Biomaterials & Bioengineering
Received July 21, 2010; Last revision September 9, 2010;
Accepted October 4, 2010
A supplemental appendix to this article is published elec-
tronically only at http://jdr.sagepub.com/supplemental.
© International & American Associations for Dental Research
A. Tezvergil-Mutluay1,2*, K.A. Agee2,
T. Uchiyama3, S. Imazato4,
M.M. Mutluay1, M. Cadenaro5,
L. Breschi5,6, Y. Nishitani7, F.R. Tay2,8,
and D.H. Pashley2
1Department of Prosthodontics and Turku Clinical
Biomaterials Center, University of Turku, Institute of
Dentistry, Lemminkaisenkatu 2, FI-20520, Turku, Finland;
2School of Dentistry, Medical College of Georgia, Augusta,
GA, USA; 3Department of Renascent Dentistry, Nihon
University School of Dentistry at Matsudo, Chiba, Japan;
4Department of Biomaterial Science, Osaka University
Graduate School of Dentistry, Osaka, Japan; 5Department of
Biomedicine, Unit of Dental Sciences and Biomaterials,
University of Trieste, Italy; 6Department of Biomedicine,
University of Trieste, Trieste, Italy, and IGM-CNR, Unit
of Bologna c/o IOR, Bologna, Italy;
Operative Dentistry, Okayama University Graduate School
of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama, Japan; and 8Department of Endodontics, School
of Dentistry, Medical College of Georgia, Augusta, GA,
USA; *corresponding author, email@example.com
J Dent Res 90(4):535-540, 2011
Matrix metalloproteinases (MMPs) bound to dentin contrib-
ute to the progressive degradation of collagen fibrils in
hybrid layers created by dentin adhesives. This study evalu-
ated the MMP-inhibiting potential of quaternary ammonium
methacrylates (QAMs), with soluble rhMMP-9 and a
matrix-bound endogenous MMP model. Six different QAMs
were initially screened by a rhMMP-9 colorimetric assay.
For the matrix-bound endogenous MMPs, we aged demin-
eralized dentin beams for 30 days in calcium- and zinc-
containing media (CM; control), chlorhexidine, or QAMs in
CM to determine the changes in dry mass loss and solubili-
zation of collagen peptides against baseline levels. The
inhibitory effects of QAMs on soluble rhMMP-9 varied
between 34 and 100%. Beams incubated in CM showed a
29% decrease in dry mass (p < 0.05), whereas beams incu-
bated with QAMs showed only 0.2%-6% loss of dry mass.
Significantly more solubilized collagen was detected from
beams incubated in CM (p < 0.05). It is concluded that
QAMs exhibited dentin MMP inhibition comparable with
that of chlorhexidine, but required higher concentrations.
KEY WORDS: dentin, hydroxyproline, MDPB, matrix
metalloproteinase, quaternary ammonium methacrylates.
layers formed during dentin bonding. Dentin matrix contains MMP-2, 8, 9, and
20 (Sulkala et al., 2007). During the acid-etching phase of bonding, MMPs
that are normally bound to mineralized collagen fibrils become exposed and
activated (Mazzoni et al., 2006; Tay et al., 2006). Inhibition of host-derived
MMPs may retard the degradation of resin-dentin bonds over time (Pashley
et al., 2004; Hebling et al., 2005; Carrilho et al., 2007a,b). Chlorhexidine
(CHX) has broad anti-MMP activity (Gendron et al., 1999) in addition to its
antimicrobial activity. When demineralized dentin was incubated with 0.2 wt%
CHX, collagen degradation was almost completely blocked (Pashley et al.,
2004; Breschi et al., 2010). However, CHX is water-soluble and may leach out
of hybrid layers and compromise its long-term anti-MMP effectiveness.
Quaternary ammonium compounds possess antimicrobial properties
(Pernak et al., 2001) and have been incorporated into dental resins (Imazato
et al., 1997; Xiao et al., 2008; Imazato, 2009; Namba et al., 2009). Similar to
CHX, these compounds are water-soluble and may leach out of bonded inter-
faces. The use of polymerizable quaternary ammonium methacrylates (QAMs)
such as 12-methacryloyloxydodecylpyridinium bromide (MDPB) (Imazato
et al., 1997, 2007; Imazato, 2009; Li et al., 2009) is advantageous in that they
can copolymerize with adhesive monomers. Since cationic CHX inhibits
MMPs, we speculate that cationic quaternary ammonium methacrylates
would also inhibit the endogenous dentin MMPs.
