Journal of Materials Science: Materials in Medicine (2018)29:107

https://doi.org/10.1007/s10856-018-6116-7

SPECIAL ISSUE: ESB 2017

Original Research

Examination of 2-methacryloyloxyethyl phosphorylcholine polymer

coated acrylic resin denture base material: surface characteristics
and Candida albicans adhesion
1
İrem Türkcan ●
A. Dilek Nalbant2 Erhan Bat3 Gülçin Akca4
● ●

Received: 21 November 2017 / Accepted: 5 June 2018

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
The aim of this study is to evaluate the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coating with
various concentrations onto acrylic resin denture base material on surface characteristics such as contact angle and surface
roughness and on Candida albicans adhesion which is the major factor of denture stomatitis. Specimens, prepared from heat-
polymerized acrylic denture base material, were divided into control and three test groups, randomly. Surfaces of the
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specimens in test groups were coated with poly(MPC) (PMPC) by graft polymerization of MPC in different concentrations
(0.25 mol/L; 0.50 mol/L and 0.75 mol/L), while no surface treatment was applied to the control group. Contact angles and
surface roughness were examined, and chemical composition of the surfaces was analyzed by Attenuated Total Reflectance-
Fourier Transform Infrared Spectroscopy (FTIR) to verify the presence of PMPC coatings. Then, specimens were incubated
with C. albicans for 18 h and the number of adhered cells was determined. Upon PMPC coating, the contact angle values
statistically decreased, but no difference was found in surface roughness values. A statistically significant decrease was
observed in C. albicans adhesion in parallel with the increase in the MPC polymer concentration. There was no significant
difference between 0.50 mol/L and 0.75 mol/L groups in terms of adhesion. These findings indicated that graft
polymerization of MPC on acrylic denture base material reduces the adhesion of C. albicans, and may be evaluated as a
coating for prevention of denture stomatitis.

1 Introduction

Acrylic resin based conventional or implant supported

removable dentures are frequently preferred when fixed
dentures are not indicated, particularly in elderly patients.
This report was presented as an “oral presentation” in 22nd BaSS
Although patients are mostly satisfied regarding esthetics
Congress that was held on 4–6 May 2017 in Makedonia Palace Hotel,
Thessaloniki, Greece. and function, denture plaque, formed by oral micro-
organisms on poly(methyl methacrylate) (PMMA) based
Electronic supplementary material The online version of this article
acrylic denture base, is one of the major problems. Den-
(https://doi.org/10.1007/s10856-018-6116-7) contains supplementary
material, which is available to authorized users. ture plaque is known to be a risk factor for opportunistic
infections especially in elderly or immune-suppressed
* İrem Türkcan patients. In clinical applications, non-polished surfaces of
irem.turkcan@hotmail.com
dentures, also in contact with mucosa, are deprived of the
1
Özel Çankaya Hikmet Bozyel Oral and Dental Health Policlinic, washing effect of saliva resulting in a convenient envir-
Ankara, Turkey onment for the formation of denture plaque. Due to the
2
Faculty of Dentistry, Department of Prosthodontics, Gazi overgrowth of yeast cells in the space between the denture
University, Ankara, Turkey and palatal mucosa, there is a high risk of denture sto-
3
Department of Chemical Engineering, Middle East Technical matitis in maxilla [1, 2].
University, Ankara, Turkey Chronic atrophic (erythematous) candidiasis, also known
4
Faculty of Dentistry, Department of Microbiology, Gazi as “denture stomatitis” or “denture sore mouth”, is char-
University, Ankara, Turkey acterized by localized chronic erythema and edema of the
107 Page 2 of 9 Journal of Materials Science: Materials in Medicine (2018)29:107
mucosa in contact with the fitting surface of the denture. Its
primary etiology is the overgrowth of opportunistic Can-
dida in the area between the palate and the denture surface
where natural salivary flow is restricted [3, 4]. Poor oral
hygiene and poorly fitting dentures also promote the for-
mation of denture stomatitis [5], and it is usually observed
in patients using worn dentures and who do not remove
their dentures at night [6]. Lesions usually occur on the
palate and maxilla, and the mucosa beneath mandibular
dentures is almost never involved [3, 4]. According to
Akpan and Morgan [3] denture stomatitis is quite common
with an incidence rate up to 65% reported. Treatment of this
clinical condition, which can lead to health problems and
decrease the quality of life in elderly, is problematic because
of the patients’ inadequate disinfection of the surfaces,
resulting in rapid recolonization [7]. Therefore, inhibition of
adhesion and accumulation of microorganisms is an effec-
tive method for suppression of the formation and maturation
of denture plaque.
In order to prevent candidal colonization on denture base
materials, many studies have been conducted using various
materials and methods. To benefit from the oligodynamic
effect silver nanoparticles had been incorporated into
PMMA, and both promising results [8] and inefficient
findings [9] regarding Candida albicans adhesion were
reported. Physical or chemical surface modifications on
PMMA like bonding of antifungal agents [7, 10–12],
alteration of surface charge [13, 14], and increasing surface
wettability and decreasing surface energy [15–18] were
examined in several studies and significantly lower results
were obtained in C. albicans adhesion. Recent studies have
evaluated whether experimental photopolymerized coatings
containing zwitterionic or hydrophilic monomers would
reduce C. albicans adhesion on denture base acrylic resins
and encouraging results were reported for further investi-
gations [15–17].
