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Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.

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Vol. 63, No. 8, August 2011, pp 2385–2395
DOI 10.1002/art.30385
© 2011 American College of Rheumatology
Sclerostin Antibody Treatment Enhances Bone Strength but
Does Not Prevent Growth Retardation in
Young Mice Treated With Dexamethasone
M. Marenzana, K. Greenslade, A. Eddleston, R. Okoye, D. Marshall,
A. Moore, and M. K. Robinson
Objective. Exposure to supraphysiologic levels of
glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients
with sclerosteosis lack the bone regulatory protein
sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to
dexamethasone (DEX).
Methods. Young mice were concomitantly treated
with DEX (or vehicle control) and Scl-AbI antibody
(or isotype-matched control antibody [Ctrl-Ab]) in 2
independent studies. Linear growth, the volume and
strength of the bones, and the levels of bone turnover
markers were analyzed.
Results. In DEX-treated mice, Scl-AbI had no
significant effect on linear growth when compared to
control treatment (Ctrl-Ab). However, in mice treated
with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total
volume ⴙ117% versus Ctrl-Ab–treated mice) and in the
width and volume of the cortical bone at the femoral
diaphysis (ⴙ24% and ⴙ20%, respectively, versus Ctrl-
Ab–treated mice) was noted. Scl-AbI treatment also
improved mechanical strength (as assessed by 4-point
bending studies) at the femoral diaphysis in DEXtreated mice (maximum load ⴙ60% and ultimate
strength ⴙ47% in Scl-AbI–treated mice versus Ctrl-Ab–
treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid
phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab.
Conclusion. Scl-AbI treatment does not prevent
the detrimental effects of DEX on linear growth, but the
antibody does increase both cortical and trabecular
bone and improves bone mechanical properties in
DEX-treated mice.
Glucocorticoid (GC)–based drugs have potent
immunosuppressive and antiinflammatory properties
and have assumed an important role in the treatment of
many types of inflammatory and autoimmune conditions. However, drugs of this type are associated with
a range of well-known side effects (1). One of the most
serious problems associated with GC exposure is a
deleterious effect on bone, which leads to a high proportion of patients who, after receiving long-term GC
therapy, develop GC-induced osteoporosis and are susceptible to bone fractures (2). The detrimental effect of
GCs on bone strength has been reported to involve
many different mechanisms, including inhibition of osteoblastic bone formation, increased osteoclastic bone
resorption, changes in calcium balance, and inhibition
of the osteoanabolic action of sex steroids (3). More
recently, it has also been proposed that GC exposure not
only may cause changes to bone mass and bone architecture, but also may alter the localized material properties of bone (4).
When administered to children or to growing
Supported by UCB.
M. Marenzana, PhD (current address: Imperial College,
London, UK), K. Greenslade, BSc, A. Eddleston, MSc, R. Okoye,
MSc, D. Marshall, PhD, A. Moore, PhD, M. K. Robinson, PhD: UCB
Celltech, Slough, UK.
Ms Okoye and Drs. Marshall, Moore, and Robinson hold
stock or stock options in UCB Celltech. Dr. Robinson is a named
inventor on a number of UCB patents, including ones related to
Address correspondence to M. K. Robinson, PhD, Department of Inflammation Biology, UCB Celltech, 216 Bath Road, Slough
SL1 4EN, UK. E-mail:
Submitted for publication August 19, 2010; accepted in
revised form March 29, 2011.
animals, GCs not only have detrimental effects on bone
strength, but also cause linear growth retardation (5–7).
The inhibitory effects of GCs on growth are primarily
mediated by disruption of chondrogenesis at the growth
plate (8). This occurs via direct effects of GCs on chondrocyte growth and apoptosis (9,10), as well as through
changes to factors such as those in the insulin-like
growth factor (11) and vascular endothelial growth
factor pathways (12), which disturb normal growth plate
Sclerostin is a glycoprotein that is produced by
osteocytes and negatively regulates osteoblastic bone
formation (13). The gene encoding sclerostin was identified by molecular and genetic analysis of the rare
inherited condition sclerosteosis, a condition in which
the gene is known to be defective (14,15). Patients with
sclerosteosis are typically of large stature and show
excessive bone formation (16). Sclerostin has been reported to antagonize signaling in the bone morphogenetic protein pathway (17), but it is currently believed
that inhibition of the Wnt pathway is more important for
the activity of sclerostin on bone formation in vivo (18).
Antibodies to sclerostin increase bone strength, bone
formation, and bone healing in a range of animal models
(19–22), and a monoclonal antibody to sclerostin was
recently evaluated for its clinical effects (23).
