Abstract
Background:
Effective root canal irrigation removes the smear layer for optimal sealer penetration. While 17% ethylenediaminetetraacetic Acid (EDTA) is effective, concerns about dentin erosion exist. Alternatives like 7% maleic acid (MA) and 20% N-acetylcysteine (NAC) show promise with fewer adverse effects.
Aim:
To compare the effects of 20% NAC, 7% MA, and 17% EDTA as final irrigating solutions on the depth of sealer penetration into dentinal tubules at coronal, middle, and apical thirds of root canals using confocal laser scanning microscopy (CLSM).
Materials and Methods:
Sixty-six single-canal mandibular premolars free of caries, fractures, or prior treatment were selected. The teeth were decoronated to 14 mm root length using a diamond disk under water spray. Working length was determined by inserting a size 10 K-file until visible at the apical foramen, subtracting 1 mm. Root canals were instrumented up to F3 using ProTaper Universal rotary files with 1 mL of 2.5% NaOCl irrigation between files. Based on the final irrigation protocol, samples were divided into three groups (n = 22): Group 1–20% NAC, Group 2–7% MA, and Group 3–17% EDTA. Each group was irrigated with 5 mL of the respective irrigant, followed by a final rinse with 10 mL of distilled water. AH Plus sealer with 0.1% Rhodamine B was applied using a #25 Lentulo, and an F3 gutta-percha cone coated with the sealer was placed to working length, trimmed, and sealed with Cavit. Samples were incubated at 37°C and 100% humidity for 7 days to allow sealer setting. Roots were sectioned at 2, 5, and 8 mm from the apex to obtain 1 mm thick sections. Sealer penetration into dentinal tubules was evaluated using CLSM at ×10 magnification, measuring the penetration depth in micrometers from the canal wall to the point of maximum sealer infiltration using ImageJ software, measuring the longest penetration depth from the canal wall to the point of deepest sealer infiltration.
Results:
Sealer penetration was greatest in the coronal third, followed by the middle, with the least in the apical third (P < 0.0001). NAC demonstrated the highest mean in the coronal region (829.35 ± 85.36), while MA exhibited superior performance in the middle (522.92 ± 112.32) and apical (361.76 ± 49.03) regions. Intergroup comparisons showed superior penetration with 7% MA in the apical region (P < 0.0001). NAC and EDTA demonstrated comparable penetration across regions.
Conclusion:
While all irrigants enhanced sealer penetration, 7% MA was most effective in the apical region. Both 7% MA and 20% NAC can serve as alternatives to 17% EDTA for final irrigation.
Keywords: Confocal laser scanning microscopy, dentinal tubules, ethylenediaminetetraacetic acid, maleic acid, N-acetylcysteine, sealer, smear layer
INTRODUCTION
Mechanical instrumentation during cleaning and shaping generates a smear layer.[1] This smear layer poses a significant obstacle to optimal root canal disinfection, by harboring bacteria, blocking disinfectant penetration, and creating a physical barrier that compromises the effectiveness of root canal sealers. Therefore, its removal is crucial.[2]
Chelating agents such as ethylenediaminetetraacetic acid (EDTA) are widely used to eliminate the smear layer by breaking down inorganic components. At a 17% concentration and neutral pH, EDTA reacts with calcium ions to decalcify dentin, penetrating up to 20–30 μm. However, this process may lead to dentin erosion, diminishing microhardness, and potentially causing structural changes.[3]
Malic acid (MA), another chelating agent, demonstrates the ability to efficiently eliminate the smear layer during endodontic treatment. Using a 7% malic acid solution as a final 1-min irrigation has been shown to be superior to 17% EDTA solution in eliminating the smear layer.[4]
Recent research indicates that NAC, a glutathione precursor, demonstrates similar effectiveness to EDTA in eliminating the smear layer, with substantially less harm to dentin microhardness.[5] Furthermore, 20% NAC demonstrates potent antioxidant and antimicrobial capabilities, proving effective against the bacterium Enterococcus faecalis and exhibiting the ability to diminish biofilm formation.[6]
Achieving an effective seal is essential for successful endodontic treatment, primarily through thorough sealer penetration into dentinal tubules during obturation. This infiltration entombs bacteria and debris, preventing reinfection, while also enhancing retention through mechanical locking. Therefore, the degree of sealer penetration indicates the efficiency of smear layer elimination following final irrigation with various chelating agents.[7]
This study evaluates and compares the effects of NAC, MA, and EDTA, as final irrigants on the penetration depth of an epoxy resin-derived sealer within dentinal tubules across the apical, middle, and coronal regions of root canals, utilizing confocal laser scanning microscopy (CLSM). Given the established efficacy of NAC and MA in smear layer removal, this investigation addresses the lack of comparative research on their impact on sealer penetration depth relative to EDTA.