2-methacryloyloxyethyl phosphorylcholine (MPC) is a
widely used biocompatible and hydrophilic biomedical
polymer [1, 19]. Subsequent to the introduction of the
synthesis and purification processes of MPC in 1990 by
Ishihara et al. [20] in a refined and complete manner, sig-
nificant progress has been made in the development of the
MPC polymer (PMPC) as a biomaterial. It has been shown
to have substantial antithrombogenicity because of its high
resistance to cell adhesion and its reaction with endogenous
proteins and cells [21–24]. Coating of medical devices with
PMPC has been reported to be very useful in reducing the
number of adherent bacteria [1, 25–29]. The efficacy and
safety of MPC polymers as a biomaterial is well established
and approved by FDA [1, 30–34]. Several medical devices
with PMPC coating, like intravascular stents and soft con-
tact lenses, have been developed and are in clinical use [26,
27, 32, 35].
Despite the use of MPC polymer in medicine, there is no
usage of MPC in dental practice yet. There are few dental
studies [1, 36–42] regarding MPC, but no studies con-
cerning C. albicans adhesion on PMPC coated PMMA have
been found through the literature search. Based on the
medical studies which have proved MPC polymer to reduce
microorganism and cell adhesion, this study was designed
to evaluate the effects of MPC polymer on C. albicans
adhesion onto PMMA which is a commonly used dental
prosthetic material usually at 36.5–37 °C for oral use.
The purpose of this study is to evaluate the effects of
PMPC coating with various concentrations onto acrylic
resin denture base material on surface characteristics such as
contact angle and surface roughness, and on C. albicans
adhesion which is a common factor for denture stomatitis.
2 Materials and methods
2.1 Preparation of acrylic specimens
Using pink modelling wax (Berrwax Modelling Wax, Berr
Dental, Turkey), heat polymerized acrylic resin (Meliodent
Heat Cure, Heraeus Kulzer, Germany) disks with a thick-
ness of 2.0 mm and a diameter of 6.0 mm, were con-
ventionally fabricated following the manufacturer’s
instructions. In order to standardize the surfaces, both sides
of each specimen were rubbed conventionally with no.
600 silicon carbide abrasive paper (Waterproof Silicon
Carbide Paper, English Abrasives Ltd., London, England)
until the final thickness of the disks were 1.5 mm [10].
2.2 Graft polymerization of MPC on the acrylic resin
specimens
Specimen surfaces were cleaned with hexane and ethanol in
an ultrasonic bath for 5 and 30 min, respectively, and dried
at room temperature. Then, they were divided into four
groups randomly. One group was randomly chosen as
control and no surface treatment was applied. Specimens to
be coated were immersed in an ethanol solution containing
10 mg/mL benzophenone for 30 s and were dried in the dark
at room temperature to remove the ethanol [1].
MPC, purchased from Sigma-Aldrich, Saint Louis, USA,
was dissolved in degassed pure water to obtain aqueous
solutions of the monomer at different concentrations:
0.25 mol/L, 0.50 mol/L and 0.75 mol/L. Acrylic resin spe-
cimens that had been coated with benzophenone were
immersed in the MPC solutions. Photo-induced graft
polymerization of MPC on acrylic resin specimens was
carried out at 70 °C for 90 min under ultraviolet-ray(UV)-
irradiation using an ultraviolet ray source (White/Ultraviolet
Transilluminator TLW-20, UVP, CA, USA) with a
Journal of Materials Science: Materials in Medicine (2018)29:107
wavelength of 365 nm and power of 8 watts which was
placed 20 cm away from the specimens. After polymeriza-
tion, PMPC-grafted PMMA specimens were removed,
washed with pure water to remove residual monomers, and
dried at room temperature [1, 25, 28, 32, 43].
2.3 Surface analysis of the specimens
To evaluate the wettability of the surfaces, static contact
angles were measured by sessile drop method using an
optical tensiometer (KSV Attension Theta Lite, Biolin
Scientific AB, Stockholm, Sweden). Drops of pure water
(2 µL) were deposited on the surfaces and after 5 s the
contact angles were measured with an optical camera.
Measurements were performed twice for each specimen (n
= 3), and the average values were recorded [9, 15].
Surface roughness measurements were performed using
a mobile roughness measuring instrument (MarSurf M
300C, Mahr GmbH, Göttingen, Germany) with a drive
speed (Vt) of 0.2 mm/s and movement length (Lt) of
1.75 mm. After the calibration of the device, specimens
were fixed on a metal plate provided by the manufacturer,
and the measurements were performed under manu-
facturer’s instructions. Measurements were performed three
times for each specimen (n = 3), and the average Ra (µm)
values were recorded [44, 45].
To confirm the MPC polymerization on specimens, the
chemical compositions of the surfaces were examined by
Fourier-transform infrared (FTIR) spectroscopy with atte-
nuated total reflection (ATR) equipment. For two specimens
randomly selected from each group, ATR-FTIR spectra
were obtained in 10 scans over the range of 550–4000 cm−1
using an FTIR analyzer (FT-IR Spectrometer Spectrum
Two, PerkinElmer Inc., Waltham, MA, USA) at a resolution
of 4.0 cm−1 [46].
2.4 Adhesion assay and analysis
C. albicans (ATCC #90028) was incubated at 37 °C for
48 h in Sabourraud dextrose agar (SDA) (LabM, Lanca-
shire, England) medium. In Sabourraud dextrose broth
(SDB) (Merck, Darmstadt, Germany) medium, using 0.5
McFarland test standards, yeast cell concentration was
adjusted to 1.5 × 108 cfu/mL, turbidometrically. One hun-
dred µL of this suspension was diluted to an optical density
of 600 (OD600) at 450 nm using an ELISA reader (ELx800
Absorbance Reader, BioTek, USA). After being disinfected
by immersing in ethanol for 10 min, acrylic specimens (n =
12) were immersed individually in 2 mL of SDB medium in
sterile Eppendorf tubes. 100 µL of C. albicans suspension
was added into each tube, and incubated at 37 °C for
overnight (18 h), representing the duration of daily pros-
thesis usage, after shaking several times.
