Eur J Oral Sci 2010; 118: 494–501  2010 Eur J Oral Sci

DOI: 10.1111/j.1600-0722.2010.00758.x European Journal of

Printed in Singapore. All rights reserved
Oral Sciences

Jan De Munck1, Atsushi Mine1,

Enzymatic degradation of adhesive– Philippe E. Van den Steen2, Kirsten
L. Van Landuyt1, Andr Poitevin1,
dentin interfaces produced by mild Ghislain Opdenakker2, Bart Van
Meerbeek1

self-etch adhesives 1
Department of Conservative Dentistry, Leuven
BIOMAT Research Cluster, School of Dentistry,
Oral Pathology and Maxillo-facial Surgery,
Catholic University of Leuven, Leuven,
Belgium; 2Laboratory of Immunobiology, Rega
De Munck J, Mine A, Van den Steen PE, Van Landuyt KL, Poitevin A, Opdenakker G, Institute for Medical Research, Catholic
Van Meerbeek B. Enzymatic degradation of adhesive–dentin interfaces produced by mild University of Leuven, Leuven, Belgium
self-etch adhesives.
Eur J Oral Sci 2010; 118: 494–501.  2010 Eur J Oral Sci

Endogenous matrix metalloproteinases (MMPs) released by adhesive procedures may

degrade collagen in the hybrid layer and so compromise the bonding effectiveness of
etch-and-rinse adhesives. In this study, endogenous enzymatic degradation was eval-
uated for several simplified self-etch adhesives. In addition, primers were modified by
adding two MMP inhibitors: chlorhexidine, a commonly used disinfectant, but also a Dr Jan De Munck, Department of Conservative
Dentistry, Leuven BIOMAT Research Cluster,
non-specific MMP inhibitor; and SB-3CT, a specific inhibitor of MMP-2 and MMP-9. School of Dentistry, Oral Pathology and
Gelatin zymography of fresh human dentin powder was used to identify the enzymes Maxillo-Facial Surgery, Catholic University of
released by the adhesives. Micro-tensile bond strength (lTBS) testing was used to Leuven, Kapucijnenvoer 7, 3000 Leuven,
assess the mechanical properties of resin–dentin interfaces over time. In none of the Belgium
experimental groups treated with the mild self-etch adhesives was MMP-2 and/or
Telefax: +32–16–332752
MMP-9 identified. Also, no difference in the lTBS was measured for the inhibitor- E-mail: jan.demunck@med.kuleuven.be
modified and the control inhibitor-free adhesives after 6 months of water storage. It is
concluded that in contrast to etch-and-rinse adhesives, the involvement of endogenous Key words: dental adhesive; dentin; matrix
metalloproteinase; SDS–PAGE; self-etch
MMP-2 and MMP-9 in the bond-degradation process is minimal for mild self-etch
adhesives. Accepted for publication May 2010

Currently, bonding to dentin is achieved by partially Material and methods

dissolving the dentin surface and infiltrating the pro-
duced porosities with resin (1). This results in a resin-
infiltrated collagen mesh, commonly referred to as the
Ôhybrid layerÕ (2). Endogenous enzymes [mainly matrix
metalloproteinases (MMPs)], released and activated by
adhesive procedures, may provide dentin with a low
collagenolytic activity (3). These enzymes can hydrolyse
the collagen fibrils that make up the hybrid layer and so
compromise the long-term bonding effectiveness. For
two-step etch-and-rinse adhesives, it was proven that
chlorhexidine and galardine to some extent enhance
bond durability (4–6).
Etch-and-rinse adhesives treat dentin aggressively and
completely demineralize the surface up to a depth of
5 lm. Mild self-etch adhesives demineralize dentin only
partially and to a depth of < 1 lm, probably releasing a
smaller amount of enzymes and at the same time
exposing less collagen vulnerable for hydrolysis (7). The
purpose of this study was to evaluate the process of
endogenous enzymatic bond degradation for mild self-
etch adhesives. The hypotheses tested were that (i) mild
self-etch adhesives extract and activate dentinal endoge-
nous MMPs and (ii) MMP-inhibitor-modified primers/
adhesives inactivate the proteinases potentially involved
and so prevent mechanical bond degradation.
Dentin extraction and gelatin zymography
Human third molars of patients younger than 25 yr of age
(obtained with informed consent and according to a pro-
cedure approved by the Commission for Medical Ethics of
the Catholic University of Leuven) were stored at 4 C in
water containing 0.5% chloramine and used within 1 month
after extraction. The teeth were rather extensively prepared
using a gypsum trimmer, a slow-speed diamond saw, and a
diamond bur in order to produce pure coronal dentin blocks
that were free from contamination with proteins from
enamel or pulp tissue. This ÔmiddleÕ dentin (8) was pulver-
ized using a water-cooled analytical grinder (A10; IKA,
Staufen, Germany). Directly after grinding, 50 mg of dentin
powder was mixed with 0.035 ml of a 35% solution of
phosphoric acid (H3PO4). This acid etching was stopped by
dilution with 1 ml of 100 mM Tris–HCl and brief centri-
fugation (1 min). This step was repeated three times. After
re-collection of the powder, the acid-treated dentin powder
was mixed by hand with 0.035 ml of the Scotchbond 1 XT
(3M ESPE, Seefeld, Germany) resin. For the self-etch
adhesives Clearfil Protect Bond (Kuraray, Tokyo, Japan)
and G-Bond (GC, Tokyo, Japan), untreated powder was
mixed by hand with 0.035 ml of the respective self-etch
primer and adhesive. Subsequently, the adhesive monomers
were removed by dilution in pure acetone followed by brief
centrifugation (1 min); this step was repeated three times.
 Enzymatic degradation and mild self-etch adhesives 495