The purpose of this study was to screen the MMP-inhibiting potential of 6
quaternary QAMs using a soluble recombinant MMP-9 (rhMMP-9). The
effects of QAMs on matrix-bound endogenous MMPs were also evaluated in
demineralized dentin. The null hypothesis tested was that QAMs have no
effect on MMP inhibition.
atrix metalloproteinases (MMPs) bound to dentin are thought to con-
tribute to the progressive degradation of collagen fibrils in hybrid
MATERIAlS & METHODS
The chemical structures and abbreviations of the potential inhibitors are listed
in the Table. All inhibitors except MDPB were purchased from Sigma-Aldrich
The Inhibitory Effects of
Methacrylates on Soluble
and Matrix-bound MMPs
536 Tezvergil-Mutluay et al. J Dent Res 90(4) 2011
(St. Louis, MO, USA) and used as received. The MDPB was
used as a powder as received from the manufacturer (Kuraray
Medical Inc., Tokyo, Japan). All QAMs except for MDPB
were used as 30 wt% solutions for screening. The MDPB was
used as 5 wt% based on the concentration utilized in commercial
products containing this resin monomer (Imazato, 2009).
Chlorhexidine digluconate and OTX, a hydrophilic tertiary amine
photo-accelerator (Ye et al., 2008), were used at 0.2 wt%.
We used two screening levels to evaluate the inhibitory activ-
ity of QAMs on MMPs. The first level involved using soluble
human rhMMP-9 in a colorimetric assay. The second level used
matrix-bound endogenous MMPs present in completely demin-
Purified human rhMMP-9 and a generic MMP Assay kit
(Sensolyte, AnaSpec Inc., Fremont, CA, USA) were used. The
procedures involved incubating a constant concentration of
human MMP-9 with a proprietary chromogenic substrate from
the assay kit. The latter is a thiopeptolide that can be cleaved by
MMPs and collagenases to release a sulfhydryl group. The sulf-
hydryl group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to
produce a colored reaction product (2-nitro-5-thiobenzoic acid)
that can be detected spectrophotometrically at 412 nm.
The thiopeptolide substrate solution was diluted to 0.2 mM
with the assay buffer in a 1:50 volume ratio. The rhMMP-9 was
activated with trypsin (10 µg/mL) at 37°C for 2 hrs immediately
before the experiment, after which the trypsin was inactivated
with trypsin inhibitor. The assay was performed in quinta-replicates
in a 96-well plate for each experimental QAM inhibitor and
control. In each experimental well, 2 µL of rhMMP-9 (19.6 ng/
well), and 10 µL of the potential MMP inhibitor were pre-
incubated for 20 min to avoid the burst of MMP-9 activity that
occurs when all reagents are mixed together simultaneously.
After pre-incubation, additional assay buffer and 50 µL of the
Table. The Chemical Structures of the Materials Used in This Study
hydroxypropyl trimethylammonium chloride
METMAC[2-(Methacryloyloxy)ethyl] trimethylammonium chloride
MCMSMethacryloyl choline methyl sulfate
MAPTAC[3-(Methacryloylamino)propyl] trimethylammonium chloride
DDAC Diallyldimethylammonium chloride
J Dent Res 90(4) 2011 QAMs Inhibit Dentin MMPs 537
thiopeptolide solution were added to reach a total of 100 µL for
each well. We mixed the reagents completely by vibrating the
plate gently for 30 sec, and readings were taken every 10 min
for 60 min. Absorbance was measured at 412 nm by means of a
plate reader (Synergy HT, BioTek, Winooski, VT, USA).
Background absorbance of the controls was determined from
the mean corresponding absorbance readings and subtracted
from the readings of the positive control. For the QAMs, the
background of each QAM was determined separately and sub-
tracted from the wells containing the MMP-9 and thiopeptolide
substrate. The potencies of MMP-9 inhibition by the proprietary
MMP kit inhibitor (GM6001) and QAMs groups were expressed
as percentages of the adjusted absorbance of the “positive con-
trol”, which was taken to be 100% inhibition. Neither OTX nor
CHX could be used to inhibit MMP-9, because they created a
yellow color with the substrate, even in the absence of MMP-9.
