Ultrastructural study of collagen fibrils,proteoglycans and lamellae of the cornea treated with iontophoresis – UVA cross-linking and hypotonic riboflavin solution

To investigate the effects of iontophoresis–ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1), a UVA irradiance of 3 mW/cm² for 30 min; Group 2 (G2), a UVA irradiance of 10 mW/cm² for 9 min; Group 3 (G3), without UVA irradiation. Three untreated corneas were used as controls (G0). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm² for 30 min and 10 mW/cm² for 9 min was a suitable method of CXL; however, 3 mW/cm² for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.