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

Jiong Wang, Tanja Stachon,Xuef

Abstract

The purpose of our study was to determine the impact of riboflavin-UVA photodynamic inactivation (PDI) (Collagen crosslinking technique) on viability, cell cycle phase, apoptosis and proliferation of human corneal endothelial cells (HCECs), in vitro. A HCEC line was cultured in DMEM/Ham's F12 medium supplemented with 5% fetal calf serum. HCECs cultures underwent 370 nm-UVA-light illumination for 4.1 minutes during exposure to 0.05% or 0.1% riboflavin and 20% dextran containing PBS. Twenty-four hours after riboflavin-UVA-PDI, viability was determined by the Alamar blue assay, cell cycle phase and apoptosis of the cells using the APO-DIRECTTM 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 illumination and after 0.05% and 0.1% riboflavin-UVA-PDI, HCECs viability decreased significantly (P<0.01 for all) compared to controls. Twenty-four hours following riboflavin-UVA-PDI, the percentage of HCECs at the G1 cell cycle phase decreased significantly using 0.05% or 0.1% riboflavin concentration (P=0.02 and P=0.03), the percentage of HCECs at the G2/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 concentration, HCEC proliferation decreased significantly (P=0.02 for all). There was no significant difference in percentage of apoptotic HCECs at any of the treated groups compared to controls 24 hours after riboflavin-UVA-PDI (P=0.10).Crosslinking arrests HCECs at the G2/M phase, decreases viability and proliferation, however does not trigger apoptosis of human corneal endothelial cells in vitro.

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