- Email: [email protected]
15% EDTA for Root Canal Irrigation
Basic Research—Technology
Comparison of the Antimicrobial Efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for Root Canal Irrigation J. Craig Baumgartner, Stephen Johal, DMD, and J. Gordon Marshall, DMD Abstract The purpose of this study was to compare the antimicrobial efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. Twenty-six bilaterally matched pairs of human teeth were collected. The teeth were incubated with Enterococcus faecalis for 4 weeks. The teeth were divided into two experimental groups and one positive control group. The canals were instrumented and irrigated with either 5.25% NaOCl/15% EDTA or 1.3% NaOCl/BioPure MTAD. Bacterial samples were collected after instrumentation/irrigation and after additional canal enlargement. Statistical analysis of the data using the Wilcoxon Signed Rank test showed significant differences between the experimental groups. The first bacterial samples revealed growth in 0 of 20 samples with 5.25% NaOCl/15% EDTA irrigation and in 8 of 20 samples with 1.3% NaOCl/BioPure MTAD irrigation. Samples taken after additional canal enlargement revealed growth in 0 of 20 samples in 5.25% NaOCl/15% EDTA and in 10 of 20 samples in 1.3% NaOCl/BioPure MTAD group. This investigation showed consistent disinfection of infected root canals with 5.25% NaOCl/15% EDTA. The combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the canals contaminated with E. faecalis. (J Endod 2007;33:48 –51)
Key Words MTAD, root canal irrigation, sodium hypochlorite
From the Department of Endodontology, OHSU School of Dentistry, Portland, Oregon. Address requests for reprints to Dr. J. Craig Baumgartner, Department of Endodontology, OHSU School of Dentistry, 611 SW Campus Drive, Portland, OR 97201. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.08.007
O
ne of the most important objectives of endodontic therapy is the complete elimination of microorganisms from the root canal system. The positive correlation between bacteria and endodontic disease has been established (1, 2). Failure of root canal treatment is likely caused by the inability to eliminate the bacteria responsible for refractory endodontic infections (3). Facultative bacteria such as Enterococcus faecalis have been isolated from infected root canals and may be related to failure of endodontic treatment (4 – 6). Although chemomechanical preparation of the root canal system is able to reduce the number of bacteria, complete canal disinfection is difficult because of the internal complexity of root canal systems (7, 8). Thus a root canal irrigant, such as sodium hypochlorite (NaOCl), is needed to aid in the disinfection of the canals (9 –16). NaOCl effectively eliminates microbes, and its antibacterial effectiveness may be a function of concentration and contact time (13, 15, 16). Endodontic instrumentation produces a smear layer, which is a 1- to 2-m-thick amorphous, granular structure of calcified tissue mixed with fragments of odontoblastic processes, microorganisms, and necrotic materials (17). Because of its potential contamination and its ability to inhibit or delay penetration of antimicrobial agents, smear layer removal has been advocated (10, 18). A final flush with ethylenediaminetetraacetic acid (EDTA) followed by NaOCl is an effective method for removing the smear layer and superficial debris from the surfaces of instrumented root canals (19). Solutions of 15% disodium, 15% tetrasodium, or 25% tetrasodium EDTA tested in combination with NaOCl are equally effective at removing the smear layer (20). A new irrigating solution, MTAD, containing a mixture of a tetracycline isomer, an acid, and a detergent, has shown promising results (21–25). Recent investigations involving the use of MTAD have demonstrated its ability to remove the smear layer, disinfect contaminated root canals, and eradicate E. faecalis (21, 24, 25). It was initially thought that MTAD could be used as an irrigant to replace NaOCl, but it was found that the cleanest canals were obtained if NaOCl was used before a final soak and flush with MTAD (22). MTAD’s ability to remove the smear layer is not dependent on the concentration of NaOCl used as root canal irrigant (22). Therefore, MTAD is recommended as a final soak and flush of canals after the use of 1.3% NaOCl (22, 23). 1.3% NaOCl/MTAD irrigation has been shown to be significantly more effective against E. faecalis than NaOCl with and without EDTA (25). However, it has also been reported that BioPure MTAD (Dentsply, Tulsa, OK) may not be effective against E. faecalis biofilms (26). The purpose of this investigation was to compare the antimicrobial efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation.
Materials and Methods The experimental protocol was deemed exempt by the Oregon Health Science University Institutional Review Board. Twenty-six bilaterally matched pairs of extracted mature maxillary anterior human teeth were collected. Each tooth was radiographed to confirm the presence of a single root canal. The teeth were soaked in 5.25% NaOCl for 30 minutes to remove residual loose tissue and debris from the root surface. The crowns of the teeth were reduced to the cemento-enamel junction. An access opening was prepared and the pulp was removed with a barbed broach.
