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Inhibition of osteoclastic bone resorption by mechanical stimulation in vitro.

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The influence of mechanical stimulation by intermittent compressive force (ICF) of physiologic magnitude on osteoclastic bone resorption was investigated in
cultures of fetal mouse cartilaginous long bones. Exposure to ICF resulted in a significant decrease in mineral
resorption, as indicated by the decreased release of %a
and a decreased number of osteoclasts in the diaphysis.
Conditioned medium (CM) from ICF-exposed periosteum-free cultures (ICF-CM), but not from control
cultures (Co-CM), inhibited mineral resorption in fresh
bones cultured under control conditions. Co-CM increased, but ICF-CM decreased, the number of tartrate-resistant acid phosphatase-positive cells in 7-day
bone marrow cultures. Direct exposure of bone marrow
cultures to ICF yielded the same results. Thus, osteoclastic bone resorption in cartilaginous long bones is
inhibited by ICF in vitro. A soluble factor($ acting on
tartrate-resistant acid phosphatase-positive, osteoclast
precursor-like cells seems to play a role in this effect.
Mechanical forces play an important role in the
differentiation, growth, and remodeling of the skeleton. An overall decrease in the functional load exerted
on the skeleton produces mineral loss and osteoporoFrom the Department of Oral Cell Biology, ACTA-Vrije
Universiteit, Amsterdam, The Netherlands.
Jenneke Klein-Nulend, PhD (present address: Department
of Medicine, Division of Endocrinology and Metabolism, University
of Connecticut Health Center, Farmington); J. Paul Veldhuijzen,
PhD; Michael E. van Strien, DDS; Marcel de Jong; Elisabeth H.
Burger, PhD: Professor of Histology.
Address reprint requests to J. Paul Veldhuijzen, PhD,
Department of Oral Cell Biology, Academic Centre for Dentistry,
Vrije Universiteit, vd Boechorststraat 7. Amsterdam, 1081 BT, The
Submitted for publication November 9, 1988; accepted in
revised form July 10, 1989.
Arthritis and Rheumatism, Vol. 33, No. 1 (January 1990)
sis. This phenomenon is observed with immobilization
by plaster cast (1,2), paralytic poliomyelitis (3), prolonged bedrest ( 4 3 , and with simulated (6,7) or actual
(8,9) weightlessness.
Using organ cultures of mouse calvarial bone
rudiments, we have previously shown that intermittent
compressive force (ICF) generated by intermittently
compressing the gas phase above cultures in a closed
chamber had important effects on mineral metabolism.
ICF enhanced alkaline phosphatase activity and decreased the release of calcium (lo), which suggests
that ICF may inhibit bone resorption while stimulating
bone formation in vitro. The importance of ICF in
governing the activity of growth plate cartilage in
proteoglycan synthesis and in the process of matrix
calcification has also been reported (1 1,12). In organ
cultures of fetal mouse cartilaginous long bones, ICF
increased diaphyseal calcification and modulated sulfate metabolism (1 1,12).
Although the osteoclast has long been recognized to be the major effector of bone resorption, little
is known of the local mechanisms by which osteoclastic bone resorption is controlled. Other cells may
modify or even mediate the bone-resorptive response
of osteoclasts to environmental factors, such as hormones and local factors (13). An intermediate role for
osteoblasts in elaborating the bone-resorbing effect of
intraarterial parathyroid hormone and interleukin-1
has been demonstrated (14), although the identity of
the communications signal between osteoblasts and
osteoclasts remains obscure. It has also been shown
that primary cultures of fetal rat calvaria secrete
growth-promoting factor activity into the culture medium (15,16). In addition, we have shown that conditioned medium (CM) obtained from cultured cartilag-
inous fetal long bones from mice stimulates the growth
of osteoclast precursor-like cells (17,18).
In t h e present study, we explored the effect of
mechanical stimulation on mineral resorption and the
formation of osteoclasts in fetal long bones from mice.
The effect of mechanical stimulation on the release of
a factor(s) that influences bone resorption andor the
formation of t a r t r a t e - r e s i s t a n t a c i d p h o s p h a t a s e
(TRAP)-positive (osteoclast precursor-like) cells from
bone marrow was also examined. The results suggest
an intimate link between mechanical stimulation a n d
osteoclastic bone resorption, and they demonstrate
the involvement of a soluble bone factor(s) that a c t s on
osteoclast precursor cells in the response of fetal long
bones to mechanical stimulation.
