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The Antimicrobial Effect of MTAD, Sodium Hypochlorite, Doxycycline, and Citric Acid on Enterococcus faecalis
Basic Research—Technology
The Antimicrobial Effect of MTAD, Sodium Hypochlorite, Doxycycline, and Citric Acid on Enterococcus faecalis Trisha A. Krause, DMD, MS, Frederick R. Liewehr, DDS, MS, and Chin-Lo Hahn, PhD, DDS Abstract This study compared the antimicrobial effect of MTAD, two of its components, doxycycline and citric acid, and sodium hypochlorite (NaOCl) in two in vitro models on Enterococcus faecalis. In the bovine tooth model, the lumens of 30 bovine dentin discs were infected with E. faecalis for 2 weeks before treating with either one of the experimental irrigants or saline. Bacteria in the shavings were collected with two sizes of burs and enumerated after overnight culturing. Zones of inhibition were recorded in the agar diffusion model for each irrigant. In the tooth model, NaOCl and doxycycline were more effective than control in killing E. faecalis at the shallow bur depth, but at the deeper bur depth only NaOCl was superior. In the agar diffusion model, NaOCl produced less inhibition than MTAD or doxycycline. (J Endod 2007;33:28 –30)
Key Words Antimicrobial effects, citric acid, doxycycline, Enterococcus faecalis, MTAD, NaOCl
From the Virginia Commonwealth University, Richmond, Virginia. Address requests for reprints to Dr. Frederick R. Liewehr, 9244 Royal Grant Drive, Mechanicsville, GA 23116. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.08.011
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uccessful root canal therapy relies on the combination of proper instrumentation, disinfection, and obturation of the root canal (1). Of these three essential steps of root canal therapy, disinfection of the root canal is a major determinant in the healing of the periapical tissues (2). Sundqvist and associates (3) found that infection of the root canal at the time of obturation has a negative influence on the prognosis of endodontic therapy. Sodium hypochlorite (NaOCl) is bacteriocidal but is also toxic to periapical tissues (4 –7). A new irrigant, BioPure MTAD (Tulsa Dentsply, Tulsa, OK), has shown promise as an antimicrobial against Enterococcus faecalis and as a smear layer removal agent, even superior to NaOCl (8 –10). MTAD is a formulation of doxycycline, Tween-80, and citric acid. Doxycycline and citric acid exhibit antimicrobial and acid etching properties (11, 12). The primary purpose of this study was to compare the antimicrobial efficacy of MTAD, doxycycline, citric acid, and NaOCl against E. faecalis. Two different in vitro models were used to test this efficacy: a bovine tooth model and a zone of inhibition model. E. faecalis was chosen as the test bacterium as it has been cultured from obturated canals of teeth with persistent chronic apical periodontitis (3).
Materials and Methods Bovine teeth were prepared according to the description of Haapasalo and Orstavik with minor modification (13). In brief, 30 root segments of intact bovine central and lateral incisors were sectioned with a water-cooled diamond saw (Isomet, Buehler LTD, Evanston, IL). Each root segment was 5 mm in height and at least 6 mm in diameter. The pulpal lumens were standardized to a 3.5-mm diameter with an ISO 035 bur (Brasseler USA, Savannah, GA). The smear layer of the lumen was removed using 17% EDTA in an ultrasonic bath for 4 minutes followed by another 4-minute rinse with 5.25% NaOCl; root segments were autoclaved and stored in saline. Individual root segments were mounted in wells of six-well tissue culture plates (Corning Cell Wells, Corning Glass Works, Corning, NY) on wax bases approximately 6 mm tall. Overnight cultures of E. faecalis (ATCC 29212) in brain-heart infusion (BHI) broth were harvested and their bacterial concentration was determined using a spectrophotometer with an OD650. Each tooth segment received 60 l of E. faecalis (5 ⫻ 108/ml) and was then incubated overnight at 37°C in a 5% CO2 chamber. Every day for 14 consecutive days, the inoculum was replaced with fresh E. faecalis (60 l, 5 ⫻ 108 cfu/ml) under a laminar-flow hood. After 14 days of inoculation, the tooth segments were irrigated with 60 l of (1) BioPure MTAD; (2) 5.25% NaOCl (Clorox, Oakland, CA); (3) 100 mg/ml doxycycline hyclate (Sigma, St. Louis, MO); (4) 10% citric acid; or (5) saline. Each lumen received two successive 5-minute flushes with irrigant. After the second irrigation, the 5.25% NaOCl group was rinsed twice with 60 l of 5% sodium thiosulfate (Sigma) to neutralize any NaOCl remaining in the dentinal tubules (14). The other groups were not rinsed to simulate their use in a clinical setting. Two consecutive bacterial samples were immediately taken from each of the dentin cylinders with sterile round burs (bur 1: ISO 037; bur 2: ISO 040) (Brasseler USA). Dentin shavings were collected in Eppendorf tubes containing 500 l of phosphate-buffered saline. After vortexing, a 10-fold dilution was made and a 100-l sample was plated on a 5% sheep blood trypticase soy agar (TSA) plate using a spiral-plating machine (Spiral Biotech, Norwood, MA). The number of bacterial colony forming units (cfu) was counted and recorded after overnight incubation in a 5% CO2 incubator at 37°C.
