|Year : 2015 | Volume
| Issue : 2 | Page : 56-61
Comparative Scanning Electron Microscopy Evaluation of Smear Layer Removal with 17% Ethylenediaminetetraacetic Acid, 10% Citric Acid and Newer Irrigant QMix: In Vitro Study
Ankur Mahesh Banode1, Vandana Gade1, Sanjay Patil1, Jaykumar Gade2, Deepika Chandhok1, Roshan Sinkar1
1 Department of Conservative Dentistry and Endodontics, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India
2 Department of Prosthodontic Dentistry and Implantology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India
|Date of Web Publication||17-Dec-2015|
Ankur Mahesh Banode
Old Shukrawari, Shakardara Road, Near Murlidhar Temple, Nagpur - 440 009, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study was to compare the effectiveness of 17% ethylenediaminetetraacetic acid (EDTA) with that of 10% citric acid and newer irrigant QMix in the removal of smear layer from root canal wall dentin. Materials and Methods: Twenty single-rooted teeth were accessed and instrumented with crown down technique up to protaper F3. Between each instrument used, the canals were irrigated with 1 ml of 5% sodium hypochlorite (NaOCl). After instrumentation, the teeth were irrigated with distilled water and then divided into four groups according to the different surface treatment with different irrigants. The irrigants used were 1% NaOCl, 17% EDTA, 10% citric acid, and newer irrigant QMix. The samples were prepared and observed by means of scanning electron microscopy. Three photomicrographs (Χ2000) were recorded for each sample regarding the apical, middle, and cervical thirds. A score system described by Takeda et al. was used to evaluate the images.
Keywords: 10% citric acid, 17% ethylenediaminetetraacetic acid, QMix TM , scanning electron microscope
|How to cite this article:|
Banode AM, Gade V, Patil S, Gade J, Chandhok D, Sinkar R. Comparative Scanning Electron Microscopy Evaluation of Smear Layer Removal with 17% Ethylenediaminetetraacetic Acid, 10% Citric Acid and Newer Irrigant QMix: In Vitro Study. Indian J Oral Health Res 2015;1:56-61
|How to cite this URL:|
Banode AM, Gade V, Patil S, Gade J, Chandhok D, Sinkar R. Comparative Scanning Electron Microscopy Evaluation of Smear Layer Removal with 17% Ethylenediaminetetraacetic Acid, 10% Citric Acid and Newer Irrigant QMix: In Vitro Study. Indian J Oral Health Res [serial online] 2015 [cited 2019 Mar 19];1:56-61. Available from: http://www.ijohr.org/text.asp?2015/1/2/56/172030
| Introduction|| |
The success of root canal treatment depends on proper biomechanical preparation, disinfection, and three-dimensional obturation of root canal system. In endodontic practice, special emphasis is given to clean and shape the root canal system which removes pulpal remnants, dentinal fillings, and microbes from it. During biomechanical preparation, organic and inorganic dentinal debris adheres to the root canal walls, which is called as smear layer. McComb and Smith were the first researchers to describe the smear layer on the surface of instrumented root canals. It has been noted that the smear layer is superficial and is 1-5 um in thickness. , Smear layer blocks the dentinal tubules and prohibits the canal irrigants from reaching the bacteria harbor in the dentinal tubules.  This layer may also be contaminated with bacteria and necrotic tissue, and it disrupt the seal between filling material and canal wall, which may lead to endodontic failure.
For the successful root canal treatment, it is essential to use an irrigating solution for cleaning and removing the smear layer and debris from the root canals.  Sodium hypochlorite (NaOCl) is a commonly used irrigating solution. It has the ability to dissolve organic tissue; at the same time, it is an excellent antibacterial agent. However, its capacity to remove the smear layer has been proved to be lacking. If it enters the periapical tissues through apical foramen, it will have widespread cytotoxicity resulting in the necrosis and inflammation of periapical tissues. ,
So far, ethylenediaminetetraacetic acid (EDTA) being the most common chelating agent used for removal of smear layer.  The citric acid has also been suggested as a chelating agent for smear layer removal. In a previous study, it has been shown that there is no significant difference in smear layer removal between citric acid and EDTA.  However, EDTA and citric acid, both has the main disadvantage of limited antibacterial activity. 
Recently, QMix TM 2 in 1 (Dentsply Tulsa Dental, Tulsa, OK, USA), a novel irrigant for smear layer removal with added antimicrobial agent, has been developed. It contains a mixture of bisbiguanide antimicrobial agent, a polyaminocarboxylic acid calcium-chelating agent, and a surfactant. QMix is a clear solution, ready to mix with no chairside mixing. It has been found to be effective against bacterial biofilms. 
Smear layer can be identified using the electron microprobe with scanning electron microscope (SEM) attachment was first reported by Eick et al., ( 1970).  Numerous investigation have been performed since then to evaluate the cleanliness of root canals using SEM.
