J Dent Res 77(2): 387-392, February, 1998
Threshold for Bone Resorption
Induced by Continuous and Intermittent
Pressure in the Rat Hard Palate
T. Sato*, T. Hara, S. Mori, H. Shirai, and S. Minagi
Department of Removable Prosthodontics, Okayama University Dental School, 2-5-1, Shikata-cho, Okayama 700, Japan; *corresponding
author
Abstract. Osteoclastic bone resorption under the denture
base has been considered to be influenced by mechanical
force exerted through the denture base. However, no
scientific evidence has been shown concerning whether
osteoclastic bone resorption is a pressure-threshold-
regulated phenomenon or merely a proportionally pressure-
dependent phenomenon. In this study, a clear statistical
criterion as an objective standard for the evaluation of
histopathological bone resorption was established, and the
relationship between the amount of continuous or
intermittent compressive pressure exerted on the rat hard
palate and induced osteoclastic bone resorption was studied
based on this criterion. Eighty-five male rats of the Wistar
strain, 15 weeks of age, were fitted with experimental
dentures which were designed to exert a defined amount of
continuous or intermittent compressive pressure on the
intermolar region of the hard palate. No bone resorption
was observed when continuous compressive pressure was <
1.96 kPa or when intermittent compressive pressure was <
9.8 kPa. In contrast, continuous compressive pressure - 6.86
kPa or intermittent compressive pressure > 19.6 kPa caused
significant bone resorption in all rats studied. From these
results, it was revealed that osteoclastic bone resorption
under the denture base was a pressure-threshold-regulated
phenomenon, and that the thresholds exist for the induction
of bone resorption under the denture base by both
continuous and intermittent compressive pressure,
respectively. It was also clearly revealed from the results
that the threshold for continuous compressive pressure is
lower than that for intermittent compressive pressure.
Key words: threshold, osteoclastic bone resorption,
continuous compressive pressure, intermittent compressive
pressure.
Received January 2, 1997; Last Revision June 12, 1997;
Accepted June 21, 1997
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Introduction
Mechanical pressure transmitted continuously and/or
intermittently through the denture prosthesis has been
considered to be one of the causative factors of bone
resorption in denture-supporting tissues (Lytle, 1959;
Heartwell and Rahn, 1986). However, despite the frequent
discussions about the possible existence of a threshold for
osteoclastic bone resorption (Pendleton, 1951; Weinmann
and Sicher, 1955), no definitive evidence showing whether
osteoclastic bone resorption is a pressure-threshold-
regulated phenomenon or merely a proportionally pressure-
dependent phenomenon has been reported to date. This
lack of scientific evidence is a result of studies which up to
this point have been only qualitatively oriented.
Multi-factorial causation is another feature of the
histopathological change in denture-supporting tissue which
has made the approach to the study of this tissue more
complicated. Although most histopathological studies of
denture-supporting tissue have not avoided this complexity of
causative factors (Ostlund, 1958; Kapur and Shklar, 1963;
Turck, 1965; Nedelman et al., 1970; Jani and Bhargava, 1976;
Cook, 1991), a few researchers have succeeded in simplifying
the conditions for an experimental denture base (Nakashima et
al., 1994; Hara et al., 1996; Mori et al., 1997). The well-controlled
and simplified conditions reported by Nakashima et al. (1994),
in particular, could be utilized as a stable platform for the
statistical analysis of histopathological findings.
The purpose of this study was to investigate the nature of the
pressure dependency of osteoclastic bone resorption in the rat
hard palate, by means of bone resorption induced by continuous
or intermittent compressive mechanical pressure. A statistical
analysis of the histopathological results was adopted in this
study to establish an objective criterion for standardization of
