Impact of crosslinking/riboflavin-UVA-photodynamic inactivation on viability, apoptosis and activation of human keratocytes in vitro.

ABSTRACT

Riboflavin-UVA photodynamic inactivation is a potential treatment alternative in therapy resistant infectious keratitis. The purpose of our study was to determine the impact of riboflavin-UVA photodynamic inactivation on viability, apoptosis and activation of human keratocytes in vitro. Primary human keratocytes were isolated from human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 10% fetal calf serum. Keratocytes underwent UVA light illumination (375 nm) for 4.10 minutes (2 J/cm²) during exposure to different concentrations of riboflavin. Twenty-four hours after treatment, cell viability was evaluated photometrically, whereas apoptosis, CD34 and alpha-smooth muscle actin (α-SMA) expression were assessed using flow cytometry. We did not detect significant changes in cell viability, apoptosis, CD34 and α-SMA expression in groups only treated with riboflavin or UVA light. In the group treated with riboflavin-UVA-photodynamic inactivation, viability of keratocytes decreased significantly at 0.1% riboflavin (P<0.01) while the percentage of CD34 (P<0.01 for both 0.05% and 0.1% riboflavin) and alpha-SMA positive keratocytes (P<0.01 and P<0.05 for 0.05% and 0.1% riboflavin, respectively) increased significantly compared to the controls. There was no significant change in the percentage of apoptotic keratocytes compared to controls at any of the used riboflavin concentrations (P=0.09 and P=0.13). We concluded that riboflavin-UVA-photodynamic-inactivation decreases viability of myofibroblastic transformation and multipotent haematopoietic stem cell transformation; however, it does not have an impact on apoptosis of human keratocytes in vitro.