Archs oral Bid Vol. 25, pp.

175 to 179
Pergamon Press Lfd 1980. Prrnted in Great Britam

MAMMALIAN PLATELET DAMAGE IN VITRO BY

AN ULTRASONIC THERAPEUTIC DEVICE
A. R. WILLIAMSand B. V. CHATER*
Department of Medical Biophysics, University of Manchester, Manchester, Ml3 9PT and
Central Toxicology Laboratory, ICI Limited, Alderley Park, Macclesfield, Cheshire, England

Summary-Human blood platelets were aggregated in vitro within the ultrasonic field gener-
ated by a Cavitron@ dental descaling device. The aggregation increased as the power output of
the device, or the time of exposure, was increased. Rabbit platelets, which contain high concen-
trations of histamine, released their histamine when irradiated by means of this device in vitro.
The results show that mammalian platelets are susceptible to damage by the forces associated
with ultrasonic cavitation; if such damage occurred within the vasculature of a tooth pulp,
thrombosis could occur. The ability of the device to disrupt platelets in uiuo was investigated by
applying it over the ear arteries of anaesthetized rabbits at full power; no increase in plasma
histamine levels was detected.

INTRODUCTION ultrasonic power is conducted into the pulp cavity

Hand-held ultrasonic instruments are employed in
dentistry for the removal of calculus and bacterial
plaque (Balamuth, 1963). Examination of descaled
teeth using the scanning electron microscope has
shown that both manual and ultrasonic techniques
cause some form of damage to the tooth surface but
that ultrasonic devices remove less tooth structure
and produce a burnished surface (Pameijer, Stallard
and Hiep, 1972; Jones, Lozdan and Boyde, 1972).
Despite the inherently destructive nature of the ultra-
sonic technique, no adverse sequelae have been
reported (Goldman, 1961; Zach, Morrison and
Cohen, 1959) provided that the temperature rise
within the pulp cavity is minimized by cooling (Frost,
1977).
Human platelets are extremely susceptible to
damage by the hydrodynamic shear forces associated
with even the most gentle form of ultrasonic cavita-
tion (Williams, 1974, 1977). When cavitation occurs in
blood or plasma containing platelets, those platelets
close to the cavitation sites are either disrupted or
induced to undergo the release reaction. Materials
liberated from the platelets initiate a self-perpetuating
cycle of aggregation and further release in the
remainder of the platelet population (Chater and Wil-
liams, 1977; Williams et al., 1978). The resulting
aggregates would adhere to a vessel wall or occlude
small blood vessels and so participate in thrombus
formation. If this sequence of events were to occur
within the microvasculature of the pulp, pulp death
could occur.
The platelet aggregation and release studies de-
scribed above employed frequencies of the order of
one megahertz (MHz) to generate the destructive
cavitation. However, cavitation occurs more readily,
and at lower power levels, when the ultrasonic fre-
quency is decreased to about 20 to 30KHz (Esche,
1952). Thus, ultrasonic descaling devices (which oper-
ate at about 25 KHz) could damage the tooth if the
*Author for correspondence.
175
where it could induce cavitation within the blood
vessels.
We investigated the effects of a commercially avail-
able dental descaling device (CAVITRON@) on the
function of human and rabbit platelets in vitro. Other
experiments were designed to detect platelet damage
in oiuo following the irradiation of intact cutaneous
blood vessels in rabbits.
MATERIALS AND METHODS
Exposure system and dosimetry
A Dentsply-Cavitron model 700-l 1A ultrasonic
descaler (Cavitron Corp., L.I. City, N.Y.) was used in
conjunction with a solid hook probe. The device
operated at a nominal frequency of 25 KHz and had a
30Watt power output which was continuously vari-
able from zero. The output control had 3 graduations
(designated 1, 2 and 3) the intervals between which
were sub-divided by us into units of one quarter so as
to allow more precise positioning of the control knob.
The longitudinal displacement of the probe tip was
measured using a microscope at overall magnification
of x 100; its micrometer eyepiece was calibrated with
a standard calibrated slide. Figure 1 presents the dis-
placement amplitude of the probe tip as a function of
the power output setting.
Blood
Samples of human blood were collected from
healthy adult volunteers who had not consumed any
known anti-platelet drugs (e.g. aspirin) during the pre-
vious 7 days. Nine volumes of blood were removed by
venepuncture of the antecubital vein and anticoagu-
lated with one volume of 3.8 per cent trisodium
citrate. Rabbit blood was collected from the central
