Basic ResearchTechnology
Effect of EDTA, Sodium Hypochlorite, and Chlorhexidine
Gluconate with or without Surface Modifiers
on Dentin Microhardness
Eda E. Aslantas, DDS, Hatice Dogan Buzoglu, DDS, PhD, Emre Altundasar, DDS, PhD,
and Ahmet Serper, DDS, PhD
Abstract
Introduction: This study aimed to evaluate the effects
of root canal irrigants on the microhardness of root canal
dentin in the presence and absence of surface-modifying
agents. Methods: Forty-eight root halves were pre-
pared by longitudinal splitting of the distal roots of 24
freshly extracted mandibular human third molars and
embedded in autopolymerizing acrylic resin, leaving
the dentin surface exposed. After polishing, the micro-
hardness values of the untreated dentin surfaces were
recorded by using Vickers tester at the mid-root level.
The root halves were randomly assigned to 6 groups
composed of 8 samples each and treated for 5 minutes
with one of the following irrigants: 17% EDTA, REDTA,
2% chlorhexidine gluconate (CHX), 2% CHX with sur-
face modifiers (CHX-Plus), 6% NaOCl, or 6% NaOCl
with surface modifiers (Chlor-XTRA). After surface treat-
ment, dentin microhardness values were recorded at
close proximity to the initial indentation areas. Experi-
mental data were statistically analyzed by using the t
test and one-way analysis of variance, followed by Tu-
key honestly significant difference test at a = 0.05. Re-
sults: EDTA, REDTA, NaOCl, and Chlor-XTRA
significantly decreased the microhardness of root dentin
compared with intact controls (P < .05). Conclusions:
The addition of surface modifiers to the irrigants did
not affect the microhardness of the samples. (J Endod
2014;40:876879)
Key Words
Chlorhexidine gluconate, dentin, EDTA, microhardness,
sodium hypochlorite, surface modifier
From the Department of Endodontics, Faculty of Dentistry,
Hacettepe University, Ankara, Turkey.
Address requests for reprints to Dr Hatice Dogan Buzoglu,
Department of Endodontics, Faculty of Dentistry, Hacettepe
University, 06100 Sihhiye, Ankara, Turkey. E-mail address:
hdogan67@hotmail.com
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.10.041
876 Aslantas et al.
R oot canal preparation aims to remove bacteria from the root canal system, primarily
through the use of irrigating solutions. Irrigation flushes away loose, necrotic, and
contaminated materials before they are pushed deeper in the apical direction (1).
Because of its solvent activity, sodium hypochlorite (NaOCl) has been recommended
to remove necrotic and vital tissues from root canals. Chlorhexidine gluconate
(CHX), which has antibacterial effects and no cytotoxicity, is another popular irrigant
for chemomechanical debridement (2, 3). Chelating agents, such as different
concentrations of EDTA, are also suggested to improve chemomechanical
debridement of root canals by facilitating the removal of the smear layer (3, 4).
None of these irrigants can easily reach target areas within the intricate structure of
the root canal system. To achieve deeper penetration of irrigants into the dentinal tu-
bules and lateral canals, surface-active agents have been added to the irrigant to reduce
surface tension (58). Surface-active agents, which are also called surface modifiers or
surfactants, may act as detergents, wetting agents, emulsifiers, foaming agents, or dis-
persants (9, 10). Previous reports indicated that an EDTA solution with added
surfactant demonstrated effectiveness similar to plain EDTA for both smear removal
(11) and Ca+2 ion release (12). However, the addition of surfactant enhanced bacte-
ricidal effectiveness and may improve clinical performance (1315). The addition of a
low concentration of surfactant to NaOCl increased the antibacterial effectiveness of the
solution (15). Similarly, surface modifier addedCHX (CHX-Plus) killed bacteria faster
than 2% CHX alone (16).
The structural properties of dentin, such as microhardness, permeability, and sol-
ubility, may change after the use of chemical irrigants, which are capable of altering the
proportion of organic and inorganic components (17). Microhardness is considered
indirect evidence of mineral changes in root canal dentin; such changes could affect the
adhesive properties of the dentin surface (1820). Previous studies demonstrated that
NaOCl and EDTA solutions decreased the microhardness of root dentin, whereas
solutions containing different concentrations of CHX had varying effects on
microhardness (2026). In a recent study, the surfactant agent cetrimide decreased
dentin microhardness to a similar extent as EDTA alone. However, EDTA solution
with cetrimide did not cause additional microhardness alteration of root dentin
when compared with EDTA alone (26). Despite interest in the effects of irrigation so-
lutions on root canal dentin, limited information is available concerning the role of sur-
face modifiers on irrigant performance. This study aimed to evaluate the effects of root
canal irrigants on the microhardness of root canal dentin in the presence and absence
of surface modifiers.
