J Dental 2018 01 019

DENTAL-3101; No.
of Pages 10
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Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.intl.elsevierhealth.com/journals/dema
Nano-graphene oxide incorporated into PMMA

resin to prevent microbial adhesion
Jung-Hwan Lee a,b , Jeong-Ki Jo b , Dong-Ae Kim b,c , Kapil Dev Patel a,d ,
Hae-Won Kim a,b,d , Hae-Hyoung Lee a,b,∗
a Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South Korea
b Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, South Korea
c Department of Dental Hygiene, Kyungwoon University, Gumi-si, South Korea
d Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine
Research Center, Dankook University, Cheonan 330-714, South Korea
a r t i c l e i n f o a b s t r a c t
Article history: Objective. Although polymethyl methacrylate (PMMA) is widely used as a dental mate-
Received 24 September 2017 rial, a major challenge of using this substance is its poor antimicrobial (anti-adhesion)
Received in revised form effects, which increase oral infections. Here, graphene-oxide nanosheets (nGO) were incor-
6 January 2018 porated into PMMA to introduce sustained antimicrobial-adhesive effects by increasing the
Accepted 16 January 2018 hydrophilicity of PMMA.
Available online xxx Methods. After characterizing nGO and nGO-incorporated PMMA (up to 2 wt%) in terms of
morphology and surface characteristics, 3-point flexural strength and hardness were eval-
Keywords: uated. The anti-adhesive effects were determined for 4 different microbial species with
Sustained antimicrobial-adhesive experimental specimens and the underlying anti-adhesive mechanism was investigated
effect by a non-thermal oxygen plasma treatment. Sustained antimicrobial-adhesive effects were
Graphene oxide nanoparticle characterized with incubation in artificial saliva for up to 28 days.
PMMA Results. The typical nanosheet morphology was observed for nGO. Incorporating nGO into
Hydrophilicity PMMA roughened its surface and increased its hydrophilicity without compromising flexural
Non-thermal oxygen plasma strength or surface hardness. An anti-adhesive effect after 1 h of exposure to microbial
species in artificial saliva was observed in nGO-incorporated specimens, which accelerated
with increasing levels of nGO without significant cytotoxicity to oral keratinocytes. Plasma
treatment of native PMMA demonstrated that the antimicrobial-adhesive effects of nGO
incorporation were at least partially due to increased hydrophilicity, not changes in the
surface roughness. A sustained antimicrobial-adhesive property against Candida albicans
was observed in 2% nGO for up to 28 days.
Significance. The presence of sustained anti-adhesion properties in nGO-incorporated PMMA
without loading any antimicrobial drugs suggests the potential usefulness of this com-
pound as a promising antimicrobial dental material for dentures, orthodontic devices and
provisional restorative materials.
© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, South Korea.
E-mail address: haelee@dku.edu (H.-H. Lee).
https://doi.org/10.1016/j.dental.2018.01.019
0109-5641/© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee J-H, et al. Nano-graphene oxide incorporated into PMMA resin to prevent microbial adhesion. Dent Mater
(2018), https://doi.org/10.1016/j.dental.2018.01.019
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incubation. This study is the first to show sustained, long-

