Efficacy of different instrumentation techniques on reducing Enterococcus faecalis infection in experimentally infected root canals

Journal of Dental Sciences (2014) 9, 23e28 Available online at www.sciencedirect.com journal homepage: www.e-jds.com ORIGINAL ARTICLE Efficacy of different instrumentation techniques on reducing Enterococcus faecalis infection in experimentally infected root canals Ebru Özsezer Demiryürek a, Elif Kalyoncuoglu a *, Esra Duran a, Ahmet Yılmaz Çoban b, Yeliz Tanrıverdi Çaycı b a Department of Endodontics, Faculty of Dentistry, Ondokuz Mayıs University, Samsun, Turkey b Department of Microbiology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey Received 29 November 2011; Final revision received 8 March 2012 Available online 14 February 2013 KEYWORDS antimicrobial activity; Enterococcus faecalis; root canals; rotary instruments Abstract Background/purpose: Enterococcus faecalis is the bacterium most commonly isolated from infected root canals. This study evaluated the effects of four instrumentation techniques on reducing E. faecalis in curved root canals. Materials and methods: Sixty maxillary first molar teeth were used. After mesiobuccal canals had been instrumented with nickeletitanium (NiTi) K-files up to size 25, the teeth were autoclaved, immersed in a broth inoculated with E. faecalis, and incubated for 48 hours. The teeth were divided into four experimental groups (n Z 15). The mesial root canals were instrumented using ProTaper, RaCe, and Mtwo rotary instruments, and hand-operated NiTi files. Irrigation was performed using sterile saline solution. For each root canal, three samplings were performed: twice before preparation and once after preparation. After serial dilutions, samples were incubated, and the colony-forming units were counted. Results: All instrumentation techniques reduced E. faecalis infection in curved root canals. However, there were statistically significant differences among the instrumentation techniques regarding the numbers of residual bacteria (P < 0.01). The ProTaper and RaCe NiTi rotary instruments were more effective than Mtwo and hand-operated NiTi instruments. Conclusion: This study indicates that instruments with a greater taper play an important role in maximizing the effectiveness of mechanical preparation. However, since using mechanical * Corresponding author. Department of Endodontics, Faculty of Dentistry, Ondokuz Mayıs University, Kurupelit, Samsun 55139, Turkey. E-mail address: elif_kalyoncu@hotmail.com (E. Özsezer Kalyoncuoglu). 1991-7902/$36 Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jds.2012.03.024

24 E. Özsezer Demiryürek et al instrumentation alone is insufficient to completely eliminate root canal infection, the use of complementary antibacterial compounds is necessary. Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved. Introduction Since pulpal infection plays a critical role in the development of periradicular lesions, 1,2 endodontic treatment must be directed towards eliminating bacteria, their products, and substrates from root canal systems. 3,4 It has been demonstrated that eradicating endodontic infection enhances the success rate of endodontic therapy. 5,6 Microorganisms are present in all parts of the root canal system, including fins and anastomoses. They may be found at depths of up to 300 mm within dentinal tubules. 7 There also appear to be regional variations in the extent to which dentine is invaded: cervical tubules are invaded to a greater extent than mid-root tubules, which are invaded more than those in the apical region. 8 Enterococcus faecalis is the bacterium most commonly isolated from root canals presenting with post-treatment disease. 9 Pinheiro et al 10 found E. faecalis in 52.94% of canals with bacterial growth. This microorganism can survive in an environment in which there are scant available nutrients and in which commensality with other bacteria is minimal. 11 Enterococcus faecalis can invade dentinal tubules, 12 and it is therefore probable that cells in the dentinal tubules that survive chemomechanical instrumentation and intracanal medication are able to colonize the tubules and reinfect obturated root canals. 13 Reducing the bacterial count in infected root canals is accomplished by a combination of mechanical instrumentation, various irrigation solutions, and antibacterial medicaments or dressings placed in the canal. Mechanical instrumentation is often the first means of bacterial reduction during endodontic treatment of infected root canals. 14 In the last 15 years, several new automated instrumentation systems based on rotary nickeletitanium (NiTi) instruments have been developed for root canal preparation. More recently, advanced instrument designs, including noncutting tips, different cross-sections, and varying tapers, have been developed to improve working safety, shorten working times, and create a greater flare within preparations. 