Creating Hydrogel Tissue Mimics to Better Understand Congenital Heart Disease

创建水凝胶组织模拟物以更好地了解先天性心脏病

• 批准号: 9908506
• 负责人: Deanna Bousalis
• 金额: $ 3.97万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-21 至 2022-02-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908506
• 关键词: 3-Dimensional; Affect; Beds; Behavior; Biophysics; Cardiac; Cardiac Myocytes; Cell Culture System; Cell membrane; Cell physiology; Cells; Characteristics; Contracts; DNA Sequence Alteration; Data; Development; Disease; Disease Progression; Elements; Embryo; Embryonic Ventricle; Engineering; Extracellular Matrix; Extracellular Space; Fibroblasts; Gene Expression; Genes; Genetic; Goals; Heart; Heart Abnormalities; Heart Diseases; Human; Hyaluronic Acid; Hydrogels; In Vitro; Inherited; Live Birth; Mechanics; Methods; Modulus; Morphology; Motivation; Mus; Mutation; Myocardial; Myocardial Infarction; Phenotype; Play; Proteomics; Reporter Genes; Role; Supporting Cell; System; Techniques; Testing; Time; Tissue Model; Tissues; Ventricular; Western Blotting; Wild Type Mouse; Work; base; biophysical properties; blood pump; cardiac repair; cell growth; cellular imaging; congenital heart disorder; disease phenotype; genome wide screen; in vitro Model; in vitro testing; insight; interest; mechanical properties; migration; mutant; mutant mouse model; novel; protein expression; scaffold; tandem mass spectrometry; three dimensional cell culture

PROJECT SUMMARY/ABSTRACT In this proposal, I aim to develop natural materials-based hydrogel cardiac tissue mimics that can serve as in vitro test beds for congenital heart disease (CHD). The motivation for this work is the lack of three- dimensional in vitro models for CHD that currently exist, and the little information known about the biophysical inputs of disease progression. CHD affects 1 percent of all live births worldwide. Numerous genetic mutations resulting in cardiac maldevelopment exist, a common one of which is a mutation in the Nkx2-5 gene. Preliminary data in an Nkx2-5 mutant mouse model from collaborator Dr. Kasahara revealed that approximately 20 genes are downregulated in the ventricles of diseased versus healthy hearts, two thirds of which are localized to the cell membrane or extracellular space. As the extracellular matrix (ECM) has been shown to play a role in many cellular processes such as migration, differentiation, and proliferation, this data warrants further exploration. Additionally, mutant hearts were shown to have ventricular noncompaction, a phenotype in which the heart’s trabecular layer is spongy rather than firm, limiting the heart’s capacity to pump blood effectively. Hence, the overall hypothesis for this project is that the Nkx2-5 mutation influences the biophysical properties of embryonic cardiac tissue, which in turn dynamically regulate developing cardiac cells. Replicating this disease phenotype in vitro could provide an effective method for studying the mechanisms of cardiac maldevelopment. An ECM component of interest in this project is hyaluronic acid (HA) because it is found ubiquitously throughout the ECM of the body, including cardiac tissue, and provides an excellent base for the development of hydrogel tissue mimics. The Schmidt lab has engineered HA-based hydrogels to mimic the mechanical properties of various ECM landscapes, independent of composition. These techniques will be applied to the creation of hydrogel scaffolds in this project. To this end, I propose 3 aims to accomplish the tasks of creating my ECM-based hydrogel tissue mimics. In Aim 1, I will determine the biophysical properties of cardiac tissue affected by the Nkx2-5 mutation. To do this I will assess ECM composition of healthy and diseased hearts through tandem mass spectrometry and Western blotting, and mechanical properties through indentation. In Aim 2, I will develop ECM-based hydrogel scaffolds to mimic the biophysical properties of healthy and diseased cardiac tissues and establish these scaffolds as 3D cell culture systems. To accomplish this, I will create HA- based hydrogel scaffolds with ECM compositions and mechanical properties matched to those of diseased and healthy heart tissue. In Aim 3, I will evaluate the biophysical properties that modify the fate of developing myocardial cells. For these studies, cardiomyocytes isolated from healthy and diseased hearts will be cultured in mutant and wild type test beds. The assumption of cell noncompaction phenotype as well as gene and protein expression will be assessed, and hydrogels will be indented to assess potential mechanical remodeling by cells.

项目总结/摘要 在这个提案中，我的目标是开发基于天然材料的水凝胶心脏组织模拟物， 作为先天性心脏病（CHD）的体外试验床。这项工作的动机是缺乏三个- 目前存在的CHD的三维体外模型，以及关于生物物理学的信息很少。 疾病进展的输入。 CHD影响全世界所有活产婴儿的1%。许多基因突变导致心脏 存在发育不良，其中一个常见的是Nkx 2 -5基因突变。Nkx 2 -5中的初步数据 合作者Kasahara博士的突变小鼠模型显示，大约20个基因下调 在患病心脏与健康心脏的心室中，其中三分之二位于细胞膜上， 细胞外间隙由于细胞外基质（ECM）已被证明在许多细胞过程中发挥作用， 如迁移、分化和增殖，这些数据值得进一步探讨。此外，突变 心脏显示有心室致密化不全，这是一种心脏小梁层 海绵状而非坚实，限制了心脏有效泵血的能力。因此，总体假设 Nkx 2 -5突变影响胚胎心脏组织的生物物理特性， 进而动态调节发育中的心肌细胞。 在体外复制这种疾病表型可以为研究其发病机制提供一种有效的方法。 心脏发育不良在该项目中感兴趣的ECM组分是透明质酸（HA），因为它是 在身体的ECM中无处不在，包括心脏组织，并为 水凝胶组织模拟物的发展。施密特实验室设计了HA基水凝胶， 机械性能的各种ECM景观，独立的组成。这些技术将 应用于这个项目中的水凝胶支架的制作。为此，我提出了三个目标，以完成任务 创造我的ECM水凝胶组织模拟物。在目标1中，我将确定心脏的生物物理特性， Nkx 2 -5突变的组织。为此，我将评估健康和患病心脏的ECM组成 通过串联质谱法和蛋白质印迹法，以及通过压痕的机械性能。在Aim中 2，我将开发基于ECM的水凝胶支架，以模拟健康和患病的生物物理特性 心脏组织，并建立这些支架作为3D细胞培养系统。为了实现这一目标，我将创建HA- 基于水凝胶的支架，其具有ECM组成和与患病的和 健康的心脏组织在目标3中，我将评估生物物理特性，这些特性改变了发展中国家的命运。 心肌细胞在这些研究中，将培养从健康和患病心脏中分离的心肌细胞 在突变体和野生型试验床中。细胞致密化不全表型与基因、蛋白质的假设 将评估表达，并且将使水凝胶凹进以评估细胞的潜在机械重塑。