Accepted Manuscript

Title: Evaluation of antibiofilm and mechanical properties of

new nanocomposites based on acrylic resins and silver
vanadate nanoparticles

Author: Denise T. Castro Mariana L.C. Valente Cláudia H.L.

Silva Evandro Watanabe Renato L. Siqueira Marco A.
Schiavon Oswaldo Luiz Alves Andréa C. Reis

PII: S0003-9969(16)30059-0
DOI: http://dx.doi.org/doi:10.1016/j.archoralbio.2016.03.002
Reference: AOB 3565

To appear in: Archives of Oral Biology

Received date: 21-4-2015

Revised date: 9-12-2015
Accepted date: 12-3-2016

Please cite this article as: Castro Denise T, Valente Mariana LC, Silva Cláudia HL,
Watanabe Evandro, Siqueira Renato L, Schiavon Marco A, Alves Oswaldo Luiz, Reis
Andréa C.Evaluation of antibiofilm and mechanical properties of new nanocomposites
based on acrylic resins and silver vanadate nanoparticles.Archives of Oral Biology
http://dx.doi.org/10.1016/j.archoralbio.2016.03.002

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Evaluation of antibiofilm and mechanical properties of new nanocomposites based on acrylic
resins and silver vanadate nanoparticles

Denise T. Castro(a), Mariana L. C. Valente(a), Cláudia H. L. Silva(b), Evandro Watanabe(b), Renato

L. Siqueira(c), Marco A. Schiavon(d), Oswaldo Luiz Alves(e) and Andréa C. Reis(b)

(a)
Postgraduate student, Department of Dental Materials and Prosthesis, School of Dentistry of

Ribeirão Preto, USP - University of São Paulo, Ribeirão Preto, SP, Brazil.

(b)
Professor, Department of Dental Materials and Prosthesis, School of Dentistry of Ribeirão Preto,

USP - University of São Paulo, Ribeirão Preto, SP, Brazil.

(c)
Postgraduate student, Postgraduate Program in Materials Science and Engineering - PPG-CEM ,

Federal University of São Carlos - UFSCAR, São Carlos, SP, Brazil.

(d)
Professor, Department of Natural Sciences, GPQM - Research Group of Materials Chemistry,

Federal University of São João del Rei - UFSJ, São João del Rei, MG, Brazil.

(e)
Professor, Laboratory of Solid State Chemistry, State University of Campinas (UNICAMP),

Campinas, Brazil.

Address for correspondence: Andréa Cândido dos Reis, Department of Dental Materials and

Prosthesis – School of Dentistry of Ribeirão Preto - FORP-USP, Av. do Café, s/nº, CEP: 14040-

904; Ribeirão Preto; São Paulo - Brazil. E-mail: andreare@forp.usp.br

 Highlights

 The nanostructured silver vanadate (β-AgVO3) was synthesized and characterized.

• Antibiofim activity promoted by adding β-AgVO3 in acrylic resins.
• We evaluate the dispersion pattern of β-AgVO3 in polymeric matrix.
• We examined the mechanical properties of the modified resins.

Abstract

Objective: The purpose of this study was evaluate, for the first time, the impact of incorporation of

nanostructured silver vanadate (β-AgVO3) in antibiofilm and mechanical properties of dental

acrylic resins (poly(methyl methacrylate), PMMA).

Design: The β-AgVO3 was synthesized and characterized by X-ray diffraction (XRD), Fourier

transform infrared spectroscopy (FTIR), scanning electron microscopy, and microanalysis

(SEM/EDS). Resins specimens were prepared with 0-10% wt.% β-AgVO3 and characterized by

SEM, XRD and optical microscopy. The antibiofim activity of the samples against Candida

albicans and Streptococcus mutans was investigated by XTT reduction test, colony-forming units

(CFUs), and confocal laser scanning microscopy (CLSM). The flexural strength, hardness, and

surface roughness of the samples containing β-AgVO3 were compared with the pure PMMA

matrix.

Results: The incorporation of 10% β-AgVO3 significantly reduced the metabolic activity of C.

albicans and S. mutans (p<0.05). There was a reduction in microbial load (CFU/mL) of

microorganisms for the different concentrations used (p<0.05), which was confirmed by confocal

microscopy. The addition of β-AgVO3 did not change the mechanical properties of hardness and

surface roughness of the resins (p>0.05). However, flexural strength decreased with the addition

of amounts greater than 1% (p<0.05).

Conclusions: β-AgVO3 additions in dental acrylic resin may have an impact on inhibition of biofilm

of main microorganisms associated with dental prostheses. However, the viability of clinical use

should be evaluated in function of changed promoted in some mechanical properties.

Keywords: Nanomaterials; Silver vanadate; Acrylic resin; Antibiofilm properties; Mechanical

behavior.

Introduction

The formation of biofilm on the surfaces of provisional crowns and fixed/removable

prostheses plays an important role in the development of caries, periodontal disease and mucositis

(Rupf et al., 2012; Queiroz et al., 2013). These factors limit the longevity of rehabilitative treatment

and constitute a risk of opportunistic infections, reducing quality of life and generating additional

costs to patients (Larazin et al., 2013). The colonization process on the surface of dental prostheses

is characterized by various steps (Rupf et al., 2012) and occurs because acrylic resins have porosity,

an absence of ionic charge on the surface, roughness, and a capacity to absorb fluids, all of which

lead to the accumulation of microorganisms (Santos, Pithon, Carvalho, Ramos & Romanos, 2013;

Sivakumar et al., 2014). The development of dental acrylic resins capable of inhibiting biofilm

formation is therefore critical in the control of oral disease (Wang, Shen & Haapasalo, 2014).

