Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro

Abstract Solutions of 0.2% and 2% chlorhexidine, 0.2% and 2% sodium hypochlorite (NaOCl) and normal saline were tested for their efficacy in disinfecting dentinal tubules following root canal irrigation in vitro. Freshly extracted bovine incisor roots were prepared as cylindrical shapes, 4 mm high and 5 mm wide with a lumen 2.1 mm wide. After mechanical removal of the cementum and elimination of the smear layer on the dentine surface with EDTA and NaOCl, the root sections were autoclaved and the dentinal tubules infected with E. faecalis (NCTC 775) by incubating in yeast extract glucose broth for 1 week. The root canals were irrigated with 20 ml of an irrigant solution using a syringe. Each irrigant was used in six specimens. Dentine was removed from the canal wall by sterile burs of increasing diameter to give samples 100, 100–300 and 300–500 μm deep. The dentine samples were then cultured to determine the presence and quantity of remaining micro-organisms. The results indicated that chlorhexidine and NaOCl were equally effective antibacterial agents at similar concentrations against the test microorganism. They significantly reduced the bacterial counts in the first 100 μm of dentinal tubules, however up to 50% of dentine samples remained infected following use of both agents.     

Extension of Bactericidal Effect of Sodium Hypochlorite into Dentinal Tubules

Introduction

The aim of this study was to determine the extent of disinfection mediated by 2 concentrations of sodium hypochlorite for bacteria-laden dentin wall of a root canal.

Methods

A dual-species film, Enterococcus faecalis and Porphyromonas gingivalis, was cultivated on root canal dentin for 7 days. The canal wall was irrigated with 0.5% or 3% sodium hypochlorite (n = 8 each), with a control group (n = 2) irrigated with sterile saline. The roots were then split horizontally at 5-, 7-, and 9-mm levels from root apex and examined under confocal laser scanning microscope and then scanning electron microscope. The proportions of viable cells situated on different depths into root canal dentin were compared at a significance level of P = .05.

Results

Both concentrations of sodium hypochlorite significantly reduced the amount of live bacteria in the most superficial layer (first 0.1 mm) of root canal dentin, compared with the control; however, the difference was not significant between the 2 concentrations. For the next 2 layers (0.1–0.3 mm into dentinal tubules), irrigation with 3% hypochlorite resulted in significantly lower amounts of viable bacteria than 0.5% hypochlorite or saline. No significant difference between 0.5% or 3% sodium hypochlorite and the positive control was observed in deeper (>0.3 mm) regions into the root canal wall.

Conclusions

Increasing the concentration of sodium hypochlorite improves the penetration depth of its antibacterial action into dentinal tubules, but that seemed unable to completely eradicate bacteria residing there. Total elimination of bacteria from dentinal tubules cannot be achieved by irrigation with sodium hypochlorite alone.     

Effect of endodontic irrigation on bonding of resin cement to radicular dentin

The influence of endodontic irrigation on shear bond strengths of resin cement to radicular dentin was investigated. Human radicular dentin blocks were divided into four groups and subjected to one of four endodontic irrigations: ethylenediaminetetraacetic acid (EDTA) group, 17% EDTA for 60 s; EDTA/sodium hypochlorite (NaOCl) group, 17% EDTA for 60 s followed by 10 ml of 5% NaOCl for 15 s; NaOCl group, 10 ml of 5% NaOCl for 15 s; and control group, no treatment. Morphological changes of dentin surface after endodontic irrigation were observed by scanning electron microscopy (SEM). A resin block was bonded to the radicular dentin after irrigation using resin cement with either wet-bonding (Uni-Etch/One-Step; Bisco) or self-etching (Tyrian SPE/One-Step Plus; Bisco) adhesives. Shear bond strengths were measured and the penetration of resin tags into dentinal tubules at resin-dentin interface was observed by SEM. With the wet-bonding system, the shear bond strengths for the EDTA/NaOCl group, in which dentinal tubules openings and uniform resin tag penetration into dentinal tubules were observed, were significantly higher than the EDTA and control groups. With the self-etching system, the shear bond strengths were significantly lower in the EDTA group compared with the NaOCl and control groups. The effects of endodontic irrigation on the bonding of resin cement to radicular dentin depended on the dentin bonding system used.     

