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| Smile Dental Journal | Volume 6, Issue 4 - 2011
Abstract
This study investigated adherence of Candida albicans to denture base acrylic resins
and denture soft liners with varying surface roughness.
Materials and Methods: Two denture base acrylic resins (heat cured resin and cold
cured resin) and two commercial soft liners (one is heat polymerized acrylic resin
based and one is room temperature polymerized silicon based) having dimensions of
10X10X1.5mm for each specimen. Each material was divided into two groups: one is
processed against glass slide surface and the other is processed against dental stone
(10 samples for each group). Surface roughness measurements were made using a
profilometer where a stylus traverses across the layer of the surface. Human saliva
was collected from volunteers and the specimens were stored in human saliva which
was contaminated with yeast suspension of approximately 106 Candida albicans per
milliliter and incubated for 24hrs at 37C. After incubation, fixation of the attached
cells was done by treating the specimens with 100% ethanol for 3s and left to dry in
sterile plates. Specimens were stained using sterilized, fixated Methylene Blue stain for
1min and subsequently evaluated under optical microscope (Olympus, Japan) at X400
magnification. Visible measurement field was calculated in mm
2
and the obtained data
were expressed in cell/mm
2
.
Results: The materials processed against glass surface showed a very high significant
difference in surface roughness values than those processed against dental stone
surface (student t test, P<0.0001). The adherence of Candida albicans to rough
surface was very highly significant than the adhesion on smooth surface (student t
test, P<0.0001). There was no statistically significant difference in Candida albicans
adhesion among the materials processed against glass surface (One-way ANOVA,
P>0.05), while for those processed against dental stone surface, there was a very high
statistically significant difference in adhesion (One-way ANOVA, P<0.001) and there
was no significant difference in Candida albicans adhesion between cold cured denture
base acrylic resin and Heat-polymerized acrylic resin-based resilient liner (LSD test,
P>0.05).
Conclusion: Rough surfaces of the denture base and soft liner promote the adhesion of
Candida albicans in vitro.
Keywords: Candida albicans, Surface roughness, Denture base, Soft liner.
Introduction
The base of complete denture is largely responsible for providing the prosthesis with
retention, stability, and support by being closely adapted to the oral mucosa. The
material most commonly used for denture bases is acrylic resin
1
but on occasions, soft
lining materials are used to provide a cushion between the hard denture base and
supporting tissue.
2
The adherence of Candida albicans to host cells or polymers such
as, denture acrylic resin and soft lining materials is an essential and necessary first
step in successful colonization and the development of pathogenesis and infection.
3

Adhesion of Candida Albicans to Denture
Base and Denture Liners with Different
Surface Roughness
An In-vitro Study
Zahraa Nazar Al-Wahab
BDS, MSc
Lecturer, Department of
Dental Technologies, College
of Health and Medical
Technologies, Foundation of
Technical Education
Baghdad, Iraq
zahraawahab@yahoo.com
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The presence of Candida albicans on the upper fitting
surface of the denture is a major causative factor
in denture-associated chronic atrophic candidosis
(denture stomatitis), the most common form of oral
candidosis.
2
Candida albicans is a dimorphic fungus that
is commensal in the gastrointestinal and reproductive
tracts of healthy individuals. Under certain predisposing
conditions, Candida albicans can convert into a
pathogen capable of causing a variety of oral infections
including pseudomembranous candidiasis, erythematous
candidiasis and hyperplastic candidiasis, as well as
Candida-associated denture stomatitis, Candida
associated angular cheilitis, rhomboid glossitis and
chronic mucocutaneous candidiasis.
4
Denture stomatitis is an erythematous pathogenic
condition of the denture bearing mucosa and is mainly
caused by microbial factors, especially Candida albicans.
5

