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

2 2
method [5}9], they often showed yellow or brown colour
It is widely known that materials containing silver owing to existence of silver colloid particles.
show antibacterial property [1}3]. Recently, various in- In the present study, silver-containing silica glasses
organic antibacterial materials containing silver have with various Al/Ag atomic ratios were prepared by
been developed and some of them are in commercial use a sol}gel method. The release of the silver ion and other
[4]. Colorless and more chemically durable materials constituent ions from the glasses were examined in water
which slowly release silver ion for a long period are, at 373C. The results were discussed in terms of the state
however, desirable for medical applications such as com- of the silver in the glasses. Antibacterial property of
posite resin for dental restoration. composites of the obtained powder with Bis-GMA/
Silica glass containing silver is expected to be a candi- TEGDMA was also examined.
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. Materials and methods
2 2
glasses by the conventional melt-quenching method. Al-
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 C H OH AgNO Al(NO ) ) 9H O

2 54 2 3 2 5 3 33 2
molar ratio

0 1 8 0.01 2 0.023 0
1 1 8 0.01 2 0.023 0.023
2 1 8 0.01 2 0.023 0.046

1/0.023 were prepared. The compositions of the solu- carbon+151 ppb, Zn(0.1 ppb, SiO +1.3 ppb, Cl~(
2
tions are given in Table 1. First, tetraethoxysilane 0.5 ppb, SO2~(1 ppb) in a polypropylene bottle at
4
(Si(OC H ) ; LS-2430, Shinetsu Chemical Co. Ltd., To- 373C and rotated with a diameter of 3 cm and a fre-
2 54
kyo, Japan) was mixed with half of given amount of quency of 120 rpm. After 1}14 days of rotation, the ion
99.5% ethanol (C H OH; nacalai tesque, Kyoto, Japan) concentrations of silicon, aluminum and silver in the
2 5
to form Solution A. Second, aluminum nitrate nonahy- water were measured by an inductively coupled plasma
drate (Al(NO ) ) 9H O; nacalai tesque, Kyoto, Japan) atomic emission spectrometer (SPS-1500 VR, Seiko In-
33 2
and silver nitrate (AgNO ; nacalai tesque, Kyoto, Japan) struments Inc., Tokyo, Japan). In this study, only one
3
were dissolved in a mixed solution of water (H O), 1 N- sample was subjected to the chemical durability test, with
2
nitric acid (HNO ; nacalai tesque, Kyoto, Japan) and respective Al/Ag ratio.
3
another half of given amount of ethanol. This formed
Solution B. Solution B was slowly added to Solution 2.4. Antibacterial property test
A under vigorous stirring at room temperature. After
stirring for approximately 30 min, the obtained tetra- Composite resin 3 mm thickness]4 mm diameter was
ethoxysilane-based solutions were poured into a polysty- prepared by mixing the powders (Al/Ag"1) heat-treated
rene box with 180]90]45 mm3 in size, and kept at 403C at 10003C with Bis-GMA/TEGDMA (Bis-GMA/
for 7 d for gelation and drying. The dried gels were TEGDMA"50/50 in weight ratio) in 70 : 30 weight
ball-milled into "ne powders with average partcle size of ratio. The bacterium Streptococcus mutans ATCC 25175,
about 10 lm (tPulverisette 7, Fritsch Japan Co. Ltd., which is known as a major bacterium inducing caries
Tokyo, Japan). The powders were then placed in a sin- [10,11], was used in this study to test the antibacterial
tered alumina dish, and sintered in the range 900}10003C property of the samples. This organism was anaer-
at a heating rate of 1003C h~1 in a Ni}Cr electric furnace, obically incubated in 5 ml Trypticase Soy Broth (BBL,
and then soaked at the highest temperature for 2 h. Cockeysvill, MD, USA) containing 0.5% of yeast ex-
tract (Difco Laboratories, Detroit, MI, USA, TSBY)
2.2. Structure analysis at 373C for 12 h. The growth term of tested bacteria
was 24 h. The "nal concentration of the bacterial cell
The color of the obtained powders was measured by an was adjusted to of 1]106 cells ml~1 with reduced
Ultraviolet-Visible (UV-Vis) di!usive re#ection spec- transport #uid [12] of pH 7.2. The composite resin was
trometer (Lambda 19, The Perkin}Elmer Corp., CT, then immersed in this suspension and anaerobically incu-
USA). In this measurement, the powders were "lled in bated at 373C for 2, 6 and 12 h. The surviving bacteria
a rectangular quartz cell and magnesium oxide (MgO) was then estimated by culturing on TSBY agar plates
powder was used as the reference. either undiluted or diluted into tenfold, and incubated at
The structures of the obtained powders were charac- 373C for 4 days. A plate with approximately 100 discrete
terized by a powder X-ray di!ractometer (RAD-C, colonies was chosen from the serial agar cultures, and the
Rigaku-Denki Co., Tokyo, Japan) using Ni-"ltered Cu- exact number of colonies was counted at each sampling
K radiation. 40 kV and 40 mA of voltage and current time.
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 3. Results
Joint Committee on Powder Di!raction Standards.
Fig. 1 shows the UV-Vis di!usive re#ection spectra of
2.3. Chemical durability test the heat-treated powders with various Al/Ag atomic ra-
tios. An absorption peak assigned to silver colloids [13]
The 0.1 g powder was soaked in 20 ml of distilled water was observed at about 410 (.) nm in wavelength for the
(electric conductivity+0.093 mS m~1 at 253C, organic 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- the powder heat-treated at 9503C. At Al/Ag*1, the
treated powders with the compositions Al/Ag*1. release of silicon ions was hardly detected at both heat
Fig. 2 shows the powder XRD patterns of the heat- treating temperatures, 9003C and 10003C.
treated powders with various Al/Ag atomic ratios. In the Fig. 4 shows the release of aluminum ions as a function
case of the composition Al/Ag"0, the powders were of soaking time at 373C in water. At Al/Ag*1, the
coagulated at the heating temperature of 10003C. Thus, release of aluminum ions was extremely low in spite of
the heat treatment temperature for this composition was the change of heating temperatures.
controlled at 9503C. Besides a glassy phase around 233, Fig. 5 shows the release of silver ions as a function of
three peaks assigned to silver metal at 38, 44 and 643 were soaking time at 373C in water. At Al/Ag"0, it was
observed at both 900 and 9503C for this composition. detected that a large amount of silver ions was rapidly
However, for the compositions with Al/Ag*1, only released into water. However, at Al/Ag*1, silver ions
a glassy phase was detected at both 900 and 10003C. were gradually released with increase of soaking time at
Fig. 3 shows the release of silicon ions released as both studied temperatures.
a function of soaking time at 373C in water. At Al/Ag" The comparisons of the cell-forming unit among three
0, a large amount of silicon ions was rapidly released at media, with composite resin, with pure Bis-GMA/
both heating temperatures, 9003C and 9503C, although TEGDMA resin, and without any foreign substances,
a relatively lower silicon releasing rate was detected for 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.

