Assessment of Corneal Fibroblast Biomechanical Behavior

项目总结 细胞和细胞外基质(ECM)之间的机械相互作用推动基本过程，如 生物工程组织的形态发生、伤口愈合和组织。我们的研究重点是如何 这些相互作用通过发展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，以及使用体外、原位和体内的多维成像方法。 前期使用3-D培养模型的研究表明，基质成分、硬度和 结构可以影响角膜基质细胞对伤口愈合的机械行为和模式 细胞因子与Rho/Rac活化的变化。此外，使用我们定制修改的体内HRT-RCM 共聚焦显微镜结合体外荧光和二次谐波成像 首次证实了冷冻损伤(FI)或板层角膜切削术(LK)后， 在受伤的间质内迁移的成纤维细胞形成了通常平行运行的相互连接的长流， 这些细胞流的排列与胶原板的排列高度相关。相比之下，细胞 随着LK在基质顶部迁移，形成一个随机排列的、相互连接的网络。 诱导肌成纤维细胞转化和纤维组织生成的生化因素 损伤或屈光手术后的间质已被广泛研究。然而，人们对此知之甚少。 调节基质内角质细胞行为的生化和生物物理信号。的层状结构 角膜，结合强大的体内和体外成像能力，为我们提供了独特的 有机会评估调控细胞分化、迁移和构图的生物物理因素 组织。AIM 1将在兔体内使用共聚焦显微镜和原位荧光/倍频成像来：a) 首次对间质内和间质外细胞分化进行全面比较 准分子激光屈光性角膜切削术(PRK)后的模式，以及b)调查基质内和基质外的 间质表型受到不同的调控。目标2将调查ECM结构的变化和 比较刚性对基质再生过程中细胞构型和机械表型的影响 两种不同体内损伤模型的迁移机制。ECM组织和力学性能有 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。为了分离出 调节这些体内过程的特定因素，Aim 3将评估细胞因子和下游Rho/Rac如何 信号对角膜基质细胞构型、机械分化、纤维连接蛋白沉积和细胞外基质的影响 利用多种新的体外实验模型进行重组。通过这种方法，我们希望找出关键 区分破坏性和非破坏性细胞模式的生化和生物物理信号通路 3-D矩阵内的行为，这可能导致在体内调节细胞行为的新策略。

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.