Rational Design of a Cardiac Tissue Engineering Scaffold

心脏组织工程的最终目标是通过使用细胞、生物材料支架、生长因子和生物反应器来再生功能失调的心肌。这一领域的进展一直面临着两个主要的成功障碍：(i)组织工程心脏移植物（TECG）的组织和机械功能不足；（ii）心脏发生不足（即心肌细胞（CM）的存活、排列和分化不足）。这些缺点部分是由于以前的支架和通常用于制造TECG的经验方案的性能不理想。本研究的假设是合理设计结构和力学特征的三维支架可以增强TECG的功能组装。该项目利用了最近的研究成果，展示了使用建模和实验研究来设计具有模拟心脏结构和机械性能的支架和TECG，并使用灌注生物反应器来提高CM的存活率和TECG的收缩性。在Aim 1中，我们将从一个由聚甘油脂酸酯（PGS）制成的手风琴状蜂窝支架开始，我们最近证明了该支架在生理状态下与天然心肌的平面内机械反应相匹配，并指导培养CM的定向。我们将使用预测模型来确定是否一个特定的支架孔布局与细胞负载的水凝胶相结合产生仿生移植物。如果模型预测可行，那么将采用激光微烧蚀法制备250 μm厚的具有开放孔布局的PGS支架，并将其用作心脏细胞培养支架。所得的TECG将评估CM的方向、分化、收缩性和机械性能，这些数据将用于进一步建模以优化支架设计。具体来说，我们将通过系统研究平面内支架的力学性能和不同PGS固化条件、孔布局、PGS表面拓扑结构和载细胞水凝胶特征的CM分化来优化TECG的收缩性。在目标2中，我们将通过灌注生物反应器培养心脏细胞在具有合理设计的全3D孔网络的PGS支架上，扩大到全3D TECG。这些支架将通过结合激光微消融和膜层压技术，将心脏细胞包埋在水凝胶中，并在灌注生物反应器中培养。TECG的发展和收缩性将通过生物反应器操作条件的系统研究来量化和优化，包括流动方案和流体动力剪切，这些将根据CM的存活、分化、收缩性以及TECG的整体结构、电气和机械性能来选择。广泛而长期的项目目标是合理设计组织工程心脏移植物，以提高心肌修复手术的成功率。拟议的TECG有望通过以下方式促进心肌再生：(1)提供仿生机械特性，帮助恢复心脏机械功能；(2)提高细胞递送的规模和效率，促进移植物的存活和整合。

The ultimate goal of cardiac tissue engineering is the regeneration of dysfunctional myocardium by using cells, biomaterial scaffolds, growth factors, and bioreactors. Progress in this field consistently faces two major barriers to success: (i) insufficient organization and mechanical function of tissue engineered cardiac grafts (TECG) and (ii) inadequate cardiogenesis (i.e. inadequate survival, alignment, and differentiation of cardiomyocytes (CM)). These shortcomings are due in part to sub-optimal properties of previous scaffolds and the empirical schemes typically used to create TECG. The hypothesis of this work is that a three dimensional (3D) scaffold with rationally designed structural and mechanical features can enhance the functional assembly of TECG. The project leverages recent work demonstrating the use of modeling and experimental studies to design scaffolds and TECG with cardiac-mimetic structural and mechanical properties, and the use of perfusion bioreactors to improve CM survival and TECG contractility. In Aim 1 we will start with an accordion-like honeycomb scaffold made of poly(glycerol sebacate) (PGS) that we recently demonstrated matches in-plane mechanical responses of native myocardium in the physiologic regime and guides orientation of cultured CM. We will use predictive modeling to determine if a particular scaffold pore layout in combination with a cell-laden hydrogel yields a biomimetic graft. If modeling predicts feasibility, then 250 μm thick PGS scaffolds with open pore layouts will be made by laser microablation and used as scaffolds for heart cell culture. Resulting TECG will be assessed for CM orientation, differentiation, contractility, and mechanical properties, and these data will be used with further modeling to optimize scaffold design. Specifically, we will optimize TECG contractility by systematic studies of in-plane scaffold mechanical properties and CM differentiation by varying PGS curing conditions, pore layout, PGS surface topology, and characteristics of the cell-laden hydrogel. In Aim 2, we will scale-up to a fully 3D TECG by perfusion bioreactor culture of heart cells on a PGS scaffolds with rationally designed, fully 3D pore networks. These scaffolds will be produced by combining laser microablation and membrane lamination technologies, seeded by entrapping heart cells in hydrogel, and cultured in a perfusion bioreactor. The development and contractility of TECG will be quantified and optimized by systematic studies of bioreactor operating conditions, including flow regimen and hydrodynamic shear, that will be selected based on CM survival, differentiation, contractility, and the overall TECG structural, electrical and mechanical properties. The broad, long-term project objective is the rational design of tissue engineered cardiac grafts that can improve the success of myocardial repair procedures. The proposed TECG are expected to enhance myocardial regeneration by (i) providing biomimetic mechanical properties to help restore cardiac mechanical function and (ii) improving the scale and efficacy of cell delivery to promote graft survival and integration.