Effect of experimental photopolymerized coatings on the
hydrophobicity of a denture base acrylic resin and on Candida
albicans adhesion

Andréa Azevedo Lazarin a, Ana Lucia Machado a,*, Camila Andrade Zamperini a,
Amanda Fucci Wady a, Denise Madalena Palomari Spolidorio b, Carlos Eduardo Vergani a

aAraraquara Dental School, UNESP - Univ. Estadual Paulista, Department of Dental Materials and Prosthodontics, Araraquara, São Paulo, Brazil
bAraraquara Dental School, UNESP - Univ. Estadual Paulista, Department of Physiology and Pathology, Araraquara, São Paulo, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 9

a r t i c l e i n f o

Article history:

Accepted 7 October 2012

Keywords:

Adhesion

Candida albicans

Hydrophilic

Prosthesis

Surface modification

a b s t r a c t

Objective: This study investigated the effect of experimental photopolymerized coatings,

containing zwitterionic or hydrophilic monomers, on the hydrophobicity of a denture base

acrylic resin and on Candida albicans adhesion.

Methods: Acrylic specimens were prepared with rough and smooth surfaces and were either

left untreated (control) or coated with one of the following experimental coatings: 2-

hydroxyethyl methacrylate (HE); 3-hydroxypropyl methacrylate (HP); and 2-trimethylam-

monium ethyl methacrylate chloride (T); and sulfobetaine methacrylate (S). The concen-

trations of these constituent monomers were 25%, 30% or 35%. Half of the specimens in each

group (control and experimentals) were coated with saliva and the other half remained

uncoated. The surface free energy of all specimens was measured, regardless of the

experimental condition. C. albicans adhesion was evaluated for all specimens, both saliva

conditioned and unconditioned. The adhesion test was performed by incubating specimens

in C. albicans suspensions (1 � 107 cell/mL) at 37 8C for 90 min. The number of adhered yeasts

were evaluated by XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[{phenylamino}carbo-

nyl]-2H-tetrazolium-hydroxide) method.

Results: For rough surfaces, coatings S (30 or 35%) and HP (30%) resulted in lower absorbance

values compared to control. These coatings exhibited more hydrophilic surfaces than the

control group. Roughness increased the adhesion only in the control group, and saliva did

not influence the adhesion. The photoelectron spectroscopy analysis (XPS) confirmed the

chemical changes of the experimental specimens, particularly for HP and S coatings.

Conclusions: S and HP coatings reduced significantly the adhesion of C. albicans to the acrylic

resin and could be considered as a potential preventive treatment for denture stomatitis.

# 2012 Elsevier Ltd. 

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Open access under the Elsevier OA license. 
1. Introduction

In spite of its multifactorial etiology, Candida albicans infection

has often been associated with denture-induced stomatitis.1
* Corresponding author at: Departament of Dental Materials and Pros
Paulista, Rua Humaita, 1680, CEP: 14801-903, Araraquara, SP, Brazil. T

E-mail addresses: cucci@foar.unesp.br, almachado98@uol.com.br 

0003–9969      # 2012 Elsevier Ltd.   

http://dx.doi.org/10.1016/j.archoralbio.2012.10.005
Open access under the Elsevier OA license. 
In addition to its high incidence in denture wearers, there is a

concern that Candida species from the oral cavity may colonize

the upper gastrointestinal tract in immunosuppressed

patients and lead to septicemia.2
thodontics, Araraquara Dental School, - UNESP - Univ. Estadual
el.: +55 16 33016547; fax: +55 16 33016406.

(A.L. Machado).

http://dx.doi.org/10.1016/j.archoralbio.2012.10.005
mailto:cucci@foar.unesp.br
mailto:almachado98@uol.com.br
http://www.sciencedirect.com/science/journal/00039969
http://dx.doi.org/10.1016/j.archoralbio.2012.10.005
http://www.elsevier.com/open-access/userlicense/1.0/
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a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 92
Candida spp. are more frequently isolated from the fitting

surface of dentures when compared to the corresponding

region of the oral mucosa.1 Therefore, the treatment of

denture-induced stomatitis should include denture cleansing

and disinfection in addition to topic or systemic antifungal

drugs. Although these treatments do show some efficacy, they

aim at inactivating the microorganisms after denture surface

colonization. As the adhesion of microorganisms to denture

surfaces is a prerequisite for microbial colonization,3,4 the

development of methods that can reduce C. albicans adhesion

may represent a significant advance in the prevention of

denture-induce stomatitis.

