Thesis Abstract


AN IN-VITRO SEM STUDY COMPARING THE DEBRIDEMENT EFFICACY OF THE ENDOACTIVATOR SYSTEM VERSUS THE ULTRASONIC BYPASS SYSTEM FOLLOWING HAND-ROTARY INSTRUMENTATION

Steve W. Binkley, DMD

INTRODUCTION: Removal of all microorganisms, microbial by-products, and vital and necrotic tissue is the goal of endodontic therapy. This is accomplished during the chemo-mechanical preparation of the canal space. Due to the intricate nature and inherent complexities associated with canal anatomy, this goal is never completely achieved. Sufficient disinfection of these irregularities, fins, isthmuses, webs, and anastomoses cannot be accomplished with traditional instruments alone. Activation of the irrigation solutions utilized during the cleaning and shaping phase of therapy has shown to increase canal cleanliness and decrease viable bacterial counts in infected canals.

PURPOSE: It is the aim of this study to determine if there is any difference in debridement efficacy between sonic and ultrasonic activation of canal irrigation solutions.

METHODS & MATERIALS: Sixty teeth were instrumented to one millimeter from the canal terminus using standard hand-rotary instrumentation techniques. Twenty teeth were then treated for one minute with the EndoActivator, the Ultrasonic Bypass System, and the final twenty were used as controls with no activation of the irrigation solutions. Each tooth was then sectioned and examined under Scanning Electron Microscope in the coronal, middle, and apical three millimeters of each root. Two dental professionals rated and scored each section of each root based on a standard scoring regimen. A total of one hundred eighty images were recorded (sixty from each group). Intra-examiner repeatability and inter-examiner agreement of the debris removal scores were assessed using two-way contingency tables and weighted kappa statistics. Using the consensus scores separately for each of the three locations, the three methods were compared for differences in debris removal scores using a Kruskal-Wallis test, which determined if there were any differences among the three groups. When the overall test was significant, Wilcoxon Rank Sum tests were used to compare each pair of groups.

RESULTS: The repeatability in specimen scoring for each examiner was acceptable (weighted kappa of 0.83 for the first examiner and 0.84 for the second examiner). Both the EndoActivator™ and Ultrasonic Bypass™ groups had a smaller percentage of canal space occupied by smear layer and debris when compared to the control group at all three levels. This difference was statistically significant for the Ultrasonic Bypass™ System when compared to the control at both the coronal and middle thirds of the samples evaluated. This difference was not statistically significant in the apical third. When compared to the EndoActivator™, the Ultrasonic Bypass™ System produced cleaner canals in the coronal and middle thirds, with the difference being statistically significant in the middle third only.

CONCLUSION: These results of this research support the use of either of these two devices when compared to the controls. Smear layer removal and debridement efficacy was greatly increased when using either sonic or ultrasonic activation of sodium hypochlorite.

AN IN-VITRO STUDY EVALUATING THE EFFICACY OF THE ULTRASONIC BYPASS SYSTEM, USING DIFFERENT INTRACANAL IRRIGATING SOLUTIONS

Jason P. Barney, DDS

INTRODUCTION: Passive ultrasonic irrigation is an important adjunct in endodontic therapy that assists in smear layer removal, better debridement of fins and isthmuses, and bacterial elimination. The Ultrasonic Bypass unit functions by expressing a constant flow of irrigating solutions in conjunction with ultrasonic vibrations. Both of which can greatly cleanse the smear layer from the canal walls.

PURPOSE: Of interest in this study is the examination of the Ultrasonic Bypass system with the common irrigation solutions often used in chemo/mechanical debridement of root canals, such as 6% NaOCl and 17% EDTA, and to study the effectiveness of smear layer removal. If the findings of this study are significant, clinicians can implement the use of a specific irrigation regimen to improve smear layer removal.

METHODS AND MATERIALS:  Eighty maxillary and mandibular anterior single canal teeth were selected. Four groups were randomly divided between the teeth, with each group having 20 teeth. All the teeth were instrumented using the same protocol with all them. A working length was established by reducing back 1mm from where a #10 file is visible at the apex. Each tooth was worked up to #35/0.06 taper nickel-titanium rotary files. Following the manufacturer’s guidelines for use, the Ultrasonic Bypass System was used on each group with different irrigating solutions. Group one no irrigating solution was used. In group two, 6% NaOCl was used alone. In group three, 17% EDTA alone was used. In Group four 6% sodium hypochlorite and 17% EDTA were used. After this all teeth were sectioned longitudinally into two halves and then the half showing the canal from the apex to the coronal portion of the canal were examined under SEM. Intra-examiner repeatability and inter-examiner agreement of the debris removal scores were assessed using two-way contingency tables, percent agreement, and weighted kappa statistics. Using the consensus scores separately for each of the three locations, the four groups were compared for differences in debris removal scores using a Kruskal-Wallis test, which determines if there are any differences among the four groups. If the overall test were significant, Wilcoxon Rank Sum tests was used to compare each pair of groups.