In an effort to add antimicrobial activity to these materials, studies have used additives such

as nystatin, miconazole, chlorhexidine, modified monomers, zirconium dioxide nanoparticles and

aluminum borate (Redding et al., 2009; Alcântara et al., 2012; Han et al., 2015). The antimicrobial

properties of silver (Ag) dates to 3000 years ago, and the mechanism is based on the interaction of

silver with thiol groups of enzymes involved in bacterial cell metabolism thus causing cell death
(Kwakye-Awuah, Williams, Kenward & Radecka, 2008). Due to this property, silver ions and

silver nanoparticles (AgNPs) have also been introduced in dental materials (Bürgers et al., 2009;

Yang, Zhang, Yu, Zhen & Huang, 2014). For AgNPs these properties appear to be even better in

function to their nanoscale dimensions, interact extensively with microorganisms (Fan et al., 2011;

Holtz, Lima, Souza Filho, Brocchi & Alves, 2012; Kumar, Jolivalt, Pulpytel, Jafari & Arefi-

Khonsari, 2013; Hojati et al., 2013). However, in addition to great aesthetic inconvenience (color),

there is the difficulty of stabilizing nanoparticles when used alone (Holtz, Lima, Souza Filho,

Brocchi & Alves, 2012). AgNPs tend to cluster, which may negatively influence in their

antimicrobial effect by reducing the surface contact area (Shameli et al., 2011).

A possible alternative to overcome this problem was recently proposed and is to support

AgNPs on Ag nanowires vanadate (β-AgVO3) (Holtz et al., 2010). This nanomaterial has

antimicrobial property as it the silver and the vanadium elements act synergistically and interact

with microorganisms' cell membranes. In addition, this compound has a high dispersion of silver

nanoparticles on silver vanadate nanowires, maintaining high surface contact with the

microorganisms (Holtz et al., 2010; Holtz, Lima, Souza Filho, Brocchi & Alves, 2012). Previous

studies have shown the possibility of using this hybrid nanomaterial as an antimicrobial additive

for water-based paint for domestic or hospital environment in order to improve sanitary conditions

and reduce the number of hospital infections once it exhibits antibacterial activity 30 times larger

than that of Oxacillin (Holtz et al., 2010) and as an additive for acrylic resin for using in dental

applications, as it functionalized samples with this nanomaterial showed inhibition zone against

the growth of the main microorganisms in the oral cavity demonstrating the need for studies on

biofilm models as well as the mechanical properties (de Castro et al., 2014).

The objective of this study was therefore to evaluate the antibiofilm activity of two acrylic

resins containing β-AgVO3 against C. albicans and S. mutans, the main microorganisms associated
with dental prostheses. The hardness, surface roughness, and flexural strength of the resins were

also evaluated, as these mechanical properties, together with antibiofilm action, are factors that are

directly related to the effectiveness of the material in the oral cavity.

Materials and methods

Synthesis of nanostructured silver vanadate

Nanostructured silver vanadate (β-AgVO3) was synthesized through a precipitation

reaction between silver nitrate (AgNO3, Merck 99.8%) and ammonium metavanadate (NH4VO3,

Merck 99%) according to the methodology described by Holtz et al (2010). Initially, 1.3569 g of

AgNO3 and 0.9736 g of NH4VO3 were each solubilized in 200 mL of distilled water. The solutions

were stirred separately on a 65 °C heated surface for 10 min. Next, the silver nitrate solution was

added dropwise using a burette into the ammonium metavanadate solution under constant stirring

at 65 °C. The precipitate obtained was washed with distilled water and absolute ethanol several

times, filtered and then dried in a vacuum line for 10 h.

Characterization of β-AgVO3

The silver vanadate was characterized to ensure standardization of the resulting materials.

For this purpose, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR),

scanning electron microscopy, and microanalysis (SEM/EDS) were used. XRD analysis was

performed at room temperature using a Shimadzu XRD-7000 diffractometer operating with Cu Kα

radiation (λ = 1.5406 Å) at 40 kV and 30 mA. Data were collected continuously from 10° < 2θ <

70° with a velocity of 1°/min to identify the crystal structure. The crystallinity index (IC) of the

sample was determined (Krimm & Tobolsky, 1951). The IC (%) was calculated from the
ratio between the crystalline diffracted area (AC) and the total area of diffraction (AT =

crystalline + amorphous), as shown in Equation 1:

(1)

Infrared spectra with Fourier transform were collected on an FTLA 2000 – 102

spectrometer using transmittance mode in the region of 4000-400 cm-1 to monitor β-AgVO3

synthesis. Finally, the morphological analysis of β-AgVO3 was performed by scanning electron

microscopy (SEM) on a Magellan 400L FEI microscope coupled with an energy dispersive X-ray

spectrometer (EDS), which allowed for qualitative chemical analysis.

