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Effect of the Controlled Delivery of Chelating Agents on the Pulp Tissue Dissolution Ability of Fresh Sodium Hypochlorite Solutions

Open AccessPublished:December 24, 2022DOI:https://doi.org/10.1016/j.joen.2022.12.009

      Abstract

      Introduction

      The purpose of this study was to evaluate pulp tissue dissolution ability of sodium hypochlorite (NaOCl) when mixed with tetrasodic etidronate (Na4HEBP) and disodic ethylenediaminetetraacetate (Na2EDTA) under controlled flow.

      Methods

      Bovine pulp tissue was extracted from the lower incisors of 10 bovine jaws. Pulp specimens were standardized in size (1 × 3 × 10 mm), blotted dry, and weighed (initial weight [T0]: mean (SD) = 31.98 (1.18) mg). Specimens from the same jaw were randomly assigned to 2 control (n = 3 each) and 4 experimental groups (n = 10 each): NC (negative control/distilled water), PC (positive control/6%NaOCl), G1 (3%NaOCl), G2 (3%NaOCl-17% Na2EDTA), G3 (3%NaOCl-18% Na4HEBP), and G4 (3%NaOCl-9% Na4HEBP). Distilled water and NaOCl were provided using a delivery pump under a continuous controlled rate (1 mL/min). A second pump alternately delivered either Na2EDTA or Na4HEBP at the same rate with a 30-second programmed interval. Percentage of tissue weight loss was calculated at 2, 5, and 10 minutes (T2, T5, and T10) and compared among groups with analysis of variance. Free available chlorine and pH were controlled at T0 and T10.

      Results

      No tissue remained in PC at T5. No dissolution occurred in NC. There were no significant differences in the percentage of weight loss among experimental groups at any point of time. Some remnant tissue was found in G3 (1.4% ± 2.4) and G4 (1.6% ± 2.3) at T10, whereas nothing was left in G1 and G2.

      Conclusions

      The controlled delivery of Na2EDTA and Na4HEBP did not alter tissue dissolution ability of NaOCl when fresh solutions were mixed in the root canal.

