journal of dentistry 37 (2009) 807–812

available at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

The incorporation of chlorhexidine in a two-step self-etching

adhesive preserves dentin bond in vitro

Jianfeng Zhou a, Jianguo Tan a,*, Li Chen a, Deli Li b, Yao Tan a

a
Department of Prosthodontics, Peking University School and Hospital of Stomatology, 22 Zhongguancun Nandajie, Haidian District,
Beijing 100081, PR China
b
Department of General Dentistry, Peking University School and Hospital of Stomatology 2nd dental center, 66 Anli Avenue,
Chaoyang District, Beijing 100101, PR China

article info abstract

Article history: Objectives: To investigate whether the incorporation of chlorhexidine in a two-step self-
Received 1 May 2009 etching adhesive can preserve dentin bond strengths.
Received in revised form Methods: Different amounts of 20% chlorhexidine digluconate were added directly to the
17 June 2009 primer of Clearfil SE Bond to prepare mixtures of four different concentrations of chlorhex-
Accepted 18 June 2009 idine: 0.05%, 0.1%, 0.5% and 1.0%. Sixteen extracted third molars were randomly divided into
4 groups. Each group corresponded to one of the four chlorhexidine concentrations. Each of
the 16 teeth was sectioned into two halves. One half was bonded with Clearfil SE Bond
Keywords: without chlorhexidine, and the other half was bonded with Clearfil SE Bond containing
Chlorhexidine different concentrations of chlorhexidine. Specimens were stored in 0.9% NaCl containing
Bond degradation 0.02% sodium azide at 37 8C. Microtensile bond strengths were tested 24 h after specimen
Matrix metalloproteinases preparation or 12 months later. The modes of fractures were examined under a stereo-
Self-etching adhesives microscope.
Microtensile bond test Results: Twelve-month storage resulted in significant bond strength reduction of all control
groups ( p < 0.05). When incorporated in SE Bond primer, chlorhexidine preserved dentin
bond in the 0.1%, 0.5% and 1.0% chlorhexidine group ( p < 0.05); in the 0.05% group, there is
no statistical difference of bond strength between control group and experimental group
tested at the 12-month period ( p > 0.05).
Conclusions: When incorporated in the primer of Clearfil SE Bond, chlorhexidine can pre-
serve dentin bond as long as the concentration of chlorhexidine in the primer is higher than
or equal to 0.1%.
# 2009 Elsevier Ltd. All rights reserved.

1. Introduction
The major shortcoming of contemporary dentin adhesives is
their limited durability.1–5 The incorporation of increasing
concentrations of ionic and hydrophilic resin monomers in
total-etch and self-etch adhesives arises from the need to
bond to wet dentine substrates. However, such objectives are
accomplished at the expense of creating potentially perme-
able, unstable resin matrices that are prone to water sorption,
resin leaching and hydrolysis over time.6–10 Apart from these
extrinsic factors, intrinsic, host-derived matrix metallopro-
teinases (MMPs) also appear to be involved in the breakdown
of hybrid layers.11
MMPs are a class of zinc- and calcium-dependent endo-
peptidases capable of degrading all extracellular matrix
components. Human dentin contains at least MMP-2, MMP-

* Corresponding author. Tel.: +86 10 62179977x2347; fax: +86 10 62173402.

E-mail address: kqtanjg@bjmu.edu.cn (J. Tan).
0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2009.06.011
808 journal of dentistry 37 (2009) 807–812

Table 1 – The composition of the Clearfil SE Bond and the chlorhexidine digluconate application mode.
The composition of the Clearfil SE Bond Concentration Chlorhexidine
of CHX digluconate
application mode

Primer (pH 1.8) Bond

10-methacryloyloxydecyl dihydrogen phosphate
(MDP), 2-hydroxyethyl methacrylate (HEMA),
hydrophilic dimethacrylate, di-camphoroquinone,
N,N-diethanol-p-toluidine, water
10-methacryloyloxydecyl dihydrogen 0.05% 2.5 ml CHX digluconate
phosphate (MDP), bis-phenol A (20%) + 997.5 ml SE B primer
diglycidylmethacrylate (bis-GMA),
2-hydroxyethyl methacrylate (HEMA),
hydrophobic dimethacrylate,
di-camphoroquinone, N,N-diethanol-p-
toluidine, silanated colloidal silica
0.1% 5.0 ml CHX digluconate
(20%) + 995 ml SE B primer
0.5% 25 ml CHX digluconate
(20%) + 975 ml SE B primer
1.0% 50 ml CHX digluconate
(20%) + 950 ml SE B primer

CHX, Chlorhexidine; SE B, Clearfil SE Bond.

