No-ozone cold plasma can kill oral pathogenic microbes in H2O2-dependent and independent manner

Abstract

To apply the sterilisation effect of low-temperature plasma to the oral cavity, the issue of ozone from plasma must be addressed. In this study, a new technology for generating cold plasma with almost no ozone is developed and is named Nozone (no-ozone) Cold Plasma (NCP) technology. The antimicrobial efficacy of the NCP against four oral pathogens is tested, and its specific mechanism is elucidated. The treatment of NCP on oral pathogenic microbes on a solid medium generated a growth inhibition zone. When NCP is applied to oral pathogens in a liquid medium, the growth of microbes decreased by more than 10⁵ colony forming units, and the bactericidal effect of NCP remained after the installation of dental tips. The bactericidal effect of NCP in the liquid medium is due to the increase in hydrogen peroxide levels in the medium. However, the bactericidal effect of NCP in the solid medium depends on the charged elements of the NCP. Furthermore, the surface bactericidal efficiency of the dental-tip-installed NCP is proportional to the pore size of the tips and inversely proportional to the length of the tips. Overall, we expect this NCP device to be widely used in dentistry in the near future.

Figure 1

The NCP device can mount dental tips and produces argon plasma with low ozone. (A) The photographs of the NCP device developed and used in this study. This device can mount most of dental tips in the market. (B) A schematic diagram describing the structure of NCP generating part of the device. (C) The result of OES analysis of the plasma from the NCP device without dental tips. (D) The results of the measurement for the O₃ level from NCP device. The measurements was performed for 10 min, and the O₃ level was detected at every min. The distance between the gas inlet of O₃ detector and the end of the NCP outlet (regardless of with or without capillary tip) was maintained at 1 cm.

Figure 2

NCP treatment can kill 4 kinds of oral microbes on solid medium. The representative photographs showing the surface bactericidal effect of NCP against S. mutans, E.faecalis, C.albicans and P.gingivalis (left). The photographs were taken at 24 h after the NCP treatment for 1, 3 and 5 min. The distance between the end of NCP device and solid medium inoculated with oral microbes were maintained at 1 cm during the treatment. The diameter of inhibition zone were measured and presented in a table (right).

Figure 3

The mounting of dental tips reduced the bactericidal effect of NCP. The representative photographs showing the effect of dental tip (capillary and microcapillary tip) mounting on the bactericidal activity of NCP against C.albicans (upper panel). The photographs were taken at 24 h after the NCP treatment for 1, 3 and 5 min. The distance between the end of NCP outlet and solid medium were maintained at 1 cm during the treatment regardless of dental tip mounting. The diameter of inhibition zone were measured and presented in a table (bottom panel).

Figure 4

The bactericidal effect of NCP against 4 oral microbes in liquid medium. The results of experiments testing the sterilsation effect of NCP on S. mutans, E.faecalis, C.albicans and P.gingivalis in liquid medium. The medium containing oral microbes were subjected to 1, 3, and 5 min of NCP treatment respectively. After 5 h (A) and 24 h (B) of additional incubation in liquid medium, the samples were undergone a serial dilution, and plated on solid medium. At 24 h of further incubation, the representative photographs were taken (upper panel), and the number of colony was counted, and the results was presented as a graph (bottom panel). Data shown are the representatives of three independent experiments, *p < 0.05, **< 0.01, ***< 0.001.

Figure 5

The effect of dental tip mounting on the bactericidal activity of NCP in liquid medium. Four kinds of oral microbes (S.mutans, E.faecalis, C.albicans and P.gingivalis) inoculated in the liquid medium were subjected to NCP treatment in the condition of unmounted or mounted with capillary or microcapillary tips. The treatment was performed for 1, 3 and 5 min, and the samples were incubated for 24 h further in the liquid medium. The samples were diluted serially, and plated on the solid medium. After 24 h, the colony forming units (CFU) were counted, and presented as a graph. Data shown are the average of three independent experiments, *p < 0.001.