1. Introduction term antimicrobial-adhesive effects without compromising
mechanical properties using carbon-based nanomaterials
Polymethyl methacrylate (PMMA) has been clinically utilized
(nGO). This work provides informative ideas for the devel-
for over 70 years as a biomaterial in the dental/medical fields
opment of future clinically available antimicrobial-adhesive
for removable or implantable devices (e.g., denture base resin,
PMMA. The null hypothesis of this study is that there is no dif-
provisional restorative materials, bone cement, facial pros-
ference between the antimicrobial-adhesive effects of PMMA
theses) due to its desirable mechanical/biological properties,
with and without nGO incorporation.
easy fabrication, and economic cost [1–3]. However, its poor
antimicrobial property still remains a major drawback that can
cause infection when in contact with tissues or when being 2. Materials and methods
implanted for restoration [4,5]. One of the most intriguing
approaches recently used to address this problem is incorpo- 2.1. Synthesis of nGO-incorporated denture resin
rating nano-additives in the form of nanospheres, nanosheets,
nanofibers, or nanotubes [6–10]. nGO powder (Graphene Supermarket, Calverton, NY, USA) was
Mesoporous silica nanoparticles (MSNs), bioactive glass washed with 70% ethanol for cleaning and sterilization. Chem-
nanoparticles, gold nanoparticles, titanium nanoparticles and ically activated PMMA products (Orthocryl resin, Dentaurum,
other metallic (oxide) nanoparticles have been explored, each Ispringen, Germany) were selected to avoid thermal damage to
with their own various advantages as additives in dental the nGO during heat-induced polymerization. nGO was added
materials, including high stability, excellent biocompatibil- in quantities of 0.25, 0.5, 1.0, or 2.0% by weight relative to
ity, therapeutic biological activity, mechanical reinforcement PMMA powder. After each of these nGO concentrations were
properties or drug (or ion) loading/releasing capacity [11,12]. homogeneously dispersed in liquid MMA under sonication for
Recently, MSNs were introduced into PMMA as a potential 1 h according to other literatures [24,25]. PMMA powder was
nano-additive to prevent microbes from adhering to the PMMA mixed into the liquid at a powder (g) to liquid (ml) ratio of 1.2:1.
when more than 2.5 wt% is incorporated, which was mainly After low-temperature polymerization (40 ◦ C, 2.2 bars, 30 min,
due to increased hydrophilicity. In addition, the antimicrobial- MultiCure, Vertex, Zeist, Netherlands), disk (h = 1.5 mm and
adhesive effects of the MSNs were synergistically enhanced d = 11.5 mm) specimens were cut, polished with SiC papers
after loading drugs (i.e., silver sulfadiazine or amphotericin B) up to 1200 grit, and used for all investigations unless oth-
into the MSN-incorporated PMMA [13–15]. erwise mentioned. Bar-shaped (1.4 × 3.0 × 18 mm) specimens
Antimicrobial-adhesive surfaces of dental biomaterials are were specifically prepared for 3-point flexural testing and were
usually generated by one of two strategies: introducing either polished with up to 2400 grit SiC paper. All specimens for bio-
hydrophilic or zwitterionic surfaces, which generate a hydra- logical testing were sterilized with ethylene oxide (EO) gas
tion layer on the surface; this tightly bound water layer creates according to a previously described method [26].
a physical or energetic barrier that prevents microbial adhe-
sion [16,17].
2.2. Characterization of nGO-incorporated denture
Carbon-based nanomaterials such as graphene oxide
resin
nanosheets (nGO) and carbon nanotubes (CNTs) have been
reported to exert antimicrobial effects when placed in direct
After nGO was visualized by transmission electron microscopy
contact with microbes [18–20]. Moreover, when incorpo-
(TEM; 7100, JEOL, Peabody, MA, USA), nGO-incorporated den-
rated into a scaffold, antimicrobial-adhesive effects were
ture resin was characterized by attenuated total reflectance-
observed with (functionalized) nGO or CNTs, which increased
Fourier transform infrared spectroscopy (ATR-FTIR; Varian
the hydrophilicity of the native materials [21,22]. However,
640-IR, Australia) in the range of 4000–400 cm−1 according
whether these hydrophilic carbon-based nanomaterials have
to a previously reported method [15,27]. Baseline correction
antimicrobial-adhesive effects when incorporated into PMMA
after getting data from FTIR machine was performed for nGO
remains unknown.
by Magicplot student software (ver. 2.7.2, Magicplot systems,
Here, we focused on incorporating pristine nGO, which
Saint Petersburg, Russia). Other data were used as given. Rep-
has many hydroxyl (–OH) and carboxyl (–COOH) groups
