Abstract
Background and aimKeratoconus is a slowly progressive and non-inflammatory disease of eye of unknown etiology characterized by significant structural degeneration, thinning and protrusion of cornea. Collagen cross linking using Riboflavin/ultraviolet A, paraformaldehyde and glutaraldehyde has been widely used to treat keratoconus, but these techniques have several clinical complications. This study aimed to develop a rapid, safer, less toxic and more effective method to increase the corneal stiffness hence treating keratoconus by using EDC/NHS coupling with pimelic acid (PA) as cross-linkers. MethodsThe viability of corneal epithelial and endothelial cells was examined using Alamar blue. The corneal collagen and tissue integrity were evaluated with Masson and hematoxylin and eosin stains respectively. Uniaxial tensile testing was conducted to determine the tissue stiffness. We further assessed the effective penetration depth of cross-linking by fluorescence dye and confocal microscopy. TUNEL assay was performed to detect the damaged DNA fragments in the presence of PA. ResultsThe effect of PA solution on corneal cross-linking showed a significant increase in corneal rigidity without affecting the epithelium integrity under neutralized pH condition, while DEC/NHS alone did not induce the mechanical property. The penetration depth showed consistent results with increased stiffness, which could be affected by the parameters including pH condition, coupling with or without PA. In addition, PA significantly decreased the percentage of TUNEL-positive cells compared to that of EDC/NHS only condition. The cytotoxic effect to endothelium was considered minimal and protected in the presence of PA. ConclusionsWe demonstrated PA as a potential alternative for cross-linking in the model of whole eye with intact epithelium and its protective role on endothelium. The administration of this chemical cross-linker stands as an extremely promising technique for the treatment of corneal keratoconus.
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