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

修复先天性心脏缺陷：胎儿心脏 Extracel 的作用

• 批准号: 8396432
• 负责人: Corin Williams
• 金额: $ 4.92万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396432
• 关键词: Adult; Affect; Anatomy; Animals; Biocompatible Materials; Biological Assay; Birth; Bromodeoxyuridine; Cardiac; Cardiac Myocytes; Cell Survival; Cell physiology; Child; Childhood; Clinical; Collagen; Congenital Heart Defects; Cues; Development; Embryo; Engineering; Environment; Extracellular Matrix; Eye; Fetal Heart; Fetus; Fibrin; Future; Gel; Goals; Growth; Harvest; Heart; Histone H3; Human; Hypoplastic Left Heart Syndrome; Implant; In Vitro; Individual; Infant; Infiltration; Injectable; Investigation; Lead; Left; Left ventricular structure; Life; Ligands; Measures; Mechanics; Mus; Natural regeneration; Neonatal; Operative Surgical Procedures; Patients; Pilot Projects; Polystyrenes; Proliferating; Property; Rattus; Reconstructive Surgical Procedures; Regenerative Medicine; Resected; Site; Staining method; Stains; Stem cells; Techniques; Testing; Tissue Engineering; Tissues; Translations; Work; Zebrafish; aurora B kinase; base; cell behavior; cell growth; crosslink; density; design; fetal; functional restoration; heart cell; in vitro testing; in vivo; mortality; polyacrylamide gels; regenerative; repaired; tissue culture; tissue regeneration

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.

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）。左心发育不全综合征（HLHS）是一种罕见的冠心病，需要多次大的重建手术(3)。然而，重建的心脏不能复制正常的解剖结构和功能，许多患者在一生中都会出现严重的继发性并发症(4)。心脏组织工程在治疗HLHS方面尤其有前景，因为它可以创造出与儿童一起生长的功能性活心脏组织。然而，在体外创造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（CMs）大多是非增殖的(5)。虽然胚胎和胎儿CM具有高度增殖能力，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在很大程度上仍然未知。阐明促进CM增殖的因素将极大地影响组织工程和再生治疗儿童冠心病的方法。细胞外基质（ECM）调节多种细胞功能，包括增殖(8)，有证据表明，心脏的ECM成分和硬度在发育和成熟过程中发生变化（9-12）。这个项目的假设是胎儿ECM环境的重现会促进CMs的增殖；具体来说，我们预计胎儿心脏ECM促进CM增殖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测试，以确定ECM组成和硬度在2D和3D培养中的单独和联合影响，而在体内，作为未来临床转化ECM组成研究的第一步，将对天然胎儿和成年大鼠心脏进行脱细胞处理以获得ECM，然后将其溶解并吸附到组织培养皿上。原代新生大鼠CMs将被播种到心脏ecm包被的基质上，并检测其增殖情况。与此同时，基材刚度的影响也将被确定。脱细胞心脏和原生心脏将进行力学测试，以确定ECM的刚度。聚丙烯酰胺（PAAm）凝胶的硬度将模仿胎儿和成人的心脏。在这些研究中，I型胶原将以相同的配体密度用于胎儿和成人的僵硬凝胶中，以使成分与僵硬分离。为了研究ECM成分和僵硬对CM增殖的联合影响，将溶解的心脏ECM掺入PAAm凝胶中。导致CM增殖最高的ECM/刚度组合将用于指导可注射ECM基生物材料的设计。3D凝胶将在体外进行表征和优化，并将揭示2D与3D培养对CM增殖的影响是否不同。体外细胞活力、浸润和增殖能力最强的ECM凝胶将在体内测试其对新生大鼠CM增殖的刺激作用。这项工作的结果将通过开发和表征促进CM增殖和心脏组织再生的基于ecm的生物材料，对未来的心脏组织工程方法产生重大影响。