After re-collection of the powder, the enzymes were inhibitors. The primer of Clearfil Protect Bond and the
extracted from the dentin powder with 60 ll of a sodium adhesives of G-Bond and Scotchbond 1 XT were mixed with
dodecyl sulfate (SDS) solution [100 mM Tris–HCl (pH 6.8), chlorhexidine diacetate powder (Fluka Sigma-Aldrich,
4% SDS, 20% glycerol, and 200 lg ml)1 of Bromophenol St Louis, MO, USA) to a final concentration of 0.05%, or
Blue as the tracking dye]. This solution was used as the with SB-3CT (Alexis Biochemicals, Lausen, Switzerland)
electrophoresis loading buffer for the sodium dodecyl solution (200 lM in ethanol) to a final concentration of
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) 10 lM. Additional specimens were stored for 3 months in
zymography that was carried out according to Masure water, and then stored for 60 min in 10% sodium hypo-
et al. (9). Briefly, samples were separated on 7.5% poly- chlorite (NaOCl); this non-specific deproteinizing treatment
acrylamide gels to which 0.1% (weight/volume) gelatin was served as a reference, with the maximum amount of organic
added and copolymerized. Stacking gels were 5% (weight/ interface material destroyed and removed, as a worst-case
volume) polyacrylamide and did not contain gelatin sub- scenario (15). Therefore, for each adhesive, three conditions
strate. Samples were separated by electrophoresis at 4 C (control, chlorhexidine-modified, and SB-3CT-modified) at
for approximately 16 h at 85 V. After electrophoresis, the four time-points (0, 3, 6, and 12 months of water storage)
gels were washed with Triton X-100 to remove SDS, then were obtained and subsequently analyzed using two-way
incubated for development of enzyme activity, stained with analysis of variance (anova) and Tukey–Kramer multiple-
Coomassie Brilliant Blue, and destained in methanol/acetic comparison tests. NaOCl-degraded groups were excluded
acid. Gelatinase activity was detected as unstained bands on from this analysis because they only served as reference
a blue background. Along with each test, an MMP-9 stan- values.
dard with known molecular weights (10, 11) was included to
identify the molecular weights of the test samples. Addi-
tionally, the respective primers were mixed with activated
MMP-2, to evaluate, by gel electrophoresis, the effect of the Results
primers on active MMP-2. Therefore, 5 ll of MMP-2
Identification of gelatinases in adhesively treated
solution (containing 1 ng of enzyme) was mixed with 1 ll of
primer, 9 ll of 100 mM Tris–HCl, and 15 ll of SDS loading dentin
buffer before SDS–PAGE. Gelatinase A (or MMP-2) was identified in the control
dentin powder and in dentin treated with H3PO4
Micro-tensile bond strength testing (Fig. 1A). Matrix metalloproteinase-9 was not found in
any of the samples. The enzymatic activity of the H3PO4-
Midcoronal dentin surfaces with a standardized smear treated dentin and of the solution containing active
layer were prepared using a high-speed medium-grit enzyme was retained in the presence of Scotchbond 1 XT.
(100 lm) diamond bur (12). Next, the adhesive was ap-
plied (see further) and a resin composite build-up (APX;
Kuraray, Kurashiki, Japan) was made. After 1 wk of
water storage at 37 C, the teeth were sectioned perpen-
dicular to the adhesive–tooth interface using an auto-
mated water-cooled diamond saw (accutom-50; Struers,
Ballerup, Denmark) to obtain 1 · 1 mm resin–dentin
sticks. After the assigned storage period (1 wk, or 3 or
6 months), two to four sticks from each tooth were at-
tached to a notched BIOMAT jig (13) and stressed at a
crosshead speed of 1 mm min)1, until failure, in an LRX
testing device (LRX; Lloyd, Leicester, UK) to determine
the micro-tensile bond strength (lTBS). The mode of
failure was determined light-microscopically at a magnifi-
cation of up to 50· using a stereo-microscope (MSA
166305; Wild Heerbrugg, Heerbrugg, Switzerland). Some
representative fracture surfaces were processed for
scanning electron microscopy (Feg-SEM, Philips XL30;
Philips, Eindhoven, the Netherlands), using common
scanning electron microscopy (SEM) specimen-processing
techniques, including fixation in 2.5% glutaraldehyde in
cacodylate buffer solution, dehydration in ascending
concentrations of ethanol, chemical drying using hexa-
methyldisilazane, and gold-sputter coating (14).
A B