JOE — Volume 33, Number 1, January 2007
Basic Research—Technology
Results In the bovine tooth model, doxycycline and NaOCl produced a significantly greater antimicrobial effect than the saline control, citric acid, or MTAD against E. faecalis in the more superficial dentin sample (ISO 037) (Fig. 1). At the deeper bur depth (ISO 040), NaOCl remained significantly more antimicrobial than the control, whereas none of the other irrigants was more effective than the saline control. Interestingly, MTAD and citric acid performed similarly to the saline control at both bur depths. When comparing the antibacterial effect at the two bur depths for each irrigant, there was no statistical significant difference. In all three dilutions (1:1, 1:5, and 1:10), doxycycline produced a larger zone of inhibition than MTAD, NaOCl, or citric acid (Fig. 2). MTAD yielded the second largest zone of inhibition for all three of the dilutions and was significantly more effective than NaOCl or citric acid. At the 1:10 dilution, the zones of inhibition for NaOCl and citric acid were not statistically different from the saline control. Citric acid produced the smallest zone of inhibition at the 1:1 concentration, and at the 1:5 and 1:10 dilutions it was no different from the saline control, which exhibited no inhibition zone.
Discussion Bovine incisors were used in this experiment because their dentinal tubules are similar to those in human teeth in quantity, size, diameter, morphology, and density. In addition, these teeth are more readily obtained and are larger in size, thus making them easier to manipulate in the laboratory (13). In our model, 5.25% NaOCl, was not able to render the dentinal shavings sterile but was significantly more effective in killing E. faecalis than MTAD, which is different from the results reported by Shabahang and Torabinejad (8). They found that the combination of 1.3% NaOCl irrigation and an MTAD final treatment eliminated all E. faecalis from human tooth cementum and dentin samples. The better bacteriocidal effect of MTAD in their experiment may have been because of a carryover effect of the doxycycline in the MTAD
Figure 1. Antimicrobial effect of five irrigants on bovine dentinal blocks.
JOE — Volume 33, Number 1, January 2007
1:10 Concentration
The spiral-plating machine was used to spread a lawn of E. faecalis on 5% sheep blood TSA plates on which sterile paper discs 6 mm in diameter were then placed. Three dilutions (1:1, 1:5, and 1:10) of the following test irrigants (20 l per disc) were added to the sterile paper discs in triplicate: 5.25% NaOCl, doxycyline 100 mg/ml, 10% citric acid, BioPure MTAD, and saline. The plates were incubated at 37°C in a CO2 incubator, and the zones of inhibition for each group were measured after 24 hours. A two-way analysis of variance (ANOVA) was used for statistical evaluation of the data in the bovine tooth model, and a one-way ANOVA was used in the agar diffusion model. The Tukey-Kramer test was run in both experiments for multiple comparisons. Statistical significance for both experiments was set at p ⬍ 0.05.