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JOE — Volume 33, Number 1, January 2007
Basic Research—Technology 26 bilaterally matched extracted human maxillary anterior teeth
Negative Control -----3 pairs of bilaterally matched teeth stored in BHI and examined for turbidity throughout experimental period
Teeth reduced to CEJ, pulp contents removed & sterilized
23 pairs of bilaterally matched teeth cultured with E. faecalis for 4 weeks
Group A (Experimental) – 20 teeth (one of each paired teeth) cleaned, shaped and irrigated with 5.25% NaOCl, followed by 15% EDTA and 5.25% NaOCl final rinse
Group B (Experimental) – 20 teeth (one of each paired teeth) cleaned, shaped and irrigated with 1.3% NaOCl, followed by MTAD soak and rinse
1st Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological sampling
1st Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological sampling
Group C (Positive Control) ----3 pairs of bilaterally matched teeth cleaned, shaped and irrigated with saline
1St Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological Sampling
Figure 1. Flowchart of the methodology.
A customized model was fabricated for each tooth that allowed handling of the tooth during the instrumentation sequence of the experiment. One end of a 2-in segment of surgical tubing was injected with polyvinyl-siloxane impression material (Dentsply Caulk, Milford, DE) to a depth of 1 in. The root of each tooth was inserted into the tubing to the coronal one-third of the root before setting of the impression material so that the impression material molded around the root. Each tooth, along with a 20-ml scintillation vial (Wheaton, Millville, NJ) and its corresponding customized tooth model were steam autoclaved for 30 minutes under 15 psi pressure at 121°C. Each tooth was then placed in a sterilized vial, immersed in sterile brain heart infusion (BHI) broth (Difco, Detroit, MI), and sealed. As a sterility check, the sealed specimens were incubated for 48 hours at 37°C and inspected daily to ensure that the BHI broth showed no signs of turbidity. Three pairs of bilaterally matched teeth stored in sterile BHI broth served as negative controls and were examined throughout the experimental period to ascertain the effectiveness of the sterilization procedures. From this stage forward, all samples were processed using strict aseptic protocols. Figure 1 illustrates the methodology used. A 24-hour pure culture of Enterococcus faecalis (ATCC 19433) verified by polymerase chain reaction was grown in BHI broth. Five milliliters of this culture was added to the 20-ml vial containing BHI broth and the sterilized tooth. The teeth were incubated with E. faecalis for 4 weeks under aerobic conditions at 37°C. Fresh media was added every seventh day. Random sampling and Gram staining were done to confirm the viability and purity of the E. faecalis culture.
JOE — Volume 33, Number 1, January 2007
After 4 weeks the teeth were removed from the broth, and the working length was established for each tooth by using a #10 K-file to penetrate the apical foramen and then pulling back to its minor diameter as visualized with a surgical operating microscope (Global Surgical Corp., St. Louis, MO). With the #10 K-file inserted at the working length, the apex of each tooth was sealed with cyanoacrylate. Each tooth was then inserted into its customized model, and the interface between the outer tooth surface and impression material was sealed with cyanoacrylate. Twenty pairs of matching teeth were randomly assigned into the experimental groups, that is, one tooth to group A and the other to group B. Three pairs of matching teeth were placed into the positive control group (group C). The root canals were then instrumented in the following manner. Coronal flaring was accomplished using #1 to #3 Gates Glidden drills (Dentsply Tulsa Dental, Tulsa, OK). The canals were prepared to working length in a crown-down manner using Profile 0.04 Series 29 rotary files (Dentsply Tulsa Dental) to an apical size #6 Series 29 (tip diameter 0.360 mm). In group A, 1 ml of 5.25% NaOCl delivered through a 30-gauge ProRinse needle (Dentsply Tulsa Dental) was used after each file. After instrumentation, the prepared canals received a final irrigation sequence of 5 ml of 15% tetrasodium EDTA, followed by 5 ml of 5.25% NaOCl. The total irrigation time was 2 minutes. The canals were then dried with sterile paper points. In group B, 1 ml of 1.3% NaOCl delivered through a 30-gauge ProRinse needle was used after each file. The canal was then filled using a syringe containing 5 ml of BioPure MTAD (Dentsply Tulsa Dental). With the needle passively placed 1 to 2 mm
EDTA for Root Canal Irrigation
