Efficacy of glycolic acid at different concentrations for smear layer removal with various activation systems

Abstract

Background

This study aimed to evaluate the effects of different irrigation solutions and activation systems on smear layer removal.

Methods

Ninety mandibular canines were instrumented using T-Endo Must files (#40.04) and randomly assigned to nine groups (n = 10) according to the final irrigation protocol: Group 1a (17% EDTA, Needle), Group 1b (5% GA, Needle), Group 1c (10% GA, Needle), Group 2a (17% EDTA, EDDY), Group 2b (5% GA, EDDY), Group 2c (10% GA, EDDY), Group 3a (17% EDTA, XP-F), Group 3b (5% GA, XP-F), and Group 3c (10% GA, XP-F). Smear layer removal was evaluated using scanning electron microscopy. A three-way analysis of variance with Bonferroni correction was performed to assess the effects of the independent variables and their interactions, and Tukey’s post hoc test was used for pairwise comparisons. Statistical significance was set at p < 0.05.

Results

Root section, irrigation solution, and activation system each significantly affected smear scores (p < 0.0001). A significant two-way interaction was observed between root section and irrigation solution (p = 0.025). Smear scores increased from the coronal to the apical third of the root canal. Both 5% and 10% GA significantly reduced smear scores compared with 17% EDTA (p < 0.05), and activation using EDDY and XP-F resulted in significantly lower smear scores compared with needle irrigation (p < 0.05).

Conclusion

Both 5% and 10% GA resulted in lower smear scores, while irrigation activation with EDDY and XP-F significantly enhanced smear layer removal.

Introduction

The smear layer is an amorphous structure composed of both inorganic and organic components that forms on root canal walls during chemomechanical instrumentation. It contains bacteria, bacterial by-products, and necrotic tissue, serving not only as a reservoir for microorganisms but also facilitating their deeper penetration and colonization within dentinal tubules [1]. The presence of the smear layer can negatively affect root canal disinfection by restricting the penetration of irrigants and medicaments into dentinal tubules. Furthermore, it acts as a barrier between the canal wall and filling materials, potentially compromising the seal of the root canal filling [2]. Consequently, the removal of the smear layer is widely regarded as essential.

Various irrigation solutions, including etidronic acid, citric acid and ethylene diamine tetraacetic acid (EDTA) are used to remove the smear layer, with EDTA being the most commonly preferred solution. At a concentration of 17% and an application time of approximately one minute, EDTA can effectively remove the smear layer [3]. However, prolonged exposure may cause excessive calcium ion loss from root dentin and erosion of tubular dentin, negatively affecting sealer adhesion and reducing dentin microhardness [4–6]. Additionally, the metal complexes formed by EDTA are not readily biodegradable, raising concerns about its potential long-term negative environmental impact [7, 8]. Given these limitations, the search continues for irrigation solutions and activation systems that can effectively remove the smear layer without compromising dentin integrity or harming the environment.

Glycolic acid (GA) is a weak organic acid and the smallest member of the alpha-hydroxy acid family, is both colorless and odorless. Owing to its biodegradability, GA has been proposed as an alternative chelating agent to EDTA [9]. Previous studies have demonstrated that GA is as effective as EDTA in removing the smear layer [10], while inducing less alteration in the collagen/apatite ratio compared with EDTA [11]. Several studies [10–12] have explored GA at concentrations ranging from 5% to 17%; however, no consensus has been reached regarding the optimal concentration.

The sonic activation device EDDY (VDW, Munich, Germany) features a flexible polyamide tip with a size of 25/0.04 and operates at a frequency of 5,000–6,000 Hz, generating acoustic streaming and three-dimensional irrigant movement within the canal to enhance cleaning efficiency [13]. The XP-Endo Finisher (XP-F; FKG, LCF, Switzerland) is a specially heat-treated NiTi file that is martensitic below 37 °C and straight in shape but transforms into an austenitic phase and adopts a helical form at body temperature. Unlike sonic devices that vibrate to agitate the irrigant, the XP-F employs a unique whipping motion designed to contact previously untouched canal walls and improve debridement[14].. Both sonic activation [15] and XP-F [16] have been reported to improve the removal of the smear layer and debris when used for irrigation activation. However, to the best of our knowledge, the effects of GA in combination with sonic activation and XP-F have not yet been investigated. Therefore, the aim of this study was to evaluate the effects of root section, irrigation solution, and activation system on smear layer removal. The null hypothesis was that there would be no significant difference among the evaluated parameters.

