ARTHRITIS & RHEUMATISM
Vol. 65, No. 3, March 2013, pp 721–731
© 2013, American College of Rheumatology
Sclerostin Is Expressed in Articular Cartilage but Loss or
Inhibition Does Not Affect Cartilage Remodeling
During Aging or Following Mechanical Injury
Martine Roudier,1Xiaodong Li,2Qing-Tian Niu,2Efrain Pacheco,2James K. Pretorius,2
Kevin Graham,2Bo-Rin P. Yoon,1Jianhua Gong,2Kelly Warmington,2
Hua Z. Ke,2Roy A. Black,1Joanne Hulme,1and Philip Babij2
Objective. Sclerostin plays a major role in regu-
lating skeletal bone mass, but its effects in articular
cartilage are not known. The purpose of this study was
to determine whether genetic loss or pharmacologic
inhibition of sclerostin has an impact on knee joint
Methods. Expression of sclerostin was deter-
mined in articular cartilage and bone tissue obtained
from mice, rats, and human subjects, including patients
with knee osteoarthritis (OA). Mice with genetic knock-
out (KO) of sclerostin and pharmacologic inhibition of
sclerostin with a sclerostin-neutralizing monoclonal
antibody (Scl-Ab) in aged male rats and ovariectomized
(OVX) female rats were used to study the effects of
sclerostin on pathologic processes in the knee joint. The
rat medial meniscus tear (MMT) model of OA was used
to investigate the pharmacologic efficacy of systemic
Scl-Ab or intraarticular (IA) delivery of a sclerostin
antibody–Fab (Scl-Fab) fragment.
Results. Sclerostin expression was detected in
rodent and human articular chondrocytes. No differ-
ence was observed in the magnitude or distribution of
sclerostin expression between normal and OA cartilage
or bone. Sclerostin-KO mice showed no difference in
histopathologic features of the knee joint compared to
age-matched wild-type mice. Pharmacologic treatment
of intact aged male rats or OVX female rats with Scl-Ab
had no effect on morphologic characteristics of the
articular cartilage. In the rat MMT model, pharmaco-
logic treatment of animals with either systemic Scl-Ab
or IA injection of Scl-Fab had no effect on lesion
development or severity.
Conclusion. Genetic absence of sclerostin does
not alter the normal development of age-dependent OA
in mice, and pharmacologic inhibition of sclerostin with
Scl-Ab has no impact on articular cartilage remodeling
in rats with posttraumatic OA.
Osteoarthritis (OA) is a common degenerative
disease of the joints and a major healthcare burden in
today’s aging population (1–3). All structures within the
joint may be affected during progression of the disease,
but the underlying causes are not well understood (4).
Many factors contribute to the disease, including age,
alterations in joint mechanical stability, body mass index,
inflammation, and genetic heritability estimates that
vary depending on the affected site (5–7).
Loss of cartilage and subsequent narrowing of the
joint space are features of worsening OA (8,9). Degra-
dation of the articular cartilage extracellular matrix is
associated with changes in the anabolic and catabolic
functions of embedded chondrocytes following exposure
to multiple signals (10,11). The potential role of the Wnt
signaling pathway in the pathogenesis of OA has been
reviewed (12–14), and recent studies suggest that
changes in the activity of this pathway can occur in OA
Supported by Amgen and UCB Pharma.
1Martine Roudier, MD, PhD, Bo-Rin P. Yoon, BS, Roy A.
Black, PhD, Joanne Hulme, PhD: Amgen, Seattle, Washington;
2Xiaodong Li, MD, PhD, Qing-Tian Niu, MD, Efrain Pacheco, BS,
James K. Pretorius, MSc, Kevin Graham, MSc, Jianhua Gong, MD,
Kelly Warmington, MS, Hua Z. Ke, MD, Philip Babij, PhD: Amgen,
Thousand Oaks, California.
Drs. Roudier and Li contributed equally to this work.
All authors are or were Amgen employees and own stock or
stock options in Amgen. Mr. Graham and Ms Warmington are listed
as inventors on an Amgen, Inc. and UCB Pharma patent for sclerostin
Address correspondence to Philip Babij, PhD, Amgen, De-
partment of Metabolic Disorders, One Amgen Center Drive, Thou-
sand Oaks, CA 91320. E-mail: firstname.lastname@example.org.
Submitted for publication June 27, 2012; accepted in revised
form November 15, 2012.
A critical role for canonical Wnt signaling is also
well established in the process of osteogenesis and in the
control of bone mass (18,19). Several soluble inhibitors
of Wnt signaling show profound effects on bone mass,
including the effects of sclerostin (20). Mutations in the
human SOST gene lead to sclerosteosis (21,22), and
deletion of sclerostin in mice results in a high bone mass
phenotype (23). Preclinical studies (24,25) and recent
human clinical trials with sclerostin-neutralizing mono-
clonal antibody (Scl-Ab) therapy have shown beneficial
effects on bone mineral density (BMD) and bone for-
mation and resorption markers (26).
Recently, it was demonstrated that sclerostin is
expressed in articular cartilage, and in cell-based studies,
it was proposed to play a chondroprotective role in
response to catabolic signals (27). In the current study,
we also demonstrate expression of sclerostin in articular
chondrocytes. However, neither genetic deletion of
sclerostin in mice nor pharmacologic inhibition of
sclerostin with Scl-Ab therapy in rats showed any major
effects on articular cartilage.
