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 Table of Contents  
Year : 2018  |  Volume : 30  |  Issue : 1  |  Page : 55-61

Comparison of new irrigating solutions on smear layer removal and calcium ions chelation from the root canal: An in vitro study

Department of Conservative Dentistry and Endodontics, Inderprastha Dental College, Sahibabad, Uttar Pradesh, India

Date of Web Publication4-Jun-2018

Correspondence Address:
Prof. Anika Mittal
Department of Conservative Dentistry and Endodontics, Inderprastha Dental College, Sahibabad, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_71_17

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Background: The action of endodontic instruments leads to the formation of smear layer during biomechanical preparation. Smear layer removal not only enhances the three-dimensional sealing of the root canal system but also improves the dentinal tubule disinfection.
Aim: With the help of scanning electron microscope (SEM), assessing the effectiveness of smear layer removal from the root canal wall using various final irrigating solutions, and to quantify, the concentration of calcium ions in these solutions after irrigation using atomic absorption spectrophotometry with flame.
Materials and Methods: Forty human maxillary canines were selected and prepared and the final irrigation was performed to quantify the concentration of calcium ions released with 0.2% chitosan, apple cider vinegar, and 15% ethylenediaminetetraacetic acid (EDTA), which were then composed and analyzed using atomic absorption spectrometry. From the middle and apical thirds of the root canal, the smear layer removal was evaluated using SEM.
Results: There was statistically significant difference between 0.2% chitosan and the other solutions with regard to smear layer removal. The highest concentrations of calcium ions were obtained with apple cider vinegar followed by 0.2% chitosan and 15% EDTA.
Conclusion: Nearly 0.2% chitosan showed greater smear layer removal as compared to apple cider vinegar which enhanced the release of the highest concentrations of calcium ions than the other solutions verified.

Keywords: Apple cider vinegar, calcium, chitosan, ethylenediaminetetraacetic acid, scanning electron microscope, smear layer

How to cite this article:
Mittal A, Dadu S, Yendrembam B, Abraham A, Singh NS, Garg P. Comparison of new irrigating solutions on smear layer removal and calcium ions chelation from the root canal: An in vitro study. Endodontology 2018;30:55-61

How to cite this URL:
Mittal A, Dadu S, Yendrembam B, Abraham A, Singh NS, Garg P. Comparison of new irrigating solutions on smear layer removal and calcium ions chelation from the root canal: An in vitro study. Endodontology [serial online] 2018 [cited 2023 Jan 30];30:55-61. Available from: https://www.endodontologyonweb.org/text.asp?2018/30/1/55/233749

  Introduction Top

The successful endodontic treatment depends on comprehensive cleaning and shaping of root canal system. After the root canal treatment, more than 35% of the root canal surface can be left without preparing with the help of advanced technology such as nickel titanium files.[1] Thus, it is critical to have an irrigation system or intervention as part of the conventional root canal treatment. The main objective of the irrigation is for cleansing that does not take place with biomechanical preparation.[2] Accurate debridement of root canals is recommended in most endodontic treatment.[3] Currently, various methods have been introduced to remove the smear layer which includes chemical, ultrasonic, and laser techniques, neither of them has been accepted universally nor they have proved to be more operative.[4]

Based on a literature review of current endodontic treatment and irrigating interventions, the irrigants are most likely recommended due to their capability to dissolve broad spectrum antibioticand necrotic pulp.[5] These irrigants also help in dissolving inactive endotoxins which are critical in endodontic treatment and are also known to inhibit or completely dissolve the smear layer.[3]

Chitosan is a natural polysaccharide, which has gained popularity in the field of dentistry because of its properties (biodegradability, biocompatibility, bioadhesion, and no toxicityetc).[6] Due to its acidic pH, it shows significant chelating capacity for different metal ions, which validates its use in various industries.[7] It is the most abundant substance innature after cellulose; making its use more eco-friendly.[8] Chitosan possesses the high chelating capacity for various metal ions, including Zinc, Cobalt, Iron, Magnesium, and Copper ions (Zn2þ, Co2þ, Fe2þ, Mg2þ, and Cu2þ, respectively) in acid conditions.[9] Due to these properties, chitosan was used in various dental treatments such as in cases of direct pulp capping, in the treatment of dentinal tubule infection,[10] and in tissue regeneration in pulp wounds.[11] Applications for this substance is been seen mainly in the areas of medicine and pharmaceuticals, biotechnology, environment, agriculture, cosmetics, and food.[12] The antifungal property of a 2% chitosan gel which contains 0.1% chlorhexidine, against Candida albicans has been demonstrated in dentistry,[13] and its addition to calcium hydroxide paste as an intracanal medication has been shown to promote prolonged calcium ion release.[14]

