Dental Materials Journal 2014; 33(2): 268274

Effect of silver ion coating of fixed orthodontic retainers on the growth of oral
pathogenic bacteria
Yusuke MORITA1, Susumu IMAI2, Ai HANYUDA1, Khairul MATIN2, Nobuhiro HANADA2 and Yoshiki NAKAMURA1
Department of Orthodontics, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan
Department of Translational Research, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501,
Japan
Corresponding author, Susumu IMAI; E-mail: imai-s@tsurumi-u.ac.jp
1
2

Titanium and stainless steel wires used for retainers in orthodontic procedures were coated with Ag ions and the effects of the coating
on common oral pathogens and their pathogenicity were investigated. Two species of cariogenic and three species of periodontopathic
bacteria were assessed. Biofilms of Streptococcus sobrinus and two VSC gases produced by P. gingivalis were also examined. Ag ioncoated wires showed marked antibacterial activities compared with uncoated wires; in most cases, the differences were statistically
significant (p<0.05). All Ag ion-coated wires (Ti+ and SS+ wires) displayed more than 2-mm diameter bacteria growth-resistant zones
around them in radial diffusion tests. Ag ion release was 0.0430.005 ppm in 24 h that didnt show cytotoxicity. Thus, these results
suggest that a simple Ag ion coating on pure titanium and stainless steel wires can restrict growth and pathogenic activities of oral
pathogenic bacteria, even in the early stages of culture.
Keywords: Fixed orthodontic retainers, Titanium and stainless steel wires, Ag ion coating, Oral pathogenic bacteria, Biofilm

INTRODUCTION
Retention after active orthodontic treatment is one of the
most important procedures in orthodontics. It prevents
the relapse of teeth in dental arches; orthodontic
retainers are typically used to retain teeth in the arches.
In particular, fixed retainers are often applied to lower
anterior teeth (canine-to-canine fixed retainer) to prevent
relapse of the teeth (Fig. 1). The retainer ensures a
steady and stable retention effect in the treatment and
it causes little discomfort to patients, because it consists
of a piece of thin wire attached to the lingual surfaces
of the lower anterior teeth with orthodontic resin1).
However, fixed retainers have some side effects; the
wire is a gathering site for bacteria, biofilm, and dental
calculus that cause caries and periodontal disease2,3).
The initial bacterial adhesion to the wire is a key
step that induces a biofilm4-7). The subsequent growth
of these initially adherent bacteria is difficult to remove
completely by tooth brushing, and consequently can lead
to the formation of pathogenic oral biofilms, which are
key factors in dental caries and periodontal disease8-11).
Thus, it is important to prevent pathogenic bacteria
from adhering to the wire in the first place.
Silver is recognized as a strong disinfectant,
having a broad bactericidal spectrum12-14). An important
characteristic is that bacteria do not build up strong
resistance against Ag13). Even at low concentrations,
silver ions are effective against bacteria and fungi
in aqueous solution. Recently, Ag ion and silver
nanoparticles have been used to prevent the initial
bacterial adherence to the dental materials by

Color figures can be viewed in the online issue, which is available at J-STAGE.
Received Aug 7, 2013: Accepted Jan 29, 2014
doi:10.4012/dmj.2013-216 JOI JST.JSTAGE/dmj/2013-216

incorporating them into the materials. This has reduced

biofilm formation. However, to the best of our knowledge
there is no study report that describes antibacterial
activities of surface coated fixed orthodontic retainers.
Thus, we sought to examine the effects of silver ion (Ag
ion) coating of the orthodontic wires on the inhibition of
growth of oral pathogenic bacteria.

MATERIALS AND METHODS

Orthodontic wires
Commercially available stainless steel (SS) and
titanium (Ti) wires, typically used for orthodontic fixed
retainers (canine-to-canine fixed retainers), were used.
For all experiments, 0.016 inch (0.04 cm) diameter wires
were cut into different lengths according to the needs of
the experiments before applying the coat as described
below. Briefly, the surfaces of SS and Ti wires were
cleaned with alcohol and dried before applying the coat.
A modified microwave plasma process was employed to
coat the wires that doesnt require air-vacuum, can be
done at a low temperature (below 50C) in short period
of time. A microwave machine; EM-1900 (SANYO
Electric Co., Ltd., Osaka, Japan) at 570 w was used to
generate plasma15). The cleaned surfaces of the wires
were coated with silver ions (Ag ions) by immediately
dipping into a solution containing Ag ion (5,400 ppm)
and generating plasma inside the microwave machine
for 90 s limiting the risk of any damage that may occur.
After 90 s, the wires were washed with distilled water
and dried to obtain a uniform coat. With this method an
average of 14.8172.163 m coating ranging from 10.672
m to 19.01 m was obtained (Pika Power Technology
Ltd. Co., Hyogo, Japan). Equal numbers of uncoated