Page 3 of 9 107
To control the viability of C. albicans suspensions, cul-
tivation was performed from each sample into SDA med-
ium. Specimens were removed and washed with 1 mL
sterile phosphate buffered saline (PBS, pH = 7.4) (Oxoid
Limited, Hampshire, England) twice. Except for two spe-
cimens from each group (in total 8 specimens which were
examined by scanning electron microscope (SEM)), each
specimen was placed into Eppendorf tubes containing 1 mL
sterile PBS, and vortexed (Vortex V-1 plus, Biosan Ltd.,
Riga, Latvia) for 2 min to detach adhered yeast cells. 100 µL
of yeast suspension of each detached specimen was culti-
vated individually in SDA medium and incubated at 37 °C
for 24 h. Proliferated colonies were counted, and by cal-
culating dilution ratio, values were recorded in terms of
colony forming unit per mL.
C. albicans cells adhered onto the specimens which were
reserved for SEM examination, were fixed with methanol.
After drying in vacuum, specimens were coated with gold
and examined with SEM (Zeiss GeminiSEM, Carl Zeiss
Microscopy GmbH, Jena, Germany).
2.5 Statistical analysis
The results were statistically analyzed using SPSS 17.0 for
Windows software. Shapiro-Wilk test was used to examine
the distribution of the continuous numeric variables, and
Levene test was used to determine the homogeneity of the
variances. Descriptive statistics for contact angles and C.
albicans adhesion values were demonstrated as mean and
standard deviation, and for surface roughness values median
(interquartile range) was used. Since C. albicans adhesion
values were not normally distributed logarithmic transfor-
mation was performed.
In terms of mean contact angles and C. albicans adhesion
values after logarithmic transformation, the significance of
the differences between groups were evaluated by One Way
Variance Analysis (One-Way ANOVA). For surface
roughness, the significance of the differences between
groups were compared with Kruskal-Wallis test. In con-
sequence of significant One-Way ANOVA results, the
condition(s) causing the differences were identified by post
hoc Tukey test. A statistical significance level of p < 0.05
was used.
3 Results
In this study, we investigated the surface characteristics of
PMPC coated PMMA and the effects of PMPC coating with
various concentrations on C. albicans adhesion. After graft
polymerization of MPC on PMMA had been completed,
contact angle values for all groups were examined to
determine the changes in surface hydrophilicity. According
107 Page 4 of 9 Journal of Materials Science: Materials in Medicine (2018)29:107
Fig. 1 Mean (standard deviation) of contact angle values for non-
treated and PMPC coated specimens. Equal letters denote statistical
similarity between the factors evaluated (One-way ANOVA, p <
0.001)
Fig. 2 Median of mean surface roughness (Ra) values for non-treated
and PMPC coated specimens. No significant differences were found
between groups according to Kruskal-Wallis test (p = 0.190)
to One-Way ANOVA, statistically significant differences
were determined between at least two of the groups with
regard to contact angle values (F = 7.229 and p < 0.001).
Compared to control group, a statistically significant
decrease was observed in 0.25 mol/L MPC and 0.75 mol/L
MPC groups (p < 0.001 and p = 0.026, respectively). No
significant differences were found among other groups (Fig.
1).
With the increase in the concentration of MPC grafting, a
slight increase in surface roughness was observed. How-
ever, the median Ra values were not statistically different
between the groups, according to Kruskal-Wallis test (p =
0.190) (Fig. 2).
Figure 3 shows the ATR-FTIR spectra of untreated and
PMPC-grafted PMMA with different initial MPC con-
centrations in the polymerization solution. Different from
the control group, between 3624 and 3080 cm−1 wave
numbers a broad peak and then increase was observed in
direct proportion to the PMPC-graft concentration which is
attributed to the OH group, and it refers to the water
absorbed by the hydrophilic PMPC layer. With the increase
in PMPC concentration, it was observed that the intensity of
the peaks also increased likely due to increase in the water
absorbed on the surface, which is considered as an increase
Fig. 3 ATR-FTIR spectra of non-treated and PMPC coated specimens.
B: benzophenone
in surface hydrophilicity. Transmission peaks were
observed for the test groups at 1595, 1577, 705 and 639 cm
−1
which are attributed to aromatic C=C and C–H stretches
and refers to the presence of benzophenone on the surface.
As characteristic transmission peaks of the phosphate
group (P–O) in MPC at 1240 and 970 cm−1 were not
observed due to the overlaps of transmission peaks of the
groups in PMMA, peak at 1080 cm−1 was observed for 0.50
and 0.75 mol/L MPC groups slightly. Therefore, the inten-
sity of C=O and C–O bonds, which are found in different
ratios in MPC and PMMA, were compared and the intensity
of C–O peak was higher than C=O in PMPC-grafted
groups indicating the surface coating with PMPC.
Table 1 shows the descriptive statistics for C. albicans
adhesion numbers. Since C. albicans adhesion numbers
were not normally distributed, logarithmic transformation
was performed. According to the One-Way ANOVA, sta-
tistically significant differences were determined between at
least two of the groups with regard to C. albicans adhesion
numbers (F = 19.502 and p < 0.001). When all groups were
compared with one another, all groups were found to be
significantly different except 0.50 mol/L MPC and
0.75 mol/mL MPC groups in terms of C. albicans adhesion
(p = 0.999) (Fig. 4).