Preparation of functionalized resins with β-AgVO3

To prepare the specimens, Dencor Lay autopolymerizable (SC) and Clássico

thermopolymerizable (TR) (Clássico Artigos Odontológicos®) acrylic resins were used. Six

experimental groups functionalized with β-AgVO3 at concentrations of 0.5, 1, 2.5, 5 and 10%

(wt.%) and a control group without additive were formed. The β-AgVO3 was added to the polymer

particle, and after homogenization, the monomer was added at a ratio of three parts powder

(polymer) to one part liquid (monomer) according to the manufacturer's recommendations. During

the plastic phase, the resin was placed in holes prepared in metal muffles with appropriate

dimensions for each test and was cured according to the manufacturer's recommendations. For the

antibiofilm tests, surface roughness was standardized at 0.2 µm using a Surftest SJ-201P (Mitutoyo

Corporation).
Characterization of functionalized resins

The characterization of the samples with β-AgVO3 was performed in comparison with

control samples without nanomaterial by XRD and SEM order to identify the changes introduced

by the incorporation and the pattern of dispersion of the particles. These techniques were performed

as described previously for the characterization of β-AgVO3. The morphology of resins was also

analyzed by optical microscopy using a Leica MZ7.5 stereomicroscope coupled with a DFC-

490 camera.

Antibiofilm experiment

Two standard strains were used in the study: Candida albicans (ATCC 10231) and

Streptococcus mutans (ATCC 25175). For inoculum preparation, the microorganisms were seeded

in Petri dishes with specific culture media (Sabouraud Dextrose Agar for yeast and modified SB-

20 Agar for bacteria). After incubation at 37°C for 48 h, standardized S. mutans (108 CFU / mL)

and C. albicans (106 CFU / mL) microbial inocula were obtained in 0.85% saline using a PCB 687

spectrophotometer (BYK Gardner). The biofilm was formed in 24-well polystyrene plates. Sterile

resin samples were placed individually in each well, and 1000 µL of the inoculated culture medium

was transferred to each hole. The plates were incubated at 37°C for 1 h 30 min with stirring at 750

rpm in a bacteriological incubator (Incubator Shaker Mod. - EC-320, Cienlab) for adherence of the

biofilm. After the adhesion period, the specimens were rinsed with 2000 µL of 0.85% saline to

remove non-adherent cells, to buffer the medium, and to remove metabolites. Next, 1000 µL of

sterile culture medium was added to each well to provide more nutrients for the microorganisms.

The plates were incubated at 37°C with 750 rpm stirring for 48 h for biofilm maturation.

Biofilm viability was evaluated using the XTT reduction test ([2,3-bis (2-methoxy-4-nitro-

5-sulfophenyl)-2H-tetrazolio- 5-carboxanilide]). Specimens with 9 mm in diameter and 2 mm in

height were prepared and n = 8 was used for each group and microorganism. After biofilm

formation, each specimen was washed once with saline, inserted into a polypropylene tube with

3000 µL of PBS, and sonicated (40 Hz) for 20 min to detach the biofilm. Then, 100 µL aliquots of

homogenized tube suspension and 50 µL of XTT solution were transferred to each well of a 96-

well plate. The plates were incubated in the dark at 37 ° C for 24 h to measure the formation of

XTT-formazan via spectrophotometry at 492 nm using a Synergy II microplate reader (BioTek

Instruments). The number of viable cells was quantified in terms of colony forming units per

milliliter (CFU/mL) after sowing 25 µL of the raw and diluted aliquots (n = 8) in specific culture

media and incubating at 37 °C for 24 h. Qualitative analyses of the biofilms (48 h) were performed

for the control group and for samples containing 2.5, 5 and 10% β-AgVO3. The specimens were

stained using a Live Dead/Back Light Kit (Molecular Probes, Eugene) and incubated for 15 min

under light at room temperature. An image of each group was captured with a confocal laser

scanning microscope (Leica Microsystems CMS) at 63x magnification.

Mechanical tests

For mechanical testing, n = 10 was used for each group. The three-point flexural strength

test was conducted on an EMIC DL 2000 universal testing machine at a speed of 5 mm/min and a

load of 20 kgf according to the ISO 20795-1:2008 standard, using specimens with dimensions 65

mm x 10 mm x 3.3 mm. The analysis of surface roughness was carried out according to technical

standard NBR ISO 4287:2002 using Surftest SJ-201P (Mitutoyo Corporation); the analyzer tip

touched the piece and covered a distance of 4 mm, making three measurements on each specimen

in the direction of its larger diameter.

 Finally, surface hardness analysis was performed using a Shimadzu HMV 2000 micro

hardness tester. Three randomly equidistant measurements were performed on each specimen using

a Knoop type indenter with a load of 25 gf for 5 sec.

Statistical analysis

For microbiological and mechanical variables, 2 variation factors were considered:

the concentration of β-AgVO3 (on 6 levels: 0%, 0.5%, 1%, 2.5%, 5%, and 10%) and acrylic

resin (on 2 levels: SC and TR). After the normal (Shapiro-Wilk test) and homogeneous

(Levene test) distribution of data was verified, 2-way ANOVA, followed by the Tukey test

(α= 5%) was used. Statistical analysis was performed with software (SPSS v17.0; SPSS Inc).