Chen et al (24) reported that inhibition of signaling through ␤-catenin (a pivotal component of the
Wnt signaling pathway) led to decreased width of
the proliferating and hypertrophic zones of the growth
plate, and this was associated with decreased skeletal
growth. In adolescent humans, sclerostin has been reported to be expressed by both osteocytes and mineralized hypertrophic chondrocytes in the growth plate. It
has been speculated that sclerostin may play a role in
regulating chondrocyte proliferation, and that this could
explain the tall stature of patients with sclerosteosis
The negative regulatory role of sclerostin in bone
formation and Wnt signaling, coupled with its reported
expression close to the growth plate, suggest that its
inactivation with a monoclonal antibody might both
stimulate bone formation and prevent growth retardation in young, GC-treated animals. In the present study,
we describe the effects of a monoclonal antibody to
sclerostin (Scl-AbI) in young mice being concomitantly
treated with dexamethasone (DEX). The young age of
the mice makes this model most relevant to the inhibition of the bone growth (or bone rarefaction) seen in
children treated with GCs (5,6).
Antibodies and drugs. DEX and methylcellulose were
obtained from Sigma UK. The Scl-AbI antibody and isotypematched control antibody (Ctrl-Ab) have been previously
described (19).
Animals and experiment outline. BALB/c mice (obtained from Charles River UK) were ⬃7 weeks of age at the
start of the studies. Mice were maintained and studied in a
manner in compliance with UK Home Office regulations. Two
similar experiments were performed with DEX (prepared in a
0.5% methylcellulose solution), which was administered at a
daily dose of 3 mg/kg by oral gavage for either 6 weeks or
9 weeks. In both experiments, 4 groups of mice were treated, as
follows: 1) vehicle (0.5% methylcellulose solution) and CtrlAb, 2) DEX and Ctrl-Ab, 3) vehicle and Scl-AbI, or 4) DEX
and Scl-AbI. The sclerostin or control antibodies were administered subcutaneously at a dosage of 25 mg/kg twice weekly.
The body weight of the animals was monitored weekly.
Measurement of serum markers. Serum levels of
type 5b tartrate-resistant acid phosphatase (TRAP5b) and
N-terminal type I procollagen propeptide (PINP) were measured using mouse sandwich enzyme-linked immunosorbent
assay kits from ImmunoDiagnostic Systems. Serum levels of
osteocalcin were measured using Luminex kits obtained from
Millipore. All kits were used according to the manufacturer’s
Measurement of areal bone mineral density (BMD).
Mice were anesthetized with 2% isofluorane inhalation. After
being placed under general anesthesia, the mice were scanned
on a Lunar PIXImus (GE Medical Systems). BMD measurements were determined at baseline and at 3, 6, and 9 weeks
Microfocal computed tomography (micro-CT) analysis. Microstructural analysis was performed on bone samples
at the end of the 6-week dosing experiment. Dissected femurs were scanned at high resolution (7 ␮m pixel size) in a
micro-CT scanner (Scanco ␮CT 35). The Scoutview images
were used to measure femur length and to position the imaging
window. Analysis was performed in 2 regions: 1) the middiaphysis cortical bone, a 0.7-mm–long segment (or 100 tomograms) centered over the femoral midline, and 2) the distal
femoral metaphysis, a region starting 0.29 mm from an anatomic landmark in the growth plate and extending 1.2 mm (or
170 tomograms) proximally. The vertebral bodies of L5 vertebra, including upper and lower disks, were selected from the
Scoutview images of the whole mouse spine and scanned at a
resolution of 6 ␮m (pixel size). The longitudinal length of the
vertebral body was the difference between the top and bottom
tomograms. Trabecular bone regions of interest were manually
drawn, with exclusion of the cortex.
Two- and three-dimensional morphometric measurements were obtained using Scanco analysis software, with a
fixed threshold of 500 mg/cm3 to segment bone from the
background. Cortical volumetric BMD and bone mineral content were also obtained, after calibrating the absorption values
against known density phantoms. The width of the growth
plate in the distal femur was measured in reconstructed scans
by generating 3 coronal sections (1 central and 2 symmetrically
placed 100 ␮m apart) from the stack of cross-sectional tomograms, and then enclosing the growth plate and a thin layer of
bone tissue along its length. An inverse threshold was applied
to this region (i.e., everything below 400 mg/cm3) to select
nonmineralized cartilage and exclude bone. The average width
and other geometric features of the segmented regions were
obtained using Scanco analysis software. Results from 10
age-matched mice that were killed at the start of the study were
used for the baseline values.
Immunohistochemistry. Femurs were fixed in neutral
buffered formalin before decalcification in EDTA, and were
then embedded in paraffin wax. Five-micrometer sections
were cut, followed by proteinase K antigen retrieval and
blocking of endogenous peroxidase. Sections were incubated
with goat polyclonal anti-mouse sclerostin antibody (AF1589,
used at 1:100 dilution; R&D Systems) followed by biotinylated
donkey anti-goat secondary antibody (used at 1:500 dilution;
Stratech Scientific). The signal was visualized using a peroxidase–
streptavidin and diaminobenzidine substrate–chromogen system (Dako UK). Sections were washed and counterstained
with hematoxylin.
