Acta of Bioengineering and Biomechanics Original paperVol. 18, No. 2, 2016 DOI: 10.5277/ABB-00232-2014-04

Preparation and characterizationof ZnO-PMMA resin nanocomposites for denture bases

MARIUSZ CIERECH1*, JACEK WOJNAROWICZ2, DARIUSZ SZMIGIEL3, BOHDAN BĄCZKOWSKI1,ANNA MARIA GRUDNIAK4, KRYSTYNA IZABELA WOLSKA4, WITOLD ŁOJKOWSKI2, 5,

ELŻBIETA MIERZWIŃSKA-NASTALSKA1

1 Department of Prosthetic Dentistry, Medical University of Warsaw, Poland.2 Institute of High Pressure Physic, Polish Academy of Sciences, Warsaw, Poland.

3 Division of Silicon Microsystem and Nanostructure Technology, Institute of Electron Technology, Warsaw, Poland.4 Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Poland.

5 Faculty of Management, Białystok University of Technology, Białystok, Poland.

Purpose: The aim of the paper was to investigate the antifungal activity of zinc oxide nanoparticles (ZnONPs) against Candida albi-cans. Some attempts have been made to find out the best way to introduce ZnONPs into polymethyl methacrylate (PMMA) resin mate-rial and to determine some parameters of a newly formed composite. Material and methods: Zinc oxide nanoparticles were manufacturedand their basic physical parameters were determined (average particle size, density, specific surface area). Minimal inhibitory concentration(MIC) of ZnONPs was determined for the Candida albicans standard strain. The average size of ZnO conglomerates in the monomersolution of PMMA resin was measured using a dynamic light scattering instrument. PMMA resin samples with incorporated ZnONPswere produced. The morphology of nanopowder and the newly formed composite was examined under a scanning electron microscope(SEM). In addition, the roughness parameter of PMMA resin material was investigated before and after ZnONPs modification. Results:Nanopowder with the average particle size of 30 nm, density of 5.24 g/cm3 and surface area of 39 m2/g was obtained. MIC was deter-mined at the level of 0.75 mg/mL. The average size of ZnO conglomerates in the monomer solution of acrylic resin dropped by 11 timesafter ultrasound activation. SEM examination of a newly formed composite showed a successful introduction of ZnONPs confirmed bythe energy dispersive X-ray spectroscopy (EDS) analysis. There were no statistically significant differences in the biomaterial roughnessbefore and after the modification of ZnONPs. Conclusion: Zinc oxide nanoparticles were successfully incorporated into acrylic resinused for the production of denture bases. The presence of nanoparticles with sizes below 100 nm was confirmed. Nevertheless a newlycreated composite needs to be further investigated to improve its homogeneity, and to check its microbiological properties, strength andbiocompatibility prior to its possible clinical use.

Key words: ZnO nanoparticles, Candida albicans, acrylic resins

1. Introduction

Denture stomatitis is defined as all types of in-flammatory lesions in oral cavity, directly caused byprosthetic appliances. The main causes of its devel-opment comprise mechanical injury, denture plaquerelated to poor hygiene and fungal infection (24). The

etiology of this phenomenon is therefore multifacetedand epidemiological studies indicate its occurrenceeven in 70% of denture wearers (11). Polymethylmethacrylate (PMMA) resin is the most often usedmaterial in dental prosthodontics, especially in pros-thetic rehabilitation of edentate or nearly edentatepatients. In such cases the reduced efficiency of theimmune system must be taken into consideration,

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* Corresponding author: Mariusz Cierech, Department of Prosthetic Dentistry, Medical University of Warsaw, Poland,Nowogrodzka 59, 02-006 Warsaw, Poland. Tel: +48 225021886, e-mail: mariusz.cierech@wp.pl

