Lens Epithelial Cell Response to Biomaterial Interfaces

项目摘要 白内障仍然是世界范围内致盲的主要原因，每年有超过300万例白内障摘除手术 仅在美国。在白内障手术期间，通过一个称为“晶状体囊”的装置移除透镜囊内的内容物。 前透镜囊中的孔，并且将聚合物眼内透镜（IOL）放置在囊中。领导 当残留的透镜上皮细胞 (LEC)从前部迁移到后透镜囊或IOL上，并经历上皮细胞- 间充质转化（EMT）。虽然影响机械生物学和上皮细胞反应的几个因素 以前已经研究过，对粘弹性的影响没有清楚的了解， 曲率对LEC行为的影响。该项目的总体目标是使用聚合物和水凝胶基质， 分别模拟植入物和透镜微环境，以更好地分析机械影响 性质对LEC响应和EMT的影响。据推测， 在去除透镜组织和放置IOL后，微环境改变，促进LEC中的EMT。 在目标1中，将使用可调聚合物基底和水凝胶来研究刚度的影响， 粘弹性对LEC响应和EMT的影响。假设基底比透镜囊更硬，并且 具有较低损耗角正切的衬底将驱动LEC中的EMT。在目标2中，衬底曲率的影响将是 使用相同的聚合物和水凝胶基质进行研究。主导假设是LEC迁移 和EMT是由IOL植入后透镜囊变平引起的较大曲率半径驱动的。 将使用具有不同曲率半径的微图案化聚合物来评估曲率效应。玻璃 将各种尺寸的微珠嵌入水凝胶制剂中，模拟透镜中的变化 手术后的胶囊形状。在这两个目标中，将使用相关的体外和离体模型。LEC增殖， 迁移和EMT标志物。TGF-β和雷帕霉素将用作阳性和阴性 EMT的诱导剂。RT-PCR将定量基因表达，蛋白质表达的变化将被定量。 使用Western印迹和免疫荧光进行评价。将被评估的特定基因和蛋白质 包括SMAD信号蛋白、α-SMA、Slug、Snail、纤连蛋白、E-钙粘蛋白和雅普。这个项目的目标 是为了确定基板的机械性能，即粘弹性和曲率，如何有助于LEC EMT的行为和诱导。这将大大提高我们对LEC机械生物学的认识， 这些因素在EMT中的作用，提出了预防病理性EMT的策略。结果将导致未来 研究设计材料以预防植入后EMT和纤维化，特别是预防PCO。

PROJECT SUMMARY Cataract remains the leading cause of blindness worldwide with over 3 million extractions performed each year in the United States alone. During cataract surgery, the contents inside the lens capsule are removed through a hole in the anterior lens capsule and a polymeric intraocular lens (IOL) is placed in the capsule. The leading vision-threatening complication, posterior capsule opacification (PCO), occurs when residual lens epithelial cells (LEC) migrate from the anterior to the posterior lens capsule or onto the IOL and undergo epithelial-to- mesenchymal transition (EMT). While several factors impacting mechanobiology and epithelial cell response have been previously investigated, there is not a clear understanding of the impact of viscoelasticity and curvature on LEC behavior. The overall objective of this project is to use polymer and hydrogel substrates that mimic the implants and lens microenvironment, respectively, to better analyze the influence mechanical properties have on LEC response and EMT. It is hypothesized that the physical and mechanical properties of the microenvironment are altered after the removal of the lens tissue and IOL placement, facilitating EMT in LEC. In Aim 1, tunable polymer substrates and hydrogels will be used to investigate the impact of stiffness and viscoelasticity on LEC response and EMT. It is hypothesized that substrates stiffer than the lens capsule, and substrates with lower loss tangent will drive EMT in LEC. In Aim 2, the effect of substrate curvature will be investigated using the same polymer and hydrogel substrates. The governing hypothesis is that LEC migration and EMT are driven by larger radius of curvature caused by flattening of the lens capsule after IOL implantation. Curvature effects will be evaluated using polymers micropatterned with different radii of curvature. Glass microbeads of various sizes will be embedded in hydrogel formulations, mimicking the changes in the lens capsule shape following surgery. In both aims, relevant in vitro and ex vivo models will be used. LEC proliferation, migration, and markers for EMT will be assessed. TGF-β and rapamycin will be used as positive and negative inducers of EMT, respectively. RT-PCR will quantify gene expression, and changes in protein expression will be evaluated using Western blot and immunofluorescence. Specific genes and proteins that will be evaluated include SMAD signaling proteins, α-SMA, Slug, Snail, fibronectin, E-cadherin, and YAP. The goal of this project is to determine how substrate mechanical properties, namely viscoelasticity and curvature, contribute to LEC behavior and induction of EMT. This will significantly enhance our knowledge of LEC mechanobiology and the role of these factors in EMT, suggesting strategies to prevent pathological EMT. The results will lead to future research on design of materials to prevent EMT and fibrosis after implantation, particularly for preventing PCO.