Four-point bending test of the femoral shaft. Mechanical testing was performed on the right femurs of the mice. The
femurs had been excised from the mice at the termination of
the 9-week study, and were kept frozen at ⫺70°C until the day
of the test. For mechanical testing, the 4-point bending test was
used (26). This test allows the calculation of a number of bone
mechanical properties, including resistance to bending under
load (stiffness), the maximum load that a bone can sustain
(maximum load), and the maximum stress that a bone can
sustain and the amount of energy the bone can absorb before
failure (toughness). The mechanical testing was carried out at
MDS Pharma, as previously described (19).
Statistical analysis. Statistical analysis of the data was
performed using one-way analysis of variance with a Bonferroni post hoc test to adjust for multiple comparisons. P values
less than 0.05 were considered significant.
Distribution of sclerostin-positive cells in the
growing mouse epiphysis. Immunohistochemical analysis of the mouse femurs using Scl-AbI confirmed previ-
ous findings indicating that the osteocyte is the major
cellular source of sclerostin in the mouse epiphysis
(Figure 1A). It was also clear that the basal layer of the
articular cartilage contained a number of sclerostinpositive cells, which appeared to be hypertrophic chondrocytes, similar to observations recently reported in a
study by van Bezooijen et al (25). Although the growth
plate itself did not contain sclerostin-positive cells, there
were nests of cells adjacent to the growth plate that were
positive for sclerostin expression (Figure 1B). The results of immunohistochemistry revealed no obvious differences in the distribution of sclerostin expression or
the intensity of sclerostin staining when mice were
treated with DEX (results not shown).
Lack of effect of Scl-AbI treatment on DEXinduced changes in body weight, bone length, or growth
plate thickness. As shown in Figures 2A and B, the
pattern of changes in body weight in the groups of mice
in 2 separate experiments, involving treatment of growing mice with DEX for either 6 weeks or 9 weeks, was
very similar in both experiments. In each case, the
vehicle/Ctrl-Ab–treated mice showed a normal pattern
of weight gain, whereas DEX/Ctrl-Ab–treated mice
failed to gain weight over the course of the experiment.
The weight of the mice in the DEX/Scl-AbI treatment
group was not significantly different from that of those
in the DEX/Ctrl-Ab group, indicating that Scl-AbI had
no influence on the suppressive effects of DEX on body
Femurs from the mice in the 6-week experiment
were examined to determine bone length. Micro-CT was
used to determine the thickness of the femoral distal
epiphyseal growth plate and L5 vertebrae. The vehicle/
Ctrl-Ab–treated mice showed a significant increase in
Figure 1. Immunocytochemical localization of sclerostin in the femoral head of mice. The results of immunohistochemistry in a representative mouse femur reveal A, sclerostin-positive cells
(in dark brown, with nuclei counterstained in blue) in the articular surface (AS) of the epiphysis,
growth plate (GP), and metaphysis, and B, nests of sclerostin-positive cells adjacent to the growth
plate (arrowheads). The image in B is a higher-power magnification of the boxed area in A.
Figure 2. Effects of dexamethasone (DEX) and sclerostin monoclonal antibody (Scl-AbI) treatment on body weight and bone growth of mice. A and B, Body weight was assessed after treatment
of mice in 2 separate experiments, with durations of 9 weeks (A) and 6 weeks (B). C and D, Femoral
length, as measured from microfocal computed tomography (micro-CT) Scoutview images (C), and
growth plate thickness, as measured from micro-CT reconstruction images (D), were assessed in
mice after 6 weeks of treatment. Values are the mean with 95% confidence intervals in 5–10 mice
per group, including a satellite group of 5–10 age-matched mice from which samples were obtained
at baseline (BSLN). ⴱ ⫽ P ⬍ 0.05 versus vehicle/isotype-matched control antibody (Ctrl-Ab);
ⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab, DEX/Scl-AbI, and BSLN; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus
vehicle/Ctrl-Ab and BSLN; † ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab and DEX/Scl-AbI.