the phenomenon of osteoclastic bone resorption.
Materials and methods
Experimental animals
Eighty-five male rats of the Wistar strain, 15 wks of age (Japan
SLC Inc., Shizuoka, Japan), were used in this study. The animal
387
388 Sato et al.
Figure 1. Schematic diagram of the experimental denture for
continuous compressive pressure (N = 45). (A) Occlusal view of the
experimental denture in position. (B) Frontal section at the antero-
posterior middle of the experimental denture (chain line) in
position. M = fixed metal framework; RD = resin denture base; TM
= orthodontic tube in fixed metal framework for insertion of the
rigid resin denture base (diameter equal to that of orthodontic
wire); TD = orthodontic tube in denture base for threading the
orthodontic wire, and for insertion of the rigid resin denture base
(diameter equal to that of orthodontic wire); W = orthodontic wire.
use protocol was reviewed and all the experiments were
conducted according to the animal experimental guide
approved by the Animal Experiment Committee, Okayama
University Dental School. The rats were divided into two test
groups: continuous-pressure group (n = 45) and intermittent-
pressure group (n = 40).
Experimental denture
Two types of experimental denture were constructed for the
application of continuous or intermittenit pressure on the
intermolar region of the rat hard palate, respectively. Each
experimental denture consisted of a fixed metal framework part
and a removable denture base, as shown in Figs. I and 2. The
fixed metal (Ag-Au-Pd alloy) framework was made to fit the
maxillary molars of each individual rat and was attached to the
molars with dental adhesive (PanaviaO EX, Kuraray Co., Ltd.,
Osaka, Japan) according to the method described by Nakashima
ct al. (1994).1n the continuous-pressure group, a prescribed
intensity of continuous compressive pressure was exerted
against the left side of the intermolar region of the hard palate
by way of the removable denture base (Fig. 1). The intensities of
continuous compressive pressure applied to the mucosal
surface were 0.49, 0.98, 1.96, 2.94, 3.92, 4.90, 5.88, 6.86, and 7.84
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I Dcnt Rcs 77(2) 1998
B
,M TM
C
Figure 2. Schematic diagram of the experimental denture for
intermittent compressive pressure (N = 40). (A) Occlusal view of
the experimental denture in position. (B) Frontal section at the
antero-posterior middle of the experimental denture (chaini line) in
the resting position (left), and in the TM region of the experimental
denture (dotted line) in the resting position (right). The palatal
tissue was covered with the denture base without any pressure. (C)
Frontal section at the antero-posterior middle of the experimental
(chain line) denture in occlusion (left), and in the TM region of the
experimental denture (dotted line) under pressure in occlusion
(right). We transmitted a defined amount of intermittent pressure
to the denture-supporting tissue by allowing the denture base to
subside a defined amount into palatal mucosa. M = fixed metal
framework; MD = metal framework of the denture base; RD = resin
denture base; TM = orthodontic tube in fixed metal framework to
permit subsidence of the resin denture base (the inner diameter is
thicker than threaded orthodontic wire); TD = orthodonitic tube in
the denture base for threading the orthodontic wire (diameter
equal to that of orthodontic wire); W orthodontic wire.
kPa, whiclh were defined by the use of pressure-loading
equipment as reported by Mori ct ol. (1997). Five rats were used
for each pressure condition. The denture base was composed of
heat-cured acrylic resin (Acron, GC Corp., Tokyo, Japan).
Orthodontic tubes were embedded in the removable denture
base and the fixed metal framework, and the denture base was
designed to be inserted rigidly into the metal framework. We
applied the pressure by threading orthodontic wire through the
tubes and subsequently applying additional acrylic (see Mori ot
ol., 1997), as shown in Fig. 1. The right side of the intermolar
I Deiit Res 77(2) 1998 Threshold of Pressure for Bone Resorption3 389

