Bioactive Hydrogel Niches for 3D VIC Culture

描述（由申请人提供）：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）的三维培养和随后的细胞微环境操作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，我们相信支架将被确定，这将有助于功能瓣叶的再生。具体来说，我们建议确定特定基质相互作用（如纤维连接蛋白、肝素）和可溶性细胞因子（如bFGF、TGF-¿1）对2D中VIC功能的影响（目的1）。然后，这些结果将用于开发高度调控的生物材料龛，以控制VIC功能和3D基质生产（目标2和3）。我们计划设计3d支架化学物质，使VIC能够存活和增殖，并促进VIC的表达和激活，形成肌成纤维细胞表型，这在瓣膜重塑和发育过程中很普遍（目标2）。随后，我们将操纵降解依赖的支架特性，以支持细胞外基质沉积和功能性组织再生（目标3）。目标2和目标3的实验方法是将VIC光封装在聚乙二醇（PEG）和透明质酸（HA）基共聚物水凝胶中，这些共聚物水凝胶将被基质组分系统地修饰以支持VIC相互作用。此外，bFGF和TGF-¿1将通过散装和局部递送的方法引入到细胞-凝胶结构中。共聚焦显微镜将用于直接观察细胞活力随时间的变化。通过实时RT-PCR、免疫染色和原位杂交测定BRDU掺入和基因随时间的表达，将用于评估VIC增殖和肌成纤维细胞分化。vic的功能活动将通过测量细胞间通讯、细胞外基质分泌和进化的机械特性来评估。凝胶化学，特别是基质成分和细胞因子的引入，对VIC功能的影响将通过体外培养这些细胞负载的水凝胶来筛选。这些目标的结果将用于鉴定允许VIC功能的水凝胶配方，促进受控的肌成纤维细胞分化，并促进基质形成。这些体外实验结果将为未来在适当设计的生物反应器中开发功能瓣膜结构提供选择配方的基础。本课题旨在制备包含信号的生物材料微环境，积极促进心脏瓣膜细胞的组织再生功能，并通过提供更仿生的心脏瓣膜细胞三维培养系统，提高该领域对心脏瓣膜功能的认识。如果成功，这种策略将延长以组织为基础的瓣膜置换术的持续时间和功能，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.