Basic Research—TechnologyDisinfection of Dentinal Tubules with 2% Chlorhexidine,2% Metronidazole, Bioactive Glass when Com...
Basic Research—TechnologyTABLE 1. Mean Colony Counts for Different Intracanal Medicaments at 200 and 400 m Depths at Diffe...
Basic Research—Technology                                                                                when compared wit...
Basic Research—Technology 2. Sirén EK, Haapasalo MPP, Ranta K, Salmi P, Kerosuo ENJ. Microbiological findings             ...
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Disinfection of dentinal tubules with 2% chlorhexidine,

  1. 1. Basic Research—TechnologyDisinfection of Dentinal Tubules with 2% Chlorhexidine,2% Metronidazole, Bioactive Glass when Compared withCalcium Hydroxide as Intracanal MedicamentsJogikalmat Krithikadatta, MDS,* Rajamani Indira, MDS,† and Alfred Leo Dorothykalyani, MSc‡AbstractThorough disinfection of the root canal system is es-sential for the success of root canal therapy. This re-quires the use of an intra-canal medicament. Entero- M icroorganisms play a fundamental role in the etiology of pulp and periapical diseases. Their control and elimination are important during endodontic treat- ment (1). Aerobic and facultative anaerobic microorganisms are usually minor con-coccus faecalis is the most frequently found species in stituents of primary infections and are found in higher frequency in endodontic flare-persistent/secondary intracanal infection associated ups and in failed cases. Enterococcus faecalis is more likely to be found in failed casesendodontic treatment failure. This study evaluates the than in primary infection (2), and starvation increases the resistance of E. faecalisdisinfection of dentinal tubules using 2% chlorhexidine 1000-fold to 10,000-fold (3). It is probable that the physiologic state of the cells,gel, 2% metronidazole gel, bioactive glass (S53P4) in particularly in retreatment cases, is closest to the starvation phase (4).comparison with calcium hydroxide. The antibacterial Cleaning and shaping of the root canal reduce the bacterial population but do notefficacy of the four medicaments against Enterococcus completely eliminate them. One possible reason for persistent endodontic infectionfaecalis was assessed in vitro using extracted premolar might be due to the retention of microorganisms in the dentinal tubules of the root canalteeth at the depths of 200 m and 400 m. The overall (5). Hence, the use of an intracanal medicament helps in the elimination of bacteria thatpercentage inhibition of bacterial growth (at 200 m remain even after cleaning and shaping, thereby providing an environment conduciveand 400 m depth) was 100% with 2% chlorhexidine for periapical tissue repair (6).gel. The inhibition of growth was moderate with 2% Calcium hydroxide is the most widely used intracanal medicament, requiring ametronidazole gel (86.5%), followed by bioactive glass disinfection period of 7 days (7). The high pH of calcium hydroxide formulations alters(62.8%) and calcium hydroxide (58.5%). It can be the biologic properties of bacterial lipopolysaccharides in the cell walls of gram-neg-concluded from the present study that 2% chlorhexi- ative species and inactivates membrane transport mechanisms, resulting in bacterialdine gel alone was most effective against E. faecalis cell toxicity (8). However, E. faecalis has been reported to be resistant to this effect aswhen compared to other medicaments tested. (J Endod a result of its ability to penetrate the dentinal tubules and adapt to changing environment2007;33:1473–1476) (9). The search for a better alternative has lead to the introduction of newer antimi-Key Words crobial agents like chlorhexidine, metronidazole, and particulate bioactive glass.Bioactive glass, calcium hydroxide, chlorhexidine, den- Hence, this study was undertaken to evaluate the disinfection of dentinal tubules con-tin tubule disinfection, Enterococcus faecalis, metroni- taminated with E. faecalis by using 2% chlorhexidine gel, 