      Key Words

      Contrary to the findings from studies that immersed tissue in premixed solutions of NaOCl and chelating agents, the controlled delivery of EDTA and etidronate did not alter tissue dissolution ability of NaOCl when fresh solutions were mixed in the root canal.
      The combination of mechanical preparation and chemical disinfection is necessary for a successful endodontic therapy
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      Changes in root canal geometry after preparation assessed by high-resolution computed tomography.
      . For this reason, manual or rotary root canal preparation needs to be combined with irrigating solutions. Since the ideal irrigant solution has not been described yet, a combination of irrigants is needed for proper tissue dissolution, reduction of biofilm, cleaning of unprepared areas and removal of debris, and the smear layer produced on the dentinal tubules by the action of endodontic shaping instruments
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      • Zehnder M.
      Root canal irrigants.
      . It is a strong oxidizing agent with an alkaline pH that relieves free available chlorine in solution. NaOCl is used in endodontics at varying concentrations from 1%–6%
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      Root canal irrigants.
      . The properties of NaOCl depend on its concentration, temperature, pH, frequency and intensity of mechanical agitation, and solution refreshment. Importantly, it is also affected by the surface area of the exposed tissue
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      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      .
      Chelating agents, such as different salts of ethylendiaminetetraacetic acid (EDTA), are used to remove the inorganic matter exerting a minimal tissue dissolution capacity and antimicrobial activity
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      • Bhat K.S.
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      . EDTA is a polyprotic acid, normally used in endodontics at concentrations varying from 10%–17%. Different forms of the salt of EDTA have been described: disodium (Na2EDTA), trisodium (Na3EDTA), and tetrasodium EDTA (Na4EDTA)
      • Tartari T.
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      • et al.
      Analysis of the effects of several decalcifying agents alone and in combination with sodium hypochlorite on the chemical composition of dentine.
      .
      Etidronate (also known as 1-hydroxyethylidene-1, 1-bisphosphonate or Na4HEBP) is a weak and biocompatible chelator with a pH varying from 10–12
      • Zehnder M.
      • Schmidlin P.
      • Sener B.
      • Waltimo T.
      Chelation in root canal therapy reconsidered.
      . Its use in combination with NaOCl has been suggested to reduce the formation of smear layer and debris accumulation during mechanical preparation of the root canal; however, it requires longer contact times to achieve a similar chelating effect as EDTA (5 minutes vs 1 minute)
      • De-Deus G.
      • Zehnder M.
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      • et al.
      Longitudinal co-site optical microscopy study on the chelating ability of etidronate and EDTA using a comparative single-tooth model.
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      Reduction of hard-tissue debris accumulation during rotary root canal instrumentation by etidronic acid in a sodium hypochlorite irrigant.
      .
      At the same time, chelators, like EDTA, interacted with NaOCl affecting drastically its tissue dissolution ability by reducing active chlorine content
      • Rossi-Fedele G.
      • Doramac E.J.
      • Guastalli A.R.
      • et al.
      Antagonistic interactions between sodium hypochlorite, chlorhexidine, EDTA, and citric acid.
      • Prado M.
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      • Rezende C.M.
      • et al.
      Interactions between irrigants commonly used in endodontic practice: a chemical analysis.
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      • Grande N.M.
      • Plotino G.
      • Falanga A.
      • et al.
      Interaction between EDTA and sodium hypochlorite: a nuclear magnetic resonance analysis.
      • Clarkson R.M.
      • Podlich H.M.
      • Moule A.J.
      Influence of ethylenediaminetetraacetic acid on the active chlorine content of sodium hypochlorite solutions when mixed in various proportions.
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      . For this reason, the mixture and alternate use of NaOCl and EDTA during root canal preparation was discouraged, and the use of chelators was only recommended at the end of the shaping procedure instead
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      . This effect was not observed with Na4EDTA or HEBP that, when combined with NaOCl, the solution promoted smear layer removal and organic matter dissolution
      • Tartari T.
      • Oda D.F.
      • Zancan R.F.
      • et al.
      Mixture of alkaline tetrasodium EDTA with sodium hypochlorite promotes in vitro smear layer removal and organic matter dissolution during biomechanical preparation.
      ,
      • Biel P.
      • Mohn D.
      • Attin T.
      • Zehnder M.
      Interactions between the tetrasodium salts of EDTA and 1-hydroxyethane 1, 1-diphosphonic acid with sodium hypochlorite irrigants.
      . Moreover, NaOCl did not affect the calcium chelation ability either of EDTA or HEBP
      • Grande N.M.
      • Plotino G.
      • Falanga A.
      • et al.
      Interaction between EDTA and sodium hypochlorite: a nuclear magnetic resonance analysis.
      ,
      • Biel P.
      • Mohn D.
      • Attin T.
      • Zehnder M.
      Interactions between the tetrasodium salts of EDTA and 1-hydroxyethane 1, 1-diphosphonic acid with sodium hypochlorite irrigants.
      .
      Nevertheless, the most published research on the dissolution effect of root canal irrigants has been carried out in static models with mixed solutions at different ratios
      • Cullen J.K.T.
      • Wealleans J.A.
      • Kirkpatrick T.C.
      • Yaccino J.M.
      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      ,
      • Prado M.
      • Santos Júnior H.M.
      • Rezende C.M.
      • et al.
      Interactions between irrigants commonly used in endodontic practice: a chemical analysis.
      ,
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      ,
      • Clarkson R.M.
      • Podlich H.M.
      • Moule A.J.
      Influence of ethylenediaminetetraacetic acid on the active chlorine content of sodium hypochlorite solutions when mixed in various proportions.
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      • Tartari T.
      • Oda D.F.
      • Zancan R.F.
      • et al.
      Mixture of alkaline tetrasodium EDTA with sodium hypochlorite promotes in vitro smear layer removal and organic matter dissolution during biomechanical preparation.
      ,
      • Tartari T.
      • Guimarães B.M.
      • Amoras L.S.
      • et al.
      Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.
      in which the active chlorine content and tissue dissolution properties were altered when submerging different tissue specimens in previously mixed solutions. The specimens used in the different studies varied from porcine
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      ,
      • Naenni N.
      • Thoma K.
      • Zehnder M.
      Soft tissue dissolution capacity of currently used and potential endodontic irrigants.
      ,
      • Estevez R.
      • Conde A.J.
      • Valencia de Pablo O.
      • et al.
      Effect of passive ultrasonic activation on organic tissue dissolution from simulated grooves in root canals using sodium hypochlorite with or without surfactants and EDTA.
      , to bovine
      • Stojicic S.
      • Zivkovic S.
      • Qian W.
      • et al.
      Tissue dissolution by sodium hypochlorite: effect of concentration, temperature, agitation, and surfactant.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      ,
      • Tartari T.
      • Guimarães B.M.
      • Amoras L.S.
      • et al.
      Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.
      and human
      • Cullen J.K.T.
      • Wealleans J.A.
      • Kirkpatrick T.C.
      • Yaccino J.M.
      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      ,
      • De-Deus G.
      • De Berredo Pinho M.A.
      • Reis C.
      • et al.
      Sodium hypochlorite with reduced surface tension does not improve in situ pulp tissue dissolution.
      tissues, as well as rats
      • Abou-Rass M.
      • Oglesby S.W.
      The effects of temperature, concentration, and tissue type on the solvent ability of sodium hypochlorite.
      and rabbits
      • Moorer W.R.
      • Wesselink P.R.
      Factors promoting the tissue dissolving capability of sodium hypochlorite.
      . However, these models differ from the clinical situation in which NaOCl solution is delivered under a controlled flow and is continuously refreshed during the shaping procedure. It is, therefore, relevant to analyze the interaction between the irrigation solutions and the organic matter under a more realistic model that reproduces the delivery and refreshment of irrigants. The purpose of this study was to evaluate pulp tissue dissolution ability of refreshed NaOCl when mixed with Na4HEBP (both at 9% and 18%) and Na2EDTA (17%) under a controlled flow.