MATERIALS AND METHODS
Histologic analysis of articular cartilage. Knee joints
were obtained from male sclerostin-knockout (KO) mice and
wild-type (WT) littermates and from both male and female
Sprague-Dawley rats. For cartilage and bone assessment at the
femoral diaphysis and tibial plateau, individual femurs and
tibias were dissected and fixed in 10% neutral buffered forma-
lin (NBF). Two undecalcified frontal sections were evaluated
per animal for cartilage histology and for determination of
bone formation parameters. To ensure that similar regions
were compared for analysis, the shape of the growth plate was
used as a landmark for orientation of sections. Quantitative
analysis of the dimensions of the mouse and rat cartilage was
performed by one unblinded observer (QTN) using Osteomea-
sure bone-analysis software (Osteometrics). The cartilage area
included the entire region between the 2 cartilage surfaces
from the medial to the lateral aspect. The thickness of the
cartilage reflected the average distance measured between the
2 cartilage surfaces. The percentage length of the cartilage
surface from the medial to lateral aspect was calculated by
dividing the length of the cartilage surface by the length of the
For Mankin assessment of the severity of cartilage
lesions (28,29), both knee joints with intact femur and tibia
from WT and sclerostin-KO mice were positioned at a stan-
dardized 90° angle and fixed in 10% NBF. Tissues were
decalcified and frontal serial sections (n ? 2 per animal) were
stained with hematoxylin and eosin and Safranin O. Mankin
scoring included analysis of the structure, cells, and matrix
staining of the articular cartilage, as described previously
(28,29). The femur and tibia were scored independently, with
the medial and lateral aspects combined for each tissue, and a
final additive score for both tissues was generated for compar-
ison between WT and sclerostin-KO mice.
Immunohistochemical and in situ hybridization analy-
ses were performed on tissue specimens from male WT and
sclerostin-KO mice (age 12 weeks, n ? 3 per group) and male
Sprague-Dawley rats (age 12 weeks, n ? 3). Mankin scoring for
the severity of histopathologic features of the mouse knee
joints was performed in a blinded manner by an independent
pathologist (MR). Immunohistochemical staining of human
knee joint sections was also scored in a blinded manner. In the
rat medial meniscus tear (MMT) studies (see below), a second
independent pathologist performed the histologic scoring, also
in a blinded manner.
Procurement of human tissue. Human tissue samples
were obtained from the tissue bank at Articular Engineering.
The samples comprised knee joint tissue obtained from pa-
tients undergoing total knee replacement for OA (n ? 5 men
and n ? 7 women, mean ? SD age 65 ? 3 years [range 60–69
years]) and from clinically normal cadaveric donors (n ? 3 men
and n ? 3 women, mean ? SD age 58 ? 11 years [range 39–70
Slabs of human femoral condyle and tibial plateau
tissue, measuring ?30 mm in length, 20 mm in width, and 10
mm in depth, with overlying articular cartilage were fixed in
10% formalin for 48 hours. A circular saw was used to cut two
4-mm–thick slices, one centered on normal-appearing cartilage
and the second on the worst-appearing OA cartilage lesion.
Slices were decalcified and sections were stained with both
hematoxylin and eosin and Safranin O. The severity of the
histopathologic features of the cartilage was scored based on
the Mankin protocol.
A semiquantitative assessment was performed to de-
termine the number of sclerostin-positive cells in the human
knee joint specimens showing cartilage and bone. The entire
articular cartilage area contained within the section was used
as the region of interest, and the percentage of sclerostin-
positive chondrocytes was scored in increments of 10, on a
scale from 0 to 100%, in the superficial, intermediate, deep,
and calcified zones of the cartilage.
A separate pool of normal (n ? 16) and human OA
(n ? 16) cartilage biopsy tissue samples was obtained for
preparation of total RNA. Real-time polymerase chain reac-
tion (PCR) assays were performed for messenger RNA
(mRNA) expression of sclerostin (normal group n ? 11 [7 men
and 4 women, mean ? SD age 54 ? 14 years]; OA group n ?
12 [6 men and 6 women, mean ? SD age 60 ? 6 years]) and
DKK1 (normal group n ? 11 [6 men and 5 women, mean ? SD
age 56 ? 14 years]; OA group n ? 12 [5 men and 7 women,
mean ? SD age 60 ? 6 years]).
Immunohistochemistry protocol. Immunohistochemi-
cal analyses to assess the expression of sclerostin were per-
formed using standard protocols. In these experiments, 5-?m
sections of knee joint tissue were formalin fixed and immuno-
stained with a goat anti-mouse sclerostin antibody (AF1589;
R&D Systems) at concentrations of 0.5–4 ?g/ml.
In situ hybridization protocol. Isotopic in situ hybrid-
ization analyses of sclerostin expression in the knee joint tissue
were performed using standard protocols. For this procedure,
specific complementary DNA templates for mouse (NM_024449,
nucleotides 43–678), rat (AF32674.1, nucleotides 21–252), and
human (NM_025237, nucleotides 11–771) sclerostin were used.