Intermittent compressive force. The experimental setup to apply ICF to metatarsal rudiments in vitro has been
described in detail previously ( 12). Briefly, ICF was generated by intermittently compressing the gas phase (5% CO, in
air) within a closed culture chamber (98% humidity, 37°C)
that contained the culture dishes. The pressure magnitude
was 130 mbar above ambient, and the pulse frequency was
0.3 Hz. The pressure applied to the metatarsal rudiments
was 132 gdcm'. which is similar to the calculated maximal
physiologic pressure in vivo (12).
Calcium release studies. Cartilaginous long bones
from Swiss albino mouse embryos were labeled in vivo with
"Ca by injecting the mother with 100 pCi of "CaClz
(specific activity 1 mCi/mmole; Amersham. Buckinghamshire, UK), intraperitoneally, I day prior to harvesting.
Rudiments of the metatarsus of 17-day-old embryos were
aseptically harvested, without removing the periosteum. and
precultured for 1 day to remove freely exchangeable %a.
Tissues were placed in 24-well po1,ystyrene culture
dishes (I5 mm; Falcon, Oxnard, CA) containing 300 pI of
culture medium per well, and incubated at 37°C in a humidified incubator (98% humidity) in an atmosphere of 5% C 0 2
in air. The culture medium consisted of *minimum essential
medium ( a - M E M ) without nucleosides (Gibco, Paisley.
Scotland) supplemented with 10%. heat-inactivated rat serum
and 10% embryonic extract of 8-day-old chick embryos
(Gibco), pH 7.4. After preculture, tissues were transferred to
fresh culture medium and incubated for an additional 5 days
in the absence (controls) or presence of ICF. Culture medium was changed once, on day 3.
The amount of mobilized mineral was determined by
assessing the release of 45Ca into the medium. The amount of
Ca released was defined as a percentage of the initial
radioactivity. This value was calculated as the sum of the
radioactivity in the medium on days 3 and .5 plus that in the
5% (volume/volume) formic acid-soluble mineral fraction of
the rudiment after culture.
Histologic and histomorphometric analyses. Nonradiolabeled 17-day-old long bone rudiments were fixed before
or after 5 days of culture (medium change after 3 days) for
1.5 hours at 4°C in 5% paraformaldehyde solution supplemented with 4% glucose in 0.1M phosphate buffer. After
routine treatment for histologic examination, methacrylateembedded (Historesin; Kulzer, Hamburg, FRG) longitudinal
sections (4 pm thick) were stained for acid phosphatase, as
described below.
The osteoclast content was assessed by counting the
number of TRAP-positive cell profiles (i.e., cross-sections)
and nuclei in the periosteum and bone collar, as well as in the
excavating cartilaginous bone center. A Zeiss I11 microscope, at 2 5 0 ~magnification, was used to examine each
sample (10 longitudinal sections at 15-pm intervals).
Acid phosphatase staining. The technique described
by Van de Wijngaert and Burger (19), using naphthol AS-BI
phosphate as the substrate and pararosaniline hexazonium
salt as the coupler, was used. The tartrate-resistant acid
phosphatase activity in histologic sections was demonstrated
after preincubation with tartrate before application of substrate, and in bone marrow cell cultures by the addition of 20
mM sodium potassium tartrate (Sigma, Munich, FRG) to the
substrate solution.
Fetal bone conditioned medium. Nonradiolabeled,
17-day-old metatarsal rudiments were aseptically harvested,
cleaned of adherent periosteum (20) by rolling them on a
glass slide in a drop of collagenase solution (2 mdml of 156
units/mg, type I1 collagenase; Worthington, Freehold, NJ),
and rinsed briefly in Puck's saline solution supplemented
with 10% heat-inactivated rat serum (TNO, Zeist, The
Netherlands). The rudiments were cultured in 24-well, ISmm (inner diameter) polystyrene culture dishes (6 rudiments
in 500 pl of culture medium per well) at 37°C in a humidified
(98% humidity) incubator (5% CO, in air). The culture
medium consisted of a - M E M without nucleosides, supplemented with 10% heat-inactivated rat serum. pH 7.4. The
rudiments were cultured with or without applying intermittent compressive force, as described above.
