Basic Research—Technology
Influence of the Dentinal Wall on the pH of Sodium Hypochlorite during Root Canal Irrigation Ricardo Gomes Macedo, DMD, MSc,* Noemi Pascual Herrero, DDS,* Paul Wesselink, PhD,* Michel Versluis, PhD,†‡§ and Luc van der Sluis, DDS, PhDjj Abstract Introduction: The purpose of this study was to evaluate the influence of dentin on the pH levels of different concentrations of sodium hypochlorite (NaOCl) solutions over time and to evaluate if preconditioning of dentin with 17% EDTA or agitation of the NaOCl solution influences these pH levels. Methods: A novel clinically representative model that scales with the ratio of the irrigant volume to the dentin surface area of a human root canal was used. Three standardized bovine dentin bars (2 2 10 mm) were placed in a plastic test tube. A total of 150 tubes were distributed in 29 groups. In the first experiment, the pH of various NaOCl solutions, with different concentrations (3%, 6%, and 9%) and starting pH levels (5 and 12), was monitored during exposure to dentin between 10 and 300 seconds. In a second experiment, the effect of agitation (45 Hz) and pretreatment of dentin with 17% EDTA on the pH levels of various NaOCl solutions was studied after 30 seconds of exposure to dentin. The short-term chemical stability of the tested solutions was assessed for both the concentration and the pH. Results: The exposure time (P < .001) and concentration of the NaOCl solution (P < .011) significantly influence the pH level after exposure to dentin. However, the change in pH is too small to induce a change in the irrigant antimicrobial/tissue dissolution capacity. Conclusions: Agitation of the irrigant and preconditioning of the dentin did not alter the pH (P > .05). Both the pH 5 and pH 12 solutions were chemically stable for 1 hour. (J Endod 2014;-:1–4)
Key Words Agitation, buffer, dentin, EDTA, hypochlorite, hypochlorous acid, pH, stability From the *Department of Cariology, Endodontology and Pedodontology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University, Amsterdam, The Netherlands; †Physics of Fluids Group, ‡MIRA Institute for Biomedical Technology and Technical Medicine, and §MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands; and jjCenter for Dentistry and Oral Hygiene, University Medical Center Groningen, Groningen, The Netherlands. Address requests for reprints to Dr Ricardo Gomes Macedo, Department of Cariology, Endodontology and Pedodontology, Academic Center for Dentistry Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.12.018
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S
odium hypochlorite (NaOCl) is widely used as the primary root canal irrigant of choice (1) because of its unpaired action against microorganisms (2) and biofilm (3) and its unique capacity to efficiently dissolve pulp tissue (4) and organic components of the smear layer (5). The reaction of NaOCl with these components in the root canal system, including the root canal wall, will reduce the available chlorine (6). The reaction rate (chemical efficacy) and/or tissue dissolution capacity of NaOCl are significantly influenced by several parameters including concentration, exposure time, laser or ultrasonic energy applied (6), contact area (7, 8), temperature (4), interaction with other chemicals (9–11), and pH level (12). The pH of the solution determines the equilibrium of the freely available chlorine, the hypochlorite ion (OCl), and the hypochlorous acid (HOCl) (13) (Fig. 1). The equilibrium will influence the biological effect of NaOCl, which can be defined as its tissue-dissolving capacity and antimicrobial effect. In alkaline solutions (pH > 7.5), OCl prevails, which has a powerful oxidative effect and therefore a higher tissuedissolving capacity than HOCl (13). On the other hand, HOCl is more abundant in acidic solutions (3 < pH < 7.5). It has a powerful bactericidal effect probably because it is a smaller uncharged molecule, which can relatively easily penetrate the bacterial membrane. After penetration, it can result in protein degradation (14). However, contradicting findings have been reported in the endodontic literature. A reduction of the pH down to 5 did not show a decrease in the tissue dissolution of a NaOCl solution (15) nor did it show a change in its reaction rate when exposed to dentin (6). The buffer capacity of dentin (16) or any other component of the root canal system such as pulp tissue (12) could be an explanation for these results. Therefore, it is important to know the buffer capacity of the different components of the root canal system. The influence of the buffer capacity of dentin on the pH of a NaOCl solution has never been evaluated; therefore, the first aim of this study was to evaluate the influence of dentin on the pH levels of different concentrations of NaOCl solutions over time. The second aim was to evaluate if preconditioning of dentin with 17% EDTA or agitation of the NaOCl solution would influence the pH levels of NaOCl solutions. A clinically representative model that scales with the ratio of the irrigant volume to the dentin surface area of a human root canal was used. The null hypothesis was that there is no change in the pH level of NaOCl solutions with the concentration of the NaOCl solution, the agitation of it, or the preconditioning of dentin with EDTA.
