doi:10.1111/j.1365-2591.2011.01959.xAntimicrobial effect of endodontic solutions usedas final irrigants on a dentine biofilm...
The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al.    duced by the action of endodontic instruments o...
Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm modelmicrobes (Shen et al. 2009). The concentration of t...
The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al.             (a)             (b)                   ...
Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model                                             (a)   ...
The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al.       (a)                                         ...
Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model  single-rooted human teeth. International Endodont...
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International endodontic journal 2011 ordinola zapata

  1. 1. doi:10.1111/j.1365-2591.2011.01959.xAntimicrobial effect of endodontic solutions usedas final irrigants on a dentine biofilm modelR. Ordinola-Zapata1, C. M. Bramante1, B. Cavenago1, M. S. Z. Graeff2, I. Gomes de Moraes1,M. Marciano1 & M. A. H. Duarte11 Department of Endodontics, Dental School of Bauru, University of Sao Paulo, Bauru; and 2Integrated Research Center (CIP), ˜Dental School of Bauru, University of Sao Paulo, Bauru, Brazil ˜Abstract biofilm thickness (lm) and substratum coverage (%) of the treated biofilms were determined using nonpara-Ordinola-Zapata R, Bramante CM, Cavenago B, Graeff metric statistical tests (P < 0.05).MSZ, Gomes de Moraes I, Marciano M, Duarte MAH. Results Similar values of biovolume total, biovolumeAntimicrobial effect of endodontic solutions used as final of live subpopulations and substratum coverage wereirrigants on a dentine biofilm model. International Endodontic found in 2% chlorhexidine, 10% citric acid, 17% EDTAJournal. and distilled water-treated biofilms (P > 0.05). TheAim To evaluate the residual biovolume of live lower values of the studied parameters were found inbacterial cells, the mean biofilm thickness and the 1% NaOCl-treated dentine (P < 0.05) with the excep-substratum coverage found in mixed biofilms treated tion of the mean biofilm height criteria that did notwith different endodontic irrigant solutions. reveal significant differences amongst the irrigantMethodology Twenty-five bovine dentine specimens solutions (P > 0.05).were infected intraorally using a removable orthodontic Conclusions One per cent sodium hypochlorite wasdevice. Five samples were used for each irrigant solution: the only irrigant that had a significant effect on biofilm2% chlorhexidine, 1% sodium hypochlorite (NaOCl), viability and architecture.10% citric acid, 17% EDTA and distilled water. The Keywords: confocal laser scanning microscopy,solutions were used for 5 min. The samples were stained dentin, irrigant solutions, oral biofilms.using the Live/Dead technique and evaluated using aconfocal microscope. Differences in the amount of total Received 28 April 2011; accepted 28 August 2011biovolume (lm3), number of surviving cells (lm3), mean criteria proposed by Parsek & Singh (2003) to define aIntroduction biofilm-associated disease as: bacteria associated to aBiofilms can be defined as bacterial cells attached to a substratum, bacteria encased in a matrix of bacterial orsurface embedded in an exopolysaccharide matrix that host constituents, infection localized and resistant tofills the space between the cells (Costerton 2007). antibiotic therapy.Biofilms are present in necrotic pulp canal spaces of Although the eradication of intraradicular biofilmsprimary and secondary root canal infections (Ricucci should be ideally performed by mechanical methods,et al. 2009). A previous study (Ricucci & Siqueira the instrumentation process cannot directly access a2010) has reported that apical periodontitis meets the considerable amount of infected tissue owing to the irregularities of the root canal system (Paque et al. 2009, 2010). For this reason, antimicrobial com-Correspondence: Ronald Ordinola-Zapata, Faculdade de pounds are used during instrumentation and as a final ´Odontologia de Bauru, USP, Octavio Pinheiro Brisolla, 9-75,CEP 17012-901, Bauru, Sao Paulo, Brazil (Tel.: +55- ˜ rinse before canal filling. Several irrigant solutions have1432358344; fax: +55-1432358344; e-mail: ronaldordin been proposed for use during the final rinse. EDTA citric acid are used to eliminate the smear layer pro-ª 2011 International Endodontic Journal International Endodontic Journal 1