The etiology is multifactorial consisting of either ill-fitting
prostheses leading to mechanical irritation or poor
hygiene leading to chronic infection, regardless of the
initiating process Candida ablicans is the main cause of
fungal origin in denture stomatitis.
6
The first step implicated in denture stomatitis is adherence
of Candida to acrylic or to salivary pellicles adsorbed on
the surface of dental prosthesis. This is considered the
most important event in the ability of Candida albicans to
colonize dentures in the mouth.
4
The aim of this study is
to assess the ability of Candida albicans adherence to two
types of acrylic resin and two types of soft lining materials
with different surface roughness.
Materials and Methods
Two commercially available denture base acrylic resins
were used, one is heat cured (HC) and the other is
cold cured (CC). Two liners were used, one is heat
polymerized acrylic resin based (V) and the other is room
temperature polymerized silicone based (M). All of these
materials were listed in Table 1.
Preparation of the specimens
Pink modeling wax forms (10X10X1.5) mm were
punched from a sheet of wax. Stone was mixed
according to the manufacturers instruction in the lower
half of the flask. Two types of mold were prepared in
such a manner that in the first type, one part of the mold
was dental stone and the other is glass surface, while
in the second type, both parts of the mold were dental
stone. To produce the specimens against the glass,
a glass microscope slide was pressed onto the stone
mixture in the lower part of the flask. After the stone has
set, wax specimens were placed on the top of the glass
slide surface. The upper part of the flask was placed in
position and the dental stone was poured over the wax
specimens. The flasks were separated and boiled out,
and the cover glass was degreased with liquid detergent.
The surface of the investing dental stone was lubricated
with separating media before packing of the materials.
All the tested materials were processed according to the
manufacturers instructions.
Sample grouping
HC1: Heat cured denture base acrylic resin processed
against glass
CC1: Cold cured denture base acrylic resin processed
against glass
V1: Heat polymerized acrylic resin-based resilient liner
processed against glass
M1: Room temperature polymerized silicone-based
resilient liner processed against glass
HC2: Heat cured denture base acrylic resin processed
against dental stone
CC2: Cold cured denture base acrylic resin processed
against dental stone
V2: Heat polymerized acrylic resin-based resilient liner
processed against dental stone
M2: Room temperature polymerized silicone-based
resilient liner processed against dental stone
Estimation of surface roughness of the specimens
The surface roughness of the specimens was measured
with profilometer (Talysurf 4, Taylor Hasbon, UK), where
a stylus traverses across the layer of the surface. Three
readings were taken for every specimens and the
average was calculated. The average surface roughness
values for all tested specimens are presented in Table 2.

Methods of saliva collection
Whole unstimulated saliva samples were collected and
pooled from 5 healthy male volunteers to eliminate
sample variation,
4
aged 18 22, (mean 20 years). The
Type of Material Trade Name Manufacturer Batch Number
Heat polymerized denture base acrylic resin Major base 2 (HC) Italy
ISO 1567, type I class I ADA
no.12
Room temperature polymerized denture base acrylic resin Major repair 2 (CC) Italy
ISO 1567, type II class I ADA
no.12
Heat-polymerized acrylic resin-based resilient liner Vertex Soft (V)
Vertex-Dental BV, Zeist,
The Netherlands
100001
Room temperature polymerized silicone-based resilient liner Mollosil (M)
Detax, GmbH & Co. KG,
Germany
03008
(Table 1) Materials used in this study
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| Smile Dental Journal | Volume 6, Issue 4 - 2011
saliva was collected between 9.00 and 10.00 am and
the volunteers had not eaten that morning. They were
not taking any drugs or medications known to affect
saliva production, composition, or flow within the last
three months. They were not taking any antibiotics or
antifungal agents.
8,7,4
Saliva was centrifuged at 14000g
for 15min and then it was used immediately.
8
Obtaining Candida albicans
Candida albicans strain ATCC 2091 was obtained as a
stock culture (from Pathological Analysis Department of the
College of Health and Medical Technologies, Baghdad,
Iraq), and incubated on Sabouraud dextrose agar slope
at 37C for 48
h
. Standard amounts of this culture were
inoculated into 2ml of liquid Sabouraud dextrose agar
and incubated at 37C for 24
h
. The culture was then
centrifuged (Function Line, Labofuge 400 R, Hereaus
Instruments, Germany) at 3,000 rpm for 10min and the
resultant cell pellet was washed twice with phosphate-
buffered saline solution (0.15 M, pH 7.3). After dilution
with this solution, a final yeast suspension of approximately
106 Candida albicans per milliliter was prepared.
8
Adherence assay
Specimens were sterilized in autoclave at 121C for
20min. They were then placed in separate sterile tubes
and incubated with 1.5ml natural saliva (pH=7) that
was contaminated with Candida albicans for 24hrs.
9

After incubation, in order to remove the unattached cells,
the specimens were gently removed from the tubes and
rinsed by dipping them into phosphate buffer solution
with 0.89% NaCl at pH 7.2 for approximately 75s.
Then for fixation of the attached cells, the specimens
were treated with 100% ethanol for 3s and left to dry in
sterile plates. Specimens were stained using sterilized,
fixated Methylene Blue stain for 1min and subsequently
evaluated under optical microscope (Olympus, Japan)
at X400 magnification. Visible measurement field was
calculated in mm
2
and the obtained data were expressed
in cell/ mm
2
.
9
(as in Table 3)
Results
Surface roughness measurements
Mean and standard deviation (SD) of surface roughness
values of all tested groups are illustrated in Table 2. The
highest surface roughness value was obtained with M2
(7.480.345), while the lowest value was obtained with
HC1 (1.310.166). In Table 4, student t test compared
the mean surface roughness values of each material
according to the type of surface processing. There was
a very high statistically significant differences between
the groups (P<0.0001), and this indicates that, the type
of surface processing (against glass or dental stone)
significantly affected the surface roughness values of the
tested materials.

