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The Antimicrobial Effect of MTAD: An In Vitro Investigation
Printed in U.S.A. VOL. 29, No. 6,JUNE2003
JOURNAL OF ENKIDONTICS Copyright 0 2003 by The American Association of Endodontists
The Antimicrobial Effect of MTAD: An In Vitro Investigation Mahmoud Torabinejad, DMD, MSD, PhD, Shahrokh Shabahang, DDS, MS, PhD, Raydolfo M. Aprecio, OD, and James D. Kettering, PhD
Pulp and periradiculardiseases are of microbial origin. To effectively clean the root canal system a disinfecting agent must be able to penetrate into difficult-to-reach areas and kill microorganismswith minimal damage to the host tissues. The purpose of this investigationwas to test the a b i l i of a mixture of a tetracycline isomer, an acid, and a detergent (MTAD) to kill Entemwcxus faecalis and compare its efficacy to that of sodium hypochlorite (NaOCI) and ethylene diamine tetraacetic acid (EDTA). The zones of inhibition and minimum inhibitory concentdons were measured for these solutions.Measurement of zones of inhibition and determination of the minimum inhibitory concentrations showed that MTAD is as effective as 5.25% NaOCl and significantly more effective tJ-ian EDTA (p c 0.OOOl). Furthermore, MTAD is significantly more effective in killing E. faecalis than NaOCl when the solutions are diluted (p c 0.OOOl). Measurement of the minimum inhibitory concentrations demonstrated that atthough MTAD is still effective in killing €. faecalis at 2OOx dilution, NaOCl ceases to exert its antibacterial activity beyond32x dilution. EDTA did not exhibit any antibacterial activity. Based on the results of this study, it seems that MTAD is an effective solution in eradicating E. faecalis.
The main objectives of root canal therapy are cleaning and shaping, obturating the root canal system in three dimensions, and preventing reinfection. Investigations in animal studies have demonstrated the importance of microorganisms in the pathogenesis of pulpal and periradicular pathosis (1, 2). To determine the influence of infection at the time of root canal therapy on the treatment outcome of patients, Sjogren et al. (3) compared the success rate of teeth with negative cultures at the time of obturation to those that had positive bacterial cultures before the same procedure. They reported a significantly higher success rate when teeth were free of bacteria (94% versus 68%). The results are consonant with the findings of Bystrom et al. (4)who also showed improved clinical success rates after effective disinfection of root canals.
Pataky et al. (5) infected the root canals of extracted teeth with Enterococcusfueculis and examined the number of remaining bacteria after preparation with various techniques using sterile saline as an intracanal imgant. Their results show a reduction in bacterial population after mechanical preparation. They found no statistical differences among the efficacy of the test techniques or files used in the study. According to Bystrom and Sundqvist (6), current regimens in chemomechanical debridement using instrumentation and irrigation with sodium hypochlorite (NaOCl) are not predictably effective in root canal disinfection. These findings are not surprising because the root canal morphology is complex and contains numerous ramifications and anatomical irregularities (7). The microorganisms in root canals not only invade the anatomic irregularities of the root canal system but also are present in the dentinal tubules (8). Because of the limitations of current root canal cleaning techniques, some investigators recommend placement of antimicrobial dressings such as calcium hydroxide in the root canals before obturation (4,9).However, when treatment is completed in one visit, no intracanal medications other than intracanal imgants are used. The efficacy of intracanal inigants and calcium hydroxide against certain bacterial species such as E. fueculis has been questioned (10). An ideal intracanal inigant or medication should be able to disinfect the dentin and its tubules in one visit. In addition, it should have sustained antimicrobialeffect after use. FuI.thermore, it must be biccompatible with live host tissues (8). Studies have shown that a mixture of a tetracycline A D [A patent application has been isomer, an acid, and a detergent W fded for this technology]) as a final rinse is capable of removing the smear layer with minimal erosive changes on the surface of dentin (1 1, 12). In another study Beltz et al. (13) investigated the amount of tissue loss after exposing the bovine pulp and dentin to various concentrations of NaOCI, EDTA, or MTAD. Their results showed that various concentrations of NaOCl removed organic components of pulp and dentin effectively. The solubilizing effects of EDTA on pulp and dentin were somewhat similar to those of MTAD. The major difference between the actions of these solutions is a high binding affiity of doxycycline present in MTAD for the dentin. The purpose of this investigation was to compare the antimicrobial properties of MTAD with those of NaOCl and EDTA.