Results

Synthesis and characterization of β-AgVO3

The X-ray diffraction pattern demonstrates the formation of β-AgVO3, indexed by

crystallographic card JCPDS 29-1154. The crystallinity index of the sample was estimated to

be 67% according to the background displayed by XRD (Krimm & Tobolsky, 1951). This

result indicates that the sample is not completely crystalline and contains 33% amorphous phase.

The diffraction peaks located at 2θ of 37.79 and 44.10 correspond to the presence of nanoparticles

of metallic silver, Ag0 (JCPDS 87-597), which formed over the entire surface of the crystals (Figure

1).

The main bands observed are located between 400-1000 cm-1, most of them attributed to

bonds between the oxygen and vanadium atoms. In the spectrum, the band located at 1633 cm-1

corresponds to the vibration of the −OH bond of the water molecule (Figure 2).
 The strong band observed at 1409 cm-1 in ammonium vanadate is attributed to N−H

symmetric stretching. This band completely disappears in the β-AgVO3 spectrum, indicating its

formation, which is also illustrated by the intensification of the band at approximately 849 cm-1

associated both with the vibration of V−O stretching and with the vibration mode of the Ag−O−V

bond. The bands located at 965, 925, 896 and 653 cm-1 in the spectrum of β-AgVO3 are attributed

to V=O stretching, while the band located at 501 cm-1 may be associated with the V−O vibration

mode. The bands in the region 940-775 cm-1 also suggest the presence of polymeric vanadate

groups. As with water, the presence of silver in the system also affects the vibration mode of the

metal-oxygen bonds, contributing to the displacement of certain bands in relation to the ammonium

vanadate spectrum. This effect could be observed especially for the bands in the 700 cm -1 region.

It can be observed that the β-AgVO3 showed acicular crystals, with a mean diameter of 150

nm and length in the order of micrometers. The EDS spectrum reveals the presence of the elements

silver, vanadium and oxygen, which is consistent with the established formulation for the synthesis

of the material. The peak located at 0.28 keV is attributed to elemental carbon (C), used in tape

form only to fix the sample on the microscope support.

Characterization of acrylic resins functionalized with β-AgVO3

Comparing the resins with different β-AgVO3 content, it appears that regardless of

the content and type of resin, there are always β-AgVO3 domains throughout the matrix,

following a standard circular dispersion (Figure 4). The dispersion mode presented by β-

AgVO3 can be related to the particle size presented by the polymer powder used in the

preparation of the resins. The β-AgVO3 crystals appears to have been confined to the area

surrounding the larger polymer particles during the curing stage.

 Figure 5 shows images obtained by optical microscopy of samples containing 5 and

10% β-AgVO3. Is observed the presence of clusters, which indicates that the method used to

prepare the resins was unable to promote good dispersion of β-AgVO3 in the polymeric

matrix.

X-ray diffraction of the resins revealed that the incorporation of β-AgVO3 in percentages

above 1% resulted in segregation in the system and that it is possible to identify the vanadate

diffraction peaks in the polymeric matrix. This phenomenon becomes more evident when the β-

AgVO3 content increases from 2.5 to 5 and 10% (Figure 6).

Analysis of antibiofilm activity

The XTT reduction and count of colony-forming units per milliliter (CFU/mL) analyses

showed that the metabolic activity of C. albicans and Streptococcus mutans biofilms decreased

significantly as the β-AgVO3 content in the resins increased. The absorbance values obtained using

XTT on C. albicans were reduced significantly compared to that of the control group for both resins

(p = 0.368) when concentrations of 5 and 10% were used (p < 0.05). However, for S. mutans, a

greater reduction was observed only with the addition of 10% (p = 0.023) (Figure 7).

With the incorporation of 10% β-AgVO3, the number CFUs of C. albicans was reduced

compared to that of the control group (p = 0.000) for both resins (p = 0.431). For S. mutans, this

reduction was observed with the incorporation of 2.5% followed by 5 to 10% (Figure 8).

The images obtained by confocal laser scanning microscopy (CLSM) of the metabolic

activity and amount of C. albicans and S. mutans biofilm formed on the surface of certain TR

specimens. Greater cell density and a higher proportion of green cells were observed in the control

group than in the other groups for both micro-organisms. A change in cell viability (increased ratio

of red/green fluorescence) in the samples containing β-AgVO3 could be observed, as well as a

reduction in cell number, with a higher number of dark spaces between the cells; therefore, in the

samples containing 10% additive, almost complete inhibition was observed (Figure 9).

Mechanical properties

For SC, the incorporation of 0.5% β-AgVO3 resulted in a significant increase in surface

hardness values (26.14 ± 5.13) compared to those of the other groups (p=0.000). The surface

hardness of the TR remained unchanged after the incorporation of different fractions of β-AgVO3

(p=1.000). When comparing the two kind of resins, a significant difference could be observed only

at concentrations of 0.5 and 10% (p=0.023) (Table 1).

The flexural strength of both resins was reduced when a 2.5% concentration was

incorporated (p=0.446), followed by concentrations of 5 and 10% β-AgVO3, compared to the

control group (p=0.000), which, in turn, showed similar results to the groups containing

concentrations of 0.5% (p=0.847) and 1% (p=1.000) (Figure 10).

