1 Department of Orthopaedics, 2 Department of Pathology, and 3 Department of Physiology and Biophysics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106-5000
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ABSTRACT |
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Conditionally immortalized
murine calvarial (CIMC) cells that support differentiation of
precursors into mature osteoclasts were isolated. All six CIMC cell
lines supported osteoclast differentiation in response to
1,25-dihydroxyvitamin D3 or interleukin (IL)-11. CIMC-4
cells also supported osteoclast differentiation in response to tumor
necrosis factor (TNF)-, IL-1
, or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and
formed resorption lacunae on mineralized surfaces. CIMC-4 cells,
therefore, establish an osteoclast differentiation assay that is
responsive to many cytokines and does not rely on isolation of primary
stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells.
Osteoclast differentiation induced by all stimuli other than
TNF-
was completely blocked by osteoprotegerin, whether the
stimulators were examined alone or in combination. Moreover, study of
precursors that lack TNF-
receptors showed that TNF-
induces
osteoclast differentiation primarily through direct actions on
osteoclast precursors, which is a distinct mechanism from that used by
the other bone-resorptive agents examined in this study.
conditionally immortalized murine calvarial (CIMC-4) cells; cytokines; osteoclast differentiation; RANKL; tumor necrosis factor-
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INTRODUCTION |
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BONE RESORPTION BY OSTEOCLASTS is carefully regulated to prevent changes in bone mass that would lead to osteoporosis or osteopetrosis. One mechanism responsible for this is precise control of osteoclast differentiation. For example, estrogen inhibits bone resorption primarily by reducing osteoclast differentiation (40, 49). Altered osteoclast differentiation is also a main cause of altered bone turnover in disuse osteoporosis (6, 62), osteopetrosis (51), hyperparathyroidism/humoral hypercalcemia of malignancy (3, 66), Paget's disease (15), tumor-induced osteolysis (8, 13), rheumatoid arthritis (21), Gorham-Stout disease (18), orthopedic implant loosening (9), periodontitis (4), McCune-Albright syndrome (63), and chronic alcohol ingestion (14). Thus knowledge of the mechanisms that regulate osteoclast differentiation may shed light on pathogenesis of a number of different diseases as well as on the physiological regulatory mechanisms that maintain bone balance in the absence of disease.
Osteoclast differentiation is stimulated by a wide variety of hormones
and cytokines, including 1,25-dihydroxyvitamin D3
(1,25-D3), parathyroid hormone (PTH), tumor necrosis factor
(TNF)-, interleukin (IL)-1, IL-6, and IL-11 (50, 51).
Most of these agents are thought to primarily stimulate osteoclast
differentiation indirectly by inducing stromal cells that support
osteoclast differentiation to increase production of receptor activator
of nuclear factor (NF)-
B ligand (RANKL) and/or decrease production
of osteoprotegerin (OPG) (39, 41, 52, 56, 65). RANKL is a
cell surface member of the TNF superfamily that, along with macrophage
colony-stimulating factor (M-CSF), is necessary and sufficient for
osteoclast differentiation even in the absence of stromal cells
(32, 65). OPG is a decoy receptor that binds to RANKL and
prevents it from interacting with its receptor, which is known as RANK
(52, 56).
In most physiological systems, cytokines function together in networks
in which the stimulators often act synergistically. However, it is
unknown whether this is also true for osteoclast differentiation.
Consistent with this possibility, others (28, 38, 48) have
shown synergistic stimulation of bone resorption induced by TNF-,
IL-1, IL-6, leukemia inhibitory factor, the soluble form of the IL-6
receptor
-chain, and prostaglandins. However, it is not known
whether the effects observed in those organ culture studies are due to
stimulation of osteoclast activity, osteoclast differentiation,
osteoclast survival, or a combination of these processes. More
recently, it was shown (53) that submaximal levels of IL-1
and IL-6 act cooperatively to stimulate osteoclast differentiation. The
concentrations of the cytokines used in that study, however, prevented
a clear demonstration of synergism.