Materials and Methods Dentin bars were prepared from the roots of bovine incisors immediately after extraction using a saw microtome (SP1600; Leica Microsystems, Glattbrugg, Switzerland) to a cross-section of 2 2 mm; the length of the bars was controlled using a stereomicroscope (Stemi SV6; Carl Zeiss, G€ottingen, Germany) and grinded to 10 mm. Two dentin bars were collected per root, always at the same location, from both sides of the root canal. For all the dentin bars, the orientation of the tubuli was perpendicular to the long axis of the bar. All biological material used was stored overnight in sterile MilliQ water (Millipore Corp, Billerica, MA) at 4 C to prevent bacterial growth. A total of 450 dentin bars (2 2 10 mm) were prepared, weighed, and distributed to 150 plastic sample tubes (volume = 1.5 mL, diameter = 2.5 mm, taper = 4%, length = 25 mm) (Thermo Fisher Scientific, Waltham, MA). Three dentin bars were
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Basic Research—Technology
Figure 1. Free available chlorine in solution with respect to pH. Temperature = 25 C (after Baker 1959 [28]).
included in each sample tube, and they had an average dentin surface area of 264 mm2 and a weight of 527.7 9.9 mg. The tubes were randomly distributed within 29 groups. Figure 2 represents the distribution of samples per groups. Three different concentrations of NaOCl solutions and 2 pH values were tested: 12% and 6% at pH 12 and 3% at pH 12 and 5. The pH 12 solutions were obtained by a dilution of 10%–15% NaOCl reagent grade (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) with Milli-Q water. The pH 5 solutions were obtained by titration of perchloric acid (HClO4 70%–72%) (Merck KGaA, Darmstadt, Germany). The pH and the concentration were measured before and after each test using a Sentron pH meter (Sentron Europe BV, Roden, The Netherlands) with a Sentron microelectrode (Topac Inc, Cohasset, MA) and a standard iodine/thiosulfate titration assay (17), respectively. The dentin bars placed in the sample tubes were irrigated with 0.6 mL of the NaOCl solution using a micropipette (Finnpipettes; MTX Lab Systems, Vienna, VA) for a time ranging from 10–300 seconds (Fig. 2). In a second experiment, the influence of agitation of the NaOCl solutions or preconditioning of dentin with 17% EDTA on the pH of the tested solutions was investigated (Fig. 2 [Experiment B]). During these tests, the dentin bars were exposed to a 3% and 6% NaOCl solution with a pH of 12 and 3% NaOCl solution with a pH of 5 for a time period of 30 seconds. Negative control groups without agitation were included for all solutions tested. To determine the effect of agitation, a vortex shaker was used at 45 Hz (Vortex Genie 2; The Lab Warehouse, London, UK). To
precondition the dentin surface with EDTA, the bars in the sample tube were immersed for 1 minute in 0.6 mL EDTA 17% (18). The dentin bars were immediately rinsed with Milli-Q water to stop the chelation, dried using absorbent papers (Kleenex, Milsons Point, NSW, Australia), and placed in new sample tubes before the experiments. The short-term chemical stability of the previously described solutions in 1.5-mL plastic tubes (Thermo Fisher Scientific) was assessed by measuring the amount of available chlorine using the iodometric titration assay. The pH of the solution was measured over time (10, 30, 45, and 60 minutes) using a pH meter and an electrode. Three measurements were performed for each solution at room temperature (21 C). The relation of the pH and free chlorine levels of the different NaOCl solutions was monitored using the Spearman rank correlation coefficient. Kruskal-Wallis 1-way analysis of variance was performed to assess the differences in the final pH levels between groups with different concentrations (3%, 6%, and 12%). Mann-Whitney U tests were performed to compare the effect of agitation and pretreatment of dentin with EDTA on the pH levels of different NaOCl solutions. For all tests, P values .05). The chemical stability assay showed that lowering the pH of the NaOCl solution to 5 using HClO4 reduces approximately 50% of the initial concentration of 6%. After preparing the solutions, the concentration of 3% and pH of 5 were kept constant for 1 hour at 21 C; therefore, the turnover of the freshly made NaOCl solutions used in this study was 1 hour.