      Materials and Methods

      Pulp Specimen Preparation

      Bovine pulp tissue was extracted from the 6 mandibular incisors of 10 bovine jaws (30–70 months old) dissected after being slaughtered for food production. A total of sixty intact fresh bovine anterior mandibular teeth were extracted within 48 hours after slaughtering and stored in glass vials with 0.1% thymol solution. Crowns were removed at the cementum–enamel junction and roots sectioned at 3 mm from the apex with a microtome (0.2 mm/D64, Leica Microsystem, Barcelona, Spain). Pulp tissue was then carefully removed with a periodontal probe and cotton pliers and rinsed with distilled water to remove debris and excess of blood (Fig. 1).
      Figure thumbnail gr1
      Figure 1A schematic representation of specimen preparation and arrangement of needles and pumps: (A) Bovine jaw and incisor; (B) root sectioning, pulp tissue removal, and standardization; (C) introduction of tissue sample in centrifuge tube with inner filter to collect the supernatant and needles fixation; (D) delivery pumps attached.
      All samples were individually stored in 1.5 mL Eppendorf tubes (Deltalab, S.L., Barcelona, Spain) with 1 mL distilled water at -20°C until further use. Before the test, the tubes with the specimens were maintained at room temperature for 30 minutes and later placed in a hot water bath at 37°C (Precisterm Selecta, Barcelona, Spain) for 15 minutes to simulate clinical conditions.
      Pulp specimens were then standardized in size (1 × 3 × 10 mm) under magnification (loupes 3.8, ExamVision Akura Medical, NovaMed Concepts S.L., Madrid, Spain) using a millimeter graph paper vector sheet, a stainless-steel surgical scalpel blade nº15 (Braun, Tuttlingen, Germany), and a digital gauge (Mitutoyo, Guipuzcoa, Spain) (Fig. 1). Specimens were blotted dry for 30 seconds and weighed using a hermetic precision electronic balance (Sartorius AG, Göttingen, Germany) before the test to determine baseline weight (T0) of the samples.

