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Item Effectiveness of ozonated water irrigation against an established Enterococcus faecalis biofilm in root canal treated teeth in vitro(2020) Broady, Adam B.; Spolnik, Kenneth J.; Duarte, Simone; Gossweiler, Ana; Bringas, Josef S.; Ehrlich, YgalIntroduction: One of the main objectives of endodontic therapy is to reduce microbes and remove inflamed pulpal tissue within the root canal system (RCS). This is accomplished through chemomechanical debridement of the RCS using hand and rotary instrumentation along with an antimicrobial irrigant. Today, the most commonly used irrigant is sodium hypochlorite (NaOCl), often at concentrations toxic to human cells. The use of ozone as an endodontic irrigant is a novel technique that has been proven to be antimicrobial against several microorganisms. However, independent research is lacking on ozone’s efficacy against an established endodontic biofilm. If ozone’s efficacy against biofilms is confirmed, the use of toxic and potentially dangerous sodium hypochlorite could be replaced in some clinical situations (i.e., regeneration, immature teeth, resorption) with a safer and effective alternative. Objective: The aim of the current study was to evaluate the anti-biofilm activity of different concentrations of ozonated water compared to various concentrations of NaOCl against an established endodontic biofilm of Enterococcus faecalis in root canal treated teeth in vitro. Materials and Methods: The crowns of similarly sized, maxillary anterior teeth were removed, and the roots cut to a standard length (12 mm). All root canals were instrumented to a standard size. Specimens were sterilized and then inoculated with E. faecalis, which were allowed to grow for two weeks to form an established biofilm. There were six treatment groups: 1) 6% NaOCl; 2) 1.5% NaOCl; 3) 16µg/mL ozonated water; 4) 25µg/mL ozonated water; 5) 50µg/mL ozonated water, and 6) saline. Following treatment, samples were collected, plated, and incubated for two days. The number of CFU/mL were determined, and samples visualized using confocal imaging. The effect of treatment group on bacterial counts was made using one-way ANOVA followed by pair-wise comparisons. Null Hypothesis: Endodontically treated teeth irrigated with ozonated water will not demonstrate a statistically significant decrease in the E. faecalis biofilm compared to those treated with sodium hypochlorite Results: CFUs were converted to log10 and compared using Fisher’s Exact tests or one-way ANOVA followed by pair-wise tests. In all observations utilizing NaOCl irrigation, no colonies formed following treatment. The two NaOCl groups, with 0 CFU/mL, were significantly different than the other four groups (p=0.009). Saline showed a trend towards higher CFU/mL than 50 µg/ml O3 (p=0.068). None of the other comparisons approached statistical significance (p=0.453 25 µg/ml O3, p=0.606 16 µg/ml O3, p=0.999 25 µg/ml O3 vs 50 µg/ml O3, p=0.990 16 µg/ml O3 vs 50 µg/ml O3, p=1.000 16 µg/ml O3 vs 25 µg/ml O3). Confocal imaging helped illustrate effects of irrigation and confirm CFU findings. Conclusion: The results of this study failed to reject the null hypothesis. There was a statistically significant difference in the E. faecalis biofilm remaining in the groups treated with ozonated water compared to those treated with NaOCl. However, there was a trend towards higher CFU/mL in the saline group compared to the 50µg/mL ozonated water group. According to this finding, future studies should evaluate the effects of higher concentrations of ozonated water against an established E. faecalis biofilm. In addition, other follow-up studies might include ozonated water’s effect on human cells, such as the stem cells of the apical papilla that are so critical to the success of regenerative endodontic procedures. Due to university and laboratory closures caused by the COVID-19 pandemic, this project was stopped short and an insufficient sample size did not allow for proper statistical power. Additional occasions should be run upon the university’s re-opening to allow for proper statistical power.Item An in-vitro SEM study comparing the debridement efficacy of the EndoVac® system versus the Canal CleanMax® following hand-rotary istrumentation(2010) Ricketts, Benjamin P. (Benjamin Paul), 1980-; Vail, Mychel; Legan, Joseph J.; Moore, B. Keith; Spolnik, Kenneth Jacob, 1950-; Zunt, Susan L., 1951-This in-vitro, prospective, randomized study microscopically compared the debridement efficacy of negative pressure irrigation with the EndoVac (Discus Dental, Culver City, CA) versus the Canal CleanMax (Maximum Dental, Inc., Secaucus, NJ). Sixty extracted human canines were instrumented using a combination of hand-instrumentation with Lexicon K-type files and rotary instrumentation with ProTaper files. All canals were irrigated with 6.0-percent sodium hypochlorite and 17- percent ethylenediaminetetraacetic acid (EDTA). However, the irrigation/aspiration techniques differed among three