Original article

A study of the efficacy of ultrasonic waves in removing biofilms

Takeshi Nishikawa1, Akihiro Yoshida2, Amit Khanal1, Manabu Habu1, Izumi Yoshioka1,
Kuniaki Toyoshima3, Tadamichi Takehara2, Tatsuji Nishihara4, Katsuro Tachibana5 and
Kazuhiro Tominaga1
1
Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu,
Japan; 2Division of Community Oral Health Science, Department of Health Promotion, Kyushu Dental College, Kitakyushu, Japan; 3Division of
Oral Histology and Neurobiology, Department of Biosciences, Kyushu Dental College, Kitakyushu, Japan; 4Division of Infections and Molecular
Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu, Japan; 5Department of Anatomy, Fukuoka University School
of Medicine, Fukuoka, Japan

doi:10.1111/j.1741-2358.2009.00325.x
A study of the efficacy of ultrasonic waves in removing biofilms
Objective: The removal of adherent biofilms was assessed using ultrasonic waves in a non-contact mode.
Materials and Methods: In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to
ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0–90%), and exposure
times (1–3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the
effect of adding a contrast medium, such as micro bubbles (Sonazoid), was examined. The spatial dis-
tribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined
via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments,
using a custom-made oral cleaning device.
Results: When a 280 kHz probe was used, the biofilm-removing effect increased significantly compared to
1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50–90%
and a 3 minutes exposure time. The maximum biofilm-removing effect was observed with a duty cycle of
80%. Furthermore, the addition of micro bubbles enhanced this biofilm-removing effect. In in vivo
experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes.
Conclusions: This study demonstrated that ultrasonic wave exposure in a non-contact mode effectively
removed adherent biofilms composed of S. mutans in vitro.

Keywords: ultrasonic wave, Streptococcus mutans, biofilm, micro bubble.

Accepted 21 April 2009

At present, the oral healthcare system is inade-

Introduction
The geriatric population world-wide has increased
markedly because of advances in medical science
and technology1. However, the death rate owing to
simple ailments, such as pneumonia, has also
increased. Indeed, in Japan, pneumonia is the
fourth leading cause of death, and people over
65 years of age account for 92% of pneumonia
fatalities2. Many studies have found a positive
relationship between the risk of mortality and oral
health and hygiene in elderly patients with pneu-
monia, and many such cases are caused by silent
aspiration3–5. Aspiration pneumonia is not only
damaging the patient’s health but also imposes a
major medical insurance/financial burden.
quate to provide necessary services, such as routine
oral cleaning, to bedridden, handicapped and
geriatric patients; this is due to a lack of caregivers
with appropriate technical skills, scarce equipment
and economic factors. Thus, the significance of
good oral care and hygiene cannot be overstated in
patients who are susceptible to aspiration pneu-
monia, such as geriatric or bedridden patients3.
Accordingly, we are seeking to develop a simple,
safe and inexpensive oral cleaning device that can
be used by anyone for effective dental cleaning.
The removal of dental plaque is the most
important step in oral cleaning. Routine oral
hygiene, such as tooth brushing and cleaning, is
one of the most effective ways to control plaque6–8.