We analyzed the data statistically to examine the effects of
QAMs on MMP-9 inhibition. Since the normality and homosce-
dasticity assumptions of the data appeared to be valid, % inhibi-
tion in the six groups was analyzed by one-way ANOVA and
Tukey multiple comparison tests at α = 0.05.
Matrix-bound MMPs in Demineralized Dentin
Ninety extracted unerupted human third molars were obtained from
18- to 21-year-old patients (Martin-de-las Heras et al., 2000a,b)
with their informed consent under a protocol approved by the
Human Assurance Committee of the Medical College of Georgia.
Ninety percent of the teeth had completely formed roots. None of
the teeth was carious. The teeth were stored at 4°C in 0.9% NaCl
supplemented with 0.02% NaN3 to prevent bacterial growth and
were used within 1 mo of extraction. The enamel and superficial
dentin of each tooth were removed from the crown by means of an
Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water cool-
ing. Dentin beams with dimensions 6 x 2 x 1 mm were sectioned
from the middle of each dentin disk (i.e., 90 beams).
The beams were completely demineralized in 10 wt% H3PO4
(pH 1) for 18 hrs at 25°C. We used digital radiography to con-
firm the absence of residual minerals. They were randomly
divided into 9 groups (N = 10). The groups included 6 QAMs
(ATA, MCMS, MAPTAC, METMAC, DDAC, 30 wt% each and
5 wt% MDPB), as well as 0.2 wt% CHX or OTX prepared in
a calcium- and zinc-containing complete storage medium
(CM), or CM only. The CM contained 5 mM HEPES, 2.5 mM
CaCl2.H2O, 0.05 mM ZnCl2, and 0.3 mM NaN3 (pH 7.4). Each
beam was placed in 1.0 mL of respective incubation medium in
individually labeled polypropylene tubes. The control groups
were incubated in CM only. The tubes were incubated in a
shaker-water bath (60 cycles/min) at 37°C for 30 days.
Matrix-bound MMP Activity Assessment
loss of Dry Mass over Time
This attribute was used as an indirect measure of MMP-
induced hydrolysis of endogenous matrix collagen (Carrilho et
al., 2009; Tezvergil-Mutluay et al., 2010). After incubation,
the beams were transferred to individually labeled, uncapped
polypropylene tubes and placed in a sealed desiccator contain-
ing anhydrous calcium sulfate (Drierite, W.A. Hammond
Company, Xenia, OH, USA). They were desiccated to a con-
stant weight within 8 hrs. The initial dry mass was measured
to the nearest 0.001 mg. After dry mass determination, the
desiccated dentin beams were rehydrated with water for 1 hr
(Agee et al., 2006) to their original dimensions before return-
ing to the corresponding polypropylene tubes containing the
original incubation medium. After 30 days of incubation, dry
mass was re-measured under the same conditions. Since the
normality and homoscedasticity assumptions of the data had
been violated, loss of dry mass from the demineralized dentin
beams was evaluated by Kruskal-Wallis one-way ANOVA and
Dunn’s multiple comparison tests at α = 0.05.
Solubilized Collagen Peptides
The other index of matrix degradation was determined by mea-
surement of the quantity of collagen peptide fragments that were
solubilized over the 30-day incubation period. The collagen in
demineralized dentin is insoluble type I collagen (Carrilho et al.,
2009; Tezvergil-Mutluay et al., 2010). Demineralization of the
mineralized dentin matrix with 10% phosphoric acid exposes and
activates endogenous MMPs, even though they remain bound to
the collagen (Martin-de-las Heras et al., 2000a). At the end of the
incubation period, a 400-µL quantity of the medium was col-
lected from each vial and placed in an individually labeled
ampule, diluted with an equal volume of 12 N HCl to give a final
concentration of 6 N HCl. The ampules were sealed (Ampulmatic,
Biosciences Inc., Allentown, PA, USA), and the media were
hydrolyzed at 120°C in an oil bath for 18 hrs. After hydrolysis,
the ampules were opened and placed in glass desiccators contain-
ing anhydrous Drierite and NaOH pellets to trap the HCl vapor
released from the hydrolysates as they were evaporated to dry-
ness. The hydroxyproline content of each hydrolysate was ana-
lyzed spectrophotometrically at 558 nm by the method of Jamall
et al. (1981). The hydroxyproline content was used to estimate
the percent of degraded collagen, assuming that 90% of the dry
mass of demineralized dentin consists of type I collagen, and that
dentin collagen contains 9.6 mass% of hydroxyproline (Butler,
2000). For each specimen, the solubilized collagen was expressed
as micrograms of hydroxyproline/mg of the dry mass of the
demineralized dentin before incubation. Since the normality and
homoscedasticity assumptions of the data had been violated, the
data were evaluated by Kruskal-Wallis one-way ANOVA and
Dunn’s multiple comparison tests at α = 0.05.