The aim of this in vitro study was to evaluate the effectiveness of 17% EDTA, 10% citric acid, and newer irrigant QMix in the removal of smear layer from root canal wall dentin using an SEM.
| Materials and methods|| |
Twenty freshly extracted human single-rooted sound mandibular first premolars were selected for this study. Selected teeth were thoroughly cleaned and examined under a Dental operating microscope at ×12 magnifications (Seiler) to rule out any teeth with cracks or craze line. In all acceptable teeth, soft tissue and calculus were removed using an ultrasonic scaler and the teeth were stored in 10% formalin. Teeth were handled according to the Occupational Safety and Health Administration guidelines.
Access preparations were made by round diamond burs and patency of apical foramen was determined with a size 10 K file (MANI, INC., Dental Products, Japan). Working length was established by subtracting 1 mm, from the length of inserted #10 K file with its tip visualized at the apical foramen under the dental operating microscope. Root canals was then prepared with the ProTaper universal Nickel-Titanium rotary system (Dentsply-Maillefer Switzerland), starting with SX for improving the straight line access. Each canal was prepared up to an apical preparation till #F 3 . Between each file size, irrigation was performed with 1 ml of 5.25% NaOCl (Neelkanth Health Care (P) LTD., India). Apical patency was maintained by passing a size 10 K file through the apical foramen. A 5 ml flush of distilled water was used as a final rinse.
The specimen will be then randomly divided into 4 groups (n = 20), five samples in each groups, according to dentin surface treatment.
In Group 1 (n = 5): Final irrigation with 1% sodium hypochlorite
Teeth irrigated with 1% of NaOCl and rinsed with 10 ml of distilled water for 1 min.
In Group 2 (n = 5): Final irrigation with 10% citric acid
Teeth irrigated with 5 ml of 5% NaOCl and rinsed with distilled water for 1 min followed by 1 ml of 10% citric acid for 1 min, which is followed by 10 ml of distilled water for 1 min.
In Group 3 (n = 5): Final irrigation with 17% ethylenediaminetetraacetic acid
Teeth irrigated with 5 ml of 5% NaOCl and rinsed with distilled water for 1 min followed by 1 ml of 17% EDTA for 1 min, which is followed by 10 ml of distilled water for 1 min.
In Group 4 (n = 5): Final irrigation with QMix
Teeth irrigated with 5 ml of 5% NaOCl and rinsed with distilled water for 1 min followed by 1 ml of QMix for 1 min.
Scanning electron microscopy
After the removal of smear layer, two parallel longitudinal grooves were prepared with a diamond disc in low-speed rotation on the buccal and lingual surfaces of each root without penetrating the canal. The roots were then split into two halves using hammer and chisel. For each root, the half containing the most visible part of the apex was selected for the study.
After that, the samples were coated with gold sputtered and examined with the SEM. The images were performed without the knowledge of the group tested. First, a photograph of each sample was made at ×200 magnification for each group. Then the representative areas of each third of each sample were photographed at ×1000 magnification. After that, each ×1000 images were scanned, and the most representative areas were magnified at ×2000 of each third, respectively. The images magnified at ×2000 magnification were taken for observation. Therefore, three images of each sample were obtained, i.e., one image each third. In the end, each group (n = 5) had 15 images for coronal, middle, and apical thirds.
Scanning electron microscope evaluation
The images were magnified at ×2000 magnification were evaluated for the presence of a smear layer. The scoring system described by Takeda et al. was used.  It is briefly described as below [Figure 1].
|Figure 1: Representative photomicrographs of scoring system used to analyze the SEM result. (a) Score 1: No smear layer, with all tubules cleaned and opened. (b) Score 2: Few areas covered by smear layer with, most tubules cleaned and opened. (c) Score 3: Smear layer covering almost all the surface, with few tubules opened. (d) Score 4: Smear layer covering all the suerface|
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Score 1: No smear layer, opened and cleaned dentinal tubules
Score 2: Moderate smear layer, most tubules cleaned and opened
Score 3: Smear layer covering almost all the surface, with few tubules opened
Score 4: Heavy smear layer covering all the surfaces.
The degree of evaluation was scored in a blind manner by five independent observers.
Krushal-Wallis test was used to determine if there was significant difference between groups [Table 1]. Accordingly, pairwise comparison of median scores among groups for cervical, middle, and apical region was performed using Wilcoxon rank sum test [Table 2].
|Table 1: Mean, SD, and median of scores obtained for smear layer for samples in each group|
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|Table 2: Pair wise comparison of groups for cervical, middle and apical third of root canal|
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| Results|| |
The Krushal-Wallis test showed good agreement between observers. [Figure 1] shows representative images of the scores. The results of the smear layer scores for each group are listed in groups.
