In Vitro Evaluation of Adhesion of Candida albicans

on CAD/CAM PMMA-Based Polymers

Sema Murat, DDS, PhD,1 Gülce Alp, DDS, PhD ,2 Canan Alatalı, MSc,3 & Meltem Uzun, MSc, PhD3
1
Department of Prosthodontics, Ankara University Faculty of Dentistry, Ankara, Turkey
2
Department of Prosthodontics, Okan University Faculty of Dentistry, Istanbul, Turkey
3
Department of Medical Microbiology, Faculty of Medicine, Istanbul University, Istanbul, Turkey

Keywords
C. albicans; CAD/CAM; denture base
materials; PMMA; surface properties.
Correspondence
Sema Murat, Department of Prosthodontics,
Faculty of Dentistry, Ankara University,
Besevler, Istanbul 06500, Turkey. E-mail:
smurat@ankara.edu.tr.
Previously presented at the 23rd Scientific
Congress of Turkish Prosthodontics and
Implantology, 09–12 November, 2017,
Dalaman/Turkey
The authors claim no conflict of interest in
this study.
Accepted April 26, 2018
doi: 10.1111/jopr.12942
Abstract
Purpose: To compare the amount of adherent Candida albicans to different
CAD/CAM poly(methyl methacrylate) (PMMA)-based polymers and conventional
heat-polymerized PMMA after long-term thermal cycling.
Materials and Methods: The specimens were subjected to 10,000 thermal cycles (5-
55°C) and divided into two groups, uncoated and pellicle-coated. Surface roughness
and contact angles of the specimens were measured. The surface morphology was
observed with scanning electron microscopy (SEM). An adhesion test was performed
by incubating the disk specimens in C. albicans suspensions at 37°C for 2 hours, and
the adherent cells were counted under an optical microscope. The data were analyzed
statistically using a variance analysis and Tukey HSD post hoc comparison test. The
correlation between measurements was tested using a Pearson correlation analysis
(α = 0.05).
Results: CAD/CAM polymers generally showed statistically significant lowest Ra
and contact angle values, whereas conventional PMMA showed the highest Ra and
contact angle values in the uncoated group (p < 0.05). Pellicle coating essentially
increased contact angle of all materials and reduced the differences in a number of
Candida cells on the materials (p < 0.05). Candida adhesion was statistically signif-
icantly greatest on conventional PMMA when compared to CAD/CAM polymers. A
strong positive correlation was found between the surface roughness of the specimens
(p < 0.05) and the amount of adhered cells, whereas no correlation was found between
hydrophobicity of the specimens and the amount of adhered cells (p > 0.05).
Conclusions: CAD/CAM PMMA-based polymers may be preferable to reduce
Candida-associated denture stomatitis in long-term use.

Contemporary technological advancements in digital den-
tistry have enabled the introduction of new materials that
improve mechanical and biological characteristics for clin-
ical usage. Recently, in order to produce digital complete
dentures with computer-assisted design/computer-assisted
manufacturing (CAD/CAM), poly(methyl methacrylate)
(PMMA)-based polymers were presented by manufacturers
as an alternative to conventional heat-polymerized PMMA.1-7
CAD/CAM PMMA-based materials have different chem-
ical structures, mechanical properties, and polymerization
techniques. Highly crossed-linked PMMA-based polymers
are industrially polymerized under standardized conditions
at high temperature and pressure. This produces more bio-
hygienic materials with lower porosity and fewer residual
monomers.8-10
Prevention of the accumulation of microorganisms on pros-
thetic materials preserves the health of the oral tissues. It is
known that microorganisms are more prone to accumulate
on restorations when compared to natural tissues. Therefore,
dental materials should be resistant to adhesion to reduce
plaque accumulation.11 Denture stomatitis is an erythematous
pathogenic condition of the denture-bearing mucosa, which
is caused primarily by microbial factors, particularly Candida
albicans. Studies have found that the fitting surface of the max-
illary denture is the main reservoir of C. albicans and related
Candida species.12,13
The presence of saliva, serum, and other microorganisms,
as well as differences in surface topography and physicochem-
ical properties of the polymers, may also contribute to their
adhesion, further complicating the adherence process.13-17 Nu-
merous studies have investigated the relationship between a ma-
terial’s surface roughness and Candida colonization; however,
few studies have assessed the interactions between saliva pro-
teins, C. albicans, and the surface properties of polymers.18-22