resentative images and results are shown for independent
capable of introducing hydrophilicity [23] into PMMA, to
experiments performed in triplicate. The surface character-
induce long-term antimicrobial effects. First, after charac-
istics were visualized by scanning electron microscopy (SEM,
terizing the morphology and surface characteristics of nGO
Sigma 500, ZEISS, Jena, Germany). Surface roughness (n = 5,
and PMMA incorporated with nGO in varying amounts
Ra, SJ-400, Mitutoyo, Japan) and static contact angles (n = 5,
(0.25, 0.5, 1, or 2 wt%), we examined the resulting mechan-
Phoenix 300, SEO, Suwonsi, Gyunggido, Korea) were mea-
ical properties and antimicrobial-adhesive effects against
sured using distilled water and ethylene glycol, as described
4 different representative microbes related to denture- or
in detail elsewhere [15,28]. Surface energy was calculated by
provisional-appliance-induced infection. Furthermore, non-
Owens–Wendt methods equipped in Image XP software (ver
thermal oxygen plasma treatment was used to demonstrate
5.9., SEO), which uses non-polar (dispersive) and polar com-
that the anti-adherence mechanism of the nGO-incorporated
ponents for analysis [28,29]. Total surface tension and their
PMMA was due to increases in hydrophilicity, not to changes
sub-categorized force (dispersive and polar force) of ethylene
in the surface roughness. Finally, long-term antimicrobial-
glycol and water were set at 48.3, 29.3, 19.0 and 72.8, 21.8,
adhesive effects were investigated with artificial saliva
51.0 mJ/m2 , respectively. The room temperature was set at
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20 ◦ C. To check dispersion ability of nGO, nGO (20 mg) was 2.6. Non-thermal oxygen plasma treatment effects
functionalized with PEG-amine (NH2 -PEG-NH2 , Sigma, 5 mg)
and form nGO-PEG. Then nGO-PEG was labeled by fluorescein To elucidate nGO’s anti-adhesive effects against microbes
isothiocyanate (FITC, Sigma). In brief, the solution of nGO- on denture resin, disk specimens (0% or 2% nGO) were
PEG (approximately 1.5 mg/mL) was mixed with 0.1 mL FITC treated with non-thermal oxygen plasma (Cute, Femto Sci-
(10 mM) dissolved in DMSO and then stirred overnight at room ence, Hwasungsi, Kyounkyido, Korea) for 60 s at 50 W with
temperature. The resulting mixtures labeled with FITC was 30 standard cubic centimeters per minute of oxygen flow
washed with distilled water several times to remove the excess rate, which generates super-hydrophilicity without altering
and loosely bounded/unbounded FITC and then lyophilized. the surface topology. Surface roughness, surface energy, and
The obtained nGO-PEG-FITC (FITC-nGO) was re-dispersed in anti-adhesive effects against C. albicans were investigated
distilled water and monomer solution for checking disper- before and after the plasma treatment according to the
sion. Conjugation of FITC-nGO was performed according to above-described methodology. C. albicans was selected as a
aforementioned protocols. Florescence image was taken by representative microbial species among the four microbial
fluorescence microscope (IRIS, Logos biosystems, Anyang-si, species. To confirm the hydrophilicity effects on acrylic den-
Korea). The whole procedures were operated in the dark place. ture resin, SEM (Sigma 500) was performed after 1 h incubation
with C. albicans following fixation with 4% paraformaldehyde
2.3. Mechanical properties for 10 min.
After the bar-shaped specimens were incubated for 48 h at 2.7. Sustained anti-adhesive effects after cleaning
37 ◦ C in DW, they were positioned on an Instron 5966 machine
(500-N load cell, Norwood, MA, USA) for 3-point flexural tests To analyze the duration of the anti-adhesive effects, disk
at a span length of 14 mm. The flexural strength and elastic specimens cultured with C. albicans were cleaned as follows:
modulus were measured at a 1.0 mm/min crosshead speed washing twice with PBS, sonication for 20 min at 4◦ C [39],
(n = 10) [30]. A Vickers hardness machine (HM-221, Mitutoyo, spraying with ethanol for 10 s, immediately washing twice
Tokyo, Japan) was used to determine the hardness with 300 gf with PBS, drying in a sterilized hood at room temperature for
(2.94 N) for 30 s; an average value was calculated from five dif- 4 h, and incubating in sterilized artificial saliva in a humidified
ferent locations on each specimen (n = 5). incubator at 37◦ C with 5% CO2 for the indicated days between
anti-adhesion tests. Antimicrobial adhesion testing against C.
albicans (n = 5) was repeated in the same manner as described
2.4. Antimicrobial study
in Section 2.4 at various time points over 28 days.
Escherichia coli (E. coli, ATCC 25922), Candida albicans (C. albi-
cans, ATCC 10231), Staphylococcus aureus (S. aureus, ATCC 6583), 2.8. Statistical analysis