The use of polymers containing zwitterionic groups such as

phosphatidylcholines and sulfobetaines,5–10 which originate

from the simulation of biomembranes,9,11 has been proposed

to modify the surface of biomaterials.12–14 A significant

reduction in protein adsorption has been demonstrated5,8–

10,12–18 and attributed to the formation of a hydration layer on

the material surface5–7,9–14,16,17,19 that prevents the conforma-

tional alteration of these proteins.9,11,13,14,19 Previous research-

ers7,13,16,20,21 reported that sulfobetaine application on

substrate surfaces reduced bacterial adhesion. These results

suggest that sulfobetaine-based polymers may be used to

modify the surface of acrylic materials used in the fabrication

of removable dentures and reduce microbial adhesion.6

However, the effectiveness of this surface modification on

C. albicans adhesion remains to be investigated.

Surface modification by deposition of polymer coatings

such as parylene has been reported to improve the wettability

of a silicone elastomer and reduce C. albicans adhesion and

aggregation on its surface.22 Hydrophilic polymers have also

been investigated in biomaterial research.19,23,24 The hydra-

tion state of hydrophilic polymers is different from that of

zwitterionic polymers, and the free water fraction on polymer

surface is lower in the former.19 Despite these differences,

hydrophilic polymers have been used to modify the surface of

biomaterials and reduce bacterial adhesion.23,24 The adsorp-

tion of proteins to neutral hydrophilic surfaces is relatively

weak, while their adsorption to hydrophobic surfaces tends to

be very strong and practically irreversible.25,26 Therefore,

altering the characteristics of the inner surfaces of dentures by

increasing their hydrophilicity could reduce colonization by

pathogenic microorganisms, including Candida spp. It has

been reported that substratum surface properties, such as

surface free energy, may influence C. albicans adhesion to

polymers, where hydrophobic interactions play a role.27–29

The purpose of this study was to evaluate the effect of

experimental photopolymerized coatings, containing zwitter-

ionic or hydrophilic monomers, on the hydrophobicity of a

denture base acrylic resin and on C. albicans adhesion. The

hypotheses were that the coating application would decrease

the surface hydrophobicity and reduces C. albicans adhesion,

and that there would be differences among coatings.

2. Material and methods

2.1. Specimen fabrication

Disc-shaped silicone patterns (13.8 mm � 2 mm) were

obtained from metallic matrices. Half of the silicone patterns
were inserted between two glass plates and the other half

were inserted in dental flasks directly in contact with the

stone. These two methods of specimen preparation were used

to obtain smooth and rough surfaces that simulate the outer

and inner surfaces of the dentures, respectively. The silicone

patterns were then removed, and the surfaces were coated

with a layer of separating medium (Vipi Film; VIPI Indú stria e

Comércio Exportação e Importação de Produtos Odontológicos

Ltda Pirassununga, SP, Brazil). A colourless microwave-

polymerized denture base acrylic resin (Vipi Wave; VIPI

Indú stria e Comércio Exportação e Importação de Produtos

Odontológicos Ltda., Pirassununga, SP, Brazil) was mixed

according to the manufacturer’s instructions at a mixing ratio

of 1 g powder to 0.47 mL of liquid for each specimen. The

moulds were filled with the acrylic resin, a trial pack was

completed, and excess material was removed. A final pack was

performed and held for 15 min. The denture base acrylic resin

was processed in a 500 W domestic microwave oven (Bras-

temp; Brastemp da Amazô nia SA, Manaus, AM, Brazil) for

20 min at 20% power followed by 5 min at 90% power. After

polymerization, the flasks were allowed to cool at room

temperature, the specimens were deflasked, and the excess

was trimmed with a sterile bur (Maxi-Cut; Lesfils de August

Malleifer SA, Ballaigues, Switzerland). A total of 468 disc-

shaped specimens were fabricated by a single operator

wearing a mask, gloves and protective clothing.

2.2. Surface roughness measurements

Considering the possible influence of roughness on the

adhesion of microorganisms to substrate surfaces,3,30 the

surface roughness of the specimens was measured using a

profilometer (Mitutoyo SJ 400; Mitutoyo Corporation, Tokyo,

Japan) accurate to 0.01 mm. The cutoff length was 0.8 mm, the

transverse length was 2.4 mm, the stylus speed was 0.5 mm/s

and the diamond stylus tip radius was 5 mm. Four measure-

ments were made on the surface of each specimen and

averaged to obtain the Ra value (mm). All measurements were

recorded by a single operator.

2.3. Experimental photopolymerized coatings

After roughness reading, the specimens were randomly

assigned to 13 groups of 36 specimens each; 18 specimens

had smooth surfaces and 18 specimens had rough surfaces. In

the control group (C), the specimens did not receive any

surface treatment. In each experimental group, all specimen

surfaces were coated with a layer of one of the experimental

photopolymerized coatings. Four coating formulations were

evaluated: 3 coatings containing hydrophilic monomers: 2-

hydroxyethyl methacrylate (HEMA) – HE, 2-hydroxypropyl

methacrylate (HPMA) – HP, and 2-trimethylammonium ethyl

methacrylate chloride (TMAEMC) – T, and 1 coating containing

a zwitterionic monomer (sulfobetaine methacrylate) – S.

