JOURNAL OF ENDODONTICS

Copyright 2004 by The American Association of Endodontists

Printed in U.S.A.
VOL. 30, NO. 11, NOVEMBER 2004

Evaluation of the Effect of Endodontic Irrigation

Solutions on the Microhardness and the Roughness
of Root Canal Dentin
Hale Ari, DDS, PhD, Ali Erdemir, DDS, PhD, and Sema Belli, DDS, PhD

irrigating systems to flush loosened materials away, and chemicals

to dissolve contents from inaccessible regions. Irrigation is presently the best method for the removal of tissue remnants and dentin
debris during instrumentation. The simple act of irrigation flushes
away loose, necrotic, contaminated materials before they are inadvertently pushed deeper into the canal and apical tissues. Irrigation solutions also provide gross debridement, lubrication, and
destruction of microbes and dissolution of tissues. NaOCl, H2O2,
combination of NaOCl and H2O2 have been widely used as an
irrigant in endodontic therapy for many years, especially because
of their substantive antimicrobial properties (1). Also EDTA solutions have been used to assist in removing the smear layer (2, 3).
During irrigation, radicular and coronal dentin and the enamel
are exposed to solutions deposited in the pulp chamber. This may
cause alterations on dentin and enamel surface and effect their
interaction with materials used for obturation and coronal restoration, as well as inhibit resistance to bacterial ingress and permitting
coronal leakage (4).
It was important to test the effect of the irrigation solutions on
all dentin tissues, because contact may occur during irrigation
procedures. The effect of some medicals, such as EDTA, NaOCl,
fluorides, trichloracetic acid, and bleaching agents, on dentin hardness were previously evaluated (4 9); however, the effect of 0.2%
chlorhexidine gluconate on microhardness of root canal dentin has
not been evaluated. Therefore, this study was designed to evaluate
the effect of 0.2% chlorhexidine gluconate on the hardness and
roughness of root canal dentin compared with widely used irrigation solutions.

The purpose of this study was to evaluate the effect

of 0.2% chlorhexidine gluconate on the microhardness and roughness of root canal dentin compared
with widely used irrigation solutions. Ninety, mandibular, anterior teeth extracted for periodontal reasons
were used. The crowns of the teeth were removed at
the CEJ. The roots were separated longitudinally into
two segments, embedded in acrylic resin, and polished. A total of 180 specimens were divided into 6
groups of 30 teeth at random according to the irrigation solution used: group 1: 5.25% NaOCl for 15
min; group 2: 2.5% NaOCl for 15 min; group 3: 3%
H2O2 for 15 min; group 4: 17% EDTA for 15 min; group
5: 0.2% chlorhexidine gluconate for 15 min; and
group 6: distilled water (control). Each group was
then divided into 2 subgroups of 15 specimens:
groups 1a, 2a, 3a, 4a, 5a, and 6a were submitted to
Vickers microhardness indentation tests; groups 1b,
2b, 3b, 4b, 5b, and 6b were used for determination of
the roughness of root dentin. The data were recorded as Vickers numbers and Ra, m for roughness test. The results were analyzed statistically by
using one-way ANOVA and Tukey tests. The results
indicated that all the irrigation solutions except chlorhexidine significantly decreased microhardness of
root canal dentin (p < 0.05); 3% H2O2 and 0.2% chlorhexidine gluconate had no effect on roughness of
the root canal dentin (p > 0.05). Although there are
many other factors for irrigation solution preference,
according to the results of this study, 0.2% chlorhexidine gluconate seems to be an appropriate endodontic irrigation solutions because of its harmless effect
on the microhardness and roughness of root canal
dentin.