2% metronidazole gel, anddazole bioactive glass (S53P4) when compared with calcium hydroxide as intracanal medica- ments. From the *Department of Conservative Dentistry & End- Materials and Methodsodontics, Meenakshi Ammal Dental College, Maduravoyal; The model proposed by Haapasalo and Orstavik (10) was modified for this study,† Department of Conservative Dentistry & Endodontics, RagasDental College & Hospitals, Uthandi; and ‡Mohan Diabetes and freshly extracted single rooted human premolar teeth were selected.Specialty Center, Gopalapuram, Chennai, Tamilnadu, India. Address requests for reprints to Dr Jogikalmat Krithika-datta, No. 32, Arumuga Nainar Street, Thirunagar, Valasara- Preparation of the Blocksvakkam, Chennai 600087, Tamilnadu, India. E-mail address: A rotary diamond disk was used to decoronate the teeth 5 mm below the toenamel junction. The remaining root was then sectioned such that 6 mm of the middle0099-2399/$0 - see front matter Copyright © 2007 by the American Association of third of the root was obtained. Cementum was removed from the root surface to stan-Endodontists. dardize the external diameter to 4 mm (11). The internal diameter was standardized todoi:10.1016/j.joen.2007.08.016 Gates Glidden drill no.3 (Mani Inc, Tachigi-ken, Japan) in a slow speed handpiece (NSK, Tokyo, Japan). Organic and inorganic debris was removed by treating the blocks in an ultrasonic bath of 17% ethylenediaminetetraacetic acid for 5 minutes followed by 3% sodium hypochlorite for 5 minutes. The blocks were immersed in an ultrasonic bath of distilled water for 5 minutes to remove all traces of the chemicals used and sterilized in an autoclave at 121°C. The blocks were subjected to a second cycle of sterilization, with the blocks immersed in 1 mL of tryptone soya (TS) broth (HiMedia, Mumbai, India) in individual microcentrifuge tubes. This allows better penetration of the broth into the dentinal tubules (12).JOE — Volume 33, Number 12, December 2007 Disinfection of Dentinal Tubules 1473
  2. 2. Basic Research—TechnologyTABLE 1. Mean Colony Counts for Different Intracanal Medicaments at 200 and 400 m Depths at Different Time Intervals Mean (Standard Deviation) Colony Count ( 105) GROUPS Day 1 Day 3 Day 5 200 m 400 m 200 m 400 m 200 m 400 m Saline 3.26 (0.26) 3.46 (0.26) 3.12 (0.56) 3.38 (0.48) 2.8 (0.53) 3.16 (0.42) Calcium hydroxide 0.85 (0.31) 0.98 (0.36) 1.73 (0.77) 1.86 (0.78) 1.2 (0.26) 1.30 (0.17) Bioactive glass 1.31 (0.22) 1.38 (0.21) 1.06 (0.42) 1.34 (0.65) 1.0 (0.20) 1.07 (0.20) Chlorhexidine 2% gel 0.00 0.00 0.00 0.00 0.00 0.00 Metronidazole 2% gel 0.39 (0.05) 0.44 (0.08) 0.36 (0.15) 0.41 (0.13) 0.38 (0.09) 0.54 (0.19)Contamination of the Blocks and 400 m) by using Gates Glidden drills nos. 4 and 5 (Mani Inc), E. faecalis was used as the test organism in this study. This gram- respectively, and collected in 1 mL of sterile TS broth and incubated inpositive facultative anaerobic bacterium is the most common isolate found an anaerobic environment at 37°C for 24 hours. After the incubationin endodontically failed cases. Isolated 24-hour colonies of pure culture of period, the content of each microcentrifuge tube was serially diluted,E. faecalis (ATCC 29212) grown on tryptone soya agar were suspended in 100 L of broth in 100 L of normal saline for 5 times. Five microliters5 mL of TS broth and incubated for 4 hours at 37°C. The culture suspen- of this diluted sample was plated on TS agar plates and incubated for 24sion was adjusted to match the turbidity equivalent to 0.5 McFarland hours. Colonies were counted, and readings were tabulated.standard. Fifty microliters of the inoculum was transferred to presteril-ized individual microcentrifuge tubes containing 1 mL of the TS brothand dentin block. The dentin blocks were transferred to fresh broth Statistical Analysiscontaining E. faecalis every second day. All the procedures were carried The data were statistically analyzed with one-way analysis of vari-out under laminar flow (Clean Air, Mumbai, India).The purity of the ance