Antimicrobial and mechanical properties of acrylic resins with ...

Original articleAntimicrobial and mechanical properties of acrylic resins withincorporated silver–zinc zeolite – part ILuciana Assirati Casemiro 1,2 , Carlos Henrique Gomes Martins 1 , Fernanda de Carvalho PanzeriPires-de-Souza 2 and Heitor Panzeri 21 Dentistry College, University of Franca, Franca, SP, Brazil; 2 Department of Dental Materials and Prosthesis, Ribeirão Preto Dentistry College,University of São Paulo, Ribeirao Preto, SP, BrazilGerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.xAntimicrobial and mechanical properties of acrylic resins with incorporated silver–zinc zeolite – part IObjective: The purpose of this in vitro study was to evaluate the antimicrobial activity of acrylic resinscontaining different percentages of silver and zinc zeolite, and to assess whether the addition of zeolitealters the flexural and impact strength of the resins.Background: The characteristics of acrylic resins support microorganism development that can threatenthe health of the dentures user.Material and methods: A microwave-polymerised (Onda-Cryl) and two heat-polymerised (QC20 andLucitone 550) acrylic resins were used. The materials were handled according to the manufacturers’instructions. Fifty rectangular-shaped specimens (8 · 10 · 4mm) were fabricated from each resin andassigned to 5 groups (n = 10) according to their percentage of Irgaguard B5000 silver–zinc zeolite (0% –control, 2.5%, 5.0%, 7.5% and 10%). Flexural strength and Izod impact strength were evaluated. Theantimicrobial activity against two strains of Candida albicans and two strains of Streptococcus mutans wasassessed by agar diffusion method. Data were analysed statistically by one-way ANOVA and Tukey’s test at5% significance level.Results: The addition of 2.5% of Irgaguard B5000 to the materials resulted in antimicrobial activityagainst all strains. Flexural strength decreased significantly with the addition of 2.5% (QC20 and Lucitone550) and 5.0% (Onda-Cryl) of Irgaguard B5000. The impact strength decreased significantly with theaddition of 2.5% (Lucitone 550) and 5.0% (QC20 and Onda-Cryl) of zeolite.Conclusion: The addition of silver–zinc zeolite to acrylic resins yields antimicrobial activity, but may affectnegatively the mechanical properties, depending on the percentage of zeolite.Keywords: acrylic resin, zeolite, antimicrobial activity, strength.Accepted 4 August 2007IntroductionIt is well-known that removable denture basesfabricated from heat-polymerised acrylic resinsmay act as a reservoir for microorganisms andcontribute to re-infection in denture wearers 1,2 .Biofilm deposition on the surface of acrylic denturebases is enhanced by the characteristics of thematerial, especially its porosity, irregularity andabsorption 3 .Oral candidiasis is the most common infectioninvolving oral mucosal tissues in complete denturewearers 4 . It is estimated that it affects about 72% ofthis population 5 . In spite of its multifactorial etiology5 , denture biofilm components, such as Candidaalbicans yeast, play a fundamental role in thedevelopment of Candidiasis 6 . The treatment ofcandidiasis includes denture repair or replacement,adoption of prophylactic measures by the patientand the prescription of antifungal drugs 7,8 . Forelderly and institutionalised patients with limitedmotor skills and special needs, this treatment isfurther complicated because of factors such as lossof memory, difficulty in rendering appropriatecleaning of the denture and following a strict routineof topical application of an antifungal agent 9 .These shortcomings have stimulated the developmentof other methods of drug elution, such as theÓ 2008 The Gerodontology Association and Blackwell Munksgaard Ltd, Gerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.x 1