Received: November 15th, 2014Accepted for publication: July 9th, 2015

M. CIERECH et al.32

primarily due to advanced age, systemic diseases andrelated medication. The denture base then becomesa significant risk factor for the development of denturestomatitis complicated by fungal infection. Candidaalbicans is the microorganism most frequently iso-lated in these cases. It occurs as a saprophytic organ-ism in a healthy oral cavity, but due to environmentalchanges and microbial imbalances under a denturebase, it may lead to the formation of inflammatorylesions (1). Complete denture creates specific condi-tions in oral cavity by impairing natural flow of saliva,exerting mechanical compression, increasing the tem-perature or decreasing the light access, especiallywhen the denture, against dentist’s advice, is wornwithout night-breaks (24). In these cases, Candidastrains are present not only on the mucosa, but theyalso form a biofilm structure on the acrylic denture.This structure is difficult to remove and increases theresistance to antifungal treatment (7). Using antifun-gal agents designed to eradicate Candida it is neces-sary to consider their toxicity and possible develop-ment of multiresistant strains. Besides, it is impossibleto eradicate Candida from the previously infecteddenture, which results in oral mucosa recontaminationand disease recurrence (7). It is, therefore, essential toconduct research aimed at modifying PMMA resin toimpede or prevent the process of forming a bacterialand fungal biofilm. Currently, the research is aimed tomodify the surface of the polymerized PMMA resinby forming a layer impeding biofilm formation. Thislayer is designed inter alia to smooth all the mi-croroughness and change free surface energy byexerting effect on the hydrophilic/hydrophobic prop-erties of the material. For this purpose, photopolym-erized coatings (14), titanium dioxide (3), mannan ormannose (23) are designed to disrupt non-specific andspecific interactions between the microbes and thedenture base, thus preventing their adhesion to thebiomaterial. This is a simple and fast method in clini-cal practice, however the biocompatibility of the ap-plied coating gives rise to doubts, as well as its dura-bility owing to mechanical and chemical agents usedfor cleaning dentures. This provides the rationale formaking an attempt to modify the entire chemicalcomposition of PMMA resin before the polymeriza-tion process. Due to the constant progress in the fieldof nanotechnology, some attempts have been made toadd nanoparticles to PMMA resin, i.e. silver (15) orplatinum (20) to make use of their microbiologicalproperties. The best method known to date is themodification of the material by silver nanoparticleswith a proven antibacterial activity for both Grampositive and Gram negative or strains resistant to con-

ventional antibiotics (13). Silver nanoparticles showa lower cytotoxicity and genotoxicity compared withsubstances containing silver in the scale of the mi-crometer and are less prone to the formation of strainsresistant to treatment compared with antibiotics (8).Li et al. (15) evidenced a reduction of bioactivity andbiomass of biofilm with increasing concentrations ofnano-Ag solution. Anti-adhesive properties againstCandida showed PMMA resin modified by a 5% con-tent of nanoparticles. Microscopic analysis revealedthat the 2% silver content samples already showeddisorder in the formation of biofilm, while at 5% theanalyzed average thickness of the biofilm and thenumber of live cells showed only a few cells of Can-dida. It is worth mentioning that the authors of thisstudy are not familiar with reports on the clinical useof biomaterials containing silver nanoparticles fordenture bases.

The aim of the paper was to investigate antifungalactivity of zinc oxide nanoparticles (ZnONPs) againststandardized strain of Candida albicans. Some at-tempts have been made to find out the best way tointroduce ZnONPs to PMMA resin material and todetermine some parameters of newly formed nano-composites.

2. Material and methods

Preparation of ZnO nanopowder

The materials used for the ZnONPs synthesiswere: zinc acetate dihydrate (Zn(CH3COO)2·2H2O,pure for analysis, CHEMPUR, Poland) and ethyleneglycol (C2H4(OH)2, pure for analysis, CHEMPUR,Poland). Zinc oxide was obtained by dissolving zincacetate in ethylene glycol. For ZnONPs synthesis themicrowave solvothermal synthesis (MSS) technologywas used (17). After 45 min of additional stirring, thereaction solution was transferred into a capped teflonvessel and heated using microwave radiation. Themicrowave reactor MSS2 (IHPP, ITeE – PIB, ERTEC,Poland) (16) was operated at 2.45 GHz and at a powerdensity adjusted to approximately 10 W/ml. The du-ration of the reactions was 12 min under a constantpressure of 3 bars at a microwave power of 3 kW.After the synthesis, the obtained powder was sedi-mented, washed three times with deionized water(1 class, HLP 20 UV, Hydrolab, Poland) centrifuged(MPW-350, MPW Med Instruments, Poland) anddried in freeze dryer (Lyovac GT-2, SRK System-technik GmbH, Germany).