RESULTS: Intra-examiner repeatability: Intra-examiner repeatability analysis for examiner 1 resulted in a weighted kappa=0.71, with disagreements usually due to a lower score on the repeat evaluation. Intra-examiner repeatability analysis for examiner 2 resulted in a weighted kappa=0.60, with disagreements usually due to a higher score on the repeat evaluation. Inter-examiner agreement: The inter-examiner agreement analysis showed that disagreements were usually caused by lower scores given by examiner 1 than by examiner 2, with the weighted kappa slightly lower than the intra-examiner kappas as expected. The Ultrasonic Bypass System when used with the combination of 6% NaOCl and 17% EDTA after hand/rotary instrumentation significantly removed smear layer at the coronal, middle, and apical areas of a tooth when compared to the following groups: (Hand/rotary instrumentation alone, Hand/rotary + Ultrasonic Bypass with 6% NaOCl. Hand/rotary + Ultrasonic Bypass with 17% EDTA). In the coronal and middle thirds of the tooth, the one minute addition of the Ultrasonic Bypass with either 6% NaOCl alone or 17% EDTA alone significantly removed more smear debris than the control. There was no significant difference when the Ultrasonic Bypass system was used with NaOCl compared with EDTA, except in the middle third where PUI with EDTA was significantly more effective. In the apical third the combination of NaOCl and EDTA with the Ultrasonic Bypass System was significantly more effective in smear removal than any other group.

CONCLUSION: A one-minute PUI with the Ultrasonic Bypass System combined with NaOCl and EDTA is significantly better in smear removal and ultimately will result in better biofilm elimination and cleaner canal walls.

AN IN-VITRO SEM COMPARISON OF DEBRIDEMENT EFFICACY USING THE ENDOVAC® SYSTEM (DISCUS DENTAL®) VS CANAL CLEANMAX® (MAXIMUM DENTAL®) FOLLOWING HAND AND ROTARY INSTRUMENTATION

Ben Ricketts, DDS

INTRODUCTION: Multiple studies have suggested that bacteria and debris remain within the root canal system even after meticulous chemo-mechanical debridement. However, some studies have suggested that negative pressure irrigation and aspiration techniques can increase efficacy of debris and smear layer removal in all areas of the canal system.

PURPOSE: The purpose of this study was to examine human anterior teeth after hand and rotary instrumentation to determine the effectiveness of debridement after irrigation and aspiration via EndoVac® (Discus Dental®, Culver City, CA) versus Canal CleanMax® (Maximum Dental®, Inc., Secaucus, NJ).

METHODS AND MATERIALS: Sixty extracted anterior teeth were instrumented using a combination of hand-instrumentation with K-files and rotary instrumentation with ProTaper® files (Tulsa Dental Products, Tulsa, OK). All canals were irrigated with six-percent sodium hypochlorite and 17- percent ethylenediamine tetra-acetic acid (EDTA). However, the irrigation/aspiration techniques differed among three groups of 20 randomly selected teeth. Group one (control) was irrigated and with only a 12-milliliter monojet syringe via 30-gauge side-vent 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. The 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 amount of debris and/or smear layer present. These scores were statistically analyzed to determine differences between groups. Intra-examiner repeatability and inter-examiner agreement of the debris removal scores were assessed using two-way contingency tables, percent agreement, and weighted kappa statistics. If the two examiners disagreed, they reached a 'forced consensus' after discussing the case. Using the consensus scores separately for each of the three locations of the canal, the three methods were compared for differences in debris removal scores using a Kruskal-Wallis test, which determined if there were any differences among the three groups. If the overall test was significant, Wilcoxon Rank Sum tests were used to compare each pair of groups.

RESULTS: For the coronal aspect, there were significant differences in debris scores among groups (p=0.0327), with significantly lower scores for EndoVac® than Control (p=0.0119) and no significant difference between Canal CleanMax® and Control (0.3965) or Canal CleanMax® and Endovac® (p=0.1196). For the middle section of canal wall there was not a significant difference in debris scores among groups (p=0.3877), and none of the pairwise differences were significant. For the apical location of canal there was not a significant difference in debris scores among groups (p=0.8619), and none of the pairwise differences were significant.

CONCLUSION: Root canal walls exhibited less debris and/or smear layer when irrigated with the EndoVac® system after hand and rotary instrumentation, but only in the coronal third of the root canal system.

ANTIBACTERIAL EFFICACY OF 0.12% AND 2% CHLORHEXIDINE GLUCONATE AT 37 DEGREES CELCIUS AND 46 DEGREES CELCIUS AGAINST ENTEROCOCCUS FAECALIS

Craig Thiessen, DDS

INTRODUCTION: Various irrigation solutions and intracanal medicaments are used during and after root canal preparation to reduce debris, necrotic pulp tissue, and microorganisms. While sodium hypochlorite (NaOCl) is the most commonly used endodontic irrigation solution, other irrigation solutions such as chlorhexidine gluconate (CHX) have proven to be effective during root canal therapy. There have been several studies stating the antibacterial properties of CHX as an endodontic irrigation solution. This antibacterial efficacy is dependent on the concentration of the irrigation solution used. A unique property of CHX is its substantivity effect up to 72 hours after its removal. Several studies have reported that increasing the temperature of irrigation solutions can enhance their antimicrobial efficacy.