both femoral length (Figure 2C) and L5 vertebral length
(⫹4.4%) (Table 1) when compared with the baseline
values from the satellite group of mice that were killed at
the start of the experiment, which was consistent with
Table 1. Microstructural parameters of trabecular bone in the distal femur metaphysis and L5 vertebral body of young mice concomitantly treated
with dexamethasone (DEX) (or vehicle control) and an antibody to sclerostin (Scl-AbI) (or isotype-matched control antibody [Ctrl-Ab])*
Distal femur
BV/TV, %
TbTh, mm
TbSp, mm
Vertebra L5
BV/TV, %
TbTh, mm
TbSp, mm
Longitudinal length
13.69 (12.76–14.62)
0.028 (0.027–0.029)
0.179 (0.167–0.191)
4.861 (4.593–5.128)
13.56 (12.71–14.41)
0.025 (0.025–0.026)§
0.163 (0.154–0.172)
5.344 (5.108–5.581)§
31.33 (29.50–33.17)†
0.054 (0.051–0.056)†
0.118 (0.113–0.124)†
5.811 (5.655–5.967)¶
29.38 (26.94–31.82)‡
0.046 (0.045–0.048)‡
0.113 (0.104–0.122)‡
6.304 (6.016–6.592)‡
12.79 (11.97–13.62)
0.026 (0.026–0.027)
0.180 (0.170–0.190)
4.873 (4.647–5.099)
18.85 (18.15–19.54)
0.033 (0.032–0.034)
0.141 (0.136–0.147)
5.753 (5.562–5.943)
3.385 (3.320–3.450)
18.38 (17.49–19.27)
0.030 (0.030–0.031)§
0.136 (0.130–0.141)
6.026 (5.841–6.211)
3.191 (3.142–3.240)§
40.99 (39.98–42.01)†
0.066 (0.064–0.067)†
0.094 (0.092–0.097)†
6.257 (6.170–6.344)§
3.373 (3.326–3.420)#
28.84 (27.63–30.05)‡
0.048 (0.048–0.049)‡
0.120 (0.113–0.126)‡
5.976 (5.755–6.197)
3.232 (3.163–3.301)§
17.98 (17.14–18.82)
0.030 (0.029–0.030)§
0.136 (0.130–0.143)
6.035 (5.813–6.258)
3.241 (3.190–3.292)§
* Values are the mean (95% confidence interval) measurements in 5–10 mice per group. Baseline values were obtained from a group of 5–10
age-matched mice that were killed at the start of the study. BV/TV ⫽ femoral trabecular volume fraction expressed as bone volume/total volume;
TbTh ⫽ trabecular bone thickness; TbSp ⫽ trabecular bone spacing; TbN ⫽ trabecular number.
† P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, DEX/Scl-AbI, and baseline.
‡ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, and baseline.
§ P ⬍ 0.05 versus vehicle/Ctrl-Ab.
¶ P ⬍ 0.05 versus vehicle/Ctrl-Ab and DEX/Ctrl-Ab.
# P ⬍ 0.05 versus DEX/Ctrl-Ab, DEX/Scl-AbI, and baseline.
Figure 3. Effects of DEX and Scl-AbI treatment of mice on femoral bone mineral density (BMD)
and bone mechanical strength. A and B, Changes to areal BMD over 9 weeks (A) or 6 weeks (B)
of treatment were assessed by dual x-ray absorptiometry. C and D, Biomechanical properties of the
excised mouse femurs after 9 weeks of treatment were assessed using the 4-point bending test,
which tested for maximum load to failure (C) and ultimate strength (D). Values are the mean with
95% confidence intervals in 3–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍
0.05 versus vehicle/Ctrl-Ab and DEX/Ctrl-Ab; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab. See Figure 2
for other definitions.
normal growth. Both groups of mice receiving DEX had
significantly shorter femurs (Figure 2C) and vertebrae
(Table 1) than did those receiving vehicle. There was no
significant difference in the values for femoral and
vertebral length between the mice in the DEX/Ctrl-Ab
and DEX/Scl-AbI treatment groups (Table 1 and Figure
Measurements of the femoral epiphyseal growth
plates showed that the thickness of the growth plate in
vehicle/Ctrl-Ab–treated mice was significantly less than
that in mice at baseline (probably due to a slowing in
bone growth as the mice aged). In DEX/Ctrl-Ab–treated
mice, the growth plates were significantly thinner than
those in vehicle/Ctrl-Ab–treated mice, indicating a negative effect of DEX on the growth plate (Figure 2D). In
vehicle/Scl-AbI–treated mice, the thickness of the
growth plate was not significantly different from that in
the vehicle/Ctrl-Ab group. When Scl-AbI was administered to DEX-treated mice (DEX/Scl-AbI group), the
growth plate thickness was not significantly different
from that in mice treated with DEX/Ctrl-Ab (Figure
2D). These findings indicate a significant inhibitory
effect of DEX on growth in young mice, which is
consistent with the results from previous studies (7).
Moreover, the findings show that Scl-AbI treatment is
unable to counter this effect.
Prevention of DEX-induced changes to areal
BMD by Scl-AbI. Assessments of areal BMD of the
femur in both the 6-week study and the 9-week study
indicated that, from the start of the experiment, there
was a gradual increase in areal BMD in vehicle/Ctrl-Ab–
treated mice (Figures 3A and B). These changes were
consistent with the age of the mice and their continuing
skeletal growth. In the DEX/Ctrl-Ab treatment group,
there was no significant increase in the areal BMD of
these mice over the course of the experiments, in either
the femur (Figures 3A and B) or the spine (results not
shown). By the end of the experiments (6 and 9 weeks),
the values for areal BMD in the DEX/Ctrl-Ab group
were significantly lower than those in the vehicle/
Ctrl-Ab group.