Doxycycline
1:5
MTAD NaOCl Citric Acid
1:1
0
5
10
15
20
25
millimeters
Figure 2. Average zone of inhibition measurements (millimeters) with three dilutions of root canal irrigants against E. faecalis. Note: the saline group (control) produced no measurable zones of inhibition and so is not included.
preparation, because all tooth samples were soaked in MTAD. Although teeth were vortexed for 15 seconds in BHI and transferred to fresh medium to minimize this carryover effect before bacterial sampling, the reversible binding between doxycycline and dentin requires over 160 minutes of elution with water for complete removal from dentin (15). In our study we merely filled the lumens of the samples to more closely simulate clinical use, and found that MTAD was no more effective than the saline control. When the antimicrobial property of the four root canal irrigants was examined using an agar diffusion method, NaOCl was significantly less effective in creating a zone of inhibition than doxcycycline and MTAD in all dilutions, which is in contrast to our tooth model data. We reason that the ability of NaOCl to denature protein (16) might affect its diffusibility and bacteriocidal property on agar plates. In addition, the doxycycline concentration used in our study (10%) was much higher than the 3% concentration in Biopure MTAD (17), which could explain a larger inhibition zone produced by doxycycline. Torabinejad and associates also reported that MTAD produced a significantly larger zone of inhibition than 5.25% NaOCl at 1:5 and 1:10 dilutions; however, they found no significant difference at 1:1 concentration (10). The difference we observed at the 1:1 concentration may be because of our use of a different strain of experimental E. faecalis. Dissimilar strains of E. faecalis may exhibit different susceptibility or resistance patterns to NaOCl or MTAD (18). Clewell and Weaver (19) described strains of intestinal E. faecalis that harbor plasmids that determine antibiotic resistance, and Kayaoglu and Orstavik (20) believe that this property may be possessed by root canal microbiota as well. In addition, Torabinejad and associates (10) mixed their MTAD according to an undisclosed proprietary protocol. Our MTAD, packaged as BioPure MTAD, was prepared according to the manufacturer’s instructions and may have differed in doxycycline concentration from that used in their study. The concentration of doxycycline in the MTAD solution would likely affect its antimicrobial activity and effectiveness against E. faecalis. Our results suggest that doxycycline, and not citric acid, provides MTAD’s antimicrobial effect, because citric acid was not significantly bactericidal in either the bovine tooth or agar diffusion model. Citric acid is commonly used in periodontal therapy to remove the smear layer (21), and it has also been investigated for use as an intracanal smear layer removal agent (22). The smear layer may protect bacteria and prevent irrigants from exerting an antimicrobial effect in the tubules (23); these protected bacteria may contribute to endodontic failure (13). In the MTAD preparation, citric acid may serve to remove the smear layer, thus allowing doxycycline to penetrate the dentinal tubules and exert an antimicrobial effect. In our experimental model, the smear layer was removed before infection of the dentinal tubules, and our
Comparing the Antimicrobial Effect of MTAD and NaOCl on E. faecalis
29
Basic Research—Technology results showed that doxycycline effectively penetrated the tubules and was more antimicrobial than the control in the shallow dentin sample. The difference between the two bur sizes was approximately 250 m, but bacterial recovery from the two depths was not different for any irrigant. It is unlikely that the superior antimicrobial effect of NaOCl at both bur depths was caused by a carryover effect because the samples were rinsed twice with sodium thiosulfate to neutralize it before sampling. Although the actual depth of penetration by E. faecalis into the tubules was not verified in our study, our data suggest that NaOCl has the ability to penetrate into the dentinal tubules, which is consistent with previous studies (24, 25). In conclusion, in the bovine tooth model, NaOCl and 100 mg/ml doxycycline were significantly more effective in killing E. faecalis than MTAD, citric acid, or the saline control to a depth of approximately 100 m into the dentinal tubules. NaOCl was significantly more effective than the other experimental irrigants at approximately 250 m into dentinal tubules. In the agar diffusion model, doxycycline was significantly more effective than MTAD, NaOCl, or citric acid. The results in the bovine tooth model, which may more closely simulate in vivo conditions, emphasize the fact that none of the experimental groups were able to render the canal sterile; therefore, further research is indicated to find an intracanal irrigant that can completely eradicate E. faecalis from root canal dentin.