The preparation of root canal using the most widely used irrigant for smear layer removal is ethylenediaminetetraacetic acid (EDTA) which is achieved by acting on an inorganic material.[15],[16],[17] Its reaction with calcium ions in dentine results in calcium chelation, promoting decalcification of dentine at approximate depths of 20–30 μm within 5 min.[18] For the complete elimination of the smear layer, irrigant that has been recommended was EDTA in combination with different concentrations of sodium hypochlorite (NaOCl).[17] Few literatures reveal that EDTA can cause harm to the periapical tissues with its increasing frequency of usage.[5] EDTA can also cause erosion to dentin, depending on its application time and concentration.[19] Thus, the search of more biocompatible substance than EDTA continues. 1%, 5%, 10%, and 15% of EDTA when used for 1 min caused erosion of the root dentin as reported by Sen et al.[20] Another drawback is that since EDTA is not found originally in nature, it is considered to be a pollutant.[17]

This substance is a weak acid with chelating action and concomitant protein denaturing,[21] which promotes the increase in dental permeability facilitating the action of the intracanal medication [22] and the bond between dentin and endodontic cements,[23] in addition to being biocompatible.[24]

Some studies suggested with the objective of using chelating solutions other than EDTA that would be more proficient and biocompatible with the organic structures. Therefore, citric acid [25],[26] and apple vinegar [17] have also been studied. Apple cider vinegar has proven antimicrobial action, reduces dentinal microhardness,[27] in addition to removing the smear layer. It is a combination of acetic, citric, formic, lactic, succinic (succinate), and tartaric acids with a lesser amount of alcohol, helps in reducing the surface tension of the solution. However, the highest acid concentrations of the vinegar are represented by the acetic (5%) and malic (0.35%) acids.[28] The use of apple vinegar as a secondary solution in the preparation of root canals has also been evaluated and deserves contemplation due to the positive results attained when compared to EDTA and NaOCl.[29]

The purpose of this study was to assess the various final irrigants such as 0.2% chitosan, apple cider vinegar and 15% EDTA using scanning electron microscopy (SEM) for smear layer removal efficacy, and to enumerate the calcium ion concentration using atomic absorption spectrophotometry with flame (AASF) after root canal irrigation.

  Materials and Methods Top

The study included forty extracted human maxillary canines. The teeth were submerged for 15 min in 2.5% NaOCl solution. In 0.9% saline solution mixed with thymol the residual tissue and debris eliminated from the root surface were then stored. The selected teeth did not have caries, fracture, or cracks and were not previously restored or treated endodontically. The samples were then distributed into four groups.

Preparation of the root canal

The sound teeth were decoronated at the cementoenamel junction. The working length was established using a K-file #10 (DentsplyMaillefer), which was introduced into the root canal of each tooth up to the point that it was visible at the apical foramen and 1 mm of length was subtracted from it. The canals were enlarged with rotary nickel-titanium instruments – ProTaper Universal (DentsplyMaillefer), using the crown-down technique according to the manufacturer's protocol. With different irrigants used in between the files, each sample group was irrigated, respectively. In Group I (n = 10), 15% EDTA was used, Group II (n = 10) 0.2% chitosan (for preparation of the 0.2% chitosan solution, 0.2 g of chitosan material was diluted in 1% acetic acid of 100 ml, and then the sample was stirred using a magnetic stirrer for 2 h), and in Group III (n = 10) apple cider vinegar was used.

Specimen preparation

Each tooth was placed in a 15-ml falcon tube, and the tube lid was perforated in such a way that the tooth could be placed with the crown portion outside and the root of the tooth inside the tube. Then, using a needle of 0.45 mm × 13 mm of the needle 5 ml of the respective chelating solution were inserted into the root canal during 3 min, passing through the entire root canal and exiting through the patent foramen into the collection tube. The needle was held in position using Top Dam Blue ® light-cured resin (FGM/Dentscare, Joinvile, SC, Brazil), which sealed the canal entrance to prevent flow back of the solution. These procedures were repeated for all teeth in each group. The total quantity of the solution accumulated in the tubes was used for quantification of calcium ion concentration by AASF.

Absorption spectrophotometry with flame analysis

After collection of the solutions, the teeth were removed from the tubes and separated. For the spectrometric determination of calcium ion concentration within the liquid, new lids were placed on the tubes and were labeled and forwarded. Ten tubes were used in each group (one for each tooth). Individual values were obtained, and a mean value was calculated for each group. An Analyst 400 atomic absorption spectrometer, which features a combined flame furnace, was used. The following parameters were employed: hollow calcium cathode lamps and an acetylene gas were used for absorbance measurement, and a standard calcium solution with 100 mg/L concentration (Ultra Scientific) was used for curve calibration adjustment for calcium ions. In deionized water both EDTA and apple vinegar solution were diluted before being analyzed while the chitosan solution was diluted in a 0.1% lanthanum solution (by mass/volume) to avoid the interference of chitosan polymeric matrix with calcium ion quantification.