PURPOSE: The aim of this study is to evaluate the antibacterial efficacy of 0.12% and 2.0% CHX on eliminating E. faecalis from dentinal tubules, and whether this antibacterial effect is enhanced by heat.

METHODS AND MATERIALS:  Ninety-five human extracted, single rooted, maxillary, central, lateral, and canine teeth were used to prepare dentin disk specimens. After proper sterilization, a 2.5mm ISO sized diameter lumen was prepared, then these canals were filled with brain-heart infusion (BHI) broth and infected with Enterococcus faecalis and incubated for 72 hours to allow sufficient time for growth and adherence to the lumen wall. The BHI was removed and the specimens in equally divided groups were rinsed with sterile saline and filled with either, saline, 0.12% CHX or 2.0% CHX at ambient temperature (24°C) or experimental temperature (46°C) and incubated at oral temperature (37°C) or the experimental temperature (46°C), respectively. The specimens were frozen at -70?C, pulverized in liquid nitrogen, serial dilutions prepared and spiral plated on BHI agar plates, incubated, and the number of bacterial colonies recorded 24 hours later for data analysis. Comparisons of E. faecalis CFU were performed using two-way analysis of variance (ANOVA), with factors for solution, solution temperature, and the solution-by-temperature interaction. Pair-wise comparisons between groups were examined for significance using the Fisher’s Protected Least Significant Differences Method.

RESULTS: The interaction between solution and solution temperature was not significant (p=0.66), so the solution comparisons can be generalized for any temperature and the temperature comparisons can be generalized for any solution. E. faecalis CFU differed by solution (p<0.0001): Saline had significantly higher E. faecalis CFU than 0.12 percent CHX (p=0.00181) and 2.0 percent CHX (p<0.0001), and 0.12 percent CHX had significantly higher E. faecalis CFU than 2.0 percent CHX (p=0.0001). Solution temperature did not have a significant effect on E. faecalis CFU (p=0.44).

CONCLUSION: The antibacterial efficacy of a higher concentration of CHX was more effective against E. faecalis compared to a lower concentration. The antibacterial effects of heat made no difference in eliminating E. faecalis within the dentinal tubules.

AN IN-VITRO COMPARISON OF BACTERIAL MICROLEAKAGE OF ZINC-OXIDE EUGENOL AND BRASSELER BIOCERAMIC SEALER

Joyce Nazzal, DDS

INTRODUCTION: Root canal sealers are an important component in sealing the interface between the dentinal walls and the obturation material. The goal is to obtain a hermetic seal after adequate cleaning and shaping of the canal. This hermetic seal cannot be obtained without the use of a sealer because gutta-percha as mentioned previously does not bond to the dentin walls.Early sealers were modified zinc oxide-eugenol cements based on Grossman and Rickert’s. These sealers are still used today. New sealers have been placed on the market to improve the property of this hermetic seal and decrease the gap between the gutta-percha and dentin wall. An ideal endodontic sealer should, in part, adhere firmly both to dentin and gutta-percha. Differences in the adhesive properties of endodontic sealers may be expected because their interaction with either dentin or gutta-percha may vary with their chemical composition. No specific interaction either with dentin or gutta-percha is expected from the setting reaction of calcium hydroxide based sealers and the epoxy-based sealers. In contrast, the zinc oxide-eugenol sealer should firmly bond to dentin and gutta-percha. The setting reaction of the zinc oxide-eugenol mixtures is a chelation reaction occurring with the zinc ion of the zinc oxide.

PURPOSE: To evaluate the sealing properties of EndoSequence BC sealer, a bioceramic sealer, and Roth’s sealer using gutta-percha with warm vertical condensation and using the single cone technique with the EndoSequence BC sealer only.

METHODS AND MATERIALS: Eighty-five single-rooted maxillary anterior teeth were used for this study.  Endodontic cleaning and shaping of each root canal system was accomplished using K-type hand files and NiTi Rotary files. Group A consisted of 27 anterior teeth, which weres obturated using EndoSequence BC sealer and gutta-percha with System B and Obtura. Another 27 anterior teeth forming Group B were obturated using Roth’s sealer and gutta-percha with System B and Obtura. In the final group, Group C, 27 teeth were obturated using the single-cone technique by means of a single gutta-percha point with EndoSequence BC sealer.  Two teeth were used as a positive control group, and two other teeth as a negative control group. A microbial leakage apparatus was constructed using a similar two-chamber method.  The test bacterium used to determine microleakage was E. faecalis ATCC 29212.

The outcome of interest (bacterial turbidity) and time-to-leakage (in days), was determined for each of the samples. Survival analysis was used to compare the two groups, with a Kaplan-Meier plot to visualize the results and a nonparametric log-rank test for the group comparison

RESULTS:  No microleakage was observed in the negative control or Group B. Microleakage was observed in all positive controls. Group A and C had a significantly higher proportion of samples with microleakage than group B (p < 0.0001), but Group A and Group C were not significantly different from each other (p = 0.50). Time to microleakage was also significantly lower in Group A and Group C than Group B (p < 0.0001), but group A and C were not significantly different from each other (p = 0.37).

