Construction of Conjunctival Equivalents Using Molecular Deposition Techniques

摘要 A1-R01 资助名称：利用分子沉积构建结膜等效物 技巧 具有杯状、复层鳞状和未分化细胞的强健、功能性结膜对于结膜至关重要。 透明的角膜和健康的前眼。眼表疾病，包括传染性（沙眼）、 自身免疫性疾病（眼部瘢痕性类天疱疮）和遗传性疾病，以及烧伤和史蒂文斯-约翰逊病 综合征，可通过疤痕损害眼表，尤其是结膜。虽然结膜 具有巨大的修复潜力，这些疾病是痛苦的炎症性疾病，患者 迫切需要更好的治疗方法。目前的治疗方法包括使用多余的结膜或羊膜 膜。新型生物相容性材料的开发正在为以下领域提供改进的治疗方法 再生医学。对于这个提案，我们将利用新开发的分子层 沉积（MLD）技术构建生物活性材料涂层。我们的总体目标是开发一个 由三种类型的人类结膜组成的可移植和功能性结膜结构 上皮细胞（未分化、复层鳞状和杯状）在我们的新型表面上生长。表面是 基于沉积在丝素蛋白基质上的 MLD 薄膜，易于进行手术移植。 对于 SA1，我们将表征在 MLD 丝结构上生长的结膜上皮细胞的表型，并 将其与羊膜上生长的细胞进行比较。对于SA2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，并且 将它们与羊膜上生长的细胞进行比较。对于 SA3，我们将测量修复响应 与羊膜相比，MLD 丝结构上的细胞机械损伤。细胞计数， 分子和免疫荧光显微镜技术将用于评估 细胞生长。物理技术将用于确定结构强度。 Ca2 成像、ELISA MUC5AC 分泌和其他生理技术将用于确定细胞的功能 构建。免疫荧光显微镜和多细胞因子测量将用于评估伤口 治愈。在 MLD 丝上生长的三种类型的结膜上皮细胞的最终产品将具有功能性，并且 可移植并且非常适合修复患病和受损的结膜。

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.