MATERIALS AND METHODS Doxycycline, citric acid, and Tween-80 were all purchased from Sigma-Aldrich Company, St. Louis, MO. TWOmethods were used
Vol. 29, No. 6, June 2003
Antimicrobial Effects
401
TABLE 1. Zones of inhibition (mm) and standard deviations for NaOCI, MTAD, and EDTA Sample
Undiluted
1:5
1:lO
NaOCl MTAD EDTA
34.3755 1.19 34.75 2 0.71 16.88 ? 0.64
15.752 0.71 30.15 5 1.89
11.252 0.71 29.63 -+ 3.81 0
to determine the extent of antimicrobial activity of MTAD and to compare it with those of NaOCl and EDTA. In the first test we measured the zone of inhibition on plates inoculated with E. faecalis. An overnight culture of E. ,faecalis (ATCC 4082) was standardized to 0.1 1 optical density (OD) measured at 570 nm. One hundred microliters of the microorganism was spread onto a trypticase soy agar (TSA) plate with the use of a sterile L-shaped glass rod. One-quarter-inch sterile S & S filter paper (Schleicher & Schuell) was placed in each of the four quadrants of the TSA plate. Twenty microliters of sterile saline (B. Braun Medical, Inc., Irvine, CA), 5.25% NaOCl (All Purpose Bleach, Mission Laboratories, Los Angeles, CA), MTAD (freshly prepared before each use according to a proprietary protocol at Loma Linda University), or 17% EDTA (Roth International, Chicago, IL) were added onto the filter papers. Eight replicates were prepared for each of the sample solutions. The plates were incubated overnight at 37°C for 24 h, and the zones of inhibition were measured in millimeters. In the second experiment we used the minimum inhibitory concentration (MIC) method. E. faecalis was cultured overnight and the concentration was adjusted to 0.1 1 OD at 570 nm. The test solutions were serially diluted from 1:2 up to 1:2048 dilutions. One milliliter of 2X trypticase soy broth (TSB) and the same amount of test solutions were mixed in various tubes. One hundred microliters of the standardized E. faecalis was added into each of the test tubes and incubated overnight at 37°C. The presence or absence of turbidity was determined the next day. To determine whether the test solutions had inhibitory or bactericidal effect, a high concentration of bacteria (1 X 10' colonyforming units of E. faecalis) was exposed to 2 ml of undiluted and 1:2 dilution of MTAD or NaOCl for 2 or 5 min. Samples were plated on TSA media to determine viability of any remaining bacteria.
RESULTS No zone of inhibition was observed adjacent to the filter papers saturated with sterile saline. The means and standard deviations of the zones of inhibition for the other test solutions are presented in Table 1. Figure 1 shows the comparison of the average areas of inhibition for the three test solutions. When the undiluted solutions were tested, the average zones of inhibition for 5.25% NaOCl and MTAD were measured at 34 and 35 mm, respectively. The zone of inhibition of undiluted EDTA was 17 m. Statistical analysis using Kruskal-Wallis ranks test and Mann-Whitney U test (a = 0.05) showed no statistical difference between 5.25% NaOCl and MTAD (p = 0.442). However, the zones of inhibition for the two solutions were significantly larger than those seen around the filter papers saturated with undiluted EDTA (p < 0.0001 for both). Dilution of the solutions reduced the zones of inhibition for all solutions, albeit at different rates. At 5- and 10-fold dilution, MTAD created a zone of inhibition that was significantly larger in diameter than that seen with NaOCl (p < 0.0001). EDTA demonstrated no bactericidal properties when diluted 1:5 or 1:10. Further analysis of the effects of dilution on the bactericidal properties of
0
FIG 1. The differences between the averages of zones of inhibition between NaOCI, MTAD, and EDTA measured at three dilutions.
