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Doi:10.1016/j.tripleo.2007.03.02

The effect of EDTA, EGTA, EDTAC, and tetracycline-HCl with
and without subsequent NaOCl treatment on the
microhardness of root canal dentin

Taner Cem Sayin, DDS, PhD,a Ahmet Serper, DDS, PhD,b Zafer C. Cehreli, DDS, PhD,c andHarika G. Otlu, BSc,d Ankara, TurkeyHACETTEPE UNIVERSITY Objective. The purpose of this study was to evaluate the effect of single and combined use of ethylenediamine tetra
acetic acid (EDTA), ethylene glycol bis [b-aminoethylether] N,N,N=,N=-tetraacetic acid (EGTA), EDTA plus Cetavlon
(EDTAC), tetracycline-HCl, and NaOCl on the microhardness of root canal dentin.
Study design. The crowns of 30 single-rooted human teeth were discarded at the cementoenamel junction and the
roots were bisected longitudinally to obtain root halves (N ϭ 60). The specimens were embedded in autopolymerizing
acrylic resin, leaving the root canal dentin exposed. Dentin surfaces were prepared for microhardness test by grinding
and polishing. The reference microhardness values of untreated specimens were recorded using a Vicker’s
microhardness tester at the apical, midroot, and cervical levels of the root canal. Thereafter, the specimens treated
with single (test solution only) or combined (test solution, followed by 2.5% NaOCl) versions of the irrigants for 5
minutes. Posttreatment microhardness values were obtained as with initial ones. Statistical comparisons between the
test groups and among single and combined treatments were carried out using 2-way ANOVA with repeated measures
(P ϭ .05). Comparisons within each group with respect to application regions were made with Friedman’s
nonparametric 2-way analysis of variance at the same level of significance.
Results. All treatment regimens except distilled water significantly decreased the microhardness of the root canal
dentin (P Ͻ . 05). The single and combined use of EDTA decreased the microhardness of the root canal dentin
significantly more than all other treatment regimens (P Ͻ .05). Compared with their single-treatment versions, all
combined treatment regimens decreased the mean microhardness values significantly (P Ͻ .05). A comparison of
single and combined treatment regimens revealed significant decreases only for EDTA and EDTA ϩ NaOCl in the
coronal region and for EDTAC and EDTAC ϩ NaOCl in the apical and middle regions of the root canal (P Ͻ .05).
Conclusions. The use of EDTA alone or prior to NaOCl resulted in the maximum decrease in dentin microhardness.
The softening effect of subsequent NaOCl treatment was both material and region dependent. However, for combined
treatment regimens, subsequent use of NaOCl levels the statistical differences between the regional microhardness
values obtained after treatment with EGTA, EDTAC, and tetracycline-HCl. (Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 2007;104:418-24)

The success of root canal treatment depends on the ment. However, accumulating evidence suggests the root canal system being thoroughly cleansed and importance of removing the smear layer because it disinfected, followed by obturation of this space.
can result in a more thorough disinfection of the root Since the first description of the smear layer in canal system and the dentinal tubules, which would ensure a better adaptation between the obturation there is an ongoing debate regarding the influence of this layer on the success rate of endodontic treat- created during root canal instrumentation is com-posed of dentin structure and some nonspecific inor-ganic The organic components may Received from the Faculty of Dentistry, Hacettepe University.
consist of reacted coagulated proteins, necrotic or aFormerly, Research Assistant, Department of Endodontics. Cur- viable pulp tissue, odontoblastic processes, and mi- rently, Assistant Professor, Nova Southeastern University, College of Dental Medicine, Fort Lauderdale, FL, USA.
b Different solutions have been used to remove the Professor, Department of Endodontics.
cAssociate Professor, Department of Pediatric Dentistry.
smear layer. Sodium hypochlorite (NaOCl) in a 1% dResearch Assistant, Department of Biostatistics.
to 5.25% concentration is an irrigant solution widely Received for publication Oct 12, 2006; returned for revision Mar 7, used in root canal treatment because of its bacteri- 2007; accepted for publication Mar 19, 2007.