followed by Tukey multiple comparison means to check the dif-culture was checked by subculturing 5 L of broth from the incubated ferences in bacterial inhibition between groups (P .01). The paireddentin block in TS broth on tryptone soya agar plates (HiMedia). The t test was used to check for differences in growth at different timedentin blocks were contaminated during a period of 21 days. Five intervals within groups and for differences at the 2 depths (P .01).blocks were picked randomly and assessed for the depth of penetrationof E. faecalis by using light microscopy and scanning electron micros-copy. ResultsAntimicrobial Assessment The current study showed that all 4 medicaments studied exerted After the incubation period, the blocks were irrigated with 5 mL of antibacterial activity. The scanning electron microscopy and light mi-sterile saline to remove the incubation broth. The dentin blocks were as- croscopy evaluation of 5 dentin blocks showed invasion of the bacteriasigned to the following groups (n 45 dentin blocks): group 1, saline within the dentinal tubules. Infection of the dentin blocks was confirmed(negative control); group 2, calcium hydroxide (Ref no.23,923-2; Sigma- when debris samples harvested from the saline group (negative con-Aldrich, Mumbai, India); group 3, bioactive glass S53P4 (AbminDent; Ab- trol) yielded positive growth. Table 1 shows the antibacterial activity,min Technologies Ltd, Turku, Finland); group 4, 2% chlorhexidine gel (Ref measured at 2 depths (200 and 400 m) and at 3 time intervals (1, 3,no.24800; Sigma-Aldrich); and group 5, 2% metronidazole gel (Ref and 5 days). The inhibition of growth in all the groups was statisticallyno.M3761-5G; Sigma-Aldrich). significant in comparison to the control group (saline). Group 4 (2% According to Fava and Saunders (13), the antibacterial activity of chlorhexidine gel) was the most effective against E. faecalis to the depthintracanal medicaments is enhanced by the vehicle used. Hence, appro- of 400 m on all days of incubation. Intergroup comparison showedpriate vehicles were chosen for the individual medicament as described that the inhibition in group 5 (2% metronidazole gel) was statisticallybelow. significant compared with groups 2 and 3 (calcium hydroxide and Bioactive glass S53P4 with a particle size of less than 45 m bioactive glass, respectively). Intergroup comparison of inhibition be-enables the obtainment of a paste-like consistency when mixed with tween groups 2 and 3 showed no statistical difference.saline. The bioactive glass S53P4 powder used in this study is composed The inhibition of growth of E. faecalis at 200 and 400 m wasof 53% SiO2 (wt/wt), 23% Na2O, 20% CaO, and 4%P2O5 (12). Group1 uniform with no statistical difference.received no medicament. In groups 2 and 3, the powder was mixed with The inhibition of growth of E. faecalis at the end of day 1, day 3,sterile saline in the ratio of 1.5:1 (wt/vol) to obtain a paste-like consis- and day 5 was varied with different medicaments. Calcium hydroxidetency. This was carried into the canal by using a plastic instrument andcondensed with a hand plugger. Polyethylene glycol 400 (HiMedia) was showed an increased antibacterial activity on day 3, which declined byused as a vehicle in group 4. In a pilot study, metronidazole was found day 5. Bioactive glass showed a gradual increase in antibacterial activityto dissolve better in glycerin than in polyethylene glycol. Hence, purified at the end of day 5. Two percent metronidazole gel showed a decreaseglycerin (HiMedia) was used as the vehicle in group 5. Methyl cellulose in antibacterial activity at the end of day 5. However, the differences inwas used as a thickening agent in both groups. All blocks after medica- antibacterial potential of the medicaments during different time inter-tion were sealed above and below with paraffin wax and incubated in an vals were not statistically significant. Two percent chlorhexidine gelanaerobic environment at 37°C. showed complete inhibition of E. faecalis