Scanning electron microscope analysis

Horizontal grooves were made on both buccal and lingual surfaces of the root using a diamond disk without penetration of the canal. With the help of a chisel, the roots were then separated into two halves. The half portion of each root was coded and chosen, containing the most visible part of the apex and the whole canal length. Each specimen was measured lengthwise with a digital caliper from the apex to the cementoenamel junction for delimitation of the root thirds. Then, starting from the apex, the points corresponding to ½ and 1/6 of the root length were separated to specify the half of the middle and apical thirds, respectively. These areas were used for the SEM analysis. With respect to the quantity of smear layer remaining on the dentinal walls, SEM micrographs with a magnification of 1.5Kx [Figure 1] were obtained and investigated by three endodontic specialists using a scanning electron microscope. Scores from 1 to 5 were attributed according to the following scoring system modified from Takeda et al.:[30] (i) complete surface covered with smear layer, (ii) smear layer partially covering few visible tubules and the surface (iii) about half covered with open tubules and remaining of the surface with smear layer, (iv) a small amount of surface and visible tubules covered with smear layer, (v) no smear layer seen on the surface.
Figure 1: Scanning electron microscope micrographs of the root canal walls after irrigation (original magnification, ×1.5 K). Smear layer removal of a root canal after final irrigation with (1) 0.2% chitosan. (2) Apple cider vinegar. (3) Ethylenediaminetetraacetic acid (better smear layer removal with chitosan than apple vinegar and ethylenediaminetetraacetic acid)

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Statistical analysis

For analysis of calcium ion concentration, Tukey–Kramer test and one-way analysis of variance and were used. The Kruskal–Wallis test was used for analysis of remaining smear layer. A significance level of 5% was adopted.

  Results Top

According to the present study, the highest calcium ion concentrations was observed with apple vinegar (0.110 mg/L) followed by chitosan (0.079 mg/L) and EDTA (0.018 mg/L).

[Table 1] presents the means and standard deviations of calcium ion concentration for each chelating solution.
Table 1: Means and standard deviations of the calcium ion concentration of the solutions (mg/L)

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Tukey's test revealed that 0.2% chitosan, apple cider vinegar and 15% EDTA were significantly different from each other (P< 0.05) [Table 2].
Table 2: Comparison of means of calcium ion concentration of three groups by one-way ANOVA

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SEM analysis showed a comparability of results in Group I (EDTA), Group II (Apple cider vinegar), and Group III (Chitosan). The highest smear layer removal was observed in Group III (0.2% chitosan).

[Table 2] presents the distribution of mean ± standard deviation of scores of smear layer removal of three groups in middle and apical.

Kruskal–Wallis test

The dentinal tubules covered by smear layer at middle and apical root canal levels were compared. SEM analysis revealed that, 0.2% chitosan had better smear layer removal in middle and apical third, followed by apple cider vinegar and 15% EDTA. Hence statistically, the P value in middle third is statistically significant (P< 0.05) and in apical third is not statistically significant (P > 0.05).

According to the Kruskal–Wallis test, there were statistically significant differences between the middle and apical thirds for the comparison of individual specimens within groups.

  Discussion Top

The analysis of calcium ion concentrations in the chelating solutions used for final irrigation after root canal instrumentation revealed that apple vinegar and 0.2% chitosan values to be greater than those of 15% EDTA. SEM analysis showed that 0.2% chitosan even in such a low concentration, was able to remove smear layer as compared to 15% EDTA and apple cider vinegar. This study showed better smear layer removal in the middle third (P< 0.05) than an apical third (P > 0.05) [Table 3], [Table 4], [Table 5]. The apical region did not show much effectiveness in the apical region as reported by most studies.[31],[32],[33]
Table 3: Distribution of mean±standard deviation of scores of smear layer removal of three groups in middle

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Table 4: Distribution of mean standard deviation of scores of smear layer removal of three groups in apical

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Table 5: Mean of the smear scores at the middle and apical levels

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Chelating agent depends application time, pH, concentration and amount of the solution.[21] In addition, the link between the concentration of the chelating solution and the application time seems to be essential since it was found that highly concentrated solutions applied for a long period, cause roughness of dentin surface.[34] However, the application time of final irrigants and its concentration were not consistent in this study. These were chosen according to the manufacturers instructions and the findings of previous researchers.[4],[34],[35],[36] Based on the studies by Marques et al.[15] and Spanó et al.[17] the combined use of AASF and SEM results were verified.

