Inactivation of Matrix-bound Matrix
Metalloproteinases by Cross-linking

Agents in Acid-etched Dentin

DLS Scheffel � J Hebling � RH Scheffel
K Agee � G Turco � CA de Souza Costa

D Pashley

Clinical Relevance

Cross-linking agents used in clinically applicable periods of time are capable of inactivating
matrix-bound matrix metalloproteinases (MMP) in demineralized dentin. Such treatment
may render the hybrid layer less prone to degradation over time and produce long-lasting
resin-dentin bonds.

SUMMARY

Objectives: Published transmission electron
microscopy analysis of in vitro resin-dentin
bonds shows that, after 44 months, almost 70%

of collagen fibrils from the hybrid layer disap-
pear. Matrix metalloproteinases (MMPs) play
an important role in that process and are
thought to be the main factor responsible for
the solubilization of dentin collagen. There-
fore, this study aimed to evaluate the inacti-
vation of matrix-bound MMPs by two different
cross-linking agents, carbodiimide (EDC) or
proanthocyanidin (PA), or the MMP-inhibitor,
chlorhexidine (CHX), on acid-etched dentin
using a simplified MMP assay method.

Materials and Methods: Dentin beams (23136
mm) were obtained from mid-coronal dentin of
sound third molars and randomly divided into
six groups (G) according to the dentin treat-
ment: G1: Deionized water (control); G2: 0.1 M
EDC; G3: 0.5 M EDC; G4: 0.5 M EDC + 35%
hydroxyethyl methacrylate (HEMA); G5: 5%
PA; and G6: 2% CHX. The beams were etched
for 15 seconds with 37% phosphoric acid,
rinsed, and then immersed for 60 seconds in
one of the treatment solutions. The data were
expressed both in absorbance values at 412 nm
and in MMP-9 activity equivalents. The total
MMP activity of dentin was analyzed for one

Debora Lopes Salles Scheffel, DDS, PhD student, Araraquara
School of Dentistry, Sao Paulo, Brazil

Josimeri Hebling, DDS, PhD, FOAR UNESP, professor,
Department of Orthodontics and Pediatric Dentistry, Rua
Humaitá, Araraquara, Brazil

Regis Henke Scheffel, DDS, private practice, Sao Paulo, Brazil

Kelli Agee, BS, Laboratory Manager, Georgia Regents
University, Augusta, GA, USA

Gianluca Turco, BS, PhD (Mechanical Engineering), School of
Dentistry, University of Trieste, Trieste, Italy

Carlos de Souza Costa, DDS, PhD, professor and chair,
Department of Oral Biology, Ararquara School of Dentistry,
Sao Paulo, Brazil

*David Pashley, DMD, PhD, emeritus regents’ professor,
Department of Oral Biology, Georgia Regents University,
College of Dental Medicine, Augusta, GA, USA

*Corresponding author: 1120 15th Street, CL2112, College
of Dental Medicine, Augusta, GA 30912-1129, USA; e-mail:
dpashley@gru.edu

DOI: 10.2341/12-425-L

�Operative Dentistry, 2014, 39-2, 152-158



hour by colorimetric assay (Sensolyte). Data
were submitted to Wilcoxon nonparametric
test and Mann-Whitney tests (p.0.05).

Results: All experimental cross-linking solu-
tions significantly reduced MMP activity from
79.8% to 95.2% when compared to the control
group. No difference was observed among 0.1
M EDC (84.8%), 5% PA (87.6%), and 2% CHX
(79.8%). Addition of 35% HEMA to 0.5 M EDC
produced inactivation (95.2%) that was similar
to that of 0.5 M EDC alone (92.7%).

Conclusion: Dentin treatment with cross-link-
ing agents is effective to significantly reduce
MMP activity. Mixing 0.5 M EDC and 35%
HEMA did not influence EDC inhibitor poten-
tial.