CONCLUSION:  Using E. faecalis as our test bacteria, the microleakage of canals obturated with gutta-percha and Roth sealer was significantly less than in canals obturated with gutta-percha and EndoSequence BC sealer.

AN IN-VITRO COMPARISON Of THE MICROLEAKAGE OF REALSEAL/RESILON AND REALSEAL SELF-ETCH/RESILON ROOT CANAL OBTURATION SYSTEM

Haris Iqbal, DDS

INTRODUCTION: Currently in endodontics, one of the resin obturation systems using dentin adhesive technology is RealSeal (SybronEndo, Orange, CA), formerly known as Epiphany (Pentron Clinical Technologies, Wallingford, CT). The two-step self-etching (sixth generation) adhesive system utilizes an acidic primer applied to the dentin surface.The acid and primer have been combined that eliminates one step in the process. After the acidic primer is dried, RealSeal sealer, a dual-cured resin cement, is applied to the root canal system and polymerized.

Recently, a new version of the RealSeal sealer, based on the self-adhesive cement concept (seventh generation) was introduced with the promise of optimizing clinical performance with a simplified one-step application procedure. Thus, the manufacturers have stated that the new RealSeal Self-Etch sealer (RealSeal SE; Pentron Clinical Technologies LLC, Wallingford, CT) could bond simultaneously to both radicular dentin and Resilon points.

PURPOSE: To evaluate and compare microleakage of teeth obturated using either RealSeal/Resilon or RealSeal Self-Etch/Resilon systems. The goal was to determine whether a significant difference in microleakage exists between these two groups. To date, no study has been done comparing the microleakage of root canal systems obturated with using RealSeal/Resilon versus RealSeal SE/Resilon.

METHODS AND MATERIALS: Sixty-two human, single-rooted, anterior teeth were accessed and instrumented for non-surgical root canal therapy. Teeth were randomly assigned to two experimental groups of 27 teeth each. Group I consisted of teeth obturated with the RealSeal/Resilon system, whereas Group II consisted of teeth obturated with the RealSeal SE/Resilon system. In addition, two control groups containing four teeth each served as positive and negative controls, Group (+) and Group (-), respectively. The teeth were then evaluated for microleakage using a dual-chamber microleakage model. Visual turbidity in the lower chamber denoted microleakage within the experimental groups observed for 33 days.

RESULTS: RealSeal SE Group II had a significantly higher proportion of samples than Real Seal Group I. Time to microleakage was also significantly lower in RealSeal SE Group II than in Real Seal Group I. No microleakage was observed in the negative control and microleakage was observed in all four samples in the positive control.

DISCUSSION/CONCLUSION: To date, this is the first study comparing the microleakage of RealSeal/Resilon and RealSeal SE/Resilon systems. The higher microleakage associated with RealSeal SE is attributed to the higher pH of the self-etch (SE) sealer in comparison with the self-etch primer of RealSeal. The self-etching potential of the sealer system is particularly critical in areas inaccessible to calcium chelating agents such as EDTA in root canal systems. Further research needs to be done to corroborate the microleakage results from this study.

The microbial leakage apparatus devised in this study, which used a selective growth medium with streptomycin, has also been validated by the results of the study. The bacterial leakage apparatus has been considered to be clinically relevant and acceptable by the Journal of Endodontics. Thus, the modified dual-chambered microleakage apparatus with a selective growth medium used in this research can be replicated easily in future microleakage studies.     

AN IN-VITRO COMPARISON OF MICROLEAKAGE WITH E. FAECALIS IN TEETH WITH ROOT-END FILLINGS OF PROROOT MTA AND BRASSELER'S ENDOSEQUENCE ROOT REPAIR PUTTY

Beau Brasseale, DDS

INTRODUCTION:  Mineral trioxide aggregate (MTA) has been thoroughly investigated in a variety of clinical endodontic applications. No dental material previously available to endodontists has demonstrated such a desirable combination of biocompatibility, hydrophilicity, sealability, strength, and antibacterial action. MTA clinical applications include direct pulp capping, apexogenesis, apexification, regenerative endodontics, root perforation repair, and surgical root-end filling. The clinical success of MTA in these applications is well-studied, but many authors describe the poor handling characteristics of MTA and the resulting technique sensitivity of its application as the major disadvantage of this outstanding material.

EndoSequence Root Repair Material (ERRM) (Brasseler; Savannah GA) is stated by the manufacturer to bond to adjacent dentin, to have no shrinkage, and to be highly biocompatible, hydrophilic, radiopaque, and antibacterial due to a high pH during setting. Brasseler?s ERRM comes premixed from the manufacturer in a jar as putty and in preloaded syringes as a flowable paste and sets within 30 minutes. The major advantages of this material are improved handling characteristics over traditional MTA and the delivery of a consistent product with each application. The current research on ERRM is limited and warrants further investigation. ERRM is composed of calcium silicates, monobasic calcium phosphate, zirconium oxide, tantalum oxide, proprietary fillers, and thickening agents.