MATERIALS AND METHODS

Ninety, human, mandibular, anterior teeth extracted for periodontal reasons were used. The teeth were immediately stored at
37C in buffered saline and examined under a dissecting microscope to eliminate teeth with cracks or hypoplastic defects. The
crowns were removed at the CEJ using a high-speed bur under
water-cooling.
The roots were separated longitudinally into two segments using
a diamond saw under water cooling, horizontally embedded in
polymerizing acrylic resin exposing the root-dentin surfaces, and
ground polishing with a water-cooled carborundum paper. A total
of 180 specimens were divided into 6 groups at random according

In root canal treatment, cleaning is the removal all contents of the

root canal system before and during shaping. Successful cleaning
entails the use of instruments to physically remove substances,
792

Vol. 30, No. 11, November 2004

Effect of Endodontic Irrigation Solutions on Root Canal Dentin

TABLE 1. The means and standard deviations of the root

dentin microhardness values for endodontic irrigation solution
treatment groups and control group
Endodontic Irrigation Solution

Vickers Microhardness
Values (Mean SD)

Group 1, 5.25% NaOCl

Group 2, 2.5% NaOCl
Group 3, 3% H2O2
Group 4, 17% EDTA
Group 5, 0.2% chlorhexidine
gluconate
Group 6, distilled water (control)

15
15
15
15
15

51.74 6.03b
50.86 5.08b
53.57 5.52b
53.66 3.87b
61.58 4.18a

15

61.86 11.70a

793

TABLE 2. The means and standard deviations of the root

dentin roughness values for endodontic irrigation solution
treatment groups and control group
Endodontic Irrigation Solution

Roughness Values
(Mean SD)

Group 1, 5.25% NaOCl

Group 2, 2.5% NaOCl
Group 3, 3% H2O2
Group 4, 17% EDTA
Group 5, 0.2% chlorhexidine
gluconate
Group 6, distilled water (control)

15
15
15
15
15

0.25 0.11b
0.26 0.19b
0.15 0.04ab
0.27 0.14b
0.14 0.05ab

15

0.11 0.10a

Different letters indicate significant differences (p 0.05). All the irrigation solutions
except chlorhexidine decreased microhardness of root canal dentin significantly (p 0.05).

Different letters indicate significant differences (p 0.05). 3% H2O2 and 0.2% chlorhexidine gluconate have not effect on roughness of root canal dentin (p 0.05).

to the irrigation solution to be used. Exposed dentin surfaces of the

specimens were treated with irrigation solutions in glass plate at
37C as follows:

The mean and standard deviation of the root-dentin roughness values of the groups and control group are listed in Table
2. 3% H2O2 and 0.2% chlorhexidine gluconate had no effect on
roughness of root canal dentin (p 0.05). Treatment with 2.5%
to 5.25% NaOCl and 17% EDTA was significantly increased
root-dentin roughness when compared with the control group (p
0.05).

Group
Group
Group
Group
Group
Group

1:
2:
3:
4:
5:
6:

5
5
5
5
5
5

ml,
ml,
ml,
ml,
ml,
ml,

5.25% NaOCl for 15 min

2.5% NaOCl for 15 min
3% H2O2 for 15 min
17% EDTA for 15 min
0.2% chlorhexidine gluconate for 15 min
distilled water (control)

At the end of active treatment period (15 min), the samples

were rinsed with distilled water and dried. Midroot region was
used during the measurements. Every group was then divided
into 2 subgroups of 15 specimens each. Groups 1a, 2a, 3a, 4a,
5a, and 6a were used to determinate the surface hardness of the
root dentin with a Vickers Hardness Tester (Matsuzawa
MHT2, High Quality Microhardness Tester, Matsuzawa SEIKI
Co; Ltd, Tokyo, Japan). Indentations were made with a Vickers
diamond indenter a minimum of three widely similarly positioned locations. The indentations were made on the top surface
of each specimen using 300 g and a dwell time of 20 s. The three
values were averaged to produce one hardness value for each
specimen. These measurements were converted into Vickers
numbers.
Groups 1b, 2b, 3b, 4b, 5b, and 6b were used for determination
of the roughness (Ra, m) of root dentin with a computerized
roughness tester (Mitutoyo Surftest Analyser, Matsuzawa SEIKI
Co; Ltd, Tokyo, Japan). Three tracings at different locations on
each of all specimens were made. The mean and standard deviation
of Ra were determined. Ra parameter describes the overall roughness of a surface and can be defined as the arithmetical average
value of all absolute distances of the roughness profile from the
centerline within the measuring length.
The data were analyzed statistically using one-way analysis of
variance (ANOVA), and the comparison of means was conducted
using Tukey multiple comparison test. The testing was performed
at the 95% level of confidence (p 0.05).
RESULTS
The means and standard deviations of the root-dentin microhardness values for the irrigation solution treatment groups and
control group are listed in Table 1. All the irrigation solutions
except chlorhexidine decreased microhardness of root canal dentin
significantly (p 0.05); 0.2% chlorhexidine gluconate did not
effect microhardness of root canal dentin (p 0.05).