Mice in the vehicle/Scl-AbI treatment group had
significantly higher areal BMD after 6 and 9 weeks of
treatment, both in the femur (Figures 3A and B) and in
the spine (results not shown), when compared with mice
in the vehicle/Ctrl-Ab group. Mice in the DEX/Scl-AbI
group had areal BMD values that were significantly
higher than those in the DEX/Ctrl-Ab group at 6 and 9
weeks, in the femur (Figures 3A and B) as well as in the
lumbar spine and caudal vertebrae (6-week mean BMD
of lumber spine 0.093 gm/cm2, 95% confidence interval
[95% CI] 0.091–0.094 gm/cm2 in DEX/Scl-AbI group
versus 0.074 gm/cm2, 95% CI 0.072–0.075 gm/cm2 in
DEX/Ctrl-Ab group; 6-week mean BMD of caudal
vertebrae 0.059 gm/cm2, 95% CI 0.057–0.062 gm/cm2 in
DEX/Scl-AbI group versus 0.051 gm/cm2, 95% CI
0.049–0.052 gm/cm2 in DEX/Ctrl-Ab group). The areal
BMD values observed in the femurs of the DEX/SclAbI–treated mice were not significantly different from
those in the vehicle/Ctrl-Ab group, but these levels did
not reach the values seen in mice in the vehicle/Scl-AbI
group (Figures 3A and B).
Prevention of DEX-induced changes in bone mechanical strength by Scl-AbI. High-level DEX exposure
is known to reduce both bone formation and bone
mechanical properties (4). To investigate the effects of
Scl-AbI treatment on bone mechanical properties in
DEX-treated mice, the femurs of mice in the 9-week
experiment were removed at the end of the experiment
and subjected to 4-point bending tests. Data on the
mechanical strength of the femurs are shown in Figures
3C and D. Compared with vehicle/Ctrl-Ab–treated mice,
mice in the DEX/Ctrl-Ab group had weaker bones, as
evidenced by significantly reduced maximum load (Figure 3C) and stiffness (mean 183.4 N/mm, 95% CI
161.9–204.8 N/mm in vehicle/Ctrl-Ab group versus 105
N/mm, 95% CI 77.7–132.4 N/mm in DEX/Ctrl-Ab
group) (both are extrinsic bone properties), as well as
significantly reduced ultimate strength (Figure 3D) and
toughness (mean 8.21 MJ/m3, 95% CI 5.96–10.46 MJ/m3
in vehicle/Ctrl-Ab group versus 3.73 MJ/m3, 95% CI
2.68–4.78 MJ/m3 in DEX/Ctrl-Ab group) (both are
intrinsic bone properties).
Mice in the vehicle/Scl-AbI group had improved
bone mechanical properties, with significantly increased
values for maximum load (Figure 3C), stiffness (mean
252.8 N/mm, 95% CI 225.1–280.5 N/mm), toughness
(mean 15.93 MJ/m3, 95% CI 12.38–19.48 MJ/m3), and
ultimate strength (Figure 3D) in comparison with the
values observed in mice in the vehicle/Ctrl-Ab group.
Intrinsic and extrinsic mechanical properties of the
femurs in the mice in the DEX/Scl-AbI–treated group
were significantly improved (maximum load and ultimate strength shown in Figures 3C and D; stiffness and
toughness, mean 172.3 N/mm, 95% CI 155.2–189.5
N/mm and mean 8.19 MJ/m3, 95% CI 4.62–11.76 MJ/m3,
respectively) when compared with those in the DEX/
Ctrl-Ab group, and the values were not significantly
different from those in the vehicle/Ctrl-Ab group. However, the values for intrinsic and extrinsic bone properties of the femurs in the DEX/Scl-AbI–treated mice
were not as high as the values in those receiving vehicle/
These findings show that the dose of DEX used
in these studies produced profound changes in growth,
body weight, areal BMD, and bone mechanical strength
in Ctrl-Ab–treated mice. Importantly, Scl-AbI treatment
was able to prevent some of the detrimental changes that
compromised the bone in DEX-treated mice.
Prevention of DEX-induced changes in cortical
and trabecular bone by Scl-AbI. Femurs removed from
the mice in the 6-week dosing experiment were subjected to further analysis by micro-CT. The cortical bone
volume (CtV) and average cortical bone width (CtW) of
the femoral mid-shaft diaphysis were significantly higher
in the vehicle/Ctrl-Ab–treated group than in mice killed
at baseline (Figure 4A). In contrast, mice in the DEX/
Ctrl-Ab group showed no significant change in CtV from
baseline over the course of the experiment, whereas
there was a small, but significant, reduction in average
CtW when compared with baseline values (Figure 4B).
