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Article

Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique

by
Anna Mikheikina
1,*,†,
Ksenia Babina
1,†,
Maria Polyakova
1,
Vladlena Doroshina
1,
Alexandr Zaytsev
2,
Irina Makeeva
1,‡ and
Nina Novozhilova
1,‡
1
Department of Therapeutic Dentistry, I.M. Sechenov First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia
2
Institute of Linguistics and Intercultural Communication, I.M. Sechenov First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors contributed equally to this work.
Appl. Sci. 2024, 14(18), 8422; https://doi.org/10.3390/app14188422
Submission received: 15 August 2024 / Revised: 16 September 2024 / Accepted: 16 September 2024 / Published: 19 September 2024
(This article belongs to the Special Issue Modern Applications for Dentistry and Oral Health)
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Abstract

:
The study aimed to assess the effect of two etidronate-containing irrigants and EDTA on the ability of sodium hypochlorite (NaOCl) to remove the smear layer and dissolve organic tissues. This study evaluated the following solutions: distilled water, 3% NaOCl, 3% NaOCl + EDTA, and 3% NaOCl mixed with etidronate powder from two manufacturers [Dual Rinse, (DR); IsraDent, (ID)] to obtain 9%, 15%, and 18% solutions. To assess the proteolytic activity, bovine muscle tissue samples (56.1 ± 5.4 mg; n = 10 per group) were immersed in the tested solutions for 15 min. Absolute weight changes and percentages of weight changes (wt%) were calculated. To assess smear layer removal, the root canals of human wisdom teeth were instrumented, irrigated with the tested solutions (n = 10 per group), and evaluated using SEM. Statistical analysis employed an ANOVA with a post-hoc Tukey test and paired t-test, the Kruskal–Wallis test with a pairwise Wilcoxon rank sum test, and the Friedman test with a pairwise Wilcoxon signed-rank test. The mean weight loss in the NaOCl group comprised 17.3 mg (32 wt%). Sequential use of NaOCl and EDTA resulted in a significant increase in proteolytic activity of the former (57 wt%), while mixing these solutions led to a reduction of proteolytic activity (17 wt%). All NaOCl/DR groups exhibited a significantly greater dissolution activity than that of NaOCl alone, with the mean weight loss ranging from 23.3 mg (40 wt%) to 24 mg (41 wt%). ID9% and ID15% significantly decreased the proteolytic activity of NaOCl. In most groups, the apical thirds of the root canals demonstrated a significantly heavier smear layer compared to the middle and coronal thirds. The most effective smear layer removal was observed after irrigation with NaOCl combined with EDTA or DR (all concentrations); however, in the NaOCl + EDTA and DR18% groups, it was accompanied by moderate to severe erosion. Irrigation with ID did not result in smear layer removal or dentin erosion. In conclusion, the smear layer removal and pulp dissolving effects of continuous chelation using etidronate were manufacturer/composition-dependent. DR9% combined with NaOCl demonstrated the most promising results.