2 L.A. Casemiro et al.incorporation of antimicrobials into denture basematerials 8,10–16 .The addition of antimicrobial agents to dentalmaterials is part of a current trend 8,10–20 . Amongthe advantages of this process is the possibility ofeluting the agents from the base materials, thuspreventing or reducing bacterial, fungal and yeastcontamination.Antimicrobial zeolites have been incorporated toserve such a purpose 10,11,13–15,21,22 . Zeolites arealuminum silicate crystalline structures that presentvoid spaces measuring 3–10 angstroms in theirstructure. Antimicrobial cations, such as silver andzinc, may be lodged within the void spaces of thezeolites and be exchanged over time with othercations from their environment 14,20–22 . As this ionavailability occurs, the free cations come into contactwith the environmental microorganisms, suppressingtheir development by inactivating vitalmicrobial enzymes, interrupting RNA replicationand blocking their respiration by an oxidativeprocess 23–31 . The efficacy of antimicrobial zeolitesagainst aerobic and anaerobic bacteria and fungihas been demonstrated 20,24,32,33 .Because of their characteristics, including prolongedantimicrobial activity, low toxicity and lackof odour or flavour, zeolites have a large range ofapplications. They have been incorporated, amongothers, into polymers for packaging of food, paint,tissue, catheter and orthopedic prostheses, dentalrestorative materials and oral hygiene products13,14,20,22,34–45 . The dental materials most frequentlyassociated with antimicrobial zeolites arethe tissue conditioners. The favourable outcome ofthis association from a microbiological standpoint14,20 as well as their good viscoelastic properties15 and biocompatibility 11 , indicate that zeolitesmight also be successfully associated with othermaterials used for denture fabrication. Althoughthis procedure might potentially bring direct benefitsfor denture wearers, there have only beenlimited investigations into the addition of antimicrobialagents to acrylic resin denture bases 16,46 .The purposes of this in vitro study were to evaluatethe antimicrobial activity of acrylic resinscontaining different percentages of silver–zincantimicrobial zeolites, and to assess whether theaddition of zeolites alters the flexural and impactstrength of the denture bases.Material and methodsThe study was conducted under climate-controlledconditions (23 ± 2°C; 50 ± 10% relative airhumidity).Specimen fabricationThe tested materials with their compositions,specifications and manufacturers are listed inTable 1. Stainless steel rectangular-shaped patterns(8 · 10 · 4mm) were invested in flasks with dentalstone. After the setting of the stone, the flasks wereopened and the patterns removed, leaving rectangular-shapedcavities that were used as matrixes forthe fabrication of heat-polymerised acrylic resinspecimens.For each assay, 50 specimens were fabricatedfrom each material [a microwave-polymerised(Onda-Cryl) and two heat-polymerised (QC20 andLucitone 550) acrylic resins], and assigned to fivegroups (n = 10) according to their percentage ofsilver–zinc zeolite (Irgaguard B5000; Ciba SpecialtyChemicals Corporation, Tarrytown NY, USA): 0%(control); 2.5%; 5.0%, 7.5% and 10.0% by drywet). For this, the materials were weighted in ananalytical balance (Fisher Scientific, Pittsburgh PA,USA) and Irgaguard B5000 (zeolite) was incorporated,in pre-defined percentages, to the polymerpowder of the acrylic resins. These were mixedmanually for 3 min, by the same operator, toobtain an adequate particle distribution. TheTable 1 Tested materials.Material Manufacturer Handling dose Type of polymerisationQC20High impactLucitone 550Onda-CrylDentsply Indústria e Comércio Ltda,Petrópolis, RJ, BrazilDentsply Indústria e Comércio Ltda,Petrópolis, RJ, BrazilClássico Artigos Odontológicos Ltda.,São Paulo, SP, Brazil23g:10 ml Heat-polymerised (immersion inboiling water for 20 min)21g:10 ml Heat-polymerised (immersion inwater at 73°C for 90 min andboiling water for 30 min)7g:21 ml Microwave-polymerised with 500Wpower (3 min at 40% power, 4 min rest,then 3 min at 90% power)Ó 2008 The Gerodontology Association and Blackwell Munksgaard Ltd, Gerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.x

