Impact of riboflavin-UVA-photodynamic inactivation ( PDI ) ( collagen crosslinking technique ) on viability , cell cycle phase , apoptosis and proliferation of human corneal endothelial cells

The purpose of our study was to determine the impac t of riboflavin-UVA photodynamic inactivation (PDI) (Collagen crosslinking technique) on viability, cell cycle ph ase, apoptosis and proliferation of human corneal e ndothelial cells (HCECs), in vitro. A HCEC line was cultured in DMEM /Ham's F12 medium supplemented with 5% fetal calf s erum. HCECs cultures underwent 370 nm-UVA-light illuminat on for 4.1 minutes during exposure to 0.05% or 0.1 % riboflavin and 20% dextran containing PBS. Twenty-f our hours after riboflavin-UVA-PDI, viability was d etermined by the Alamar blue assay, cell cycle phase and apop tosis of the cells using the APO-DIRECT TM Kit, and two and twenty-four hours after PDI, HCECs proliferation by the BrdU Cell Proliferation Assay Kit. Twenty-four hours after the use of 0.1% riboflavin concentration without il lumination and after 0.05% and 0.1% riboflavin-UVAPDI, HCECs viability decreased significantly (P<0.01 for all) compared to controls. Twenty-four hours follo wing riboflavin-UVA-PDI, the percentage of HCECs at the G1 cell cycle phase decreased significantly using 0.0 5% or 0.1% riboflavin concentration (P=0.02 and P=0.03), the percentage of HCECs at the G 2/M phase increased significantly using 0.05% riboflavin concentration (P=0.03), compared to controls. Two and twenty-four hours after riboflavin-UVA-PDI using 0.05% or 0.1% riboflavin c oncentration, HCEC proliferation decreased signific antly (P=0.02 for all). There was no significant differen ce in percentage of apoptotic HCECs at any of the t reated groups compared to controls 24 hours after riboflavin-UVAPDI (P=0.10).Crosslinking arrests HCECs at the G 2/M phase, decreases viability and proliferation, however does not trigger apoptosis of human corneal endothelial cel s in vitro.