Vehicle/Scl-AbI–treated mice showed significant increases in the CtV and CtW as compared with both the
baseline and vehicle/Ctrl-Ab groups. When Scl-AbI was
administered to DEX-treated mice (DEX/Scl-AbI
group), the CtV and CtW were both significantly higher
than the values observed in mice in either the DEX/
Ctrl-Ab group or the baseline group (Figures 4A and B).
The values for CtV and CtW in the DEX/Scl-AbI–
treated group were not significantly different from the
values found in mice in the vehicle/Ctrl-Ab group (Figures 4A and B).
The changes in cortical thickness seen in mice
treated with DEX, Scl-AbI, or their combination were
mostly endosteal, since we found no differences in the
periosteal perimeter or cross-sectional area of the femoral midshaft. However, we did observe significant changes
in the bone marrow area between the groups (mean
0.604 mm2, 95% CI 0.555–0.654 mm2 in vehicle/Ctrl-Ab
group versus 0.725 mm2, 95% CI 0.692–0.758 mm2 in
DEX/Ctrl-Ab group; mean 0.526 mm2, 95% CI 0.499–
0.554 mm2 in vehicle/Scl-AbI group versus 0.612 mm2,
95% CI 0.567–0.658 mm2 in DEX/Scl-AbI group).
The secondary spongiosa of the distal femoral
metaphysis was also scanned by micro-CT at the end of
the experiment (week 6). The values for femoral trabecular volume fraction (bone volume/total volume
[BV/TV]) and trabecular thickness (TbTh) in the
vehicle/Ctrl-Ab–treated group were not significantly different from those obtained in mice killed at baseline
(Table 1 and Figures 4C and D). Similarly, the femoral
BV/TV values at week 6 were not significantly different
between the vehicle/Ctrl-Ab group and the DEX/
Ctrl-Ab group; the values for TbTh were marginally
lower in the DEX/Ctrl-Ab group compared with the
vehicle/Ctrl-Ab group.
Figure 4. Effects of DEX and Scl-AbI treatment of mice on cortical and trabecular bone.
Microfocal computed tomography measurements were made after 6 weeks of treatment, to
determine the femoral cortical bone volume (A), femoral cortical width (B), trabecular bone
fraction (bone volume/total volume [BV/TV]) of the femoral metaphysis (C), and trabecular
thickness (TbTh) of the femoral metaphysis (D). Values are the mean with 95% confidence
intervals in 9–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍ 0.05 versus
vehicle/Ctrl-Ab, DEX/Ctrl-Ab, DEX/Scl-AbI, and BSLN; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab and
BSLN; † ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and BSLN; †† ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab,
DEX/Ctrl-Ab, and BSLN. See Figure 2 for other definitions.
Mice receiving vehicle/Scl-AbI treatment had significantly higher femoral BV/TV and TbTh values than
were found in the vehicle/Ctrl-Ab group (Table 1 and
Figures 4C and D). When Scl-AbI administration was
combined with DEX treatment (DEX/Scl-AbI), femoral
BV/TV and TbTh values were significantly higher than
those in mice treated with DEX/Ctrl-Ab, but were not
significantly different from the values obtained from the
vehicle/Scl-AbI group (Table 1 and Figures 4C and D).
Changes in the L5 vertebral trabecular bone showed
patterns similar to those found in the trabecular bone of
the femoral metaphysis (Table 1). These findings indicate a detrimental effect of DEX on cortical and trabecular bone structure that was prevented by concurrent
Scl-AbI treatment.
Serum markers of bone turnover. The levels of
bone turnover markers in mice from the 6-week experiment are shown in Figure 5. At 3 weeks of treatment, vehicle/Scl-AbI–treated mice, compared to
vehicle/Ctrl-Ab–treated mice, had significantly elevated
levels of osteocalcin (Figure 5A) and also had elevated
levels of PINP, although the difference was not significant (Figure 5C). These findings are consistent with the
increased bone found in vehicle/Scl-AbI–treated mice.
However, by week 6, neither of these bone formation
markers was expressed at higher levels in the vehicle/
Scl-AbI group compared to the vehicle/Ctrl-Ab group
(Figure 5B) (for PINP at 6 weeks, mean 41.7 ng/ml, 95%
CI 34.6–48.9 ng/ml in vehicle/Ctrl-Ab group versus 44.0
ng/ml, 95% CI 37.0–50.9 ng/ml in vehicle/Scl-Ab group).
In DEX/Ctrl-Ab–treated mice, the levels of osteocalcin
and PINP were significantly and profoundly depressed
relative to the levels in mice treated with vehicle/CtrlAb, at week 3 (Figures 5A and C) and at week 6 (Figure
5B and results not shown). Treatment with Scl-AbI did
not elevate the level of either of these markers in mice
receiving DEX at either the 3-week or 6-week time point.