PURPOSE: To compare the microbial leakage of Enterococcus faecalis in teeth with root-end fillings using ProRoot MTA and Brasseler?s ERRM in a dual-chamber bacterial leakage model as described by Torabinejad and colleagues. The aim of this investigation was to compare the bacterial microleakage of these two root-end filling materials exists.

METHODS AND MATERIALS: Sixty-two human, single-rooted, mandibular premolars in which extraction was indicated were accessed and instrumented in an orthograde fashion with hand and rotary files. Root resection of the apical 3 mm was then completed and root-end retropreparations were created for placement of root-end filling material. Twenty-seven of these premolars had root-end fillings using ProRoot MTA and 27 had root-end fillings using ERRM. Two teeth were used as a positive control group with no root-end filling, and two other teeth were used as a negative control group and were sealed and coated with dentin bonding agent. The teeth were then evaluated for microleakage using a dual-chamber bacterial microleakage model for 40 days as described by Torabinejad and colleagues. Microleakage was determined by the presence of turbidity in the lower chamber of the apparatus and was assessed each day. Fresh samples of E. faecalis were used every three days to inoculate the apparatus and serve as a bacterial challenge for the materials. Results were recorded every day for 30 days. The outcome of interest (bacterial turbidity) and time-to-leakage (in days) were determined for each of the samples. Survival analysis was used to compare the two groups with a Kaplan-Meier plot to visualize the results and a nonparametric log-rank test for the group comparison.

RESULTS:The microleakage of ERRM was not statistically different (p > 0.05) than leakage of ProRoot MTA when subjected to E. Faecalis over the 40 day observation period. Both groups had a small number o fearly failures (4 days) and no leakage was observed for the remaining 40 days of the study. Therefore, the null hypothesis was rejected. 

CONCLUSION: The results of this research support the use of either of these two materials when compared with the controls. The microleakage of Brasseler?s EndoSequence Root Repair Material was at least as good as ProRoot Mineral Trioxide Aggregate when tested with E. faecalis.

ANTIMICROBIAL PROPERTIES OF DRUG-CONTAINING ELECTROSPUN SCAFFOLDS

John Jeppson, DDS

INTRODUCTION:  Endodontic treatment of the infected immature tooth has undergone a dramatic change. Conventional endodontic treatment can control infection, but root development usually remains impaired. A novel regenerative endodontic procedure- the revascularization method can now control the infection and enable such teeth to continue root development. This is done by creating a fibrin-matrix scaffold in the antibiotic treated root canal space (RCS).  Dental stem cells and growth factors have been able to continue root development in such an environment. The fibrin-matrix scaffold is dependent on the induction of a blood clot into the RCS and this cannot always be predictably induced.  PDS is a biocompatible material that can be electrospun to provide a matrix for cells and growth factors and perhaps improve on the blood clot induced fibrin scaffold by incorporating metronidazole as an adjuvant antimicrobial.  A metronidazole containing electrospun PDS scaffold was examined in vitro using a turbidimetric test, the modified direct contact test.  This scaffold significantly inhibited growth of an anaerobic primary endodontic pathogen Porphyromonas gingivalis. This scaffold may improve the treatment of the infected immature tooth by providing a designed matrix for root regeneration while serving simultaneously as an antibiotic drug delivery device to disinfect the RCS.

PURPOSE: The aim of this study is to evaluate in vitro the property of a synthetic scaffold to function as an antibacterial drug delivery device.

MATERIALS AND METHODS: PDS*II (polydioxanone) suture was obtained from Ethicon, INC. (Somerville, NJ) and was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP (Sigma Aldrich). Three different scaffolds were electrospun onto an aluminum foil background; (1) control scaffold with no antibiotic incorporated, (2) scaffold with 5% metronidazole incorporated and (3) 25% metronidazole incorporated. All scaffolds were cut using a 4mm diameter biopsy punch under aseptic conditions and removed from foil, control scaffold n=64, scaffold containing 5% metronidazole n=32, and scaffold containing 25% metronidazole n=32. Experimental scaffolds were placed in a 96 well sterile flat bottom microtiter plate. Porphymonas gingivalis a known primary endodontic pathogen was grown in 5ml of BHI + YE with 0.25 microliters of vitamin K with incubation at 37°C under anaerobic conditions for 48 hours. Microplates were sterilized before inoculation with Pg with 400 microliters of 70% EtOH for a minimum of 30 minutes then pipetted out. After sterilization the microwells were washed with 400 microliters of sterile water and pipetted out. Group 1 (negative control) microwells n=8 contained control scaffold and 190 microliters of broth only. Group 2 (positive control) microwells n=8 contained 190 microliters of broth and Pg only. Group 3 microwells n=8 contained control scaffold, 190 microliters of broth, and 10 microliters of Pg inoculum. Group 4 microwells n=8 contained scaffold with 5% metronidazole, 190 microliters of broth, and 10 microliters of Pg inoculum. Group 5 microwells n=8 contained scaffold with 25% metronidazole, 190 microliters of broth, and 10 microliters of Pg inoculum. Group 6 contained 190 microliters of uninnoculated broth for spectrophotometer calibration. Sterile microplate lids were used to isolate microwells from the surrounding environment. Microplates were incubated at 37°C under anaerobic conditions for 48 hours. After 48 hours the microplates were read by using an endpoint reading in the spectrophotometer. This was repeated four times.

