2009 The Authors.

Journal compilation  2009 Eur J Oral Sci

Eur J Oral Sci 2009; 117: 587596

Printed in Singapore. All rights reserved

European Journal of
Oral Sciences

Influence of chlorhexidine digluconate

concentration and application time on
resindentin bond strength durability

Alessandro D. Loguercio1, Rodrigo

Stanislawczuk1, Luceli G. Polli1,
Jully A. Costa1, Milton D. Michel2,
Alessandra Reis1
1

Department of Restorative Dentistry, School of

Dentistry, Universidade Estadual de Ponta
Grossa, PR, Brazil; 2Department of Mechanical
Engineering, School of Mechanical
Engineering, University Estadual de Ponta
Grossa, Ponta Grossa, PR, Brazil

Loguercio AD, Stanislawczuk R, Polli LG, Costa JA, Michel MD, Reis A. Inuence of
chlorhexidine digluconate concentration and application time on resindentin bond
strength durability. Eur J Oral Sci 2009; 117: 587596.  2009 The Authors. Journal
compilation  2009 Eur J Oral Sci
Although it is known that chlorhexidine application may preserve resindentin bonds
from degradation, the lowest optimal concentration and application time have yet to
be established. This study evaluated the eects of dierent concentrations of chlorhexidine digluconate and dierent application times on the preservation of resin
dentin bonds formed using two etch-and-rinse adhesives. In experiment 1, after acid
etching, the occlusal demineralized dentin was rewetted either with water or with
0.002, 0.02, 0.2, 2, or 4% chlorhexidine for 60 s. In experiment 2, the surfaces were
rewetted with water, or with 0.002% or 2% chlorhexidine for 15 or 60 s. After this,
both adhesives and composite resin were applied and light-cured. Bonded sticks
(0.8 mm2) were tested under tension (0.5 mm min)1) immediately or after 6 months of
storage in water. Two bonded sticks from each tooth were immersed in silver nitrate
and analyzed quantitatively using scanning electron microscopy. Reductions in microtensile bond strengths and higher silver nitrate uptake were observed for both
adhesives when the rewetting procedure was performed with water. Stable bonds were
maintained for up to 6 months under all chlorhexidine conditions tested, irrespective
of the chlorhexidine concentration and application time. The use of 0.002% chlorhexidine for 15 s seems to be sucient to preserve resindentin interfaces over a 6month period.

Recent studies have pointed out that dentin bondstrength values measured immediately after formation of
the bond do not always correlate with long-term bond
stability (1), as degradation throughout the dentin
bonding interface occurs rapidly (i.e. within 6 months)
(2, 3). Bonding is created by impregnating the dentin
substrate with blends of resin monomers, and the stability of the bonded interface relies on the creation of a
compact and homogenous hybrid layer. In the etchand-rinse strategy, after preliminary etching to demineralize the substrate, resin monomers impregnate the
porous etched substrate (4, 5) and thus stable bonds may
be achieved if the etched substrate is fully inltrated by
the adhesive (6).
However, when incompletely inltrated zones occur
along the bottom of hybrid layers within the acid-etched
dentin, a decreasing gradient of resin monomer diusion,
with denuded collagen brils (79), is likely to occur.
This region is susceptible to hydrolytic degradation in
the long term, leading to reductions in bond strength
(10). Moreover, the elution of unpolymerized monomers
from the hybrid layer in contact with oral uids is
responsible for the accelerated process of bond interface
degradation. This leaves more collagen brils within the
hybrid layer exposed and susceptible to the degradation

Alessandro D. Loguercio, Universidade

Estadual de Ponta Grossa Mestrado em
Odontologia, Rua Carlos Cavalcanti, 4748,
Bloco M, Sala 64A Uvaranas, Ponta Grossa,
Paran 84030-900, Brazil
Telefax: +554232203741
E-mail: aloguercio@hotmail.com
Key words: adhesive system; application time;
chlorhexidine concentration; dentin; longevity
Accepted for publication May 2009

process (11). The combined degradation by hydrolysis of

resin and/or collagen weakens the physical properties of
the resindentin bond interface (11).
Studies have revealed the contribution of host-derived
proteinases, responsible for the breakdown of collagen
matrices, in the pathogenesis of dentin caries (12, 13),
periodontal disease (14), and in the degradation of dentin
bonding (15). It has been reported that mineralized
dentin contains collagenolytic and gelatinolytic enzyme
activities derived from the action of matrix metalloproteinases (MMPs) (1618). Both in vitro and in vivo
studies have shown that 2% chlorhexidine digluconate
(CHX), applied for 60 s on demineralized dentin, postpones the resindentin degradation of adhesive interfaces, when compared with interfaces to which no CHX
is applied (1922). Despite these advantages, the use of
2% CHX for 60 s demands more chair-time during the
adhesive procedure and this contrasts with the clinicians
needs for simplication (23).
It is also worthwhile mentioning that CHX solutions
with concentrations (close to 0.01%) are highly cytotoxic
to cultured cells (24, 25). Obviously, when CHX solution
is applied as a cavity cleanser there is enough dentin
structure to act as a barrier and protect the pulp cells
against the cytotoxic eects of dental materials or rinsing