Acknowledgment This research was made possible by funding from the VCU Alexander Fellowship. The authors thank Dr. Al Best for assistance in planning the statistical aspects. In addition, the authors thank the Leona Meat Plant of East Troy, PA, for kindly providing the bovine jaws used in this study.
References 1. Ingle JI. Root canal obturation. J Am Dent Assoc 1956;53:47–55. 2. 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 1965;20:340 –9. 3. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93. 4. Ringel AM, Patterson SS, Newton CW, Miller CH, Mulhern JM. In vivo evaluation of chlorhexidine gluconate solution and sodium hypochlorite solution as root canal irrigants. J Endod 1982;8:200 – 4.
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5. Hales JJ, Jackson CR, Everett AP, Moore SH. Treatment protocol for the management of a sodium hypochlorite accident during endodontic therapy. Gen Dent 2001; 49:278 – 81. 6. Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of NaOCl on vital tissue. J Endod 1985;11:525– 8. 7. Neaverth EJ, Swindle R. A serious complication following the inadvertent injection of sodium hypochlorite outside the root canal system. Compendium 1990;11:474, 476, 478 – 81. 8. Shabahang S, Torabinejad M. Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth. J Endod 2003;29:576 –9. 9. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 10. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400 –3. 11. Smith JJ, Wayman BE. An evaluation of the antimicrobial effectiveness of citric acid as a root canal irrigant. J Endod 1986;12:54 – 8. 12. Demirel K, Baer PN, McNamara TF. Topical application of doxycycline on periodontally involved root surfaces in vitro: comparative analysis of substantivity on cementum and dentin. J Periodontol 1991;62:312– 6. 13. Haapasalo M, Orstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375–9. 14. Fouad AF, Barry J. The effect of antibiotics and endodontic antimicrobials on the polymerase chain reaction. J Endod 2005;31:510 –3. 15. Ciarlone AE, Johnson RD, Tomaselli DL, Jr., Sue Seale N, Pashley DH. The quantitative binding of tetracycline to dentin. J Endod 1988;14:494 – 6. 16. Hannig M. Effect of Carisolv solution on sound, demineralized and denatured dentin: an ultrastructural investigation. Clin Oral Investig 1999;3:155–9. 17. Tay FR, Pashley DH, Loushine RJ, et al. Ultrastructure of smear layer-covered intraradicular dentin after irrigation with BioPure MTAD. J Endod 2006;32:218 –21. 18. Portenier I, Waltimo T, Orstavik D, Haapasalo M. The susceptibility of starved, stationary phase, and growing cells of Enterococcus faecalis to endodontic medicaments. J Endod 2005;31:380 – 6. 19. Clewell DB, Weaver KE. Sex pheromones and plasmid transfer in Enterococcus faecalis. Plasmid 1989;21:175– 84. 20. Kayaoglu G, Orstavik D. Virulence factors of Enterococcus faecalis: relationship to endodontic disease. Crit Rev Oral Biol Med 2004;15:308 –20. 21. Vanuspong W, Eisenburger M, Addy M. Cervical tooth wear and sensitivity: erosion, softening and rehardening of dentine; effects of pH, time and ultrasonication. J Clin Periodontol 2002;29:351–7. 22. Abou-Rass M, Patonai FJ, Jr. The effects of decreasing surface tension on the flow of irrigating solutions in narrow root canals. Oral Surg Oral Med Oral Pathol 1982;53:524 – 6. 23. Baker NA, Eleazer PD, Averbach RE, Seltzer S. Scanning electron microscopic study of the efficacy of various irrigating solutions. J Endod 1975;1:127–35. 24. 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. 25. Gutierrez JH, Jofre A, Villena F. Scanning electron microscope study on the action of endodontic irrigants on bacteria invading the dentinal tubules. Oral Surg Oral Med Oral Pathol 1990;69:491–501.
