Or i gi nal ar t i cl e

Candida albicans adhesion on reinforced polymethylmethacrylate

denture resin: effect of bre architecture and exposure to saliva
Buket Akaln-Evren
1
, Yasemin Kulak-O

zkan
1
, Mutlu O

zcan
2
and Tanju Kadir
3
1
Department of Prosthodontics, Faculty of Dentistry, University of Marmara, Istanbul, Turkey;
2
Center for Dental and Oral Medicine, Clinic
for Fixed and Removable Prosthodontics and Dental Materials Science, Dental Materials Unit, University of Zurich, Zurich, Switzerland;
3
Department of Microbiology, Faculty of Dentistry, University of Marmara, Istanbul, Turkey
Gerodontology 2012; doi: 10.1111/ger.12024
Candida albicans adhesion on reinforced polymethylmethacrylate denture resin: effect of bre
architecture and exposure to saliva
Background and objectives: Fiber-reinforced composites (FRC) are used to reinforce the prosthetic
and restorative appliances. This may result in the exposure of the FRCs which may affect the adherence
of microorganisms. This study evaluated the adhesion of Candida albicans to a denture base resin
(N = 48), reinforced with E-glass FRCs with different architectures [unidirectional (n = 16) and woven
(n = 16)], when exposed to either saliva or distilled water.
Materials and methods: Specimens without FRC reinforcement served as control (n = 16). After ber
surfaces were exposed, half of the specimens (n = 8/per group) were treated with saliva, the other half
was stored in distilled water prior to C. albicans adhesion. The adhered microorganisms were counted
under an optical microscope and the data were analyzed.
Results: When exposed to distilled water or saliva, specimens with unidirectional (5403.4 cells/cm
2
and
5013.4 cells/cm
2
, respectively) (p = 0.202), woven bers (4053.5 cells/cm
2
and 3726.6 cells/cm
2
, respec-
tively) (p = 0.283) and specimens without bers (2250 cells/cm
2
and 2006.8 cells/cm
2
, respectively)
(p = 0.423) showed C. albicans adhesion, being not signicant. In general, all the specimens exposed to
saliva showed 3582.2 cells/cm
2
C. albicans adhesion, while specimens exposed to distilled water showed
3902 cells/cm
2
C. albicans adhesion, yet being not signicant (p = 0.436). Regarding ber type, C. albicans
adhesion was signicantly affected by the exposed FRC architecture with more favorable results for
woven bers (p < 0.001).
Conclusion: Polymethylmethacrylate without FRCs collected less C. albicans. The presence of saliva
seems to reduce the adhesion.
Keywords: Acrylic resin, Candida, Saliva
Accepted 10 October 2012
Introduction
Since its introduction in 1937, polymethylmethac-
rylate (PMMA) has been the most commonly
used denture base material because of its easy
handling and low cost
1
. Despite their popularity,
the mechanical strength of denture resins is not
sufcient
2
. The longevity of the removable den-
tures is highly affected either due to fractures
3,4
or microbial colonisation
5,6
.
One approach to reinforce such materials is
the incorporation of the bre-reinforced compos-
ites (FRC) during processing dentures
7,8
. The use
of FRC materials became a routine application in
frameworks of anterior or posterior direct and
indirect xed-dental-prosthesis (FDP)
9,10
, posts
11
,
periodontal splints
12
and removable dentures
13
.
All the commonly used FRC types in clinical
dentistry are carbon, aramid, ultra-high-molecu-
lar weight polyethylene and R-, S- or E-glass
14
18
. Among these FRC types, glass bres are gen-
erally preferred for reinforcement of dentures
because of their superior biological properties
and the possibility of their silanization and
impregnation with resin monomers
19
. Preim-
pregnation of glass bres particularly increases
2012 The Gerodontology Society and John Wiley & Sons A/S 1
wettability and adhesion of the relatively high-
viscose denture base resins
20,21
.
Candida albicans is the most common virulent
opportunistic fungal pathogen in the oral cavity,
and therefore, in the case of removable dentures,
it is considered as the primary microbial factor
playing a role in denture stomatitis
2225
. The inci-
dence of denture stomatitis has been shown to
range between 27 and 65% in clinical stud-
ies
22,23,25
. The ability of C. albicans adhesion to
polymeric surfaces is usually as a consequence
of London, van der Waals and electrostatic
forces
26,27
. In fact, these forces are only important
in the initial adherence phase of the yeasts. The
presence of saliva, serum and other microorgan-
isms as well as differences in surface topography
of the polymers may further contribute to their
adhesion that surely makes the adherence process
more complicated
2830
.
