Rational Design of a Cardiac Tissue Engineering Scaffold

心脏组织工程的最终目的是利用细胞、生物材料支架、生长因子和生物反应器等手段使功能障碍的心肌再生。该领域的进展始终面临两个主要的成功障碍：（i）组织工程化心脏移植物（TECG）的组织和机械功能不足和（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先前支架的次优性质和通常用于创建TECG的经验方案。这项工作的假设是，一个三维（3D）支架与合理设计的结构和机械功能，可以提高TECG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心脏模拟结构和机械特性的支架和TECG，以及使用灌注生物反应器来改善CM存活和TECG收缩性。在目标1中，我们将从一个手风琴样的蜂窝状支架制成的聚（甘油癸二酸酯）（PGS），我们最近证明匹配的平面内的机械反应的天然心肌在生理制度和指导方向的培养CM。我们将使用预测模型来确定特定的支架孔布局与载有细胞的水凝胶组合是否产生仿生移植物。如果模型预测可行性，则将通过激光微消融制备具有开放孔布局的250 μm厚的PGS支架，并将其用作心脏细胞培养的支架。将评估所得TECG的CM方向、分化、收缩性和机械特性，这些数据将用于进一步建模以优化支架设计。具体而言，我们将通过系统研究平面内支架机械性能和CM分化，通过改变PGS固化条件，孔布局，PGS表面拓扑结构和载细胞水凝胶的特性来优化TECG收缩性。在目标2中，我们将通过在具有合理设计的全3D孔网络的PGS支架上灌注生物反应器培养心脏细胞来扩大到全3D TECG。这些支架将通过激光微消融和膜层压技术相结合来生产，通过将心脏细胞包埋在水凝胶中来接种，并在灌注生物反应器中培养。将通过系统研究生物反应器操作条件（包括流动方案和流体动力学剪切）来量化和优化TECG的发育和收缩性，所述条件将基于CM存活、分化、收缩性以及总体TECG结构、电气和机械特性来选择。广泛的，长期的项目目标是合理设计组织工程心脏移植物，可以提高心肌修复手术的成功率。预期所提出的TECG通过以下方式增强心肌再生：（i）提供仿生机械特性以帮助恢复心脏机械功能，以及（ii）改善细胞递送的规模和功效以促进移植物存活和整合。

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.