rapid communication
Osteocyte hypoxia: a novel mechanotransduction pathway
J. S. DODD,1 J. A. RALEIGH,2 AND T. S. GROSS1
of Orthopaedic Surgery, University of Cincinnati, Cincinnati, Ohio 45267-0212;
and 2Radiation Oncology and Toxicology, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27599
1Department
tissue (16), form gap junctions with adjacent osteocytes
and lining cells (8), and are extremely sensitive to
alterations in their local physical environment (23).
Physiologically, mechanical loading greatly enhances
nutrient exchange and diffusion within bone (17, 25).
Because osteocytes reside distant from the blood supply, their metabolic needs are satisfied by a combination of passive diffusion and enhanced diffusion arising
when the skeleton is loaded during functional activity.
Therefore, we hypothesized that depriving a bone of
functional loading would rapidly induce osteocyte hypoxia. Here, we use an in vivo model of bone adaptation
to demonstrate that osteocyte hypoxia is rapidly induced when bone is unloaded. Further, we present
preliminary data indicating that osteocytes can be
rescued from this fate by a brief loading regimen. When
coupled with the known cellular consequences of oxygen deprivation, these results suggest that osteocyte
oxygen metabolism may function as a novel mechanotransduction pathway within bone tissue.
disuse; bone loss; bone adaptation; oxygen metabolism
METHODS
MECHANOTRANSDUCTION IS THE process by which cells
within a living tissue perceive physical stimuli and
respond with biochemical signals. Mechanotransduction is integral to the successful functioning of both
organ systems (7) and whole organisms such as Caenorhabditis elegans (14) and plants (11). The vital
weight-bearing role of the skeleton and the sensitivity
of its cellular populations to physical stimuli (12, 24)
make bone a unique model in which to study mechanotransduction. Although the rapid loss of bone precipitated when the skeleton is unloaded emphasizes that
mechanical stimuli are required to maintain bone mass
in the adult skeleton, the pathway by which deprivation of bone loading (i.e., disuse) is transduced into
biochemical signals is unknown.
In bone, the osteocyte is an ideal cellular candidate to
initiate biochemical responses culminating in tissue
adaptation. Osteocytes are ubiquitous through the
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C598
In vivo model. Ten adult (1–1.5 yr) male turkeys were
included in the study. Animals were randomly assigned to
negative control (n ⫽ 4), disuse (n ⫽ 4), or disuse ⫹ loading
(n ⫽ 2) groups. The left ulna of each turkey in the disuse and
disuse ⫹ loading groups was isolated from functional loading
via parallel metaphyseal osteotomies (21). The exposed diaphyseal bone ends were covered with methacrylate-filled
stainless steel caps. In the disuse ⫹ loading group, transcutaneous Steinmann pins were passed through mating holes in
the caps and parallel holes drilled through the bone ends via a
surgical template. The exposed pins were then clamped to
inhibit noncontrolled loading. Each animal within the disuse
and disuse ⫹ loading groups was injected 18 h postsurgery
and 6 h before death with 40 mg/kg pimonidazole (Hyproxyprobe-1; Natural Pharmacia International, Research Triangle Park, NC). Negative control animals had no surgery
and did not receive pimonidazole. Pimonidazole is distributed
to all tissues in the body. In hypoxic cells, pimonidazole is
reduced via the nitroreductase pathway. The reduced form
binds to protein adducts in hypoxic cells and is detected using
a monoclonal antibody (3). In the disuse ⫹ loading group,
loading was conducted 18 h postsurgery and just before
pimonidazole injection. Pin clamps were removed and the
ulna was loaded for 100 cycles in bending at 1 Hz with a
trapezoidal shaped waveform. This loading induced a normal
strain distribution similar in orientation to that developed
during maximal wing flap (2). Next, 100 cycles of torsion were
applied at 1 Hz with a trapezoidal waveform (20). Previous
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
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Dodd, J. S., J. A. Raleigh, and T. S. Gross. Osteocyte
hypoxia: a novel mechanotransduction pathway. Am. J.