The culture medium was collected daily for 4 days,
pooled, and stored at -20°C until tested. Conditioned
medium obtained from control cultures (Co-CM) or from
ICF-exposed cqltures (ICF-CM) was added with 50%
fresh medium to prelabeled rudiments to examine their
effects on the release of 45Ca, and to use as a source of
osteoclast growth-regulating activity in the experiments
described below.
Bone marrow cultures. The technique used for culturing TRAP-positive cells has been described previously
(21.22). Briefly, nucleated bone marrow cells were flushed
from the femoral diaphysis of 10-12-week-old male Swiss
albino mice with 2 ml of culture medium consisting of
a - M E M supplemented with 20% heat-inactivated rat serum.
Cells were cultured in 24-well. 15-mm culture dishes with
13-mm plastic coverslips (Thermanox; Miles Laboratories,
Naperville, 1L) at 5 x lo5 nucleated bone marrow cells per
well in 500 pl of culture medium, or in 6-well. 35-mm culture
dishes (Falcon) at 3 x lo6 nucleated bone marrow cells per
well in 1,,500 pI of culture medium. Cells were incubated for
7 days at 37°C .in's humidified (98% humidity) atmosphere of
5% CO, in air, with no change of medium.
l o study the effect of ICF on the growth of TRAPpositive cells. bone marrow cells were cultured in the
not subjected to ICF (-35% on day 3 and -33% on
day 5).
Histologic examinations of noncultured 17day-old fetal long bones revealed that the diaphysis
was still intact (19) and consisted of a core of calcified
cartilage surrounded by a thin calcified bone collar.
TRAP-positive cells were present, but were mostly
Figure 1. Effect of intermittent compressive force (ICF) on the
release of 45Cafrom prelabeled fetal mouse long bones in vitro. 45Ca
release was assessed after 3 days and 5 days of culture in the
absence (control; Co) or presence of ICF. Bars show the mean 2
SEM, n = 12, from 2 experiments. * = P < 0.001.
absence (control) or presence of ICF for the entire culture
To study the effects of Co-CM and ICF-CM, bone
marrow cells were cultured without ICF in culture medium
containing 50% Co-CM or ICF-CM. The final concentration
of rat serum was always kept at 20%.
After culture, bone marrow cells were fixed in 4%
formalin containing 0.1% CaCl, in dextran 70,000 (Macrodex; Organon Teknika, Oss, The Netherlands) for 15 minutes at room temperature. Subsequently, cultures were
stained for TRAP as described above. The number of cells
expressing TRAP activity was monitored by using a Zeiss I11
microscope at 250x magnification (21,22).
Statistical analysis. Data were analyzed for statistical significance using Student’s t-test, unless otherwise
There was strong inhibition of mineral resorption in fetal long bones exposed to intermittent compressive force during culture (Figure I). There was
significantly lower release of 45Ca from the ICFtreated long bones compared with that from controls
Figure 2. Histologic sections of the central area of metatarsal
rudiments obtained from Swiss albino mouse embryos. A, After 5
days of culture in the absence of intermittent compressive force
(control), several tartrate-resistant acid phosphatase (TRAPj
positive osteoclasts (double arrows) can be seen in the calcified
center (CC), which also contains many other cells, as well as
TRAP-positive cells (large horizontal arrows) in contact with the
bone collar (small vertical arrows), adjacent to the periosteum (P).B,
After 5 days of culture in the presence of intermittent compressive
force, TRAP-positive cells (large horizontal arrow and large vertical
arrow) are found only in the periosteum or in contact with the
bone collar (small vertical arrows), suggesting that invasion has
not occurred. (Hematoxylin counterstained, original magnification
x 500.)
Figure 3. Effect of intermittent compressive force (ICF) on the
distribution of tartrate-resistantacid phosphatase-positive (TRAcPPOS) cells in fetal mouse long bones in vitro. Percentage distribution
was determined after 5 days of culture in the absence (control; CO)
or presence of ICF. Bars show the mean 2 SEM, n = 1 1 . * = P <
0.001, by two-way analysis of variance.
calcified cartilaginous center than was found in the
control samples (Figures 2 and 3).