1. Introduction

A biological aim of endodontic treatment is the elimination of bacterial biofilms alongside a rigorous removal of pulpal remnants and smear layers from the root canal walls, followed by the hermetic sealing of the root canals [1,2,3,4,5,6,7,8]. For endodontic treatment success, it is specifically important that the radicular system is regarded as a three-dimensional entity, including not only the main canal but also lateral canals, ramifications, loops, isthmuses, and deltas [1,8,9]. These root canal complexities may give refuge to biofilms, pulp remnants, and dentin debris that may become a source of re-infection after root canal treatment is completed [10,11,12]. Thus, an important step of endodontic treatment is root canal chemo-mechanical preparation, i.e., irrigation and instrumentation aimed to reach difficult-to-access areas to the maximum possible extent [1,9,13,14].
At the same time, instrumentation results in the formation of an amorphous layer consisting of dentin debris, remnants of odontoblastic processes, pulp tissue, and bacteria known as the smear layer [15,16]. The surface smear layer is usually 1–2 μm thick, but it may form smear plugs in the dentinal tubules up to 40 μm deep [6,16]. These plugs decrease dentin permeability and prevent the penetration of antiseptic and dissolving agents into the dentinal tubules [15,16,17]. The removal of this layer enhances the antibacterial effectiveness of chemo-mechanical treatment by allowing antiseptic solutions to penetrate deeper into the dentin and also provides stronger bonding of sealer to the root canal walls [15,18,19].
Smear layer elimination is mainly achieved by meticulous irrigation of the root canal system and may be enhanced by the use of different activation techniques [20,21,22,23,24]. In order to effectively remove the smear layer, the irrigation protocol should include medicaments affecting its organic and inorganic components [18,19,25]. Sodium hypochlorite (NaOCl) in different concentrations is considered to be a gold standard in root canal irrigation due to its antimicrobial and tissue-dissolving properties [26,27,28,29]. However, it provides limited activity against the inorganic part of the smear layer [25,30,31,32]. Therefore, smear layer removal should be accomplished by irrigation with NaOCl combined with decalcifying (chelating) substances [33,34]. Chelating agents react with calcium ions in the dentin wall and form soluble calcium compounds, which may be easily rinsed off [35].
Sodium hypochlorite’s proteolytic activity is directly proportional to its concentration [1,29]. It can also be potentiated by laser-assisted irrigation and ultrasonic activation [2] and frequent refreshing of the solution [29]. On the other hand, the presence of dentin powder [36] and the use of some chelating substances have been reported to hamper sodium hypochlorite’s proteolytic activity [37,38,39,40,41,42]. Various chelating substances have demonstrated different inhibiting potentials towards the NaOCl effect [4,43,44]. Ethylenediaminetetraacetic acid (EDTA) is the most widely used chelating agent [45,46,47,48]; however, the use of NaOCl combined with EDTA has been found to cause demineralization and collagen rearrangement of root canal dentin, i.e., peritubular and intertubular dentin erosion [41,49,50]. Moreover, a protocol involving alternating use of EDTA and NaOCl alters the ability of the latter to dissolve organic matter. EDTA, being a strong chelator, actively reacts with NaOCl, leading to a decrease in its proteolytic activity due to the loss of free available chlorine (hypochlorite ion and hypochlorous acid) that is responsible for protein breakdown [14,37,38,39,40,51]. Other strong chelating agents, such as citric [41] and maleic [52,53] acids, demonstrated similar inhibiting effects when combined with NaOCl.
To overcome the aforementioned issue, a “continuous chelation” approach was proposed. This approach employs mixing NaOCl with weak chelating agents that consume less free available chlorine than EDTA and other strong chelators [37,38]. The studies report on the potential use of clodronate [35], tetrasodium EDTA [54], or etidronic acid (HEDP) [38] for this purpose. A mixture of tetrasodium EDTA and NaOCl was shown to alter the content of free available chlorine to a greater extent compared with clodronate and etidronate [32]. Clodronate demonstrated promising results as a potential irrigant for continuous chelation due to its minor influence on the content of free available chlorine and wide therapeutic window [35,44,55,56]. However, to the best of our knowledge, currently, there are no commercially available irrigants based on clodronate. HEDP is a non-nitrogen-containing bisphosphonate that binds with calcium [57]. HEDP is highly compatible with NaOCl [34,58]; however, the findings of the previous studies on smear layer removal [4,7,58,59,60] and proteolytic activity [58,61,62] of NaOCl/HEDP mixtures are controversial.
To the best of our knowledge, no previously published articles reported on the effect of different etidronate formulations and concentrations on the proteolytic activity of NaOCl and smear layer removal. Therefore, the aim of our study was to compare the effect of two etidronate-containing irrigants and EDTA on the ability of NaOCl to remove the smear layer and dissolve organic tissues.

2. Materials and Methods

The use of extracted teeth for research purposes was approved by the Local Ethics Committee (Sechenov University, Moscow, Russia, protocol No. 22-02). This study utilized wisdom teeth extracted in the context of a treatment plan; written informed consent from the patients was obtained.
This study comprised two parts. First, the proteolytic activity of the irrigating solutions was assessed using the method described by Tartari et al. [62]. Second, the effectiveness of smear layer removal was evaluated using scanning electron microscopy (SEM).

2.1. Solutions Preparation

All irrigating solutions were mixed immediately before the experimental procedures. NaOCl (Hypochloran-3, Omegadent, Moscow, Russia) and etidronate mixtures were prepared as described in the manufacturers’ instructions. Etidronate from two manufacturers was used: Dual Rinse (Medcem, Weinfelden, Switzerland) and HEBP Etidronic acid (Isradent, Tyumen, Russia). The weight of the HEDP powder was adjusted using hermetic precision electronic balance (Sartorius AG, Göttingen, Germany). Other irrigants included EDTA (MD-Cleanser, Metabiomed, Cheongju, Republic of Korea) and distilled water (VeryGoods, Chelyabinsk, Russia). The concentrations of the solutions and preparation details are shown in Table 1.

2.2. Tissue Dissolution Assessment

Bovine muscle tissue was used to simulate the pulp [29,43,54]. Rounded samples of tissue (diameter 8 mm, thickness 1 mm) [n = 100] were cut using a microtome Rotmik 2A (Orion Medik, St. Petersburg, Russia) and puncher (Apexmed Slim 8.0 mm, Apexmed International, Amsterdam, The Netherlands). After weighing on an electronic balance (Sartorius AG, Göttingen, Germany) with an accuracy of 0.1 mg, each sample was randomly assigned to one of the experimental groups (n = 10). The groups were as follows: DW, 3% NaOCl, 3% NaOCl + EDTA (mixed), 3% NaOCl + EDTA (sequential), 3% NaOCl/ID9%, 3% NaOCl/ID15%, 3% NaOCl/ID18%, 3% NaOCl/DR9%, 3% NaOCl/DR15%, and 3% NaOCl/DR18% (Table 1). A computer-generated schedule prepared by a third-party person was used for randomization. The mean weight of the samples was 56.1 ± 5.4 mg. Each specimen was immersed in a plastic vial containing 10 mL of the tested solution for 15 min [44]. For the NaOCl + EDTA (sequential) group, the specimens’ 15 min immersion in NaOCl was followed by a 1 min immersion in the EDTA solution. Then, the specimens were rinsed with distilled water to eliminate the irrigants, blotted dry, and weighed again [63,64]. The absolute weight changes and percentages of weight changes (wt%) were calculated [2].