DISCUSSION
In this study, endodontic irrigation solutions were applied on
root canal dentin surface for 15 min, and the surface microhardness
and roughness tests were used to determine changes on dentin
surface. Although 3% H2O2 and 0.2% chlorhexidine gluconate had
no effect on surface roughness of root canal dentin, a significant
increase on surface roughness was found in 2.5% to 5.25% NaOCl
and 17% EDTA-treated groups (p 0.05). All the irrigation
solutions except chlorhexidine were decreased microhardness of
root canal dentin significantly. This results show that all the irrigation solutions except chlorhexidine may have an effect on the
components of dentin structure.
In previous studies, Knoop indenter microhardness test (10 12)
and Vickers indenter method (7, 13, 14) were used to measure the
hardness of dentin. Microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues
(15). Previous investigations have shown the suitability and practicality of Vickers microhardness test for evaluating surface
changes of dental hard tissues treated with chemical agents (7, 13,
14, 16). Although Knoop hardness test was used for evaluating
surface changes of dental hard tissues in some other studies (10
12), Vickers microhardness test was preferred in this study because
of the methods suitability.
Pashley et al. (10) reported an inverse correlation between
dentin microhardness and tubular density. The degree of mineralization and amount of hydroxyapatite in the intertubular substance
are considerable factors in determining the intrinsic hardness profile of dentin structure (17). In a previous study, 5% NaOCl
treatment significantly reduced bond strength to adhesive resin
(18). Grigoratos et al. (19) and Sim et al. (20) found that NaOCl
reduced the modulus of elasticity and flexural strength of dentin.
Slutzky-Goldberg et al. (9) demonstrated that instrumentation and
irrigation with NaOCl changes the biomechanical properties of
dentin. Results of White et al. (21) indicated that root dentin was
weakened after 5 weeks of exposure to calcium hydroxide, mineral
trioxide aggregate, or sodium hypochlorite. ODriscool et al. (22)
observed that the weight loss of dentin after immersion in 0.5% and

794

Ari et al.