Figure 4 shows the effect of a 50% concentration of conditioned medium from periosteum-free rudiments on the release of radiolabeled calcium from
labeled, nonstripped bones. CM obtained from rudiments exposed to ICF, but not control CM, caused a
significant decrease in 45Carelease compared with that
in cultures without CM.
To determine if the observed inhibition of 45Ca
release by intermittent compressive force was related
to an effect on the release of soluble factors stimulating
TRAP-positive cell growth (17,18), mouse bone marrow cells were incubated in the presence of Co-CM or
ICF-CM, or in the absence of CM. Figure 5 shows that
at a 50% concentration, Co-CM obtained from metatarsal rudiments significantly increased the number of
TRAP-positive cells after a 7-day culture. These findings were similar to those of previous studies (17,18).
ICF-CM did not produce this increase in TRAPpositive cells; ICF-CM actually caused a reduction in
the number of TRAP-positive cells compared with that
in the tissues cultured without CM. Moreover, when
ICF was applied directly to the bone marrow cultures
mononuclear (19). Eighty-eight percent of these cells
were found in the periosteum, not in contact with the
bone surface, 9% were found in direct contact with the
bone collar, and only 3% had invaded the calcified
cartilaginous center of the rudiment. These findings
indicated that invasion and resorption of the calcified
cartilage had only recently begun. After 5 days of
culture, many TRAP-positive multinucleated osteoclasts were present in the resorbing diaphysis (Figures
2A and B). There was no significant difference in the
number of TRAP-positive profiles or nuclei in control
and ICF-exposed bone rudiments (mean ? SEM 47.0
f 8.8 profiles and 33.9 ? 7.7 nuclei in control tissues
and 42.1 2 10.9 profiles and 29.3 f 9.4 nuclei in
ICF-treated tissues, n = 11). The number of multinucleated TRAP-positive profiles was also unchanged by
ICF treatment (results not shown). However, the
distribution of TRAP-positive cells over calcified cartilage and periosteum was significantly different in
ICF-exposed rudiments. There were more TRAPpositive cells in the periosteum and fewer within the
Figure 4. Effect of conditioned medium (CM) on the release of 45Ca
from prelabeled fetal mouse long bones in vitro. 45Ca release was
assessed after 3 days and 5 days of culture in the absence (Non-CM)
or presence of 50% CM obtained from cultures exposed to intermittent compressive force (ICF-CM) or not exposed to ICF (Co-CM).
Bars show the mean ? SEM, n = 20. * = P < 0.001.
(without addition of CM), there was also a significant
reduction in the number of TRAP-positive cells compared with that in the control cultures not exposed to
ICF (Figure 6).
The maintenance of normal skeletal metabolism
is strongly dependent upon, among other factors, such
mechanical forces as the pull of muscles and the
bearing of weight. When the influence of these mechanical forces is decreased or is absent, bone mass
decreases and osteoporosis ensues (1-9). To explain
these observations, it has been widely accepted that
the mechanical environment somehow stimulates osteoblastic activity and thus leads to new bone formation in an apparent attempt to strengthen the bone.
Absence of mechanical stresses is therefore presumed
to lead to a decrease in osteoblastic activity. It has
been further assumed that bone resorption continues
unchanged; hence, the resulting imbalance between
formation and resorption leads to a net reduction in
2 20a
Figure 5. Effect of 50% conditioned medium (CM) obtained from
fetal mouse long bone rudiments cultured without mechanical stimulation (CO-CM) or with intermittent compressive force (ICF-CM),
on the number of tartrate-resistant acid phosphatase-positive
(TRAcP-POS) cells from whole mouse bone marrow after 7 days of
culture. CM without bone rudiments (NON-CM) was used as a
control. Bars show the mean 2 SEM, n = 6. * = P < 0.001 versus
Figure 6. Effect of intermittent compressive force (ICF) on the
number of tartrate-resistant acid phosphatase-positive (TRAcPPOS) cells from whole mouse bone marrow after 7 days of culture.
CO = control; cultures not exposed to ICF. Bars show the mean 2
SEM. n = 6. * = P < 0.001.
bone mass. However, direct proof of either the dependence of osteoblastic activity on stress or the possible
constancy of bone resorption under such circumstances has been lacking.