the solutions indicated that undiluted NaOCl is significantly more effective than 5X and 1OX diluted NaOCl (p < 0.0001). Similar evaluation of the efficacy of MTAD showed that although undiluted MTAD was significantly more effective against E. faecalis than 5X or 1OX diluted MTAD, no significant difference was noted in the diameter of the zones of inhibition of 1:5 dilution or 1:lO dilution (p = 0.083). The results of the minimum inhibitory concentration method are listed in Table 2. Figure 2 shows the comparison of the maximum dilution of each solution with continued antimicrobial activity. Using this technique although EDTA demonstrated no antibacterial effects against E. faecalis, NaOCl and MTAD proved to be antibacterial to varying degrees. NaOCl continued to exert its efficacy up to 32X dilutions. On the other hand, MTAD was effective in killing E. faecalis up to 200X dilutions. Furthermore, although a 2- and 5-min exposure of E. faecalis to undiluted or 1:2 dilution of NaOCl did not result in total killing of the bacteria, exposure of the same bacterial species to undiluted or 1:2 diluted MTAD resulted in a completely negative culture after 2 or 5 min of exposure.
DISCUSSION Despite the presence of a controversy regarding the effect of the smear layer on the quality of instrumentation and obturation, the smear layer itself may be infected and may protect the bacteria already present in the dentinal tubules (8). Because of these concerns, one may deem it prudent to remove the smear layer in infected root canals and allow penetration of intracanal medications into the dentinal tubules in these teeth. To completely disinfect the root canal system, including the dentinal tubules, the disinfecting solution must be able to penetrate all aspects of the root canal system. To date, there is no single solution used in endodontics that simultaneously removes the smear layer and disinfects the entire root canal system. The effectiveness of MTAD in removing the smear layer has been described in previous publications ( 1 1, 12). MTAD is more effective than undiluted or
402
Journal of Endodontics
Torabinejad et al. TABLE 2. MIC dilutions for E. faecalis exposed to NaOCI, MTAD, and EDTA Solution
End Point
Control
NaOCl (Undiluted, 1:2 dilution up to 1:2048, Control) MTAD (Undiluted, 1:2 dilution up to 1:2048, Control) EDTA (Undiluted, 1:2 dilution up to 1:2048, Control)
No growth up to 1:32 dilution No growth up to 1:200 dilution
Growth Growth Growth
250 200 150
100 50
NaOCl
MTAD
EDTA
FIG 2. The maximum dilution of each solution with continued antimicrobial activity.
diluted NaOCl in removing the smear layer from the surface of instrumented root canals (11). The ability of MTAD is enhanced as a final rinse when low concentrations of NaOCl are used as intracanal irrigants (12). Compared with EDTA, MTAD removes the smear layer with significantly less erosion of the dentinal tubules (11, 12). In this investigation, we evaluated the ability of MTAD to kill E. faecalis in an in vitro model without the presence of the variables that are encountered in a tooth model. Standard microbiological methods were used to determine the antimicrobial properties of MTAD in Comparison to NaOCl and EDTA. E. faecalis was selected because this bacterium has been identified as one of the most difficult bacterial species to eradicate from infected root canals (10). The persistence of E. faecalis may be attributed to the ineffectiveness of currently used chemicals such as sodium hypochlorite (14), potassium iodide (13, or calcium hydroxide (16). The results of this study demonstrated the superior efficacy of MTAD in killing E. faecalis compared with the two most commonly used imgants in root canal therapy: NaOCl and EDTA. Two microbiological techniques were used to compare the antimicrobial properties of MTAD to these solutions. Determination of the zones of inhibition provided quantitative data, which allowed us to perform statistical analysis. Our findings