cidal properties and ability to dissolve organic tis- However, it has been shown to be ineffective 2007 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2007.03.021 in removing the entire smear layer when used 418
419
Thus, the use of chelating agents and acids MATERIAL AND METHODS
have been suggested to remove the smear layer from Thirty periodontally involved, human maxillary the root canal, because the components of this incisor and mandibular premolar teeth were extracted loosely bound structure are very small particles with and stored in distilled water at 4°C for a maximum of a large surface-mass ratio that makes them very 2 months. Before experiments, soft tissues covering the root surfaces were removed with gauze and a fine lating agents are based on different concentrations of brush. The crowns were removed at the cementoe- namel junction by using a high-speed bur under water cooling. Thereafter, the roots were bisected addition of a quaternary ammonium bromide (Cetavlon) longitudinally in the buccolingual direction to obtain increased the action of EDTA by reducing its surface root halves (N ϭ 60), after which the pulp tissue was tension, because EDTA solutions act only through direct removed with a toothbrush. The root halves were contact with the This combination, known as embedded in autopolymerizing acrylic resin, leaving EDTA plus Cetavlon (EDTAC), was shown to be very the dentin surface exposed. Then, the specimens effective in smear layer removal and increasing the diam- were ground flat on a circular grinding machine with eter of the opened dentinal Recently, Çalt and ascending grades of SiC abrasive papers (500, 800, reported that ethylene glycol-bis [b-aminoethyl- 1000, and 1200 grit) under constant water irrigation, ether]-N,N,N=, N=-tetraacetic acid (EGTA) was also ef- and further polished with fine alumina suspension fective in removing the smear layer, without inducing dentinal erosion commonly caused by EDTA. Tetracy- The following irrigation solutions were tested in the cline-hydrochloride (HCl) has also been proposed as a present study: 2.5% NaOCl, 17% EDTA (ethylenedi- root canal irrigant. In addition to its antimicrobial effect, aminetetraacetic acid), 15% EDTAC (EDTA ϩ 0.1% tetracycline-HCl acts as a calcium chelating agent due to cationic surfactant, Cetavlon [cetyltrimethylammonium its low One percent tetracycline HCl has been bromide]), 17% EGTA (ethylene glycol bis[2-amin- shown to be as effective as 50% citric acid in the removal oethylether]-N,N,N=N=-tetraacetic acid), and 1% tetra- of smear layer, while causing less demineralization in cycline hydrochloride. All chemicals except NaOCl were obtained from Sigma Chemical Co. (St. Louis, It has been reported that some chemicals used for MO). The test solutions were freshly prepared in lab- endodontic irrigation are capable of causing alterations in oratory conditions. The pH of EDTA, EDTAC, EGTA, the chemical composition of Any change in and tetracycline-HCl solutions was adjusted to 7.5 by the Ca/P ratio may alter the original proportion of organic and inorganic components, which in turn change the mi- Prior to application of test solutions, the Vicker’s crohardness, permeability, and solubility characteristics of hardness values of the specimens were measured on a Indeed, studies have shown that different con- Zwick-type 3212002 microhardness tester (Zwick centrations of EDTA, EDTAC, and EGTA are capable of GMBH, Ulm, Germany) and recorded. Accordingly, 3 decreasing the microhardness of root canal dentin, separate indentations, each using 200 gram load and 20 and that this effect can increase by extended application second dwell time were made along the central axis of Changes in the mineral content of superficial the root canal at the apical, midroot, and cervical levels.
dentin may also adversely affect the sealing ability and The samples were then randomly distributed into the adhesion of dental materials such as resin-based cements following treatment groups (n ϭ 10/group): group 1, 2.5% NaOCl; group 2, 17% EDTA; group 3, 17% For effective removal of both organic and inorganic EGTA; group 4, 15% EDTAC; group 5, 1% tetracy- components of the smear layer, it is generally recom- cline-HCl; and group 6, distilled water (negative con- mended to use endodontic chelator solutions followed by Although NaOCl is not a chelating agent, The specimens were immersed for 5 minutes in a it can significantly decrease the Ca/P ratio of superficial magnetic stirrer bath that contained 10 mL of each test solution. Following treatment with the chelating on the concentration of the solution. To date, the effect agents (groups 2 to 5), the same specimens were of NaOCl on dentin microhardness following initial treated with NaOCl (combined treatment). Thus, irrigation with chelating solutions has not been inves- each specimen served as its own control. In groups 2 tigated. Consequently, the aim of this study was to to 5, the specimens received a final flush of 10-mL evaluate the effect of single or combined use of NaOCl, distilled water immediately after treatment, to avoid EDTA, EGTA, EDTAC, and tetracycline-HCl on the the prolonged effect of chelating solutions. The same microhardness of human root canal dentin.
procedure was carried out after treatment with 420
Table I. Mean of changes (apical ϩ middle ϩ coronal)
in the microhardness values of the root canal dentin
following treatment with the test solutions
Min, minimum; Max, maximum; EDTA, ethylenediamine tetra-aceticacid; EGTA, ethylene glycol-bis [b-aminoethylether]-N,N,N=, N=-tetraacetic acid; EDTAC, EDTA plus Cetavlon.
