dental materi als 2 5 ( 2 0 0 9 ) 424430

avai l abl e at www. sci encedi r ect . com

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Antibacterial effect of bactericide immobilized
in resin matrix
Naoko Namba
a
, Yasuhiro Yoshida
b,
, Noriyuki Nagaoka
c
, Seisuke Takashima
d
,
Kaori Matsuura-Yoshimoto
a
, Hiroshi Maeda
a
, Bart Van Meerbeek
e
,
Kazuomi Suzuki
b
, Shogo Takashiba
a
a
Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Science, 2-5-1 Shikata-cho, Okayama 700-8525, Japan
b
Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science,
2-5-1 Shikata-cho, Okayama 700-8525, Japan
c
Laboratory for Electron Microscopy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
2-5-1 Shikata-cho, Okayama 700-8525, Japan
d
Department of Orthopaedic Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
e
Leuven BIOMAT Research Cluster - Department of Conservative Dentistry, School of Dentistry, Oral Pathology and Maxillo-Facial
Surgery, Catholic University of Leuven, Kapucijnenvoer 7, B-3000 Leuven, Belgium
a r t i c l e i n f o
Article history:
Received 13 May 2008
Accepted 27 August 2008
Keywords:
Adhesive resin
Antibacterial effect
Bacteria
Bactericide
Cetylpyridinium chloride
CPC
Immobilization
Streptococcus mutans
a b s t r a c t
Objective. Biomaterials with anti-microbial properties are highly desirable in the oral cav-
ity. Ideally, bactericidal molecules should be immobilized within the biomaterial to avoid
unwanted side-effects against surrounding tissues. They may then however loose much of
their antibacterial efciency. The aimof this study was toinvestigate howmuchantibacterial
effect an immobilized bactericidal molecule still has against oral bacteria.
Methods. Experimental resins containing 0, 1 and 3% cetylpyridinium chloride (CPC) were
polymerized, and the bacteriostatic and bactericidal effects against Streptococcus mutans
were determined. Adherent S. mutans on HAp was quantitatively determined using FE-
SEM and living cells of S. mutans were quantied using real-time RT-PCR. The amount of
CPC released from the 0%-, 1%- and 3%-CPC resin sample into water was spectrometrically
quantied using a UVvis recording spectrophotometer.
Results. UV spectrometry revealed that less than 0.11ppm of CPC was released from the
resin into water for all specimens, which is lower than the minimal concentration generally
needed to inhibit biolm formation. Growth of S. mutans was signicantly inhibited on the
surface of the 3%-CPC-containing resin coating, although no inhibitory effect was observed
on bacteria that were not in contact with its surface. When immersed in water, the antibac-
terial capability of 3%-CPC resin lasted for 7 days, as compared to resin that did not contain
CPC.
Signicance. These results demonstrated that the bactericidal molecule still possessed sig-
nicant contact bacteriostatic activity when it was immobilized in the resin matrix.
2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +81 86 235 6666; fax: +81 86 235 6669.
E-mail address: yasuhiro@md.okayama-u.ac.jp (Y. Yoshida).
0109-5641/$ see front matter 2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2008.08.012
dental materi als 2 5 ( 2 0 0 9 ) 424430 425
1. Introduction
Cetylpyridinium chloride (CPC) is a well-known and effective
antibacterial agent, of which its wide use as an OTC drug and
inoral hygiene aids is regulatedbythe FoodandDrug Adminis-
tration (FDA) [14]. The mechanism of antibacterial activity of
CPC is ascribed to the positive charge of the pyridiniumgroup.
This group attracts the negatively charged cell membrane of
bacteria, by which the cell membrane loses its electrical bal-
ance, and eventually the bacteria explode under their own
osmotic pressure, similar to a bursting soap bubble (a process
called bacteriolysis) (Fig. 1).
Several experiments have been conducted to incorporate
