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Item The ability of new intracanal medicaments to prevent the formation of multi-species biofilm on radicular dentin(2017) Jacobs, Jordon C.; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Yassen, Ghaeth; Bringas, JosefThe residual antibacterial effects of antimicrobials used in endodontic regeneration against biofilm bacteria obtained from immature and mature teeth Jordon C. Jacobs DDS, Richard L Gregory PhD, Ygal Ehrlich DMD, Kenneth Spolnik DDS, MS, Josef S. Bringas DMD, DDS, MS, and Ghaeth Yassen BDS, MSD, PhD We explored the residual antibacterial properties of dentin pretreated with low concentrations of double antibiotic paste (DAP) against biofilm bacteria obtained from different clinical sources. Dentin blocks were sterilized and randomized into 4 treatment groups and 2 control groups (n=20). Blocks from treatment groups were pretreated with DAP (1 or 5 mg/ml) loaded into methylcellulose, calcium hydroxide (Ca(OH)2), or methylcellulose paste. After one week, the treatment pastes were removed and all blocks were immersed in PBS. The dentin blocks from treatment groups and one of the control groups were then inoculated with bacterial isolates obtained from immature or mature teeth with pulpal necrosis(n=10). The remaining control group received no bacteria and was used as a sterile control. Blocks were then incubated anaerobically for 3 weeks. Biofilm disruption assays were conducted for all samples. Two-way ANOVA and pair-wise comparisons were used for statistical analyses. The residual antibacterial effect of dentin pretreated with 5 mg/ml of DAP was significantly higher than all other groups regardless of the source of biofilm. Dentin pretreated with 1 mg/ml of DAP demonstrated significantly higher residual antibacterial effects in comparison to dentin pretreated with placebo paste and Ca(OH)2 only in bacterial isolates obtained from mature teeth with pulpal necrosis. Dentin pretreated with Ca(OH)2 did not demonstrate any residual antibacterial effects. Dentin pretreated with 1 or 5 mg/ml of DAP demonstrated significantly better residual antibacterial effects against biofilm bacteria obtained from mature teeth with pulpal necrosis in comparison to bacterial isolates obtained from immature teeth with pulpal necrosis.Item The antibacterial effect of new intracanal medicaments against established mutlispecies biofilm(2017) Troxel, Alex; Spolnik, Kenneth J.; Gregory, Richard; Ehrlich, Ygal; Bringas, Josef; Zunt, Susan L.; Yassen, GhaethWe investigated the antibacterial effect of low concentrations of double antibiotic paste (DAP) loaded into a methylcellulose system against bacterial biofilms obtained from mature and immature teeth with necrotic pulps. Standardized radicular dentin specimens were randomly divided into six experimental groups (n = 20). Group 1: 5mg/mL DAP treatment. Group 2: 1mg/mL DAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose. Group 5: No treatment. Group 6: No bacteria or treatment. Clinical bacterial isolates were obtained from mature and immature teeth with necrotic pulps indicated for endodontic regeneration or routine endodontic treatment, respectively. Specimens in each group were inoculated with either bacterial isolates (n = 10) and incubated anaerobically for 3 weeks. Specimens were then treated for one week with the assigned group treatment. Treatments were rinsed with sterile saline and biofilms were detached and spiral plated using biofilm disruption assays. Wilcoxon Rank Sum tests followed by pair-wise comparisons were used for statistical analyses. Treatment of infected dentin with 1 mg/ml of DAP, 5 mg/mL of DAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste. Furthermore, 1 mg/mL of DAP caused complete eradication of biofilm obtained from mature tooth with necrotic pulp. However, the same concentration was not able to completely eradicate biofilm obtained from the immature tooth with necrotic pulp. Low concentrations of DAP (1-5 mg/mL) loaded into a biocompatible methylcellulose system demonstrated significant antibacterial effects against biofilm obtained from both mature and immature teeth with necrotic pulps.Item Antibiotic Treatment of Pseudomonas aeruginosa Biofilms Stimulates Expression of mgtE, a Virulence Modulator(2012-08-07) Redelman, Carly Virginia; Anderson, Gregory G.; Blazer-Yost, Bonnie.; Bauer, Margaret.Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.Item The antimicrobial efficacy of innovative 3D triple antibiotic paste-mimic tubular scaffold against actinomyces naeslundii(2015) Azabi, Asma Abulqasem; Bottino, Marco C.; Gregory, Richard L.; Spolnik, Kenneth J.; Cook, Norman Blaine, 1954-; Chu, Tien-Min GabrielBackground: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.Item Bactericidal Efficacy of EdgePRO Er,Cr:YSGG Laser-Activated Irrigation Against a Mature Endodontic Multispecies Biofilm Using an in vitro Infected Tooth Model(2024) Patterson, Samuel B.; Spolnik, Kenneth J.; Gregory, Richard; Ehrlich, Ygal; Movila, AlexandruIntroduction: Treatment goals of non-surgical root canal therapy (nsRCT) include the removal of all organic tissue material, bacterial biofilm and their by-products, and debris materials, in order to disinfect the canal system to a level compatible with healing and to further prevent infection. Standard chemo-mechanical protocols have several well-documented shortcomings and subsequent areas for improvement regarding their disinfection abilities. In recent years, emerging laser technology and its application in root canal therapy