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ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 6  |  Page : 156-163  

The evaluation of dentin microhardness after use of 17% EDTA, 17% EGTA, 10% citric acid, MTAD used as chelating agents combined with 2.5% sodium hypochlorite after rotary instrumentation: An in vitro SEM study


1 Department of Conservative Dentistry and Endodontics, Chettinad Dental College and Research Institute, Kanchipuram, India
2 Department of Conservative Dentistry and Endodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, India
3 Deptartment of Conservative Dentistry and Endodontics, Rajas Dental College, Tirunelveli, Tamil Nadu, India
4 Deptartment of Conservative Dentistry and Endodontics, Noorul Islam College of Dental Sciences, Neyyattinkara, Kerala, India
5 Deptartment of Periodontics, Noorul Islam College of Dental Sciences, Neyyattinkara, Kerala, India

Date of Web Publication28-May-2019

Correspondence Address:
Dr. Manu Unnikrishnan
Flat 405, Block 4C, Doshi Etopia-2, Panchayat 3rd Link Road, Perungudi, Chennai 600096, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JPBS.JPBS_282_18

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   Abstract 

Introduction: The smear layer removed during root canal instrumentation enables penetration of irrigant solution and adhesion of root canal sealants providing a fluid tight seal of obturated root canals, necessitating its removal. The aim of the study was to compare the effect of smear layer removal by 17% Ethylene diamine tetraacetic acid (EDTA), 17% Ethylene glycol tetraacetic acid (EGTA),10% Citric acid and (MTAD solution) a Mixture of tetracycline isomer, an acid and a detergent applied as final rinse , when used along with 2.5% (NaOCl) Sodium hypochlorite and its effect on dentin microhardness. Materials and Methods: Sixty single-rooted human mandibular premolars with minimum curvature (<5°) were instrumented using rotary instrumentation till apical enlargement size 35 RaCe file 0.04 taper. During instrumentation, the canals were irrigated with 2-mL 2.5% NaOCl. After instrumentation, teeth were rinsed with distilled water and were divided into 5 groups (n = 12) according to final rinse for 1min with 5-mL 17% EDTA followed by 5-mL 2.5% NaOCl (group 1, control group), 17% EGTA (group 2), MTAD solution (group 3), 10% citric acid (group 4), and 17% EDTA (group 5). Teeth were split through the groove prepared, and one-half of specimen were evaluated for smear layer removal using scanning electron microscopy. A score system was followed to evaluate the images and comparisons among groups were performed using Kruskal–Wallis one-way analysis. Multiple comparisons were performed using Mann–Whitney U test. The other half was evaluated for dentin microhardness using Vickers microhardness testing machine. Microhardness values were statistically analyzed using parametric one-way analysis of variance test. Conclusion: Irrigation regimen following the use of 2.5% NaOCl during instrumentation followed by application of 5-mL 17% EDTA solution for 1min resulted in efficient smear layer removal and less decrease in dentin microhardness compared with 17% EGTA, 10% citric acid, and MTAD solution.

Keywords: Chelating solutions, dentin erosion, dentin microhardness, smear layer


How to cite this article:
Unnikrishnan M, Mathai V, Sadasiva K, Santakumari RS, Girish S, Shailajakumari AK. The evaluation of dentin microhardness after use of 17% EDTA, 17% EGTA, 10% citric acid, MTAD used as chelating agents combined with 2.5% sodium hypochlorite after rotary instrumentation: An in vitro SEM study. J Pharm Bioall Sci 2019;11, Suppl S2:156-63

How to cite this URL:
Unnikrishnan M, Mathai V, Sadasiva K, Santakumari RS, Girish S, Shailajakumari AK. The evaluation of dentin microhardness after use of 17% EDTA, 17% EGTA, 10% citric acid, MTAD used as chelating agents combined with 2.5% sodium hypochlorite after rotary instrumentation: An in vitro SEM study. J Pharm Bioall Sci [serial online] 2019 [cited 2019 Jun 18];11, Suppl S2:156-63. Available from: http://www.jpbsonline.org/text.asp?2019/11/6/156/258825




