Assessment of Corneal Fibroblast Biomechanical Behavior

角膜成纤维细胞生物力学行为的评估

• 批准号: 10211772
• 负责人: W MATTHEW PETROLL
• 金额: $ 40.19万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211772
• 关键词: 3-Dimensional; 4D Imaging; Anisotropy; Area; Behavior; Biochemical; Biomechanics; Biophysics; Cell Differentiation process; Cells; Cellular biology; Clinical; Collagen; Cornea; Data; Development; Disease; Elements; Engineering; Environment; Event; Extracellular Matrix; F-Actin; Fibrin; Fibroblasts; Fibronectins; Fibrosis; Filament; Funding; Growth Factor; In Situ; In Vitro; Injury; Integrins; Keratoconus; Knowledge; Mechanics; Modeling; Morphogenesis; Morphology; Myofibroblast; Natural regeneration; Nature; Normal Cell; Operative Surgical Procedures; Oryctolagus cuniculus; Pattern; Penetrating Keratoplasty; Photorefractive Keratectomy; Play; Procedures; Process; Publishing; Recovery; Regulation; Research; Resolution; Role; Signal Pathway; Stromal Cells; Structure; Surgical incisions; System; Testing; Thick; Time; Tissue Engineering; Tissues; Vimentin; base; biophysical properties; cell behavior; cell motility; clinically relevant; corneal epithelial wound healing; corneal scar; crosslink; healing; imaging approach; in vivo; insight; mechanical behavior; mechanical properties; mechanotransduction; migration; models and simulation; novel; reflectance confocal microscopy; response; restoration; second harmonic generation imaging; three dimensional cell culture; tissue regeneration; tool; wound; wound bed; wound healing

PROJECT SUMMARY Mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as morphogenesis, wound healing, and organization of bioengineered tissues. Our research focuses on how these interactions regulate corneal keratocyte behavior through development of culture models that mimic the 3-D tissue environment, and use of multi-dimensional imaging approaches in vitro, in situ, and in vivo. In the previous funding period, we used these tools to perform a comprehensive assessment of the differentiation and patterning of corneal keratocytes following photorefractive keratectomy (PRK) surgery in the rabbit. These studies provided novel insights into the nature of the transition between native stromal and fibrotic tissue, and how cells use the collagen lamellae as a template for tissue remodeling and regeneration. Subsequent studies combining superficial phototherapeutic keratectomy (PTK) and UV cross-linking (CXL) showed that CXL induces a disruption in normal cell patterning within the stroma, and appears to block the development of fibrosis on top of the stroma. These studies highlight the profound impact that changes in corneal structure and stiffness can have on overall corneal wound healing responses. However, the large size and overall symmetry of standard CXL procedures extends the normal time course of healing after PTK, and limits the scope of biomechanical insights that can be made. Using our in vitro 3D culture models, we also performed mechanistic studies on how cell spreading and collective migration are regulated in fibrin matrices, and identified key roles for fibronectin, 51 integrin, and local cell-induced matrix reorganization in this process. We also demonstrated for the first time, that inhibition of vimentin filament organization alters corneal fibroblast spreading, morphology and motility in 3-D matrices. Vimentin has been associated with myofibroblast transformation of corneal keratocytes in vivo, and recent studies in other systems suggest it can play a central role in regulating key aspects of cell mechanical behavior, including mechanosensing, polarization, and directional migration. Based on these and other published and pilot data, we now propose to: 1) Investigate the effects of tissue stiffness and anisotropy on corneal wound healing following PTK, by applying UV cross-linking in specific patterns determined from finite element modeling simulations, 2) Establish the time course of cell differentiation and cell/matrix patterning during the development and resolution (remodeling) of fibrosis following full thickness incisional injury, and determine the effects of modulating the wound boundary conditions and mechanical environment on these processes, and 3) Apply our established 3-D culture models and engineered 2-D substrates to investigate the role of vimentin in regulating corneal fibroblast differentiation, patterning and mechanical behavior. Given the general importance of cell mechanics in numerous fields of cell biology, these findings could have broad significance.

项目摘要 细胞和细胞外基质（ECM）之间的机械相互作用驱动基本过程， 形态发生、伤口愈合和生物工程组织的组织化。我们的研究重点是如何 这些相互作用通过培养模型的发展来调节角膜基质细胞的行为， 三维组织环境，以及在体外、原位和体内使用多维成像方法。 在上一个资助期间，我们使用这些工具对 准分子激光屈光性角膜切除术（PRK）后角膜细胞的分化和模式化 兔子这些研究提供了新的见解之间的天然基质和过渡的性质， 纤维化组织，以及细胞如何使用胶原蛋白层作为组织重塑和再生的模板。 随后的研究结合了浅表光疗性角膜切除术（PTK）和UV交联（CXL） 表明CXL诱导基质内正常细胞模式的破坏，并似乎阻断了细胞的增殖。 基质上纤维化的发展。这些研究强调了变化的深刻影响， 角膜结构和硬度可能对整个角膜伤口愈合反应有影响。但 标准CXL手术的大尺寸和整体对称性延长了正常的时间进程 PTK后的愈合，并限制了生物力学的见解，可以作出的范围。 使用我们的体外3D培养模型，我们还对细胞如何扩散和增殖进行了机制研究。 集体迁移在纤维蛋白基质中受到调节，并确定了纤连蛋白、β 5 β 1整合素和 在这个过程中局部细胞诱导的基质重组。我们还首次证明， 波形蛋白丝组织的改变角膜成纤维细胞的蔓延，形态和运动在3-D矩阵。 波形蛋白与体内角膜基质细胞的肌成纤维细胞转化有关， 最近在其他系统中的研究表明，它可以在调节细胞的关键方面发挥核心作用。 机械行为，包括机械感测、极化和定向迁移。 基于这些和其他已发表和试点数据，我们现在建议：1）调查组织的影响， 在PTK后角膜伤口愈合的刚度和各向异性，通过在特定的 从有限元建模模拟确定的模式，2）建立细胞分化的时间过程 以及在全厚度后纤维化的发展和消退（重塑）期间的细胞/基质图案化 切口损伤，并确定调节伤口边界条件和机械 3）应用我们建立的三维培养模型和工程化的二维培养模型， 研究波形蛋白在调节角膜成纤维细胞分化、图案形成和 机械性能鉴于细胞力学在细胞生物学的许多领域中的普遍重要性， 这些发现可能具有广泛的意义。