15 Although it was demonstrated that these newer instruments and techniques improved the shaping of root canals, few studies have evaluated their ability to reduce the number of microorganisms within canal roots and dentinal tubules. Such studies reported that rotary and hand instrumentation techniques were equally effective in reducing intracanal bacteria. 5,14 A literature review revealed no studies comparing the effects of the ProTaper, Mtwo, and RaCe rotary instruments on reducing bacterial populations in root canals. Therefore, this study evaluated the efficacy of four instrumentation techniques on reducing E. faecalis levels in curved root canals. Materials and methods In total, 60 extracted human maxillary first molar teeth were selected. Mesiobuccal root canals of maxillary molars were used in this study because they usually present an accentuated curvature. The teeth were stored in tap water prior to the experimental procedures. At the beginning of the study, teeth were examined for the number of roots, the canal configuration, and the presence of additional mesiobuccal canals (MB2). The teeth were attached to a digital X-ray sensor (Planmeca Dixi3; Planmeca, Helsinki, Finland) with soft wax and were aligned so that the long axis of the root was parallel and as close as possible to the surface of the digital X-ray sensor. All radiographs were taken in both the buccolingual and mesiodistal directions. To eliminate superposition in the buccolingual direction, the MB and MB2 canals were radiographed separately. Teeth with open apices, resorption, calcified canals, or the presence of MB2 canals were excluded from the study. After access cavity preparation, using hand instruments and copious amounts of 2.5% sodium hypochlorite irrigation, an attempt was made to remove the contents of the pulp chamber and locate all canal orifices. The distobuccal and palatal canal orifices were then sealed with glass ionomer cement (Photac Fil Quick Aplicap; 3M ESPE AG, Seefeld, Germany) to allow easy mesiobuccal canal preparation. The working length was established by subtracting 1 mm from the actual root length as determined by introducing a #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) until it was visible through the apical foramen. Radiographs were also taken to evaluate the mesiobuccal root curvature. Mesiobuccal roots with curvatures of 30e45 degrees as assessed by Schneider s method 16 were included in the study (Fig. 1). The roots were coated with two layers of nail polish to prevent any extrusion or leakage of material during root canal preparation and sample collection. All mesiobuccal canals were instrumented with NiTi K-files (DiaDent Group International, Burnaby, BC, Canada) up to size 25. The canals were irrigated with a total volume of 10 ml of 0.85% sterile saline solution, using separate 3-mL plastic syringes with a 23-gauge needle for each root canal. To make handling and identification easier, teeth were mounted vertically in resin blocks, which were then placed inside sterile plastic bags and sterilized by autoclaving at 121 C for 15 minutes. A single operator, using aseptic techniques, carried out the preparation and sampling procedures on each specimen in a laminar air flow cabinet to prevent airborne bacterial contamination. For each root canal, three samplings were performed; twice before preparation and once after preparation. The first intracanal sampling before preparation

Efficacy of different instrumentation techniques 25 Figure 1 Technique used for determining the mesiobuccal root canal curvature in the clinical view. The angle of curvature (A, 30 degrees) was measured according to the method described by Schneider. 16 was called the S1 sample and was checked for sterility after autoclaving the samples (S1 served as the negative control group). To conduct the first sampling (S1), root canals were filled with a 0.85% sterile saline solution. This was delivered into the canals using a 3-mL plastic syringe with a 23-gauge needle. Three paper points were used to carefully absorb the fluid from the canal in order not to touch the outer surface of the canal. The points were then transferred into tubes containing 1 ml of a 0.85% saline solution and vortexed for 30 seconds. Following serial dilution, aliquots of 0.1 ml were taken and plated onto braineheart infusion agar (Difco Laboratories Inc., Detroit, MI, USA). The labeled plates were incubated for 24 hours at 37 C. The external surface of the root specimen was then washed with 3 ml of sodium hypochlorite, and the blocks were again placed inside sterile plastic bags. A suspension was prepared by adding 1 ml of a pure culture of E. faecalis (ATCC 29212), grown in braineheart infusion broth (Difco Laboratories Inc.) for 24 hours. A McFarland standard number 1.0 was used to evaluate the broth to ensure that the number of bacteria was 1.5 10 8 colony-forming