Physiol. 277 (Cell Physiol. 46): C598–C602, 1999.—Bone is a
unique tissue in which to examine mechanotransduction due
to its essential role in weight bearing. Within bone, the
osteocyte is an ideal cellular mechanotransducer candidate.
Because osteocytes reside distant from the blood supply, their
metabolic needs are met by a combination of passive diffusion
and enhanced diffusion, arising when the tissue is loaded
during functional activity. Therefore, we hypothesized that
depriving a bone of mechanical loading (and thus eliminating
diffusion enhanced by loading) would rapidly induce osteocyte hypoxia. Using the avian ulna model of disuse osteopenia, we found that 24 h of unloading results in significant
osteocyte hypoxia (8.4 ⫾ 1.8%) compared with control levels
(1.1 ⫾ 0.5%; P ⫽ 0.03). Additionally, we present preliminary
data suggesting that a brief loading regimen is sufficient to
rescue osteocytes from this fate. The rapid onset of the
observed osteocyte hypoxia, the inhibition of hypoxia by brief
loading, and the cellular consequences of oxygen deprivation
are suggestive of a novel mechanotransduction pathway with
implications across organ systems.
OSTEOCYTE HYPOXIA: A NOVEL MECHANOTRANSDUCTION PATHWAY
RESULTS
Acute deprivation of bone loading induced significant
osteocyte hypoxia compared with intact contralateral
bones from the same animals (8.4 ⫾ 1.8% vs. 1.1 ⫾
0.5%; P ⫽ 0.03; Figs. 1 and 2). Hypoxic and nonhypoxic
osteocytes were located a similar distance from the
nearest Haversian canal (42.3 ⫾ 2.8 vs. 43.6 ⫾ 2.3 µm).
As a negative control for the assay, bones from animals
that did not receive injections of pimonidazole demonstrated minimal positive staining (1.0 ⫾ 0.2%). As a
positive control, a high percentage of osteocytes demonstrated strong staining for actin (72 ⫾ 9.0%). In a
preliminary attempt to demonstrate a cause and effect
Fig. 1. Mean ⫾ SE percentage of hypoxic osteocytes. Acute disuse (24
h) significantly elevated osteocyte hypoxia compared with negative
(Neg) control bones (animals not injected with pimonidazole; * P ⫽
0.03) and intact control bones (contralateral ulna of animals injected
with pimonidazole; P ⫽ 0.03).
relation between loading of bone and osteocyte oxygen
homeostasis, we supplemented 24 h of disuse with a
single brief external loading protocol. The brief regimen (⬍4 min in duration) completely rescued osteocytes from disuse-induced hypoxia (1.3 ⫾ 0.4%; Fig. 3).
DISCUSSION
The surgery required to deprive the avian ulna of
mechanical loading holds the potential to confound this
study. Two observations mitigate this possibility. First,
we have reported that middiaphyseal bone blood flow in
this model is not altered by an invasive sham surgery
(10), suggesting that passive oxygen exchange derived
via diffusion from the bone’s blood supply is not diminished by the procedure. Second, the brief external
loading regimen was successful in counteracting disuseinduced osteocyte hypoxia. This result would be improbable if the observed hypoxia was induced by the surgical procedure. Additionally, the avian ulna model is
unique among in vivo models of bone adaptation, in
that it facilitates either unloading or controlled external loading of a bone without alterations in the experimental procedure. As such, the model was highly suited
for this study.
The technique we applied to detect osteocyte hypoxia
was originally developed to assess tumor resistance to
radiation therapy (13). The ability of 2-nitroimidazoles,
such as pimonidazole, to detect hypoxia rests on their
ability to bind proteins in cells at oxygen tensions of
⬍10 mmHg (3). Pimonidazole and other nitroimidazoles with similar binding characteristics have been
used to detect hypoxia in a variety of intact tissues (3,
15, 30). Low tissue oxygen tension alone is not sufficient
to impose a state of hypoxia on cells within the tissue,
because chondrocytes within normal cartilage are not
hypoxic (22). When used to study osteocytes, the assay
is highly specific, because, under normal conditions,
only osteocytes are present within lacunae. At this
time, however, we are unable to apply this technique to
examine cells on either the periosteal or endocortical
surfaces, due to fluorescence saturation at the bone
edge. Although the oxygen status of individual osteocytes that reside in individual lacunae is not known, it
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calibrations indicated that the external loading protocols
induced peak compressive strains of ⫺1,000 µ⑀ (during
bending) and a peak shear of 1,000 µ⑀ (during torsion). These
strain magnitudes are well below those induced during daily
activity.