NaOCl. Posttreatment indentations were made on Fig 1. Changes in the microhardness values (percentage) each specimen adjacent to the initial specimens in the with respect to single and combined treatment regimens.
same manner, and the microhardness values were recorded. For each specimen, the change (percent-age) in microhardness values was calculated as fol-lows: Table II. Changes in microhardness values with re-
spect to apical root canal dentin following treatment where M ϭ initial microhardness and M ϭ posttreat- Statistical comparisons between the test groups and among single and combined treatments were carried out using 2-way ANOVA with repeated measures (P ϭ .05). Comparisons within each group with respect to application regions were made with Friedman’s non- parametric 2-way analysis of variance at the same level Min, minimum; Max, maximum; EDTA, ethylenediamine tetra-acetic acid; EGTA, ethylene glycol-bis [b-aminoethylether]-N,N,N=, N=- Posttreatment changes in the microhardness values tetraacetic acid; EDTAC, EDTA plus Cetavlon.
(percentage) of the entire root canal dentin (mean ofapical, middle, and coronal regions) are presented in Changes in the microhardness values (per-centage) with respect to single and combined treat- 3, 4, and 5; P Ͻ .05). However, there was no signif- icant difference between the microhardness values of microhardness values with respect to the apical, mid- EGTA, EDTAC, tetracycline-HCl, and NaOCl (P Ͼ .05). For single-solution treatments, a statistical and respectively. All treatment regimens except ranking for the change in microhardness was ob- distilled water significantly decreased the microhard- ness of the root canal dentin (P Ͻ .05). Ethylenedi- EDTA Ͼ EGTA ϭ EDTAC ϭ tetracycline-HCl amine tetra-acetic acid decreased the overall micro-hardness of the root canal dentin significantly more A comparison of combined treatment regimens than the other single-solution treatments (groups 1, showed that EDTA ϩ NaOCl induced significantly 421
Table III. Changes in microhardness values with re-
NaOCl and IV; P Ͻ .05). For all single- spect to middle root canal dentin following treatment solution treatments, a statistical ranking for the change in regional microhardness values was obtained as fol- Min, minimum; Max, maximum; EDTA, ethylenediamine tetra-acetic In all regions, combined use of EDTA and NaOCl acid; EGTA, ethylene glycol-bis [b-aminoethylether]-N,N,N=, N=- decreased the microhardness of the root canal dentin tetraacetic acid; EDTAC, EDTA plus Cetavlon.
significantly more than EGTA ϩ NaOCl, EDTAC ϩNaOCl, and tetracycline ϩ NaOCl (P Ͻ .05). A com-parison of single and combined treatment regimens Table IV. Changes in microhardness values with re-
revealed significant decreases only for EDTA and spect to coronal root canal dentin following treatment EDTA ϩ NaOCl in the coronal region and for EDTAC and EDTAC ϩ NaOCl in the apical and middle regions of the root canal (P Ͻ .05). In all regions, the same statistical ranking was obtained for changes in micro- hardness values achieved with combined treatment reg- ϭ EDTAC ϩ NaOCl ϭ tetracycline-HCl ϩ NaOCl DISCUSSION
Current concepts of chemomechanical preparation im- ply that chemicals should be applied on instrumented root Min, minimum; Max, maximum; EDTA, ethylenediamine tetra-acetic canal surfaces in order to remove the smear Such acid; EGTA, ethylene glycol-bis [b-aminoethylether]-N,N,N=, N=- procedures may induce considerable changes in the tetraacetic acid; EDTAC, EDTA plus Cetavlon.
surface morphology of dentin, which may also exertchanges in its mechanical and physical proper-Moreover, alteration of the inorganic more reduction in microhardness than EGTA ϩ phase of dentin surfaces by acidic pretreatments mod- NaOCl, EDTAC ϩ NaOCl, and tetracycline ϩ NaOCl ifies their surface properties, and undoubtedly, their (P Ͻ .05). When compared with their single-treatment versions, all combined treatment regimens decreased correlation between hardness and the mineral content of the mean microhardness values significantly (P Ͻ .05).
