Dental Materials (2006) 22, 527–532

www.intl.elsevierhealth.com/journals/dema

In vitro antibacterial effects of the dentin primer

of Clearfil Protect Bond
Satoshi Imazatoa,*, Akiko Kuramotoa, Yusuke Takahashia,
Shigeyuki Ebisua, Mathilde C. Petersb
a
Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry,
1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
b
Cariology, Restorative Sciences and Endodontics, School of Dentistry, The University of Michigan, 1011 N.,
D2361 Ann Arbor, MI, USA

Received 21 March 2005; accepted 11 May 2005

KEYWORDS Summary Objectives. This study aimed to investigate the antibacterial effects of
Protect bond; the dentin primer of a commercially available self-etching adhesive system, Clearfil
Antibacterial activity; Protect Bond, which contains antibacterial monomer 12-methacryloyloxydodecyl-
Adhesive system; pyridinium bromide (MDPB).
MDPB; Methods. Inhibitory effects against Streptococcus mutans, Lactobacillus casei, or
Self-etching primer; Actinomyces naeslundii were examined by an agar-disc diffusion method using the
Cavity disinfecting Clearfil Protect Bond primer containing 5% MDPB and an acidic adhesion-promoting
effects monomer MDP, the primer only with MDP, and the primer with 1% cetylpyridinium
chloride. The minimum inhibitory/bactericidal concentrations (MIC/MBC) of each
primer for the three bacterial species were determined by serial microdilution
assays. For testing the bactericidal effects seen in dentin, the primer was applied to
demineralized dentin blocks in which S. mutans had been impregnated, and numbers
of viable bacteria were counted.
Results. For all three bacteria, the sizes of the inhibition zones produced by Clearfil
Protect Bond primer were significantly greater than for the other primers (p!0.05,
ANOVA and Scheffe’s F-test). The MIC/MBC values of Clearfil Protect Bond primer
were less than those of the primer without MDPB, and comparable to those of the
primer containing cetylpyridinium chloride. No bacterial recovery was obtained after
application of Clearfil Protect Bond primer to the bacteria-impregnated dentin,
although the primer without MDPB showed some bactericidal effect.
Significance. Clearfil Protect Bond primer has strong antibacterial activity based
upon MDPB against S. mutans, L. casei and A. naeslundii, and the capability to
disinfect cavities containing residual bacteria.
Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: C81 6 6879 2928; fax: C81 6 6879 2929.
E-mail address: imazato@dent.osaka-u.ac.jp (S. Imazato).

