Basic Research—Technology
Effects of Irrigation Solutions on Dentin Wettability and
Roughness
Xiaoli Hu, DDS, MS,* Junqi Ling, DDS, MS, PhD,* and Yan Gao, DDS, MS†
Abstract
Introduction: Irrigation solutions are used to disinfect
the root canal, flush out debris, and dissolve pulp tissue.
It is hypothesized that irrigation solutions might influ-
ence the physicochemical properties of human root
canal dentin including the wettability and roughness.
Methods: The roots of 20 human permanent anterior
teeth were sectioned longitudinally into dentin slices
and randomly divided into 4 groups with different treat-
ments as follows: (1) 17% ethylenediaminetetraacetic
acid (EDTA) for 10 minutes, (2) 5.25% NaOCl for
10 minutes, (3) 3% H2O2 for 10 minutes, and (4) distilled
water as control. Wettability was evaluated by contact
angle measurement. Atomic force microscopy was
used to measure the surface roughness (Ra). Results:
Water contact angles significantly decreased from
54.35 degrees (control) to 22.04 and 46.35 degrees on
the dentin treated by 5.25% NaOCl and 3% H2O2,
respectively, with the wettability increasing accordingly
(both P < .05). The roughness of dentin surfaces (Ra)
was increased from 85.39 (control) to 121.90 and
140.49 nm by 5.25% NaOCl and 17% EDTA, respec-
tively (both P < .05). Conclusions: These findings
indicated that all the irrigation solutions could alter
the physicochemical characteristics of dentin surfaces
and might influence bacterial adhesion. (J Endod
2010;36:1064–1067)
Key Words
Atomic force microscopy, irrigation, roughness, wetta-
bility
From the *Department of Operative Dentistry and
Endodontics and †Department of VIP, Guanghua College of
Stomatology, Sun Yat-sen University, Guangzhou, China.
Address requests for reprints to Junqi Ling, PhD, Professor,
Department of Operative Dentistry and Endodontics, Guanghua
College of Stomatology, Sun Yat-sen University, Guangzhou,
China. E-mail address: lingjq@mail.sysu.edu.cn.
0099-2399/$0 - see front matter
Copyright ª 2010 American Association of Endodontists.
doi:10.1016/j.joen.2010.03.007
1064 Hu et al.
T he success of root canal therapy depends on the quality of several factors, including
the instrumentation, irrigation, disinfection, and 3-dimensional obturation of the
root canal (1). Mechanical and chemical effects of the irrigant solutions are crucial
during irrigation. Mechanical effects are generated by the flow and backflow of
irrigant solutions in the root canal (2). The irrigant solutions with antimicrobial
effects demonstrate superior effectiveness in bacteria elimination (3). Sodium
hypochlorite (NaOCl), hydrogen peroxide (H2O2), and combination of NaOCl and
H2O2 have been widely used in endodontic therapy for many years because of their
antimicrobial properties (4). Ethylenediaminetetraacetic acid (EDTA) solutions have
been used to assist in removing the smear layer (5, 6). NaOCl dissolves proteins and
removes other organic components from biologic materials. In recent studies,
researchers found that collagen exposed on the dentin surface can be quickly
removed by NaOCl in a deproteination process (7).
Irrigation with H2O2, NaOCl, or EDTA significantly decreased the microhardness of
root canal dentin (8, 9). NaOCl and EDTA also increased the roughness of dentin (9).
Irrigation solutions might also change other surface properties of radicular and coronal
dentin such as wettability, which might influence the adhesion of bacteria (10) and the
interaction between the dentin and restorative materials (11). Because wettability of the
surface is a crucial factor for adhesion, it is of interest to examine the effect of irrigation
on dentin surface wettability. Wettability is strongly dependent on chemical composi-
tion, roughness, and hydration state and could be influenced by tubule density (12).
Currently, there is no report about the effect of irrigation on dentin wettability.
Roughness is an important factor in adhesion and has been related to wettability in
the Wenzel equation. Wenzel equation applies to equilibrium angles on rough surfaces
and is expressed as follows (13): r = cos q1/cos q2, where r is the ratio of the actual to
apparent or projected area, and the contact angles q1 and q2 refer to the roughened
surface and smooth surface contact angles, respectively.
Roughness can be measured by different instruments such as a stylus profilom-
eter, a computerized roughness tester, or an atomic force microscope (9, 14).
Atomic force microscopy (AFM) emerged as a powerful imaging tool in 1986
(15). AFM works by scanning a very tiny tip mounted at the end of a flexible
microcantilever in gentle touch with the sample in a raster fashion. AFM has some
advantages. It can work in air and liquid, requires little or no sample preparation,
and provides high-resolution imaging of 3-dimensional surface topography (16).