Study design Fig. 1. Release and inactivation of gelatinases by primer/

The study methodology was applied to two ÔmildÕ self-etch
adhesives: a two-step self-etch adhesive (Clearfil Protect
Bond) and a one-step self-etch adhesive (G-Bond). A two-
step etch-and-rinse adhesive (Scotchbond 1 XT) served as a
control because it has been used in most studies investi-
gating the effect of MMPs on adhesion to dentin (4, 5). For
lTBS testing, the adhesives were also modified with MMP
adhesives. (A) Gelatin zymography of dentin powder and
dentin powder exposed to phosphoric acid (H3PO4) and the
three adhesive primers. Matrix metalloproteinase-2 (MMP-2)
was identified in the dentin, H3PO4, and Scotchbond 1 XT
groups. (B) Gelatin zymography of a known active MMP-2
solution mixed with phosphoric acid and the three adhesive
primers. Phosphoric acid and both self-etch adhesives inacti-
vated the MMP-2 solution.
496 De Munck et al.
Fig. 2. Micro-tensile bond strength (lTBS) in MPa: error bars denote 95% confidence intervals. Groups connected by the same
horizontal line are not significantly different. CHX, chlorhexidine.
When a mild self-etch primer was mixed with solution
containing active enzyme, both primers completely
inactivated the gelatinolytic activity (Fig. 1B).
Micro-tensile bond strength testing
The lTBS and respective failure analyses are summarized
in Figure 2 and Table 1. Adding an MMP inhibitor (either
chlorhexidine or SB-3CT) to the primer did not affect the
short-term lTBS for any of the adhesives tested.
For the Scotchbond 1 XT specimens, bond strengths
decreased over time (P = 0.0015). After 6 months of
water storage, the bond strengths were still somewhat
higher than the specimens treated with the non-specific
deproteinizing NaOCl solution. After 12 months of
water storage, the bond strengths were similar to (for the
control) or lower than (for the experimental groups)
those treated with NaOCl (Fig. 1). The decrease in
bonding effectiveness was different for each inhibitor
group (interaction factor P = 0.0188) and the highest
values after 12 months of water storage were observed in
the control group (20.7 MPa).
The bond strengths of the Clearfil Protect Bond
specimens showed a decrease over the 12-month storage
period (P < 0.0001). However, even after 12 months of
water exposure, the bond strengths of the Clearfil Protect
Bond specimens were still considerably higher than those
from the specimens deproteinized with NaOCl. Modi-
fying the primer with MMP inhibitors had no effect on
the lTBS (P = 0.3948).
For G-Bond, the bonding effectiveness was much lower
than for the multi-step adhesives. Moreover, the lTBS
decreased over time (P = 0.0002) and the largest decrease
was observed between the 3- and 6-month time-points.
After 12 months of water storage, the lTBS values were in
the same range as those measured for the specimens de-
proteinized with NaOCl. Modifying the primer with MMP
inhibitors had no effect on the lTBS (P = 0.0919).
Fracture analysis
For the Scotchbond 1 XT specimens, failure occurred
mostly mixed at baseline (Table 1), most often involving
failure in the adhesive resin or resin composite. After
1 wk of water storage, many collagen fibrils could be
observed in the areas of the fracture surface where the
hybrid layer was exposed. After 6 months of water
storage, mostly interfacial failures were observed
(Fig. 3A), irrespective of inhibitor modification. Failure
occurred mostly at the bottom of the hybrid layer and,
especially in the control group, the collagen fibrils in the
hybrid layer appeared to be degraded after 6 and
12 months of water storage.
For Clearfil Protect Bond, most failures occurred at
the resinous part of the interface for the baseline
specimens (Table 1), and even after 12 months of water
storage many fractures involved failures of the adhesive
resin. Over time, however, the interfacial failures
showed a greater tendency to occur towards the bottom
of the hybrid layer (Fig. 4D), leaving a deproteinized
surface similar to those in the NaOCl-treated groups.
Also, when viewed using a very high magnification, the
hybrid layer itself was not morphologically affected by
long-term water storage (Fig. 4E,F), although the
adhesive resin showed some signs of degradation.
Treatment with NaOCl resulted in a typical failure
pattern; at the outer rim of the fracture surface, the
specimen failed at the interface, exposing the hybrid
layer, while at the central portion, the specimen failed
within the adhesive resin.
For G-Bond, most failures occurred at the adhesive–
dentin interface, irrespective of the experimental condi-
tion (Table 1). After 1 wk, many collagen fibrils could
be observed in the exposed hybrid layer. After 3 months
of water storage, mostly no differences in failure mode
and ultra-morphology were observed compared with the
baseline group. After 6 and 12 months of water storage,
 Enzymatic degradation and mild self-etch adhesives 497