0109-5641/$ - see front matter Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2005.05.009
528
Introduction
One problem regarding the treatment of caries in
clinical situations is the lack of a comprehensive
and precise diagnosis of the extent of dentinal
caries. Although there are some subjective and
objective methods to diagnose carious lesions and
to differentiate between ‘infected’ and ‘affected’
dentin, clinical criteria that reflect the existence of
cariogenic bacteria or their virulence in dentin have
not been established. Although advocated for
almost a century, traditional complete caries
removal failed to render caries-free cavities [1]. A
classic study showed that after traditional com-
plete caries removal carious dentin was left in 72%
of cavities, which were deemed to be caries free
[2]. More recently, after complete removal of soft
caries at the dentino–enamel junction samples of
the stained but hard dentin still showed a low level
of infection [3].
The available evidence no longer supports the
concept of complete removal of carious dentin
during cavity preparation [4]. In many cases, a
surgical approach can be replaced by a minimally
invasive tissue-saving approach [5]. By saving more
affected tissue it can be expected that minimally
excavated lesions inadvertently will harbor more
residual bacteria [6,7]. With some pioneer bacteria
to be found in the remineralizable affected layer
after minimally invasive caries removal, the sub-
sequent use of materials that have an antibacterial
or bactericidal effect provide an adjunct treatment
contributing to suppression of residual infection
and increasing the survival of the restored tooth.
Etching of the dentinal surface with an acidic
solution, such as phosphoric acid, during the
bonding procedures may be effective to reduce
the number of residual bacteria in a cavity [8].
However, the cleansing effect by the acid followed
by water rinsing is limited and should not be
regarded as reliable [9]. With self-etching/priming
systems in which the smear layer is not washed
away, residual bacteria can be anticipated. There-
fore, adhesive systems that possess antibacterial
activity may be useful for eliminating harmful
effects caused by bacteria, and contribute to better
prognoses for minimal restorative treatments of
dental caries.
We have reported the achievement of an
antibacterial adhesive system by incorporation of
the new monomer 12-methacryloyloxydodecylpyr-
idinium bromide (MDPB), that has strong bacteri-
cidal activity against oral bacteria [10,11], into the
primer of two-step self-etching/priming system
Clearfil Liner Bond 2 [12]. The primer incorporating
S. Imazato et al.
MDPB has been shown to be promising for inactivat-
ing residual bacteria in cavities in in vitro and
in vivo studies [12–14]. Based on the results
obtained for this experimental material, a new
single-bottled 5% MDPB-containing primer was
developed, and the adhesive system employing
this primer was commercialized as Clearfil Protect
Bond.
This study assessed the intrinsic antibacterial
activity of the dentin primer of the Clearfil Protect
Bond system using an agar disc-diffusion test and by
determining the minimum inhibitory/bactericidal
concentrations (MIC/MBC). The hypothesis that the
Protect Bond primer shows cavity disinfecting
effects was also examined by in vitro tests using
dentin blocks containing bacteria.
Materials and methods
Primers
The materials used are listed in Table 1. Clearfil
Protect Bond primer (PB; Kuraray Medical, Tokyo,
Japan) is a single-bottle self-etching/priming sol-
ution with the antibacterial monomer MDPB incor-
porated at 5 (w/w)%. It also contains
2-hydroxyethylmethacrylate (HEMA), water, and
an acidic adhesion-promoting monomer 10-metha-
cryloyloxydecyldihydrogen phosphate (MDP). To
investigate the net contribution of MDPB to
antibacterial activity, a PB-based primer without
MDPB [PB(K/C)] and one with neither MDPB nor
MDP [PB(K/K)] were prepared and examined. A
primer prepared by incorporation of 1 (w/w)%
cetylpyridinium chloride (CPC; Sigma Chemical,
St Louis, USA) into PB(K/K) was also included as
a positive control. The bactericide CPC is
Table 1 Materials used in this study.
Code Inclusion of
MDPBa/MDPb
Clearfil Protect PB C/C
Bond primer
Primer without PB(K/C) K/C
MDPB
Primer without PB(K/K) K/K
MDPB and MDP
Primer contain- CPC-primer K/K
ing 1% CPCc
a
12-methacryloyloxydodecylpyridinium bromide.
b
10-methacryloyloxydecyldihydrogen phosphate.
c
Cetylpyridinium chloride (CPC) was added at 1% to