      Groups and Solutions

      The pulp specimens obtained from the 6 anterior teeth of the same bovine jaw were randomly distributed into 4 experimental groups (n = 10) and 2 control groups (n = 3) according to the irrigation protocols as follows:
      G1—Continuous delivery of 3% NaOCl (CanalPro 3% NaOCl, Coltène/Whaledent, GmbH & Co KG, Langenau, Germany).
      G2—Continuous delivery of 3% NaOCl + alternate delivery of 17% Na2EDTA (CanalPro EDTA 17%, Coltène/Whaledent).
      G3—Continuous delivery of 3% NaOCl + alternate delivery of 18% Na4HEBP (Na4HEBP, Cublen K8514 GR; Zschimmer & Schwarz, Moshsdorf GmbH & Co KG, Burgstädt, Germany).
      G4—Continuous delivery of 3% NaOCl + alternate delivery of 9% Na4HEBP (Na4HEBP, Cublen K8514 GR; Zschimmer & Schwarz).
      NC (negative control)–Distilled water.
      PC (positive control)–Continuous delivery of 6% NaOCl (CanalPro 6% NaOCl, Coltène/Whaledent).
      HEBP solutions were freshly prepared before the tests. The white granules of HEBP tetrasodium salt (85% active salt) were dissolved in distilled water to obtain 18% and 9% HEBP solutions.

      Tissue Disolution Method

      Each sample was introduced into a centrifuge tube that contained an inner filter (Ultrafree-MC-HV, Merck Millipore, Germany). The filter was placed in a 50 mL plastic tube (Cellstar graduated polypropylene tubes, Greiner Bio-one8, VWR, Pennsylvania, USA) to collect the supernatant.
      Both distilled water (in the NC group) and fresh NaOCl (in PC group and experimental groups) were continuously delivered with a pump (CADD-Prizm VIP, model 6101, Smiths Medical ASD, Inc, Minnesota, USA) and a 24G/9 mm needle under a continuous controlled rate (1 mL/min). A second pump and a second needle alternately delivered either EDTA (in G2) or HEBP (18% Na4HEBP in G3 and 9% Na4HEBP in G4) at the same rate with a 30-second programmed interval. The delivery of NaOCl with the first pump was not interrupted when the second pump was activated. Both needles were fixed to the cap of the plastic tube, allowing the individual solutions not to contact before the test (Fig. 1).
      The pumps were stopped after 2 minutes of application of the irrigant solutions. Samples were then washed with 2 mL of distilled water for 30 seconds to neutralize the effects of the different solutions, blotted dry again and reweighed to evaluate remnant tissue (T2). The same procedure was repeated after 5 (T5) and 10 (T10) minutes.

      Available Chlorine, pH and Temperature

      Three samples in each group were randomly selected for further registration of free available chlorine and pH at both T0 and T10. A standard iodine/thiosulfate titration method
      American Public Health Association
      Standard Methods for the Examination of Water and Wastewater.
      was used to calculate available chlorine content in the NaOCl solutions at T0 and T10 in all study groups. Initial and final pHs of each solution were registered with a calibrated pH-meter (MicropH 2001, CRISON, Hach Lange, Spain, S.L.U.). Final temperature of supernatant solutions was also registered.

      Statistical Analysis

      Percentage of remnant tissue was calculated at T2, T5, and T10 using the formula: 100-([(initial weight-final weight)/initial weight] ∗100).
      After confirming the assumption of normal distribution of data, a factorial repeated measures analysis of variance (ANOVA) was used to detect any overall difference in the percentage of remnant tissue across time points and groups. A Greenhouse–Geisser correction was applied to the repeated-measure ANOVA because the data violated the assumption of sphericity. Tukey post hoc test was used for pairwise comparisons when repeated measures ANOVA rendered significant differences. Descriptive statistics was used for the rest of the variables: available chlorine, pH, and temperature of the different solutions.

