Engineering biomimetic corneal constructs

描述（由申请人提供）：在美国，每年进行超过45000例角膜移植。由于角膜组织中的免疫反应相对温和，这种手术的成功率相当高（两年后为90%）。目前，供体组织的获取是足够的，然而，供体组织的质量差异很大，影响手术结果。此外，LASIK手术的激增使角膜不符合移植目的，这可能会减少未来供体角膜的可用性。最后，在世界其他地方，有600多万角膜失明病例，获得捐赠组织的机会受到严重限制。 近年来，通过组织工程学方法制备角膜替代物的尝试值得称赞。这些构建体已经证明了这样的概念，即类似于上皮、角膜细胞和内皮的三层细胞可以在胶原基质上和胶原基质内培养延长的时间。然而，这种结构不能用于临床，主要是因为不能复制为角膜提供机械强度和透明度的基质基质， 该提议采用基质中心方法来产生人工角膜构造。角膜基质包括排列的35 nm胶原原纤维的多层（薄片），所述胶原原纤维一个堆叠在另一个上，在每个连续的薄片中纤维取向旋转90度。最近，使用微流体方法，已经通过在表面上自组装胶原原纤维来产生人工从头“laminate”，在所述表面上存在胶原单体的极薄流动膜。单独使用该技术或作为组织工程的模板，将尝试产生用于角膜修复或置换的合适的基质支架。 第一种方法提出通过将许多人工产生的从头“层”彼此正交地堆叠以产生厚的基质构建体来产生基质支架。第二种方法使用一些这些从头laminate的影响，通过接触指导，抗坏血酸刺激角膜成纤维细胞产生的基质的结构。在通过每种方法生成厚基质支架之后，将上皮和内皮在工程化基质上培养以产生人工角膜。将对这两种结构进行测试，以确定其作为角膜替代物的潜在效用。

DESCRIPTION (provided by applicant): Every year in the United States, over 45000 corneal transplants are performed. The success rate for this procedure, owing to the relatively modest immune response in corneal tissue, is fairly high (90% after two years). Currently, access to donor tissue is adequate, however, the quality of donor tissue varies significantly, affecting surgical outcomes. In addition, the proliferation of LASIK procedures, which disqualifies a cornea for transplantation purposes, threatens to reduce the availability of donor corneas in the future. Finally, access to donor tissue elsewhere in the world, where there are over six million cases of corneal blindness, is severely limited. In recent years, laudable attempts have been made to produce corneal equivalents by tissue engineering. These constructs have proven the concept that three layers of cells, resembling the epithelium, keratocytes and endothelium may be cultured onto and within a collagen matrix for extended periods. However, such constructs cannot be used in the clinic primarily because the stromal matrix, which provides the cornea with mechanical strength and transparency cannot be reproduced, This proposal takes a stromal centric approach to generation of an artificial corneal construct. The corneal stroma comprises multiple layers (lamellae) of aligned 35 nm collagen fibrils stacked one upon another with fiber orientation rotating 90 degrees in each successive lamella. Recently, using microfluidics methods, artificial de novo "lamellae" have been generated by self-assembling collagen fibrils on a surface over which there is an extremely thin flowing film of collagen monomers. Using this technique alone or as a template for tissue engineering, the generation of a suitable stromal scaffolding for use in corneal repair or replacement, will be attempted. The first approach proposes to generate a stromal scaffolding by stacking many artificially generated, de novo "lamellae" orthogonally to one another to produce a thick stromal construct. The second approach uses a few of these de novo lamellae to influence, through contact guidance, the structure of matrix produced by ascorbic acid stimulated corneal fibroblasts. Following generation of thick stromal scaffolds by each method, epithelium and endothelium will be cultured on to the engineered stroma to produce an artificial cornea. The two constructs will be tested to determine their potential utility as corneal replacements.