Antimicrobial acrylic resins 3materials were then proportioned, mixed, packedand polymerised following the manufacturers’instructions. The specimens were ground withwater-cooled silicon carbide papers of decreasingabrasiveness (400-, 500- and 600-grit) and measuredto obtain rectangles of standard dimensions.Assessment of antimicrobial activityThe specimens were autoclaved and then immersedin sterile artificial saliva (School of PharmaceuticalSciences of Ribeirão Preto, University of São Paulo,SP, Brazil) at 37°C for 7 days. Thereafter, the salivawas discarded and the specimens subjected to theantimicrobial activity assays.The agar plate diffusion method was performedin triplicate using the well technique 47 . Forobtaining the inoculum, the reference bacteriastrains (C. albicans ATCC 28366 and Streptococcusmutans ATCC 25175) and the clinical isolates (C.albicans and S. mutans) (Laboratory of Microbiology,University of Franca, Franca, SP, Brazil) were replicatedin Mueller Hinton Broth (C. albicans) orBrain Heart Infusion (for S. mutans). After incubationat 37°C for 24 hours, the turbidity of C. albicansand S. mutans inoculums was adjusted to tubes 1and 0.5 McFarland standard respectively. To formthe base layer, 25 ml of Mueller Hinton Medium orBrain Heart Infusion-Agar at 50°C were depositedon sterile Petri plates (25 · 125mm). After solidification,12.5ml of the culture media inoculatedwith the test organisms (2.5ml) were added to formthe seeding layer. Four-millimetre-diameter wellswere then perforated to receive the cylindricalspecimens (4.0 · 4.0mm). The plates were kept atroom temperature for 120 min for diffusion of theantimicrobial agents 48 and then inoculated inaerobiosis or microaerophilia at 37°C for 24 hours.The zone of inhibition diameters were measuredwith sliding callipers (mm) and this measurementindicated the microbial susceptibility to the material.Data were analysed statistically by one-wayANOVA and Tukey’s post hoc test at 5% significancelevel.Flexural strengthThe specimens were submitted to a three-pointbending test using a universal testing machine(Instron 5569, Norwood, MA, USA) calibrated toprovide a crosshead speed of 5mm/min with a500kgf load cell. Testing was performed in compliancewith the specifications of ISO 178:2003standard (Plastics. Determination of flexural properties).Means (in MPa) were obtained and the datawere analyzed statistically by one-way ANOVA andTukey’s post hoc test at 5% significance level.Izod impact strengthThe specimens were taken to an impact testingmachine (Resil 25, Charlotte, NC, USA) and weresubjected to a 5.5J impact delivered by a swingingpendulum positioned at height of approximately30cm. From the energy absorbed by the specimenduring fracture the attrition value (0.022J) wassubtracted and the impact strength was determined.Testing was performed in compliance withthe specifications of ISO 180:2001 standard (Plastics.Determination of Izod impact strength) Means(in MPa) were obtained and the data were analysedstatistically by one-way ANOVA and Tukey’s post hoctest at 5% significance level.ResultsAssessment of antimicrobial activityThe means of zone of inhibition diameters (in mm)of the heat-polymerised acrylic resins containingdifferent percentages of silver–zinc zeolite areshown in Table 2.None of the control groups showed antimicrobialactivity. For all acrylic resins, the addition of 2.5%of zeolite was enough to assign antimicrobialactivity against the test strains. For QC20, thisactivity was significantly increased when the percentageof zeolite incorporated into the resin was5% (against S. mutans clinical isolate), 7.5%(against S. mutans ATCC 25175) and 10% (againstC. albicans ATCC 28366 and clinical isolate). Lucitonehad a significant increase of its antimicrobialactivity with the addition of 5% of zeolite (againstS. mutans