and Streptococcus mutans (S. mutans, ATCC 25175) were grown
according to the manufacturer’s protocols [14]. Then, 100 ␮l The data are expressed as the representative mean ± SD of
(106 microbes), consisting of 90 ␮l artificial saliva [31] and 10 ␮l three independent experiments, unless otherwise noted. Sta-
microbial suspension media containing 108 microbes/ml in tistical significance was determined by one-way analysis of
the log phase of growth, was seeded exclusively on the surface variance with a post hoc test (Tukey) using SPSS (SPSS 21.0,
of a disk specimen. After 1 h of culturing at 37 ◦ C in 5% CO2 to Chicago, IL, USA). A p-value < 0.05 was considered statistically
allow adherence to the specimen, the non-adherent microbes significant.
were washed away twice with PBS. After the disk specimens
were further cultured with 10% Prestoblue (Molecular probes,
3. Results
USA) in microbial culture media for the desired times, 100 ␮l of
culture media was transferred to a 96-well plate at each time
3.1. Characterization of nGO-incorporated acrylic resin
point to measure the optical density (OD 570–600 nm) using
a microplate reader (BioTek, Winooski, VT, USA), which was
The morphological characteristics of nGO were visualized
normalized to a control (bacteria only, n = 5). The procedure is
with ∼90 nm of diameter (longest axis) of very little black col-
described in detail elsewhere [32,33].
ored fragment (Fig. 1A), indicating few layers of nanosized
sheets according to other literatures [40,41]. FTIR analysis
2.5. Cytotoxicity testing showed varying increases in the C O, C O C and C O peaks
representing nGO (Fig. 1B, 1724, 1140, and 1084 cm−1 respec-
Immortalized human oral keratinocytes (IHOKs) were selected tively), depending on the nGO level in the PMMA [42]. Increases
to represent human keratinocytes [34,35]. After cells were in OH-related broad peaks approximately 3550–3200 cm−1
seeded (n = 6, 1 × 104 cells) in 96-well plates and incubated in were also observed in the same manner (not shown). SEM
a humidified atmosphere at 37 ◦ C with 5% CO2 and 95% air, images of nGO-incorporated acrylic resin (Fig. 2A–E) showed
specimen extract (3 cm2 /ml in DW) was added to 2X DMEM/F- similar surface morphology with the increasing nGO incor-
12 (3:1) supplemented with 20% fetal bovine serum (Gibco), 2% poration. But, highly magnified SEM images revealed several
penicillin/streptomycin (Invitrogen, Waltham, MA, USA). After sheet-like particles (possibly nGOs or nGOs-PMMA complex,
24 h of incubation, a WST assay was performed according to red arrow) on the surface, which were not detected in acrylic
previously described methods (OD 450 nm) [36–38]. resin without nGO (Fig. 2F). The surface roughness was
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Fig. 1 – Characteristics of nGO and nGO-incorporated PMMA. TEM images of (A) graphene-oxide nanosheets (nGO) and FTIR
peaks from nGO and nGO-incorporated PMMA, with varying incorporation amounts up to 2%.
evaluated (Fig. 2G), which showed more significant increases 2 wt% nGO (p < 0.05, Fig. 2H). The surface energy calculated
in surface roughness (Ra ) in the 2 wt% nGO group than that in from the contact angle results using DW and ethylene gly-
the 0 wt% nGO group. Water contact angle testing using DW col revealed an approximately threefold increase in surface
showed a noticeable decrease in the water contact angle with energy in the 2 wt% nGO specimen relative to that of the 0 wt%
Fig. 2 – Surface characteristics of nGO-incorporated PMMA. SEM images of nGO-incorporated PMMA (A–E, 0, 0.25, 0.5, 1, 2%)
and (F) highly magnified SEM images confirming the morphological presence of nGO in PMMA (red arrow from F) with the
inset showing no detected nGO structures in 0% nGO. (G) Surface roughness, (H) water contact angle, and (I) surface energy
calculated by the Owens–Wendt method using water and ethylene glycol contact angles. Asterisks (*) indicate statistical
significance compared to 0% (n = 5, P < 0.05). Representative means and SDs are shown for independent experiments
performed in triplicate.
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Fig. 3 – Three-point flexural and hardness testing. (A) Black boxes indicate flexural strength, and white boxes show the
flexural modulus (n = 10). (B) Vickers hardness was measured in five spots per specimen and averaged (n = 5). Asterisks (*)
indicate statistical significance compared to 0% nGO (p < 0.05).
nGO specimen (p < 0.05, Fig. 2I), which was primarily due to flexural test, whereas more than 0.5 wt% nGO incorporation
polar tension. significantly increased the surface hardness (Fig. 3B, p < 0.05).
3.3. Immediate antimicrobial-adhesive effects

