AATEX 14, Special Issue, 655-658
Proc. 6th World Congress on Alternatives & Animal Use in the Life Sciences
August 21-25, 2007, Tokyo, Japan
Effects of experimental alloys containing indium for dental use in vitro embryotoxicity test
Koichi Imai and Masaaki Nakamura
Department of Biomaterials, Osaka Dental University
Corresponding author: Koichi Imai Ph.D
Department of Biomaterials, Osaka Dental University
8-1 Kuzuha Hanazono-cho, Hirakata, Osaka 573-1121, Japan
Phone: +(81)-6-864-3056, Fax: +(81)-6-864-3156, [email protected]
Abstracts
The purpose of this study was carried out by the embryotoxicity risk assessment of the dental alloys
containing indium. We produced Ag-In alloy, Ag-Pd-Au-Cu alloy and gold alloy for porcelain bonding. For
the extraction of these alloy components into the ES-D3 cell culture medium, each alloy was reduced to a
powder using a diamond point and sterilized after rinsing. In order to facilitate the extraction, each alloy
powder was subjected to extraction together with diamond powder using a rotary stirrer. Two-fold diluted
test medium was used to estimate the presence of embryotoxicity from the differentiation rate of EBs in the
extract. Our test results this time suggest that 35Ag-30Pd-20Au-15Cu alloys and gold alloys for porcelain
bonding involve virtually no embryotoxicity risk. On the other hand, the alloy of silver and 20% indium may
require further examination.
Keywords: ES cell, embryotoxicity, Ag-In alloy, Ag-Pd-Au-Cu alloy, gold alloy for porcelain bonding
Introduction
The results of animal experiments have indicated
the embryotoxicity of indium compounds (Nakajima
et al, 2000). We also conducted an experiment as per
the EST protocol, which uses ES-D3 cells for testing
embryotoxicity, and obtained different embryotoxicity
levels according to the type of indium compound
tested (Imai et al, 2003). Currently, many indiumadded alloys are used as dental alloys, but no evidence
of their embryotoxicity risk has been reported.
Among the dental alloys which contain indium, Ag-In
alloy, Ag-Pd-Au-Cu alloy and gold alloy for porcelain
bonding are known. Therefore, by way of trial, we
produced the above three types of alloys with three
different rates of added indium. For the extraction of
these alloy components into the cell culture medium,
each alloy was reduced to a powder using a diamond
point and sterilized after rinsing. In order to facilitate
the extraction, each alloy powder was subjected to
extraction together with diamond powder using a
rotary stirrer. Two-fold diluted test medium was used
to estimate the presence of embryotoxicity from the
differentiation rate of EBs in the extract.
Materials and method
1. The trial production of indium alloys
By referring to the rates of indium contained in
dental alloys for the clinical use, we produced by way
© 2008, Japanese Society for Alternatives to Animal Experiments
of trial Ag-In alloys with indium additive ratios of
10%, 15% and 20%, 5Ag-30Pd-20Au-15Cu alloys
with indium additive ratios of 2%, 4% and 6%, and
gold alloys for porcelain bonding with the same
indium additive ratios as for the 5Ag-30Pd-20Au15Cu alloys.
2. Cells and culture medium
We used embryonic stem cells from the D3 mouse
cell line (ES-D3 cell). For ES-D3 cells, preheat
treated 20% (v/v) fetal calf serum (FCS) (Hyclone,
USA) was added to 1% (v/v) nonessential amino
acids (Gibco, USA), 0.1 mM β -mercaptoethanol
(Sigma, USA), 2 mM L-glutamine (Gibco), and
Dulbecco's Modified Eagle Medium (DMEM) (Gibco)
with penicillin/streptomycin (Gibco). Except during
tests, 1,000 U/mL of mouse leukemic inhibitory
factor (mLIF) was added to inhibit natural cell
differentiation.
