.hrna/ ofDentisfry Vol. 23. No. 3. pp. 177-181.1995 Elsevier Science Ltd Printed in Great Britain 0300-5712195 $10.00 + 0.00 Antibacterial incorporated S. Imazato*, *Department Tyne, UK activity of MDPB polymer in dental resin R. R. B. Russell* and J. F. McCabet of Oral Biology and tDental Materials Science Unit, The Dental School, University of Newcastle upon ABSTRACT Objectives: Previously, we have reported that dental composite incorporating the new monomer methacryloyloxydodecylpyridinium bromide (MDPB) showed no release of antibacterial components after being cured but still exhibited antibacterial activity against Streptococcus mu fans on its surface. In this study, in order to elucidate the mechanism of the antibacterial effect of immobilized MDPB, the bactericidal activity of MDPB polymer in water-soluble and -insoluble form was investigated, and the effect of MDPB polymer on bacterial attachment was estimated. Methods: Solutions of homo-polymer of MDPB and co-polymer of MDPB with acrylamide were prepared and the viability of seven major oral streptococci was determined after incubation with each polymer solution. For the estimation ofbactericidal activity of insolubilized MDPB polymer, bacteria were kept in contact with cured unfilled Bis-GMA-based resin discs with or without MDPB, and the recovery of viable cells was measured. Attachment of streptococci to cured resin discs with or without MDPB was also compared using radiolabelled bacteria. Results: Water-soluble homo-polymer of MDPB and co-polymer with acrylamide showed bactericidal activity against oral streptococci. However, cured resin incorporating MDPB, which is in water-insoluble form, had little bactericidal activity. Attachment of streptococci, including species which are early colonizers in dental plaque formation, to the cured resin containing MDPB was significantly less than to the control without MDPB. Conclusions: These results indicate that the bactericidal activity of MDPB polymer is reduced after immobilization, but MDPB on the surface of a resin-based material still shows a bacteriostatic effect and antiadhesion property against oral streptococci. KEY WORDS: J. Dent. 1994) 1995; Antibacterial 23: 177-181 activity, Polymer, Dental resin, Oral streptococci 1994; reviewed 10 May 1994; accepted 20 June (Received 31 March Correspondence should be addressed to: Dr S. Imazato, Department Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan. of Operative Dentistry, Osaka University INTRODUCTlON reports have described experiments in which an antibacterial agent was incorporated into filling materials, in order to inhibit bacterial attachment and plaque accumulation on their surfaces’-j. However, the antibacterial activity of these materials is dependent upon release of the agent and this is associated with some Several disadvantages including influence on mechanical propertiesz,3, loss of effectiveness*, toxic effects and disruption of microbial homeostasis. In order to compensate for such disadvantages we have developed a new monomer, methacryloyloxydodecylpyridinium bromide (MDPB), which has antibacterial activity against mutans streptococci4”. MDPB is a compound of the antibacterial agent dodecylpyridinium bromide and a methacryloyl group, and it is able to copolymerize with other dental monomers. Therefore, after the resin-based material containing MDPB has been cured, the antibacterial agent is chemically bound to the resin matrix and is not released.Composite incorporating this monomer has been shown to have an inhibitory effect on the growth of Streptococcus mutans on its surface without releasing the antibacterial componenF6. Although the antibacterial effect of composite incorporating MDPB is thought to be ascribable to bacterial contact with the immobilized agent, this has not yet been 4 28. Purified MDB was then converted to MDPB by reaction with pyridine at 100°C for 30 min and purified. Structure of homo-polymer of MDPB with acrylamide. . PBS solution without polymer served as controls and tests were performed in triplicate for all species. S. Then. Structure of MDPB. After the reaction.m.0 MDPB. After incubation.Japan). 2. Osaka. c . 3 Table 1. 