3.2. Mechanical properties
Based on the previous results, the 0.25% nGO group was
Among the acrylic resin specimens containing different excluded from further analysis, as it was expected to show
amounts of nGO (0.25, 0.5, 1, 2%) relative to the PMMA powder reduced effects against microbial adhesion because less nGO
by mass, incorporating only 0.5% significantly enhanced the was exposed. Thus, other nGO-incorporated groups (0.5, 1, and
flexural strength (Fig. 3A, p < 0.05), as shown by the 3-point 2%) were chosen to further study the antimicrobial-adhesive
Fig. 4 – Antimicrobial-adhesive effects of nGO-incorporated PMMA against four different microbial species. After microbial
species (C. albicans, E. coli, S. aureus, and S. mutans) were seeded and cultured on native PMMA or PMMA incorporating nGO
for 1 h in artificial saliva, the levels of attachment for each species were determined by Prestoblue assay with 3–9 h of further
incubation in microbial media. Asterisks (*) indicate statistical significance compared to 0% nGO (n = 5, p < 0.05).
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3.5. Mechanistic analysis of GO-induced

antimicrobial-adhesive effects using non-thermal plasma
To determine the mechanism underlying the GO-induced

antimicrobial-adhesive effects after incorporation into acrylic
resin, non-thermal oxygen plasma was used to treat the 0%
nGO and 2% nGO specimens. Plasma treatment induced a sig-
nificant increase in the surface energy (approximately 10-fold
and 5-fold,), primarily due to polar energy, in the 0% nGO and
2% nGO groups, respectively (p < 0.05, Fig. 6A),while the sur-
face roughness did not significantly change (p > 0.05, Fig. 6B).
Interestingly, the plasma-treated 0% nGO group also showed
a significant decrease in C. albicans adhesion compared to
the untreated 0% nGO group ( p< 0.05, Fig. 5C). Furthermore,
Fig. 5 – Cytotoxicity testing against human oral the plasma-treated 2% nGO specimen demonstrated a greater
keratinocytes using extracts from specimens. After IHOKs reduction in the adhesion of C. albicans than the untreated 2%
were seeded, they were cultured for 24 h with extract and nGO specimen (p < 0.05, Fig. 6C).
analyzed by WST assay to determine cell viability (n = 6).
3.6. Sustained anti-adhesive effects
Fig. 7 shows sustained anti-adhesive effects against C. albicans

after prolonged incubation in sterilized artificial saliva. The 2%
nGO and plasma-treated 2% nGO specimens showed greater
effects. A better anti-adhesion effect against C. albicans, E. anti-adhesion effects against C. albicans than the 0% nGO spec-
coli, S. aureus, and S. mutans after 1 h of attachment in arti- imens for up to 28 days of aging (Fig. 7, p < 0.05), although the
ficial saliva was observed in samples with incorporated nGO anti-adhesive effects decreased with prolonged artificial saliva
(0.5, 1, and 2%) than that in samples without nGO, except incubation. Interestingly, the 2% nGO specimen continuously
for 0.5% nGO against E. coli and S. mutans (p < 0.05, Fig. 4). demonstrated extremely good anti-adhesive effects against C.
The same amount of bacteria for all anti-adhesion test- albicans, with less than 10% remaining adhered.
ing on specimens was cultured as in the positive control
(black line, bacteria only). Specimens with more nGO showed
stronger anti-adhesion effects against all microbial species: 4. Discussion
2% nGO ≤ 1% nGO ≤ 0.5% nGO ≤ 0% nGO.
The present study utilized nGO as a novel additive in
PMMA to induce antimicrobial-adhesive effects via increased
3.4. Cytocompatibility testing against oral hydrophilicity. We demonstrated that incorporating nGO into
keratinocytes PMMA increased the peak intensities related to hydroxyl and
carboxy groups, which are unique peaks for nGO, and can
Compared to the control, specimen extracts incorporating therefore be expected to enhance multiple aspects of PMMA.
nGO (0, 0.25, 0.5, 1, and 2%) did not significantly decrease the SEM analysis showed that nGO was more exposed in the
viability of oral keratinocytes (Fig. 5, p > 0.05). outer surface of 2% nGO than with other nGO levels, which
Fig. 6 – Mechanism of antimicrobial effect of nGO-incorporated PMMA against C. albicans. After confirming (A) the increase
in hydrophilic surface energy (n = 5, polar part) after oxygen plasma treatment (B) regardless of surface roughness (n = 5),
amounts of C. albicans adhering to the 0% and 2% nGO samples were evaluated with and without plasma treatment.
Adherence significantly decreased after plasma treatment in both groups, partially indicating an anti-adhesive mechanism
of nGO against C. albicans. Different letters in A and B indicate statistical significance between the corresponding samples
(n = 5, p < 0.05). Asterisks (*) in each color indicate a statistically significant decrease in C. albicans adhesion after plasma
treatment compared to the level before plasma treatment (p < 0.05).
ARTICLE IN PRESS
or polymer composites, such as gold, copper, silver, titanium

