Engineering thick human cardiac tissue constructs with patterned, perfusable microvessels for treatment of myocardial infarction

项目总结 每年导致1700多万人死亡的主要原因是心血管疾病 世界范围内的死亡。由心肌梗死引起的进行性心力衰竭，或因以下原因导致的心脏组织死亡 冠状动脉血管系统的堵塞是造成这一负担的主要原因。在心脏病发作期间，多达25%的 左心室的收缩细胞可能丢失，导致重塑反应，使心壁变薄 伤痕累累，极大地降低了心脏功能。除了全心脏移植外，现有的治疗方法 选择只能减缓疾病的进展并提供姑息治疗。因此，有一个很好的临床 需要通过恢复肌肉来预防或治疗心肌梗死的破坏性后果的疗法 组织和支持肌肉组织所需的复杂的血管系统。植入型细胞化心脏 贴片已经成为治疗心肌梗死的一种潜在方法，因为复杂的组织可以 通过调整架构和让组织成熟来创造。尽管组织工程和组织工程的进展 多能干细胞技术已经导致心脏组织显示出改善心脏功能的希望， 由于营养供应不足，目前可植入的细胞化心脏补片仍然不够薄。 以往使心脏组织血管化的方法严重依赖于内皮细胞的自组装 网络，使其最少可灌装，或者使用不适当平衡僵硬的支架材料 所需的血管完整性和重塑能力所需的支持心肌细胞偶联和快速 有效的体内灌流需要宿主血管的渗透。为了满足这一需求，我们建议设计 通过将图案化的、可灌流的血管系统结合到 用于植入梗塞心脏的胶原蛋白支架。我们的团队之前已经开发出一种技术 结合注射成型和软光刻以产生嵌入其中的可灌流的血管 胶原基质和证明，内皮细胞将容易地重塑基质，而可灌流 血管系统保持完好。在这项提议中，我们将利用这项技术来产生包含以下内容的心脏组织 干细胞来源的心肌细胞和干细胞来源的内皮细胞的可灌流网络。我们 假设将模式化的、可灌流的内皮网络整合到心脏组织中将 增强心肌细胞在体外的存活和功能，促进宿主血管快速整合 以及植入后的组织存活率。为了验证这一假设，我们将使用多层堆叠技术 用三维血管网络生成3毫米厚的心脏组织，以使更大的组织 灌流。我们将评估这些组织和类似组织中的组织存活、成熟和功能 可灌流的血管网络。然后，我们将把厚厚的心脏组织植入梗塞的大鼠心脏，并研究 预灌流的内皮网络是否能改善厚组织的长期存活率。如果成功，该平台 将使比以前更厚的心脏组织能够长期存活。

PROJECT SUMMARY Causing the death of more than 17 million people every year, cardiovascular disease is the leading cause of death worldwide. Progressive heart failure caused by myocardial infarction, or death of cardiac tissue due to a blockage in the coronary vasculature, is a major contributor to this burden. During a heart attack, up to 25% of the left ventricle’s contractile cells may be lost, leading to a remodeling response that leaves the heart wall thin and scarred and greatly reduces cardiac function. Besides whole heart transplantation, available treatment options can only slow the progression of disease and provide palliative care. As such, there is a great clinical need for therapies that prevent or treat the ruinous aftermath of myocardial infarction by restoring both the muscle tissue and the complex vasculature that is needed to support the muscle tissue. Implantable cellularized cardiac patches have emerged as a potential approach to treat myocardial infarction because complex tissues can be created by tuning architecture and allowing the tissues to mature. Although advances in tissue engineering and pluripotent stem cell technology have resulted in cardiac tissues that show promise for improving heart function, current implantable cellularized cardiac patches remain insufficiently thin due to inadequate nutrient supply. Previous approaches of vascularizing cardiac tissues have heavily relied on self-assembly of endothelial networks, leaving them minimally perfusable, or utilize scaffold materials that inadequately balance the stiffness required for vascular integrity with the remodeling capacity needed support cardiomyocyte coupling and rapid host vascular infiltration needed for efficient perfusion in vivo. To address this need, we propose to engineer thick, densely cellularized cardiac patches by incorporating a patterned, perfusable vasculature into a collagen scaffold for implantation onto infarcted hearts. Our group has previously developed a technique incorporating injection molding and soft lithography to generate perfusable vasculatures embedded within collagen matrices and demonstrated that endothelial cells will readily remodel the matrix while the perfusable vasculature remains intact. In this proposal, we will utilize this technique to generate cardiac tissues containing stem cell-derived cardiomyocytes and perfusable networks of stem cell-derived endothelial cells. We hypothesize that incorporating patterned, perfusable endothelial networks into cardiac tissues will enhance cardiomyocyte survival and function in vitro as well as promote rapid host vascular integration and tissue survival after implantation. To test this hypothesis, we will utilize a multilayer stacking technique to generate 3-mm thick cardiac tissues with a three-dimensional vascular network to enable greater tissue perfusion. We will assess tissue survival, maturation, and function in these tissues and similar tissues without perfusable vascular networks. We will then implant thick cardiac tissues onto infarcted rat hearts and investigate whether pre-perfused endothelial networks improve long-term survival of thick tissues. If successful, this platform will enable long-term survival of thicker cardiac tissues than has previously been achieved.