units (CFU)/mL. Each mesial root canal was completely filled with the E. faecalis suspension using a sterile 1-mL tuberculin syringe. Sterile K-type #15 files were used to spread the bacterial suspension along the entire root canal length. The blocks were then placed inside sterile plastic bags and incubated at 37 C for 48 hours. The second intracanal sample (S2) was obtained by the same procedure from the canal of each specimen. The rationale was to determine the presence of viable E. faecalis within the canals (S2 serving as the positive control group). S2 samples were vortexed, and aliquots of 0.1 ml were taken and plated onto braineheart infusion agar. The plates were incubated for 24 hours at 37 C, and E. faecalis concentrations in the S1 and S2 (negative and positive control) samples were determined as CFU/mL. Contaminated roots were divided into four experimental groups, and the preparations were completed using different techniques. In group A, 15 root canals were instrumented using ProTaper NiTi instruments (Dentsply Maillefe) with a convex triangular cross-sectional design and an advanced flute design that combines multiple tapers within the shaft. ProTaper instruments were used in a crown-down manner, according to the manufacturer s instructions. The instrumentation sequence was: (1) an S1 file (shaping file #1; taper 02-0.11; size 17) was used to one-third of the working length; (2) an SX (auxiliary shaping file; taper 035-0.19; size 19) was used to one-half of the working length; (3) an S1 file was used from one-half to two-thirds of the working length; (4) an S2 file (shaping file #2; taper 04-0.115; size 20) was used to two-thirds of the working length; (5) an F1 file (finishing file #1; taper 07-0.55; size 20) was used to the full working length; (6) an F2 file (finishing file #2; taper 08-0.55; size 25) was used to the full working length; and (7) an F3 file (finishing file #3; taper 09-0.5; size 30) was used to the full working length. In group B, 15 root canals were instrumented using the reamer with an alternating cutting edge (RaCe) rotary system (FKG Dentaire, La Chaux-de-Fonds, Switzerland). RaCe instruments have a triangular cross-sectional design with alternating cutting edges. The RaCe instruments were also used in a crown-down manner, according to the manufacturer s instructions. The instrumentation sequence was: (1) a 0.10 taper, size 40 instrument was used to onethird of the working length; (2) a 0.08 taper, size 35 instrument was used from one-third to one-half of the working length; (3) a 0.06 taper, size 30 instrument was used from one-half to two-thirds of the working length; (4) a 0.04 taper, size 25 instrument was used to two-thirds of the working length; (5) a 0.02 taper, size 25 instrument was used to the full working length; and (6) a 0.02 taper, size 30 instrument was used to the full working length for apical preparation. In group C, 15 root canals were instrumented using the Mtwo rotary system (VDW, Munich, Germany). Mtwo instruments have an S-shaped cross-sectional design and are characterized by a positive rake angle with two cutting edges. Moreover, the pitch length increases from the tip to the shaft. All Mtwo instruments were used to the full length of the canals, according to the manufacturer s instructions and using a gentle in-and-out motion. The instrumentation sequence was: (1) a 0.04 taper, size 10 instrument was used to the full working length; (2) a 0.05 taper, size 15 instrument was used to the full working length; (3) a 0.06 taper, size 20 instrument was used to the full working length; (4) a 0.06 taper, size 25 instrument was used to the full working length; and (5) a 0.05 taper, size 30 instrument was used to the full working length. In group D, 15 root canals were hand-instrumented using 0.02 taper NiTi K files (DiaDent Group International). A #15 file was placed to the working length. A combination of a filing action and a reciprocal reaming action was used until it fitted comfortably in the canal. This was repeated with successively longer files until the apical portion of the canal was instrumented to a size 30 file. The last samples (S3) were collected from each specimen after preparation and were used to calculate numbers