Because a sample does not require extensive preparation to be analyzed by AFM,
it can be mapped many times, acting as its own control. Furthermore, because
AFM is software-driven, a high mathematical accuracy of surface characterization
might be obtained, and image properties (such as roughness, peaks, etc) can be
determined quantitatively. Although studies involving AFM have been conducted in
a number of fields of dentistry (17–20), no reports are available describing the
application of this technique to roughness measurement of dentin after irrigation.
Therefore, the 2-fold aim of this study was to (1) evaluate changes in average
roughness (Ra) after irrigation by using AFM and (2) determine dentin wettability by
contact angle measurement.
Materials and Methods
Preparation of Dentin Specimens
Twenty human fully developed permanent anterior teeth were used in this study.
They were recently extracted for periodontal reasons from male and female patients
JOE — Volume 36, Number 6, June 2010

ranging in age from 30–45 years. The teeth were selected on the
basis of dimension, similarity in morphology, and absence of any
crack or carious defects, especially within the root portion. Debris
and soft tissue remnants on the root were removed with a sharp
scalpel. All teeth were stored in phosphate-buffered saline at 4 C
until used.
The tooth crowns were removed at the cementoenamel junction
by using a high-speed bur under water coolant. Each root was
sectioned longitudinally into 2 parallel dentin slices by a Microslice
2 (Metals Research Limited, Cambridge, England). Forty dentin slices
were ground smooth through 400-, 600-, 800-, and 1200-grit polish-
ing papers under distilled water to remove any surface scratches. At
each step, dentin samples were rinsed and ultrasonicated in purified
water.
The 40 dentin samples were randomly divided into 4 groups of
10 samples each and prepared as follows: group 1, treatment with
17% EDTA (Sigma-Aldrich Inc, St Louis, MO) for 10 minutes; group
2, treatment with 5.25% NaOCl (Sigma-Aldrich Inc) for 10 minutes;
group 3, treatment with 3% H2O2 (Sigma-Aldrich Inc) for 10 minutes;
and group 4 (controls), treated with deionized and distilled water for
10 minutes.
Dentin samples were rinsed copiously by the deionized and
distilled water after immersion into the above irrigating solutions.
Contact Angle Measurements
Water contact angles (q) were measured by sessile drop tech-
nique on dentin surfaces by using a VCA Optima Surface Analysis
System (AST Products, Billerica, MA). Two drops (0.0002 mL/
drop) of distilled water were deposited on each dentin surface with
a microsyringe. Images were captured immediately after deposition
within a microvideo system. The acquired images were analyzed by
VCA-2500XE software (AST Products), and contact angles were
provided. The values were averaged to produce 1 water contact angle
for each dentin specimen.
AFM Analysis
A Digital Instruments Nanoscope atomic force microscope
(Veeco, Santa Barbara, CA) was operated in contact mode to image
the dentin surfaces by using pyramidal tips made of Si3N4 (DNP-20;
Veeco). It was controlled with the aid of the included software
V614r1. Three fields of view of 10  10 mm2 for each sample were
obtained, and the roughness (Ra) was measured. The values were
averaged to produce a single Ra value for each dentin specimen.
Statistical Analysis
Water contact angles and roughness values were statistically
analyzed by one-way analysis of variance, followed by a post hoc
Tukey test for pair-wise comparison. Statistical significance was set
at P <.05.
Results
The means and standard deviations of water contact angles of
dentin surfaces for the treatment and control groups are listed in
Table 1. The values for contact angles were significantly reduced after
the dentin samples were treated with 5.25% NaOCl and 3% H2O2.
Hence, wettability was increased (P < .05). Seventeen percent EDTA
did not influence the wettability of root dentin surface after 10 minutes
(P > .05).
The means and standard deviations of roughness values (Ra) of
dentin surfaces for the treatment and control groups are listed in
Table 2. The roughness values were significantly increased by 5.25%
JOE — Volume 36, Number 6, June 2010
Basic Research—Technology
TABLE 1. Means and Standard Deviations of Water Contact Angles of Dentin
Surfaces for Treatment and Control Groups (n = 10/group)
Water contact angle (degrees)
Group (mean ± standard deviation)*
17% EDTA 54.99  4.06a
5.25% NaOCl 22.04  3.07b
3% H2O2 46.35  4.58c
Control 54.35  4.25a
*Different letters in the column indicate a statistically significant difference (P < .05). Same letters in
the column indicate the differences are not significant (P > .05).
NaOCl and 17% EDTA (P < .05). There was no effect on the roughness
of root dentin surface by 3% H2O2 (P > .05). Figure 1 shows the AFM
images for typical dentin surfaces after treatment with 17% EDTA,
5.25% NaOCl, 3% H2O2, and distilled water (control).
