Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules

Abstract The effect of endodontic irrigants and dressings was tested on bacteria in bovine dentin specimens experimentally infected with Enterococcus faecalis, Streptococcus sanguis, Escherichia coli, or Pseudomonas aeruginosa. Standardized, cylindrical dentin test pieces were prepared and cleaned by ultrasonic treatment with EDTA and sodium hypochlorite. The specimens were infected with the test organism for periods up to 14 days, and the degree of infection into the tubules was monitored using Brown & Brenn stain, scanning electron microscopy, and culturing of dentin dust from sequential bur samples starting from the pulpal side. E. faecalis rapidly infected the whole length of the tubules; S. sanguis required up to 2 weeks for complete infection; E. coli only penetrated to some 600 μm, even after prolonged incubation periods. P. aeruginosa infected dentin quickly, but apparently in very low numbers. E. faecalis persisted for at least 10 d after withdrawal of nutrient support, whereas the other 3 organisms died within 4 to 48 h. Endodontic medicaments were applied to infected specimen for comparison of antibacterial potency. Camphorated p-monochlorophenol was generally more efficient than Calasept, and of the irrigants tested, iodine potassium iodide appeared more potent than sodium hypochlorite or chlorhexidine. The presence of a smear layer delayed, but did not eliminate, the effect of the medicaments.     

In vitro disinfection of dentinal tubules by various endodontics irrigants

Effectiveness of endodontic irrigants within dentinal tubules of human teeth was evaluated. Mid-sections of single-rooted teeth were prepared into dentin wedges. The pulpal sides of the sections were exposed to Micrococcus luteus or Bacillus megaterium that grew into the tubules. Irrigants used in the study included: 0.525% NaOCl, 0.12% chlorhexidine, RC Prep, 0.5% betadine iodine, and sterile H2O (as a control). Pulpal surfaces were exposed to an irrigant and then rinsed in sterile water. The samples were then cracked, exposing a fresh surface. Culture of the exposed dentin surfaces showed that selected irrigants reached to the far ends of the dentinal tubules in a concentration sufficient to kill 100% of the M. luteus. However B. megaterium was neither killed nor apparently inhibited by any irrigant. We conclude that endodontic irrigants permeate throughout dentinal tubules, but their effectiveness is dependent on the type of bacteria found within the tubules.     

Scanning electron microscope study on the action of endodontic irrigants on bacteria invading the dentinal tubules

Three groups of 10 maxillary anterior teeth each, which had been exposed to oral media, were flushed with 9 ml of tap water, physiologic saline solution, and alternating irrigation with NaOCl and hydrogen peroxide respectively. Residual caries, necrotic tissue remnants, intertubular cavitation, and dentinal tubule invasion by bacteria were searched for by means of the scanning electron microscope. Cuboidal sodium chloride crystals were very frequently found deposited in the main root canal, in lateral canals, and inside the dentinal tubules. This study discusses the important role NaOCl may play as a solvent of dental plaque and necrotic tissue remnants in the clinical endodontic treatment of infected teeth that had been exposed to oral media.     

Antibacterial activity of Propolis versus conventional endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules

Introduction

The antibacterial activity against Enterococcus faecalis of 2 propolis samples was investigated in a dentin block model, and their effectiveness was compared with that of established endodontic disinfectants, chlorhexidine (CHX) and calcium hydroxide [Ca(OH)₂].

Methods

Standardized dentin blocks were infected with E. faecalis ATCC 29212. The root canal space was filled with one of the ethanolic extracts of propolis (Artvin or Tekirda? mix [TM]), CHX 2%, Ca(OH)₂, or ethanol or phosphate-buffered saline for control. Canal dentin was sampled after 1 or 7 days by using a standard-size bur. The dentinal shavings were vortexed vigorously in phosphate-buffered saline, and aliquots were cultured on tryptone soy agar plates. Colonies were counted after 2 days of incubation. Statistical significance was set to 5%.

Results

All experimental agents significantly reduced the number of the cultivable bacteria. CHX was the most potent disinfectant at both times. Compared with the ethanol control, no significant reduction in the number of colonies was found for the propolis extracts at day 1; however, significant reduction was found at day 7. The 2 propolis samples were statistically similar to each other and to Ca(OH)₂, but the TM sample was also similar to CHX at day 7. This has been linked to the greater concentration of flavonoids, a group of antibacterially active compounds, in the TM sample as determined by gas chromatography-mass spectrometry analysis.

Conclusions

The antimicrobial activity of the propolis samples tested in this study was between Ca(OH)₂ and CHX. Both propolis samples were antimicrobially effective; however, their activity did not exceed CHX.     