   Introduction Top


The smear layer interferes with the adhesion of sealer against canal walls and prevents tubular penetration of sealer, thus resulting in increased chances for leakage. If smear layer acts as a substrate for growth and survival of bacteria and alters dentin permeability leading to ineffective disinfection of dentinal tubules, then its removal contributes to successful root canal treatment.[1] Partial removal and not complete removal of smear layer is obtained with use of acids and chelators. The smear layer components are small particles with large surface–mass ratio; these particles are highly soluble in acid.[2] The quantity of smear layer removed by a material is related to its pH and time of exposure. A number of chemicals have been investigated as irrigants to remove smear layer. A working solution is the one which is used to clean the canal, and an irrigation solution is one which is essential to remove debris and smear layer created by instrumentation process.[3] Dentin microhardness depends on the amount of calcified matrix per square millimeter and its determination provides indirect evidence of mineral loss or gain in the dental hard tissues. Surface changes evaluation of dental hard tissues for alteration in calcium –phosphorous ratio has been carried out by methods such as microhardness measurement, microradiographic assessments, scanning electron microscopic methods, energy dispersive spectrometric analysis, and surface roughness testing.[1] The microhardness measurement was one of the simplest nondestructive mechanical characterization methods. The suitability and practicality of Vickers hardness test for evaluating surface changes is adopted in this study.[1]


   Objective Top


The study evaluated and compared smear layer removal property of 17% ethylenediaminetetraacetic acid (EDTA), 17 % Ethylene glycol tetra acetic acid (EGTA), 10% citric acid, and MTAD solution for 1min as final irrigant along with 2.5% sodium hypochlorite (NaOCl) from coronal, middle, and apical portions of rotary nickel– titanium instrumented root canals and the effect of the irrigation regimen on dentin microhardness


   Materials andMethods Top


An in vitro experimental design was followed in the study, conducted in a tertiary care setting. A total of 60 single-rooted recently extracted human mandibular premolars teeth with minimum curvature (less than 5°) without caries or extensive restoration were selected. Single-rooted mandibular premolars extracted for orthodontic treatment were selected having approximate length of 20–22mm and curvature less than 5mm, which was confirmed by buccal and proximal radiographs according to Schneider to avoid anatomic variation as suggested by Wu et al.[4]

The teeth were stored in 0.2% thymol solution until use. Sixty teeth were randomly divided into 5 groups. Root apex was covered with sticky wax to prevent extrusion of irrigants. Each group contained 12 teeth and one positive control group. A flat occlusal surface was made as a reference for determining working length (WL), and pulp chamber of each tooth was accessed. The WL was determined by K-file (Dentsply Maillefer, Ballaigues, Switzerland). The file was introduced into canal till visualized at the apex and pulled back 1mm. The working diameter of the canal at WL was determined by introducing K-files of successively larger diameters and the instrument showing resistance to removal at WL was recorded. And cervical pre-flaring was performed with stainless-steel LA Axxess bur (Sybron Endo Corporation, Orange, CA). A nickel– titanium rotary system RaCe was used to prepare the canal by crown down technique. (1) 0.08 tapered #35 instrument was used to one-half of the WL, (2) 0.06 tapered #25 instrument was used to two-thirds of the WL, (3) 0.04 tapered #30 instrument was used to full WL, and (4) 0.04 tapered #35 instrument used to full WL. During the preparation, root canal was irrigated with 2-mL 2.5% NaOCl (Vensons, India) at each change of file in all the study groups. After completion of instrumentation, the teeth were irrigated with distilled water, dried with paper points, and then divided into groups according to time and substance used. A final irrigation of 5-mL 17% EDTA for 1min using conventional needle irrigation and for 60s irrigant was left undisturbed, followed by 5-mL 2.5% NaOCl for 1min as final rinse for the following:

  • Group 1, positive control group—17% EDTA followed by 2.5% NaOCl
  • Group 2, 5-mL 17% EGTA for 1min as final rinse using conventional needle irrigation and for 60s irrigant was left undisturbed

    Group 3, 5-mL MTAD solution for 1min as final rinse using conventional needle irrigation, and for 60s irrigant was left undisturbed

    Group 4, 5-mL 10% citric acid for 1min as final rinse using conventional needle irrigation, and for 60s irrigant was left undisturbed

    Group 5, 5-mL 17% EDTA for 1min as final rinse using conventional needle irrigation, and for 60s irrigant was left undisturbed