  2. 2. The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al. duced by the action of endodontic instruments on root treated with irrigant solutions used for final rinse canal walls (Violich & Chandler 2010, Prado et al. purposes do not result in different amounts of live cells, 2011). Others irrigants, such as 2% chlorhexidine, substratum coverage and mean biofilm thickness. have been proposed to enhance antimicrobial control at the end of the chemo-mechanical process (Zamany Materials and methods et al. 2003). Although EDTA and citric acid have been reported to have no or only a slight antimicrobial effect The irrigant solutions used were: 1% sodium hypo- (Siqueira et al. 1998, Arias-Moliz et al. 2009), previous ´ chlorite (CloroRio, Sao Jose do Rio Preto, Brazil), 10% ˜ reports have concluded that EDTA can remove 83% of Citric Acid (Farmacia Especıfica, Bauru, Brazil), 17% ´ a Streptococcus gordonii biofilm (Chavez de Paz et al. EDTA (Biodinamica, Ibipora, Brazil) and 2% chlorhex- ˜ 2010). Whilst a final rinse with an antimicrobial idine (Villevie, Joinville, Brazil). Distilled water was used compound is highly desirable, it is not clear how for control purposes. beneficial it would be on residual biofilms that were Twenty-five dentine sections (3 · 3 · 2 mm) were obviously not affected by the primary chemo-mechan- obtained from previously sterilized bovine root dentine. ical treatment. The samples were treated with 1% sodium hypochlorite Dentine is a mineralized connective tissue that is (NaOCl) for 15 min and 17% EDTA for 3 min to porous because of the presence of dentinal tubules. eliminate organic debris and the smear layer produced Microorganisms are usually able to adhere to its surface during the sectioning process. Five dentine samples and to enter into root dentinal tubules (Love & were fixed in each experimental procedure on a Hawleys Jenkinson 2002). For this reason, infected dentine is orthodontic device to produce biofilm. To standardize commonly found in primary and secondary root canal the rate of biofilm development, one healthy volunteer infections (Ricucci et al. 2009). The use of dentine as a used the intraoral device for 48 h except during regular substrate to induce microbial biofilms has been used oral hygiene practices. The ethical human committee commonly to test the effect of endodontic procedures approved the protocol (CEP-134/2010). After the mainly in combination with scanning electron micros- intraoral infection process, each sample was incubated copy or culture techniques (George et al. 2005, Clegg in 2 mL of brain heart infusion (BHI) at 37 °C for 12 h et al. 2006, Bhuva et al. 2010). However, SEM meth- using 24-well tissue culture plates. ods do not provide appropriate three-dimensional After the incubation period, the dentine samples quantification of the residual biomass, and culture were washed with 1 mL of distilled water to eliminate techniques depend on a representative sample that is the culture medium and nonadherent cells. The contact taken after dispersing the microbial cells (Costerton test was performed by immersing the dentine sample 2007). Direct comparisons addressing three-dimen- for 5 min in 1 mL of the irrigant solutions tested. sional quantification of the affected biomass attached Twenty-four-well tissue culture plates were used for the to dentine after exposure to common irrigants solutions contact test. One additional dentine sample was treated are unknown. with 1 mL distilled water for 5 min to serve as control. Direct observation techniques using confocal micros- Five samples were used for each irrigant solution. copy can provide data on bacterial quantification After the contact test, the 1% NaOCl samples were (Lawrence et al. 1991, Costerton 2007). Previous treated with 100 lL of 5% sodium thiosulfate and a research has shown that in situ microscopic quantifi- final rinse of distilled water was used. Chlorhexidine, cation of the live bacterial biomass is better represented 10% citric acid and 17% EDTA-treated samples were in comparison with UFC quantification using culture washed with distilled water. A pilot study revealed that techniques (Shen et al. 2010), and this is an important sodium thiosulfate did not have a dissolution ability task when the limits of