Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro

Purpose Collagen crosslinking using ultraviolet-A (UVA) - irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone. Methods Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm(2) and with UVA alone between 2 and 9 mW/cm(2) for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypanblue and Yopro-fluorescence staining. Results An abrupt cytotoxic irradiance level was found at 0.5 mW/cm(2) for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/ cm 2 after UVA-irradiation alone. Conclusions A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm(2), which is reached in the clinical setting in human corneas down to a depth of 300 mm using the standard surface UVA-irradiance of 3 mW/cm(2).