An additional SEM imaging was performed on PMPC
grafted and non-incubated specimens. When comparing the
incubated and non-incubated specimen surfaces, no marked
difference in surface topography was observed between the
specimens (Supplementary Fig. 1). SEM images of the C.
albicans adhered specimens from each group are shown in
Fig. 5. For the surface coating, no differences were
observed between control group and PMPC-grafted test
groups. In all groups, the rough surface of PMMA was
observed and C. albicans cells were clearly identified. With
the increase in MPC concentration, in parallel with loga-
rithmic C. albicans adhesion statistics, a relative decrease
was observed in the number of adhered yeast cells. Due to
Journal of Materials Science: Materials in Medicine (2018)29:107
Table 1 Descriptive statistics
Groups
according to the groups for C.
albicans adhesion Control
0.25 mol/mL MPC
0.50 mol/mL MPC
0.75 mol/mL MPC
a
cfu/mL
Fig. 4 Mean (standard deviation) of adhered C. albicans numbers after
logarithmic transformation. Equal letters denote statistical similarity
between the factors evaluated (One-Way ANOVA, p < 0.001)
the 18 h-incubation period, at 37 °C incubator, early stage of
biofilm formation was examined, and yeast, any hyphae
formation or three-dimensional biofilm structure was not
observed.
4 Discussion
Especially in removable partial and total dentures, acrylic
resins have a wide area of use in prosthodontics. Despite
being biocompatible for long-term oral usage, being sus-
ceptible to microbial adhesion is an important disadvantage
of acrylic resins, compared to other dental materials. Den-
ture plaque is known to be a significant risk factor for
opportunistic infections particularly in elder patients with
attenuated hand skills for denture maintenance, patients
with underlying systemic diseases or immunocompromised
individuals. Denture stomatitis is the most common situa-
tion among denture plaque-related infections. With the
cofactors like poor oral hygiene, weakness in the immune
system, usage of incompatible old dentures, the most
common etiology for denture stomatitis is the overgrowth of
C. albicans. Instead of treating infections induced by den-
ture plaque, inhibition of denture plaque formation is more
efficient and long-termed. For the prevention of C. albicans
adhesion on acrylic resins, physical or chemical surface
modification like increasing surface wettability and
decreasing surface energy [13, 16–18], and bonding of
antifungal agents on PMMA [10, 11] were examined in
several studies and significantly lower results were obtained
in C. albicans adhesion.
Page 5 of 9 107
n Meana Standard deviation Minimuma Maximuma
10 7931 5435.0 3670 21,680
10 3809 3877.9 330 12,340
10 852 637.5 150 2190
10 766 380.5 280 1520
In recent studies hydrophilic polymers were frequently
used in surface modification [16, 17]. Especially known for
its antithrombogenic effect, MPC polymer is one of the
most common biocompatible and hydrophilic polymers
which used in medical devices like contact lenses and
artificial joints, and FDA approves the biosafety of MPC
[21–24, 27, 34]. In many studies, MPC polymer coating has
been shown to be effective in decreasing adhesion of pro-
teins, platelets, fibroblasts, bacteria and fungi to the surface
[1, 21, 23, 24, 29, 30, 34, 47–50]. Since it is not used in
dentistry yet, dental studies including MPC polymer are
rare. Therefore, in this study it was aimed to evaluate the
effects of MPC polymer coating with various concentrations
onto a commonly used acrylic resin denture base material
on C. albicans adhesion.
Several MPC coating methods have been used in the
studies with surface modification with MPC polymer.
Coating of substrate surfaces [24, 29, 30, 34, 49], plasma
treatment [48] and graft polymerization [21, 47, 50] are
most frequently used methods. Modified surfaces prepared
by graft polymerization were found to be quite stable due to
the covalent bonds between polymer and substrate [1, 51].
Graft polymerization is performed using one of these
methods: (1) surface-initiated graft polymerization termed
the “grafting from” method in which poly-MPC (PMPC) is
grafted onto substrate via covalent bonds; (2) adsorption of
the polymer to the substrate surface termed the “grafting to”
methods including dipping, cross-linking, or reaction of the
end groups of the ready-made polymers with the functional
groups of the substrate. Since a high-density polymer brush
interface with a multifunctional polymer is formed in
“grafting from” method, it has an advantage over the
“grafting to” method, resulting in an efficient function and
higher strength [1, 28]. Therefore, “grafting from” method
was preferred in this study.
After surface modification with MPC, alterations in
surface characteristics of PMMA were examined. For the
evaluation of wettability, contact angle measurement is
frequently used in the literature [1, 15, 27, 43, 52, 53]. It
was reported that static contact angle values were sig-
nificantly decreased with MPC grafting of PMMA surfaces
[1]. The most important factor which led MPC polymer to
be used in biomedical devices is its high hydrophilic
property. MPC polymer coating of materials such as
107 Page 6 of 9 Journal of Materials Science: Materials in Medicine (2018)29:107
Fig. 5 SEM images of
specimens from each group
(arrows are indicating adhered
yeast cells); a control, b
0.25 mol/L MPC, c 0.50 mol/L
MPC, d 0.75 mol/L MPC
PMMA, PEEK cross-linked polyethylene (CLPE) and
polyethylene (PE) has resulted in significantly decreased
static contact angle [1, 25–28, 32, 36]. Results of our study
also indicate that MPC polymer increases surface wett-
ability of PMMA, similar to previous studies.
To prevent C. albicans adhesion, Park et al. [13] mod-
ified the surface of PMMA by copolymerization of methyl
methacrylate acid with various concentrations resulted in a
negative charge and increased the hydrophilicity of the
surface. As a result, it was reported that C. albicans adhe-
sion decreased with the increase in the incorporated
methacrylic acid ratio. Similar to this result, as the con-
centration of MPC increased, C. albicans adhesion sig-
nificantly decreased in our study.
Yodmongol et al. [18] coated PMMA with silane-SiO2
nanocomposite films and examined the effects on the sur-
face characteristics and C. albicans adhesion. An alteration
in surface chemical composition was shown by FTIR, while
no significant change in surface roughness or surface free
energy was reported. Nanocomposite film coating led to a
significant decrease in C. albicans adhesion while the sur-
face free energy was insignificantly reduced. In our study,
an alteration was observed in surface chemical composition,
but differences in surface roughness were not statistically
significant. Although contact angle values of 0.50 mol/L
MPC group were not significantly lower, a quite sharp
decrease in C. albicans adhesion was observed when
compared to the control group.