antimicrobial compounds need and improved the quality of the staining process for to be determined. Thus, this study aimed to evaluate sodium hypochlorite-treated dentine. the residual biovolume of live cells, mean biofilm height After the contact test, the samples were stained using and the substratum coverage found in mixed biofilms the Syto-9/Propidium iodide technique (Live/Dead, treated with irrigant solutions used commonly during Bacligth; Invitrogen, Eugene, OR, USA); SYTO-9 is a final rinse procedures such as: 2% chlorhexidine, 17% green fluorescent nucleic acid stain, generally labelling EDTA, 10% citric acid and distilled water. The effect of both live and dead microorganisms. PI is a red fluo- 1% sodium hypochlorite was also tested for compara- rescent nucleic acid stain and penetrates only the cells tive purposes. The null hypothesis was that: biofilms with damaged membranes, thus visualizing dead2 International Endodontic Journal ª 2011 International Endodontic Journal
  3. 3. Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm modelmicrobes (Shen et al. 2009). The concentration of the pictures in the reflection mode were also taken tofluorochromes was adjusted during a pilot study. To observe the surfaces of dentine after treatment with theimprove the contrast between the biofilm cells and irrigant solutions. Tridimensional models were recon-dentine, 1 lL of each dye was added to 1 mL of distilled structed using the OsiriX software (http://www.osirix-water. From this working solution, 200 lL was added running under Snow Leopard Mac OSXto the treated dentine samples and controls for 20 min 10.5.8 software (Apple, Cupertino, CA, USA).at room temperature in a dark environment. This dye Preliminary analysis of the data did not show aconcentration avoids the staining of dentine allowing normal distribution. Thus, the Kruskal–Wallis andan appropriate quantification using specially dedicated Dunn tests were used for multiple comparisonsbiofilm software. amongst the groups. The level of significance was set All the samples were observed using a confocal laser at P < 0.05, and Prisma 5.0 (GraphPad Software Inc,scanning microscope (CLSM Leica TCS-SPE; Microsys- La Jolla, CA, USA) was used as the analytical tool.tems GmbH, Mannheim, Germany). Four confocalpictures were obtained from each sample using the Results40X oil lens and a 1 lm step size in a format of512 · 512 pixels. The sequential frame scan mode was A total of 100 confocal ‘stacks’ were evaluated for allused to prevent crosstalk. At least 7 lm of the scanning the irrigant solution tested (20 for each irrigantprocess included the subsurface level of the dentine. solution). In all the biofilms evaluated, the maximumEach 40X biofilm picture represented an area of biofilm thickness ranged from 20–30 lm without275 · 275 lm2. For quantification purposes the bioi- statistical significances amongst the groups (data notmage_L software ( was shown). The median, mean and range values ofused, which produced information on the total biofilm biovolume total, biovolume of the green sub-populationpopulation as well as the independent subpopulations expressed in lm3 and substratum coverage (%) arerepresented by red and green fluorescent colours presented in Table 1 and Fig. 1. No statistical differ-(Chavez de Paz 2009). The biofilm analysis tool of the ences were found for these parameters tested when thesoftware was used to evaluate the four stacks obtained biofilms were treated with distilled water, 10% citricfor each sample. The data obtained in the biovolume/ acid, 17% EDTA or 2% chlorhexidine (P > 0.05). Thestack analysis of five independent experiments were lower values of the studied parameters were found inpooled as a single column to provide a single mean 1% NaOCl-treated dentine (P < 0.05) with the excep-representative of the 20 evaluated stacks in each tion of mean biofilm height that did not show statisticalirrigant solution. The parameters evaluated were bio- differences amongst the irrigant solutions (P > 0.05).volume of total and green population (live cells) in lm3, (See Fig. 1). Chlorhexidine-treated biofilms showed amean biofilm height in lm and substratum coverage discrete reduction of the viable cells in comparison withthat was expressed in terms of percentage. The biovo- the distilled water group but the difference was notlume is the volume occupied by microorganisms in a significant.3D space. The mean biofilm height calculates the mean Although some clean dentine surfaces were observedvertical expansion and the substratum coverage shows in the 1% NaOCl-treated specimens, residual biofilmshow efficiently the organism colonizes the dentine appeared firmly attached into the dentine structure.