722 ROUDIER ET AL
Real-time PCR. Real-time PCR was performed on
purified human cartilage RNA using reagents for human
sclerostin (forward primer GAA-TGA-TGC-CAC-GGA-
AAT-CAT, reverse primer CGG-TTC-ATG-GTC-TTG-TTG-
TTC-TC, probe 6-FAM–AC-CCC-GAG-CCT-CCA-CCG-
GAG–TAM), while for DKK1, RT2Profiler PCR arrays
specific for the Wnt signaling pathway (PAHS-043A; SA
Biosciences) were used.
Sclerostin-KO mouse studies and knee joint assess-
ment. All animal protocols and procedures were approved by
the Institutional Animal Care and Use Committee of Amgen.
Male sclerostin-KO mice and WT littermates (n ? 12 per
group) at 12 months of age were used for dynamic histomor-
phometric analysis of the distal femoral epiphysis and for
histologic analysis of the articular cartilage. A separate cohort
of older (age 16 months) male sclerostin-KO mice and WT
littermates (n ? 16 per group) was used for histologic analysis
of the whole knee joints and for lesion assessment based on the
Mankin scoring protocol.
Pharmacologic studies and analysis of articular carti-
lage after treatment. In one pharmacologic study, 16-month-
old male Sprague-Dawley rats were treated with Scl-Ab (25
mg/kg subcutaneously [SC], twice per week) or vehicle control
(n ? 10 per group) for 5 weeks. In a second study, 14-month-
old ovariectomized (OVX) female Sprague-Dawley rats (7
months post-OVX) were treated with Scl-Ab (15 mg/kg SC,
twice per week) or vehicle control (n ? 9–10 per group) for 12
weeks. In both studies, dynamic histomorphometry and carti-
lage analyses were performed on the epiphyseal region of the
Histomorphometric analysis of bone. The knee joints
of mice and rats were labeled with calcein, as previously
described (23,24). Subchondral bone and articular cartilage
were evaluated at the epiphyseal region of either the distal
femur or proximal tibia. Undecalcified frontal sections (4 ?m
in thickness) were used to measure bone parameters, including
the bone volume (calculated as bone volume/total volume, in
%), mineralizing surface (calculated as mineralized surface/
bone surface, in %), and bone formation (calculated as bone
formation rate/bone surface, in ?m3/?m2/day), across the
entire epiphyseal region.
Preparation of a sclerostin antibody–Fab (Scl-Fab)
fragment. Sclerostin antibody fragment Scl-Fab (MW 48 kd)
and, as control, keyhole limpet hemocyanin (KLH)–derived
Fab (KLH-Fab; MW 48 kd) were produced using standard
cloning techniques. Fab fragments were tested for endotoxin
contamination, and test results confirmed that the fragments
contained ?0.06 endotoxin units/mg. Based on the final for-
mulation, the maximum amount of Fab that could be injected
intraarticularly (IA) in a volume of 50 ?l was 385 ?g.
MC3T3-E1-STF assay for Scl-Ab activity. Stable
MC3T3-E1-SuperTopFlash (STF) osteoblast cells were grown
in ?-minimum essential medium (?-MEM; Gibco BRL) con-
taining 10% fetal bovine serum and 1 ?g/ml puromycin for
selection. Cells were switched to differentiation medium con-
taining ?-MEM, 50 ?g/ml ascorbic acid (Sigma), and 10 mM
?-glycerophosphate (Sigma), with daily changes of medium for
4 days prior to evaluation of cell activity in luciferase assays.
Rat sclerostin protein (0.2 ?g/ml) was preincubated with
Scl-Ab or Scl-Fab (at concentrations of 0.5, 1, and 2 ?g/ml) at
37°C for 1 hour prior to the addition of complexes to the cells
for 24 hours. Cells were treated with lysis buffer (Promega) for
measurement of luciferase activity.
Ex vivo dual x-ray absorptiometry (DXA). BMD of the
whole femurs with intact epiphyses was assessed in 2 treatment
groups of rats (vehicle-treated n ? 15, Scl-Ab–treated n ? 20)
in the rat MMT study. BMD was determined using DXA, with
the Piximus II system (GE/Lunar Medical Systems).
Pharmacologic studies in the rat MMT model of OA.
Studies using the rat MMT model of OA were performed at
Bolder BioPATH in Colorado. These studies were approved
by the Bolder BioPATH Institutional Animal Care and Use
Committee. MMT surgery was performed as described previ-
In one study, male Lewis rats weighing 260–286 grams
(mean 276 grams) on day ?1 prior to MMT surgery were
injected with either Scl-Ab (25 mg/kg SC, twice per week) or
vehicle control (n ? 20 per group) beginning on the day of
surgery and continuing until termination at 3 weeks postsur-
gery. At necropsy, the left (unoperated) femurs (n ? 15 per
group) were transferred to 70% ethanol for ex vivo DXA
analysis. In a second study, male Lewis rats weighing 281–330
grams (mean 310 grams) on day ?3 prior to MMT surgery
were injected with either Scl-Fab (385 ?g/50 ?l IA, twice per
week), KLH-Fab (385 ?g/50 ?l IA, twice per week), or vehicle
control (n ? 20 per group) beginning 3 days prior to surgery
and continuing until termination at 3 weeks postsurgery. It was
thought that the smaller-sized Scl-Fab fragment would facili-
tate its movement into the articular cartilage.