the tooth. The determination of microhardness can thus For changes in microhardness values achieved with provide valuable evidence of mineral loss (or gain) in combined treatment regimens, the following statistical dental hard with special regard to the effects In the present study, all specimens were subjected to a 5-minute contact with the test solutions. Currently, there is a lack of consensus on the duration a decalci- EDTAC ϩ NaOCI ϭ tetracycline Ϫ HCI ϩ NaOCI fying agent must be in contact with the root canal to With respect to the region being compared (apical, middle, or coronal), treatment with EDTA resulted in a herein, De-Deus et limited the contact time of 3 significantly higher decrease in dentin microhardness chelator solutions (EDTA, EDTAC, and citric acid) to compared with EGTA, EDTAC, tetracycline-HCl, and 5 minutes, stating that this duration is more realistic in 422
terms of clinical Other researchers have sug- effective in reducing the surface tension at the apical gested extending the application time to 10 to 15 min- region than in the middle and coronal It could be expected that the removal of the inorganic content of reported that EDTA can remove the smear layer in 1 dentin would reduce more its microhardness than re- In addition to contact time, the concentration move the organic portion. Unlike what is commonly of the irrigation solution needs to be considered as accepted, the treatment of dentin with NaOCl may not another determinant in the posttreatment microhardness only remove the organic matrix but also some of the values of dentin. On the basis of the results obtained, inorganic content that ultimately renders dentin much EDTA decreased the microhardness of dentin by weaker than The precise mechanism of this 17.33% to 29.48%, and this effect was significantly phenomenon is unknown, leaving room for speculation.
greater than that achieved with both the test and control With special regard to the combined treatment regi- solutions. Although EDTA and EDTAC had similar mens tested, subsequent application of NaOCl may concentrations (17% vs. 15%), the efficacy of EDTAC facilitate further exposure of the inorganic material on was significantly lower than that of EDTA. This finding decalcified dentin substrate through removal of the corroborates previous work,showing that reduc- ing its surface tension does not improve the effective- izing effect that would eventually decrease the dentin ness of EDTA. According to De-Deus et the lesser microhardness. Nevertheless, this effect can be material efficiency of EDTAC to remove calcium ions from and/or region dependent. For instance, compared with dentin could be responsible for this finding. Although their respective single-treatment versions, significant this explanation could be reasonably extended to the microhardness reductions in the combined treatment findings obtained with EGTA and tetracycline-HCl, groups were observed only when NaOCl was used after there is currently no published study to support this EDTA in the coronal third and after EDTAC in the assumption, especially when all solutions are adjusted apical and middle thirds of the root canal.
to the same concentration and/or pH.
Microhardness tests have been traditionally em- The relative softening effect on dentinal walls ex- ployed to evaluate materials, presenting a certain ho- erted by chemical irrigants could be of clinical benefit Biological materials such as dentin are far since it permits rapid preparation and facilitates nego- less homogenous, with dentin tubule density increasing tiation of small tight but these alterations from cervical to apical resulting in an inverse also affect the sealing ability and adhesion of sealers to correlation between dentin microhardness and tubule This may lead to deviations in the results solution removes the entire smear within the same because of differences in adjacent regions of the dentin period of time, lower concentrations of EDTA should This is clearly confirmed in the present study be preferred to reduce its adverse (softening) effect on by the differences in the statistical ranking of single- root dentin. In this regard, the tested concentrations of solution treatments with EGTA, EDTAC, NaOCl, and EGTA and EDTAC can be considered less detrimental tetracycline-HCl at the apical, middle, and coronal re- to dentin. However, the efficacy of lower concentra- gions of root canal dentin. However, following subse- tions of EGTA and EDTAC merits further evaluation, quent treatment with NaOCl, the statistical ranking for because these 2 solutions also significantly decreased all three regions was the same. This indicates that regional differences in microhardness are leveled in the ing solutions also needs to be considered as another combined treatment groups in a similar pattern ob- important factor. However, since the pH of all test served in the general mean ranking of all 3 regions solutions were adjusted to 7.5, comparisons cannot be suggests that the combination of tetracycline-HCl and Results obtained within the experimental conditions NaOCl appears to yield the least softening (adverse) of the present study indicate that the single use of NaOCl significantly reduces the microhardness of rootcanal dentin compared with control. Further, despite the CONCLUSION
lack of significant differences, comparison of numerical On the basis of the results obtained and experimental data has shown that the use of NaOCl alone can also conditions of the present study, the use of EDTA alone induce more reduction in microhardness in comparison or prior to NaOCl resulted in the maximum decrease in with EDTAC and tetracycline-HCl in the middle and dentin microhardness. The softening effect of subse- coronal root canal dentin. NaOCl was not as effective quent NaOCl treatment was both material and region in the apical region as it was in the coronal and middle dependent. However, for combined treatment regimens, thirds, probably because it has been shown to be less subsequent use of NaOCl levels the differences be- 423
tween the microhardness values obtained after treat- of citric acid solutions on the calcium and phosphorus contents of ment with EGTA, EDTAC, and tetracycline-HCl.
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