Investigating the usage of MPC polymers in dentistry is
still very new, and studies are mostly about dental com-
posite resins, adhesives and cements. Based on the results of
the studies including the incorporation of MPC into dental
composite resins or coating the surface of composite with
MPC incorporated adhesive, MPC polymer significantly
decreased protein adsorption and microorganism adhesion
onto materials [36–40]. As a result of MPC polymer
incorporation into resin-modified glass ionomer cement
(RMGIC) a reduction in biofilm formation was indicated
[41, 42].
Except for dental composite resins and cements, one
study was found about PMMA among dental literature.
Takahashi et al. [1], modified PMMA surfaces by coating
and grafting methods of MPC polymer with various con-
centrations (0.125 mol/L; 0.25 mol/L; 0.50 mol/L; 0.75 mol/
L) and evaluated S. mutans biofilm formation before and
after 500 brushing cycles. It has been reported that S.
mutans biofilm formation was increased five times in coated
group after brushing, and there was no significant difference
in biofilm formation in the grafted group. Consequently, it
was indicated that grafting method is more durable than
coating method. In addition, S. mutans biofilm formation
decreased about one-tenth of the control group in both
coated and grafted groups. Similar to these results, a decline
about one-tenth of the C. albicans adhesion values of the
control group in 0.50 mol/L and 0.75 mol/L MPC groups
was observed in our study.
Journal of Materials Science: Materials in Medicine (2018)29:107
Some studies associate the surface roughness and adhe-
sion [54–56]. In a study investigating surface roughness of
dental composite resins coated with dental adhesive, in
which MPC was incorporated with various concentrations, a
significant decrease in Ra values was reported parallel to
increased MPC concentration [36]. In other studies, MPC
was incorporated into dental adhesives or composite resins,
and the bonding strength or flexural strength of materials
were evaluated, not the surface properties [37–40]. In
another research, the contact angle of PMMA-grafted with
MPC-was evaluated but surface roughness was not exam-
ined [1].
According to the results obtained in our study, a slight
increase was observed in surface roughness with the
increase in MPC polymer concentration. However, differ-
ences between groups were not statistically significant,
consequently it is considered that the PMPC layer may be
very thin when compared to the surface roughness of the
PMMA. Surface roughness values for all groups (surface
roughness median values were 0.51; 0.50; 0.57 and 0.61 µm
for control; 0.25 mol/L MPC; 0.50 mol/L MPC and
0.75 mol/L MPC groups, respectively) were quite lower
than Zissis et al. [45] (3.4 and 3.8 µm) and Ulusoy et al. [57]
(2.53 µm) reported for heat-polymerized acrylic denture
base resins without any surface treatment, and similar with
the results of Azevedo et al. [44] (0.40–0.70 µm) who
applied abrasive paper to the acrylic surfaces.
In many studies conducted by Kyomoto et al. [28, 32, 35,
43, 46, 58], FTIR analysis with ATR was utilized for the
surface analysis of various materials, such as CLPE and
PEEK, coated with graft polymerization of MPC. At 1240,
1080 and 970 cm−1 absorption peaks were observed which
correspond to the phosphate group (P–O) in the MPC unit.
Absorption peaks were reported at 1460 and 1720 cm−1
which were attributed to the methylene (CH2) and carbonyl
(C=O) groups, respectively. In our study, distinct peaks
were not observed at values mentioned above. This situation
is thought to have occurred due to the difference in the
materials coated with MPC, between the studies. No other
study performed ATR-FTIR analysis on PMMA coated
with graft polymerization of MPC, was found in the
literature.
It has been proven that surface roughness of acrylic
denture base resins has a significant effect on C. albicans
adhesion rather than surface free energy [54, 59, 60]. To
prevent yeast adhesion, acrylic resin surfaces were coated
using various methods, resulting in the reduction of surface
roughness and C. albicans adhesion [55, 56]. Although
there was no statistically significant difference between
control and test groups regarding surface roughness, a
reduction in C. albicans adhesion was determined in our
study.
Page 7 of 9 107
Mayahara et al. [55] reported that surface roughness
increased the adhesion of mycelial C. albicans but it had no
apparent effect on the adhesion of yeast-form C. albicans.
In SEM examinations, adhesion of yeast-form C. albicans
was observed in the 9-h-culture group while adhesion of
mycelia-form C. albicans was found in the 24-h-culture
group. In our SEM analyses, strong adhesion of C. albicans
cells was observed in control group, and a significant
reduction in C. albicans adhesion was observed in test
groups in parallel with the increase in MPC concentration.
Although it was reported that expression of hyphae could be
observed in short periods of time depending on the culture
medium and incubation temperature [61], candidal hyphae
were not observed under the experimental conditions of this
study.
In our study, a decrease was observed in C. albicans
adhesion with the MPC coating, inversely proportional to
concentration. The differences between control group
(8.82 ± 0.55) and test groups (7.73 ± 1.13 for 0.25 mol/L
MPC; 6.47 ± 0.83 for 0.50 mol/L MPC; and 6.52 ± 0.54 for
0.75 mol/L MPC) were statistically significant. Since the
groups concerning surface roughness were slightly different
but the difference is not statistically significant, this
decrease in adhesion was considered to be related to the
increase in surface hydrophilicity of test groups. The C.
albicans adhesion differences between test groups were
statistically significant except 0.50 mol/L MPC and
0.75 mol/L MPC groups. Based on these results, graft
polymerization of MPC onto PMMA surface was deter-
mined to decrease C. albicans adhesion significantly at all
tested concentrations of MPC when compared to the control
group. We concluded that to reduce C. albicans adhesion, a
monomer density of 0.50 mol/L would be enough for
PMMA surface modification with MPC.