Preparation and characterization of ZnO-PMMA resin nanocomposites for denture bases 33

ZnONPs characteristics1. Powder X-ray diffraction

The X-ray diffraction (XRD) patterns were collectedin the 2Θ range of 10°–100° at room temperature, witha step of 0.02° using an XRD (X’Pert PRO diffractome-ter, Cu Kα1 radiation, PANalytical BV, Almelo, TheNetherlands). The average crystallite size was deter-mined using the Scherrer formula. XRD peak profileanalysis was performed using analytical formula forpolydispersive powders [X, Y]. While Scherrer methodprovides a single size parameter, this technique providesfour parameters: (a) average crystallite size, (b) error ofthe average crystallite size, (c) dispersion of size and(d) error of dispersion of sizes. Hence, one obtains fullcrystallite size distribution curve and estimation of“thickness” of this curve (error bars). On-line toolscience24.com/xrd is a webpage, where diffraction filescan be directly dropped. Files are processed on server toextract crystallite size distribution for XRD peaks. Un-like standard fitting, the tool does not act in reciprocalspace at all, but solves sets of equations in few auxiliaryspaces simultaneously. This allows us to analyze XRDdata with heavily convoluted reciprocal space peaks.

2. Density and specific surface area (SSA) measure-mentsDensity measurements were carried out using a he-

lium pycnometer (AccuPyc II 1340, Micromeritics,USA) using an in-house procedure (25). This methodenabled the density of ZnONPs to be measured withan accuracy of 0.01 g/cm3. The specific surface areaof the powder was measured by the Brunauer–Emmett–Teller (BET) method (Gemini 2360, Micromeritics,USA). The powder was subjected to desorption at150 °C for 2 h prior to the measurement. The averagediameter of the particles was determined on the basisof SSA and density; identity and sphericity of theparticles were assumed (25).3. Morphology analysis

The morphology of the nanopowder was investi-gated with a scanning electron microscope (SEM)(Ultra Plus; Carl Zeiss Meditec AG, Jena, Germany)and ZnONPs thin layer of carbon using a sputter coater(SCD 005/CEA 035, BAL-TEC, Switzerland). Imag-ing was performed using an InLens detector. WithInLens detector superficial contaminations becomevisible. It also allows the quality and purity of nano-particles to be specified.

4. The transmission electron microscope (TEM) andhigh-resolution TEM (HRTEM) investigationsThe specimens for the TEM observations were

prepared by dropping the methanol particle disper-

sion, created by an ultrasonic technique, on a carbonfilm supported on a 300 mesh copper grid. In addition,TEM studies were used to determine the nanoparticlessize distribution. The particle size histograms wereobtained by considering a region of a sample consist-ing of ca. 200 spherically-shaped nanocrystals. Theobtained histograms were fitted to either normal orlog-normal distributions (Chi2 test and Person’s coef-ficient).

5. Determination of minimal inhibitory concentration(MIC)The overnight cultures of Candida albicans 14053

(obtained from the collection of the Department ofDental Microbiology, Medical University of Warsaw,Poland) were adjusted to 104 viable cells per mL.Prior to adjustment, they were incubated for 24 h inSabouraud medium (DifcoTM, USA ) containingvarious amounts of ZnONPs. The density of inocu-lums was standardized to obtain 104 colony formingunits (CFU) per spot on the agar. The lowest ZnONPsconcentration resulting in the invisibility of turbiditywas taken as the minimal inhibitory concentration(MIC). Sabouraud medium plates were prepared asrecommended by the manufacturer. A dilution seriesof antimicrobial agents ZnONPs (from 31.2 µg/ml to2 mg/ml) was produced, including a drug-free control.These were followed by adding 19 ml of Sabouraudagar to each container, mixed thoroughly, and placedon prelabeled sterile Petri dishes at a level surface.Plates were incubated at 35–37 °C for 18 h (9, 10).

