d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 424–430

avai lab le at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls /dema

Antibacterial effect of bactericide immobilizedin resin matrix

Naoko Nambaa, Yasuhiro Yoshidab,∗, Noriyuki Nagaokac, Seisuke Takashimad,Kaori Matsuura-Yoshimotoa, Hiroshi Maedaa, Bart Van Meerbeeke,Kazuomi Suzukib, Shogo Takashibaa

a Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and PharmaceuticalScience, 2-5-1 Shikata-cho, Okayama 700-8525, Japanb Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science,2-5-1 Shikata-cho, Okayama 700-8525, Japanc Laboratory for Electron Microscopy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,2-5-1 Shikata-cho, Okayama 700-8525, Japand Department of Orthopaedic Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japane Leuven BIOMAT Research Cluster - Department of Conservative Dentistry, School of Dentistry, Oral Pathology and Maxillo-FacialSurgery, 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 efficiency. The aim of this study was to investigate how much antibacterial

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 quantified using real-time RT-PCR. The amount of

CPC released from the 0%-, 1%- and 3%-CPC resin sample into water was spectrometrically

quantified using a UV–vis recording spectrophotometer.

Results. UV spectrometry revealed that less than 0.11 ppm of CPC was released from the

resin into water for all specimens, which is lower than the minimal concentration generally

needed to inhibit biofilm formation. Growth of S. mutans was significantly 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.

Significance. These results demonstrated that the bactericidal molecule still possessed sig-

nificant 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. Pudoi:10.1016/j.dental.2008.08.012

blished by Elsevier Ltd. All rights reserved.

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. Introduction

etylpyridinium chloride (CPC) is a well-known and effectiventibacterial agent, of which its wide use as an OTC drug andn oral hygiene aids is regulated by the Food and Drug Adminis-ration (FDA) [1–4]. The mechanism of antibacterial activity ofPC is ascribed to the positive charge of the pyridinium group.his group attracts the negatively charged cell membrane ofacteria, by which the cell membrane loses its electrical bal-nce, and eventually the bacteria ‘explode’ under their ownsmotic pressure, similar to a bursting soap bubble (a processalled bacteriolysis) (Fig. 1).

Several experiments have been conducted to incorporaten antibacterial agent into dental filling materials such asesin composites and glass-ionomers, in order to inhibit bac-erial attachment and thus plaque accumulation on theirurfaces [3,5–9]. However, the antibacterial activity is con-idered to largely depend upon release of the antibacterialgent [3,5–9], and a consensus on the antibacterial potential ofmmobilized bactericides has still not been reached. Imazatot al. reported on the antibacterial potential of a bactericidemmobilized within resin composite [10–13]. A unique den-al adhesive (used to bond resin composite to tooth enamelnd dentin) with anti-microbial activity has recently beenommercialized by Kuraray (Tokyo, Japan) as Clearfil Protectond. This adhesive contains the antibacterial monomer 12-ethacryloyloxydodecylpyridinium bromide (MDPB), which

hanks to its quaternary ammonium salt group has in aonomer state (before being polymerized) strong antibacte-

ial activity against oral bacteria [14].Biomaterials with anti-microbial properties are highly

esirable in the oral cavity. Ideally, bactericidal moleculeshould be immobilized within the biomaterial to avoidnwanted side-effects against surrounding tissues. They mayhen however loose much of their antibacterial efficiency. Theim of this study was to investigate how much antibacterialctivity an immobilized bactericidal molecule still has againstral bacteria. The null hypothesis tested was that antibac-

erial activity resulted only from bactericidal molecules thatere released from the resin material and that the bacterici-al molecules, once immobilized within the resin, lost theirntibacterial activity.

ig. 1 – The antibacterial action mechanism of CPC. Notehat CPC has a positive charge, and attracts the negativelyharged bacteria and subsequently destroys their cellembrane through disturbing its electric balance.

( 2 0 0 9 ) 424–430 425

2. Materials and methods

2.1. Specimens

In order to immobilize bactericide in resin we used anexperimental light-curable resin containing a mixture of10-methacryloyloxydecyl dihydrogen phosphate (MDP),2-hydroxyethyl methacrylate (HEMA), triethylene gly-col dimethacrylate (TEGDMA) and hydrophobic aromaticdimethacrylate in a weight ratio of 5:45:25:25. Cam-phorquinone (1 wt.%) and ethyl-4-dimethylaminobenzoate(1 wt.%) were added as a photosensitizer and a reducing agentinitiator, respectively. As immobilized bactericide, CPC wasadded at a concentration of 0, 1 and 3% (hereafter abbreviatedas 0%-, 1%- and 3%-CPC resin, respectively).

