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

项目摘要 角膜基质细胞位于角膜基质中的胶原层之间， 用于分泌维持正常角膜结构和透明度所需的ECM成分。 通过与细胞外基质（ECM）的相互作用，基质角膜细胞在角膜上皮细胞的增殖和分化中发挥着重要作用。 基本的生物过程，如发育形态发生和伤口愈合。等 相互作用在组织工程领域中也很重要，其中需要 调节细胞和ECM图案化以产生特定的基质结构。虽然我们知道很多 关于生长因子对刚性2-D刚性基质上的角膜细胞分化的影响， 角膜细胞存在于体内复杂的ECM环境中，其包括机械， 地形和生化线索。ECM硬度、形貌和蛋白质组成都具有 已经显示出调节角膜细胞分化、收缩性和图案形成。但 在我们对多种刺激如何整合以产生 特定的细胞表型。该提案的总体目标是开发和测试新的2D和3D 实验平台，这将使我们能够系统地将可溶性和 不溶的线索 在具体目标1中，我们将确定基质弹性如何调节角膜细胞对 通过在机械强度范围内制造可调聚丙烯酰胺底物 硬度值和使用牵引力显微镜，定量免疫荧光成像和 生物化学测定以评价角膜细胞分化的变化。在目标2中，我们将确定如何 基质形貌和组成调节角膜细胞对生物化学的反应， 使用具有与其他关键ECM组合的对齐的纤维状胶原的基底的生物力学线索 proteins.在目标3中，我们将确定基底维度（2-D与3-D）如何调节 角膜细胞对生化、生物力学和地形学线索的反应 将这些线索呈现给细胞的腹侧和背侧表面的三明治结构。 这些新的实验平台可用于识别，分离和研究关键的作用。 生物物理信号通路对角膜细胞分化（例如静止，迁移， 再生和纤维化表型）。获得的知识可以帮助发展有针对性的 抗纤维化治疗，以及指导组织工程方法来发展基质组织 替代品重要的是，开发的实验模型可以广泛应用于 已知生物物理线索和机械转导具有深刻意义的其他领域 对细胞模式和行为的影响，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.