Bioactive Hydrogel Niches for 3D VIC Culture

用于 3D VIC 培养的生物活性水凝胶生态位

• 批准号: 7782692
• 负责人: KRISTI S. ANSETH
• 金额: $ 32.71万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7782692
• 关键词: 3-Dimensional; Apoptosis; Biochemical; Biocompatible Materials; Biological; Biomimetics; Bioreactors; Bromodeoxyuridine; Cell Communication; Cell Culture Techniques; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chemicals; Chemistry; Child; Confocal Microscopy; Cues; Cultured Cells; Deposition; Development; Diffusion; Drug Formulations; Encapsulated; Engineering; Environment; Ethylene Glycols; Evolution; Extracellular Matrix; Fibroblast Growth Factor 2; Fibronectins; Film; Foreign Bodies; Foundations; Future; Gel; Gene Expression; Heart Valves; Heparin; Histologic; Hyaluronan; Hydrogels; In Situ Hybridization; In Vitro; Life; Matrix Metalloproteinases; Measures; Mechanics; Metabolic; Methods; Myofibroblast; Natural regeneration; Phenotype; Principal Investigator; Production; Property; Relative (related person); Replacement Therapy; Reverse Transcriptase Polymerase Chain Reaction; Risk; Signal Transduction; Structure; Surface; System; Time; Tissue Engineering; Tissues; base; copolymer; cytokine; design; ethylene glycol; extracellular; improved; interstitial cell; programs; repaired; scaffold; tissue regeneration; valve replacement

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.

描述（由申请人提供）：该提案的重点是开发一种生物材料平台，该平台将能够进行瓣膜间质细胞 (VIC) 的 3D 培养，并随后操纵细胞微环境以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助于功能性瓣膜小叶再生的支架。具体来说，我们建议确定特定基质相互作用（例如纤连蛋白、肝素）和可溶性细胞因子（例如 bFGF、TGF-¿1）对 2D 中 VIC 功能的影响（目标 1）。然后，这些结果将用于开发高度监管的生物材料利基，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 3-D 支架化学物质，使 VIC 具有活力和增殖，并促进 VIC 表达和激活成肌成纤维细胞表型，这在瓣膜重塑和发育过程中很常见（目标 2）。随后，我们将操纵降解依赖性支架特性来支持细胞外基质沉积和功能性组织再生（目标 3）。目标 2 和 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，该水凝胶将用基质成分进行系统修饰以支持 VIC 相互作用。此外，bFGF 和 TGF-¿1 将通过批量和局部递送方法引入细胞凝胶构建体中。共聚焦显微镜将用于直接观察细胞随时间的活力。通过实时 RT-PCR、免疫染色和原位杂交测定的 BRDU 掺入和基因表达随时间的变化将用于评估 VIC 增殖和肌成纤维细胞分化。 VIC 的功能活性将通过测量细胞间通讯、细胞外基质分泌和不断变化的机械特性来评估。将通过体外培养这些充满细胞的水凝胶来筛选凝胶化学，特别是基质成分和细胞因子的引入对 VIC 功能的影响。这些目标的结果将用于鉴定允许 VIC 功能、促进受控肌成纤维细胞分化并促进基质形成的水凝胶制剂。这些体外结果将为未来在适当设计的生物反应器中开发功能性瓣膜结构的配方选择奠定基础。该提案旨在制备包含信号的生物材料微环境，以积极促进心脏瓣膜细胞的组织再生功能，并通过为心脏瓣膜细胞提供更具仿生性的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术的持续时间和功能，特别是对于儿童。