RESULTS: Comparisons between the groups for differences in optical density as a measure of bacterial growth were made using mixed-model ANOVA, with a fixed effect for group and a random effect for experimental run. Pair-wise group comparisons were performed using Tukey's multiple comparisons procedure to control the overall significance level at 5%. The analyses were performed using the ranks of the data. Broth had significantly lower OD than all other groups (p<0.0001). Broth+Pg and Broth+Pg+Scaffold had significantly higher OD than 5% Metro (p<0.0001) and 25% Metro (p<0.0001), but Broth+Pg and Broth+Pg+Scaffold were not significantly different from each other (p=0.97). 5% Metro and 25% Metro were not significantly different from each other (p=0.24).

CONCLUSIONS: From the results of our study we concluded that the 5 and 25% metronidazole containing scaffolds significantly inhibited bacterial growth and could be effectively utilized for the endodontic regeneration procedure.

 

AN IN-VITRO EVALUATION ON THE BIODEGRADABILITY OF RESILON

Jenny Whatley, DMD

INTRODUCTION:  Resilon is a resin-based obturation material that claims to create a monoblock through bonding of RealSealsealer to the dentin walls and to the core material. Resilon is comprised of a biodegradable polymer, polycaprolactone, and inorganic fillers. Resilon has been shown to undergo enzymatic hydrolysis by bacterial enzymes such as lipase.

PURPOSE:  This study aims to demonstrate if bacteria found within the infected root canal system are capable of degrading Resilonutilizing an agar disc hydrolysis method. 

MATERIALS AND METHODS:  A 0.1-percent Resilon emulsion and a gutta-percha emulsion were prepared with Tryptic Soy Agar in plates. Several bacterial species were inoculated in eight spots each on the Resilon and gutta-percha agar plates and the plates were observed for the formation of hydrolytic halos surrounding bacteria signifying their ability to degrade the material.  The bacterial enzyme Lipase PS served as a positive control.  P. intermedia, P. aeruginosa, P. assacharoylitica, S. epidermidis and S. aureus all demonstrated hydrolytic halos, clear zones, at each of the eight inoculation locations (100%, 95%CI 63%-100%) on the Resilon plates. The halos were similar to those seen in the positive lipase control. No halos were seen with E. faecalis, F. nucleatum, S. mutans, S. sanguis, or P. gingivalis at any of the eight inoculation spots (0%, 95%CI 0%-37%) on the Resilon plates. No hydrolytic halos were seen around any bacterial colonies or the Lipase PS on the gutta-percha plates. 

RESULTS:  The results of this study indicate that bacteria found in endodontic infections can hydrolize Resilon dispersed into an emulsion. The potential exists for Resilon degradation after its use as an obturation material in infected root canal systems.

CONCLUSION:  Given that root canal therapy does not render a canal void of microorganisms, it is prudent to obturate the root canal system with a material that cannot be degraded by bacteria and their enzymes.

BIODEGRADABILITY OF RESILON, A RESIN-BASED ROOT CANAL OBTURATING MATERIAL, BY TYPICAL ENDODONTIC

Ashleigh Rexford, DMD

INTRODUCTION:  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 effect.  Furthermore, it has been shown that Resilon is susceptible to alkaline and enzymatic hydrolysis as well as bacterial degradation.  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. 

 PURPOSE:  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.

MATERIALS AND METHODS: 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. 

RESULTS: The overall results were inconclusive; and using these methods it cannot be determined that the selected bacteria can degrade Resilon. 

CONCLUSION:  The overall findings of this study do not support or contradict the null hypothesis.  The results were inconclusive due to a variety of factors.  However, one notable finding was that Resilon turned black when exposed to bacteria. Future studies are needed to evaluate the significance of this color change as well as Resilon’s susceptibility to degradation by endodontic pathogens.  Ultimately it was concluded that this experimental design is an ineffective way to evaluate degradation of Resilon and gutta-percha.

EFFECTS OF DYNAMATRIX® MEMBRANE ON ANGIOGENIC CYTOKINE EXPRESSION FROM HUMAN DENTAL PULP STEM CELLS

Ryan W. Baker, DMD

INTRODUCTION: Historically, treatment protocols for infected, immature teeth involved disinfection of the root canal space (RCS) followed by creation of an apical barrier and subsequent obturation.  While generally effective at removing the source of infection, these methods resulted in teeth with thin, underdeveloped roots that were prone to fracture over time.                                           

An alternative to the conventional methods is now available in the form of regenerative endodontic procedures.  These are currently performed to treat infected immature teeth utilizing the “revascularization method.” This method includes two steps: disinfection of the root canal followed by the laceration of apical tissues to induce blood flow into the root canal space.  The blood clot formed in the canal serves as a fibrin scaffold for the accumulation of stem cells and other cells with dentin/root formation capability.  This protocol has resulted in continued root formation (in length and width) in certain clinical cases.  However, the adequacy of hemorrhage induced in the apical tissues has a significant impact on the quality of the regenerated dental structures.  This phenomenon indicates that the blood clot itself may play an important role in continued root formation by locally releasing molecular signals, which induce angiogenesis.  Successful angiogenesis is then crucial for the vitality of the newly regenerated pulp tissues and for the initiation of continued root development.  Therefore, the production of angiogenic growth factors from the local surviving cells or the effective delivery of exogenous angiogenic growth factors to the desired area is one of the key components in regenerative endodontic treatment.                 

