IU Indianapolis ScholarWorks :: Browsing by Author "Bringas, Josef S."

Browsing by Author "Bringas, Josef S."

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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, Ygal
Introduction: 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.
The effects of electromagnetic wave stimulation (EMS) on osteoblast differentiation and activity
(2020-06) Pauly, Katherine L.; Spolnik, Kenneth; Bruzzaniti, Angela; Ehrlich, Ygal; Bringas, Josef S.
Introduction: The goal of nonsurgical root canal therapy is to reduce the bacterial load within an infected root canal system, and the subsequent objective is to prevent or treat apical periodontitis. Clinical studies have shown more expedient healing of apical periodontitis treated with electromagnetic wave stimulation (EMS) as compared to apical periodontitis not treated with EMS. Stimulation of osteoblasts and growth factors has been shown when EMS was applied to rat calvaria, resulting in increased bone healing. Objective: The purpose of this vitro study was to evaluate the effects of EMS on the proliferation and differentiation of osteoblasts. Using primary neonatal calvaria osteoblast-lineage cells, the effects of different EMS regimens on proliferation, alkaline phosphatase (ALP) activity, and mineral deposition were determined. Materials and Methods: EMS regimen included currents of 0mA, 0.1mA, 1mA, and 10mA delivered for five consecutive 1s pulses per day for one, two, and three days. Cell proliferation was assayed after 1 or 2 days using an MTS assay. Alkaline phosphatase activity and mineral deposition were assayed after culturing the cells in osteogenic media containing ascorbic acid and -glycerol phosphate for 7 days. Comparisons were performed using analysis of variance, with a 5% significance level. Results: There was no statistically significant differences noted in MTS proliferation and mineral deposition between the experiment EMS treatment groups of 0.1, 1.0, and 10.0 mA compared to the control group of 0 mA current on calvaria-derived osteoblast. While there were no statistically significant differences noted in ALP activity in the 0.1, and 1.0 mA EMS groups, compared to 0 mA control, alkaline phosphatase activity was significantly increased in the 10 mA EMS group. Conclusion: There was no significant differences in MTS proliferation and mineral deposition of the EMS group compared to the control group. However, 10 mA EMS favored increased ALP activity suggesting EMS can promote matrix maturation by osteoblasts. Additional in vitro experimental studies, including different stem cell populations, culture duration and EMS treatment regimens are needed to understand the mechanism of action of EMS for future applications in regenerative endodontics.
The Effects of Nano-Hydroxyapatite in a Double Antibiotic Paste-Loaded Methycellulose Carrier on Dental Pulp Stem Cells
(2019) Everhart, Adam R.; Spolnik, Kenneth J.; Bruzzaniti, Angela; Bringas, Josef S.; Ehrlich, Ygal; Gregory, Richard L.
The effects of hydroxyapatite in a DAP-loaded MC carrier on dental pulp stem cells Introduction: Regenerative endodontic procedures (REP) require disinfection techniques to eliminate bacteria from the infected immature root canal system and promote new growth of the pulp-dentin complex. Double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole, has shown efficacy in doing so while minimizing cytotoxicity on dental pulp stem cells (DPSC). Stem cells, scaffolding, and growth factors are necessary in the maturation, proliferation, and differentiation of mesenchymal stem cells into the root canal system. Nano-hydroxyapatite (n-HA) has a history of biocompatibility and, in addition, has shown promising effects as a tissue bioengineering material. Objective: The aim of this in vitro study was to investigate the proliferation and mineralization of DPSC in the presence of 1% DAP and methylcellulose (MC) with varying concentrations of nano-hydroxyapatite. Materials and Methods: DPSC were plated in 24-well plates containing culture media. The next day, semi-permeable 0.1 mm Transwell chambers were inserted into the wells to separate the reservoirs for medicaments. Treatment paste composed of methylcellulose containing 1% DAP with either 0.25%, 0.50%, or 1.0% nano-hydroxyapatite was added along with culture media. Methylcellulose alone and calcium hydroxide (Ultracal) were used as control groups. After 3 days, cells were evaluated for cytotoxic effects using an MTS proliferation assay (n = 10, in triplicate). DPSCs were also cultured with these medicaments for 7 days in osteogenic media and evaluated for alkaline phosphatase (ALP) activity and mineralization activity (n = 13, in triplicate). Comparisons between groups for differences in mineralization, BSA, and ALP activity were performed using analysis of variance (ANOVA), with different variances allowed for each group and a random effect included in the model to account for correlation within each of the three trials. A simulation-based model was used to adjust for multiple comparisons. Results: Addition of n-HA treatment groups increased mineralization significantly greater than calcium hydroxide, with MC alone and MC+DAP+0.5% HA providing the greatest effect. Regarding ALP, all HA concentrations performed significantly greater than MC and DAP concentrations. Proliferation demonstrated similar metabolic activity in all experimental groups with few comparisons significant. Conclusion: The challenge in REPs is to maintain survival, and preferably promote the proliferation and development of DPSCs into the pulp-dentin complex with a consistent treatment outcome. The combination of DAP with hydroxyapatite may allow for both disinfection and improved mineralization and cellular differentiation. This contribution has shown significant ability to increase stem cell differentiation into an osteogenic lineage as well as calcium deposition, indicating end goal results of regenerative procedures.
