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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 ACCURACY OF GUIDED ENDODONTICS IN ENDODONTIC MICROSURGERY(2024-06) Grayson, Michelle Sarah; Spolnik, Kenneth; Ehrlich, Ygal; Dutra, Vinicius; Hine, Charlie; Warner, NedItem Accuracy of Guided Endodontics in Simulated Perforated Teeth with Calcified Canals(2023) Eidelstein, Dyana M.; Spolnik, Kenneth; Ehrlich, Ygal; Dutra, Vinicius; Hine, Charles; Warner, NedIntroduction: Endodontic treatment in teeth following iatrogenic accidents can be challenging and difficult to correct. Guided Endodontic Access (GEA) has been used as an adjunct in endodontic treatment to in in treating teeth with complex anatomy and may assist in treating teeth with procedural accidents. The accuracy of GEA stents in teeth with a simulated procedural accident will be assessed. Objective: This in vitro study will utilize a 3D model of a tooth with a simulated procedural accident: deviation and perforation during root canal treatment(RCT). GEA stents will be fabricated designed to overcome and compare the previous ledge formation and perforation. The influence on the degree of deviation of the designed access path from the prepared path will be determined by assessing the degree of angle of deviation and amount of deviation in millimeters. Materials and Methods: A 3-D printed maxillary model of an anonymous patient will be used. The deviated path and perforation will be simulated in a 3D printed tooth #4 using the meshmixer software tooth at two levels: apical and mid-root. The stent extends from tooth #3 to tooth #14 and was designed using coDiagnostiX software over tooth #4 with a simulated perforation. 15 GEA stents will have guides for the mid root perforation, 15 GEA stents will have guides for the canals not in contact with the perforation, and 15 GEA stents will have guides for canals apical to the perforation. All cases will be accessed with a 1.0 mm drill that is planned to fit the access sleeve. Results: The distal( base), the distal(tip) and the vestibular (tip) for the guided access in which the canal was in contact with the deviated path had no significant difference in deviation. The angle was significantly deviated in all models, but the model in which the canal was in contact with the deviated canal had a significantly lower angle, distal base, and vestibular base that both models in which the canal was not in contact with the deviated path and at different heights. The degree of deviation for all samples ranged from 1.40° to 10.60°. The largest degree of deviation corresponds with the increased depth of the original canal system. Conclusion: In conclusion, our study revealed that the utilization of GEA in calcified teeth with PCO and a deviated path demonstrated greater effectiveness in canals located in closer proximity compared to those located farther away from the deviated path. Therefore, we can infer that the use of a GEA stent in calcified teeth with PCO and a deviated path is favorable only under certain conditions.Item The antibacterial effect of a radiopaque double antibiotic paste against both an established multispecies and a single enterococcus faecalis biofilm(2019) Haslam, Bryce S.; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Bringas, Josef; Warner, Ned A.For regenerative endodontic procedures (REPs) to be successful an elimination of bacteria from the root canal system must be accomplished. Many different medicaments with antibacterial properties have been used to obtain complete disinfection. Double antibiotic paste (DAP) containing a mixture of ciprofloxacin and metronidazole has been shown to be a promising intracanal medicament. The addition of a radiopaque filler such as zirconium oxide to DAP may affect the antibacterial properties of DAP as well as allow precise placement and radiographic visualization of its position in the canal system. The aim of the proposed study was to evaluate the direct antibacterial properties of zirconium oxide radiopacifier combined with DAP (RoDAP) against a multispecies biofilm from a bacterial isolate from an infected immature tooth with a necrotic pulp and a known single species biofilm. 4x4 mm radicular dentin specimens (n = 112) obtained from human extracted teeth were used prepared and sterilized prior to use. A multispecies clinical bacterial isolate from an immature tooth with a necrotic pulp and a single species Enterococcus faecalis isolate were obtained. These bacterial isolates were used to inoculate dentin slabs and grown for 3 weeks. The dentin slabs were treated for 1 week with 1.0-mg/mL and 10- mg/mL RoDAP, 1.0-mg/mL DAP, and two placebo pastes consisting of methyl cellulose (MC) and methyl cellulose combined with zirconium oxide (RoMC), respectively, as well as two no-treatment controls. Following treatment, the grown biofilm was detached and spiral plated. The plated biofilm cells were cultured for 24 hours and each group examined using a colony counter to determine bacterial numbers (CFUs/mL). Data analysis, using a 5.0-percent significance level was conducted using one-way ANOVA followed by pair-wise group comparisons. Both 1.0-mg/mL and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from an immature tooth with a necrotic pulp as well as an E. faecalis isolate. The precise application of RoDAP confirmed radiographically with its direct antibacterial properties may be beneficial for intracanal disinfection during REPs.