Discussion This study contributes to the quantification of the effect of the buffering capacity of dentin on the pH of an NaOCl solution during root canal irrigation. It is known that the contact of NaOCl with dentin causes
Figure 2. Distribution of the samples per group. In experiment A, the buffering effect of dentin on various NaOCl solutions was monitored over time. In experiment B, the buffering effect of dentin was compared with that with agitation of the solution and by dentin preconditioning with 17% EDTA.
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Figure 3. The effect of the buffering capacity of dentin on the pH of NaOCl. The average pH levels of various NaOCl solutions as a function of the contact time with dentin (n = 3). The dashed line represents the pKa of HOCl (pH = 7.5) where both forms of active chlorine (OCl and HOCl) exist in equilibrium. The Spearman rank correlation coefficient shows a correlation coefficient of 1.00 and P values .05). Nonetheless, even with the extended exposure times, which are far from typical in endodontic practice, the pH levels were kept below 6.5 for the 3% NaOCl solution at a pH level of 5 and above 9.5 for NaOCl solutions initially at a pH level of 12. At these pH levels, the freely available chlorine is expected to be predominantly (>90%) HOCl for the acidic solutions and OCl for the basic counterparts (Fig. 1). Thus, we can conclude that although dentin had a significant buffering effect on the pH level for different NaOCl solutions, it will most likely not influence the biological effect. For a typical root canal with a working length of 15 mm, prepared until a master apical file size of 0.3 mm and a taper of 0.06, the volume is 7.42 mm3, and the dentin surface area is 35.4 mm2. This results in a volume to surface area ratio of 0.21 mm. The model used in this study is composed of 3 dentin bars with a dimension of 2 2 10 mm inside an inert plastic sample tube. The irrigant volume used was 0.6 mL, which was chosen to guarantee an optimum surface coverage of the bars within the liquid and to ensure a clinically realistic volume to surface ratio, which was 0.227 mm in the present study. Previous studies neglected the typical clinical conditions exposing large amounts of NaOCl with relatively small amounts of substrate to be dissolved (4, 8). Consequently, the expected deactivation of the irrigant solution (6) and the eventual buffering effect did not occur. NaOCl solutions with a lower pH have a higher antimicrobial effect when planktonic microorganisms are concerned (20). However, in the root canal, microorganisms are present in a biofilm (21). Characteristic of a biofilm is that the microorganisms are protected by a highly hydrated extracellular matrix (22). Therefore, the dynamics of the antimicrobial effect can be completely different for microorganisms that are contained in a biofilm. It has been suggested that penetration of chlorine in a biofilm follows a diffusion-reaction pattern in which chlorine will be reduced by the matrix material (23). A large variation of penetration of JOE — Volume -, Number -, - 2014
chlorine into the biofilm was detected depending on the structure of the biofilm. A study by Bremer et al (20) showed that a reduction of pH of NaOCl to 6.5 resulted in a better bactericidal effect compared with the use of the same concentration at a pH level of 12. This work was performed in a 2-species biofilm, with the results given for 1 of the 2 species of microorganisms present in the biofilm. Moreover, the effects on a root canal biofilm are not known. In contrast, OCl has proven to be more efficient in tissue dissolution than HOCl (14). The same authors suggested to alkalize NaOCl solutions using NaOH to compensate for the buffering effect that may occur whenever the solution is in contact with organic tissue inside the root canal. The contribution of dentin to such a buffering effect does not justify this extra step. Nonetheless, the influence of other substances present in the root canal in early stages of the treatment (ie, pulp tissue and biofilm) or retreatment (ie, sealer and filling material) were not evaluated here and should be the subject of further studies. For the later stages of the root canal treatment when the bulk of pulp tissue has been removed, alkalinization does not seem to be necessary. The initial