Treatment with DEX/Ctrl-Ab resulted in significantly higher serum levels of TRAP5b than were found
in the vehicle/Ctrl-Ab group at week 6, indicating a
higher level of bone resorption in the mice exposed to
DEX. At this time point, TRAP5b levels in vehicle/SclAbI–treated mice were not significantly different from
those in the vehicle/Ctrl-Ab group. Mice in the DEX/
Scl-AbI group had significantly lower TRAP5b levels
than did those in the vehicle/Ctrl-Ab and DEX/Ctrl-Ab
groups (Figure 5D), suggesting that Scl-AbI treatment
reduced bone resorption in the presence of DEX.
GC treatment is known to have negative effects
on both bone strength and linear bone growth, but the
Figure 5. Effects of DEX and Scl-AbI treatment of mice on bone serologic markers. The markers
assessed were the levels of osteocalcin after 3 weeks (A) and 6 weeks (B) of treatment, as well as
the levels of N-terminal type I procollagen propeptide (PINP) after 3 weeks of treatment (C) and
type 5b tartrate-resistant acid phosphatase (TRAP5b) after 6 weeks of treatment (D). Values are
the mean with 95% confidence intervals in 8–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus
vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, and DEX/Scl-AbI; ⴱⴱⴱ ⫽ P
⬍ 0.05 versus DEX/Ctrl-Ab and DEX/Scl-AbI; † ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and
DEX/Ctrl-Ab. See Figure 2 for other definitions.
exact mechanism of these detrimental effects remains
unclear. One pathway that is believed to play an important role in regulating many aspects of bone formation is
the Wnt signaling pathway (27). GC treatment has been
shown to up-regulate the expression of a number of
molecules involved in inhibiting Wnt signaling (28–31).
In addition, Wang et al (31) showed that downregulation of Dkk-1 (a Wnt antagonist) with antisense
oligonucleotides protected bone from the effects of GC
Sclerostin is a relatively newly reported regulator
of Wnt signaling, whose expression is largely restricted
to bone and cartilage tissue (14). The immunohistologic
findings described in the present study are consistent
with those from previous studies describing sclerostin
expression as being primarily osteocytic, but they also
confirm a recent report describing sclerostin expression
in cells in the vicinity of the growth plate (25). Based on
the expression pattern of sclerostin and the large stature
of individuals with a defective sclerostin gene, it has
been postulated that sclerostin may help to regulate
linear growth in humans (25). The results presented
herein show that neutralization of sclerostin by monoclonal antibody treatment does not prevent DEXinduced inhibition of linear growth in mice. However,
differences in the patterns of sclerostin expression
around the growth plate (25) make it difficult to be
certain of the relevance of this finding in humans.
At least some of the positive effects of parathyroid hormone on bone formation have been attributed
to its ability to moderately down-regulate sclerostin
(32–34). The inability of Scl-AbI to prevent DEXinduced inhibition of growth is therefore consistent with
the findings from a study by van Buul-Offers et al (35),
in which they showed that parathyroid hormone was also
ineffective at reducing GC-mediated growth inhibition
Scl-AbI treatment of mice receiving DEX had
beneficial effects on bone volume and bone strength.
Mice exposed to DEX that were concomitantly treated
with Scl-AbI showed significantly higher areal BMD in
both the femur (⫹22% at week 6) and the lumbar spine
(⫹26% at week 6) than did those treated with DEX and
Ctrl-Ab (Table 1 and Figure 3). In addition, micro-CT
analysis showed that mice in the DEX/Scl-AbI group
had significantly more cortical bone volume (CtV
⫹20%) and cortical bone width (CtW ⫹24%) than did
those receiving DEX/Ctrl-Ab (Figures 4A and B). In
DEX-treated mice, the Scl-AbI antibody did not significantly increase the periosteal perimeter but did produce
a decrease in marrow area, which is consistent with bone
formation occurring predominantly on the endosteal
Scl-AbI treatment was less effective at increasing
bone in the presence of DEX than was achieved in the
absence of DEX, suggesting that the presence of DEX
somewhat blunted the effects of Scl-Ab. A similar blunting effect has also been seen on the activity of parathyroid hormone in DEX-treated animals (36).
Micro-CT analysis of trabecular bone in the
femoral metaphysis showed no significant differences in
trabecular bone volume between mice in the DEX/
Ctrl-Ab group and the vehicle/Ctrl-Ab group. DEX/SclAbI treatment had a very positive effect on trabecular
bone in DEX-treated mice, with significant increases at
the femoral metaphysis in both trabecular thickness
(TbTh ⫹84%) and trabecular volume (BV/TV ⫹117%)
when compared with mice in the DEX/Ctrl-Ab group
(Figures 4C and D). Similar responses were also seen
in trabecular bone in the lumbar spine (BV/TV ⫹57%
in DEX/Scl-AbI group versus DEX/Ctrl-Ab group)
(Table 1). Generally, the changes in trabecular bone in
response to Scl-AbI appeared smaller in the presence of
DEX than in its absence, again suggesting a slight
blunting effect of the Scl-AbI effect by DEX.
