Ozonated Water Inhibits Effects of Lipopolysaccharide on Odontoblast-Like Cells

Objective: We previously demonstrated antimicrobial effects of ozonated water on oral microorganisms through the destruction of bacterial cell wall. Recently, we established a rat clonal dental pulp cell line, KN-3 cells showing odontoblastic properties. In the present study, we examined the effects of ozonated water on lipopolysaccharide (LPS) to clarify the mechanisms of antimicrobial effects. We also examined whether ozonated water is a useful disinfectant for the treatment of exposed pulp. Methods: After the exposure of KN-3 cells to LPS, ozonated water, and LPS treated with ozonated water, we examined the cell viability by MTT assay, the phosphorylation of p38 by Western blot, and mRNA expression of TNF-α by RT-PCR. To analyze the calcification ability of KN-3 cells, we examined ALP activity and von Kossa staining in the presence of ascorbic acid and β-glycerophosphate in in vitro calcification assay. Results: Ozonated water showed no effects on the cell viability, ALP activity and the formation of calcification nodules by KN-3 cells. However the treatment of ozonated water activated the p38 MAP kinase, expression of TNF-α. LPS did not inhibit the cell viability of KN-3 cells. However, LPS induced the phosphorylation of p38 MAP kinase and TNF-α expression. LPS also suppressed the formation of calcification nodules by KN-3 cells without the inhibition of ALP activity. Interestingly, we found that the exposure of LPS to ozonated water down-regulated the phosphorylation of p38 MAP kinase, the expression of TNF-α, and the suppression of calcification nodule formation induced by LPS. Conclusion: We found that ozonated water inhibited the inflammatory responses and calcificaiton ability of KN-3 cells through the neutralization of LPS activities. Supported by Grants (18592094, 18209057) from The Ministry of Education, Science, and Culture of Japan, Tokyo, Japan.