MPC polymer usage in dentistry is quite recent and
studies on this subject are limited. Within the limitations of
our study, modification of PMMA surface with graft poly-
merization of MPC is found to have an inhibitory effect on
C. albicans adhesion due to the significant increase in
surface hydrophilicity and may be evaluated as a coating for
prevention of denture stomatitis. However, to bring MPC
polymer into use in dental applications, further studies
should be performed with various methods evaluating its
mechanical and biological characteristics, and its durability
on PMMA surface.
5 Conclusion
Within the limitations of this study, the following conclu-
sions can be drawn:
107 Page 8 of 9 Journal of Materials Science: Materials in Medicine (2018)29:107
1. Surface modification of PMMA by graft polymerization
of MPC, provided a statistical decrease in contact angle
in 0.25 mol/L MPC and 0.75 mol/L MPC groups.
Although contact angle values were also decreased in
0.50 mol/L MPC group, it was not statistically
significant when compared to the control group.
2. Graft polymerization of MPC does not cause a
statistically significant change in surface roughness
of PMMA.
3. Graft polymerization of MPC significantly decreased
C. albicans adhesion onto PMMA surface.
Acknowledgements Special thanks to Professor Hatice Mehtap Kutlu
and Assistant Professor Nesil Ertorun from Department of Biology,
Faculty of Science, Anadolu University, Eskişehir, Turkey and Öznur
Doğan from Department of Chemical Engineering, Middle East
Technical University for their kindness and valuable assistance in
scanning electron microscopy imaging. This research did not receive
any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
1. Takahashi N, Iwasa F, Inoue Y, Morisaki H, Ishihara K, Baba K.
Evaluation of the durability and antiadhesive action of 2-
methacryloyloxyethyl phosphorylcholine grafting on an acrylic
resin denture base material. J Prosthet Dent. 2014;112:194–203.
2. Kulak-Ozkan Y, Kazazoglu E, Arikan A. Oral hygiene habits,
denture cleanliness, presence of yeasts and stomatitis in elderly
people. J Oral Rehabil. 2002;29:300–4.
3. Akpan A, Morgan R. Oral candidiasis. Postgrad Med J.
2002;78:455–9.
4. Samaranayake LP, Keung Leung W, Jin L. Oral mucosal fungal
infections. Periodontol 2000. 2009;49:39–59.
5. Düzgüneş N. Medical microbiology and immunology for den-
tistry. 1st ed. Batavia, IL: Quintessence Publishing Co, Inc.; 2016.
6. Cannon RD, Firth NA. Fungi and fungal infections of the oral
cavity. In: Lamont RJ, Hajishengallis GN, Jerkinson HF, editors.
Oral microbiology and immunology. 2nd ed. Washington DC:
ASM Press; 2014. pp. 343–59.
7. Edgerton M, Raj PA, Levine MJ. Surface-modified poly(methyl
methacrylate) enhances adsorption and retains anticandidal
activities of salivary histatin 5. J Biomed Mater Res.
1995;29:1277–86.
8. Acosta-Torres LS, Mendieta I, Nuñez-Anita RE, Cajero-Juárez M,
Castaño VM. Cytocompatible antifungal acrylic resin containing
silver nanoparticles for dentures. Int J Nanomed. 2012;7:4777–86.
9. Wady AF, Machado AL, Zucolotto V, Zamperini CA, Berni E,
Vergani CE. Evaluation of Candida albicans adhesion and biofilm
formation on a denture base acrylic resin containing silver nano-
particles. J Appl Microbiol. 2012;112:1163–72.
10. Vukosavljevic D, Custodio W, Del Bel Cury AA, Siqueira WL.
The effect of histatin 5, adsorbed on PMMA and hydroxyapatite,
on Candida albicans colonization. Yeast. 2012;29:459–66.
11. Redding S, Bhatt B, Rawls HR, Siegel G, Scott K, Lopez-Ribot J.
Inhibition of Candida albicans biofilm formation on denture
material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.
2009;107:669–72.
12. Etienne O, Gasnier C, Taddei C, Voegel JC, Aunis D, Schaaf P,
et al. Antifungal coating by biofunctionalized polyelectrolyte
multilayered films. Biomaterials. 2005;26:6704–12.
13. Park SE, Periathamby AR, Loza JC. Effect of surface-charged
poly(methyl methacrylate) on the adhesion of Candida albicans. J
Prosthodont. 2003;12:249–54.
14. Park SE, Blissett R, Susarla SM, Weber H-P. Candida albicans
adherence to surface-modified denture resin surfaces. J Prostho-
dont. 2008;17:365–9.
15. Lazarin AA, Zamperini CA, Vergani CE, Wady AF, Giampaolo
ET, Machado AL. Candida albicans adherence to an acrylic resin
modified by experimental photopolymerised coatings: an in vitro
study. Gerodontology. 2014;31:25–33.
16. Lazarin AA, Machado AL, Zamperini CA, Wady AF, Spolidorio
DMP, Vergani CE. Effect of experimental photopolymerized
coatings on the hydrophobicity of a denture base acrylic resin and
on Candida albicans adhesion. Arch Oral Biol. 2013;58:1–9.
17. Izumida FE, Moffa EB, Vergani CE, Machado AL, Jorge JH,
Giampaolo ET. In vitro evaluation of adherence of Candida
albicans, Candida glabrata, and Streptococcus mutans to an acrylic
resin modified by experimental coatings. Biofouling.
2014;30:525–33.