To address this issue, we originally attempted to employ the cell
culture model of osteoclast differentiation that involves coculture of
osteoclast precursors with ST2 mesenchymal support cells (50,
59). However, although this is an excellent model for
examination of osteoclast differentiation in response to
1,25-D3, the ST2 coculture system does not respond to
TNF-, IL-1
, or IL-6 (see RESULTS). Similarly, UMR106
cells support osteoclast differentiation in response to
1,25-D3 and M-CSF but not in response to TNF-
, IL-1,
IL-6, or IL-11 (20). Moreover, although conditionally immortalized mesenchymal cells isolated from bone marrow support osteoclast differentiation in response to 1,25-D3 and PTH
(10, 17, 35, 37), they do not support osteoclast
differentiation in response to TNF-
or IL-1
(A. A. Ragab and
J. E. Dennis, unpublished observations). In contrast, primary
calvarial cells support osteoclast differentiation in response to the
cytokines (50, 51). Therefore, we hypothesized that
conditionally immortalized calvarial cells would also perform this
function. To test this possibility, we isolated six distinct
conditionally immortalized murine calvarial (CIMC) cell lines from
transgenic mice that express an interferon-inducible and
temperature-sensitive version of the SV40 large T antigen (29). Using cocultures of the CIMC cells and spleen cells
as a source of osteoclast precursors, we found that osteoclast
differentiation is induced by 1,25-D3, TNF-
, IL-1
,
IL-6, and IL-11. Moreover, synergistic stimulation was observed when
low concentrations of the cytokines (TNF-
, IL-1
, IL-6, and IL-11)
were added to the cultures simultaneously.
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METHODS |
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All animals were treated in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals under the supervision of the Case Western Reserve
University Institutional Animal Care and Use Committee. CIMC cells were
prepared from calvaria of 3-day-old mice that express an
interferon-inducible, temperature-sensitive, SV40 large T antigen
transgene (29). Three groups of CIMC cells were obtained
from the calvaria by harvesting cells after either the first or second
hour of collagenase digestion [0.1% collagenase (Wako Pure Chemical,
Richmond, VA) in PBS containing 100 U/ml penicillin (Mediatech,
Herndon, VA) and 100 mg/ml streptomycin (Mediatech); 37°C] or after
11 days of culture (5% CO2, 37°C) in collagen gel (2.3%
rat tail type I collagen; Becton Dickinson, Bedford, MA) as described
previously (54). The three groups of CIMC cells were then
plated at 10,000 cells/cm2 and either cultured directly
under permissive conditions [33°C, 100 U/ml interferon (IFN)-;
GIBCO, Gaithersburg, MD] or, as suggested by Dennis and Caplan
(16), switched to permissive conditions after 1 wk under
nonpermissive conditions (37°C, no IFN-
). This protocol resulted
in isolation of six distinct cell lines: CIMC-1 (second hour of
collagenase digestion, directly in permissive conditions), CIMC-2
(collagen gel, 1 wk in nonpermissive conditions), CIMC-3 (1st hour of
collagenase digestion, 1 wk in nonpermissive conditions), CIMC-4 (2nd
hour of collagenase digestion, 1 wk in nonpermissive conditions),
CIMC-5 (collagen gel, directly in permissive conditions), and CIMC-6
cells (1st hour of collagenase digestion, directly in permissive
conditions). CIMC cell lines were maintained under permissive
conditions in phenol red-free
MEM (GIBCO) containing 10% fetal
bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine (Mediatech), 100 U/ml penicillin (Mediatech), and 100 mg/ml
streptomycin (Mediatech). Before experiments, CIMC cells were cultured
for 1 wk under nonpermissive conditions in phenol red-free basal MEM (GIBCO) containing the supplements described above. All media, sera, and additives were from lots that contained the lowest
concentration of endotoxin available. CIMC cell cultures were
consistently negative when assayed for mycoplasma contamination by
solution hybridization to a probe complementary to mycoplasma rRNA
(Gen-Probe, San Diego, CA).
Primary calvarial cells were isolated from 2- to 3-day-old C57BL/6
neonates, and cells were collected from the second hour of collagenase
treatment as described for the conditionally immortalized cells.