X- -, * ;IO
tZ-;Lr yrX 4tt . , .wJR
* -i,#-., 'w; 20f: . ttN % t q.*.X.
*,
.t -
u:
f:P-UL Xr
.##
.tk,s
, .s -'r $;L-
I! ,*#Ia-
*m'-
: ½§X- P-- '

1. ' <-4 t e '-. ,

,t:M,j',
,- 4 +. - D 4n,5
Figure 3. Typical histopathological finding in the continuous-pressure (7.84 kPa) group rat. Several osteoclasts (arrows)
and Howship's lacunae (arrowheads) are observed on the bone surface of the pressure-loaded region (P-L). In contrast, no
osteoclasts or Howship's lacunae are observed on the pressure-unloaded control region (P'-UL). This section was
recognized to have a significantly bone-resorbed status in the pressure-loaded region. E epithelium, LP = lamina propria
mucosae, B = bone. Hematoxylin and eosin stain. Bar = 100 p.m.

region of the hard palate of each rat served as the pressure-
unloaded control region.
In the intermittent-pressure group, occlusal force was
transmitted as an intermittent compressive pressure to the
whole intermolar region of the hard palate through the
experimental denture base (Fig. 2). The removable denture base
consisted of an Ag-Au-Pd alloy metal framework and heat-
cured acrylic resin. The removable denture base was installed to
cover the palatal mucosa without any pressure unless occlusal
force was exerted by occlusion. The denture base was permitted
to subside into the palatal mucosa utilizing the difference of the
diameter of the threading orthodontic wire and the inner
diameter of the orthodontic tube which was embedded in the
fixed metal framework. In occlusion, the metal framework of
the denture base came into contact with the fixed metal
framework to regulate the subsidence of the denture base,
consequently regulating the amount of pressure transmitted to
the denture-supporting tissue. The intensities of intermittent
compressive pressure were regulated to be 4.9, 9.8, 14.7, 19.6,
24.5, 29.4, 34.3, and 39.2 kPa, utilizing the pressure-loading
equipment reported by Mori et al. (1997). Five rats were used for
each pressure condition.
The experimental denture was worn for 6 days. The palate
and the denture base were cleaned at 3 days after insertion. The
rats were given free access to water and food (MF, Oriental
Yeast Co., Ltd., Tokyo, Japan).
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Histopathological observation
The animals were killed under anesthesia 6 days after the
insertion of the denture base. The excised palatal tissue was
immediately immersed in 101k neutral buffered formalin for 14
days. The formalin-fixed tissues were then decalcified in I 0lP
EDTA (pH 7.4) for 21 days, and were embedded in paraffin.
Fifty sections for each rat were cut frontally through the first
and second molar regions of the hard palate under the denture
base to a 6- Lm thickness at an intersectional interval of about 60
p.m. The sections were stained with hematoxylin and eosin. For
the intermittent-pressure group, only the left half of the palate
was used for histopathological observation.
Statistical evaluation of osteoclastic bone resorption
Fifty sections for each rat were examined between the median
palatine suture and the palatine groove under a light
microscope. The number of sections which showed any
osteoclasts and/or Howship's lacunae on the bone surface was
counted in the continuous pressure-loaded region, the
intermittent pressure-loaded region, and the pressure-unloaded
control region, respectively. The fitness of a Poisson distribution
to the distribution of the number of sections counted in the
pressure-unloaded control region was evaluated to give
statistical bases for the baseline activity of osteoclastic bone
resorption. According to the count distribution for the pressure-
unloaded control region, the minimum integer count with the
390 Sato et al. j Dciit Rcs 77(2) 1998
390 Sato et al. I Dent Re. 77(2) 1998
30
27
25 -

expected frequencies by
20 ,/ Poisson distribution
co (m=0.533) significant.
bone
sv 15 *:observed frequencies resorption
-0 0

13
10
4
5 19
1
It 0 BC 10
...E. ...

........ ..
1I -r .-::~t.... ....
0
0 1 2 3 4 5 6
Number of the sections with N=45
osteoclasts and/or Howship's lacunae
Figure 4. The number of sections with osteoclasts and/or Howship's lacunae in the unloaded control region for 45 rats. The agreement
between the observed frequencies and expected frequencies, calculated by Poisson distribution, was satisfactory.