an antibacterial agent into dental lling materials such as
resin composites and glass-ionomers, in order to inhibit bac-
terial attachment and thus plaque accumulation on their
surfaces [3,59]. However, the antibacterial activity is con-
sidered to largely depend upon release of the antibacterial
agent [3,59], and a consensus on the antibacterial potential of
immobilized bactericides has still not been reached. Imazato
et al. reported on the antibacterial potential of a bactericide
immobilized within resin composite [1013]. A unique den-
tal adhesive (used to bond resin composite to tooth enamel
and dentin) with anti-microbial activity has recently been
commercialized by Kuraray (Tokyo, Japan) as Clearl Protect
Bond. This adhesive contains the antibacterial monomer 12-
methacryloyloxydodecylpyridinium bromide (MDPB), which
thanks to its quaternary ammonium salt group has in a
monomer state (before being polymerized) strong antibacte-
rial activity against oral bacteria [14].
Biomaterials with anti-microbial properties are highly
desirable in the oral cavity. Ideally, bactericidal molecules
should be immobilized within the biomaterial to avoid
unwanted side-effects against surrounding tissues. They may
then however loose much of their antibacterial efciency. The
aim of this study was to investigate how much antibacterial
activity an immobilized bactericidal molecule still has against
oral bacteria. The null hypothesis tested was that antibac-
terial activity resulted only from bactericidal molecules that
were released from the resin material and that the bacterici-
dal molecules, once immobilized within the resin, lost their
antibacterial activity.
Fig. 1 The antibacterial action mechanism of CPC. Note
that CPC has a positive charge, and attracts the negatively
charged bacteria and subsequently destroys their cell
membrane through disturbing its electric balance.
2. Materials and methods
2.1. Specimens
In order to immobilize bactericide in resin we used an
experimental light-curable resin containing a mixture of
10-methacryloyloxydecyl dihydrogen phosphate (MDP),
2-hydroxyethyl methacrylate (HEMA), triethylene gly-
col dimethacrylate (TEGDMA) and hydrophobic aromatic
dimethacrylate in a weight ratio of 5:45:25:25. Cam-
phorquinone (1wt.%) and ethyl-4-dimethylaminobenzoate
(1wt.%) were added as a photosensitizer and a reducing agent
initiator, respectively. As immobilized bactericide, CPC was
added at a concentration of 0, 1 and 3% (hereafter abbreviated
as 0%-, 1%- and 3%-CPC resin, respectively).
A slight amount of 0%-, 1%- and 3%-CPC resin was next
dropped onto a at 10mm10mm synthetic hydroxyap-
atite plate (HAp; APP-101, Pentax, Tokyo, Japan) with a 2mm
thickness. Then, a at 110mm110mm polyethylene sheet
(GC Polyethylene Films, GC) with a 0.025mm thickness was
pressed on top, after which the resultant polyethylene-resin-
HAp sandwich was thinned with gentle hand pressure onto a
at glass plate to a uniform lm, followed by light-curing for
1min using the -Light II light-curing device (Morita, Saitama,
Japan). The slide glass and the top polyethylene sheet were
then removed, leaving a thin lm of cured resin attached to
HAp. After trimming the HAp sample to its original shape by
removing the resin that set outside the sample edges, it was
again cured for 1min using -Light II.
2.2. Bacterial strain and culture conditions
Streptococcus mutans 854S, an erythromycin-resistant strain
[15], was kindly donated from Dr. Akihiro Yoshida (Kyusyu
University, Japan) and was used in this study. S. mutans
was cultivated under aerobic condition in tryptic soy broth
(Becton, Dickinson and Company, Sparks, MD, USA), sup-
plemented with 0.5% yeast extract (Becton, Dickinson and
Company, Sparks, MD, USA) and 10g/ml of erythromycin
(TSBYbroth). The bacterial cells were inthe exponential phase
harvested by centrifugation (4