A slight amount of 0%-, 1%- and 3%-CPC resin was nextdropped onto a flat 10 mm × 10 mm synthetic hydroxyap-atite plate (HAp; APP-101, Pentax, Tokyo, Japan) with a 2 mmthickness. Then, a flat 110 mm × 110 mm polyethylene sheet(GC Polyethylene Films, GC) with a 0.025 mm thickness waspressed on top, after which the resultant polyethylene-resin-HAp sandwich was thinned with gentle hand pressure onto aflat glass plate to a uniform film, followed by light-curing for1 min using the �-Light II light-curing device (Morita, Saitama,Japan). The slide glass and the top polyethylene sheet werethen removed, leaving a thin film of cured resin attached toHAp. After trimming the HAp sample to its original shape byremoving the resin that set outside the sample edges, it wasagain cured for 1 min 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 (KyusyuUniversity, Japan) and was used in this study. S. mutanswas cultivated under aerobic condition in tryptic soy broth(Becton, Dickinson and Company, Sparks, MD, USA), sup-plemented with 0.5% yeast extract (Becton, Dickinson andCompany, Sparks, MD, USA) and 10 �g/ml of erythromycin(TSBY broth). The bacterial cells were in the exponential phaseharvested by centrifugation (4 ◦C, 900 × g, 15 min) and weresuspended in TSBY broth containing 5% sucrose. The colonyforming units (CFU) of the bacterial suspension was adjustedto 1 × 105 CFU/ml and was used in the experiments I, II and IV,described hereafter.

2.3. Experiment I—antibacterial activity of free CPC insolution

To investigate the antibacterial activity of free CPC, seriallydiluted CPC (ranging from 0.1 to 300 ppm at final concen-tration) was added to the bacterial suspension. The 0%-CPCresin placed in a well of a 12-well dish (Corning Inc., Corn-ing, NY, USA) was inoculated with a 4-ml bacterial suspensioncontaining free CPC. After incubation at 37 ◦C for 12 h, a S.

mutans biofilm formed on the 0%-CPC resin plate were rinsedtwice with 0.01 M sodium cacodylate/0.15 M NaCl buffer at pH7.0 for 10–20 min, and fixed with 1% glutaraldehyde in 0.01 Msodium cacodylate/0.15 M NaCl buffer (pH 7.0) for 1–2 h at room

l s 2

426 d e n t a l m a t e r i a

temperature. Then, the samples were rinsed twice in 0.01 Msodium cacodylate/0.15 M NaCl buffer for 15 min, dehydratedin ascending grades of ethanol (50% for 15 min, 70% for 10 min,90% for 15 min, 95% for 15 min, and 100% for 15 min with 2changes), 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 filmof Pt-Pd in a vacuum evaporator (Eiko IB-3 Ion Coater, Eikoengineering, Ibaraki, Japan), and quantitatively analyzed usingFE-SEM (Topcon DC-720, Tokyo, Japan).

2.4. Experiment II

2.4.1. Antibacterial activity of immobilized CPCHAp with/without a CPC-resin coating was placed in a 12-welldish and was again inoculated with a 4-ml bacterial suspen-sion (without CPC). After incubation at 37 ◦C for 12 h, adherentS. mutans on HAp was quantitatively determined using FE-SEMand living cells of S. mutans were quantified using real-timeRT-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%-CPCresin was immersed in 2 ml Molecular Biology Grade (MBG)water at 37 ◦C for 7 days with MBG water being changedevery day. A bacterial suspension of 4 ml per well was then

Fig. 2 – SEM images of S. mutans biofilms formed on 0%-CPC resiinoculated with S. mutans in TSBY containing 5% sucrose and frerevealed biofilm formation on the resin coating. Note that a S. muconcentration of free CPC in suspension was less than 0.3 ppm.

5 ( 2 0 0 9 ) 424–430

inoculated on top of the 0%-, 1%- and 3%-CPC resin sam-ples in a 12-well dish. After incubation at 37 ◦C for 12 h, thesurface of specimens was quantitatively analyzed using Fe-SEM.