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Basic Research—Technology from working length, 1 ml of BioPure MTAD solution was slowly injected using an up and down motion. The BioPure MTAD was left in the canal for 5 minutes, after which the canal was flushed with the remaining BioPure MTAD. Preparation of BioPure MTAD and its use followed manufacturer’s recommendations. In group C (positive control), 1 ml of saline delivered through a 30-gauge ProRinse needle was used after each instrument size. Both teeth within each matched pair were instrumented to an identical master apical file size with the same amount of total irrigation volume during instrumentation. After treatment, a standardized procedure was used for all bacterial samples (positive controls, group A and group B). The root canal of the each tooth was dried with sterile paper points and filled with reduced transport fluid (27). A #15 file was placed into the canal to within 1 mm of working length and the canal was circumferentially filed for 10 seconds. Three consecutive sterile coarse paper points were introduced into the canal to absorb the reduced transport fluid. The paper points were transferred to a test tube containing 1.0 ml of reduced transport fluid. After collection of the first bacterial sample, the canal was refilled with reduced transport fluid. Profile 0.04 Series 29 rotary files size #7 (tip diameter 0.465) and #8 (tip diameter 0.600) were then used to instrument the canal to two sizes larger than the original master apical file. Dentin chip shavings collected on the rotary files were transferred to a second test tube containing 1.0 ml of reduced transport fluid. A #15 K-file was then placed into the canal to within 1 mm of working length and the canal was circumferentially filed for 10 seconds. The canal contents were sampled with paper points as described earlier and placed into the test tube containing the dentin chip shavings and reduced transport fluid. All collected samples were vortexed for 10 seconds and 10-fold dilutions were prepared. Aliquots of 0.1-ml suspensions were plated onto BHI agar plates and incubated at 37°C for 48 hours. Colony-forming units (CFUs) per 1 ml were enumerated. The Wilcoxon Signed Rank Test with significance set at p ⬍ 0.01 was used to analyze differences in data obtained.
Results All samples in the negative control showed the absence of turbidity throughout the experimental period, whereas all samples in the positive control showed the presence of bacterial growth on BHI plates. The first bacterial samples from the experimental groups showed a significant difference (p ⬍ 0.01) between the total number of CFUs of E. faecalis. The teeth treated with 5.25% NaOCl/15% EDTA produced 0 CFU/ml, while those treated with 1.3% NaOCl/BioPure MTAD produced a mean of 19 ⫾ 15 CFU/ml. None of the 20 samples in group A showed any growth, while 8 of 20 samples in group B yielded growth. The second bacterial samples, taken after additional instrumentation, showed a significant difference (p ⬍ 0.01) between the total number of CFUs of E. faecalis. The teeth treated with 5.25% NaOCl/ 15% EDTA produced 0 CFU/ml, while those treated with 1.3% NaOCl/ BioPure MTAD produced a mean of 293 ⫾ 306 CFU/ml. None of the 20 samples in group A showed any growth, while 10 of 20 samples in group B yielded growth. Statistical analysis of the data using the Wilcoxon Signed Rank test showed significant differences in the ability of 5.25% NaOCl/ 15% EDTA to disinfect the canals compared with 1.3% NaOCl/BioPure MTAD (z ⫽ 2.67, p ⬍ 0.01). Significant differences in the total number of CFUs were also seen within the 1.3% NaOCl/BioPure MTAD treated groups when comparing instrumentation to the original master file size versus additional canal enlargement (z ⫽ 2.75, p ⬍ 0.01). Figure 2 shows an example of the CFUs obtained by the experimental groups. 50
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Figure 2. Colony forming units obtained by the experimental groups.
Discussion The intent of this study was to determine whether irrigation with 1.3% NaOCl/BioPure MTAD was more effective than 5.25% NaOCl/15% EDTA in eradicating E. faecalis from root canals of bilaterally matched extracted human teeth cultured with this microbe. The use of bilaterally matched teeth pairs provided a way to allow direct comparison of the antibacterial activities of these irrigants. E. faecalis was chosen as the test organism because it has been associated with persistent apical inflammation in clinical situations (4, 5). It was also chosen because recent studies claimed that MTAD is an effective final irrigating solution for eradication of E. faecalis (22, 24). Furthermore, E. faecalis has been found suitable for experimental penetration into dentinal tubules, and 3 weeks of incubation with E. faecalis has been shown to produce a dense infection of the dentinal tubules (28). In the present study, the teeth were incubated with E. faecalis for 4 weeks to ensure adequate penetration of the bacteria into the dentinal tubules. The lack of turbidity of the BHI by the negative control group demonstrated that the sterilization procedures utilized were effective. The results from the samples in the positive control group confirm the presence of E. faecalis within the root canal system. Two microbial samples were collected from each tooth. The apices of all sampled teeth were sealed with cyanoacrylate to prevent any contamination from the outer tooth surface during the sampling procedure. The first sample was collected to determine the effectiveness of disinfection by the test irrigants when teeth were prepared to a size #6 Profile 0.04 Series 29, which has a tip diameter of 0.360 mm. The results of this study showed that 5.25% NaOCl/15% EDTA significantly reduced intracanal bacteria levels compared with the use of 1.3% NaOCl/BioPure MTAD. None of the 20 samples in group A showed any growth, while 8 of 20 samples in group B showed growth.
