Biomaterials 21 (2000) 393}398

Antibacterial silver-containing silica glass prepared

by sol}gel method
M. Kawashita!,*, S. Tsuneyama!, F. Miyaji!, T. Kokubo!, H. Kozuka", K. Yamamoto#
!Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
"Department of Materials Science and Engineering, Faculty of Engineering, Kansai University, Suita, Osaka 564-8680, Japan
#Department of General and Operative Dentistry, School of Dentistry, Asahi University, Hozumi-cho, Motosu-gun, Gifu-ken 501-0200, Japan
Received 15 May 1999; accepted 22 August 1999

Abstract

Recently, various inorganic antibacterial materials containing silver have been developed and some of them are in commercial use.
Colorless and more chemically durable materials which slowly release the silver ion for a long period are, however, desirable to be
developed for medical applications such as composite resin for dental restoration. In the present study, Si(OC H ) ,
2 54
Al(NO ) ) 9H O, AgNO , HNO , C H OH and H O solutions with various Al/Ag atomic ratios under a constant Si/Ag atomic
33 2 3 3 2 5 2
ratio of 1/0.023 were kept at 403C for gelation and drying. Thus obtained gels were pulverized into "ne powders with average particle
size of approximately 10 lm and then heat-treated at 900}10003C for 2 h. For the composition Al/Ag"0, a yellow-colored glass was
formed, since the silver existed in the form of metallic colloids in the glass. However, for the compositions Al/Ag*1, colorless glasses
were successfully obtained, since the silver existed in the form of Ag` ions in the glasses. For the composition Al/Ag"0, the silver ions
got released rapidly into the water, whereas, for the compositions Al/Ag*1, they gradually got released into the water at a controlled
rate. A composite of the obtained powders with Al/Ag atomic ratio of 1 with Bis-GMA/TEGDMA in 70 : 30 weight ratio showed
excellent antibacterial property. The sol}gel derived silica glass powders containing silver with compositions Al/Ag*1 are believed
to be useful as an antibacterial material for medical applications such as "ller of composite resin for dental restoration. ( 2000
Published by Elsevier Science Ltd. All rights reserved.

Keywords: Silver; Silica glass; Sol}gel method; Chemical durability; Antibacterial property

1. Introduction though Ag O}SiO glasses can be prepared by sol}gel

It is widely known that materials containing silver
show antibacterial property [1}3]. Recently, various in-
organic antibacterial materials containing silver have
been developed and some of them are in commercial use
[4]. Colorless and more chemically durable materials
which slowly release silver ion for a long period are,
however, desirable for medical applications such as com-
posite resin for dental restoration.
Silica glass containing silver is expected to be a candi-
date of such antibacterial material for medical applica-
tions, since it is assumed to show high chemical
durability. However, it is di$cult to prepare Ag O}SiO
2 2
glasses by the conventional melt-quenching method. Al-
2 2
method [5}9], they often showed yellow or brown colour
owing to existence of silver colloid particles.
In the present study, silver-containing silica glasses
with various Al/Ag atomic ratios were prepared by
a sol}gel method. The release of the silver ion and other
constituent ions from the glasses were examined in water
at 373C. The results were discussed in terms of the state
of the silver in the glasses. Antibacterial property of
composites of the obtained powder with Bis-GMA/
TEGDMA was also examined.
2. Materials and methods
2.1. Preparation of glasses

Tetraethoxysilane-based solutions with various Al/Ag

* Corresponding author. Fax: #81-75-753-4824. atomic ratios under a constant Si/Ag atomic ratio of

