Intraoral applicability of plasma-activated water: antibacterial activity, cytotoxicity, and effects on tooth structure

This study investigated the intraoral applicability of plasma-activated water (PAW) by evaluating its antibacterial efficacy, cytotoxicity, and effects on tooth structure. PAW was generated using a microwave air plasma system and compared with 0.12% chlorhexidine (CHX) and distilled water (DW). Antibacterial activity was tested against major oral pathogens (Streptococcus mutans, Enterococcus faecalis, Porphyromonas gingivalis, and Fusobacterium nucleatum) and beneficial bacteria (Streptococcus salivarius and Lactobacillus acidophilus) at different pH levels. Cytocompatibility was evaluated in normal oral cells, human gingival fibroblasts (HGFs) and immortalized oral keratinocytes (HOK-16B), using the Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) assays. Extracted human teeth were immersed in PAW, CHX, or DW under cyclic (1- and 2-week) and continuous (1- or 4-week) conditions to assess color, surface morphology, and microhardness. Statistical analyses were performed using one-way ANOVA or nonparametric tests (Mann–Whitney U or Kruskal–Wallis), with significance set at P < 0.05. At pH 3, PAW significantly reduced the viability of S. mutans and E. faecalis (P < 0.01) while largely preserving the beneficial species. PAW showed lower cytotoxicity than CHX in both HGFs and HOK-16B, with cell viability above 70% and minimal ROS or LDH elevation. Cyclic exposures to PAW gradually increased tooth brightness (ΔL* = 1.82 ± 1.02; ΔE00 = 1.21 ± 0.68) after 1 week, whereas CHX caused marked darkening after 2 weeks (ΔL* = − 2.85 ± 0.75; ΔE00 = 2.07 ± 0.44). After cyclic exposure for 1 week, microscopic analyses revealed increased surface roughness, enamel prism erosion, and dentinal tubular exposure, which intensified over time. After 4 weeks of continuous immersion, energy-dispersive X-ray spectroscopy showed elevated calcium signals on the enamel surfaces. PAW exhibited species-selective antibacterial activity and lower cytotoxicity than CHX, while preserving the viability of beneficial oral bacteria. When applied to teeth, PAW induced noticeable brightening but also showed erosive potential with reduced surface hardness, highlighting the need for controlled exposure to ensure safe clinical use.