8, MMP-9 and MMP-20.12–15 Some of these MMPs (e.g., MMP-8)
attack collagen, while others (e.g., MMP-2 and -9) attack
gelatine. They are trapped within the mineralized dentine
matrix during tooth development and play strategic roles in
tooth development16 and dentinal caries.17 The majority of
MMPs are produced as latent zymogens (pro-MMPs). Mild acids
have been shown to be able to activate MMPs.18,19 Both etch-
and-rinse adhesives and self-etching adhesives have mild
acidity, therefore can release and activate endogenous MMPs
during dentin bonding.20–22 These activated MMPs can slowly
hydrolyze unprotected collagen fibrils of dentin hybrid layers
which are thought to be responsible for the manifestation of
thinning and disappearance of collagen fibrils from incom-
pletely infiltrated hybrid layers in aged, bonded dentin.4,5,11
The low but persistent endogenous collagenolytic activity
can be completely inhibited by the use of protease inhibitors,11
indicating that MMP inhibition might be beneficial in the
preservation of hybrid layers. Chlorhexidine is reported to
have a broad-spectrum MMP-inhibitory effect even at very low
concentrations.23 Recent studies have demonstrated that the
use of chlorhexidine significantly improved the integrity of the
hybrid layers and bond strength created by simplified etch-
and-rinse adhesives.24–27 However, whether chlorhexidine
can be applied in self-etching adhesives to preserve dentin
bonding is unclear. We were trying to apply chlorhexidine in a
two-step self-etching adhesive (Clearfil SE Bond; Kuraray,
Osaka, Japan) to preserve dentin bonding in recent years. To
simplify the bonding procedure, chlorhexidine was added
directly into the primer of Clearfil SE Bond. It has been
confirmed that the addition of chlorhexidine to Clearfil SE
Bond primer has no adverse effect on the immediate resin–
dentin bond strength when the chlorhexidine concentration
in the adhesive primer is no higher than 1.0%.28 However, to
prove that chlorhexidine can definitely preserve dentin
bonding when incorporated in the primer of Clearfil SE Bond,
an aging experiment had to be carried out.
This in vitro study was designed to test whether chlorhex-
idine can preserve bond strength over 1-year of aging time
when incorporated in the primer of Clearfil SE Bond. The null
hypothesis tested was that when incorporated in the primer of
Clearfil SE Bond, chlorhexidine had no effect on the bond
strength over 1-year of aging time.
2. Materials and methods
2.1. Preparation of a self-etching adhesive primer
containing chlorhexidine
A two-step self-etching primer adhesive, Clearfil SE Bond
(Kuraray, Osaka, Japan), was used for this study. Different
amounts of 20% chlorhexidine digluconate (Mingfeng,
Quzhou, Zhejiang, China) were added directly to the Clearfil
SE Bond primer to prepare mixtures containing four
different concentrations of chlorhexidine: 0.05 wt%,
0.1 wt%, 0.5 wt% and 1.0 wt%. The composition of the
Clearfil SE Bond and the chlorhexidine digluconate applica-
tion mode see Table 1.
2.2. Tooth preparation
Sixteen unerupted, caries-free third molars were collected
from patients from whom informed consent was obtained.
The study protocol was reviewed and approved by the
Ethics Committee for Human Studies, Peking University,
Beijing, China. These molars were stored in 0.9% NaCl
containing 0.02% sodium azide at 4 8C for no more than one
month. The 16 teeth were randomly divided into 4 groups.
Each group corresponded to one of the following four
concentrations of chlorhexidine: 0.05%, 0.1%, 0.5%, and
1.0%.
The occlusal enamel of each tooth was removed. Each
tooth was hemisected facio-lingually into two halves using a
slow-speed water-cooled saw equipped with a diamond-
impregnated disk (Isomet 1000; Buehler Ltd., Lake Bluff, IL,
USA). Each half was randomly assigned to control group or
experimental group, respectively. The teeth were polished on
a wet 600-grit silicon carbide paper by hand for 30 s to create a
realistic smear layer on the surface of the occlusal mid-coronal
dentin.
 journal of dentistry 37 (2009) 807–812
2.3. Bonding procedures
The control group teeth were bonded with Clearfil SE Bond
without chlorhexidine according to the manufacturer’s
instructions. The experimental group teeth were bonded
with Clearfil SE Bond containing different concentrations of
chlorhexidine. Five or 6 increments of resin composite