In vitro and in vivo research of atmosphere pressure nonequilibrium plasmas on root canal disinfection: implication for alternative strategy for irrigation

Objective

To investigate an intracanal disinfection methodology of APNPs (atmosphere pressure nonequilibrium plasmas) or modified APNPs in root canal treatment and evaluate the antimicrobial efficiency against in vitro infected dentinal tubules and in vivo experimental apical periodontitis.

Materials and methods

Dentine specimens were centrifugated with Enterococcus faecalis to generate 1-day-old and 3-week-old biofilms, and were treated with 2% chlorhexidine (Chx), APNP or modified APNP for 3 and 10 min (n=4). LIVE/DEAD staining was employed to analyze the ratio of deactivated bacteria. Experimental apical periodontitis in beagles was induced. Root canal therapy with APNPs or modified APNPs was performed and the antimicrobial effect was evaluated by histological and radiographical analyses.

Results

APNP deactivated 1-day-old and 3-week-old E. feacalis in dentinal tubules as much as 2% Chx irrigating. Modified APNP significantly deactivated more E. faecalis biofilms in dentinal tubules for 3-min and 10-min treatments, without thermal damage or dentinal destruction being observed. In beagles’ apical periodontitis, significantly increased BV/TV and decreased lesion volume of apical bone were found in modified APNP group than 2% Chx irrigation group according to μCT. Fewer inflammatory cells and bacterial residual in dentine were observed in modified APNP-treated apical tissue by histology staining compared with those in the 2% Chx irrigation group.

Conclusion

The antimicrobial effect of APNP jet irradiation was comparable to that of 2% Chx irrigation. No structural damage in dentine or tissue necrosis at the periapical region was induced upon treatment. The modified APNP demonstrated an increased antimicrobial efficacy compared with 2% Chx irrigation both in vitro and in vivo.

Clinical relevance

The modified APNPs can be used as an alternative intracanal disinfection strategy.

     

Final Endodontic Irrigation with 70% Ethanol Enhanced Calcium Hydroxide Removal from the Apical Third

IntroductionThe purpose of this study was to evaluate the cleanliness of root canal walls and dentinal tubules after attempting to remove the calcium hydroxide dressing with different irrigant solutions and the use of nonactivated irrigation or passive ultrasonic irrigation (PUI).MethodsAfter root canal instrumentation, 80 single-rooted teeth were filled with calcium hydroxide mixed with propylene glycol and 0.1% rhodamine B dye and inserted into canals with a Lentulo spiral. The calcium hydroxide dressing was initially removed with 10 mL saline solution and reinstrumentation with the master apical file. Then, the samples were randomly assigned into 8 experimental groups (n = 10) according to the irrigant solution with or without PUI: 2.5% sodium hypochlorite, 17% ethylenediaminetetraacetic acid + 1.25% sodium lauryl ether sulfate (EDTA-T), 37% phosphoric acid, or 70% ethanol. A final flush with 5 mL saline solution was performed. The percentage of clean root canal walls and the depth of clean dentinal tubules were measured with images of confocal laser scanning microscopy. The groups were compared using the 2-way analysis of variance test with the Bonferroni post hoc test for depth analysis and the Kruskal-Wallis with Dunn post hoc test for the perimeter analysis.ResultsIrrigation with 70% ethanol presented a significantly higher percentage of clean root canal walls and a higher depth of clean dentinal tubules when compared with irrigation with 2.5% sodium hypochlorite and 17% EDTA-T for both irrigation methods (P < .05). No differences were observed between nonactivated irrigation or PUI protocols (P > .05).ConclusionsSeventy percent ethanol enhanced calcium hydroxide removal from the apical root third compared with 2.5% sodium hypochlorite or 17% EDTA-T.     

Dentinal tubule invasion and adherence by Enterococcus faecalis

Aim To investigate dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. Methodology The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured orthodentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2‐2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 μm². Results The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (anova , P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P = 0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate‐buffered saline (anova , P < 0.001). Conclusions Although E. faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.     

