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Antimicrobial Activity of Triantibiotic Paste, 2% Chlorhexidine Gel, and Calcium Hydroxide on an Intraoral-infected Dentin Biofilm Model

Published:November 12, 2012DOI:https://doi.org/10.1016/j.joen.2012.10.004

      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.

      Key Words

      Infection 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 (
      • Trope M.
      Treatment of the immature tooth with a non-vital pulp and apical periodontitis.
      ). Classically, calcium hydroxide has been used to treat necrotic immature teeth in a procedure called apexification (
      • Mohammadi Z.
      • Dummer P.M.
      Properties and applications of calcium hydroxide in endodontics and dental traumatology.
      ). 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 (
      • Garcia-Godoy F.
      • Murray P.E.
      Recommendations for using regenerative endodontic procedures in permanent immature traumatized teeth.
      ).
      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 (
      • Trope M.
      Treatment of the immature tooth with a non-vital pulp and apical periodontitis.
      ,
      • Ding R.Y.
      • Cheung G.S.
      • Chen J.
      • et al.
      Pulp revascularization of immature teeth with apical periodontitis: a clinical study.
      ). This treatment includes root canal disinfection and placement of a matrix for cell ingrowth and a coronal seal to avoid recontamination (
      • Banchs F.
      • Trope M.
      Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol?.
      ,
      • Jung I.Y.
      • Lee S.J.
      • Hargreaves K.M.
      Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series.
      ). 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 (
      • Banchs F.
      • Trope M.
      Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol?.
      ,
      • Jung I.Y.
      • Lee S.J.
      • Hargreaves K.M.
      Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series.
      ,
      • Gomes J.E.
      • Duarte P.C.T.
      • de Oliveira C.B.
      • et al.
      Tissue reaction to a triantibiotic paste used for endodontic tissue self-regeneration of nonvital immature permanent teeth.
      ).
      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 (
      • Cehreli Z.C.
      • Isbitiren B.
      • Sara S.
      • Erbas G.
      Regenerative endodontic treatment (revascularization) of immature necrotic molars medicated with calcium hydroxide: a case series.
      ). 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 (
      • Barthel C.R.
      • Zimmer S.
      • Zilliges S.
      • et al.
      In situ antimicrobial effectiveness of chlorhexidine and calcium hydroxide: gel and paste versus gutta-percha points.
      ,
      • Dametto F.R.
      • Ferraz C.C.
      • Gomes B.P.
      • et al.
      In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis.
      ,
      • Gomes B.P.
      • Souza S.F.
      • Ferraz C.C.
      • et al.
      Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro.
      ,
      • Wang C.S.
      • Arnold R.R.
      • Trope M.
      • Teixeira F.B.
      Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria.
      ). It presents low toxicity and effective antimicrobial activity especially when compared with calcium hydroxide (
      • Gomes B.P.
      • Souza S.F.
      • Ferraz C.C.
      • et al.
      Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro.
      ,
      • Manzur A.
      • Gonzalez A.M.
      • Pozos A.
      • et al.
      Bacterial quantification in teeth with apical periodontitis related to instrumentation and different intracanal medications: a randomized clinical trial.
      ). Despite previous reports addressing the antimicrobial activity of triantibiotic paste (
      • Windley 3rd, W.
      • Teixeira F.
      • Levin L.
      • et al.
      Disinfection of immature teeth with a triple antibiotic paste.
      ) or 2% chlorhexidine gel (
      • Wang C.S.
      • Arnold R.R.
      • Trope M.
      • Teixeira F.B.
      Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria.
      ), its effect against microbial biofilms in comparison with commonly used antimicrobial dressings such as calcium hydroxide has not been 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, Ibiporã, Paraná, Brazil), 2% chlorhexidine gel (Biodinamica), and triantibiotic 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, São Paulo, Brazil), minocycline (minocycline cloridrate, 100 mg; Ranbaxy, Jacksonville, FL), and ciprofloxacin (ciprofloxacin cloridrate, 250 mg; Neoquimica, Anapolis, Goiás, Brazil) were used to prepare the triantibiotic medication. Five 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) (
      • Ordinola-Zapata R.
      • Bramante C.M.
      • Cavenago B.
      • et al.
      Antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model.
      ,
      • 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.
      ); 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-μm step size, and a format of 512 × 512 pixels. At least 5 μm 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 (
      • Chavez de Paz L.E.
      Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms.
      ) 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.
      Table 1Median 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 (%)Green 24 h (%)
      Saline (control)93.36 (45.87–98.98)a82.69 (35.49–97.74)a
      CaOH2 + DW63.71 (9.20–99.23)ab83.88 (55.46–96.51)a
      2% CHX28.33 (0.00–78.32)b18.70 (5.53–74.69)b
      TRIMIX1.37 (0.00–13.18)c1.57 (0.00–25.68)c
      CHX, chlorhexidine; DW, distilled water; TRIMIX, triantibiotic paste.
      Different superscript letters in each column represent statistical significance (P < .05).
      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 (Mann-Whitney 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 1AD.
      Figure thumbnail gr1
      Figure 1Confocal 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 × 275 μm. Bars represents 20 μm.