Table 1
Micro-tensile bond strength (lTBS) values for the different experimental groups

lTBS (MPa) Failure analysis

Storage
Group (months) Mean ± SD (n*) (MPa) D I R
a,b
Scotchbond 1 XT Control 0 41 ± 12.2 (16) 24 20 56
(no inhibitor) 3 32.7 ± 14.2 (8)a,b,c 0 45 55
n = 6à 6 23.4 ± 25.5 (14)b,c,d 21 70 9
12 20.7 ± 18 (18)c,d 12 70 18
NaOCl 16.8 ± 8.4 (7) 0 67 33
Two-way anova CHX n = 7à 0 28.5 ± 13.9 (18)a,b,c 8 63 29
Inhibitor P = 0.0015 3 23.7 ± 23.8 (10)b,c,d 0 75 25
Storage P < 0.0001 6 16.7 ± 17.2 (19)c,d 9 69 22
Interaction P = 0.0188 12 6 ± 6.6 (21)d 0 100 0
NaOCl 6.7 ± 9 (4) 0 100 0
SB-3CT n = 4à 0 50.3 ± 16.8 (10)a 0 33 67
3 17.5 ± 11.8 (10)c,d 0 100 0
6 11.8 ± 17.3 (10)c,d 0 100 0
12 3.2 ± 4.2 (10)c,d 0 100 0
Clearfil Protect Bond Control 0 55.3 ± 21.3 (12)I,II 34 0 66
(no inhibitor) 3 55.4 ± 14.2 (10)I,II,III 15 33 52
n = 4à 6 43.7 ± 14.3 (12)I,II,III,IV 10 60 30
12 36.8 ± 17.1 (12)II,III,IV 13 54 33
NaOCl 19.8 ± 7.2 (10) 0 83 17
Two-way anova CHX n = 4à 0 60.5 ± 16.6 (12)I 8 0 92
Inhibitor P = 0.3948 3 49.3 ± 10.2 (9)I,II,III,IV 10 23 67
Storage P < 0.0001 6 40.5 ± 17.5 (13)I,II,III,IV 0 63 37
Interaction P = 0.806 12 31.3 ± 16.9 (12)III,IV 0 67 33
NaOCl 17.9 ± 5.2 (10) 0 70 30
SB-3CT n = 5à 0 57.2 ± 19 (13)I,II 8 6 86
3 50.4 ± 22.8 (10)I,II,III 13 34 53
6 39.8 ± 19.6 (13)I,II,III,IV 5 32 63
12 24.3 ± 8.2 (12)IV 0 82 18
G-Bond Control 0 14.2 ± 8.5 (16)A,B,C 3 95 2
(no inhibitor) 3 12.8 ± 9.6 (10)A,B,C 0 75 25
n = 4à 6 6.8 ± 6.4 (19)B,C 0 95 5
12 4.8 ± 4.8 (17)B,C 0 100 0
NaOCl 6.5 ± 4.3 (15) 0 97 3
Two-way anova CHX n = 6à 0 13.9 ± 12.8 (16)A,B,C 4 93 3
Inhibitor P = 0.0919 3 23.9 ± 26.3 (8)A 0 69 31
Storage P < 0.0001 6 9 ± 10.4 (12)A,B,C 7 90 3
Interaction P = 0.2835 12 2.9 ± 2.7 (14)C 0 100 0
NaOCl 7 ± 2.9 (4) 0 90 10
SB-3CT n = 5à 0 20.4 ± 7.5 (12)A 3 89 8
3 19.2 ± 12.2 (10)A,B 5 85 10
6 7.7 ± 5.6 (12)A,B,C 0 100 0
12 8 ± 7.1 (12)A,B,C 0 100 0