Preparation of denture base PMMA resin samplescontaining ZnONPs

1. Average size of ZnONPs in the suspension ofPMMA resin monomerAverage size measurements in suspension were car-

ried out using a dynamic light scattering (DLS) instru-ment (Zetasizer Nano-ZS, Malvern Instruments, Ltd.,England) according to the ISO 22412:2008 standard.Measurements were made for 0.01wt% solution ofZnONPs in PMMA resin monomer (Superacryl Plus,Spofa Dental, Czech Republic). The mixture was shakenfor 10 min in a Vortex shaker VX-200 and additionallysonicated for 60, 120 or 240 s in an Elmasonic S 10/(H)(30W, Elma Schmidbauer GmbH, Germany).

2. Nanocomposite preparationA thermally polymerized PMMA resin Superacryl

Plus was used to manufacture the denture bases. Therecommended mixing ratio was 22 g of powder poly-mer and 10 ml of liquid monomer, which representsa volume ratio of 3:1. Zinc oxide nanopowder was

M. CIERECH et al.34

suspended in liquid monomer of PMMA resin. Themixture was shaken in a Vortex VX-200 shaker (Labnet,USA) for 10 min. Then an appropriate amount ofPMMA resin powder was added, so that the final 2%mass concentration of ZnONPs could be obtained.This was preceded by calculating the weight loss ofPMMA-nanocomposite after polymerization. Theexperimental evaluation involved weighing each ofthe mixture components before a 10-min stirring andsonication process. Weight loss resulted from therelease of free monomer as well as evaporation ofa volatile liquid monomer while stirring and sonica-tion of solution (based on the experiment it was des-ignated as 4.5%). Furthermore, ZnONPs in the solu-tion of liquid monomer after the evaporation processincreases its weight – 1 g of ZnONPs absorbs into itsinterior 0.1 g of monomer, which must also be takeninto account, however, it is of less importance com-pared with weight loss during the nanocompositepreparation. Finally, the mixture consisted of 0.61 gZnONPs, 9.5 g liquid monomer and 22 g of PMMApowder. PMMA resin without adding ZnONPs was usedas the control group. Originally, samples were preparedwith a model wax Vertex Regular (Vertex-Dental BV,The Netherlands), of size 10 × 10 × 2 mm, applyinga standard procedure for conversion of wax to PMMAresin with hard gypsum class III (Stodent, Zhermack,Italy). The material was conventional heat-polymerizedin the polymerization integral machine (PS-2, P.E.M.,Poland) according to the manufacturer’s instructions.After cooling and removal of the samples from a po-lymerization can, only necessary treatments were ap-plied to remove residual gypsum with silicon rubber,without subjecting the samples to the polishing process.This procedure was designed to get the texture of thesurface as similar as possible to the mucosal surface ofthe denture base. Thirty samples were made for bothstudy and control groups. The samples were used in theroughness test and SEM examination. Due to the re-sults obtained in the DLS examination the procedurewas additionally modified. ZnONPs solution in liquidmonomer of PMMA was additionally sonicated for 240s using an Elmasonic S 10/(H) (30 W, Elma Schmid-bauer GmbH, Germany). The rest of the procedure wasthe same as described previously. The obtained nano-composite was used in the SEM examination.

3. Roughness test of nanocompositeThe surface roughness was examined using a stylus

profiler Dektak XT. Linear and aerial measurementswere made. The former measures a single line on a sam-ple surface, the latter measures an area of the surface.Linear scans, 5 mm long, were taken step by step every

1 mm for both reference and modified samples. More-over, a 10 micron resolution 3D map of the representativemodified surface was recorded for an area of 4 mm2. Instatistical analysis of the roughness test results Student’st-test for independent samples was used. Data wereevaluated for normal distribution using Kolmogorov–Smirnov test and homogeneity of variance was foundusing two independent assays, Brown–Forsythe andLevene tests. The level of significance was established atp = 0.05. All data were computed using the Statistica10.0 program (StatSoft, Inc. Tulsa, OK, USA).