Journal of Prosthodontics 0 (2018) 1–7 

C 2018 by the American College of Prosthodontists 1
Adhesion of C. albicans on CAD/CAM PMMA-Based Polymers
Preliminary research found no published studies on the ad-
hesion of C. albicans to CAD/CAM PMMA-based polymers
used for digital complete dentures. Therefore, this study aimed
to investigate the correlation between surface parameters and
C. albicans adhesion and to evaluate initial adhesion of C. al-
bicans to artificially aged CAD/CAM PMMA-based polymers
in the absence and presence of a salivary pellicle.
The null hypotheses were (1) the amount of adherent C.
albicans on CAD/CAM PMMA-based polymers is not differ-
ent from conventional heat-polymerized PMMA, (2) pellicle
coated with saliva does not affect C. albicans adhesion on
denture base resins, and (3) the surface roughness and hy-
drophobicity of CAD/CAM PMMA-based polymers do not
affect Candida adhesion.
Materials and methods
Three commercially available CAD/CAM PMMA-based poly-
mers (M-PM-disc [M]; Pink CAD/CAM disc [P]; AvaDent
Puck Disc [A]), and one conventional heat-polymerized PMMA
(Promolux [C]) were assessed in this in vitro study (Table 1).
Forty disc-shaped specimens (10 × 2 mm) were prepared with
each material. All specimens were subjected to 10,000 ther-
mal cycling processes (5-55°C; 30-second dwell times) and
divided into two groups (n = 20): uncoated and pellicle coated.
Ten randomly selected specimens from each group were used
to evaluate the surface roughness and hydrophobicity of the
resins (n = 10 uncoated group; n = 10 coated group).
Specimen preparation
Heat-polymerized acrylic resin was prepared according to the
manufacturer’s instructions and fabricated into disc-shaped
specimens. To prepare CAD/CAM PMMA-based specimens,
the virtual design of discs with the dimensions 10 mm (d) ×
25 mm (h) was completed using a computer program (Yenadent
CAM 5.1; Yenadent Ltd., Istanbul, Turkey) and then con-
verted into a stereolithography (STL) file. Following the milling
process (Yenadent D14; Yenadent Ltd.), CAD/CAM polymer
sheets were sectioned with a precision cutter (Secotom 10
Isomet; Struers A/S, Ballerup, Denmark) to obtain 10 mm
(d) × 2 mm (h) standard specimens for each CAD/CAM
PMMA-based polymer. All were finished with silicon car-
bide abrasive papers (400-, 600-, and 800-grit) using an auto-
matic grinder, and their dimensions were checked with a digital
caliper (IP54; Shan, Guiline, China). All resin specimens were
then stored in distilled water that was exchanged daily for 6 days
prior to the experiments to minimize the influence of residual
monomers or toxic constituents on cell viability. Then all spec-
imens were placed in a thermal cycling apparatus (MTE-101;
Moddental, Esetron Smart Robotecnologies, Ankara, Turkey)
and cycled in distilled water between 5°C and 55°C for 10,000
cycles with a dwell time of 30 seconds at each temperature.
Examination of surface roughness and surface
topography
The surface roughness value (Ra , μm) was determined at three
spots (one at a central position, two at the margins) on each
of the ten specimens from each material using a profilometric
2 Journal of Prosthodontics 0 (2018) 1–7 
Murat et al
contact surface measurement device (Perthometer M2; Mahr,
Göttingen, Germany). The mean value was calculated to obtain
general surface characteristics of the specimens.
To assess the surface topography of the specimens, two spec-
imens were randomly selected from each tested group, gold
sputtered, and subjected to microscopic surface analyses, using
a scanning electron microscope (SEM) (5000× magnification)
(Quanta FEG 400; FEI, Hillsboro, OR).
Saliva collection
To stimulate the influence of a salivary pellicle on surface hy-
drophobicity and Candida adhesion, unstimulated whole saliva
was collected from two healthy volunteers after they had pro-
vided written formal consent, as required by the Local Ethical
Committee for Research (protocol 064/2015). The collected