JOE — Volume 33, Number 1, January 2007
Basic Research—Technology The second sample was collected after additional instrumentation of the canal space to a size #8 Profile 0.04 Series 29, which has a tip diameter of 0.600 mm. It has been shown that invasion of dentinal tubules by E. faecalis usually occurs to a depth of 50 to 100 m (29). Culturing of the dentin chip shavings and canal contents at a greater depth allowed determination of the efficacy to the test irrigants at penetrating and disinfecting deeper layers of dentin. The results of this study showed that 5.25% NaOCl/15% EDTA significantly reduced intracanal bacteria levels compared with the use of 1.3% NaOCl/BioPure MTAD. None of the 20 samples in group A showed any growth, whereas 10 of 20 samples in group B showed growth. The methodology used in the present study was developed to simulate a clinical setting and was based on the methodology used by Shabahang and Torabinejad (25) to test the efficacy of BioPure MTAD on E. faecalis. The effectiveness of 1.3% NaOCl/BioPure MTAD to consistently disinfect root canals in this study disagrees with the results of their investigation. Differences between the results may be attributed to the methodology and microbial sampling procedures utilized. The present study used techniques to sample the canal contents immediately after debridement, while Shabahang and Torabinejad (25) assessed for turbidity in a growth media after a 1-week incubation period. Furthermore, they soaked the entire tooth in the test irrigants for 5 minutes after debridement. This part of the methodology was excluded in this study to more closely simulate a clinical setting. In conclusion, the results of this in vitro investigation showed consistent disinfection of infected root canals with 5.25% NaOCl/15% EDTA. The combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the canals contaminated with E. faecalis.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340 – 8. 2. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg 1994;78:522–30. 3. 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;30:297–306. 4. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endondontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93. 5. Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7. 6. Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endondontic treatment in a North Am population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579 – 86. 7. Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321– 8.
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8. Siqueira JF, Lima KC, Magalhaes FAC, Lopes HP, de Uzeda M. Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques. J Endod 1999;25:332–5. 9. Byström A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5% sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55:307–12. 10. Byström A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18:35– 40. 11. Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antibacterial endodontic irrigants. J Endod 1994;20: 276 – 80. 12. Buck RA, Eleazer PD, Staat RH, Scheetz JP. Effectiveness of three endodontic irrigants at various tubular depths in human dentin. J Endod 2001;27:206 – 8. 13. Gomes BPFA, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424 – 8. 14. Siqueira JF, Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro. Int Endod J 1997;30:279 – 82. 15. Siqueira JF, Rocas IN, Faviera A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1.0, 2.5, and 5.25% sodium hypochlorite. J Endod 2000;26:331– 4. 16. Radcliffe CE, Potouridou L, Qureshi R, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A naeslundii, Candida albicans, and Enterococcus faecalis. Int Endod J 2004;37:438 – 46. 17. Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic investigation of the smeared layer on the root canal walls. J Endod 1984;10:477– 83. 18. Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:142–9. 19. Yamada RS, Armas A, Goldman M, Peck SL. A scanning electron microscopic comparison of a high volume flush with several irrigating solutions: part 3. J Endod 1983;9:137– 42. 20. O’Connell MS, Morgan LA, Beeler WJ, Baumgartner JC. A comparative study of smear layer removal using different salts of EDTA. J Endod 2000;26:739 – 43. 21. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 22. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233–9. 23. Torabinejad M, Shabahang S, Aprecio R, Ketting JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400 –3. 24. Shabahang S, Pouresmail M, Torabinejad M. In vitro antimicrobial effect of MTAD and NaOCl. J Endod 2003;29:450 –2. 25. Shabahang S, Torabinejad M. Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth. J Endod 2003;29:576 –9. 26. Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against E. faecalis biofilm. J Endod 2005;31:218. (abstract) 27. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbiol 1972;24:638 – 44. 28. Haapasalo M, Ørstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375–9. 29. Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J Endod 1990;16:207–10.