on all time intervals. Antibacterial assessment was performed at the end of 1, 3, and 5 To summarize the results, the overall percentage inhibition at 2days, with 15 blocks from each group for every time interval. The blocks depths and different time intervals was 100% with 2% chlorhexidine gel,were washed with 5 mL of sterile saline combined with ultrasonics to 86.5% with 2% metronidazole gel, followed by 62.8% with bioactiveremove the medicament. Dentin debris was harvested at 2 depths (200 glass and 58.5% with calcium hydroxide (Figure 1).1474 Krithikadatta et al. JOE — Volume 33, Number 12, December 2007
  3. 3. Basic Research—Technology when compared with gram-positive bacteria (21). On the contrary, it was found in the present study that E. faecalis, a gram-positive bacte- rium, was found to be resistant to the effects of bioactive glass. This strange phenomenon calls for further exploration. Antibacterial activity of bioactive glass increases with time (12), and similar observations were seen in this study. Metronidazole is known to be more effective against obligate an- aerobic bacteria than on aerobic and facultative anaerobic bacteria (22). But the present study showed it to have antibacterial effect even on E. faecalis, a facultative anaerobic bacterium. Anaerobic bacteria con- tain electron transport components such as ferrodoxin that have a suf- ficiently negative redox potential to donate electrons to metronidazole. This single electron transfer forms a highly reactive nitro radical anion that kills susceptible organisms by radical-mediated mechanism that targets DNA and other biomolecules (22). A complete elimination of E. faecalis might be achieved by increasing the concentration of met-Figure 1. Percentage reduction in bacterial growth at 200 and 400 m for ronidazole (23).different medicaments. In the present study, 2% chlorhexidine gel provided 100% inhibi- tion of E. faecalis at the depths of 200 m as well as 400 m from day 1 to day 5. The plausible reason could be the bactericidal dosage of 2% Discussion and increased diffusion of the medicament into the dentinal tubules. This article reports on the disinfection potential of 4 intracanal Basrani et al (11) observed that 2% chlorhexidine gel produced a bettermedicaments. The model proposed by Haapasalo and Orstavik (10) has antimicrobial action when compared with 0.2% chlorhexidine gel orbeen modified for this study. Human permanent teeth were used instead calcium hydroxide mixed with 0.2% chlorhexidine. In another study, aof the bovine teeth as suggested by Basrani et al (11, 14). The canal 10-minute irrigation with 2% chlorhexidine before obturation of thelumens of the bovine blocks were 3 times larger than those of human root canal resulted in complete elimination of E. faecalis (24). Twoblocks, thus influencing the antimicrobial activity of certain medica- percent chlorhexidine gel has been reported to have sustained antibac-ments (14). In addition, studies with human dentin blocks would def- terial action beginning from the first day of medication (25). Similarinitely be more suitable to simulate the clinical scenario. results were found in this study. Basrani et al (26) found lower contact The results of the present study revealed significant information on angle in preparations containing chlorhexidine, enabling better diffu-the newer intracanal medicaments against E. faecalis. On average, com- sion into the tubules.plete inhibition of E. faecalis at both depths (200 and 400 m) was Portenier et al (27) and Haapasalo et al (16) have shown inobserved with 2% chlorhexidine gel, followed by 86.5 % reduction with independent studies that dentin matrix and collagen type I have inhibi-2% metronidazole gel. However, bioactive glass and calcium hydroxide tory effect on chlorhexidine. But both studies tested a concentration ofhad an overall reduction of only 62.8% and 58.5%, respectively. 