Materials and Methods
Sample Preparation
Freshly extracted, caries-free human mandibular third molars from patients be-
tween 20 and 33 years of age were obtained by using a protocol approved by the Human
Subjects Ethical Review Committee (project no: GO 13/246). After removal of sur-
rounding soft tissue and debris, 24 teeth were stored at 4 C in an aqueous solution
of 0.5% chloramine-T until use. The crowns were removed at the cementoenamel junc-
tion under water cooling. The distal roots were split longitudinally into the buccal and
JOE Volume 40, Number 6, June 2014

lingual segments with a low-speed diamond disk and embedded in
acrylic resin, leaving the dentin surface exposed. The resulting
48 root halves were ground and polished with silicon carbide abrasive
papers (180, 320, and 600 grit) and 0.25-mm diamond polishing pa-
pers (Metkon, Bursa, Turkey) under distilled water to remove any sur-
face scratches.
Determination of Dentin Microhardness
The dentin microhardness of the root samples was initially
measured by using a Durascan 20 Vickers microhardness tester
(Emco Test, Kuchl, Austria) at a magnification of 250 and a depth
of 300 mm from the pulp-dentin interface. Three separate indentations
were made by using a 300-g load and a 20-second dwell time at the mid-
root level of the root dentin samples before surface treatment.
Sample Treatment
The samples were randomly divided into 6 groups (n = 8) and
treated for 5 minutes with 5 mL of one of the following irrigants:
17% EDTA (Vista Dental, Racine, WI), REDTA (17% EDTA containing
0.84 g cetrimide) (Sigma-Aldrich, Munich, Germany), 6% NaOCl
(ACE, Proctor & Gamble, Gebze, Turkey), 6% NaOCl with surface mod-
ifiers (Chlor-XTRA) (Vista Dental), 2% CHX (Klorhex, Drogsan,
Turkey), or CHX-Plus (Vista Dental).
After surface treatment, the samples were dried, and the dentin
microhardness of each sample was determined as described above at
locations in close proximity to the initial indentation areas.
Statistical Analysis
Differences between pretreatment and post-treatment microhard-
ness were statistically analyzed by using the t test with a P value of .05.
Differences between pretreatment and post-treatment values of root
dentin were calculated as a percentage. Comparisons between experi-
mental groups were performed by using one-way analysis of variance,
followed by Tukey honestly significant difference test at a = 0.05.
Results
Descriptive statistical results concerning alterations in root canal
dentin after treatment with the experimental solutions are presented in
Table 1. The statistical comparison of pretreatment and post-treatment
microhardness values demonstrated that treatment with EDTA, REDTA,
NaOCl, and Chlor-XTRA significantly decreased the microhardness of
root dentin compared with intact controls (P < .05). The comparison
of the alteration percentages, which were calculated by using the differ-
ences between pretreatment and post-treatment values, revealed signif-
icant differences among the experimental groups (P < .001). The
pair-wise comparison (Table 1) indicated that REDTA treatment signif-
icantly decreased the microhardness of root dentin when compared
with CHX, CHX-Plus, NaOCl, and Chlor-XTRA (P < .05). EDTA treatment
also significantly decreased the microhardness value of the root dentin
surface when compared with CHX and CHX-Plus (P < .05). However,
the addition of a surfactant agent to EDTA, NaOCl, or CHX failed to signif-
icantly alter microhardness when compared with the use of those irri-
gants alone (P > .05).
