title: Effect of Keratocyte Supernatant on Epithelial Cell Migration and Proliferation After Corneal Crosslinking (CXL).
abstract: To evaluate the effect of keratocyte supernatant (harvesting time, riboflavin concentration and UV-A-light illumination) on migration and proliferation of human corneal epithelial cells (HCECs) by CXL, in vitro.
  
  
  
  Primary human keratocytes isolated from 8 normal and 6 keratoconus corneas were cultured. Thereafter, keratocytes in 0%, 0.05% or 1% riboflavin solution were split into samples without and with 370 nm UVA-light-illumination. After removal of the riboflavin solution, keratocytes were incubated in the mentioned keratocyte culture medium at 37 °C and keratocyte supernatant was harvested after 5 and 24 hours. Keratocyte supernatant without riboflavin and UVA treatment, was used as control. HCECs were cultured until reaching confluence, the HCEC culture medium was replaced by the keratocyte supernatant and HCEC migration was analyzed using the wound-healing assay. HCEC proliferation was determined by the cell proliferation ELISA BrdU (colorimetric) kit. Statistical analysis was performed using a linear mixed model in the framework of a Generalized Estimating Equations (GEE) approach to analyze the effect of harvesting time, riboflavin concentration and UV-A-light illumination using IBM-SPSS version 22.
  
  
  
  Riboflavin concentration, UVA-light illumination and harvesting time of normal or keratoconus keratocyte supernatant had no significant impact on HCEC proliferation (p > 0.10). Riboflavin concentration did not show significant impact on HCEC migration using normal or keratoconus keratocyte supernatant (p > 0.10), however, longer harvesting time of normal or keratoconus keratocyte supernatant significantly increased (p = 0.01 for both) and UVA-light illumination of keratoconus keratocyte supernatant (p < 0.001) significantly decreased HCEC migration.
  
  
  
  Harvesting time, riboflavin concentration and UV-A-light illumination of normal and keratoconus keratocyte cultures has no impact on proliferation of HCECs, in the short term. However, 24 hours harvesting time (both for normal and keratoconus keratocytes) increases and UVA-light-illumination of keratoconus keratocyte cultures decreases HCEC migration.
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Ming-Feng
authors.initials: MF
authors.lastname: Wu
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Tanja
authors.initials: T
authors.lastname: Stachon
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .|b Department of Ophthalmology , The First Affiliated Hospital of Zhengzhou University , Zhengzhou , China .
authors.forename: Jiong
authors.initials: J
authors.lastname: Wang
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Xuefei
authors.initials: X
authors.lastname: Song
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Sarah
authors.initials: S
authors.lastname: Colanesi
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Berthold
authors.initials: B
authors.lastname: Seitz
authors.affiliation: c Institute of Medical Biometry, Epidemiology and Medical Informatics, Saarland University Medical Center , Homburg/Saar , Germany .
authors.forename: Stefan
authors.initials: S
authors.lastname: Wagenpfeil
authors.affiliation: d Department of Experimental Ophthalmology , Saarland University , Homburg/Saar , Germany and.
authors.forename: Achim
authors.initials: A
authors.lastname: Langenbucher
authors.affiliation: a Department of Ophthalmology , Saarland University Medical Center , Homburg/Saar , Germany .|e Department of Ophthalmology , Semmelweis University , Budapest , Hungary.
authors.forename: Nóra
authors.initials: N
authors.lastname: Szentmáry
mesh_terms: D000368:Aged; D017209:Apoptosis; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology*; D060527:Corneal Keratocytes / Q000473:pathology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000473:pathology*; D005260:Female; D006801:Humans; D007640:Keratoconus / Q000188:drug therapy / Q000473:pathology*; D008297:Male; D010778:Photochemotherapy / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D003094:Collagen
keywords: Corneal crosslinking; human corneal epithelial cells; human keratocytes; keratoconus; riboflavin