Ozone therapy stimulates cell migration in vitro

Objectives: Due to its oxidative potential and antimicrobial effects, ozone has been used in medicine for over 100 years. In dentistry, it is applied as treatment for diseases such as periodontitis and caries, as well as in endodontics and maxillofacial surgery. Intending to understand how cells react to ozone, we evaluated its cytotoxicity and analyzed its effects in wound healing.
Methods: Cell Culture: A keratinocyte (HaCat) and a mouse fibroblast (L-929) cell lines, both maintained according to ATCC recommendations.
Cell Viability assay: 5x10³cells/well seeded in 96-well plates. After 24h, treatment with ozonized PBS [1.25-40μg/mL], chlorhexidine 0.2% or PBS. MTT assay was performed 24h and 48h after treatment.
Scratch assay: 1x10⁶cells/well seeded in 6-well fibronectin-coated-plates. After 24h, scratch and treatment with ozonized PBS [40μg/mL]. Wound closure was photographically registered in different periods.
Transwell assay: 2x10⁴cells/transwell seeded in upper chamber. After 24h, treatment with ozonized PBS [40μg/mL]. 72h later, migrated cells were fixed, stained and photographed.
Results: Chlorhexidine resulted in considerable viability decrease. Ozone slightly increased viability in HaCat after a 24h-treatment and the [40µg/mL] resulted in the highest viability (130%). None of these results was significant, though (OneWay Anova, Tukey’s). Ozone reduced viability in fibroblasts (L-929), except in the [40µg/mL] (104%). Wound healing was observed in both control and treatment groups, although no statistical difference was determined (TwoWay Anova, Bonferroni’s). In the migration assay, no difference was noticed on keratinocytes (HaCat). However, migration in ozone-treated fibroblasts was three times the observed in control group (309%) (T-test, p<0.01).
Conclusions: Ozone was not found to reduce cell viability and did not accelerate wound healing. In fibroblasts though, it substantially increased cell migration, which is an important process in physiological wound repair. Added to its antimicrobial effects, the induction of cell migration might be a significant property of ozone, and must be further studied.