1. triethyleneglycoldimethacrylate.3 23. then cured by light irradiation for 320 s with a light activation unit (Quick Light VL-I. MDPB is a compound of quaternary ammonium dodecylpyridinium bromide and a methacryIoy1 group. UK) overnight. a 100~1 aliquot from the suspension was spread onto BHI agar plates.mutans were adjusted to 1 X lo3 CFU ml-l in PBS and 1 ml of bacterial suspension was maintained in contact with the control or MDPB disc specimen for 4 h at 37°C Br-. and dried at room temperature for 24 h. The purpose of this study was to determine the antibacterial activity of MDPB polymer in water-soluble and -insoluble form against major species of oral streptococci and to elucidate the mechanism of its antibacterial activity. 1. MDPB homopolymer solution wasadjusted to 100. TEGDMA. Hampshire. Polymerization of MDPB or co-polymerization of MDPB with acrylamide was carried out using 2. proved and it is necessaryto investigate the direct effect of polymerized MDPB against bacteria.). ” Methacrylovloxydodecylpyridinium bromide (MDPEI Homo-polymer Of MDPB Co-polymer of MDPB with scrykunide Fig.-O- C-A= hi CH.m. Morita. to remove uncured monomer. and synthesized as follows: 12-methacryloyloxydodecyl bromide (MDB) was synthesized by reaction of 12-bromo-l-dodecanol and methacrylic acid for 16 h at 90°C. of MDPB Fig. Configuration of the product was confirmed with ‘H-NMR. Specimens were placed into stainless steel moulds (10 mm diameter. MATERIALS Structure AND METHODS Bis-GMA. N+- (CH. polymers were purified repeatedly and dissolved in distilled water at 1 mg ml-‘. and top and bottom surfaces were covered with polyester matrix. I shows the structure of MDPB.178 J. Bacterial strains used in this study Table II. Bactericidal activity of MDPB polymer in water-soluble form Bactericidal activity of the homo-polymer of MDPB and co-polymer with acrylamide (50 : 50) (Fig.Z-bis[4-(3-methacrloxy-2-hydroxypropoxy)phenyl] propane.p. Dent. 23: No. Composition w/w) of monomers of unfilled resin (% Streptococcus oralis miris sanguis gordonii murans Streptococcus sobrinus Streptococcus salivarius Streptococcus Streptococcus Streptococcus Streptococcus NCTC7864 NCTCI 0712 NCTC7863 NCTC7868 NCTCI 0449 SL-1 NCTC8618 Control Bis-GMA TEGDMA MDPB 67. cured disc specimenswere immersed in 5 ml of 100%methanol and shaken for 3 h at 200 r..02M phosphate-buffered saline (PBS.33mm thick). pH = 6. sanguis or S. 2) against seven speciesof streptococci was determined.ugml-l or 50 1-18 ml-l in 0. oralis. After removing from the mould. Bacteria were incubated in Brain Heart Infusion (BHI) broth (Oxoid.p. Z. mitis. Bacteria Representative strains of seven major species of oral streptococci were selected for the study (Table I).8) and co-polymer solution to 100ug ml-l. of MDPB and co-polymer . then incubated for 2 h at 37“C with shaking at 200 r. Monomer composition of resins used is given in Table II. J.2’-azobis(2-amidinopropane) dihydrochloride as initiator at 60°C for 8 h. methacryloyloxydodecylpyridinium bromide. Fig. Plates were incubated anaerobically for 24 h and subsequently for 24 h aerobically. S. Bactericidal activity of MDPB polymer in water-insoluble form MDPB was incorporated in unfilled dental resin and bactericidal activity of the specimen after curing against four species of streptococci was determined. Both control and experimental resin specimens contain camphorquinone and catalysts. Overnight cultures of S. 1995. and the number of viable cells in the suspension was determined.9 0 Experimenral 53.1 20. diluted with PBS and added to the polymer solution to give approximately 1 X 105 CFU ml-l. 0.000 1. S.p. .ir S. Control disc.000 = / T E 3 5 -0 it E $ L * f 0 10. MDPB homo-polymer (100 pg/ml). oralis S. T 100. After incubation for 24 h at Statistical analysis Data were analysed by one-way analysis of variance (ANOVA) and Student’s t-test. Hampshire. onto the cured resin disc incorporating MDPB was estimated. LKB Wallac. NY. @. The disc.1 at 550 nm (approximately 10s cells ml-l).p. ting 20% number of viable cells after contact with polymer bar represents the standard deviation of nine W.e. n . Cured disc specimens prepared as described above were immersed in 1 ml of