C, 900g, 15min) and were

suspended in TSBY broth containing 5% sucrose. The colony
forming units (CFU) of the bacterial suspension was adjusted
to 110
5
CFU/ml and was used in the experiments I, II and IV,
described hereafter.
2.3. Experiment Iantibacterial activity of free CPC in
solution
To investigate the antibacterial activity of free CPC, serially
diluted CPC (ranging from 0.1 to 300ppm at nal concen-
tration) was added to the bacterial suspension. The 0%-CPC
resin placed in a well of a 12-well dish (Corning Inc., Corn-
ing, NY, USA) was inoculated with a 4-ml bacterial suspension
containing free CPC. After incubation at 37

C for 12h, a S.
mutans biolm formed on the 0%-CPC resin plate were rinsed
twice with 0.01M sodium cacodylate/0.15M NaCl buffer at pH
7.0 for 1020min, and xed with 1% glutaraldehyde in 0.01M
sodiumcacodylate/0.15MNaCl buffer (pH7.0) for 12hat room
426 dental materi als 2 5 ( 2 0 0 9 ) 424430
temperature. Then, the samples were rinsed twice in 0.01M
sodium cacodylate/0.15M NaCl buffer for 15min, dehydrated
in ascending grades of ethanol (50%for 15min, 70%for 10min,
90% for 15min, 95% for 15min, and 100% for 15min with 2
changes), replaced with 3-methylbutyl acetate solution, fol-
lowed by drying using a critical point dryer (TCPD-5, JEOL,
Tokyo, Japan). The surfaces were next coated with a thin lm
of Pt-Pd in a vacuum evaporator (Eiko IB-3 Ion Coater, Eiko
engineering, Ibaraki, Japan), andquantitativelyanalyzedusing
FE-SEM (Topcon DC-720, Tokyo, Japan).
2.4. Experiment II
2.4.1. Antibacterial activity of immobilized CPC
HAp with/without a CPC-resin coating was placed in a 12-well
dish and was again inoculated with a 4-ml bacterial suspen-
sion (without CPC). After incubation at 37

C for 12h, adherent

S. mutans onHAp was quantitatively determined using FE-SEM
and living cells of S. mutans were quantied using real-time
RT-PCR (see below). Duplicate analyses were carried out indi-
vidually three times (6 plates/group).
To investigate the durability of antibacterial activity of
immobilized CPC, a plate sample of 0%-, 1%- and 3%-CPC
resin was immersed in 2ml Molecular Biology Grade (MBG)
water at 37

C for 7 days with MBG water being changed

every day. A bacterial suspension of 4ml per well was then
inoculated on top of the 0%-, 1%- and 3%-CPC resin sam-
ples in a 12-well dish. After incubation at 37

C for 12h, the

surface of specimens was quantitatively analyzed using Fe-
SEM.
2.5. Quantication of living S. mutans (real-time
RT-PCR)
2.5.1. RNA extraction and cDNA synthesis
The plates were washed with phosphate-buffered saline
(PBS) solution (pH 7.2) to remove unattached cells, and total
RNA was extracted from the remaining S. mutans attached
onto HAp and the resin coatings using Trizol LS Reagent
(Invitrogen, Life Technologies, Carlsbad, CA, USA) according
to the manufacturers instructions. Contaminated genomic
DNA was removed by DNase I (Takara Bio, Shiga, Japan)
with RNase Inhibitor (Invitrogen, Life Technologies, Carlsbad,
CA, USA). First-strand cDNA synthesis was performed using
SuperScript
TM
II RT (Invitrogen, Life Technologies, Carlsbad,
CA, USA) with random primers in accordance with the manu-
facturers instructions.
2.6. Real-time RT-PCR
Living S. mutans was quantied by real-time RT-PCR.
GeneAmp
R
5700 Sequence Detection System (PE Applied
Fig. 2 SEM images of S. mutans biolms formed on 0%-CPC resin in the presence of free CPC. The 0%-CPC resin was
inoculated with S. mutans in TSBY containing 5% sucrose and free CPC ranging from 0.1 to 300ppm. After 12h, SEM
revealed biolm formation on the resin coating. Note that a S. mutans biolm covered the total resin surface when the
concentration of free CPC in suspension was less than 0.3ppm.
dental materi als 2 5 ( 2 0 0 9 ) 424430 427
Biosystems, Foster City, CA, USA) was used for monitoring the
uorescence from dsDNA-binding SYBR Green I. Quantitative
PCR was performed using universal primers for bacterial 16S
rRNA gene, as described previously [16]. Briey, the PCR mix-
ture was prepared to contain 2 SYBR Green PCR Master Mix
(PE Applied Biosystems, Foster City, CA, USA), 20pmol of for-
ward and reverse primer and 2.5l of synthesized cDNA. The
thermo-cycling program was 40 cycles of 95

