Background. The use of low-temperature plasma (LTP) is a novel approach to treating peri-implantitis. LTP disrupts the biofilm while conditioning the surrounding host environment for bone growth around the infected implant. The main objective of this study was to evaluate the antimicrobial properties of LTP on newly formed (24 h), intermediate (3 days), and mature (7 days) peri-implant-related biofilms formed on titanium surfaces. Methods. Actinomyces naeslundii (ATCC 12104), Porphyromonas gingivalis (W83), Streptococcus oralis (ATCC 35037), and Veillonella dispar (ATCC 17748) were cultivated in brain heart infusion supplemented with 1% yeast extract, hemin (0.5 mg/mL), and menadione (5 mg/mL) and kept at 37°C in anaerobic conditions for 24 h. Species were mixed for a final concentration of ~105 colony forming units (CFU)/mL ( OD = 0.01 ), and the bacterial suspension was put in contact with titanium specimens (7.5 mm in diameter by 2 mm in thickness) for biofilm formation. Biofilms were treated with LTP for 1, 3, and 5 min at 3 or 10 mm from plasma tip to sample. Controls were those having no treatment (negative control, NC) and argon flow under the same LTP conditions. Positive controls were those treated with 14 μg/mL amoxicillin and 140 μg/mL metronidazole individually or combined and 0.12% chlorhexidine ( n = 6 per group). Biofilms were evaluated by CFU, confocal laser scanning microscopy (CLSM), and fluorescence in situ hybridization (FISH). Comparisons among bacteria; 24 h, 3-day, and 7-day biofilms; and treatments for each biofilm were made. Wilcoxon signed-rank and Wilcoxon rank sum tests were applied ( α = 0.05 ). Results. Bacterial growth was observed in all NC groups, corroborated by FISH. LTP treatment significantly reduced all bacteria species compared to the NC in all biofilm periods and treatment conditions ( p ≤ 0.016 ), and CLSM corroborated these results. Conclusion. Within the limitation of this study, we conclude that LTP application effectively reduces peri-implantitis-related multispecies biofilms on titanium surfaces in vitro.

Peri-implantitis is a bacteria-initiated infection that as yet has no effective treatment. A novel approach to treat peri-implantitis is the use of low-temperature plasma (LTP). LTP disrupts the biofilm while conditioning the surrounding host environment for bone growth around the infected implant. The goal of this study was to evaluate the antimicrobial properties of LTP on newly formed (24-h) and mature (7-days) peri-implant-related biofilms. Biofilm was composed of Actinomyces naeslundii (ATCC 12104), Porphyromonas gingivalis (W83), Streptococcus oralis (ATCC 35037), and Veillonella dispar (ATCC 17748). They were cultivated in brain heart infusion supplemented with 1% yeast extract, hemin (0.5 mg/mL), and menadione (5 mg/mL) and kept at 37⁰C in anaerobic conditions for 24-h. The species were mixed for a final concentration of ~105 colony forming units (CFU)/mL (OD=0.01), and the bacterial suspension was transferred to 24-well plates containing titanium specimens. Biofilms were treated with LTP for 1, 3, and 5 min at 3 or 10 mm from plasma-tip to sample. Controls were no treatment (Negative control=NC) and argon-flow at the same LTP conditions. Positive controls were 14 g/mL amoxicillin and 140 µg/mL metronidazole individually or combined, and 0.12% chlorhexidine. Biofilms were evaluated by CFU, confocal laser scanning microscopy (CLSM), and Fluorescence in situ Hybridization (FISH). Wilcoxon Signed-Rank and Wilcoxon Rank Sum tests were applied (α = 0.05). Bacterial growth was observed in all no-treatment groups corroborated by FISH. LTP treatment significantly reduced all bacteria species when compared to the NC in both tested periods and in all treatment combinations (p≤0.016), these results were corroborated by CLSM. There were no significant differences during biofilm development, between 24-h, 3, and 7 days within each LTP treatment, or among the bacteria within each LTP treatment (p≥0.05). LTP application is effective to reduce peri-implantitis-related multispecies biofilms on titanium surfaces.