In clinical applications, FRC materials are placed
at the tensile side of the dentures or FDPs, and
they are covered with either particulate ller
composites or denture base polymers. During
placement of such reconstructions in the mouth,
usually adjustments are needed that may result in
the exposure of the FRCs to oral mucosa, saliva
and microorganisms
27,31,32
. Adhesion of C. albicans
and other yeasts on denture base polymers has
been widely studied in dentistry
30,33,34
. Reinforce-
ment of denture bases with FRCs having semi-
interpenetrating polymer matrix (IPN) has been
studied in vitro and clinically proven to perform
well from the mechanical aspect
13,30
. However,
information on the microbiological perspective to
FRC reinforced denture resins is scarce
27,31,32
.
Fibre-reinforced composite materials are avail-
able in different architectures namely, unidirec-
tional, woven (bidirectional), prepegs or bre mat.
When total reinforcement is needed for the remov-
able dentures, the entire denture base is reinforced
with woven FRCs but for partial reinforcement,
only the weakest part of the PMMA is reinforced
using unidirectional FRCs
35,36
. Hence, both types
of materials are indicated. It can be hypothesised
that due to larger surface area covering the PMMA,
woven FRCs in the form of a net would lead to
more microbial adhesion when compared with
unidirectional ones. The objectives of this in vitro
study therefore, were (i) to investigate the adhe-
sion of C. albicans to a heat-polymerised denture
base resin, reinforced with E-glass FRCs having dif-
ferent architectures and (ii) to study the effect of
contamination with either distilled water or saliva
on the adherence levels.
Materials and methods
Specimen preparation
The brand names, abbreviations, types, chemical
compositions, batch numbers and manufacturers
of the materials used for the experiments are
listed in Table 1. A heat-polymerised denture base
resin (Paladent 20, Heraeus Kulzer GmbH&Co.,
Hanau, Germany) was reinforced using either
unidirectional (everStick C&B, StickTech Ltd,
Turku, Finland) or woven (everStick Net, Stick-
Tech Ltd, Turku, Finland) E-glass FRCs.
Pink modelling wax (N = 48, n = 16 per group),
(10 9 10 9 1 mm) (Dental Wax, Cavex Set Up
Modelling Regular, Cavex, Harleem, the Nether-
lands) was placed in dental plaster (Moldano, He-
raeus Kulzer GmbH&Co., Hanau, Germany) in a
two-part mould using a standard dental ask. The
moulds were prepared in such a manner that both
parts of the moulds were lled with vacuum mixed
hard dental plaster under vibration (Bego GmbH &
Co., Bremen, Germany). After the boil out process,
the wax was eliminated under running hot water,
and plaster surfaces were sealed with two coats of
Table 1 Materials tested in the study.
Brand name and
Abbreviations Type Chemical composition Batch number Manufacturer
Paladent 20
(PMMA)
Heat-polymerised
polymethylmethacrylate
Powder: Polymethylmethaxrylate
Liquid: Methylmethacrylate,
dimethacrylate
A1333B-2
012121
Heraeus Kulzer
GmbH&Co.,
Hanau, Germany
everStick
C&B (U)
Unidirectional
E-glass bre
PMMA, bis-GMA impregnated,
silanised E-glass bres
2031119-EK-027 StickTech Ltd.
Turku, Finland
everStick
Net (W)
Bidirectional
(woven)
E-glass bre
PMMA, bis-GMA impregnated,
silanised bidirectional weave
E-glass bre
2031119-EK-027 StickTech Ltd.
Turku, Finland
PMMA, Polymethylmethacrylate; bis-GMA, Bisphenol A diglycidylmethacrylate; TEGDMA, Triethyleneglycol
dimethacrylate.
2012 The Gerodontology Society and John Wiley & Sons A/S
2 B. Akaln-Evren et al.
isolation medium (Isolant CMS, Dentsply, Surrey,
UK) using a clean brush each time.
Specimen preparation with FRCs
The unidirectional E-glass FRC was cut with ster-
ile straight scissors into 10 mm length through
the silicon mould where they were stored. They
were then placed into the spaces in the ask with
special metal hand instruments only. FRC was
ared to cover the 100 mm space. As this FRC
was readily impregnated, no additional adhesive
was applied.