radiolabelled cell suspension and incubated for 4 h at 37°C with gentle agitation at 200 r. The bar represents the standard deviation of three replicates.: Antibacterial activity of MDPB polymer 179 800 . ES.m.m. The antibacterial activity of MDPB homo-polymer varied among species.. a 100 ul aliquot from the suspension was spread onto BHI agar plates.m. Results are expressed as adhesion rate for each disc: Adhesion rate = (c. q .ssli”ari”s S. Aylesbury. S. experimental disc incorporating 20% MDPB. RESULTS Bactericidal water-soluble activity form of MDPB polymer in Fig. sanguis or S.mutans Fig. oralis S. i. The specimens were then washed gently with PBS and the radioactivity on the disc was counted in a liquid scintillation counter (1211RackBeta. Farmingdale. sanguis S. UK). mitis S. mitis.000 100 10 1 S. S. This procedure resulted in a population of single cells and short chains of streptococci. 4.OdiS xmiris s.7 MBq) [methyl 3H] thymidine (Amersham International. Control. The bar represents the standard deviation of three replicates.mutans Fig. Turku. MD.smg. Then. Then. 37“C. 5. the suspension was sonicated for 10 s at a power output of 20% with an XL2010 sonicator (Heat System Inc. and resuspended in PBS.gordonii s. USA) supplemented with 0.5% yeast extract (Oxoid. Fig. MDPB homopolymer (50 fig/ml). or-ah. of 1 ml of cell suspension) X lOO(%) Three determinations were made for each bacterium. USA) and diluted with PBS to give an optical density of 0. co-polymer with acrylamide (100 pg/ml). S. Finland). mutans. with constant agitation at 200 r. Bacterial adhesion to MDPB polymer in water-insoluble form Adhesion of four species of streptococci. Cockeysville.m.sobrh”r S. Adhesion of bacteria to polymer disc. the bacterial suspension was centrifuged at 4000g for 20 min. washed twice with PBS.p. R.p. Control disc. UK) and 20 uCi ml-l (total 3. The number of viable cells after contact with MDPB homo-polymer or co-polymer solution.mutens S. mitis Ssanguis S. 3. n . on the disc/c. 3 shows the number of viable cells (CFU ml-i) recovered after contact with each polymer solution.lmazato et a/. Tests were repeated nine times for each species. Each species was inoculated into 5 ml of Trypticase Soy Broth (BBL. experimental disc incorporaMDPB. and the number of viable cells determined after incubation anaerobically for 24 h and subsequently for 24 h aerobically. The replicates. The numbers of all species of bacteria were reduced after incubation with MDPB homo-polymer. It was also reported that S. which is a linear. In comparison with homo-polymer. mutans and S. but co-polymer showed a different activity for some species. it should be noted that there are also reports describing the killing activity of an immobilized bactericide on some carrierslO+ and the results obtained in this study do not necessarily imply that insoluble MDPB polymer has no bactericidal activity at all. mutans.There is thus no consistent influence of co-polymerization on the bactericidal activity of MDPB. in some species.17. Bacterial adhesion to MDPB polymer Fig. but contrary results were obtained with other species.sanguis and S. Bactericidal water-soluble activity form of MDPB polymer in The number of viable cells after contact with control or experimental resin discs is shown inFig. mitis or S. mitior (an old species designation which would have included most isolates now recognized as S. On the other hand.01). Hence these species are of particular interest in the bacterial adhesion assay involving cured resin. S. 3). oralis and S. Interestingly.The different characteristics of BVP and MDPB for bacterial adhesion are thought to arise from the difference in structure. although the mean value of viable cells after contact with MDPB disc was lower than that after contact with control. 5). 