concentration of the NaOCl solution was another factor that influenced the final pH. The lower the concentration, the higher the reduction of pH after exposure. This effect is significant (P < .01) for the highest concentration (12%), whereas there was no difference for the 3% and 6% NaOCl solutions. Clinically, concentrations between 0.5% and 6% have been recommended; however, the optimal clinical concentration is still a subject of controversy (24). In this work, extending the concentration range up to 12% does not suggest its use in endodontics but contributes to a full characterization of the influence of the concentration. Agitation of a solution inside the root canal stimulates the refreshment of the free chlorine available at the interface with organic tissues, whereas pretreatment of dentin with 17% EDTA removes the inorganic component of dentin and smear layer (5), exposing more collagen fibers. However, the effect of both variables does not influence the final pH of the NaOCl solution after being exposed to dentin for 30 seconds (P > .05). The exposure time was selected according to the best evidence presently available, which should govern the clinical procedures (25). The intermittent flush method with 3 cycles of refreshment with 2 mL NaOCl solution followed by 20 seconds of ultrasonic activation was found to be the optimum irrigation protocol to remove dentin debris from an oval-shaped canal (26). If the flow rate of the refreshment is 0.22 mL/s, as reported by Boutsioukis et al (27), then 30 seconds is the approximate clinical time of each cycle. It can be concluded that the exposure time with dentin and the concentration of the NaOCl solution significantly influence the pH of the solution (P < .001 and P < .01, respectively). However, the observed change in the pH level is too limited to induce a biological effect. Agitation of the irrigant and preconditioning of the dentin did not change the pH (P > .05). Both the solutions with a pH of 5 and 12 were chemically stable for 1 hour.
Acknowledgments The authors thank M. Hoogenkamp, I. Persoon (ACTA, The Netherlands), and P. Viana (APA, Portugal) for their valuable critical appraisal of the setup and I. Aartman (ACTA) for her advice on the statistics. The authors deny any conflicts of interest related to this study.
References 1. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod 2012;38:37–40.
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Basic Research—Technology 2. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 1999;12:147–79. 3. Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garcıa M, et al. Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod 2009;25:711–4. 4. Sirtes G, Waltimo T, Schaetzle M, et al. The effects of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. J Endod 2005;31:669–71. 5. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 6. Macedo R, Wesselink P, Zaccheo F, et al. Reaction rate of NaOCl in contact with bovine dentine: effect of activation, exposure time, concentration and pH. Int Endod J 2010;43:1108–15. 7. Rosenfeld EF, James GA, Burch BS. Vital pulp tissue response to sodium hypochlorite. J Endod 1978;4:140–6. 8. Moorer W, Wesselink P. Factors promoting the tissue dissolving capability of sodium hypochlorite. Int Endod J 1982;15:187–96. 9. Shiozawa A. Characterization of reactive oxygen species generated from the mixture of NaClO and H2O2 used as root canal irrigants. J Endod 2000;26:11–5. 10. Zehnder M, Kosicki D, Luder H, et al. Tissue-dissolving capacity and antibacterial effect of buffered and unbuffered hypochlorite solutions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:756–62. 11. Zehnder M, Schmidlin P, Sener B, et al. Chelation in root canal therapy reconsidered. J Endod 2005;31:817–20. 12. Jungbluth H, Marending M, De-Deus G, et al. Stabilizing sodium hypochlorite at high pH: effects on soft tissue and dentin. J Endod 2011;37:693–6. 13. Baker R. Studies on the reaction between sodium hypochlorite and proteins: 1. Physico-chemical study of the course of the reaction. Biochem J 1947;41:337. 14. Winter J, Ilbert M, Graf PC, et al. Bleach activates a redox-regulated chaperone by oxidative protein unfolding. Cell 2008;135:691–701. 15. Christensen CE, McNeal SF, Eleazer P. Effect of lowering the pH of sodium hypochlorite on dissolving tissue in vitro. J Endod 2008;34:449–52.