Histologic lesion assessment in the rat MMT model of
OA. In rats with MMT-induced OA, 3 frontal sections were cut
from each operated knee at ?200-?m steps, followed by
staining of the sections with toluidine blue. All 3 sections of
each knee were analyzed microscopically. A number of criteria
were used to quantitatively assess the lesion severity, including
1) the cartilage degeneration width, 2) the cartilage degener-
ation score, 3) the lesion depth ratio, and 4) the osteophyte
Weight-bearing assessment in the rat MMT model of
OA. The extent of weight-bearing of the rat knee joints
following MMT surgery (left [unoperated] knee versus right
[operated] knee) was recorded in each treatment group on day
20 or day 17 postsurgery, depending on the study, using an
incapacitance meter. The difference in force (left knee minus
right knee force) and the right-paw force (as a percentage of
the total force exerted by both paws) were determined and
compared between treatment groups, as was the percentage of
the total body weight that was carried on the hind legs (32).
Statistical analysis. For human OA studies, two-way
analysis of variance (ANOVA) was used for comparisons of
the percentage of sclerostin-positive cells between the normal
and OA cartilage groups and within different cellular compart-
ments. For mouse KO and pharmacologic studies, Student’s
unpaired 2-tailed t-test was used for comparisons between 2
groups. One-way ANOVA followed by Tukey’s post hoc test
was used for statistical comparisons among 3 groups. For
pharmacologic studies in the rat MMT model, data were
analyzed using Student’s t-test or Mann-Whitney U test (for
nonparametric data). For comparisons of the ex vivo DXA
findings, data were analyzed by Student’s one-tailed t-test using
a 2-sample equal variance assumption. Significance for all tests
was set at P values less than 0.05.
SCLEROSTIN INHIBITION IN ARTICULAR CARTILAGE723
Sclerostin expression in rodent articular chon-
drocytes. In the long bones of 12-week-old WT mice,
strong immunoreactivity for sclerostin was observed not
only in cortical and cancellous bone osteocytes, as
expected, but also in chondrocytes associated with the
articular cartilage (Figure 1A). In sclerostin-KO mice,
no expression of sclerostin was detected in the bone or
cartilage by immunohistochemical staining (results not
shown). In the WT mice, intense staining was apparent
in the deep chondrocyte layer approaching the tidemark,
whereas less staining was generally evident in the super-
ficial zone chondrocytes. Strong expression of sclerostin
in the deep layers of the WT mouse articular cartilage
was confirmed by in situ hybridization (Figure 1B).
Similar results were observed in the articular cartilage of
12-week-old rats (Figure 1B).
Sclerostin expression in normal and OA human
articular cartilage. Analysis of sclerostin immunoreac-
tivity in normal human knee joint specimens clearly
demonstrated the presence of sclerostin protein in the
articular cartilage (Figure 1A). Staining was also evident
in the underlying subchondral bone (results not shown).
Within the normal human cartilage, sclerostin protein
appeared to show variable expression, extending from
chondrocytes located within the superficial zone to those
throughout the deeper layers of the articular cartilage.
Generally, it appeared that the least-intense staining for
sclerostin was associated with cartilage regions of the
middle zone. Sclerostin expression in normal human
articular cartilage was confirmed by in situ hybridization
Sclerostin immunoreactivity was also evident in
articular cartilage from patients with knee OA (Figure
1A). Similar to our observations in normal human
cartilage specimens, there appeared to be variation in
the intensity and location of the sclerostin expression
across the different chondrocyte zones. Furthermore,
sclerostin staining was also detected in the chondrocyte
clusters that are often observed to be present in OA
articular cartilage (Figure 1A). Sclerostin expression in
Figure 1. Expression of sclerostin in the articular cartilage of adult
mouse, rat, and human knee joints. A, Articular cartilage samples from
a 12-week-old normal male mouse, 12-week-old normal male rat,
39-year-old normal human female subject, and 64-year-old male
patient with knee osteoarthritis (OA) were immunostained for sclero-
stin. In the mouse and rat cartilage, staining is evident in hypertrophic
chondrocytes (arrows), with less staining in the superficial zone
(arrowheads). Staining is also evident in subchondral bone osteocytes.
In normal human cartilage, sclerostin immunoreactivity is seen in
superficial zone chondrocytes as well as in deeper cell layers (arrows),
with a similar pattern seen in human OA cartilage, including staining
in chondrocyte clusters within the superficial zone (arrows). Lack of
staining of isotype controls is shown in insets. Original magnifica-
tion ? 20. B, Sclerostin expression was assessed by in situ hybridization
in the articular cartilage from a 12-week-old normal male mouse,
12-week-old normal male rat, 66-year-old normal human male subject,
and 52-year-old female patient with knee OA. In the mouse and rat
cartilage, sclerostin expression is strongest in hypertrophic chondro-
cytes within the deeper cell zones (arrows). Expression is also evident
in subchondral bone osteocytes (arrowheads). The pattern of sclero-
stin expression in both the normal and the OA human articular
cartilage is consistent with immunoreactivity in the superficial and
deeper chondrocyte layers (arrows). In A and B, representative images
Figure 2. Semiquantitative analysis of sclerostin immunoreactivity
and quantitative analysis of SOST and DKK1 mRNA expression in
human knee specimens from normal subjects and patients with knee
osteoarthritis (OA). A, Normal and OA articular cartilage was divided
into 4 cellular zones, and the number of SOST-positive cells in each
area was counted separately; staining in subchondral bone osteocytes
was also counted. Values are the mean ? SD cell proportions in
cartilage samples from 6 normal subjects and 12 OA patients. B, The
proportion of sclerostin-positive cells in each area of the normal or OA
articular cartilage was assessed for correlations with the severity of OA
based on Mankin scoring categories of normal, mild OA, and moder-
ate OA. C and D, SOST (C) and DKK1 (D) mRNA expression was
assessed in articular cartilage samples from separate groups of 11
normal subjects and 12 OA patients from the subject pool. Symbols
represent individual data points; horizontal lines show the mean.