2.3. Smear Layer Removal Assessment (SEM)

2.3.1. Sample Preparation for SEM

Ninety human caries-free wisdom teeth with complete root formation extracted in the context of a treatment plan were stored in 0.1% thymol solution for 1 week [7]. After disinfection, the samples were kept in distilled water until the start of the experiment. Each tooth was decoronated to obtain a uniform working length of 13 mm, and the roots were separated with a diamond bur using a high-speed handpiece [65]. The roots were randomly assigned to the following experimental groups (n = 10): DW, 3% NaOCl, 3% NaOCl + 17% EDTA (sequential), 3% NaOCl/ID9%, 3% NaOCl/ID15%, 3% NaOCl/ID18%, 3% NaOCl/DR9%, 3% NaOCl/DR15%, and 3% NaOCl/DR18% (Table 1). A computer-generated schedule prepared by a third-party person was used for randomization.
Root canals were negotiated with hand N10 K-files (MANI, Inc., Utsunomiya, Japan) and instrumented with the Mtwo® system (VDW GmbH, Munich, Germany) up to size 35.04. Each step of preparation was followed by rinsing with the assigned irrigating solutions using a syringe with a 30-gauge endodontic needle (NaviTip; Ultradent, South Jordan, UT, USA). Chelating agents were used for sequential irrigation (NaOCl + EDTA group) or continuous chelation (in all HEDP groups).
In the NaOCl group, irrigation during instrumentation was performed with NaOCl alone (10 mL overall); the final rinse was performed with 5 mL of NaOCl solution [66].
In the NaOCl + EDTA group, irrigation during instrumentation was performed with NaOCl (a total of 10 mL); the final rinse was performed with 2 mL of EDTA followed by 5 mL of NaOCl.
In all HEDP groups, irrigation during instrumentation was performed with a mixture of HEDP and NaOCl (a total of 10 mL); the final rinse was performed with 5 mL of the same mixture.
After the final irrigation, the root canals were dried with paper points (MTwo Absorbent Paper Points, VDW GmbH, Munich, Germany). Two groves were prepared along the long axis of the roots using a high-speed diamond disk [4] with a 6 mm diameter (MonAliT, Moscow, Russia). Then, the roots were vertically split into halves using a chisel and a hammer [67] and cleaned for 1 min in an ultrasonic bath filled with distilled water (UltraEst-M, JSC GEOSOFT DENT, Moscow, Russia) to remove debris loosely attached to the root canal walls. The specimens were dehydrated in ascending concentrations of ethyl alcohol (30–100%), mounted on coded stubs, dried by air, placed in a vacuum chamber (Bal-Tec SCD 005, Balzers, Lichtenstein), and coated with a platinum layer in argon atmosphere (0.1–0.2 mbar) for 130 s [68].

2.3.2. SEM Analysis

The samples were examined under a scanning electron microscope (LEO-1430 VP, Carl Zeiss, Oberkochen, Germany) with a 20 kV beam at 500–4000× magnification. To obtain the smear layer and dentin erosion scores, microphotomicrographs were randomly coded and evaluated independently by two blinded and calibrated (Cohen’s Kappa > 80%) researchers (A.M. and M.P.). The inconsistencies were resolved by a third party (K.B.). The images were assessed for the presence of a smear layer (at ×2000 magnification) and dentin erosion (at ×4000 magnification) at the coronal, middle, and apical thirds of the root canal walls.
The smear layer was graded on a scale from 1 to 5 using the modification of a scoring system proposed by Hülsmann et al. [69]. The criteria for the scoring were as follows:
1—no smear layer; all tubules are completely open;
2—small amount of smear layer; most tubules are completely open, some partially open;
3—moderate amount of smear layer; some tubules are completely open, some are partially open, some are closed;
4—moderate amount of smear layer; the majority of dentinal tubules are closed or partially open;
5—severe homogeneous smear layer covering the dentin surface with few or no dentin tubules open (Figure 1).
The scores for dentin erosion were attributed using a modified rating system proposed by Torabinejad et al. (2003) [70] as follows:
1—no erosion (all tubules look normal in appearance and size);
2—moderate etching/erosion (peritubular dentin is eroded, dentinal tubules are slightly enlarged, <10% of tubules are connected);
3—severe etching/erosion (intertubular dentin is destroyed, and >10% of tubules are connected to one another) [Figure 2].