588

Loguercio et al.

solutions. However, no data have so far been published

concerning the capacity of CHX molecules to diuse
through dentin tubules and reach the pulpal cells.
The use of very low concentrations of CHX has been
shown to strongly inhibit the collagenolytic activity of
pure MMPs (26) and dentin matrix-bound MMPs (27).
Specic host-derived MMPs, responsible for the breakdown of the collagen matrices (MMP-2, MMP-8, and
MMP-9), can be inhibited by low concentrations of
CHX (0.00010.02%) (26). Fortunately, no cytotoxic
eect on odontoblast-like cells has been reported for
CHX at these concentrations (25). However, to the best
of the authors knowledge, no study has so far evaluated
the eects of CHX concentrations on the durability of
resindentin bonding.
The application time of MMP inhibitors is still a
matter to be addressed. In a recent investigation,
Stanislawczuk et al. (28) showed that when 2% CHX
containing phosphoric acid was applied for 15 s the
durability of the resindentin bonds was preserved. This
seems to indicate that even a short period of CHX in
contact with the demineralized dentin appears to be
sucient to inhibit the action of specic host-derived
proteinases. Therefore, the ideal situation would be to
apply CHX at a low concentration, and also for a short
period of time, as part of the strategy to simplify the
bonding protocol.
Therefore, the aim of this study was to evaluate the
eect of dierent concentrations of CHX and dierent
application times on the durability of the dentin bonds.
To do so, the study was divided into two experiments.
The objective of the rst experiment was to evaluate the
early and 6-month resindentin bond strength and silver
nitrate uptake of two-step etch-and-rinse adhesives
rewetted with dierent CHX concentrations (0.002, 0.02,
0.2, 2, and 4%) applied for 60 s. The second experiment
examined the early and 6-month resindentin bond
strength and the silver nitrate uptake (SNU) pattern of
two-step etch-and-rinse adhesives after rewetting the
demineralized dentin with two CHX concentrations
(0.002 and 2%) applied for two dierent application
times (15 and 60 s). It was hypothesized that the lowest
concentration of CHX applied for only 15 s would be

sucient to prevent reductions in resindentin bond

strength and deposition of SNU within adhesive and
hybrid layers after 6 months of water storage.

Material and methods

Preparation of teeth

One-hundred and twenty extracted, caries-free human

third molars were used. The teeth were collected after
obtaining the patients informed consent. The Ponta
Grossa State University Review Board approved this
study. Teeth were disinfected in 0.5% chloramine, stored
in distilled water, and used within 6 months of extraction.
A at and supercial dentin surface was exposed on each
tooth after wet grinding the occlusal enamel on #180-grit
silicon carbide (SiC) paper. The enamel-free, exposed
dentin surfaces were further polished on wet #600-grit SiC
paper for 60 s to standardize the smear layer.
Microtensile bond strength (lTBS) test Two dierent
solvent-based, etch-and-rinse adhesive systems were tested: Adper Single Bond (SB; 3M ESPE, St Paul, MN,
USA), an ethanol/water-based system; and Prime &
Bond 2.1 (PB; Dentsply De Trey, Konztanz, Germany),
an acetone-based system (Table 1).
For the rst experiment, 60 teeth were divided into 12
groups (n = 5) according to the combination of the
main factors adhesive (two levels) and rewetting
solution (six levels). The exposed dentin surfaces were
conditioned with the respective phosphoric acids of each
adhesive system for 15 s, rinsed o (15 s), air-dried
(Table 1), and rewetted either with water (control group)
or with aqueous solutions of 0.002, 0.02, 0.2, 2, or 4%
CHX (Fleming drugstore, Ponta Grossa, PR, Brazil). All
rewetting procedures were performed for 60 s. Afterwards, the adhesive systems were applied in accordance
with the manufacturers directions (Table 1). The aim of
this rst experiment was to select two extreme CHX
concentrations that had benecial eects on the preservation of the resindentin bonds over a period of
6 months.