Adherence of Streptococcus gordonii to smeared and nonsmeared dentine

The purpose of this study was to investigate the adherence of Streptococcus gordonii to smeared and non-smeared dentine and to assess the influence of patent dentinal tubules on bacterial retention. In order to examine bacterial adherence, 10 non-smeared (group 1) and 10 smeared samples (group 2) of outer root dentine were prepared from teeth exhibiting dentine sclerosis. Ten non-smeared samples from inner coronal root dentine that did not exhibit sclerosis were prepared in order to study the influence of patent tubules on bacterial retention (group 3). Cells of the bacterium were radioactively labelled and an adhesion assay was performed. The number of bacteria adhering to the dentine surface was determined by scintillation counting. The results show that the number of bacteria adhering to both smeared and non-smeared outer sclerotic dentine was low (0.3% of inoculum), and there was no significant difference between the groups (P > 0.3). A significantly higher number of bacteria was retained on the inner non-smeared coronal root dentine (P < 0.0001) compared to groups 1 or 2. The results suggest that dentinal smear layers do not enhance or impede bacterial adherence to the dentine matrix. Dentinal surfaces with patent dentinal tubules retain more bacteria than a smeared surface.     

Invasion of dentinal tubules by root canal bacteria

Bacterial invasion of dentinal tubules and the clinical consequences have been recognized for over a century. However, while many components of the infected dentinal tubule microflora have been identified, it is likely that there are etiological agents involved in endodontic infections that have not yet been recognized. Bacterial invasion of coronal dentinal tubules occurs when the dentine is exposed to the oral environment and of radicular dentinal tubules subsequent to infection of the root canal system or as a consequence of periodontal disease. The content and architecture of a dentinal tubule can influence bacterial invasion, with tubule patency being important. This can account for regional variations in bacterial invasion and is particularly seen with dentinal sclerosis, where more advanced sclerotic changes in apical radicular tubules, especially in elderly individuals, limit bacterial invasion in this area. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria are involved in invasion of dentinal tubules. Gram‐positive organisms dominate the tubule microflora in both carious and non‐carious dentine. The relatively high numbers of obligate anaerobes present, such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp., suggests that the environment favors the growth of these bacteria. Gram‐negative obligate anaerobic rods, e.g. Porphyromonas spp., are less frequently recovered; however, with time, fastidious obligately anaerobic bacteria become established as principal components of the microflora and can be found within the deep dentine layers. In the early stages of infection, Gram‐positive bacteria dominate the microflora. The identification of adhesins that mediate these initial interactions of bacteria with dentine is important for understanding the development of tubule infection and in designing adhesion‐blocking compounds. Recent evidence suggests that streptococci and enterococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra‐tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate invasion of dentine by select bacteria. It is important therefore that the mechanisms of invasion and inter‐bacterial adhesion are understood to assist development of novel control strategies.     

Laser-activated irrigation within root canals: cleaning efficacy and flow visualization

Aim  To test ex vivo the efficiency of laser-activated irrigation in removing dentine debris from the apical part of the root canal and to visualize in vitro the fluid dynamics during the activation of the irrigant by laser, using high-speed imaging at a relevant timescale.
Methodology  Root canals with a standardized groove in one canal wall filled with dentine debris were irrigated with syringe irrigation, ultrasonically or laser-activated irrigation (LAI) using 2% sodium hypochlorite as irrigant. The quantity of dentine debris after irrigation was determined. Visualization of the fluid dynamics during activation was achieved using a high-speed camera and a glass model.
Results  Laser-activated irrigation was significantly more effective in removing dentine debris from the apical part of the root canal than passive ultrasonic irrigation or hand irrigation when the irrigant was activated for 20 s.
Conclusions  The in vitro recordings suggest that streaming, caused by the collapse of the laser-induced bubble, is the main cleaning mechanism of LAI.     

Evaluation of Glyde File Prep in combination with sodium hypochlorite as a root canal irrigant

The purpose of this study using a scanning electron microscope was to investigate the efficacy of four different irrigation techniques after canal preparation with Profile Ni-Ti rotary instruments. A modified technique for the use of Glyde File Prep is proposed. Forty anterior teeth were divided into four groups, instrumented, and irrigated as follows: physiological solution (group A), 2.5% sodium hypochlorite (group B), 2.5% sodium hypochlorite and Glyde File Prep (group C), and 2.5% sodium hypochlorite and Glyde File Prep applied at the end of the preparation with sterile paper points (group D). After scanning electron microscopic evaluation at three different levels, debris, smear layer, and dentinal tubules were scored. Groups A and B had significantly more smear layer and less open tubules on the canal walls compared with the group C and group D samples. Differences in the mean amount of debris between group A samples and other irrigation regimes were statistically significant.     