      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 (
      • Ding R.Y.
      • Cheung G.S.
      • Chen J.
      • et al.
      Pulp revascularization of immature teeth with apical periodontitis: a clinical study.
      ,
      • Jung I.Y.
      • Lee S.J.
      • Hargreaves K.M.
      Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series.
      ). 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 (
      • Dametto F.R.
      • Ferraz C.C.
      • Gomes B.P.
      • et al.
      In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis.
      ) because of its effective antimicrobial activity (
      • Gomes B.P.
      • Souza S.F.
      • Ferraz C.C.
      • et al.
      Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro.
      ,
      • Lenet B.J.
      • Komorowski R.
      • Wu X.Y.
      • et al.
      Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles.
      ). However, a previous clinical study showed a limited ability of this medication to kill bacteria (
      • Malkhassian G.
      • Manzur A.J.
      • 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.
      ). 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 (
      • Portenier I.
      • Haapasalo H.
      • Rye A.
      • et al.
      Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin.
      ,
      • 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.
      ) 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 (
      • Nakajo K.
      • Nakazawa F.
      • Iwaku M.
      • Hoshino E.
      Alkali-resistant bacteria in root canal systems.
      ,
      • Chavez de Paz L.E.
      • Bergenholtz G.
      • Dahlen G.
      • Svensater G.
      Response to alkaline stress by root canal bacteria in biofilms.
      ,
      • van der Waal S.V.
      • van der Sluis L.W.
      • Ozok A.R.
      • et al.
      The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study.
      ,
      • Distel J.W.
      • Hatton J.F.
      • Gillespie M.J.
      Biofilm formation in medicated root canals.
      ). 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 (
      • Greenstein G.
      • Tonetti M.
      The role of controlled drug delivery for periodontitis. The Research, Science and Therapy Committee of the American Academy of Periodontology.
      ,
      • Quirynen M.
      • Teughels W.
      • De Soete M.
      • van Steenberghe D.
      Topical antiseptics and antibiotics in the initial therapy of chronic adult periodontitis: microbiological aspects.
      ). Previous studies have shown the antimicrobial activity of these medications against oral bacteria, and its ability to sterilize infected dentin has been confirmed (
      • Windley 3rd, W.
      • Teixeira F.
      • Levin L.
      • et al.
      Disinfection of immature teeth with a triple antibiotic paste.
      ,
      • 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.
      ,
      • 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.
      ). 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 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 rich-medium 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 (
      • Lenet B.J.
      • Komorowski R.
      • Wu X.Y.
      • et al.
      Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles.
      ,
      • Baker P.J.
      • Evans R.T.
      • Slots J.
      • Genco R.J.
      Susceptibility of human oral anaerobic bacteria to antibiotics suitable for topical use.
      ,
      • Baker P.J.
      • Evans R.T.
      • Coburn R.A.
      • Genco R.J.
      Tetracycline and its derivatives strongly bind to and are released from the tooth surface in active form.
      ). 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 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 (
      • Kim J.H.
      • Kim Y.
      • Shin S.J.
      • et al.
      Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report.
      ). In addition, an adequate antibiotic concentration that avoids toxicity to host stem cells has not been completely addressed yet (
      • Ruparel N.T.F.
      • Ferraz C.
      • Diogenes A.
      Direct effect of intracanal medicaments on survival of stem cells of the apical papilla.
      ). 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.

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