saliva was purified in a centrifuge (Labofuge 400R; Heraeus
Instruments, Hanau, Germany) at 2400 × g for 15 minutes
and kept at –20°C until use. Specimens were then horizontally
placed in presterilized 24-well plates and incubated at 37°C
with 2 mL of the saliva on an orbital shaker for 2 hours. There-
after, specimens were washed in distilled water to remove the
surplus of salivary fluid and loosely bound proteins.
Contact angle measurement
Surface hydrophobicity of the resin specimens was evaluated
using the contact angle measurement method with an auto-
mated contact angle measurement device equipped with a video
camera and an image analyzer (OCA 15 plus; Dataphysics In-
struments GmbH, Filderstadt, Germany). The contact angle
(degree) was calculated by dispensing a droplet (2 μL) of
distilled water onto the specimens. The angle of the tangent
to the surface of a liquid droplet was measured and repeated
three times on different areas of the surface of each specimen,
and the average contact angle was calculated. Measurements
were performed on ten specimens from each group.
C. albicans adherence assay
The C. albicans ATCC 2091 strain was provided from a lo-
cal laboratory (Istanbul University, Faculty of Medicine, De-
partment of Medical Microbiology). A standard inoculum for
C. albicans biofilm formation was prepared as previously
described.23
Prior to the Candida adhesion experiment, specimens were
cleaned with an ultrasonic cleaner for 15 minutes and then
disinfected with 70% alcohol, and ultrasonically cleaned with
purified sterile water for 20 minutes to remove any contami-
nants and residues from the surface. In the pellicle-coated group
(n = 10), the specimens were immediately used following incu-
bation with the whole saliva at 37°C for 2 hours. Subsequently,
all specimens were incubated in 2 mL of yeast suspension in
24-well plates at 37°C for 2 hours (initial adherence) on an
orbital shaker device. The surfaces were then rinsed by immer-
sion in PBS buffer under constant stirring at 650 to 700 rpm for
10 minutes to remove non-adherent cells. Following the wash-
ing procedure, the adherent cells were fixed with methanol for
20 minutes at room temperature, then washed with distilled
water and stained with sterile methylene blue (Sigma-Aldrich
C 2018 by the American College of Prosthodontists
Murat et al
Table 1 Denture base polymers tested
Material Code Polymer type
Promolux C Conventional PMMA
M-PM-disc M CAD/CAM PMMA-based polymer
Polident, pink CAD/CAM disc P CAD/CAM PMMA-based polymer
AvaDent Puck Disc A CAD/CAM PMMA-based polymer
Co., St. Louis, MO) for microscopic evaluation. The stained
specimens were washed again with phosphate-buffered saline
(0.01 M PBS; pH 7.4), and air-dried for 30 minutes. They
were then kept in sterile experiment cups until microscopic
evaluation.37,38
Adherent yeast cells on each specimen were counted under
light microscopy (Olympus CH2; Olympus Co., Ltd., Tokyo,
Japan) at 40 × 10 magnification by one researcher. Fifteen
fields were counted, and the mean values were calculated. The
results were expressed as cell/field.
Statistical analysis
Data were exported to Minitab Release 15 (Minitab Inc., State
College, PA), a software package for statistical analysis. A
variance analysis was conducted to determine the effects of
material and pellicle coating on surface properties and Can-
dida adhesion and to assess the interaction between the two
factors.
Tukey HSD post hoc test was used to determine differences
in measurements between different denture base materials. The
correlation between measurements was tested using a Pearson
correlation analysis. For all comparisons, statistical signifi-
cance was declared if the p-value was ˂0.05.
Results
One-way ANOVA revealed material type affected surface
roughness. According the Tukey results, there were statistically
significant differences in the surface roughness between the
conventional PMMA and CAD/CAM PMMA-based polymers
(p < 0.05). CAD/CAM PMMA-based polymers had the lowest