Disinfection efficacy of photon-induced photoacoustic streaming on root canals infected with Enterococcus faecalis: An ex vivo study

BackgroundIn 2010, one of the authors proposed that lasers could be used to enhance the decontaminating action of sodium hypochlorite (NaOCl). The authors conducted a study to compare the disinfection efficacy of laser-activated irrigation (LAI) by using a photon-induced photoacoustic streaming (PIPS) tip with conventional irrigation and specifically LAI's ability to remove bacterial film formed on root canal walls.MethodsThe authors shaped 26 human anterior teeth to a master apical file size of International Organization for Standardization 25/06 (size 25 tip and size .06 taper) and then sterilized the teeth, infected them with Enterococcus faecalis and incubated them for four weeks. The authors used two irrigation protocols. Group A received two cycles of 30 seconds each of 5 percent NaOCl laser activation and one cycle of 30 seconds with laser activation involving the use of 17 percent ethylenediaminetetraacetic acid (EDTA). The erbium:yttrium-aluminum-garnet (Er:YAG) laser's settings were 20 millijoules, 15 hertz, 50-microsecond pulse duration, and it had a 600-micrometer PIPS tip. Group B received two cycles of 30 seconds each of 5 percent NaOCl and 17 percent EDTA irrigation alone, delivered via a syringe with a 25-gauge needle.ResultsThe authors found that group A had significantly better disinfection compared with group B (P < .05). The results of cultures obtained after 48 hours showed that disinfection was maintained better in group A compared with group B (P < .0001). Scanning electron microscopic images showed absence of bacterial biofilm remaining after LAI using PIPS.ConclusionsEr:YAG laser activation of 5 percent NaOCl and 17 percent EDTA was more effective than conventional irrigation for eradicating E. faecalis and preventing new bacterial growth ex vivo. Additional clinical studies are needed to clarify the effect on endodontic treatment outcomes.Practical ImplicationsPIPS appears to be effective in enhancing the effect of the irrigants commonly used in endodontics.     

Sodium hypochlorite penetration into dentinal tubules after manual dynamic agitation and ultrasonic activation: a histochemical evaluation

The aim of this study was to compare the effects of Manual Dynamic Agitation and Passive Ultrasonic Irrigation on sodium hypochlorite (NaOCl) penetration into dentinal tubules using its bleaching ability. Thirty-four single-rooted teeth with round-shaped root canals were distributed in two homogeneous groups and one control group, characterized by different NaOCl activation systems: Manual Dynamic Agitation and Passive Ultrasonic Irrigation. After instrumentation, all root canals were stained with 10% copper sulphate solution followed by 1% rubeanic acid alcohol solution under vacuum. Final irrigation was performed with 5 mL of 5.25% NaOCl solution for 1 min and activated with Manual Dynamic Agitation or Passive Ultrasonic Irrigation for another 1 min depending on the treatment group. The teeth were transversely sectioned at the middle portion of the apical, middle, and coronal thirds and observed under light microscope. NaOCl solution penetration was evaluated by measuring the percentage of bleached circumference of the root canal relative to the stained circumference, bleached areas, mean, and maximum penetration depth. No differences in the evaluated parameters were observed between groups (p?>?0.05). Within groups, an increase of values was recorded from apical to coronal direction as for percentage of staining, percentage of bleaching and bleached area. NaOCl penetration into dentinal tubules did not significantly vary among the three levels. No significant differences in penetration of sodium hypochlorite into dentinal tubules when activated by means of Manual Dynamic Agitation or Passive Ultrasonic Irrigation were observed in the apical, middle, and coronal thirds of teeth with single straight round root canals.     

Antimicrobial Activity of a Sodium Hypochlorite/Etidronic Acid Irrigant Solution

Introduction

The aim of this study was to evaluate the antimicrobial activity of a 2.5% sodium hypochlorite (NaOCl)/9% etidronic acid (HEBP) irrigant solution on Enterococcus faecalis growing in biofilms and a dentinal tubule infection model.