3. Preparation of assay medium
After 0.5g of each of the powdered Ag-In alloys,
35Ag-30Pd-20Au-15Cu alloys and gold alloys for
porcelain bonding was poured into a separate silicone
mold of the stirrer shown in Fig. 1 together with
0.5g of diamond powder with a particle diameter of
1-2μm, 1 ml DMEM was poured into each mold, and
the mixture was stirred for an hour. Each test medium
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Koichi Imai and Masaaki Nakamura
was diluted 100, 200, 400 and 800 times with culture
medium, and each diluted test medium was filtered
using a membrane filter with a pore diameter of
0.22µm and sterilized to make each assay medium.
No specimen was added to the control.
Fig. 1 A motor stirrer
An indium alloy particle is agitated with a diamond particle, and
an ingredient element is extracted.
4. Differentiation assay
ES-D3 cells were diluted in each test solution to
a final concentration of 3.75 x 10 4 cells/mL using
a hemacytometer, and a 20µL cell suspension was
dropped 60-70 times onto the inside of the lid of
a 10 cm diameter Petri dish using a micropipette.
Five ml of sterilized phosphate buffered saline
(PBS) was poured into the Petri dish, and the lid
was quickly reversed and placed on the dish before
the cell suspension on the lid could flow down.
Suspension culture was carried out for three days
in a CO 2 incubator (5% CO 2 and 95% air; 37ºC)
(Sanyo, Japan). Drops of the cell suspension on the
inside of the Petri dish lid were then collected into a
6 cm diameter Petri dish for germiculture. The test
solutions were replaced with new ones using a pipette
and each test solution was subjected to reaction for
two days in the above listed incubator. Subsequently,
two 24-well multidishes were used for every test
Fig. 2 The results of cell differentiation rate with the Ag-In
alloys.
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solution. Each of the one embryoid bodies (EBs)
formed were placed in the well with a micropipet,
and the EBs were cultured statically for five days.
The presence of beating myocardial cells in each well
was examined under an inverted phase difference
microscope (IX-70, Olympus, Japan). The number
of wells in which beating cells were observed at
each concentration was examined, and the ID50 was
calculated from the ratio of the number of the above
wells to the number of wells in which EBs were
successfully disseminated.
Results
Fig. 2 shows the results for the Ag-In alloys. In the
medium diluted 100 times, no myocardial pulsation
caused by the differentiation of EBs was observed.
For the alloy of silver and 20% indium, virtually
no myocardial pulsation was observed even in the
medium diluted 200 or 400 times. However, for the
alloy of silver and 10% indium and that of silver
and 15% indium, pulsation rates of 40-60% were
observed in the medium diluted 200 or 400 times. For
the alloy of silver and 10% indium and that of silver
and 20% indium, pulsation rates of about 70% were
observed in the medium diluted 800 times, whereas
for the alloy of silver and 20% indium, pulsation
rates of only about 50% were observed at the same
medium dilution rate.
Fig. 3 shows the results for the 35Ag-30Pd-20Au15Cu alloys. The pulsation rates for these alloys
were higher than those for the Ag-In alloys, and they
Fig. 3 The results of cell differentiation rate with the 35Ag30Pd-20Au-15Cu alloys.
Fig. 4 The results of cell differentiation rate with the gold alloy
for porcelain bonding.
were more dependent on the dilution rate than on
the additive ratio of indium. More specifically, at the
dilution rate of 800 times, the pulsation rates for the
three alloys with different additive ratios of indium
were about 90%, similarly to those of the control
group, whereas, at the dilution rate of 100 times, the
pulsation rates were around 50%.
Fig. 4 shows the results for the gold alloys for
porcelain bonding. The pulsation rates for these
alloys were even higher than those for the 35Ag30Pd-20Au-15Cu alloys. Similarly to the results for
the 35Ag-30Pd-20Au-15Cu alloys, the pulsation rates
were more dependent on the dilution rate than on the
additive ratio of indium.