2.4. Statistical Analysis

The sample size for this experiment was determined based on the previous studies [4,44]. For sample size calculation in the tissue dissolution experiment, the effect size for the mean weight losses was estimated to be 2.04 (“large”) [44]. To detect significant differences in 10 groups using a one-way ANOVA with alfa = 0.05 and 80% power, the required total sample size was 20 specimens (2 specimens per group). However, as it was an in vitro study and the specimens were easily acquired, it was decided to use 10 specimens per group (a total sample size of 100) as in other studies [44,61,62,64]. For sample size calculation in the smear layer removal experiment, the effect size for the mean percentages of weight losses was estimated to be 1.6 based on the scores for the smear layer in the middle third of the root canal [4]. To detect significant differences between the groups using the Wilcoxon rank-sum test with alfa = 0.05 and 80% power, the required total sample size was 10 specimens per group (90 total).
The outcome variables of the study were weight loss of the specimens and smear layer and dentin erosion scores. All collected data were subjected to statistical analysis using the Shapiro–Wilk test to verify the assumption of normality.
The weights of the specimens were normally distributed and showed homogeneity of variance according to Levene’s test. Therefore, the differences between the weights of the specimens were analyzed using a repeated-measures ANOVA. The differences between the groups at the same time points (absolute weights, weight losses, and percent weight losses) were assessed using a one-way ANOVA with a post-hoc Tukey test; a paired t-test was used to compare the weights of the specimens before and after the immersion in the tested solutions.
For inter-group comparisons of smear layer and dentin erosion scores data, the Kruskal–Wallis test and Benjamini–Hochberg adjusted pairwise Wilcoxon rank-sum test was used since the variables were not normally distributed. The Friedman test and Benjamini–Hochberg adjusted pairwise Wilcoxon signed-rank test was used to compare scores within the apical, middle, and coronal thirds for each irrigating solution.
A two-sided p-value less than 0.05 was considered significant. All analyses were performed using R version 4.2.3 2023-03-15 (R Development Core Team, Columbia University, New York, NY, USA) in RStudio software version 2023.03.0+386 (Posit Software, PBC, Boston, MA, USA).

3. Results

3.1. Organic Tissue Dissolution

The repeated-measures ANOVA revealed that the “Irrigant” (p = 0.025), “Timepoint” (p < 0.001), and interaction of “Irrigant” and “Timepoint” factors (p < 0.001) had a significant impact on the proteolytic activity of the tested solutions, as measured by weight loss.
The weights of the bovine muscle specimens before and after immersion in the studied irrigating solutions are presented in Table 2. There were no significant differences in the samples’ weights among the groups at baseline (p = 0.838). Immersion in all the tested solutions resulted in a significant loss of the specimens’ weights (p < 0.001). The mean weight loss in the NaOCl group was 17.3 mg (32 percent by weight [wt%]). Sequential use of NaOCl and EDTA resulted in a significant increase in proteolytic activity of the former (57 wt%), while mixing these solutions led to a reduction of proteolytic activity (17 wt%). The immersion in DW resulted in a minor decrease in the specimens’ weights, which, however, differed significantly from that of NaOCl and NaOCl + EDTA (mixed). All NaOCl/DR groups exhibited a significantly greater dissolution activity, with the mean weight loss ranging from 23.3 mg (40 wt%) to 24 mg (41 wt%), while ID9% and ID15% significantly decreased the proteolytic activity of NaOCl.

3.2. Smear Layer Removal

Cohen’s kappa for interobserver agreement was 0.87 ± 0.04 (CI95% 0.78–0.95) for smear layer assessment and 0.84 ± 0.07 (CI95% 0.73–0.95) for dentin erosion assessment, indicating almost perfect agreement. The repeatability of the smear layer grading was 0.88 ± 0.04 (CI95% 0.81–0.95) and 0.86 ± 0.04 (CI95% 0.79–0.94) for observers 1 and 2, respectively, while the repeatability of dentin erosion grading was 0.88 ± 0.05 (CI95% 0.79–0.98) and 0.91 ± 0.04 (CI95% 0.83–0.98) for observers 1 and 2, respectively.
In all groups, apart from the NaOCl and ID18% ones, the apical thirds of the root canals demonstrated significantly heavier smear layers compared to the middle and coronal thirds (Table 3). The apical thirds showed a moderate to severe amount of smear layer in all studied groups: the median scores ranged between 3 and 5 (Figure 3). A relatively small amount of smear layer was observed in the DR9%, DR18%, and ID9% groups.
In the middle third, complete removal of the smear layer was achieved either by alternating irrigation with NaOCl and EDTA solutions or by continuous chelation with NaOCl mixed with DR (all concentrations). The rest of the groups demonstrated a significantly worse smear layer-removing ability, with a median score of 4 (Figure 4). Similar results were found in the coronal third, with one exception: ID9% also removed the smear layer completely (Figure 5).

3.3. Erosive Potential of the Studied Irrigants

No dentin erosion was found in the DW and ID groups (all concentrations) (Table 4). In the other groups, the dentin erosion scores were significantly lower in the apical thirds than in the coronal thirds.
In the apical third, a more prominent erosion was observed after the irrigation with NaOCl and EDTA as well as after continuous chelation with NaOCl/ID18% mixture, with no differences between these groups (Figure 4). Also, moderate erosion developed in the DR9% group.
In the middle third, continuous chelation with NaOCl and DR18% resulted in severe erosion, while irrigation with NaOCl and EDTA and NaOCl mixed with DR15% resulted in moderate erosion. The use of NaOCl alone and NaOCl mixed with DR9% caused no or minimal erosion.
NaOCl and EDTA irrigation induced the most intense dentin erosion in the coronal third (median score 3), followed by DR18% (median score 2.5) and pure NaOCl, DR9%, and DR15% (median score 2).

