In Vitro Cytotoxicity of Maxillofacial Silicone

Elastomers: Effect of Accelerated Aging

 ükran Yılmaz2
Bilge Turhan Bal,1 Handan Yılmaz,1 Cemal Aydın,1 Seçil Karakoca,1 S
1
Department of Prosthodontics, Faculty of Dentistry, Gazi University, Ankara, Turkey

2
ap) Institute, Ankara, Turkey
Department of Cell and Virus Bank, Foot and Mouth Disease (S

Received 11 February 2008; revised 6 May 2008; accepted 3 June 2008

Published online 19 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31194

Abstract: The purpose of this in vitro study was to evaluate the cytotoxicity of three
maxillofacial silicone elastomers at 24, 48, and 72 h on L-929 cells and to determine the effect
of accelerated aging on the cytotoxicity of these silicone elastomers. Disc-shaped test samples of
maxillofacial silicone elastomers (Cosmesil, Episil, Multisil) were fabricated according to
manufacturers’ instructions under aseptic conditions. Samples were then divided into three
groups: (1) not aged; (2) aged for 150 h with an accelerated weathering tester; and (3) aged for
300 h. Then the samples were placed in Dulbecco’s Modified Eagle Medium/Ham’s F12
(DMEM/F12) for 24, 48, and 72 h. After the incubation periods, cytotoxicity of the extracts to
cultured fibroblasts (L-929) was measured by MTT assay. The degree of cytotoxicity of each
sample was determined according to the reference value represented by the cells with a control
(culture without sample). Statistical significance was determined by repeated measurement
ANOVA (p < 0.01) followed by Duncan’s test (p < 0.05). All test materials in each group
demonstrated high survival rates in MTT assay (Episil; 93.84%, Multisil; 88.30%, Cosmesil;
87.50%, respectively); however, in all groups, Episil material demonstrated significantly
higher cell survival rate after each of the experimental incubation periods (p < 0.05).
Accelerated aging for 150 and 300 h had no significant effect on the biocompatibility of
maxillofacial silicone elastomers tested (p > 0.05). ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res
Part B: Appl Biomater 89B: 122–126, 2009