(Chavez de Paz et al. 2010). Representative confocal Complete cleaning of dentine was not observed in anyTable 1 Median, mean and range values of total and green biovolume after the contact with the experimental solutions 1% NaOCl 17% EDTA 10% Citric acid 2% Chlorhexidine Distilled water 3Biovolume total (lm ) Median 3.04 · 105 5.30 · 106 5.39 · 106 7.26 · 106 5.42 · 106 Mean 7.88 · 105 6.36 · 106 6.37 · 106 7.33 · 106 7.68 · 106 Range 1.60 · 103–4.35 · 106 1.02 · 106–1.79 · 107 1.23 · 106–1.36 · 107 7.11 · 105–1.68 · 107 2.72 · 106–1.64 · 107 3Live cells (lm ) Median 5.98 · 103 4.23 · 106 4.44 · 106 2.14 · 106 4.69 · 106 Mean 3.24 · 105 4.60 · 106 4.87 · 106 2.76 · 106 6.67 · 106 Range 0–2.30 · 106 3.84 · 105–1.20 · 107 2.53 · 105–1.20 · 107 1.92 · 104–7.71 · 106 1.69 · 106–1.52 · 107ª 2011 International Endodontic Journal International Endodontic Journal 3
  4. 4. The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al. (a) (b) Figure 1 Box-plots of mean biofilm height in lm (a) and substratum cover- age values in terms of percentage (b) after 5 min of contact with the irrigant solutions. of the tested solutions, neither in confocal fluorescence identify variables in which bacteria can survive appli- or the reflection picture. In all samples an intense cation of strong antibacterial compounds. One of these bacterial colonization was observed at the entrance of variables appears to be the presence of anatomical dentinal tubules. Representative pictures of the treated irregularities. The presence of a fissure or fin extending biofilms are shown in Figs 2 and 3. into the dentine structure not only can increase the vertical dimension in which the biomass can grow but also can limit the antimicrobial action (Deng & ten Cate Discussion 2004). Other interesting variables to be tested are if the The use of intraorally infected dentine and mixed surviving cells or new colonizers can attach to the biofilm models formed from subgingival plaque have residual biofilm or if ultrasonic activation has an been applied in previous studies to test the effect of additive effect on biofilm dissolution (Harrison et al. endodontic antimicrobial compounds as irrigants and 2010). Although a detailed description of the oral intracanal dressings (Barthel et al. 2002, Virtej et al. bacterial species that survive antimicrobial stress is 2007, Shen et al. 2009). Because dead oral bacteria are beyond the scope of this study, future research is commonly an integral part of oral biofilms (Auschill needed on this topic. et al. 2001, Arweiler et al. 2004), the samples of this The use of dentine as a substrate allows the study were additionally incubated for 12 h under rich identification of the presence of bacteria cells several supplement conditions (BHI). Using this method at least micrometres (7 lm) above the surface. However, the 85% of the biomass was found to be viable when the detection of viable bacteria in the deep layers of the biofilm was treated with a nonantimicrobial compound dentine remains restricted by the limited penetration of (water). the laser during the scanning process. Retamozo et al. Despite the clear advantages of the biofilm model (2010) showed that 40–60 min of 5.25% NaOCl are used in the present study, it is difficult to reproduce the necessary to obtain a negative culture in infected real clinical conditions in which the antimicrobial dentine and thus confirmed the challenge of eliminat- agent diffuses into the root canal system at the apical ing infection in the deep dentine layers. Future level. The use of the present biofilm model can serve to improvements of the CLSM experimental method may4 International Endodontic Journal ª 2011 International Endodontic Journal