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16. 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. 17. Vogel AI. A Text-book of Quantitative Inorganic Analysis. London, UK: Longman; 1962. 18. Lottanti S, Gautschi H, Sener B, et al. Effects of ethylenediaminetetraacetic, etidronic and peracetic acid irrigation on human root dentine and the smear layer. Int Endod J 2009;42:335–43. 19. Macedo R, Verhaagen B, Wesselink P, et al. Influence of refreshment/activation cycles and temperature rise on the reaction rate of sodium hypochlorite with bovine dentine during ultrasonic activated irrigation. Int Endod J 2014;47:147–54. 20. Bremer P, Monk I, Butler R. Inactivation of Listeria monocytogenes/Flavobacterium spp. biofilms using chlorine: impact of substrate, pH, time and concentration. Lett Appl Microbiol 2002;35:321–5. 21. Ricucci D, Siqueira JF Jr. Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings. J Endod 2010;36: 1277–88. 22. Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8:623–33. 23. De Beer D, Srinivasan R, Stewart PS. Direct measurement of chlorine penetration into biofilms during disinfection. Appl Environ Microbiol 1994;60:4339–44. 24. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98. 25. Liang YH, Jiang LM, Jiang L, et al. Radiographic healing following root canal treatments performed in single-rooted teeth with and without ultrasonic activation of the irrigant: a randomized controlled trial. J Endod 2013;39:1218–25. 26. van der Sluis LW, Vogels MP, Verhaagen B, et al. Study on the influence of refreshment/activation cycles and irrigants on mechanical cleaning efficiency during ultrasonic activation of the irrigant. J Endod 2010;36:737–40. 27. Boutsioukis C, Lambrianidis T, Kastrinakis E, et al. Measurement of pressure and flow rates during irrigation of a root canal ex vivo with three endodontic needles. Int Endod J 2007;40:504–13. 28. Baker RJ. Types and significance of chlorine residuals. J Am Water Works Assoc 1959;51:1185–90.
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Influence of the Dentinal Wall on the pH of Sodium Hypochlorite during Root Canal Irrigation Ricardo Gomes Macedo, DMD, MSc,* Noemi Pascual Herrero, DDS,* Paul Wesselink, PhD,* Michel Versluis, PhD,†‡§ and Luc van der Sluis, DDS, PhDjj Abstract Introduction: The purpose of this study was to evaluate the influence of dentin on the pH levels of different concentrations of sodium hypochlorite (NaOCl) solutions over time and to evaluate if preconditioning of dentin with 17% EDTA or agitation of the NaOCl solution influences these pH levels. Methods: A novel clinically representative model that scales with the ratio of the irrigant volume to the dentin surface area of a human root canal was used. Three standardized bovine dentin bars (2 2 10 mm) were placed in a plastic test tube. A total of 150 tubes were distributed in 29 groups. In the first experiment, the pH of various NaOCl solutions, with different concentrations (3%, 6%, and 9%) and starting pH levels (5 and 12), was monitored during exposure to dentin between 10 and 300 seconds. In a second experiment, the effect of agitation (45 Hz) and pretreatment of dentin with 17% EDTA on the pH levels of various NaOCl solutions was studied after 30 seconds of exposure to dentin. The short-term chemical stability of the tested solutions was assessed for both the concentration and the pH. Results: The exposure time (P < .001) and concentration of the NaOCl solution (P < .011) significantly influence the pH level after exposure to dentin. However, the change in pH is too small to induce a change in the irrigant antimicrobial/tissue dissolution capacity. Conclusions: Agitation of the irrigant and preconditioning of the dentin did not alter the pH (P > .05). Both the pH 5 and pH 12 solutions were chemically stable for 1 hour. (J Endod 2014;-:1–4)
Key Words Agitation, buffer, dentin, EDTA, hypochlorite, hypochlorous acid, pH, stability From the *Department of Cariology, Endodontology and Pedodontology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University, Amsterdam, The Netherlands; †Physics of Fluids Group, ‡MIRA Institute for Biomedical Technology and Technical Medicine, and §MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands; and jjCenter for Dentistry and Oral Hygiene, University Medical Center Groningen, Groningen, The Netherlands. Address requests for reprints to Dr Ricardo Gomes Macedo, Department of Cariology, Endodontology and Pedodontology, Academic Center for Dentistry Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.12.018