INTRODUCTION

Since the introduction of the total-etching concept by
Fusayama1 in 1980, the effects of acid-etching of
dentin have been subject to many studies. Etching
dentin with 32-37% phosphoric acid removes the
mineral content of the top 10 lm of dentin and
exposes the collagen fibrils of the matrix, thereby
creating space for monomer infiltration to achieve
micromechanical retention of adhesive resins.2 Al-
though acid-etching of dentin provides satisfactory
initial bond strength, those bond strengths fall over
time, raising concerns about the long-term stability
of adhesive-resin restorations.3

Resin/dentin bond degradation is a complex
process that is not completely understood, involving
the hydrolysis of both the resin and the collagen
component of the hybrid layers. Acid-etched dentin
contains bound matrix metalloproteinases (MMPs)-
2, -3, -8, -9, and -20 and cathepsins4,5 in their active
forms. These enzymes are exposed and activated by
acid-etching and can slowly degrade collagen fi-
brils6-9 within the hybrid layer, resulting in a
significant bond strength loss of 36-70% between
12 and 14 months.10,11

In order to reduce the activity of these proteases
and preserve the long-term integrity of adhesive
interfaces, chlorhexidine (CHX) has been used as a
nonspecific inhibitor of MMPs.6-9,11-13 CHX is also an
effective inhibitor of cysteine cathepsins.14 However,
this substance is water-soluble and may undergo
leaching from the hybrid layer, which impairs its
long-term anti-MMP effectiveness.13

A new alternative to the inhibition of proteases by
inhibitors is the treatment of demineralized dentin
with cross-linking agents that can inactivate the

catalytic site of these enzymes.15 The cross-linker 1-
ethyl-3-[3-dimethylaminopropyl] carbodiimide
(EDC) is capable of forming covalent peptide bonds
between proteins by activating the free carboxyl
groups of glutamic and aspartic acids present in
protein molecules.16,17 This results in the formation
of a O-acylisourea intermediate that reacts with the
epsilon amino group of lysine or hydroxylysine in an
adjacent polypeptide chain to form a stable, covalent
amide bond. The only by-product of the reaction is
urea,18,19 which is water-soluble and easily removed
from dentin by water rinsing. Furthermore, 0.5 M
EDC shows no transdentinal cytotoxicity on odonto-
blast-like cells (Scheffel and others, unpublished
data) and is able to increase the mechanical
properties of the collagen matrix.20

Other cross-linking agents, such as the proantho-
cyanidins (PAs), are polyphenolic natural products
composed of flavan-3-ol subunits linked mainly
through C4-C8 (or -C6) bonds.21 This substance is
widely present in fruits, vegetables, nuts, seeds,
flowers, and barks and shows numerous biological
activities, such as antioxidant capacity,22 antimicro-
bial effects,23 anti-inflammatory properties,24 posi-
tive effects on cardiovascular diseases,25 and
antiallergic activity.26

Thus, the purpose of this study was to evaluate the
inactivation of matrix-bound MMPs by topical
application of cross-linking agents on acid-etched
dentin. The null hypothesis was that cross-linker–
treated and –untreated dentin do not differ regard-
ing MMP activity.

MATERIALS AND METHODS

Thirty extracted human third molars were obtained
from 18-21-year-old patients with informed consent
under a protocol approved by the Georgia Regents
University. The teeth were stored frozen until
required. After thawing, the enamel and superficial
dentin were removed using an Isomet saw (Buehler
Ltd, Lake Bluff, IL, USA) under water cooling. One
1-mm-thick dentin disk was produced from the
midcoronal dentin of each tooth. Then 60 dentin
beams (23136 mm) were sectioned from the dentin
disks. One such beam was placed in each well. This
represents 40 mm2 of dentin, which is equivalent to a
Class I cavity prepared in a mandibular first molar 2
mm into the dentin and 3 3 4 mm in dimension. The
beams were etched by dipping them into 37%
phosphoric acid (pH, �0.5) for 15 seconds and then
copiously rinsing with deionized water for 15
seconds. The beams were randomly divided into six
groups (n=10) according to the dentin treatment, as