0.2% chlorhexidine, which is much lower than the concentration used There was no increase in antimicrobial effect of calcium hydroxide in the present study. The inhibitory effect of dentin on chlorhexidine canwhen left for longer periods in the root canal because the hydroxyl ions be overcome by increasing the concentration (16). This appears to bedo not pass through patent dentinal tubules to alkalize the medium true, because the present study used a higher concentration andsurrounding the teeth (15). Calcium hydroxide has a pH of 12, but achieved complete inhibition. However, caution must be exercisedbecause of the buffering effect of dentin, it is unlikely that this high pH when drawing conclusions to in vivo situations. There is plausibility ofwould be maintained within the dentinal tubules, enabling E. faecalis to negative interactions between endodontic disinfecting agents and thesurvive and replicate (16, 17). The results of our study also show no various compounds present in the root canal environment. This mightincrease in the inhibition over time and thus corroborate these findings. have a vital role in deciding the clinical effectiveness of antibacterialEvans et al (18) demonstrated that the proton pump activity of E. fae- agents (28, 29).calis offers resistance to high pH of calcium hydroxide. The ability of E. Under the limitations of the present study it can be concluded thatfaecalis to penetrate deep into the tubules is attributed to Ace, a bacte- 2% chlorhexidine was most effective against E. faecalis.rial adhesin (19). In this study, the results of antimicrobial activity of bioactive glassagainst E. faecalis were moderate. However, its mechanism of action is Acknowledgmentsnot completely understood. An increase in pH of the aqueous bioactive The authors thank Dr V. Mohan, Professor and Chairman, Ma-glass has been the principle behind the observed effects (20, 21), and dras Diabetes Research Foundation, for financial help for the study;this increase was found to be brought about by the surface deterioration Dr. Deepa Raj and Ms. M. Deepa, Research scientist, Madras Diabetesof glass, which releases sodium oxide (12). The smaller the particle size Research Foundation, for assistance with the statistics; and Dr Mat-of the glass, the greater is the surface deterioration (21). thais Zehnder, Professor, Division of Endodontology, Department of According to Zehnder et al (12), a mixture of bioactive glass and Preventive Dentistry, Periodontology and Cariology, University ofdentin increased the antibacterial efficacy of bioactive glass. They hy- Zurich Center for Dental Medicine, for providing the bioactive glass.pothesized that dentin might serve as a source of Ca and P ions, whichallows bioactive glass to mineralize the bacterial cell wall. However,even though the dentin block model was used in the present study, the Referencesoverall percentage reduction of E. faecalis was only 62.2%. 1. Kakehashi S, Stanley HR, Fitzgerald. The effects of surgical exposure of dental pulps Gram-negative bacteria have an efficient permeability barrier that in germfree and conventional laboratory rats. J South Calif Dent Assoccan explain the relative resistance of these bacteria to bioactive glass 1966;34:449 –51.JOE — Volume 33, Number 12, December 2007 Disinfection of Dentinal Tubules 1475
  4. 4. Basic Research—Technology 2. Sirén EK, Haapasalo MPP, Ranta K, Salmi P, Kerosuo ENJ. Microbiological findings 17. Cwikla SJ, Bélanger M, Giguère S, Fox A P, Vertucci FJ. Dentinal tubule disinfection and clinical treatment procedures in endodontic cases selected for microbiological using three calcium hydroxide formulations. J Endod 2005;31:50 –2. investigation. Int Endod J 1997;30:90 –5. 18. Evans M, Davies JK, Sundquist G, Figdor D. Mechanisms involved in the resistance of 3. Portenier I, Waltimo T, Ø=rstavik D, Haapasalo M. The susceptibility of starved Enterococcus faecalis to calcium hydroxide. Int Endod J 2002;35:221– 8. stationary phase, and growing cells of Enterococcus faecalis to endodontic medi- 19. Kowalski WJ, Kasper EL, Hatton JF, Murry BE, Nallappareddy SR, Gillespie MJ. En- caments. J Endod 2005;31:380 –5. terococcus faecalis adhesin, ace, mediates attachment to particulate dentin. J Endod 4. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root 2006;32:634 –7. canal treatment failure and current concepts in retreatment. J Endod 2005;32:93– 8. 20. Allan I, Newman H, Wilson. Antibacterial activity of particulate bioglass against supra- 5. Safavi KE, Spångberg SW, Langeland K. Root canal dentinal tubule disinfection. and subgingival bacteria. Biomaterials 2001;22:1683–7. J Endod 1990;16:207–10. 21. Stoor P, Söderling E, Salonen JI. Antibacterial effects of a bioactive glass plate on oral 6. Chong BS, Pittford TR. The role of intracanal medication in root canal treatment. Int Endod J 1992;25:97–106. microorganisms. Acta Odontol Scand 1998,56:161–5. ´ 7. Sjögren U, Figdor D, Spångberg L, Sunquist G. The antimicrobial effect of calcium 22. Brunton LL, Lazo JS, Parker KL. Chemotherapy of protozoal infections. In: Goodman hydroxide as a short-term intracranial dressing. Int Endod J 1991;24:119 –25. and Gillman’s the pharmacological basis of therapeutics. 11th ed. New York: McGill- 8. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: Hill Companies, Inc, 2006:1021–70. critical review. Int Endod J 1999;2:361–369. 23. Crook J, Nandakumar R, Fouad AF. Molecular- and culture-based comparison of the 9. George S, Kishen A, Song KP. The role of environmental changes on monospecies effects of antimicrobial agents on bacterial survival in infected dentinal tubules. J biofilm formation on root canal wall by Enterococcus faecalis. J Endod Endod 2007;33:690 –2. 2005;31:867–72. 24. Gomes BPFA, Souza SFC, Ferraz CCR, et al. Effectiveness of 2% chlorhexidine gel and10. Haapasalo M, Ø=rstavik D. In vitro infection and disinfection of dentinal tubules. J calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Dent Res 1987;66:1375–9. Endod J 2003;36:267–75.11. Basrani B, Tjäderhane L, Santos JM, et al. Efficacy of chlorhexidine- and calcium 25. Siqueira JF, Uzeda M. Intracanal medicaments: evaluation of the antibacterial effects hydroxide- containing medicaments against Enterococcus faecalis in vitro study. of chlorhexidine, metronidazole and calcium hydroxide associated with three vehi- Oral Surg Oral Med Oral Pathol Radiol Endod 2003;96:618 –24. cles. J Endod 1997;23:167–9.12. Zehnder M, Söderling E, Salonen J, Waltimo T. Preliminary evaluation of bioactive 26. Basrani B, Ghanem A, Tjäderhane L. Physical and chemical properties of chlorhexi- glass S53P4 as an endodontic medication in vitro. J Endod 2004;30:220 – 4. dine and calcium hydroxide- containing medications. J Endod 2004;30:413–7.13. Fava LRG, Saunders WP. Calcium hydroxide pastes: classification and clinical indi- 27. Portenier I, Haapasalo H, Ø=rstavik D, Yamauchi M, Haapasalo M. Inactivation of the cations. Int Endod J 1999;32:257– 82. antibacterial activity of iodine potassium iodide and chlorhexidine digluconate14. Basrani B, Santos MJ, Tjäderhane L, et al. Substantive antimicrobial activity in chlor- hexidine-treated human root dentine. Oral Surg Oral Med Oral Pathol Radiol Endod against Enterococcus faecalis by dentin, dentin matrix, type-I collagen and heat 2002;94:240 –5. killed microbial whole cells. J Endod 2002;9:634 –7.15. Molander A, Reit C, Dahlén G. The antimicrobial effect of calcium hydroxide in root 28. Portenier I, Waltimo T, Orstavik D, Haapasalo M. Killing of Enterococcus faecalis by canals pretreated with 5% iodine potassium iodide. Endod Dent Traumatol MTAD and chlorhexidine digluconate with or without cetrimide in the presence or 1999;15:205–9. absence of dentine powder or BSA. J Endod 2006;32:138 – 41.16. Haapasalo HK, Siren EK, Waltimo TMT, Ø=rstavik D, Haapasalo MPP. Inactivation of local root 29. Haapasalo M, Qian W, Portenier I, Waltimo T. Effects of dentin on the antimicrobial canal medicaments by dentin: an in vitro study. Int Endod J 2000;33:126–32. properties of endodontic medicaments. J Endod 2007;33:917–25.1476 Krithikadatta et al. JOE — Volume 33, Number 12, December 2007