Results are expressed relative to the values for the housekeeping genes
HPRT-1 or GAPDH. ? ? P ? 0.05 versus normal. NS ? not
724ROUDIER ET AL
OA cartilage specimens was confirmed by in situ hybrid-
ization (Figure 1B).
When the fraction of sclerostin-immunoreactive
cells was counted in normal human knee specimens and
compared to that in knee specimens from OA patients,
there did not appear to be any significant differences in
the percentages of sclerostin-positive cells across any of
the chondrocyte layers, including the subchondral bone
compartment (Figure 2A). Moreover, when the severity
of the OA lesions was taken into account using Mankin
scoring, there was still no dramatic difference in the
percentage of sclerostin-positive cells across the differ-
ent cartilage zones (Figure 2B).
Quantitative real-time PCR analysis of sclerostin
mRNA expression in cartilage tissue showed no differ-
ence in expression between normal and OA human
cartilage, despite more variation in the mRNA levels in
OA cartilage specimens (Figure 2C). These results were
consistent with the findings from regional immunohisto-
chemical analysis of sclerostin protein expression in the
OA cartilage (as shown in Figure 2A). In contrast,
however, a significant increase in DKK1 mRNA expres-
sion was observed in cartilage from patients with OA
compared to normal human cartilage, and the presence
of OA was also associated with greater variation in
DKK1 mRNA levels when compared to that in normal
samples (Figure 2D). This increase in DKK1 mRNA
expression in OA human cartilage is consistent with
findings in previous studies (33,34).
Histomorphometric analysis of the knee joints
from 12-month-old male sclerostin-KO mice. The re-
sults of bone histomorphometric analysis demonstrated
a 65% increase in bone volume and 58% increase in
bone formation rate in the distal femoral epiphysis of
12-month-old male sclerostin-KO mice compared to WT
control mice (Figure 3A), consistent with previous find-
ings (23). The bone marrow area was significantly de-
Figure 3. Articular cartilage integrity of the knee joints from
sclerostin–knockout (KO) mice. A, The knee joints of 12-month-old
sclerostin-KO mice and wild-type (WT) mice were assessed for bone
mass using bone histomorphometric parameters (bone volume/total
volume [BV/TV], bone marrow area, and bone formation rate/bone
surface [BFR/BS] in the distal femoral epiphysis) and for measure-
ment of the articular cartilage area and thickness and percentage of
joint surface occupied by the articular cartilage (Art Cart). Values are
the mean ? SD of 12 mice per group. ? ? P ? 0.05; ??? ? P ? 0.001,
versus WT mice. B, The knee joints of 16-month-old sclerostin-KO
mice (n ? 13) and WT mice (n ? 14) were assessed with immunohis-
tologic staining for subchondral bone sclerosis (asterisk) and normal
age-dependent changes, including loss of proteoglycans (arrows) and
osteophyte formation. Original magnification ? 20. C, The severity of
changes in the knee joints of 16-month-old WT and sclerostin-KO
mice was assessed by Mankin scoring for cartilage lesions. Symbols
represent individual data points; horizontal lines show the mean.
Figure 4. Articular cartilage integrity following pharmacologic treat-
ment of Sprague-Dawley rats with the sclerostin monoclonal antibody
(Scl-Ab). In pharmacologic studies, 16-month-old intact male Sprague-
Dawley rats were treated with Scl-Ab or vehicle for 5 weeks (A) and
14-month-old ovariectomized (OVX) female Sprague-Dawley rats
were treated with Scl-Ab or vehicle for 12 weeks (with sham-operated
female rats as control) (B). After treatment, bone parameters (bone
volume/total volume [BV/TV], mineralized surface/bone surface [MS/
BS], and bone formation rate/bone surface [BFR/BS] in the proximal
tibial epiphysis) were determined and changes to the articular cartilage
(Art Cart) were measured. Values are the mean ? SD of 10 rats per
group in A and 9–10 rats per group in B. ??? ? P ? 0.001 versus
vehicle; a ? P ? 0.05 versus sham-operated female rats; b ? P ? 0.05
versus vehicle-treated OVX female rats.
SCLEROSTIN INHIBITION IN ARTICULAR CARTILAGE725
creased in sclerostin-KO mice compared to WT mice.
However, detailed quantitative histologic analysis of the
articular cartilage compartment indicated that both the
cartilage area and the cartilage thickness were not
significantly different between WT and sclerostin-KO
mice (Figure 3A). In addition, the percentage of carti-
lage surface covering the joint surface was also un-
sclerostin-KO mice. The femoral condyles of 16-month-
old WT mice showed loss of articular cartilage, rough-
ening of the articular surface, and substantial loss of
proteoglycans as shown by Safranin O staining (Figure
3B). In addition, the knee joints showed evidence of
osteophyte formation, consistent with indications of
age-dependent onset of OA. Similar features were also
observed in age-matched sclerostin-KO mice. However,
a major difference in the sclerostin-KO mice was the
increased amount of subchondral bone, which is a
known feature associated with the high bone mass
phenotype in these mice. However, despite the in-
creased mass of subchondral bone in the sclerostin-KO
mice, essentially all other joint features were similar to
those in WT mice.