Penetration of dentinal tubules by endodontic sealer cements in extracted teeth and in vivo

AIM: To compare the depth and consistency of penetration of three different root canal sealer cements into dentinal tubules in extracted teeth and to measure the penetration of an epoxy resin-based sealer cement in vivo. METHODOLOGY: Root canals of 50 extracted human pre-molar teeth were prepared and obturated using three different sealer cements based on epoxy resin (AH26), zinc oxide eugenol (Pulp Canal Sealer EWT) and methacrylate resin (EndoREZ). Five teeth filled without sealer were used as controls. Teeth were sectioned and prepared for observation using scanning electron microscopy. A further 12 teeth with a history of successful root filling and subsequent extraction were collected and sectioned. The depth of sealer penetration into dentinal tubules was measured and the consistency and appearance of the sealer within the tubules observed. RESULTS: AH26 demonstrated the deepest penetration (1337 microm), followed by EndoREZ (863 microm) and Pulp Canal Sealer EWT (71 microm). The difference in penetration between all sealer groups was found to be statistically significant (P < 0.05). The resin-based sealers appeared to penetrate tubules more consistently. In the clinical cases, all teeth demonstrated sealer penetration to varying depths (98-1490 microm). CONCLUSIONS: The depth and consistency of dentinal tubule penetration of sealer cements appears to be influenced by the chemical and physical characteristics of the materials. Resin-based sealers displayed deeper and more consistent penetration. Penetration depths observed for the epoxy resin-based sealer in vivo were consistent with that found in the experimental model.     

Effect of irrigants and cementum injury on diffusion of hydroxyl ions through the dentinal tubules

This study measured hydroxyl ion diffusion through dentinal tubules into a bathing solution. Eighty single-canal, instrumented teeth were divided into 8 groups. Control groups 1 and 3 were irrigated with 10 mL 0.9% saline and 10 mL 6% sodium hypochlorite (NaOCl), respectively. Control groups 5 and 7 were irrigated with 3 mL and 1 mL 17% ethylenediaminetetraacetic acid (EDTA) and then 10 mL 6% NaOCl, respectively. Experimental groups 2, 4, 6, and 8 were irrigated as groups 1, 3, 5, and 7, followed by placement of calcium hydroxide (Ca(OH)2) into canals. Bathing solution pH was recorded for 30 days, a cementum defect was made, and then pH was recorded for another 30 days. With a paired difference test, average pH during steady state was statistically different and higher after the defect (P < .001). With Tukey multiple comparisons, post-defect pH for group 6 was found to be significantly greater (P < .01) than in other groups. This study indicated final canal irrigation with 3 mL 17% EDTA and 10 mL 6% NaOCl before Ca(OH)2 placement allowed the greatest hydroxyl ion diffusion to the root surface.     

Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM

Abstract The aim of this study was to observe the root canal flora and possible penetration of microorganisms into dentinal tubules in teeth with necrotic pulps. Ten infected maxillary and mandibular molars with periapical lesions were extracted and fixed in 2.5% phosphate-buffered glutaraldehyde solution for nine days. After separation from the crowns, longitudinal grooves were cut in the roots, and they were split into two halves. The specimens were prepared for SEM. The root canals and the dentinal tubules of the fractured dentin were scanned systematically from the cervical to the apical area of the root. Cocci and rods were seen in 6 specimens. Penetration of bacteria into the dentinal tubules ranged from 10 to 150 μm. In 4 specimens, the root canals were heavily invaded by yeasts. The antimicrobial effect of routinely used endodontic disinfectants also on yeasts may be considered in persistent root canal infections.     

Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules.

The objective of this study was to evaluate the antimicrobial activity of calcium hydroxide in infected dentinal tubules. Four microorganisms, strains of ATCC (Streptococcus faecalis (ATCC-29212), Staphylococcus aureus (ATCC-6538), Bacillus subtilis (ATCC-6633), and Pseudomonas aeruginosa (ATCC-27853)) and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. Sixty-three human maxillary central incisors were prepared and sterilized by autoclaving. Five groups of 12 teeth each were contaminated for 28 days using new 24-h cultures every 72 h, prepared and adjusted to tube 2 of the MacFarland scale (6 x 10(8) cells/ml). Root canals were then irrigated with 5 ml of saline, dried, and completely filled with calcium hydroxide paste. At intervals of 0, 48, and 72 h, and 7 days, dressings were removed and teeth were immersed in 5 ml of BHI and incubated at 37 degrees C for 48 h to observe the growth and multiplication of the microorganisms. Three uninoculated teeth were maintained in a humid environment as an aseptic control. These teeth were immersed in BHI and maintained at 37 degrees C for 7 days to determine microbial growth. Bacterial growth was shown by turbidity of the culture medium and confirmed by seeding these broths on BHI agar at 37 degrees C for 24 h. The positive BHI tubes were selected, and inoculum was spread on the surface of BHI agar, followed by the same incubation conditions. Gram stain was conducted from BHI growth and from colonies growing on solid medium. Calcium hydroxide in infected dentinal tubules showed no antimicrobial effect on S. faecalis, S. aureus, B. subtilis, P. aeruginosa, or on the bacterial mixture used throughout the experiment.     