Materials and methods

A priori power analysis for sample size was performed using the G*Power 3.1.9.4 software (Heinrich Heine University, Dusseldorf, Germany) within the F-test family. The calculation was based on an effect size of 0.40 (f = 0.40) [17], 5% α error (α = 0.05), and 80% power (1-β = 0.80), resulting in a total sample size of 80. Therefore, a total of 90 samples were included, with 10 samples in each group.

The local ethics committee approved the study design (Protocol Number: 22/04/2024-02).

Sample selection

Ninety extracted human mandibular canines with single, oval canals (long-to-short diameter ratio ≥ 2.5) were selected for this study. The teeth were obtained within six months of extraction from individuals aged 18–45 years, primarily for periodontal reasons. Canal dimensions were confirmed using periapical radiographs taken 5 mm from the root apex in both buccolingual and mesiodistal views [18]. Teeth exhibiting root caries, resorption, calcifications, immature root formation, previous root canal treatment, cracks, or fractures were excluded. Canal curvature was verified to be < 5° according to Schneider’s method based on radiographic evaluation. All specimens were stored in 0.1% thymol solution until use and decoronalized to a standardized root length of 14 mm.

Root canal instrumentation and irrigation procedures

Access cavities were prepared using a high-speed round diamond bur #801 (016) (Frank Dental, Germany). Apical patency was confirmed with #10 and #15 K-files (VDW), and teeth with an apical diameter larger than a #15 K-file were excluded. Working length (WL) was determined visually by subtracting 1.0 mm from the length at which the file tip extended beyond the major apical foramen.

To simulate a closed canal system, all root apices were coated with adhesive, as previously described [19]. Root canal instrumentation was performed using T-Endo Must reciprocal files (#40.04, Dentac, Istanbul, Turkey) with a pecking motion of 2–3 mm. After every three pecking motions, canals were irrigated with 2.5% sodium hypochlorite (NaOCl; Wizard, RehberKimya, Istanbul, Turkey). Irrigation was delivered using a 30-G side-vented needle (C-K ENDO, CK Dental, China) positioned 1 mm short of the WL. A total of 10 mL of NaOCl was used, followed by 5 mL of distilled water to neutralize residual NaOCl. The specimens were randomly assigned to nine groups (n = 10) according to the final irrigation protocol:

Group 1a (17% EDTA-Needle): Root canals were irrigated with 3 mL of 17% EDTA (Coltene, Altstätten, Switzerland) for 1 min using a 30-G side-vented needle, followed by 5 mL of distilled water, and dried with paper points (VDW).

Group 1b (5% GA-Needle): The same protocol as Group 1a was applied, but 5% glycolic acid (GA; Doa Chemistry, İzmir, Turkey) was used instead of EDTA.

Group 1c (10%GA-Needle): The same protocol as Group 1a was applied, but 10% GA was used.

Group 2a (17% EDTA-EDDY): Root canals were irrigated with 1 mL of 17% EDTA, activated for 20 s using an EDDY 25/.04 tip, and this cycle was repeated three times. Afterward, 5 mL of distilled water was used, and canals were dried with paper points.

Group 2b (5% GA- EDDY): The same protocol as Group 2a was applied, but 5% GA was used instead of EDTA.

Group 2c (10%GA- EDDY): The same protocol as Group 2a was applied, but 10% GA was used.

The EDDY tip was positioned 1 mm short of the WL and operated with an in-and-out motion of 4–5 mm amplitude

Group 3a (17% EDTA-XP-F): Root canals were irrigated with 1 mL of 17% EDTA and activated for 20 s using XP-F. This cycle was repeated three times. Afterward, 5 mL of distilled water was used, and canals were dried with paper points.

Group 3b (5% GA- XP-F): The same protocol as Group 3a was applied, but 5% GA was used instead of EDTA.

Group 3c (10%GA- XP-F): The same protocol as Group 3a was applied, but 10% GA was used.

For XP-F activation, the instrument was positioned 1 mm short of the WL and operated with in-and-out strokes of 7–8 mm amplitude at 1000 rpm and 1 Ncm torque. Before intracanal use, the XP-F file was immersed in distilled water preheated to 37 °C until it assumed its helical shape; its ability to maintain this helical configuration during activation was confirmed.

The pH of the GA solutions was adjusted to 1.2 using a pH meter and was consistent across all GA groups.

