Reconstructed corneas: effect of three-dimensional culture, epithelium, and tetracycline hydrochloride on newly synthesized extracellular matrix

PURPOSE: To evaluate the influence of the 3-dimensional collagen-glycosaminogycan-chitosan (CGC 3D) scaffold, epithelialization, and the addition of tetracycline hydrochloride on the ultrastructural organization, measured by the diameter and spacing of newly synthesized collagen I fibrils. METHODS: Little is known about the role of interactions between epithelial cells and fibroblasts in controlling the extracellular matrix of the cornea. We developed a hemicornea from a CGC 3D matrix cocultured with keratocytes and human epithelial cells. The keratocytes colonized this substrate, proliferated, and synthesized the extracellular matrix, reproducing a living stroma equivalent. RESULTS: Without a 3D scaffold, the collagen fibrils produced had an average diameter that was 42.7 nm and sigma = 16.9 nm. In the CGC 3D scaffold, the fibrils had an average diameter of 33.4 nm, with little dispersion (sigma = 6.7 nm), suggesting a greater regulation. The epithelium permitted a significant reduction in fibril diameter and interfibrillar spacing. Tetracycline hydrochloride had no effect on spacing but did have a significant effect on fibril diameter. We found positive interactions between the epithelium and tetracycline hydrochloride on the regulation of collagen fibrils but not on spacing. The presence of epithelium led to the increased formation of collagens I and V in the subepithelial area of the newly formed matrix. Type VI collagen was localized around the keratocytes throughout the matrix. CONCLUSIONS: Epithelialization and the 3D scaffold had a great influence on the diameter of collagen I fibrils.