The severity of the joint phenotype was assessed
quantitatively using the Mankin scoring system. Com-
pared to WT mice, the sclerostin-KO mice did not show
any significant difference in lesion severity (Figure 3C).
Pharmacologic treatment with Scl-Ab and effects
on articular cartilage in aged male rats and OVX female
rats. In aged male rats and OVX female rats, treatment
with Scl-Ab increased the bone volume and bone forma-
tion rate at the proximal tibial epiphysis (Figures 4A and
B). However, despite these observations of acute (non-
Figure 5. Systemic effects of the sclerostin monoclonal antibody (Scl-Ab) on articular cartilage in the rat medial meniscus tear (MMT) model of
osteoarthritis. A, The bone mineral density (BMD) of the whole femur was determined in male Lewis rats after treatment with Scl-Ab or vehicle
for 3 weeks. B, Histologic analysis of the rat articular cartilage following MMT surgery shows the typical medial meniscal cartilage lesions observed
after treatment. Representative samples from a vehicle-treated and Scl-Ab–treated rat are shown. Original magnification ? 50. C, The diagram
depicts the critical parameters used for lesion scoring in each zone (Z1, Z2, and Z3) of the articular cartilage from the rat medial tibial joints.
D, These parameters were used to quantitatively assess the severity of cartilage damage and presence of osteophytes in the medial tibial (M tibial)
joints of vehicle-treated and Scl-Ab–treated rats. E, Incapacitance testing of the animals at the end of the MMT study was used to determine the
weight-bearing activity in each treatment group, assessed as the mean weight exerted on the right (operated/injured) hind leg compared to the left
(unoperated) hind leg and the percentage of total weight carried by the right (R) hind leg. Values are the mean ? SD of 20 rats per group. ? ?
P ? 0.05 versus vehicle-treated rats or versus the left (unoperated) leg within a treatment group.
726 ROUDIER ET AL
genetic) enhancement of bone mass in both male and
female rats, no morphologic changes to the articular
cartilage were noted after treatment. Both the area and
the thickness of the cartilage were unchanged by treat-
ment with Scl-Ab compared to that with vehicle control,
suggesting that in aged male rats or osteopenic, OVX
female rats, treatment with Scl-Ab will not alter the
integrity of the articular surface.
Systemic effects of Scl-Ab in the rat MMT model
of OA. Results of the ex vivo DXA analysis of the intact
(uninjured) left femurs of rats in the MMT model of OA
confirmed that a significant increase in BMD occurred
following treatment with Scl-Ab (Figure 5A), consistent
with previous findings (24). As evident in the represen-
tative histologic images shown in Figure 5B, the knee
joints from both treatment groups of rats showed devel-
opment of typical joint damage in this MMT model of
OA, with the greatest effects seen on the medial tibial
plateau. However, no major differences in gross lesions
were observed between the vehicle-treated and Scl-Ab–
treated groups. In the Scl-Ab–treated animals, bone
sclerosis was observed in both the medial and the lateral
tibial subchondral bone compartments, as would be
expected based on the known effects of Scl-Ab treat-
ment in rodents.
The major criteria used to assess lesion severity
in the rat knee joints are shown diagrammatically in
Figure 5C. None of the key parameters of cartilage
lesion severity was significantly different between the
vehicle-treated and Scl-Ab–treated rats (Figure 5D).
Figure 6. Intraarticular effects of the sclerostin antibody–Fab (Scl-Fab) fragment on articular cartilage in the rat medial meniscus tear (MMT)
model of osteoarthritis. A, In MC3T3-E1-STF cells, the presence of rat sclerostin protein decreased luciferase (Luc) activity, whereas preincubation
of the cells with Scl-Ab or Scl-Fab dose-dependently neutralized sclerostin and restored luciferase activity. The control keyhole limpet
hemocyanin–Fab (KLH-Fab) fragment had no effect. The half triangles represent the descending concentrations (2, 1, and 0.5 ?g/ml) of each
treatment. B, Histologic analysis of the rat articular cartilage following MMT surgery shows the typical medial meniscal cartilage lesions observed
after treatment with vehicle, Scl-Fab, or control KLH-Fab. Original magnification ? 50. C, The critical parameters for lesion scoring in the articular
cartilage, as well as each zone (Z1, Z2, and Z3) of the articular cartilage, were used to quantitatively assess the severity of cartilage damage and
presence of osteophytes in the medial tibial (M tibial) joints of vehicle-treated, Scl-Fab–treated, and KLH-Fab–treated rats. D, Incapacitance testing
at the end of the MMT study was used to determine the weight-bearing activity of the rats in each treatment group, assessed as the mean weight
exerted on the right (operated/injured) hind leg compared to the left (unoperated) hind leg and the percentage of total weight carried by the right
(R) hind leg. Values are the mean ? SD of 20 rats per group. ? ? P ? 0.05 versus the left (unoperated) hind leg.
SCLEROSTIN INHIBITION IN ARTICULAR CARTILAGE 727
Moreover, no changes in lesion scores were noted,
despite the development of bone sclerosis in rats follow-
ing 3 weeks of Scl-Ab treatment.