Dent Mater J 2014; 33(2): 268274

269

plates were then cultured under anaerobic conditions at

37C for 48 h in an anaerobic box (AnaeroPack System,
Mitsubishi Gas Chemical Co. Inc, Tokyo, Japan) for the
cariogenic group or in a complete anaerobic box (10% H2,
10% CO2 and 80% N2) for the periodontopathic group.
For the growth of periodontopathic bacteria hemin
(Wako Pure Chemical Industries Ltd., Osaka, Japan)
and vitamin K (Wako Pure Chemical Industries Ltd.)
were added to all culture media. After incubation for
48 h, the diameters of the clear zones around the wires
were measured with a slide caliper.
Fig. 1

Fig. 2

Orthodontic canine-to-canine fixed retainer.

Photograph showing the non-coated (Ti) and Ag

coated (Ti+) wires.
Clearly, Ti+ looks thicker than Ti and surface
appearance also changed due to the application of
the coat.

wires were used as controls (Fig. 2).

Bacteria used
Two laboratory strains, Streptococcus mutans MT8148
(S. mutans) and Streptococcus sobrinus 6715 (S.
sobrinus), were used as the cariogenic bacteria.
Antibacterial activities of the Ag ion-coated wires were
examined using the radial diffusion test method16,17).
Porphyromonas gingivalis ATCC33277 (Pg), Prevotella
intermedia ATCC25611 (Pi), and Fusobacterium
nucleatum ATCC25586 (Fn) were also used as
periodontopathic bacteria.
Radial diffusion test
Bacteria were collected from a log-phase Tryptic Soy (TS;
Becton, Dickinson and Company, Sparks, MD, USA)
culture medium. Radial diffusion tests were performed
by installing the 5-mm long wires vertically into the
first layer of TS (6 mg in 10 mL DW) agar (with 100 mg
agarose) containing each bacterium at a concentration of
OD540=2.0 (100 L). Then, the second layer of TS (600 mg
in 10 mL DW) agar (with 100 mg agarose) medium was
poured onto the first layer in such a manner that the
tips of the standing wires were covered completely. The

Effect on biofilm formation

The inhibitory effect on biofilm formation was examined
on the surfaces of the wires using a S. sobrinus
suspension. Five pieces of 50 mm long wire from each
group were incubated in TSs medium with a suspension
of S. sobrinus (TS without dextrose but containing 1%
sucrose) for 8, 12, or 24 h. Wires were then gently shaken
in chilled PBS to remove loose biofilm. Biofilm retained
attached on the wires was then stained with BacLight. A
LIVE/DEAD BacLight Bacterial Viability Kit (Molecular
Probes, Invitrogen Detection Technologies, Carlsbad,
CA, USA) was used to evaluate the bactericidal effects
of the Ag ion coat. S. sobrinus biofilms attached to
the wires were stained with 0.5 L BacLight stain (a
mixture of SYTO 9 and propidium iodide) in separate
microtubes. In this staining system, viable bacterial cells
exhibit green fluorescence, whereas nonviable bacterial
cells show red fluorescence. The selective dye uptake
depends upon cell membrane integrity, allowing dead
bacteria to be distinguished readily from viable bacteria.
The excitation/emission wavelengths of the dyes were
approximately 480/530 nm for SYTO 9 (green signals)
and 520/580 nm for propidium iodide (red signals).
Bacteria cells were evaluated using a fluorescence
microscope (CKX41, Olympus, Tokyo, Japan).
Water-insoluble glucan measurement
The bacterial cell suspension of S. sobrinus (OD540=0.7)
was cultured in 7.5 mL of TSs medium with 50-mm
long pieces of wires under anaerobic condition to form
biofilms on the surfaces of wires for 8 or 16 h. The
water-insoluble glucan (WIG) in the retained biofilms
was measured after separating the bacterial cells
from WIG on the wire. Each piece of wire was gently
washed in PBS to wash away culture medium and then
transferred carefully in 1 mL of 0.5 M sodium hydroxide
solution, incubated for 15 min on ice, vortexed, and
centrifuged (5,000 rpm, 10 min) to separate the WIG
from the bacterial cells in the biofilms. The amount of
dissolved WIG was measured colorimetrically using
the phenol-H2SO4 method using a Bio-Rad iMark
Microplate reader (Bio-Rad, Tokyo, Japan) at 492 nm.
The 500 L of WIG solution from each sample was
reacted with 5% phenol and H2SO4, and then 200 L of
each reaction mixture was used to measure the amount
of WIG (g/mL). To obtain a standard curve, 0, 25, 50, 75,
100, 150, and 200 g/mL of glucose were also measured
using the same method. The experiments were repeated

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Dent Mater J 2014; 33(2): 268274

three times for reproducibility.