Table 1
Adhesive systems: composition, groups, and application mode
Adhesive systems
Prime & Bond 2.1 (PB)
(Dentsply De Trey,
Konstanz, Germany)
Adper Single Bond (SB)
(3M ESPE, St Paul, MN, USA)

Composition

Application mode*

1. Caulk Tooth Conditioner Gel 34% phosphoric acid

2. Adhesive UDMA, Bis-GMA, PENTA,
butylated hydroxitoluene, 4-ethyl dimethyl aminobenzoate,
cetilamine hydrouoride, initiator, and acetone
1. Scotchbond Etchant: 37% phosphoric acid
2. Adhesive Bis-GMA, HEMA, dimethacrylates,
polyalkenoic acid copolymer, initiators, water, and ethanol

a, b, c, d, e1, f, g

a, b, c, d, e2, f, g

*a, acid-etch (15 s); b, rinse (15 s); c, air-dry (30 s); d, dentin rewetted with water or chlorhexidine digluconate (CHX) solution for 15
or 60 s; e1, two coats of adhesive were applied for 20 s; e2, one coat of adhesive was applied for 10 s; f, air-dry for 10 s at 20 cm; g,
light-cure (10 s; 600 mW cm)2).
Bis-GMA, bisphenol A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; PENTA, dipentaerythritol pentacrylate
monophosphate; UDMA, urethane dimethacrylate.

Chlorhexidine on dentin adhesion

For the second experiment, 60 teeth were divided into

12 groups (n = 5) according to the combination of the
main factors adhesive (two levels), rewetting solution
(three levels), and application time (two levels). The
dentin surfaces were acid etched with phosphoric acid,
rinsed o, air-dried, and rewetted either with water
(control group) or with aqueous solutions of 0.002 or 2%
CHX (Fleming drugstore). In this case, all rewetting
procedures were performed for 15 or 60 s. Afterwards,
thee adhesive systems were applied according to the
manufacturers instructions.
For both experiments, the light-curing procedure was
performed by means of a quartz-tungsten-halogen light
(VIP; Bisco, Schaumburg, IL, USA; 600 mW cm)2) for
the recommended time (10 s). Resin-composite build-ups
(Opallis; FGM, Joinville, SC, Brazil) were placed on the
bonded surfaces (three increments of approximately
1.5 mm each) and individually light activated for 40 s
each. All bonding procedures were carried out by a single
operator, and at 24C and 50% relative humidity.
After the bonded teeth had been stored in distilled
water at 37C for 24 h, they were longitudinally sectioned in both x and y directions across the bonded
interface using a diamond saw in a Labcut 1010 machine
(Extec, Eneld, CT, USA), under water cooling at
300 r.p.m., to obtain bonded sticks with a cross-sectional
area of approximately 0.8 mm2. The number of prematurely debonded sticks (D) per tooth during specimen
preparation was recorded. The remaining dentin thickness (RDT) was measured with a caliper and recorded
(Absolute Digimatic, Mitutoyo, Tokyo, Japan). The
cross-sectional area of each stick was measured with the
digital caliper to the nearest 0.01 mm and recorded for
subsequent calculation of the lTBS. The bonded sticks
that originated from the same teeth were randomly
divided and assigned to be tested immediately, or after
6 months of storage in distilled water at 37C. The
storage solution was not changed and its pH was
monitored monthly.
Each bonded stick was attached to a jig in the universal testing machine (Emic, Sao Jose dos Pinhais, PR,
Brazil) with cyanoacrylate resin (Zapit; Dental Ventures
of North America, Corona, CA, USA) and subjected to
a tensile force of 0.5 mm min)1. The failure modes were
evaluated at 400 magnication (microhardness HMV-2;
Shimadzu, Tokyo, Japan) and classied as cohesive
(failure exclusively within the dentin or composite; C),
adhesive (failure at resin/dentin interface; A), or
adhesive/mixed (failure at the resindentin interface that
included cohesive failure of the neighboring substrates;
A/M).
SNU measurement under scanning electron microscopy
For both experiments, the amount of SNU into the
hybrid layer and the adhesive layer was measured. Two
bonded sticks from each tooth for each storage period
were coated with two layers of nail varnish applied up to
within 1 mm of the bonded interfaces. The specimens
were rehydrated in distilled water for 10 min before
immersion in the tracer solution for 24 h. Ammoniacal
silver nitrate was prepared according to the protocol