C for 15s and

60

C for 1min with an initial cycle of 95

C for 10min. A dis-

sociation curve (melting curve) was constructed in the range
of 6095

C, and the data were analyzed using the GeneAmp

5700 SDS software. Duplicate measurements were performed
for each sample.
2.7. Statistical analysis
The mean values of total bacterial 16S rRNA in each condition
were calculated and the difference was analyzed by one-way
factorial ANOVA, followed by Scheffes multiple comparison
analysis at a signicance level of p<0.05.
2.8. Experiment IIIquantication of released CPC
A plate sample each of 0%-, 1%- and 3%-CPC resin was
immersed in 2ml MBG water at 37

C for 12h, and for 1, 3,

5 and 7 days with MBG water being changed every day. Then,
the amount of CPC released from the 0%-, 1%- and 3%-CPC
resin sample into MGB water solutions was spectrometrically
quantied using a UVvis recording spectrophotometer (UV-
160A, Shimadzu, Kyoto, Japan). Standard samples with a CPC
concentration of 0.11, 0.27, 0.90, 2.0, 3.9, 6.5 and 8.9ppm were
prepared by dilution with distilled water, using the weighing
method by electric balance (detecting limit; 10
4
g). A cali-
bration curve was then obtained in the concentration range
of 0.118.90ppm by least square linear regression analysis of
CPC concentration versus the peak height from an arbitrary
base line at 260nm attributed to the pyridinium ring struc-
ture of CPC [17]. Three samples were employed per condition
and reproducibility was guaranteed by three UV spectrometry
measurements.
2.9. Experiment IVantibacterial activity of resin
with and without immobilized CPC
A plate sample of 0%- and 3%-CPC resin was immersed in 2ml
MBG water at 37

C for 1, 3, 5 and 7 days with MBG water being

changed every day. The bacterial suspension was inoculated
on the top of both the 0%- and 3%-CPCresin plates in the same
well of a 6-well dish. After incubation at 37

C for 12h, the

surface of specimens was observed using Fe-SEM.
3. Results
3.1. Experiment Iantibacterial activity of free CPC in
solution (Fig. 2)
When the concentration of free CPC was less than 0.3ppm in
suspension, 0%-CPC resin surfaces were totally covered with
a S. mutans biolm (Fig. 2). The resin surface was partially cov-
Fig. 3 (a) Viable cell counts of S. mutans on HAp with or
without CPC-resin coating. Total RNA was extracted from
the biolm formed on HAp, 0%-CPC resin, 1%-CPC resin and
3%-CPC resin, and the amount of viable cells were
quantied by real-time RT-PCR. (b) SEM images of S. mutans
biolms formed on HAp with or without CPC-resin coating
S. mutans was incubated on HAp or CPC-resin (0%, 1% or
3%) coated HAp in TSBY containing 5% sucrose at 37

