Antimicrobial Effect of Ozonated Water against Bacteria in Dentinal Tubules

Objective: Ozonated water is known as a powerful antimicrobial agent against bacteria, fungi, protozoa and viruses. Recently, we have demonstrated that ozonated water reduced the viability of oral microorganisms, suggesting that ozonated water might be useful to control oral infection. In the present study, we established in vitro root canal model, and examined whether ozonated water has antimicrobial effect on bacteria invading into root dentinal tubules. Methods: Standardized bovine dentin blocks were infected with Enterococcus faecalis or Streptococcus mutans, and irrigated with ozonated water or other irrigants such as 2.5% sodium hypochlorite (NaOCl). Dentin tips were then scraped from the pulpal surface of BHI broth. Each suspension of dentin tips including bacteria was cultured on BHI agar, and colony-forming unit (CFU) was counted. Irrigated dentin blocks were also sectioned and stained with LIVE/DEAD^®BacLight^TM Bacterial Viability Kit (Molecular Proves) to detect the viability of S. mutans invading into dentinal tubules. We further examined the cytotoxicity of ozonated water against L-929 mouse fibroblasts by MTT assay. Results: The viability of E. faecalis and S. mutans invading into dentinal tubules significantly decreased by the irrigation with ozonated water. Furthermore, the treatment with ozonated water in combination with ultrasonication had a strong antimicrobial effect, which was almost same with that of 2.5% NaOCl. Ozonated water had almost no cytotoxicity on fibroblasts, while 2.5% NaOCl significantly decreased the viability of fibroblasts in vitro culture system. Conclusion: These results indicated that ozonated water has the antimicrobial effect on oral bacteria invading into dentinal tubules in endodontic therapy. This investigation was supported in part by Grants-in-Aid for Scientific Research (14207081, 14207082, 15592025) from The Ministry of Education, Culture, Sports, Science and Technology of Japan.