EPITHELIALIZATION OF TISSUE ENGINEERED CORNEAS

描述(改编自申请人的摘要)：尝试增加和 用合成材料替代角膜遇到了有限的困难 成功主要是因为这些设备一直无法支持和 保持正常的复层上皮。根本的问题是 宿主上皮细胞将聚合物表面识别为 间质或“自我”，因此，异物反应的程度是 在植入物的整个生命周期中都存在。尽管许多曲面都有 被测试允许一些上皮细胞生长，由此产生的上皮细胞 一般是不规则的，缺乏正常的三层外观。 尝试吸附生长因子(GFS)和细胞外基质 这些材料表面的蛋白质(ECMP)并没有改善 上皮细胞的反应是因为1)这些生物聚合物的吸附 对合成材料是非特定的，因此他们躺在 许多不同构象的表面，2)一些生物聚合物 不可逆地结合到人造表面，以及3)生物聚合物- 涂层表面，当放置在眼睛中时，本身就是由非特异性涂层 泪液中的蛋白质。申请者提议调查 这些问题是否可以通过捆绑细胞外基质来避免 蛋白质(ECMP)和生长因子(GFS；层粘连蛋白、纤维连接蛋白、 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，这种拴系应该 显著增强上皮细胞对以下各项的反应 理由。系绳(或将ECMP和GFS固定到 水凝胶表面)将使生物物质远离 聚合物表面因此保持其天然构象状态。 此外，系绳将使ECMP和GFS远离水凝胶 表面；它们不会被非特异性蛋白结合所遮挡 放置在撕裂环境中。专门的实验旨在 验证1)系绳改性水凝胶表面可以开发为 保持系留分子的细胞黏附活性，以及2) 系绳修饰的表面增强了上皮细胞的粘附性，并 在水凝胶表面迁移。方法包括衰减 全反射傅里叶变换红外光谱(ATR-FTIR) 为表征水凝胶表面，酪氨酸免疫化学 磷酸化和F-肌动蛋白，喷射撞击以确定细胞 层粘连蛋白、大疱性类天疱疮的黏附和免疫组织化学 抗原和IV型胶原。这些研究的结果将提供 深入了解角膜手术成功或失败的过程 人工植入物的增强和替换。

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.