      Results

      The mean percentages and standard deviations of remnant tissue over time for each group are shown in Table 1. The mean initial weight (T0) of the pulp specimens was 31.98 mg (±1.18).
      Table 1Mean Percentages and SD of Remnant Tissue in the Different Groups Over Time
      Remnant tissue (%)
      Initial (T0)2 min (T2)5 min (T5)10 min (T10)
      NC: Distilled water10098.3 ± 0.4111.1 ± 5.1123.1 ± 6.3
      PC: 6% NaOCl10013.2 ± 2.300
      G1: 3% NaOCl10049.6 ± 14.312.24 ± 9.30
      G2: 3% NaOCl-17% Na2EDTA10052.3 ± 7.819.4 ± 6.80
      G3: 3% NaOCl-18% Na4HEBP10058 ± 10.718.5 ± 9.81.4 ± 2.4
      G4: 3% NaOCl-9% Na4HEBP10062.3 ± 7.923.2 ± 8.11.6 ± 2.3
      NC, negative control; PC, positive control.
      No tissue was remnant in PC at T5. Tissue dissolution was not observed in NC.
      There was a significant reduction in the percentage of remnant tissue over time in all groups (P < .001). No significant differences in the percentage of weight loss among experimental groups were obtained at any point time, except for G4 that showed significant more remnant tissue at both T2 and T5 (P < .001) than G1, although no significant differences were detected when compared to G2 and G3. At the same time there were no significant differences among groups at T10; however, some remnant tissue was found in G3 (1.4% ± 2.4) and G4 (1.6% ± 2.3) at T10, whereas nothing was left in G1 and G2 (Table 1).
      Values of available chlorine and pH for each group at T0 and T10 are shown in Table 2. Available chlorine did not change in the supernatant for both PC and G1; however, a reduction in the available chlorine was observed after 10 minutes (T10) in the rest of the groups.
      Table 2Available Chlorine and pH of the Different Solutions at T0 and T10, and the Mean Final Temperature
      GroupComponentsAvailable chlorinepHTemperature (°C)
      InitialFinal% remnantInitialFinalFinal
      PC6% NaOCl6.46.410012.8912.9225
      G13% NaOCl3.93.897.412.8112.7725.33
      G23% NaOCl3.912.81
      17% Na2EDTA8.57
      NaOCl + EDTA1.743.69.4735
      G33% NaOCl3.612.81
      18% Na4HEBP10.93
      NaOCl + HEBP2.466.712.7026.67
      G43% NaOCl3.612.81
      9% HEBPNa411.13
      NaOCl + HEBP2.261.112.6327.33
      PC, positive control.
      Other reactions were also observed in the supernatant of G2: a decrease in the initial pH, the presence of bubbles due to gas formation, and an increase in the temperature (35°C). This exothermic reaction was not observed in the rest of the groups (Table 2). Moreover, at the end of the experiment the filter containing the pulp specimen presented salt precipitate in G2.