Means with the same superscript letter are not significantly different (P < 0.05, Tukey–Kramer multiple comparisons).
anova, analysis of variance; CHX, chlorhexidine; D, dentin; I, adhesive–dentin interface; NaOCl, sodium hypochlorite; R, resin
(adhesive resin or composite); SD, standard deviation.
*Number of sticks used for this group.
Percentage of failure at this location.
àNumber of teeth used for this group.

by contrast, failure occurred mostly at the base of the

hybrid layer (Fig. 5B), although at many sites the hybrid
layer detached from the resin part, exposing the adhesive
resin and its characteristic droplets (Fig. 5C). The hy-
brid layer degraded over time; collagen fibrils disap-
peared and fracture surfaces appeared that were similar
to those of the NaOCl deproteinized groups. Ultra-
morphologically, however, no significant changes were
observed, apart from filler plug-out from the adhesive
resin.
Discussion
The first hypothesis, that mild self-etch adhesives
can extract endogenous MMPs from dentin, was
rejected, as in none of the dentin specimens treated
with mild self-etch adhesives MMP-2 or MMP-9
was detected (Fig. 1A). The second hypothesis, that
MMP-inhibitor-modified primers reduce the mechanical
deterioration of the interface, was also rejected, as no
difference between SB-3CT-modified or chlorhexidine-
498 De Munck et al.
A B
C D
E F
Fig. 3. Representative fracture surfaces of specimens bonded
with Scotchbond 1 XT. (A) Scanning electron photomicrograph
of a sodium hypochlorite (NaOCl)-treated micro-tensile bond
strength (lTBS) specimen. A typical fracture pattern was
observed; at the outer rim, the failure occurred mostly at the
interface, while in the inner part (dotted line), which had greater
protection against the deproteinizing solution, failure occurred
more in the adhesive resin. (B) Higher magnification of the
outer rim of (A). The specimen failed at the bottom of the
hybrid layer, leaving a deproteinized surface, although at some
locations, hybrid layer remnants can be observed. (C) After
6 months of water storage, most failures occurred at the
interface (C, insert), in contrast to mostly mixed failures at
baseline, often involving failure in the adhesive resin or resin
composite. After water storage, failure occurred more specifi-
cally at the top or the bottom of the hybrid layer, creating an
island-like pattern. (D) After 12 months of water storage, fail-
ure at the bottom of the hybrid layer was the predominant
finding, covered with some remnants of the hybrid layer, similar
to the NaOCl-exposed specimens. (E) Transmission electron
photomicrograph of a 12-month-stored SB-3CT-modified
lTBS beam. The specimen was stained to highlight the collagen
fibrils. Interfibrillar spaces appear somewhat widened, but the
staining was uniform and collagen banding can be easily
observed (F) Transmission electron photomicrograph of a
12-month-stored control lTBS beam. The specimen was stained
to highlight the collagen fibrils. In this section a gradient can be
observed, where the top part appears to be stained more
strongly than the bottom part. In the hybrid layer also some
zones were observed where the stain reacted more weakly with
the collagen substrate (open arrow). Closer to the bottom of the
hybrid layer (hand pointer), some areas were observed where
the collagen fibrils are considerably thinner, another sign of
collagen degradation. Ar, adhesive resin; Hb, bottom of the
hybrid layer; Ht, top of the hybrid layer; Hy, hybrid layer; I,
resin–dentin interface; Rt, resin tag.
modified primers and their respective controls was
observed after 12 months of water storage.
Matrix metalloproteinase-2 is deposited in dentin
during tooth development and is still present in mature
dentin (16). Recently, MMP-9 was also found in the
A B
C D
E F
Fig. 4. Representative fracture surfaces of specimens bonded
with Protect Bond. (A) Scanning electron photomicrograph of
an SB-3CT-modified baseline micro-tensile bond strength
(lTBS) specimen. At baseline almost no failures occurred at the
interface. (B) For all groups, the failure pattern shifted over
time towards more interfacial failures, as in this 6-month-stored
chlorhexidine-modified specimen (composite side of the beam).
In the grooves created by the diamond bur, some dentin frag-
ments can be observed on the fracture surface (hand-pointer).
(C) High magnification of a chlorhexidine-modified baseline
specimen that failed partly at the interface. As a result of the
numerous amounts of collagen fibrils visible, the specimen
failed within the hybrid layer. (D) Higher magnification of (B),
showing a more deproteinized surface than the baseline speci-
men (C), but not as deproteinized as the specimens exposed for
60 min to sodium hypochlorite (NaOCl). (E) Transmission
electron photomicrograph of a 12-month-stored control lTBS
beam. The specimen was stained to highlight the collagen fi-
brils. A Ôshag carpetÕ appearance of collagen fibrils extending
into the adhesive resin can be observed (white arrows). In the
bottom part of the adhesive resin, the nanofillers (but not the
larger filler particles, hand pointer) were plugged out during
ultra-microtomy, which should be attributed to hydrolysis of
the filler–matrix coupling (35). (F) Transmission electron pho-
tomicrograph of a 12-month-stored SB-3CT-modified lTBS
beam. Despite the high magnification, no ultra-morphological
differences with the baseline specimens were observed. Ar,
adhesive resin; CHX, chlorhexidine; D, dentin; Hb, bottom of
hybrid layer; Hy, hybrid layer.
dentin matrix, using gelatin zymography and immuno-
histochemical SEM/transmission electron microscopy
(TEM) analysis (17, 18). In contrast to MMP-2, MMP-9
is typically an inducible metalloproteinase, present in, for
example, neutrophils. This might have been the origin of
MMP-9 in the former study, as the zymographs suggest a
band at 120 kDa, which is probably the neutrophil
gelatinase-associated lipocalin (NGAL)–Gelatinase B
complex typical for neutrophils (19). As MMP-2 and
MMP-9 are the main proteolytic enzymes present in
dentin, we opted to use a gelatin zymography test (20)