Figure 6

The NCP-mediated increase of H₂O₂ level in the medium is crucial for the bactericidal effect of NCP in liquid medium. (A) The results of the H₂O₂ concentration measurement. NCP was treated on 1 ml of liquid medium for 0.5, 1, 3, 5 min, and right after the treatment, the H₂O₂ level of the medium was detected using Amplex red assay kit. (B) The liquid medium (1 ml) samples were subjected to un-mounted NCP, NCP mounted with capillary or microcapillary tips for 5 min. After the treatment, H₂O₂ level in the medium was detected. (C) The results of nitrite level detection using Griess assay kit. The liquid medium (1 ml) samples were subjected to the NCP treatment for 1, 3, 5 min, and the NO²⁻ level in the medium was detected right after the treatment. (D) The results of the experiments monitoring the effect of NAC on the NCP-mediated bactericidal effect against S.mutans. The liquid medium samples containing S. mutans were treated with NCP for 5 min in presence or absence of NAC at concentration of 0.2, 0.4, and 1 mM respectively. The treated samples were incubated for 24 h further, and plated on solid medium. At 24 h after the additional incubation, CFU was counted. Data shown are the average of three independent experiments, **p < 0.05, **< 0.01, ***< 0.001.

Figure 7

The effect of the dental tip length on the bactericidal effect of NCP mounted with dental tips. (A) The capillary tips (originally 4.5 cm in length) were cut into 1.5, 2, 2.5 and 3.5 cm, and mounted on NCP device. S. mutans inoculated on solid medium was treated with the NCP for 1, 3, 5 min. At 24 h after the treatment, the photographs showing a clear zone were taken, and the size of a clear zone was measured. (B) The microcapillary tips (originally 4 cm in length) were cut into 3 and 3.5 cm, and mounted on NCP device. The S.mutans on solid medium was subjected to NCP treatment for 1, 3 and 5 min. At 24 h after the treatment, the photographs showing a clear zone were taken, and the size of a clear zone was measured.

Figure 8

The charged particles of NCP is critical for the NCP-mediated bactericidal in solid medium. (A) Upper panel The photographs showing the 3 different NCP treatment conditions. NCP-D.E represents the NCP treatment condition placing a dielectric mesh between the target and NCP device, and NCP-E.G. means a grounded electric meshes were placed during the treatment. Bottom panel: The photographs showing the bactericidal effect of NCP in 3 different treatment conditions. The distance between NCP device and the S.mutans inoculated on solid medium in all three treatment conditions were maintained as 1 cm. At 24 h after the 5 min of NCP treatment, the photographs were taken. (B) The effect of 3 different NCP treatment methods on H₂O₂ level in the liquid medium. 1 ml of liquid medium was treated with NCP for 5 min using 3 different treatment methods described in (A). Data shown are the average of three independent experiments, **< 0.01. (C). S.mutans inoculated on solid medium was subjected to NCP using two treatment methods. Fixed treatment means NCP device was fixed at the middle of the plate during 5 min of treatment as usual, whereas moving treatment means the NCP was treated in moving manner during 5 min on the circle sized in 3 cm in diameter, keeping 1 cm distance. The photoghraphs showing a clear zone were taken at 24 h after the NCP treatment.

Figure 9

The bactericidal effect of NCP mounted with dental tips is dependent on the pore size and the length of tips. (A) Total 7 kinds of dental tips that can be mounted on NCP device were prepared. Left panel a photograph showing the appearance of 7 tips. Right panel a table showing specific information of the tips. (B) S.mutans samples on solid medium were subjected to NCP treatment for 5 min in unmounted or mounted with 7 kind of tips respectively. Upper panel A photographs showing a clear zone formed by 5 min of NCP treatment. Bottom panel: a graph showing the size of a clear zones. (C) The H₂O₂ level change of 2 ml of liquid medium after NCP treatment mounted with 7 kinds of dental tips. Data shown are the average of three independent experiments, **< 0.01.