There was no statistically significant difference in the surface roughness of the acrylic resins

(p=0.209) when the samples containing β-AgVO3 were compared to the control group (p=0.751).

This result indicates that the polishing process of functionalized resin samples provides a surface

similar to conventional resin and does not promote an uneven surface that would encourage the

accumulation of microorganisms (Figure 11).

Discussion

This study demonstrated that nanostructured silver vanadate (β-AgVO3) can be easily

synthesized by the precipitation reaction between silver nitrate and ammonium metavanadate. The

β-AgVO3 formed had acicular morphology with a mean crystal diameter of 150 nm and length in

the order of micrometers, similar to the pattern observed in the images obtained by

transmission electron microscopy (STEM) by Castro et al (2015). The sample was not fully

crystalline and approximately 33% was in the amorphous phase.

Upon completion of the synthesis and confirmation of the formation of β-AgVO3 by X-ray

diffraction and FTIR spectrum, different amounts of β-AgVO3 were incorporated into two acrylic

resins used in the manufacture of dental prostheses. The antibiofilm action and mechanical

properties of the resulting materials were then evaluated, as these factors are directly related to the

material's effectiveness in the oral cavity.

As microbial species colonization on acrylic resin surfaces is a significant clinical problem

in dentistry practice (Larazin et al., 2013; Sato, Ohshima, Maeda & Ohkubo, 2013), this study used

CFU, XTT reduction, and CLSM to evaluate the modified resins' antibiofilm activity. As in studies

involving other compounds, the antimicrobial effect was dose dependent (Fan et al., 2011; Shameli

et al., 2011; de Castro et al., 2014; Han et al., 2015). In the analysis of cellular metabolism using

the XTT method, a decline in the metabolism of C. albicans was observed with the incorporation

of 5 and 10% β-AgVO3. For S. mutans, this decline was clearly evident only at a concentration of

10%. The CFU count showed a significant reduction in the number of viable microorganisms when

vanadate was incorporated at percentages of 2.5, 5 and 10% for S. mutans and 10% for C albicans.

Note that there was no complete correlation between the methodologies used in this research

because of the difference in sensitivity of each technique as well as the morphological and

metabolic characteristics of each microorganism. However, in general, the higher the concentration
of β-AgVO3 incorporated into the resins, the higher the antibiofilm activity observed. CLSM

analysis qualitatively complemented the previous analyses, showing a reduction in the amount of

biofilm as well as its viability with increasing concentrations of β-AgVO3.

There is a consensus that the adhesion of microorganisms on a surface is the

prerequisite for the colonization of dental prostheses which will function as a reservoir of

infection. Thus, the resin surface topography is critical for the biofilm accumulation,

interfering with the success of rehabilitation treatment and quality of life of patients

(Nevzatoğlu, Ozcan, Kulak-Ozkan & Kadir, 2007; Regis et al., 2011).

In dental literature, the surface roughness values of acrylic resins can range from

0.02 to 7.6 µm. Smooth and highly polished surfaces are important not only for the patient's

comfort, but also for denture longevity, good aesthetic results and base plate retention

(Akalın-Evren, Kulak-Özkan, Ozcan & Kadir, 2014). The presence of antimicrobial agents

may be useless if the tested acrylic resin is able to retain greater amounts of debris. The

values of surface roughness in this study were close to 0.2 µm, regardless of the concentration

of the nanomaterial incorporated in resins. This value which can be considered a value below

which microbial adhesion is not expected (Bollen, Lambrechts & Quirynen, 1997). These

results demonstrate that the incorporation of β-AgVO3 did not impossible to obtain a smooth

and polished surface.

In order to predict the performance of the materials when exposed to the forces of the oral

cavity, hardness and flexural strength tests performed. Hardness in the case of dental resins, may

be associated with forces arising from occlusion and mechanical cleaning as well as contact with

oral fluids that can alter the surface properties of the prosthesis (Ayaz & Durkan, 2013; Goiato et

al., 2013). The results showed that the surface hardness of SC increased significantly with the

incorporation of 0.5% β-AgVO3, suggesting that vanadate had no negative effect on the degree of
resin polymerization. This phenomenon is observed because, in general, hardness shows a positive

correlation with the degree of polymer conversion (Faltermeier, Rosentritt & Müssig, 2007). At

concentrations above 0.5% β-AgVO3 and for TR, hardness remained practically unchanged.

Finally, flexural strength is an important property for dental resins, as the major cause of

prosthesis fracture in the oral cavity is related to stresses caused by repetitive chewing movements

(Ayaz & Durkan, 2013). According to ISO 20795-1:2008 (ISO, 2008), the flexural strength of

polymeric materials must be at least 50 MPa. Based on the flexural strength values obtained,

incorporating β-AgVO3 at concentrations above 1% reduced this property significantly compared

to the control group. The incorporation of 10% promoted results below those required by this

standard, the average being 48.03 MPa. Castro et al (2015) found that incorporation of 5 and

10% β-AgVO3 causes a reduction in impact strength of acrylic resins which may increase the

possibility of fracture of dental prostheses in cases of accidental falls, repeated masticatory

forces and stress concentration areas.