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 The antibacterial effects of radiopaque double antibiotic pastes against clinical bacterial isolates from mature and immature teeth with necrotic pulps(2018) Ibrahim, Carolin Francis; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Zunt, Susan; Bringas, Josef; Yassen, GhaethLow concentrations (1-10mg/mL) of double antibiotic paste (DAP) have demonstrated antibacterial properties in regenerative endodontics. The aim of this study was to evaluate if DAP made radiopaque (RoDAP) with barium sulfate has antibacterial effects against bacterial isolates from a mature and immature tooth with necrotic pulp. Clinical bacterial isolates were obtained from the canals of mature and immature teeth with necrotic pulps during root canal therapy or a regenerative procedure, respectively. Bacterial isolates were grown anaerobically for three weeks on 4x4mm dentin specimens prepared from extracted human teeth (n=48 per biofilm type). The dentin specimens were allocated into six groups and treated as follows: 1mg/mL RoDAP, 10mg/mL RoDAP, calcium hydroxide (UltraCal), placebo (barium sulfate in methylcellulose), no treatment, and no bacteria or treatment (sterile control). After one week of treatment the biofilm was detached and biofilm disruption assays were conducted to determine the bacterial numbers (CFUs/mL). The data was analyzed using Wilcoxon Rank Sum tests followed by pairwise comparisons. 1 and 10 mg/mL RoDAP as well as calcium hydroxide demonstrated significant antibacterial effects against the tested bacterial isolates. The placebo paste did not demonstrate any significant antibacterial effects. No significant difference in antibacterial effects was found against isolates from both mature and immature teeth regardless of the type of treatment. Both 1 and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from mature and immature teeth with necrotic pulps. RoDAP can be beneficial clinically since its adequate placement within the canal system can be confirmed radiographically.Item The antibacterial stability of a new radiopaque double antibiotic paste(2018-06) Epkey, Kathryn Eileen; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Zunt, Susan; Bringas, Josef; Yassen, GhaethWe evaluated the antibacterial stability (shelf life) of a new radiopaque double antibiotic paste (RoDAP) loaded in a methylcellulose system with 30% w/v barium sulfate against biofilm collected from an immature tooth with necrotic pulp. Uniform radicular dentin specimens were infected with bacterial biofilm obtained from an immature tooth with a necrotic pulp and incubated anaerobically for three weeks. These samples were randomly divided into 6 experimental groups (n=7) and treated for 1 week at three time points of aged radiopaque DAP: 0 months, 3 months, and 6 months. Group 1: 1mg/mL RoDAP treatment. Group 2: 10 mg/mL RoDAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose with barium sulfate. Group 5: No treatment. Group 6: No bacteria or treatment. The samples were rinsed with sterile saline to detach biofilms and then spiral plated using a biofilm disruption assay. Statistical analyses were performed using Wilcoxon rank-sum tests and Wilcoxon signed rank tests with fixed effects for treatment, time, and the treatment-by-time interaction. Treatment of infected dentin with 1 mg/mL RoDAP, 10 mg/mL RoDAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste after 0, 3, and 6 months of aging. Calcium hydroxide, however, showed slightly less antibiofilm activity after 6 months of aging when compared to 0 months and 3 months of aging. This difference was statistically significant (p > 0.05). In conclusion, both concentrations of RoDAP maintained full antibacterial efficacy after 6 months of aging, while calcium hydroxide lost some antibacterial activity after a shelf life of 6 months.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 Biodegradability of resilon, a resin based root canal obturating material, by typical endodontic pathogens(2012) Rexford, Ashleigh M.; Spolnik, Kenneth Jacob, 1950-; Vail, Mychel Macapagal, 1969-; Hara, Anderson T.; Ehrlich, Ygal; Zunt, Susan L., 1951-; Gregory, Richard L.; Legan, Joseph J.Root canal therapy is a recommended treatment for apical periodontitis. Root canal failure can occur as a result of microbial leakage. Resilon, a resin based root canal obturating cone material introduced in 2004 attempts to minimize leakage by a unique bonding method of the resin sealer to both the core material and to the dentin of the canal walls. Resilon has no bactericidal or antimicrobial effect15. Furthermore, it has been shown that Resilon is susceptible to alkaline and enzymatic hydrolysis as well as bacterial degradation.73, 184-186 