The objective of this study was to evaluate the effects of glycolic acid (GA) (with pH 1.2 and 5) and ethylenediaminetetraacetic acid (EDTA) on the chemical and mechanical properties of dentin to investigate the potential use of GA as final irrigant in the root canal therapy. Specifically, changes in microhardness, smear layer removal, erosion, mineral content distribution, apatite/collagen ratio and flexural strength of mineralized dentin treated with GA were assessed. Saline solution was used as a negative control. Knoop microhardness (KHN) was measured on the root canal lumen of root segments. Dentin beams were used for 3-point flexural strength (σ) test. Scanning electron microscopy (SEM) images of root sections were obtained for evaluation of smear layer removal and dentin erosion on root segments and energy dispersive X-ray spectroscopy (EDS) was used for mineral content distribution. The apatite/collagen ratio (A/C) in dentin powder were examined by Fourier transform infrared (FTIR) spectroscopy. KHN, σ and A/C results were statistically analyzed with ANOVA and Tukey tests (α = 0.05). Smear layer and dentin erosion scores were analyzed with Kruskal-Wallis and Dunn tests (α = 0.05). Root dentin treated with EDTA and GA presented similar KHN regardless of the pH (p > 0.05). However, KHN was significantly reduced in EDTA and GA groups when compared to control group (p<0.001). GA showed the same ability to remove the smear layer and to cause dentin erosion as EDTA. EDS results showed that the GA and EDTA solutions did not alter the dentin mineral content distribution. The apatite/collagen ratio reduced with all irrigant solution and was the lowest with GA pH 5 (p<0.001), while σ was not significantly affected by the experimental solutions (p = 0.559). It can be concluded that GA has similar ability to remove the smear layer than EDTA. GA does not affect negatively the chemical/mechanical properties and it does not increase dentin erosion. The use of GA with low pH seems to promote less change in collagen/apatite ratio, but further studies are needed to establish an ideal clinical protocol. Therefore, this study supports the potential use of GA as an alternative final irrigation solution for root canal preparation.
The main purpose of the root canal preparation is cleaning and disinfection by using endodontic files and root canal irrigants
However, an effective final irrigant solution must act only on superficial dentin removing the smear layer without causing damage to the internal portion of the root dentin. Although EDTA has good capacity of smear layer removal
The glycolic acid (GA) is an alpha hydroxy acid (AHA) extracted from sugar cane and other sweet vegetables. It is uncolored, odorless, has only two carbons in its molecular structure, and can be easily dissolved in water. GA is commonly used in dermatology for applications that range from skin moisturizing to deep chemical peeling, a common esthetic procedure. As the smallest AHA, GA has great penetration potential and its absorption on skin and mineral surfaces is faster than other AHA
Previously published studies from our research group suggest the use of GA as a final irrigant in the root canal therapy, since it demonstrated ability to remove the smear layer without negatively affecting the dentin properties when used at low pH (around 2)
Tables Microhardness, Apatite/Collagen Ratios and Flexural Strength Values. NaOCl: sodium hypochlorite. EDTA: ethylenediaminetetraacetic acid. GA: glycolic acid. Means followed by different letters in the same column present statistically significant difference (p < 0.001). Median (Md) and first (Q1) and third (Q3) quartiles of the scores of smear layer removal for the experimental groups. NaOCl: sodium hypochlorite. EDTA: ethylenediaminetetraacetic acid. GA: glycolic acid. *Medians followed by different lowercase letters in the same column are statistically different (p < 0.001). *Medians followed by different uppercase letters in the same row are statistically different (p < 0.05). Median (Md) and first (Q1) and third (Q3) quartiles of the erosion scores of the experimental groups. NaOCl: sodium hypochlorite. EDTA: ethylenediaminetetraacetic acid. GA: glycolic acid. *Medians followed by different lowercase letters in the same column are statistically different (p < 0.001). *Medians