A B
C
D
E F
Fig. 5. Representative photomicrographs of specimens bonded
with G-Bond. (A) Transmission electron photomicrograph of a
baseline control micro-tensile bond strength (lTBS) beam. At
the area of interaction with dentin, mostly the resin-impreg-
nated smear and only a tiny Ônano-interactionÕ zone were
observed (36). Note the abundance of nanofillers (arrow) in the
adhesive resin. For all G-Bond-treated specimens, failure
occurred mostly at the adhesive–dentin interface. After 1 week,
many collagen fibrils could be observed in the exposed hybrid
layer; some hybridized smear plugs were also apparent. (B)
After 6 months of water storage, the hybrid layer showed
extensive changes and failure occurred mostly at the base of the
hybrid layer. (C) At many sites the hybrid layer detached from
the resin part, exposing the adhesive resin and its characteristic
droplets, as shown on this composite part of a 12-month stored
chlorhexidine-modified specimen. (D) Sodium hypochlorite
(NaOCl) treatment resulted in typical fracture surfaces, with
different failure patterns at inner and outer parts of the fracture
surface (similar to Fig 3A). At the outer rim, the failure was
located at the base of the hybrid layer, as observed at high
magnification of this chlorhexidine-modified specimen. There-
fore, the appearance changed mainly between 3 and 6 months
of water storage; failure occurred more at the bottom of the
hybrid layer and collagen fibrils were no longer observed. This
resembled the specimens that were deproteinized with NaOCl.
(E) Transmission electron photomicrograph of a 12-month
stored control lTBS beam. In the bottom part of the adhesive
resin, most nanofillers were plugged out during ultra-microto-
my, a sign of hydrolytic degradation of the bond between filler
and resin (35). (F) Transmission electron photomicrograph of a
12-month-stored chlorhexidine-modified lTBS beam. Despite
the high magnification, no ultra-morphological differences with
the baseline specimens were observed. Ar, adhesive resin; CHX,
chlorhexidine; Ud, unaffected dentin; I, adhesive–dentin inter-
face; Hb, bottom of the hybrid layer.
that is very sensitive for gelatinases (up to 10 pg of
MMP-2 or MMP-9 can be detected). No enzyme activity
was detectable in the samples treated with mild self-etch
adhesives. However, stronger acidic treatments with
aH3PO4 solution, or with a strong self-etch adhesive
Enzymatic degradation and mild self-etch adhesives 499
(Adper Prompt L-Pop; 3M ESPE, data not shown),
resulted in the release of MMP-2 from dentin.
Nishitani et al. (21) measured gelatinolyic activity
using a fluorescent gelatin-conversion assay. When
dentin powder was treated for 15 s with H3PO4, the
gelatinolytic activity decreased. This finding is in contrast
to the results of our study, which demonstrated an
increase in MMP-2 activity. Moreover, when dentin
powder was treated for 5 min with self-etch adhesives,
the gelatinolytic activity increased, which again is in
contrast to the decrease in MMP-2 activity observed in
our study. These contrasting findings may be explained
by the fact that different fractions of MMPs exist in
dentin (16). The gelatinolytic activity of these fractions
might differ significantly, depending on the activation
state of the enzymes, explaining the paradoxical differ-
ence between activity analyses and zymographic tests.
In this study, two MMP inhibitors were used: SB-3CT
and chlorhexidine. SB-3CT is a specific inhibitor for
MMP-2 and MMP-9, which, by a mechanism-based
action, and thus very specifically, binds covalently to the
enzymeÕs active site and so blocks all gelatinolytic activity
(22). Because of its specificity, the gelatinolytic activity of
other enzymes, including other MMPs, will not be
inhibited by treatment with SB-3CT; this is in contrast to
treatment with galardine, which inhibits all MMPs and
even some other enzymes (6). SB-3CT is a potent inhib-
itor with a minimum inhibitory concentration (MIC) in
the nanomolar range. In this study, it was mixed with the
self-etch primer to a final concentration of 10 lM, high