PB(K/K).
Antibacterial effects of Protect Bond primer
a quaternary ammonium that has an analogical
chemical composition as does MDPB.
Agar disc-diffusion test
Three bacterial species; Streptococcus mutans
NCTC10449, Lactobacillus casei ATCC4646, and
Actinomyces naeslundii (formerly viscosus)
ATCC19246, were used. Each bacterium from
stock cultures stored in 50% glycerol at K20 8C
was cultivated in Brain Heart Infusion (BHI; Becton
Dickinson, Sparks, USA) broth at 37 8C, and a loopful
inoculum was transferred to 10 ml of BHI broth.
After incubation for 24 h, for S. mutans, or 48 h, for
L. casei or A. naeslundii, 350 ml of bacterial
suspension was spread onto a BHI agar plate. A
20 ml portion of each of the four primers was
impregnated into a sterile paper disc (diameter:
6 mm, thickness: 1.5 mm) and placed on a plate
inoculated with a bacterial suspension. A 20 ml
volume was chosen as the optimum for impreg-
nation into a paper disc without overflow of the test
solution. Plates were incubated aerobically for 48 h
at 37 8C, and the size of the inhibition zones for
each material calculated from the diameters of the
halo of inhibition produced and of the specimen as
follows.
Size of inhibition zoneZ(diameter of haloK
diameter of specimen)!1/2.
Three specimens were tested for each primer,
and the results subjected to ANOVA and Scheffe’s
F-test at a significance level of p!0.05.
MIC and MBC measurements
MIC and MBC of PB, PB(K/C) and CPC-primer for
the three bacterial species were determined by
serial microdilution assays. Fifty microlitres of the
primer solution was added to the wells of micro-
plates (Corning, New York, USA) each containing
50 ml of BHI broth, and serial two-fold dilutions
were made into the 50 ml volumes of BHI broth in
the wells. Cultures of S. mutans NCTC10449,
L. casei ATCC4646, or A. naeslundii ATCC19246
were adjusted to 2!106 CFU/ml of suspension in
BHI broth, and 50 ml was inoculated into each well
containing the diluted primers. After incubation
anaerobically for 24–48 h, the MIC value was
determined from visual examinations as being the
lowest concentration of the primers in the wells
with no bacterial growth. Subcultures were made
by spreading on BHI agar plates the content from
the wells that showed no visible growth of bacteria.
Each plate was incubated anaerobically for 48 h,
and the MBC value was given as the lowest
529
concentration of primer solution which produced
no colonies on the plate. Tests were repeated in
triplicate.
Assessment of bactericidal effects in dentin
The bactericidal effects of PB against bacteria in
dentin were assessed by a method described
elsewhere [15]. Human extracted molars were
obtained from patients under informed consent,
and dentin blocks (4 mm x 4 mm) were cut from the
crown. Thicknesses of the blocks were adjusted to
200 mm using 1500-grit silicone carbide abrasive
paper. The block was then demineralized by
immersion in 0.2 M sodium acetate buffer at pH
3.0 for 4 weeks with the solution being changed
every week, and washed in 0.02 M phosphate
buffered saline (PBS; pH 6.8) for 1 week. A 2 ml
aliquot of S. mutans NCTC10449 suspension in PBS
(approximately 1!106 CFU) was put on the surface
of the demineralized specimen and left for 10 min
to simulate bacterial colonization in carious dentin.
PB or PB(K/C) was applied to the specimens and
left in place for 20 s, the block was then cut into
small pieces and homogenized in 500 ml of PBS. The
homogenized solution was diluted immediately
with BHI broth, and a 100 ml aliquot was inoculated
on Mitis salivarius agar (Becton Dickinson) plates.
The plates were incubated anaerobically for 48 h,
and the number of viable bacteria (CFU) assessed by
counting the colonies formed. The number of CFU
recovered when the bacteria-impregnated speci-
men was homogenized with no treatment was used
as a negative control, and three specimens were
tested for each primer.
Results
Agar disc-diffusion test
Table 2 shows the sizes of the inhibition zones
produced by each primer. PB and CPC-primer
showed clear inhibition against all three bacteria,
but PB produced significantly greater inhibition
zones than the CPC-primer. PB(K/C) and PB(K/K)
did not inhibit S. mutans and L. casei. Although
A. naeslundii was inhibited by PB(K/C) and
PB(K/K), inhibition zones produced by these
primers were significantly smaller than those with PB.
MIC and MBC measurements
The MIC and MBC values for the three species
determined as a percentage of the original solution
530 S. Imazato et al.