Scanning electron microscopy evaluation

The roots were longitudinally split in the buccolingual direction using a precision cutting device, and one half of each root was selected for evaluation. Reference lines were drawn to delineate the coronal, middle, and apical thirds of the canal. After gold–palladium coating, scanning electron microscopy (SEM) images of these thirds were captured at ×2000 magnification (Fig. 1). Unprepared areas of the canal and regions beyond the WL were excluded from the analysis. Within each third, the area presenting the greatest amount of debris and smear layer was consistently selected for evaluation. The smear layer was assessed by two calibrated examiners using a five-point rating scale described previously [13]. Both examiners were blinded to the experimental groups. The scoring criteria were defined as follows:

1. Smear layer is not present. Fully open dentinal tubules are visible.

2. A minimal quantity of smear layer exists, with some dentinal tubules open.

3. A mostly uniform smear layer covers the canal wall, with very few open dentinal tubules.

4. There are no open dentinal tubules. The canal walls are coated with a consistent smear layer.

5. A dense, uniform smear layer completely covers the canal wall.

Fig. 1.

Images obtained at 2000x magnification using a scanning electron microscope

Statistical analysis

Cohen’s kappa test was used to analyze interobserver reliability between two practitioners. The normality of the data derived from the smear scores was tested using the Shapiro-Wilk test, and the homogeneity of variance was assessed with Levene’s test. Three-way analysis of variance with Bonferroni correction was used to analyze the effects of the independent variables—root level, irrigation solution, and activation system—as well as their interactions. Tukey’s post hoc test was performed for multiple comparisons. Statistical analyses were performed using the Statistical Package for the Social Sciences 22 software program (IBM, Chicago, USA). The results were evaluated at a 95% confidence interval, with significance set at p < 0.05.

Results

The kappa value for interobserver reliability was 0.89. The statistical analysis revealed that the root section (F = 31.424, p < 0.0001), irrigation solution (F = 29.229, p < 0.0001), and activation system (F = 24.670, p < 0.0001) each had a significant effect on the smear scores. Significant two-way interactions were observed between the root section and the irrigation solution (F = 2.828, p = 0.025). However, the interactions between the root section and the activation system, as well as between the irrigation solution and the activation system, were not significant (p > 0.05). Additionally, no significant three-way interaction was found among the variables studied (p > 0.05) (Table 1).

Table 1.

Tests of between-subjects effects. Dependent variable: smear score

Source	Type III Sum of Squares	df	Mean Square	F	Sig.	Partial Eta Squared
Root section	41.356	2	20.678	31.424	0.000***	0.205
Irrigation solution	38.467	2	19.233	29.229	0.000***	0.194
Activation	32.467	2	16.233	24.670	0.000***	0.169
Root section * Irrigation solution	7.444	4	1.861	2.828	0.025*	0.044
Root section * Activation	3.244	4	0.811	1.233	0.298	0.020
Irrigation solution * Activation	1.133	4	0.283	0.431	0.786	0.007
Root section * Irrigation solution * Activation	2.289	8	0.286	0.435	0.899	0.014

df Degree of freedom, *** extremely significant (p < 0.001), ** highly significant (0.001 < p < 0.01), * significant (0.01 < p ≤ 0.05); ns non-significant (p  > 0.05)

The means and standard deviations of the smear scores for the root section, irrigation solution, and activation system are presented in Table 2. Post hoc multiple comparisons revealed a significant difference among the apical, middle, and coronal thirds, with smear scores decreasing from the apical to the coronal third. Furthermore, compared with 17% EDTA, both 5% GA and 10% GA significantly reduced smear scores (P < 0.05). Similarly, compared with needle, both sonic activation and XP-F significantly reduced smear scores (P < 0.05) (Table 3).

Table 2.

Mean and standard deviation of smear score based on root section, irrigation solution, and activation system

Irrigation solution	Activation system	Coronal		Middle		Apical
		Mean	Std. Deviation	Mean	Std. Deviation	Mean	Std. Deviation
%17 EDTA	Needle	3.30	0.675	3.80	0.789	4.20	0.789
	EDDY	2.20	0.789	2.90	0.568	4.00	0.667
	XP-F	2.30	0.675	2.90	0.738	3.90	0.738
%5 GA	Needle	2.60	0.966	3.40	1.075	3.10	1.197
	EDDY	2.20	0.632	2.20	0.919	2.70	0.823
	XP-F	2.20	0.789	2.60	0.966	2.80	0.789
%10 GA	Needle	2.50	0.707	3.00	0.667	3.40	1.075
	EDDY	1.70	0.675	1.90	0.738	2.60	0.699
	XP-F	1.80	0.789	1.80	0.789	2.70	0.823

Table 3.

