Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis

AIM: To investigate the antimicrobial activity of root canal sealers on Enterococcus faecalis, either allowing or avoiding direct contact between sealers and bacteria. METHODOLOGY: Filter paper discs were immersed in standardized E. faecalis suspensions and exposed to freshly mixed sealers (MCS, AH Plus, Grossman's sealer, Sealapex, Apexit) in teflon wells for 30 min, with or without a filter membrane placed between filter paper discs and sealers (membrane-restricted contact test and direct contact test, respectively). After exposure, the filter paper discs were transferred to vials containing phosphate-buffered saline (PBS) and glass beads, and vigorously vortexed. PBS with resuspended bacterial cells was serially diluted and 25 microL droplets were seeded on TSA plates. The plates were incubated in air at 37 degrees C for 24 h and colony-forming units were counted. Using alpha = 0.05 as level for statistical significance, the data obtained were analysed using Student's t-test. RESULTS: In the direct contact test, MCS and AH Plus killed the bacteria to a level below the detection limit. They were followed in decreasing order of efficacy by Grossman's sealer, Sealapex and Apexit. In the membrane-restricted contact test, the sealers ranked: MCS, AH Plus, Grossman's sealer, Apexit and Sealapex, in descending order of antibacterial potency. MCS, AH Plus and Grossman's sealer significantly reduced the number of viable bacteria in both tests. Sealapex and Apexit were not statistically different from control. CONCLUSIONS: MCS, AH Plus and Grossman's sealer were effective in reducing the number of cultivable cells of E. faecalis. Calcium hydroxide-based sealers, Sealapex and Apexit were ineffective in this short-term experiment.     

利用粪肠球菌感染根管模型评价不同根管封闭剂的抗菌效果

目的 体外建立粪肠球菌根管感染模型,比较3种生物陶瓷类根管封闭剂的抗菌性能.方法 选择人单直根管的离体前牙48颗,接种粪肠球菌并孵育4周以构建体外根管感染模型.完成根管成形和清理后,随机将样本分为3个实验组和2个对照组,每组按照如下方法进行根管充填:A组,Biodentine+牙胶;B组,iRoot BP+牙胶;C组,...     

Solubility and biocompatibility of calcium hydroxide-containing root canal sealers

Abstract The solubility and biocompatibility of 2 commercially available calcium hydroxide-containing root canal sealers, CRGS and Sealapex, were studied. Four dogs were anesthetized and a sulcular incision was made to expose the vestibular surface of the mandble. Two rows of holes, 2 mm deep, 2 mm wide and 10 mm apart, were drilled into the mandible. Teflon cups with the same dimensions as the holes were filled with the experimental materials and placed in the cavities. For the purpose of comparison, Teflon cups filled with a traditional zinc oxide-eugenol sealer and a paste of calcium hydroxide and saline were implanted as well. After 90 days the animals were killed. The mandibles were dissected free, fixed in formalin and demineralized. Bone specimens containing 1 Teflon cup were then embedded in paraffin, sectioned longitudinally, and the sections were stained with hematoxylin and eosin and examined in the light microscope. The cups which originally contained the paste of calcium hydroxide and saline were completely filled with bone. Sealapex was partly missing from the cups and was generally replaced by ingrowth of connective tissue. Large amounts of the material were seen in macrophages and tissue outside the cups. CRCS and the traditional zinc oxide-eugenol sealer generally filled the cups well after 3 months. Tissue fluids were seen in the superficial layers of these materials, but ingrowth of tissue had not occurred. No macrophage reaction was seen with either of these materials, but a stronger inflammatory reaction was caused by CRCS than by the traditional zinc oxide-eugenol cement. CRCS appeared to be the most stable of the calcium hydroxide-containing sealers tested.     

Recovery of Enterococcus faecalis after single- or multiple-visit root canal treatments carried out in infected teeth ex vivo