clinical isolate) and 7.5% (againstC. albicans ATCC 28366 and clinical isolate andS. mutans ATCC 25175). Onda-Cryl had its antimicrobialactivity significantly increased with theincorporation of 7.5% (against C. albicans ATCC28366) and S. mutans ATCC 25175 and clinicalisolate) and 10% (against C. albicans clinical isolate)of silver–zinc zeolite.Flexural strengthFlexural strength results are shown in Fig. 1.Compared to the control groups, no statisticallysignificant differences (p > 0.05) were observedamong the tested acrylic resins [QC20(91.9 ± 7.5 MPa); Lucitone 550 (99.4 ± 6.7 MPa)and Onda-Cryl (0.31 ± 0.1 MPa)].Ó 2008 The Gerodontology Association and Blackwell Munksgaard Ltd, Gerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.x

4 L.A. Casemiro et al.Table 2 Inhibition halos of the acrylic resins with incorporation of different percentages of Irgaguard B5000 silver–zinczeolite (values given in mean ± SD mms).Strains 0% 2.5% 5.0% 7.5% 10.0%Percentage of silver–zinc zeolite added to QC20Candida albicans ATCC 28366 0.0 ± 0.0 12.3 ± 0.4 13.5 ± 0.7 15.49 ± 0.8 16.43 ± 0.7Candida albicans (clinical isolate) 0.0 ± 0.0 11.6 ± 0.8 12.4 ± 0.4 14.74 ± 0.4 16.78 ± 0.2Streptococcus mutans ATCC 25175 0.0 ± 0.0 10.9 ± 0.3 11.7 ± 0.6 13.76 ± 0.8 15.65 ± 0.4Streptococcus mutans (clinical isolate) 0.0 ± 0.0 11.4 ± 0.8 14.1 ± 0.2 15.53 ± 0.7 16.03 ± 0.1Percentage of silver–zinc zeolite added to Lucitone 550Candida albicans ATCC 28366 0.0 ± 0.0 11.5 ± 0.3 13.6 ± 0.4 14.3 ± 0.5 16.65 ± 0.9Candida albicans (clinical isolate) 0.0 ± 0.0 10.7 ± 0.6 12.7 ± 0.3 14.8 ± 0.7 15.12 ± 0.1Streptococcus mutans ATCC 25175 0.0 ± 0.0 9.8 ± 0.8 10.3 ± 0.1 13.48 ± 0.4 15.76 ± 0.2Streptococcus mutans (clinical isolate) 0.0 ± 0.0 11.6 ± 0.4 13.5 ± 0.8 14.07 ± 0.3 16.32 ± 0.1Percentage of silver–zinc zeolite added to Onda-CrylCandida albicans ATCC 28366 0.0 ± 0.0 9.12 ± 0.7 10.25 ± 0.5 12.52 ± 0.3 13.41 ± 0.7Candida albicans (clinical isolate) 0.0 ± 0.0 10.44 ± 0.5 12.79 ± 0.6 13.21 ± 0.4 14.65 ± 0.6Streptococcus mutans ATCC 25175 0.0 ± 0.0 10.90 ± 0.9 11.28 ± 0.5 13.69 ± 0.1 15.97 ± 0.1Streptococcus mutans (clinical isolate) 0.0 ± 0.0 9.89 ± 0.2 11.69 ± 0.1 13.42 ± 0.2 15.70 ± 0.2Flexural strength (MPa)1251007550250QC20 Lucitone 550 Onda-CrylPercentage of Irgaguard B50000%2.5%5.0%7.50%10%Figure 1 Flexural strength test values of the acrylicresins with incorporation of the different percentages ofIrgaguard B5000 silver–zinc zeolite (values given inmean ± SD MPa).For QC20, the addition of zeolite in percentagesgreater than or equal to 2.5% resulted in decreaseof flexural strength compared to the controlgroup (p < 0.05). No statistically significantdifferences (p > 0.05) were observed between thefollowing pairs of groups: 2.5% and 5%; 2.5%and 7.5%; 2.5% and 10%; 5% and 7.5%; 7.5%and 10%. For Lucitone 550 and QC20, theaddition of antimicrobial zeolite in any of thepercentages under study resulted in statisticallysignificant decrease of flexural strength(p < 0.05). Nevertheless, there were no significantdifferences (p > 0.05) between the groups withaddition of 2.5% and 5% of zeolite or betweenthe groups with addition of 7.5% and 10% ofzeolite. The incorporation of 2.5% of zeolite toOnda-Cryl did not yield statistically significantdecrease in flexural strength (p > 0.05). However,when a percentage of zeolite ‡5% was added tothe resin, there was decrease in flexural strengthwhen compared with the control group(p < 0.05). There was no significant differencebetween the groups to which 5% and 7.5% ofzeolite had been added.Izod impact strengthImpact strength results are shown in Fig. 2. Comparedto the control groups, no statistically significantdifferences (p > 0.05) were observed betweenQC20 (0.4 ± 0.2 MPa) and Lucitone 550(0.4 ± 0.1 MPa). The other interactions [Lucitone550 X Onda-Cryl (0.3 ± 0.1 MPa) and QC20 XOnda-Cryl differed significantly (p < 0.05).The addition of 5%, 7.5% and 10% zeolite toQC20 decreased its impact strength (p < 0.05).There was no statistically significant difference(p > 0.05) between the control and the 2.5%Impact strength (MPa)0.50.40.30.20.10.0QC20 Lucitone 550 Onda-CrylPercentage of Irgaguard B50000%2.5%5.0%7.5%10.0%Figure 2 Impact strength values of the acrylic resinswith incorporation of the different percentages forIrgaguard V5000 silver–zinc zeolite (values given inmean ± SD MPa).Ó 2008 The Gerodontology Association and Blackwell Munksgaard Ltd, Gerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.x