Ra values, whereas conventional PMMA-C had the highest Ra
values (p < 0.05; Table 2). No statistically significant differ-
ence was found between the three CAD/CAM PMMA-based
polymers (p > 0.05). Representative SEM images of polymers
used in this study are presented in Figure 1.
The surface hydrophobicity of each denture base material is
shown as the contact angle against water in Table 2. The vari-
ance analysis revealed that both material and pellicle coating in-
fluenced the hydrophobicity (p < 0.05) and that interaction oc-
curred between the two variables (p < 0.05; Fig 2). According to
the Tukey results, in the uncoated group, conventional PMMA-
C had a significantly higher contact angle, and consequently
higher hydrophobicity when compared to CAD/CAM PMMA-
based polymers (p < 0.05); however, no statistically significant
differences were found among CAD/CAM PMMA-based poly-
mers (p > 0.05). In the pellicle-coated group, the contact an-
Journal of Prosthodontics 0 (2018) 1–7 
C 2018 by the American College of Prosthodontists
Adhesion of C. albicans on CAD/CAM PMMA-Based Polymers
Manufacturer
Merz Dental GmbH, Lutjenburg, Germany
Merz Dental GmbH, Lutjenburg, Germany
Polident d.o.o, Draga, Poland
Avadent Global Dental Science LLC, Tilburg, The Netherlands
gle of CAD/CAM PMMA-based polymer P and conventional
PMMA-C was also found to be statistically significantly higher
than those of CAD/CAM PMMA-based M and A polymers
(p < 0.05); however, no statistically significant differences were
found between CAD/CAM PMMA-based M and A polymers
(p > 0.05). Statistical analysis revealed that the coated group
had a significantly higher contact angle than the non-coated
group for all materials assessed (p < 0.05) (Fig 3).
The number of adherent C. albicans on the uncoated and
pellicle-coated specimens for each denture base polymer is
shown in Table 2. Variance analysis revealed that the amount of
Candida adhesion varied depending upon the materials and the
presence of pellicle and that interaction occurred between the
two variables (p < 0.05; Fig 4). On uncoated specimens, Can-
dida adhesion was statistically significantly greatest on con-
ventional PMMA-C, followed by CAD/CAM PMMA-based
A, P, and M polymers, respectively (p < 0.05). CAD/CAM
PMMA-based A showed statistically significantly higher Can-
dida adhesion than CAD/CAM PMMA-based P and M poly-
mers, whereas no statistically significant differences between
CAD/CAM PMMA-based P and M polymers were found. Anal-
ysis of the effect of pellicle coats on initial Candida adhesion
revealed no statistically significant differences between mate-
rials (p > 0.05). Pellicle coats reduced the differences in the
number of Candida cells on the materials (p < 0.05). Further-
more, a strong positive correlation (r = 0.823) between the Ra
values of the materials and the amount of Candida adhesion
on specimens (p < 0.05) was found; however, a weak nega-
tive correlation (r = –0.274) was found between the amount
of Candida adhesion on uncoated or pellicle-coated specimens
and contact angle measurements (p < 0.05).
Discussion
Based on these findings, CAD/CAM PMMA-based poly-
mers showed less initial Candida adhesion than conventional
heat-polymerized PMMA. Therefore, the first null hypothe-
sis, that the amount of adherent C. albicans on CAD/CAM
PMMA-based polymers is not different from conventional heat-
polymerized PMMA, was rejected. Moreover, the second hy-
pothesis, that pellicle coated with saliva does not affect C.
albicans adhesion on denture base resins, was also rejected, as
the pellicle coating with saliva had a significant influence on C.
albicans adhesion and the hydrophobicity of denture base poly-
mers. However, the third hypothesis, that the surface roughness
and hydrophobicity of CAD/CAM PMMA-based polymers do
not affect Candida adhesion, was partially accepted. No signifi-
cant negative correlation between C. albicans adhesion and the
3
Adhesion of C. albicans on CAD/CAM PMMA-Based Polymers Murat et al