No differences in the mean weight supported by
the hind limbs (left [unoperated] versus right [operated]
legs) were observed following treatment with either
vehicle or Scl-Ab (Figure 5E). In addition, no difference
in the total percentage of hind-limb force supported by
the operated (injured) leg was observed following treat-
ment with either vehicle or Scl-Ab (Figure 5E).
Intraarticular effects of Scl-Fab in the rat MMT
model of OA. The sclerostin-neutralizing activity of
Scl-Fab was confirmed to be as effective as that of the
parent Scl-Ab IgG in the rat MMT model of OA (Figure
6A). In contrast, preincubation with KLH-Fab had no
effect on the Wnt inhibitory activity of sclerostin. Similar
to our observations following treatment with Scl-Ab,
there were no overt gross histologic differences between
the control-treated groups and Scl-Fab–treated group
(Figure 6B). No subchondral bone sclerosis was ob-
served following local delivery of Scl-Fab. Furthermore,
quantitative assessment of lesion severity revealed no
significant effect of Scl-Fab on cartilage destruction or
osteophyte formation when compared to that in the
control groups (Figure 6C).
The dose of Scl-Ab used, when administered
systemically, results in a maximum concentration (Cmax)
of ?200 ?g/ml and ?10,000-fold coverage of the target
in the circulation (results not shown). The estimated
Cmaxfor Scl-Fab, when administered via the IA route, is
?7.7 mg/ml, suggesting that the Scl-Fab is present in
very large excess to satisfy local target coverage. How-
ever, we do not have any data on the local concentration
of sclerostin protein and the potential of the Scl-Fab to
penetrate damaged cartilage as occurs in the rat MMT
Similar to the results reported above for Scl-Ab,
the effects of Scl-Fab treatment on weight-bearing
showed no difference when compared to either the
vehicle-treated controls or the KLH-Fab–treated con-
trols (Figure 6D). Moreover, there were no differences
in gait between the treatment groups (results not
The present study provides evidence of the ex-
pression of sclerostin mRNA and protein in rodent and
human articular chondrocytes, including human OA
cartilage. However, in contrast to the in vitro chondro-
protective effects of sclerostin that have been reported
previously (27), the current in vivo studies showed that
neither genetic loss of sclerostin in mice nor pharmaco-
logic inhibition of sclerostin in intact male or OVX
female rats had an impact, implying that sclerostin may
not have a critical role in articular cartilage. Further-
more, pharmacologic treatment of rats with Scl-Ab or
Scl-Fab following MMT surgery also did not appear to
affect either the histopathologic features of the knee
joints or the development of cartilage lesions.
Expression of sclerostin is typically associated
with osteocytes, particularly the more mature osteocytes
that are surrounded by a mineralized matrix (35,36).
Nevertheless, some studies have demonstrated sclerostin
expression in other cell types, such as hypertrophic
chondrocytes in the growth plate and cementocytes
(37–39). A recent study provided strong evidence for
sclerostin expression in cartilage, particularly in the
deeper chondrocyte cell layers of murine articular carti-
lage (27), and results of the present study support this
finding. Species differences in the local regulation of
sclerostin expression could explain the apparent regional
differences observed in sclerostin expression between
rodent and human cartilage. Since mechanical loading is
known to influence sclerostin expression in bone, and
loading is known to affect signal transduction in articular
cartilage, it is possible that unique loading signals asso-
ciated with different zonal regions in the articular carti-
lage may contribute to species-specific regulation of
In addition to the observations in normal human
chondrocytes, strong sclerostin staining was also evident
in the chondrocyte clusters that are often observed in
damaged OA articular cartilage. This finding also sup-
ports the recent report describing the expression of
sclerostin in some, but not all, human OA cartilage
samples examined (27). Wnt signaling may play a role in
this pathogenic adaptive response, as was demonstrated
by the increased ?-catenin activity observed in the
Hartley guinea pig model of OA (40) and also in human
OA cartilage (17,41,42). In addition, constitutively active
?-catenin expression has been shown to accelerate car-
tilage destruction and the onset of OA in mice (17).
However, in contrast to the activation of ?-catenin,
previous studies also showed that inhibition of ?-catenin
can lead to destruction of articular cartilage and in-
creased chondrocyte apoptosis (43,44).
When sclerostin expression was correlated with
OA lesion severity in human OA cartilage specimens,
there was no obvious relationship suggesting a role for
sclerostin in the pathogenesis of OA. Previously, it was
reported that sclerostin expression was modestly re-
728 ROUDIER ET AL
duced in bone from lumbar zygapophyseal joints (45)
and in femoral neck biopsy samples from human OA
specimens (46), but a similar response was not observed
in the human knee joint specimens in the current study.
In agreement with our present findings, one study found
that sclerostin expression appeared to be unchanged in
the bone tissue of patients with OA, despite changes in
other Wnt pathway genes (47). Our findings, based on a
combination of real-time PCR, in situ hybridization, and
immunohistochemistry, are in contrast to the recent
findings from a microarray analysis of human OA carti-
lage, which showed a 14-fold increase in sclerostin
mRNA expression in OA cartilage biopsy specimens
(48). It is not clear why those findings with regard to
sclerostin mRNA expression differed from ours, but the
differences could be related to patient heterogeneity,
cartilage biopsy sampling differences, or differences in
the assay methods used.