Discussion
For the Ag-In alloys, the differentiation rate of
EBs to myocardial cells differed greatly depending
on the additive ratio of indium. Above all, for the
alloys of silver and 20% indium, the differentiation
rate of EBs to myocardial cells was lower than under
other conditions. However, for the 35Ag-30Pd-20Au15Cu alloys and gold alloys for porcelain bonding,
the differentiation rate of EBs to myocardial cells was
not very dependent on the additive ratio of indium;
it was more dependent on the dilution rate of the test
medium. We poured powdered alloy and the same
amount of diamond powder into the silicone mold of
the stirrer with the aim of accelerating the extraction
by stirring, and the above results may suggest that the
fine diamond powder was not sufficiently filtered by
the membrane filter (pore diameter: 0.2 µm) and was
absorbed by the cells. After the filtered test medium
was left alone in a test tube for a long time, a slight
amount of precipitate was observed on the bottom.
Regarding the effect of the nano-level or near-nanolevel fine powder that penetrated into the cells on cell
differentiation, the mechanism is quite complicated
and we expect a future elucidation thereof.
The general toxicity of indium itself has been
reported widely (Wiltrout et al., 1978, Ferluga et al.,
1979, McCullough et al., 1981, Lockshin et al., 1984,
Blann et al., 1985, Ghaleb et al., 1985, Nakajima et
al., 1997). The acute toxicity value of indium oxide
is 10g/kg (LD50) for oral administration to rats and
5g/kg (LD50) for intraperitoneal administration to
mice (Castronovo et al., 1973). However, virtually no
study has been conducted on the effects of indium on
human development. Indium is added to dental silver
alloys in order to improve corrosion resistance and
sulfur resistance, as well as to overcome the fragility
typical of silver alloys (Kakuta et al., 2002). JIS
defines Class I silver alloys as those containing less
than 5% indium and no platinum group elements, and
Class II silver alloys as those containing 5% or more
indium and 10% or less platinum group elements. The
applications of these alloys are metal cores, inlays,
etc. Indium may be added in small amounts to certain
amalgam alloys, solders, etc. Indium was not always
added to the alloys tested by us this time; we only
tested frequently used alloys.
Our test results this time suggest that 35Ag-30Pd20Au-15Cu alloys and gold alloys for porcelain
bonding involve virtually no embryotoxicity risk. On
the other hand, the alloy of silver and 20% indium
may require further examination. Regarding the
embryotoxicity of indium, Nakajima, et al. reported
that, after administering indium compounds to rats
and mice, anomalies were observed in the embryos
(cleft palates and malformed tails); they concluded
that the malformations were caused by the inhibition
of tail growth as a result of relatively prolonged
apoptosis. Furthermore, in the embryotoxicity testing
of indium compounds conducted by us as per the EST
method, InCl3, In(OH)3 and InPO4 were determined to
be non-embryotoxic, whereas In(NO3)3 and In2(SO4)3
were determined to be weakly embryotoxic, and the
embryotoxicity level differed according to the type of
compound (Imai et al, 2003). The test results this time
also showed that the differentiation rate of ES-D3
cells to myocardial cells was high, except in the case
of Ag-In alloys, and the embryotoxicity risk was
low. However, the fact that the differentiation rate
decreased according to the additive ratio of indium
suggests that indium ions are in fact embryotoxic,
albeit weak. This means that, when designing the
alloy composition, any immoderate increase in the
additive ratio of indium should be avoided.
Acknowledgements
The authors would like to thank Prof. Horst
Spielmann, BfR - Federal Institute for Risk
Assessment, Germany. This present study was
partly supported by the Grant-in-Aid for General
Scientific Research (17592055) from the Ministry of
Education, Science, Sports and Culture in Japan, and
a Grant for Child Health and Development (19C-4)
from the Ministry of Health, Labour and Welfare.
This study was carried out using the Institute of
Dental Research, Osaka Dental University.
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