Controlling Cell Phenotype in a Tissue-Engineered Corneal Model

描述(申请人提供)：一个可行的、透明的角膜模型可以为研究新的眼科药物和新的基因输送方法提供基础。此外，目前临床上需要更好地了解角膜创伤愈合机制，以解决与LASIK手术和角膜移植结果相关的角膜混浊问题。了解影响角膜伤口愈合表型表达的因素可以帮助理解如何控制这些变化。这项拟议项目的总体目标是了解和控制角膜细胞行为与组织工程角膜模型结果透明度之间的关系。对电池的一些输入信号进行了表征，包括化学信号、机械信号、电磁信号和地形信号。这些研究的结果综合起来表明，物理信号比化学信号在控制角膜成纤维细胞的伤口愈合反应方面更有希望。所提出的研究旨在了解几种信号的差异，也许更重要的是，几种信号的协同效应，以便确定在我们迈向三维角膜模型的最终目标时，哪些信号或信号组合最成功。蛋白质水平将通过蛋白质印迹分析，mRNA水平将通过定量实时聚合酶链式反应(QR-T PCR)分析，光散射程度将通过光学相干显微镜(OCM)确定。具体地说，我们将评估与伤口愈合反应相关的信号、整合素信号、α-SMA、TKT、ALDH1和基质重塑标记物，以及与本建议详细说明的各种治疗条件下角质细胞表型变化相关的基质重塑标记物。将对细胞行为进行评估，以了解(1)基质排列和基质组成的相对重要性；(2)基质硬度的影响；(3)光和机械应变输入信号的潜在协同效应。通过了解这些输入信号的相对强度，我们可以更智能地构建透明角膜矩阵和模型。总体而言，这些研究将有助于更好地理解生物仿生学在组织工程系统设计和实施中的作用。此外，它们还将有助于理解成纤维细胞中的伤口愈合。

DESCRIPTION (provided by applicant): A viable, transparent corneal model can provide the basis for studying novel ophthalmic drugs and new gene delivery approaches. Moreover, there is currently a clinical need for improved understanding of corneal wound-healing mechanisms in order to solve corneal haze problems associated with LASIK procedures and outcomes in corneal transplants. An understanding of the factors that contribute to the expression of the wound-healing phenotype in the cornea can lead to an understanding of how to control these changes. The overall goal of this proposed project is to understand and control the relationship between corneal cell behavior and resulting transparency in a tissue-engineered corneal model. A number of input signals to the cells have been characterized, including chemical, mechanical, electromagnetic, and topographical signals. The results of these studies taken together indicate that physical signals are more promising than chemical signals for the control of the wound-healing response in corneal fibroblasts. The proposed studies aim at understanding the differential, and perhaps more importantly, the synergistic effects of several signals in order to determine which signals or signal combinations are most successful as we move toward our ultimate goal of a three-dimensional corneal model. Cells will be analyzed for protein levels by western blot, mRNA levels will be analyzed by quantitative real-time PCR (qR-T PCR), and the extent of light scattering will be determined by optical coherence microscopy (OCM). Specifically, we will assess signaling associated with the wound healing response, integrin signaling, alpha-SMA, TKT, ALDH1, and matrix remodeling markers associated with changes in the keratocyte phenotype under the various treatment conditions detailed in this proposal. Cell behavior will be assessed to understand (1) the relative importance of matrix alignment and matrix composition; (2) the effect of matrix stiffness; and (3) the potentially synergistic effectsof light and mechanical strain input signals. By understanding the relative strengths of these input signals, we can more intelligently construct a transparent corneal matrix and model. Overall, these studies will lead to a better understanding of the role of biomimicry in the design and implementation of tissue-engineered systems. In addition, they will contribute to the understanding of wound healing in fibroblastic cells.