Discussion
Wettability is one of the most important physicochemical proper-
ties. Contact angle measurements determine the wettability of the
substratum. Low contact angles are an indication of high surface free
energy, whereas high contact angles indicate low surface free energy
(21). Interfacial surface free energy has been postulated as a driving
force for initial adhesion of microorganisms to solid surface.
To date there is no report about the effect of irrigation solutions on
the wettability of dentin surfaces. Our study indicated that both 3% H2O2
and 5.25% NaOCl significantly increased the wettability of dentin
surfaces. Some groups have found that hydrophilic materials were
more resistant to bacterial adhesion than hydrophobic materials
(22, 23). However, An et al (24) found that hydrophobic UHMWPE
disks attracted much less Staphylococcus epidermidis compared
with the hydrophilic metal surface. Furthermore, there were significant
increases in adhesion force of Enterococcus faecalis to NaOCl- and
EDTA-treated dentin, whereas NaOCl alone did not significantly influ-
ence the adhesion force (25). Hence, adhesion of bacteria is not just
related to surface wettability but might also relate specifically with the
species of bacteria.
It is well-known that H2O2 is a strong oxidant and is also acidic.
Hydrogen peroxide affects the inorganic components of dentin
through acidic demineralization and attacks the organic-rich intertub-
ular dentin by collagen denaturation (26). It is likely that the
increased wettability of the dentin surface is the result of this acidic
property of H2O2. In support of this view, Rosales et al (12) reported
that the values of water contact angles of superficial dentin were
reduced significantly after their specimens were etched by 10% phos-
phoric acid for 15 seconds, thus increasing wettability. Because of the
enlargement of the fluid-filled dentin tubules by the acid-etching
action, the volume of water significantly increased. Consequently,
this highly hydrated surface became more hydrophilic (27). Our
TABLE 2. Means and standard deviations of roughness values of dentin
surfaces for treatment and control groups (n = 10/group)
Roughness values,
Group Ra (nm) (mean ± SD)*
17% EDTA 140.49  28.09b
5.25% NaOCl 121.90  27.63b,c
3% H2O2 99.22  31.11a,c
Control 85.39  26.45a
*Different letters in the column indicate a statistically significant difference (P < .05). Same letters in
the column indicate the differences are not significant (P > .05).
Effect of Irrigation on Dentin Wettability and Roughness 1065
Basic Research—Technology
Figure 1. AFM images of 10  10 mm area of dentin surfaces after treatment with (A) 17% EDTA, (B) 5.25% NaOCl, (C) 3% H2O2, and (D) distilled H2O
(control).
study also found that 5.25% NaOCl increased the wettability of dentin
significantly. Toledano et al (28) found that contact angles on deep
dentin etched by 35% phosphoric acid and then deproteinized by
5% NaOCl were lower than those of etched only dentin. Morhonine
(14.3% EDTA containing a surfactant 0.084% cetrimide) spread
rapidly over the dentin surfaces, resulting in the inability to measure
the contact angle of these surfaces after conditioning with NaOCl solu-
tion (29). NaOCl is frequently used to clean, disinfect, and nonspecif-
ically deproteinize the root canal environment during endodontic
treatment. Recently, NaOCl was used to remove the collagen fibrils
(30). The removal of collagen results in dentin surfaces similar to
those of etched enamel (31, 32). An increase in wettability after
NaOCl treatment can be explained because deproteination leads to
a hydrophilic surface (33).
In our study, NaOCl also increased the roughness of dentin. This
finding that increased roughness resulted in a decrease in contact
angle is in agreement with the Wenzel equation (13). The roughness
of dentin (Ra) after irrigation was evaluated by AFM. Ra values
increased significantly after treatment by 5.25% NaOCl and 17%
EDTA. This increase agrees with the findings of Ari et al (9), who
used a computerized roughness tester (Mitutoyo Surftest Analyser;
Matsuzawa Seiki Co, Ltd, Tokyo, Japan) to measure the surface rough-
ness after treatment with the same irrigation solutions. The chelating
action of EDTA softens root canal wall by the dissolution of its inor-
ganic components. Dentin tubules became patent and the surface
roughness increased as a result of the smear layer removal by EDTA
(1). The organic-dissolving properties of NaOCl on the collagen
components of dentin (34) might explain how this irrigation solution
increased the surface roughness. Rough surfaces could be a clinical
benefit as in the case of micromechanical bonding of the adhesive
materials that need the irregularities on the surface. However, rough
surfaces will promote bacterial adhesion, which might lead to plaque
formation (35).
All the irrigation solutions in this study influenced the physico-
chemical properties of dentin surfaces, such as wettability and rough-
ness. Hence, the effects of the changes of physicochemical properties
on the bacterial adhesion and restorative adhesive procedure should
be further studied. Due consideration should be given to these effects
as the irrigants are used during root canal treatment.
1066 Hu et al.
Acknowledgments
This study was supported by Key Clinical Program of the
Ministry of Health, China.
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