has been gaining popularity as a safe and promising tool for advancing endodontic treatment. The newest FDA-approved laser for endodontic application is the EdgePRO Erbium,Chromium-doped:Yttrium-Scandium-Gallium-Garnet (Er,Cr:YSGG) infrared laser operating at a 2780 nm wavelength. Previous in vitro studies using Er,Cr:YSGG lasers have demonstrated their ability to enhanced canal debridement, cleaning, smear layer removal, and bacterial disinfection. Additionally, a few in vivo trails have been completed using this laser type as an adjunct in RCT procedures, which have yielded safe and highly successful results in the clinical setting. However, research specifically using the EdgePro device as well as a standardized protocol for optimal clinical usage of the laser is lacking. Objectives: The aim of this study was to evaluate the bactericidal and biofilm dissolution effects of laser-activated irrigation using the EdgePro laser against a mature multispecies biofilm in an infected tooth model and to assess the potential increased disinfection and cleaning ability compared to a standard needle irrigation protocol. Materials and Methods: Single rooted teeth (n=36) were decoronated to a standardized length of 16mm. The root canals were endodontically prepared using a standard irrigation, hand-filing, and rotary protocol to a final size of ISO 25.06 while maintaining a fully patent apical foramen. An irrigation solution reservoir was created in the coronal 4 mm of the canal space. Sterile specimens were inoculated with multispecies bacterial sample containing E. faecalis. The mixed bacteria was grown anaerobically for 10 days to form a mature biofilm using a previously established protocol. The teeth were divided into a negative control group (saline rinse, n=12), positive control group (standard needle irrigation – SNI, n=12), and an experimental group (laser-assisted treatment protocol, n=12). The positive control and experimental laser groups utilized the same irrigation solutions of 2 mL 17% EDTA followed by 5 mL 3% NaOCl using a standard 27-gauge side-vented irrigation needle placed as far apically as possible without binding. The experimental group underwent additional laser activation using laser tip #2 (350 m diameter) and settings of: 15 mJ, 0.75 W, 50 Hz, 0% air, and 0% water spray (Mid-Root Solutions 1 preset). The laser tip was inserted halfway into the irrigation filled canals (8 mm from orifice and apex) and fired upon withdrawal at a speed of 0.8 mm/sec, which comprised a single lasing cycle of 10 seconds. Three lasing cycles were completed with EDTA first followed by NaOCl, for a total of six lasing cycles with 60 seconds of irradiation time per tooth. A final rinse of sterile saline was used in all tooth samples prior to bacterial sample collection via Versa-brushes and sterile paper points. The samples were transferred to a laboratory setting where they underwent ultrasonic agitation, serial dilution, spiral plating on blood-agar, and two days of anaerobic incubation for assessment of bacterial growth. Colony forming units (CFUs/mL) were counted as a means of quantitative analysis. Results: The negative control group yielded the highest level of bacterial growth with an average of 934,771 CFUs/mL. The positive control group displayed a statistically significant lower amount of bacterial growth with an average of 4,698 CFUs/mL and yielded 1 sample with no bacterial growth. The experimental laser group had statistically significant lower bacterial growth present compared to both the positive and negative control groups and produced all negative bacterial samples with none of the 12 agar plates demonstrating CFU growth and averaged 0 CFUs/mL.. Conclusion: Within the scope of this study, laser-activated irrigation (LAI) using the EdgePro Er,Cr:YSGG laser was capable of producing no detectable bacterial samples in an in vitro infected tooth model. EdgePro LAI displayed statistically significant superior cleaning and disinfection of infected canal space compared to teeth treated with standard needle irrigation alone. The EdgePro laser system indeed shows promise as an adjunctive tool in clinical root canal treatment procedures. Further investigation is warranted using similar protocols in teeth with more complicated anatomy and with supplemental methods for analyzing bactericidal potential.Item Combined Effects of Soda Drinks and Nicotine on Streptococcus Mutans Metabolic Activity and Biofilm Activity(2019) Mokeem, Lamia Sami; Gregory, Richard; Cook, Norman Blaine; Windsor, Jack; Eckert, GeorgeItem Dual Functions of the Protein MgtE in Pseudomonas aeruginosa(2012-07-03) Coffey, Barbara M.; Anderson, Gregory G.; Marrs, James A.; Randall, Stephen K.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.Item Effect of nicotine on biofilm formation of streptococous mutans isolates from smoking versus non-smoking human subjects(2017) El-ezmerli, Nasreen Farouk; Gregory, Richard L.; Hara, Anderson T.; Cook, Norman Blaine; Windsor, Jack L.Tooth decay is a complex dieto-bacterial disease with an association of social, behavioral and biological factors. Streptococcus mutans plays a major role in tooth decay. This endogenous oral microorganism adheres to tooth surfaces and grows and develops into micro-communities that mature and form dental biofilm. Development of cariogenic biofilm is one of the major factors associated with the tooth decay process. The use of tobacco is considered a great risk factor for oral diseases. Several studies demonstrated the association of tooth decay and the use