Scheffel & Others: Matrix-bound MMP Inactivation of Acid-etched Dentin 153



follows: G1: Deionized water (positive control) (pH
6.73); G2: 0.1 M EDC (pH 6.07); G3: 0.5 M EDC (pH
6.24); G4: 35 vol% hydroxyethyl methacrylate (HE-
MA) in water þ 0.5 M EDC (pH 6.34); G5: 5% PA
(Polyphenolics Inc, Madera, CA, USA) (pH 5.2) in
phosphate-buffered saline (pH 6.0); and G6: 2 vol%
CHX digluconate (negative control) in water (pH
6.43). All beams were dipped in the treatment
solutions for 60 seconds and rinsed with distilled
water for 10 seconds, except for 2% CHX, in which
case the beams were only blot dried. After the
treatment, each beam was placed in a 200 lL/well
containing generic MMP substrate (Sensolyte Ge-
neric MMP colorimetric assay kit; catalog No. 72095,
AnaSpec Inc, Fremont, CA, USA) for 60 minutes at
258C in a 96-well plate. At the end of 60 minutes, the
total MMP activity was determined by measuring
the absorbance of the wells at 412 nm in a plate
reader (Synergy HT microplate reader, BioTek
Instruments, Winooski, VT, USA) against appropri-
ate blanks. All chemicals were purchased from
Sigma/Aldrich Chemical Co. The generic MMP assay
uses a proprietary thiopeptide to assay MMP-1, -2,
-3, -7, -8, -9, -12, -13, and -14. Thus, the kit measured
the total endogenous MMP activity of dentin, with
the exception of MMP-20 (enamelysin). A standard
curve of absorbance of the substrate vs rh MMP-9
activity (ng) was constructed to permit expression of
total MMP activity in MMP-9 equivalents. The rh
MMP-9 was activated using trypsin at a final
concentration of 10 lg/mL, pH 7.4, at 378C for two
hours. Then the trypsin was inactivated by addition
of trypsin inhibitor at a final concentration of 100 lg/
mL. Human recombinant MMP-9 was purchased
from Calbiochem (catalog No. PF038; Billerica, MA,
USA). Its specific activity was 1300 pmoles/mg.

Statistical Analysis

For determination of MMP activity, the absorbance
data set was submitted to Wilcoxon nonparametric
test and Mann-Whitney test at the 5% level of
significance. The percentage of MMP activity inhi-
bition was calculated based on the water control
group MMP activity and MMP-9 equivalents (ng/
well) based on the rh MMP-9 curve.

RESULTS

When mineralized dentin beams were dipped in 37
wt% phosphoric acid for 15 seconds and then rinsed
with water, the top 8-10 lm of the beams were
completely demineralized (Figure 1). When etched
dentin beams were dipped in water (control) and
then dropped into the generic MMP substrate, the

absorbance at 412 nm gradually increased to 0.51

(60.138) over 60 minutes. That value was considered

to represent 100% of the total MMP activity in the

etched dentin, and it was used to calculate the

Figure 1. Confocal laser scanning microscope (CLSM) images of the
etched layer shown dyed green on the surface of a dentin beam.
Dentin beams were etched in 37% phosphoric acid for 15 seconds
and then rinsed with deionized water for 60 seconds and labeled for
five hours, respectively, with 1% w/v fluorescein isothiocyanate (FITC)
in anhydrous dimethyl sulfoxide (DMSO) and 1% w/v xylenol orange
(XO) in water. The two fluorochromes selectively label collagen (FITC)
and the mineralized matrix (XO), respectively. Prior to CLSM
observation, the slabs were rapidly blotted with absorbent paper to
remove the excess of fluorochrome, mounted on glass slides, and
promptly examined. Samples were scanned in two-channel fluores-
cence mode with both 488 nm excitation–525 nm emission (green
channel) and 546 nm excitation–580 nm emission (red channel),
respectively, for FITC and XO labeling. (A) 103 projection of 53
images (final Z-stack thickness: 346 lm); the sample was intentionally
tilted to highlight the peripheral distribution of demineralized collagen.
(B) 1003 image of the border between demineralized surface collagen
fibrils (etched layer) and underlying mineralized dentin matrix.

154 Operative Dentistry



percentage of MMP activity inhibition of the inves-

tigated cross-linking agents and CHX. A standard

curve of substrate absorbance at 60 minutes vs ng of

rh MMP-9 is shown in Figure 2. All cross-linking

agents significantly reduced MMP activity in acid-

etched dentin after 60 seconds of topical treatment

(Table 1). The percentage of MMP inhibition for the

EDC solutions, PA, and CHX ranged from 79.8% for

2 wt% CHX to 95.2% for 0.5 M EDC þ 35% HEMA

(Table 1). There was no statistical difference in MMP

activity when 0.1 M EDC, 5% PA, and 2% CHX were

compared (Table 1). When 0.5 M EDC was mixed

with 35% HEMA to simulate the composition of an

adhesive primer, the HEMA did not interfere with

that cross-linker in inactivating the total MMP

activity of acid-etched dentin. When the absorbances

of the MMP activity of acid-etched dentin were

expressed in MMP-9 activity equivalents, the total

MMP activity of acid-etched dentin was equivalent

to 6.10 (61.93) ng of MMP-9 per 23136 mm of acid-

etched dentin.