Inhibition of Soluble MMP-9
The inhibitory effects of QAMs on soluble rhMMP-9 varied
between 34 and 100% (Fig. 1). The inhibitory effects of
ATA, MCMS, and METMAC were from 97 to 100%, whereas
MAPTAC and DDAC inhibited MMP-9 by only 34 and 55%,
respectively. Inhibition by MDPB was approximately 89%,
which was comparable with the kit inhibitor, GM 6001
538 Tezvergil-Mutluay et al. J Dent Res 90(4) 2011
Inhibition of Matrix-bound MMPs
Loss of dry mass (Fig. 2) and solubilization of collagen peptides
(Fig. 3) over the 30-day incubation period showed significant
differences between the CM control and the QAMs. Dentin
beams incubated with QAM showed a 0.2-6% decrease in dry
mass, compared with a 29% decrease in the CM control. If one
assumes that the loss of dry mass is due to the collagenolytic
action of endogenous MMPs, the QAMs inhibited those enzymes
79.3- 99.3% compared with the CM control.
A similar trend was observed with the dissolution of collagen
peptides determined by hydroxyproline analysis. The beams
stored in the CM medium liberated 35 µg hydroxyproline/mg
dentin, whereas beams incubated in media containing inhibitors
liberated 0.2-1.5 µg hydroxyproline/mg dentin. Thus, over the
incubation period, endogenous MMPs released 39.9% of the
total hydroxyproline available in the control beams, which is
greater than one-third of the total insoluble collagen present in
those beams (Appendix). Conversely, the inhibitors exhibited
98.6-99.4% inhibition of the insoluble, matrix-bound MMPs.
In our previous papers, we have reported that acid-etching den-
tin powder with 37 wt% phosphoric acid (PA) has produced
65% (Pashley et al., 2004) or 98.1% inhibition (Mazzoni et al.,
2006) in collagenolytic activity. We speculated, at that time, that
those results indicated that although 37% PA uncovered the col-
lagen matrix and exposed MMPs, it may have denatured the
enzymes as soon as they were exposed. Later, work by Nishitani
et al. (2006) using self-etching adhesives showed that they too
exposed MMPs and activated them without denaturing the
enzymes, because the pH was only 2-3 instead of -0.37 for 37%
PA (Pashley et al., unpublished observations).
Preliminary unpublished experiments by Nishitani on the
effects of 37% PA on the gelatinolytic activity of mineralized
dentin powder revealed an initial enzyme activity of 126 ± 15
RFU/80 mg/24 hrs. When that dentin powder was acid-etched
with 37% PA for 15 sec, the enzyme activity fell to 61 ± 28
RFU/80 mg/24 hrs. He continued to incubate the acid-etched
dentin powder in pH 7.4 buffer at 37°C for 1, 2, and 4 wks. The
enzyme activity slowly rose from 61 ± 28 to 369 ± 58 to 873 ±
126 and to 962 ± 115 RFU/80 mg/24 hrs over the ensuing 4 wks
(n = 22 wells per time period). Clearly, acid-etching with PA
initially lowered MMP activity but later increased it 16-fold. We
now believe that acid-etching dentin with 37% PA raises the
local calcium ion concentrations so high that it forms insoluble
reaction products (i.e., CaHPO4) that precipitate as fine crystals
over exposed collagen and MMPs, thereby blocking access of
fluorescent substrates to the enzyme. Within days, these rela-
tively soluble reaction products can dissolve, thereby allowing
substrates access to the enzymes. Thus, the notion that 37% PA
denatures all MMPs may not be correct. Gelatinase A (MMP-2)
Loss of dry mass %
Figure 2. The loss of dry mass (%) from demineralized dentin beams
incubated over a 30-day period in the control (CM) vs. QAM-, CHX-,
or OTX-containing media. The loss of dry mass from each beam was
calculated as a percentage of the dry mass of that beam at baseline.