Gas-chromatography
after
inoculation
with
Porphyromonas gingivalis
A gas chromatography method (Shimadzu GC-2010;
Shimadzu Corporation, Kyoto, Japan) was used to
evaluate the effect of Ag ion-coated fixed orthodontic
appliance by measuring the volumes of two major volatile
sulfur compounds (VSCs); hydrogen sulfide (H2S) and
methyl mercaptan (MMC) gases from overnight cultures
of periodontopathic bacteria; Porphyromonas gingivalis
(P. gingivalis) in this study. These gases are responsible
for the offensive odor commonly known as bad breath
or oral malodor, which is most commonly attributed to
periodontal pathogens, including P. gingivalis, as the
chemical end products of bacterial putrefaction16-18). Ti
and Ti+ wires used in this experiment; two pieces of
50-mm long wire were incubated for 0, 8, or 16 h under
anaerobic conditions in P. gingivalis (ATCC33277)
suspensions (OD540=1.0). Each time, the total gas from
each culture tube was collected using a 2.5cc disposable
syringe and a 23-G needle. Then, VSCs was measured
using a gas chromatography unit (Shimadzu GC-2010
GC, Kyoto, Japan). After 24 h, the concentrations of the
gases were high and thus were diluted 500-fold with pure
O2 and then measured. Bacterial OD also was measured
by the method mentioned above.
Measurement of released Ag ions
The release of Ag ion from the coating Ti wire was
measured with inductively coupled plasma atomic
emission spectrometer (ICP-AES, system, CIROS-120,
Rigaku, Japan). The quantity of Ag ion released after
24 h from the specimens (50 mm long wire) was
expressed as the amounts (ppm) of Ag ion in PBS
solution (10 mL). Ti wire without Ag ion coating were
also prepared as controls. Three specimens from each
group were measured.
Cell cytotoxicity
To test the effect of Ag ion released from specimens on
human gingival fibroblast cell (HGF Gin-1, DS Pharma

(a)
Fig. 3

(b)

Biomedical, Japan) were used. The cytotoxicity of Ag ion

was tested by using a standard MTT assay described
elsewhere with a minor modification19). Briefly, HGF
cells were plated in 96-well plates at a density of
approximately 7.0104 cells/mL in medium DMEM with
10% FBS, 24 h after plating above mentioned solutions
(PBS with Ag ion and without Ag ion) were added. The
Ag ion solution added to the cell culture medium at
10%, 1%, 0.1% concentrations separately. After 48 h of
incubation, the medium was replaced with MTT solution
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium
bromide] (Roche Diagnostics Corporation, Indianapolis,
IN) dissolved at a final concentration of 1 mg/mL in
serum-free, Phenol red-free RPMI (Biochrom KG,
Germany), for a further 4 h incubation. Then, the MTT
formazan reaction was measured at a wave length of 540
nm and a reference wave length of 655 nm using a BioRad iMark Microplate reader (Bio-Rad, Tokyo, Japan).
Statistical analysis
All numerical data were analyzed using the SPSS
software (ver. 11 for Windows; SPSS Inc., Chicago, IL,
USA). In all experiments minimum of three samples
(e.g.; for bacterial OD 6 sample-data, total WIG 4
sample-data, for VSC 4 sample-data, for MTT assay 6
sample-data, for coat thickness 10 measurements, for
others 3 sample-data) were used for each group and the
bacteriological experiments were repeated three times
under the same conditions to ensure reproducibility. For
comparisons, the Mann-Whitney U-test was used. The
levels of WIG/mm2 were analyzed by one-way analysis of
variance (ANOVA) and Tukeys HSD with a confidence
level of 95%.

RESULTS
Effects on bacterial growth
The radial diffusion test demonstrated that most of the
Ag ion-coated samples (Ti+ and SS+ wires) showed a
clear zone of more than 2 mm diameter around them
with both the cariogenic and periodontopathic bacteria
(Figs. 3, 4). In contrast, almost no detectable clear zone

(c)

A photograph of culture plates showing the results of radial diffusion tests (a). All coated samples
demonstrated bacterial growth inhibition zones around the wires. Two cariogenic bacteria used in this
study showed similar result (b, c). The asterisks indicate statistically significant differences (p<0.05).