Group 1: Sodium hypochlorite (control group)
The smear layer was not removed by NaOCl, heavy smear layer was present, and outlines of tubules are obliterated [Figure 2].
|Figure 2: Scanning electron microscope images of NaOCl irrigation at ×2000 magnification (a) coronal 1/3rd (b) middle 1/3rd (c) apical 1/3rd|
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Group 2: 10% citric acid (test group)
The root canal wall was cleaned, and the smear layer was removed from the cervical and middle third. However, apical third was not completely cleaned, smear plugs could be observed in the opening of dentinal tubules [Figure 3].
|Figure 3: Scanning electron microscope images 10% of citric acid irrigation at ×2000 magnification (a) coronal 1/3rd (b) middle 1/3rd (c) apical 1/3rd|
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Group 3: 17% ethylenediaminetetraacetic acid (test group)
The root canal wall was cleaned, and the smear layer was removed completely from the cervical and middle third similar to the samples treated with 10% citric acid. Dentinal tubules were more cleaned and opened in this group. However, the apical part was not completely cleaned a seen in 2 nd group [Figure 4].
|Figure 4: Scanning electron microscope images of 17% ethylenediaminetetraacetic irrigation at ×2000 magnification (a) coronal 1/3rd (b) middle 1/3rd (c) apical 1/3rd|
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Group 4: QMix TM (test group)
Most of the smear layer was removed from the cervical and middle third but in some specimens, moderate smear layer could be observed. However, the apical third is not completely cleaned as shown in 2 nd and 3 rd groups [Figure 5]. The test suggests that final flush with 17% EDTA, 10% citric acid, and QMix did not produce the expected smear free surface in the apical third of the canals. In the cervical region, the difference between the median score of test groups in comparison to the control group was statistically significant with P < 0.05. The median score for 10% citric acid, 17% EDTA and QMix was significantly smaller than NaOCl. Similar results were found in middle and apical regions. There is insignificant difference in the median scores of 17% EDTA, 10% citric acid, and QMix as observed through pairwise comparison (P > 0.05) [Bar Chart 1 [Additional file 1]],[Bar Chart 2 [Additional file 2]],[Bar Chart 3 [Additional file 3]].
|Figure 5: Scanning electron microscope images of QMixTM irrigation at ×2000 magnification (a) coronal 1/3rd (b) middle 1/3rd (c) apical 1/3rd|
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| Discussion|| |
Whenever there is hand or rotary instrumentation of the root canals, smear layer is formed consisting of dentine, remnants of odontoblastic processes, pulp tissue, and bacteria. Smear can be described as "organic matter trapped within translocated inorganic matter."  It's thickness was generally 1-2 um and it has two parts, i.e., first, the superficial layer and second, the material packed in dentinal tubules.  Smear layer contains bacteria, their by-product, and may act as a substrate for bacteria.  Removal of smear layer can allow optimum penetration of disinfecting agent which cause a better disinfection procedure. Smear layer can act as barrier between the filling materials and canal walls, therefore, compromise the coronal and apical seal, which may result in micro-leakage and endodontic failure. 
A number of chemicals have been investigated as root canal irrigants to remove the smear layer. NaOCl is one of the most commonly used irrigant and its ability to dissolve organic tissues is well-known.  however its weakness include its inability to remove smear layer of the instrumented root canal because of its lack of effect on inorganic material. 
EDTA is the most common chelating agent used to remove the smear layer. In recent SEM evaluation, Spanó et al. verified smear layer removal with several root canal chelators and found that citric acid was as effective as EDTA.  However, their antimicrobial effectiveness appear to be lacking.
Newer irrigant QMix TM was used for this study as it has been proven to have antibacterial properties, and it can also remove smear layer. 
In this study, there was a significant difference between the test groups (17% EDTA, 10% citric acid, and QMix TM ) and control group (NaOCl) in removing the smear layer from coronal, middle, and apical third of root canal. NaOCl was not effective in removing the smear layer. This result was in agreement with previous study. 
The result of this study confirms previous report that final flush with 17% EDTA and 10% citric acid did not produce the expected smear free surface in the apical third of the canal.  Similar results were found in the previous investigation done by Gade et al. in which smear layer was not completely removed in apical third using conventional needle irrigation.  QMix TM was also not able to remove the smear layer from the apical third in this study. The possible reason may be the access of instruments and chemical solutions to the cervical and middle third is easier when compared to apical third.
When the contribution from different canal levels was taken into consideration, 17% EDTA and 10% citric acid was equally effective in removing the smear layer as found in previous studies as well.  QMix TM also removes the smear layer effectively but not as effective as other two test groups, but the difference is found to be insignificant in this study. Similar results were found by Stojicic et al., in that ability to remove smear layer by QMix was comparable to EDTA. 