589

previously described by Tay et al. (29). The sticks were

placed in ammoniacal silver nitrate in the dark for 24 h,
rinsed thoroughly in distilled water, and immersed in
photo developing solution for 8 h under uorescent light
to reduce silver ions into metallic silver grains within
voids along the bonded interface.
All sticks were wet-polished with 600 SiC paper to
remove the nail varnish. After this, the specimens were
placed inside an acrylic ring, which was attached to a
double-sided adhesive tape, and embedded in epoxy
resin. After the epoxy resin had set, the thickness of the
embedded specimens was reduced to approximately half
by grinding with SiC papers under running water.
Specimens were polished with a 1,000-grit SiC paper and
1 and 0.25 lm diamond paste (Buehler, Lake Blu, IL,
USA) using a polishing cloth. Specimens were ultrasonically cleaned and demineralized in a 50% phosphoric
acid solution for 3 s followed by immersion in 1%
NaOCl for 10 min. Next, specimens were air-dried for
24 h, mounted on aluminum stubs, and sputtered with
gold (Sputter Coater IC 50; Shimadzu, Tokyo, Japan).
Resindentin interfaces were analyzed using a scanning
electron microscope (SSX-550; Shimadzu) operated in
the back-scattering electron model. The working distance
was 10 mm and the accelerating voltage (ACCV) was
15 Kv.
Three pictures were taken of each specimen. The rst
picture was taken in the center of the stick. The other
two pictures were taken 0.3 mm to the left and right of
the rst picture. As two sticks per tooth were evaluated
and a total of ve teeth were used for each experimental
condition, 30 images were evaluated for each group.
They were all taken by a technician who was blinded to
the experimental conditions under evaluation. The relative percentage of SNU within the adhesive and hybrid
layer areas was measured in all pictures using the
uthscsa ImageTool 3.0 software (Department of Dental
Diagnostic Science at The University of Texas Health
Science Center, San Antonio, TX, USA) by an author
blind to the test and control samples. First of all, the
total area of the adhesive layer plus the hybrid layer was
recorded. Then the area occupied by the silver nitrate
deposits was delimitated by a software tool, summed,
and the relative ratio between the total area vs. the
impregnated areas was calculated to give the percentage
of SNU within each specic bonding interface.
The experimental unit in this study was the tooth half,
because half of the sample was tested immediately and
the other half was tested after 6 months. The lTBS
values of all sticks from the same tooth half were averaged for statistical purposes. The prematurely debonded
specimens were included in the tooth half mean. The
average value attributed to specimens that failed
prematurely during preparation was arbitrary and
corresponded to approximately half of the minimum
lTBS that could be measured in this study (ca. 5.0 MPa).
For SNU (%), the mean SNU of all pictures originated
from sticks from the same tooth half was averaged for
statistical purposes. The SNU of every test group was
expressed as the mean of the ve tooth halves used per
group and is expressed in per cent.

590

Loguercio et al.

Before submitting the data for analysis using the

appropriate statistical test, the KolmogorovSmirnov
test was performed to assess whether the data followed a
normal distribution, and the Barletts test for equality of
variances was performed to determine if the assumption
of equal variances was valid (30). After observing the
normality of the data distribution and the equality of the
variances, the lTBS (MPa) and SNU (%) data were
subjected to appropriate statistical analysis.
In the rst experiment, the lTBS data and SNU were
subjected to a two-way repeated-measures analysis of
variance (chlorhexidine concentration vs. storage time)
for each adhesive. The repeated measure was the storage
time. The post hoc test (Tukeys test at a = 0.05) was
used for pairwise comparisons. In the second experiment,
the lTBS and SNU data were subjected to a three-way
repeated-measures analysis of variance (chlorhexidine
concentration vs. application time vs. storage time) for
each adhesive. The repeated measure was the storage
time. A post hoc test (Tukeys test at a = 0.05) was used
for pairwise comparisons.

Results
The mean cross-sectional area ranged from 0.78 to
1.02 mm2 and no signicant dierence among groups
was detected (P > 0.05). The RDT for all specimens
ranged from 2.7 to 3.3, indicating that the interfaces were
located in mid-dentin (31).
lTBS

For the rst experiment, the cross-product interaction

CHX concentration vs. storage time for each adhesive
system was statistically signicant (P < 0.05). Signicant reductions in the lTBS were observed in the control
group (i.e. when water was used instead of CHX as the
rewetting agent) (Table 2). The two etch-and-rinse
adhesives tested had approximately the same overall
failure mode rates under similar experimental conditions
(Table 3).
For the second experiment, only the cross-product
CHX concentration vs. storage time for both adhesives

was statistically signicant (P < 0.05). Signicant

reductions in resindentin bond-strength values were
observed for both adhesives when the demineralized
dentin was rewetted with water, irrespective of the
application time. By contrast, rewetting with CHX in the
dierent concentrations and application times yielded
stable resindentin bonds after 6 months of water storage (Table 4). The two etch-and-rinse adhesives tested
had approximately the same overall failure mode rates
under similar experimental conditions (Table 5).
SNU