Generally, composite materials with lower mechanical properties that of the matrix are

obtained when the filler content is below the critical concentration or when it forms clusters that

weaken the system. It is believed that for the system in question, the non-improvement in resistance

with the addition of low concentrations of β-AgVO3 and the significant reduction observed with

concentrations of 5 to 10% was caused by the dispersion mode and the formation of agglomerates.

Scanning electron micrographs revealed that the β-AgVO3 dispersion along the polymeric

matrix follows a circular mode, with an increase in the number of domains in the resins with higher

β-AgVO3 content. This distribution can be explained by the fact that when β-AgVO3 is added to

the mixture to prepare the resin, it disperses in the monomer, being confined mainly around the

large polymer particles to the extent that polymerization of the system occurs. As the polymer

particles already existed, vanadate dispersion of the resin was limited. It should also be noted that
increasing the β-AgVO3 fraction from 2.5 to 5 and 10% caused the formation of clusters in the

system, a result that could be enhanced by X-ray diffraction of the resins and optical microscopy.

As the β-AgVO3 was confined to the area surrounding preexisting polymer particles during

the curing stage, there was no possibility of better dispersion, and the increase of its fraction in the

system caused the formation of agglomerates. These agglomerates can act as a stress center and

adversely affect the mechanical properties of resins, especially properties involving more complex

forces, such as bending resistance (Han, Kiat-Amnuay, Powers & Zhao, 2008; Sodagar et al.,

2012). These results are consistent with the findings of the studies of Sodagar et al (2012) and

Shibata et al (2007), when adding silver nanoparticles to acrylic resins, reported that they can act

as impurities in the internal structure of the polymerized material, weakening the system.

The attempt to functionalizing the resin with β-AgVO3 through a simplistic method of

manual manipulation of easy access to the dentist in clinical practice and low processing costs,

proposal in the present study was unable to promote effective dispersion of inorganic

particles in polymeric matrix. The reduced chemical affinity between the inorganic filler of

hydrophilic nature and the polymer, predominantly hydrophobic, may be responsible for the

spatial confinement of the polymer chains. As an approach to overcome this problem other

methods of synthesis and the incorporation should be proposed, among which attempting to

improve the β-AgVO3 compatibility with the polymeric matrix by means of chemical surface

modification may be effective (Bershtein et al., 2002). The dispersion of β-AgVO3 by a

polymer solution followed by solvent removal (Utracki, 2008), use of mixers such as amalgam

mixer (Hamedi-Rad, Ghaffari, Rezaii & Ramazani, 2014) or extruder (Soares et al., 2015)

may also provide different results and should be better studied.

Considering the method proposed in this study, it may be concluded that acrylic resins

functionalized with β-AgVO3 antibiofilm exhibit significant activity against C. albicans and S.
mutans. The development of new methodologies to improve the dispersion pattern of β-AgVO3 in

a polymer matrix may provide the materials with better mechanical properties and further expand

their antibiofilm action by exposing a larger surface area. Furthermore, analyses that evaluate the

possible cytotoxicity of these materials are required to ascertain the viability of their use.

Conclusions

In this work we have successfully demonstrated that the addition of different concentrations

of β-AgVO3 to dental acrylic resins can promote antibiofilm activity against C. albicans and S.

mutans. This makes the material promising to use for fabrication of dental devices. The dispersion

of β-AgVO3 in the resins suggests that the mixing procedure adopted in this work was not efficient

enough to disperse the resins evenly in the system. Therefore, new methodologies are currently

under study to optimize this parameter toward reproducibly and efficiency.

Conflict of interest

The authors declare that they have no conflict/s of interest related to the present study.

Acknowledgements

We thank the São Paulo Research Foundation – FAPESP (process numbers: 2012/09124-

1 and 2012/09347-0) for funding the study and the Solid State Chemistry Laboratory - UNICAMP

and Oral Rehabilitation Research Laboratory FORP / USP for the facilities.
References

Akalın-Evren, B., Kulak-Özkan, Y., Ozcan, M., & Kadir, T. (2014). Candida albicans adhesion

on reinforced polymethylmethacrylate denture

resin: effect of fibre architecture andexposure to saliva. Gerodontology, 31, 194-201.

Alcântara, C.S., Macêdo, A.F.C., Gurgel, B.C.V., Janaina, H.J., Neppelenbroek, K.H., et al.

(2012). Peel bond strength of resilient liner modified by the addition of antimicrobial agents to

denture base acrylic resin. The Journal of Applied Oral Science, 20, 607-612.

Ayaz, E.A., & Durkan, R. (2013). Influence of acrylamide monomer addition to the acrylic denture-

base resins on mechanical and physical properties. International Journal of Oral Science, 5,229-

235.

Bershtein, V.A., Egorova, L.M., Yakushev, P.N., Pissis, P., Sysel, P., et al. (2002). Molecular

dynamics in nanostructure polyimide-silica hybrid materials and their thermal stability. Polymer

Science, 40, 1056-1069.

Bollen, C.M., Lambrechts, P., & Quirynen, M. (1997). Comparison of surface roughness of oral

hard materials to the threshold surface roughness for bacterial plaque retention: a review of the

literature. Dental Materials, 13, 258-269.