oxide, zinc oxide, iron oxide, poly-N-vinyl carbazole, chitosan,
polyurethane and poly-l-lysine [48–51].
Antimicrobial-adhesive surfaces are usually obtained on
PMMA via two strategies: introducing either hydrophilic
or zwitterionic surfaces, which help generate a hydration
layer on the surface that creates a tightly bound water
layer; this layer presents a physical or energetic barrier
to prevent microbial adhesion [16,17]. When nGO comes
into direct contact with microbes, the generally suggested
mechanism for its antimicrobial effects include phospho-
lipid extraction, membrane cutting, and isolation of microbes
by wrapping [51]. In contrast, when nGO was incorporated
into a composite, forming a hydration layer on the surface
was shown to be one of the primary antimicrobial-adhesion
mechanisms, as the tightly bound water layer provides a
physical or energetic barrier [48]. Although the increased
Fig. 7 – Sustained antimicrobial-adhesive effects of hydrophilicity was demonstrated on the nGO-incorporated
nGO-incorporated PMMA. PMMA, which supports the above hypothesis, non-thermal
The long-term antimicrobial effects against C. albicans over oxygen plasma treatment was used to further confirm the
28 days of incubation in artificial saliva were evaluated hydrophilicity-induced anti-adhesive effects via nGO incorpo-
(n = 5). Different letters indicate statistical significance ration.
between the corresponding samples for each incubation Possible toxic reactions are of concern when nGO incor-
time point (p < 0.05). porated into PMMA encounters the oral mucosa in clinical
settings. As such, cytotoxicity testing was performed using
oral keratinocytes, as most of the outer mucosal layer consists
of keratinocytes [52]. Cytotoxicity was not revealed in any of
increased both the surface roughness and the hydrophilic- the specimens compared to the control, systemic toxic effects
ity. In agreement with previous studies, increased roughness in humans from fully polymerized PMMA products have not
and consequent decreased water contact angles were corre- been reported [8]. However, future in vivo studies are nec-
lated with increases in microbial adherence on biomaterials essary to confirm the biocompatibility of nGO-incorporated
including PMMA [43–45]. However, nGO incorporation showed PMMA.
interesting surface characteristics; increase of roughness as Soft non-thermal plasma, including oxygen plasma treat-
well as increase of hydrophilicity, which was majorly due to ment, has been known to induce significant hydrophilicity
exposed nGO on the outer surface after polishing and their without changes in roughness, which was also confirmed
unique roles of surface modification. by surface energy and roughness experiments in this study
Before investigating antimicrobial-adhesive effects, the [28,33,53]. As a representative of the four microbial species,
mechanical properties were analyzed, including 3 point flex- C. albicans was selected for testing the anti-adhesion prop-
ural strength and surface hardness. Neither parameter was erties, due to its pathogenic role in oral candidiasis in
compromised after incorporating nGO into PMMA. Interest- denture-wearing patients, which represents a major PMMA-
ingly, 3 point flexural strength was slightly increased for 0.5% induced disease from dental appliances. Unexpectedly, great
nGO incorporation and Vickers hardness was also increased antimicrobial-adhesive effects were observed even on native
for nGO incorporation levels greater than 0.5% relative to those PMMA after the plasma treatment, similar to those in the
of the control; nGO is known to play the role of crack deflec- 2% nGO specimen (∼20% compared to tissue culture plate
tion and bridging when incorporated into a matrix, which control), and more synergistic antimicrobial-adhesive effects
can have potential to enhance the mechanical properties of were observed with the plasma-treated 2% nGO specimen
PMMA [46,47]. However, this innate nGO did not dramatically (∼0%) than those observed with the 2% nGO specimen