Table 2 Sizes of inhibition zones produced against S. mutans, L. casei and A. naeslundii.
Inhibition zone size (mm)
S. mutans L. casei A. naeslundii
PB 8.31 (0.33) 6.37 (0.40) 12.67 (0.94)
PB(K/C) 0 0 2.15 (0.23)
PB(K/K) 0 0 5.63 (0.53)
CPC-primer 3.89 (0.06) 4.78 (0.32) 5.78 (0.40)
( ): SD of three replicates.

Table 3 MIC and MBC values of each primer for S. mutans, L. casei and A. naeslundii expressed as a percentage of
the original solution.
MIC/MBC (%)
S. mutans L. casei A. naeslundii
PB 0.156/1.25 0.156/0.625 0.156/0.313
PB(K/C) 25.0/25.0 25.0/25.0 0.625/1.25
CPC-primer 0.078/0.313 0.078/0.156 0.078/0.156

are shown in Table 3. In all tests, the same endpoint
was obtained for each of three replicates. For
S. mutans and L. casei, PB was demonstrated to
have much smaller MIC and MBC values compared
with PB(K/C). The MIC/MBC values of PB(K/C)
for A. naeslundii were smaller than those for other
two species, but PB was still more effective than
PB(K/C). CPC-primer was the most bacterio-
static/bactericidal against the three bacteria
among the three primers tested, but the differ-
ences in MIC/MBC values between PB were only one-
or two-step dilution levels.
Assessment of bactericidal effects in dentin
Table 4 demonstrates the number of viable
S. mutans recovered after application of PB or
PB(K/C) to the bacteria-impregnated dentin. By
application of PB(K/C), bacterial recovery was
reduced to approximately 1% of the negative
control, in which the specimen was not treated
with any primer. On the contrary, no formation of
bacterial colony was found in all three cases for PB,
resulting in bacterial recovery at !10 CFU for this
group.
Discussion
Many dental clinicians routinely use a cavity
disinfectant, such as chlorhexidine or peroxide, in
the treatment of dentinal caries as they cannot be
sure that the lesion has been completely removed.
By incorporation of the antibacterial monomer
MDPB into the primer solution, Clearfil Protect
Bond has been developed as a self-etching/priming
system with cavity disinfecting effects. Clearfil
Protect Bond primer (PB) also contains an acidic
adhesion-promoting monomer MDP, with a pH value
of 2.0. Antibacterial activity of dentin bonding
agents depends upon several factors [16–19], but
acidity of the self-etching primers has been
recognized as one of the major factors to inhibit
bacteria [19–23]. Therefore, in this study, the
antibacterial effects of PB were compared with
those of a primer containing only MDP to elucidate
the net contribution of MDPB.
Agar-disc diffusion tests clearly demonstrated
that PB could inhibit the three bacteria that have
been reported to be associated with dentinal caries
[24,25]. These findings coincided with previous
results reported for a prototype antibacterial
primer containing 5% MDPB based upon the Liner
Bond 2 system [12]. PB(K/C) produced no
inhibition against S. mutans and L. casei, similarly
to PB(K/K) which contains neither MDPB nor MDP.
In addition, the inhibitory effects against
Table 4 Number of viable S. mutans recovered after
application of primer.
Materials Number of bacteria
(CFU)
Controla 3.82 (1.09)!105
PB !10b
PB(K/C) 1.27 (1.57)!103
( ): SD of three replicates.
a
Control means recovery without application of any primer
solution.
b
No colony formation on plates inoculated with a 100 ml
aliquot from 1 ml of suspension.
Antibacterial effects of Protect Bond primer
A. naeslundii by PB(K/C) and PB(K/K) were less
compared with those of PB. The results imply that
the acidity of the primer derived from MDP was not
effective in this test method. The results of agar-
disc diffusion tests reflect a combination of
antibacterial activity and diffusivity of the com-
ponents from the materials [17,26]. Diffusivity of
MDPB and MDP in the agar is suspected to be similar
as the hydrophilicity and molecular weights of both
components are not so different. MDP eluted from
the primer may have been neutralized by the
buffering action of the medium, so that very limited
inhibition was obtained after 48 h of incubation.
A quaternary ammonium CPC is a strong bactericide
frequently used for mouth rinses or dentifrices
[27,28]. MDPB synthesized from dodecylpyridinium
is an analogue of CPC, and both PB and CPC-primer
could inhibit the growth of all bacteria. It is possible
that the greater inhibition zones observed for PB
than the CPC-primer is merely due to amount of
