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Basic Research—Technology

Antimicrobial Activity of Triantibiotic Paste,
2% Chlorhexidine Gel, and Calcium Hydroxide
on a...
Basic Research—Technology
previously addressed. Thus, the aim of this study was to evaluate the
antimicrobial activity of ...
Basic Research—Technology

Figure 1. Confocal laser scanning microscopy of biofilms treated with (A) saline solution, (B) c...
Basic Research—Technology
permeability without disturbing the attached cells. This evaluation
procedure in which the cell ...
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Pd_f_s_en22_journal_of_endodontics_2013_ordinola_zapata(1)

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Pd_f_s_en22_journal_of_endodontics_2013_ordinola_zapata(1)

  1. 1. Basic Research—Technology Antimicrobial Activity of Triantibiotic Paste, 2% Chlorhexidine Gel, and Calcium Hydroxide on an Intraoral-infected Dentin Biofilm Model Ronald Ordinola-Zapata, DDS, MSc,* Clovis M. Bramante, DDS, PhD,* Paloma Gagliardi Minotti, DDS, MSc,* Bruno Cavalini Cavenago, DDS, MSc,* Roberto Brand~ o Garcia, DDS, PhD,* Norberti Bernardineli, DDS, PhD,* a David E. Jaramillo, DDS,† and Marco A. Hungaro Duarte, DDS, PhD* Abstract Introduction: The purpose of this study was to evaluate the antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste (ie, metronidazole, minocycline, and ciprofloxacin) by using an intraorally infected dentin biofilm model. Methods: Forty bovine dentin specimens were infected intraorally using a removable orthodontic device in order to induce the biofilm colonization of the dentin. Then, the samples were treated with the medications for 7 days. Saline solution was used as the control. Two evaluations were performed: immediately after the elimination of the medication and after incubation in brain-heart infusion medium for 24 hours. The Live/Dead technique (Invitrogen, Eugene, OR) and a confocal microscope were used to obtain the percentage of live cells. Nonparametric statistical tests were performed to show differences in the percentage of live cells among the groups (P < .05). Results: Calcium hydroxide and 2% chlorhexidine gel did not show statistical differences in the immediate evaluation. However, after application of the brain-heart infusion medium for 24 hours, 2% gel chlorhexidine showed a statistically lesser percentage of live cells in comparison with calcium hydroxide. The triantibiotic paste significantly showed a lower percentage of live cells in comparison with the 2% chlorhexidine gel and calcium hydroxide groups in the immediate and secondary (after 24 hours) evaluations. Conclusions: The triantibiotic paste was most effective at killing the bacteria in the biofilms on the intraorally infected dentin model in comparison with 2% chlorhexidine gel and calcium hydroxide. (J Endod 2013;39:115–118) Key Words Antimicrobials, bacteria, biofilms, dentin, root canal dressings I nfection of the pulp canal space of immature teeth is considered a challenging and complex problem. The thin dentinal walls of these teeth limit the mechanical instrumentation with traditional techniques, and the disinfection process is only dependent on the antimicrobial properties of the irrigant solution and the intracanal medications (1). Classically, calcium hydroxide has been used to treat necrotic immature teeth in a procedure called apexification (2). This procedure aims to create an environment that favors the placement of the root filling materials by the induction of an apical calcified barrier. However, this therapy does not favor the formation of dentin deposition on the radicular walls of immature teeth, which can lead to an increased risk of radicular fracture (3). Several case reports have shown that dentin deposition in root canal walls and continued root development of immature necrotic teeth can be possible by a treatment-denominated revascularization (1, 4). This treatment includes root canal disinfection and placement of a matrix for cell ingrowth and a coronal seal to avoid recontamination (5, 6). In these cases, the aim of the intracanal medication is to only promote disinfection. In order to support revascularization, sodium hypochlorite disinfection and the use of a triantibiotic paste have been recommended based on clinical results and biologic biocompatibility (5–7). The use of calcium hydroxide is commonly used for the apexification technique or the standard 2-visit treatment of necrotic mature teeth, but it is not indicated for revascularization procedures, except in isolated clinical reports (8). In order to evaluate the antimicrobial activity of triantibiotic paste against biofilms, the evaluation of medications such as calcium