Bürgers, R., Eidt, A., Frankenberger, R., Rosentritt, M., Schweikl, H., et al. (2009). The anti-

adherence activity and bactericidal effect of microparticulate silver additives in composite resin

materials. Archives of Oral Biology, 54, 595-601.

de Castro, D.T., Valente, M.L.C., Agnelli, J.A.M., da Silva, C.H.L., Watanabe, E., et al. (2015). In

vitro study of the antibacterial properties and impact strength of dental acrylic resins modified with

a nanomaterial. The Journal of Prosthetic Dentistry, Epub Ahead of Print.

de Castro, D.T., Holtz, R.D., Alves, O.L., Watanabe, E., Valente, M.L., et al. (2014). Development

of a novel resin with antimicrobial properties for dental application. The Journal of Applied Oral

Science, 22, 442-449.

Faltermeier, A., Rosentritt, M., & Müssig, D. (2007). Acrylic removable appliances: comparative

evaluation of different postpolymerization methods. American Journal of Orthodontics and

Dentofacial Orthopedics, 131, e16-22.

Fan, C., Chu, L., Rawls, H.R., Norling, B.K., Cardenas, H.L., et al. (2011). Development of an

antimicrobial resin–a pilot study. Dental Materials, 27, 322–328.

Goiato, M.C., Santos, D.M., Baptista, D.T., Moreno, A., Andreotti, A.M., et al. (2013). Effect of

termal cycling and disinfection on microhardness of acrylic resin denture base. Journal of Medical

Engineering & Technology, 37, 203-207.

Hamedi-Rad, F., Ghaffari, T., Rezaii, F., & Ramazani, A. (2014). Effect of nanosilver

on thermal and mechanical properties of acrylic base complete dentures. Journal of Dentistry

(Tehran, Iran), 11, 495-505.

Han, Y., Kiat-Amnuay, S., Powers, J.M., & Zhao, Y. (2008). Effect of nano-oxide concentration

on the mechanical properties of a maxillofacial silicone elastomer. The Journal of Prosthetic

Dentistry, 100, 465-473.

Han, Z., Zhu, B., Chen, R., Huang, Z., Zhu, C., et al. (2015). Effect of silver-supported materials

on the mechanical and antibacterial properties of reinforced acrylic resin composites. Materials &

Design, 65, 1245–1252.

Hojati, S.T., Alaghemand, H., Hamze, F., Babaki, F.A., Rajab-Nia, R., et al. (2013). Antibacterial,

physical and mechanical properties of flowable resin composites containing zinc oxide

nanoparticles. Dental Materials, 29, 495-505.

Holtz, R.D., Lima, B.A., Souza Filho, A.G., Brocchi, M., & Alves, O.L. (2012). Nanostructured

silver vanadate as a promising antibacterial additive to water-based paints. Nanomedicine, 8, 935-

940.

Holtz, R.D., Souza, A.G., Brocchi, M., Martins, D., Duran, N., et al. (2010). Development of

nanostructured silver vanadates decorated with silver nanoparticles as a novel antibacterial agent.

Nanotechnology, 21, 185102.

International Organization for Standardization. Dentistry, base polymers, part 1: denture base

polymers. ISO 20795-1:2008.

Krimm, S., & Tobolsky, A.V. (1951). Quantitative x-ray studies of order in amorphous and

crystalline polymers. Quantitative x-ray determination of crystallinity in polyethylene. Journal of

Polymer Science, 7, 57-76.

Kumar, V., Jolivalt, C., Pulpytel, J., Jafari, R. & Arefi-Khonsari, F. (2013). Development of silver

nanoparticle loaded antibacterial polymer mesh using plasma polymerization process. Journal of

Biomedical Materials Research. Part A, 101, 1121-1132.

Kwakye-Awuah, B., Williams, C., Kenward, M.A., & Radecka, I. (2008). Antimicrobial action

and efficiency of silver-loaded zeolite X. Journal of Applied Microbiology, 104, 1516–1524.

Lazarin, A.A., Machado, A.L., Zamperini, C.A., Wady, A.F., Spolidorio, D.M., et al. (2012). Effect

of experimental photopolymerized coatings on the hydrophobicity of a denture base acrylic resin

and on Candida albicans adhesion. Archives of Oral Biology, 58, 1-9.

Nevzatoğlu, E.U., Ozcan, M., Kulak-Ozkan, Y., & Kadir, T. (2007). Adherence of Candida

albicans to denture base acrylics and silicone-based resilient liner materials with diferente surfasse

finishes. Clinical of Oral Investigations, 11, 231-236.

Queiroz, J.R., Fissmer, S.F., Koga-Ito, C.Y., Salvia, A.C., Massi, M., et al. (2013). Effect of

diamond-like carbon thin film coated acrylic resin on Candida albicans biofilm formation. Journal

of Prosthodontics, 22, 451-455.

Redding, S., Bhatt, B., Rawls, H.R., Siegel, G., Scott, K., et al. (2009). Inhibition of Candida

albicans biofilm formation on denture material. Oral Surgery, Oral Medicine, Oral Pathology,

Oral Radiology, and Endodontics, 107,669-672.

Regis, R.R., Zanini, A.P., Della Vecchia, M.P., Silva-Lovato, C.H., Paranhos, H.F.O., et al. (2011).