corroborated the findings of D’Arcangelo et al. (17) who have shown that the majority of NaOCl dilutions have some degree of antibacterial activity when placed in direct contact with bacteria. Our study also confirmed the findings of Harrison and Hand (18) who have shown that NaOCl is an effective bactericidal agent when it is used undiluted. Another observation in this investigation was the lack of strong antimicrobial activity of EDTA. Dilution of 17% EDTA completely eliminated its efficacy even when placed in direct contact with E. faecalis. Our results are somewhat similar to the findings of Heling and Chandler (19) who showed no antimicrobial effect in undiluted EDTA, similar to that of sterile saline. A disadvantage of determining the antimicrobial effect of a solution by measuring the zone of inhibition is that this technique does not account for the variation in the diffusion rate of the
Growth
medication through the agar. To address this concern, a second technique was used to verify the results of the first experiment. The MIC method uses serial dilutions of a solution to determine the minimum concentration that would still exert antimicrobial properties. In this experiment, the MIC method confirmed that EDTA possesses little or no bactericidal activity. A significant observation was that MTAD continued to have bactericidal properties even when it was diluted 200 times. This finding was in contrast to the bactericidal properties of NaOCI, which ceased to exist beyond 32X dilution. In addition to its decreased antibacterial effect, dilution of NaOCl results in a significant decrease in its ability to dissolve necrotic tissue as demonstrated by Hand et al. (20) and Beltz et al. (13). Although dilution reduced the zone of inhibition for all imgants tested, the results of both microbiological methods demonstrated that MTAD maintains its bactericidal properties significantly more than NaOCl or EDTA. Another significant finding in our study was the capacity of MTAD to kill E. faecalis after an exposure of 2 or 5 min. This ability was not observed with NaOCl or EDTA. This finding strengthens the results of inhibition and dilution tests, showing that MTAD has superior antibacterial effect cornpared with NaOCl or EDTA even in a short contact time. Based on the results of this and previous investigations (1 1-13), it seems that MTAD has the ability to remove most of the smear layer and it possesses superior bactericidal activity compared with NaOCl or EDTA when tested against E. faecalis. These results are significant because they demonstrate the efficacy of an irrigant to remove most of the smear layer and kill a bacterial strain that has been shown to be resistant to many commonly used intracanal imgants and dressings. The antimicrobial activity of MTAD continues to exist even after dilution of the solution. A limitation of this in vitro study is that it does not account for the penetration ability of test irrigants in root canals. Studies are in progress to examine the efficacy of MTAD as a final rinse in disinfecting experimentally infected human root canals. The authors thank Dr. Jay Kim for his expertise and assistance in completing the statistical analysis for this project. Dr. Torabinejad is professor and program director, Department of Endodontics; Dr. Shabahang is associate professor, Department of Endodontics; Mr. Aprecio is affiliated with the Department of Microbiology and Molecular Genetics; and Dr. Kettering is professor, Department of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA.
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 1965;20:340. 2. Mbller AJ, Fabricius L, Dahlen G, Ohman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981;89:475-84. 3. Sjogren 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. Bystrom A, Happonen R-P. Sjijgren U, Sundqvist G. Healing of periapica1 lesions of pulpless teeth after endodontic treatment with controlled asepsis. Endod Dent Traumatol 1987;3:58-63.