groups of 20 randomly selected teeth. Group one (control) was irrigated with only a 12-ml Monoject syringe via 30-gauge side-vented, closed-end needle. Group two was irrigated with the EndoVac system. Group three was irrigated similar to group one, but with the adjunct of the Canal CleanMax system. All teeth were sectioned longitudinally, and the more intact sections were divided into coronal, middle, and apical thirds. Each portion of the canal was photographed with a scanning electron microscope (SEM). The photographs were scored by two independent examiners according to relative amount of debris and/or smear layer present, as well as relative number of patent dentinal tubules. These scores were statistically analyzed using a Krustal-Wallis test and Wilcoxon Rank Sum tests to determine differences between groups. The coronal aspect of root canal walls irrigated with the EndoVac system exhibited significantly less debris and/or smear layer present when compared to the coronal aspect of root canals irrigated with only a standard 12-ml Monoject syringe equipped with 30-gauge ProRinse side-vented, closed-end needle (control). There were no other significant differences in scores between any groups at any location. For all locations combined, the EndoVac system produced significantly cleaner root canal walls as compared to the control. No significant differences were seen between the Canal CleanMax and Control or Canal CleanMax and EndoVac. This study suggested negative pressure irrigation delivery with the EndoVac system during and after hand-rotary instrumentation is more effective in removal of debris and smear layer from the coronal third and combined thirds of root canal walls compared to irrigation with a standard 12-ml Monoject syringe equipped with 30-gauge ProRinse side-vented, closed-end needle.Item Use of electromagnetic stimulation on an Enterococcus faecalis biofilm in root canal treated teeth in vitro(2019) Kindler, Justin K.; Spolnik, Kenneth J.; Duarte, Simone; Gregory, Richard L.; Ehrlich, Ygal; Bringas, Josef S.Introduction: Nonsurgical root canal therapy procedures aim to reduce the total microbial load within an infected root canal system through chemomechanical debridement of the root canal system via instrumentation in conjunction with an antibacterial irrigating solution. The most commonly used irrigant is sodium hypochlorite, often at concentrations toxic to human cells. Electromagnetic wave irradiation is a novel method of disinfection that has been shown to be bactericidal against planktonic microorganisms in solution, but its efficacy against an established biofilm is unknown. Pilot studies have demonstrated a synergistic killing effect with sodium hypochlorite through a process termed electromagnetic stimulation (EMS). If confirmed, lower concentrations of the current gold standard of 6.0-percent sodium hypochlorite could be used to irrigate infected root canals during endodontic treatment, resulting in less toxicity to human cells. There are also regenerative implications as EMS could be used to disinfect the root canals of immature teeth using 1.5-percent sodium hypochlorite, as recommended by the American Association of Endodontists. Objectives: The purpose of this in-vitro study was to evaluate the anti-biofilm effect of EMS against an established biofilm of Enterococcus faecalis. Materials and Methods: Single rooted teeth were cut to a standardized length (12 mm) and instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with E. faecalis, which grew for two weeks to form an established biofilm. There were five treatment groups: 1) 6.0-percent sodium hypochlorite; 2) 1.5-percent sodium hypochlorite; 3) 1.5-percent sodium hypochlorite with EMS; 4) 0.9-percent saline with EMS and 5) 0.9-percent saline. Samples were collected, plated, and incubated for two days. The number of CFUs/mL was determined and converted to log10. The effect of treatment group on bacterial counts was made using Wilcoxon Rank Sums Test. One sample per group was scored and split for confocal imaging. Null Hypothesis: Teeth treated with EMS in combination with 1.5-percent sodium hypochlorite or 0.9-percent saline will not demonstrate a significant anti-biofilm effect in comparison to those treated with 6.0-percent sodium hypochlorite alone. Results: 0.9-percent saline and 0.9-percent saline with EMS were significantly higher than 6.0-percent NaOCl, 1.5-percent NaOCl, and 1.5-percent NaOCl with EMS. 0.9-percent saline was significantly higher than 0.9-percent saline with EMS. The three groups that included treatment with NaOCl were not significantly different from each other. Confocal imaging confirmed the CFU findings. Conclusion: Because there was no growth in any of the NaOCl groups, the null hypothesis cannot be rejected. However, there was an antibiofilm effect when comparing the two saline groups, demonstrating that EMS has an antibiofilm effect. Future studies should focus on determining what concentration of NaOCl is most effective in combination with EMS.