ear artery and similarly anticoagulated. The blood
was at all times allowed to come into contact only
with plastic or siliconized materials; this minimized
the activation of platelets during collection and prep-
aration. Platelet-rich plasma (PRP) was prepared
176 A. R. Williams and B. V. Chater
.i“\I
1
10
( with the skin overlying the blood vessel and activated
0 I I I
0 1.0 2.0
Meter reading
Fig. 1. The longitudinal displacement amplitude of the tip
of the probe as a function of the output setting (power
control knob).
from the human blood by differential centrifugation
at 2,250 g for 35 s, the blood being contained in 10 ml
plastic tubes. Platelet-poor plasma (PPP) was pre-
pared by further centrifuging the blood remaining
after the PRP had been removed, for 15 min at
2,250 g.
Platelet aggregation
Conventional platelet aggregometry (Born, 1962;
O’Brien, 1962) was performed using a Peyton dual
channel aggregometer linked to a Vitatron (Fisons)
twin channel pen recorder. Platelet-rich plasma was
adjusted with PPP to give a final cellular concen-
tration of approximately 250 x lo9 cells/l, the aggreg-
ometer being calibrated in a standard manner
(Chater, 1976). Samples (2 ml) of PRP were incubated
at 37°C in 15 mm diameter siliconized glass tubes
within the aggregometer for 3 min prior to the addi-
tion of 0.20ml of 1OOmM adenosine diphosphate
(ADP) (Sigma) in 0.9 per cent NaCl which had been
stored at -20°C before use. The platelet suspensions
were continuously stirred at l,OOOrev/min during
aggregation by means of a magnetically-driven stirrer.
No ADP was added to the platelet suspensions
which were to be subjected to ultrasonic irradiation.
Here, the PRP samples were pre-incubated for 3 min
at 37°C and then the tip of the probe was immersed
so that it just interrupted the light path through the
stirred sample. The probe was not allowed to touch
the wall of the sample tube. Immediately after ex-
posure (timed with a stop watch), the probe was with-
drawn and washed while the platelets were allowed to
aggregate.
Histamine release
Platelet damage in whole blood was quantified by
the measurement of plasma histamine levels. Rabbit
blood (2.0ml) was maintained at 37°C and stirred
within the aggregometer. The sample was exposed to
ultrasonic irradiation as described above and then
stirred at 37°C for 2 min before adding 200~1 of
EDTA-theophylline to inhibit the platelet-release
reaction. The free-histamine content of each blood
sample was subsequently assayed by the automated
fluorimetric assay of Evans et al. (1973).
The in vivo experimental system
Fifteen adult New Zealand white rabbits (body
weight 2.5-3.5 Kg) were anaesthetized by the intra-
venous administration of Sagittal@ (pentobarbital) at
a dosage of 0.5 ml/Kg. The ears were shaved to reveal
the central ear artery, and a control 2.5ml blood
sample was collected by means of a syringe fitted with
a 21 g Luer hypodermic needle.
Aquasonic@ coupling gel was applied to the shaven
skin overlying the central ear artery at a site just
upstream of the point where the test sample was to be
collected. The ultrasonic probe was placed in contact
3.0 at setting number 3 (i.e. maximum displacement
amplitude) for approx. 25 s. The test sample of blood
was collected while the ultrasound was being applied.
All blood samples were immediately anticoagulated
with EDTA-theophylline and the histamine content
of their plasma was assayed as described above.
RESULTS
Two superimposed aggregation traces obtained
with 2 aliquots from the same blood sample are
presented in Fig. 2. The traces were lined up so that
the 5-s irradiation using the Cavitron device co-
incided with the addition of the ADP in the other
sample. Both traces are similar in shape but the
ultrasonically-induced sample appears to have a
longer delay before the appearance of the hump in-
dicative of platelet shape change.
Human PRP was irradiated as described above for
2 s at various output settings. Five representative
aggregation traces are given in Fig. 3. Trace A was
obtained at setting number 1 and shows that little
aggregation had occurred. Above this value, the
extent of aggregation increased with increasing power
output up to a maximum at about 2.0 (trace D).
Above this value, the initial rate of aggregation was
increased, but the overall extent (i.e. the maximum
lo-
Cavitron
A.tI.P.
50-J , I I I 1 1
0 1 2 3 4 5
Time in minutes
Fig. 2. Comparison of the aggregation traces obtained at
37°C with aliquots from the same human PRP suspension
stimulated by a final concentration of 1OmM of added
ADP or a 5 s exposure to ultrasound at an output setting
of 2.0.
 In vitroultrasonic platelet damage 177