Discussion
The degree of mineral content and the amount of hydroxyapatite in
the intertubular substance are important factors in the intrinsic hard-
ness profile of a dentin structure. Previous studies reported a positive
correlation between hardness and mineral content in teeth. Conse-
quently, the measurement of microhardness can provide indirect evi-
JOE Volume 40, Number 6, June 2014
Basic ResearchTechnology
dence of mineral loss or gain in dental hard tissues (1820). In this
study, the effect of root canal irrigants on root dentin microhardness
in the presence and absence of surface modifiers was evaluated by
using a Vickers hardness tester. The results demonstrated that the
addition of surface modifiers to irrigants did not significantly alter the
hardness of root canal dentin when compared with the irrigants
alone. Surface modifiers are reported to bind to membranes in a
detergent-like manner, causing membrane disruption and solubiliza-
tion and lowering the surface tension of a liquid (27). Surface tension
is the force between molecules that generates a tendency for the surface
area of a liquid to decrease. When this intramolecular attraction is de-
stroyed, the surface tension decreases. This effect can be achieved by
using heat or a surfactant (57). Surfactants bind primarily to the
Ca++ and Mg++ ions in water, which decreases detergency and foam
(10, 27). In the present study, cetrimide was added to an EDTA
solution (REDTA) as a surfactant. Cetrimide is both a disinfecting
agent and a cationic surfactant, which forms micelles in aqueous
solutions. The addition of cetrimide reduces the surface tension of
the irrigant (7, 12, 28), improves antibacterial effectiveness (13, 14),
facilitates penetration of the irrigant to the dentin surface (6), and in-
creases the wettability of the dentin surface (28). In addition, cetrimide
may alter the structure of hydroxyapatite nanorods in a concentration-
dependent manner (29). It was previously reported that a 0.5% cetri-
mide solution decreased dentin microhardness to a similar extent as 5%
EDTA, potentially altering the physical properties of dentin (26). In
contrast, Zehnder et al (12) demonstrated that the use of chelators
with wetting agents did not increase calcium removal from root canals.
This study demonstrated that EDTA and REDTA displayed similar poten-
tial for reducing dentin microhardness. Chelating agents cause dentin
softening by removing calcified components of dentin; hence, a reduc-
tion in microhardness is expected as shown by the literature (4, 20, 23).
The measured dentin hardness value may vary on the basis of the
location of the measurement. The microhardness value decreases if the
tested indentations are in close vicinity to the pulp. This finding may be
due to the increased number of widely opened dentinal tubules near the
pulp, which provides less resistance for the testing indenter (24, 30). In
this study, the dentin microhardness of the samples was measured at a
depth of 300 mm from the pulp-dentin interface at the mid-root level,
and the obtained microhardness measurements were consistent with
the literature (21).
The results of this study demonstrated that irrigation with 6% NaOCl
and Chlor-XTRA for 5 minutes significantly decreased dentin microhard-
ness. Treatment with different concentrations of NaOCl for 15 minutes
and treatment with 6% NaOCl for 10 minutes have been previously
demonstrated to decrease root dentin microhardness (21, 22, 24).
However, it has also been reported that even a 5-minute treatment with
a 2.5% NaOCl solution containing EDTA caused an excessive decrease
in microhardness (23). NaOCl is known to dissolve both collagen com-
ponents of dentin and magnesium and phosphate ions, while increasing
the amount of dentinal carbonate (23, 31). In addition, it was reported
that NaOCl treatment significantly altered the Ca/P ratio of the root dentin
surface (32). Because the degree of dentin mineralization may affect the
hardness profile of the dentin structure (18), changes in mineral content
after NaOCl treatment could be responsible for changes in dentin micro-
hardness. According to the manufacturer, Chlor-XTRA contains an un-
known wetting agent, proprietary surface modifiers, and alkylating
agents. In recent studies, Chlor-XTRA showed antibacterial effectiveness
similar to 6% NaOCl treatment (13, 14, 33). The results obtained in the
current study also indicated similar microhardness alterations for both
Chlor-XTRA and NaOCl treatment.
In this study, treatment with CHX and CHX-Plus for 5 minutes did not
affect the microhardness of root canal dentin. This finding is consistent
Effect of EDTA, NaOCl, and Chlorhexidine Gluconate on Dentin Microhardness 877
Basic ResearchTechnology
TABLE 1. Mean Vickers Microhardness Values of Root Dentin Specimens with Respect to the Type of Treatment
Treatment Mean Standard deviation Minimum Maximum Median Percentage decrease
EDTA
Control* a, b 66.01 5.51 59.39 72.75 65.94 14.45
Post-treatment* 56.76 8.05 44.91 67.32 57.39
REDTA
Control* c, d, 59.76 3.28 56.4 64.68 58.8 15.5
Post-treatment* e, f 50.44 4.23 42.6 55.2 51.6
NaOCl
Control* c 68.47 1.96 66.64 72.04 66.73 6.07
Post-treatment* 64.3 1.66 62.63 67.32 62.96
Chlor-XTRA
Control* d 58.71 3.71 53.38 64.85 58.31 5.16
Post-treatment* 56.66 4.27 49.14 61.75 55.87
CHX
Control a, e 65.09 3.9 60.92 72.24 63.6 3.11
Post-treatment 62.86 1.57 61.19 65.41 62.42
CHX-Plus
Control b, f 60.26 1.91 57.48 62.88 60.34 0.42
Post-treatment 60.04 4.80 52.41 65.41 61.17
For each pair of values with asterisks, pretreatment and post-treatment Vickers microhardness numbers R measurements are significantly different at P = .05 (paired t test). Subgroups connected with brackets
represent the % change between pretreatment and post-treatment readings, with same letters showing significant differences among all pair-wise comparisons (Tukey honestly significant difference test, P = .05).