4. SEM examination and elemental composition ofnanocompositesThe morphology of the obtained nanocomposites

was investigated with a scanning electron microscope(SEM) (Ultra Plus; Carl Zeiss Meditec AG, Jena, Ger-many). An Energy Dispersive X-ray (EDS) (Quantax400, Bruker, Billerica, Massachusetts, USA) detectionsystem built in the SEM instrument was used to recordthe X-ray emission spectra of the samples interactingwith the electron beam. For the SEM and EDS analysesbreakthroughs of nanocomposites made in liquid nitro-gen were used. Breakthroughs were sprayed with a thinlayer of carbon using sputter coater. The aim of thestudy was to obtain a qualitative microscopic charac-teristics of the obtained polymer nanocomposites. Forimaging two detectors, ESB and SE2, were used. Theenergy selective backscattered (ESB) detector is suit-able for clear compositional contrast. The ability todetect back-scattered electrons (BSE) makes the sub-surface information and nano-scale composition visi-ble. The SE2 image contrast reveals both topographicaland compositional information due to the greater sam-ple interaction depth of SE2 electrons. Pores and frac-tures appear dark in the SE2 image, therefore, porosityinformation can be readily characterized with the SE2electrons. In this paper, we present the results of thenanocomposite surface.

3.Results

ZnONPs characteristics

The obtained ZnONPs had a density equal to5.24 g/cm3 (Table 1), which is 6.6% less than thevalue given in the literature, 5.61 g/cm3 (5). It wasobserved that the presence of nanoparticles caused thematerial density reduction, probably due to the sig-nificant contribution of the surface layers, which areless densely packed than the bulk. The material spe-cific surface area was 39 m2/g, and the average parti-

Preparation and characterization of ZnO-PMMA resin nanocomposites for denture bases 35

cle diameter was 30 nm; the calculation was based onSSA. The equation to calculate the particle diameter isas follows

ρ⋅=

SSAND

where D – is a particle size, N – factor dependenton the shape of the particles for sphere equal to 6,SSA – specific surface area, and ρ – density.

Fig. 1. XRD pattern of ZnONPs synthesizedusing microwave radiation

The XRD analysis for ZnONPs is presented in Fig. 1.The results confirmed that ZnONPs is a pure wellcrystallized hexagonal wurtzite structure with nano-

sized particles ca. 25 nm in diameter (Table 1). Noindication of the presence of other crystalline phasesor any amorphous component was found.

Figure 2 presents the results of the SEM andTEM investigations. As can be seen ZnONPs exhibitstrong homogeneity. The average particle size of thesynthesized ZnONPs was estimated to be 25–30 nm.Figure 3 shows an image of the spherical particlestaken in the dark field. According to TEM investiga-tions, the average particle size was ≈26 nm (Fig. 4).According to XRD investigations, the average crys-talline size was 26 nm (Fig. 5). Determination ofminimal inhibitory concentration (MIC) is widelyused in the comparative testing of new agents. Thelowest concentration of antimicrobial agent inhibit-ing the visible growth of a microorganism is knownas the MIC. For the standardized strain of Candidaalbicans14053 MIC of ZnONPs was established at0.75 mg/mL level.

Nanocomposite characteristics

The analysis of ZnONPs (Cp = 0.01 wt%) suspen-sion in a liquid monomer of PMMA resin showed theaverage ZnO conglomerates of size 1608 nm withpolydispersion index (Pdl) 0.597. After activating thesolution with ultrasounds, an eleven-fold decrease inthe average size of particles (down to 141 nm) withPdl = 0.173 was observed (Table 2).