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odium hypochlorite (NaOCl) is widely used as the primary root canal irrigant of choice (1) because of its unpaired action against microorganisms (2) and biofilm (3) and its unique capacity to efficiently dissolve pulp tissue (4) and organic components of the smear layer (5). The reaction of NaOCl with these components in the root canal system, including the root canal wall, will reduce the available chlorine (6). The reaction rate (chemical efficacy) and/or tissue dissolution capacity of NaOCl are significantly influenced by several parameters including concentration, exposure time, laser or ultrasonic energy applied (6), contact area (7, 8), temperature (4), interaction with other chemicals (9–11), and pH level (12). The pH of the solution determines the equilibrium of the freely available chlorine, the hypochlorite ion (OCl), and the hypochlorous acid (HOCl) (13) (Fig. 1). The equilibrium will influence the biological effect of NaOCl, which can be defined as its tissue-dissolving capacity and antimicrobial effect. In alkaline solutions (pH > 7.5), OCl prevails, which has a powerful oxidative effect and therefore a higher tissuedissolving capacity than HOCl (13). On the other hand, HOCl is more abundant in acidic solutions (3 < pH < 7.5). It has a powerful bactericidal effect probably because it is a smaller uncharged molecule, which can relatively easily penetrate the bacterial membrane. After penetration, it can result in protein degradation (14). However, contradicting findings have been reported in the endodontic literature. A reduction of the pH down to 5 did not show a decrease in the tissue dissolution of a NaOCl solution (15) nor did it show a change in its reaction rate when exposed to dentin (6). The buffer capacity of dentin (16) or any other component of the root canal system such as pulp tissue (12) could be an explanation for these results. Therefore, it is important to know the buffer capacity of the different components of the root canal system. The influence of the buffer capacity of dentin on the pH of a NaOCl solution has never been evaluated; therefore, the first aim of this study was to evaluate the influence of dentin on the pH levels of different concentrations of NaOCl solutions over time. The second aim was to evaluate if preconditioning of dentin with 17% EDTA or agitation of the NaOCl solution would influence the pH levels of NaOCl solutions. A clinically representative model that scales with the ratio of the irrigant volume to the dentin surface area of a human root canal was used. The null hypothesis was that there is no change in the pH level of NaOCl solutions with the concentration of the NaOCl solution, the agitation of it, or the preconditioning of dentin with EDTA.
Materials and Methods Dentin bars were prepared from the roots of bovine incisors immediately after extraction using a saw microtome (SP1600; Leica Microsystems, Glattbrugg, Switzerland) to a cross-section of 2 2 mm; the length of the bars was controlled using a stereomicroscope (Stemi SV6; Carl Zeiss, G€ottingen, Germany) and grinded to 10 mm. Two dentin bars were collected per root, always at the same location, from both sides of the root canal. For all the dentin bars, the orientation of the tubuli was perpendicular to the long axis of the bar. All biological material used was stored overnight in sterile MilliQ water (Millipore Corp, Billerica, MA) at 4 C to prevent bacterial growth. A total of 450 dentin bars (2 2 10 mm) were prepared, weighed, and distributed to 150 plastic sample tubes (volume = 1.5 mL, diameter = 2.5 mm, taper = 4%, length = 25 mm) (Thermo Fisher Scientific, Waltham, MA). Three dentin bars were
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Figure 1. Free available chlorine in solution with respect to pH. Temperature = 25 C (after Baker 1959 [28]).
