Construction of Conjunctival Equivalents Using Molecular Deposition Techniques

摘要A1-R 01资助题目：使用分子沉积构建结膜等效物 技术 具有杯状、复层鳞状和未分化细胞的强健、功能性结膜对于眼结膜炎的发生至关重要。 透明的角膜和健康的前眼。眼表疾病，包括感染性（沙眼）， 自身免疫性（眼瘢痕性类天疱疮）和遗传性疾病，以及烧伤和Stevens-Johnson 综合征，可以损害眼表，特别是结膜，通过疤痕。虽然结膜 具有相当大的修复潜力，这些疾病是痛苦的炎症性疾病， 渴望更好的治疗目前的治疗方法包括使用多余的结膜或羊膜 膜的新型生物相容性材料的开发正在提供治疗领域的改进。 再生医学对于这个建议，我们将利用新开发的分子层 沉积（MLD）技术来构建生物活性材料的涂层。我们的总体目标是发展一个 由三种类型的人结膜组成的可移植的功能性结膜构建体 上皮细胞（未分化、复层鳞状和杯状）生长在我们的新表面上。表面 基于沉积在丝素蛋白基质上的MLD膜，其易于操作用于外科移植。 对于SA 1，我们将表征在MLD丝构建体上生长的结膜上皮细胞的表型， 与生长在羊膜上的细胞相比。对于SA 2，我们将评估三种类型的功能 上皮细胞在我们的MLD丝结构上生长，以响应细胞功能的生理刺激， 与生长在羊膜上的细胞进行比较。对于SA 3，我们将测量对 与羊膜相比，MLD丝构建体上细胞的机械损伤。细胞计数， 分子和免疫荧光显微镜技术将用于评估的数量和表型， 细胞生长将使用物理技术确定结构强度。Ca 2+成像，ELISA用于 MUC 5AC分泌和其他生理学技术将用于确定细胞在肿瘤细胞上的功能。 构建体将使用免疫荧光显微镜和多种细胞因子测量来评价伤口 治愈在MLD丝上生长的三种类型的结膜上皮细胞的最终产物将具有功能， 可移植的并且对于患病和受损的结膜的修复是极好的。

Abstract A1-R01 Grant Title: Construction of Conjunctival Equivalents Using Molecular Deposition Techniques A robust, functional conjunctiva with goblet, stratified squamous, and undifferentiated cells is critical for a transparent cornea and a healthy anterior eye. Ocular surface disorders, including, infectious (trachoma), autoimmune (ocular cicatricial pemphigoid), and genetic diseases, as well as burns and Stevens-Johnson syndrome, can damage the ocular surface, especially the conjunctiva, by scarring. Although the conjunctiva has substantial repair potential, these diseases are painful inflammatory diseases and the patients are desperate for better treatments. Current treatments include use of redundant conjunctiva or amniotic membrane. Development of novel biocompatible materials is providing improved treatments in the field of regeneration medicine. For this proposal we will take advantage of the newly developed molecular layer deposition (MLD) technique to build coatings of bioactive materials. Our overall goal is to develop a transplantable and functional conjunctival construct consisting of the three types of human conjunctival epithelial cells (undifferentiated, stratified squamous, and goblet) growing on our novel surface. The surface is based on an MLD film deposited on a silk fibroin matrix that is easily manipulated for surgical transplantation. For SA1 we will characterize the phenotype of conjunctival epithelial cells growing on MLD silk construct and compare it with cells growing on amniotic membrane. For SA2 we will evaluate the function of the three types of epithelial cells growing on our constructs of MLD silk in response to physiological stimuli of cell function and compare them with cells growing on amniotic membrane. For SA3 we will measure the repair response to a mechanical injury of the cells on constructs of MLD silk compared to amniotic membrane. Cell counting, molecular, and immunofluorescence microscopy techniques will be used to evaluate amount and phenotype of cell growth. Physical techniques will be used to determine construct strength. Ca2+ imaging, ELISAs for MUC5AC secretion, and other physiological techniques will be used to determine function of cells on the construct. Immunofluorescence microscopy and multi-cytokine measurements will be used to evaluate wound healing. The final product of three types of conjunctival epithelial cells grown on MLD silk will be functional, and transplantable and excellent for repair of the diseased and damaged conjunctiva.