In this study, the calcium ion concentration was proved to be more effective in apple cider vinegar due to its chelating property than 0.2% chitosan, 15% EDTA. In a study conducted by Silva et al.,[37] the evaluation of calcium ion concentrations in the chelating solutions, which are used for final irrigation after root canal instrumentation (15% EDTA, 0.2% chitosan, 10% citric acid, and 1% acetic acid), revealed greater values of 15% EDTA and 0.2% chitosan than 10% citric acid, which showed higher calcium ion concentration values than 1% acetic acid.

The presence of malic acid gives the biocompatibility action to apple cider. In this process, the ethyl alcohol produced is converted and oxidized into acetic acid under the presence of specific microorganisms. This procedure is called acetification.[28] The total amount of calcium ion found in the apple vinegar solution is due to the action of H+ ions present. The more the concentration of H+ ions the more efficient the attack of the acid would be.[38] Furthermore, the apple cider vinegar has a medicinal potential due to its rich mineral content such as potassium, phosphorus, and magnesium. Despite fully knowing its mechanism of action, it is believed that adsorption, ionic exchange and chelation are responsible for the elimination of dentin calcium ions.[39]

The better chelating agent was 0.2% chitosan when compared to all tested chelating agents. Application of 0.2% of chitosan solution for 3–5 min as the most viable combination for use on the root dentin was revealed by a study.[40] A study reported, the properties of chitosan to be better and also enhanced cleansing and chelating.[42] Researchers have theorized two theories based on the mechanism involved in chelating with chitosan. The bridge model first theory and it is based on, the mechanism where two or more amino groups on the chitosan chain bounds the same metal ion.[42] The second theory claims that only one of the amino groups on the chitosan chain is anchored to the metal ion.[43] Further analysis revealed that chitosan and metal ion complexes are a result of ion exchange, chelation, and adsorption.[41] The chelating interaction depends on factors such as the type of interaction, pH of the solution, and the structure of chitosan.[44] Based on a recent study, chitosan is known to have conditioning effects on radicular dentin.

The dentin reducing properties of 0.2% chitosan at 3.2% pH has been revealed by researchers.[45] Thus, it can be concluded that chitosan citrate is also an ideal conditioner for radicular dentin.[45] In this study, atomic AASF analysis of 0.2% chitosan revealed a calcium ion concentration of 0.079 mg/L, with a significant difference when compared with apple vinegar and 15% EDTA [Graph 1]. The groups irrigated with chitosan showed better smear removal than EDTA and apple vinegar [Graph 2] and [Graph 3]. When there are economic concerns, then the less concentrated solution should be favored if both solutions have a similar effect of chelation. The most abundant substance in nature to be considered after cellulose is the precursor to chitosan, chitin polysaccharides.

The effectiveness of EDTA in removing the smear layer was proposed by Silva et al.[37] Spanó et al.showed the greatest concentration of calcium ions by using atomic absorption spectroscopy and SEM.[17] As compared to NaCl and NaOCl, EDTA was more effective in opening the dentinal tubule and also smear layer removal was reported by Gu et al.[46] EDTA is effective at a neutral pH and does not depend on a high hydrogen ion concentration to accomplish decalcification. There is also decrease in pH due to the exchange of calcium from dentin by hydrogen. This decrease in pH leads to decrease in the efficacy of EDTA over time. When applied for 3 min, 0.2% chitosan was the most proficient for removal of smear layer and smear plug as compared to other solutions with minimal erosion. EDTA can be used as an alternative to 0.2% chitosan.[21]

Atomic AASF analysis showed that all solutions were able to remove calcium ions from the root canal walls. It is important to emphasize that decalcification of the inorganic structure of the smear layer does not lead to the presence of calcium in the solution after irrigation. Chelating and demineralizing solutions act on the hydroxyapatite calcium matrix of the dentine, with subsequent collagen exposure and reduction of microhardness.[47] Analyzing the results of both studies, there seems to be a direct relationship between smear layer removal ability and the amount of calcium ions removed from root canals. This relationship was also observed by Marques et al.,[15] who found that 17% cethylenediaminetetraacetic acid plus Cetavlon and 17% cyclohexane diamine tetra-acetic acid in addition to promoting a more efficient cleaning than 17% ethylene glycol-bis(β-aminoethyl) ether tetraacetic acid also had the highest concentrations of calcium ions after spectrometric analysis.

In the present study, the most efficient solution for removing the smear layer and smear plug with minimal erosive effect was 0.2% chitosan, and apple vinegar was the most effective in calcium ion concentration.

  Conclusion Top

Under the experimental conditions and within the limitations of this investigation, 0.2% chitosan, apple cider vinegar and 15% EDTA, removed smear layer from the middle and less in apical thirds of root canals. In addition, 0.2% chitosan, apple cider vinegar were associated with the greatest effect on the root dentine demineralization, followed by 15% EDTA.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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