included in each sample tube, and they had an average dentin surface area of 264 mm2 and a weight of 527.7 9.9 mg. The tubes were randomly distributed within 29 groups. Figure 2 represents the distribution of samples per groups. Three different concentrations of NaOCl solutions and 2 pH values were tested: 12% and 6% at pH 12 and 3% at pH 12 and 5. The pH 12 solutions were obtained by a dilution of 10%–15% NaOCl reagent grade (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) with Milli-Q water. The pH 5 solutions were obtained by titration of perchloric acid (HClO4 70%–72%) (Merck KGaA, Darmstadt, Germany). The pH and the concentration were measured before and after each test using a Sentron pH meter (Sentron Europe BV, Roden, The Netherlands) with a Sentron microelectrode (Topac Inc, Cohasset, MA) and a standard iodine/thiosulfate titration assay (17), respectively. The dentin bars placed in the sample tubes were irrigated with 0.6 mL of the NaOCl solution using a micropipette (Finnpipettes; MTX Lab Systems, Vienna, VA) for a time ranging from 10–300 seconds (Fig. 2). In a second experiment, the influence of agitation of the NaOCl solutions or preconditioning of dentin with 17% EDTA on the pH of the tested solutions was investigated (Fig. 2 [Experiment B]). During these tests, the dentin bars were exposed to a 3% and 6% NaOCl solution with a pH of 12 and 3% NaOCl solution with a pH of 5 for a time period of 30 seconds. Negative control groups without agitation were included for all solutions tested. To determine the effect of agitation, a vortex shaker was used at 45 Hz (Vortex Genie 2; The Lab Warehouse, London, UK). To
precondition the dentin surface with EDTA, the bars in the sample tube were immersed for 1 minute in 0.6 mL EDTA 17% (18). The dentin bars were immediately rinsed with Milli-Q water to stop the chelation, dried using absorbent papers (Kleenex, Milsons Point, NSW, Australia), and placed in new sample tubes before the experiments. The short-term chemical stability of the previously described solutions in 1.5-mL plastic tubes (Thermo Fisher Scientific) was assessed by measuring the amount of available chlorine using the iodometric titration assay. The pH of the solution was measured over time (10, 30, 45, and 60 minutes) using a pH meter and an electrode. Three measurements were performed for each solution at room temperature (21 C). The relation of the pH and free chlorine levels of the different NaOCl solutions was monitored using the Spearman rank correlation coefficient. Kruskal-Wallis 1-way analysis of variance was performed to assess the differences in the final pH levels between groups with different concentrations (3%, 6%, and 12%). Mann-Whitney U tests were performed to compare the effect of agitation and pretreatment of dentin with EDTA on the pH levels of different NaOCl solutions. For all tests, P values .05). The chemical stability assay showed that lowering the pH of the NaOCl solution to 5 using HClO4 reduces approximately 50% of the initial concentration of 6%. After preparing the solutions, the concentration of 3% and pH of 5 were kept constant for 1 hour at 21 C; therefore, the turnover of the freshly made NaOCl solutions used in this study was 1 hour.
Discussion This study contributes to the quantification of the effect of the buffering capacity of dentin on the pH of an NaOCl solution during root canal irrigation. It is known that the contact of NaOCl with dentin causes
Figure 2. Distribution of the samples per group. In experiment A, the buffering effect of dentin on various NaOCl solutions was monitored over time. In experiment B, the buffering effect of dentin was compared with that with agitation of the solution and by dentin preconditioning with 17% EDTA.