Calvarial cells were plated (10,000 cells/cm2) in phenol
red-free MEM containing the supplements described above, and
aliquots were frozen when the cultures reached 80% confluence (after
4-5 days at 5% CO2, 37°C).
Osteoclast precursors were obtained from spleens of 6- to 16-wk-old
female C57BL/6 mice. In selected experiments, spleen cells were
obtained from double knockout mice lacking both TNF receptor-1 and TNF
receptor-2 (B6 129S-Tnfrsf1atm1/mx
Tnfrsf1btm1/mx, stock no. 003243; Jackson Labs, Bar
Harbor, ME) (43). Control spleen cells were from wild-type
mice matched for age, sex, and genetic background (B6129SF2/J, stock
no. 101045; Jackson Labs). Differentiation of osteoclast precursors was
assessed in cocultures containing CIMC cells, freshly thawed primary
calvarial cells, or ST2 cells (RIKEN Cell Bank, Tsukuba Science City,
Japan) plated at a density of 10,000 cells/cm2 in phenol
red-free basal MEM containing the supplements described above plus
nonessential amino acids (Mediatech). After CIMC cells, calvarial
cells, or ST2 cells were cultured for 1 day (5% CO2, 37°C), osteoclast precursors were added (500,000 nucleated spleen cells/cm2). All cocultures were performed in phenol
red-free basal MEM with 10% FBS, nonessential amino acids,
L-glutamine, antibiotics, and freshly added 100 nM
dexamethasone (Sigma, St. Louis, MO) and 50 µg/ml ascorbic acid
(GIBCO) as previously described (45). Osteoclast
differentiation was stimulated with the indicated concentrations of
1,25-D3 (Biomol Research Labs, Plymouth Meeting, PA),
bovine PTH-(1-34) (Bachem, Torrance, CA), murine TNF-
(R&D Systems, Minneapolis, MN), murine IL-1
(R&D Systems), murine
IL-6 (R&D Systems), or human IL-11 (Peprotech, Rocky Hill, NJ) in the
presence or absence of murine OPG/Fc chimera (R&D Systems). All of the stimulators were screened for endotoxin contamination by using the
high-sensitivity version of the colorimetric Limulus amoebocyte lysate
assay (QCL-1000; Whittaker Bioproducts, Walkersville, MD) as we have
previously described (22). Endotoxin levels were <0.0007
EU/ml for the highest concentrations used of the stimulators. Cocultures received fresh media, cytokines, and other additives on
days 3 and 6 and were stained for
tartrate-resistant acid phosphatase (TRAP) on day 9 by using
a commercially available kit (no. 387-A; Sigma) with the previously
described modifications (45). TRAP-positive multinucleated
cells (TRAP-positive MNCs) containing three or more nuclei were counted
by using an inverted microscope with video attachments.
In selected experiments, formation of resorption lacunae was determined by performing the CIMC cell/spleen cell cocultures on slices of elephant ivory. For this purpose, both CIMC cells (30,000 cells/cm2) and spleen cells (500,000 cells/cm2) were plated on round slices of ivory, which covered virtually the entire bottom of individual wells in a 96-well plate. Cocultures were performed as described in the previous paragraph. After 9 days, the number of TRAP-positive MNCs were counted on the ivory slices and the extent of resorption was determined by using toluidine blue staining as we have previously described (23).
Figures 1-4 are representative of multiple experiments. All
numerical results are reported as means ± SE. Statistical
analysis was by ANOVA performed on Super ANOVA software (Abacus
Concepts, Berkeley, CA). The Bonferroni-Dunn (control) post hoc test
was used when groups were compared with a single control group (Figs. 1
and 2), and Fisher's protected least significant difference post hoc
test was used when multiple groups were compared (Figs. 3 and 4).
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RESULTS |
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We wanted to determine whether osteoclast differentiation is
synergistically stimulated by TNF-, IL-1
, IL-6, and IL-11. However, as described in the Introduction, none of the available cell
lines support osteoclast differentiation in response to all of these
agents. We reasoned that CIMC cells might fulfill these criteria
because primary calvarial cells have been shown to have this capability
(50, 51). Therefore, we developed six distinct CIMC cell
lines and tested their ability to support osteoclast differentiation.
