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

项目摘要 心血管疾病是导致每年超过1700万人死亡的主要原因 世界范围内的死亡心肌梗死引起的进行性心力衰竭，或由于 冠状血管系统中的阻塞是这种负担的主要原因。在心脏病发作期间， 左心室的收缩细胞可能会丢失，导致重塑反应，使心脏壁变薄 留下疤痕，大大降低心脏功能。除了全心脏移植， 选择只能减缓疾病的发展，提供姑息治疗。因此，有一个伟大的临床 需要通过恢复肌肉和心脏功能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脉管系统。植入式细胞化心脏 由于复杂的组织可以被 通过调整结构和允许组织成熟而产生。虽然组织工程学的进展和 多能干细胞技术已经产生了显示出改善心脏功能的前景的心脏组织， 由于营养供应不足，目前的可植入细胞化心脏补片仍然不够薄。 以前的心脏组织血管化方法严重依赖于内皮细胞的自组装 网络，使它们最低限度地灌注，或利用不充分平衡刚度的支架材料， 血管完整性所需的重塑能力所需的支持心肌细胞耦合和快速 体内有效灌注所需的宿主血管浸润。为了满足这一需求，我们建议设计 通过将图案化的、可灌注的脉管系统结合到一个厚的、密集的细胞化的心脏补片中， 用于植入梗死心脏的胶原支架。我们的团队以前开发了一种技术 结合注射成型和软光刻技术，生成嵌入其中的可灌注血管 胶原基质，并证明内皮细胞将很容易重塑基质，而可灌注的 血管系统保持完整在这个建议中，我们将利用这种技术来产生心脏组织， 干细胞衍生的心肌细胞和干细胞衍生的内皮细胞的可灌注网络。我们 假设将图案化的、可灌注的内皮网络并入心脏组织将 增强体外心肌细胞存活和功能以及促进快速宿主血管整合 和移植后的组织存活率。为了验证这一假设，我们将利用多层堆叠技术 以生成具有三维血管网络的3毫米厚的心脏组织， 灌注。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， 可灌注的血管网然后，我们将在梗塞的大鼠心脏上植入厚的心脏组织， 预灌注的内皮网络是否改善厚组织的长期存活。如果成功，这个平台 将使较厚的心脏组织能够比以前实现的长期存活。

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.