C for
12h, after which the biolm formation on the surface was
registered (immediately). In addition, samples of HAp with
or without CPC-resin coating were immersed in MBG water
for 7 days, and again analyzed for biolm formation (after 7
days).
ered with a biolm for a CPC concentration at 1ppm. On the
contrary, a visible S. mutans biolm was seldom observed on
resin surfaces in a suspension containing CPC with a concen-
tration of more than 3ppm.
3.2. Experiment IIantibacterial activity of
immobilized CPC (Fig. 3)
Total RNA extracted from the biolm formed onto uncoated
HAp and resin-coated HAp with and without CPC in Fig. 3a
revealed that the amount of living S. mutans on HAp sig-
nicantly decreased by coating the surface with 3%-CPC
resin. The living S. mutans amount on 1%-CPC resin was
slightly smaller than that on uncoated HAp and on 0%-CPC
resin-coated HAp, although the differences were not sig-
nicant. Dissociation curves of all RT-PCR products showed
a sharp peak at the expected T
m
, implicating that there
428 dental materi als 2 5 ( 2 0 0 9 ) 424430
Fig. 4 (a) UV spectra of standard samples with a CPC concentration of 0.11, 0.27, 2.0 and 6.5ppm. (b) The calibration curve
obtained in the concentration range of 0.118.90ppm. (c) The absorbance peaks of CPC released in water during 12h, 1 day
and 7 days.
was no contamination with other bacterial species (data not
shown).
In accordance with the results of real-time RT-PCR, Fe-SEM
demonstrated that S. mutans biolm formation was consider-
ably inhibited by 3%-CPC resin as compared to uncoated HAp
and 0%- and 1%-CPC resin-coated HAp (Fig. 3b: immediately).
Samples immersed in MBG water for 7 days revealed that
biolm formation was still inhibited on 3%-CPC resin (Fig. 3b:
after 7 days).
3.3. Experiment IIIstability of immobilized CPC
Fig. 4 shows the UV spectra of standard samples with a
CPC concentration of 0.11, 0.27, 2.0 and 6.5ppm in (a), the
calibration curve obtained in the concentration range of
0.118.90ppm in (b), and the absorbance peaks of released
CPC in water after 12h, 1 and 7 days of immersion in water
in (c). The resultant calibration curve has the highest linear-
ity (R
2
=0.9971), up to the very low concentration of CPC at
0.11ppm (b). A tiny peak at 260nm that must be attributed
to the pyridinium ring structure of CPC, was detected for the
12-h and 1-day immersed samples (arrow). These peaks were
less intense than that of the standard sample with a concen-
tration of 0.11ppm, indicating that less than 0.11ppm of CPC
was releasedinwater. Incase of the 7-day immersion, the peak
at 260nm could also slightly be detected, but its intensity was
weaker than that of the 260-nm peaks for the 12-h and 1-day
immersed samples.
3.4. Experiment IVantibacterial activity of resin
with and without immobilized CPC
Fig. 5 shows SEM images of S. mutans biolm incubated on
0%- and 3%-CPC resins in the same well, after immersion in
2ml water at 37

C for 1, 3, 5 and 7 days. Fe-SEM revealed that

3%-CPCresinhas antibacterial activity incomparisonwith0%-
CPC resin for all conditions.
4. Discussion
Dental caries has plagued humans since the beginning of civi-
lization and still constitutes one of the most common human
infectious diseases. Therefore, several trials to produce com-
posites with antibacterial potential have been reported [3,59],
including attempts in which the materials showed antibac-
terial activity by releasing antibacterial agents. However,
leaching of anti-microbials frommaterials has disadvantages,
dental materi als 2 5 ( 2 0 0 9 ) 424430 429
Fig. 5 SEM images and schematic drawing of S. mutans biolm formation on 0%- and 3%-CPC resin in the same well, after
immersion in 2ml water at 37