26 E. Özsezer Demiryürek et al of surviving bacteria. S3 samples were vortexed for 30 seconds, and 0.01-mL aliquots were taken and plated onto braineheart infusion agar. The labeled plates were incubated for 24 hours at 37 C, and the concentration of bacteria in each sample was determined as CFU/mL. Since the data used in the present study were continuous, a one-way analysis of variance (ANOVA) was used to compare the means of the four different instruments. Duncan s multiple-range test was also used to determine mean differences between pairs of instruments. Results The autoclave sterilization of negative control group specimens (S1) proved to be 100% effective, as all tests for microbial growth proved negative. In the contaminated group (S2), it was determined that an adequate amount of E. faecalis contamination was present in all root canals. There were no statistically significant differences among the groups of the concentration of microorganisms before root canal preparation (P > 0.05). All instrumentation techniques (S3) reduced the concentration of E. faecalis in the curved root canals, and ANOVA showed no statistically significant differences among instrumentation techniques (P > 0.05; F Z 2.035). Bacterial elimination success rates of the Pro Taper, RaCe, and Mtwo rotary instruments were around 99%, while that of the hand-operated NiTi was 98% (Table 1). In the present study, in order to reveal differences in the residual bacteria in root canals among the different instruments, samples with 100% efficient instrumentation (i.e., no residual bacteria were found) were eliminated from all the groups because the focus was on the number of residual bacteria in the root canal after preparation. In order to explore differences among the four different instruments by examining only contaminated samples, ANOVA and Duncan s multiple-range test were applied. Results of the ANOVA revealed significant differences in the number of residual bacteria among the instrumentation techniques (P < 0.01; F Z 13.054). The ProTaper and RaCe rotary instruments had 4.2 10 3 and 5.5 10 3 CFU/mL of surviving bacteria, respectively, while numbers for Mtwo rotary and hand-operated NiTi files were 1.4 10 5 and 2.3 10 5 CFU/mL, respectively. According to Duncan s multiple-range test, the use of the ProTaper and RaCe rotary instruments resulted in lower numbers of residual bacteria (P < 0.05). However, the difference between the Table 1 Elimination success of instrumentation techniques. Instrumentation technique Mean Standard deviation Group A (ProTaper) 0.9965 0.0042 Group B (RaCe) 0.9966 0.0059 Group C (MTwo) 0.9936 0.1180 Group D (hand NiTi) 0.9858 0.1686 *No statistically significant differences among instrumentation techniques were detected (analysis of variance, F Z 2.035; P > 0.05). ProTaper and RaCe rotary instruments was not significant (P > 0.05). The least effective instruments in terms of eliminating bacteria were the hand-operated NiTi files (P < 0.05) (Table 2). Discussion One of the most important objectives of root canal instrumentation is the removal of vital and/or necrotic pulp tissues, infected dentine, and dentine debris in order to eliminate most of the microorganisms from the root canal system. 17,18 There are several reported methods to evaluate endodontic instrumentation, including microscopic observation of the remaining debris, a morphometric analysis, and a bacteriologic assessment. 4,19e21 A bacteriologic assessment was selected for the present study because of the importance of canal disinfection in successfully treating apical periodontitis. The sampling method used in the present study has not been previously employed. Also, studies of the efficiency of current rotary instruments in eliminating microorganisms in root canals are very rare. Therefore, the present study attempted to compare the efficiencies of the ProTaper, Mtwo, and RaCe rotary instruments, the clinical use of which has become increasingly widespread. In the current study, the use of instrumentation techniques and irrigation with saline solution removed over 95% of the bacterial cells from the root canal, agreeing with findings of Siqueira et al. 5 A nonbactericidal saline solution was used as an irrigant in order to limit its bacterial reduction effect so as to reveal only the effects of mechanical preparation. In addition, it was thought that mechanical preparation results would be affected to a minimal extent if the saline solution was given by the same operator in the same quantity and at the same pressure. Enterococcus faecalis was chosen as the test microorganism because it can cause persistent endodontic infections and periradicular inflammation, 10 and can readily infect dentinal tubules. 12 In addition, E. faecalis is commonly found in root canals in which endodontic treatment has failed. Peciuliene et al 9 examined the microbiologic status of 40 root-filled teeth with periradicular lesions and found enterococci in 64% of them. In similar studies, Pinheiro et al 10 reported a 52.94% occurrence and Sundqvist et al 11 reported a 38% occurrence of enterococci. Table 2 Numbers of surviving bacteria (after eliminating no residual bacteria samples from all groups). Instrument Number of surviving bacteria (colony-forming units/ml) Group A (Pro Taper) 4.2 10 3a Group B (RaCe) 5.5 10 3a Group C (MTwo) 1.4 10 5b Group D (hand NiTi) 2.3 10 5c Values with different superscript letters are statistically significantly different according to an analysis of variance with Duncan s multiple-range test (F Z 13.054; P < 0.01). NiTi Z nickeletitanium.

