Effect of NecroX-5, a novel necrosis inhibitor, on the survival of cultivated oral mucosal epithelial sheets

Abstract

Purpose: Cell sheets are exposed to ischemic damages during the interval from procurement to attachment after transplantation. We intended to investigate the effect of NecroX-5,a novel necrosis inhibitor that shows scavenger activity against mitochondrial reactive oxygen species, on the survival of cultivated oral mucosal epithelial sheets after procurement.

Methods: Human oral mucosal epithelial cells were cultivated to make sheets for 2 weeks. Sheets were harvested and divided into 5 groups. One was stained with trypanblue(TB) immediately after detachment from the bottom of the culture well as a negative control. Another one was kept in the media at room temperature for 3.5 hours after procurement as a positive control. The other sheets were immersed in 10µm, 20µm, and 30µm of NecroX-5 for 30 minutes after detachment, and were re-suspended in culture media for 3 hours. In vivo, six limbal deficient rabbits received cultivated oral mucosal epithelial sheet transplant(COMET) after the sheet had been immersed in 10 µm NecroX-5 for 30 minutes (group 1), and the other six limbal deficient rabbits received COMET as control (group 2). 7 days after transplantation, periodic-acid-Schiff (PAS) staining and immunohistochemistry for P63 was performed.

Results: Negative control showed a live cell percentage of 80.5%, whereas positive control showed 100% of cell death. Live to dead cell ratio was 51.5% vs 48.5% in the 10 µm NecroX-5 treated sheet. There were no goblet cells on the transplanted corneal epithelial cells in bothgroups. The expression of P63 showed no significant difference between groups.

Conclusions: Our results suggest that immersion in 10µm NecroX-5 may increase the cell survival of cultivated oral mucosal cell sheets.

Keywords: 482 cornea: epithelium