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Fig. 4

Fig. 5

(a)

(b)

(c)

(d)

Photographs indicating the clear zone in radial diffusion test in Fn (a).

Antibacterial activities of SS and Ti wires against Fn (b), Pg (c), and Pi (d) are
shown. The asterisks indicate statistically significant differences (p<0.05).

(a)

(b)

(c)

(d)

Fluorescence photomicrographs of S. sobrinus biofilms stained with the BacLight Bacterial Viability Kit.
Clusters of S. sobrinus biofilm adhered on the surfaces of the different wires: SS (a), SS+ (b), Ti (c), and
Ti+ (d). Viable bacterial cells exhibit green fluorescence, whereas nonviable bacterial cells exhibit red
fluorescence.

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apparently inhibited by the presence of Ag ions.
The amounts of bacteria and WIG in the retained
biofilm on the wire surfaces are summarized in Fig.
6. From both results, both the coated wires (SS+ and
Ti+) showed significantly lower susceptibility to biofilm
formation compared with the uncoated wire samples
(p<0.05) after 24 h specifically. It appeared that a
maximum amount of biofilms was formed on SS and a
minimum on Ti+.

was observed around uncoated SS or Ti wires with the

cariogenic (Fig. 3) or periodontopathic (Fig. 4) bacteria.
The diameters of the growth-resistant zones of the Ag
ion-coated groups were significantly larger than those of
the uncoated groups. The effect on the cariogenic bacteria
was stronger than on the periodontopathic bacteria.
Effects on formation of S. sobrinus biofilms
Ag ion-coated wires showed bactericidal effects using
the BacLight (Fig. 5). Live S. sobrinus cells are
visualized with green fluorescence and dead cells with
red in the same microscopic location on a slab surface
by changing only the emission filters. In the uncoated
groups, only live cells were observed on the slab surface.
However, in the coated group, both dead and live cells
were present. When bacterial cells were incubated
with uncoated wires, large numbers of S. sobrinus cells
remained alive in the biofilm clusters (Figs. 5a, 5c). There
was no marked difference between the control groups.
In reverse, more dead than live cells were observed in
all the coated groups. Biofilm clusters were not formed
in the normal way; most bacteria appeared to have
suffered damage and some died even before secreting
glucan (Figs. 5b, 5d). The growth of biofilms was

Effects on the production of oral malodor gases

The cell growth of Pg was reduced in the presence
of Ti+ compared with Ti and control (Fig. 7a). Gas
chromatography showed that production of hydrogen
sulfide (H2S) and methylmercaptan (CH3SH) gases
by Pg were reduced significantly in the coated groups
compared with uncoated groups in the 8-h culture.
Lower amounts of H2S and CH3SH gases were produced
in the 16-h culture in the presence of Ag ion coated on Ti
wire surfaces (Figs. 7b, 7c).
Amount of released Ag ion
The Ag ions released from the specimens were
measured by ICP-AES. After 24 h of incubation the

(a)
Fig. 6

(a)
Fig. 7

(b)

The amount of bacterial cells (a) and WIG (b) after S. sobrinus biofilm formation with
different types of wires for 5, 10, or 24 h are shown.
The asterisks indicate statistically significant differences (p<0.05).

(b)

(c)

Bacterial cell viability of Pi after 8 and 16 h cultures with Ti wires (a).

The amounts of H2S (b) and CH3SH (c) are shown. The asterisks indicate statistically
significant differences (p<0.05).

Dent Mater J 2014; 33(2): 268274

Table 1 Silver ion release from the titanium wire
Wire immersion (24 h)

Silver ion release (ppm)

Control (Ti)

Ti+

Fig. 8

0.0430.005

Activity of human gingival fibroblast (HGF) cells

acquired from MTT assay shows that addition of
Ag ion didnt cause any change compared with
the control (grown without Ag ion) at any stages.
Growth activity was same with the control even
when the solution (released Ag ion from Ti+ in
PBS) was added at 10% concentration in the cell
culture medium (DMEM).

average concentrations of detected Ag ion were as

follows: 0 ppm for Ti wire, 0.0430.005 ppm for Ti+ wire;
shown in Table 1.
Cell cytotoxicity
Results of MTT assay show that there were no
significant differences in detection of formazan
concentrations between the experimental group and
the control group (Fig. 8). That indicates that cellular
activities were kept at almost the same levels at all
these experimental conditions. Even cell activity was
same when Ag ion concentration was 10% and also when
cells were cultured for 24 h.