4. Discussion

This study assessed the influence of two etidronate-containing irrigants and EDTA on the proteolytic activity of NaOCl and its ability to remove the smear layer. We found that the effect of HEDP-containing irrigants on the tissue-dissolving ability of NaOCl was manufacturer(formulation)-dependent. ID9% and ID15% significantly decreased the proteolytic activity of NaOCl, showing no significant differences from EDTA (mixed), while the sequential use of NaOCl and EDTA and all DR concentrations significantly increased the proteolytic activity of the former. The most effective smear layer removal was observed after irrigation with NaOCl combined with EDTA or DR (all concentrations); however, in the NaOCl + EDTA and DR18% groups, it was accompanied by moderate to severe erosion. Irrigation with ID did not result in smear layer removal or dentin erosion.
Although the exact compositions of the irrigants used in this study are not disclosed, it may be hypothesized that the differences in the effects of etidronates provided by different manufacturers can be explained by the differences in their formulations. This may also explain the controversy in the previous studies regarding the tissue-dissolving and smear layer-removing properties of HEDP.
A number of studies have assessed the proteolytic effect of HEDP combined with NaOCl. Ballal et al. assessed the ability of NaOCl alone or combined with DR9% to dissolve shrimp tissue after immersion in the irrigants for 30 s [10]. The authors found that 9% HEDP (DR) did not hamper the tissue-dissolving ability of a 2.5% NaOCl solution. The effect of a continuous chelation protocol with the use of 18% HEDP was assessed by Tartari et al. In their study, a 15 min immersion in 2.25% NaOCl alone or in a mixture of 5% NaOCl with 18% HEDP provided a more prominent proteolytic activity compared to that of Na4EDTA–NaOCl mixture and saline [71]. In a study by Wright et al., the mean percentage weight reductions of specimens prepared from porcine palatal mucosa were 60.5% and 46.8% for 5% NaOCl and 5% NaOCl mixed with etidronate, respectively [44]. As it was mentioned above, in our study, the effect of etidronate on the ability of NaOCl to degrade organic components was manufacturer-dependent. All concentrations of ID decreased this activity of NaOCl, although, for ID18%, the differences did not reach the level of statistical significance. At the same time, the DR of all concentrations promoted the tissue-dissolving capability of NaOCl. The increase in the proteolytic activity in the HEDP/NaOCl group was also reported by Tartari et al. [62]. The authors demonstrated that 2.25% NaOCl alone dissolved 27% of bovine muscle tissue by weight, while 5% NaOCl combined with 18% HEDP dissolved 41% of organic tissue.
Previously, it has been shown that EDTA may decrease the ability of NaOCl to degrade organic components. However, there is a controversy in the literature regarding the proteolytic activity of the NaOCl + EDTA mixture. In some studies, mixtures of 5% NaOCl with 17% EDTA produced no tissue-dissolving effect at all [43,62], while de Almeida et al. reported that EDTA significantly reduced the capability of NaOCl to dissolve pulp tissue [72]. In the latter study, the percent weight losses were 57% in the 2.5% NaOCl group and 33% in the 2.5% NaOCl + EDTA group after a 15 min immersion. Similarly, in our study, we observed a two-fold decrease in the organic tissue degrading capacity of NaOCl mixed with EDTA as compared with NaOCl alone. However, these solutions are not used as a mixture in clinical practice; instead, they are used sequentially. We found that the sequential use of NaOCl and EDTA resulted in a significantly higher proteolytic activity compared with NaOCl used alone or mixed with EDTA. On the other hand, it is next to impossible to completely avoid contact between these solutions in root canal complexities where pulp dissolving activity is of particular importance.
In most studies assessing the ability of irrigating solutions to dissolve pulp tissue remnants, saline was used as a negative control. The immersion in this solution produced no [64,73] or a non-significant [62,71] decrease in the weights of the specimens. Kumar et al. used distilled water as a negative control. They found no decrease in the weights of the specimens in the control group [74]. In our study, we found a slight decrease in the samples’ weights after a 15 min immersion in distilled water. However, these changes were insignificant.
For the assessment of SEM images, we modified the Hülsmann scale of the smear layer [69] based on the preliminary analysis of the images. In our scale, we described the smear layer removal pattern by focusing on the degree of dentinal tubule closure and not on the thickness of the smear layer. Dentinal tubules were graded as completely open, partially open, or closed. All cases where all tubules were completely closed were graded with the same score (score 5).