Candida albicans adhesion
Mean and standard deviation of Candida albicans
adhesion to the smooth surfaces (materials processed
against glass) and to the rough surfaces (materials
processed against dental stone) for all tested materials
are presented in Table 3. The highest value of adhesion
was observed in M2 (7916.5306.68), while the lowest
value of adhesion was in HC1 (1273.7220.82). In
Table 5, one way ANOVA test compared the Candida
albicans adhesion among materials processed against
glass surface. There was no statistically significant
difference among them (P>0.05).

In Table 6, one way ANOVA test compared the Candida
albicans adhesion among materials processed against
dental stone surface. There was a very high statistically
significant difference among them (P<0.0001). In order
to show where the significance is occurred, Least significant
difference (LSD) test was performed. In Table 7, Least
Mean and SD Group Mean SD Group
2.50.253 HC2 1.31 0.166 HC1
5.59 0.272 CC2 1.570.125 CC1
4.110.272 V2 1.40.221 V1
7.480.345 M2 1.50.290 M1
(Table 2) Mean surface roughness values (Ra) and standard
deviation (SD) of all tested groups (in m)
Mean and SD Group Mean SD Group
3426.7 118.3 HC2
1273.7
220.82
HC1
5371.8223.45 CC2
1580.8
117.46
CC1
5189.9127.87 V2
1588.77
152.12
V1
7916.5306.68 M2
1590.36
452.17
M1
(Table 3) Mean and Standard deviation of Candida albicans
adhesion to all groups in cells/mm
2
Groups t value P value Group
HC1 & HC2 2.9748 P<0.0001 HC1
CC1 & CC2 1.74102 P<0.0001 CC1
V1 & V2 3.00601 P<0.0001 V1
M1 & M2 2.15696 P<0.0001 M1
(Table 4) Studenttest comparing the mean surface roughness
values for each material according to the type of surface processing
P<0.0001: Very high significant
Groups F value P value
Significance
HC1 3.843 P>0.05 NS
CC1
V1
M1
(Table 5) One-way ANOVA test comparing Candida albicans
adhesion among materials processed against glass surface
NS: Non significant
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significant difference test (LSD) revealed that, there is a
very high significant difference in comparing HC2 versus
CC2, or in comparing HC2 versus V2, or in comparing
HC2 versus M2, or in comparing CC2 versus M2, or
in comparing V2 versus M2 (P<0.001); while there is
non significant difference in comparing CC2 versus
V2 (P>0.05). In Table 8, student t test comparing the
Candida albicans adhesion of all groups according to
the type of surface processing. It revealed that there is
a very high significant difference between the Candida
albicans adhesion to smooth surfaces and the adhesion
of Candida albicans to rough surfaces (P<0.0001).
Discussion
Candida albicans can produce biofilms on natural
surfaces, such as teeth, and foreign surfaces, such as
prostheses. These biofilms are normally resistant to
common antimicrobial therapy, an increasing problem
in clinics.
10
Candida species are generally ubiquitous
commensal microorganisms that are part of the normal
mucosa microflora. If, however, the balance of the
normal flora is disrupted or the immune defenses are
compromised, Candida yeasts can invade mucosal
surfaces and cause diseases, such as Candida-
associated denture stomatitis.
7
Saliva is the biologic fluid that bathes all oral surfaces
and acts as a defense against microorganisms present
in the oral cavity,
4
and in this study, the specimens were
saliva coated in order to simulate the oral environment
9