Post-hoc comparisons

			Mean Difference	Std. Error	Sig.	95% Confidence Interval
						Lower Bound	Upper Bound
Root section
Coronal		Middle	− 0.41	0.121	0.002*	− 0.70	− 0.13
		Apical	− 0.96	0.121	0.000*	−1.24	− 0.67
Middle		Coronal	0.41	0.121	0.002*	0.13	0.70
		Apical	− 0.54	0.121	0.000*	− 0.83	− 0.26
Apical		Coronal	0.96	0.121	0.000*	0.67	1.24
		Middle	0.54	0.121	0.000*	0.26	0.83
Irrigation solution
%17 EDTA	%5 GA		0.63	0.121	0.000*	0.35	0.92
	%10 GA		0.90	0.121	0.000*	0.61	1.19
%5 GA	%17 EDTA		− 0.63	0.121	0.000*	− 0.92	− 0.35
	%10 GA		0.27	0.121	0.072	− 0.02	0.55
%10 GA	%17 EDTA		− 0.90	0.121	0.000*	−1.19	− 0.61
	%5 GA		− 0.27	0.121	0.072	− 0.55	0.02
Activation system
Needle	EDDY		0.77	0.121	0.000*	0.48	1.05
	XP-F		0.70	0.121	0.000*	0.41	0.99
EDDY	Needle		− 0.77	0.121	0.000*	−1.05	− 0.48
	XP-F		− 0.07	0.121	0.846	− 0.35	0.22
XP-F	Needle		− 0.70	0.121	0.000*	− 0.99	− 0.41
	EDDY		0.07	0.121	0.846	− 0.22	0.35

*Statistically significant (p < 0.05)

Discussion

This study aimed to evaluate the effects of the root section, irrigation solution, and activation system on smear layer removal. The findings demonstrated that all three variables had a significant influence on smear layer scores, leading to the rejection of the null hypothesis.

In this study, both 5% and 10% GA demonstrated similar effectiveness in smear layer removal, with both concentrations producing lower smear scores than 17% EDTA. Dal Bello et al. [10] reported no significant differences among 5%, 10%, and 17% GA in terms of smear layer removal, and their efficacy was comparable to that of 17% EDTA. The differences between studies may be attributed to variations in apical preparation size (40.02) and irrigation volume (5 mL) used in their study on mandibular incisors. Furthermore, authors noted that higher GA concentrations (17%) exhibited increased cytotoxicity and had adverse effects on dentin microhardness and surface roughness. Another study [20] reported a progressive reduction in the hydroxyapatite/collagen ratio as GA concentration increased from 5% to 10% and 17%. Marafiga et al. [12] observed that 17% EDTA and 17% GA demonstrated similar effects on fracture strength and were both inferior to 10% GA, which had fewer negative effects on the mechanical properties of dentin. Souza et al. [21] also demonstrated that the effects of 10%, 17%, and 25% GA on dentin microhardness were similar to those of distilled water, suggesting that 10% GA may be a viable alternative due to its lower cytotoxicity. The cytotoxicity of GA has been shown to be dose-dependent, with 10% GA being less cytotoxic than 17% EDTA. Based on these findings, the present study focused on GA concentrations of 5% and 10%.

The effectiveness of irrigation solutions depends on factors such as concentration, surface tension, and pH. Although GA has a larger molecular size than EDTA, its surface tension is similar [20]. The use of GA at lower pH (pH 1.2 vs. 5) has been associated with more superficial changes in the collagen/apatite ratio [11], therefore, in the present study, the pH was adjusted to 1.2. GA is a naturally occurring acid derived from plant sources such as grapes, sugarcane, and beets [22], and it has been shown to induce collagen synthesis and promote fibroblast proliferation [23, 24].

Glycolic acid, owing to its low pH as an α-hydroxy acid, induces a proton-mediated acidic dissolution mechanism [25]. As a result, the inorganic components of the smear layer are disrupted by proton attack, and the released calcium ions are simultaneously complexed, preventing their reprecipitation [26]. In contrast, EDTA primarily functions as a strong chelating agent in a neutral or alkaline environment and does not induce pH-driven acidic dissolution. Moreover, as calcium ions are displaced from dentin by hydrogen ions, the gradual decrease in pH reduces the chelating efficiency of EDTA over time [27]. These mechanistic distinctions likely underlie the differences observed in our experimental findings.