0142-9612/00/$ - see front matter ( 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 0 1 - X
394 M. Kawashita et al. / Biomaterials 21 (2000) 393}398
Table 1
Compositions of starting solutions (molar ratio)
Al/Ag atomic Si(OC H ) H O HNO
2 54 2 3
molar ratio
0 1 8 0.01
1 1 8 0.01
2 1 8 0.01
1/0.023 were prepared. The compositions of the solu-
tions are given in Table 1. First, tetraethoxysilane
(Si(OC H ) ; LS-2430, Shinetsu Chemical Co. Ltd., To-
2 54
kyo, Japan) was mixed with half of given amount of
99.5% ethanol (C H OH; nacalai tesque, Kyoto, Japan)
2 5
to form Solution A. Second, aluminum nitrate nonahy-
drate (Al(NO ) ) 9H O; nacalai tesque, Kyoto, Japan)
33 2
and silver nitrate (AgNO ; nacalai tesque, Kyoto, Japan)
3
were dissolved in a mixed solution of water (H O), 1 N-
2
nitric acid (HNO ; nacalai tesque, Kyoto, Japan) and
3
another half of given amount of ethanol. This formed
Solution B. Solution B was slowly added to Solution
A under vigorous stirring at room temperature. After
stirring for approximately 30 min, the obtained tetra-
ethoxysilane-based solutions were poured into a polysty-
rene box with 180]90]45 mm3 in size, and kept at 403C
for 7 d for gelation and drying. The dried gels were
ball-milled into "ne powders with average partcle size of
about 10 lm (tPulverisette 7, Fritsch Japan Co. Ltd.,
Tokyo, Japan). The powders were then placed in a sin-
tered alumina dish, and sintered in the range 900}10003C
at a heating rate of 1003C h~1 in a Ni}Cr electric furnace,
and then soaked at the highest temperature for 2 h.
2.2. Structure analysis
The color of the obtained powders was measured by an
Ultraviolet-Visible (UV-Vis) di!usive re#ection spec-
trometer (Lambda 19, The Perkin}Elmer Corp., CT,
USA). In this measurement, the powders were "lled in
a rectangular quartz cell and magnesium oxide (MgO)
powder was used as the reference.
The structures of the obtained powders were charac-
terized by a powder X-ray di!ractometer (RAD-C,
Rigaku-Denki Co., Tokyo, Japan) using Ni-"ltered Cu-
K radiation. 40 kV and 40 mA of voltage and current
a
settings were used. All samples were examined at 2h
angles from 3 to 703. The crystalline phases precipitated
in the powders were identi"ed by referring to data of
Joint Committee on Powder Di!raction Standards.
2.3. Chemical durability test
The 0.1 g powder was soaked in 20 ml of distilled water
(electric conductivity+0.093 mS m~1 at 253C, organic
C H OH AgNO Al(NO ) ) 9H O
2 5 3 33 2
2 0.023 0
2 0.023 0.023
2 0.023 0.046
carbon+151 ppb, Zn(0.1 ppb, SiO +1.3 ppb, Cl~(
2
0.5 ppb, SO2~(1 ppb) in a polypropylene bottle at
4
373C and rotated with a diameter of 3 cm and a fre-
quency of 120 rpm. After 1}14 days of rotation, the ion
concentrations of silicon, aluminum and silver in the
water were measured by an inductively coupled plasma
atomic emission spectrometer (SPS-1500 VR, Seiko In-
struments Inc., Tokyo, Japan). In this study, only one
sample was subjected to the chemical durability test, with
respective Al/Ag ratio.
2.4. Antibacterial property test
Composite resin 3 mm thickness]4 mm diameter was
prepared by mixing the powders (Al/Ag"1) heat-treated
at 10003C with Bis-GMA/TEGDMA (Bis-GMA/
TEGDMA"50/50 in weight ratio) in 70 : 30 weight
ratio. The bacterium Streptococcus mutans ATCC 25175,
which is known as a major bacterium inducing caries
[10,11], was used in this study to test the antibacterial
property of the samples. This organism was anaer-
obically incubated in 5 ml Trypticase Soy Broth (BBL,
Cockeysvill, MD, USA) containing 0.5% of yeast ex-
tract (Difco Laboratories, Detroit, MI, USA, TSBY)
at 373C for 12 h. The growth term of tested bacteria
was 24 h. The "nal concentration of the bacterial cell
was adjusted to of 1]106 cells ml~1 with reduced
transport #uid [12] of pH 7.2. The composite resin was
then immersed in this suspension and anaerobically incu-
bated at 373C for 2, 6 and 12 h. The surviving bacteria
was then estimated by culturing on TSBY agar plates
either undiluted or diluted into tenfold, and incubated at
373C for 4 days. A plate with approximately 100 discrete
colonies was chosen from the serial agar cultures, and the
exact number of colonies was counted at each sampling
time.
3. Results
Fig. 1 shows the UV-Vis di!usive re#ection spectra of
the heat-treated powders with various Al/Ag atomic ra-
tios. An absorption peak assigned to silver colloids [13]
was observed at about 410 (.) nm in wavelength for the
heat-treated powders with the composition Al/Ag"0,
 M. Kawashita et al. / Biomaterials 21 (2000) 393}398 395

Fig. 1. UV-Vis di!usive re#ection spectra of the heat-treated powders with various Al/Ag atomic ratios.

Fig. 2. Powder X-ray di!raction patterns of the heat-treated powders with various Al/Ag atomic ratios.