Vol. 29, No. 6, June 2003 5. Pataky L, lvanyi I, Grigar A, FazekasA. Antimicrobial efficacy of various root canal preparation techniques: an in vitro comparative study. J Endodon 2002;28:603-5. 6. Bystram A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8. 7. Vertucci FJ. Root canal anatomy of the human permanent teeth. Oral Surg Oral Med Oral Pathol 1984;58:589-99. 8. Torabinejad M, Handysides R, Khademi A, Bakland LK. Clinical implications of the smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:658-66. 9. Sjogren U, Figdor D, Spangberg L, Sundqvist G. The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing. Int Endod J 1991:24:119-25. 10. Sundqvist G, Figdor D, Persson S,Sjogren U. Microbiologic analysis of teeth with failed endcdontic treatment and the outcome of conservative retreatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93. 11. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endodon 2003;29:170-5. 12. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochloriteon the ability of MTAD to remove the smear layer. J Endodon 2003;29:233-9. 13. Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of sol-
Antimicrobial Effects
403
ubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endodon 2003;29:334-7. 14. Molander A, Reit C, Dahlen G. The antimicrobial effect of calcium hydroxide in root canals pretreated with 5% iodine potassium iodide. Endod Dent Traumatol 1999;15:205-9. 15. Siqueira JF Jr, 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. 16. Estrela C, Pimenta FC, Ito IY, Bammann LL. Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules. J Endodon 1999;25:416-8. 17. D’Arcangelo C, Varvara G, De Fazio P. An evaluation of the action of different root canal irrigants on facultative aerobic-anaerobic, obligate anaerobic, and microaerophilic bacteria. J Endodon 1999;25:351-3. 18. Harrison JW, Hand RE. The effect of dilution and organic matter on the antibacterial property of 5.25% sodium hypochlorite. J Endodon 1981;7:12832. 19. Heling I, Chandler NP. Antimicrobial effect of irrigant combinations within dentinal tubules. Int Endod J 1998;31:8-14. 20. Hand RE, Smith ML, Harrison JW. Analysis of the effect of dilution on the necrotic tissue dissolution property of sodium hypochlorite. J Endodon 1978;4:60-4.
JOURNAL OF ENKIDONTICS Copyright 0 2003 by The American Association of Endodontists
The Antimicrobial Effect of MTAD: An In Vitro Investigation Mahmoud Torabinejad, DMD, MSD, PhD, Shahrokh Shabahang, DDS, MS, PhD, Raydolfo M. Aprecio, OD, and James D. Kettering, PhD
Pulp and periradiculardiseases are of microbial origin. To effectively clean the root canal system a disinfecting agent must be able to penetrate into difficult-to-reach areas and kill microorganismswith minimal damage to the host tissues. The purpose of this investigationwas to test the a b i l i of a mixture of a tetracycline isomer, an acid, and a detergent (MTAD) to kill Entemwcxus faecalis and compare its efficacy to that of sodium hypochlorite (NaOCI) and ethylene diamine tetraacetic acid (EDTA). The zones of inhibition and minimum inhibitory concentdons were measured for these solutions.Measurement of zones of inhibition and determination of the minimum inhibitory concentrations showed that MTAD is as effective as 5.25% NaOCl and significantly more effective tJ-ian EDTA (p c 0.OOOl). Furthermore, MTAD is significantly more effective in killing E. faecalis than NaOCl when the solutions are diluted (p c 0.OOOl). Measurement of the minimum inhibitory concentrations demonstrated that atthough MTAD is still effective in killing €. faecalis at 2OOx dilution, NaOCl ceases to exert its antibacterial activity beyond32x dilution. EDTA did not exhibit any antibacterial activity. Based on the results of this study, it seems that MTAD is an effective solution in eradicating E. faecalis.
The main objectives of root canal therapy are cleaning and shaping, obturating the root canal system in three dimensions, and preventing reinfection. Investigations in animal studies have demonstrated the importance of microorganisms in the pathogenesis of pulpal and periradicular pathosis (1, 2). To determine the influence of infection at the time of root canal therapy on the treatment outcome of patients, Sjogren et al. (3) compared the success rate of teeth with negative cultures at the time of obturation to those that had positive bacterial cultures before the same procedure. They reported a significantly higher success rate when teeth were free of bacteria (94% versus 68%). The results are consonant with the findings of Bystrom et al. (4)who also showed improved clinical success rates after effective disinfection of root canals.