Groups with the same lower-case letter are not statistically significant
(p > 0.05) by Kruskal-Wallis and Dunn’s multiple comparisons. See the
Table for definition of abbreviations.
µgHYP/mg of dentin
Figure 3. Hydroxyproline contents derived from the aging media
(QAMs, CHX, and OTX) and the control (CM) after the 30-day
incubation period. For each specimen, the dissolved collagen from the
demineralized dentin beam was expressed as micrograms of
hydroxyproline/mg of the dry mass of the baseline demineralized
dentin. Groups with the same upper-case letter are not statistically
significant (p > 0.05) by Kruskal-Wallis and Dunn’s multiple
comparisons. See the Table for definition of abbreviations.
ATA 30%DDAC 30% GM 6001
Soluble MMP-9 inhibition (%)
Figure 1. A bar chart comparing the percentage inhibition of rhMMP-9
by the control (GM 6001) and 6 potential MMP inhibitors. Values are
means and standard deviations. Groups with the same letters on top
of the bars are not statistically different (p > 0.05). See the Table for
definition of abbreviations.
J Dent Res 90(4) 2011 QAMs Inhibit Dentin MMPs 539
is known to resist extremes in temperature and pH (Sulkala
et al., 2007).
This is why we can successfully completely demineralize
mineralized dentin beams in 10 wt% PA without denaturing
MMPs. If they were denatured, the beams would not lose dry
mass over time. They would not slowly solubilize collagen pep-
tides over time. However, beams incubated in simulation body
fluids did lose dry mass and did solubilize collagen. Beams
incubated in MMP inhibitors like chlorhexidine (Carrilho et al.,
2009) or polyvinylphosphoric acid (Tezvergil-Mutluay et al.,
2010) do prevent loss of dry mass and solubilization of collagen.
Thus, this simple in vitro model is very useful in such studies.
We may have altered the spectrum of MMPs by inadvertently
denaturing some MMPs, but sufficient activity remains to
screen a wide variety of potential MMP inhibitors.
Since the experimental QAMs inhibit both soluble rhMMP-9
and matrix-bound MMPs in the experimental model, the null
hypothesis that QAMs have no effect on MMP inhibition has to
be rejected. This is the first report on antibacterial QAMs being
effective in inhibiting MMPs. The ability of QAMs to inhibit
micro-organisms and MMPs at similar concentrations makes
them very attractive from a therapeutic perspective.
Quaternary ammonium compounds (QACs) and the related
biguanide CHX are cationic (i.e., they have positive charges).
Most QACs have only one positive charge, while biguanides
such as CHX have two fixed charges. Both mineralized and
demineralized dentin substrates have net negative charges, due to
the presence of trivalent phosphates in apatite and carboxylic
groups in the collagen molecules, respectively. Thus, when cat-
ionic QACs are applied to dentin, they bind electrostatically to
dentin and give it a net positive charge (Markowitz and
Rosenblum, 2010). This changes the 3-D configuration of pro-
teins, which rely, in part, on electrostatic attractions and repul-
sions to stabilize their ternary structure. We speculate that
non-specific binding of QACs and biguanides alters the configu-
ration of the active site of MMPs, making them unable to accept
the complementary peptide sequence for collagen. That QAM-
MMP complex remains inactive as long as the QAC is bound to
the MMPs and the insoluble collagen. The substantivity of CHX
has recently been shown to be greater than one might expect
(Kim et al., 2010). In a recent study, binding of 0.2% CHX to
acid-etched dentin resulted in the retention of 98% of the CHX
for up to 8 wks (Carrilho et al., 2010). Presumably, QAMs mixed
with adhesive monomers and polymerized in situ after their infil-
tration into demineralized dentin will be retained for years. That
assumption requires further validation with long-term studies.