In experiment 1, the cross-product interaction CHX

concentration vs. storage time was statistically signicant
for both adhesive systems (P < 0.05). For both adhesives, a lower percentage of SNU immediately after
treatment was observed for the interfaces treated with
CHX, irrespective of its concentration, when compared
with the control group immediately after treatment
(P = 0.01) (Table 6). After 6 months of water storage, a
signicantly higher SNU was observed for both adhesives under the experimental conditions; however, this
uptake was much less pronounced for the CHX groups
when compared with the controls (P = 0.001) (Table 6).
Observe in Table 6 that among the dierent CHX concentrations used, 4% CHX showed the highest SNU,
which diered statistically from that of the lower CHX
concentrations (P = 0.01).
In experiment 2, only the cross-product CHX concentration vs. storage time for each adhesive system was
statistically signicant (P < 0.05). For both adhesives, a
lower percentage of SNU immediately after treatment
was observed for the interfaces treated with CHX, irrespective of its concentration, when compared with the
control group immediately after treatment (P = 0.01)
(Table 7). After 6 months of water storage, signicantly
higher SNU was observed for both adhesives under the
experimental conditions; however, this uptake was much
less pronounced for the CHX-treated groups than for the
controls (P = 0.001) (Table 7).
Representative back-scattering scanning electron
microscopy images captured at the resindentin interfaces from experiment 2 are depicted in Figs 14.

Table 2
Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition in
experiment 1*
Prime & Bond 2.1
Conditions
Control
0.002% CHX
0.02% CHX
0.2% CHX
2% CHX
4% CHX

Immediate
32.0
28.2
29.9
30.9
34.2
26.7

3.2 A
3.4 A
5.3 A
4.7 A
5.1 A
4.7 A,B

Adper Single Bond

6 months

21.3
25.1
30.1
27.4
31.3
21.1

2.4
3.2
4.2
4.6
4.1
3.5

B
A,B
A
A
A
B

Immediate
34.1
35.2
30.2
34.2
32.4
26.3

4.6
6.2
4.3
4.1
6.1
4.2

a
a
a
a
a
a,b

6 months
24.2
31.1
27.3
36.2
28.3
24.3

5.4
5.3
5.1
4.0
3.5
4.2

b
a
a,b
a
a
b

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter
are not signicantly dierent (P > 0.05).
CHX, chlorhexidine digluconate.

591

Chlorhexidine on dentin adhesion

Table 3

Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens from each
experimental condition from experiment 1*
Prime & Bond 2.1
Immediate

Pattern
fracture
Control
0.002% CHX
0.02% CHX
0.2% CHX
2% CHX
4% CHX

A/M
24
38
38
35
39
30

(75.0)
(77.6)
(67.9)
(87.5)
(75)
(88.2)

0
9
0
0
1
0

Adper Single Bond

6 months

Debonded

A/M

Immediate
Debonded

(0)
8 (25.0) 34 (80.9) 0 (0)
(18.4) 2 (4.0) 33 (71.7) 5 (10.9)
(0)
18 (32.1) 36 (75.0) 11 (22.9)
(0)
5 (12.5) 32 (94.1) 0 (0)
(2.5)
9 (22.5) 36 (94.4) 0 (0)
(0)
4 (11.8) 25 (76)
0 (0)

8
8
1
2
2
6

(19.1)
(17.4)
(2.1)
(5.9)
(5.6)
(24)

A/M
26
36
26
32
37
38

(81.2)
(73.5)
(46.4)
(80)
(84.1)
(77.6)

0
7
0
3
2
4

6 months

Debonded

A/M

(0)
6 (18.8) 29 (69)
(14.3) 6 (12.2) 35 (76.1)
(0)
30 (53.6) 36 (75)
(7.5)
5 (12.5) 30 (88.2)
(4.5)
5 (11.4) 34 (97.1)
(8.2)
7 (14.2) 33 (70.2)

C
3
5
7
2
0
7

Debonded

(7.2) 10 (23.8)
(10.9) 6 (13)
(14.6) 5 (10.4)
(5.9)
2 (5.9)
(0)
1 (2.9)
(14.9) 7 (14.9)

*A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debonded
specimens.

Table 4
Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition from
experiment 2*
Prime & Bond 2.1
Immediate
Application
time

15 s

Adper Single Bond

6 months

60 s

Immediate

15 s

60 s

15 s

6 months

60 s

15 s

60 s

Control
28.3 4.3 A
32.4 5.4 A 20.1 4.2 B
21.2 3.8 B
39.2 5.4 a 41.5 6.4 a 27.9 6.2 b 25.4 4.1 b
0.002% CHX 25.7 2.4 A,B 29.2 3.4 A 23.2 4.1 A,B 27.0 3.6 A,B 41.4 4.8 a 43.2 6.1 a 37.2 6.1 a 40.1 3.7 a
2% CHX
33.1 6.5 A
31.3 5.1 A 27.3 4.2 A,B 28.1 4.4 A
43.5 4.1 a 41.2 4.2 a 40.1 5.7 a 37.6 3.3 a

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter
are not signicantly dierent (P > 0.05).
CHX, chlorhexidine digluconate.