 It is believed that an appropriate exogenous scaffold could aid the endogenous elements (fibrin meshwork and molecular signals in blood clot) and result in more predictable regenerative endodontic procedures.  DynaMatrix® is a biological extracellular matrix (ECM) scaffold product that is marketed to dental practitioners.  In addition to structural and functional proteins (collagens types I, III, IV, and VI, glycosaminoglycans, glycoproteins, proteoglycans) DynaMatrix contains biological signals such as cytokines.  Its track record of successful use in periodontal procedures encourages investigation of the material for use in regenerative endodontic procedures.  Specifically, DynaMatrix may be placed within the root canal system of the immature tooth to facilitate the continued development of the entire length of the root from the apical foramen to the level of the Dentin Enamel Junction (DEJ).  As a scaffold containing biological signals, DynaMatrix has the potential to induce angiogenesis and encourage the migration of cells that are involved in the continued development of the root canal system.                    

PURPOSE: The aim  was to determine if the exposure of human dental pulp stem cells (HDPSC) to the DynaMatrix membrane will result in an increased production of angiogenic growth factors that are critical for pulp/root regeneration. 

MATERIALS & METHODS:  This study utilized human dental pulp cells (HDPSC) in vitro.   Three different groups were tested as follows: (1) control group 1- Human Dental Pulp Cell (HDPC) culture only; (2) control group 2 – DynaMatrix®membranes placed in culture media without any cells present and (3) experimental group- HDPSC seeded on DynaMatrix® membranes.  The conditioned media from the various groups was collected and tested for the expression of specific cytokines using angiogenesis cytokine arrays which have been established as a viable method for assessing expression of angiogenic cytokines.                            

To determine the level of cytokine expression, the optical densities of the visible dots on the membrane were measured by Quantity One 1-D Analysis Software (Bio-Rad). For each membrane, the density of each dot was adjusted for the background by subtracting the average value of the negative controls on each membrane and then normalized by dividing by the average of the positive controls.  The experiment was run three times resulting in three membranes for each group.  This generated 4-6 densitometry readings (two per membrane, some statistical outliers removed) for each cytokine in each group.  Group comparisons were performed using one-way ANOVA.

RESULTS: The cell-only group (C) had significantly lower ENA-78 (p=0.0166), leptin (p=0.0208), PDGF-BB (p=0.0034), VEGF (p=0.0174), VEGF-D (p=0.0230), and bFGF (p=0.0047) compared to the DynaMatrix membrane-only group (M). C had significantly higher IL-6 (p=0.0268), IL-8 (p=0.0153), MCP-1 (p=0.0021), TIMP-1 (p=0.0027), and TIMP-2 (p=0.0105) compared to M.

C had significantly lower PDGF-BB (p=0.0182) and bFGF (p=0.0009) compared to the DynaMatrix membrane + dental pulp stem cell group (M+C). Control had significantly higher IL-6 (p=0.0203), IL-8 (p=0.0097), MCP-1 (p=0.0019), TIMP-1 (p=0.0006), and TIMP-2 (p=0.0086) compared to M+C. C and M+C did not have significantly different ENA-78 (p=0.78), leptin (p=0.42), VEGF (p=0.42), or VEGF-D (p=0.07).

M had significantly lower bFGF (p=0.0452) compared to M+C. M had significantly higher ENA-78 (p=0.0178), TIMP-1 (p=0.0363), and VEGF (p=0.0117) compared to M+C. M and M+C did not have significantly different IL-6 (p=0.79), IL-8 (p=0.63), leptin (p=0.06), MCP-1 (p=0.92), PDGF-BB (p=0.10), TIMP-2 (p=0.81), or VEGF-D (p=0.34).                                            

CONCLUSION:  The results of this study demonstrate that HDPSC can grow successfully on DynaMatrix in vitro, and that DynaMatrix may contribute to success in regenerative endodontic procedures by providing a predictable mechanical scaffold for cellular and vascular in-growth into the RCS.  Additionally, it may improve angiogenesis by increasing the quantity of angiogenic growth factors present in the microenvironment via release of angiogenic cytokines present in DynaMatrix, as well as possible interactions with the cells involved in regeneration that result in greater expression of angiogenic cytokines from these cells.

