Effects of Na-DNA mouthwash solutions on oral soft tissues. A bioreactor-based reconstituted human oral epithelium model

Purpose: To investigate whether the addition of sodium-DNA (Na-DNA) to chlorhexidine (CHX)-containing mouthwash influenced morphology and viability of a reconstituted human oral epithelium (ROE), and protects ROE against oxidative stress. Methods: Multi-layered 0.5 cm(2) ROE specimens were positioned inside a continuous flow bioreactor and grown air-lifted for 24 hours. They were treated with phosphate-buffered saline (PBS) (n= 16) or 1 vol% H2O2 for 1 minute (n= 16). Then, they were treated for 5 (n= 8) or 30 minutes (n= 8) with the experimental mouthwash solutions containing: 0.2 wt% CHX, 0.2 wt% CHX + 0.2 wt% Na-DNA, 0.2 wt% Na-DNA, PBS. After 60 minutes washout specimens were subjected to tetrazolium-based viability assay (MTT) confocal laser-scanning microscopy (CLSM), and histological evaluation using optical microscopy and transmission electron microscopy (TEM). Results: ROE treated with Na-DNA for 30 minutes revealed significantly higher viability than PBS, and CHX + Na-DNA showed higher viability after 30-minute treatment than after 5 minutes, suggesting a significant protective activity of Na-DNA. Moreover, the protective effect of Na-DNA on cell viability was higher after the induction of oxidative stress. After treatment with CHX, CLSM revealed cell stress, leading to cell death in the outer layer. On the contrary, specimens treated with Na-DNA showed a much lower number of dead cells compared to PBS, both in the absence or presence of oxidative stress. Histological examination showed that the protective action of Na-DNA formulations reached more in-depth into the epithelium exposed to oxidative stress, due to intercellular spaces opening in the outer epithelium layers, giving way to Na-DNA to the inner parts of the epithelium. It can be concluded that Na-DNA had a topical protective activity when applied for 30 minutes unless the epithelium barrier is damaged, allowing it to act more in-depth.