and the exposure of specimens to natural saliva was for
24 hours because, prolong period may lead to possible
cell death.
11
To compare the material performance
in terms of surface roughness and Candida albicans
adherence, the test materials have been processed
on glass surfaces to achieve surfaces as smooth as
possible
12
and in this study, roughness measurements
were undertaken to determine of surface texture of the
tested materials. The surface roughness of a material
used for a removable prosthesis is an important since
it affects, directly or indirectly, retention, staining
resistance, plaque accumulation, as well as oral
tissue health and patients comfort.
9
For all the four
tested materials, There was an adhesion of Candida
albicans on rough surfaces in greater number than
that on smooth surfaces and cells were observed in
surface irregularities after the washing process. This is
in agreement with,
13
a study which stated that, the pits
observed in the surface of tested materials processed
against dental stone are presumably caused by the
crystalline structure of the surface of the stone since these
pits were often filled with yeast cells. In vivo, such surface
defects would provide an ideal protective area for
microorganisms and the potential for a focus from which
outgrowth and infection might proceed.
13
Materials with
rough surfaces make the cleaning of the prosthesis and
mechanical removal of the microorganisms difficult,
also, they cause discoloration of the denture base
materials.
12
Another study
13
stated that the differences
in surface topography affects the attachment of
microorganisms to a surface, with higher number of cells
retained on rough surfaces and surface irregularities
would increase the likelihood of microorganisms
remaining on the surface. This result agrees with.
8,9,12,14-18

A study of
9
found that, during microbial colonization,
cells produce acidic substances as an outcome of their
natural metabolisms that affects the pH of the surface
they can interact with.
In this study, the highest value of adhesion of Candida
albicans was on soft liner molosil processed against
dental stone and the lowest value of adhesion was on
heat cured denture base acrylic resin processed against
glass surface. This agrees with
8
, a study found a greater
adherence of Candida albicans to silicone soft liner than
denture base acrylic and agrees with
12
which explained
this result on the bases of polymerization. Such study
stated that, heat polymerization would increase the cross
linking of residual monomer and the electrochemical
reaction that occurs between Candida albicans and the
resilient silicon liner leads to such adhesion. The study
explained such adhesion on silicon due to the fact that
acrylic resin materials are considered hydrophobic than
silicon based resilient liners and Candida albicans
Significance P-value F-value Group
VHS P<0.0001 82.16
HC2
CC2
V2
M2
(Table 6) One-way ANOVA test comparing Candida albicans
adhesion among materials processed against dental stone surface
VHS: Very high significance
Significance P value
Differences
between means
Group
VHS P<0.001 -1945.1 HC2 & CC2
VHS P<0.001 -1763.2 HC2 & V2
VHS P<0.001 -4489.8 HC2 & M2
NH P>0.05 181.90 CC2 & V2
VHS P<0.001 -2544.7 CC2 & M2
VHS P<0.001 -2726.6 V2 & M2
(Table 7) LSD comparing Candida albicans adhesion among
materials processed against dental stone surface
P<0.0001: Very high significant VHS: Very high significance
t value P value Group
P<0.0001 4.57642 HC1 & HC2
P<0.0001 2.27974 CC1 & CC2
P<0.0001 7.91177 V1 & V2
P<0.0001 2.3467 M1 & M2
(Table 8) Student t-test comparing the Candida albicans adhesion
for each material according to the type of surface processing
P<0.0001: Very high significant
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| Smile Dental Journal | Volume 6, Issue 4 - 2011
adhesion on hydrophobic material was low. This result
also agrees with
16
and
19
, these studies stated that silicon
soft liner are more susceptible to Candida albicans
adhesion than acrylic resin since surface porosity, texture
and biologic and physical / chemical affinity between the
materials and microbial cells may be an important factor.
The results of the present study showed that molosil
soft liner processed against dental stone showed
significantly higher adhesion than vertex soft liner and
this agrees with
20
, a study stated that heat polymerized
soft liner showed lower adhesion than room temperature
polymerized soft liner. This finding is in agreement with
3
,
a study which explained this result due to the presence
of porosities inside the matrix of the room temperature
polymerized material which facilitates the penetration
of blastospores. This finding agrees with
5,3
and
19
.
This finding also agrees with
9
, a study found that the
adherence of Candida albicans on room temperature
polymerized surfaces is related to the polymerization
method of the material tested.
In this study, there was no statistically significant
difference in Candida albicans adhesion between
cold cured denture base acrylic resin (CC2) and heat
polymerized acrylic resin based liner (V2) polymerized
against dental stone surface. This is because the
chemical composition of (V2) is similar to that of the
polymethyl methacrylate of cold cured denture base
acrylic resin polymer.
21

Conclusion
Rough surfaces of the denture base and soft liner
promote the adhesion of Candida albicans in vitro and
the surfaces that are as smooth as possible are more
desirable in terms of cleanability and prevention of
fungal disease. The selection of appropriate material
for a given function and their fabrication may affect the
performance of the material.
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