QMix TM is combinational product that contains EDTA, chlorhexidine (CHX) and a detergent (surface active agent). It is recommended as final rinse after NaOCl. EDTA is the most commonly used chelating agent which allows deeper penetration of irrigant in dentinal tubules and CHX have an excellent antimicrobial property along with its substantivity (long-term continued effect). The underlying principle of adding a surface active agent in QMix is because of its ability to lower surface tension of solution and increase their wettability and consequently enhance the flow of the irrigation solution in the root canal wall. ,[ 20],
This study revealed that QMix TM endodontic irrigant removed smear layer as effectively as 17% EDTA and 10% citric acid.
| Conclusion|| |
Based on the result of this study, 17% EDTA, 10% citric acid, and QMix TM all chelating agent removes smear layer effectively from cervical and middle parts of canal as compared to apical third. In future, QMix TM may act as a promising chelating agent as well as antimicrobial irrigant. However, further investigations are required to determine the efficacy of QMix TM as root canal chelating agent.
| References|| |
Hargreaves KM, Cohen S, Berman LH, editors. Cohen′s pathways of the pulp. 10 th
ed. St. Louis, Mo: Mosby Elsevier; 2011. p. 952.
McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975;1:238-42.
Zand V, Mokhtari H, Lotfi M, Rahimi S, Sohrabi A, Badamchi Zadeh S, et al.
A scanning electron microscope study on the effect of an experimental irrigation solution on smear layer removal. Iran Endod J 2014;9:131-6.
Arruda MP, Carvalho Junior JR, Miranda CE, Paschoalato C, Silva SR. Cleaning of flattened root canals with different irrigating solutions and nickel-titanium rotary instrumentation. Braz Dent J 2009;20:284-9.
Hülsmann M, Hahn W. Complications during root canal irrigation - Literature review and case reports. Int Endod J 2000;33:186-93.
Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of NaOCl on vital tissue. J Endod 1985;11:525-8.
Calt S, Serper A. Smear layer removal by EGTA. J Endod 2000;26:459-61.
Di Lenarda R, Cadenaro M, Sbaizero O. Effectiveness of 1 mol L-1 citric acid and 15% EDTA irrigation on smear layer removal. Int Endod J 2000;33:46-52.
Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: Mode of action and indications for their use. Int Endod J 2003;36:810-30.
Dai L, Khechen K, Khan S, Gillen B, Loushine BA, Wimmer CE, et al.
The effect of QMix, an experimental antibacterial root canal irrigant, on removal of canal wall smear layer and debris. J Endod 2011;37:80-4.
Eick JD, Wilko RA, Anderson CH, Sorensen SE. Scanning electron microscopy of cut tooth surfaces and identification of debris by use of the electron microprobe. J Dent Res 1970;49:1359-68.
Takeda FH, Harashima T, Kimura Y, Matsumoto K. A comparative study of the removal of smear layer by three endodontic irrigants and two types of laser. Int Endod J 1999;32:32-9.
Lester KS, Boyde A. Scanning electron microscopy of instrumented, irrigated and filled root canals. Br Dent J 1977;143:359-67.
Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic investigation of the smeared layer on root canal walls. J Endod 1984;10:477-83.
Foster KH, Kulild JC, Weller RN. Effect of smear layer removal on the diffusion of calcium hydroxide through radicular dentin. J Endod 1993;19:136-40.
Goldman M, Goldman LB, Cavaleri R, Bogis J, Lin PS. The efficacy of several endodontic irrigating solutions: A scanning electron microscopic study: Part 2. J Endod 1982;8:487-92.
McComb D, Smith DC, Beagrie GS. The results of in vivo
endodontic chemomechanical instrumentation - A scanning electron microscopic study. J Br Endod Soc 1976;9:11-8.
Spanó JC, Silva RG, Guedes DF, Sousa-Neto MD, Estrela C, Pécora JD. Atomic absorption spectrometry and scanning electron microscopy evaluation of concentration of calcium ions and smear layer removal with root canal chelators. J Endod 2009;35:727-30.
Stojicic S, Shen Y, Qian W, Johnson B, Haapasalo M. Antibacterial and smear layer removal ability of a novel irrigant, QMiX. Int Endod J 2012;45:363-71.
Giardino L, Ambu E, Becce C, Rimondini L, Morra M. Surface tension comparison of four common root canal irrigants and two new irrigants containing antibiotic. J Endod 2006;32:1091-3.
Gade VJ, Sedani SK, Lokade JS, Belsare LD, Gade JR. Comparative evaluation of debris removal from root canal wall by using EndoVac and conventional needle irrigation: An in vitro
study. Contemp Clin Dent 2013;4:432-6.
Elnaghy AM. Effect of QMix irrigant on bond strength of glass fibre posts to root dentine. Int Endod J 2014;47:280-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]