These monomers were used at concentrations of 25%, 30%

and 35% of the total composition in mmol which resulted in 12

experimental coatings (HE25; HE30; HE35; HP25; HP30; HP35;

T25; T30; T35; S25; S30; S35). In addition to the above

monomers, all coatings contained the monomer methyl

methacrylate, two crosslinking agents (triethylene glycol



a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 9 3
dimethacrylate (TEGDMA) and bisphenol-A-glycidyl methac-

rylate (Bis-GMA)) and an initiator agent (4-methyl benzophe-

none). For the coating S, amino propyl methacrylate was also

added. The monomer methyl methacrylate causes the

polymer surface to swell,31 and the adhesion is obtained by

interdiffusion of the coatings into the swollen denture base

polymer structure, photopolymerization, and formation of

interpenetrating polymer network.

The application of the 12 coatings on the specimen surfaces

was performed in a sterile laminar flow chamber followed by a

4 min polymerization on each surface in an EDG oven

(Strobolux, EDG, São Carlos, São Paulo, SP, Brazil). For the S

coating, propane sultone was brushed on specimen surfaces,

and the specimens were maintained in an oven at 80 8C for 2 h.

Thereafter, all specimens were stored individually in properly

labelled plastic bags containing sterile distilled water for 48 h

at room temperature for release of uncured residual mono-

mers.32

2.4. Exposure of the specimens to human saliva

Half of the specimens in each group (control and experi-

mentals) were exposed to saliva. For this purpose, non-

stimulated saliva was collected from 50 healthy male and

female adults. Ten millilitres of saliva from each donor were

mixed, homogenized and centrifuged at 5000 � g for 10 min at

4 8C. The saliva supernatant was prepared at 50% (v/v) in

sterile PBS33 and immediately frozen and stored at �70 8C. The

specimens were incubated with the prepared saliva at room

temperature for 30 min.34,35 The other half of the specimens

was not exposed to saliva. The research protocol was

approved by the Research Ethics Committee of Araraquara

Dental School, and all volunteers signed an informed consent

form.

2.5. Surface free energy

To characterize the hydrophobicity of the surfaces, the

surface free energy of all specimens, regardless of the

experimental condition, was calculated from contact angle

measurements using the sessile drop method and a contact

angle measurement apparatus (System OCA 15 PLUS; Data-

physics). This device has a CCD camera that records the drop

image (15 mL) on the specimen surface, and image-analysis

software determines the right and left contact angles of the

drop after 5 s. The wettability and surface energy of the

specimens were evaluated from data obtained in the contact

angle measurements. In these analyses, deionized water was

used as the polar liquid and diiodomethane (Sigma–Aldrich,

St. Louis, MO, USA) as the dispersive (non-polar) compound.36

Surface free energy components were evaluated by the

Owens–Wendt method based on the contact angles of two

test liquids with different polarities.37 For each liquid, both

the left and right sides of two drops (on different locations)

were obtained for all specimens, and the average was

calculated.

The specimens were packed in sealed sterile plastic bags

with sterile distilled water and ultrasonicated for 20 min. Then

all specimen surfaces were exposed to ultraviolet light in a

laminar flow chamber for 20 min for sterilization.38
2.6. Microorganism, growth conditions and adhesion to
the specimen surface

C. albicans adhesion was evaluated for all specimens, both

saliva conditioned and unconditioned. For the preparation of

the inoculum, the yeast C. albicans ATCC 90028 was seeded in

an agar YEPD culture medium (1% yeast extract, 2% peptone,

2% dextrose, 2% agar) and incubated for 48 h at 37 8C. After this

period, two loops of the cultivated yeast were transferred to

20 mL of the YNB (yeast nitrogen base) medium (Difco, Detroit,

MI, USA) with 50 mM glucose. After incubation for 21 h at

37 8C, the cells were washed twice with sterile phosphate-

buffered saline solution (PBS) (pH 7.2) by agitation and

centrifugation at 5000 � g for 5 min. After washing, the cells

were re-suspended in 20 mL of YNB broth with 100 mM sterile

glucose. C. albicans suspensions were standardized to a

concentration of 1 � 107 cell/mL, spectrophotometrically. An

aliquot of 3 mL of the standardized C. albicans suspension was

added to each well of a 12-well microplate containing the

specimens and maintained for 90 min at 37 8C in the adhesion

phase.39 Thereafter, the specimens were carefully washed

twice with 3 mL of PBS to remove the non-adhered cells.

Negative controls were sterile specimens immersed in YNB

broth supplemented with glucose at 100 mM. All experiments

were performed in triplicate on three different occasions.

2.7. XTT assay

The viability of the C. albicans cells adhering to acrylic

specimen surfaces was evaluated by XTT (2,3-bis(2-meth-

oxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide)-

reduction assay, which measures the cell metabolic activity.

