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，严重的病例可导致完全角膜混浊和失明。在这些眼中，如果不首先重建结膜穹窿，通过角膜移植进行视力康复常常失败。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，但会减少局部炎症。然而，在具有持续性炎症（例如粘膜类天疱疮）的眼睛中，由于蛋白水解酶的存在和干细胞的缺乏，AM移植物通常迅速溶解，导致缺乏上皮再形成。AM对酶消化的生物力学强度和抗性可以通过用戊二醛交联其胶原基质而显著增加，并且可以使用上皮化AM来增强上皮再生。本课题的目的是建立一种模拟人结膜生物力学特性的AM交联方法，并在体外对交联AM上的结膜、口腔和鼻腔上皮进行组织工程实验，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，并对交联AM的生物力学特性进行评价。并重新测试上皮化结构的生物力学性能。该项目的最终目标是获得一种生物力学稳定的结膜结构用于临床，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.