2.5. Quantification of living S. mutans (real-timeRT-PCR)

2.5.1. RNA extraction and cDNA synthesisThe plates were washed with phosphate-buffered saline(PBS) solution (pH 7.2) to remove unattached cells, and totalRNA was extracted from the remaining S. mutans attachedonto HAp and the resin coatings using Trizol LS Reagent(Invitrogen, Life Technologies, Carlsbad, CA, USA) accordingto the manufacturer’s instructions. Contaminated genomicDNA was removed by DNase I (Takara Bio, Shiga, Japan)with RNase Inhibitor (Invitrogen, Life Technologies, Carlsbad,CA, USA). First-strand cDNA synthesis was performed usingSuperScriptTM II RT (Invitrogen, Life Technologies, Carlsbad,CA, USA) with random primers in accordance with the manu-facturer’s instructions.

2.6. Real-time RT-PCR

Living S. mutans was quantified by real-time RT-PCR.GeneAmpR 5700 Sequence Detection System (PE Applied

n in the presence of free CPC. The 0%-CPC resin wase CPC ranging from 0.1 to 300 ppm. After 12 h, SEMtans biofilm covered the total resin surface when the

2 5 ( 2 0 0 9 ) 424–430 427

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Fig. 3 – (a) Viable cell counts of S. mutans on HAp with orwithout CPC-resin coating. Total RNA was extracted fromthe biofilm formed on HAp, 0%-CPC resin, 1%-CPC resin and3%-CPC resin, and the amount of viable cells werequantified by real-time RT-PCR. (b) SEM images of S. mutansbiofilms formed on HAp with or without CPC-resin coatingS. mutans was incubated on HAp or CPC-resin (0%, 1% or3%) coated HAp in TSBY containing 5% sucrose at 37 ◦C for12 h, after which the biofilm formation on the surface wasregistered (immediately). In addition, samples of HAp withor without CPC-resin coating were immersed in MBG water

d e n t a l m a t e r i a l s

iosystems, Foster City, CA, USA) was used for monitoring theuorescence from dsDNA-binding SYBR Green I. QuantitativeCR was performed using universal primers for bacterial 16SRNA gene, as described previously [16]. Briefly, the PCR mix-ure was prepared to contain 2× SYBR Green PCR Master MixPE Applied Biosystems, Foster City, CA, USA), 20 pmol of for-ard and reverse primer and 2.5 �l of synthesized cDNA. The

hermo-cycling program was 40 cycles of 95 ◦C for 15 s and0 ◦C for 1 min with an initial cycle of 95 ◦C for 10 min. A dis-ociation curve (melting curve) was constructed in the rangef 60–95 ◦C, and the data were analyzed using the GeneAmp700 SDS software. Duplicate measurements were performedor each sample.

.7. Statistical analysis

he mean values of total bacterial 16S rRNA in each conditionere calculated and the difference was analyzed by one-way

actorial ANOVA, followed by Scheffe’s multiple comparisonnalysis at a significance level of p < 0.05.

.8. Experiment III—quantification of released CPC

plate sample each of 0%-, 1%- and 3%-CPC resin wasmmersed in 2 ml MBG water at 37 ◦C for 12 h, and for 1, 3,and 7 days with MBG water being changed every day. Then,

he amount of CPC released from the 0%-, 1%- and 3%-CPCesin sample into MGB water solutions was spectrometricallyuantified using a UV–vis recording spectrophotometer (UV-60A, Shimadzu, Kyoto, Japan). Standard samples with a CPConcentration of 0.11, 0.27, 0.90, 2.0, 3.9, 6.5 and 8.9 ppm wererepared by dilution with distilled water, using the weighingethod by electric balance (detecting limit; 10−4 g). A cali-

ration curve was then obtained in the concentration rangef 0.11–8.90 ppm by least square linear regression analysis ofPC concentration versus the peak height from an arbitraryase line at 260 nm attributed to the pyridinium ring struc-ure of CPC [17]. Three samples were employed per conditionnd reproducibility was guaranteed by three UV spectrometryeasurements.