with the literature (21). It was previously reported that CHX is not capable
of dissolving necrotic tissues or removing the smear layer. The remaining
smear layer may act as a barrier, which allows for only minimal changes
in microhardness by limiting irrigant contact with dentin (34). In
contrast, it was reported that CHX treatment at concentrations of 0.2%
and 2% for 15 minutes decreased the Ca and P levels and the microhard-
ness of root dentin (24, 35). It was suggested that this change in
microhardness might be dependent on the application time of CHX.
The softening effect of chemical irrigants on dentinal walls could
be of clinical benefit because it permits rapid preparation and facilitates
the negotiation of root canals with small dimensions (24). However,
these alterations may affect the adhesion profile of sealers to treated
dentin surfaces. Further studies are needed to clarify the effects of sur-
factant addition to irrigation solutions on the surface properties and ad-
hesive characteristics of dentin.
The results of this study demonstrated that EDTA and NaOCl signif-
icantly decreased the microhardness of the root dentin surface in the pres-
ence and absence of surfactant. In addition, EDTA in the presence and
absence of surfactant caused a larger reduction of microhardness than
NaOCl and CHX. However, the addition of surfactants to the irrigation so-
lutions did not significantly alter their effect on root dentin microhardness.
Acknowledgments
The authors deny any conflicts of interest related to this study.
References
1. West JD, Roane JB. Cleaning and shaping the root canal system. In: Cohen S,
Burns RC, eds. Pathways of the Pulp, 7th ed. St Louis: CV Mosby; 1998:20357.
2. Jeansonne MJ, White RR. A comparison of 2.0% of chlorhexidine gluconate and
5.25% sodium hypochloride as antimicrobial endodontic irrigants. J Endod
1994;20:2768.
3. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root
canal irrigation regimens. J Endod 1987;13:14757.
4. Hulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment:
mode of action and indications for their use. Int Endod J 2003;36:81030.
5. Cameron JA. The effect of a fluorocarbon surfactant on the surface tension of the
endodontic irrigant, sodium hypochlorite: a preliminary report. Aust Dent J
1986;31:3648.
6. Abou-Rass M, Patonai FJ Jr. The effects of decreasing surface tension on the flow of irri-
gating solutions in narrow root canals. Oral Surg Oral Med Oral Pathol 1982;53:5246.
7. Ylmaz Z, Aktemur S, Buzoglu HD, Gumusderelioglu M. The effect of temperature
and pH variations on the surface tension of EDTA solutions. J Endod 2011;37:
8257.
8. Palazzi F, Morra M, Mohammadi Z, et al. Comparison of the surface tension of
5.25% sodium hypochlorite solution with three new sodium hypochlorite-based
endodontic irrigants. Int Endod J 2012;45:12935.
9. Schreier S, Malheiros SV, de Paula E. Surface active drugs: self association and inter-
action with membranes and surfactantsphysicochemical and biological aspects.
Biochim Biophys Acta 2000;1508:21034.
10. Holmberg K, Jonsson B, Kronberg B, Lindman B. Surfactants and Polymers in
Aqueous Solution, 2nd ed. West Sussex, England: John Wiley & Sons; 2002:
166.
11. Scelza MF, Antoniazzi JH, Scelza P. Efficacy of final irrigation: a scanning electron
microscopic evaluation. J Endod 2000;26:3558.
12. Zehnder M, Schicht O, Sener B, Schmidlin P. Reducing surface tension in endodon-
tic chelator solutions has no effect on their ability to remove calcium from instru-
mented root canals. J Endod 2005;31:5902.
13. Rossi-Fedele G, Prichard JW, Steier L, de Figueiredo JA. The effect of surface tension
reduction on the clinical performance of sodium hypochlorite in endodontics. Int
Endod J 2013;46:4928.
14. Ferrer-Luque CM, Conde-Ortiz A, Arias-Moliz MT, et al. Residual activity of chelating
agents and their combinations with cetrimide on root canals infected with Entero-
coccus faecalis. J Endod 2012;38:8268.
15. Wang Z, Shen Y, Ma J, Haapasalo M. The effect of detergents on the antibacterial
activity of disinfecting solutions in dentin. J Endod 2012;38:94853.
