Rational Design of a Cardiac Tissue Engineering Scaffold

心脏组织工程的最终目标是利用细胞、生物材料支架、生长因子和生物反应器来再生功能不全的心肌。这一领域的进展一直面临着两大成功障碍：(1)组织工程心脏移植物的组织和机械功能不足；(2)心脏生成不足(即心肌细胞(CM)的存活、排列和分化不足)。这些缺陷部分归因于以前支架的次优性能和通常用于创建TCG的经验方案。这项工作的假设是，具有合理设计的结构和力学特征的三维支架可以增强经颅心电的功能组装。该项目利用最近的工作，展示了使用建模和实验研究来设计具有仿心结构和机械性能的支架和经心电信号，并使用灌流生物反应器来提高CM存活率和经心电信号的收缩能力。在目标1中，我们将从一种手风琴状的蜂窝支架开始，这种支架由我们最近证实的聚癸二酸甘油(PGS)制成，在生理状态下与天然心肌的平面机械反应相匹配，并指导培养的CM的取向。我们将使用预测模型来确定特定的支架孔道布局与细胞水凝胶相结合是否产生仿生移植物。如果模型预测的可行性，那么250μm厚的PGS支架将通过激光微消融制成具有开放孔布局的支架，并用作心脏细胞培养的支架。生成的TECG将评估CM的取向、分化、收缩和机械性能，这些数据将用于进一步的建模以优化支架设计。具体地说，我们将通过对平面内支架力学性能和CM分化的系统研究，通过改变PGS固化条件、孔布局、PGS表面拓扑和细胞水凝胶的特性来优化TCG的收缩性能。在目标2中，我们将通过灌流生物反应器将心脏细胞培养在具有合理设计的全3D孔道网络的PGS支架上，从而放大到全3D TCG。这些支架将通过激光微消融和膜层压技术相结合的方式产生，将心肌细胞包裹在水凝胶中种植，并在灌流生物反应器中培养。通过对生物反应器操作条件的系统研究，包括流动方案和流体动力剪切，将根据CM的存活率、分化、收缩能力以及整体TCG的结构、电气和机械性能来选择TCG，从而量化和优化TCG的开发和收缩性能。广泛的、长期的项目目标是合理设计组织工程心脏移植物，以提高心肌修复程序的成功率。通过(I)提供仿生机械特性以帮助恢复心脏机械功能和(Ii)改善细胞输送的规模和效率以促进移植物的存活和整合，拟议中的经皮冠状动脉心动过速有望促进心肌再生。

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.