Engineering Biomimetic Corneal Constructs

描述（由申请人提供）：在美国，每年有超过33,000例角膜移植手术。该手术的成功率相当高（两年后达到90%），尽管目前获得供体组织是足够的，但组织质量差异很大，这影响了手术结果。此外，LASIK手术的普及使角膜不适合移植，在不久的将来可能会减少角膜供体的可用性。近年来，通过组织工程制造角膜等效物的尝试值得称赞。这些结构证明了类似于上皮细胞、角质细胞和内皮细胞的三层细胞可以培养成胶原基质的概念。然而，这种结构只遇到了有限的成功，因为基质基质，它提供了角膜独特的（和至关重要的）机械和光学特性，还没有被复制。随机导向的胶原蛋白凝胶代表了组织工程角膜的典型起点，但对于临床应用来说，它可能不够牢固或不够清晰。此外，期望有显著的体内重塑反应来整合部分功能的人工角膜是不可接受的。人工构造物在植入时应具有功能。基于这些原因，我们提出了一种以基质为中心的人工角膜生成方法。我们首先通过跟踪人类成纤维细胞在长期实时成像培养系统中产生基质的过程，精确地研究成纤维细胞如何在体外产生有组织的组织。然后结合生物工程、生物学、生物力学和生物化学，尝试制造仿生基质薄片（人造角膜的基本材料）。该方法需要使用已经“编码”到胶原蛋白三螺旋中的智能，只需通过浓缩单体来诱导液晶结构的形成和纤维的形成，就可以产生有组织的原纤维阵列。由此产生的有组织的原纤维阵列将作为全面研究基质分子对胶原原纤维形态和间距的作用的起点。一旦我们的阵列被充分表征，人类角膜成纤维细胞和人类脐带血来源的干细胞将被植入其中，并暴露在机械刺激下。我们期望在两种细胞群中诱导分化。这项应用的完成将为理解角膜基质发育的基础科学提供见解，并实现我们的最终目标，即从天然成分中产生功能性的仿生人工角膜。公共卫生相关性：这项应用的完成将为理解角膜基质发育的基础科学提供见解，并实现我们的最终目标，即从天然成分中产生功能性仿生人工角膜。鉴于生物材料工程和干细胞研究的最新进展（在本应用中结合），我们期望最终提高临床医生的能力，为患有严重疾病的患者提供基于工程组织的可行替代治疗方案。

DESCRIPTION (provided by applicant): Every year in the United States, over 33,000 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. We start by investigating precisely how fibroblastic cells produce organized tissue in vitro by tracking human fibroblasts live as they produce matrix on a long-term live imaging culture system. Then by combining bioengineering, biology, biomechanics and biochemistry an attempt will be made to produce biomimetic stromal lamellae (the building blocks for an artificial cornea). The method entails using the intelligence already "encoded" into the collagen triple helix which produces organized arrays of fibrils simply by concentrating the monomers to induce liquid crystalline structure formation and fibrillogenesis. The resulting organized arrays of fibrils will be used as starting point for comprehensive investigation into the role of matrix molecules on collagen fibril morphology and spacing. Once our arrays are well-characterized, human corneal fibroblasts and human cord blood derived stem cells will be seeded into them and exposed to mechanical stimulation. We expect to induce differentiation in both populations of cells. Completion of this application will provide insight both to the basic science of understanding corneal stromal development and to achieving our ultimate goal, which is the ex vivo generation of a functional, biomimetic artificial cornea from natural components. PUBLIC HEALTH RELEVANCE: Completion of this application will provide insight both to the basic science of understanding corneal stromal development and to achieving our ultimate goal, which is the ex vivo generation of a functional, biomimetic artificial cornea from natural components. Given recent advances in biomaterials engineering and stem cell research (which are combined in this application) we expect to ultimately enhance the ability of clinicians to offer patients with significant morbidity viable alternative treatment options based on engineered tissue.