    Irrigants were passively delivered using side-vented 27 gauge needle to within 1mm from the WL in each canal, and small apico-coronal movement of needle was maintained during final irrigation. The specimens were fixed using glutaraldehyde and the fixed specimens were rinsed three times with a sodium cacodylate–buffered solution (concentration 0.1, pH 7.2), incubated in osmium tetroxide for 2h, dehydrated with ascending concentrations of ethyl alcohol, and placed in a desiccator for 24h. The canals were dried with absorbent paper points and the entrance to each of the canals was protected with a cotton pellet to prevent penetration of the dentinal debris into the canals during decoronation. The teeth were decoronated using diamond discs with water, the crown was removed at the cement–enamel junction, and deep grooves were made on the buccal and palatal surfaces of the roots without perforating the canal. The roots were then split longitudinally using a chisel. One-half of each root was selected for examination under a scanning electron microscopy. Each specimen was mounted on aluminum stub, coated with 30 µm of gold palladium, and examined under scanning electron microscope (SEM).


       Evaluation ofOutcome Top


    The outcome variables were smear layer removal property and dentine microhardness. SEM evaluation was performed to assess the smear layer removal property and microhardness testing was performed by Vickers microhardness tester.


       SEM Evaluation Top


    The root canals were prepared by one operator whereas SEM evaluation were performed by two other examiners and were blinded to experimental groups. The smear layer on the surface of the root canal or in the dentinal tubules at the cervical, middle, and the apical portion of each canal was evaluated according to the following criteria used by Torabinejad et al.[5]

    1. No smear layer: no smear layer on the surface of the root canals; all tubules were clean and open.
    2. Moderate smear layer: no smear layer on the surface of the root canal, but tubules contained debris.
    3. Heavy smear layer: smear layer covered the root canal surface and the tubules.[5]


    A scoring system was followed to evaluate the images. The scores were discrete variables and hence comparison among groups was performed using Kruskal–Wallis one-way analysis and chi-square values were determined. Multiple comparisons of two groups taken at a time were performed using Mann–Whitney U test.


       Dentine Microhardness Top


    The other half of longitudinally split portions was ground polished with water-cooled carborundum disc. Final polishing was carried out in felt cloth and buff using 0.05 µm size aluminum oxide powder mixed with distilled water. A plastic ring was then taken and poured with a mixture of cold cure resin. Specimens were embedded on the resin with polished surface facing outside. After curing of the resin, the ring was removed and repolishing of specimens was carried out to remove excess material present on the tooth surface. Microhardness testing was performed by mounting specimens on the stage of Vickers microhardness tester. The mid-root portion halfway from the outer surface was focused for testing. Indentations were made with Vickers diamond indenter using 300g load with a dwell time of 15s. These indentations were measured and converted into Vickers hardness number. Microhardness values across the groups were compared using parametric one-way analysis of variance test, and significance was tested using F test. For all comparisons, P values were fixed as <5%


       Results Top


    Comparison of smear layer removal property of selected irrigation regimens and its effect on dentine microhardness are presented later.

    [Table 1] shows mean, median, and standard deviation values for smear layer removal from cervical, middle, and apical portions for five groups. Group 5 (17% EDTA) as final rinse showed better smear layer removal than (group 1) NaOCl as final rinse and 17% EGTA (group 2), MTAD solution (group 3), and 10% citric acid (group 4) [Figure 1] A, B and C. Multiple comparisons of two groups taken at a time are presented in [Table 2]. The smear layer removal was more for 10% citric acid from cervical, middle, and apical portions of root canal when compared with 17% EDTA, 17% EGTA, and MTAD solution [[Figure 2] A, B and C. In the apical portions of root canal, 17% EDTA showed better smear layer removal (P value 0.039) when compared to 17% EGTA and MTAD solution [[Figure 5C]. The control group (group 1) when NaOCl was used as final rinse showed statistically significant value 0.0008 when compared with 10% citric acid at apical portions of root canal, indicating more removal of peritubular and intertubular dentin from apical portions of root canal [Figure 3]C. MTAD solution and 17% EGTA showed no statistical significance (P value 0.090), indicating less efficient in removing smear layer from apical portions of root canal when compared to 17% EDTA.
    Table 1: Mean, median, and standard deviation of smear layer removal scores and one-way analysis using Kruskal–Wallis test

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    ,
    Figure 1: Scanning electron microscope images of group 5, (17% EDTA) at coronal third (A), middle third (B), and apical third (C)

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    ,
    Table 2: Multiple comparisons of smear layer removal scores using Mann-Whitney U Test of Apical third of root canal

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    ,
    Figure 2: Scanning electron microscope images of group 4, (10% citric acid) at coronal third (A), middle third (B), and apical third (C)