Efficacy of different instrumentation techniques 27 Microorganisms are present in all parts of the root canal system, including fins and anastomoses, and may be found within the dentinal tubules at varying depths of up to 300 mm from the pulpal end. 7 This finding has been supported by Haapasalo and Ørstavik 12,22 who found penetration of E. faecalis to a depth of 300e400 mm in bovine dentinal tubules after 1 day. Enterococcus faecalis is the most commonly used microorganism for experimental bacterial penetration into dentinal tubules. 12,23,24 Since incubation depth was not measured in the present study, the incubation period was taken to be 48 hours, based on studies from Siqueira et al, 5,25 Berber et al, 26 and Chuste- Guillot et al 27 in order to increase the depth of invasion in dentinal tubules. Most previous studies on reducing bacterial levels in root canals have focused on the efficient application of instruments. 5,14,25e27 However, most instruments solely reduced the number of bacteria in the root canals, rather than eliminated the bacteria completely. This study, therefore, concentrated on the number of residual bacteria in the root canals and compared four different instruments. In addition, a further statistical analysis showed that the use of the ProTaper and RaCe rotary instruments left fewer residual bacteria in the root canals, compared to the Mtwo and hand-operated NiTi instruments. Since most microorganisms in root canals are in the coronal one-third, 28 the initial preparation of this section can help reduce the number of microorganisms. In addition, large preparations can incorporate more anatomic irregularities and allow the removal of a substantial number of bacterial cells from the root canal. 5,27 Removal of 200 mm of dentine from canal walls has been suggested to provide sufficient preparation in molars. 29 The efficacy of rotary instruments with a greater taper in preparing oval root canals was studied by Elayouti et al, 30 who found that instruments with a greater taper were more efficient than hand-operated NiTi files. In addition, they stressed that, based on their measurements, ProTaper instruments made a more aggressive cut in terms of the remaining dentine wall thickness. ProTaper instruments left a smaller thickness (0.5 mm) of the dentine wall after root canal preparation than did Mtwo instruments. It was chosen to leave 0.5 mm of the remaining dentine because this value represents removal of more than 50% of the dentine wall in narrow roots. In other words, ProTaper instruments removed more dentine wall. In the present study, different instrumentation techniques that removed different amounts of dentine were selected. Unlike the Mtwo system, both file systems of ProTaper and RaCe, in which preparations were made in accordance with the crown-down system and began with the orifice sharper files (ProTaper: SX; size 19, taper 0.19; RaCe: size 40, taper 0.10), helped to enlarge the coronal one-third of the root. It may be that the taper of the Pro- Taper and RaCe instruments, being greater than that of the Mtwo instruments, resulted in removal of more of the coronal dentine, and hence eliminated more bacteria and led to lower bacterial counts in the final samples. Foschi et al 20 and Schäfer et al 21 reported that the use of Mtwo rotary instruments required less time and was more convenient for preparing narrow roots due to their 10 and 15 size instruments, and the ability to cut close to the original form of the root. In spite of these advantages of Mtwo rotary instruments, the results of the present study show that an increase in taper and size of instruments and working with the crown-down technique eliminated more bacteria. The results of the present study therefore support the findings of Chuste-Guillot et al 27 that, when using a greater taper and crown-down technique, more infected dentine appeared to have been eliminated from the coronal onethird, which contains the largest bacterial population. It should be noted that, in the present study, the apical preparation was standardized to a size 30 in all groups; although the Mtwo system also has size 35 (0.04 taper) and 40 (0.04 taper) instruments, they were not used in this study. Within the limitations of this study, the ProTaper and RaCe instruments were more effective than the Mtwo and hand-operated NiTi files in reducing bacterial numbers in the root canals. The results of this study therefore support the contention that instruments with a greater taper can play an important role in maximizing the effectiveness of mechanical preparation. Due to technologic development, more studies can be conducted with new canal preparation instruments and techniques, including lasers, which are becoming more popular in endodontic procedures. However, despite modern instrumentation and techniques, the complete disinfection of root canal systems cannot be achieved by mechanical preparation alone. Since bacteria surviving instrumentation pose a risk to successful root canal treatment, an antimicrobial irrigation solution and intracanal medication are still recommended. Conflicts of interest The authors have no conflicts of interest relevant to this article. Acknowledgments The author thanks Gregory T. Sullivan of OYDEM at Ondokuz Mayis University for editing the English in an earlier version of this manuscript. Thanks also go to Professor Dr Vedat Ceyhan of the Faculty of Agriculture, Ondokuz Mayis University, for his help with the statistical analysis and valuable suggestions during manuscript preparation. References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1965;20:340e9. 