Corneal collagen crosslinking in vitro: Inhibited regeneration of human limbal epithelial cells after riboflavin–ultraviolet-A exposure

Purpose To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet-A (UVA) may have a toxic effect on human limbal epithelial cells. Setting Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ulleval, Oslo, Norway. Design Experimental study. Methods In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin–UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki-67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay. Results The mean outgrowth from the biopsies was 2.25 mm 2 ± 6.90 (SD) in the riboflavin–UVA group, 181.4 ± 94.8 mm 2 in the riboflavin-only group, 128.5 ± 129.5 mm 2 in the UVA-only group, and 176.2 ± 114.0 mm 2 in the control group. There were no statistically significant between-group differences in the number of cell layers except in the riboflavin–UVA group, in which no cells were found. Detection of apoptosis with the TUNEL-assay was found in the riboflavin–UVA group only (4/5 sections). The proliferation marker Ki-67 was positive in some sections in all groups. Conclusion Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin–UVA treatment in vitro, emphasizing the importance of avoiding riboflavin–UVA on the limbus during CXL. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.