Keywords: cell culture; cytotoxicity; maxillofacial; silicone(s)/PDMS

INTRODUCTION
Maxillofacial prostheses are used to transform congenital,
developmental, and acquired defects of the head and neck
into natural-appearing reproductions of the missing parts;
thus, providing an acceptable appearance and improved
function.1 Modern materials for external prostheses include
vinyl plastisols, polymethylmethacrylates, polyurethanes,
latex, and silicone elastomers. Because of the materials’
clinical inertness, strength, durability, and ease of manipu-
lation, silicone elastomers have become the material of
choice for maxillofacial prostheses.2 A wide variety of
maxillofacial silicone elastomers are commercially avail-
able and are clinically used. Some investigators have listed
the qualities and requirements of an ideal maxillofacial ma-
terial.3–5 With regard to ideal quality features (chemical,
physical, or mechanical), the material should be compatible
Correspondence to: Dr. B. T. Bal (e-mail: bilgeturhan@gmail.com)
Contract grant sponsor: Scientific Research Project, Gazi University Rectorate
(GÜ-BAP)
' 2008 Wiley Periodicals, Inc.
122
with human tissues and should cause no irritation. The ma-
terial must not be capable of initiating an inflammatory or
foreign body reaction, and it should be noncarcinogenic.6
Biocompatibility is the ability of a material to elicit an
appropriate biological response in a given application in
the body.7,8 Biological and toxicological properties of den-
tal materials are important in relation to their clinical
usage.9 In the literature various test methods have been
proposed to determine the biocompatibility of dental mate-
rials.6,10–13 The principal ones are cytotoxicity test con-
ducted in vitro on cell or tissue cultures,6,12,14 and
subcutaneous connective tissue or bone implantation meth-
ods in experimental animals.7,13,15 In vitro cytotoxicity tests
are a necessary screening step in the testing of new materi-
als used in humans.9,16 Testing of dental materials by cell
culture methods are relatively simple to perform, reproduci-
ble, and cost-effective, and they can be carefully controlled.
These tests may be more suitable as an alternative to the
costly, controversial animal experiments, which may also
have several uncontrollable variables.8,16,17 In addition,
these tests generally avoid the ethical and legal issues that
surround the use of animals and humans for testing.7 Dif-
 IN VITRO CYTOTOXICITY OF MAXILLOFACIAL SILICONE ELASTOMERS
TABLE I. Maxillofacial Silicone Elastomers
Material Processing Procedure
8
Cosmesil 1 h at 100 C in mold
Multisil 30 min at 608C in mold
Episil 1 h at 608C in mold
ferent parameters, such as inhibition of cell growth, cytoly-
sis, effects on membrane or cytoplasmic markers, and
changes in metabolic activity, are used to monitor cytotoxic
effects of dental materials.18 The analysis of the metabo-
lism of yellow methyltetrazolium salt (MTT) by mitochon-
drial dehydrogenases of active cells into blue formazan
crystals is a biologic assay commonly used for cytotoxicity
testing.19–23
The use of a wide variety of materials in the construc-
tion of maxillofacial prostheses makes biocompatibility
testing a necessity.6 However, the dental literature contains
few reports of biocompatibility testing of maxillofacial
prosthetic materials.6,12–14 Furthermore a review of the
available dental literature reveals lack of information about
the effects of accelerated aging on the biocompatibility of
these materials. The purpose of the present study is to eval-
uate the cytotoxic profiles of three commercially available
maxillofacial silicone elastomers by MTT assay before and
after the accelerated aging.
MATERIALS AND METHODS
Cells
The cells used for the experiments were L-929 mouse
fibroblasts (L-929, 95030802, HÜKÜK, S AP Enstitüsü,
Ankara, Turkey). The cells were grown as monolayer cul-
tures in T-25 flasks (Costar, Cambridge, MA), subcultured
three times a week at 378C, in an atmosphere of 5% CO2
in air and 100% relative humidity, and maintained at third
passage. The culture medium was Dulbecco’s Modified
Eagle Medium (DMEM) (Biochrom, Berlin, Germany) sup-
plemented with 10% (v/v) foetal bovine serum (FBS; Bio-
chrom, Berlin, Germany) without antibiotics. Adherent
cells were detached with a mixture of 0.025% trypsin
(Sigma) and 0.02% ethylenediaminetetraacetic acid
(EDTA; Sigma), incubated for 2–5 min at 378C and used
for cell inoculation.
Sample Preparation
Three different maxillofacial silicone elastomers were
tested (Table I). Totally 45 disc-shaped (14 mm diameter,
1.2 mm thickness) samples were prepared for each test ma-
terial. The samples were fabricated and polymerized in
ADA type IV dental stone (Amberok, Anadolu dental prod-
_ Statistical Analysis
ucts, Istanbul, Turkey) molds according to the manufac-
turers’ instructions. In each of the three materials the
samples were randomly divided into three different groups
with 15 samples in each group. Group 1 samples served as
Journal of Biomedical Materials Research Part B: Applied Biomaterials
123
Type Manufacturer
Addition curing Principality Medical Ltd, Newport, UK
Addition curing Bredent, Senden, Germany
Addition curing Dreve-Dentamid GmbH, Unna, Germany
control and not aged. Group 2 samples were aged with an
accelerated weathering tester (Q-U-V, Q-Panel Campony,
Cleveland, OH) for 150 h and group 3 samples were aged
for 300 h. All samples were prepared by the same operator
and previously autoclaved at 1208C for 15 min to prevent
bacterial contamination.
The ratio of the surface area of the disc samples to the
extraction volume was 3 cm2 mL21 in the present study,
which is in line with ISO 10993-5:1997.24 The samples
were placed in DMEM/F12 with 10% FBS and incubated
at 378C in an atmosphere of 5% CO2 in air without agita-
tion for 24, 48, and 72 h periods. After the incubation peri-
ods the extracts were filtered through 0.22-lm cellulose
acetate filters (Milipore; Sigma) and then they were used to
evaluate cytotoxicity.
Cytotoxicity Testing (MTT Assay)
The L-929 cell suspension with DMEM/F12 with 10% FBS
and 1% antibiotic was prepared at a concentration of 3 3
104 cells mL21 and inoculated onto 96-well cluster cell
culture plates (100 lL per well). The multiwell plates were
incubated at 378C, 5% CO2 in air for 24 h, the culture
medium was removed from the wells and equal volumes
(100 lL) of the extracts were added into each well. In con-
trol wells, 100 lL DMEM/F12 with 10% FBS and %1 anti-
biotic was added. Then 96-well cluster cell culture plates
were incubated for 24 h at 378C. Following removal of the
test extracts, 100 lL per well fresh medium with 10% FBS
and 13-lL MTT (tetrazolium salt 3-[4,5 dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide) were added to each
well. The medium also was changed in the control wells.
Culture plates were covered with aluminum foil to protect
from light and cells were incubated in a dark environment
for 4 h at 378C. After incubation 96-wells were checked
for formazan crystals with inverted tissue culture micro-
scope. MTT was aspirated and 100 lL per well of isopro-
panol (Merck, Darmstadt, Germany) was added to each
well. Subsequently, the absorbance at 570 nm was meas-
ured using a UV-visible spectrophotometer (Molecular
Devices, USA). Survival rates of the controls were set to
represent 100% proliferation. The control wells consisted
of untreated cell cultures. MTT assay were repeated in
three separate experiments.
Statistical analysis of the data was performed by using
repeated measurement ANOVA (p \ 0.01). If a significant
difference among means was found Duncan’s test was per-
124 BAL ET AL.