(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
Fig. 4. Concentration of aluminum released from the heat-treated amount of the silver ion released from the powders was in
powders with various Al/Ag atomic ratios into the water at 373C as
a function of soaking time.
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
surviving cells remarkably decreased with increasing cul- shown in Fig. 3. As a result, metallic silver colloids were
ture period, and no surviving cell was observed after 12 h exposed to water and then easily released. In contrast,
of soaking in the medium containing composite resin, at Al/Ag*1, silica ions did not get released easily from
whereas the amount of surviving cells was only slightly the powders. As a result, silver ions could only be re-
decreased after 12 h of soaking in both media containing leased from the powders via ion exchange between Ag`
no foreign substance and the pure resin. It was noticed ion and hydronium (H O`) ion from surrounding water.
3
that the composite resin showed no cytotoxicity and the The release of Ag` ion is controlled by the rate of
e!ect of carry over of silver on the antibacterial property interdi!usion of these ions within the solid powders.
was negligible, since the concentration of silver released It is well known that when the release is controlled by
from the composite resin was extremely low (0.0608 ppm the interdi!usion within a solid, the amount of the re-
for one block of composite resin). It has been reported leased ions being in proportion to the square root of
that the low concentration of released silver does not time [15,16].
cause cytotoxicity [14]. 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
4. Discussion was cultured, the concentration of surviving cells remark-
ably decreased with increasing culture period, and no
The heat-treated powders with the composition Al/ survival of S. mutans was observed after 12 h of soaking
Ag"0 were yellow, whereas those with the compositions (see Fig. 6). These results indicate that thus prepared
Al/Ag*1 were colorless as indicated in Fig. 1. The composite shows a high antibacterial activity.
yellow color of the powders at Al/Ag"0 was attributed In conclusion, colorless and chemically durable silica
to the presence of the silver colloids in these powders, glass powders which release silver ion slowly at a control-
since the metallic silver was detected by powder XRD led rate can be obtained, when tetraethoxysilane-based
 M. Kawashita et al. / Biomaterials 21 (2000) 393}398 397

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. 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.

Fig. 6. CFU of S. mutans cultured for various periods in the medium Acknowledgements
containing composite of the powder (Al/Ag"1) with Bis-GMA/
TEGDMA in 70 : 30 weight ratio, in comparison with those in the We thank Dr. T. Tanaka, Department of Molecular
media containing no foreign substance (control) and pure resin. Engineering, Faculty of Engineering, Kyoto University,
for the UV-Vis di!usive re#ection spectroscopic mea-
surement.

gels with the compositions Al/Ag*1 under a constant

Si/Ag ratio of 1/0.023 are heated at either 900 or 10003C. References

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