5% NaOCl was greater in the higher concentration. In this study,

both 2.5% and 5.25% NaOCl irrigation solutions effected hardness
and the roughness of root canal dentin.
Pecora et al. (23), Chng et al. (24), and Lewinstein et al. (7)
reported that high concentration of hydrogen peroxide caused the
great decrease in dentin microhardness. The significant alteration
in dentin hardness after irrigation treatment indicates direct effects
of these chemical solutions on the components of dentin structure
(25, 26). Hydrogen peroxide affects the inorganic parts of dentin
through acidic demineralization and attacks the organic-rich intertubular dentin by collagen denaturation action (7). Organic-dissolving properties of sodium hypochlorite on the collagen component of dentin (25, 26) explains how the alternated irrigation with
these two solutions effect the hardness of dentin. Although 3%
H2O2 had no effect on surface roughness of root canal dentin, a
significant decrease on surface microhardness was shown in this
study. This result probably may be related to the materials low
concentration.
Chelating agents have been suggested to improve chemomechanical debridement during root canal treatment by removing the
smear layer from the root canal as well as demineralizing and
softening. The most commonly used chelating agents are based on
different concentrations of EDTA (2, 3). Cruz-Filho et al. (27)
showed that the chelating solutions significantly reduced dentin
microhardness. Similar to our study, Saleh and Ettman (4) demonstrated that irrigation with H2O2/NaOCl or EDTA decreased the
microhardness value of root dentin. Although Knoop hardness test
was used for evaluating surface changes of dental hard tissues at
their study, the results of the both studies are similar.
In this study, chlorhexidine did not effect microhardness of root
canal dentin (p 0.05). In a previous study (28), the teeth treated
with chlorhexidine solution showed the highest bond strength
values compared with other irrigants. Oncag et al. (29) found that
Cetrexidin (0.2% chlorhexidine gluconate plus 0.2% cetrimide)
and 2% chlorhexidine gluconate were more effective, had more
residual antibacterial effect, and lower toxicity than 5.25% NaOCl
solution. Vianna et al. (30) reported that the timing required for
1.0% and 2.0% chlorhexidine gluconate to eliminate all microorganisms was the same required for 5.25% NaOCl. Chlorhexidine
was significantly more effective against E. faecalis than Ca(OH)2,
and there were no significant differences between the modes of
medication or concentrations of chlorhexidine (31). In another in
vitro microbiological study, White et al. (32) and Leonardo et al.
(33) demonstrated that chlorhexidine absorbs by dentin and releases from dentin as long as 48 to 72 h after instrumentation.
Chlorhexidine absorption by dentin might have produced a positive effect. This positive effect can be probably an explanation for
this result.
Although the relative softening effect exerted by chemical irrigant on the dentinal walls could be of clinical benefit, because it
permits rapid preparation and facilitates negotiation of small tight
root canals, these alterations affect the adhesion and sealing ability
of sealers to the treated dentin surfaces (4). Perfect obturation of
root canal system is required for a successful root canal treatment
to inhibit resistance to bacterial ingress and prevent leakage. A
harmless irrigation solution seems to be more appropriate to provide a perfect obturation. According to the results of this study,
0.2% chlorhexidine gluconate seems to be an appropriate irrigation
solution because of its harmless effect on the microhardness and
roughness of root canal dentin.

Journal of Endodontics
Dr. Ari is assistant professor, Dr. ErdemIr is research assistant, and Dr. BellI
is associate professor and chair, Selcuk University Faculty of Dentistry, Department of Endodontics, Konya, Turkey.
Address requests for reprints to Dr. Hale Ari, Selcuk University Faculty of
Dentistry, Department of Endodontics, 42079-Konya, Turkey. E-mail:
Hale29tr@yahoo.com.