Table 2 Materials, mean surface roughness (Ra), contact angle (°), cell count per field results

Material Group Mean Ra μm Mean contact angle (°) Mean cell/field SD Ra μm SD (°) SD cell/field
a a a
C Uncoated 0.34 73.43 279.06 0.06 17.82 3.34
C Pelicle coated 86.51c 24.86d 11.07 5.44
M Uncoated 0.18b 71.31b 18.30b 0.04 6.94 2.39
M Pelicle coated 79.56d 21.16d 5.06 5.90
P Uncoated 0.21b 69.63b 22.44b 0.04 4.85 4.64
P Pelicle coated 86.19c 18.40d 5.82 6.99
A Uncoated 0.20b 69.72b 60.28c 0.05 10.57 5.59
A Pelicle coated 74.98a,e 15.50d 4.28 4.75

Different superscript letters indicate statistical difference inside the respective column p < 0.05.

Figure 1 Surface morphology of polymers (1000× magnification). A: Conventional heat-polymerized PMMA; B: CAD/CAM PMMA-based polymer M;
C: CAD/CAM PMMA-based polymer P; D: CAD/CAM PMMA-based polymer A.

hydrophobicity of denture base polymers was found, whereas
a strong positive correlation between C. albicans adhesion and
the surface roughness of denture base polymers was found.
Several researchers21,24-28 suggested that the surface rough-
ness of the materials is the most important factor affecting
the adhesion of C. albicans. Light and electron microscopy
studies11,24-28 reveal that initial adhesion of microorganisms
generally begins in the pits and fissures of rough surfaces,
which are known to provide protection against shear forces
and provide time for irreversible adhesion of microbial cells to
the surface. In this study, the conventional laboratory polishing
procedure with rubber polishers, abrasives, and pumice stones
was not applied on the specimens to mimic the fitting surface
of dentures. Therefore, the surface roughness values of all resin
materials were higher than the threshold values (Ra = 0.2 μm)
reported by Bollen et al.29 It was also observed that surface
roughness values of CAD/CAM PMMA-based polymers
were significantly lower than conventional heat-polymerized
PMMA. According to our SEM observations, conventional
PMMA exhibited a more porous surface with multi dots and