Effect of dentinal tubules and resin-based endodontic sealers on fracture properties of root dentin

ObjectiveTo investigate the role of dentinal tubules in the fracture properties of human root dentin and whether resin-filled dentinal tubules can enhance fracture resistance.Materials and methodsCrack propagation in human root dentin was investigated in 200 μm thick longitudinal samples and examined by light and scanning electron microscopy. 30 maxillary premolar teeth were prepared for work of fracture (Wf) test at different tubule orientations, one perpendicular and two parallel to dentinal tubules. Another 40 single canal premolars were randomly divided into four groups of 10 each: intact dentin, prepared but unobturated canal, canal obturated with epoxy rein (AH Plus?/gutta percha), or with UDMA resin sealer (Resilon^®/RealSeal^®). The samples were prepared for Wf test parallel to dentinal tubules. Wf was compared under ANOVA with statistical significance set at p < 0.05.ResultsDentinal tubules influenced the path of cracks through dentin, with micro-cracks initiated in peritubular dentin of individual tubules ahead of the main crack tip. A significant difference (p < 0.001) was found between Wf perpendicular to tubule direction (254.9 J/m²) vs. parallel to tubule direction from inner to outer dentin (479.4 J/m²). Neither canal preparation nor obturation using epoxy- or UDMA-based resins as sealer cements substantially influenced fracture properties of root dentin, despite extensive infiltration of dentinal tubules by both sealer cements.     

The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin

Abstract

Objectives. To evaluate if the incorporation of antimicrobial compounds to chelating agents or the use of chelating agents with antimicrobial activity as 7% maleic acid and peracetic acid show similar disinfection ability in comparison to conventional irrigants as sodium hypochlorite or iodine potassium iodide against biofilms developed on dentin. Materials and methods. The total bio-volume of live cells, the ratio of live cells and the substratum coverage of dentin infected intra-orally and treated with the irrigant solutions: MTAD, Qmix, Smear Clear, 7% maleic acid, 2% iodine potassium iodide, 4% peracetic acid, 2.5% and 5.25% sodium hypochlorite was measured by using confocal microscopy and the live/dead technique. Five samples were used for each irrigant solution. Results. Several endodontic irrigants containing antimicrobials as clorhexidine (Qmix), cetrimide (Smear Clear), maleic acid, iodine compounds or antibiotics (MTAD) lacked an effective antibiofilm activity when the dentin was infected intra-orally. The irrigant solutions 4% peracetic acid and 2.5–5.25% sodium hypochlorite decrease significantly the number of live bacteria in biofilms, providing also cleaner dentin surfaces (p < 0.05). Conclusions. Several chelating agents containing antimicrobials could not remove nor kill significantly biofilms developed on intra-orally infected dentin, with the exception of sodium hypochlorite and 4% peracetic acid. Dissolution ability is mandatory for an appropriate eradication of biofilms attached to dentin.     

Detoxification of endotoxin by endodontic irrigants and calcium hydroxide

The effects of endodontic irrigants and calcium hydroxide on lipopolysaccharide (LPS; endotoxin) were analyzed using the highly selective technique of mass spectrometry/gas chromatography with selected ion monitoring. An aqueous solution of LPS was mixed with one of a variety of endodontic irrigants for 30 min. Because it is a commonly used interappointment dressing, calcium hydroxide was also applied to LPS for 1, 2, or 5 days. LPS inactivation was measured by quantitation of free fatty acid release. Water, EDTA, ethanol, 0.12% chlorhexidine, chlorhexidine + sodium hypochlorite, and sodium hypochlorite alone showed little breakdown of LPS. Long-term calcium hydroxide--as well as 30-min exposure to an alkaline mixture of chlorhexidine, ethanol, and sodium hypochlorite--did detoxify LPS molecules by hydrolysis of ester bonds in the fatty acid chains of the lipid A moiety.     