Table 1. Density, specific surface4 area and average particle size of ZnONPs

Sample SSA,[as ± σ.m2/g]

Density,[ρ ± σ.g/cm3]

Averageparticle size

from SSA BET,[d ± σ.nm]

Average crystallitesize, Scherer’s for-

mula, basedon XRD,[dc.nm]

Average crystallitesize, NanopowderXRD ProcessorDemo, based on

XRD,[d ± σ.nm]

Averageparticle sizefrom TEM,[d ± σ.nm]

ZnONPs 39 ± 1 5.24 ± 0.05 30 ± 2 25 25 ± 7 26 ± 1

Fig 2 Images of ZnONPs: left image – SEM right image – TEM

M. CIERECH et al.36

Mapping of zinc content in the nanocompositewithout sonication process (Fig. 6) showed the pres-ence of ZnO clusters in the polymer, which indicatedsome deficiencies in the nanocomposite preparationmethod. Clusters of even a few micrometers indiameter were visible in the whole volume of thesample. Nevertheless, large areas of well dispersednanoparticles were visible. After 240 s of the sonica-

tion process it was still possible to find areas withlarge clusters (Fig. 7), but in the whole volume of thesample ZnO was better dispersed in polymer matrix(Fig. 8). The SEM taken scans at a magnificationof 50k× for both nanocomposites without (Fig. 9)and with (Fig. 10) the sonication process confirmedthat the material also contained particles smaller than100 nm.

Table 2. Average diameter of ZnONPs in methyl methacrylate suspension

SampleZnONPs/MMA,Cp = 0.01 wt%

Size by DLS,Z-Average [d ± σ.nm]

Polydispersion index,Pdl

Mixing for 10 min 1608 ± 145 0.597 ± 0.054Mixing for 10 minand ultrasonic mixing for 60 s 447 ± 23 0.471 ± 0.024

Mixing for 10 minand ultrasonic mixing for 120 s 144 ± 3 0.201 ± 0,022

Mixing for 10 min andultrasonic mixing for 240 s 141 ± 2 0.173 ± 0,016

Cp = 0.01 wt%, DLS – dynamic light scattering; ZnONPs – zinc oxide nanoparticles.

Fig. 3. The dark fieldTEM image of ZnONPs Fig. 4. Histogram of the ZnO particle size distribution

Fig. 5. Crystallize size distribution of ZnONPs,obtained in the web program

Nanopowder XRD Processor Demo pre α ver.0.0.8,© Pielaszek Research, http://science24.com/xrd

Preparation and characterization of ZnO-PMMA resin nanocomposites for denture bases 37

Fig. 6. Representative element map of PMMA resin speciments with ZnONPs,Cp = 2 wt% for C and Zn using EDS on SEM

(nanocomposite manufactured without sonication process)

Fig. 7. Representative SEM image of nanocomposite (ZnONPs Cp = 2 wt%)manufactured using the sonication method

Fig. 8. Element map of nanocomposite(sonication method)

for Zn (ZnONPs Cp = 2 wt%) using EDS on SEM

Fig. 9. SEM images of cross section area of acrylic resin materialmodified with ZnONPs, Cp = 2 wt%

(nanocomposites manufactured without sonication process)

M. CIERECH et al.38

Fig. 10. SEM image of cross section areaof nanocomposite (ZnONPs, Cp = 2wt%) manufactured

using the sonication method

Roughness test

The study of the roughness of acrylic samplesmodified by the addition of 2% ZnONPs showed the

Fig. 11. The roughness parameter (Ra) of acrylic resin materialbefore and after modification of ZnONPs, Cp = 2 wt%

Ra parameter of 3.26 microns (SD = 0.96 microns),while for non-modified materials, Ra was 3.38 microns(SD = 1.06 microns). Normal distributions, confirmedby the Kolmogorov–Smirnov test, were obtained forboth the study and control groups (d = 0.12021,p > 0.2 and d = 0.20844, p < 0.15, respectively) andhomogeneity of variance was found using two inde-pendent assays, Brown–Forsythe (F = 0.146664,p = 0.702975) and Levene (F = 0.761664, p =0.385972). Thus the conditions for the use of paramet-ric Student’s t-test for independent samples (t =–0.503941, p = 0.615981) were met. The survey re-sults revealed no statistically significant differencesbetween the study and control groups (Fig. 11). Allthe analysis was performed for the significance levelof p = 0.05. 3D mapping of the representative samplemodified with ZnONPs (Ra = 3.05 microns) is shownin Fig. 12a and 12b).