followed by different uppercase letters in the same row are statistically different (p < 0.05). FTIR spectra of dentin powder after irrigation with the experimental solutions for 1 min. GA 1.2: glycolic acid at pH 1.2. GA 5.0: glycolic acid at pH 5.0. Saline: saline solution used in the control group. EDTA: ethylenediaminetetraacetic acid. Phosphate ( Representative scanning electron micrographs of all groups and root thirds and EDS results with the respective atomic ratios (at. %) of calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), and magnesium (Mg).Groups KHN Flexural strength (MPa) Apatite/collagen ratio Saline (negative control) 40.3 ± 3.7a 3.4 ± 0.8a 2.7 ± 0.1a 2.5% NaOCl + 17% EDTA 21.2 ± 4.5b 4.1 ± 1.2a 0.9 ± 0.0b 2.5% NaOCl + 17% GA pH 1.2 24.0 ± 2.5b 3.7 ± 1.6a 1.0 ± 0.1b 2.5% NaOCl + 17% GA pH 5 25.1 ± 3.7b 4.1 ± 1.5a 0.3 ± 0.0c EXPERIMENTAL GROUP CORONAL MIDDLE APICAL p value Md Q1 Q3 Md Q1 Q3 Md Q1 Q3 Saline 5.0 a A 5.0 5.0 5.0 a A 5.0 5.0 5.0 a A 5.0 5.0 1.000 2.5% NaOCl + 17% EDTA 2.0 b B 2.0 2.5 2.0 b AB 2.0 2.5 3.0 b A 2.0 3.0 0.007 2.5% NaOCl + 17% GA pH 1.2 1.5 b B 1.0 2.0 2.0 b AB 1.5 3.0 3.0 b A 2.0 3.0 <0.001 2.5% NaOCl + 17% GA pH 5.0 2.0 b B 2.0 2.0 2.0 b AB 2.0 3.0 3.0 b A 2.0 3.0 0.009 P value <0.001 <0.001 <0.001 EXPERIMENTAL GROUP CORONAL MIDDLE APICAL p value Md Q1 Q3 Md Q1 Q3 Md Q1 Q3 Saline 0.0 b A 0.0 0.0 0.0 b A 0.0 0.0 0.0 b A 0.0 0.0 1.000 2.5% NaOCl + 17% EDTA 1.0 a A 0.5 1.0 1.0 a A 1.0 1.0 0.5 a A 0.0 1.0 0.133 2.5% NaOCl + 17% GA pH 1.2 1.0 a A 1.0 1.0 1.0 a AB 0.0 1.0 0.5 a B 0.0 1.0 0.006 2.5% NaOCl + 17% GA pH 5.0 1.0 a A 1.0 1.0 1.0 a AB 0.0 1.0 0.0 ab B 0.0 1.0 0.010 P value <0.001 <0.001 0.002
The mean and standard deviation of the KHN values, flexural strength and apatite/collagen ratio of the experimental groups are presented in Table
Figure
Table
The erosion scores for the experimental groups are summarized in Table
Figure
In order to evaluate whether the irrigation with GA at acidic or neutral pH is effective in removing the smear layer and its potential effects on dentin surface integrity and chemical/mechanical properties when compared to EDTA, the pH values of 1.2 and 5.0 for GA were tested in the present study. The ability to remove the smear layer of EDTA was similar to GA at both pH values in each root third evaluated, so the first null hypothesis was accepted. Considering the chemical and mechanical properties evaluated in the present study, EDTA and GA irrigants caused significant reduction on the dentin microhardness, showed similar potential to promote erosion of root dentin walls, and no clear differences were noted in mineral composition on dentin surface. However, when comparing the collagen/apatite ratio, the most significant change was observed for GA at pH 5. Therefore, the second null hypothesis was rejected.
The decrease of dentin microhardness values promoted by EDTA compared to control (saline) is in agreement with other studies
However, the irrigant solutions EDTA or GA did not negatively affected the dentin flexural strength, as reported by Bello
Both EDTA and GA showed similar ability to remove smear layer. Recent studies showed that GA has the same ability to remove dentin smear layer than etching and irrigant solutions such as PA, EDTA and citric acid
Likewise, erosion scores were similar between EDTA and GA at both pH values in the coronal and middle thirds. The erosion caused on the wall of the root canal could modify some of the dentin properties
In addition, no major differences were noted in the chemical composition of dentin when comparing the saline solution with the EDTA and GA groups, thus Ca and P contents remained stable. Na and Cl were detected in all groups, possibly associated with saline solution and NaOCl residue. A vestige of Mg was also found, which has been considered to influence the crystal growth in mineralization
In conclusion, our results support the use of GA at neutral or acidic pH as an alternative irrigant in the root canal therapy. GA has the ability to remove the smear layer without promoting more erosion or negative effects on dentin mechanical properties. The use of GA at a lower pH seems to promote less change in collagen/apatite ratio, but future studies should clarify the effects of the pH on other dentin properties for the development of an ideal clinical protocol.