enough to inactivate all MMP-2 and MMP-9 activities,
but low enough to maintain its specificity.
Chlorhexidine is an amphipathic molecule that binds
to several proteins by a cation-chelating mechanism. In
this way, it can act on ferritin, an iron-storage protein, to
destruct the cellÕs protection from oxyradicals (23). This
mechanism accounts for the excellent antiseptic proper-
ties of chlorhexidine. Through a similar cation-chelating
mechanism, chlorhexidine prevents the binding of metal
ions, such as zinc or calcium, to the MMP and so
inhibits its catalytic activity. However, at higher con-
centrations of chlorhexidine, the MMPs are probably
inactivated by protein denaturation. In the present
study, chlorhexidine was mixed with the self-etch prim-
ers to a final concentration of 0.05%. The concentration
of chlorhexidine was kept relatively low, to minimize
interference with the well-balanced monomer cocktail of
the self-etch adhesives. However, the chlorhexidine
concentration was still higher than the minimal amount
of chlorhexidine needed for complete inhibition
(0.0001% and 0.002% for MMP-2 and MMP-9,
respectively) (24). Another factor that might play a role
in this inhibition process is the release of calcium salts
through demineralization of the dentin surface. In con-
trast to etch-and-rinse adhesives, calcium salts are not
removed from the surface by rinsing, but are incorpo-
rated inside the primer/adhesive layer. A large amount
of calcium salts can prevent the chelation-mediated
inhibition of a low-concentrate chlorhexidine solution
(24), which might explain the inability of chlorhexidine
to prevent degradation in this study.
500 De Munck et al.
In most other studies, achlorhexidine solution is
applied to the etched dentin surface as a pretreatment
before resin infiltration (4, 5, 25, 26), which is less feasible
for self-etch adhesives because of the concurrent demin-
eralization and infiltration. Clinically, this protocol is
also less favorable because it introduces an additional
step to the procedure, thus increasing valuable chair-time.
Typically, chlorhexidine is used as a 0.2 or 2% solution in
water, which is applied to the etched dentin and dried, but
not rinsed off (4, 5, 25). The resultant concentration in the
hybrid layer is dependent on the specific technique used,
but is probably always much higher than in our study, as
a concentration of 0.002% prevents interface degradation
for up to 6 months (26). Acid etching of dentin produces
calcium salts and these are known to prevent the chela-
tion-mediated inhibition of a low-concentrate chlorhexi-
dine solution (24). This might interfere with the lower
concentration of chlorhexidine used in this study.
Scotchbond 1 XT is a two-step etch-and-rinse adhesive,
so the chemical composition is more challenging because
the infiltration, solvent removal, and polymerization to a
strong resin all have to be accomplished in a single step.
On top of this, Scotchbond 1 XT contains a high-
molecular-weight polyalkenoic acid copolymer that
accumulates on top of the hybrid layer (27, 28). This layer
hinders further adequate resin interdiffusion, leading to
hybrid layers consisting of collagen fibrils mainly infil-
trated by the low-molecular-weight 2-hydroxyethyl-
methacrylate (HEMA), polymerized to linear poly-
HEMA chains, and of residual water (solvent) that can be
removed only insufficiently (and/or kept in situ because of
the presence of HEMA). This very hydrophilic interface
composite is prone to rapid degradation, as observed in
the present study (Table 1) and in others (29–31). The
combination of poor infiltration of the adhesive (and
resultant low chlorhexidine concentration), the initial
lower concentration of chlorhexidine, and the partial
inhibition by residual calcium salts from acid etching,
might explain our unfavorable results when compared
with other studies employing this adhesive (5, 25).
The TEM observations in our study, by contrast, do
corroborate the literature to some extent (3, 5). After 6 and