Scl-AbI treatment of mice dosed with DEX not
only had positive effects on areal BMD and bone
volume, but also resulted in femurs with significantly
better mechanical properties (both intrinsic and extrinsic) than were observed in mice dosed with DEX and
Ctrl-Ab. Differences in bone geometry in Scl-AbI–
treated mice may have contributed to improvements in
the maximum load values, but increases in ultimate
strength suggest that improvements in bone quality may
also have occurred. Volumetric BMD was not affected
by DEX treatment (mean 1,094 mg/cm3, 95% CI 1,078–
1,109 mg/cm3 in vehicle/Ctrl-Ab group versus 1,081 mg/
cm3, 95% CI 1,062–1,099 mg/cm3 in DEX/Ctrl-Ab group),
suggesting that changes in mineralization played only a
minor role in the DEX-induced decline of bone mechanical properties in this model.
It is possible that the reduced body weight of the
DEX-treated mice may have contributed to the failure
of the antibody treatment to increase areal BMD during
the course of the experiments. However, similar trends
were seen in the weight-bearing femur and in the
non–weight-bearing lumbar spine, as well as in the
non–weight-bearing caudal vertebrae.
Osteocalcin and PINP are well-recognized markers of osteoblast activity, and the levels of both markers
were elevated at week 3 (although the increase in PINP
levels did not reach significance) in the mice in the
vehicle/Scl-AbI group when compared with mice in the
vehicle/Ctrl-Ab group (Figures 5A and C). Osteocalcin
levels (Figure 5B) and PINP levels (results not shown)
were measured again at week 6 and, by this point, they
were no longer elevated. We have previously noted that
the rate at which BMD increases in Scl-AbI–treated
animals declines with time (19), which may help explain
the failure to see elevated bone formation markers at the
later time point in this study. In both DEX treatment
groups, osteocalcin and PINP levels were significantly
lower than those in mice not receiving DEX. The failure
of Scl-AbI to elevate the levels of these osteoblast
markers is surprising, in light of our findings showing
more trabecular and cortical bone in Scl-AbI–treated
mice. It is possible to speculate that the DEX effect on
osteocalcin levels is a direct effect on the promoter (37),
and that Scl-AbI could stimulate bone formation in the
absence of osteocalcin, since this gene product is not essential for bone formation (38). Circulating PINP is
derived from both osseous and nonosseous tissues, and
this may complicate interpretation of changes in
this marker in steroid-treated animals. Further work is
needed to investigate the mechanisms of bone formation
in animals treated with both DEX and Scl-AbI.
Analysis of circulating levels of TRAP5b, a
marker of osteoclast numbers, showed that at week 6,
TRAP5b levels were elevated in DEX/Ctrl-Ab–treated
mice when compared to mice receiving vehicle/Ctrl-Ab,
consistent with a GC-induced increase in osteoclast
activity. DEX/Scl-AbI–treated mice had significantly
lower TRAP5b levels than did DEX/Ctrl-Ab–treated
mice. This suggests that one of the positive effects of
Scl-AbI in DEX-treated animals is a reduction of bone
resorption. It has previously been observed that Scl-AbI
can reduce circulating TRAP5b levels in mice (19). This
observation was supported by the findings from a recent
study in human volunteers (23), in which individuals
treated with an antibody to sclerostin showed a dosedependent reduction in serum C-telopeptide (CTX)
levels, a marker of bone resorption. In addition, it was
recently suggested that sclerostin might have an important role in mediating GC-induced bone resorption. This
suggestion was based on measurements of serum CTX in
a GC-treated patient with van Buchem’s disease (a
condition in which expression of sclerostin is defective in
adults) (39).
In summary, although Scl-AbI treatment does
not prevent the GC-mediated inhibition of linear
growth, it does prevent some of the detrimental effects
of GCs on bone formation and bone strength. The
molecular mechanisms by which Scl-AbI treatment generate improved bone strength and architecture in GCtreated animals will require further investigation.
We thank the sclerostin team members from Amgen
Inc. and UCB for their support of these studies.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Robinson had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Marenzana, Greenslade, Marshall,
Moore, Robinson.
Acquisition of data. Marenzana, Greenslade, Eddleston, Okoye.
Analysis and interpretation of data. Marenzana, Greenslade, Okoye,
Marshall, Moore, Robinson.
UCB Celltech employed all of the authors at the time of the
study. The study design, execution, data analysis, and the decision to
submit the manuscript for publication were solely the responsibility of
the authors. In addition, the content of the manuscript was decided
solely by the authors. Publication of this article was not contingent
upon approval by UCB Celltech.
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