hydroxide that are not commonly used for revascularization procedures is also necessary for comparative purposes. Another common antimicrobial substance used in endodontic therapy includes 2% chlorhexidine. This antimicrobial substance in its gel form has been proposed as an intracanal dressing (9–12). It presents low toxicity and effective antimicrobial activity especially when compared with calcium hydroxide (11, 13). Despite previous reports addressing the antimicrobial activity of triantibiotic paste (14) or 2% chlorhexidine gel (12), its effect against microbial biofilms in comparison with commonly used antimicrobial dressings such as calcium hydroxide has not been From the *Department of Endodontics, Bauru Dental School of Bauru, University of S~o Paulo, Brazil; and †Department of Endodontics, Loma Linda School of a Dentistry, Loma Linda, California. Supported by the Brazilian Funding Agency FAPESP (grant no. 2010/16002-4). Address requests for reprints to Dr Ronald Ordinola-Zapata, Faculdade de Odontologia de Bauru–USP, Al Octvio Pinheiro Brisolla, 9-75-CEP 17012-901, Bauru, S~o a a Paulo, Brazil. E-mail address: ronaldordinola@usp.br 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.004 JOE — Volume 39, Number 1, January 2013 Antimicrobial Activity of Intracanal Medications 115
  2. 2. Basic Research—Technology previously addressed. Thus, the aim of this study was to evaluate the antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste on the intraorally infected dentin biofilm model. Material and Methods The evaluated medications were calcium hydroxide (Biodinamica, Ibipor~, Paran, Brazil), 2% chlorhexidine gel (Biodinamica), and tria a antibiotic paste. The medications were left in contact with the infected dentin for 7 days. A saline solution was used for 7 days for control purposes. Forty sterile bovine dentin sections (2  2  2 mm) were used (n = 10). The samples were treated with 17% EDTA for 3 minutes to eliminate the smear layer produced during the sectioning process. To test the antimicrobial activity against oral bacteria, an in situ model of intraorally dentin infection was selected. The dentin samples were fixed into the cavities of a Hawley’s orthodontic device with sticky wax. The dentin surface in contact with the oral cavity was fixed 1 mm above the surface to favor the accumulation of plaque. The device was used by 1 volunteer for 72 hours in order to induce the plaque formation and the subsequent surface infection of dentin. Regular oral hygiene practices were maintained (Human Committee for Ethical Research, CEP134/2010). After the intraoral infection process, each sample was incubated in 2 mL brain-heart infusion (BHI) medium at 37 C for 24 hours in aerobic conditions. Then, 1 mL saline solution was used to eliminate the culture medium and the nonadherent cells. For the direct contact test, the infected dentin samples were immersed in the medications using 12-well tissue culture plates for 7 days at 37 C. The calcium hydroxide paste was prepared by using 1 g calcium hydroxide powder mixed with 1 mL distilled water. For the 2% chlorhexidine gel medication, 1 mL was used. Metronidazole (250 mg; Flagyl Aventis Pharma, S~o Paulo, Brazil), minocycline a (minocycline cloridrate, 100 mg; Ranbaxy, Jacksonville, FL), and ciprofloxacin (ciprofloxacin cloridrate, 250 mg; Neoquimica, Anapolis, Gois, Brazil) were used to prepare the triantibiotic medication. Five a hundred milligrams of each antibiotic was homogenized to obtain a uniform powder. Propylene glycol was added to the antibiotic powder in an approximate proportion of 7:4 powder/propylene glycol and then mixed to obtain a paste-like consistency. The saline solution was used for 7 days for control purposes. Ten dentin blocks were used to test each medication and the control; 40 blocks were analyzed. After 7 days, the medications were washed with saline solution for 5 minutes. Five treated dentin blocks were immediately evaluated by confocal analysis, and 5 blocks were immersed in 2 mL fresh BHI and incubated at 37 C. These last samples were evaluated after 24 hours of the elimination of the antimicrobial substances. These samples were used to test if the residual microflora has the ability to recover from the antimicrobial stress. After 24 hours, the samples were washed with the saline solution, stained, and evaluated in a similar fashion to the first samples. The analysis of biofilm viability was performed by using the SYTO 9/propidium iodide technique (Live/Dead, Bacligth, Invitrogen, Eugene, OR) (15, 16); SYTO 9 is a green-fluorescent stain that labels both live and dead microorganisms. Propidium iodide is a red-fluorescent nucleic acid stain and only penetrates the cells with