_
TABLE II. Statistical Comparisons of Incubation Periods TABLE IV. Statistical Comparisons of Silicone
Elastomers Tested
Incubation
Periods N Mean SE Mean SD Minimum Maximum Silicone
elastomers N Mean SE Mean SD Minimum Maximum
24 h 27 96.06 A 1.85 9.59 82.40 116.40
48 h 27 85.92 B 1.16 6.02 72.15 94.09 Cosmesil 27 87.50 B 1.61 8.38 72.15 107.47
72 h 27 87.67 B 1.25 6.51 77.74 101.90 Episil 27 93.84 A 1.29 6.71 82.09 111.77
Multisil 27 88.30 B 1.84 9.56 77.42 116.40
Vertically, means with identical capital letters were not significantly different
(p [ 0.05). Vertically, means with identical capital letters were not significantly different
(p [ 0.05).

formed to identify differences between any of the groups at DISCUSSION

a significance level of p \ 0.05.
RESULTS
In the MTT assay, exposure of L-929 cells to the test
materials in all groups (1, 2, 3) resulted in a high cell sur-
vival rate (defined as ratio (%) of OD values of extract
treated cells to the control cells by MTT assay) at 24-h
incubation period; however, the cell survival rates
decreased after 48 and 72 h of incubations. According to
repeated measurement ANOVA, significant differences
were found between the incubation periods (p \ 0.01).
There were no significant differences at 48 and 72 h incu-
bation periods (p [ 0.05) whereas the differences between
24 and 48 h incubation periods were found to be statisti-
cally significant among all groups. Furthermore significant
differences were found between 24 and 72 h incubation
periods (p \ 0.05) (Table II).
Table III shows the mean and standard error mean of
aging times. According to repeated measurement ANOVA,
no significant difference was found between the accelerated
aging times (p [ 0.01). However, the differences among
means of silicone elastomers were found to be statistically
significant (p \ 0.01); therefore, Duncan’s test was per-
formed to identify these differences. According to Dun-
can’s test, in all groups1–3 Episil material demonstrated
significantly higher cell survival rates when compared with
Cosmesil and Multisil materials after all experimental incu-
bation periods (p \ 0.05). Furthermore there were no sig-
nificant differences between Cosmesil and Multisil
materials (p [ 0.05) (Table IV). Figures 1, 2, and 3 show
the distribution of cell survival rates (%) of silicone elasto-
mers in each accelareted aging time after 24, 48, and 72 h
of incubation.
Biocompatibility of dental materials has been evaluated in
a variety of ways. The MTT assay is a good indicator of
cell viability. The dimethlthiazol diphenyltetrazolium
(MTT) test can be used to indicate cytotoxic effects by
assessing the functional state of the cell mitochondria after
exposure to chemicals or devices. Mitochondrial dehydro-
genases in living cells reduce the yellow tetrazolium salt,
MTT to blue MTT formazan, which is retained in the
cell.12,25 Formation of the formazan product has been found
to correlate well with the number of viable cells.26 The test
results reflect not only the cell number but also the cell
metabolic level. Consequently the MTT method is consid-
ered as a sensitive index to evaluate the cytotoxicity of
dental materials.27–29 In the present study, the cytotoxicity
of maxillofacial silicone elastomers was assessed by MTT
assay, which is a well-established method for dental mate-
rial testing.30–32
In vitro cytotoxicity tests were developed to simulate
and predict biological reactions to the materials placed into
or on human tissues. Considering this aim, particular care
should be taken to select cell types and experimental condi-
tions.33 These tests are not normally performed on cultured
cell actually taken from the oral environment, but more
commonly, are conducted using established cell lines that
are commercially available, which allows comparison of
testing performed for different materials using nearly iden-
tical cloned cells.34 Continuous cell lines, like 375 or L-

TABLE III. The Mean and Standard Error Mean of Aging Times

Aging
Times N Mean SE Mean SD Minimum Maximum
0 27 87.67 A 1.32 6.86 72.15 105.80
150 h 27 90.63 A 1.79 9.28 77.74 116.40
300 h 27 91.34 A 1.83 9.51 77.42 114.52
Figure 1. Cell survival rates (ratio (%)of OD values of extract treated
Vertically, means with identical capital letters were not significantly different cells to the control cells) and standard error mean of silicone elasto-
(p [ 0.05). mers before accelareted aging (C, cosmesil; E, episil; M, multisil).