Methods

The antimicrobial activity of the solutions 2.5% NaOCl and 9% HEBP alone and associated was evaluated on E. faecalis biofilms grown in the Calgary biofilm model (minimum biofilm eradication concentration high-throughput device). For the dentinal tubule infection test, the percentage of dead cells in E. faecalis–infected dentinal tubules treated with the solutions for 10 minutes was measured using confocal laser scanning microscopy and the live/dead technique. Available chlorine and pH of the solutions were also measured. Distilled water was used as the control. Nonparametric tests were used to determine statistical differences.

Results

The highest viability was found in the distilled water group and the lowest in the NaOCl-treated dentin (P < .05). Both NaOCl solutions killed 100% of the E. faecalis biofilms and showed the highest antimicrobial activity inside dentinal tubules, without statistical differences between the 2 (P < .05). The HEBP isolated solution killed bacteria inside dentinal tubules but did not present any significant effect against E. faecalis biofilms. The incorporation of HEBP to NaOCl did not cause any loss of available chlorine within 60 minutes.

Conclusions

HEBP did not interfere with the ability of NaOCl to kill E. faecalis grown in biofilms and inside dentinal tubules.     

Penetration depth of a dye marker into dentine using a novel hydrodynamic system (RinsEndo)

AIM: To investigate the efficiency of a hydrodynamic irrigation system compared with conventional cleansing techniques in root canals. METHODOLOGY: Forty-five freshly extracted single-rooted teeth were de-coronated and their root canals were enlarged to size 30 at the apex. The teeth were randomly divided into three groups (n = 15) for the final rinsing sequence using 2% NaOCl plus acid fuchsin: group I: static application of irrigant, 3 min; group II: flushing with a syringe; 5-mL NaOCl, 1 min; group III: RinsEndo-system; 5-mL NaOCl, 50 s. Apical extrusion was documented photographically. The roots were sectioned at 2, 4, 6 and 8 mm from their apices and the penetration depths of dye into dentine measured, using a stereomicroscope. Wilcoxon's test and Pearson's chi-squared test were employed to prove statistic relevance. RESULTS: Greater dye penetration depth into the dentinal tubules was achieved when employing hydrodynamic rinsing procedures. Using this technique, 23% of the specimens were penetrated for more than 50% of their dentine thickness, whereas the results for flushing with a syringe were 12% (static application, 7%). No penetration of dentine occurred in 63% of specimens with static application, 39% flushing with a syringe and 15% using the hydrodynamic system (P < 0.05 Pearson's chi-squared test). Apical extrusion occurred more frequently after hydrodynamic rinsing (extruded specimens: RinsEndo = 80%; static application/flushing with a syringe = 13%; P < 0.05 Pearson's chi-squared test). CONCLUSIONS: Hydrodynamic rinsing demonstrated an improvement over conventional methods in terms of dentine penetration of a dye marker. A higher risk of apical extrusion with the RinsEndo-system was evident.     

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy‐starvation stress and to explore the potential mechanisms. Roots from single‐rooted human teeth were infected with E. faecalis under alkaline and energy‐starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule‐penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real‐time quantitative PCR. Culture of E. faecalis under alkaline and energy‐starvation stress conditions resulted in a marked reduction of tubule‐penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down‐regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose‐starvation stress conditions, possibly because of the increase of hydrophobicity and down‐regulation of some adhesion genes.     

In vivo study of bacterial invasion in root planed and citric acid treated radicular surfaces of periodontally involved human teeth