18. Yodmongkol S, Chantarachindawong R, Thaweboon S, Thawe-
boon B, Amornsakchai T, Srikhirin T. The effects of silane-SiO2
nanocomposite films on Candida albicans adhesion and the sur-
face and physical properties of acrylic resin denture base material.
J Prosthet Dent. 2014;112:1530–8.
19. Iwasaki Y, Aiba Y, Morimoto N, Nakabayashi N, Ishihara K.
Semi-interpenetrating polymer networks composed of biocompa-
tible phospholipid polymer and segmented polyurethane. J
Biomed Mater Res. 2000;52:701–8.
20. Ishihara K, Ueda T, Nakabayashi N. Preparation of phospholipid
polymers and their properties as polymer hydrogel membranes.
Polym J. 1990;22:355–60.
21. Tateishi T, Kyomoto M, Kakinoki S, Yamaoka T, Ishihara K.
Reduced platelets and bacteria adhesion on poly(ether ether
ketone) by photoinduced and self-initiated graft polymerization of
2-methacryloyloxyethyl phosphorylcholine. J Biomed Mater Res
A. 2014;102:1342–9.
22. Ishihara K, Oshida H, Endo Y, Watanabe A, Ueda T, Nakabayashi
N. Effects of phospholipid adsorption on nonthrombogenicity of
polymer with phospholipid polar group. J Biomed Mater Res.
1993;27:1309–14.
23. Ishihara K, Ziats NP, Tierney BP, Nakabayashi N, Anderson JM.
Protein adsorption from human plasma is reduced on phospholipid
polymers. J Biomed Mater Res. 1991;25:1397–407.
24. Ishihara K, Ishikawa E, Iwasaki Y, Nakabayashi N. Inhibition of
fibroblast cell adhesion on substrate by coating with 2-
methacryloyloxyethyl phosphorylcholine polymers. J Biomater
Sci Polym Ed. 1999;10:1047–61.
25. Moro T, Kawaguchi H, Ishihara K, Kyomoto M, Karita T, Ito H,
et al. Wear resistance of artificial hip joints with poly (2-metha-
cryloyloxyethyl phosphorylcholine) grafted polyethylene: com-
parisons with the effect of polyethylene cross-linking and ceramic
femoral heads. Biomaterials. 2009;30:2995–3001.
26. Moro T, Takatori Y, Ishihara K, Konno T, Takigawa Y, Mat-
sushita T, et al. Surface grafting of artificial joints with a bio-
compatible polymer for preventing periprosthetic osteolysis. Nat
Mater. 2004;3:829–36.
27. Moro T, Takatori Y, Ishihara K, Nakamura K, Kawaguchi H.
Frank Stinchfield Award grafting of biocompatible polymer for
longevity of artificial hip joints. Clin Orthop Relat Res.
2006;453:58–63.
Journal of Materials Science: Materials in Medicine (2018)29:107
28. Kyomoto M, Ishihara K. Self-initiated surface graft polymeriza-
tion of 2-methacryloyloxyethyl phosphorylcholine on poly(ether
ether ketone) by photoirradiation. ACS Appl Mater Interfaces.
2009;1:537–42.
29. Sibarani J, Takai M, Ishihara K. Surface modification on micro-
fluidic devices with 2-methacryloyloxyethyl phosphorylcholine
polymers for reducing unfavorable protein adsorption. Colloids
Surf B Biointerfaces. 2007;54:88–93.
30. Hirota K, Murakami K, Nemoto K, Miyake Y. Coating of a sur-
face with 2-methacryloyloxyethyl phosphorylcholine (MPC) co-
polymer significantly reduces retention of human pathogenic
microorganisms. FEMS Microbiol Lett. 2005;248:37–45.
31. Nakabayashi N, Iwasaki Y. Copolymers of 2-
methacryloyloxyethyl phosphorylcholine (MPC) as biomaterials.
Biomed Mater Eng. 2004;14:345–54.
32. Kyomoto M, Moro T, Takatori Y, Kawaguchi H, Nakamura K,
Ishihara K. Self-initiated surface grafting with poly(2-methacry-
loyloxyethyl phosphorylcholine) on poly(ether-ether-ketone).
Biomaterials. 2010;31:1017–24.
33. Yumoto H, Hirota K, Hirao K, Miyazaki T, Yamamoto N,
Miyamoto K, et al. Anti-inflammatory and protective effects of 2-
methacryloyloxyethyl phosphorylcholine polymer on oral epithe-
lial cells. J Biomed Mater Res A. 2015;103:555–63.
34. Hirota K, Yumoto H, Miyamoto K, Yamamoto N, Murakami K,
Hoshino Y, et al. MPC-polymer reduces adherence and biofilm
formation by oral bacteria. J Dent Res. 2011;90:900–5.
35. Kyomoto M, Moro T, Yamane S, Hashimoto M, Takatori Y, Ishi-
hara K. Effect of UV-irradiation intensity on graft polymerization of
2-methacryloyloxyethyl phosphorylcholine on orthopedic bearing
substrate. J Biomed Mater Res A. 2014;102:3012–23.
36. Lee J. Inhibition of bacterial adhesion by poly(2-methacryloy-
loxyethyl phosphorylcholine) coating on dental composite resin.
Seoul: Yonsei University; 2013.
37. Zhang N, Ma J, Melo MaS, Weir MD, Bai Y, HHK Xu. Protein-
repellent and antibacterial dental composite to inhibit biofilms and
caries. J Dent. 2015;43:225–34.
38. Zhang N, Chen C, Melo MAS, Bai Y-X, Cheng L, Xu HHK. A novel
protein-repellent dental composite containing 2-methacryloyloxyethyl
phosphorylcholine. Int J Oral Sci. 2015;7:103–9.