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    ,
    Figure 5: Scanning electron microscope images of group 3, (MTAD) at coronal third (A), middle third (B), and apical third (C)

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    ,
    Figure 3: Scanning electron microscope images of group 1, (17% EDTA + 2.5% NaOCl) at coronal third (A), middle third (B), and apical third (C)

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    [Table 3] shows mean, median, and standard deviation value for five groups. 17% EDTA (group 5) and 17% EGTA (group 2) as final rinse resulted in less reduction in dentin microhardness as compared to NaOCl as final rinse (group 1), MTAD solution (group 3), and 10% citric acid (group 4). Multiple comparisons of microhardness values between groups were evaluated using post hoc Tukey analysis and presented in [Table 4]. Group 4, 10% citric acid, showed statistically significant value (P value 0.0001) when compared with group 5 (17% EDTA). Group 1 (17% EDTA followed by final rinse with 2.5% NaOCl) showed statistically significant value (P value 0.0001) when compared to group 5 (17% EDTA) used as final rinse. Group 2 17% EGTA (72.6 knoop hardness value [KHV]) and 17% EDTA group 5 (72.02 KHV) caused less reduction in dentin microhardness when compared to group 1, group 4 (10% citric acid), and group 3 (MTAD solution).
    Table 3: Parametric one way analysis of microhardness values using ANOVA test

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    ,
    Table 4: Multiple comparison of microhardness values using post hoc test by Tukey honest significant difference analysis

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       Discussion Top


    The purpose of our study was to evaluate the efficacy of four chelating agents using 5-mL 17% EDTA, 17% EGTA, 10% citric acid, and MTAD solution, when used as final rinse for 1min, along with 2-mL 2.5% NaOCl as working solution on smear layer removal from instrumented root canal dentine. The effect of these irrigants when used as final rinse along with 2.5% NaOCl for smear layer removal on dentin microhardness was assessed. The irrigation regimen of using 5-mL 17% EDTA for 1min as final rinse was effective in removing smear layer from cervical, middle, and apical portions of instrumented root canal when compared to 5-mL 10% citric acid, 17% EGTA, and MTAD solution [Figure 2]AB, [Figure 4]AB, and [Figure 5]AB. The use of 5-mL 10% citric acid for 1min resulted in loss of peritubular and intertubular dentin and decrease in dentin microhardness. The irrigation protocol of using 2ml of 2.5 % of sodium hypochlorite during instrumentation of root canals as a working solution facilitated debridement, lubrication, tissue dissolution, disinfection, collagen layer removal and dentin dehydration. When 17 % Ethylenediaminetetraacetic acid (EDTA) was used as final rinse after instrumentation of root canals results in dentin demineralization and facilitates excellent cleaning of the canal walls. This helped in canal disinfection by penetration of chemical substances and provided an intimate contact of the filling materials with radicular dentin. EDTA acts on the inorganic components of the smear layer, causing decalcification of the peri- and intertubular dentin. EDTA also covalently bonds to metal ions and sequesters calcium ions present in hydroxyapatite dentin. In this study, the irrigation protocol following 2.5% NaOCl functioned as working solution and 5-mL 17% EDTA used as final rinse for 1min gave successful removal of smear layer and less reduction of dentin microhardness. Five milliliters of 2.5% NaOCl used as final rinse after 17% EDTA in the positive control group (group 1) resulted in considerable reduction in dentin microhardness [Figure 3] A and B. The reduction in dentin microhardness caused was more than with 5-mL 10% citric acid, indicating severe dentinal erosion. The use of 5-mL 17% EGTA and MTAD solution was less effective in removing smear layer from apical portions of instrumented root canals [Figure 4]C and [Figure5]C. The application of 5-mL 17% EGTA caused less reduction in dentin microhardness whereas 10% citric acid resulted in dentin erosion producing reduction in dentin microhardness in instrumented radicular dentin walls. There is no definite time a chelating agent must be in contact within root canal to remove smear layer. In this study, a 1-minute time of application was adopted in accordance with various other studies.[6],[7] Medici and Frener also conducted a study by SEM analysis and observed better cleaning using 1% NaOCl followed by 17% EDTA, which is related to the fact that the association between the halogen and a chelating agent acts on both organic and inorganic portions of the smear layer.[8],[9] However the association between EDTA and NaOCl might cause dentinal erosion as observed by Qin et al.[10] These authors found remarkable dentinal loss when EDTA irrigation was followed by final irrigation with 6% NaOCl, because EDTA facilitates decalcification of the inorganic component and hypochlorite promotes dissolution of the organic matrix. The factors influencing demineralization capacity of chelating agents include contact time, pH, concentration, and amount of available solution. Flame atomic absorption spectrophotometry to measure the concentration of left out calcium ions in chelating solutions after root canal irrigation can be performed to evaluate the effect of demineralization on root canal dentin.[11] Gorus et al, reported when Er.YAG laser was used for smear layer removal, there was no signigicant difference in dentin microhardness between teeth treated with lasers and conventional irrigation method.[12] The various other factors such as age of the tooth, degree of dentin mineralization, testing conditions, and complex root canal anatomy in apical third with few open tubules that limits to reproduce the in vivo biological conditions are the limitations of this in vitro experimental study design.
    Figure 4: Scanning electron microscope images of group 2 , (17% EGTA) at coronal third (A), middle third (B), and apical third (C)