Pataky et al. (5) infected the root canals of extracted teeth with Enterococcusfueculis and examined the number of remaining bacteria after preparation with various techniques using sterile saline as an intracanal imgant. Their results show a reduction in bacterial population after mechanical preparation. They found no statistical differences among the efficacy of the test techniques or files used in the study. According to Bystrom and Sundqvist (6), current regimens in chemomechanical debridement using instrumentation and irrigation with sodium hypochlorite (NaOCl) are not predictably effective in root canal disinfection. These findings are not surprising because the root canal morphology is complex and contains numerous ramifications and anatomical irregularities (7). The microorganisms in root canals not only invade the anatomic irregularities of the root canal system but also are present in the dentinal tubules (8). Because of the limitations of current root canal cleaning techniques, some investigators recommend placement of antimicrobial dressings such as calcium hydroxide in the root canals before obturation (4,9).However, when treatment is completed in one visit, no intracanal medications other than intracanal imgants are used. The efficacy of intracanal inigants and calcium hydroxide against certain bacterial species such as E. fueculis has been questioned (10). An ideal intracanal inigant or medication should be able to disinfect the dentin and its tubules in one visit. In addition, it should have sustained antimicrobialeffect after use. FuI.thermore, it must be biccompatible with live host tissues (8). Studies have shown that a mixture of a tetracycline A D [A patent application has been isomer, an acid, and a detergent W fded for this technology]) as a final rinse is capable of removing the smear layer with minimal erosive changes on the surface of dentin (1 1, 12). In another study Beltz et al. (13) investigated the amount of tissue loss after exposing the bovine pulp and dentin to various concentrations of NaOCI, EDTA, or MTAD. Their results showed that various concentrations of NaOCl removed organic components of pulp and dentin effectively. The solubilizing effects of EDTA on pulp and dentin were somewhat similar to those of MTAD. The major difference between the actions of these solutions is a high binding affiity of doxycycline present in MTAD for the dentin. The purpose of this investigation was to compare the antimicrobial properties of MTAD with those of NaOCl and EDTA.
MATERIALS AND METHODS Doxycycline, citric acid, and Tween-80 were all purchased from Sigma-Aldrich Company, St. Louis, MO. TWOmethods were used
Vol. 29, No. 6, June 2003
Antimicrobial Effects
401
TABLE 1. Zones of inhibition (mm) and standard deviations for NaOCI, MTAD, and EDTA Sample
Undiluted
1:5
1:lO
NaOCl MTAD EDTA
34.3755 1.19 34.75 2 0.71 16.88 ? 0.64
15.752 0.71 30.15 5 1.89
11.252 0.71 29.63 -+ 3.81 0
to determine the extent of antimicrobial activity of MTAD and to compare it with those of NaOCl and EDTA. In the first test we measured the zone of inhibition on plates inoculated with E. faecalis. An overnight culture of E. ,faecalis (ATCC 4082) was standardized to 0.1 1 optical density (OD) measured at 570 nm. One hundred microliters of the microorganism was spread onto a trypticase soy agar (TSA) plate with the use of a sterile L-shaped glass rod. One-quarter-inch sterile S & S filter paper (Schleicher & Schuell) was placed in each of the four quadrants of the TSA plate. Twenty microliters of sterile saline (B. Braun Medical, Inc., Irvine, CA), 5.25% NaOCl (All Purpose Bleach, Mission Laboratories, Los Angeles, CA), MTAD (freshly prepared before each use according to a proprietary protocol at Loma Linda University), or 17% EDTA (Roth International, Chicago, IL) were added onto the filter papers. Eight replicates were prepared for each of the sample solutions. The plates were incubated overnight at 37°C for 24 h, and the zones of inhibition were measured in millimeters. In the second experiment we used the minimum inhibitory concentration (MIC) method. E. faecalis was cultured overnight and the concentration was adjusted to 0.1 1 OD at 570 nm. The test solutions were serially diluted from 1:2 up to 1:2048 dilutions. One milliliter of 2X trypticase soy broth (TSB) and the same amount of test solutions were mixed in various tubes. One hundred microliters of the standardized E. faecalis was added into each of the test tubes and incubated overnight at 37°C. The presence or absence of turbidity was determined the next day. To determine whether the test solutions had inhibitory or bactericidal effect, a high concentration of bacteria (1 X 10' colonyforming units of E. faecalis) was exposed to 2 ml of undiluted and 1:2 dilution of MTAD or NaOCl for 2 or 5 min. Samples were plated on TSA media to determine viability of any remaining bacteria.