39. Zhang N, Melo MAS, Bai Y, Xu HHK. Novel protein-repellent
dental adhesive containing 2-methacryloyloxyethyl phosphor-
ylcholine. J Dent. 2014;42:1284–91.
40. Zhang N, Weir MD, Romberg E, Bai Y, Xu HH. Development of
novel dental adhesive with double benefits of protein-repellent and
antibacterial capabilities. Dent Mater Acad Dent Mater.
2015;31:845–54.
41. Zhang N, Zhang K, Melo MAS, Chen C, Fouad AF, Bai Y, et al.
Novel protein-repellent and biofilm-repellent orthodontic cement
containing 2-methacryloyloxyethyl phosphorylcholine. J Biomed
Mater Res B Appl Biomater. 2016;104:949–59.
42. Zhang N, Chen C, Weir MD, Bai Y, Xu HHK. Antibacterial and
protein-repellent orthodontic cement to combat biofilms and white
spot lesions. J Dent. 2015;43:1529–38.
43. Kyomoto M, Moro T, Miyaji F, Hashimoto M, Kawaguchi H,
Takatori Y, et al. Effect of 2-methacryloyloxyethyl phosphor-
ylcholine concentration on photo-induced graft polymerization of
polyethylene in reducing the wear of orthopaedic bearing surface.
J Biomed Mater Res A. 2008;86:439–47.
44. Azevedo A, Machado AL, Vergani CE, Giampaolo ET, Pavarina
AC, Magnani R. Effect of disinfectants on the hardness and
roughness of reline acrylic resins. J Prosthodont. 2006;15:235–42.
45. Zissis AJ, Polyzois GL, Yannikakis SA, Harrison A. Roughness
of denture materials: a comparative study. Int J Prosthodont.
2000;13:136–40.
Page 9 of 9 107
46. Kyomoto M, Moro T, Yamane S, Watanabe K, Hashimoto M,
Takatori Y, et al. Poly(2-methacryloyloxyethyl phosphorylcho-
line) grafting and vitamin E blending for high wear resistance and
oxidative stability of orthopedic bearings. Biomaterials.
2014;35:6677–86.
47. Wang JJ, Liu F. Photoinduced graft polymerization of 2-
methacryloyloxyethyl phosphorylcholine on silicone hydrogels
for reducing protein adsorption. J Mater Sci Mater Med.
2011;22:2651–7.
48. Huang X-D, Yao K, Zhang H, Huang X-J, Xu Z-K. Surface
modification of silicone intraocular lens by 2-
methacryloyloxyethyl phosphoryl-choline binding to reduce Sta-
phylococcus epidermidis adherence. Clin Exp Ophthalmol.
2007;35:462–7.
49. Fujii K, Matsumoto HN, Koyama Y, Iwasaki Y, Ishihara K,
Takakuda K. Prevention of biofilm formation with a coating of 2-
methacryloyloxyethyl phosphorylcholine polymer. J Vet Med Sci.
2008;70:167–73.
50. Ishihara K, Iwasaki Y, Ebihara S, Shindo Y, Nakabayashi N.
Photoinduced graft polymerization of 2-methacryloyloxyethyl
phosphorylcholine on polyethylene membrane surface for
obtaining blood cell adhesion resistance. Colloids Surf B Bioin-
terfaces. 2000;18:325–35.
51. Iwasaki Y, Ishihara K. Cell membrane-inspired phospholipid
polymers for developing medical devices with excellent bioin-
terfaces. Sci Technol Adv Mater. 2012;13:64101.
52. Compagnoni MA, Pero AC, Ramos SMM, Marra J, Paleari AG,
Rodriguez LS. Antimicrobial activity and surface properties of an
acrylic resin containing a biocide polymer. Gerodontology.
2014;31:220–6.
53. Chandra J, Patel JD, Li J, Zhou G, Mukherjee PK, McCormick
TS, et al. Modification of surface properties of biomaterials
influences the ability of Candida albicans to form biofilms. Appl
Environ Microbiol. 2005;71:8795–801.
54. Quirynen M, Marechal M, Busscher H, Weerkamp A, Darius PL,
Vansteenberghe D. The influence of surface free energy and
surface roughness on early plaque formation: an in vivo study in
man. J Clin Periodontol. 1990;17:138–44.
55. Mayahara M, Kataoka R, Arimoto T, Tamaki Y, Yamaguchi N,
Watanabe Y, et al. Effects of surface roughness and dimorphism
on the adhesion of Candida albicans to the surface of resins:
scanning electron microscope analyses of mode and number of
adhesions. J Investig Clin Dent. 2014;5:307–12.
56. Pereira T, Del Bel Cury AA, Cenci MS, Rodrigues-Garcia RCM.
In vitro Candida colonization on acrylic resins and denture liners:
influence of surface free energy, roughness, saliva, and adhering
bacteria. Int J Prosthodont. 2007;20:308–10.
57. Ulusoy M, Ulusoy N, Aydin AK. An evaluation of polishing
techniques on surface roughness of acrylic resins. J Prosthet Dent.
1986;56:107–12.
58. Kyomoto M, Moro T, Konno T, Takadama H, Kawaguchi H,
Takatori Y, et al. Effects of photo-induced graft polymerization of
2-methacryloyloxyethyl phosphorylcholine on physical properties
of cross-linked polyethylene in artificial hip joints. J Mater Sci
Mater Med. 2007;18:1809–15.
59. Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of
Candida albicans to denture-base materials with different surface
finishes. J Dent. 1998;26:577–83.
60. Radford DR, Challacombe SJ, Walter JD. Denture plaque and
adherence of Candida albicans to denture-base materials in vivo
and in vitro. Crit Rev Oral Biol Med. 1999;10:99–116.
61. Sudbery PE. Growth of Candida albicans hyphae. Nat Rev
Microbiol. 2011;9:737–48.