(Clearfil AP-X; Kuraray, Osaka, Japan) were added to the
bonded surfaces and individually light-cured for 20 s using a
halogen light-curing unit with an output of 700 mW/cm2.
The teeth were then stored in distilled water at 37 8C for a
week.
2.4. Microtensile bond testing
The teeth were longitudinally sectioned across the bonded
interface in sections perpendicular to the bonded surfaces
with a diamond saw (Isomet 1000; Buehler Ltd., Lake Bluff, IL,
USA), to produce a series of 0.9 mm  2 mm  8 mm beams.
Eight to 10 beams were obtained from each preparation.
These beams were then trimmed into hourglass-shaped
specimens with a cross-sectional area of approximately
0.6 mm2 and were stored in 0.9% NaCl containing 0.02%
sodium azide at 37 8C. Four specimens were tested after 24 h.
Another four specimens were tested after 12 months. Each
specimen was individually fixed to a custom-made testing jig
with a cyanoacrylate glue (Universal Instant Adhesive;
Henkel Adhesives Co. Ltd., Shantou, China). The specimen
was then subjected to tensile load at a crosshead speed of
1.0 mm/min until failure (Autograph DCS-5000, Shimadzu,
Tokyo, Japan).
2.5. Debond pathway determination
Both surfaces of each fractured specimen were observed
under a stereomicroscope (Olympus 220670; Tokyo, Japan)
with 80 magnification to record the failure modes. The
fracture modes were classified as follows: (1) cohesive failures
in the composite resin or adhesive resin; (2) failures in the
adhesive joint; (3) cohesive failures in dentin; and (4) mixed
failures. For failure modes that could not be accurately
established under the stereomicroscope, the surfaces were
examined in a scanning electron microscope (JEOL-5600 LV,
Tokyo, Japan).
Table 2 – In vitro microtensile bond strengths (MPa).
Concentration of chlorhexidine Control/experimental group
0.05% Control
Chlorhexidine
0.10% Control
Chlorhexidine
0.50% Control
Chlorhexidine
1.00% Control
Chlorhexidine
Values are mean (SD) n = 16 per subgroup. Values identified by different letters are significantly different (P < 0.05).
809
2.6. Statistical analysis
Two-way analysis of variance (ANOVA) and post hoc Tukey
tests was used to compare the effects of the dentin treatments
(control vs. chlorhexidine) and storage time (24 h vs. 12
months) on bond strengths, and to compare the distribution
of failure modes after 24 h and 12 months of storage time.
Student’s t test was used to compare the effects of the
treatment modes on the distribution of failure modes.
Statistical significance was pre-set at a = 0.05. The statistical
unit was beams, not teeth.
3. Results
3.1. Microtensile bond strengths
From a total of 260 specimens, only 4 specimens failed during
the pre-test phase, and they were statistically treated as
missing data. The means and standard deviations of the
microtensile bond strengths (mTBS) are summarized in
Table 2. Chlorhexidine did not affect in vitro bond strength
of specimens tested after 24 h in every group with different
chlorhexidine concentrations ( p > 0.05). Twelve-month sto-
rage resulted in significant bond strength reduction of all
control groups (18.7%, 16.1%, 21.8% and 15.6%, respectively)
and the experimental group containing 0.05% chlorhexidine
(28.8%). In the 0.05% chlorhexidine group, there is no statistical
difference of bond strength between control and experimental
groups tested at the 12-month period ( p > 0.05). After 12
months of aging time, there is no significant reduction of bond
strength in the three experimental groups containing 0.1%,
0.5% and 1.0% chlorhexidine.
3.2. Distribution of the failure mode
Table 3 summarizes the distribution of the failure modes. At
the 24 h testing period, most failure modes were cohesive
failures in the composite resin or adhesive observed in both
the control and experimental groups, followed by cohesive
failures in the dentin and mixed failures. There were few
adhesive joint failures. All mixed failures were the combina-
tion of cohesive failures in resin and failures in the adhesive
joint.
mTBS (SD) immediately testing mTBS (SD) 12 mos
64.89 (9.24) a 52.75 (20.12) b
62.81 (9.36) a 44.71 (13.51) b
68.60 (11.87) a 57.65 (15.94) b
67.88 (15.52) a 68.39 (12.28) a
67.41 (14.40) a 52.73 (16.93) b
68.59 (12.69) a 64.53 (13.08) a
63.05 (12.75) a 53.21 (13.11) b
66.51 (14.39) a 64.60 (11.90) a
810 journal of dentistry 37 (2009) 807–812