AIM: To assess the presence of Enterococcus faecalis after root canal treatment in single or multiple visits in an ex vivo model. METHODOLOGY: Forty-five premolar teeth were infected ex vivo with E. faecalis for 60 days. The canals were then prepared using a crowndown technique with System GT and Gates-Glidden burs and irrigated with 2% chlorhexidine gel. The specimens were divided into five groups (G1, G2, G3, G4 and G5) according to the time elapsed between chemical-mechanical preparation and root canal filling, the irrigant solution used and the use or nonuse of a calcium hydroxide intra-canal medicament. The teeth were then root-filled and incubated for 60 days at 37 degrees C. Dentine chips were removed from the canal walls with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing Brain-Heart Infusion broth. These samples were placed onto agar plates and colony forming units were counted after 24 h at 37 degrees C. Data were ranked and analysed using the Kruskal-Wallis statistical test. RESULTS: Enterococcus faecalis was recovered from 20% (three of 15 specimens) of G1 (chlorhexidine irrigation and immediate root filling in a single visit), 25% (four of 15 specimens) of G2 (chlorhexidine irrigation and filling after 14 days use of a calcium hydroxide dressing in multiple visits), 40% (two of five specimens) of G3 (chlorhexidine irrigation and filling after 7 days), 60% (three of five specimens) of G4 (saline irrigation and filling after 7 days) and from 100% (five of five specimens) of G5 (saline irrigation and immediate filling without sealer). CONCLUSIONS: Neither single- nor multiple-visit root canal treatment ex vivo, eliminated E. faecalis completely from dentinal tubules. Up to 60 days after root filling, E. faecalis remained viable inside dentinal tubules. When no sealer was used, E. faecalis presented a higher growth rate.     

In vivo sealing ability of calcium hydroxide-containing root canal sealers

Abstract It was the purpose of this study to determine the sealing ability of 2 commercially available calcium hydroxide-containing root canal sealers, CRCS and Sealapex. One hundred sixty single-rooted human teeth were used. The clinical crowns were removed and the roots were cleaned mechanically and by immersion for 24 h in 5% sodium hypochlorite. The root canals were then instrumented to size 80 at the apical foramen and the roots were sterilized by γ-radiation. Under aseptic conditions the root canals were filled with a gutta-percha master cone size 70 and Sealapex (48 roots), CRCS (48 roots), and a zinc oxide-eugenol type sealer, Roth 801 (50 roots). The coronal aspects of the root canals were then sealed with zinc oxide-eugenol cement. In order to bring the roots and root filling materials in contact with tissue and tissue fluids, the roots were implanted subcutaneously in rabbits for 90 days and 1 year. Leakage as demonstrated by penetration of India ink was evaluated using a stereomicroscope. The best results were obtained with CRCS. Significantly less leakage occurred with both calcium hydroxide-containing sealers than with the traditional zinc oxide-eugenol sealer.     

评价两种低温等离子体对粪肠球菌生物膜作用的体外实验研究

目的 比较辉光放电和介质阻挡放电两种低温等离子体装置产生的大气压低温等离子体对根管内粪肠球菌生物膜的杀菌效果。方法 在120颗离体牙的根管内部培养粪肠球菌生物膜,培养时间为7 d。将离体牙随机分为12个组,其中,10组分别接受介质阻挡放电和辉光放电这两种大气压低温等离子体装置处理离体牙根管,每种装置各处理5组,每组处理时间分别为2、4、6、8、10 min;另外2组为两种不同装置的单纯气体对照组。采用菌落形成单位计数法比较两种装置对根管内生物膜的杀菌效果,通过光谱测量仪分析两种装置的等离子体活性成分。结果 介质阻挡放电装置比辉光放电装置对根管内粪肠球菌生物膜的杀菌效果更好,不同时间段二者存活的细菌数量均有统计学差异（P<0.05）,而且随着处理时间延长优势更加明显。发射光谱显示两种装置的低温等离子体活性物质成分一致,但激发态Ar原子的群峰总体上表现为介质阻挡放电装置是辉光放电装置的2倍。结论 介质阻挡放电装置产生的低温等离子体杀灭根管内粪肠球菌生物膜更具优势。     

不同消毒方法对感染根管内粪肠球菌抗菌性的研究现状

粪肠球菌属于人体的正常菌群,但在根管治疗后持续感染和再感染的根管中常被检出,此菌是导致根管治疗失败的主要致病菌。目前,根管治疗中常规的机械预备和根管冲洗方法并不能完全清除已定植于根管中的粪肠球菌。本文就几种临床根管消毒方法对粪肠球菌的抑菌作用方面做一综述。     

Antibacterial activity of various root canal sealers and root-end filling materials in dentin blocks infected ex vivo with Enterococcus faecalis

