Acellular Tissue Matrix for Bioprosthetic Corneas

描述（由申请人提供）：供体组织的有限性限制了全世界数百万人获得角膜移植的机会。人们正在努力制造用于移植的人造角膜和生物工程角膜，然而，目前还没有理想的材料作为基质。合成基质不能很好地融入眼环境，人工胶原蛋白凝胶缺乏足够的强度和透明度。该项目的长期目标是开发由活角膜细胞组装而成的假角膜。这个初始阶段测试了一个假设，即无细胞的非人类角膜基质将提供一个有效的支架，在这个支架上，类似的角膜可以在体外组装。该项目将确定牛基质组织的脱细胞和提取程序，以消除细胞碎片，最大化角质细胞的组织再生，并支持角质细胞分化功能的表达。在日益复杂的生物环境中，将有三个特定的目标来测试基质的这些特性：(1)研究通过各种方法制备的脱细胞基质对体外角质细胞再生和分化功能的影响。角膜将通过冷冻、低渗酵解、酶消化和洗涤剂萃取的组合来脱细胞。相关的组织抗原和生长因子将通过各种提取程序去除。然后将这些脱细胞组织用作原代和传代角质细胞的培养基质。角化细胞分化表型标志物的表达将被检测。(2)评估上皮细胞和内皮细胞在决定细胞分化功能中的作用

DESCRIPTION (provided by applicant): Restricted availability of donor tissue limits access to corneal transplantation for millions of individuals worldwide. Efforts are under way to produce artificial keratoprostheses and bio-engineered corneas for transplantation, however, currently there is no ideal material to serve as stroma. Synthetic substrates do not integrate well into the ocular environment and artificial collagen gels lack adequate strength and transparency. The long term goals of the project are to develop prosthetic cornea assembled from living corneal cells. This initial phase tests the hypothesis that acellular non-human corneal stromal matrix will provide an effective scaffolding on which a corneal equivalent can be assembled invitro. The project will identify decellularization and extraction procedures for bovine stromal tissue that eliminates cellular debris, maximize tissue repopulation by keratocytes, and supports expression of keratocyte differentiated function. Three specific aims will test these properties of stromal matrix in increasingly complex biological environments: (1) Examine the influence of acellular stromal matrix prepared by various procedures in promotion of repopulation and differentiated function of keratocytes in vitro. Corneas will be decellularized by a combination of freezing, hypotonic lysis, enzyme digestion, and detergent extraction. Associated tissue antigens and growth factors will be removed by a variety of extraction procedures. These decellularized tissues will then be used as culture matrix for primary and passaged keratocytes. Expression of markers of keratocyte differentiated phenotype will be examined. (2) Assess the role of epithelial and endothelial cells in the determining the differentiated functions of keratocytes in acellular stromal matrix. Matrix populated with keratocytes will be assembled into a three dimensional corneal equivalent with primary epithelial and endothelial cells. Stromal deturgescence, epithelial differentiation and basal lamina formation will be examined in addition to keratocyte function. (3) Compare the re-cellularization, transparency, and tissue rejection of acellular stromal matrix preparations in interlamellar grafts in vivo in mice. Acellular matrix successful in previous procedures will be transplanted in a mouse interlamellar model. The transplants will be examined for tissue rejection, recellularization, and transparency. These three aims will establish the suitability of stromal matrix as a scaffold for prosthetic corneas and determine the most effective methods for its preparation.