五种冲洗液对根管内粪肠球菌抗菌效果的体外实验

目的评价五种冲洗液对根管内粪肠球菌的杀菌效果。方法建立粪肠球菌根管内感染模型,将40个感染根管标本随机分为5组,每组8颗牙。器械预备时分别采用5.25%NaClO（A组）、2.5%NaClO（B组）、1%NaClO（C组）、17%EDTA（D组）、17%EDTA＋1%NaClO（E组）冲洗根管。冲洗前、冲洗后即刻及冲洗后72h分别取样,37℃下CO2孵育箱中培养,48h后计数菌落数（CFU/ml）。结果冲洗后5组根管内的粪肠球菌量均显著下降（P〈0.05）,其中A组与其余各组间差异均有显著性（P〈0.01）;B组与C组、D组间差异有显著性（P〈0.05）,与E组间差异无显著性（P〉0.05）;C组与D组、E组间差异有显著性（P〈0.05）;D组与E组间差异有显著性（P〈0.05）。根管冲洗后培养72h均有细菌生长。结论5.25%NaClO抗菌效果最强;17%EDTA＋1%NaClO的抗菌效果优于1%NaClO,与2.5%NaClO抗菌效果相近似。     

Control of microorganisms in vitro by endodontic irrigants

The aim of this study was to determine the minimum inhibitory concentration (MIC) and antimicrobial effectiveness by the direct exposure test of 4 endodontic irrigants [1% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 1% calcium hydroxide (Ca(OH)2; prepared with 1 g of Ca(OH)2 and 100 mL of sterile distilled water), a solution of Ca(OH)2 + detergent (HCT20)] for S. aureus, E. faecalis, P. aeruginosa, B. subtilis, C. albicans and a mixed culture. Microbial growth was analyzed by two methods: turbidity of the culture medium that was confirmed by Gram stain and subculture in a specific nutrient broth. In the dilution test, NaOCl solution showed MIC equal to 0.1% for S. aureus, E. faecalis, P. aeruginosa and C. albicans and equal to 1% for B. subtilis and the mixed culture. CHX (2%) presented MIC equal to 0.000002% for S. aureus, 0.02% for E. faecalis, B. subtilis, C. albicans and the mixed culture and 0.002% for P. aeruginosa. Ca(OH)2 solution (1%) showed MIC greater than 1% for all the microorganisms except P. aeruginosa for which it was equal to 1%. Calcium hydroxide solution + detergent showed MIC equal to 4.5 mL for S. aureus, P. aeruginosa, B. subtilis, C. albicans and the mixed culture and greater than 4.5 mL for E. faecalis. In the direct exposure test, NaOCl had better antimicrobial effectiveness for all microorganisms at all times. CHX (2%) was effective for S. aureus, E. faecalis and C. albicans at all times, and ineffective for P. aeruginosa, B. subtilis and the mixed culture. The other solutions showed the worst results.     

Nanostructural changes in dentine caused by endodontic irrigants

Objective: To study nanostructural dentinal changes produced by endodontic irrigants. Study Design: Experimental study. Nanoindentations were performed on peritubular (PD) and intertubular dentine (ID) with an atomic force microscopy. Stiffness and adhesion force were determined before and after application of 5.25% sodium hypochlorite (NaOCl) and 17% ethylenediaminetetraacetic acid (EDTA). Normalized differences before and after treatment for stiffness and adhesion forces were calculated. A paired T-test was used to compare stiffnes and adhesion force before and after irrigants application. Results: After treatment with EDTA there was a 29.80% reduction in stiffness in ID and a 63.53% reduction in PD. Adhesion force was reduced by 21.22% and 8.21% respectively. After treatment with 5.25% NaOCI stiffness was reduced by 2.49% in ID and increased by 15.01% in PD. Adhesion force increased by 25.11% and 23.97% respectively. Conclusions: 17% EDTA reduced stiffness and adhesion force in ID and PD. Treatment with NaOCI at 5.25% had no significant effect on stiffness but did affect adhesion force in ID and PD. Key words:Atomic force microscope, stiffness, adhesion force, peritubular dentine, intertubular dentine.     

四种冲洗剂对根管内粪肠球菌清除效果的体外实验

目的比较常用的根管冲洗剂对根管内粪肠球菌感染的清除效果。方法建立粪肠球菌根管内感染模型，实验组用4种常用的化学冲洗剂、对照组用0．9％NaCl溶液冲洗根管。冲洗前、后计数根管内的细菌量，检测残余细菌并观察72h细菌复苏情况。结果化学冲洗剂的杀菌效果明显好于0．9％NaCl溶液（P〈0．05），2．5％次氯酸钠及2％氯己定明显好于3％H2O2（P〈0．05）。结论2％氯己定、2％氯胺-T的杀菌效果与2．5％次氯酸钠相似，3％H2O2杀菌效果较弱。     

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.     

Efficacy of Ledermix paste in eliminating Staphylococcus aureus from infected dentinal tubules in vitro

Abstract The efficacy of Ledermix paste in disinfection of dentinal tubules was studied in a model developed by Haapasalo and Ørstavik with some modifications. Ledermix and 3% Tetra-cycline in a hydrous base were effective in reducing the amount of Staphylococcus aureus in dentinal tubules after 7 days of incubation and also after recontamination. They were not effective after 24 h.