DISCUSSION
The surfaces of dental appliances act as passive
surfaces and are prone to bacterial adhesion that can
eventually develop into material-associated infection20).
To prevent the adhesion of bacteria on biomaterials
surfaces several surface coating materials and surface
coating techniques are being tested21,22). Among them,
the antibacterial and antifungal effects of silver ions
have long been known. For centuries silver has been
known to have bactericidal properties. As early as 1000
B.C., the antimicrobial properties of silver in rendering

273

water potable were appreciated22). Silver compounds

have been exploited for their medicinal properties
for centuries as well23-25). Avicenna, in 980 AD, used
and prescribed silver as a blood purifier for heart
palpitations and for offensive breath23). Silver
was combined with arsephenamine, because of its
antimicrobial properties, and was used to treat syphilis
during the early part of the 20th century. From this
historical background, we sought to inhibit bacterial
growth and activities on the surfaces of coated wires.
In this study, Ag ion coating on the wire showed
anti-bacterial effects against the most common species
of oral pathogenic bacteria. In radial diffusion tests,
clear zones were obviously produced around the coated
wires, indicating that Ag ions on the coated wires were
diffusing into the gel and inhibited bacterial growth.
Clear zones caused by the coated wire were significantly
larger than those of the uncoated samples. There were
some differences in the susceptibility to Ag ions between
cariogenic and periodontopathic pathogens. The size of
clear zone in the gel with S. mutans was larger than
that with S. sorbinus. The effect of Ag ions on cariogenic
pathogens was stronger than on the periodontopathic
pathogens. This may be because anaerobic bacteria
absorb a lower amount of the ions during metabolic
activity.
Microscopy with the LIVE/DEAD BacLight
Bacterial Viability Kit revealed that Ag ions have
bactericidal activity on cariogenic cells as well as
antibacterial activity. Although not investigated in this
study, these antibacterial and bactericidal behaviors
of Ag ions may result from the following mechanisms:
inhibition of DNA replication and mitosis, effects of the
permeability of the cell membrane, and control of the
oxidation of glucose as reported by other researchers26).
The antibacterial and bactericidal effects of the Ag
ion-coated wires were confirmed in measuring waterinsoluble glucan (WIG), the main component of biofilm
formed by cariogenic bacteria. The coated wires showed
a significant reduction of WIG on the wire after 24 h.
This indicates that the Ag ion coat inhibited the bacteria
from producing the biofilm through its antibacterial
and bactericidal effects. The same effects of the Ag ioncoated wires with the cariogenic bacteria were confirmed
in the measurements of H2S and CH3SH, two major
components of the VSCs produced by periodontopathic
bacteria. VSCs were significantly reduced after 8 h
culture with the Ag ion-coated wire. Although that
effect was not significant at 16 h but reduced production
of VSCs was observed. In the present culture protocols
the antibacterial effect of Ag ion on periodontopathic
bacteria appeared to be weaker than that on the
cariogenic bacteria. More specifically, small amount of
S. sobrinus produced biofilms were collected directly
from the surfaces of the wires for analysis. While,
proportionally large volume of gases produced by Pg
were collected for VSCs analysis; might be due to the
availability of culture medium for excessive Pg growth
even after 8 h. That may have made the differenceneeds
further investigations with an improved experimental

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Dent Mater J 2014; 33(2): 268274

protocol.
The level of released Ag ion in PBS was 0.043
(0.005) ppm in this study, which is well below the mark
that WHO allows for drinking water disinfection (up to
0.1 mg/L)27). Furthermore, it seemed that this level of
Ag ion didnt cause detectable cytotoxic effect on HGF
cells. Therefore, it can be suggested the Ag ion coated
orthodontic wires would serve better by maintaining
low toxicity for mammalian tissue when used clinically.
In summary the results of this study suggest that
Ag ion coating on pure titanium and stainless steel
wires inhibited the growth and pathogenic activities
of oral cariogenic and periodontopathic bacteria,
important evidence for the future application of simple
Ag ion-coated dental materials, including orthodontic
appliances. Remarkable resemblance could also be
observed with the data reported in some recently
published articles, most of them showing enough
promises needing little more improvement19-25). In
contrast, the effect of Ag ion coating on two types of
orthodontic wires (stainless steel and titanium) was
investigated by analyzing antibacterial potency against
five species of oral pathogenic bacteria including
measurements of S. sobrinus biofilms and VSC gases
produced by Pg in this study. In addition, this study
opens a wide range of possibility for coating the surfaces
of various types of materials without limiting on the
orthodontic wires only that also may serve better
while antibacterial Ag-ion is applied. However, further
studies on the long-term anti-bacterial effects of the
Ag ion coat are required, along with clinical trials.

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