The purpose of this study was to investigate whether plaque bacteria invade the exposed radicular dentin after root planing or chemical root treatment in vivo. Eighteen caries-free human periodontally involved teeth with hopeless prognoses were studied. Fourteen teeth were scaled and root planed with hand curette type scalers. The proximal surface of each treated tooth was designated as the RP surface. The remaining half of the proximal surface was treated with citric acid (pH 1.0) for 3 minutes and was designated as the CA surface. Four untreated teeth served as controls. After 4 weeks, the teeth were extracted, and were processed for light microscopy and for scanning electron microscopy concerning bacterial invasion into the supragingival radicular dentin. The following results were obtained. 1. Radicular cementum was present on most untreated tooth surfaces. However, bacteria were never seen in the dentinal tubules. 2. Bacterial invasion into the dentinal tubules was observed in five of the 10 proximal surfaces (50% of the RP surfaces) and in nine of the 10 proximal surfaces (90% of the CA surfaces). 3. The depth (9.5 +/- 24.1 microns vs 84.6 +/- 136.3 microns) and percentage (0.8 +/- 2.1% vs 20.3 +/- 17.3%) of bacterial invasion in the dentinal tubules of the RP surfaces was lower than that of the CA surfaces. 4. Cocci and short rods were present in the supragingival dentinal tubules. 5. Since CA surfaces may accelerate bacterial invasion the citric acid treatment might be harmful in patients with inadequate plaque control.     

Effect of Ultrasonics on Enterococcus faecalis Biofilm in a Bovine Tooth Model

Introduction

The purpose of this study was to evaluate in vitro the effect of the ultrasonic irrigation of sodium hypochlorite and EDTA in root canals of bovine teeth infected with Enterococcus faecalis.

Methods

Eighty-four bovine incisors were inoculated with E. faecalis, remaining in culture for 50 days for biofilm formation. The teeth were divided into four groups: the control group, which received no treatment; the ultrasonic + distilled water group; the conventional irrigation with sodium hypochlorite + EDTA group; and the passive ultrasonic irrigation with sodium hypochlorite + EDTA group. Microbiological tests and analysis in scanning electron microscopy (SEM) were performed.

Results

In microbiological testing, groups using sodium hypochlorite did not show bacterial growth. There were significant differences between the control group and the ultrasonic + distilled water group and between these groups and groups using sodium hypochlorite. In SEM analysis, at the canal wall area, there was no significant difference between the groups using sodium hypochlorite, but these were different from the others groups. The control group was significantly different from the ultrasonic + distilled water group. At the exposed tubule area, there was no significant difference between the groups.

Conclusions

Passive ultrasonic irrigation can be an aid in cleaning the root canal; however, the main role in bacteria elimination is played by the irrigant.     

A Horizontal Sequential Cutting Method to Estimate the Effectiveness of Dentin Disinfection by Using Confocal Laser Scanning Microscopy

IntroductionThis study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin.MethodsCylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20°C, 37°C, and 45°C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 μm along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test.ResultsHorizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test.ConclusionsThe horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion.     

Effects of root canal irrigations on intracanal medication with calcium hydroxide effects in root external resorption models

Objective

The objective of the present in vitro study was to evaluate the influence of endodontic irrigation systems on the removal of smear layer and ion diffusion of calcium hydroxide through dentinal tubules in root external resorption models.

A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy

Introduction

The present study was designed to develop a standardized model for quantification of the effectiveness of dentin disinfection by different antibacterial solutions including a new root canal irrigant, Qmix.

Methods

Dentinal tubules from the root canal side in semicylindrical dentin specimens were infected with Enterococcus faecalis by centrifugation of the bacterial suspension into the tubules. Scanning electron microscopy (SEM) was used to verify the presence of bacteria in dentin. The outer side of dentin pieces was closed, and the specimens were subjected to 1-minute and 3-minute exposure to sterile water, 1%, 2%, 6% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Qmix. Confocal laser scanning microscopy (CLSM) and viability staining were used to quantitatively analyze the proportions of dead and live bacteria inside dentin.

Results

A heavy invasion by E. faecalis was detected by both SEM and CLSM throughout the dentinal tubules. The amount of dead cells in dentin increased with increasing NaOCl concentration and time of exposure (P < .05). Qmix was equally effective in killing bacteria in dentin as 6% NaOCl; more than 40% and 60% of the bacteria were killed by both at 1 minute and 3 minutes, respectively. One percent and 2% NaOCl and 2% CHX killed 20%–30% and 30%–40% bacteria after 1 and 3 minutes of exposure, respectively, with no statistically significant differences among the 3 agents (P > .05). In the control group, which was treated with sterile water, only 4%–6% of the bacteria were dead.

Conclusions

Centrifugation helped to create a heavy, evenly distributed infection deep into the dentinal tubules. The new model made it possible to compare the effectiveness of several disinfecting solutions in killing bacteria inside dentin by a noninvasive CLSM method.