The woven E-glass FRCs were obtained in
square sheets (100 mm) and placed in the same
manner as described above. The denture resin
was placed in the asks and heat-polymerised
according to the manufacturers instructions.
After deasking, the specimens were nished
with a rotary instrument (Kavo, Kavo Dental
GmbH&Co. KG, Biberach, Germany) using a ne
grit cross-cut tungsten carbide bur (Komet, Paris,
France; Batch no. H79EFL), and the plaster side
of all the specimens, where the FRCs were placed,
were roughened using 800- and 2400-grit silicon
carbide abrasive papers (3M ESPE, St Paul, MN,
USA) in sequence under water cooling until the
bre-rich regions were exposed. As all of the
bres (both unidirectional and woven) were
placed on the plaster side of the specimens, they
were easily exposed after a few abrasions and
could be seen clearly with naked eye. Before
treating with saliva, surface roughness should be
measured to make sure that all specimen surfaces
had similar roughness. This is one of the limita-
tions of our study. All test specimens were stored
in distilled water for 24 h at 37C. They were
then rinsed in 70% ethanol for 5 min in sterile
glass beakers and placed in phosphate-buffered
saline (PBS) solution (pH 7.3) (Delta Medical and
Chemical Materials Trading, Aurora, IL, USA)
prior to treating with saliva. The specimens were
randomly divided into two groups. While half of
each group was kept in distilled water, the other
half was treated with saliva.
Collection of saliva
Unstimulated mixed saliva was collected from ve
healthy individuals (aged between 30 and 40, and
having 2832 teeth) using a collection cup. The
donors had not taken any medication, and they had
no active periodontal disease or active caries. The
collected saliva was puried in centrifuge (Function
Line, Labofuge 400R, Heraeus Instruments, Hanau,
Germany) at 2400 g for 15 min and kept at 20C
prior to the experiment. Specimens were pretreated
with the saliva solution at room temperature for
10 min to obtain a pellicle on the surface.
Preparation of the Candida albicans
Candida albicans strain was obtained (Istanbul Uni-
versity, Faculty of Medicine, Department of
Microbiology) and incubated on Sabouraud dex-
trose agar slope at 37C for 48 h. The culture was
centrifuged (Function Line, Labofuge 400R) at
1750 g for 10 min, and the resultant cell pellet
was washed twice with PBS solution. Finally, a
yeast suspension of approximately 10
6
C. albicans
per ml was prepared
37,38
.
Contamination procedure
Specimens (n = 8) from each group were placed
into sterile experiment cups that were lled with
30 ml C. albicans suspension and incubated at 37
C for 1 h. They were then washed with PBS solu-
tion to remove the non-adherent cells. Following
the washing procedure, the specimens were placed
in methanol for the xation of the adherent cells
and dyed with sterile methylene blue for the
microscopical evaluations. The dyed specimens
were washed again with PBS, allowed to dry in air
for 30 min. They were then kept is sterile experi-
ment cups until microscopical evaluation
37,38
.
Microscopical evaluation
C. albicans contaminated specimens were examined
under an optical microscope (Olympus CH2,
Olympus Optical Co., Ltd., Tokyo, Japan) at a
magnication of 9100. Number of cells was
counted at 30 microscopical areas (1 microscope
area = 0.25 mm)
38
by one researcher. For each
specimen in total an area of 7.5 mm (30 9
0.25 mm) was examined. The total number of
cells (C) on each specimen (10 mm 9
10 mm = 100 mm = 1 cm
2
) was calculated by
the following formula:
C
100mm
2
number of cells in 7:5mm
2
7:5mm
2
Statistical analysis
The statistical analysis was performed with the
SPSS software package (version 11.5; SPSS,
Chicago, IL, USA). Mean ranks obtained from
adherence assay were evaluated with two-way
2012 The Gerodontology Society and John Wiley & Sons A/S
Candida albicans adherence on ber-reinforced PMMA 3
ANOVA. As signicant differences were found
between or within groups, Least Signicant Dif-
ference (LSD) post hoc tests were used to deter-
mine the differences. Furthermore, one-way
ANOVA was used to distinguish the differences
between distilled water and saliva-treated groups.
In all comparisons, statistical signicance was
declared if the p-value was <0.05.