damaged membranes (dead microbes). A confocal laser scanning microscope (Leica TCS-SPE; Leica Biosystems CMS, Mannheim, Germany) was used to perform the analysis. The respective absorption and emission wavelengths were 494/518 nm for SYTO 9 and 536/617 nm for propidium iodide. Four confocal ‘‘stacks’’ from random areas were obtained from each sample using a 40 oil lens, a 1-mm step size, and a format of 512  512 pixels. At least 116 Ordinola-Zapata et al. 5 mm of the scanning included the subsurface level of the dentin. In total, 20 stacks (4 operative fields  5 samples) were obtained for each medication immediately after the removal of the medication, and 20 stacks were obtained after 24 hours of incubation with BHI. For quantification purposes, bioImage_L software (www.bioImageL.com) was used (17) to calculate the percentage of the volume of live cells found after the antimicrobial treatment. Statistical analysis of the percentage of live cells in the samples evaluated after 7 days of contact or after an additional 24 hours of reincubation was performed using the nonparametric Kruskal-Wallis and Dunn tests (P .05) by the absence of a normal distribution confirmed in the preliminary analysis. The Mann-Whitney U test was used to compare the effect of an immediate and a 24-hour evaluation. Prisma 5.0 software (GraphPad Software Inc, La Jolla, CA) was used as the analytic tool. Results The median and range of the percentage of live cells in the evaluated groups are shown in Table 1. Overall, calcium hydroxide showed the weakest antimicrobial activity, and the triantibiotic mix showed the highest with or without 24 hours of additional reincubation in BHI. Calcium hydroxide and 2% chlorhexidine gel did not show statistical differences (P .05) in the immediate evaluation; however, after the application of BHI for 24 hours, 2% chlorhexidine gel showed a statistically (P .05) lesser percentage of live cells in comparison with calcium hydroxide. This effect was observed because the calcium hydroxide–treated dentin significantly increased the percentage of live cells from 63.71% to 83.88% after the additional BHI treatment (MannWhitney U test, P .05). The additional BHI treatment did not show any effect on 2% chlorhexidine gel or in the triantibiotic paste–treated dentin (Mann-Whitney U test, P .05). The triantibiotic paste showed the lowest percentage of live cells in comparison with the 2% chlorhexidine gel and the calcium hydroxide groups in the immediate and secondary (after 24 hours) evaluations (P .05). Representative pictures of the evaluated samples are shown in Figure 1A–D. Discussion Disinfection of immature teeth can be considered a challenge that needs specific disinfection procedures when compared with conventional endodontic treatment. In this context, the use of triantibiotic paste has been previously reported for the treatment of necrotic teeth with open apexes (4, 6). For methodologic purposes, this study was designed to test the isolated effect of 3 root canal medications used for disinfection purposes in endodontic therapy. However, during clinical situations intracanal dressings are only one of the steps to treat endodontic infections. The antimicrobial control stage also includes the use of irrigant solutions and mechanical instrumentation to treat the previously infected dentinal biofilm. Two percent chlorhexidine gel has been proposed as an intracanal dressing (10) because of its effective antimicrobial activity (11, 18). TABLE 1. Median and Range Values of the Percentage of Live Cells after Contact with the Experimental Medicaments for 1 Week Evaluated Immediately (Green) and after an Additional 24 Hours of Incubation in BHI (Green 24 h) Green (%) Saline (control) CaOH2 + DW 2% CHX TRIMIX Green 24 h (%) a 93.36 (45.87–98.98) 63.71 (9.20–99.23)ab 28.33 (0.00–78.32)b 1.37 (0.00–13.18)c 82.69 (35.49–97.74)a 83.88 (55.46–96.51)a 18.70 (5.53–74.69)b 1.57 (0.00–25.68)c CHX, chlorhexidine; DW, distilled water; TRIMIX, triantibiotic paste. Different superscript letters in each column represent statistical significance (P .05). JOE — Volume 39, Number 1, January 2013