In our study, we assessed smear layer removal with the help of different irrigating solutions at different levels of the root canals. A number of previous studies have underlined a greater amount of the residual smear layer in the apical root canal third compared to the middle and coronal root canal thirds [7,58,59,60]. We also found less cleanliness of the root canal walls in the apical part.
Previous studies evaluating the removal of the smear layer observed inconsistent results on whether the combination of NaOCl with HEDP was more effective than the standard irrigating sequence (NaOCl and EDTA). In a study by Castagnola et al., the amount of the smear layer in the apical and coronal thirds did not differ significantly between the 6% NaOCl/HEDP (DR) and 6% NaOCl and 17% EDTA groups. However, NaOCl and EDTA removed significantly more of the smear layer in the middle thirds of the root canals [4]. Kfir et al. reported that the 9% HEDP-based (DR) continuous irrigation did not differ from alternating irrigation with 3% NaOCl, followed by EDTA, in terms of the ability to remove the smear layer [59]. According to Aoun et al., 9% HEDP (DR) in 3% NaOCl improved smear layer elimination at the apical level of the root canal compared with 3% NaOCl and EDTA alternating irrigation [7]. Similar results were reported by Ulusoy et al., who found that irrigation with 9% and 18% HEDP was significantly more effective for the removal of the smear layer in the apical third of the root canal than 17% EDTA [75]. In a study by Morago et al., the use of a 2.5% NaOCl/9% HEDP mixture freed 95% of the dentinal tubules from the smear layer [76].
Although chelating agents remove the smear layer, thus enhancing the antibacterial effectiveness of irrigation and providing stronger adhesion of sealer to the root canal walls [15,18,19], their use may potentiate excessive removal of organic and inorganic content of the dentin, resulting in erosion of its peritubular and intertubular parts [59,65,77]. The sequential use of EDTA and NaOCl has been found to induce the erosion of dentinal walls [78,79]. HEDP has also been shown to exhibit erosive potential [58,59,65].
With regard to the erosive potential of EDTA and HEDP, there is still no consensus in the literature. Kfir et al. found that irrigation with the NaOCl/HEDP (DR) mixture did not differ from the sequential irrigation with 3% sodium hypochlorite and EDTA, with no or minimal erosion in both groups [59]. Similar results were obtained by Kadulkar et al. The authors observed moderate erosion of the radicular dentin in the coronal third, whereas the middle and apical thirds showed no erosion after final irrigation with 17% EDTA or 9% HEDP, with no differences between the groups [65]. On the other hand, Ulusoy et al. identified destruction of the intertubular dentin in the middle and coronal thirds after the combined use of NaOCl and HEDP, whereas the sequential use of EDTA and NaOCl resulted in no erosive areas [58]. In contrast, according to the results of a study by Zarean et al., EDTA produced deeper erosion of the root canals (45.75, 41.79, and 32.25 μm) than HEDP (DR) [20.25, 16.40, and 15.96 μm] in the cervical, middle, and apical thirds, respectively [80].
In our study, complete removal of the smear layer was strongly associated with dentinal wall damage; some degree of dentin erosion was observed in all the areas free of the smear layer. In the NaOCl + EDTA group, the middle and coronal thirds of the root canals presented with completely open dentinal tubules, while in the apical thirds, there were some areas with completely closed dentinal tubules; all the thirds demonstrated moderate to severe dentin erosion. In the ID groups (all concentrations), the smear layer covered most of the root canal surfaces, and the majority of the dentinal tubules were completely or partially closed. No signs of dentin erosion were found. In all the DR groups, irrespective of the concentrations, the middle and coronal thirds had the majority of dentinal tubules open, while the apical thirds had some dentinal tubules closed. The most prominent dentin erosion comparable to that in the NaOCl + EDTA group was observed in the DR18% group.
The generalizability of the results obtained in our study is subject to certain limitations. For instance, the in vitro design did not allow the estimation of the clinical relevance of the findings. The issue that was not addressed in this study was whether the detected degree of dentin erosion could significantly affect the mechanical characteristics of the root and sealer bonding. The use of bovine muscle tissue for testing the proteolytic activity of the irrigants made these findings less generalizable to human pulp tissue. Finally, the scope of this study was limited to the comparison of the tested irrigants, which is why the activating techniques were not applied. Moreover, it could be interesting to test smear layer removal in combination with other recently introduced treatments [81,82].