JOE — Volume 33, Number 1, January 2007
The Antimicrobial Effect of MTAD, Sodium Hypochlorite, Doxycycline, and Citric Acid on Enterococcus faecalis Trisha A. Krause, DMD, MS, Frederick R. Liewehr, DDS, MS, and Chin-Lo Hahn, PhD, DDS Abstract This study compared the antimicrobial effect of MTAD, two of its components, doxycycline and citric acid, and sodium hypochlorite (NaOCl) in two in vitro models on Enterococcus faecalis. In the bovine tooth model, the lumens of 30 bovine dentin discs were infected with E. faecalis for 2 weeks before treating with either one of the experimental irrigants or saline. Bacteria in the shavings were collected with two sizes of burs and enumerated after overnight culturing. Zones of inhibition were recorded in the agar diffusion model for each irrigant. In the tooth model, NaOCl and doxycycline were more effective than control in killing E. faecalis at the shallow bur depth, but at the deeper bur depth only NaOCl was superior. In the agar diffusion model, NaOCl produced less inhibition than MTAD or doxycycline. (J Endod 2007;33:28 –30)
Key Words Antimicrobial effects, citric acid, doxycycline, Enterococcus faecalis, MTAD, NaOCl
From the Virginia Commonwealth University, Richmond, Virginia. Address requests for reprints to Dr. Frederick R. Liewehr, 9244 Royal Grant Drive, Mechanicsville, GA 23116. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.08.011
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Krause et al.
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uccessful root canal therapy relies on the combination of proper instrumentation, disinfection, and obturation of the root canal (1). Of these three essential steps of root canal therapy, disinfection of the root canal is a major determinant in the healing of the periapical tissues (2). Sundqvist and associates (3) found that infection of the root canal at the time of obturation has a negative influence on the prognosis of endodontic therapy. Sodium hypochlorite (NaOCl) is bacteriocidal but is also toxic to periapical tissues (4 –7). A new irrigant, BioPure MTAD (Tulsa Dentsply, Tulsa, OK), has shown promise as an antimicrobial against Enterococcus faecalis and as a smear layer removal agent, even superior to NaOCl (8 –10). MTAD is a formulation of doxycycline, Tween-80, and citric acid. Doxycycline and citric acid exhibit antimicrobial and acid etching properties (11, 12). The primary purpose of this study was to compare the antimicrobial efficacy of MTAD, doxycycline, citric acid, and NaOCl against E. faecalis. Two different in vitro models were used to test this efficacy: a bovine tooth model and a zone of inhibition model. E. faecalis was chosen as the test bacterium as it has been cultured from obturated canals of teeth with persistent chronic apical periodontitis (3).
Materials and Methods Bovine teeth were prepared according to the description of Haapasalo and Orstavik with minor modification (13). In brief, 30 root segments of intact bovine central and lateral incisors were sectioned with a water-cooled diamond saw (Isomet, Buehler LTD, Evanston, IL). Each root segment was 5 mm in height and at least 6 mm in diameter. The pulpal lumens were standardized to a 3.5-mm diameter with an ISO 035 bur (Brasseler USA, Savannah, GA). The smear layer of the lumen was removed using 17% EDTA in an ultrasonic bath for 4 minutes followed by another 4-minute rinse with 5.25% NaOCl; root segments were autoclaved and stored in saline. Individual root segments were mounted in wells of six-well tissue culture plates (Corning Cell Wells, Corning Glass Works, Corning, NY) on wax bases approximately 6 mm tall. Overnight cultures of E. faecalis (ATCC 29212) in brain-heart infusion (BHI) broth were harvested and their bacterial concentration was determined using a spectrophotometer with an OD650. Each tooth segment received 60 l of E. faecalis (5 ⫻ 108/ml) and was then incubated overnight at 37°C in a 5% CO2 chamber. Every day for 14 consecutive days, the inoculum was replaced with fresh E. faecalis (60 l, 5 ⫻ 108 cfu/ml) under a laminar-flow hood. After 14 days of inoculation, the tooth segments were irrigated with 60 l of (1) BioPure MTAD; (2) 5.25% NaOCl (Clorox, Oakland, CA); (3) 100 mg/ml doxycycline hyclate (Sigma, St. Louis, MO); (4) 10% citric acid; or (5) saline. Each lumen received two successive 5-minute flushes with irrigant. After the second irrigation, the 5.25% NaOCl group was rinsed twice with 60 l of 5% sodium thiosulfate (Sigma) to neutralize any NaOCl remaining in the dentinal tubules (14). The other groups were not rinsed to simulate their use in a clinical setting. Two consecutive bacterial samples were immediately taken from each of the dentin cylinders with sterile round burs (bur 1: ISO 037; bur 2: ISO 040) (Brasseler USA). Dentin shavings were collected in Eppendorf tubes containing 500 l of phosphate-buffered saline. After vortexing, a 10-fold dilution was made and a 100-l sample was plated on a 5% sheep blood trypticase soy agar (TSA) plate using a spiral-plating machine (Spiral Biotech, Norwood, MA). The number of bacterial colony forming units (cfu) was counted and recorded after overnight incubation in a 5% CO2 incubator at 37°C.
