title: 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.
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar 66424, Germany. tanja.stachon@uks.eu.
authors.forename: Tanja
authors.initials: T
authors.lastname: Stachon
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar 66424, Germany.|Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, Hubei 43060, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar 66424, Germany.|Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin 130041, China.
authors.forename: Xufei
authors.initials: X
authors.lastname: Song
authors.affiliation: Experimental Ophthalmology, Saarland University, Homburg, Saar 66424, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Langenbucher
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar 66424, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Seitz
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar 66424, Germany.
authors.forename: Nóra
authors.initials: N
authors.lastname: Szentmáry
keywords: CD34; apoptosis; crosslinking; human keratocytes; viability; α-SMA