active components included in the primer.
From the results of agar-disc diffusion tests, it is
not possible to distinguish whether the materials
exhibit bactericidal or bacteriostatic effects, as the
production of inhibition zones on the agar plate
indicates only that bacterial growth was hindered.
To investigate the intrinsic antibacterial activity of
PB, therefore, MIC and MBC values were deter-
mined. The MIC/MBC values of PB for all of three
species were smaller than PB(K/C), indicating
that inclusion of antibacterial monomer MDPB is
effective to provide the dentin primer with reliable
bactericidal activities. Although the differences in
the values were of only one- or two-step dilutions,
the CPC-primer demonstrated smaller MIC/MBC
values than PB. The mechanism of the antibacterial
effect of quaternary ammonium compounds is
believed to be due to the cationic and hydrophobic
binding to cell wall components that disturbs
membrane function and subsequently induces
leakage of cytoplasmic material [28,29]. With
agar-disc diffusion methods, components gradually
leach out from materials and a certain period is
needed for the antibacterial agents to reach a
concentration that will disturb bacterial cells.
Contrarily, in MIC/MBC determination using sus-
pended bacteria, active components in sufficient
amounts to suppress or disrupt bacteria are able to
quickly come in contact. The solubility of CPC in
water is greater than the monomer MDPB, and the
immediate contact of CPC with a greater affinity to
cells resulted in smaller MIC/MBC values for the
CPC-primer compared with PB.
PB was confirmed to be effective for eradicating
bacteria in dentin in our experiments using bac-
teria-impregnated dentin blocks. That there was no
531
recovery of bacteria (!10 CFU of detection limit by
cultural method) for PB indicates that almost all the
bacteria inoculated to the cavity were killed by
MDPB while some bacteria were left as viable using
PB(K/C). We preliminarily found that bacteria
placed on the upper surface of a demineralized
block penetrated deeply into the specimen via
dentinal tubules or intertubular spaces (Fig. 1). The
limited antibacterial effects of PB(K/C) observed
in this test, in addition to previous findings for agar-
disc diffusion methods and MIC/MBC determination,
proved that the acidity of a self-etching primer is
not effective by itself to eliminate bacteria in
carious dentin. Soon after application to the
dentinal surface, acidic monomer reacts with Ca
to produce salts, and so the pH value of the primer
increases [30]. The antibacterial effects of acidic
monomer, therefore, can be compromised by this
buffering action of dentin. The present findings
support that PB, the antibacterial effects of which
are not dependant upon acidity but the incorpor-
ated MDPB, is advantageous for eliminating bac-
teria in infected dentin.
The self-etching adhesive system Clearfil Protect
Bond employing antibacterial primer has been
demonstrated to show high bond strength to dentin
and enamel [31–33]. Since MDPB can polymerize and
be immobilized in polymer, the bonding interface
of Clearfil Protect Bond is considered to be stably
maintained even after long-term clinical service, in
contrast to the incorporation of soluble antibacter-
ial agents in dentin bonding systems. Furthermore,
cured primer incorporating MDPB exhibits inhibition
of bacterial growth on its surface by immobilized
antibacterial components [34]. It is, therefore,
expected that Clearfil Protect Bond is effective to
inhibit invading bacteria through gaps at the
Figure 1 SEM images of the surface of a dentin block in
which S. mutans was impregnated.
532
bonding interface after restoration placement,
leading to inhibition of secondary caries. This
possible advantage is now under investigation
in vitro as well as an assessment of the clinical
performance of PB.
Acknowledgements
This study was supported by a Grant-in-aid for
Scientific Research (15209066, 16390545) from the
Japan Society for the Promotion of Science, and the
21st Century COE entitled ‘Origination of Frontier
BioDentistry’ at Osaka University Graduate School
of Dentistry supported by the Ministry of Education,
Culture, Sports, Science and Technology. A
research grant from Daiwa Securities Health Foun-
dation also supported this investigation.
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