The Effect of Riboflavin–UV-A Treatment on Corneal Limbal Epithelial Cells—A Study on Human Cadaver Eyes

PURPOSE:To determine the effect of riboflavin–UV-A treatment on the corneal limbal epithelial cells during a corneal collagen cross-linking (CXL) procedure. METHODS:Thirty freshly enucleated human cadaveric eyeballs were subjected to a CXL procedure, mimicking the clinical protocol. During the UV-A exposure, one half of the limbus (sector A) was left unprotected, whereas the other half (sector B) was covered by a metal shield. Limbal biopsies from both sectors before and after the procedure were analyzed. Each strip of tissue was divided into 3 segments, for cell count of viable cells, for cultivation on human amniotic membrane (HAM), and for stem cell and differentiated corneal epithelial cell marker studies using reverse transcriptase–polymerase chain reaction. RESULTS:Compared with the cell count before CXL, there was a statistically significant drop in the mean number of viable cells after CXL in sector A but not in sector B. Biopsies from both sectors before CXL and from sector B after CXL showed good growth on HAM. Biopsies from sector A after CXL showed no growth on HAM. The putative stem cell marker ABCG2 was absent in all samples and p63 was absent in 3 of 10 samples taken from sector A after CXL. All markers were present in all samples from sector B after CXL. CONCLUSIONS:Riboflavin–UV-A treatment can result in damage to limbal epithelial cells, particularly the stem cells. Covering the limbal region with a metal shield effectively prevents this damage.