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Comparison of the Antimicrobial Efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for Root Canal Irrigation J. Craig Baumgartner, Stephen Johal, DMD, and J. Gordon Marshall, DMD Abstract The purpose of this study was to compare the antimicrobial efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. Twenty-six bilaterally matched pairs of human teeth were collected. The teeth were incubated with Enterococcus faecalis for 4 weeks. The teeth were divided into two experimental groups and one positive control group. The canals were instrumented and irrigated with either 5.25% NaOCl/15% EDTA or 1.3% NaOCl/BioPure MTAD. Bacterial samples were collected after instrumentation/irrigation and after additional canal enlargement. Statistical analysis of the data using the Wilcoxon Signed Rank test showed significant differences between the experimental groups. The first bacterial samples revealed growth in 0 of 20 samples with 5.25% NaOCl/15% EDTA irrigation and in 8 of 20 samples with 1.3% NaOCl/BioPure MTAD irrigation. Samples taken after additional canal enlargement revealed growth in 0 of 20 samples in 5.25% NaOCl/15% EDTA and in 10 of 20 samples in 1.3% NaOCl/BioPure MTAD group. This investigation showed consistent disinfection of infected root canals with 5.25% NaOCl/15% EDTA. The combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the canals contaminated with E. faecalis. (J Endod 2007;33:48 –51)
Key Words MTAD, root canal irrigation, sodium hypochlorite
From the Department of Endodontology, OHSU School of Dentistry, Portland, Oregon. Address requests for reprints to Dr. J. Craig Baumgartner, Department of Endodontology, OHSU School of Dentistry, 611 SW Campus Drive, Portland, OR 97201. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.08.007
O
ne of the most important objectives of endodontic therapy is the complete elimination of microorganisms from the root canal system. The positive correlation between bacteria and endodontic disease has been established (1, 2). Failure of root canal treatment is likely caused by the inability to eliminate the bacteria responsible for refractory endodontic infections (3). Facultative bacteria such as Enterococcus faecalis have been isolated from infected root canals and may be related to failure of endodontic treatment (4 – 6). Although chemomechanical preparation of the root canal system is able to reduce the number of bacteria, complete canal disinfection is difficult because of the internal complexity of root canal systems (7, 8). Thus a root canal irrigant, such as sodium hypochlorite (NaOCl), is needed to aid in the disinfection of the canals (9 –16). NaOCl effectively eliminates microbes, and its antibacterial effectiveness may be a function of concentration and contact time (13, 15, 16). Endodontic instrumentation produces a smear layer, which is a 1- to 2-m-thick amorphous, granular structure of calcified tissue mixed with fragments of odontoblastic processes, microorganisms, and necrotic materials (17). Because of its potential contamination and its ability to inhibit or delay penetration of antimicrobial agents, smear layer removal has been advocated (10, 18). A final flush with ethylenediaminetetraacetic acid (EDTA) followed by NaOCl is an effective method for removing the smear layer and superficial debris from the surfaces of instrumented root canals (19). Solutions of 15% disodium, 15% tetrasodium, or 25% tetrasodium EDTA tested in combination with NaOCl are equally effective at removing the smear layer (20). A new irrigating solution, MTAD, containing a mixture of a tetracycline isomer, an acid, and a detergent, has shown promising results (21–25). Recent investigations involving the use of MTAD have demonstrated its ability to remove the smear layer, disinfect contaminated root canals, and eradicate E. faecalis (21, 24, 25). It was initially thought that MTAD could be used as an irrigant to replace NaOCl, but it was found that the cleanest canals were obtained if NaOCl was used before a final soak and flush with MTAD (22). MTAD’s ability to remove the smear layer is not dependent on the concentration of NaOCl used as root canal irrigant (22). Therefore, MTAD is recommended as a final soak and flush of canals after the use of 1.3% NaOCl (22, 23). 1.3% NaOCl/MTAD irrigation has been shown to be significantly more effective against E. faecalis than NaOCl with and without EDTA (25). However, it has also been reported that BioPure MTAD (Dentsply, Tulsa, OK) may not be effective against E. faecalis biofilms (26). The purpose of this investigation was to compare the antimicrobial efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation.
Materials and Methods The experimental protocol was deemed exempt by the Oregon Health Science University Institutional Review Board. Twenty-six bilaterally matched pairs of extracted mature maxillary anterior human teeth were collected. Each tooth was radiographed to confirm the presence of a single root canal. The teeth were soaked in 5.25% NaOCl for 30 minutes to remove residual loose tissue and debris from the root surface. The crowns of the teeth were reduced to the cemento-enamel junction. An access opening was prepared and the pulp was removed with a barbed broach.