  5. 5. Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model (a) (b) (c) (d) (e) (f)Figure 2 Representative three-dimen- (g) (h)sional constructions (left) and confocalsections of dentine surfaces (right) afterthe treatment with: distilled water (a–b),17% EDTA (c–d), 10% citric acid (e–f)and 2% chlorhexidine (g–h). Infection ofdentinal tubules is visible in all theconfocal sections. Bars in confocal sec-tions represent 40 lm. Three-dimen-sional constructions represent an area of275 · 275 lm2.address the viability of bacteria inside dentinal tubules horizontal dimensions is difficult using only 5 minmore precisely (Zapata et al. 2008). contact time. Longer decontaminating periods were The significantly lower values of substratum cover- avoided in this study because EDTA, citric acid and 2%age found in 1% NaOCl-treated biofilms may be chlorhexidine are generally used for short time periodsexplained by the dissolution effect that decreased the during root canal treatment.biovolume in the horizontal dimension. However, the Antimicrobial activity is desirable for all the irrigantmean biofilm height was not significantly different solutions used during root canal treatment includingamongst the irrigant solutions. This results show that chelating agents. However, the antimicrobial limits ofcomplete biofilm dissolution in both vertical and endodontic irrigants should be recognized. A contactª 2011 International Endodontic Journal International Endodontic Journal 5
  6. 6. The effect of irrigants on a dentin biofilm model Ordinola-Zapata et al. (a) time of 5 min of 17% EDTA and 10% citric acid had no effect on the biofilm viability results that are in agreement with Arias-Moliz et al. (2009). The effect of 2% chlorhexidine on the biofilm viability was also limited. Limited anti-biofilm activity of this compound in comparison with NaOCl has been found in previous studies (Clegg et al. 2006, Dunavant et al. 2006, Hope et al. 2010, Chavez de Paz et al. 2010). Under clinical conditions, the antimicrobial activity of 2% chlorhex- idine could be expected only in well-cleaned root canal walls; on the other hand, there may be no effect of this compound on the architecture or viability on biofilms situated in areas unaffected by the primary chemo- (b) mechanical process. In addition, the strong antimicro- bial effect of 1% NaOCl on biofilms attached to dentine was shown in this study confirming the results of others (Bryce et al. 2009, Hope et al. 2010, Bhuva et al. 2010). Overall, 1% sodium hypochlorite-treated bio- films removed 90% of the total biovolume and killed more bacteria in comparison with the other irrigant solutions. Thus, rejecting the null hypothesis. Conclusion One per cent sodium hypochlorite was the only irrigant that showed a significant effect on the biofilm viability and architecture. (c) Acknowledgements This work was supported by FAPESP (2010/16002-4). References Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garcia M, Baca P (2009) Enterococcus faecalis biofilms eradication by root canal irrigants. Journal of Endodontics 35, 711–4. Arweiler NB, Hellwig E, Sculean A, Hein N, Auschill TM (2004) Individual vitality pattern of in situ dental biofilms at different locations in the oral cavity. Caries Research 38, 442–7. Auschill TM, Artweiler NB, Netuschil L, Brecx M, Reich E, Sculean A (2001) Spatial distribution of vital and dead microorganisms in dental biofilms. Archives of Oral Biology 46, 471–6. Barthel CR, Zimmer S, Zilliges S, Schiller R, Gobel UB, Roulet Figure 3 The effect of 5 min of 1% NaOCl on biofilm structure JF (2002) In situ antimicrobial effectiveness of chlorhexidine can be seen in (a) and (b). After 5 min, there is a partial and calcium hydroxide: gel and paste versus gutta-percha disruption of the biofilm structure and some viable cells points. Journal of Endodontics 28, 427–30. (green) are still covering the dentine (a). However, the Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Mannocci F confocal reflection picture shows that biofilm layers are still (2010) The effectiveness of passive ultrasonic irrigation on covering partially the dentine structure (c) intraradicular Enterococcus faecalis biofilms in extracted6 International Endodontic Journal ª 2011 International Endodontic Journal
