Bioactive Hydrogel Niches for 3D VIC Culture

描述(申请人提供)：本提案的重点是开发一种生物材料平台，该平台将使瓣膜间质细胞(VIC)的三维培养和随后对细胞微环境的操纵能够促进或抑制选定的细胞功能。通过这种类型的三维培养系统，我们相信将鉴定出有利于功能瓣叶再生的支架。具体地说，我们建议在2D(目标1)中确定特定的基质相互作用(例如，纤维连接蛋白、肝素)和可溶性细胞因子(例如，碱性成纤维细胞生长因子、转化生长因子β1)对VIC功能的影响。这些结果将被用来开发高度受监管的生物材料，以控制3D中的VIC功能和基质生产(目标2和3)。我们计划设计允许VIC存活和增殖的3D支架化学物质，并促进VIC的表达和激活为肌成纤维细胞表型，这种表型在瓣膜重塑和发育过程中很常见(目标2)。随后，我们将操纵依赖于降解的支架属性来支持细胞外基质沉积和功能组织再生(目标3)。AIMS 2和AIMS 3的实验方法将是将VIC光包裹在聚乙二醇基和透明质酸基共聚物水凝胶中，这些水凝胶将被基质组分系统地修饰以支持VIC相互作用。此外，碱性成纤维细胞生长因子和转化生长因子-1将通过批量和局部给药的方式引入细胞凝胶构建物中。共聚焦显微镜将被用来随着时间的推移直接观察细胞的存活情况。通过实时定量RT-PCR、免疫染色和原位杂交检测BRDU掺入和基因表达随时间的变化，将用于评估VIC的增殖和肌成纤维细胞的分化。VICS的功能活性将通过测量细胞间的通讯、细胞外基质的分泌和演变的机械性能来评估。凝胶化学的影响，特别是基质成分和细胞因子的引入，将通过体外培养这些细胞载水凝胶来筛选对VIC功能的影响。这些目标的结果将用于确定允许VIC功能、促进受控的肌成纤维细胞分化和促进基质形成的水凝胶配方。这些体外结果将为在适当设计的生物反应器中选择未来开发功能性瓣膜结构的配方提供基础。这项建议旨在准备包含信号的生物材料微环境，以积极促进心脏瓣膜细胞用于组织再生的功能，并通过为心脏瓣膜细胞提供更仿生的3D培养系统来提高该领域对心脏瓣膜功能的理解。如果成功，这一策略将延长基于组织的瓣膜置换的持续时间和功能，特别是对儿童。

DESCRIPTION (provided by applicant): The focus of this proposal is to develop a biomaterial platform that will enable the 3-D culture of valvular interstitial cells (VICs) and subsequent manipulation of the cellular microenvironment to promote or suppress selected cell functions. Through this type of three-dimensional culture system, we believe that scaffolds will be identified that will facilitate the regeneration of functional valve leaflets. Specifically, we propose to determine the effect of specific matrix interactions (e.g., fibronectin, heparin) and soluble cytokines (e.g., bFGF, TGF-¿1) on VIC function in 2D (aim 1). These results will then be used to develop highly regulated biomaterials niches to control VIC function and matrix production in 3D (aims 2 & 3). We plan to design 3-D scaffold chemistries that will permit VIC viability and proliferation, as well as promote expression and activation of VICs to a myofibroblast phenotype, which is prevalent during valve remodeling and development (aim 2). Subsequently, we will manipulate the degradation- dependent scaffold properties to support extracellular matrix deposition and functional tissue regeneration (aim 3). The experimental approach for aims 2 and 3 will be to photoencapsulate VICs in poly (ethylene glycol) (PEG) and hyaluronan (HA)-based copolymer hydrogels that will be systematically modified with matrix components to support VIC interactions. In addition, bFGF and TGF-¿1 will be introduced into the cell-gel constructs through bulk and localized delivery methods. Confocal microscopy will be used to directly visualize cell viability over time. BRDU incorporation and gene expression with time, as determined by real time RT-PCR, immunostaining, and in situ hybridization will be used to assess VIC proliferation and myofibroblast differentiation. Functional activity of VICs will be assessed by measuring cell- cell communication, extracellular matrix secretion, and evolving mechanical properties. The effects of gel chemistry, especially the introduction of matrix components and cyotokines, on VIC function will be screened by culturing these cell-laden hydrogels in vitro. Results from these aims will be used to identify hydrogel formulations that permit VIC function, promote controlled myofibroblast differentiation, and facilitate matrix formation. These in vitro results will then provide the foundation to select formulations for future development of functional valve structures in appropriately designed bioreactors. This proposal aims to prepare biomaterial microenvironments that incorporate signals to actively promote the function of heart valve cells for tissue regeneration and to improve the field's understanding of heart valve function by providing a more biomimetic 3D culture system for heart valve cells. If successful, this strategy will prolong the duration and function of tissue-based valve replacements, especially for children.