4 Journal of Prosthodontics 0 (2018) 1–7 

C 2018 by the American College of Prosthodontists
Murat et al
Figure 2 Results of the variance analysis for the contact angle of the
surfaces of polymers. Symbols (*) indicate significant differences (p <
0.05).
Figure 3 Contact angle means of the uncoated and pellicle coated spec-
imens. C: Conventional heat-polymerized PMMA; M: CAD/CAM PMMA-
based polymer M; P: CAD/CAM PMMA-based polymer P; A: CAD/CAM
PMMA-based polymer A.
Figure 4 C. albicans adherence to the uncoated and pellicle coated
specimens. C: Conventional heat-polymerized PMMA; M: CAD/CAM
PMMA-based polymer M; P: CAD/CAM PMMA-based polymer P; A:
CAD/CAM PMMA-based polymer A.
Journal of Prosthodontics 0 (2018) 1–7 
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Adhesion of C. albicans on CAD/CAM PMMA-Based Polymers
surface irregularities than CAD/CAM PMMA-based poly-
mers. Scratch lines resulting from the grinding procedure were
detected on the surface of all resin specimens. This study
found a significant strong positive correlation between the
surface roughness values of resins and the amount of ad-
hered cells on the uncoated specimens consistent with previous
reports; however, our results are in disagreement with other
researchers,17,20,30-32 who have established no correlation be-
tween surface roughness of the resins and C. albicans adhesion.
Surface free energy is another factor affecting initial
microbial adhesion and biofilm formation on material
surfaces.18,21,28,30,33,34 In principle, materials with lower
surface free energy (hydrophobic) exhibit a higher water
contact angle.35 In this study, conventional PMMA-C showed
a significantly higher contact angle, and consequently higher
hydrophobicity when compared to CAD/CAM PMMA-based
polymers in the uncoated groups. This finding disagrees with
specifications provided by the manufacturers. According to
a manufacturer’s report,36 the contact angles of CAD/CAM
PMMA-based polymer A (96.3°) were higher than previously
reported values for conventional heat-polymerized PMMA
(generally ranging between 68.7° and 83.8°).36,37 This may be
attributed to the effect of thermal cycling used in this study. It
is likely that 10,000 thermal cycles reduced the hydrophobicity
of CAD/CAM PMMA-based polymers. In line with this
scenario, Hahnel et al38 reported that the hydrophobicity
of denture base polymers decreased after thermal cycling;
however, unlike Hahnel et al we also speculated that thermal
cycling likely caused an increase in the hydrophobicity of
conventional resin. This phenomenon is likely due to the
large number of thermal cycles performed, which could have
modified the residual components of conventional PMMA,
enhancing its hydrophobic properties.39 In addition, long-term
thermal cycling may also affect the surface roughness of
resin materials, which is related to the surface hydrophobicity
of resins. Nevertheless, the effects of thermal cycling on
surface properties should be critically evaluated in future
research.
Inconsistent results have been reported regarding the effect
of the pellicle on surface hydrophobicity of materials. Some
researchers,37,40,41 have established that surface hydrophobic-
ity of materials decreases after pellicle coating with saliva. This
study’s findings are consistent with those of Cavalcanti et al,22
Sipahi et al,35 and Gocke et al,42 who found that the hydropho-
bicity of specimens significantly increased due to the presence
of saliva; however, the hydrophobicity changes reported in these
previous studies were likely related to different pellicle types
on the denture base polymers.22
Contradictory reports have been published regarding the role
of surface free energy of materials on the degree of microor-
ganism adhesion. Authors of several studies14,17,21,28,34,41 have
revealed that higher Candida adhesion was observed on hy-
drophilic surfaces with higher surface energy. Additionally, sev-
eral researchers17,19,42 have reported that an increase in the sur-
face free energy of materials resulted in a decrease in Candida
adhesion; however, in our study, surface hydrophobicity seemed
to have no direct influence on the adhesion of C. albicans, which
is in agreement with other studies16,27,28,32,38,44 that found no
clear relationship between hydrophobicity and the number of
5
Adhesion of C. albicans on CAD/CAM PMMA-Based Polymers
adhered yeast cells. Therefore, it is possible that the adsorption
of proteins has a homogenizing effect and masks originally
distinct differences in substratum surface properties,38 which
could explain the lack of a relationship between surface prop-
erties and biofilm formation.
According to our findings, a significant decrease in the
amount of Candida adhesion to pellicle-coated specimens was
demonstrated, in agreement with several studies.13,17,21,28,39,43
Reducing adhesion of C. albicans to pellicle-coated specimens
could be due to the anti-Candida salivary components that
whole saliva contains, such as secretory immunoglobulin A
(sIgA), which provides an inhibitory effect on the adherence of
C. albicans cells to material surfaces.45 Inconsistent with our
results, Nikawa et al44 showed increased adherence rates with
saliva coating. Further investigation is needed to understand
the effects of the surface properties of the material on free sur-
face energy and the absorption of the saliva proteins along with
surface properties of C. albicans.
Finally, it should be remembered that the adherence mecha-
nism is a complex and multifactorial process, and is thus dif-
ficult to simulate through in vitro conditions. We aimed to
compare the effect of different denture base materials on the
adhesion of C. Albicans leading to Candida-associated denture
stomatitis. C. albicans biofilm formation is affected by the pres-
ence of other microorganisms in the dynamic environment of
the oral cavity. It should also be noted that the design of the
present in vitro study included the formation of single-species
Candida. Further investigation on multispecies biofilms and
saliva-coating conditions is needed. It would be also more con-
tributive to the literature to repeat the work with clinical isolates
and to perform further tests under in vivo conditions to increase
the clinical relevance of the research. In this study, we observed
the initial attachment of cells after a 2-hour incubation period
and obviously, the findings will not necessarily relate to a long-
term study where biofilm formation occurs; however, initial
attachments are best monitored over a short period, enabling
cell-substratum interactions to be visualized and recorded.
Thereby, affinity or tendency of biomaterials to biofilm forma-
tion prior to long-term clinical usage could be easily assessed in
vitro.
Conclusions
Within the limitations of this in vitro study, it can be concluded
that:
1. CAD/CAM PMMA-based polymers showed less surface
roughness, hydrophobicity, and Candida adhesion when
compared to conventional heat-polymerized PMMA in
uncoated groups.
2. Pellicle coating increased the hydrophobicity of all the
materials tested and decreased the differences in the num-
ber of Candida cells on the materials.
3. Surface roughness influenced adhesion, as Candida ad-
hesion was greatest on conventional PMMA-C, which
showed the highest surface roughness value, whereas
surface hydrophobicity did not have a significant impact
on Candida adhesion.
6 Journal of Prosthodontics 0 (2018) 1–7 
Murat et al
Acknowledgment
The authors are grateful to Tümer Arıtürk of SPAC Consulting,
Ankara, Turkey, for statistical analysis.
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