 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2010; 27: 199–206 199
200 T. Nishikawa et al.
The manual toothbrush has been the most com-
monly used device for removing dental plaque
since ancient times. Recently, various types of
powered and ultrasonic toothbrushes have been
developed with the aim of removing plaque more
efficiently, and their clinical efficacy has been
reported7–12. Indeed, studies have demonstrated
that powered and ultrasonic toothbrushes are
more efficient than manual toothbrushes in this
regard7–9,13,14. However, the plaque-removing
effect is limited to the areas that are accessible to
the bristle tips. Thus, it is impossible for these types
of toothbrushes to contribute significantly to
improving oral health and hygiene in bedridden,
geriatric or handicapped patients.
Ultrasonic waves are cyclic sound pressure waves
with a frequency greater than the upper limit of
human hearing (i.e. 20 kHz). The mechanisms of
ultrasonic wave function consist of cavitation,
rectilinear flow and acceleration of the medium,
which act synergistically15,16. The cavitation effect
is one of the main by-products of ultrasonic waves.
This phenomenon, when applied in a fluid, pro-
duces equal portions of positive and negative
pressure, alternately, where the negative pressure
splits the fluid and produces a cavity15,17. When
fluid is pressurised, the cavity is crushed and
destroyed instantly, during which the molecules
collide with each other, causing local shock
waves18. Such shock waves have recently been
used in gene and drug delivery systems19–21. Fur-
thermore, this cavitation effect is widely used in
washing precision instruments, such as lenses,
jewellery, hard disks, quartz oscillators, solar bat-
teries and medical equipment.
The aim of this study was to exploit the cavita-
tion effect of ultrasonic waves to develop a new and
efficient plaque control method. Biofilms composed
of Streptococcus mutans, an early colonising organism
that is responsible for acid production and the
progression of caries, were prepared in vitro. Simi-
larly, ultrasonic waves were also applied to the
biofilm in a non-contact mode in a clinical exper-
iment (in vivo) to identify the optimal conditions for
biofilm removal.
Materials and methods
In vitro experiments
Bacterial strains A monoclonal strain of S. mutans
(UA159) was used. A frozen stock of S. mutans
UA159 was cultured in 5 ml of brain heart infusion
(BHI) broth (Difco Laboratories, Detroit, MI, USA)
at 37C under anaerobic conditions for 24 h.
 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2010; 27: 199–206
Biofilm growth Biofilms were prepared in 24-well
polystyrene microplates (Iwaki Brand; Asahi
Techno Glass, Tokyo, Japan). Each microplate was
partitioned into four groups of six wells; three
groups of wells were used for the experiment and
the remaining group was used to create a calibra-
tion curve.
Growth was initiated by inoculating individual
wells with 2 ll of a microbial suspension in 1 ml of
BHI broth, supplemented with 0.5% sucrose. The
microplates were incubated at 37C under anaer-
obic conditions for 48 h without agitation.
Biofilm staining After the 48-h incubation, the
liquid medium was removed from the plate. The
wells were rinsed gently with fresh water, stained
with a plaque-disclosing solution (DENT Liquid
Plaque Tester; Lion Co. Ltd, Tokyo, Japan), and
rinsed twice with fresh water to remove excess dye.
Then, 3 ml of sterile phosphate-buffered saline
(PBS) was added to each well. Of this, 1 ml from
each well was removed and designated ‘sample 1’.
Exposure to ultrasonic waves The ultrasonic trans-
ducer used here was a Sonopore KTAC-3000
(Nepa Gene Co. Ltd, Chiba, Japan), which is nor-
mally used for gene transfection purposes. The
frequency of the various detachable ultrasonic
probes was adjustable, ranging from 200 kHz to
3 MHz. A concave ultrasonic probe (6 mm in
diameter) was used at various frequencies
(280 kHz, 1 MHz or 2 MHz). The duty ratio
(0–90% duty cycle), ultrasonic exposure time
(0–99 s), burst rate (0.1–99.9 Hz) and intensity
output (0–3.00 W) were also adjustable. Optimal
conditions for burst rate and intensity depended
on the frequency used.
Streptococcus mutans biofilms in 18 wells contain-
ing 2 ml of PBS were exposed to ultrasonic waves
at various frequencies (280 kHz, 1 MHz or 2 MHz),
duty ratios (0–90%) and exposure times (1–3 min).
The distance between the ultrasonic probe and the
plate bottom was maintained at 5 mm. Further-
more, the effect of adding micro-bubbles
(Sonazoid; Daiichi-Sankyo, Tokyo, Japan; GE
Healthcare, Milwaukee, WI, USA) at a frequency of
280 kHz and a 1-min exposure time was examined,
while the duty ratio was varied. Ultrasonic wave
exposure dispersed and sheared biofilms; a sample
of PBS containing this sheared biofilm was col-
lected and designated ‘sample 2’.
Assay of biofilm removal effect The absorbance ratios
of samples 1 and 2 at OD600 were measured with a
dual-wavelength spectrophotometer (Ultrospec