Table 5
Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens of each
experimental condition from experiment 2*
Prime & Bond 2.1
Immediate
Pattern fracture

A/M

Debonded

Adper Single Bond

6 months

A/M

Control (15 s)
27 (69.2) 02 (5.1) 10 (25.7) 38 (84.5)
Control (60 s)
35 (85.4) 2 (4.9) 4 (9.7) 37 (84.1)
0.002% CHX (15 s) 42 (75.0) 0 (0)
14 (25.0) 39 (79.6)
0.002% CHX (60 s) 38 (82.6) 1 (2.2) 7 (15.2) 33 (82.5)
2% CHX (15 s)
41 (87.2) 2 (4.3) 4 (8.5) 39 (88.6)
2% CHX (60 s)
36 (94.7) 2 (5.3) 0 (0)
40 (88.9)

C
2
3
3
3
4
0

(4.4)
(6.8)
(6.1)
(7.5)
(9.1)
(0)

Immediate

Debonded
5
4
7
4
1
5

(11.1)
(9.1)
(14.3)
(10.0)
(2.3)
(11.1)

A/M
26
33
32
39
35
38

(74.3)
(76.7)
(80.0)
(83.0)
(85.4)
(90.5)

C
2
7
6
2
1
3

(5.7)
(16.3)
(15.0)
(4.3)
(2.4)
(7.1)

6 months

Debonded
7
3
2
6
5
1

(20.0)
(7.0)
(5.0)
(12.7)
(12.2)
(2.4)

A/M
35
36
40
36
42
37

(85.4)
(87.8)
(95.2)
(92.3)
(85.7)
(90.2)

C
4
2
1
0
3
4

(9.7)
(4.9)
(2.4)
(0)
(6.1)
(9.8)

Debonded
2
3
1
3
4
0

(4.9)
(7.3)
(2.4)
(7.7)
(8.2)
(0)

*A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debonded
specimens.

Discussion
The results of this study conrm previously published
ndings that resindentin interfaces bonded with simplied etch-and-rinse adhesives can degrade after short
periods of time (2, 3). One mechanism of degradation
proposed in the literature is the incomplete impregnation
of resin into the collagen network and hybrid layer itself
(1, 11).

It has recently been speculated that the autodegradation of dentin collagen brils is responsible for such
ndings (15). This mechanism is initiated from beneath
the bonded interface, with the breakdown of aciddemineralized collagen matrices by host-derived MMPs.
The low, but persistent, endogenous collagenolytic
activity can be completely inhibited by the use of protease inhibitors (15). Chlorhexidine digluconate has the
potential to minimize the reductions in the resindentin

592

Loguercio et al.
Table 6

Overall mean percentage of silver nitrate uptake (%) within hybrid layer and adhesive layer and the respective standard deviations
(in MPa) obtained in each experimental condition from experiment 1*
Prime & Bond 2.1
Conditions

Immediate

Control
0.002% CHX
0.02% CHX
0.2% CHX
2% CHX
4% CHX

26.3
12.1
12.2
10.3
9.8
14.6

4.6
3.3
4.2
2.9
3.2
5.6

Adper Single Bond

6 months

B,C
A
A
A
A
A

42.3
24.6
25.4
22.1
27.8
32.4

5.6
4.3
4.1
3.8
3.9
4.6

Immediate

D
B
B
B
B,C
C

24.5
10.1
11.1
9.6
12.4
16.2

3.6
2.3
3.5
2.7
4.3
3.6

6 months

b,c
a
a
a
a
a

38.1
22.9
21.4
22.5
24.5
27.4

6.5
4.2
2.4
3.2
5.4
3.6

d
b
b
b
b,c
c

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter
are not signicantly dierent (P > 0.05).
CHX, chlorhexidine digluconate.