Table 3 – Distribution of the failure mode.

Control/experimental group Failure mode

Cohesive failures in Failures in the Cohesive failures in Mixed failures

the composite resin adhesive joint dentin
or adhesive resin

24 h 12 months 24 h 12 months 24 h 12 months 24 h 12 months

Control 12/16 7/16 0/16 5/16 2/16 1/16 2/16 3/16

Chlorhexidine (0.05%) 13/16 7/16 0/16 1/16 1/16 0/16 2/16 8/16

Control 15/16 12/16 0/16 2/16 1/16 0/16 0/16 2/16

Chlorhexidine (0.1%) 11/16 9/16 0/16 6/16 4/16 1/16 1/16 0/16

Control 13/16 15/16 0/16 0/16 1/16 0/16 2/16 1/16

Chlorhexidine (0.5%) 11/16 11/16 1/16 1/16 2/16 0/16 2/16 4/16

Control 10/16 8/16 1/16 1/16 2/16 0/16 3/16 7/16

Chlorhexidine (1.0%) 14/16 7/16 1/16 4/16 1/16 1/16 0/16 4/16

Total
Control 50/64 42/64 1/64 8/64 6/64 1/64 7/64 13/64
Chlorhexidine 49/64 34/64 2/64 12/64 8/64 2/64 5/64 16/64

xx/XX: xx = numbers of sticks tested reporting the indicate failure mode; XX = total sticks tested.