3000 UV/Visible Spectrophotometer; Pharmacia
Biotech Ltd, Cambridge, UK). To obtain a calibra-
tion curve, 25, 50, 75 and 100% of the biofilm
adhering to bottom of four control wells was
mechanically removed with sterile toothpicks,
whereas the two remaining wells were left intact as
unscrubbed controls. This assay was repeated using
four separate plates. The absorbance ratio before
and after the procedure was measured and a
calibration curve prepared. The absorbance ratio in
each condition was converted into a biofilm
removal ratio based on the calibration curve gen-
erated in each plate. Then, the biofilm-removing
effects of the procedure were assessed.
Scanning electron microscopy The spatial distribution
of S. mutans biofilms before and after ultrasonic
wave exposure was examined via scanning elec-
tron microscopy (Hitachi S-4300 FE; Hitachi Co.
Ltd, Tokyo, Japan). Individual wells of a 24-well
microplate were filled with 1 ml of BHI broth
supplemented with 0.5% sucrose. A sterile, poly-
styrene disc was added to each well, and each well
was then inoculated with 2 ll of the previously
described microbial suspension in 1 ml of BHI
broth supplemented with 0.5% sucrose. The plates
were incubated at 37C under anaerobic conditions
for 48 h without agitation. Then, the disks to which
the biofilm had adhered were divided into two
groups; the non-exposed (A) and ultrasonic wave-
exposed groups (B), rinsed briefly with PBS, and
transferred to another 24-well microplate contain-
ing 2 ml PBS in each well. Group B was exposed to
ultrasonic waves using 280 kHz and 1 MHz probes
at an 80% duty cycle for 2 min. The disks were
rinsed briefly with PBS and then fixed with 2.5%
glutaraldehyde in 0.1 M sodium cacodylate buffer
(pH 7.3) for 30 min. Following dehydration
through a graded series of ethanol solutions, the
samples were freeze-dried with t-butyl alcohol and
sputter-coated with platinum, approximately 2 nm
thick. The samples were then examined under a
scanning electron microscope.
Measurement of thermal changes caused by ultrasonic
waves A temperature sensor (Ray Temp Infrared
and Probe Thermometer; Electronic Temperature
Instruments Ltd, Worthing, West Sussex, UK) was
inserted into a 15 ml centrifuge tube containing
2 ml PBS, and suspended in a water bath main-
tained at 37C. Then, thermal changes caused by
ultrasonic exposure from the 6-mm diameter probe
at 280 kHz, 1 MHz and 2 MHz were measured
every minute for a 10-min period. The temperature
rise was less than 0.3C at all time points.
 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2010; 27: 199–206
Ultrasonic waves in removing biofilms 201
In vivo experiments
Three healthy volunteers without any decayed or
filled anterior maxillary teeth took part in the study
and this part of the study was performed with the
approval of the ethics committee of Kyushu Dental
College and informed consent was obtained from
each subject. The bilateral maxillary lateral incisors
and canine teeth were examined. The subjects re-
ceived oral prophylaxis, during which plaque and
calculus were removed and the teeth were pol-
ished. They then refrained from brushing for 72 h.
After 72 h, 2 oz of plaque-disclosing solution
(2Tone Solution; Young Dental Mfg. Co. Ltd, Earth
City, MO, USA) was applied to the labial surfaces of
eight teeth: those to be sonicated and the adjacent
teeth (central incisors and first premolars). The
subjects were asked to rinse out their mouth gently
with water, and photographs of the labial surfaces
of the stained teeth were taken using a specialised
digital camera for dentistry (Eye Special C-1; Shofu
Inc., Kyoto, Japan).
A custom-made oral cleaning device consisted of