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Figure 3. The effect of the buffering capacity of dentin on the pH of NaOCl. The average pH levels of various NaOCl solutions as a function of the contact time with dentin (n = 3). The dashed line represents the pKa of HOCl (pH = 7.5) where both forms of active chlorine (OCl and HOCl) exist in equilibrium. The Spearman rank correlation coefficient shows a correlation coefficient of 1.00 and P values .05). Nonetheless, even with the extended exposure times, which are far from typical in endodontic practice, the pH levels were kept below 6.5 for the 3% NaOCl solution at a pH level of 5 and above 9.5 for NaOCl solutions initially at a pH level of 12. At these pH levels, the freely available chlorine is expected to be predominantly (>90%) HOCl for the acidic solutions and OCl for the basic counterparts (Fig. 1). Thus, we can conclude that although dentin had a significant buffering effect on the pH level for different NaOCl solutions, it will most likely not influence the biological effect. For a typical root canal with a working length of 15 mm, prepared until a master apical file size of 0.3 mm and a taper of 0.06, the volume is 7.42 mm3, and the dentin surface area is 35.4 mm2. This results in a volume to surface area ratio of 0.21 mm. The model used in this study is composed of 3 dentin bars with a dimension of 2 2 10 mm inside an inert plastic sample tube. The irrigant volume used was 0.6 mL, which was chosen to guarantee an optimum surface coverage of the bars within the liquid and to ensure a clinically realistic volume to surface ratio, which was 0.227 mm in the present study. Previous studies neglected the typical clinical conditions exposing large amounts of NaOCl with relatively small amounts of substrate to be dissolved (4, 8). Consequently, the expected deactivation of the irrigant solution (6) and the eventual buffering effect did not occur. NaOCl solutions with a lower pH have a higher antimicrobial effect when planktonic microorganisms are concerned (20). However, in the root canal, microorganisms are present in a biofilm (21). Characteristic of a biofilm is that the microorganisms are protected by a highly hydrated extracellular matrix (22). Therefore, the dynamics of the antimicrobial effect can be completely different for microorganisms that are contained in a biofilm. It has been suggested that penetration of chlorine in a biofilm follows a diffusion-reaction pattern in which chlorine will be reduced by the matrix material (23). A large variation of penetration of JOE — Volume -, Number -, - 2014
chlorine into the biofilm was detected depending on the structure of the biofilm. A study by Bremer et al (20) showed that a reduction of pH of NaOCl to 6.5 resulted in a better bactericidal effect compared with the use of the same concentration at a pH level of 12. This work was performed in a 2-species biofilm, with the results given for 1 of the 2 species of microorganisms present in the biofilm. Moreover, the effects on a root canal biofilm are not known. In contrast, OCl has proven to be more efficient in tissue dissolution than HOCl (14). The same authors suggested to alkalize NaOCl solutions using NaOH to compensate for the buffering effect that may occur whenever the solution is in contact with organic tissue inside the root canal. The contribution of dentin to such a buffering effect does not justify this extra step. Nonetheless, the influence of other substances present in the root canal in early stages of the treatment (ie, pulp tissue and biofilm) or retreatment (ie, sealer and filling material) were not evaluated here and should be the subject of further studies. For the later stages of the root canal treatment when the bulk of pulp tissue has been removed, alkalinization does not seem to be necessary. The initial concentration of the NaOCl solution was another factor that influenced the final pH. The lower the concentration, the higher the reduction of pH after exposure. This effect is significant (P < .01) for the highest concentration (12%), whereas there was no difference for the 3% and 6% NaOCl solutions. Clinically, concentrations between 0.5% and 6% have been recommended; however, the optimal clinical concentration is still a subject of controversy (24). In this work, extending the concentration range up to 12% does not suggest its use in endodontics but contributes to a full characterization of the influence of the concentration. Agitation of a solution inside the root canal stimulates the refreshment of the free chlorine available at the interface with organic tissues, whereas pretreatment of dentin with 17% EDTA removes the inorganic component of dentin and smear layer (5), exposing more collagen fibers. However, the effect of both variables does not influence the final pH of the NaOCl solution after being exposed to dentin for 30 seconds (P > .05). The exposure time was selected according to the best evidence presently available, which should govern the clinical procedures (25). The intermittent flush method with 3 cycles of refreshment with 2 mL NaOCl solution followed by 20 seconds of ultrasonic activation was found to be the optimum irrigation protocol to remove dentin debris from an oval-shaped canal (26). If the flow rate of the refreshment is 0.22 mL/s, as reported by Boutsioukis et al (27), then 30 seconds is the approximate clinical time of each cycle. It can be concluded that the exposure time with dentin and the concentration of the NaOCl solution significantly influence the pH of the solution (P < .001 and P < .01, respectively). However, the observed change in the pH level is too limited to induce a biological effect. Agitation of the irrigant and preconditioning of the dentin did not change the pH (P > .05). Both the solutions with a pH of 5 and 12 were chemically stable for 1 hour.
Acknowledgments The authors thank M. Hoogenkamp, I. Persoon (ACTA, The Netherlands), and P. Viana (APA, Portugal) for their valuable critical appraisal of the setup and I. Aartman (ACTA) for her advice on the statistics. The authors deny any conflicts of interest related to this study.
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