1. To investigate the effects of non-thermal atmospheric plasma as a root canal disinfectant. Antimicrobial Effects of Non-thermal Atmospheric Plasma as a Novel Root Canal Disinfection Method M HABIB, L HONG, T HOTTEL, F GARCIA-GODOY, F YANG M HABIB, L HONG, T HOTTEL, F GARCIA-GODOY, F YANG Bioscience Research Center, University of Tennessee, Memphis, TN, USA Bioscience Research Center, University of Tennessee, Memphis, TN, USA ABSTRACT Objectives: This study aimed to investigate the effects of non-thermal plasma on E. faecalis biofilm within root canals of extracted human teeth. Methods: Twenty-four anterior single rooted extracted human teeth were collected and prepared using standard endodontic technique. The roots were autoclaved and divided into three groups. E. faecalis bacterial suspension containing 1x10 9 cells/ml was injected into root canals and was incubated at 37 C for 48 hours in a sealed humidified chamber. Group I was flushed three times with sterile media (TSB; 0.5 ml). Group II was similarly flushed with hypochlorite (6%). Group III was subjected to plasma treatment for 2 minutes and then injected with 0.5 ml of media. All media was collected and plated on blood agar plates to enumerate the viable bacteria. MTT assays were conducted to determine the viability of test bacteria and XTT assay for bacterial metabolic activity. Results: Average bacterial counts were 6.4x10 6 (SD 0.73x10 6 ) for PBS control group, 0 for hypochlorite group, and 1.47 (SD 2.07) for the plasma group. Plasma had a killing rate of 99.999% when compared to the PBS control group. MTT assay showed a significant reduction in the viability of bacteria by plasma treatment with a reduction rate of 98.939%. XTT assay also indicated that plasma treatment reduced metabolic activity of bacteria by 99.7%. Conclusions: Non-thermal atmospheric plasma was shown to have a significant antimicrobial effect and is as effective as 6% hypochlorite. Thus it could be used as an innovative, non-invasive method for root canal disinfection. Further studies are needed. This study was supported by the UTHSC Alumni Endowment Fund and the Tennessee Dental Association Fund CONCLUSIONS Non-thermal atmospheric plasma was shown to have significant antimicrobial effects, with nearly all bacterial activity reduced. Therefore, plasma could be used as a non-invasive method for root canal disinfection among other things, but further studies are needed. This study was funded by the UTHSC Alumni Endowment Fund and the Tennessee Dental Association Fund. MATERIALS AND METHODS Low temperature atmospheric Argon plasma is operated under very low electrical power and close to human body temperature making it easy to handle. 24 extracted human anterior teeth were selected; all were trimmed with a slow-speed handpiece to a uniform length of 10-12 mm. The teeth were split into 3 groups depending on treatment: sterile media, 6% hypochlorite and low temperature atmospheric plasma laser. Enterococcus faecalis was cultured, then diluted and placed in the canals of all the teeth. All media was collected and plated on blood agar plates to enumerate the viable bacteria. MTT assay was conducted to determine bacterial viability while XTT assay was conducted to determine bacterial metabolic activity. PURPOSE RESULTS Instrumented tooth root Table 1. Bacterial Loads from Agar Plates Treatment # of viable bacteria/ml (mean) Killing rate ANOVA test* PBS (negative control) 6.4x10 6 ± 0.74 ReferenceA 6% hypochlorite0100%B Plasma (2 minutes)1.47±2.0999.9999%B Table 2: Bacterial Loads from MTT Assay TreatmentO.D. at 540 nmPercent reductionANOVA test* PBS (negative control) 1.98 ± 0.52 ReferenceA 6% hypochlorite 0.018 ± 0.0011 99.991%B Plasma (2 minutes)0.021 ± 0.00398.939%B Table 3: Metabolic Activity by XTT Assay TreatmentO.D. at 540 nmMetabolic activity reductionANOVA test PBS (negative control) 2.78±0.094 ReferenceA 6% hypochlorite0100%B Plasma (2 minutes)0.008 ±0.00299.7%B Bacterial activity was measured initially on agar plates, with PBS control group having 6.4x10 6 bacteria/ ml. No bacteria were viable with hypochlorite and 1.47 bacteria/ ml for plasma. When plated on MTT assay to determine bacterial viability, 6% hypochlorite had reduced bacterial loads by 99.991% while low atmospheric plasma brush reduced bacterial loads by 98.939%. The last agar measured bacterial metabolic activity with an XTT assay. Bacterial metabolic activity was reduced 100% with 6% hypochlorite and 99.7% with plasma brush. Injection and incubation of bacteria In roots Plasma treatmentControlCold Plasma