AN IN-VITRO COMPARISON OF BACTERIAL MICROLEAKAGE OF GUTTA-PERCHA AND THE GUTTACORE CROSSLINKED GUTTA-PERCHA CORE OBTURATOR

Abigail C. Edds, DMD

INTRODUCTION:   A new obturation material by Dentsply Tulsa, GuttaCore crosslinked gutta-percha core obturator, has been recently introduced that replaces the plastic core with a crosslinked gutta-percha core.  The manufacturer states removal of the obturation material and core is fast and easy.  To date, no microleakage studies have been done to test this newer obturation material.

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is useful as a bacterial label because the fluorescent marker can be exhibited in the bacterial host without having to use stains.  A plasmid that encodes for a copy of the green fluorescent variant gene is transferred into the Enterococcus faecalis. The marker glows green under a standard fluorescence microscope and has been used successfully to evaluate microleakage.

PURPOSE  The purpose of this investigation is to evaluate the sealing ability of a new obturation material, GuttaCore, to determine if there will be a significant decrease in microleakage of AH Plus with GuttaCore Obturator versus AH Plus with gutta-percha. 

MATERIALS & METHODS:   Sixty-two human, single-rooted premolars extracted for periodontal considerations were accessed and instrumented for non-surgical root canal therapy.  Hand and rotary instrumentation was accomplished to a MAF size of 40 .04, and irrigation was accomplished with 6% NaOCl and 17% EDTA with use of EndoActivator. Teeth were randomly assigned to two experimental groups of 27 teeth each.  Group I (conventional method) teeth were obturated with gutta-percha and AH Plus sealer using warm vertical condensation, and Group II (test method) teeth were obturated with GuttaCore and AH Plus sealer.  Two control groups containing four teeth each served as positive and negative controls.  The positive and negative control groups ensured that the microleakage model was working correctly. 

The teeth were evaluated for microbial microleakage of Enterococcus faecalis green fluorescent protein (GFP) construct using a dual chamber leakage model.  If turbidity is observed in the lower chamber, it will indicate microleakage and an inadequate seal of the obturation method.  The teeth were sectioned and viewed with a standard fluorescence microscope to determine the depth of microleakage utilizing the inherent fluorescence of the E. faecalis GFP construct. 

 RESULTS:  No microleakage was observed in the negative control groups.  Microleakage was observed in both of the gutta-percha positive control groups and 1 of the 2 GuttaCore positive control groups.  One out of 27 GuttaCore samples displayed turbidity, which occurred at day 14.  None the 26 gutta-percha samples displayed turbidity at any point.  The 95% confidence intervals (CI) for the percentage of samples with turbidity were 0.1%-19.0% for GuttaCore and 0.0%-13.2% for gutta-percha using a Fisher’s Exact Test.  The two groups did not have a significantly different percentage of samples with turbidity (p=1.00). 

CONCLUSIONS:  Within the limitations of this study, there was no significant decrease in microleakage between the GuttaCore Obturator and warm vertical condensation with gutta-percha.

EFFICACY OF PROPOLIS AGAINST FUSOBACTERIUM NUCLEATUM BIOFILM

Anthony L. Griglione, DMD

INTRODUCTION:   Research shows that after a single visit of chemomechanical debridement microbes continue to remain within the canal system.  An interappointment medication step has been advocated to maximize potential elimination of microbes within the root canal system.  Previous studies have shown propolis to be antibacterial against common endodontic microbes.  Studies have shown trends in different microbes being present in primary verus secondary endodontic infections.  The majority of literature has focused on the efficacy of propolis against Enterococcus faecalis, a microbe commonly implicated in secondary endodontic infections.  The aim of this study was to demonstrate the efficacy of propolis against Fusobacterium nucleatum, a microbe commonly found in primary endodontic infections. 

PURPOSE: To demonstrate the efficacy of propolis against a bacterium of primary endodontic infections (Fusobacterium nucleatum) as well as against microbial biofilm to further support its potential use as a novel intracanal medicament. 

MATERIALS & METHODS:  Dilutions of propolis were added to cultures of F. nucleatum in microtiter plates in a range from 390 mg/ml to 50,000mg/ml.  The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the minimum biofilm inhibitory concentration (MBIC) were determined.  The MIC was determined of the total solution (biofilm+planktonic), planktonic, and biofilm (MBIC) after a 48 hour incubation period.  The MBIC was determined by fixing biofilm to the wells and using crystal violet staining with spectrophotometry.  The MBC was examined by plating solution from each concentration test well and reading the plates after 48 hours of incubation. 

RESULTS:  MIC of the total (biofilm+planktonic) appears to occur at a concentration of 6,250 mg/ml.  The MBIC appears to occur at the concentration of 1,562.5 mg/ml.  The planktonic results exhibit no significant difference in test and control wells.  There was no MBC at any of the test concentrations.  The propolis appears to inhibit bacterial growth and biofilm formation but does not appear to be bactericidal at any of the tested concentrations. 

CONCLUSION: Propolis has an MIC and MBIC when tested in-vitro against Fusobacterium nucleatum although it does not show an MBC.  There appears to be potentially significant interaction of propolis with biofilm as displayed by the lower concentration needed to exibit inhibitory effects on biofilm formation.  This information may contribute to the ability to develop a proper concentration of propolis to use in-vivo when treating endodontic infections.