12 months of water storage, staining of the Scotchbond 1
XT control specimens was weak (Fig. 5F) and showed
other signs of degradation (Fig. 5F), while fewer signs of
degradation were noted for the inhibitor-modified speci-
mens (Fig. 5E). A concentrated chlorhexidine solution
applied in a separate application step, as employed in
other studies (4, 5, 25), might also influence the bonding
effectiveness in ways other than MMP inhibition. At high
concentrations chlorhexidine has strong antibacterial
properties. However, other properties can also alter the
hydrolytic stability of the hybrid layer indirectly. For
example, the amphipathic properties of chlorhexidine
might interfere with the resin infiltration, or its cation-
chelating properties might interact with the calcium salts
remaining from the acid-etching procedure. However,
these side effects are not considered in the literature.
In this study, hydrolytic degradation of the interface
was observed, especially for the one-step self-etch adhe-
sive (Fig. 5). This degradation is probably related to the
ingression of water that can interfere with the van der
Waals forces established at the time of bonding. At a
later stage, hydrolytic cleavage of resins, as well as
collagen, might play a role. Despite the fact that enzymes
might play a role in these hydrolytic breakdown
processes, this study suggests that the degradation
observed for mild self-etch adhesives should not to be
attributed to endogenous MMP-2 or MMP-9, for the
following reasons. (i) The amount of MMPs released by
mild self-etch adhesives is below the picogram sensitivity
of the zymographic test used (Fig. 1A). Moreover, the
enzymes released can be de-activated by the self-etch
primer itself (Fig. 1B). (ii) Inhibition with a specific
(SB-3CT) or non-specific (chlorhexidine) inhibitor had
no effect at all on the long-term bond strengths.
(iii) Released and activated MMPs are only stable and
active for a few hours; however, the degradation in our
study was most prominent between 3 and 6 months after
application (Table 1). (iv) MMP-2 and MMP-9 are both
gelatinases and cannot degrade the collagen fibrils
directly, so the initial degradation step has to be
performed by another mechanism. (v) Some authors
suggest that uncured acidic components at the bottom of
the hybrid layer can decalcify dentin long after the initial
application (32). This might explain the degradation
observed between 3 and 6 months in the present study.
However, taking into account that the primers used do
inactivate MMPs (Fig. 1B), and that MMPs are not
efficient at low pH, it is very unlikely that MMPs are
responsible for the degradation observed.
For the etch-and-rinse adhesive and both self-etch
adhesives used in this study, the concentration of
chlorhexidine appears to be too low to have a significant
effect on the long-term dentin bond strengths. In studies
employing a similar set-up, higher concentrations were
better able to stabilize bond strengths over time (25, 33).
As the MIC of chlorhexidine, as well as of SB-3CT, is
much lower than the concentration used in this study, it is
probable that enzymes other than MMP-2 and MMP-9
are involved in this degradation process. For example, the
role of MMP-8 might be more important than previously
thought, as it is less affected by chlorhexidine (24), not
affected by SB-3CT, and present in mature dentin (34).
This might explain why galardine, which inhibits almost
all MMPs, seems to be able to prevent degradation (6), as
opposed to the SB-3CT used in our study
Acknowledgements – Philippe E. Van den Steen is a post-
doctoral fellow of the Fund for Scientific Research of Flanders.
K.L. Van Landuyt is Aspirant of the Fund for Scientific
Research of Flanders. We thank the Fund for Scientific
Research of Flanders and the ÔGeconcentreerde Onderzoeks-
ActiesÕ (GOA 2007-2011) for financial support. We thank all
manufacturers for the materials provided. We would also like to
thank Ilse Van Aelst for her excellent help with the gelatin
zymography.
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