of tobacco as effects of first-hand or second- hand smoking. Nicotine has been reported to increase the biofilm growth and metabolism of S. mutans in a dose-dependent manner up to 16 mg/ml of nicotine. However, its effects on biofilm formation of S. mutans strains isolated from smokers are not known and should be investigated. Therefore, we proposed the use of an in-vitro model to better understand the effects of nicotine on biofilm formation of strains of S. mutans isolates from smokers versus non-smoking subjects. Objectives: To investigate the effects of nicotine on biofilm formation of S. mutans isolates from oral washes of smoker and non-smoker human subjects. Materials and Methods: This study was conducted using three S. mutans isolates collected from oral washes of 10 smoking subjects and 10 non-smoking subjects. The oral wash samples were stored at -80oC before S. mutans isolation. S. mutans isolates were obtained by plating on Mitis Salivarius Sucrose Bacitracin plates and species identity confirmed by carbohydrate fermentation assays. Nicotine from Sigma-Aldrich (St. Louis, MO, USA) was used. Biofilm was formed by overnight culturing of each S. mutans strain (10 μl) in 190 μl of tryptic soy broth (TSB) supplemented with 1.0-percent sucrose (TSBS) containing 0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 4.0 mg/ml, 8.0 mg/ml, 16.0 mg/ml, and 32.0 mg/ml of nicotine for 24 hours in 5.0-percent CO2 at 37oC in sterile (8 x 12) 96-well microtiter plates (Fisher Scientific, Newark, DE, USA). The absorbance values of biofilm were measured at 490 nm in a microplate spectrophotometer (SpectraMax 190; Molecular Devices, SunnyVale, CA, USA) after crystal violet staining. Null Hypotheses: 1) Nicotine will not increase biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations will not increase biofilm formation in both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) Nicotine will not produce significant differences in biofilm formation between smoker and non-smoker S. mutans isolates. Alternative Hypotheses: 1) Nicotine increases the growth of biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations increase biofilm formation of both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) However, nicotine increases biofilm formation of smoker S. mutans strains more than non-smoker S. mutans isolates. The rationale for this hypothesis is that our preliminary data indicated that S. mutans can become resistant to increased nicotine concentrations and that this resistance appears to be stable and may allow the smoker isolates to be able to respond more vigorously to higher nicotine concentrations than the non-smoker isolates. Results: There was a significant effect (p < 0.05) of both nicotine concentrations and smoking on the growth of biofilm, planktonic cells, and total absorbance, for all strains of S. mutans (p < 0.0001). Isolates from smokers had significantly more biofilm at 0 mg/ml to 16 mg/ml of nicotine compared with those from non-smokers (p-value < 0.0001). Conclusion: S. mutans smoker isolates are more affected by high nicotine concentrations than non-smoker isolates.Item Effectiveness of GentleWave CleanFlow on Multispecies Endodontic Biofilm Removal in Single Rooted Extracted Teeth(2024-06) Beswick, Adam J.; Spolnik, Kenneth J.; Movila, Alexandru; Gregory, Richard L.; Ehrlich, YgalIntroduction: One of the challenges of non-surgical root canal treatment is disinfection. Bacterial biofilms adhere to canal walls and invade the intricate anatomy present within root canal systems. Traditional irrigation methods are unable to deliver irrigation solutions to all parts of the canal system. The GentleWave system is an advanced irrigation method designed to improve irrigation and disinfection, ultimately leading to more successful root canal outcomes. Objective: The aim of this study is to evaluate the GentleWave CleanFlow posterior instrument’s ability to remove a multispecies biofilm from a single canaled extracted tooth compared to traditional irrigation techniques. Materials and Methods: Thirty-six single rooted premolar teeth with single canals were prepared to a uniform size, instrumented to size 25.06 and inoculated with a multispecies bacterial biofilm taken from an adult tooth with pulpal necrosis. Teeth were incubated and biofilm established before teeth were disinfected. Three disinfection groups included: GentleWave irrigation using the Posterior CleanFlow Procedure Instrument on the necrotic tooth cycle, standard needle irrigation with 2.5% NaOCl and 8% EDTA, and needle irrigation with sterile water. Following treatment, canals were swabbed and plated on blood agar plates and incubated for 48 hours when colony forming units were counted. Results: Both GW and standard needle irrigation demonstrated significantly lower CFU/mL than the negative control (p<0.001). However, the GW and positive control groups were not significantly different from one another (p=0.132). Conclusion: The findings of this study suggest that the GentleWave Posterior CleanFlow procedure instrument does not exhibit improved biofilm removal compared to standard needle irrigation. However, based on mixed results when comparing this study to previous GentleWave biofilm removal studies, it is clear that more research is necessary. Future studies should considering using a multispecies biofilm, the GentleWave CleanFlow procedure instrument and multiple techniques to assess biofilm removal.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.
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