Objective. The aim was to investigate the antibacterial activity of the root-end filling materials MTA and IRM, different endodontic sealers and calcium hydroxide [Ca(OH)₂] in experimentally infected dentinal tubules. Materials and methods. Ninety-four human root segments were prepared and the root canals were enlarged to ISO size 90. After smear removal, the specimens were infected with Enterococcus faecalis for 3 weeks. The roots were divided into eight groups and filled either with MTA, IRM, Ca(OH)₂, gutta-percha and EndoRez (ER)/GuttaFlow (GF)/AH Plus (AH+) or with Resilon and Epiphany (EpRe). One group of specimens was left unfilled for control. Half of the specimens were treated for 1 day and the other half for 7 days in humid conditions at 37°C. Dentin samples from each canal were collected by enlarging the canals to ISO size 150; thus a dentinal depth of 300 µm was sampled. The number of cultivable bacteria was determined for each specimen. Statistical significance was set to 5%. Results. After 1-day or 7-days of treatment, compared to control, all materials (except ER and GF at day 7) significantly reduced the number of bacteria. At day 1 and day 7, no significant difference was found between ER and GF and between Ca(OH)₂, AH+, EpRe, IRM and MTA. However, a significant difference was found between these two groups of materials (except between GF and EpRe at day 7). Significantly more bacteria were cultured in the ER, GF, EpRe and IRM groups at day 7 compared to day 1. Conclusions. All materials exerted varying degrees of antibacterial activity which generally tended to decrease with time. The most stable antibacterial effect throughout the 7-day period was for Ca(OH)₂, AH+ and MTA.     

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.     

Calcium hydroxide root canal sealers: evaluation of pH, calcium ion concentration and conductivity

The calcium hydroxide ionization of four root canal sealers (Sealapex, CRCS, Sealer 26, and Apexit) was studied by measuring conductivity and pH and by conducting atomic absorption spectrophotometry. Samples 6 mm in diameter and 15 mm long were prepared from these sealers. After setting and 48 h storage in a desiccator, five samples of each material were placed in 50 mL distilled water and analysed after 0,1,2,4, 6 and 24 h and 5, 15 and 30 days. The results showed that Sealapex was the root canal sealer showing the highest pH, ionic calcium and total calcium values ( P <0.05) throughout the experimental period, followed by CRCS, Apexit and Sealer 26.     

In vitro evaluation of the cytotoxicity of calcium hydroxide-based root canal sealers

Abstract The cytotoxicity of three calcium hydroxide-containing root canal sealers (Sealapex, CRCS and Apexit) was tested by using L929 and BHK 21/CIS cells. After setting for 24h, the sealers were covered with cell suspension. Cytotoxicity was determined by a quantitative technique at 24h, 48h and 72h. All the sealers were found to be cytotoxic. Sealapex showed the highest cytotoxicity, causing a significant decrease in cell density. CRCS was less toxic than Sealapex and more toxic than Apexit. Apexit proved to be the least toxic material.     

Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalis

Introduction:  Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections.
Methods:  Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis.
Results:  Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of Nsi I and Nde I DNA fragments from one of the Siphoviridae phage isolates (phage φEf11) indicated a genome size of approximately 41 kbp.
Conclusion:  This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.     

Survival of Enterococcus faecalis in root canals ex vivo

AIM: The hypotheses tested in this study were that: (i) Enterococcus faecalis can survive long-term entombment in root filled teeth without additional nutrients, (ii) initial cell density influences the survival of E. faecalis in instrumented root canals and (iii) gelatinase-production capacity influences the survival of E. faecalis in root canals. METHODOLOGY: The root canals of 150 extracted single canal teeth were instrumented to apical size 60 and divided into six groups of 25. Within each group 10 canals were inoculated with either gelatinase-producing E. faecalis OG1-S and the other 10 with its gelatinase-defective mutant E. faecalis OG1-X. Five canals per group were kept as uninoculated controls. The root canals in groups 1 and 2 were inoculated with 10(6) bacteria, incubated for 48 h at 37 degrees C then filled with gutta-percha and zinc-oxide eugenol sealer. Root canals were inoculated with 10(6), 10(5), 10(4) and 10(3) bacteria in groups 3-6, respectively, and left unfilled. All teeth were sealed coronally with glass-ionomer cement. After 6- (groups 1, 3-6) and 12-month (group 2) incubation at 37 degrees C in 100% humidity, root fragments were analysed for presence of E. faecalis, using culture, polymerase chain reaction and histological methods. RESULTS: Viable E. faecalis was recovered from all root filled teeth and from 95-100% of unfilled inoculated teeth. Initial cell density and gelatinase production did not influence the recovery of viable E. faecalis (P > 0.05; chi-square test). Enterococcus faecalis 16S rRNA gene products were present in all inoculated teeth and absent in all noninoculated controls. Dentinal tubule infection was evident under light microscopy in sections from inoculated teeth after 48-h, 6- and 12-month incubation. CONCLUSIONS: Enterococcus faecalis inoculated into root canals maintained viability for 12-months ex vivo. The clinical implications are that viable E. faecalis entombed at the time of root filling could provide a long-term nidus for subsequent infection.     