Discussion

Nanomaterials are defined as materials with atleast one dimension of the structural elements in therange of 1–100 nm (18). The antibacterial propertiesof zinc oxide have been used in dentistry over dec-ades. With the development of nanotechnology theopportunity to produce not only a specific size of ZnOgrains, but also to modify their shape has appeared.Potent microbial action of nanoparticles, compared totheir micrometer counterparts, is made possible due toa very large surface area relative to the volume of theparticle [18]. This was confirmed by the study carriedout by Gondal et al. [12], who determined minimalinhibitory concentration (MIC) for Aspergillus niger(5.0 mg/mL for ZnO, and 2.5 mg/mL for nano-ZnO)and for Candida (10 mg/mL and 5 mg/mL, respec-tively). Thus, in both cases, MIC for the nanoparticles

a) b)

Fig. 12. The surface topography of the sample modified with ZnONPs, Cp = 2 wt%: #d map obtained using a stylus profiler (a)and its corresponding height histogram (b)

Preparation and characterization of ZnO-PMMA resin nanocomposites for denture bases 39

used was reduced twice. Khan et al. [13] studied theeffect of ZnONPs on Gram positive (Staphylococcusaureus) and Gram negative (Escherichia coli) bacte-ria and fungi (Candida albicans) using the disk dif-fusion method. It was observed that the inhibitionof growth was evident at both concentrations of0.50 mg/mL and 0.25 mg/mL and was proportionalto the concentration of nanoparticles. Furthermore,the fungicidal activity for Candida albicans at a con-centration of 0.50 mg/mL was comparable to that ofnystatine, in the absence of the adverse effects, whichmay occur during the antibiotic treatment. Raghupathiet al. [22] have shown that microbial activity is di-rectly proportional to the concentration of ZnONPs,and inversely proportional to their diameter. The useof inorganic nanooxides has many advantages overorganic oxides, namely stability, long life and dura-bility. Comparing the nano zinc oxide with othernanooxides, such as nano CuO or nano Fe2O3, a muchstronger antibacterial activity was found [4]. Inaddition, ZnONPs are regarded as a non-toxic andbiocompatible compound, therefore, it has found appli-cation as a carrier of drugs, cosmetics, or as a compo-nent dressing for wound healing [2].

Numerous studies have attempted to determine themechanism of the antibacterial action of zinc oxidenanoparticles. One hypothesis is about activation ofreactive oxygen species (ROS), reactive moleculescontaining oxygen, such as H2O2, superoxide anion

−2O , OH– or singlet oxygen O2, which are responsible

for the destruction of DNA, lipids or bacterial proteins[26]. According to another theory ZnO joins bacteriavia the electrostatic forces and disorganization of theirwall or cell membrane, which results in direct killingof bacteria [21]. Association of ZnO can dissociateinto ions Zn2+ while changing the pH and potentialbactericidal reaction. However, the studies carried outby Raghupathi et al. [22] demonstrated first, that mi-crobial activity cannot depend only on the solutionpH, and second, that the antibacterial properties ofnanoparticles are much superior to that of Zn2+. Bray-ner et al. [6] observed the deposition of ZnONPs onthe surface of the bacteria and their accumulation inthe cytoplasm, which may damage their functions.Moreover, Raghupathi et al. [22] showed that ZnOparticles larger than 100 nm exhibit bacteriostaticproperties only, while smaller ones exhibit both bacte-riostatic and bactericidal properties. Therefore, itseems justified to make an attempt to introduceZnONPs to PMMA resin material and to make use oftheir antibacterial properties for the prevention andtreatment of denture stomatitis complicated by fungalinfection.