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JOE — Volume 33, Number 1, January 2007
Basic Research—Technology 26 bilaterally matched extracted human maxillary anterior teeth
Negative Control -----3 pairs of bilaterally matched teeth stored in BHI and examined for turbidity throughout experimental period
Teeth reduced to CEJ, pulp contents removed & sterilized
23 pairs of bilaterally matched teeth cultured with E. faecalis for 4 weeks
Group A (Experimental) – 20 teeth (one of each paired teeth) cleaned, shaped and irrigated with 5.25% NaOCl, followed by 15% EDTA and 5.25% NaOCl final rinse
Group B (Experimental) – 20 teeth (one of each paired teeth) cleaned, shaped and irrigated with 1.3% NaOCl, followed by MTAD soak and rinse
1st Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological sampling
1st Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological sampling
Group C (Positive Control) ----3 pairs of bilaterally matched teeth cleaned, shaped and irrigated with saline
1St Microbiological sampling
Canals enlarged 2 sizes larger than MAF
2nd Microbiological Sampling
Figure 1. Flowchart of the methodology.
A customized model was fabricated for each tooth that allowed handling of the tooth during the instrumentation sequence of the experiment. One end of a 2-in segment of surgical tubing was injected with polyvinyl-siloxane impression material (Dentsply Caulk, Milford, DE) to a depth of 1 in. The root of each tooth was inserted into the tubing to the coronal one-third of the root before setting of the impression material so that the impression material molded around the root. Each tooth, along with a 20-ml scintillation vial (Wheaton, Millville, NJ) and its corresponding customized tooth model were steam autoclaved for 30 minutes under 15 psi pressure at 121°C. Each tooth was then placed in a sterilized vial, immersed in sterile brain heart infusion (BHI) broth (Difco, Detroit, MI), and sealed. As a sterility check, the sealed specimens were incubated for 48 hours at 37°C and inspected daily to ensure that the BHI broth showed no signs of turbidity. Three pairs of bilaterally matched teeth stored in sterile BHI broth served as negative controls and were examined throughout the experimental period to ascertain the effectiveness of the sterilization procedures. From this stage forward, all samples were processed using strict aseptic protocols. Figure 1 illustrates the methodology used. A 24-hour pure culture of Enterococcus faecalis (ATCC 19433) verified by polymerase chain reaction was grown in BHI broth. Five milliliters of this culture was added to the 20-ml vial containing BHI broth and the sterilized tooth. The teeth were incubated with E. faecalis for 4 weeks under aerobic conditions at 37°C. Fresh media was added every seventh day. Random sampling and Gram staining were done to confirm the viability and purity of the E. faecalis culture.
JOE — Volume 33, Number 1, January 2007
After 4 weeks the teeth were removed from the broth, and the working length was established for each tooth by using a #10 K-file to penetrate the apical foramen and then pulling back to its minor diameter as visualized with a surgical operating microscope (Global Surgical Corp., St. Louis, MO). With the #10 K-file inserted at the working length, the apex of each tooth was sealed with cyanoacrylate. Each tooth was then inserted into its customized model, and the interface between the outer tooth surface and impression material was sealed with cyanoacrylate. Twenty pairs of matching teeth were randomly assigned into the experimental groups, that is, one tooth to group A and the other to group B. Three pairs of matching teeth were placed into the positive control group (group C). The root canals were then instrumented in the following manner. Coronal flaring was accomplished using #1 to #3 Gates Glidden drills (Dentsply Tulsa Dental, Tulsa, OK). The canals were prepared to working length in a crown-down manner using Profile 0.04 Series 29 rotary files (Dentsply Tulsa Dental) to an apical size #6 Series 29 (tip diameter 0.360 mm). In group A, 1 ml of 5.25% NaOCl delivered through a 30-gauge ProRinse needle (Dentsply Tulsa Dental) was used after each file. After instrumentation, the prepared canals received a final irrigation sequence of 5 ml of 15% tetrasodium EDTA, followed by 5 ml of 5.25% NaOCl. The total irrigation time was 2 minutes. The canals were then dried with sterile paper points. In group B, 1 ml of 1.3% NaOCl delivered through a 30-gauge ProRinse needle was used after each file. The canal was then filled using a syringe containing 5 ml of BioPure MTAD (Dentsply Tulsa Dental). With the needle passively placed 1 to 2 mm
