Engineering biomimetic corneal constructs

描述(申请人提供)：在美国，每年进行超过45,000例角膜移植手术。这种手术的成功率相当高(两年后为90%)，虽然目前获得供体组织的途径充足，但组织的质量差异很大，这影响了手术结果。此外，LASIK手术的扩散使角膜失去了移植资格，在不久的将来可能会减少供体角膜的供应。近年来，通过组织工程学制造角膜替代物的尝试值得称道。这些构造证明了这样一个概念，即类似于上皮、角质细胞和内皮的三层细胞可以培养成胶原基质。然而，这种结构只取得了有限的成功，因为为角膜提供独特(和至关重要的)机械和光学特性的基质基质尚未被复制。随机取向的胶原凝胶代表了组织工程角膜的典型起点，但不太可能足够坚固或足够透明，不适合临床使用。此外，预计在体内会有显著的 整合部分功能人工角膜的重塑反应是不可接受的。人造的 结构应在植入时起作用。基于这些原因，我们提出了一种以基质为中心的方法来生成人工角膜。通过将工程学、生物学、生物力学和生物化学相结合，将尝试两种不同的路线，都是为了生产仿生基质层(人造角膜的构建块)。第一种方法使用微流体来影响由胶原和蛋白多糖组成的排列整齐的薄片的自组装。这种方法是无细胞的，旨在产生一种新的仿生基质支架。第二种方法利用机械提示，如接触指导(由 通过抗坏血酸刺激的人角膜成纤维细胞来“指导”基质的合成。本课程将对人工合成刺激的反应进行广泛的表征，以获得对高度结构基质的合成、重塑和自稳的基本了解。完成这项提案的两个部分将提供对实现我们的最终目标至关重要的洞察力，即从天然成分体外生成具有功能的仿生人工角膜。

DESCRIPTION (provided by applicant): Every year in the United States, over 45,000 corneal transplants are performed. The success rate for this procedure is fairly high (90% after two years) and although current access to donor tissue is adequate, the quality of tissue varies significantly which influences surgical outcomes. In addition, the proliferation of LASIK procedures, which disqualifies a cornea for transplantation, threatens to reduce the availability of donor corneas in the near future. 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 into a collagen matrix. However, such constructs have only met with limited success because the stromal matrix, which provides the cornea with its unique (and critically important) mechanical and optical properties, has not been reproduced. Randomly oriented collagen gels, which represent the typical starting point for tissue-engineered corneas, are not likely to be strong enough or clear enough for clinical use. In addition, expecting a significant in vivo remodeling response to integrate a partially functioning artificial cornea is not acceptable. The artificial construct should be functional at the time of implantation. For these reasons, we propose a stromal-centric approach toward the generation of an artificial cornea. By combining engineering, biology, biomechanics and biochemistry, two different routes, both designed to produce biomimetic stromal lamellae (the building blocks for an artificial cornea) will be attempted. The first method employs microfluidics to influence the self-assembly of thin, aligned lamellar sheets comprising collagen and proteoglycans. This approach is acellular and is designed to produce a biomimetic stromal scaffold de novo. The second approach utilizes mechanical cues such as contact guidance (provided by the de novo lamellae) and strain to "direct" the synthesis of matrix by ascorbic acid stimulated human corneal fibroblasts. An extensive characterization of the synthetic response to the imposed stimuli will be conducted to gain a fundamental understanding of synthesis, remodeling and homoeostasis of highly structured matrix. Completion of both parts of this proposal will provide insight critical to achieving our ultimate goal, which is the ex vivo generation of a functional, biomimetic artificial cornea from natural components.