DISCUSSION

The conventional method for analyzing the total
bound MMP activity using Sensolyte Generic MMP
colorimetric assay kit includes the complete demin-
eralization of dentin beams for 18 hours with 10%
phosphoric acid.27 The current study used a simpli-
fied MMP assay method in which the dentin was
acid-etched for 15 seconds with 37% phosphoric acid.
That avoids the complete dentin demineralization
and reproduces more closely the surface demineral-
ization of dentin that is completed during etch-and-
rinse bonding procedures. The complete demineral-
ization of the dentin beam creates a much deeper
collagen area (23136 mm) to be infiltrated by the
cross-linking solutions and adhesive resins. Clinical-
ly, acid-etching of dentin by 37 wt% phosphoric acid
for 15 seconds only demineralizes dentin to a depth
of 8-10 lm (Figure 1). Such relatively thin zones of
demineralized dentin are easily saturated by test
solutions within seconds. Nevertheless, this tech-
nique does not reproduce all in vivo conditions, such
as the presence of pulpal pressure and the outflow of
dentinal fluid.

The hybrid layer is composed of 30 vol% collagen28

(primarily type I), while the other 70% corresponds
to resin and residual solvent.2 The collagen fibril
network acts as an anchorage to resin, enabling the
retention of adhesive restorations. However, trans-
mission electron microscopy analyses revealed that
almost 70% of collagen from the adhesive interface
disappears after 44 months of water storage.29

Proteases such as metalloproteinases (MMPs) and
cysteine cathepsins are thought to be responsible for
collagen fibril enzymatic degradation via hydroly-
sis.30

Exogenous MMP inhibitors have been tested in
order to reduce protease activity and prolong the
durability of resin-dentin bonds. CHX was the first

Figure 2. Standard curve of rh matrix metalloproteinase (MMP)-9
activity (ng/well) vs absorbance at 412 nm after 60 minutes.

Table 1: Absorbance, Percent Inactivation/Inhibition of Total Matrix-bound Matrix Metalloproteinase (MMP) Activity in Dentin, and
MMP-9 equivalent (ng/dentin beam)1

Demineralized Dentin Treatment Absorbance (412 nm) MMP Inhibition, % MMP-9 Equivalent, ng

Water (control) 0.515 (60.138) A 0 D 6.10 (61.93) A

0.5 M EDC 0.038 (60.014) CD 92.7 (62.6) AB 0 (60.19) CD

0.5 M EDC þ 35% HEMA 0.025 (60.016) D 95.2 (63.0) A 0 (60.22) D

0.1 M EDC 0.078 (60.042) BC 84.8 (68.2) BC 0 (60.59) BC

5% PA 0.064 (60.035) BC 87.6 (66.7) BC 0 (60.49) BC

2% CHX 0.104 (60.031) B 79.8 (66.0) C 0.37 (60.43) B

Abbreviations: CHX, 2 wt% chlorhexidine digluconate; EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; HEMA, 2-hydroxyethyl methacrylate; PA,
grape seed extract containing proanthocyanidins.
1 Values are mean (6standard deviation) absorbance, % inhibition of total MMP activity of dentin measured by SensoLyte substrate (AnaSpec Inc, Fremont, CA, USA),
and MMP-9-equivalent (ng of MMP-9/dentin beam). Within each column, groups identified by different letters are significantly different (Mann-Whitney, p,0.05).

Scheffel & Others: Matrix-bound MMP Inactivation of Acid-etched Dentin 155



MMP inhibitor proposed for such a purpose during
bonding to dentin.31 It has been largely studied as a
nonspecific MMP12 and cathepsin inhibitor.14 CHX
adsorbs on dentin and decreases hybrid layer
degradation in vitro7,32-34 and in vivo.6,8,11,13 How-
ever, this inhibitor is soluble in water and can slowly
leach from the adhesive interface over time,13 since
no chemical bond is established between the CHX
molecule and the collagen fibril.

One of the mechanisms proposed to explain how
MMPs degrade collagen is that these proteases
unwind collagen molecules when they bind to them.
By doing that, the endogenous protease’s active site
is allowed sufficient space to attack the specific
glycine-isoleucine peptide bond in peptide chains.35-

37 Cross-linking agents stiffen collagen polypeptides
so that they cannot unwind, and they can also
inactivate the catalytic site of proteases38 by creating
a new peptide bond across adjacent peptides. Hence,
it is reasonable to expect that MMP inactivation by
cross-linking agents should last much longer than
the inhibition of proteases by matrix-bound CHX.
EDC and PA were first used to increase the modulus
of elasticity of collagen and make it more difficult for
MMPs to unwind the collagen triple-helix structure.
However, EDC and PA are still not capable of
increasing the stiffness of collagen in clinically
relevant periods of times, such as 30 seconds and
60 seconds (Scheffel and others, unpublished data).