It has been suggested that Resilon may be susceptible to degradation by microorganisms found in the infected root canal space. This work focuses on the susceptibility of root canal obturating materials to be degraded by endodontic pathogens seen in root canal treated teeth with apical periodontitis. The aim of this study was to determine if Resilon could be degraded by selected pathogenic bacteria found in the infected root canal system, and if this degradation is more severe than with gutta-percha, a conventional obturating material. P. intermedia, E. faecalis and P. aeruginosa, known endodontic pathogens were inoculated on discs of obturating material (Resilon or gutta-percha) mounted on a platform and placed in wells containing TSB incubated at 37°C under aerobic conditions. The discs were polished, examined by SEM, profilometry, and elemental analysis prior to inoculation to establish a baseline, and were then re-examined by these methods one month after inoculation. The overall results were inconclusive; and using these methods it cannot be determined that the selected bacteria can degrade Resilon. An ideal future study would utilize SEM with gold coated samples as well as atomic force microscopy to evaluate for changes in topographical features of these obturating materials. A notable finding was that Resilon turns black when exposed to bacteria, and the significance of this finding should be addressed in future studies.Item Buffered vs. Unbuffered Local Anesthesia in Mandibular Molars Diagnosed with Symptomatic Irreversible Pulpitis: A Controlled, Randomized, Double-Blind Study(2022) Alena, Peter; Spolnik, Kenneth; Ehrlich, Ygal; Warner, NedIntroduction: Profound pulpal anesthesia is not always adequate in mandibular teeth after the administration of local anesthesia, especially in the presence of irreversible pulpitis. Failure to achieve anesthesia has been seen in 30–80% of patients in teeth with a diagnosis of irreversible pulpitis. Onpharma™ developed an FDA-approved device that uses sodium bicarbonate to buffer a standard local anesthetic (LA) solution so that its pH may become closer to its pKa. Claims have been made that buffering a local anesthetic increases the anesthetic’s effect. Previous studies on the anesthetic efficacy of Onpharma’s Onset buffering system were inconclusive and may be dependent on the techniques used. Objectives: The aim of this study is to determine whether a buffered local anesthetic can lead to more profound and faster pulpal anesthesia in mandibular molars diagnosed with symptomatic irreversible pulpitis as compared to a standard, unbuffered local anesthetic. Materials and Methods: 40 total subjects completed the study. Screened and eligible subjects with a mandibular molar diagnosed with symptomatic irreversible pulpitis were randomly allocated into 2 groups so 1 group received a total of 3 cartridges of a standard, unbuffered 2% lidocaine with 1:100,000 epinephrine via inferior alveolar nerve block (IANB) followed by supplemental buccal and lingual infiltrations, while the other received the equivalent yet buffered formulation. An electronic pulp tester (EPT) was used to objectively determine baseline pulpal status of the affected tooth, followed by 2-minute interval testing following the administration of all local anesthesia. The onset of pulpal anesthesia was defined by the first of 2 consecutive EPT=80 readings, and the endodontic treatment could begin. Profound pulpal anesthesia was ultimately determined if the patient reported a comfortable pulpotomy as reflected on the Wong-Baker FACES Visual Analog Scale. Null Hypothesis 1: Subjects possessing mandibular molars diagnosed with symptomatic irreversible pulpitis will not achieve pulpal anesthesia more profoundly using buffered 2% lidocaine w/ 1:100,000 epinephrine in comparison to the standard, unbuffered anesthetic formulation. Null hypothesis 2: Subjects possessing mandibular molars diagnosed with symptomatic irreversible pulpitis will not achieve pulpal anesthesia faster using buffered 2% lidocaine w/ 1:100,000 epinephrine in comparison to the standard, unbuffered anesthetic formulation. Results: We observed a local anesthetic success rate of 45% in the buffered group, 70% in the unbuffered group, and ultimately 57.5% between both groups. The findings further indicate that the VAS scores after pulpotomy is significantly different between the 2 groups (p=0.019), with the unbuffered group having a more profound mean VAS score of 1.2 (as opposed to a buffered mean of 3.1). Regarding the time of onset for pulpal anesthesia, there was no statistically significant difference noted between the buffered and unbuffered groups. Conclusion: Based on the findings of this study, the null hypothesis 1 cannot be rejected since unbuffered 2% lidocaine with 1:100,000 epinephrine had a statistically significant increase in profound pulpal anesthesia compared to the buffered equivalent. The null hypothesis 2 cannot be rejected since there was no evidence of a significant difference in the time to pulpal anesthesia between the buffered and unbuffered groups.