Regulation of Corneal Keratocyte Differentiation through the Integration of Biochemical, Biomechanical and Topographic Cues

项目摘要 角膜基质中的角质形成细胞位于胶原层之间，负责 用于分泌维持正常角膜结构和透明度所需的细胞外基质成分。 通过与细胞外基质(ECM)的相互作用，基质角质形成细胞在 基本的生物学过程，如发育形态发生和伤口愈合。是这样的 相互作用在组织工程领域也很重要，在组织工程领域，有必要 调制单元和ECM图案化以产生特定的矩阵结构。虽然我们知道的很多 关于生长因子对刚性二维刚性基质上角膜基质细胞分化的影响，角膜 角质形成细胞存在于体内复杂的细胞外基质环境中，包括机械、 地形和生化线索。ECM硬度、形貌和蛋白质组成都有 已被证明可以调节角质细胞的分化、收缩和图案化。然而，在那里 在我们关于多个刺激是如何整合在一起产生的知识上仍然存在着显著的差距 特定的细胞表型。这项计划的总体目标是开发和测试新的2D和3D 实验平台，将允许我们系统地结合可溶性和 不可溶的线索。 在特定的目标1中，我们将确定基质弹性如何调节角质形成细胞对 通过在一系列机械设备上制造可调的聚丙烯酰胺底物来实现可溶生化提示 并使用牵引力显微镜、定量免疫荧光成像和 生化分析评价角质形成细胞分化的变化。在目标2中，我们将确定如何 底物形态和组成调节角质形成细胞对生化和 使用排列的纤维胶原与其他关键细胞外基质结合的生物力学提示 蛋白质。在目标3中，我们将确定衬底尺寸(2-D与3-D)如何调制 角质形成细胞对生化、生物力学和地形图提示的反应 三明治结构将这些信号同时呈现给细胞的腹面和背面。 这些新的实验平台可以用来识别、分离和研究Key的作用 角质形成细胞分化的生物物理信号通路(如静止的，迁移的， 再生表型和纤维化表型)。所获得的知识有助于制定有针对性的 抗纤维化治疗，以及指导组织工程学方法发展间质组织 接班人。重要的是，开发的实验模型可以广泛应用于 生物物理线索和机械转导已知的其他领域具有深远的 对细胞构型和行为的影响，如发育和癌症生物学。

Project Summary Corneal keratocytes reside between the collagen lamellae in the cornea stroma, and are responsible for secreting ECM components required to maintain normal corneal structure and transparency. Through their interactions with the extracellular matrix (ECM), stromal keratocytes play a central role in fundamental biological processes such as developmental morphogenesis and wound healing. Such interactions also are also important in the field of tissue engineering, where it is necessary to either modulate cell and ECM patterning to produce specific matrix architectures. While much is known regarding the effects of growth factors on keratocyte differentiation on rigid 2-D rigid substrates, corneal keratocytes reside in a complex ECM environment in vivo, which includes a combination of mechanical, topographical and biochemical cues. ECM stiffness, topography and protein composition have all been shown to modulate keratocyte differentiation, contractility and patterning. However, there are still significant gaps in our knowledge of how multiple stimuli are integrated to produce specific cell phenotypes. The overall goal of this proposal is to develop and test novel 2D and 3D experimental platforms that will allow us to systematically incorporate combinations of both soluble and non-soluble cues. In Specific Aim 1 we will determine how substrate elasticity modulates the keratocyte response to soluble biochemical cues by fabricating tunable polyacrylamide substrates over a range of mechanical stiffness values and using traction force microscopy, quantitative immunofluorescence imaging and biochemical assays to evaluate changes in keratocyte differentiation. In Aim 2 we will determine how substrate topography and composition modulates the keratocyte response to biochemical and biomechanical cues using substrates with aligned fibrillar collagen combined with other key ECM proteins. In Aim 3 we will determine how substrate dimensionality (2-D versus 3-D) modulates the keratocyte response to biochemical, biomechanical and topographic cues by fabricating novel 3D sandwich constructs that present these cues to both the ventral and dorsal surfaces of cells. These new experimental platforms can be used to identify, isolate, and investigate the role of key biophysical signaling pathways on the differentiation of keratocytes (e.g. quiescent, migratory, regenerative and fibrotic phenotypes). Knowledge obtained could aid in the development of targeted anti-fibrosis therapies, as well as guide tissue engineering approaches to developing stromal tissue replacements. Importantly, the experimental models developed could have broad application in other fields where biophysical cues and mechanotransduction are known to have a profound impact on cell patterning and behavior, such as developmental and cancer biology.