Antimicrobial acrylic resins 5group. Interactions among the groups that hadan addition of 2.5–10% of antimicrobial zeoliteshowed statistically significant differences (p 0.05). Onda-Cryl had its impact strengthdecreased with the addition of zeolite at percentages‡5% (p < 0.05). The control and 2.5% groupsdid not differ significantly (p > 0.05). The additionof zeolite in percentages from 2.5% to 10%resulted in no statistically significant difference inthe material strength.DiscussionThe addition of silver and zinc zeolites to heatpolymerisedacrylic resins is consistent with thecurrent trend of incorporating antimicrobials intodental materials 8,10–20 . However, its feasibility isrelated to either maintenance or improvement ofthe material’s physical, mechanical and biologicalproperties. This paper presents the results of antimicrobialactivity, flexural strength and impactstrength assays performed with heat-polymerisedand micro-wave polymerised acrylic resins containingdifferent amounts of a silver–zinc zeolite.The impact strength and the flexural properties ofdenture base materials are of importance for predictingtheir clinical performance upon loading 49–51 .Other assays (sorption, solubility and elution ofsilver and zinc, assessment of the conversion degreeand biocompatibility) are currently being conductedin our laboratory and will be submitted later forpublication.The control specimens (fabricated with noincorporation of zeolite), did not have antimicrobialactivity, which agrees with the results of previousstudies that report little or no antimicrobialactivity of the tested materials. The addition of2.5% of Irgaguard B5000 to QC20, Lucitone 550and Onda-Cryl resins was enough provide antimicrobialactivity against the test strains. The higherthe percentage of zeolite, the greater the antimicrobialactivity. The smallest inhibition halowere observed for the Onda-Cryl resin with 2.5%of zeolite against C. albicans ATCC (9.12 ± 0.7 mm),while the largest inhibition halos were observed forQC20 with 10% of zeolite against C. albicans clinicalisolate (16.78 ± 0.2 mm). These results may beattributed to silver and zinc (active elements ofIrgaguard B5000), which leach out from the resins.Silver and zinc possess strong antibacterial andantifungal activity 13,14,20–26,29–35,39,42–45 . Thesefindings are similar to those reported by previousstudies that assessed the incorporation of zeolitesand other antimicrobials into the tissue conditioners8,10–15,19 . Nevertheless, the antimicrobial activityof zeolites against other microorganisms involvedin denture stomatitis, a common condition amongdenture wearers 1–5 , should be investigated. Moreover,this study evaluated the short-term (7 days)antimicrobial activity of the materials and hencetheir long-term action should also be assessed.It is important to evaluate the mechanical propertiesof acrylic resins containing zeolites becauseremovable and complete dentures are subjected torepeated flexural forces. Midline fractures are relatedto the flexural strength of the resins, and thisproperty is challenged every time the dentureundergoes cyclic functional deformation 49 . On theother hand, higher impact strength of the baseresins reduces the possibility of fracturing when theprosthesis is dropped onto a hard surface 50,51 .Therefore, if the addition of zeolite results in asignificant decrease in the flexural and impactstrengths of acrylic resins, this may increase thepossibility of a fracture