      Discussion

      The present study assessed the interaction of NaOCl with 17% Na2EDTA, and 9% and 18% Na4HEBP under a controlled flow. The dynamic model developed allowed the refreshment of irrigation solutions to simulate the delivery that occur in real conditions; and hence allowed to determine tissue dissolution ability of irrigation solutions from a realistic clinical approach.
      In the current study, bovine pulp tissue was used due to the similarity to human pulp
      • Teruel J.
      • Alcolea A.
      • Hernandez A.
      • Ruiz A.
      Comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth.
      , it is readily available and can be cut into standardized specimens in terms of size and homogeneity among groups. The age of the animals varied from 30–70 months to obtain an adequate and standardizable pulp surface area. Thus, samples were standardized in size and presented similar weights. Moreover, in order to reduce the individual variability among groups, 6 mandibular incisors of the same bovine jaw were randomly distributed to the 4 experimental and 2 control groups, so that there were pulp tissue samples of each bovine animal in all the groups. This strategy reduced the chances of individual confounding factors affecting the outcome of the study. To the authors' knowledge, no study has so far analyzed the effect of HEBP on the dissolution capacity of NaOCl in bovine pulp tissue, although Tartari et al
      • Tartari T.
      • Guimarães B.M.
      • Amoras L.S.
      • et al.
      Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.
      and Ulusoy et al
      • Ulusoy Ö.I.
      • Savur G.I.
      • Alaçam T.
      • Çelik B.
      The effectiveness of various irrigation protocols on organic tissue removal from simulated internal resorption defects.
      analyzed the dissolution of bovine muscle tissue submerged in previously mixed irrigation solutions.
      The dissolution capacity of irrigation solutions has been evaluated with different models. Indirect methods have been used to test the changes produced in irrigation solutions; for example, determination of the presence of hydroxyproline or phosphate
      • Koskinen K.P.
      • Stenvall H.
      • Uitto V.J.
      Dissolution of bovine pulp tissue by endodontic solutions.
      ,
      • Trepagnier C.M.
      • Madden R.M.
      • Lazzari E.P.
      Quantitative study of sodium hypochlorite as an in vitro endodontic irrigant.
      and the amount of available chlorine
      • Biel P.
      • Mohn D.
      • Attin T.
      • Zehnder M.
      Interactions between the tetrasodium salts of EDTA and 1-hydroxyethane 1, 1-diphosphonic acid with sodium hypochlorite irrigants.
      . However, the solutions may disturb the determinations in some biochemical assays. Since tissue dissolution capacity is related to OCl−6, the amount of available chlorine was tested in the current study in initial and final solutions through iodometric titration. Other studies used direct methods, for example the histological evaluation of the percentage of remaining pulp tissue in root cross-section of vital extracted teeth
      • De-Deus G.
      • De Berredo Pinho M.A.
      • Reis C.
      • et al.
      Sodium hypochlorite with reduced surface tension does not improve in situ pulp tissue dissolution.
      , the time needed for partial or total tissue dissolution
      • Cullen J.K.T.
      • Wealleans J.A.
      • Kirkpatrick T.C.
      • Yaccino J.M.
      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      ,
      • Abou-Rass M.
      • Oglesby S.W.
      The effects of temperature, concentration, and tissue type on the solvent ability of sodium hypochlorite.
      , and loss of tissue weight by immersing the sample in static solutions or in artificial grooves
      • Tartari T.
      • Guimarães B.M.
      • Amoras L.S.
      • et al.
      Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.
      ,
      • Estevez R.
      • Conde A.J.
      • Valencia de Pablo O.
      • et al.
      Effect of passive ultrasonic activation on organic tissue dissolution from simulated grooves in root canals using sodium hypochlorite with or without surfactants and EDTA.
      ,
      • Ulusoy Ö.I.
      • Savur G.I.
      • Alaçam T.
      • Çelik B.
      The effectiveness of various irrigation protocols on organic tissue removal from simulated internal resorption defects.
      . The current study tested the percentage of remnant tissue over time but using a pump that allowed the refreshment of irrigation solutions and a controlled delivery of irrigants at a standardized flow rate (0.017 mL sec−1). While the optimal volume of irrigant has not been determined yet, the efficacy of irrigation varied with different operators
      • Boutsioukis C.
      • Lambrianidis T.
      • Kastrinakis E.
      • Bekiaroglou P.
      Measurement of pressure and flow rates during irrigation of a root canal ex vivo with three endodontic needles.
      ; hence, it is important to standardize the flow rate in this type of studies.
      Like in previous studies
      • Stojicic S.
      • Zivkovic S.
      • Qian W.
      • et al.
      Tissue dissolution by sodium hypochlorite: effect of concentration, temperature, agitation, and surfactant.
      ,
      • Estevez R.
      • Conde A.J.
      • Valencia de Pablo O.
      • et al.
      Effect of passive ultrasonic activation on organic tissue dissolution from simulated grooves in root canals using sodium hypochlorite with or without surfactants and EDTA.
      , no pulp tissue dissolution was observed when distilled water was used and an increase in the percentage of remnant tissue occurred after 5 and 10 minutes due to tissue hydration. However, results were different for PC group in comparison with static studies
      • Cullen J.K.T.
      • Wealleans J.A.
      • Kirkpatrick T.C.
      • Yaccino J.M.