3种根管糊剂根尖封闭性能的评价

目的　体外评价iRoot SP、AH Plus、Vitapex 3种根管糊剂的根尖封闭能力。方法　 离体单根管恒牙45颗,随机分为3组,每组15颗;通过染色法微渗漏实验比较3种根管糊剂的根尖封闭能力,并通过扫描电镜观察根管壁与糊剂之间的结合特征。结果　 iRoot SP与AH Plus组的微渗漏值较小且两者无统计学差异,Vitapex糊剂组的微渗漏值最大,与其他两组有统计学差异(P<0.05)。扫描电镜下不同根管封闭剂与根管壁和牙胶间均观察到空隙存在。结论　 iRoot SP和AH Plus的根管封闭性能较Vitapex糊剂更佳。     

Vitality status of microorganisms in infected human root dentine

AIM: This experimental study was initiated to establish a method for characterizing the vitality status of bacteria in infected human root dentine by differentiating between viable and dead microorganisms. METHODOLOGY: Twenty-four root segments of extracted human teeth were infected with either Streptococcus sanguinis or Enterococcus faecalis for 8 weeks. Baseline samples from root dentine (rd) were collected after 4 weeks. These were compared with samples taken at week 8 (control group: n = 12) and with samples collected at week 12 after calcium hydroxide treatment for four weeks (test group: n = 12). After marking viable and dead bacterial cells by two fluorescent dyes, the portion of viable bacteria (PVB) was determined, as well as the number of colony-forming units (CFU). RESULTS: Viable and dead bacteria were identified in all "rd" samples. PVBrd values were lower than PVB values of the bacterial suspension in the root canal lumen. In the control group, PVBrd and CFUrd did not markedly differ at week 4 and at week 8, regardless of the strain used. In the test group, viable but non-culturable sanguinis streptococci (mean PVBrd = 27%; CFUrd = 0) were detected, despite calcium hydroxide treatment. The viability of E. faecalis was not affected by calcium hydroxide. CONCLUSIONS: Fluorescence labelling of bacteria in human root dentine gives valuable additional information about their vitality status compared to the parameter CFU. The method may be suitable for following the fate of bacteria in dentinal tubules, for example in the presence of intracanal dressings.     

再感染根管内粪肠球菌生物膜的研究进展

粪肠球菌是顽固性和继发性根管感染中最易分离到的细菌,其主要致病机制之一是形成生物膜.笔者下面就再感染根管内粪肠球菌的分离与鉴定、影响粪肠球菌生物膜形成的相关因素等作一综述.     

In vitro genotoxicity of root canal sealers

Aim  To evaluate the effect of leakage on differences in genotoxicity of root canal sealers ex vivo according to their main components using two different cytogenetic assays.
Methodology  Six materials of different composition (GuttaFlow, Epiphany, Diaket, IRM, SuperEBA and Hermetic) were tested on human peripheral blood lymphocytes using the comet assay and chromosomal aberration analysis. Prepared materials were eluted in physiological solution for 1 h, 1 day, 5 and 30 days. Thereafter cultures were treated with 8 μg, 4 μg and 2 μg of each sealer. Frequencies of chromatide and chromosome breaks and accentric fragments were determined. Comet assay was used to evaluate primary DNA damage by measuring tail length and tail intensity. Chi-square, Fisher's PLSD (Protected Least Significant Difference) and Kruskall–Wallis non parametric tests were used for statistical analysis.
Results  After 1-h elution only the highest dose of Diaket, Hermetic and SuperEBA significantly ( P = 0.035, P  = 0.048, P  = 0.037 respectively) affected the measured cytogenetic parameters. The migration ability of DNA was more strongly affected than induction of chromosomal aberrations. After elutions longer than 24 h none of the tested sealers exhibited a genotoxic effect.
Conclusion  Under the conditions used in the study all sealers had acceptable biocompatibility in terms of genotoxicity.     

Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals

INTRODUCTION: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. METHODS: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. RESULTS: The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. CONCLUSION: These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.     

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。