Although XTT is a semi-quantitative colorimetric assay,40 it

correlates well with other quantitative techniques such as ATP

and CFU assays40,41 and, thus, it has been widely used to

evaluate fungal adhesion and biofilm formation.33,40 The XTT

solution (Sigma Chemical Co., St. Louis, MO, USA) was

prepared using ultra pure water at a concentration of 1 mg/

mL, sterilized by filtration and maintained at �70 8C. The

menadione solution (Sigma Chemical Co., St. Louis, MO, USA)

was prepared in 0.4 mM acetone immediately before each

experiment. After washing, the specimens were transferred to

12-well microplates containing, in each well, 2370 mL of PBS

supplemented with 200 mM glucose, 600 mL of XTT and 30 mL

of menadione. The plates were incubated in the dark for 3 h at

37 8C. The entire contents of each well were transferred to

individual tubes and centrifuged at 5000 � g for 2 min. The

supernatant was then transferred to a 96-well microplate, and

the colour change was measured using a microplate reader

(Thermo Plate – TP Reader) at 492 nm.

2.8. X-ray photoelectron spectroscopy analysis (XPS)

The chemical composition of the specimen surfaces after the

coating application was characterized by XPS (X-ray photo-

electron spectroscopy). The XPS analysis was carried out using

a commercial spectrometer (UNI-SPECS UHV) to verify surface

chemical composition changes in the treated specimens. The

Mg Ka line was used (E = 1253.6 eV), and the analyzer pass

energy was set to 10 eV. The inelastic background of the C 1s, O



Table 1 – Mean roughness values (Ra-mm) and standard
deviations (SD) obtained in the groups (n = 18), according
to the method used for specimen fabrication.

Groups Glass Stone

Control 0.19 (0.07)a 1.95 (0.51)b

S25 0.17 (0.08) a 2.13 (0.80)b

S30 0.19 (0.09) a 2.29 (0.70)b

S35 0.18 (0.07) a 1.95 (0.74)b

HP25 0.16 (0.09) a 2.11 (0.54)b

HP30 0.20 (0.08) a 2.05 (0.69)b

HP35 0.23 (0.06) a 1.73 (0.53)b

HE25 0.23 (0.06) a 1.78 (0.56)b

HE30 0.17 (0.08) a 1.90 (0.77)b

HE35 0.17 (0.07) a 2.09 (0.61)b

T25 0.17 (0.08) a 1.93 (0.78)b

T30 0.15 (0.07) a 1.74 (0.52)b

T35 0.17 (0.08) a 1.94 (0.79)b

Kruskal–Wallis test p = 0.083 p = 0.462

Different letters indicate statistically significant difference at 5%.

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 94
1s and N 1s electron core-level spectra was subtracted using

Shirley’s method. The binding energies of spectra were

corrected using the polymer hydrocarbon component fixed

at 285.0 eV. The composition of the surface layer was

determined from the ratio of the relative peak areas corrected

by sensitivity factors of the corresponding elements. Spectra

were fitted without placing constraints using multiple Voigt

profiles. The width at half maximum (FWHM) varied between

1.6 and 2.0 eV and the accuracy of the peak positions was

�0.1 eV. In the present analysis, 1 specimen from the group

control (no surface treatment) and one specimen treated with

one of the four experimental coatings formulations were used

at the higher concentration.

2.9. Statistical analysis

The effect of the two methods used for specimen fabrication

on surface roughness was analyzed statistically by the non-

parametric Mann–Whitney test. The non-parametric Kruskal–

Wallis test was used to compare roughness among groups

within each specimen fabrication method. The surface free

energy values were analyzed statistically by the three-way

ANOVA and Tukey’s test. The metabolic activity differences

(XTT assay) between the specimens pre-treated or untreated

with saliva within each group were analyzed by the non-

parametric Kruskal–Wallis test. Since no statistically signifi-

cant difference was found, the 18 values obtained for each

group (pre-treated or untreated with saliva) were grouped and

used for group comparisons using the non-parametric

Kruskal–Wallis test. A significance level of 5% was used for

all analyses.

3. Results

Table 1 shows that the mean roughness values obtained for

specimens fabricated between glass plates (smooth surfaces)

were lower than 0.23 mm, while for those specimens fabricated

in contact with the stone (rough surfaces), the values were

significantly different ( p < 0.05) (higher than 1.73 mm). Within

each specimen fabrication method, there were no statistically

significant differences ( p > 0.05) in surface roughness among

the groups.