ultrasound range O-7.5 s. Exposure times less than 2-2.5 seconds

on off at this power level resulted in minima1 (&lo per cent)
aggregation (Fig. 5). Exposure times longer than 3.5 s
o- resulted in maximum aggregation (5CMOper cent
with this particular sample of PRP).

lo- Histamine release from rabbit platelets in vitro

6 Whole blood anticoagulated with trisodium citrate
;=
w 20- was exposed at 37°C to ultrasound at output settings
e of 1, 2 and 3, for increasing periods of time. The
B plasma levels of histamine obtained after the blood
G
i 30- was incubated at 37°C for 2 min reflect the extent of
c platelet disruption and the number of platelets subse-
S quently induced to undergo their physiological release
40
reaction. These results (Fig. 6) show that setting 1
1 caused negligible release of histamine even after I5 s
50-I, I I I I I I irradiation. At setting 2, a significant number of
0 1 2 3 4 5 6 platelets had liberated their histamine after 10 s ir-
Time in minutes radiation, whereas at setting number 3 a large propor-
Fig. 3. Five representative aggregation traces obtained
tion of the platelet population had released or been
with aliquots of the same human PRP exposed for 2 s to disrupted after only 2 s irradiation.
ultrasound at output settings of 1.0 (trace A), 1.5 (B), 1.75
(C), 2.0 (D) and 3.0 (E). 601

amount of aggregation) was decreased (e.g. trace E at

an output setting of almost 3).
The values of maximum percentage aggregation
40-r
obtained from a more extensive series of aggregation .g
'\
traces are presented graphically in Fig. 4. Having P

established that the probe itself did not induce aggre- k 30-
t
gation, it was found that output settings below 1 also ;
ii
had no effect. Above this value, the extent of aggrega-
* zo-
tion increased with increasing power up to 1.5 and
thereafter increased more rapidly with increasing
power up to 2.0. Above this value, the extent of maxi- lo-
mum aggregation began to decrease even though the
:;
ultrasonic power emitted by the device continued to
increase. 00 1.0 2.0 3.0 3.5
Metersetting
The effect of increasing exposure time
Fig. 4. The extent of maximum aggregation obtained lrom
Human PRP was irradiated with ultrasound at an traces similar to those of Fig. 3 plotted as a function of the
output setting of 1.5 for various exposure times in the output setting after a 2 s exposure at 37°C.