4. reported that the insoluble pyridinium-polymer (BVP) showed characteristics of capturing bacterial cells on its surface15. mutans was inhibited when incubated on the surface of Bis-GMA-based composite containing MDPB4*.mitis. The effect of MDPB polymer at 100ug ml-l and 50 ug ml-l was not significantly different for any species (P > 0.05). oralis. the difference was not statistically significant (P > 0.180 J. prolonged contact of bacteria with the MDPB at the disc surface may be needed for the killing effect. Adhesion of S. S. S. gordonii (P < 0. This indicates another aspect of the antibacterial activity of MDPB. Adhesion of all three species to the resin disc incorporating 20%MDPB was significantly less than that on the control disc without MDPB (Fig.05). Therefore.oralis. The MIC values of unpolymerized MDPB for the samesevenspeciesranged from 7. the antibacterial activity of MDPB polymer in the watersoluble and -insoluble form was estimated in this study. Dent.8 to 25. S. 23: No. For S. sobrinus and S.05). salivarius co-polymer showed greater bactericidal activity than homo-polymers (P < 0. 5 indicates the results of the bacterial adhesion assay. oralis were predominant streptococci in the early plaque on composite in vivor4. 1995. Since the molecule of dodecylpyridinium is much smaller than the size ofbacterial cells. It is possible that the same phenomenon occurred for insoluble MDPB polymer. 3 and S. In order to maintain mechanical and physical properties. oral&). it is thought that immobilized MDPB in Bis-GMA-based resin mainly has a bacteriostatic effect against oral streptococci. has a bactericidal activity in water-soluble form’. sanguis and S. incorporation of MDPB was limited to 20%in this study. S.05). activity was not different between homo-polymers and co-polymer (P > 0.For S. From this. We have previously reported that the growth of S. Alternatively. The sensitivity of each bacterium to co-polymer was similar to that to homopolymer.bactericidal activity was enhanced by copolymerization with acrylamide. S. Reduction of the number of viable bacteria of all spelcies was also observed by incubation with co-polymer. Solubilized co-polymer of MDPB with acrylamide also showed bactericidal activity against all speciestested(Fig. sanguis and S. sanguis onto the discs containing MDPB was significantly lessthan onto control discs (P < 0. revealed bactericidal activity against seven major species of oral streptococci. Co-polymer showed less activity for S. 3). Kawabata et al.0 ug ml-r and S. water-soluble polymer. BVP has a polymerizable . Solubilized polymer is thought to have a capability to penetrate the bacterial cell wall and hence the watersoluble form is more active in killing bacteria. but insoluble BVP produced from crosslinking is unable to penetrate the cell wall of bacteria and so interactions between the surface of bacterial cells and polymer halt at the adsorption stage7.05). though the activity varied among species (Fig. there was no clear evidence of bacterial killing when a suspension of bacteria was mixed with resin discs containing MDPB (Fig. S. sobrinus were more sensitive than other species6. oralis. the density of the active portion in the material may not be great enough to show bactericidal activity at the concentration adopted here and a greater density of the active portion on the surface may be required to enhance the antibacterial activity of the immobilized agent. N-benzyl-4vinylpyridinium bromide (designated BVP). A similar type of insoluble dodecylpyridinium polymer to MDPB has shown to be bacteriostatic against Escherichia coli8.gordonii were more sensitive than others. For all species. Homo-polymer of MDPB. mitis and S. It is reported that a pyridinium-type polymer. 9. mutans than control discs (P < 0. The activity of 100 and 50 ug ml-l of MDPB homo-polymer was the same and there seems to be no dose-response relation in the activity of MDPB homo-polymer solution above 50 ug ml-*. 4).However.The MDPB containing disc also showed less adhesion of S. mitis. there is no correlation between the sensitivity of bacteria to unpolymerized and polymerized MDPB. DISCUSSION In order to elucidate the mechanism of the antibacterial effect of immobilized MDPB in a resin matrix. sanguis are well known to be membersof ‘pioneer species’in dental plaque formation13.05). as only a slight difference in polymer structure may affect the bacterial adhesion to themr6. It is thought that the difference in activity of soluble and insoluble MDPB polymer depends upon their physical form. 11.J Dent Res 1994. Yamamoto K. Polymeric pyridinium salts with well-defined main chain structure. Immobilization antibacterial component in composite resin. 