The catalytic site of MMPs contains cysteine-rich repeats neces-
sary for substrate binding. The cysteine-rich repeat includes a glu-
tamic acid residue that is adjacent to a histidine molecule, both of
which are essential for its catalytic activity (Visse and Nagase,
2003). Since glutamic acid is a dicarboxylic acid, it contains a free
carboxylate group at physiological pH and retains a negative charge
even after formation of a peptide bond. We speculate that cationic
QAMs electrostatically bind to such negative charges. The presence
of cationic QAMs may sterically block the active site from adjacent
collagen peptides, thereby inhibiting MMP activity.
The resin monomer MDPB has strong bactericidal activity as
a monomer, and demonstrates cavity-disinfecting effects when
incorporated into a dentin primer (Imazato et al., 1997; Imazato,
2009). Previous research on adhesives containing MDPB has
shown more durable interfaces than conventional adhesives in
the oral environment (Nakajima et al., 2003; Donmez et al.,
2005). The increase in durability associated with MDPB-
containing adhesives may be partially explained by the inhibi-
tory effect of 5% MDPB on soluble and matrix-bound MMPs,
as demonstrated in the present study.
The results on soluble MMP-9 for some of the QAMs were
slightly different from those obtained with matrix-bound MMPs.
Dentin is known to contain MMPs-2, -8, and-9, and cysteine
cathepsins (Tersariol et al., 2010). The latter is a class of prote-
ases that can also hydrolyze collagen. The inhibitory potential of
QAMs may not be the same for all the endogenous enzymatic
components of dentin. This may account for the discrepancy in
results obtained for the soluble MMP-9 and the matrix-bound
MMP model. Further research is needed to identify the effects
of these inhibitors on cathepsins.
Within the limitations of this study, it may be concluded that
experimental QAMs and the commercially available QAM
(MDPB) demonstrate inhibitory effects on both soluble
rhMMP-9 and matrix-bound MMPs. However, the experimental
aliphatic QAMs require much higher concentrations to inhibit
dentin MMPs than the pyridine ring-containing MDPB. The
anti-MMP effects of QAMs on long-term durability of resin-
dentin bonds require further substantiation.
The authors are grateful to Kuraray Medical Inc. for their gener-
ous donation of MDPB. This work was supported by R01
DE015306-06 from the National Institute of Dental and
Craniofacial Research (NIDCR) to DHP (PI) and by grant
#8126472 from the Academy of Finland to AT-M (PI). The
authors thank Mrs. Michelle Barnes for her secretarial support.
Agee KA, Becker TD, Joyce AP, Rueggeberg FA, Borke JL, Waller JL, et al.
(2006). Net expansion of dried demineralized dentin matrix produced
by monomer/alcohol saturation and solvent evaporation. J Biomed Mater
Res A 79:349-358.
Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tjäderhane L, et al.
(2010). Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro
study. Dent Mater 26:320-325.
Butler WT (1984). Dentin collagen: chemical structure and role in mineral-
ization. In: Dentin and dentinogenesis. Lindhe A, editor. Boca Raton,
FL: CRC Press, pp. 40.
Carrilho MR, Carvalho RM, de Goes MF, di Hipólito V, Geraldeli S, Tay
FR, et al. (2007a). Chlorhexidine preserves dentin bond in vitro. J Dent
Carrilho MR, Geraldeli S, Tay F, de Goes MF, Carvalho RM, Tjäderhane L,
et al. (2007b). In vivo preservation of the hybrid layer by chlorhexidine.
J Dent Res 86:529-533.
Carrilho MR, Tay FR, Donnelly AM, Agee KA, Tjäderhane L, Mazzoni A,
et al. (2009). Host-derived loss of dentin stiffness associated with solu-
bilization of collagen. J Biomed Mater Res Part B: Appl Biomater 90:
Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A,
et al. (2010). Substantivity of chlorhexidine to human dentin. Dent
540 Tezvergil-Mutluay et al. J Dent Res 90(4) 2011 Download full-text
Donmez N, Belli S, Pashley DH, Tay FR (2005). Ultrastructural correlates
of in vivo/in vitro bond degradation in self-etch adhesives. J Dent Res
84:355-359; erratum in J Dent Res 85:384, 2006.
Gendron R, Grenier D, Sorsa T, Mayrand D (1999). Inhibition of the activi-
ties of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clin
Diagn Lab Immunol 6:437-439.
Hebling J, Pashley DH, Tjäderhane L, Tay FR (2005). Chlorhexidine arrests
subclinical degradation of dentin hybrid layers in vivo. J Dent Res
Imazato S (2009). Bio-active restorative materials with antibacterial effects: new
dimension of innovation in restorative dentistry. Dent Mater J 28:11-19.