RESULTS No zone of inhibition was observed adjacent to the filter papers saturated with sterile saline. The means and standard deviations of the zones of inhibition for the other test solutions are presented in Table 1. Figure 1 shows the comparison of the average areas of inhibition for the three test solutions. When the undiluted solutions were tested, the average zones of inhibition for 5.25% NaOCl and MTAD were measured at 34 and 35 mm, respectively. The zone of inhibition of undiluted EDTA was 17 m. Statistical analysis using Kruskal-Wallis ranks test and Mann-Whitney U test (a = 0.05) showed no statistical difference between 5.25% NaOCl and MTAD (p = 0.442). However, the zones of inhibition for the two solutions were significantly larger than those seen around the filter papers saturated with undiluted EDTA (p < 0.0001 for both). Dilution of the solutions reduced the zones of inhibition for all solutions, albeit at different rates. At 5- and 10-fold dilution, MTAD created a zone of inhibition that was significantly larger in diameter than that seen with NaOCl (p < 0.0001). EDTA demonstrated no bactericidal properties when diluted 1:5 or 1:10. Further analysis of the effects of dilution on the bactericidal properties of
0
FIG 1. The differences between the averages of zones of inhibition between NaOCI, MTAD, and EDTA measured at three dilutions.
the solutions indicated that undiluted NaOCl is significantly more effective than 5X and 1OX diluted NaOCl (p < 0.0001). Similar evaluation of the efficacy of MTAD showed that although undiluted MTAD was significantly more effective against E. faecalis than 5X or 1OX diluted MTAD, no significant difference was noted in the diameter of the zones of inhibition of 1:5 dilution or 1:lO dilution (p = 0.083). The results of the minimum inhibitory concentration method are listed in Table 2. Figure 2 shows the comparison of the maximum dilution of each solution with continued antimicrobial activity. Using this technique although EDTA demonstrated no antibacterial effects against E. faecalis, NaOCl and MTAD proved to be antibacterial to varying degrees. NaOCl continued to exert its efficacy up to 32X dilutions. On the other hand, MTAD was effective in killing E. faecalis up to 200X dilutions. Furthermore, although a 2- and 5-min exposure of E. faecalis to undiluted or 1:2 dilution of NaOCl did not result in total killing of the bacteria, exposure of the same bacterial species to undiluted or 1:2 diluted MTAD resulted in a completely negative culture after 2 or 5 min of exposure.
DISCUSSION Despite the presence of a controversy regarding the effect of the smear layer on the quality of instrumentation and obturation, the smear layer itself may be infected and may protect the bacteria already present in the dentinal tubules (8). Because of these concerns, one may deem it prudent to remove the smear layer in infected root canals and allow penetration of intracanal medications into the dentinal tubules in these teeth. To completely disinfect the root canal system, including the dentinal tubules, the disinfecting solution must be able to penetrate all aspects of the root canal system. To date, there is no single solution used in endodontics that simultaneously removes the smear layer and disinfects the entire root canal system. The effectiveness of MTAD in removing the smear layer has been described in previous publications ( 1 1, 12). MTAD is more effective than undiluted or
402
Journal of Endodontics
Torabinejad et al. TABLE 2. MIC dilutions for E. faecalis exposed to NaOCI, MTAD, and EDTA Solution
End Point
Control
NaOCl (Undiluted, 1:2 dilution up to 1:2048, Control) MTAD (Undiluted, 1:2 dilution up to 1:2048, Control) EDTA (Undiluted, 1:2 dilution up to 1:2048, Control)
No growth up to 1:32 dilution No growth up to 1:200 dilution
Growth Growth Growth
250 200 150
100 50
NaOCl
MTAD
EDTA
FIG 2. The maximum dilution of each solution with continued antimicrobial activity.