.9. Experiment IV—antibacterial activity of resinith and without immobilized CPC

plate sample of 0%- and 3%-CPC resin was immersed in 2 mlBG water at 37 ◦C for 1, 3, 5 and 7 days with MBG water being

hanged every day. The bacterial suspension was inoculatedn the top of both the 0%- and 3%-CPC resin plates in the sameell of a 6-well dish. After incubation at 37 ◦C for 12 h, the

urface of specimens was observed using Fe-SEM.

. Results

.1. Experiment I—antibacterial activity of free CPC inolution (Fig. 2)

hen the concentration of free CPC was less than 0.3 ppm inuspension, 0%-CPC resin surfaces were totally covered withS. mutans biofilm (Fig. 2). The resin surface was partially cov-

for 7 days, and again analyzed for biofilm formation (after 7days).

ered with a biofilm for a CPC concentration at 1 ppm. On thecontrary, a visible S. mutans biofilm was seldom observed onresin surfaces in a suspension containing CPC with a concen-tration of more than 3 ppm.

3.2. Experiment II—antibacterial activity ofimmobilized CPC (Fig. 3)

Total RNA extracted from the biofilm formed onto uncoatedHAp and resin-coated HAp with and without CPC in Fig. 3arevealed that the amount of living S. mutans on HAp sig-nificantly decreased by coating the surface with 3%-CPCresin. The living S. mutans amount on 1%-CPC resin was

slightly smaller than that on uncoated HAp and on 0%-CPCresin-coated HAp, although the differences were not sig-nificant. Dissociation curves of all RT-PCR products showeda sharp peak at the expected Tm, implicating that there

428 d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 424–430

Fig. 4 – (a) UV spectra of standard samples with a CPC concentration of 0.11, 0.27, 2.0 and 6.5 ppm. (b) The calibration curveobtained in the concentration range of 0.11–8.90 ppm. (c) The absorbance peaks of CPC released in water during 12 h, 1 day

and 7 days.

was no contamination with other bacterial species (data notshown).

In accordance with the results of real-time RT-PCR, Fe-SEMdemonstrated that S. mutans biofilm formation was consider-ably inhibited by 3%-CPC resin as compared to uncoated HApand 0%- and 1%-CPC resin-coated HAp (Fig. 3b: immediately).Samples immersed in MBG water for 7 days revealed thatbiofilm formation was still inhibited on 3%-CPC resin (Fig. 3b:after 7 days).

3.3. Experiment III—stability of immobilized CPC

Fig. 4 shows the UV spectra of standard samples with aCPC concentration of 0.11, 0.27, 2.0 and 6.5 ppm in (a), thecalibration curve obtained in the concentration range of0.11–8.90 ppm in (b), and the absorbance peaks of releasedCPC in water after 12 h, 1 and 7 days of immersion in waterin (c). The resultant calibration curve has the highest linear-ity (R2 = 0.9971), up to the very low concentration of CPC at0.11 ppm (b). A tiny peak at 260 nm that must be attributed

to the pyridinium ring structure of CPC, was detected for the12-h and 1-day immersed samples (arrow). These peaks wereless intense than that of the standard sample with a concen-tration of 0.11 ppm, indicating that less than 0.11 ppm of CPC

was released in water. In case of the 7-day immersion, the peakat 260 nm could also slightly be detected, but its intensity wasweaker than that of the 260-nm peaks for the 12-h and 1-dayimmersed samples.

3.4. Experiment IV—antibacterial activity of resinwith and without immobilized CPC

Fig. 5 shows SEM images of S. mutans biofilm incubated on0%- and 3%-CPC resins in the same well, after immersion in2 ml water at 37 ◦C for 1, 3, 5 and 7 days. Fe-SEM revealed that3%-CPC resin has antibacterial activity in comparison with 0%-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 humaninfectious diseases. Therefore, several trials to produce com-

posites with antibacterial potential have been reported [3,5–9],including attempts in which the materials showed antibac-terial activity by releasing antibacterial agents. However,leaching of anti-microbials from materials has disadvantages,

d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 424–430 429

Fig. 5 – SEM images and schematic drawing of S. mutans biofilm formation on 0%- and 3%-CPC resin in the same well, afteri thatw

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mmersion in 2 ml water at 37 ◦C for 1, 3, 5 and 7 days. Noteith 0%-CPC resin for all conditions.