The surface free energy (polar and dispersive components)

mean values and standard deviations for control and

experimental groups are presented in Table 2. Overall, the

coatings application increased the polar component of the

surface free energy with statistically significant differences for

S25 groups (smooth surface; absence of saliva), S25, S30, S35,

HP35 groups (rough surface; absence of saliva) and HP25, HP30,

HE25, T25 groups (rough surface; presence of saliva). Com-

pared to the control, the dispersive component was signifi-

cantly increased in the S35 group (presence of saliva) and

decreased in the T35 group (absence of saliva). The total

surface free energy was also higher in all the experimental

groups compared to the control; the differences were statisti-

cally significant for the S25 and S35 groups (smooth surface;

absence of saliva), S30, S35 groups (rough surface; absence of

saliva) and HP25, HP30, HP35, HE25, T25 groups (rough surface;

presence of saliva).
For the control group, Table 2 also shows that there were no

significant differences in polar and dispersive components, as

well as the surface free energy, between uncoated and saliva-

coated specimens. For the experimental groups, saliva

significantly decreased the polar component for S25 group

(smooth surface), S25, S30 and S35 groups (rough surfaces),

and significantly increased for the HP25, HP30 and HE25

groups (rough surfaces). The dispersive component signifi-

cantly increased after incubation with saliva for S35 group,

regardless of the surface roughness. The total surface free

energy of rough surfaces was significantly decreased in the

presence of saliva for the S30 group, while for HP25, HE25 and

T25 groups, a significant increase was noted.

For specimens fabricated between glass plates (smooth

surfaces), there were no statistically significant differences

( p > 0.05) in absorbance values among the groups (Table 3).

This indicates similar C. albicans initial biofilm formation. For

specimens fabricated in contact with the stone (rough

surfaces), S30, S35 and HP30 groups had significantly lower

( p < 0.05) absorbance values than the control group. When

controls were compared, a higher mean absorbance value was

observed for rough surfaces ( p < 0.05). All negative controls

exhibited no metabolic activity (data not shown).

Surface compositions evaluated by XPS analysis are shown

in Table 4. Spectra of the unmodified surfaces showed peaks

for carbon (75.3 at.%), oxygen (23.0 at.%), and silicon (0.3 at.%).

After the coatings application, the percentage of the elements

changed, particularly for HP and S coatings. HP resulted in a

decrease of C 1s and an increase of O 1s and Si 2p; a new peak

attributed to phosphor appeared. The S coating which

contains sulfobetaine resulted in an increased C 1s peak

and Si 2p and a decreased peak for O 1s. An additional peak for

the presence of sulphur (0.5 at.%) was also observed.

4. Discussion

In this study, two methods of specimen preparation were used

(between glass plates or in contact with stone), and smooth

and rough surfaces were obtained. The adhesion of C. albicans



Table 2 – Mean polar and dispersive components and surface free energy values (Dyn/cm) and standard deviations (SD)
obtained in the groups (n = 9), according to the method used for specimen fabrication.

Groups Saliva Polar component Dispersive component Total surface free energy

Glass Stone Glass Stone Glass Stone

Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD)

Control as 6.11(2.33) a 6.53 (2.94) a 36.20 (3.00) bcd 40.83 (3.02) bcd 42.31 (3.51) a 47.37 (1.97) ab

ps. 5.88 (1.65) a 6.70 (5.79) a 36.61 (4.07) abc 37.13 (2.74) abc 42.49 (4.04) a 43.82 (4.79) a

S25 as 15.80 (5.87) b 18.01 (8.04) cd 36.79 (5.08) abc 36.05 (4.45) abc 52.58 (6.07) b 54.06 (8.02) bcd

ps 7.03 (2.98) a 7.98 (4.45) ab 38.44 (3.49) cd 41.38 (2.40) cd 45.48 (2.05) ab 49.36 (4.65) abc

S30 as 11.63 (5.43) ab 20.66 (8.74) d 36.62 (4.15) abc 36.47 (4.65) abc 48.25 (5.45) ab 57.13 (7.34) d

ps 6.55 (3.39) a 5.80 (2.17) a 37.96 (3.38) cd 41.70 (1.59) cd 44.51 (3.86) a 47.51 (2.56) ab

S35 as 13.06 (4.87) ab 20.61 (5.52) d 37.34 (3.34) abc 35.15 (2.74) abc 50.40 (6.50) b 55.75 (5.55) cd

ps 6.09 (3.47) a 7.27 (2.24) ab 41.92 (2.29) d 41.24 (3.03) d 48.01 (3.47) ab 48.50 (3.46) abc

HP25 as 6.74 (2.74) a 9.34 (2.57) ab 37.56 (2.43) bcd 39.79 (3.14) bcd 44.30 (2.69) a 49.13 (2.55) abc

ps 10.87 (4.86) ab 19.02 (7.10) d 38.46 (3.89) bcd 39.85 (1.90) bcd 49.33 (2.69) ab 58.87 (6.19) d

HP30 as 7.78 (1.74) ab 8.99 (3.92) ab 39.15 (2.19) cd 39.95 (3.51) cd 46.93 (2.20) ab 48.93 (3.16) abc

ps 8.82 (3.66) ab 17.45 (8.24) cd 37.55 (3.83) abcd 38.25 (3.61) abcd 46.37 (4.56) ab 55.70 (6.32) cd

HP35 as 8.25 (3.57) ab 15.51 (2.33) bcd 37.02 (3.42) abc 37.42 (3.10) abc 45.26 (2.68) ab 52.92 (2.54) bcd

ps 7.86 (2.13) ab 13.32 (5.20) abcd 38.98 (2.81) bcd 38.95 (3.31) bcd 46.84 (3.15) ab 52.27 (5.74) bcd