MATERIALS AND METHODS
Sample preparation
Sixty-six mandibular premolars, free from caries, fractures, or prior endodontic treatment, were selected for this investigation. The teeth were thoroughly scaled to remove calculus and soft tissue using hand scalers and were preserved in a 0.1% thymol solution to prevent microbial growth and maintain hydration until experimentation. Only teeth with single canals were selected after confirming with periapical radiographs. The teeth were sectioned at cementoenamel junction with a high-speed diamond disc under constant water irrigation, standardizing the root lengths to 14 mm. The working length was determined by inserting a #10 K-file and advancing it until the tip became visible at the apical foramen, then subtracting 1 mm from the recorded length. The root canals were initially enlarged to a size 20 K-file to create a glide path and facilitate subsequent rotary instrumentation. The canals were then instrumented utilizing ProTaper Universal rotary files till F3. During instrumentation, the canals were flushed with 1 ml of 2.5% sodium hypochlorite (NaOCl) between each instrument to enhance debris removal and disinfection.
After shaping, the teeth were randomly allocated to three experimental groups as per the final irrigation protocol (n = 22).
Group 1: 20% N-acetylcysteine (NAC)
Group 2: 7% MA
Group 3:17% EDTA.
A 20% NAC solution was prepared by dissolving 0.2 g of NAC in 1 mL of distilled water. Each irrigation solution from the respective group was delivered inside the root canal using 30-gauge side-vented needle, with the needle placed 1 mm short of working length. In each group, 5 ml of the respective solution was used as the final rinse, with irrigation lasting for 1 min. Subsequently, 10 ml of distilled water was used to neutralize any remaining irrigant in the canal and prevent potential chemical reactions. In the end, sterile paper points were used to dry the canals.
Sealer penetration analysis
Rhodamine B isothiocyanate dye was incorporated into the AH Plus root canal sealer at a concentration of 0.1% by weight to facilitate imaging using CLSM. Following this, the labeled sealer was coated onto the canal walls with a #25 Lentulo to ensure even coverage. Next, a ProTaper Universal F3 gutta-percha cone, slightly layered with the labeled sealer, was placed to the working length. The excess cone was trimmed with a heated plugger. Cavit was then used to seal the coronal access. Finally, these samples were incubated at 37°C and 100% humidity for 7 days, ensuring the sealer achieved optimal setting.
A diamond disc was used to section these roots at 2, 5, and 8 mm from the apex with continuous water cooling to obtain a section of 1 mm thickness, which was then polished with silicon carbide abrasive paper and mounted on glass slides. Sealer penetration into dentinal tubules was then evaluated using CLSM at ×10 magnification with excitation and emission wavelengths of 514–561 nm for rhodamine dye [Figure 1]. Quantitative analysis was performed using ImageJ software, measuring the longest penetration depth from the canal wall to the point of deepest sealer infiltration.
Figure 1.
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Statistical analysis
The values were organized into a spreadsheet using Microsoft Excel 2021, and statistical analysis was performed using GraphPad Prism for Windows, V10.1.2 (GraphPad Software, La Jolla California USA). Shapiro–Wilk test, along with an examination of histograms, standard Q-Q plots, and box plots, all of which indicated that the data followed an approximately normal distribution. A two-way analysis of variance was conducted to evaluate the impact of irrigating solutions and the region on the average penetration depth. A P ≤ 0.05 was viewed to be statistically relevant.
RESULTS
Tables 1 and 2 represent the average (± standard deviation) penetration depth and the comparisons among the study groups at each level. The analysis highlighted significant effects of irrigating agents, regions, and their interaction (P < 0.0001). Across regions, NAC demonstrated the highest mean in the coronal region (829.35 ± 85.36), while MA exhibited superior performance in the middle (522.92 ± 112.32) and apical (361.76 ± 49.03) regions. EDTA showed consistent but lower performance across all regions. The interaction effect revealed significant differences between regions, with coronal values surpassing middle and apical values for all agents (P < 0.0001). Pairwise comparisons showed significant differences in the apical region for NAC versus MA and MA versus EDTA (P < 0.0001) [Table 2 and Figure 2]. These outcomes underscore the variability in performance across regions, with MA demonstrating a pronounced effect in the apical region.
Table 1.
Mean±standard deviation values of dentinal tubule sealer penetration (μm) at coronal, middle, and apical regions for the study groups
| Region/study groups | 20% NAC (n=22) | 7% MA (n=22) | 17% EDTA (n=22) |
|---|---|---|---|
| Coronal | 829.35±85.36 | 814.35±73.46 | 790.59±90.51 |
| Middle | 495.46±92.25 | 522.92±112.32 | 472.85±88.85 |
| Apical | 218.03±18.45 | 361.76±49.03 | 233.59±31.37 |
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n: Sample size per group, EDTA: Ethylenediaminetetraacetic acid, NAC: N-acetyl cysteine, MA: Maleic acid
Table 2.