Immunohistochemistry. At death, 4-mm thick middiaphysis cross sections were removed from the left and right ulnas
of each turkey. The sections were fixed in 10% buffered
Formalin, decalcified in EDTA (10 days at 40°C), embedded in
paraffin, sectioned at 5 µm, and mounted on charged slides.
The slides were deparaffinized (20 min at 40°C), followed by
xylene, a graded ethanol wash (100%, 95%, 90%, 70%), and
rinsing in distilled water plus 2% Brij 35 and PBS plus 2%
Brij 35. To prepare for staining, slides were blinded and
sections were digested with pronase (40 min at 40°C; Biomeda), rinsed in PBS-Brij 35, and blocked with 10% horse
serum (10 min at room temperature; Vector Laboratories).
Pronase digestion was utilized to aid antigen retrieval according to previously described protocols (18). Hypoxia was detected via incubation with a mouse IgG1 anti-pimonidazole
antibody (clone 4.3.11.3; dilution 1:50, 1 h at 37°C; Natural
Pharmacia International). Slides were rinsed and subjected
to an anti-mouse FITC secondary antibody (dilution 1:500, 30
min at room temperature; Vector Laboratories). The procedures used to detect actin were identical, with a mouse
anti-actin antibody substituted as the primary antibody
(C4D6; dilution 1:500, 30 min at room temperature). All slides
were stored at 4°C in darkness and were imaged within 3
days of staining.
Imaging. Imaging was performed with a Bio-Rad scanning
confocal microscope (MRC-600; 25 mW argon laser, 488 nm
blue filter) attached to a Nikon Diaphot inverted microscope.
Osteocyte hypoxia was examined at four repeatable midcortical locations on each cross section. At each location, a ⫻60
objective was used to obtain four adjacent fluorescent and
corresponding bright-field images. A mean of 32.2 ⫾ 1.2 (SE)
osteocyte lacunae was contained within each field. The images were overlaid, and the total numbers of osteocyte
lacunae and FITC-positive osteocyte lacunae were counted.
For each 1-day disuse section with greater than two FITCpositive osteocytes, the mean distance to the center of the
nearest Haversian canal was determined for both hypoxic
and nonhypoxic osteocytes (NIH-Image; Scion). Identical
laser intensities and gains were used for all imaging (both
hypoxia and actin). Imaging and counting were performed
with the operator blinded to the identity of the slides. Data
were expressed as the percentage of hypoxic osteocytes, and
nonparametric statistical comparisons were performed between the unloaded bones and intact control bones (Wilcoxon)
and between unloaded bones and negative control bones
(Mann-Whitney).
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OSTEOCYTE HYPOXIA: A NOVEL MECHANOTRANSDUCTION PATHWAY
Fig. 2. Combined fluorescent and bright-field confocal images illustrating positive staining for osteocyte hypoxia and actin. In normal
intact bone (A), minimal osteocyte hypoxia was detected (1.1 ⫾ 0.5%).
After 24 h of disuse (B), osteocyte hypoxia was significantly elevated
(8.4 ⫾ 1.8%). A high level of positive actin staining was evident (C; 72
⫾ 9%). Illustrated images were taken from cranial/caudal cortex of
each cross section, with A and B images from same animal (right and
left ulnas, respectively). Haversian canals (HC) and FITC-positive
osteocytes are noted (OC⫹ ).
is probable that the observed hypoxia resulted from
fluctuations in osteocyte oxygen homeostasis. This observation is supported by the hypoxia induced within
connective tissues in response to whole body hypobaric
exposure (4).