16. Shen Y, Qian W, Chung C, et al. Evaluation of the effect of two chlorhexidine prep-
arations on biofilm bacteria in vitro: a three-dimensional quantitative analysis.
J Endod 2009;35:9815.
17. Rotstein I, Dankner E, Goldman A, et al. Histochemical analysis of dental hard tis-
sues following bleaching. J Endod 1996;22:235.
18. Arends J, ten Bosch JJ. Demineralization and remineralization evaluation tech-
niques. J Dent Res 1992;71:9248.
19. Panighi M, GSell C. Influence of calcium concentration on the dentine wettability by
an adhesive. J Biomed Mater Res 1992;26:10819.
20. Cruz-Filho AM, Sousa-Neto MD, Savioli RN, et al. Effect of chelating solu-
tions on the microhardness of root canal lumen dentin. J Endod 2011;
37:35862.
21. Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solu-
tion on the microhardness and the roughness of root canal dentin. J Endod
2004;30:7925.
22. Slutzky-Goldberg I, Maree M, Liberman R, Heling I. Effect of sodium hypochloride
on dentin microhardness. J Endod 2004;30:8802.
23. Sayin TC, Serper A, Cehreli ZC, Otlu HG. The effect of EDTA, EGTA, EDTAC and
tetracycline-HCl with and without subsequent NaOCl treatment on the microhard-
ness of root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2007;104:41824.
24. Oliveira LD, Carvalho CA, Nunes W, et al. Effects of clorhexidine and sodium hypo-
chlorite on the microhardness of root canal dentin. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2007;104:e1258.

878 Aslantas et al. JOE Volume 40, Number 6, June 2014


25. De-Deus G, Paciornic S, Mauricio MH. Evaluation of the effect of EDTA, EDTAC and
citric acid on the microhardness of root dentine. Int Endod J 2006;39:4017.
26. Akcay I, Sen BH. The effect of surfactant addition to EDTA on microhardness of root
dentin. J Endod 2012;38:7047.
27. Huibers PDT, Lobanov VS, Katritzky AR, et al. Prediction of critical micelle concen-
tration using a quantitative structure property relationship approach. J Colloid
Interface Sci 1997;187:11320.
28. Yilmaz Z, Basbag B, Buzoglu HD, Gumusderelioglu M. Effect of low-surface-tension
EDTA solutions on the wettability of root canal dentin. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 2011;111:10914.
29. Tianyuan M, Zhigou X, Libing L. Effect of reaction systems and surfactants additives
on the morphology evaluation of hydroxyapatite nanorods obtained via a hydrother-
mal route. Appl Surf Sci 2011;257:43848.
Basic ResearchTechnology
30. Pashley D, Okabe A, Parham P. The relationship between dentine microhardness
and tubule density. Endod Dental Traumatol 1985;1:1769.
31. Tsuda H, Ruben J, Arends J. Raman spectra of human dentin mineral. Eur J Oral Sci
1996;104:12331.
32. Dogan H, Qalt S. Effects of chelating agents and sodium hypochlorite on mineral
content of root dentin. J Endod 2001;27:57880.
33. Williamson AE, Cardon JW, Drake DR. Antimicrobial susceptibility of monocul-
ture biofilms of a clinical isolate of Enterococcus faecalis. J Endod 2009;35:
957.
34. Naenni N, Thoma K, Zehnder M. Soft tissue dissolution capacity of currently used
and potential endodontic irrigants. J Endod 2004;30:7857.
35. Ari H, Erdemir A. Effects of endodontic irrigation solutions on mineral content of
root canal dentin using ICP-AES technique. J Endod 2005;31:1879.

ERRATUM
In the article, Revascularization Outcomes: A Prospective Analysis of 16 Consecutive Cases by Bill Kahler, Sonali Mistry, Alex Moule, Andrew K.
Ringsmuth, Peter Case, Andrew Thomson, and Trevor Holcombe (J Endod 2014;40[3]:33338) the authors inadvertently referenced the wrong
article in the following sentence:
It has been suggested, without supporting evidence, that avulsion is a contraindication for regenerative treatment.
The correct reference for this sentence is number 3 in their reference list:
3. Garcia-Godoy F, Murray PE. Recommendations for using regenerative endodontic procedures in permanent immature traumatized teeth.
Dent Traumatol 2012;28:3341.
The authors incorrectly cited number 17 in their reference list:
17. Wigler R, Kaufman AY, Steinbock N, et al. Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root
development. J Endod 2013;39:31926.
The authors regret this error.

JOE Volume 40, Number 6, June 2014 Effect of EDTA, NaOCl, and Chlorhexidine Gluconate on Dentin Microhardness 879