Results
Fibre-reinforced composite architecture showed a
signicant (p < 0.001) inuence on C. albicans
adhesion on the specimens (ANOVA, LSD) but the
contamination media did not (p = 0.072) (Table 2).
All of the specimens showed C. albicans adhesion at
varying levels. Mean ranks of C. albicans adherence
(number of adhered cells/cm
2
) per group are pre-
sented in Table 3.
Unidirectional FRC reinforced specimens showed
signicantly more C. albicans adhesion (5208.37
cells/cm
2
) than those with woven FRC (3890.06
cells/cm
2
) (p < 0.001).
When exposed to distilled water or saliva, speci-
mens with unidirectional (5403.4 cells/cm
2
and
5013.4 cells/cm
2
, respectively) (p = 0.202), woven
bres (4053.5 and 3726.6 cells/cm
2
, respectively)
(p = 0.283) and specimens without bres (2250
and 2006.8 cells/cm
2
, respectively) (p = 0.423)
showed C. albicans adhesion, being not signicant.
In general, all the specimens exposed to saliva
showed 3582.2 cells/cm
2
C. albicans adhesion,
while specimens exposed to distilled water
showed 3902 cells/cm
2
C. albicans adhesion, yet
being not signicant (p = 0.436).
Optical microscope images showed rougher sur-
faces with unidirectional FRC than with woven
ones. C. albicans adhesion was often in clusters
spread on the surface (Fig. 1ac).
Discussion
Adhesion and colonisation of microorganisms is
the primary step in the development of common
oral infectious diseases such as denture stomatitis.
Therefore, C. albicans, being the most common
virulent opportunistic fungal pathogen in the oral
cavity, that is often associated with denture sto-
matitis
5,22
, was chosen as a model organism for
this study.
Although a great plethora of FRC materials are
available, they all present some limitations com-
pared with E- or S- and R-glass ones with which
good adhesion to matrix polymers could be
achieved
8
. One of the major problems in using
FRCs for fabricating dentures is the poor impreg-
nation of bres with the dough of the denture
base polymer
19
. Poorly impregnated regions
increase the water absorption, thereby reducing
the mechanical properties of the bre-reinforced
structure
19,21
. Another problem is with the impro-
per dispersion of FRCs through the dough that
leads to latent discoloration due to penetration of
oral uids and microorganisms into the voids
15
.
The use of preimpregnated FRCs with adhesive
resins could overcome this problem, while at the
same time, higher exural strength and higher
bre volume could be achieved
20
. For these rea-
sons, a preimpregnated E-glass FRC was studied.
When the effect of FRC architecture (unidirec-
tional versus bidirectional/woven) is evaluated
on the adherence of C. albicans, specimens with
unidirectional bres showed signicantly more
C. albicans adhesion than with woven ones.
Although there were studies comparing the trans-
verse and fatigue strength of unidirectional and
woven bres
16,18
, there is no study on the colonisa-
tion of yeasts on bres with different architectures.
Table 2 Results of analysis of variance for the experi-
mental conditions.
Source DF SS MS F value p-value*
Fibre type 2 76415417 38207708 105546 0.0000
Storage
medium
1 1229120 1229120 3395 0.072
Interaction 2 43351 21675 0060 0.942
Residual 42 15204049 362001
Total 48 765112305
*p < 0.05.
Table 3 Mean ranks of Candida albicans adherence
(number of adhered cells/cm
2
) on the specimens.
Experimental
groups
Contamination medium
Distilled Water
(n = 8/per group)
Saliva
(n = 8/per group)
PMMA
(n = 16)
2250 (417.5) 2006.8 (427.2)
PMMA-U
(n = 16)*
5403.4 (803.3) 5013.4 (637.2)
PMMA-W
(n = 16)*
4053.5 (676.9) 3726.6 (553)
Total
(n = 48)
3902 (632.6) 3582.2 (539.1)
*The indicated groups show signicant differences
between the concerned groups (p > 0.001).
2012 The Gerodontology Society and John Wiley & Sons A/S
4 B. Akaln-Evren et al.
Unidirectional and woven FRC materials used in
this study have the same composition with differ-
ent congurations. Apart from architecture, unidi-
rectional single FRC materials are in larger
diameters (12 lm) than the woven ones (5 lm)
18
.