  3. 3. Basic Research—Technology Figure 1. Confocal laser scanning microscopy of biofilms treated with (A) saline solution, (B) calcium hydroxide, (C) 2% chlorhexidine gel, and (D) triantibiotic paste. Live cells are seen in green, and dead cells are seen in red. Each picture represents an area of 275 Â 275 mm. Bars represents 20 mm. However, a previous clinical study showed a limited ability of this medication to kill bacteria (19). In the present work, contrary to the results found in the triantibiotic paste, the variability of live cells found in 2% chlorhexidine and calcium hydroxide was higher. This probably could be explained by the effect of neutralizing substances (20, 21) that can be found in the biofilms as dead cells or an exopolymeric matrix. However, the antimicrobial effect of 2% chlorhexidine gel was superior to calcium hydroxide, which does not show a significant effect against the biofilm. According to previous studies, an alkaline microbial effect is not effective in killing bacteria in the form of biofilms (22–25). The findings of the current research are consistent with the cited studies. The reason for this is that the amount of hydroxyl ions reached after 1 week is probably not suitably high enough to promote antimicrobial activity. These results suggest that previous disorganization of biofilm by sodium hypochlorite treatment could be a necessary condition to enhance the effect of intracanal dressings. Triantibiotic paste includes metronidazole and minocycline. These antibiotics have been used in periodontics in order to suppress subgingival microbiota as an adjunctive therapy (26, 27). Previous studies have shown the antimicrobial activity of these medications against oral bacteria, and its ability to sterilize infected dentin has been confirmed (14, 28, 29). Our findings showed a good ability of the triantibiotic paste to kill bacteria inside the biofilms in comparison with calcium hydroxide and 2% chlorhexidine gel. The antimicrobial medications evaluated in this study could not kill 100% of the bacteria inside the biofilm. Thus, it was important to JOE — Volume 39, Number 1, January 2013 test whether the residual bacteria could recolonize the chemically treated biofilms or not. In order to reach this hypothesis, an additional evaluation of the stressed biofilm after 24 hours of incubation in a richmedium was performed (BHI). The results showed that the triantibiotic paste– and 2% chlorhexidine gel–treated dentin do not significantly increase the number of live bacteria in comparison with calcium hydroxide. It is known that minocycline (a tetracycline derivate) present in a triantibiotic paste formula and chlorhexidine presents substantivity effects (18, 30, 31). This could explain the difficulty of residual live bacteria to repopulate in the triantibiotic- and chlorhexidine-treated biofilms. In contrast, calcium hydroxide–treated dentin significantly increased the percentage of live cells from 63.71% to 83.88% after an additional BHI treatment. This result shows the following: 1. The alkaline effect can be neutralized. 2. The biofilm can increase the proportion of live cells if new nutrients are available. The inactivation of medications is essential for culture-based methods. In culture plate methodology, the antimicrobial activity is measured by the ability of surviving bacteria to produce clones. This procedure is evaluated after the detachment of the surface-associated biofilm, suspension in a transport media, and inoculation in agar plates. If residual antimicrobial compounds are introduced during these procedures, the conditions could not be permissive for the growth of new cells or clones. On the other hand, an advantage of confocal methodology is that it can evaluate in situ by direct observation the viability of microorganisms on the infected dentin by assessing the membrane Antimicrobial Activity of Intracanal Medications 117
  4. 4. Basic Research—Technology permeability without disturbing the attached cells. This evaluation procedure in which the cell viability is measured directly on the infected surface is similar to clinical conditions in which no inactivation of antimicrobial substances is performed during the endodontic treatment. Although the triantibiotic paste showed good antimicrobial activity, it presented undesirable effects such as dentin staining (32). In addition, an adequate antibiotic concentration that avoids toxicity to host stem cells has not been completely addressed yet (33). Thus, there is the necessity to search for other antimicrobial or antibiotic medications with similar useful properties to the tested triantibiotic paste but without having its associated deleterious side effects. Conclusion The triantibiotic paste was most effective at killing bacteria in the biofilms on the intraorally infected dentin model in comparison with 2% chlorhexidine gel and calcium hydroxide paste. Acknowledgments The authors deny any conflicts of interest related to this study. References 1. Trope M. Treatment of the immature tooth with a non-vital pulp and apical periodontitis. Dent Clin North Am 2010;54:313–24. 2. Mohammadi Z, Dummer PM. Properties and applications of calcium hydroxide in endodontics and dental traumatology. Int Endod J 2011;44:697–730. 