Despite the long application times that cross-
linking agents require to increase collagen stiff-
ness,20 they are effective against MMPs in 60
seconds. The results of this study require rejection
of the tested null hypothesis. All investigated
solutions significantly decreased MMP activity in
acid-etched dentin within 60 seconds. Five percent
PA, 0.1 M EDC, and 0.5 M EDC were all able to
inactivate more than 84% of the total active MMPs.
EDC activates the free carboxylic acid groups of
glutamic and aspartic acids without introducing
additional methylene groups. MMPs-2 (EC
3.4.24.24), -8 (EC 3.4.24.34), -9 (EC 3.4.24.35), and
-20 (EC 3.4.24), the MMPs reported to be in dentin
matrix, have glutamic acid in their active sites in
positions 404, 218, 402, and 227, respectively,
allowing EDC to react to those sites. Additionally,
the concentrations of EDC tested in this study did
not produce any evidence of transdentinal cytotoxic
effect on odontoblast-like cells in separate experi-
ments (Scheffel and others, unpublished data),
where they were also used at 0.1 M and 0.5 M,
making EDC safe for in vivo application. The use of
0.5 M EDC was designed to accelerate its rate of

diffusion into demineralized dentin. That EDC
concentration is far in excess of the amount of
protein in demineralized dentin. It is likely that only
1-2% of the EDC could react with proteins in 60
seconds. This would only generate 0.005-0.01 M of
urea, which is not sufficient to denature any
proteins. Denaturing concentrations of urea require
2-8 M.39,40

PA is a natural plant cross-linking agent. The
mechanism of cross-linking is not completely under-
stood. There are four different theories to explain
how PA interacts with proteins. They include
covalent,41 ionic,42 hydrogen bonding,43 and hydro-
phobic interactions.44 This substance has been
reported to increase the stiffness of demineralized
dentin45 and to inhibit the progression of artificial
root caries.46,47 Additionally, scanning electron mi-
croscopy of demineralized dentin collagen treated
with 15% PA for periods shorter than 120 seconds
showed a homogeneous and regular collagen fibril
arrangement, regardless of the surface moisture
condition.38 That result indicates that, in addition
to acting as MMP inhibitor cross-linking agents, it
can stiffen demineralized dentin sufficiently to
minimize the risk of collagen network collapse
resulting from air-drying. However, the PA solution
has a dark color, which stains the dentin despite
water rinsing. That could be a drawback for the
clinical use of this cross-linker. Its rapid, complete
inactivation of matrix-bound MMPs in dentin indi-
cates that more research should be done to try to
isolate an uncolored fraction of the PA.

When 0.5 M EDC was solubilized in 35 vol%
HEMA, there was no reduction in its ability to
inactivate all of the MMPs in dentin. That is, it was
as effective as 0.5 M EDC alone. Since HEMA is an
important component of adhesives, it may be
possible to mix EDC with HEMA and other primer
components in etch-and-rinse adhesive systems to
inactivate MMPs during bonding. However, it is not
known whether EDC influences adhesive polymeri-
zation. Further studies are still needed to demon-
strate the effects of short-time application of cross-
linking agents over time in vitro and in vivo.

CONCLUSIONS

Dentin treatment with cross-linking agents is effec-
tive in significantly reducing MMP activity; 0.5 M
EDC showed the best results. Mixing 0.5 M EDC and
35% HEMA did not influence EDC cross-linking of
MMPs, indicating that EDC could be added to
primers in adhesive systems.

156 Operative Dentistry



Acknowledgements

This study was supported, in part, by R01 DE015306 from the
NIDCR (PI. David H. Pashley) and CNPq No. 305204/2010-6
and FAPESP 2012/08866-4 (PI. Josimeri Hebling). The
authors are grateful to Mrs. Michelle Barnes for her secre-
tarial support.

Conflict of Interest

The authors of this manuscript certify that they have no
proprietary, financial, or other personal interest of any nature
or kind in any product, service, and/or company that is
presented in this article.

(Accepted 1 April 2013)

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158 Operative Dentistry