Tissue Engineering of a Biomechanically Stable Conjunctival Substitute for Fornix Reconstruction

严重的结膜和角膜瘢痕可在许多眼表疾病中发现，在严重的情况下会导致完全的角膜混浊和失明。在这些眼睛中，如果不首先重建结膜穹窿，通过角膜移植进行视力恢复的方法经常失败。羊膜（AM）已被证明是一种合适的结膜替代品，因为它薄，有弹性，耐受性好，不会引起，但可以减少局部炎症。然而，在持续炎症（如粘膜类天疱疮）的眼睛中，由于存在蛋白水解酶和缺乏干细胞，AM移植物通常会迅速溶解，导致缺乏再上皮化。通过与戊二醛交联可显著提高AM的生物力学强度和抗酶消化能力，并可增强其再上皮化。本项目的目的是建立一个优化的AM交联协议，以模拟人类结膜的生物力学特性；用交联AM对结膜、口腔和鼻上皮进行体外组织工程测试；评价交联AM对三种不同上皮细胞增殖、迁移、分化、活力和干细胞存活的影响；并重新测试上皮化结构体的生物力学特性。该项目的最终目标是获得临床使用的生物力学稳定的结膜结构，并在临床试验中对其进行测试。

Severe conjunctival and corneal scarring can be found in many ocular surface disorders and in severe cases results in complete corneal opacification and blindness. In these eyes visual rehabilitation by means of corneal transplantation frequently fails, if the conjunctival fornix is not reconstructed first. Amniotic membrane (AM) has been shown to be a suitable conjunctival substitute, as it is thin, elastic, well tolerated and does not cause, but reduces local inflammation. However, in eyes with persistent inflammation such as mucous membrane pemphigoid AM grafts often rapidly dissolves due to the presence of proteolytic enzymes and the absence of stem cells, resulting in a lack of re-epithelialization. The biomechanical strength and resistance of the AM against enzymatic digestion can be significantly increased by cross-linking its collagen matrix with glutaraldehyde and reepithelialisation can be enhanced using epithelialized AM. Aims of this project are to establish an optimized cross-linking protocol for AM to mimic the biomechanical properties of human conjunctiva; to test conjunctival, oral and nasal epithelium on cross-linked AM for tissue engineering of a conjunctival equivalent in vitro; to evaluate the effect of crosslinked AM on proliferation, migration, differentiation, viability and stem cell survival of the three different epithelia; and to retest the biomechanical properties of the epithelialized construct. The ultimate goal of the project is to obtain a biomechanically stable conjunctival construct for clinical use and to test it in a clinical trial.