increase mechanical properties compared to control due to (∼15%). Non-thermal oxygen plasma produces many radi-
lack of chemical crosslinking between nGO and PMMA matrix. cals, such as excited oxygen molecules, OH radicals, atomic
Further study is needed for optimizing salinization of nGO for oxygen, superoxide anion radicals, and singlet oxygen, and
better mechanical properties. provides high hydrophilicity to treated surfaces without ther-
Four different representative microbial species (C. albicans, mal damage (and therefore without changes to the surface)
E. coli, S. aureus, and S. mutans) were chosen for investigating [54].
the antimicrobial-adhesive effects of PMMA due to their roles Finally, the antimicrobial-adhesive effects were evaluated
in oral infection in dentistry. Against all microbial species, repeatedly against C. albicans for up to 28 days. Continuous
anti-adhesion effects were shown in the nGO-incorporated anti-adhesive effects were compared in the 2% nGO and 0%
PMMA, which increased with the increasing amount of nGO. nGO specimens, but the level of these anti-adhesive effects
These effects of nGO are consistent with the results of other decreased with time, possibly due to the residual extracel-
reports in the literature demonstrating similar anti-adhesion lular matrix (ECM) of C. albicans even after sonication and
effects against microbial species in several metal, metal oxide, alcohol spraying for 10 s [55,56]. After C. albicans was incu-
ARTICLE IN PRESS
bated on specimens for 1 h in artificial saliva, C. albicans was resin) and implantable biomaterials due to these documented
further cultured with microbial culture media for up to 9 h, antimicrobial-adhesion characteristics.
which is sufficient time for C. albicans to produce ECM [57]. In
addition, artificial saliva incubation was performed between
Acknowledgements
experiments, and storage in artificial saliva resulted in signif-
icantly higher C. albicans adherence [58].
This research was supported by the Basic Science Research
Limitation of this study is lack of dispersion of nGO
Program through the National Research Foundation of Korea
in PMMA base resin (Supplementary Fig. S1). When FITC
(NRF), funded by the Ministry of Education (NRF-2009-0093829)
tagged nGO was dispersed in water, homogenous disper-
and the Ministry of Science, ICT & Future Planning (NRF-
sion was made (Fig. S1). However, after being dispersed in
2015R1C1A1A01052127 and NRF-2015R1A2A2A01007567).
monomer solution and incorporated in PMMA, some aggre-
gation was observed from hundreds of nanometers to tens
of micrometers (Figs. S1 and S2). Along with results from Appendix A. Supplementary data
the literatures [24,25], similar well dispersion in water but
some aggregation of nGO in monomer and composite were Supplementary data associated with this arti-
observed, which might be the result of the high viscosity cle can be found, in the online version, at
of monomer solution, van der Waals forces and coulomb https://doi.org/10.1016/j.dental.2018.01.019.
attractions [25,59]. Therefore, to overcome this inhomoge-
neous dispersion of nGO in composite, new strategy such
references
as functionalization of nGO with PEG, salinization or car-
boxyl group is recommended for future studies. Within the
limitation of this lack of nGO dispersion, it was observed
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DENTAL-3101; No.

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/dema

Nano-graphene oxide incorporated into PMMA

Research Center, Dankook University, Cheonan 330-714, South Korea

incubation. This study is the first to show sustained, long-

3.3. Immediate antimicrobial-adhesive effects

3.5. Mechanistic analysis of GO-induced

To determine the mechanism underlying the GO-induced

3.6. Sustained anti-adhesive effects

Fig. 7 shows sustained anti-adhesive effects against C. albicans

or polymer composites, such as gold, copper, silver, titanium

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