The CIMC cell lines were screened for their ability to support
osteoclast differentiation in response to concentrations of
1,25-D3, PTH, TNF-
, IL-1
, IL-6, or IL-11 that other
workers have shown to be effective for this purpose. As illustrated in Table 1, 1,25-D3 and IL-11
stimulated osteoclast differentiation in cultures with any of the six
CIMC cell lines, whereas PTH had little effect in all cases. In
contrast, substantial osteoclast differentiation was induced by TNF-
only in the presence of CIMC-4 cells, by IL-1
only in the presence
of CIMC-1 or CIMC-4 cells, and by IL-6 in the presence of CIMC-4 cells
but not in the presence of CIMC-1 cells (Table 1). Thus osteoclast
differentiation is stimulated in cultures with CIMC-4 cells by all
agents that have been tested except PTH. CIMC-4 cells were therefore
used for all other experiments in this study.
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To confirm that the formation of TRAP-positive MNCs in our cultures represents authentic osteoclast differentiation, we determined whether the TRAP-positive MNCs were able to form resorption lacunae. For this purpose, CIMC-4 cell/spleen cell cocultures were performed on slices of ivory in the presence of 0.2 nM 1,25-D3 as we have previously described (9). Examination of the ivory slices showed that the TRAP-positive MNCs are closely associated with resorption lacunae. Moreover, quantitative histomorphometry demonstrated a strong correlation (r2 = 0.88) between the number of TRAP-positive MNCs (Fig. 1A) and the extent of resorption lacunae formation (Figs. 1B) induced by the cytokines. In addition, the increased resorption is primarily due to an increase in the number of resorption lacunae that are formed (Fig. 1C) rather than the size of the lacunae (Fig. 1D). This finding provides further evidence that the increased resorption is due to osteoclast differentiation.
We next examined the effect of various doses of the cytokines on
stimulation of osteoclast differentiation. Figure
2 shows that the cytokines
dose-dependently stimulated osteoclast differentiation. The lowest
concentrations of the cytokines that significantly stimulated
osteoclast differentiation in this experiment were 12 ng/ml TNF-,
0.1 ng/ml IL-1
, 10 ng/ml IL-6, and 5 ng/ml IL-11 (Fig. 2).
Most bone-resorptive agents increase osteoclast differentiation by
upregulating the RANKL/OPG ratio (41, 52, 56, 65). To test
whether stimulation of osteoclast differentiation is dependent on
the RANKL pathway, exogenous OPG was added to selected cultures. OPG
completely eliminated formation of both TRAP-positive MNCs (Fig.
3A) and TRAP-positive
mononuclear cells (data not shown) induced by 1,25-D3 and
all of the cytokines, with the notable exception of TNF-. A similar
lack of effect of OPG on stimulation by TNF-
of both TRAP-positive
MNCs and TRAP-positive mononuclear cells was observed even in the
presence of relatively low TNF-
concentrations (Fig. 3, B
and C) and when a 10-fold higher concentration of OPG was
studied (Fig. 3D). One explanation for these results is
that, whereas 1,25-D3, IL-1, IL-6, and IL-11 primarily act by increasing RANKL expression by the CIMC-4 cells, TNF-
primarily acts directly on the osteoclast precursors. To test this possibility, CIMC-4 cells were cocultured with spleen cells isolated from either double knockout mice lacking both TNF receptor-1 and TNF receptor-2 or
wild-type mice (matched for age, sex, and genetic background). Figure
3E shows that TNF-
potently stimulated differentiation of
wild-type osteoclast precursors but did not induce differentiation of
osteoclast precursors that lacked TNF-
receptors, even when they were cocultured with CIMC-4 cells that had normal TNF-
responsiveness. Specificity of this effect was demonstrated by showing
that the TNF-
receptor-deficient osteoclast precursors
differentiated normally in response to 1,25-D3 (Fig.
3F).
Concentrations of the cytokines that are low enough to have no or
minimal effects by themselves synergistically stimulate osteoclast
differentiation when added together. Thus Fig.