JOE — Volume 33, Number 1, January 2007
Basic Research—Technology
Results In the bovine tooth model, doxycycline and NaOCl produced a significantly greater antimicrobial effect than the saline control, citric acid, or MTAD against E. faecalis in the more superficial dentin sample (ISO 037) (Fig. 1). At the deeper bur depth (ISO 040), NaOCl remained significantly more antimicrobial than the control, whereas none of the other irrigants was more effective than the saline control. Interestingly, MTAD and citric acid performed similarly to the saline control at both bur depths. When comparing the antibacterial effect at the two bur depths for each irrigant, there was no statistical significant difference. In all three dilutions (1:1, 1:5, and 1:10), doxycycline produced a larger zone of inhibition than MTAD, NaOCl, or citric acid (Fig. 2). MTAD yielded the second largest zone of inhibition for all three of the dilutions and was significantly more effective than NaOCl or citric acid. At the 1:10 dilution, the zones of inhibition for NaOCl and citric acid were not statistically different from the saline control. Citric acid produced the smallest zone of inhibition at the 1:1 concentration, and at the 1:5 and 1:10 dilutions it was no different from the saline control, which exhibited no inhibition zone.
Discussion Bovine incisors were used in this experiment because their dentinal tubules are similar to those in human teeth in quantity, size, diameter, morphology, and density. In addition, these teeth are more readily obtained and are larger in size, thus making them easier to manipulate in the laboratory (13). In our model, 5.25% NaOCl, was not able to render the dentinal shavings sterile but was significantly more effective in killing E. faecalis than MTAD, which is different from the results reported by Shabahang and Torabinejad (8). They found that the combination of 1.3% NaOCl irrigation and an MTAD final treatment eliminated all E. faecalis from human tooth cementum and dentin samples. The better bacteriocidal effect of MTAD in their experiment may have been because of a carryover effect of the doxycycline in the MTAD
Figure 1. Antimicrobial effect of five irrigants on bovine dentinal blocks.
JOE — Volume 33, Number 1, January 2007
1:10 Concentration
The spiral-plating machine was used to spread a lawn of E. faecalis on 5% sheep blood TSA plates on which sterile paper discs 6 mm in diameter were then placed. Three dilutions (1:1, 1:5, and 1:10) of the following test irrigants (20 l per disc) were added to the sterile paper discs in triplicate: 5.25% NaOCl, doxycyline 100 mg/ml, 10% citric acid, BioPure MTAD, and saline. The plates were incubated at 37°C in a CO2 incubator, and the zones of inhibition for each group were measured after 24 hours. A two-way analysis of variance (ANOVA) was used for statistical evaluation of the data in the bovine tooth model, and a one-way ANOVA was used in the agar diffusion model. The Tukey-Kramer test was run in both experiments for multiple comparisons. Statistical significance for both experiments was set at p ⬍ 0.05.
Doxycycline
1:5
MTAD NaOCl Citric Acid
1:1
0
5
10
15
20
25
millimeters
Figure 2. Average zone of inhibition measurements (millimeters) with three dilutions of root canal irrigants against E. faecalis. Note: the saline group (control) produced no measurable zones of inhibition and so is not included.
