Construction of Conjunctival Equivalents Using Molecular Deposition Techniques

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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.