two separate chambers that enclosed the anterior
maxillary teeth. The ultrasonic probes were fitted
into the right chamber (experimental side) and
positioned at 5 mm away from the labial surface,
whereas the left chamber without a probe served as
control side. The device was built from two materi-
als; 0.5-mm-thick Polyester (Duran; Scheu Dental
GmbH, Burgberg, Germany) and 4.0-mm-thick
ethylene vinyl acetate (Bioplast; Scheu Dental
GmbH). The former was used as an inner layer of the
chamber while the latter was used as an outer layer.
Fresh tap water was poured into both chambers of
the device, and then applied to the maxilla. No
entrapment of air bubbles inside the chamber was
verified, making the seal air-tight (Fig. 1). Ultrasonic
wave exposure was performed using the three
specified probes at 80% duty cycles for 2 and 5 min.
After ultrasonic wave exposure, photographs of the
labial surface of maxillary lateral incisors and ca-
nines were taken as described earlier. Debris scores
were compared using the photograph taken before
and after ultrasonic wave exposure. To assess the
debris on each surface, each plaque scores were
determined using the patient hygiene performance
(PHP) method22. The plaque removal rate was cal-
culated by the following formula:
Average of debris scores using PHP after experiment
1
Average of debris scores using PHP before experiment
The plaque removal rate between experimental
side and control side was compared. The data were
analysed with Mann–Whitney U-test.
202 T. Nishikawa et al.
(a)
(b)
(c)
Figure 1 (a) Ultrasound wave source attached with a
custom made oral cleaning device. (b) Custom-made oral
cleaning device with ultrasonic probe inserted. (c) The
same device in a clinical set-up.
Results
In vitro experiments
Biofilm removal at 280 kHz, 1 MHz and 2 MHz Ultra-
sonic wave exposure at 280 kHz for 3 min removed
more than 80% of the adherent biofilm at a duty cycle
of 60–90%. However, ultrasonic wave exposure at
1 MHz for the same period resulted in a biofilm
removal rate of less than 20%. The biofilm removal
rate was similarly poor when using the 2 MHz probe
(Fig. 2).
 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2010; 27: 199–206
Figure 2 Biofilm removal rate at 280 kHz, 1 MHz and
2 MHz. In these experiments, the biofilms were exposed
to ultrasonic waves for 3 min while maintaining a
distance of 5 mm from the source [n = 6, *p < 0.01,
(ANOVA)].
Figure 3 Biofilm removal rate with or without Sona-
zoid at 280 kHz. In these experiments, the biofilms
were exposed to ultrasonic waves for 1 min while
maintaining a distance of 5 mm from the source [n = 6,
*p < 0.05, (Student’s t-test)].
Biofilm removal after Sonazoid addition After the
addition of Sonazoid, ultrasonic exposure at
280 kHz for 1 min resulted in a biofilm removal
rate of approximately 80% at a 50% duty cycle,
which was approximately equal to a 3-min expo-
sure using the same probe and duty cycle without
Sonazoid (Fig. 3).
Morphological features of Streptococcus mutans biofilms
after ultrasonic wave exposure Under a scanning
electron microscope, a thick layer of S. mutans and a
considerable amount of water-insoluble glucans
were observed on the control disk. In contrast,
ultrasonic wave exposure using the 1-MHz probe
resulted in a thinner layer of plaque compared with
the control disks; S. mutans was isolated from these
experimental disks and water-insoluble glucans
had spread. Ultrasonic wave exposure at 280 kHz
resulted in the disruption of bacterial chains, of
which few were observed, and the scattering of
water-insoluble glucans (Fig. 4).
 Ultrasonic waves in removing biofilms 203