2. Sundqvist G. Bacteriological Studies of Necrotic Dental Pulps. Umea, Sweden: Umea University;1976, Odontological Dissertations, No. 7. 3. Coldero LG, McHugh S, MacKenzie D, Saunders WP. Reduction in intracanal bacteria during root canal preparation with and without apical enlargement. Int Endod J 2002;35:437e46. 4. Aydın C, Tunca YM, Senses Z, Baysallar M, Kayaoglu G, Ørstavik D. Bacterial reduction by extensive versus

28 E. Özsezer Demiryürek et al conservative root canal instrumentation in vitro. Acta Odontol Scand 2007;65:167e70. 5. Siqueira Jr JF, Lima KC, Magalhaães FAC, Lopes HP, Uzeda M. Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques. J Endod 1999;25: 332e5. 6. Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297e306. 7. Horiba N, Mackawa Y, Matsumoto T, Nakamura H. A study of distribution of endotoxin in the dentinal wall of infected root canals. J Endod 1990;16:331e4. 8. Love MR. Regional variation in root dentinal tubule infection by Streptococcus gordonii. J Endod 1996;22:290e3. 9. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. J Endod 2001;34:429e34. 10. Pinheiro ET, Gomes BPFA, Ferraz CCR, Sousa ELR, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J 2003;36:1e11. 11. Sundqvist G, Fidgor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86e93. 12. Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:142e9. 13. Kreft B, Marre A, Schamm U, Wirth R. Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells. Infect Immun 1992;60:25e30. 14. Dalton CB, Orstavik D, Phillips C, Pettiette M, Trope M. Bacterial reduction with nickel-titanium rotary instrumentation. J Endod 1998;24:763e7. 15. Bergmans L, Van Cleynenbreugel J, Wevers M, Lambrechts P. Mechanical root canal preparation with NiTi rotary instruments: rationale, performance and safety. Status report for the American Journal of Dentistry. Am J Dent 2001;14:324e33. 16. Schneider SW. A comparison of canal preparations in straight and curved root canals. Oral Surg Oral Med Oral Pathol 1971; 32(2):271e5. 17. American Association of Endodontists. Glossary: Contemporary Terminology for Endodontics, 6th ed. Chicago, IL: American Association of Edodontists, 1998. 18. European Society of Endodontology. Consensus report of the European Society of Endodontology on quality guidelines for endodontic treatment. Int Endod J 1994;27:115e24. 19. Shuping G, Ørstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. J Endod 2000;26:751e5. 20. Foschi F, Nucci C, Montebugnoli L. SEM evaluation of canal wall dentine following use of Mtwo and ProTaper NiTi rotary instruments. Int Endod J 2004;37:832e9. 21. Schäfer E, Erler M, Dammaschke T. Comparative study on the shaping ability and cleaning efficiency of rotary Mtwo instruments. Part 2. Cleaning effectiveness and shaping ability in severely curved root canals of extracted teeth. Int Endod J 2006;39:203e12. 22. Haapasalo M, Orstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375e9. 23. Peters LB, Wesselink PR, Moorer WR. Penetration of bacteria in bovine root dentine in vitro. Int Endod J 2000;33:28e36. 24. Dametto FR, Ferraz CCR, Gomes BPFA, Zaia AA, Teixeira FB, Souza-Filho FJ. In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:768e72. 25. Siqueira Jr JF, Rôças IN, Santos SRLD, Lima KC, Magalhaães FAC, de Uzeda M. Efficacy of instrumentation techniques and irrigation regimens in reducing the bacterial population within root canals. J Endod 2002;28:181e4. 26. Berber VB, Gomes BPFA, Sena NT, et al. Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J 2006;39:10e7. 27. Chuste-Guillot MP, Badet C, Peli JF, Perez F. Effect of three nickel-titanium rotary file techniques on infected root dentin reduction. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:254e8. 28. Shovelton DS. The presence and distribution of microorganisms within non-vital teeth. Br Dent J 1964;117:101e7. 29. Weiger R, Bartha T, Kawitzki M, Löst C. A clinical method to determine the optimal apical preparation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:686e91. 30. Elayouti A, Chu AL, Kimionis I, Klein C, Weiger R, Löst C. Efficacy of rotary instruments with greater taper in preparing oval root canals. Int Endod J 2008;41:1088e92.