HE25 as 9.31 (2.93) ab 9.33 (3.46) ab 37.08 (4.09) abc 34.79 (3.55) abc 46.39 (3.36) ab 44.12 (1.78) a

ps 7.18 (3.23) a 19.37 (3.06) d 38.40 (3.28) abc 35.96 (2.36) abc 45.57 (2.18) ab 55.33 (3.00) cd

HE30 as 9.26 (3.83) ab 8.44 (2.82) ab 37.36 (4.23) abcd 38.79 (2.01) abcd 46.62 (2.82) ab 47.22 (2.47) ab

ps 10.17 (3.69) ab 10.90 (6.06) abc 38.31 (2.49) abcd 38.28 (2.71) abcd 48.48 (3.02) ab 49.17 (4.41) abc

HE35 as 12.34 (4.03) ab 10.40 (3.01) abc 36.66 (3.83) abc 38.02 (3.95) abc 49.00 (2.55) ab 48.42 (5.66) abc

ps 9.99 (3.76) ab 13.01 (6.60) abcd 37.67 (1.81) abc 36.87 (3.26) abc 47.65 (4.62) ab 49.88 (5.66) abc

T25 as 8.47 (3.05) ab 12.30 (3.75) abcd 33.73 (3.26) ab 36.34 (4.0) ab 42.21 (4.17) a 48.64 (5.40) abc

ps 12.16 (3.36) ab 18.07 (3.80) cd 37.44 (2.23) abcd 38.87 (3.06) abcd 49.61 (3.50) ab 56.94 (1.56) d

T30 as 9.06 (3.83) ab 12.56 (5.38) abcd 35.82 (4.67) ab 34.03 (4.48) ab 44.88 (5.17) ab 46.59 (4.06) ab

ps 11.33 (6.98) ab 7.55 (2.00) ab 35.59 (4.32) abcd 40.89 (3.98) abcd 46.92 (5.48) ab 48.43 (3.70) abc

T35 as 9.92 (4.33) ab 7.39 (3.06) ab 32.88 (5.88) a 35.17 (4.74) a 42.80 (3.32) a 42.56 (5.31) a

ps 11.33 (6.75) ab 10.92 (5.03) abc 36.78 (3.24) abcd 38.91 (3.58) abcd 48.11 (4.52) ab 49.83 (3.89) abc

as: absence of saliva (uncoated specimens); ps: presence of saliva (coated specimens). For polar component, dispersive component and surface

free energy, means with the same small letters within the columns are not significantly different at p � .05.

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 9 5
to the denture base acrylic resin, as determined by the XTT

assay, showed that, in control group, there was greater

adhesion of C. albicans to rough surfaces than to smooth

surfaces. This result is in agreement with other studies and

may be due to the fact that roughness increases the surface

area and may act as niches for microorganisms, thus

favouring the adhesion.3,30,42–44 For the specimens treated

with the photopolymerized coatings, significant differences

between smooth and rough surfaces were not detected.

It has been reported that the more hydrophobic the surface,

the greater is the C. albicans cell adherence by area unit.27 Thus,

a commonly used method to reduce the attachment of

microorganisms is surface modification with hydrophilic

polymers7,21,24 as attempted in the present study. For instance,

coating surfaces with a 2-methacryloyloxyethyl phosphor-

ylcholine (MPC) co-polymer decreased both water contact

angles and the adhesion of C. albicans.6 Accordingly, Yoshijima

et al.28 also observed that hydrophilic coatings of denture

acrylic surfaces reduced the adhesion of the hydrophobic C.

albicans hyphae. More recently, it has been also found that

coating a denture base material with silica nanoparticles was

effective in increasing surface hydrophilicity and decreasing
C. albicans adherence.29 Hence, in the present study, the

surface free energy of the specimens was calculated.

The total surface free energy is the sum of components

arising from dispersive and polar contributions where the

polar component describes the hydrophilic character and the

dispersive component is associated with the hydrophobic

character of the surface. While the dispersive component (or

Lifshitz–van der Waals) is influenced by the particle size or

specific surface area, the polar component is the result of

different forces/interactions such as polar, hydrogen, induc-

tive and acid–base interactions.45 Thus, while the dispersive

component is affected by the surface roughness (or specific

surface area), the polar component is dependent on the

surface activity, which is related to the surface functional

groups such as hydroxyl, carbonyl, and carboxyl.45 Generally,

in this study, the coatings application decreased the water

contact angle (data not shown) and increased the polar surface

free energy component which may have arisen from a change

in the surface polar group concentration in the coated

specimens. Only minor significant differences were observed

for the dispersive component. Therefore, although the

dispersive (or non-polar) component of the surface free energy



Table 3 – Medians (Med), minimum (Min) and maximum
(Max) absorbance values (XTT assay - 492 nm) obtained
in the groups (n = 9), according to the method used for
specimen fabrication.