1
60

M-

I - I I I I I 1
1 2 3 4 5 6 7 8
Time in seconds
Fig. 5. The effect of time of exposure on the extent of maximum aggregation of human PRP at an
output setting of 1.5 at 37°C.
178 A. R. Williams
.J”
r
5 io
Ex~suretimein seconds
Fig. 6. The concentration of histamine released into the plasma when rabbit blood was exposed
ultrasound at 37°C in uitro at output settings of 1, 2 and 3 for various times up to 15 s.
The effects ofin vivo irradiation
The sensitive fluorometric assay was used to
measure plasma histamine levels in rabbits immedi-
ately after the blood has been subjected in viuo to
ultrasound irradiation at power setting 3 from the
Cavitron device. Control and test blood samples were
collected from the central ear artery from 15 animals;
no significant differences were observed (Control
30.4 f lO.O(SD)ng/ml; Test 32.3 k 11.9 ng/ml).
DISCUSSION
The cavitating ultrasonic field generated by the
Cavitron dental descaling device induced human
platelets to aggregate in the absence of any other
stimulus (Fig. 2). Our earlier work (Williams et al.,
1978) showed that the large hydrodynamic shear
forces associated with ultrasonic cavitation can dis-
rupt adjacent platelets and liberate their contents,
which includes ADP. When enough ADP has been
released, other undamaged platelets are stimulated to
aggregate and undergo their physiological release
reaction. Thus, the prolonged delay before the soni-
cated sample begins to aggregate (Fig. 2) is the time
taken to disrupt a sufficient number of platelets.
This effect is dose-dependent (Figs 3 and 4) and
below a power setting of about 1 there was, presum-
ably, little cavitation and consequently little aggrega-
tion. The explanation for the effect at power settings
higher than 2 is that so much cavitation occurs at
these levels that a large proportion of the platelet
population is disrupted in a short time. The high
levels of ADP present result in extremely rapid aggre-
gation of the remaining platelets, but so few of these
survive that the maximum extent of aggregation is
reduced.
The results in Fig. 5 can be explained as follows:
Less than 2 s irradiation at a setting of 1.5 liberates
and B. V. Chater
is
to
too little ADP to induce the remainder of the platelet
population to aggregate, whereas irradiation for times
longer than 3 s have liberated sufficient to ensure that
aggregation can occur at maximal rate.
Rabbit platelets are physiologically less sensitive to
aggregation by ADP than human platelets (Chater,
1976). Unlike human platelets, they contain large
quantities of histamine which can be used as a con-
venient and specific marker of platelet damage and
the release reaction (Henson and Cochrane, 1969).
This low sensitivity of rabbit platelets is demonstrated
in Fig. 6 where 15 s irradiation at setting number 1,
and 7 s at setting number 2, failed to induce the
release of detectable quantities of histamine; these
conditions produced maximal aggregation of human
PRP. Under these experimental conditions, aggrega-
tion and the release reaction occur simultaneously
and are both different aspects of the same platelet
function. However, more prolonged sonication or
sonication at higher power-levels did induce the rab-
bit platelets to release and to aggregate.
The in viuo histamine release results indicate that,
under our experimental conditions, the ultrasonic ir-
radiation emitted by the Cavitron device did not dis-
rupt a detectable proportion of the platelet popula-
tion. However, several factors have mitigated against
our obtaining a positive biological result. One bio-
logical factor is the decreased sensitivity of rabbit
platelets which means that a larger stimulus of ADP
is necessary before the release reaction will occur.
Also, the short time interval between sonication in
uiuo and the inhibition of platelet function with
EDTA and theophylline would prevent the materials
liberated from any damaged platelets from inducing
the adjacent undamaged platelets to undergo their
normal release reaction and so amplify the result.
Physical factors which would tend to yield a nega-
tive result under our in uiuo sonication conditions are
the time of exposure and the geometry of the ex-
 In vitro ultrasonic platelet damage
posure arrangements. For the in uitro situation, the
gaseous micronuclei in the blood or plasma were sub-
jected to the alternating pressure field produced by
the vibrating tip for the entire duration of the ex-
posure. This would allow ample time for the micro-
nuclei to grow, fuse with each other and establish
their oscillatory patterns, i.e. undergo stable or
vaprous cavitation. Reflections from the walls of the
sample tube would assist these processes by increas-
ing the acoustic pressure amplitudes at various sites
within that tube, i.e. a partial standing wave situation.
However, any portion of the blood flowing in uiuo is
only within the alternating pressure field for 0.1 s.
This time appears to be too short to develop an
appreciable amount of intravascular cavitation at
these power levels. Also, the vessel is surrounded with
soft tissues which will only reflect a small proportion
of the incident ultrasonic energy. It is therefore more
probable that we would obtain a positive result if the
blood was flowing more slowly and/or a strong reflec-
tor of acoustic energy (e.g. bone) was behind or even
around the treated blood vessel.
Our findings show that human platelets are highly
susceptible in vitro to the acoustic cavitational fields
generated by the Cavitron descaling device at the
power levels commonly employed in the clinic. Con-
sequently, if enough of this energy enters the tooth it
could induce the platelets to aggregate and participate
in thrombosis which may result in the death of the
pulp. Consequently, the efficiency of the coupling
between the probe tip and the surface of the tooth
and the effects of tooth geometry and the trans-
mission and reflective properties of the enamel and
dentine need to be investigated to see if acoustic cavi-
tation (in any of its various forms) could be induced
within the flowing blood in uiuo.
Our preliminary in uiuo investigations have shown
that under conditions of rapid blood flow in a large
artery (approx. 3 mm diameter) in a rabbit, there is
probably no significant danger of thrombosis. How-
ever, the unusual geometry of the tooth coupIed with
the slower flow rates of the blood within the pulp
cavity suggest that pulpal thrombosis during or after
ultrasonic deszaling is still a possibility.
179
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