Physical properties of an acrylic resin after incorporation of an antimicrobial monomer. Journal of

Prosthodontics, 20, 372-379.

Rupf, S., Balkenhol, M., Sahrhage, T.O., Baum, A., Chromik, J.N., et al. (2012). Biofilm inhibition

by an experimental dental resin composite containing octenidine dihydrochloride. Dental

Materials, 28, 974-984.

Santos, R.L., Pithon, M.M., Carvalho, F.G., Ramos, A.A., & Romanos, M.T. (2013). Mechanical

and biological properties of acrylic resins manipulated and polished by different methods. Brazilian

Dental Journal, 24, 492-497.

Sato, M., Ohshima, T., Maeda, N., & Ohkubo, C. (2013). Inhibitory effect of coated mannan

against the adhesion of Candida biofilms to denture base resin. Dental Materials Journal, 32, 355-

360.

Shameli, K., Ahmad, M.B., Zargar, M., Yunus, W.M.Z.W., Rustaiyan, A., et al. (2011). Synthesis

of silver nanoparticles in montmorillonite and their antibacterial behavior. International Journal of

Nanomedicine, 6, 581-590.

Shibata, T., Hamada, N., Kimoto, K., Sawada, T., Sawada, T., et al. (2007). Antifungal effect of

acrylic resin containing apatite-coated TiO2 photocatalyst. Dental Materials Journal, 26, 437–

444.
Sivakumar, I., Arunachalam, K.S., Sajjan, S., Ramaraju, A.V., Rao, B., et al. (2014). Incorporation

of antimicrobial macromolecules in acrylic denture base resins: A research composition and

update. Journal of Prosthodontics, 23, 284-290.

Soares, I.L., Chimanowsky, J.P., Luetkmeyer, L., da Silva, E.O., Souza, D.H., et al. (2015).

Evaluation of the Influence of Modified TiO2 Particles on Polypropylene Composites. Journal of

Nanoscience and Nanotechnology, 15, 5723-5732.

Sodagar, A., Kassaee, M.Z., Akhavan, A., Javadi, N., Arab, S., et al. (2012). Effect of silver nano

particles on flexural strength of acrylic resins. Journal of Prosthodontics Research, 56, 120-124.

Utracki, L.A. (2008). Polymeric nanocomposites: compounding and performance. Journal of

Nanoscience and Nanotechnology, 8, 1582-1596.

Wang, Z., Shen, Y., & Haapasalo, M. Dental materials with antibiofilm properties. Dental

Materials, 30, e1-16.

Yang, S.X., Zhang, Y.H., Yu, J.M., Zhen, Z.C., & Huang, T.Z. (2014). Antibacterial and

mechanical properties of honeycomb ceramic materials incorporated with silver and zinc.

Materials & Design,59: 461–465.

Figure Captions

Fig. 1 X-ray diffraction pattern of β-AgVO3.

Fig. 2 FTIR spectra of ammonium metavanadate and β-AgVO3.

Fig. 3 Scanning electron micrograph and EDS spectrum of β-AgVO3.

Fig. 4 Scanning electron micrograph of the polymer used in the preparation of the resin (a)

and fracture surface of TR containing 5% β-AgVO3 (b, c and d).

Fig. 5 Optical microscopy of SC acrylic resin containing 5% β-AgVO3 (a) and TR acrylic

resin containing 10% β-AgVO3 (b).

Fig. 6 X-ray diffraction patterns of resins containing different concentrations of nanostructured

silver vanadate: TR (a); SC (b).

Fig. 7 Mean absorbance of XTT and Standard Deviation: Identical letters indicate statistical

equality for C. albicans, while equal numbers indicate equality for S. mutans.

Fig. 8 Mean number of colony forming units and Standard Deviation: Identical letters indicate

statistical equality for C. albicans, while equal numbers indicate equality for S. mutans.

Fig. 9 Confocal laser microscopy of the formed biofilm: Candida albicans (a) TR 0%; (b) TR 5%;

(c) TR 10%; Streptococcus mutans (d) TR 0%; (e) TR 5%; (f) TR 10%.

Fig. 10 Flexural strength (MPa) and Standard Deviation: Identical letters indicate statistical equality

between the groups with different levels of β-AgVO3

Fig. 11 Surface roughness (µm) and Standard Deviation: Identical letters indicate statistical

equality between groups with different β-AgVO3 content.

Fig.1

Fig.2
Fig.3
Fig.4
Fig.5

Fig.6
Fig.7
Fig.8
Fig.9
Fig.10
Fig.11
Table 1. Mean (SD) of the surface hardness Knoop (HK) of resins containing β-AgVO3

Control 0.5% 1% 2.5% 5% 10%

SC 16.88 26.14 19.23 17.85 17.05 15.65

(2.0)ABa (5.13)Ca (1.41)Aa (2.11)ABa (1.82)ABa (1.61)Ba

TR 16.62 17.87 18.06 17.88 17.89 18.51

(1.01)Aa (2.55)Ab (1.5)Aa (1.82)Aa (2.33)Aa (2.21)Ab

* SC – Autopolymerizable ; TR - Thermopolymerizable
* Lowercase letters - comparison between lines
* Uppercase letters - comparison between columns
* Equal letters - statistical equality