3. Garcia-Godoy F, Murray PE. Recommendations for using regenerative endodontic procedures in permanent immature traumatized teeth. Dent Traumatol 2008;28: 33–41. 4. Ding RY, Cheung GS, Chen J, et al. Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod 2009;35:745–9. 5. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 2004;30:196–200. 6. Jung IY, Lee SJ, Hargreaves KM. Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. J Endod 2008;34:876–87. 7. Gomes JE, Duarte PCT, de Oliveira CB, et al. Tissue reaction to a triantibiotic paste used for endodontic tissue self-regeneration of nonvital immature permanent teeth. J Endod 2012;38:91–4. 8. Cehreli ZC, Isbitiren B, Sara S, Erbas G. Regenerative endodontic treatment (revascularization) of immature necrotic molars medicated with calcium hydroxide: a case series. J Endod 2011;37:1327–30. 9. Barthel CR, Zimmer S, Zilliges S, et al. In situ antimicrobial effectiveness of chlorhexidine and calcium hydroxide: gel and paste versus gutta-percha points. J Endod 2002;28:427–30. 10. Dametto FR, Ferraz CC, Gomes BP, et al. In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:768–72. 11. Gomes BP, Souza SF, Ferraz CC, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. 12. Wang CS, Arnold RR, Trope M, Teixeira FB. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283–9. 118 Ordinola-Zapata et al. 13. Manzur A, Gonzalez AM, Pozos A, et al. Bacterial quantification in teeth with apical periodontitis related to instrumentation and different intracanal medications: a randomized clinical trial. J Endod 2007;33:114–8. 14. Windley W 3rd, Teixeira F, Levin L, et al. Disinfection of immature teeth with a triple antibiotic paste. J Endod 2005;31:439–43. 15. Ordinola-Zapata R, Bramante CM, Cavenago B, et al. Antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model. Int Endod J 2012;45:162–8. 16. Shen Y, Qian W, Chung C, et al. Evaluation of the effect of two chlorhexidine preparations on biofilm bacteria in vitro: a three-dimensional quantitative analysis. J Endod 2009;35:981–5. 17. Chavez de Paz LE. Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms. Appl Environ Microbiol 2009;75:1734–9. 18. Lenet BJ, Komorowski R, Wu XY, et al. Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles. J Endod 2000;26: 652–5. 19. Malkhassian G, Manzur AJ, Legner M, et al. Antibacterial efficacy of MTAD final rinse and two percent chlorhexidine gel medication in teeth with apical periodontitis: a randomized double-blinded clinical trial. J Endod 2009;35:1483–90. 20. Portenier I, Haapasalo H, Rye A, et al. Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin. Int Endod J 2001;34: 184–8. 21. Portenier I, Haapasalo H, Orstavik D, et al. Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells. J Endod 2002;28:634–7. 22. Nakajo K, Nakazawa F, Iwaku M, Hoshino E. Alkali-resistant bacteria in root canal systems. Oral Microbiol Immunol 2004;19:390–4. 23. Chavez de Paz LE, Bergenholtz G, Dahlen G, Svensater G. Response to alkaline stress by root canal bacteria in biofilms. Int Endod J 2007;40:344–55. 24. van der Waal SV, van der Sluis LW, Ozok AR, et al. The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study. Int Endod J 2011;44:1110–7. 25. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod 2002;28:689–93. 26. Greenstein G, Tonetti M. The role of controlled drug delivery for periodontitis. The Research, Science and Therapy Committee of the American Academy of Periodontology. J Periodontol 2000;71:125–40. 27. Quirynen M, Teughels W, De Soete M, van Steenberghe D. Topical antiseptics and antibiotics in the initial therapy of chronic adult periodontitis: microbiological aspects. Periodontol 2000 2002;28:72–90. 28. Hoshino E, Kurihara-Ando N, Sato I, et al. In-vitro antibacterial susceptibility of bacteria taken from infected root dentine to a mixture of ciprofloxacin, metronidazole and minocycline. Int Endod J 1996;29:125–30. 29. Sato I, Ando-Kurihara N, Kota K, et al. Sterilization of infected root-canal dentine by topical application of a mixture of ciprofloxacin, metronidazole and minocycline in situ. Int Endod J 1996;29:118–24. 30. Baker PJ, Evans RT, Slots J, Genco RJ. Susceptibility of human oral anaerobic bacteria to antibiotics suitable for topical use. J Clin Periodontol 1985;12:201–8. 31. Baker PJ, Evans RT, Coburn RA, Genco RJ. Tetracycline and its derivatives strongly bind to and are released from the tooth surface in active form. J Periodontol 1983; 54:580–5. 32. Kim JH, Kim Y, Shin SJ, et al. Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report. J Endod 2010; 36:1086–91. 33. Ruparel NTF, Ferraz C, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372–5. JOE — Volume 39, Number 1, January 2013

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