18. 6. Antibacterial and mechanical properties of restorative materials combined with chlorhexidines. Swanson TD. AppJ Environ Microbial 1983.: pilot studies. Capture of micro-organisms and viruses by pyridinium-type polymers and application to biotechnology and water purification. Frandsen EGV. it has been shown that Bis-GMAbased resin incorporating MDPB has little bactericidal activity but has an anti-adhesion characteristic against oral streptococci. . Tsuchitani Y. Nakagawa Y. Nishiguchi M. Another possible factor is the existence of a hydrophobic alkyl chain in the molecule of MDPB. 6: 129-133.lmazato et al. I. AppJ Environ Microbial 1978. Shibasaki I. Antibacterial activity of soluble pyridinium-type polymers. Incorporation of bacterial inhibitor into resin composite. Tazuke S. Torii M. Kourai H. the presenceof a hydrophobic alkyl chain in the molecule is thought to render the polymer less adhesive for bacteria. 36: 700-704. 17: 225-229. Kourai H. J Oral RehabiJ 1983. From this study. In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements. 17. 15. Torii M. It is also reported that the antibacterial activity of polymeric pyridinium salts was enhanced when the length of alkyl chain attached to the pyridine ring was longer r8. J Dent 1989. Shibasaki I. Removal of bacteria from water by adhesion to cross-linked poly(vinylpyridinium halide). KilianM. Ericson D. 3. 12. Makromol Chem 1986. Yamaguchi H. Kawakami M. Imazato S. 4. J Oral RehabiJ 1984. Prog PoJy Sci 1992: 17: l-34. 16. 187: 333-340. AppJ Microbial 1973. Kawabata N. However. Imazato S. 9. AppJ Microbial 1972. 8. Abbott Ek Walters PA Surface-bonded antimicrobial activity of an organosilicon quaternary ammonium chloride. Hayashi T. Abbott EA. AppJ Environ Microbial 1982. Imazato S. 2. Horie T. Walters PA. Kawabata N. Kawabata N. Antimicrobial characteristic of insoluble alkylpyridinium iodide. 72: 721. Hayashi T. McCabe JF. Noda H. References 1. Yamanano Y. Ribeiro J. J Dent Res 1992. Tsuchitani Y. 10:373-381. Matsumoto T. 25: 253-256. Tsuchitani Y. covering the surface with acquired pellicle may reduce its effectivenessand it remains to be seenwhether the antibacterial activity of immobilized MDPB is still effective after being covered with pellicle. from the fact that immobilized MDPB has little bactericidal activity. 10. D. Jedrychowski JR. Surface kinetic test method for determining rate of kill by an antimicrobial solid. Oral Microbial JmmunoJ 1991. Kawabata N. 24: 859-863. Hogg for his advice on experiments with radiolabelled bacteria. Acknowledgements We thank Dr S. Effect of structure of insoluble pyridinium-type polymer on its ability to capture bacteria in water. Algicidal activity of a surface-bonded organosilicon quaternary ammonium chloride. Ecology of viridans streptococci in the oral cavity and pharynx. Antiplaque properties of sustained release SnF. Isquith AJ. 13. Kawabata et al. The antibacterial effect of composite incorporating MDPB reported previously is therefore attributable primarily to the bacteriostatic and antiadhesive activity of immobilized MDPB. Torii M. Adsorption of Escherichia coli onto insolubilized lauryl pyridinium iodide and its bacteriostatic action. 46: 203-210. Russell RRB. Horie T. 73: 1437-1443. 47: 88-93. Kerper S. . mentioned that substitution of the benzyl group of BVP by an alkyl chain resulted in less ability to capture bacteria% l7. 99: 533-540. Adherence of oral streptococci to composite resin restorative materials. Nishiwaki M. AppJ Environ Microbial 1984. Isquith AJ. Antibacterial activity of composites containing chemically bound non-releasing antibacterial component. Pedrazzoli V. 43: 1041-1050.: Antibacterial activity of MDPB polymer 181 group next to pyridine but MDPB has it at the terminus on the alkyl chain. 14. Caputo AA. McCollum CJ. Hirayama H. 30: 63-68. Isquith J. 11: 53-63. Kimura K. TinanoffN.Accordingly. of an 5. 54: 2532-2535. 59: 2861-2863. Nakagawa Y. Dent Japan 1993. Bull Chem Sot Jpn 1986. Tawaratani T. Stand J Dent Res 1991. Ikeda T. Suzuki K. Tawaratani T. AppJ Environ Microbial 1988.