Journal of Biomedical Materials Research Part B: Applied Biomaterials

 IN VITRO CYTOTOXICITY OF MAXILLOFACIAL SILICONE ELASTOMERS
929 mouse fibroblasts are being routinely used for the test-
ing of cytotoxic properties of dental materials because of
their good reproducible growth rates and biological
responses.24,35 It is also obvious that the continuous cell
lines responded more sensitively than primary cells in most
of the studies.36,37 Thoremann et al.36 indicated that L-929
mouse fibroblasts are more sensitive than primary human
gingival fibroblasts. Therefore, in the current study the cy-
totoxicity of the silicone elastomers was evaluated by using
L-929 mouse fibroblasts.
In 1992, Wolfaardt et al.13 studied the biocompatibility
of Cosmesil maxillofacial silicone by subperiosteal, submu-
cosal, and intramuscular implantation in five Chacma
baboons. They reported that Cosmesil elastomer has accept-
able biocompatibility for its intended use and for contact
with internal tissue spaces where these occur contiguously
with external surfaces. Its acceptable biocompatibility was
reconfirmed by our cell culture test.
In 1994, Polyzois et al.12 studied the biocompatibility of
two room temperature vulcanizing (A-2186 and Silbione) and
one high temperature vulcanizing material (Mollomed). They
reported that none of the materials demonstrated any cyto-
toxic effects with the agarose overlay test. Although the prep-
aration of silicone samples, curing procedures, the type of
the silicones, and the cell culture method are different, our
results were in accordance with the study of Polyzois et al.12
In an other study, the authors studied the cytotoxic pro-
files of five room-temperature cross-linking (RTC)-silicone
elastomers by agarose overlay test and direct contact test.
They indicated that RTC-silicone elastomers adversely
affected cells in culture.6 The current study demonstrated
that none of the silicone elastomers tested were cytotoxic
after 24, 48, 72 h of incubation. In the MTT assay, expo-
sure of L-929 cells to the extracts of test materials resulted
in a high cell survival rate at 24 h incubation period; how-
ever, the cell survival rates decreased after 48 and 72 h of
incubation, and Episil silicone demonstrated higher cell sur-
Figure 2. Cell survival rates (ratio (%)of OD values of extract treated
cells to the control cells) and standard error mean of maxillofacial
silicone elastomers after 150 h of accelerated aging. (C, cosmesil;
E, episil; M, multisil).
Journal of Biomedical Materials Research Part B: Applied Biomaterials
125
Figure 3. Cell survival rates (ratio (%) of OD values of extract
treated cells to the control cells) and standard error mean of maxil-
lofacial silicone elastomers after 300 h of accelareted aging. (C,
cosmesil; E, episil; M, multisil).
vival rates than Cosmesil and Multisil silicones after 24,
48, 72 h of incubation. The difference in cell viability rates
among the maxillofacial silicone materials could be related
to the variations in their chemical composition and quantity
of chemotoxic leachables migrating from these materials.
No information was found in the available literature
about the effects of accelerated aging on the in vitro bio-
compatibility of maxillofacial silicone materials. In the
present study, the cytotoxic profiles of three silicone elasto-
mers were evaluated after 150 and 300 h of accelerated
aging. Although the oral environment is more complex,
this simulated aging treatment is useful for comparing dif-
ferent materials.38 The findings of this study revealed that
aging for 150 and 300 h did not have a significant effect
on the cytotoxicity of silicone elastomers.
Extrapolation of cell culture test results to in vivo situa-
tions must also be performed with due caution. Cell culture
techniques have been reported as being predictive for in
vivo test results.39,40 In vitro analysis provides a method of
investigating cytotoxicity in a simplified system that mini-
mizes the effect of cofounding variables.41 Within the limi-
tations of this in vitro study it can be concluded that none
of the materials in each group had any toxic effect on the
cells in MTT assay and accelerated aging for 150 and 300
h had no effect on the biocompatibility of silicone elasto-
mers tested.
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