Groups Saliva Glass Stone

Med Min Max Med Min Max

Control as 0.54 0.43 0.97 a 1.23 0.83 1.62 b*

ps 1.08 0.68 1.23 a 1.33 1.05 1.60 b*

S 25 as 0.83 0.67 1.21 a 0.94 0.46 1.13 ab

ps 0.94 0.75 1.40 a 0.87 0.66 1.52 ab

S30 as 0.69 0.45 1.34 a 0.65 0.36 1.11 a

ps 0.91 0.48 1.63 a 0.91 0.72 1.70 a

S35 as 0.80 0.57 1.14 a 0.54 0.38 0.98 a

ps 0.83 0.57 1.42 a 1.02 0.62 1.62 a

HP 25 as 0.77 0.51 1.10 a 0.80 0.45 1.12 ab

ps 1.15 0.46 1.53 a 1.16 0.71 1.32 ab

HP 30 as 0.59 0.40 0.95 a 0.72 0.40 0.94 a

ps 0.87 0.50 1.55 a 1.07 0.59 1.45 a

HP 35 as 0.66 0.31 1.03 a 0.91 0.51 1.19 ab

ps 1.00 0.61 1.46 a 1.19 0.72 1.77 ab

HE 25 as 0.77 0.45 1.03 a 0.74 0.41 0.87 ab

ps 0.90 0.56 1.31 a 1.12 0.85 1.40 ab

HE 30 as 0.77 0.55 1.02 a 0.80 0.46 1.19 ab

ps 0.91 0.58 1.33 a 1.42 0.81 1.50 ab

HE 35 as 0.79 0.33 1.21 a 0.93 0.50 1.61 ab

ps 0.82 0.55 1.47 a 1.27 0.92 1.74 ab

T 25 as 0.81 0.65 1.22 a 0.99 0.57 1.41 ab

ps 1.04 0.58 1.66 a 1.25 1.00 1.92 ab

T 30 as 0.85 0.39 1.14 a 1.10 0.82 1.31 ab

ps 1.01 0.41 1.41 a 1.27 0.85 1.70 ab

T 35 as 0.80 0.59 1.15 a 1.01 0.68 1.39 ab

ps 0.96 0.45 1.64 a 1.22 1.01 1.95 ab

Groups with the same letters in the columns did not differ

significantly at 5%.

as: absence of saliva (uncoated specimens); ps: presence of saliva

(coated specimens).
* Groups obtained between glass and stone differed significantly at

5%.

Table 4 – Elemental surface composition (at.%) of the
groups evaluated determined by XPS.

Elements (at.%) Groups

Control HE HP T S

C 1s 75.3 72.7 67.9 71.0 81.8

O 1s 23.0 24.6 23.9 23.3 15.4

Si 2p 0.3 2.0 7.6 4.6 3.1

P 2p _ 0.6 0.6 1.1 –

S 2p _ _ _ _ 0.5

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 96
is numerically higher than the polar component, the polar

component is the main factor in determining modifications of

the total surface free energy. Thus, the values of the surface

energy followed the same trend as the polar component.

Compared to the control, mean surface free energy values of

the rough surfaces coated with S30, S35 and HP30 were

significantly higher which indicates increased wettability.
These results were expected because it is known that the

contact angles are decreased (more hydrophilic) by surface

roughness for hydrophilic surfaces.46

The effect of saliva on the hydrophobicity of the surfaces

was also evaluated. The results showed that incubation with

saliva did not significantly alter the polar and dispersive

components and surface free energy for the control speci-

mens. For experimental groups, the effect of saliva on the

polar component and the total surface free energy varied

depending on type of coating, with this effect being more

significant for rough surfaces. As observed for the non-

coated specimens, significant differences were also found

mainly for the polar component of rough surfaces treated

with S and HP coatings. However, for the S coating, saliva

decreased the polar component, and the values became

similar to the polar component of the control group; for the

HP coating, an increase in the polar component was

observed after incubation with saliva. Thus, the effect of

saliva on the surface free energy varied depending on

substrate characteristics, particularly the chemical compo-

sition and surface roughness. These findings suggest that

the nature of the surface-exposed chemical groups after

coating applications may influence the formation of the

salivary pellicle (adsorbed salivary proteins). Other authors

have also reported that small differences in the chemical

composition of acrylic resins changed the adsorption of

salivary proteins and, consequently the nature of the

adsorbed salivary pellicle.47,48 In this study, this phenome-

non was particularly evident for rough surfaces due to a

larger surface area and more exposed chemical groups

available to interact with saliva.

In the present investigation, XTT assay results showed

that, for the specimens fabricated in contact with the stone,

the adhesion of C. albicans in S30, S35 and HP30 groups was

lower as compared with the control. One factor that might

have contributed to these findings would be the hydrophilicity

of the coated surfaces.21,27,28 As mentioned before, the rough

surfaces coated with S30, S35 and HP30 exhibited significantly

higher mean surface free energy values as compared with the

control group, suggesting a decreased hydrophobic character.