These data are the first, to our knowledge, to identify
in vivo cellular hypoxia in response to altered mechani-
Fig. 3. Mean ⫾ SE percent osteocyte hypoxia in experimental bones
(Exp) subjected to 24 h of disuse (n ⫽ 4) or 24 h of disuse
superimposed with a brief loading regimen (Disuse ⫹ Load; n ⫽ 2).
Osteocytes within control ulnas demonstrated minimal positive
staining for hypoxia (open bars). Disuse induced significant hypoxia
(*P ⫽ 0.03), but brief loading rescued osteocytes from this fate (solid
bars).
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cal stimuli. We have recently corroborated this observation in a preliminary study using similar immunohistochemistry and imaging techniques. In our model, we
have found that 24 h of disuse are sufficient to substantially enhance osteocyte expression of hypoxia-inducible factor 1␣, a hypoxia-dependent transcription factor
(data not shown). The precise time course of the hypoxic
response (i.e., onset, maximum, and duration) remains
to be defined.
Because hypoxia is known to induce a variety of
cellular responses, including altered ionic homeostasis
(6), activation of second messengers (28), and cytoskeletal degradation (5), we believe that osteocyte hypoxia
has the potential to directly and/or indirectly influence
bone cell dynamics. For example, many cell types are
capable of surviving extended hypoxia, but certain
conditions (e.g., prolonged hypoxia or reoxygenationinduced injury) may induce cell death. Interestingly,
osteocyte apoptosis has recently been associated with
conditions of accelerated intracortical remodeling, such
as that induced by estrogen depletion (26). The colocalization of hypoxia and apoptosis in tumors (9) suggests
that the potential relation between osteocyte hypoxia
and osteocyte apoptosis may prove a fruitful area of
study. In this model, extended hypoxia would induce
osteocyte apoptosis, followed by differentiation and
recruitment of osteoclasts from marrow precursors.
The ensuing remodeling would serve to remove cellular
debris and restore tissue viability. Altered expression of
hypoxia-induced apoptosis mediators in our model (e.g.,
p53) would corroborate evidence of this pathway in
bone. With regard to potential secondary effects on
bone cell populations, cellular hypoxia is a strong
stimulus for hyperemia at the tissue level (1). Within
this context, our identification of osteocyte hypoxia
after 24 h of disuse supports our recent report that 7
days of disuse induces bone hyperemia (10). As vasoregulatory factors are known to demonstrate potent
actions on bone cell populations (29), osteocyte hypoxia
may also exert influence on bone cell populations via an
endothelial cell mediated pathway.
It is particularly intriguing that a brief episode of
mechanical loading appears sufficient to restore osteo-
OSTEOCYTE HYPOXIA: A NOVEL MECHANOTRANSDUCTION PATHWAY
We gratefully acknowledge Dr. R. F. Hennigan for valuable advice
regarding confocal microscopy and thank Dr. J. Lessard for the gift of
the anti-actin monoclonal antibody. Drs. A. J. Crowe, T. L. Clemens,
D. R. Gross, and S. Srinivasan provided feedback on the manuscript.
This work was supported, in part, by National Institutes of Health
Grants AG-14881 and AR-45665, the University of Cincinnati Orthopaedic Research and Education Foundation, and the University of
Cincinnati Research Council.
Address for reprint requests and other correspondence: T. S. Gross,
Dept. of Orthopaedic Surgery, 231 Bethesda Ave., Univ. of Cincinnati,
Cincinnati, OH 45267-0212 (E-mail: [email protected]).
Received 15 March 1999; accepted in final form 7 June 1999.
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cyte oxygen homeostasis. Although preliminary, these
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relation between loss of loading, restoration of loading,
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The observation that osteocyte oxygen homeostasis
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In conclusion, these data emphasize that daily mechanical loading of bone is required to maintain osteocyte homeostasis. Even a brief interruption of loading
for a 24-h period is sufficient to induce osteocyte
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disuse-induced hypoxia. The extremely rapid induction
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parallel role as a mechanotransducer within the skeleton. In a broader perspective, the integral role of
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