Towards repairing congenital heart defects: The effects of fetal cardiac extracel

描述（由申请人提供）：先天性心脏缺陷（CHD）是活产婴儿死亡的主要原因（1，2）。左心发育不良综合征（HLHS）是一种罕见的CHD，需要几次重大的重建手术（3）。然而，重建的心脏不能复制正常的解剖结构和功能，许多患者在一生中遭受严重的继发性并发症（4）。心脏组织工程是特别有前途的治疗HLHS通过创造活的功能性心脏组织，可以与孩子一起成长。然而，在体外创建功能性心脏组织仍然是一个主要挑战，因为成熟的心肌细胞（CM）大多是非增殖性的（5）。虽然胚胎和胎儿CM具有高度增殖性，并且可以恢复受损或患病心脏的功能（6，7），但出生后CM增殖减少和心脏再生能力丧失的原因仍在很大程度上未知。阐明促进CM增殖的因素将极大地影响治疗儿童CHD的组织工程和再生方法。细胞外基质（ECM）调节多种细胞功能，包括增殖（8），并且有证据表明ECM组成和心脏硬度在发育和成熟期间发生变化（9-12）。该项目的假设是，胎儿ECM环境的重演将促进CM的增殖;具体而言，预计促进CM增殖的胎儿心脏ECM的特征是在成人心脏中丢失或减少的线索。该假设将在体外进行系统性测试，以确定ECM组成和刚度在2D和3D培养中的单独和组合效应，并在体内进行系统性测试，作为未来临床转化的初始步骤。对于ECM组成研究，将天然胎儿和成年大鼠心脏脱细胞以获得ECM，然后溶解并吸附到组织培养皿上。将原代新生大鼠CM接种到心脏ECM包被基质上，并测定增殖。同时，将确定基板刚度的影响。脱细胞和天然心脏将进行机械测试以确定ECM的硬度。聚丙烯酰胺（PAAm）凝胶将制成模拟胎儿和成人心脏的硬度。在这些研究中，将在胎儿与成人硬度凝胶中以相同的配体密度使用胶原蛋白I以使组合物与硬度分离。为了研究ECM组合物和硬度对CM增殖的组合作用，将溶解的心脏ECM掺入PAAm凝胶中。导致最高CM增殖的ECM/刚度组合将用于指导基于可注射ECM的生物材料的设计。3D凝胶将在体外进行表征和优化，并将揭示2D与3D培养物是否对CM增殖具有不同的影响。将在体内测试在体外导致最大细胞活力、浸润和增殖的ECM凝胶对刺激新生大鼠中CM增殖的影响。这项工作的结果将显着影响未来的心脏组织工程方法，通过开发和表征的ECM为基础的生物材料，促进CM增殖和心脏组织再生。 公共卫生相关性：该项目的长期目标是为患有先天性心脏缺陷的儿童开发新的心脏组织。本研究旨在探讨胎心基质对新生心肌细胞生长的影响。胎儿动物心脏基质可能在未来用于在实验室中生长新的心脏组织，或者可以植入心脏以促进人类新组织的生长。

DESCRIPTION (provided by applicant): Congenital heart defects (CHD) are the leading cause of mortality in live-born infants (1, 2). Hypoplastic Left Heart Syndrome (HLHS) is a rare CHD that requires several major reconstructive surgeries (3). However, the reconstructed heart does not replicate normal anatomy and function, and many patients suffer from serious secondary complications throughout life (4). Cardiac tissue engineering is especially promising for treating HLHS by creating living functional heart tissue that can grow with the child. However, creating functional heart tissue in vitro remains a major challenge, as mature cardiomyocytes (CMs) are mostly non-proliferative (5). While embryonic and fetal CMs are highly proliferative and can restore function in damaged or diseased hearts (6, 7), the causes of decreased CM proliferation and loss of cardiac regenerative capacity after birth are still largely unknown. Elucidating factor that promote CM proliferation will greatly impact tissue engineering and regenerative approaches to treating CHD in children. The extracellular matrix (ECM) modulates a variety of cell functions, including proliferation (8), and there is evidence that ECM composition and stiffness of the heart change during development and maturation (9-12). The hypothesis of this project is that recapitulation of the fetal ECM environment will promote the proliferation of CMs; specifically, it is expected that the features of fetal cardiac ECM that promote CM proliferation are the cues that are lost or diminished in the adult heart. The hypothesis will be systematically tested in vitro, to determine the individual and combined effects of ECM composition and stiffness in 2D and 3D culture, and in vivo, as an initial step towards future clinical translation For ECM composition studies, native fetal and adult rat hearts will be decellularized to obtain ECM and then solubilized and adsorbed onto tissue culture dishes. Primary neonatal rat CMs will be seeded onto cardiac ECM-coated substrates and assayed for proliferation. In parallel, the effects of substrate stiffness will be determined. Decellularized and native hearts will undergo mechanical testing to determine the stiffness of the ECM. Polyacrylamide (PAAm) gels will be made with stiffnesses that mimic fetal and adult hearts. In these studies, Collagen I will be used at the same ligand density in fetal vs. adult stiffness gels to decouple composition from stiffness To investigate the combined effects of ECM composition and stiffness on CM proliferation, solubilized cardiac ECM will be incorporated into PAAm gels. The ECM/stiffness combination that results in highest CM proliferation will be used to guide the design of an injectable ECM-based biomaterial. The 3D gel will be characterized and optimized in vitro and will reveal whether 2D vs. 3D culture has different effects on CM proliferation. The ECM gel which results in greatest cell viability, infiltration, and proliferation in vitro will be tested in vivo for itseffects on stimulating CM proliferation in neonatal rats. The results of this work will significantly impact future cardiac tissue engineering approaches through the development and characterization of ECM-based biomaterials that promote CM proliferation and regeneration of cardiac tissue. PUBLIC HEALTH RELEVANCE: The long-term goal of this project is to develop new heart tissue for children suffering from congenital heart defects. This project will investigate the effets of fetal heart matrix on neonatal cardiomyocyte (heart cell) growth. Fetal animal heart matrix could potentially be used in the future to grow new heart tissue in the lab or could be implanted in the heart to promote new tissue growth in humans.