Table 7
Overall mean percentage of silver nitrate uptake within hybrid layer and adhesive layer and the respective standard deviations (MPa)
obtained in each experimental condition from experiment 2*
Prime & Bond 2.1
Immediate
Application
time
Control
0.002% CHX
2% CHX

Adper Single Bond

6 months

Immediate

6 months

15 s

60 s

15 s

60 s

15 s

60 s

15 s

60 s

28.7 3.4 B
12.3 2.6 A
11.2 2.5 A

26.3 4.2 B
12.6 2.3 A
10.2 5.2 A

39.5 6.3 C
24.1 3.9 B
25.1 3.5 B

40.2 5.4 C
23.6 2.8 B
26.5 3.8 B

18.3 3.7 b
10.3 1.8 a
10.4 2.3 a

22.4 4.2 b
9.8 2.5 a
10.2 3.2 a

45.3 6.4 c
21.3 5.6 b
26.7 4.3 b

42.4 3.6 c
24.3 4.8 b
22.1 3.8 b

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter
are not signicantly dierent (P > 0.05).
CHX, chlorhexidine digluconate.

15 s

Adper Single Bond

Fig. 1. Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Adper Single
Bond immediately after treatment (AC) or 6 months after treatment (DF), in which the rewetting time was 15 s. In the samples
tested immediately after treatment, only a few areas of silver nitrate uptake were observed within the hybrid layer (AC), mainly in
the group in which dentin was rewetted with water (white stains indicated by the white pointer, panel A). After 6 months, silver
nitrate uptake was higher in the hybrid layer only for the control group (water) (white pointer, panel D). In panels E and F, only a
small amount of silver nitrate was taken up in the hybrid layer (white stains indicated by the white pointer, panel E) (AL, adhesive
layer; Co, composite; De, dentin; and HL, hybrid layer).

Chlorhexidine on dentin adhesion

593

Adper Single Bond

Fig. 2 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Adper Single
Bond immediately after treatment (AC) or 6 months after treatment (DF), in which the rewetting time was 60 s. In the samples
tested immediately after treatment, only a few areas of silver nitrate uptake in the hybrid layer (panels AC) were observed (white
stains indicated by the white pointer, panel A). After 6 months, silver nitrate uptake was highest in the hybrid layer and across the
entire thickness of the adhesive layer (white stains indicated by the white pointer, panel D). After 6 months, in the 0.002% and 2%
chlorhexidine digluconate (CHX) groups (panels E and F, respectively) only a small amount of silver nitrate was taken up (white
stains indicated by white pointer, panel E). (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

Prime & Bond 2.1

Fig. 3 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Prime & Bond
2.1 immediately after treatment (AC) or 6 months after treatment (DF), after rewetting for 15 s. In the samples tested immediately
after treatment, a considerable amount of silver nitrate uptake within the hybrid layer was observed in the control group (water)
(white stains indicated by the white pointer, panel A), compared with the groups rewetted with 0.002% and 2% chlorhexidine
digluconate (CHX) (panels B and C, respectively). In panel A, signs of phase separation can be observed (black star). After 6 months,
the silver nitrate uptake was higher in the hybrid layer only for the control group (water) (white stains indicated by white pointer,
panel D). In panels E and F, only a small amount of silver nitrate uptake was seen in the hybrid layer (white stains indicated by the
white pointer, panel F) (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

bond strengths commonly observed for simplied conventional adhesives after long-term water storage and
also to preserve the morphological properties of hybrid
layers by inhibiting host-derived proteases (1922, 28).
Therefore, one might consider that preventing the
degradation of these incompletely resin-inltrated collagen brils and the hybrid layer by MMPs in durability
studies is an important issue to investigate, because this
could be the key to increasing the durability of restorations that involve dentin bonding. While some in vitro

and in vivo studies have suggested that the application of

2% CHX solution, a MMP inhibitor, to acid-etched
dentin for 60 s could minimize the degradation of the
dentin bond over time, the brillar network of the hybrid
layers formed without pretreatment with CHX solution
depicts a signicant and progressive disintegration
(1922, 28). This was conrmed to some extent in the
present investigation. Signicant reductions in the lTBS
and higher SNU were seen within the adhesive and
hybrid layers after 6 months of water storage in the

594

Loguercio et al.
Prime & Bond 2.1

Fig. 4 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Prime & Bond
2.1 immediately after treatment (AC) or 6 months after treatment (DF), when the rewetting was applied for 60 s. In the samples
tested immediately after treatment, no signs of silver nitrate uptake (panels B and C), in comparison with a small amount of silver
nitrate uptake within the hybrid layer, was observed in the control group (water) (white stains indicated by the white pointer, panel
A). After 6 months, the groups rewetted with chlorhexidine digluconate (CHX) (0.002 and 2%, respectively in panels E and F)
showed preservation of the hybrid layer. Only a small amount of silver nitrate uptake was seen in panel E (white stains indicated by
white pointer). The highest amount of silver penetration occurred in the hybrid layer in the group in which demineralized dentin was
rewetted with water (white stains indicated by a white pointer, panel D) (AL, adhesive layer; Co, composite; De, dentin; and HL,
hybrid layer).