  7. 7. Ordinola-Zapata et al. The effect of irrigants on a dentin biofilm model single-rooted human teeth. International Endodontic Journal titanium rotary instruments: a micro-computed tomogra- 43, 241–50. phy study. Journal of Endodontics 36, 703–7.Bryce G, O’Donnell D, Ready D, Ng YL, Pratten J, Gulabivala K Parsek MR, Singh PK (2003) Bacterial biofilms: an emerging (2009) Contemporary root canal irrigants are able to link to disease pathogenesis. Annual Reviews in Microbiology disrupt and eradicate single- and dual-species biofilms. 57, 677–701. Journal of Endodontics 35, 1243–8. Prado M, Gusman H, Gomes BPFA, Simao RA (2011)Chavez de Paz LE (2009) Image analysis software based on Scanning electron microscopic investigation of the effective- color segmentation for characterization of viability and ness of phosphoric acid in smear layer removal when physiological activity of biofilms. Applied and Environmental compared with EDTA and citric acid. Journal of Endodontics Microbiology 75, 1734–9. 37, 255–8.Chavez de Paz LE, Bergenholtz G, Svensater G (2010) The Retamozo B, Shabahang S, Johnson N, Aprecio RM, Tora- effects of antimicrobials on endodontic biofilm bacteria. binejad M (2010) Minimum contact time and concentration Journal of Endodontics 36, 70–7. of sodium hypochlorite required to eliminate EnterococcusClegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR (2006) faecalis. Journal of Endodontics 36, 520–3. The effect of exposure to irrigant solutions on apical dentin Ricucci D, Siqueira JF Jr (2010) Biofilms and apical periodon- biofilms in vitro. Journal of Endodontics 32, 434–7. titis: study of prevalence and association with clinical andCosterton JW (2007) The biofilm primer. Berlin; New York: histopathologic findings. Journal of Endodontics 36, 1277– Springer. 88.Deng DM, ten Cate JM (2004) Demineralization of dentin by Ricucci D, Siqueira JF Jr, Bate AL, Pitt Ford TR (2009) Streptococcus mutans biofilms grown in the constant depth Histologic investigation of root canal-treated teeth with film fermentor. Caries Research 38, 54–61. apical periodontitis: a retrospective study from twenty-fourDunavant TR, Regan JD, Glickman GN, Solomon ES, Honey- patients. Journal of Endodontics 35, 493–502. man AL (2006) Comparative evaluation of endodontic Shen Y, Qian W, Chung C, Olsen I, Haapasalo M (2009) irrigants against Enterococcus faecalis biofilms. Journal of Evaluation of the effect of two chlorhexidine preparations on Endodontics 32, 527–31. biofilm bacteria in vitro: a three-dimensional quantitativeGeorge S, Kishen A, Song KP (2005) The role of environmen- analysis. Journal of Endodontics 35, 981–5. tal changes on monospecies biofilm formation on root canal Shen Y, Stojicic S, Haapasalo M (2010) Bacterial viability in wall by Enterococcus faecalis. Journal of Endodontics 31, 867– starved and revitalized biofilms: comparison of viability 72. staining and direct culture. Journal of Endodontics 36, 1820–Harrison AJ, Chivatxaranukul P, Parashos P, Messer HH 3. (2010) The effect of ultrasonically activated irrigation on Siqueira JF, Batista MMD, Fraga RC, de Uzeda M (1998) reduction of Enterococcus faecalis in experimentally infected Antibacterial effects of endodontic irrigants on black- root canals. International Endodontic Journal 43, 968–77. pigmented gram-negative anaerobes and facultative bacte-Hope CK, Garton SG, Wang Q, Burnside G, Farrelly PJ (2010) ria. Journal of Endodontics 24, 414–6. A direct comparison between extracted tooth and filter- Violich DR, Chandler NP (2010) The smear layer in endodon- membrane biofilm models of endodontic irrigation using tics – a review. International Endodontic Journal 43, 2–15. Enterococcus faecalis. Archives of Microbiology 192, 775–81. Virtej A, MacKenzie CR, Raab WH, Pfeffer K, Barthel CRLawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE (2007) Determination of the performance of various root (1991) Optical sectioning of microbial biofilms. Journal of canal disinfection methods after in situ carriage. Journal of Bacteriology 173, 6558–67. Endodontics 33, 926–9.Love RM, Jenkinson HF (2002) Invasion of dentinal tubules by Zamany A, Safavi K, Spangberg LS (2003) The effect of oral bacteria. Critical Reviews in Oral Biology & Medicine 13, chlorhexidine as an endodontic disinfectant. Oral Surgery 171–83. Oral Medicine Oral Pathology Oral Radiology and Endodontol-Paque F, Ganahl D, Peters OA (2009) Effects of root canal ogy 96, 578–81. preparation on apical geometry assessed by micro-computed Zapata RO, Bramante CM, de Moraes IG et al. (2008) Confocal tomography. Journal of Endodontics 35, 1056–9. laser scanning microscopy is appropriate to detect viabilityPaque F, Balmer M, Attin T, Peters OA (2010) Preparation of of Enterococcus faecalis in infected dentin. Journal of End- oval-shaped root canals in mandibular molars using nickel- odontics 34, 1198–201.ª 2011 International Endodontic Journal International Endodontic Journal 7