The protocols and experiments proposed in this study were approved by the Ethics Research Committee of the University of Passo Fundo (No.#2.080.284) and teeth collection were performed in accordance with relevant guidelines and regulations as per the ethical and research board committee instructions. All teeth were obtained from the teeth biorepository of the School of Dentistry of the University of Passo Fundo under informed consent of the patients. After the extraction, the teeth were stored at 4 °C in 0.9% NaCl solution and were used within 1-month period.
For the treatment of dentin samples, the GA (Natupharma, Passo Fundo, Rio Grande do Sul, Brazil) was used at pH 1.2 and pH 5.0. To obtain GA solution at higher pH (5.0), a neutralizing agent, aminomethyl propanol, was used. As a positive control, 17% EDTA (Biodinamica, Ibipora, Parana, Brazil) was selected as the conventional final endodontic irrigant. Saline solution was used to treat samples in the negative control group.
The dentin surface demineralization was indirectly assessed by the microhardness test. The smear layer removal capacity and dentin erosion were evaluated by analyzing images obtained with Scanning Electron Microscopy (SEM). The analysis of mineral dentin distribution was assessed using Energy-Dispersive X-ray Spectroscopy (EDS). The apatite/collagen ratio and flexural strength of mineralized dentin were evaluated by Fourier-transform infrared spectroscopy (FTIR) and flexural strength test, respectively.
Twenty canine human teeth were sectioned transversely below the cementum-enamel junction producing 15-mm-long root segments. Thereafter, the roots were sectioned longitudinally in two, creating 40 specimens from the buccal and lingual segments. Each root specimen was embedded in self-curing acrylic resin, leaving the root canal dentin exposed on the surface. The dentin surfaces were then flattened using silicon carbide paper (500, 800, 1000, and 1200 grit) under constant water irrigation, and polished using a suspension of 0.1-mm alumina on a rotating felt disc.
In the negative control group (10 root segments), dentin surfaces were irrigated only with a saline solution. Dentin specimens in the other three groups (30 root segments) were irrigated with 2 ml of 2.5% sodium hypochlorite (NaOCl) for 1 min, followed by irrigation with 5 ml of saline solution and randomly divided according to the final irrigant (n = 10): 17% EDTA, 17% GA at pH 1.2, and 17% GA at pH 5. All final irrigations were performed with 2 ml of solution for 1 min. Finally, all samples were rinsed with 5 ml of saline solution.
Dentin microhardness was measured using a Knoop indenter with 40× magnification (SHIMADZU HMV-2000; Shimadzu Corporation, Kyoto, Japan) under a 25-g load and a dwell time of 15 s. Three indentations were performed on each specimen along lines parallel to the edge of the root canal in the apical direction. The first indentation was performed at 1.000 µm distance from the entrance of the root canal, and the other two indentations were performed at a distance of 200 µm from each other
The chemical-mechanical preparation of 40 mandibular first premolar roots was performed using nickel-titanium rotatory instruments (ProTaper Next; Dentsply Maillefer, Ballaigues, Switzerland) following the manufacturer’s recommendations. The sequence of files used was X1, X2, and X3 at a speed of 300 rpm and torque of 2 N until the working length was achieved. At each change of instruments, the root canal was irrigated with 2 ml of 2.5% NaOCl. Thereafter, the 40 roots were randomly divided into four groups and the final irrigation was performed with 2 ml of the experimental solutions as previously mentioned. The solutions were applied inside the root canal as follows: 29-gauge needles (NaviTip Tips, Ultradent Products Incorporated, South Jordan, UT, USA) were inserted up to 3-mm short of the working length and the test solution was introduced until it fully filled the root canal. The test solution was inserted using up-down movement and remained within the root canal for one minute. Final irrigation was made with 5 ml of saline solution, and the canals were dried using absorbent paper cones
Thereafter, the 40 roots were divided into two halves resulting in 80 halves (20 in each group). The specimens were dehydrated using increasing concentrations of ethanol (25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min, and 100% for 60 min). Each half root was mounted on metal stumps, covered with palladium gold (Quorum, Laughton, East Sussex, UK), and examined using SEM (VEGA LM 3; Tescan, Libušinatř. Kohoutovice, Czech Republic). Images were obtained to observe the morphology of the surface of the canal wall at 2000× magnification and 20 kV along the coronal (10–12 mm from the apex), middle (6–7 mm from the apex), and apical (1–2 mm apex) thirds of each specimen
For the smear layer analysis the following criteria was used
Smear layer and dentin erosion data were determined to be non-parametric based on the Shapiro-Wilk Normality test (p<0.001). Therefore, data were analyzed with Kruskal-Wallis test and Dunn´s Method (α = 0.05), and results were presented using medians and quartiles (to represent data dispersion).