occurring inside or outsidethe oral cavity.Among the specimens fabricated with no additionof zeolite, Lucitone 550 controls showed thehighest flexural strength values while Onda-Crylcontrols showed the lowest but these were notsignificant. This may be attributed to the resins’compositions and polymerisation cycles, whichhave a direct influence on the mechanical properties51 . Short cycles, as for QC20, may result inincomplete polymerisation and the presence ofresidual monomer. The resulting plasticising effectreduces the molecular binding forces and allowsgreater deformation upon flexion 50,52 . The fact thatthe microwave-polymerised acrylic resin (Onda-Cryl) showed lower flexural strength than thewater-bath-polymerised resins (QC20 and Lucitone550) is consistent with those of previous studies 51 .A significant decrease of flexural strength incomparison to the control groups, was observedwith the addition of 2.5% of zeolite to QC20 andLucitone 550, as well as the addition of 5% ofzeolite to Onda-Cryl (p < 0.05). The decrease offlexural strength values is in agreement with theresults of Addy and Handlery 53 , who reported thatthe addition of a similar agent to methacrylatesnegatively affected their mechanical properties.However, it is important to realise that the flexuralstrength values obtained for those samples wherethe percentage was 5% or below were in accordanceto ISO 1567, that is 65MPa.Ó 2008 The Gerodontology Association and Blackwell Munksgaard Ltd, Gerodontology 2008; doi:10.1111/j.1741-2358.2007.00198.x

6 L.A. Casemiro et al.Regarding the impact strength of the controlgroups, QC20 and Lucitone 550 had similar results.The higher impact strength observed for Lucitone550 was already expected as this is considered to bea high-impact material. QC20 and Lucitone 550differed significantly from Onda-Cryl, which presentedthe lowest impact strength (p < 0.05). Theaddition of percentages of 5–10% of IrgaguardB5000 zeolite to QC20 and Onda-Cryl significantlyreduced their impact strength, while the addition of2.5% of silver–zinc zeolite to Lucitone 550 decreasedits impact strength in comparison to thecontrol group.The results of the mechanical tests suggest thatzeolite addition may decrease the flexural and impactstrengths of acrylic resins, which is consistentwith the findings of previous investigations. It isimportant to realise that the conversion degree ofthese materials relating to the amount of residualmonomer, may influence the values obtained 50,54 .Nevertheless, the addition of small percentages ofzeolite to polymethylmethacrylate may be effectiveagainst microorganisms and therefore its impact onmechanical properties may be less significant thanthe potential benefits, especially for patients whodo not follow an adequate denture cleaning protocol9 . The major advantages of this addition couldbe for the elderly people with restricted manualdexterity or cognitive disturbances. A further adverseresult deriving from incorporation of zeolitesto acrylic resins was to increase the opacity ofpolymethylmethacrylate; the higher the percentageof zeolite added to the resin, the greater the opacity.Further studies are required to investigate thepossibility of avoiding this decrease in translucencyof the acrylic resin base containing zeolites so as notto comprise the aesthetics of the prosthesis.ConclusionWithin the limitation of this preliminary study, itmay be concluded that the addition of low percentagesof silver–zinc antimicrobial zeolites topolymethylmethacrylate can be a valuable alternativefor reducing microbial contamination ofacrylic resin denture bases, acrylic baseplates ofremovable orthodontic appliances, myofunctionalplates or other such devices.References1. Radford DR, Challacombe SJ, Walter JD. Dentureplaque and adherence of Candida albicans todenture-base materials in vivo and in vitro. Crit RevOral Biol Med 1999; 10: 99–116.2. Keng SB, Lim M. 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