Therefore, the higher adherence of C. albicans to
unidirectional bres in comparison with woven
bres could be attributed to the mechanical reten-
tion due to the increased surface area and thereby
roughness of the bre yarns tested. Thus, the
hypothesis was rejected. Microscopic examinations
also supported this nding.
Material surface properties, such as surface
roughness
34
, surface free energy
31
and hydropho-
bicity
39
of the surface have been also shown to
inuence adhesion of microorganisms. Surface
roughness directly inuences microorganisms ini-
tial adherence to surfaces, biolm development
and Candida species colonisation. Materials with
the roughest surface usually exhibit higher yeast
counts
34,37,40
. Quirynen et al.
41
postulated a
threshold roughness value (0.2 lm) below which
no effect on the adhesion should be expected.
Smooth and highly polished surfaces are important
not only for patient comfort but also for denture
longevity, good aesthetical results and low plaque
retention. On the other hand, high-energy surfaces
are reported to collect more plaque than low
energy ones
29,32
. Dental restorative materials are
described as high-energy materials when their sur-
face energy is more than 50 mN/m. The term low
energy materials is used to describe substances that
are softer and that have low melting points and
weak intermolecular forces (i.e. waxes and poly-
mers). Materials such as PMMA have a surface
energy value of 41 mN/m. Surfaces with high-sur-
face-free energy are postulated to attract more
microorganisms than surfaces with low surface free
energy
29,31
. In a study conducted by Minagi et al.
33
,
the adherence of C. albicans and C. tropicalis to 21
different acrylic resin materials concluded that with
the increase in the surface free energy, the micro-
organism adherence increased. On the other hand,
less C. albicans adherence was observed on the
hydrophobic materials.
Waltimo et al.
27
studied the adherence of yeast
cells to the surface of an auto-polymerised denture
base polymer reinforced with unidirectional E-glass
FRCs. They found that the mean number of adher-
ent yeast cells on the surface of the polymer matrix
was signicantly higher than that on the surface of
glass bres. The number of adherent yeast cells
found at the interface between the bres and poly-
mer matrix was found to be high. The authors con-
cluded that if bres are exposed only during
polishing, the FRC material appears not to increase
the adherence but areas with permanently exposed
bres may provide mechanical retention for yeast
cells at the interface of the components. From this
conclusion, it was not clear whether the FRCs were
completely exposed. In this study, the whole FRC
surface was exposed. Although a heat-polymerised
PMMA was used and not parotid saliva only, as in
the study of Waltimo et al.
27
, all specimens exclu-
sively showed C. albicans adhesion after contamina-
tion with this microorganism. Moreover, FRC
reinforced specimens showed more C. albicans
adhesion than PMMA alone. The number of
C. albicans at the bre-PMMA interface in this
study was higher than it was reported by Waltimo
et al.
27
even though the saliva incubation time was
the same.
The presence of saliva, serum and other micro-
organisms on the surface may also affect the
C. albicans adhesion process
27,28
. Different opin-
ions exist in the dental literature on the effect of
saliva on C. albicans adhesion: several investigators
reported that a saliva coating reduces the adherence
of C. albicans in acrylic resin based materials, oth-
ers showed increased adherence rates with saliva
(a) (b) (c)
Figure 1 (a-c) Optical microscope images (9100 original magnication) of Candida albicans adherence on (a) PMMA
only, (b) PMMA with unidirectional Fibre-reinforced composites (FRC) exposure. Note the rough surface and the
C. albicans adherence in clusters spread on the surface and the larger diameters of unidirectional FRC compared with
woven FRC, (c) PMMA with woven FRC exposure. Note the narrower diameters of woven FRC when compared
with unidirectional one.
2012 The Gerodontology Society and John Wiley & Sons A/S
Candida albicans adherence on ber-reinforced PMMA 5
coating and some research groups found no effect
of a saliva coating
40,4247
. Consequently, apart
from surface properties of the materials tested,
this aspect was also considered in this study. An
acquired salivary pellicle is always present on the
denture surfaces in vivo. This was tried to be simu-
lated by exposing the specimens to saliva for 1 h.
In the present study, although not statistically sig-
nicant, less C. albicans adhesion was observed on
the specimens with saliva coating than with dis-
tilled water. These ndings are not in agreement
with several authors, stating that pellicle coating
generally results in reduced numbers of adhering
bacteria where similar number of specimens were
used
30
. However, denture-wearing subjects suffer-
ing from xerostomia or those who are on medica-
tion and eventually have less salivary ow may
be more prone to C. albicans accumulation. There-
fore, ideal saliva coating cannot be assured in
every individual.