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Basic Research—Technology from working length, 1 ml of BioPure MTAD solution was slowly injected using an up and down motion. The BioPure MTAD was left in the canal for 5 minutes, after which the canal was flushed with the remaining BioPure MTAD. Preparation of BioPure MTAD and its use followed manufacturer’s recommendations. In group C (positive control), 1 ml of saline delivered through a 30-gauge ProRinse needle was used after each instrument size. Both teeth within each matched pair were instrumented to an identical master apical file size with the same amount of total irrigation volume during instrumentation. After treatment, a standardized procedure was used for all bacterial samples (positive controls, group A and group B). The root canal of the each tooth was dried with sterile paper points and filled with reduced transport fluid (27). A #15 file was placed into the canal to within 1 mm of working length and the canal was circumferentially filed for 10 seconds. Three consecutive sterile coarse paper points were introduced into the canal to absorb the reduced transport fluid. The paper points were transferred to a test tube containing 1.0 ml of reduced transport fluid. After collection of the first bacterial sample, the canal was refilled with reduced transport fluid. Profile 0.04 Series 29 rotary files size #7 (tip diameter 0.465) and #8 (tip diameter 0.600) were then used to instrument the canal to two sizes larger than the original master apical file. Dentin chip shavings collected on the rotary files were transferred to a second test tube containing 1.0 ml of reduced transport fluid. A #15 K-file was then placed into the canal to within 1 mm of working length and the canal was circumferentially filed for 10 seconds. The canal contents were sampled with paper points as described earlier and placed into the test tube containing the dentin chip shavings and reduced transport fluid. All collected samples were vortexed for 10 seconds and 10-fold dilutions were prepared. Aliquots of 0.1-ml suspensions were plated onto BHI agar plates and incubated at 37°C for 48 hours. Colony-forming units (CFUs) per 1 ml were enumerated. The Wilcoxon Signed Rank Test with significance set at p ⬍ 0.01 was used to analyze differences in data obtained.
Results All samples in the negative control showed the absence of turbidity throughout the experimental period, whereas all samples in the positive control showed the presence of bacterial growth on BHI plates. The first bacterial samples from the experimental groups showed a significant difference (p ⬍ 0.01) between the total number of CFUs of E. faecalis. The teeth treated with 5.25% NaOCl/15% EDTA produced 0 CFU/ml, while those treated with 1.3% NaOCl/BioPure MTAD produced a mean of 19 ⫾ 15 CFU/ml. None of the 20 samples in group A showed any growth, while 8 of 20 samples in group B yielded growth. The second bacterial samples, taken after additional instrumentation, showed a significant difference (p ⬍ 0.01) between the total number of CFUs of E. faecalis. The teeth treated with 5.25% NaOCl/ 15% EDTA produced 0 CFU/ml, while those treated with 1.3% NaOCl/ BioPure MTAD produced a mean of 293 ⫾ 306 CFU/ml. None of the 20 samples in group A showed any growth, while 10 of 20 samples in group B yielded growth. Statistical analysis of the data using the Wilcoxon Signed Rank test showed significant differences in the ability of 5.25% NaOCl/ 15% EDTA to disinfect the canals compared with 1.3% NaOCl/BioPure MTAD (z ⫽ 2.67, p ⬍ 0.01). Significant differences in the total number of CFUs were also seen within the 1.3% NaOCl/BioPure MTAD treated groups when comparing instrumentation to the original master file size versus additional canal enlargement (z ⫽ 2.75, p ⬍ 0.01). Figure 2 shows an example of the CFUs obtained by the experimental groups. 50
Baumgartner et al.
Figure 2. Colony forming units obtained by the experimental groups.
Discussion The intent of this study was to determine whether irrigation with 1.3% NaOCl/BioPure MTAD was more effective than 5.25% NaOCl/15% EDTA in eradicating E. faecalis from root canals of bilaterally matched extracted human teeth cultured with this microbe. The use of bilaterally matched teeth pairs provided a way to allow direct comparison of the antibacterial activities of these irrigants. E. faecalis was chosen as the test organism because it has been associated with persistent apical inflammation in clinical situations (4, 5). It was also chosen because recent studies claimed that MTAD is an effective final irrigating solution for eradication of E. faecalis (22, 24). Furthermore, E. faecalis has been found suitable for experimental penetration into dentinal tubules, and 3 weeks of incubation with E. faecalis has been shown to produce a dense infection of the dentinal tubules (28). In the present study, the teeth were incubated with E. faecalis for 4 weeks to ensure adequate penetration of the bacteria into the dentinal tubules. The lack of turbidity of the BHI by the negative control group demonstrated that the sterilization procedures utilized were effective. The results from the samples in the positive control group confirm the presence of E. faecalis within the root canal system. Two microbial samples were collected from each tooth. The apices of all sampled teeth were sealed with cyanoacrylate to prevent any contamination from the outer tooth surface during the sampling procedure. The first sample was collected to determine the effectiveness of disinfection by the test irrigants when teeth were prepared to a size #6 Profile 0.04 Series 29, which has a tip diameter of 0.360 mm. The results of this study showed that 5.25% NaOCl/15% EDTA significantly reduced intracanal bacteria levels compared with the use of 1.3% NaOCl/BioPure MTAD. None of the 20 samples in group A showed any growth, while 8 of 20 samples in group B showed growth.