uch as only short-time effectiveness and possible toxicity tourrounding tissues [18]. In this respect, the bactericide shoulddeally be immobilized within the material in order to avoidotential side-effects against surrounding tissues, but with-ut losing its antibacterial activity. However, once immobilizedhe antibacterial effect of a bactericide is generally consid-red to be low, when compared to the antibacterial potentialf a free bactericide [19]. Consequently, much research haseen devoted to the development of materials that contain anntibacterial agent. The latter should of course not affect theaterial’s mechanical properties and may not become toxic

pon material degradation.Multiple species of bacteria colonize the human oral cavity,

nd the species most commonly associated with human cariess S. mutans [20]. Among the attributes thought to contributeo the virulence of S. mutans is its ability to produce anti-

icrobial or bacteriocin-like substances. They may help themo initially colonize or to sustain their colonization in a milieuuch as dental plaque that is densely packed with organ-sms competing each other [21,22]. Therefore, the antibacterialffect of a bactericide immobilized in a resin matrix was inves-igated using S. mutans.

The real-time RT-PCR is a popular method to quantify theopulation size of living micro-organisms [23]. However, thisechnique is relatively complicated and time-consuming inomparison with SEM. However, SEM has some disadvantages,s it for instance cannot differentiate between dead and liv-ng bacteria. Therefore, we quantified the population size of. mutans using Fe-SEM along with 16S rRNA determination.

oth combined revealed that resin that contained 3% CPC pos-essed antibacterial activity when compared to CPC-free resin.his result is in good agreement with the tendency observedsing SEM (Fig. 3a and b). Consequently, subsequent experi-

3%-CPC resin has antibacterial capability in comparison

ments were performed with 0%- and 3%-CPC resins, of whichthe antibacterial potential was quantitatively analyzed usingFe-SEM.

The antibacterial activity of CPC is based on the processof bacteriolysis or the destruction of the bacterial cell mem-brane through disturbance of its electric balance. This impliesthat the antibacterial effect of CPC must be dose-dependent.This was confirmed in Fig. 2 for free CPC. When the con-centration of free CPC was less than 0.3 ppm in suspension,a S. mutans biofilm covered the total resin surface. However,3%-CPC resin was not covered by a biofilm, confirming itsconsiderable antibacterial effect, though the amount of CPCreleased in water after 12 h was measured to be lower than0.11 ppm (Fig. 4). These results indicate that the bactericideCPC has antibacterial capability, even when immobilized ina resin matrix. We also investigated the antibacterial effect of3%-CPC resin-coated HAp plates immersed for 7 days in water.This experiment revealed that the antibacterial capability of3%-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 fromresin in water. However, the concentration of CPC released inTSBY containing 5% sucrose remains controversial. If CPC wasreleased more in TSBY containing 5% sucrose as comparedwith that released in water, this may indicate that CPC wasreleased 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 resinin TSBY containing 5% sucrose was quite difficult, 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 investigatedthe micro-organism inhibitory effect of 3%-CPC resin when it

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430 d e n t a l m a t e r i a

was immersed in the same well that contained the 0%-CPCresin-coated HAp. If in this case CPC was released from the3%-CPC resin surface in a sufficient amount, it would diffusein the solution (by convection at 37 ◦C for 12 h), and then couldaffect the S. mutans biofilm that was formed on the 0%-CPCresin surface. However, Fe-SEM demonstrated that biofilm for-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 bactericidewould have no antibacterial effect must be rejected.

The results also indicate that the immobilized bacteri-cide shows an inhibitory effect on bacteria that contactthe material surface at which the bactericide is immobi-lized, as Imazato et al. previously reported [24,25]. Thissupports the phenomenon that non-leachable, chemicallybound antibacterial agents inhibit bacterial colonization with-out a surrounding inhibition zone [24,25]. Our results in Fig. 5also revealed that the immobilized bactericide did not inhibitbacteria that did not contact the immobilized bactericide. Thismust reduce potential side-effects against surrounding tis-sues.

This study definitely proved that a bactericide when immo-bilized within a surface coating has still antibacterial activity.Further studies are now needed to examine the antibacterialeffect of materials containing an immobilized bactericide inthe oral environment.

Conflict of interest

The authors have no commercial interests in the productshereby investigated.

Acknowledgments

This study was supported in part by a Grant-in-Aid for Sci-entific Research from the Ministry of Education, Science,Sports and Culture of Japan, and by funds of Medical-Techno-Okayama (2005).

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