探讨应用生物可降解材料构建组织工程心脏瓣膜的可行性。方法:实验于2004-12/2005-10在解放军第二军医大学长海医院全军胸心外科研究所完成。①应用扫描电镜观察聚乙醇酸/聚乳酸共聚物,聚羟基丁酸/聚羟基戊酸共聚物,聚羟基丁酯3种材料的结构特点。②新西兰大白兔18只,在兔背胸腰段脊柱两侧各作3个切口,每只兔分别植入6块可吸收材料,每种材料6只。③分别于术后2,4,6,8,10和12周各取6块材料,进行大体和组织学观察,并测量纤维包裹组织的厚度。结果:15只兔全部进入结果分析。①3种材料的结构特点:扫描电镜下3种材料均呈网孔状泡沫结构,聚羟基丁酯孔径约110μm,聚羟基丁酸/聚羟基戊酸共聚物孔径约130μm,聚乙醇酸/聚乳酸共聚物孔径约170μm。②组织学检查结果:聚羟基丁酸/聚羟基戊酸共聚物和聚乙醇酸/聚乳酸共聚物2周包膜纤维疏松,与其下肌肉组织分界不清,网孔中有疏松纤维组织长入,炎细胞以中性粒细胞和淋巴细胞为主,可见多核巨细胞。10周聚羟基丁酸/聚羟基戊酸共聚物材料完全吸收,12周聚乙醇酸/聚乳酸共聚物材料完全吸收,均为胶原纤维所代替。聚羟基丁酯2周包膜结缔组织疏松,伴中性粒细胞、淋巴细胞、浆细胞浸润和成纤维细胞弥漫性增生,也可见多核巨细胞。8周时包膜增厚,以淋巴细胞浸润为主。10周材料包膜较8周薄,炎性细胞明显减少,成纤维细胞转变为梭形的纤维细胞。③包膜厚度测量结果:包埋后2,4,6,8,10周聚羟基丁酸/聚羟基戊酸共聚物和聚乙醇酸/聚乳酸共聚物材料厚度测量值均显著小于聚羟基丁酯。结论:聚乙醇酸/聚乳酸共聚物、聚羟基丁酸/聚羟基戊酸共聚物在组织相容性和降解率方面优于聚羟基丁酯,更适合于作为组织工程心脏瓣膜的支架材料。     

胶原膜、聚乙醇酸/聚乳酸共聚物在组织工程心脏瓣膜支架材料中的应用

目的将活细胞种植于可生物降解的瓣膜支架上,制造出一种组织相容性好,不需抗凝,具有修复能力,且支持患者终生的理想心脏瓣膜。方法胶原膜和聚乙醇酸/聚乳酸共聚物(PGLA)在皮下包埋和种植细胞生长做对照实验。结果胶原膜皮下包埋8周仍保持膜状结构,8～12周完全降解,且生物相容性优于聚乙醇酸/聚乳酸共聚物(PGLA)。结论胶原膜适于组织工程心脏瓣膜支架材料的应用。