Jiro Nagatomi
Department of Biomedical Engineering,
Rensselaer Polytechnic Institute,
Troy, NY 12180-3590
Bernard P. Arulanandam
1
Center for Immunology and Microbial Disease,
Albany Medical College,
Albany, NY
Dennis W. Metzger
Department of Biomedical Engineering,
Rensselaer Polytechnic Institute,
Troy, NY 12180-3590
Center for Immunology and Microbial Disease,
Albany Medical College,
Albany, NY
Alain Meunier
Faculte ´ de Me ´dicine Lariboisiere St-Louis,
Universite D.Diderot, Paris, France
Rena Bizios
Department of Biomedical Engineering,
Rensselaer Polytechnic Institute,
Troy, NY 12180-3590
e-mail: Bizios@rpi.edu
Effects of Cyclic Pressure on Bone
Marrow Cell Cultures
The present in-vitro study used bone marrow cell cultures and investigated the effects of
cyclic pressure on osteoclastic bone resorption. Compared to control (cells maintained
under static conditions), the number of tartrate resistant acid phosphatase (TRAP)-
positive, osteoclastic cells was significantly p 0.05 lower when, immediately upon
harvesting, bone marrow cells were exposed to cyclic pressure (10 – 40 kPa at 1.0 Hz). In
contrast, once precursors in bone marrow cells differentiated into osteoclastic cells under
static culture conditions for 7 days, subsequent exposure to the cyclic pressure of interest
to the present study did not affect the number of osteoclastic cells. Most important,
exposure of bone marrow cells to cyclic pressure for 1 h daily for 7 consecutive days
resulted in significantly p0.05 lower osteoclastic bone resorption and in lowered
mRNA expression for interleukin-1 (IL-1) and tumor necrosis factor- (TNF-), cytokines
that are known activators of osteoclast function. In addition to unique contributions to
osteoclast physiology, the present study provided new evidence of a correlation between
mechanical loading and bone homeostasis as well as insight into the molecular mecha-
nisms of bone adaptation to mechanical loading, namely cytokine-mediated control of
osteoclast functions. DOI: 10.1115/1.1468867
Keywords: Cyclic Pressure, Bone Marrow Cells, Osteoclasts, Bone Resorption, Cytokine
mRNA
Introduction
Clinical observations 1–4 have demonstrated that mechanical
loading on the skeleton affects bone homeostasis, a process that
requires a balance between bone formation by osteoblasts and
bone resorption by osteoclasts. Removal of loading on the skel-
eton due, for example, to extended periods of bed rest and space-
flights leads to decreased bone mineral density in healthy human
subjects 1,2. In contrast, increased high-impact loading on the
skeleton leads to increased bone mass 3,4. Despite the sur-
mounting in-vivo evidence to indicate correlations between me-
chanical loading and bone homeostasis, the underlying cellular
and molecular mechanisms of these events are yet to be eluci-
dated.
In the past, studies of the effects of mechanical stimuli on bone
cell function in vitro have focused mainly on functions of osteo-
blasts, the bone-forming cells 5–9. Numerous reports in the lit-
erature provided evidence that in-vitro osteoblasts and/or trans-
formed osteoblastic cells respond to various forms of mechanical
stimuli such as pressure 5,7,9, fluid shear stress 6, and defor-
mation of substrates 8. Since bone homeostasis requires contri-
butions from both osteoblasts and osteoclasts, in order to elucidate
the effects of mechanical stimuli on bone homeostasis at the
cellular/molecular level, it is necessary to examine the effects of
similar mechanical stimuli on the two types of these bone cells.
Availability of cell culture models made possible investigations
of the effects of mechanical forces on osteoclastic cells 10–14.
These studies provided evidence that formation of osteoclasts
from their precursors was inhibited when mouse bone marrow
cells were exposed to either sustained hydrostatic pressure 1.37
atm10 or cyclic strain 5% at 0.167 Hz11 for up to six
consecutive days; in addition, syntheses of nitric oxide and of
prostaglandin E
2
by rat osteoclastic cells increased when these
cells were exposed to fluid shear stress 16 dyne/cm
2
for 6 h 12.
Furthermore, while osmotic membrane stretching 20% resulted
in decreased actin-ring formation an index of bone resorption by
rat osteoclasts 13, exposure to cyclic strain 0.17% at 1 Hz for
24 h resulted in increased bone resorption by rabbit osteoclasts
14. These literature reports provided intriguing evidence of the
effects of physical stimuli on osteoclasts. To date, however, the
relationships between mechanical loading and osteoclast function
have not been fully explored.
It was hypothesized that cyclic loading affected osteoclast func-
tions pertinent to bone homeostasis. For this reason, a custom-
made laboratory setup and rat bone marrow cells were used to
investigate the effects of cyclic pressure on differentiation of os-
teoclasts from their precursors, on bone resorption by osteoclasts,
as well as release and mRNA expression of select cytokines.
Materials and Methods
Bone Marrow Cell Culture. Bone marrow cells, a source of
osteoclast-precursors, were harvested from the femurs of 14-day-
old Sprague-Dawley rats according to procedures reported in the
literature 15. Briefly, the femurs were excised, the epiphyses
were cut off, and the marrow cavity was flushed out with Dulbec-
co’s modified Eagle Medium DMEM; Life Technologies, Long
Island, NY, supplemented with 10% fetal bovine serum FBS;
Life Technologies and 10 nM 1,25(OH)
2
vitamin D
3
Calbio-
chem, La Jolla, CA. Released bone marrow cells were centri-
fuged at 1,700g for 5 min, suspended in fresh DMEM contain-
ing 10% FBS and 10 nM vitamin D
3
, and cultured on substrates
1
Current address: Department of Biology, University of Texas San Antonio, San
Antonio, TX 78249.
Contributed by the Bioengineering Division for publication in the JOURNAL OF
BIOMECHANICAL ENGINEERING. Manuscript received by the Bioengineering Divi-
sion October 2, 2001; revised manuscript received February 5, 2002. Associate Edi-
tor: R. Vanderby, Jr.
308 Õ Vol. 124, JUNE 2002 Copyright © 2002 by ASME Transactions of the ASME