4A shows that 0.94 ng/ml
TNF-, 0.001 ng/ml IL-1
, 3 ng/ml IL-6, and 0.5 ng/ml IL-11
synergistically stimulated osteoclast differentiation in the presence
of CIMC-4 cells. Further evidence that the four cytokines act
synergistically appears in Fig. 4B, which shows that very
similar results were also obtained when osteoclast precursors were
cocultured with primary calvarial cells rather than with CIMC-4 cells.
Moreover, addition of any three of the four cytokines also induced
synergistic stimulation of osteoclast differentiation, albeit to a
lesser extent than that induced by all four cytokines (Fig.
4C). Synergistic stimulation of osteoclast
differentiation was blocked by addition of OPG, whether CIMC-4 cells or
primary calvarial cells were used to support osteoclast differentiation (Fig. 4D). However, use of the TNF-
receptor-deficient
osteoclast precursors described above shows that TNF-
primarily
acted directly on the osteoclast precursors even in the presence of the
other cytokines (Fig. 4E). Thus the cytokine mixture
containing TNF-
induced significantly less differentiation of
TNF-
receptor-deficient osteoclast precursors than control
precursors (compare 2nd and 5th bars in Fig. 4E). Moreover,
the extent of differentiation of TNF-
receptor-deficient osteoclast
precursors that was induced by the cytokine mixture containing TNF-
is indistinguishable from that induced by the cytokine mixture lacking
TNF-
with either type of precursor (compare 5th bar in Fig.
4E with 3rd and 6th bars). Taken together with data in Fig.
3, these results show that TNF-
stimulates osteoclast
differentiation primarily by acting directly on the osteoclast
precursors even in the presence of other cytokines and
TNF-
-responsive mesenchymal support cells.
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DISCUSSION |
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This study has established an osteoclast differentiation
assay that is responsive to a wide variety of stimulators
(1,25-D3, TNF-, IL-1
, IL-6, and IL-11) and does not
rely on isolation of primary stromal support cells. Instead, we have
isolated six CIMC cell lines, all of which support osteoclast
differentiation in response to 1,25-D3 and IL-11. In
contrast, only specific CIMC cell lines support osteoclast
differentiation in response to TNF-
, IL-1
, or IL-6. For example,
CIMC-4 cells support osteoclast differentiation in response to
1,25-D3, TNF-
, IL-1
, IL-6, or IL-11; i.e., all of the
bone-resorptive agents that have been tested except for PTH.
Our results also demonstrate that TNF-, IL-1
, IL-6, and IL-11
stimulate osteoclast differentiation in a synergistic fashion. Synergistic stimulation of osteoclast differentiation by these cytokines may have important implications for a number of physiological and/or pathological conditions. Multiple cytokines appear to be involved in normal bone remodeling (26, 44, 64) as well as
in stimulation of bone resorption in many pathological conditions including estrogen deficiency (27, 38),
hyperparathyroidism/humoral hypercalcemia of malignancy
(23, 24, 61), hyperthyroidism (46),
tumor-induced osteolysis (2, 7, 12, 58), rheumatoid arthritis (19), lipopolysaccharide-induced
osteolysis (1, 11), and orthopedic implant loosening
(36). Our CIMC-4 cells provide a model of osteoclast
differentiation that should be useful for examining the precise roles
of each of the cytokines in these conditions.
Osteoclast differentiation induced in our system by
1,25-D3, IL-1, IL-6, or IL-11, acting either alone or in
combination, is blocked by addition of exogenous OPG. These results
provide further evidence that most stimulators of osteoclast
differentiation act indirectly through the RANKL pathway (39, 41,
52, 56, 65). With regard to IL-6, some mesenchymal cells do not
express adequate levels of the IL-6 receptor
-chain and, therefore,
do not upregulate RANKL expression or support osteoclast
differentiation in response to IL-6 unless the soluble form of this
molecule is also added (41, 42, 55, 60). In contrast, we
found that IL-6 induces the CIMC-4 cells to support osteoclast
differentiation. CIMC-4 cells must therefore express adequate levels of
the IL-6 receptor
-chain either constitutively or as a result of
treatment with dexamethasone, as has been demonstrated for primary
calvarial cells (60). It has been reported that IL-6 does
not increase RANKL expression by MG-63 cells even in the presence of
exogenous soluble IL-6 receptor
-chain (25). However,
because MG-63 cells have not been shown to support osteoclast
differentiation in response to IL-6, the relevance of this result is
uncertain. In contrast, primary calvarial cells upregulate RANKL
production (41) and support osteoclast
differentiation (50, 51) in response to IL-6 as well as in
response to IL-1, IL-11, and TNF-
.