5. Conclusions

Notwithstanding these limitations, this study suggests that the effects of HEDP-containing irrigants were manufacturer-dependent. ID9% and ID15% significantly decreased the proteolytic activity of NaOCl, while DR significantly increased it, irrespective of the concentration. The NaOCl + EDTA (mixed) group showed tissue-dissolving properties similar to those of the ID groups. Sequential use of NaOCl and EDTA resulted in an increased proteolytic activity of the former. Considering chelating capacity, DR9% demonstrated the most promising results, showing similar smear layer removal ability and lower erosive potential compared to EDTA.

Author Contributions

Conceptualization, N.N., K.B. and I.M.; methodology, A.M. and V.D.; software, N.N.; validation, V.D., M.P. and A.Z.; formal analysis, M.P. and K.B.; investigation, A.M. and V.D.; resources, M.P. and A.Z.; data curation, A.M.; writing—original draft preparation, all authors; writing—review and editing, A.Z. and I.M.; visualization, N.N.; project administration, K.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of I.M. Sechenov First Moscow State Medical University (Sechenov University) (Protocol No. 20-22 (20 October 2022)).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request. The data will not be publicly available until thesis defense of the first author (A.M.) due to plagiarism concerns.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 08422 g001
Figure 1. SEM smear layer scoring system (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector): Score 1—no smear layer, all tubules are completely open; Score 2—small amount of smear layer, most tubules are completely open, some partially open; Score 3—moderate amount of smear layer, some tubules are completely open, some are partially open, some are closed; Score 4—moderate amount of smear layer, the majority of dentinal tubules are closed or partially open; Score 5—severe homogeneous smear layer covering the dentin surface with few or no dentin tubules open.
Figure 1. SEM smear layer scoring system (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector): Score 1—no smear layer, all tubules are completely open; Score 2—small amount of smear layer, most tubules are completely open, some partially open; Score 3—moderate amount of smear layer, some tubules are completely open, some are partially open, some are closed; Score 4—moderate amount of smear layer, the majority of dentinal tubules are closed or partially open; Score 5—severe homogeneous smear layer covering the dentin surface with few or no dentin tubules open.
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Figure 2. SEM dentin erosion scoring system (×4000 magnification, accelerating voltage–20.23 kV, working distance–18 mm, with backscattered detector): Score 1—no erosion (all tubules look normal in appearance and size); Score 2—moderate etching/erosion (peritubular dentin is eroded, dentinal tubules are slightly enlarged, <10% of tubules are connected); Score 3—severe etching/erosion (intertubular dentin is destroyed and >10% of tubules are connected to one another).
Figure 2. SEM dentin erosion scoring system (×4000 magnification, accelerating voltage–20.23 kV, working distance–18 mm, with backscattered detector): Score 1—no erosion (all tubules look normal in appearance and size); Score 2—moderate etching/erosion (peritubular dentin is eroded, dentinal tubules are slightly enlarged, <10% of tubules are connected); Score 3—severe etching/erosion (intertubular dentin is destroyed and >10% of tubules are connected to one another).
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Figure 3. Smear layer removal by different irrigants in the apical third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
Figure 3. Smear layer removal by different irrigants in the apical third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
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Figure 4. Smear layer removal by different irrigants in the middle third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
Figure 4. Smear layer removal by different irrigants in the middle third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
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Figure 5. Smear layer removal by different irrigants in the coronal third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
Figure 5. Smear layer removal by different irrigants in the coronal third (×2000 magnification, accelerating voltage–20.23 kV, working distance–19 mm, with backscattered detector); DW—distilled water; NaOCl—pure NaOCl; EDTA—sequential irrigation with NaOCl and EDTA; DR—DualRinse HEDP; ID—IsraDent HEDP.
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Table 1. Irrigating solutions used in the study.
Table 1. Irrigating solutions used in the study.
Group NameSolutionsTrade NamePreparation Details
NaOCl3% NaOClHypochloran-3 NA