After 12-month of aging time, cohesive failures in the
composite resin or adhesive resin decreased, but were still the
most common fracture pattern. Adhesive joint failures and
mixed failures increased significantly both in the control
groups and experimental groups.
4. Discussion
As is shown in this study, when incorporated into SE Bond
primer, chlorhexidine preserved dentin bond strength when
the concentration of chlorhexidine was 0.1%, 0.5% and 1.0%;
when the concentration of chlorhexidine was 0.05%, the bond
strength decreased significantly after 1 year of aging time.
Therefore, we can conclude that when incorporated in Clearfil
SE Bond primer, chlorhexidine can preserve dentin bond
strength as long as the concentration of chlorhexidine in the
adhesive primer is higher than or equal to 0.1%.
Chlorhexidine has been widely used and studied as an
antimicrobial agent, e.g., as a cavity disinfectant before the
placement of restorations. Since Pashley et al. found in 2004
that host-derived MMPs play a key role in the breakdown of
collagen fibrils from incompletely infiltrated hybrid layers,11
many in vitro and in vivo studies have been reported to use
chlorhexidine as a MMP-inhibitor to improve dentin bond.24–30
Most of these studies applied chlorhexidine in etch-and-rinse
adhesives and used chlorhexidine as an additional primer, in
which chlorhexidine was applied after or prior to the acid-
etching step.24–27,29,30 Only a few studies applied chlorhexidine
in self-etching adhesives, in which chlorhexidine was also
used as an additional separately applied primer.31 To simplify
bonding procedures, the present study incorporated chlor-
hexidine directly into the primer of Clearfil SE Bond, a two-step
self-etching primer adhesive, and confirmed that when
applied in this manner, chlorhexidine can preserve dentin
bond strength. However, although the bond strength of the
experimental groups (0.1%, 0.5% and 1.0%) did not decrease,
adhesive joint failures and mixed failures increased signifi-
cantly, indicating that the real bond strength of experimental
groups also decreased to some extent. Therefore, longer aging
experiments should be carried out to determine the max-
imum efficiency of chlorhexidine in future studies. Never-
theless, this study provided another way of applying MMP-
inhibitors in dental adhesives; that is, by incorporating the
MMP-inhibitory agent directly into dental adhesives or
adhesive primers.
It has been reported that even at very low concentrations,
chlorhexidine possesses desirable MMP-inhibitory proper-
ties.23 Chlorhexidine at concentrations as low as 0.03% can
completely inhibit MMP-2 and MMP-9 gelatinase activity.
However, in this study 0.05% chlorhexidine could not preserve
bond strength. This is in accordance with our previous
unpublished experiment (Jianfeng Zhou et al. unpublished
observations), in which when lower concentrations of
chlorhexidine were incorporated in Clearfil SE Bond primer,
they could not inhibit MMPs. In that experiment, even higher
concentrations of chlorhexidine could only partially maintain
its inhibitory effect on MMPs when incorporated in Clearfil SE
Bond primer. This might attribute to the calcium ions released
from the underlying mineralized dentin during self-etching. It
has been previously proposed that a calcium-chelating
mechanism is involved in the inhibitory properties of
chlorhexidine on MMPs,23 since MMPs require metal ions
(namely, calcium and zinc) for their catalytic activity. Hence,
calcium ions could competitively inhibit chlorhexidine and
reverse the inhibitory effect of chlorhexidine on MMPs. It has
been reported that adding calcium chloride to the assay
mixtures that contained 0.03% chlorhexidine almost comple-
tely prevented the inhibition of MMP-9 activity.23 Therefore,
the concentration of chlorhexidine in SE Bond primer is an
important factor affecting the longevity of resin–dentin bonds.
Lower concentrations of chlorhexidine might be inhibited by
calcium ions released from dentin, while higher concentra-
tions of chlorhexidine might affect the initial resin–dentin
bond strength. Further studies with longer aging time and
more tested chlorhexidine concentrations have to be carried
 journal of dentistry 37 (2009) 807–812
out to determine the ideal concentration of chlorhexidine in
SE Bond primer.
It has been recently reported that chlorhexidine can be
released from chlorhexidine diacetate-incorporated resin
discs.32 This finding suggests that when applied in the manner
of this study, chlorhexidine might be released from cured
dentin hybrid layers over time. This might have dual effect. At
the beginning of storage period, released chlorhexidine can
continue to inhibit MMPs and protect collagen fibrils in dentin
hybrid layers. However, with the cumulative release of
chlorhexidine, the concentration of chlorhexidine will
decrease over time and its MMP-inhibitory effect will be
weakened. From this point of view, higher concentrations of
chlorhexidine should be more effective.
The incorporation of MMP-inhibitors into primers or
adhesives has several potential limitations: the incorporated
MMP-inhibitor might affect the bond strength of dentin
adhesive; the incorporated MMP-inhibitor might leach from
the cured dentin hybrid layers and its MMP-inhibitory effect
might be weakened; during acid-etching, the MMP-inhibitor
might be affected by the released calcium ions. However,
acidic monomers such as MDP form insoluble calcium salts
that may limit the amount of ionized calcium in the hybrid
layer created by SE bond primer adhesive.33–35 Even so,
incorporation of MMP-inhibitors in dentin adhesives should
be a worthwhile project to be studied, as it can simplify
bonding procedures, which is more practical and has lower
technique sensitivity. Chlorhexidine is only one of many
choices as there are hundreds of MMP-inhibitors that are used
and studied in medical field. Therefore, much additional
researches will be required to determine which MMP-
inhibitors can be incorporated in dentin adhesives and can
preserve dentin bond more efficiently and permanently.
Another limitation of this study was the use of a single
product; we only incorporated chlorhexidine into Clearfil SE
Bond. Chlorhexidine may not be beneficial to all two-step self-
etching primer adhesives because the chemistry varies for
each adhesive.
5. Conclusions
Within the limitations of this in vitro study, the conclusion can
be drawn that when incorporated in the primer of Clearfil SE
Bond, chlorhexidine can preserve dentin bond for at least 1
year as long as the concentration of chlorhexidine in the
adhesive primer is higher than or equal to 0.1%.
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