Evaluation of contact angle between root canal sealers and dentin treated with calcium hydroxide and irrigation solutions
(2018) Nakaparksin, Pranai; Platt, Jeffrey; Levon , John A.; Bringas, Josef S.; Brown, David T.
Background: Numerous studies have reported the effect of long-term use of calcium hydroxide Ca(OH)2 to dentin. Nevertheless, there is little information available about the effect of Ca(OH)2 on wettability to the dentin. Objective: To investigate the effect of Ca(OH)2 application on dentin for two and four weeks on the wettability of two root canal sealers. Methods: Polished caries-free human dentin discs (n = 156) were allocated into 12 groups; G1 and G3 had two weeks’ treatment, G4 and G6, four weeks treatment. G1 and 101 G4 were treated with sterile water. G2, G3, G5 and G6 were treated with Ca(OH)2. G1, G3, G4, and G6 were irrigated with 6.0-percent NaOCl and 17-percent EDTA while G2, and G5 were irrigated with sterile water. Then, contact angles between Tubli-Seal and the treated dentin surfaces were measured. G7 and G12 were treated in the same fashion but were treated with BC sealer. Surface morphology evaluation of G1 and G6 was carried out by scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDX). Statistics were performed using three-way ANOVA and pair-wise comparisons between groups (α = 0.05). Results: Tubli-Seal (G1 through G6) had significantly smaller values for contact angles than BC sealer (G7 through G12) (p < 0.05). For the Tubli-Seal groups (G1 through G6), G4 had the highest mean of contact angles at 104.9 ± 1.9°, whereas G5 presented the lowest mean of contact angles at 85.4 ± 15.1. For the BC sealer groups (G7 through G12), G10 had the highest mean of contact angles at 145.4 ± 1.3°, while G11 demonstrated the lowest mean of contact angles at 130.2 ± 2.6°. Groups with Ca(OH)2 treatment with water irrigation (G2, 5, 11) had significantly lower contact angle than groups with Ca(OH)2 with chemical irrigation (G3, 6, 12) (p < 0.05), except G8, 9. According to SEM and EDX, water irrigation solution showed higher remaining Ca(OH)2 than irrigation with the chemical solution while Ca(OH)2 with chemical irrigation 102 demonstrated no Ca(OH)2 remaining after irrigation, similar to the surface of the control group. Conclusion: Within the limitations of this study, Tubli-seal has better wettability on dentin than BC sealer. Remaining calcium hydroxide demonstrated a trend toward decreased contact angle between dentin and root canal sealers. Moreover, two-minute irrigation with 6-percent NaOCl and 17-percent EDTA can remove calcium hydroxide from polished dentin surfaces.
Reduction of enterococcus faecalis biofilm by blue light and sodium hypochlorite
(2017) Kwan, Daryl A.; Gregory, Richard L.; Spolnik, Kenneth J.; Bringas, Josef S.; Zunt, Susan L.; Ando, Masatoshi; Ehrlich, Ygal
Introduction: Microbial biofilms have been shown to be a cause of persistent endodontic infections. It is more resistant than planktonic bacteria to host immune defenses and antimicrobials. Studies indicate that photodynamic light therapy (PDT), which involves using light at specific wavelengths, has a potent antibacterial effect on bacterial biofilm. PDT is an antimicrobial strategy that involves the use of a nontoxic photosensitizer (PS) along with a light source. The excited PS reacts with molecular oxygen to produce highly reactive oxygen species, which induce injury or death to microorganisms. PSs have a high degree of selectivity for inhibiting microorganisms without negatively affecting host mammalian cells. PDT has been suggested as an adjuvant to conventional endodontic treatment. Studies at IUSD have shown that blue light at 380 nm to 440 nm has the ability to inactivate Streptococcus mutans biofilm without any exogenous PS. Objective: The objective of this study was to determine the effectiveness of blue light at 380 nm to 440 nm to reduce adherence of Enterococcus faecalis biofilm after NaOCl irrigation at various concentrations. Materials and Methods: E. faecalis biofilm was established for 72 hours in 96- well flat-bottom microtiter plates using Tryptic Soy Broth supplemented with 1.0-percent sucrose (TSBS). Biofilm was irradiated with blue light for 5 minutes before exposure to various concentrations of NaOCl for 30 seconds. A crystal violet biofilm assay was used to determine relative density of the biofilm. Data were analyzed with two-way ANOVA and Sidak-adjusted multiple comparisons using a 5.0-percent significance level. Null Hypothesis: Blue light and NaOCl will not have an effect against E. faecalis biofilm adherence. Results: Overall, there was a significant effect (p < 0.05) for NaOCl and a significant effect for blue light. The effects of the combination of NaOCl and blue light were also significant. Conclusion: We reject the null hypothesis and accept the alternative hypothesis that blue light when used in conjunction with NaOCl will reduce adherence of E. faecalis biofilm.
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.

Browsing by Author "Bringas, Josef S."

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