Hence, in this study, the decrease in C. albicans adhesion in the

S30, S35 and HP30 groups may be partially related to the

hydrophilicity of the rough surfaces treated with these

coatings. Changes in chemical compositions of the coated

acrylic surfaces may also have contributed to the findings as

demonstrated by the XPS analysis. There were changes in the

carbon and oxygen content with special relevance for S and HP

coatings. In addition, surfaces modified with the S coating also

exhibited an additional peak for the presence of sulphur. The S

coating contains sulfobetaine, a member of the zwitterionic

betaine family of compounds,5,10,11,13–16,18,21,49 which have a

mixture of anionic and cationic terminal groups with an

overall neutral charge. Surfaces with zwitterionic groups

resist non-specific interaction with plasma proteins and cells

via a bound hydration layer from solvation of the charged

terminal groups in addition to hydrogen bonding.13,14,17,18,21,50

As observed for the S coating, other studies have shown that

surfaces coated with zwitterionic polymers reduced E. coli, S.

aureus, Streptococcus mutans, P. aeruginosa, S. epidermidis, E.

faecalis and C. albicans attachment.5,6,21



a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 – 9 7
However, it should be noted that, in addition to S30, S35 and

HP30 coatings, other coatings also promoted changes in the

surface chemical composition and resulted in hydrophilic

surfaces but did not significantly affect the adhesion of C.

albicans to the denture base acrylic resin.

For all tested conditions, the results revealed that C.

albicans adhesion was not influenced by saliva. There is no

consensus in the literature regarding the effect of saliva in

C. albicans adhesion. Some authors4,39,51 found an increase

of C. albicans adhesion to materials covered with salivary

pellicle, while others30,32,34,52,53 observed a decrease in

adhesion. This divergence of results could be attributed to

differences among materials used as substrates to test

Candida adhesion.4,30,32–35,39,51–54 The chemical nature of

the surfaces of the biomaterials influences the formation

and composition of the acquired pellicle,47,55 and conse-

quently the adhesion and formation of biofilms.56 Further-

more, results may also be influenced by differences in

saliva-collection methods, such as the type of collected

saliva (stimulated or non-stimulated) and number of

donors, and in those procedures for saliva processing,

such as the use of filtered or non-filtered saliva, diluted or

non-diluted saliva, speed and time of centrifugation, and

incubation periods and temperatures.4,30,32,34,35,39,51–53 In

the present study, diluted saliva was prepared in the same

manner as Ramage et al.33 Diluted saliva was used for

practical reasons as the saliva volume of hundreds of mL

was required in the experiments. Although one could argue

that saliva dilution could have contributed to the lack of

effect of the pre-conditioning on Candida adhesion, other

studies where undiluted saliva was used have also shown

no significant effect on the adhesion of C. albicans.40,54

The findings of this study confirm that the interactions

among C. albicans, substrate and saliva are complex, and

that several factors such as the physicochemical properties

of the substrates (and conditioning film) and cells may

influence this process. Nevertheless, experimental photo-

polymerized S and HP coatings were able to reduce C.

albicans adherence and thus warrant further investiga-

tions.

5. Conclusions

Experimental S and HP coatings showed promising results

and significantly reduced the short-term attachment (90 min)

of C. albicans to the denture base acrylic resin under

evaluation. However, the effect of these coatings on long-

term biofilm formation remains to be investigated. In

addition, the resistance of these coatings to mechanical

(brushing) and chemical (immersion in denture cleansers)

denture cleansing methods, as well as their biocompatibility

should be analyzed before these materials can be recom-

mended for clinical use.

Competing interest

None declared.
Funding

Source of funding: FAPESP (grants 2006/00435-3 and 2006/

06842-0).

Ethical approval

The study was approved by the Ethics committee of

Araraquara Dental School, and all subjects volunteered to

participate and signed an informed consent form.

Acknowledgements

This study was supported by FAPESP (grants 2006/00435-3 and

2006/06842-0). The authors wish to acknowledge Mr. Jörg

Erxleben for preparing the coatings used in this study and Prof.

Peter Hammer for his assistance with the XPS analysis.

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	Effect of experimental photopolymerized coatings on the hydrophobicity of a denture base acrylic resin and on Candida albicans adhesion
	Introduction
	Material and methods
	Specimen fabrication
	Surface roughness measurements
	Experimental photopolymerized coatings
	Exposure of the specimens to human saliva
	Surface free energy
	Microorganism, growth conditions and adhesion to the specimen surface
	XTT assay
	X-ray photoelectron spectroscopy analysis (XPS)
	Statistical analysis

	Results
	Discussion
	Conclusions
	Competing interest
	Funding
	Ethical approval
	Acknowledgements
	References