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: 10217723
• 负责人: W MATTHEW PETROLL
• 金额: $ 8.43万
• 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 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217723
• 关键词: 3-Dimensional; 4D Imaging; Architecture; Behavior; Biochemical; Biomechanics; Biophysics; Cell Differentiation process; Cells; Clinical; Collagen; Cornea; Custom; Data; Deposition; Development; Disease; Environment; Experimental Models; Extracellular Matrix; Fibrin; Fibroblasts; Fibronectins; Fluorescence; Freezing; Generations; Grant; Growth Factor; Histone Deacetylase; Histone Deacetylase Inhibitor; In Situ; In Vitro; Individual; Injury; Lead; Mechanics; Microscope; Modeling; Morphogenesis; Morphology; Myofibroblast; Operative Surgical Procedures; Oryctolagus cuniculus; Pattern; Phenotype; Photorefractive Keratectomy; Platelet-Derived Growth Factor; Procedures; Process; Publishing; Research; Running; Signal Pathway; Signal Transduction; Stream; Stress Fibers; Stromal Cells; Structure; Testing; Time; Tissue Engineering; Tissues; Transforming Growth Factor beta; base; cell behavior; cell growth; cell motility; cell type; corneal scar; crosslink; cytokine; ex vivo imaging; imaging approach; imaging capabilities; in vitro Model; in vivo; insight; mechanical behavior; mechanical properties; migration; novel; reflectance confocal microscopy; response; rho; second harmonic generation imaging; 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. Research in the prior period using 3-D culture models demonstrated that matrix composition, stiffness and structure can influence corneal keratocyte mechanical behavior and patterning in response to wound healing cytokines and changes in Rho/Rac activation. In addition, using our custom-modified in vivo HRT-RCM confocal microscope combined with ex vivo fluorescence and second harmonic generation (SHG) imaging, we demonstrated for the first time that following freeze injury (FI) or lamellar keratectomy (LK) in the rabbit, migrating fibroblasts within the wounded stroma form long interconnected streams that often run in parallel, and that alignment of these cell streams is highly correlated with that of the collagen lamellae. In contrast, cells migrating on top of the stroma following LK form a randomly arranged, interconnected, meshwork. The biochemical factors which induce myofibroblast transformation and fibrotic tissue generation on top of the stroma following injury or refractive surgery have been studied extensively. However, little is known about biochemical and biophysical signals that regulate intra-stromal keratocyte behavior. The lamellar structure of the cornea, combined with powerful in vivo and ex vivo imaging capabilities, provides us with a unique opportunity to assess biophysical factors that regulate cell differentiation, migration and patterning within this tissue. Aim 1 will use in vivo confocal microscopy and in situ fluorescent/SHG imaging in the rabbit to: a) perform the first comprehensive comparison of intra-stromal and extra-stromal cell differentiation and patterning following photorefractive keratectomy (PRK), and b) investigate whether intra-stromal and extra- stromal phenotypes are differentially regulated. Aim 2 will investigate whether changes in ECM structure and stiffness modulate cell patterning and mechanical phenotype during stromal repopulation by comparing migration mechanisms in two distinct in vivo injury models. ECM structure and mechanical properties have become increasingly recognized as key factors in determining cell growth, differentiation and activity in a variety of cell types; thus our findings should have broad scientific impact. In order to isolate the specific factors regulating these in vivo processes, Aim 3 will assess how cytokines and downstream Rho/Rac signaling impact corneal keratocyte patterning, mechanical differentiation, fibronectin deposition and ECM reorganization using multiple novel experimental models in vitro. With this approach we hope to identify the key biochemical and biophysical signaling pathways that differentiate disruptive and non-disruptive cell patterning behavior within 3-D matrices, which may lead to new strategies to modulate cell behavior in vivo.

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 这些相互作用通过开发模拟角膜基质细胞的培养模型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。 前期使用3-D培养模型的研究表明，基质成分、刚度和 结构可以影响角膜基质细胞机械行为和响应伤口愈合的图案 细胞因子和Rho/Rac活化的变化。此外，使用我们定制的改良体内HRT-RCM 共聚焦显微镜结合离体荧光和二次谐波发生（SHG）成像，我们 首次证明了在兔冷冻损伤（FI）或板层角膜切除术（LK）后， 受伤的基质内迁移的成纤维细胞形成通常平行流动的长的相互连接的流， 并且这些细胞流的排列与胶原层的排列高度相关。相反，细胞 在LK之后迁移到基质的顶部上形成随机排列的、互连的网状结构。 诱导肌成纤维细胞转化和纤维化组织产生的生化因素， 已经广泛研究了损伤或屈光手术后的基质。然而，人们对 调节基质内角膜细胞行为的生物化学和生物物理信号。层状结构 角膜与强大的体内和体外成像能力相结合，为我们提供了独特的 有机会评估调节细胞分化，迁移和图案化的生物物理因素， 组织.目的1将在兔中使用体内共聚焦显微镜和原位荧光/SHG成像，以： 进行基质内和基质外细胞分化的首次全面比较， 光折射性角膜切除术（PRK）后的图案化，和B）研究是否基质内和基质外 基质表型受到差异调节。目标2将研究ECM结构的变化和 通过比较基质再生过程中硬度调节细胞模式和机械表型 在两种不同的体内损伤模型中的迁移机制。ECM组织和力学性能具有 越来越被认为是决定细胞生长、分化和活性的关键因素 因此，我们的发现应该具有广泛的科学影响。为了隔离 调节这些体内过程的特定因子，目标3将评估细胞因子和下游Rho/Rac 信号传导影响角膜基质细胞图案形成、机械分化、纤连蛋白沉积和ECM 重组使用多种新的体外实验模型。通过这种方法，我们希望找到关键 区分破坏性和非破坏性细胞模式的生物化学和生物物理信号通路 行为的三维矩阵，这可能会导致新的策略来调节细胞的行为在体内。