DWDistilled waterDistilled water NA
NaOCl + EDTA3% NaOClHypochloran-3 Mixing 5 mL of EDTA with 5 mL of NaOCl (for proteolytic activity testing);
 Alternating use (for smear layer removal)
17% EDTAMD-Cleanser
NaOCl/DR9%3% NaOClHypochloran-3 Mixing 0.9 mg of Dual Rinse powder with 10 mL of NaOCl
9% HEDP
NaOCl/DR15%3% NaOClMixing 1.5 mg of Dual Rinse powder with 10 mL of NaOCl
15% HEDPDual Rinse
NaOCl/DR18%3% NaOClMixing 1.8 mg of Dual Rinse powder with 10 mL of NaOCl
18% HEDP
NaOCl/ID9%3% NaOClHypochloran-3 Mixing 0.9 mg of Isradent powder with 10 mL of NaOCl
9% HEDP
NaOCl/ID15%3% NaOClMixing 1.5 mg of Isradent powder with 10 mL of NaOCl
15% HEDPHEBP Etidronic acid Isradent
NaOCl/ID18%3% NaOClMixing 1.8 mg of Isradent powder with 10 mL of NaOCl
18% HEDP
NaOCl—sodium hypochlorite, DW—distilled water, EDTA—ethylenediaminetetraacetic acid, HEDP/HEBP—1-Hydroxyethylidene-1,1-Bisphosphonate, DR—Dual Rinse, ID—Isradent, NA—not applicable.
Table 2. The mean weights of the bovine muscle specimens before and after immersion in the studied irrigating solutions.
Table 2. The mean weights of the bovine muscle specimens before and after immersion in the studied irrigating solutions.
GroupsWeight before, mgWeight after, mgWeight Loss, mgWeight Loss, %
NaOCl55.5 (6.2)37.8 (6.5) a17.3 (1.7) a32 (5) a
DW56.3 (4.7)52.3 (4.8) b4.0 (0.8) b7 (1) b
NaOCl + EDTA (mixed)55.8 (8.7)46.5 (7.6) c9.3 (2.1) d17 (3) d
NaOCl + EDTA (sequential)56.8 (6.3)30.3 (4.4) d 26.5 (4.4) c56.8 (7) e
NaOCl/DR9%60.3 (5.9)36.3 (6.7) cd24 (3.2) c40 (4) c
NaOCl/DR15%57.5 (5.9)36.3 (2.4) cd23.3 (4.3) c40 (7) c
NaOCl/DR18%56.5 (4.7)33.3 (4.3) d23.3 (1.7) c41 (4) c
NaOCl/ID9%60.0 (1.4)53.3 (2.2) b6.8 (1.5) bd11 (3) bd
NaOCl/ID15%59.8 (2.1)47.0 (1.8) b12.8 (1.0) d21 (1) d
NaOCl/ID18%58.5 (6.0)44.3 (5.3) abcd14.3 (1.0) ad24 (1) ad
p *0.838<0.0001<0.0001<0.0001
abcde different letters indicate statistically significant differences between the groups (in the columns); * among the groups according to one-way ANOVA.
Table 3. The median (q1; q3) scores for the smear layer removal after irrigation with the studied solutions.
Table 3. The median (q1; q3) scores for the smear layer removal after irrigation with the studied solutions.
GroupsApical ThirdMiddle ThirdCoronal Thirdp-Value 1
NaOCl4 (3.25; 4.75) Aa4 (3; 4) Aa2 (2; 2) Ab0.000293
DW5 (5; 5) Ba4 (4; 4) Ab4 (4; 4) Bb<0.0001
NaOCl + EDTA3 (2.25; 4.5) ACa1 (1; 1) Bb1 (1; 2) Cb0.000131
NaOCl/DR9%3 (3; 3) Ca1 (1; 1) Bb1 (1; 1) Db<0.0001
NaOCl/DR15%4 (4; 4) Aa1 (1; 1) Bb1 (1; 1) Db<0.0001
NaOCl/DR18%3 (1; 3.75) Ca1 (1; 1.75) Bb1 (1; 1) Db0.00381
NaOCl/ID9%3 (3; 3) Ca4 (4; 4) Ab1 (1; 1) Dc<0.0001
NaOCl/ID15%5 (5; 5) Ba4 (4; 4.75) Ab4 (4; 4) Bb0.000371
NaOCl/ID18%4 (4; 4) Aa4 (4; 4) Aa4 (4; 4) Bana
p *0.761<0.0001<0.0001<0.0001
ABCD different letters indicate statistically significant differences between the irrigants (in the columns) abc different letters indicate statistically significant differences between the root thirds (in the rows); * between the groups; 1 between the root canal thirds; na—not applicable.
Table 4. The median (q1; q3) scores for the dentin erosion after irrigation with the studied solutions.
Table 4. The median (q1; q3) scores for the dentin erosion after irrigation with the studied solutions.
GroupsApical ThirdMiddle ThirdCoronal Thirdp-Value 1
NaOCl1 (1; 1) Aa1 (1; 1) Aa2 (2; 2) ADb0.000335
DW1 (1; 1) A1 (1; 1) A1 (1; 1) Bna
NaOCl + EDTA3 (1.5; 3) Ba2 (2; 2.75) Ba3 (3; 3) Cb0.0302
NaOCl/DR9%1.5 (1; 2) CDa1 (1; 1.75) Aa2 (2; 2) Ab0.000255
NaOCl/DR15%1 (1; 1) Aa2 (2; 2) Bb2 (2; 2) Ab<0.0001
NaOCl/DR18%2 (1.25; 2) BCa3 (3; 3) Cb2.5 (2; 3) Db0.0000858
NaOCl/ID9%1 (1; 1) AD1 (1; 1) A1 (1; 1.75) B0.174
NaOCl/ID15%1 (1; 1) A1 (1; 1) A1 (1; 1) Bna
NaOCl/ID18%1 (1; 1) A1 (1; 1) A1 (1; 1) Bna
p *<0.0001<0.0001<0.0001
ABCD different letters indicate statistically significant differences between the irrigants (in the columns); ab different letters indicate statistically significant differences between the root thirds (in the rows); * between the groups; 1 between the root canal thirds; na—not applicable.
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MDPI and ACS Style

Mikheikina, A.; Babina, K.; Polyakova, M.; Doroshina, V.; Zaytsev, A.; Makeeva, I.; Novozhilova, N. Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique. Appl. Sci. 2024, 14, 8422. https://doi.org/10.3390/app14188422

AMA Style

Mikheikina A, Babina K, Polyakova M, Doroshina V, Zaytsev A, Makeeva I, Novozhilova N. Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique. Applied Sciences. 2024; 14(18):8422. https://doi.org/10.3390/app14188422

Chicago/Turabian Style

Mikheikina, Anna, Ksenia Babina, Maria Polyakova, Vladlena Doroshina, Alexandr Zaytsev, Irina Makeeva, and Nina Novozhilova. 2024. "Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique" Applied Sciences 14, no. 18: 8422. https://doi.org/10.3390/app14188422

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