Figure 4 Scanning electron micro-

graphs showing the spatial distribu-
tion and architecture of the
non-exposure group (A, a), the group
exposed to 1 MHz (B, b), and the
group exposed to 280 kHz (C, c). In
these experiments, the biofilms were
exposed to ultrasonic waves at an
80% duty cycle for 2 min. Magnifi-
cation: A)C, 1000¢; a)c, 3000¢.

2 min, and moderate biofilm removal was observed

In vivo experiments
after 5 min; however, a significant difference was
The effects of ultrasonic wave exposure using a not observed for both conditions (Figs 5 and 6).
custom-built device were examined in three vol-
unteers. Exposure at 1 and 2 MHz had very little
Discussion
observable effect in terms of biofilm removal.
While using a 280-kHz probe with a duty cycle of We examined the effects of ultrasonic wave expo-
80%, very little biofilm removal was observed after sure on in vitro biofilms composed of S. mutans and

US exposure side Control side

(a) (b)

Before

Figure 5 In vivo experiment using a

(c) (d)
custom-made oral cleaning device.
(a) and (b) are before ultrasonic wave
exposure, whereas (c) and (d) are After
after 5 min of exposure. Some bio-
film removal was observed on the
ultrasonic wave-exposed side (c).

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204 T. Nishikawa et al.
Figure 6 In vivo experimental right side showed slightly
higher plaque removal rate than the left control side
when 280 kHz probe was used for 5 min duration;
however, a significant difference was not observed;
[n = 12, p-value = 0.157 (Mann–Whitney U-test)].
in vivo plaque formation, with markedly superior
results in the former case. It should be noted that
the biofilms that we examined in vitro are not truly
representative of plaque, which is a much more
complex and mature collection of many interacting
species. In addition, the polystyrene microplate
surface is not an especially good model of the
tooth’s enamel surface. However, a biofilm of S.
mutans at 48 h is a rough approximation of plaque,
and similar techniques to simulate the oral micro-
environment have been used routinely. Thus, the
effects of the various parameters examined here
likely also apply to real plaque. More specifically,
our data indicate that ultrasonic wave exposure at
a frequency of 280 kHz is particularly effective
in disrupting biofilms, and that the addition of a
micro-bubble agent, such as Sonazoid, further
enhances this effect.
The ultrasonic wave source used here had many
variable settings, including frequency, duty ratio
and burst rate, and was capable of being used in
many experimental conditions. A wave pulse
(burst rate, 50%) was used to avoid increases in
temperature. Initially, we sought to mimic the
commercially available ultrasonic Ultima tooth-
brush (1.6 MHz) by using 1 and 2 MHz probes;
however, these frequencies showed poor biofilm
removal. Much improved results were obtained
when using a 280-kHz probe. The reason for this
enhanced effect at 280 kHz may be due to the
mechanism of ultrasonic cleaning, which is the
synergistic combination of cavitation, rectilinear
flow and acceleration of the medium15,16. When a
low frequency is used, the ultrasonic waves are
capable of removing adherent masses, primarily
through cavitation effects. When a high frequency
is used, primarily rectilinear flow and acceleration
of the medium are involved in removing small
 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2010; 27: 199–206
masses. Thus, an intermediate frequency of
280 kHz results in greater cavitation effects and less
rectilinear flow and acceleration of the medium. As
a result, biofilm removal was more pronounced
with the 280-kHz probe.
Furthermore, the addition of an ultrasonic con-
trast medium (Sonazoid) permitted greater biofilm
removal over a shorter time. Recently, micro-
bubbles have been used in various experiments in
ultrasonic gene transfection20, diagnostic exami-
nation23, and transcranial thrombolysis24. Sonazoid
consists of micro-bubbles of 2.3–2.9 lm in diame-
ter, which are filled with perflubutane gas and the
major biological effect is probably because of
acoustic cavitation. It was thought that the explo-
sion of micro-bubbles caused by the cavitation
effect of ultrasonic wave reinforced further biofilm
removal effect. Thus, the increase in biofilm
removal after Sonazoid addition may be the result
of enhanced cavitation.
To examine biofilm removal in vivo, we custom-
built an oral cleaning device for clinical use that
utilised ultrasound waves in a non-contact mode.
During device design, medical safety standards for
ultrasound usage were observed. Our preliminary
in vivo experiments showed slight plaque removal
after application for 5 min. Digital photographs
taken after the trial showed fading of the stained
plaque, although assessment using a plaque index
indicated that this effect was insignificant. This
apparent lack of effect may be due to the fact that
natural dental plaque consists of various bacilli and
other organisms that are able to attach much more
effectively to tooth enamel, at least in comparison
to biofilms composed of S. mutans adhering to a
smooth polystyrene plate.
Several studies have discussed the medical safety
of ultrasound13,14. The US Food and Drug Admin-
istration (FDA) has specified that, for safety rea-
sons, any ultrasound-induced temperature rise in
the soft tissues, bones or skull must be less than
1C25. At a World Federation for Ultrasound in
Medicine and Biology (WFUMB) symposium
in 2006, it was concluded that a maximum rise in
temperature of no more than 1.5C above the
normal physiological level (37C) during a diag-
nostic exposure may be used clinically without
reservation on thermal grounds26. Devices such as
ultrasonic scalars (15–40 kHz) and toothbrushes
(1.6 MHz) are commonly used in dentistry and for
daily oral prophylaxis27,28. Thus, we postulated
that any oral cleaning device with a frequency in
the range of 10 kHz to 2 MHz with a temperature
rise of <0.3C was unlikely to be hazardous to the
teeth or gingiva.

In conclusion, our in vitro experiments showed
promising results regarding the removal of mono-
strain S. mutans biofilms using ultrasonic wave
exposure. The addition of an ultrasonic wave con-
trast medium, such as Sonazoid micro-bubbles,
further enhanced the biofilm-removing effects in
in vitro experiments. However, Sonazoid was not
used in in vivo experiments because of its high cost
and less practicality at this time. Therefore, future
in vivo experiments will require various alterations
in terms of experimental design and set up.
Our ultimate goal is to create an automatic oral
cleaning device equipped with various amenities,
like water rinsing and a drainage facility, for
bedridden, geriatric patients or handicapped per-
sons as a supportive oral healthcare system. Further
research is required regarding device design and
manufacture and the development of a suitable
medium to enhance the effects of ultrasonic waves,
efficiently and safely.
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