Keratocytes are the cells of the corneal stroma which are responsible for secretion and maintenance of the strong transparent connective tissue of the cornea. In response to bacterial or viral infections, inflammation or injury, keratocytes secrete fibrotic scar tissue that scatters incident light and reduces vision for millions of individuals worldwide. Our lab is using cultured corneal cells to investigate the biology of the wound healing response. We have identified three distinct phenotypes of cultured cells that represent the quiescent keratocytes of the normal cornea, the motile fibroblasts, that repopulate healing wounds, and myofibroblasts that secrete opaque scar tissue. Each of these cell types can be generated by mitogens in cell culture. Each has a gene expression profile that exhibits characteristic molecular markers for the phenotype. One set of molecules that are markedly modified during the healing process healing are proteoglycans, large extracellular matrix components that are essential for transparent corneal tissue. A currently NIH funded project in our lab is aimed at identifying the genes involved in biosynthesis of the proteoglycans, specifically the transferases that add carbohydrates and sulfate to the high molecular weight carbohydrates of the proteoglycans We are characterizing transcription and promoter functions of these genes and examining gene function in cultured cells by manipulation gene expression using overexpression, dominant negative, and interference RNA technology. Biological roles of these genes are being examined in vivo by manipulating expression in transgenic mice.

In a second project we are committed to development of a bioprosthetic cornea. Cornea is one of the most successfully transplanted organs however predictions show that need for corneal transplants will soon outpace donor tissue. Fully synthetic non-biologic corneas are under development but currently the synthetic materials do not integrate well into the ocular environment. Corneal cells in culture show many characteristics of corneal cells in vivo, but these cultures do not secrete enough extracellular matrix to produce the transparent and strong tissue required for a functional cornea. Our lab is investigating the hypothesis that keratocytes embedded in a three dimensional scaffolding surrounded by the other cellular components of cornea will generate connective tissue similar to that made in vivo. For scaffolding we are using synthetic hydrogels generated from hydrolysable polymers and acellular tissue matrix from animal corneas. Simultaneously we are developing a source for cells in these assemblies by examining means of expanding cultured corneal cells and means of adapting stem cells to assume corneal phenotypes.