diluted NaOCl in removing the smear layer from the surface of instrumented root canals (11). The ability of MTAD is enhanced as a final rinse when low concentrations of NaOCl are used as intracanal irrigants (12). Compared with EDTA, MTAD removes the smear layer with significantly less erosion of the dentinal tubules (11, 12). In this investigation, we evaluated the ability of MTAD to kill E. faecalis in an in vitro model without the presence of the variables that are encountered in a tooth model. Standard microbiological methods were used to determine the antimicrobial properties of MTAD in Comparison to NaOCl and EDTA. E. faecalis was selected because this bacterium has been identified as one of the most difficult bacterial species to eradicate from infected root canals (10). The persistence of E. faecalis may be attributed to the ineffectiveness of currently used chemicals such as sodium hypochlorite (14), potassium iodide (13, or calcium hydroxide (16). The results of this study demonstrated the superior efficacy of MTAD in killing E. faecalis compared with the two most commonly used imgants in root canal therapy: NaOCl and EDTA. Two microbiological techniques were used to compare the antimicrobial properties of MTAD to these solutions. Determination of the zones of inhibition provided quantitative data, which allowed us to perform statistical analysis. Our findings corroborated the findings of D’Arcangelo et al. (17) who have shown that the majority of NaOCl dilutions have some degree of antibacterial activity when placed in direct contact with bacteria. Our study also confirmed the findings of Harrison and Hand (18) who have shown that NaOCl is an effective bactericidal agent when it is used undiluted. Another observation in this investigation was the lack of strong antimicrobial activity of EDTA. Dilution of 17% EDTA completely eliminated its efficacy even when placed in direct contact with E. faecalis. Our results are somewhat similar to the findings of Heling and Chandler (19) who showed no antimicrobial effect in undiluted EDTA, similar to that of sterile saline. A disadvantage of determining the antimicrobial effect of a solution by measuring the zone of inhibition is that this technique does not account for the variation in the diffusion rate of the
Growth
medication through the agar. To address this concern, a second technique was used to verify the results of the first experiment. The MIC method uses serial dilutions of a solution to determine the minimum concentration that would still exert antimicrobial properties. In this experiment, the MIC method confirmed that EDTA possesses little or no bactericidal activity. A significant observation was that MTAD continued to have bactericidal properties even when it was diluted 200 times. This finding was in contrast to the bactericidal properties of NaOCI, which ceased to exist beyond 32X dilution. In addition to its decreased antibacterial effect, dilution of NaOCl results in a significant decrease in its ability to dissolve necrotic tissue as demonstrated by Hand et al. (20) and Beltz et al. (13). Although dilution reduced the zone of inhibition for all imgants tested, the results of both microbiological methods demonstrated that MTAD maintains its bactericidal properties significantly more than NaOCl or EDTA. Another significant finding in our study was the capacity of MTAD to kill E. faecalis after an exposure of 2 or 5 min. This ability was not observed with NaOCl or EDTA. This finding strengthens the results of inhibition and dilution tests, showing that MTAD has superior antibacterial effect cornpared with NaOCl or EDTA even in a short contact time. Based on the results of this and previous investigations (1 1-13), it seems that MTAD has the ability to remove most of the smear layer and it possesses superior bactericidal activity compared with NaOCl or EDTA when tested against E. faecalis. These results are significant because they demonstrate the efficacy of an irrigant to remove most of the smear layer and kill a bacterial strain that has been shown to be resistant to many commonly used intracanal imgants and dressings. The antimicrobial activity of MTAD continues to exist even after dilution of the solution. A limitation of this in vitro study is that it does not account for the penetration ability of test irrigants in root canals. Studies are in progress to examine the efficacy of MTAD as a final rinse in disinfecting experimentally infected human root canals. The authors thank Dr. Jay Kim for his expertise and assistance in completing the statistical analysis for this project. Dr. Torabinejad is professor and program director, Department of Endodontics; Dr. Shabahang is associate professor, Department of Endodontics; Mr. Aprecio is affiliated with the Department of Microbiology and Molecular Genetics; and Dr. Kettering is professor, Department of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA.
References
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