The same specimens prepared in the previous test (smear layer removal and erosion) were used for the evaluation of surface mineral content. The entire area of the dentin matrix was visualized using SEM at a standard magnification of 200× (650×420 µm), and analyzed using EDS to determine the atomic ratio (at. %) of calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), and magnesium (Mg). Changes in the mineral levels were recorded and differences among the groups were qualitatively analyzed.
Twenty non-carious extracted human mandibular third molar teeth were selected for this test. Enamel and cementum were removed from the teeth using a diamond bur #2215 in a high-speed handpiece under refrigeration. Dentin powder (90 µm) was obtained with a high-speed handpiece and diamond bur #3145 F without refrigeration. The powder went through a 90 µm sieve, so the dentin grains were equal to or smaller than this size. The dentin powder was divided in four groups of 9 mg each. One group remained untreated (NT), and the other three groups were rinsed with the GA and EDTA solutions. For irrigation, dentin powder was placed over a filter paper and fixed on a glass Becker. Irrigation was performed with 5 ml of the experimental solutions using a 25×4 mm needle for 1 min. After irrigation, the powder was washed three times with 5 ml of deionized water to remove residue of the experimental solutions and air-dried at 37 °C
FTIR spectra of the dentin powder were collected for each group (n = 3). Spectra were obtained between 650 and 4000 cm−1 resolution, using 48 scans (Agilent Cary; 630 FTIR spectrometer, Santa Clara, USA). The IR spectrum is produced as a result of the absorption of electromagnetic radiation at frequencies that correlate to the vibration of chemical bonds within a molecule. Thereby, when IR radiation is absorbed by an organic molecule, it is converted into molecular vibration energy, and the spectrum portray the vibrational motion and usually appears in the form of bands
Apatite/collagen ratios derived from FTIR spectroscopy showed normal distribution using the Shapiro-Wilk Normality test (p = 0.882) and Equal Variance test (p = 0.419). Data were statistically analyzed using one-way ANOVA and Tukey’s post-hoc test for multiple comparison (α = 0.05).
Twenty human mandibular third molars were used for the flexural strength test. Mid-coronal dentin disks were cut perpendicular to the longitudinal axis of each tooth with a slow-speed diamond saw under constant water-cooling. The disks were trimmed to a final rectangular-shaped beam (5.0 mm length, 0.2 mm thick, 2.0 mm wide)
Flexural tests were conducted using a three-point flexure device with a 3-mm support span. The specimens were tested at a crosshead speed of 0.5 mm/min using a universal testing machine (Instron, Canton, Norwood, USA). Flexural strength (in MPa) was calculated using the following equation: 3PL/2bd
Flexural strength data was normally distributed as observed in the Shapiro-Wilk Normality test (p = 0.05) and Equal Variance test (p = 0.405). Data were statistically analyzed using one-way ANOVA and Tukey’s post-hoc test for multiple comparison (α = 0.05).
The authors deny any conflicts of interest related to this study. This study was funded by the Universidade de Passo Fundo (grant number 96301) and by the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (grant number 17/2551–0001 019–1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Contributions Barcellos, D.P.D.C. contributed to conception and design, data acquisition, statistical analysis and interpretation, drafted and critically revised the manuscript. Farina, A.P. contributed to conception and design prepared figures and critically revised the manuscript. Barcellos, R. contributed to conception and design prepared figures and critically revised the manuscript. Souza, M.A. contributed to conception and design prepared figures and critically revised the manuscript. Borba, M. contributed to statistical analysis, interpretation and critically revised manuscript. Bedran-Russo, A.K. contributed to critically revised the manuscript. Dal Bello, Y. contributed to data acquisition and interpretation, and critically revised the manuscript. Vidal, C.M.P. contributed to critically revised the manuscript Cecchin, D. conceived and coordinated the study, contributed to conception and design, statistical analysis, interpretation and critically revised manuscript. Corresponding author Correspondence to Doglas Cecchin.
The authors declare no competing interests.