In this study, nanoparticles of zinc oxide were suc-cessfully incorporated into PMMA resin serving asa material for denture bases. The only visible differ-ence between the control and the study groups afterthe polymerization process was a slight whitening ofthe material, which is fully acceptable from bothclinical and esthetic points of view. This work showsthe way of the zinc oxide powder incorporation intoPMMA resin. In the environment of liquid PMMAresin monomer ZnO shows the grains form. Uponstirring in a shaker the conglomerates with an averagesize of up to 1600 nm are created, which is a reasonfor very rapid sedimentation of particles. An eleven-time decrease in the average size of conglomeratesafter a 240-second sonication was observed, whichincreased the stability of the solution (Table 2). Boththe SEM and EDS studies confirmed the presence ofzinc compounds in polymerized PMMA resin material(Fig. 13). The examination of relevant sections (Figs. 9and 10) also confirmed the presence of ZnO particlesin the whole volume of the sample. This has importantclinical implications, as the machining removes theouter layer of the biomaterial. By modifying the mate-rial prior to the polymerization process a constantsupply of nanoparticles is provided, even after thedestruction of the outer layer of the denture. The SEMtaken scans at a magnification of 50k × (Figs. 9 and10) confirmed that the material also contains particlessmaller than 100 nm meeting the conditions requiredfor nanomaterials. In Figure 6, it can be seen that themolecules form clumps uniformly distributed through-out PMMA resin. The clustering could be the result ofthe material porosity and tendency for occupying thenatural niches by ZnO particles or due to water on thehigh surface of ZnONPs. Accumulated water on thesurface can form large clusters of ZnONPs. Priorto polymerization the sonication process decreasesZnONPs conglomerates providing more favorableconditions for better dispersion of nanoparticles inpolymer matrix. However, in some parts of nanocom-posite huge conglomerates are still visible contribut-ing to further improvement of the nanocompositemanufacturing technology.

The roughness testing of the samples performedbefore and after modification revealed no statisticallysignificant differences between the two groups. This isin agreement with the results obtained by Li et al. [8],who also revealed no significant differences betweenPMMA and PMMA with nano Ag composite. Thetexture of the denture mucosal surface varied due tothe anatomical structure of palate, as well as to themode of denture preparation. Surface roughness wasof the order of a few microns (Fig. 12b). This creates

M. CIERECH et al.40

favorable conditions for the development of microor-ganisms and makes an effective denture cleaningmore difficult. Therefore, it seems unlikely that theparticles in the nano-scale could have a significantimpact on the above-mentioned parameter. It merelyconfirms the fact that the effect of nanoparticles doesnot rely on the roughness modification, one of themain causes of denture stomatitis [24].

The study confirmed the biological activity of zincoxide nanoparticles against Candida albicans, the mostcommon pathogen of denture stomatitis. In this studyminimal inhibitory concentration value was establishedat 0.75 mg/mL while Gondal et al. [12] determinedMIC at a level of 5 mg/ml. The differences could bedue to other characteristics of ZnONPs, as well as tothe use of another strain typing.

5. Conclusions

In this study, we successfully managed to incorpo-rate ZnONPs to PMMA resin, confirming its presencein the whole volume of the sample. To the best of ourknowledge, this is the first successful attempt to pro-duce PMMA resin for bases of dentures modified withnanoparticles of zinc oxide. Nevertheless, a newlycreated nanocomposite needs to be further investi-gated to improve its homogeneity, and to check itsmicrobiological properties, strength and biocompati-

bility prior to its possible clinical use. The develop-ment of the material able to display fungistatic or fun-gicidal activity over the lifetime creates an opportunityto reduce significant clinical problems related to theprevention and treatment of denture stomatitis com-plicated by fungal infection.

Acknowledgements

We are indebted to Dr. Ewa Swoboda-Kopeć from the De-partment of Dental Microbiology, Medical University of Warsawfor supplying the Candida albicans strain.

The work was supported by a research project carried out in theyears 2014–2015, funded by a statutory grant obtained by the Fac-ulty of Medicine and Dentistry, Medical University of Warsaw.

This work was partially funded under the EU FP7 SHYMANproject (grant agreement no. FP7-NMP4-LA-2012-280983), coordi-nated by E. Lester (The University of Nottingham, United Kingdom;www.shyman.eu).

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2 4 6 8 10keV

0

2

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6

8

10

12

cps/eV

Zn Zn C O

Fig. 13. EDS analysis of the acrylic resin material modified by ZnONPs, Cp = 2 wt%

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