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       Conclusion Top


    The irrigation regimen following the final rinse with 5-mL 17% EDTA for 1min after the use of 2.5% NaOCl provided efficient removal of smear layer from coronal, middle, and apical portions of instrumented root canal walls. The amount of reduction in dentin microhardness after chemomechanical preparation was less when irrigation regimen with 1-min application of 5-mL 17% EDTA as final rinse.

    Acknowledgement

    Council of Scientific and Industrial Research, National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695 019, Kerala, India.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.

     
       References Top

    1.
    Clark-Holke D, Drake D, Walton R, Rivera E, Guthmiller JM. Bacterial penetration through canals of endodontically treated teeth in the presence or absence of the smear layer. J Dent 2003;31:275-81.  Back to cited text no. 1
        
    2.
    Lotfi M, Vosoughhosseini S, Saghiri MA, Zand V, Ranjkesh B, Ghasemi N. Effect of MTAD as a final rinse on the removal of smear layer in ten minute preparation time. J Endod 2012;38:1391-94.  Back to cited text no. 2
        
    3.
    Bansal P, Gupta H. Smear layer in endodontics—A review. Ind J Dental Sci; 2009;1:67-9.  Back to cited text no. 3
        
    4.
    Wu MK, Kast’áková A, Wesselink PR. Quality of cold and warm gutta-percha fillings in oval canals in mandibular premolars. Int Endod J 2001;34:485-91.  Back to cited text no. 4
        
    5.
    Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for removal of the smear layer. J Endodon 2003;29:170-5.  Back to cited text no. 5
        
    6.
    Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003;36:411-7.  Back to cited text no. 6
        
    7.
    Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.  Back to cited text no. 7
        
    8.
    Medici MC, Frener IC. A Scanning electron microscopic evaluation of different root canal irrigation regimens. Braz Oral Res 2006;20:235-40.  Back to cited text no. 8
        
    9.
    Teixeira CS, Felippe MC, Felippe WT. The effect of application time of EDTA and naocl on intracanal smear layer removal: An SEM analysis. Int Endod J 2005;38:285-90.  Back to cited text no. 9
        
    10.
    Qin N, Olcese R, Bransby M, Lin T, Birnbaumer L. Ca2+-induced inhibition of the cardiac ca2+ channel depends on calmodulin. Proc Natl Acad Sci U S A 1999;96:2435-8.  Back to cited text no. 10
        
    11.
    Berastegui E, Molinos E, Ortega J. To comparison of standard and new chelating solutions in Endodontics. J Dental Sci 2017;2:000131  Back to cited text no. 11
        
    12.
    Gorus Z. A comparative evaluation of smear layer removal by using different er:YAG lasers parameters: An in-vitro scanning electron microscopic study. Niger J Clin Pract 2018;21:1602-6.  Back to cited text no. 12
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        Tables

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  In this article
    Abstract
   Introduction
   Objective
   Materials andMethods
   Evaluation ofOutcome
   SEM Evaluation
   DentineMicrohardness
   Results
   Discussion
   Conclusion
   Introduction
   Objective
   Materials andMethods
   Evaluation ofOutcome
   SEM Evaluation
    Dentine Microhar...
   Results
   Discussion
   Conclusion
   Introduction
   Objective
   Materials andMethods
   Evaluation ofOutcome
   SEM Evaluation
    Dentine Microhar...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

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