upper single-tail probability less than 0.001 was defined to be
the boundary count (BC) between the baseline normal condition
and the induced bone-resorbed status. Therefore, if the
observed count in the series of 50 sections in the pressure-
loaded region of a rat was larger than BC, the region of the rat
was recognized to have a statistically significant (p < 0.001)
bone-resorbed status.
Results
A typical histopathological finding of the continuous-
pressure (7.84 kPa) group rat palate is shown in Fig. 3. On
the bone surface of the pressure-loaded region in this
section, several osteoclasts and Howship's lacunae are
observed. In contrast, no osteoclasts or Howship's lacunae
are observed in the pressure-unloaded control region in
Fig. 3.
The number of counted sections with osteoclasts and/or
Howship's lacunae in the pressure-unloaded control region
for 45 rats is summarized in Fig. 4. The expected value (m) of
observing a section with osteoclasts and/or Howship's
lacunae in one rat was calculated to be 0.533. A probability
distribution was calculated according to the Poisson
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distribution with in of 0.533, and we then obtained the
expected frequencies by multiplying the Poisson probability
distribution by n 45 (Fig. 4). The calculated BC in the
pressure-unloaded control region was found to be 4, with
the upper single-tail P values below 0.001.
The observed counts in the continuous-pressure group
and the intermittent-pressure group are shown in Tables 1
and 2, respectively. A continuous compressive pressure >
6.86 kPa or an intermittent compressive pressure > 19.6 kPa
significantly induced osteoclastic bone resorption in all rats.
In contrast, no significant bone resorption was observed in
the pressure-loaded region of any rat with a continuous
compressive pressure < 1.96 kPa or an intermittent
compressive pressure - 9.8 kPa.
Discussion
For the continuous-pressure group, the continuous
compressive pressure was exerted only on the left half of the
intermolar region of the hard palate, and the right half of the
palate was used as the pressure-unloaded control region.
Comparison of the histopathological findings between right
and left sides in each animal of the continuous-pressure
j Dent Res 77(2) 1998 Threshold of Pressure for Bone Resorption
Table 1. Bone resorption by continuous compressive pressure
Continuous Pressure (kPa) 0.49 0.98
Number of sections with osteoclasts and/or Howship's lacunae
(Each count was derived from one rat.)
0 0
0 0
0 0
2 0
3 1
a
Significant bone-resorbed status, p < 0.001.
group enabled us to confirm that the observed bone
resorption was caused by the experimental-pressure
condition. For the intermittent-compressive-pressure group,
because of the movable structure of the experimental denture
base, the whole area of the intermolar region of the hard
palate needed to be covered by the experimental denture base
to avoid possible food debris accumulation under the denture
base. Consequently, no food debris accumulation under the
experimental denture base could be observed in the
intermittent-pressure group as well as the continuous-
pressure group throughout the experimental period.
The uniformity of the pressure applied to the
experimental denture-supporting tissues is an important
condition for the observation of histopathological changes in
this study. Although the frontal section of the rat palate was
slightly concave, in general, the bone surface between the
bilateral palatine groove, which was the observation region
in this study, was almost flat and parallel laterally and
anteroposteriorly to the occlusal plane, as shown in Fig. 3.
The agreement of the thresholds for the intermittent
compressive pressure to induce the bone resorption
obtained from the data on the left half of the palate in this
study and that on the right half of the palate (data not
shown) would demonstrate that the mechanical forces
applied equally to the palate laterally, and the
histopathological homogeneity of the pressure-sensitive
changes, such as deformation and compression of the
mucosa, observed in a series of 50 sections in each rat would
also demonstrate that the forces applied equally to the
palate anteroposteriorly.
It is known that only Howship's lacunae are observed
after osteoclastic bone resorption and before the appearance
of osteoblasts or osteoid in the process of bone remodeling
(Baron, 1977). In the present study, therefore, the
appearance of only Howship's lacunae as well as osteoclasts
is realized to be representative of osteoclastic bone
resorption. Since bone remodeling can be observed in
normal bone tissue with an orderly sequence of cellular
events (Baron et al., 1984), the phenomenon of pressure-
induced bone resorption should be differentiated from
normal bone remodeling. Accordingly, statistical analysis
utilizing the Poisson distribution used in this study would
be the essential procedure to determine the presence of
pressure-induced bone resorption. The agreement between
the observed and expected frequency distributions was
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391
1.96 2.94 3.92 4.90 5.88 6.86 7.84
0 0 0 0 1 14a 18a
0 0 2 1 6a 18a 28a
1 6a 7a 22a 17a 31a 32a
2 17a 13a 27a 33a 45a 41a
3 38a 22a 40a 50a 49a 48a
examined by means of the Kolmogorov-Smirnov test. As
shown in Fig. 4, these frequency distributions showed
satisfactory agreement (p < 0.01), and the validity of
applying the Poisson distribution for the appearance
frequency of osteoclasts was established.
The wearing of a denture prosthesis has been reported to
exert many influences on denture-supporting tissues
(Stansbery, 1928). Non-pressure contact of the denture base
with palatal mucosa and the lack of physiological
stimulation for palatal tissue by tongue and/or food are
unavoidable situations for tissues under the denture base.
Covering the palatal mucosa with a denture base, without
any mechanical pressure and with the maintenance of a
clean palate and denture base, has been shown not to induce
bone resorption from 4 days to 20 wks after insertion of the
denture base (Nakashima et al., 1994). The only different
factor between the conditions in this study and the
conditions in the study of Nakashima et al. (1994) is the
transmission of continuous or intermittent compressive
pressure through experimental dentures. Therefore, the
participation of influential factors other than compressive
pressure could be subtracted, and the bone resorption
observed in this study could be realized to be caused by the
influential factor of continuous or intermittent compressive
pressure exerted through the denture base.
Osteoclastic bone resorption was markedly activated in a
rather narrow range of pressure in both groups. Regarding
the fact that the measurement error of the pressure-loading
equipment was less than 2.2% (data not shown), individual
variation among rats might be one of the possible reasons
for the observed extent of the range of threshold.
From these results, it was revealed that a pressure
threshold exists for inducing osteoclastic bone resorption for
both continuous and intermittent compressive pressures.
The threshold for the continuous-compressive pressure was
20% to 40% of that for the intermittent-compressive
pressure. The difference in the effective duration of pressure
extention might be one of the possible causes of the
observed differences in the thresholds for continuous and
intermittent pressure.
Acknowledgments
We thank Dr. Keiichiro Nakashima, the former researcher of
our department, for providing useful advice. This study was
392 Sato et al. J Dent Res 77(2) 1998

Table 2. Bone resorption by intermittent compressive pressure

Intermittent Pressure (kPa) 4.9 9.8 14.7 19.6 24.5 29.4 34.3 39.2
Number of sections with osteoclasts and/or Howship's lacunae
(Each count was derived from one rat.)
0 0 0 14a 16a 27a 22a 25a
0 0 3 24a 18a 29a 38a 38a
0 0 14a 29a 21a 43a 41a 49a
1 1 15a 32a 32a 44a 50a 50a
2 1 16a 33a 37a 46a 50a 50a
a
Significant bone-resorbed status, p < 0.001.

supported in part by Grant-in-aid for Scientific Research no.
04771604 from the Ministry of Education, Science and
Culture, Japan.
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