EPITHELIALIZATION OF TISSUE ENGINEERED CORNEAS

描述（改编自申请人摘要）：尝试增大和 用合成材料代替角膜， 成功主要是因为这些设备无法支持和 维持正常的复层上皮。根本的问题是 宿主上皮细胞将聚合物表面识别为除了 基质或“自身”，因此，一定程度的异物反应， 在植入物的整个使用寿命期间都存在。尽管许多表面具有 允许一些上皮生长，由此产生的上皮 通常是不规则的，缺乏正常的三层外观。 尝试吸附生长因子（GF）和细胞外基质 这些材料表面上的细胞外基质蛋白（ECMPs）没有得到改善 上皮细胞反应是因为1）这些生物聚合物的吸附 合成材料是非特异性的，因此他们躺在 表面有许多不同的构象，2）一些生物聚合物 与人造表面不可逆地结合，和3）生物聚合物- 当置于眼睛中时，涂层表面本身被非特异性 泪液中的蛋白质申请人建议调查 这些问题是否可以通过将细胞外基质 蛋白质（ECMP）和生长因子（GF;层粘连蛋白，纤连蛋白， P物质和胰岛素样生长因子-1（IGF-1）） 水凝胶（聚合物）表面。他们预测，这种束缚应该 显著增强上皮细胞对以下方面的反应 原因系链（或将ECMP和GF紧固到 水凝胶表面）将保持生物物质远离 聚合物表面，从而保持其天然构象状态。 此外，系绳将保持ECMP和GF远离水凝胶 它们不会被非特异性蛋白质结合所掩盖， 放置在撕裂环境中。具体的实验旨在 验证1）可以开发出栓系改性的水凝胶表面， 保留了系留分子的细胞粘附活性，和2） 系链修饰的表面增强上皮细胞粘附， 穿过水凝胶表面的迁移。方法包括衰减 全反射-傅里叶变换红外光谱（ATR-FTIR） 为了表征水凝胶表面，酪氨酸的免疫化学 磷酸化和F-肌动蛋白，射流冲击，以确定细胞 粘附和层粘连蛋白免疫组化，大疱性类天疱疮 抗原和胶原VII。这些研究的结果将提供 深入了解角膜移植成功或失败的过程 通过人工植入物来增强和替换。

DESCRIPTION (Adapted from applicant's abstract): Attempts to augment and replace the cornea with synthetic materials have met with limited success primarily because these devices have been unable to support and maintain a normal stratified epithelium. The fundamental problem is that the host epithelial cells recognize the polymer surface as other than stroma or "self" and, therefore, a degree of foreign body response is present throughout the life of the implant. Although many surfaces have been tested that allow some epithelial growth, the resulting epithelium is generally irregular, lacking the normal tri-layer appearance. Attempts to adsorb growth factors (GFs) and extracellular matrix proteins (ECMPs) onto the surfaces of these materials have not improved the epithelial cell response because 1) adsorption of these biopolymers to synthetic materials is non-specific and therefore they lay down on the surface in many different conformations, 2) some of the biopolymers bind irreversibly to the artificial surface, and 3) the biopolymer- coated surface, when placed in the eye, is itself coated by non-specific protein from the tear fluid. The applicants propose to investigate whether these problems can be avoided by tethering Extracellular matrix proteins (ECMPs) and growth factors (GFs; laminin, fibronectin, substance P, and insulin-like growth factor-1 [IGF-1]) onto synthetic hydrogel (polymer) surfaces. They predict that this tethering should significantly enhance the epithelial cell response for the following reasons. The tethers (or chains that fasten the ECMPs and GFs to the hydrogel surface) will hold the biological substances away from the polymer surface thus preserving their native conformational state. Additionally, tethers will hold the ECMPs and GFs away from the hydrogel surface; they will not be obscured by non-specific protein binding when placed in the tear environment. Specific experiments are designed to verify 1) that tether-modified hydrogel surfaces can be developed that retain the cell-adhesion activity of the tethered molecules, and 2) that tether-modified surfaces enhance the epithelial cell adhesion and migration across the hydrogel surfaces. Methodology includes Attenuated Total Reflectance-Fourier Transformed Infrared Spectroscopy (ATR-FTIR) to characterize the hydrogel surfaces, immunochemistry for tyrosine phosphorylation and F-actin, jet impingement to determine cellular adhesion, and immunohistochemistry for laminin, bullous pemphigoid antigen, and collagen VII. The results of these studies will provide insight into the processes involved in the success or failure of corneal augmentation and replacement by artificial implants.