      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      , complete pulp dissolution was observed after a 5-minute delivery of 6% NaOCl. The use of a continuous flow that allowed the refreshment of NaOCl solution reduced the time needed to half and a complete dissolution of the sample was obtained in comparison with previous studies
      • Cullen J.K.T.
      • Wealleans J.A.
      • Kirkpatrick T.C.
      • Yaccino J.M.
      The effect of 8.25% sodium hypochlorite on dental pulp dissolution and dentin flexural strength and modulus.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      .
      No significant differences in the percentage of remnant tissue among experimental groups were observed at any point of time. Again, these results differed from those obtained in previous studies that immersed tissue in premixed solutions. Tartari et al
      • Tartari T.
      • Guimarães B.M.
      • Amoras L.S.
      • et al.
      Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.
      observed that HEBP minimally affected tissue dissolution ability of NaOCl compared to EDTA. Furthermore, EDTA inactivated NaOCl and no tissue dissolution occurred according to previous studies
      • Stojicic S.
      • Zivkovic S.
      • Qian W.
      • et al.
      Tissue dissolution by sodium hypochlorite: effect of concentration, temperature, agitation, and surfactant.
      ,
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      ,
      • Clarkson R.M.
      • Podlich H.M.
      • Moule A.J.
      Influence of ethylenediaminetetraacetic acid on the active chlorine content of sodium hypochlorite solutions when mixed in various proportions.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      . When NaOCl and EDTA solutions are mixed, a reaction occurs that neutralizes NaOCl, decreasing available chlorine (OCl). This decrease was also observed in the present study when the recollected supernatant NaOCl-EDTA was analyzed. This decrease was expected since the test model used in the present study allowed the continuous refreshment of the solutions in contact with the sample tissue; however, the supernatant was collected in a closed system. The model combined a centrifuge tube with an inner filter for the pulp tissue sample and a 50 mL plastic tube, where the centrifuge tube was placed for supernatant collection. For this reason, when NaOCl and EDTA are not premixed, but simultaneously delivered and refreshed, it seemed not to meaningfully alter the available chlorine, although a reduction occurred in the collected supernatant. An exothermic reaction, the reduction in the pH and the presence of bubbles
      • Grande N.M.
      • Plotino G.
      • Falanga A.
      • et al.
      Interaction between EDTA and sodium hypochlorite: a nuclear magnetic resonance analysis.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      ,
      • Biel P.
      • Mohn D.
      • Attin T.
      • Zehnder M.
      Interactions between the tetrasodium salts of EDTA and 1-hydroxyethane 1, 1-diphosphonic acid with sodium hypochlorite irrigants.
      were also observed in the supernatant in group 2. Previous studies have also reported the increase in temperature of the mixed NaOCl-EDTA solutions at high concentrations (5%) of NaOCl
      • Prado M.
      • Santos Júnior H.M.
      • Rezende C.M.
      • et al.
      Interactions between irrigants commonly used in endodontic practice: a chemical analysis.
      ,
      • Clarkson R.M.
      • Podlich H.M.
      • Moule A.J.
      Influence of ethylenediaminetetraacetic acid on the active chlorine content of sodium hypochlorite solutions when mixed in various proportions.
      ,
      • Biel P.
      • Mohn D.
      • Attin T.
      • Zehnder M.
      Interactions between the tetrasodium salts of EDTA and 1-hydroxyethane 1, 1-diphosphonic acid with sodium hypochlorite irrigants.
      . If EDTA and NaOCl solutions would have been mixed before the test, instead of being delivered separately like in the present study, tissue dissolution could have been affected like in previous studies
      • Grawehr M.
      • Sener B.
      • Waltimo T.
      • Zehnder M.
      Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions.
      ,
      • Clarkson R.M.
      • Podlich H.M.
      • Moule A.J.
      Influence of ethylenediaminetetraacetic acid on the active chlorine content of sodium hypochlorite solutions when mixed in various proportions.
      ,
      • Irala L.E.D.
      • Grazziotin-Soares R.
      • Salles A.A.
      • et al.
      Dissolution of bovine pulp tissue in solutions consisting of varying NaOCl concentrations and combined with EDTA.
      . At the same time, free available chlorine has been shown to suffer a rapid consumption when in contact with dentin, decreasing the ability to dissolve the organic tissue
      • Tejada S.
      • Baca P.
      • Ferrer-Luque C.M.
      • et al.
      Influence of dentine debris and organic tissue on the properties of sodium hypochlorite solutions.
      . Although this has been demonstrated in previously mixed irrigation solutions without NaOCl refreshment, the absence of dentin in the present study could lead to an over estimation of the dissolution activity of the solutions. The current study demonstrated that the alternate delivery of EDTA and HEBP under controlled flow did not affect the pulp tissue dissolution ability of refreshed NaOCl. Further research should confirm its effect in the presence of dentin and preferably in human root canals
      • Boutsioukis C.
      • Arias-Moliz M.T.
      • Chávez de Paz L.
      A critical analysis of research methods and experimental models to study irrigants and irrigation systems.
      .
      With the limitations of this in vitro study, it can be concluded that the controlled delivery of EDTA and HEBP did not alter tissue dissolution ability of NaOCl when fresh solutions were mixed in the root canal.

      Acknowledgments

      The authors deny any conflicts of interest related to this study.
      The authors thank to all the staff in the research laboratory of the School of Dentistry at Complutense University.

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