control groups (rewetted with water) for both adhesive

systems tested.
However, contrasting ndings were observed for
CHX-treated groups. Very few silver nitrate deposits
were seen within the hybrid layer after 6 months and no
degradation of the resindentin bond strengths was
detected.
Interestingly, the good performance of CHX (in terms
of resindentin bond strengths) in the preservation of the
dentin bonds over time was independent of its concentration. This is in agreement with the conclusion previously reached by Gendron et al. (26), who found that
extremely low concentrations of CHX (around 0.01%)
can inhibit protease activity. It has been demonstrated
that pure MMP-2, MMP-8, and MMP-9 can be inhibited
by CHX concentrations of 0.0001, 0.02, and 0.002%,
respectively, through proteolytic action (26). Recently,
Carrilho et al. (27) evaluated the collagenolytic activity
of the dentin-bound MMPs and showed that the collagenolytic activity of demineralized dentin was signicantly reduced by pretreatment with 2% CHX in
comparison with non-treated specimens.
Gendron et al. (26) proposed two dierent mechanisms of action involved in MMP inhibition: a chelating
mechanism for the inhibition of MMP-2 and MMP-9;
and the interaction of CHX with the essential sulfhydryl
groups and/or cysteine present in MMP-active sites in
the case of MMP-8.
Therefore, it seems that 0.002% CHX is sucient to
prevent host-derived MMPs from degrading exposed
collagen brils, at least during the time-period evaluated
in the present investigation (i.e. 6 months). The use of
low CHX concentrations (0.002%) conveys a distinct
advantage in that it is not toxic to human periodontal

cells (24) or odontoblast-like cells (25); therefore, this low

CHX concentration can be regarded as a potential
chemical agent for use as a cavity cleanser.
The high substantivity of CHX may explain why the
application time did not have a signicant eect for
either the 0.002 or 2% CHX solutions. The term
substantivity has been much more correlated in the literature to the residual antibacterial activity of CHX in
human dentin (32, 33) than to the inhibitory eect of
CHX on MMP activity. Chlorhexidine digluconate is
one of the most commonly used antimicrobial agents
because it retains a therapeutic eect for a prolonged
period of time. The substantivity of CHX is related to the
release of positively charged molecules from CHXtreated surfaces and its ability to adsorb onto surfaces of
the oral cavity (33, 34). Theoretically, this can also occur
in the demineralized exposed collagen brils, and is the
explanation for the bonds being preserved after longterm water exposure.
One may suggest that CHX is likely to bind to collagen
brils at a very fast rate, and thus even short periods of
time, such as 15 s, seem to be sucient to guarantee such
binding. This hypothesis was raised on the basis of the
results of a recent investigation (28). A 2% CHX solution containing phosphoric acid was able to maintain the
stability of the resindentin bond of two simplied etchand-rinse adhesives after 6 months of water storage,
suggesting that only a few seconds of contact with CHX
might be sucient to inhibit the MMP activity, corroborating the ndings of the present investigation.
Several studies have so far evaluated the eect of CHX
in the immediate and long-term durability of dentin
bonds (1922), but among them only one has measured
the amount of silver nitrate deposits within the hybrid

Chlorhexidine on dentin adhesion

layer (28). It was previously hypothesized that CHX

could reduce the amount of SNU after 6 months but that
improvements would not be expected immediately after
treatment. Immediately after treatment with CHX, very
few silver nitrate deposits were observed within the
hybrid layer compared with the control, as shown by
Stanislawczuk et al. (28). As CHX has cationic properties it can bind to sites containing collagen, as
hypothesized by Carrilho et al. (22) and possibly to
calcium at the bottom at the hybrid layer, reducing the
nanopores within the hybrid layer to which silver nitrate
can deposit. However, this still deserves further investigation.
One cannot rule out the fact that despite the advantages of using 2% CHX, its use for 60 s after acid etching
includes another bonding step during the restorative
procedure and this works against the clinicians need for
simplication (23). In this research, the application time
of CHX was reduced (15 s) and this reduction in application time did not jeopardize the benets of CHX in the
preservation of the dentin bonds.
Therefore, the results of the present investigation
suggest that a low concentration of CHX, applied for
15 s, is sucient to preserve dentin bonds for at least
6 months under the laboratory conditions of this study.
However, further in vivo studies are needed to clarify
whether the use of a 0.002% CHX solution for 15 s is
able to preserve resindentin bonds after long-term
function.
Acknowledgements This study was partially supported by
CNPq grants 473101/2006-8 and 305870/2004-1. The authors
would like to thank FGM Dental Products for the donation of
the composite resin used in this study.

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