In an in vitro study, Tanner et al.
31
studied the
adherence of S. mutans on E-glass FRC reinforced
resin composite, denture base polymer and four
other restorative materials with and without expo-
sure to saliva. In that study again, only parotid
saliva was used and glass surfaces were the control
substrates. Their results showed that saliva coating
resulted in decreased adherence of S. mutans on all
materials except glass. In the acquired pellicle,
high-molecular weight glycoproteins (agglutinins)
were thought to be the primary promoters of
S. mutans adhesion to glass surface. In the presence
of this protein, S. mutans showed more adherence
to hard surfaces such as glass. In other studies, it
has been reported that submandibular and sublin-
gual saliva support the C. albicans adhesion due to
the musin content, while parotid and mixed saliva
reduce the adhesion related to high-molecular
weight proteins
42,43
. Although saliva from differ-
ent origins (submandibular, sublingual and paro-
tid) was used in some studies investigating the
effect of saliva on C. albicans adherence on acrylic
surfaces, the most preferred type of saliva was
unstimulated mixed saliva
4755
. So, in the present
study, unstimulated mixed saliva was used for a
better clinical approximation where all salivary
glands function simultaneously. In studies where
unstimulated mixed saliva was used, Samarana-
yake et al.
48
, Waters et al.
50
, Maza et al.
50
, McCour-
tie et al.
53
and Bosch et al.
54
observed reduced
adherence of C. albicans, Nikawa et al.
55
reported
increased adherence rates and Nikawa et al.
49
and
Jin et al.
52
observed no effect. In the present study,
the specimens exposed to saliva showed less
C. albicans adhesion, yet being not signicant. A
possible explanation for this contradictory effect
has been proposed by Elguezabal et al.
47
when
studying the adhesion of C. albicans to polym-
ethylmethacrylate, as mixed saliva decreased or
enhanced the adhesion of C. albicans to polymeth-
ylmethacrylate depending on the morphological
phase of C. albicans. Thus, it is possible that mixed
saliva plays a dual role on the adhesion of C. albi-
cans to plastic materials used to make dental pros-
thesis, decreasing adhesion of germ tubes and
enhancing the adhesion of yeast cells.
There are some limitations of this study. As sur-
face roughness directly inuences microorganisms
initial adherence to surfaces, biolm formation
and Candida species colonisation, surface rough-
ness of the specimens should be measured to make
sure that all surfaces had similar roughness prior
to the contamination procedure. This could be one
of the reasons why we had these specic outcomes
regarding adherence of Candida albicans to polym-
ethylmethacrylate. Furthermore, individual effect
of saliva from different origins and the effect of
denture cleaning regimens should be involved in
the study. Nevertheless, the results of this study
indicate that increased C. albicans adhesion could
be expected mainly dominated by the bre rough-
ness when PMMA is reinforced with FRC. There-
fore, from the clinical point of view, care should
be exercised in order not to expose the FRC mate-
rials during tting procedures of dentures, and if
the bres are exposed, the prosthetic devices
should be replaced. As the initial adhesion of Can-
dida species is inuenced by surface roughness
and surface free energy, these characteristics
should also be evaluated in in vivo-like conditions.
Conclusions
From this study, the following could be con-
cluded:
1. All specimens showed C. albicans adhesion
but PMMA without FRC exposure collected
signicantly less C. albicans.
2. C. albicans adhesion was signicantly affected
by the exposed FRC architecture with more
favourable results for woven bres than those
with unidirectional ones.
Acknowledgements
This investigation was supported by Scientic
Research Project Committee of Marmara Univer-
sity, Project No: SAG-094/081004.
2012 The Gerodontology Society and John Wiley & Sons A/S
6 B. Akaln-Evren et al.
Conict of Interest
The authors declare that they have no conict of
interest.
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Corresponding to:
Dr Buket Akaln-Evren, Faculty of Dentistry,
Department of Prosthodontics, University of Mar-
mara, Gu zelbahce Bu yu kciftlik Sok. No:6, 80200
Nisantas, Istanbul, Turkey.
Tel: +90 0 212 2319120
Fax: +90 0 212 2465247
E-mail: buketakalin@hotmail.com
2012 The Gerodontology Society and John Wiley & Sons A/S
8 B. Akaln-Evren et al.