Comparison of adjusted P values for dentinal tubule sealer penetration between study groups and regions
| Comparison Between study groups | Adjusted P value | ||
|---|---|---|---|
| Coronal | Middle | Apical | |
| NAC versus MA | 0.7957 (NS) | 0.4666 (NS) | <0.0001* |
| NAC versus EDTA | 0.2212 (NS) | 0.5958 (NS) | 0.782 (NS) |
| MA versus EDTA | 0.5646 (NS) | 0.0825 (NS) | <0.0001* |
| Between regions | NAC | MA | EDTA |
| Coronal versus middle | <0.0001* | <0.0001* | <0.0001* |
| Coronal versus apical | <0.0001* | <0.0001* | <0.0001* |
| Middle versus apical | <0.0001* | <0.0001* | <0.0001* |
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*Statistically significant (P≤0.05). NS (P>0.05). Two-way ANOVA revealed significant main effects of regions (P<0.0001) and irrigating agents (P<0.0001), as well as a significant interaction effect (P<0.0001). NS: Not significant, EDTA: Ethylenediaminetetraacetic acid, ANOVA: Analysis of variance, NAC: N-acetyl cysteine, MA: Maleic acid
Figure 2.
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DISCUSSION
Ensuring effective penetration of the final irrigation agent is crucial for antimicrobial efficacy. In addition, removing the smear layer enhances the infiltration of sealers used in endodontic treatment. Increased sealer penetration into the dentinal tubules strengthens the bond between the gutta-percha and the canal wall, creating both a physical barrier and a hermetic seal.[7] While EDTA remains the preferred chelating agent, 17% EDTA is less effective in the apical region of the root canal. In addition, it is shown to erode both peritubular and intertubular dentin resulting in reduced dentin microhardness.[8] Research has shown that NAC is as efficient as EDTA in eliminating the smear layer.[5] MA, another gentle organic acid, has also been investigated as a chelating agent in endodontic treatments. However, the high acidity of MA can substantially reduce root dentin microhardness, trigger demineralization, and potentially harm the intertubular dentin.[9]
This study sought to explore and compare the impact of 20% NAC, 7% MA, and 17% EDTA on the penetration depth of a resin-based epoxy endodontic sealer, a topic that has not been previously explored. AH Plus, an epoxy resin-derived sealer recognized as the “gold standard,” was utilized in this investigation. During the procedure, 1 ml of 2.5% NaOCl was employed between instrumentation steps to leverage its antimicrobial, tissue-dissolving, and lubricating properties. Side-vented needles were selected for their enhanced efficacy over conventional types.[10]
A CLSM method examined sealer penetration depth within dentinal tubules at various levels. This approach offers several advantages, such as minimal sample preparation, fewer artifacts, and precise control over the depth of field. This technique was further supported using Rhodamine B, a fluorescent dye used to label the sealer, which did not alter the sealer’s physicochemical characteristics.[11]
The findings of the study revealed that across all irrigant groups, including 17% EDTA, 20% NAC, and 7% MA, the depth of sealer penetration occurred most extensively in the coronal third, then the middle third, and least in the apical third, with statistically significant differences observed among these regions. This difference can be explained by the reduced number and diameter of dentinal tubules in apical areas, along with anatomical factors such as irregular secondary dentin and cementum-like tissue, which hinder the elimination of the smear layer and impede sealer infiltration.[12]
Intergroup comparisons revealed that 7% MA achieved significantly higher sealer penetration in the apical third when compared to 17% EDTA and 20% NAC. This can be attributed to MA’s superior ability to remove the smear layer and its higher acidity, with a pH of 1.05, compared to the neutral pH of EDTA and the alkaline pH of 11 exhibited by NAC, at which it is most effective against biofilm. This was supported by Ballal et al. who also noted EDTA’s lower penetration may be linked to its larger molecular size and reduced ability to decalcify dentin in the apical region. Comparable results between NAC and EDTA in the apical region suggest that NAC’s chelating and antioxidant properties facilitate smear layer removal.[4,13]
For the middle and coronal thirds, no significant differences were noted among the three irrigants, which aligns with prior findings by Kara et al.[13] and Osazir et al.[14] However, Shetty and Hegde reported increased penetration with EDTA in these sections, possibly due to its ability to form soluble calcium chelates.[15] MA and NAC also demonstrated comparable performance to EDTA in smear layer removal for these sections, which aligns with the study by Ballal et al. and Rajakumaran et al.[4,5]
The study concludes that 7% MA and 20% NAC demonstrated optimal sealer penetration, suggesting their potential as effective alternatives to 17% EDTA for final root canal irrigation. These alternative irrigants also exhibit lower cytotoxicity when extruded beyond the periapex compared to EDTA.[16,17] MA, in addition to its superior smear layer removal capability, has shown a comparable effect on dentin microhardness relative to EDTA.[18] Conversely, NAC provides similar smear layer removal efficiency as EDTA while causing a lesser reduction in dentin microhardness, further supporting its clinical applicability.[5]
However, being a laboratory-based study, the findings need validation through clinical trials to assess their impact on the success of endodontic treatments.
CONCLUSION
Considering the limitations of this research, it was observed that all three final irrigants facilitated sealer penetration inside the dentinal tubules. While sealer penetration at the coronal and middle third was comparable between the groups, 7% MA demonstrated superior penetration at the apical third compared to 20% NAC and 17% EDTA. Overall, the maximum sealer penetration occurred at the coronal third, with the middle third showing little less, with the lowest penetration observed in the apical third across all groups. These findings indicate that 7% MA and 20% NAC are effective alternatives to 17% EDTA as final irrigating agents and can be recommended for use in endodontic procedures.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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