Imazato S, Kinomoto Y, Tarumi H, Torii M, Russell RR, McCabe JF (1997).
Incorporation of antibacterial monomer MDPB into dentin primer.
J Dent Res 76:768-772.
Imazato S, Tay FR, Kaneshiro AV, Takahashi Y, Ebisu S (2007). An in vivo
evaluation of bonding ability of comprehensive antibacterial adhesive
system incorporating MDPB. Dent Mater 23:170-176.
Jamall IS, Finelli VN, Que Hee SS (1981). A simple method to determine
nanogram levels of 4-hydroxyproline in biological tissues. Anal Biochem
Kim J, Uchiyama T, Carrilho M, Agee KA, Mazzoni A, Breschi L, et al.
(2010). Chlorhexidine binding to mineralized versus demineralized
dentin powder. Dent Mater 26:771-778.
Li F, Chai ZG, Sun MN, Wang F, Ma S, Zhang L, et al. (2009). Anti-biofilm
effect of dental adhesive with cationic monomer. J Dent Res 88:372-376.
Markowitz K, Rosenblum MA (2010). The effect cationic polymer treatment
on dye staining and on the adhesion of charged particles to dentin. Arch
Oral Biol 55:60-67.
Martin-de-las-Heras S, Valenzuela A, Overall CM (2000a). The matrix metal-
loproteinase gelatinase A in human dentin. Arch Oral Biol 45:757-765.
Martin-de-las-Heras S, Valenzuela A, Overall CM (2000b). Gelatinase A in
human dentin as a new biochemical marker for age estimation. J Forensic
Mazzoni A, Pashley DH, Nishitani Y, Breschi L, Mannello F, Tjäderhane
L, et al. (2006). Reactivation of inactivated endogenous proteolytic
activities in phosphoric acid-etched dentine by etch-and-rinse adhe-
sives. Biomaterials 27:4470-4476.
Nakajima M, Okuda M, Ogata M, Pereira PN, Tagami J, Pashley DH (2003).
The durability of a fluoride-releasing resin adhesive system to dentin.
Oper Dent 28:186-192.
Namba N, Yoshida Y, Nagaoka N, Takashima S, Matsuura-Yoshimoto K,
Maida H, et al. (2009). Antibacterial effect of bactericide immobilized
in resin dentin. Dent Mater 25:424-430.
Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al.
(2004). Collagen degradation by host-derived enzymes during aging.
J Dent Res 83:216-221.
Pernak J, Rogoza J, Mirska I (2001). Synthesis and antimicrobial activities
of new pyridinium and benzimidazolium chlorides. Eur J Med Chem
Sulkala M, Tervahartiala T, Sorsa T, Larmas M, Salo T, Tjäderhane L (2007).
Matrix metalloproteinase-8 (MMP-8) is the major collagenase in human
dentin. Arch Oral Biol 52:121-127.
Tay FR, Pashley DH, Loushine RJ, Weller RN, Monticelli F, Osorio R
(2006). Self-etching adhesives increase collagenolytic activity in radic-
ular dentin. J Endod 32:862-868.
Tersariol IL, Geraldeli S, Minciotti CL, Nascimento FD, Pääkkönen V,
Martins MT, et al. (2010) Cysteine cathepsins in human dentin-pulp
complex. J Endod 36:475-481.
Tezvergil-Mutluay A, Agee KA, Hoshika T, Tay FR, Pashley DH (2010).
The inhibitory effect of polyvinylphosphonic acid on functional MMP
activities in human demineralized dentin. Acta Biomater 6:4136-4142.
Visse R, Nagase H (2003). Matrix metalloproteinases and tissue inhibitors
of metalloproteinases. Structure, function and biochemistry. Circ Res
Xiao Y-H, Chen J-H, Fang M, Xing X-D, Wang H, Wang Y-J, et al. (2008).
Antibacterial effects of three experimental quaternary ammonium salts
(QAS) monomers on bacteria associated with oral infections. J Oral Sci
Ye Q, Park JG, Topp E, Wang Y, Misra A, Spencer P (2008). In vitro performance
of nanoheterogenous dentin adhesive. J Dent Res 87:829-833.