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. Cury JA, Bragotto C, Valdrighi L. The demineralizing efficiency of EDTA
solutions on dentin. I. Influence of pH, Oral Surg Oral Med Oral Pathol
1981;52:446 8.
3. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:14757.
4. Saleh AA, Ettman WM. Effect of endodontic irrigation solutions on
microhardness of root canals dentine. J Dent 1999;27:43 6.
5. Seaman F, Shannon IL. Fluoride treatment and microhardness of dentin. J Prosthet Dent 1979;41:528 30.
6. Lewinstein I, Rotstein I. Effect of trichloracetic acid on the microhardness and surface morphology of human dentin and enamel. Endod Dent
Traumatol 1992;8:16 20.
7. Lewinstein I, Hirschfeld Z, Stabholz, Rotstein I. Effect of hydrogen
peroxide and sodium perborate on the microhardness of human enamel and
dentin. J Endod 1994;20:613.
8. Dogan H, alt S. Effect of chelating agents and sodium hypochlorite on
mineral content of root dentin. J Endod 2001;27:578 80.
9. Slutzky-Goldberg I, Liberman R, Heling I. The effect of instrumentation
with two different file types, each with 2.5% NaOCl irrigation on the microhardness of root dentin. J Endod 2002;28:3112.
10. Pashley D, Okabe A, Parham P. The relationship between dentin
microhardness and tubule density. Endod Dent Traumatol 1985;1:176 9.
11. Meredith N, Sherriff M, Setchell DJ, Swanson SA. Measurement of the
microhardness and Youngs modulus of human enamel and dentine using an
indentation technique. Arch Oral Biol 1996;41:539 45.
12. Hosoya Y, Marshall SJ, Watanabe LG, Marshall GW. Microhardness of
carious deciduous dentin. Oper Dent 2000;25:819.
13. Tulga F, zok R, Grbz A, zkan P. Effect of different types of vital
bleaching agents on microhardness of human enamel. Balkan J Stomatol
2000;4:164 6.
14. Kuramoto Junior M, Matson E, Turbino ML, Marques RA. Microhardness of Nd:YAG laser irradiated enamel surfaces. Braz Dent J 2001;12:313.
15. Arend J, ten Bosch JJ. Demineralization and remineralization evaluation techniques. J Dent Res 1992;71:924 8.
16. Lewinstein I, Grajower R. Root dentin hardness of endodontically
treated teeth. J Endod 1981;7:4212.
17. Panighi M, GSell C. Influence of calcium concentration on the dentin
wettability by an adhesive. J Biomed Mater Res 1992;26:10819.
18. Ar H, Yasar E, Belli S. Effects of NaOCl on bond strengths of resin
cements to root canal dentin. J Endod 2003;29:248 51.
19. Grigoratos D, Knowles JC, Ng YL, Gulabivala K. Effect of exposing
dentine to sodium hypochlorite and calcium hydroxide on its flexural strength
and elastic modulus. Int Endod J 2001;34:1139.
20. Sim TPC, Knowles JC, Ng YL, Shelton J, Gulabivala K. Effect of
sodium hypochlorite on mechanical properties of dentine and tooth surface
strain. Int Endod J 2001;34:120 32.
21. White JD, Lacefield WR, Chavers LS, Eleazer PD. The effect of three
commonly used endodontic materials on the strength and hardness of root
dentin. J Endod 2002;28:828 30.
22. ODriscool C, Dowker S, Anderson P, Wilson R, Gulabivala K. Effect of
sodium hypochlorite depletion of dentin [Abstract]. Int Endod J 2000;33:143.
23. Pecora JD, Cruzfilho AM, Sousaneto MD, Silva RG. In vitro action of
various bleaching agents on the microhardness of human dentin. Braz Dent J
1994;5:129 34.
24. Chng HK, Palamara JE, Messer HH. Effect of hydrogen peroxide and
sodium perborate on biomechanical properties of human dentin. J Endod
2002;28:627.
25. Cohen S, Stewart GG, Laster LL. The effects of acids, alkalies, and
chelating agents on dentine permeability. Oral Surg Oral Med Oral Pathol
Endod 1970;29:631 4.
26. Gordon TM, Damato D, Christner. Solvent effect of various dilutions of
sodium hypochlorite on vital and necrotic tissue. J Endod 1981;7:466 9.
27. Cruz-Filho AM, Sousa-Neto MD, Saquy PC, Pecora JD. Evaluation
of the effect of EDTAC, CDTA, and EGTA on radicular dentin microhardness. J Endod 2001;27:183 4.
28. Erdemir A, Ar H, Gngnes H, Belli S. Effect of medicaments for root
canal treatment on bonding to root canal dentin. J Endod 2004;30:113 6.
29. Oncag O, Hosgor M, Hilmioglu S, Zekioglu O, Eronat C, Burhanoglu D.

Vol. 30, No. 11, November 2004

Effect of Endodontic Irrigation Solutions on Root Canal Dentin

Comparison of antibacterial and toxic effects of various root canal irrigants. Int
Endod J 2003;36:42332.
30. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho
FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium
hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79
84.
31. Basrani B, Tjaderhane L, Santos JM, et al. Efficacy of chlorhexidine
and calcium hydroxide containing medicaments against Enterococcus

795

faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;
96:618 24.
32. White RR, Hays GL, Janer LR. Residual antimicrobial activity associated with a chlorhexidine endodontic irrigant used with sodium hypochlorite.
J Endod 1997;23:229 31.
33. Leonardo MR, Tanomaru Filho M, Silva LAB, Nelson Filho P, Bonifacio
KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root
canal irrigating solution. J Endod 1999;25:16771.