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Basic Research—Technology The second sample was collected after additional instrumentation of the canal space to a size #8 Profile 0.04 Series 29, which has a tip diameter of 0.600 mm. It has been shown that invasion of dentinal tubules by E. faecalis usually occurs to a depth of 50 to 100 m (29). Culturing of the dentin chip shavings and canal contents at a greater depth allowed determination of the efficacy to the test irrigants at penetrating and disinfecting deeper layers of dentin. The results of this study showed that 5.25% NaOCl/15% EDTA significantly reduced intracanal bacteria levels compared with the use of 1.3% NaOCl/BioPure MTAD. None of the 20 samples in group A showed any growth, whereas 10 of 20 samples in group B showed growth. The methodology used in the present study was developed to simulate a clinical setting and was based on the methodology used by Shabahang and Torabinejad (25) to test the efficacy of BioPure MTAD on E. faecalis. The effectiveness of 1.3% NaOCl/BioPure MTAD to consistently disinfect root canals in this study disagrees with the results of their investigation. Differences between the results may be attributed to the methodology and microbial sampling procedures utilized. The present study used techniques to sample the canal contents immediately after debridement, while Shabahang and Torabinejad (25) assessed for turbidity in a growth media after a 1-week incubation period. Furthermore, they soaked the entire tooth in the test irrigants for 5 minutes after debridement. This part of the methodology was excluded in this study to more closely simulate a clinical setting. In conclusion, the results of this in vitro investigation showed consistent disinfection of infected root canals with 5.25% NaOCl/15% EDTA. The combination of 1.3% NaOCl/BioPure MTAD left nearly 50% of the canals contaminated with E. faecalis.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340 – 8. 2. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg 1994;78:522–30. 3. 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;30:297–306. 4. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endondontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93. 5. Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7. 6. Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endondontic treatment in a North Am population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579 – 86. 7. Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321– 8.
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8. Siqueira JF, Lima KC, Magalhaes FAC, Lopes HP, de Uzeda M. Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques. J Endod 1999;25:332–5. 9. Byström A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5% sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55:307–12. 10. Byström A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18:35– 40. 11. Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antibacterial endodontic irrigants. J Endod 1994;20: 276 – 80. 12. Buck RA, Eleazer PD, Staat RH, Scheetz JP. Effectiveness of three endodontic irrigants at various tubular depths in human dentin. J Endod 2001;27:206 – 8. 13. Gomes BPFA, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424 – 8. 14. Siqueira JF, Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro. Int Endod J 1997;30:279 – 82. 15. Siqueira JF, Rocas IN, Faviera A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1.0, 2.5, and 5.25% sodium hypochlorite. J Endod 2000;26:331– 4. 16. Radcliffe CE, Potouridou L, Qureshi R, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A naeslundii, Candida albicans, and Enterococcus faecalis. Int Endod J 2004;37:438 – 46. 17. Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic investigation of the smeared layer on the root canal walls. J Endod 1984;10:477– 83. 18. Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:142–9. 19. Yamada RS, Armas A, Goldman M, Peck SL. A scanning electron microscopic comparison of a high volume flush with several irrigating solutions: part 3. J Endod 1983;9:137– 42. 20. O’Connell MS, Morgan LA, Beeler WJ, Baumgartner JC. A comparative study of smear layer removal using different salts of EDTA. J Endod 2000;26:739 – 43. 21. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 22. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233–9. 23. Torabinejad M, Shabahang S, Aprecio R, Ketting JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400 –3. 24. Shabahang S, Pouresmail M, Torabinejad M. In vitro antimicrobial effect of MTAD and NaOCl. J Endod 2003;29:450 –2. 25. Shabahang S, Torabinejad M. Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth. J Endod 2003;29:576 –9. 26. Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against E. faecalis biofilm. J Endod 2005;31:218. (abstract) 27. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbiol 1972;24:638 – 44. 28. Haapasalo M, Ørstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375–9. 29. Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J Endod 1990;16:207–10.
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