whereas no obvious peak was observed for the heat-
treated powders with the compositions Al/Ag*1.
Fig. 2 shows the powder XRD patterns of the heat-
treated powders with various Al/Ag atomic ratios. In the
case of the composition Al/Ag"0, the powders were
coagulated at the heating temperature of 10003C. Thus,
the heat treatment temperature for this composition was
controlled at 9503C. Besides a glassy phase around 233,
three peaks assigned to silver metal at 38, 44 and 643 were
observed at both 900 and 9503C for this composition.
However, for the compositions with Al/Ag*1, only
a glassy phase was detected at both 900 and 10003C.
Fig. 3 shows the release of silicon ions released as
a function of soaking time at 373C in water. At Al/Ag"
0, a large amount of silicon ions was rapidly released at
both heating temperatures, 9003C and 9503C, although
a relatively lower silicon releasing rate was detected for
the powder heat-treated at 9503C. At Al/Ag*1, the
release of silicon ions was hardly detected at both heat
treating temperatures, 9003C and 10003C.
Fig. 4 shows the release of aluminum ions as a function
of soaking time at 373C in water. At Al/Ag*1, the
release of aluminum ions was extremely low in spite of
the change of heating temperatures.
Fig. 5 shows the release of silver ions as a function of
soaking time at 373C in water. At Al/Ag"0, it was
detected that a large amount of silver ions was rapidly
released into water. However, at Al/Ag*1, silver ions
were gradually released with increase of soaking time at
both studied temperatures.
The comparisons of the cell-forming unit among three
media, with composite resin, with pure Bis-GMA/
TEGDMA resin, and without any foreign substances,
were made, as shown in Fig. 6. The concentration of
396 M. Kawashita et al. / Biomaterials 21 (2000) 393}398
Fig. 3. Concentration of silicon released from the heat-treated powders with various Al/Ag atomic ratios into the water at 373C as a function of
soaking time.
Fig. 4. Concentration of aluminum released from the heat-treated
powders with various Al/Ag atomic ratios into the water at 373C as
a function of soaking time.
surviving cells remarkably decreased with increasing cul-
ture period, and no surviving cell was observed after 12 h
of soaking in the medium containing composite resin,
whereas the amount of surviving cells was only slightly
decreased after 12 h of soaking in both media containing
no foreign substance and the pure resin. It was noticed
that the composite resin showed no cytotoxicity and the
e!ect of carry over of silver on the antibacterial property
was negligible, since the concentration of silver released
from the composite resin was extremely low (0.0608 ppm
for one block of composite resin). It has been reported
that the low concentration of released silver does not
cause cytotoxicity [14].
4. Discussion
The heat-treated powders with the composition Al/
Ag"0 were yellow, whereas those with the compositions
Al/Ag*1 were colorless as indicated in Fig. 1. The
yellow color of the powders at Al/Ag"0 was attributed
to the presence of the silver colloids in these powders,
since the metallic silver was detected by powder XRD
(see Fig. 2). The colorless powders at Al/Ag*1 can be
explained by the fact that the silver exists as Ag` ions in
these specimens. When an aluminum ion is incorporated
into the SiO network structure in a form of [AlO ]~
4 4
tetrahedra, its negative charge must be compensated by
Ag` ion. As a result, the Ag` ion is localized to the
[AlO ]~ tetrahedra and di$cult into silver colloid.
4
It can be seen from Fig. 5 that the heat-treated pow-
ders with the composition Al/Ag"0 rapidly release sil-
ver ions into water, whereas those with the compositions
Al/Ag*1 slowly released silver ions into water. The
amount of the silver ion released from the powders was in
proportion to the square root of soaking period at
Al/Ag*1, as shown in Fig. 7. This is explained as fol-
lows. In the case of the composition Al/Ag"0, silica ions
got released easily from the surfaces of the powders as
shown in Fig. 3. As a result, metallic silver colloids were
exposed to water and then easily released. In contrast,
at Al/Ag*1, silica ions did not get released easily from
the powders. As a result, silver ions could only be re-
leased from the powders via ion exchange between Ag`
ion and hydronium (H O`) ion from surrounding water.
3
The release of Ag` ion is controlled by the rate of
interdi!usion of these ions within the solid powders.
It is well known that when the release is controlled by
the interdi!usion within a solid, the amount of the re-
leased ions being in proportion to the square root of
time [15,16].
When the composite of the powders with Al/Ag atomic
ratio of 1 and heat-treated at 10003C with Bis-GMA/
TEGDMA is placed in the medium in which S. mutans
was cultured, the concentration of surviving cells remark-
ably decreased with increasing culture period, and no
survival of S. mutans was observed after 12 h of soaking
(see Fig. 6). These results indicate that thus prepared
composite shows a high antibacterial activity.
In conclusion, colorless and chemically durable silica
glass powders which release silver ion slowly at a control-
led rate can be obtained, when tetraethoxysilane-based
 M. Kawashita et al. / Biomaterials 21 (2000) 393}398
Fig. 5. Concentration of silver released from the heat-treated powders with various Al/Ag atomic ratios into the water at 373C as a function of soaking
time.
Fig. 6. CFU of S. mutans cultured for various periods in the medium
containing composite of the powder (Al/Ag"1) with Bis-GMA/
TEGDMA in 70 : 30 weight ratio, in comparison with those in the
media containing no foreign substance (control) and pure resin.
gels with the compositions Al/Ag*1 under a constant
Si/Ag ratio of 1/0.023 are heated at either 900 or 10003C.
5. Conclusion
Colorless and chemically durable silver-containing sil-
ica glass powders, which can gradually release silver ions
at a controlled rate for a long period, were successfully
prepared by the sol}gel method using tetraethoxysilane-
based solutions with the compositions Si/Ag"1/0.023
and Al/Ag*1. Thus prepared silver-containing silica
glass powders are believed to be useful as an antibacterial
397
Fig. 7. Concentration of silver released from the heat-treated powders
with various Al/Ag atomic ratios as a function of square root of soaking
period.
material for medical applications such as composite resin
for dental restoration.
Acknowledgements
We thank Dr. T. Tanaka, Department of Molecular
Engineering, Faculty of Engineering, Kyoto University,
for the UV-Vis di!usive re#ection spectroscopic mea-
surement.
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