preparation, because all tooth samples were soaked in MTAD. Although teeth were vortexed for 15 seconds in BHI and transferred to fresh medium to minimize this carryover effect before bacterial sampling, the reversible binding between doxycycline and dentin requires over 160 minutes of elution with water for complete removal from dentin (15). In our study we merely filled the lumens of the samples to more closely simulate clinical use, and found that MTAD was no more effective than the saline control. When the antimicrobial property of the four root canal irrigants was examined using an agar diffusion method, NaOCl was significantly less effective in creating a zone of inhibition than doxcycycline and MTAD in all dilutions, which is in contrast to our tooth model data. We reason that the ability of NaOCl to denature protein (16) might affect its diffusibility and bacteriocidal property on agar plates. In addition, the doxycycline concentration used in our study (10%) was much higher than the 3% concentration in Biopure MTAD (17), which could explain a larger inhibition zone produced by doxycycline. Torabinejad and associates also reported that MTAD produced a significantly larger zone of inhibition than 5.25% NaOCl at 1:5 and 1:10 dilutions; however, they found no significant difference at 1:1 concentration (10). The difference we observed at the 1:1 concentration may be because of our use of a different strain of experimental E. faecalis. Dissimilar strains of E. faecalis may exhibit different susceptibility or resistance patterns to NaOCl or MTAD (18). Clewell and Weaver (19) described strains of intestinal E. faecalis that harbor plasmids that determine antibiotic resistance, and Kayaoglu and Orstavik (20) believe that this property may be possessed by root canal microbiota as well. In addition, Torabinejad and associates (10) mixed their MTAD according to an undisclosed proprietary protocol. Our MTAD, packaged as BioPure MTAD, was prepared according to the manufacturer’s instructions and may have differed in doxycycline concentration from that used in their study. The concentration of doxycycline in the MTAD solution would likely affect its antimicrobial activity and effectiveness against E. faecalis. Our results suggest that doxycycline, and not citric acid, provides MTAD’s antimicrobial effect, because citric acid was not significantly bactericidal in either the bovine tooth or agar diffusion model. Citric acid is commonly used in periodontal therapy to remove the smear layer (21), and it has also been investigated for use as an intracanal smear layer removal agent (22). The smear layer may protect bacteria and prevent irrigants from exerting an antimicrobial effect in the tubules (23); these protected bacteria may contribute to endodontic failure (13). In the MTAD preparation, citric acid may serve to remove the smear layer, thus allowing doxycycline to penetrate the dentinal tubules and exert an antimicrobial effect. In our experimental model, the smear layer was removed before infection of the dentinal tubules, and our
Comparing the Antimicrobial Effect of MTAD and NaOCl on E. faecalis
29
Basic Research—Technology results showed that doxycycline effectively penetrated the tubules and was more antimicrobial than the control in the shallow dentin sample. The difference between the two bur sizes was approximately 250 m, but bacterial recovery from the two depths was not different for any irrigant. It is unlikely that the superior antimicrobial effect of NaOCl at both bur depths was caused by a carryover effect because the samples were rinsed twice with sodium thiosulfate to neutralize it before sampling. Although the actual depth of penetration by E. faecalis into the tubules was not verified in our study, our data suggest that NaOCl has the ability to penetrate into the dentinal tubules, which is consistent with previous studies (24, 25). In conclusion, in the bovine tooth model, NaOCl and 100 mg/ml doxycycline were significantly more effective in killing E. faecalis than MTAD, citric acid, or the saline control to a depth of approximately 100 m into the dentinal tubules. NaOCl was significantly more effective than the other experimental irrigants at approximately 250 m into dentinal tubules. In the agar diffusion model, doxycycline was significantly more effective than MTAD, NaOCl, or citric acid. The results in the bovine tooth model, which may more closely simulate in vivo conditions, emphasize the fact that none of the experimental groups were able to render the canal sterile; therefore, further research is indicated to find an intracanal irrigant that can completely eradicate E. faecalis from root canal dentin.
Acknowledgment This research was made possible by funding from the VCU Alexander Fellowship. The authors thank Dr. Al Best for assistance in planning the statistical aspects. In addition, the authors thank the Leona Meat Plant of East Troy, PA, for kindly providing the bovine jaws used in this study.
References 1. Ingle JI. Root canal obturation. J Am Dent Assoc 1956;53:47–55. 2. 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 1965;20:340 –9. 3. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93. 4. Ringel AM, Patterson SS, Newton CW, Miller CH, Mulhern JM. In vivo evaluation of chlorhexidine gluconate solution and sodium hypochlorite solution as root canal irrigants. J Endod 1982;8:200 – 4.
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JOE — Volume 33, Number 1, January 2007