Sodium hypochlorite remains a commonly used irrigant owing to its antimicrobial efficacy; however, it may reduce bond strength by forming an oxygen inhibition layer and disrupting collagen fibrils [28]. Interestingly, combining NaOCl with GA has been reported to improve fiber post bond strength compared with NaOCl–EDTA combinations [29]. Furthermore, GA has demonstrated superior antibacterial activity against Enterococcus faecalis compared with EDTA, highlighting its potential as a promising alternative irrigant in endodontic treatment [30].

This study demonstrated that, compared with conventional needle irrigation, activation of irrigants using sonic activation and XP-F significantly enhanced smear layer removal. Although various activation techniques have been shown to improve the efficacy of EDTA [16, 31], the effect of activation systems on smear layer removal with GA had not previously been investigated. Keskin et al. [32] compared 17% EDTA with 5% and 10% GA for calcium hydroxide removal and observed no significant difference between non-activated groups; however, with ultrasonic activation, 10% GA performed better than EDTA. Similarly, another study reported that ultrasonic activation of 17% EDTA and 17% GA reduced dentin microhardness but did not affect cohesive strength, flexural strength, or fracture resistance of root dentin [33]. The short exposure time (1 min) used in these studies may explain the absence of significant changes in dentin mechanical properties. Although irrigation activation with sonic activation and XP-F significantly enhanced smear layer removal in this study, the potential effects of GA activation on the mechanical properties of dentin were not evaluated.

Consistent with previous studies [34–36], this study also demonstrated lower smear scores in the coronal third than in the apical third. This result may be attributed to the reduced number and diameter of dentinal tubules as well as the increased prevalence of sclerotic dentin in the apical region [37, 38].

A primary limitation of this study is that, although a size 40.04 master apical file was selected to minimize uninstrumented surface areas in oval-shaped canals, the inherent complexity of oval canal morphology means that some canal walls, particularly in the cervical third, may have remained uninstrumented, potentially limiting smear layer formation in these areas. Smear layer evaluation was performed using SEM, as in previous studies [34, 39, 40]. While SEM provides valuable insights into dentinal tubule conditions, images captured at 2000× magnification may not fully represent the entire dentin wall, and standardization of dentinal tubule number and localization in extracted teeth remains challenging. Future studies employing high-resolution microcomputed tomography could provide a more comprehensive three-dimensional evaluation of canal wall instrumentation and smear layer removal, thereby confirming and expanding upon the present findings.

Furthermore, the study focused solely on Vertucci Type 1 teeth with straight canals, which limits generalizability, as activation techniques often compensate for inadequate wall contact by instruments in curved anatomies. Additionally, the XP-F file was immersed in distilled water at 37 °C prior to intracanal use to obtain its helical shape and its structural stability during activation was confirmed, the ambient and irrigant temperatures in this study did not fully replicate clinical conditions. This difference may have influenced the behavior of the instrument and should be considered when interpreting the results. Finally, there is no current consensus on the ideal concentration of GA; therefore, future studies comparing different GA concentrations in more complex canal anatomies using a single activation system are warranted.

Conclusion

Within the limitations of this study, both 5% and 10% GA resulted in lower smear layer scores compared with 17% EDTA. Irrigation activation with EDDY and XP-F significantly enhanced smear layer removal compared with conventional needle irrigation. Additionally, smear scores increased from the coronal to the apical third of the root canal.

Abbreviations

EDTA

Ethylene diamine tetraacetic acid

GA

Glycolic acid

XP-F

XP-Endo finisher

WL

Working length

NaOCl

Sodium hypochlorite

SEM

Scanning electron microscopy

Authors’ contributions

M.S. Conceptualization (equal); methodology (equal); investigation (equal); formal analysis (lead); writing – original draft (lead). A.A.A. Conceptualization (equal); methodology (equal); investigation (equal). P.T. Conceptualization (equal); methodology (equal); writing – review and editing (equal). B.Ç. Conceptualization (equal); methodology (equal); writing – review and editing (equal). All authors have contributed significantly to the conception or design of the work or the acquisition, analysis, or interpretation of data or the creation of new software used in the work. All authors are in agreement with the manuscript; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding 

No funding was obtained for this study.

Data availability

All data analysed during this study are included in this article.

Declarations

Ethics approval and consent to participate

This study was approved by the Hatay Mustafa Kemal University, Non-Interventional Research Ethics Committee (Protocol Number: 22/04/2022-02). The authors certify that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments (as revised in Brazil 2013). As this study did not involve human participants, the Hatay Mustafa Kemal University Non-Interventional Research Ethics Committee determined that informed consent was not required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.