C for 1, 3, 5 and 7 days. Note that 3%-CPC resin has antibacterial capability in comparison
with 0%-CPC resin for all conditions.
such as only short-time effectiveness and possible toxicity to
surrounding tissues [18]. Inthis respect, the bactericide should
ideally be immobilized within the material in order to avoid
potential side-effects against surrounding tissues, but with-
out losing its antibacterial activity. However, once immobilized
the antibacterial effect of a bactericide is generally consid-
ered to be low, when compared to the antibacterial potential
of a free bactericide [19]. Consequently, much research has
been devoted to the development of materials that contain an
antibacterial agent. The latter should of course not affect the
materials mechanical properties and may not become toxic
upon material degradation.
Multiple species of bacteria colonize the human oral cavity,
andthe species most commonlyassociatedwithhumancaries
is S. mutans [20]. Among the attributes thought to contribute
to the virulence of S. mutans is its ability to produce anti-
microbial or bacteriocin-like substances. They may help them
to initially colonize or to sustain their colonization in a milieu
such as dental plaque that is densely packed with organ-
isms competing eachother [21,22]. Therefore, the antibacterial
effect of a bactericide immobilized ina resinmatrix was inves-
tigated using S. mutans.
The real-time RT-PCR is a popular method to quantify the
population size of living micro-organisms [23]. However, this
technique is relatively complicated and time-consuming in
comparisonwithSEM. However, SEMhas some disadvantages,
as it for instance cannot differentiate between dead and liv-
ing bacteria. Therefore, we quantied the population size of
S. mutans using Fe-SEM along with 16S rRNA determination.
Both combined revealed that resin that contained 3%CPC pos-
sessedantibacterial activity whencomparedto CPC-free resin.
This result is in good agreement with the tendency observed
using SEM (Fig. 3a and b). Consequently, subsequent experi-
ments were performed with 0%- and 3%-CPC resins, of which
the antibacterial potential was quantitatively analyzed using
Fe-SEM.
The antibacterial activity of CPC is based on the process
of bacteriolysis or the destruction of the bacterial cell mem-
brane through disturbance of its electric balance. This implies
that the antibacterial effect of CPC must be dose-dependent.
This was conrmed in Fig. 2 for free CPC. When the con-
centration of free CPC was less than 0.3ppm in suspension,
a S. mutans biolm covered the total resin surface. However,
3%-CPC resin was not covered by a biolm, conrming its
considerable antibacterial effect, though the amount of CPC
released in water after 12h was measured to be lower than
0.11ppm (Fig. 4). These results indicate that the bactericide
CPC has antibacterial capability, even when immobilized in
a resin matrix. We also investigated the antibacterial effect of
3%-CPCresin-coated HAp plates immersed for 7 days in water.
This experiment revealed that the antibacterial capability of
3%-CPC resin lasted at least up to 7 days.
To investigate the concentration of free bactericide in solu-
tion, we analyzed the concentration of CPC released from
resin in water. However, the concentration of CPC released in
TSBYcontaining 5%sucrose remains controversial. If CPC was
released more in TSBY containing 5% sucrose as compared
with that released in water, this may indicate that CPC was
released from resin and that this free CPC must be respon-
sible for the antibacterial effect of 3%-CPC resin. However,
analysis of the amount of CPC released from 3% CPC resin
in TSBY containing 5% sucrose was quite difcult, because
the solution contains macromolecules, which can adsorb CPC.
Consequently, to make sure that the antibacterial capabil-
ity should be ascribed to immobilized CPC, we investigated
the micro-organism inhibitory effect of 3%-CPC resin when it
430 dental materi als 2 5 ( 2 0 0 9 ) 424430
was immersed in the same well that contained the 0%-CPC
resin-coated HAp. If in this case CPC was released from the
3%-CPC resin surface in a sufcient amount, it would diffuse
in the solution (by convection at 37

C for 12h), and then could

affect the S. mutans biolm that was formed on the 0%-CPC
resin surface. However, Fe-SEMdemonstrated that biolmfor-
mation was inhibited only on the 3%-CPC resin-coated HAp,
and not on the 0%-CPC resin-coated HAp (Fig. 5). Therefore,
the null hypothesis stating that the immobilized bactericide
would have no antibacterial effect must be rejected.
The results also indicate that the immobilized bacteri-
cide shows an inhibitory effect on bacteria that contact
the material surface at which the bactericide is immobi-
lized, as Imazato et al. previously reported [24,25]. This
supports the phenomenon that non-leachable, chemically
bound antibacterial agents inhibit bacterial colonizationwith-
out a surrounding inhibition zone [24,25]. Our results in Fig. 5
also revealed that the immobilized bactericide did not inhibit
bacteria that did not contact the immobilized bactericide. This
must reduce potential side-effects against surrounding tis-
sues.
This study denitely proved that a bactericide whenimmo-
bilized within a surface coating has still antibacterial activity.
Further studies are now needed to examine the antibacterial
effect of materials containing an immobilized bactericide in
the oral environment.
Conict of interest
The authors have no commercial interests in the products
hereby investigated.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Sci-
entic Research from the Ministry of Education, Science,
Sports and Culture of Japan, and by funds of Medical-Techno-
Okayama (2005).
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