TNF- has recently been reported to directly stimulate
differentiation of osteoclast precursors through a pathway that is not
inhibited by OPG and does not require the presence of stromal support
cells or the addition of exogenous RANKL (5, 30). However,
TNF-
also has many effects on stromal cells, and the presence of
stromal cells is required for stimulation of osteoclast activity by
TNF-
(57). Thus it was important to determine whether TNF-
primarily stimulates differentiation of osteoclast precursors directly even in the more physiological situation where stromal cells
and other cytokines are present. We found that OPG has little or no
effect on osteoclast differentiation induced by TNF-
. Moreover, TNF-
is unable to induce osteoclast differentiation when precursors lacking both TNF receptor-1 and TNF receptor-2 are cocultured with
CIMC-4 cells in the presence or absence of IL-1, IL-6, and IL-11. These
results are consistent with those of Lam et al. (33), who
performed the converse experiment and found that TNF-
stimulates osteoclast differentiation of wild-type precursors in the presence of
mesenchymal cells that lack both TNF receptor-1 and TNF receptor-2. Taken together, these results provide strong evidence that TNF-
primarily stimulates osteoclast differentiation through direct actions on osteoclast precursors, a mechanism that is distinct from
that utilized by most other bone resorptive agents that act through the
RANKL pathway. The lack of effect of OPG in our studies and those
described in Refs. 5 and 30 does not
necessarily imply that osteoclast differentiation induced by TNF-
is
completely independent of RANKL (33). In fact, TNF-
induces little osteoclast differentiation in vivo in mice that do not
express RANK (34). Thus it is likely that RANKL acts
synergistically with TNF-
to stimulate osteoclast differentiation
both in vivo (34) and in vitro (31, 33, 67,
68). Consistent with this concept, RANKL and TNF-
have
recently been shown to cooperatively activate signal transduction
pathways in osteoclast precursors (33, 47, 67).
In summary, we have isolated CIMC-4 cells that support osteoclast
differentiation in response to 1,25-D3, TNF-, IL-1
,
IL-6, and IL-11. Low concentrations of the cytokines synergistically stimulate osteoclast differentiation by osteoclast precursors cocultured with either CIMC-4 cells or primary calvarial cells. OPG
blocks osteoclast differentiation induced by all of the resorptive agents that were examined except TNF-
. In contrast to the other agents, TNF-
directly stimulates differentiation of osteoclast precursors.
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ACKNOWLEDGEMENTS |
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We are grateful to R. Van De Motter, M. Blaha, and S. Lavish for technical assistance, to N. Takahashi for advice on isolating calvarial cells by the collagen outgrowth method, to C. Cotton for providing the SV40 large T antigen transgenic mice, to T. Bettinger and A. Lewandowski (Cleveland Metroparks Zoo) for kindly providing pieces of elephant ivory, to J. Bensusan for assistance in preparing the round ivory slices, to S. Kaar for help with histomorphometry, and to C. Carlin and J. Dennis for many helpful discussions.
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FOOTNOTES |
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* A. A. Ragab and J. L. Nalepka contributed equally to this work.
This work was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-41674 and AR-43769 (to E. M. Greenfield). A. A. Ragab was supported by National Institutes of Health Institutional Training Grant AR-07505.
Address for reprint requests and other correspondence: E. M. Greenfield, Dept. of Orthopaedics, Case Western Reserve Univ., 11100 Euclid Ave., Cleveland, OH 44106-5000 (E-mail: emg3{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpcell.00421.2001
Received 31 August 2001; accepted in final form 18 April 2002.
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