We recently reported that mechanical stimulation increases matrix calcification of hypertrophic cartilage and bone collar in cultures of fetal long bone
rudiments (12). We also reported that intermittent
compressive force stimulates osteoblastic activity in
cultured fetal mouse calvaria that do not contain
cartilage (10). Osteoclastic activity in the calvaria was
also affected by ICF, but in the opposite way, leading
to diminished resorption (10).
In the present study, we observed a decrease in
the amount of 4SCa released from long bones under
conditions of ICF; this was accompanied by a decrease in the number of osteoclasts within the calcified
cartilaginous center. Compared with the control samples, more TRAP-positive, pre-osteoclasts remained
in the periosteum. In the periosteum of these 17day-old mouse long bone rudiments, there was a
restricted pool of nonproliferating osteoclast precursors, and few, if any, multinucleated osteoclasts had
been formed (19,23). Obviously, in this in vitro sys-
tem, there can be no recruitment of new osteoclast
precursors derived from the blood. Since the total
number of TRAP-positive profiles was the same in
both control and ICF-CM cultures, but the distribution
of the osteoclast-like cells was changed in those exposed to ICF, it seems that culture under ICF inhibits
the migration of osteoclast precursors from the periosteum into the calcified center of the bone rudiments.
This diminished migration and the consequently reduced number of osteoclasts in the calcified center are
consistent with the known decrease in mineral resorption. Whether treatment with ICF interferes with a
chemotactic gradient involved in the migration of
osteoclasts or whether it alters the fusion and/or
mobility of the (pre-)osteoclasts, cannot be concluded
from this study.
Evidence of the production of factors that result
in the suppression of mineral resorption activity under
ICF was obtained from the experiment in which ICFCM was added to 17-day-old long bones that had been
prelabeled with 45Ca. In those experiments, ICF-CM
inhibited mineral resorption compared with that in
cultures without CM. The experiments with cultures
of bone marrow demonstrated that ICF-CM also inhibited the formation of TRAP-positive cells; TRAPpositive cells have characteristics similar to those of
osteoclast precursors (22). Aside from some osteoblasts in the periosteum, the main body of cells in
these embryonic long bones are (hypertrophic) cartilage cells, and it is these cells that are most likely to be
involved in the synthesis of the factor(s).
There is ample evidence in the literature that
osteoblasts and osteoblast-like cells may produce factors (such as prostaglandins) that stimulate osteoclastic bone resorption in vitro (24). It has been reported
that the synthesis of prostaglandins by cultured osteoblasts isolated from rat calvaria is increased upon
stretching (25). It is not known whether ICF causes
mouse chondrocytes (the main body of the cells in a
fetal long bone) to increase their synthesis of prostaglandins. However, since prostaglandins are very
potent bone resorbers in vitro (26,27) and since ICF
has been shown to reduce osteoclastic activity, the
role of prostaglandins in the system we used should be
studied further. In addition, exposure to ICF had a
direct inhibitory effect on the growth of osteoclast
precursor-like cells, and this suggests that these cells
also have receptprs for mechanical stimuli.
It has been reported that osteoblasts and chondrocytes of long bones synthesize and deposit growth
factors into the !matrix (28,29). Transforming growth
(TGF-P) has been reported to inhibit the
formation of osteoclast-like cells in long-term cultures
of bone marrow (30,3 1). Studies to determine whether
hypertrophic chondrocytes increase their production
of TGF-P in response to ICF, resulting in decreased
resorption (less differentiated osteoclasts or decreased
movement of osteoclasts into the mineralized metaphysis), would be useful.
The results presented here indicate that mechanical stimulation of skeletal tissues may decrease
resorption by stimulating the production of a soluble
factor(s) that inhibits osteoclastic resorption. This
decreased resorption may result from a decreased
differentiation of osteoclast precursor-like cells to
mature osteoclasts and/or from a decreased ability to
reach the mineralized surfaces. This implies that the
negative bone-mineral balance reported during, for
example, prolonged periods of low loading of the
skeleton (e.g., bedrest and microgravity) may result,
at least partly, from local factors that regulate osteoclast precursor cell formation or maturation, and/or
osteoclast motility. The assumption that only bone
formation, and not bone resorption, is changed under
these conditions of disuse osteoporosis (8,9) does not
seem warranted.
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