The results of the present study indicate that
sclerostin mRNA expression was unchanged in human
OA cartilage, but DKK1 mRNA levels showed an
increase, which is similar to the findings from other
studies showing that the Wnt antagonist DKK1 was
up-regulated in OA cartilage (33,34,49). However, al-
though studies involving both inhibition of DKK1 in
vitro and systemic administration of DKK1 antisense in
vivo suggested a catabolic role for DKK1 (33,49), the
local overexpression of DKK1 specifically in the carti-
lage of mice with posttraumatic OA suggests that DKK1
has a chondroprotective role in OA (34). Interestingly,
local overexpression of the related Wnt antagonist
DKK2 had no effect in the same model, suggesting that
Wnt antagonists have potentially unique local roles in
the pathogenesis of OA.
Despite the observed increase in subchondral
bone in sclerostin-KO mice, all of the remaining joint
features associated with age progression were similar to
those observed in WT control mice. Quantitatively,
there appeared to be no differences in the knee-joint
lesion scores, suggesting that life-long genetic loss of
sclerostin does not compromise the integrity of the
cartilage within the knee joint.
In addition to genetic loss of sclerostin, short-
term pharmacologic treatment of intact, 16-month-old
male rats or osteopenic, OVX female rats with Scl-Ab
did not alter the morphologic features or integrity of
the articular cartilage. These results imply that if the
sclerostin produced by chondrocytes has an inhibitory
effect on local Wnt signaling, then genetic or acute loss
of the inhibitory activity of sclerostin, and any subse-
quent potential enhancement of Wnt signaling, is not
sufficient to alter the normal morphology and integrity
of the articular cartilage. Furthermore, it was previously
reported that degenerative osteoarthropathy was not
present in patients with sclerosteosis (50).
Our findings in sclerostin-KO mice showing that
sclerostin does not play a central role in the loss of joint
cartilage during normal aging was supported by our in
vivo pharmacologic findings, showing that short-term
treatment with Scl-Ab or Scl-Fab, administered either
systemically or IA, respectively, had no effect on lesion
development in the rat MMT model of OA. Lesion
development, characterized by substantial loss of medial
cartilage, occurs rapidly in the MMT model, but this was
not altered following treatment with Scl-Ab or Scl-Fab.
We do not know whether the Scl-Fab has the potential
to penetrate intact or damaged cartilage as occurs in the
rat MMT model, and therefore this is a limitation of the
present study. Further work will be required to investi-
gate the potential of Scl-Fab to impact chondrocytes
following IA injection, and whether this may lead to any
alteration in Wnt signaling and lesion development in
the MMT model. Moreover, the role of sclerostin in
articular cartilage could be further investigated in
sclerostin-KO mice using induced models of OA.
The efficacy of systemic Scl-Ab in terms of its
effects on subchondral bone formation was clearly evi-
dent, but despite the increase in bone mass, MMT lesion
scores and osteophyte formation were unchanged in the
Scl-Ab–treated rats compared to vehicle-treated control
rats. This suggests that the accelerated subchondral
bone formation that occurred during the 3-week time-
frame had no impact on the normal cartilaginous lesions
that develop in this model. These observations indicate
that enhanced subchondral bone formation resulting
from short-term pharmacologic inhibition or genetic loss
of sclerostin is not associated with cartilage destruction
in the knee joint. These in vivo results therefore do not
support the hypothesis that accelerated wear of articular
cartilage in OA is initiated by local subchondral bone
In summary, although we found evidence of
sclerostin expression in rodent and human articular
cartilage, genetic loss of sclerostin in mice had no effect
on cartilage integrity in aging knee joints. Furthermore,
pharmacologic treatment of rats with Scl-Ab or Scl-Fab,
even following posttraumatic knee injury, had no effect
on cartilage lesion development. These results suggest
that if sclerostin plays a role in the biology of the
articular cartilage, then its impact may be less powerful
and/or less unique compared to its clearly central regu-
latory role in the control of bone mass. One possibility is
SCLEROSTIN INHIBITION IN ARTICULAR CARTILAGE729
that in the genetic absence of sclerostin or as a result of
pharmacologic inhibition, a compensatory molecule
(e.g., another Wnt signaling inhibitor) is up-regulated in
the cartilage and masks the effects of sclerostin inhibi-
tion. Should such a compensatory molecule exist, uncov-
ering its identity could lead to important further insights
into the role of Wnt signaling in cartilage biology.
We thank Julie Hahn, Brenda Heron, and Noi Nuana-
mee for technical assistance, Alison Bendele for performing
the studies at Bolder BioPATH, and Chris Paszty for reviewing
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. Babij 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. Li, Graham, Ke, Black, Hulme, Babij.
Acquisition of data. Roudier, Li, Niu, Pacheco, Pretorius, Graham,
Yoon, Gong, Warmington, Ke, Babij.
Analysis and interpretation of data. Roudier, Li, Niu, Pretorius,
Graham, Yoon, Gong, Warmington, Ke, Black, Hulme, Babij.
ROLE OF THE STUDY SPONSORS
This study was supported by Amgen and UCB Pharma. All
authors are current or former employees of Amgen. The sponsors had
a role in the study design or in the collection, analysis, or interpretation
of the data, the writing of the manuscript, or the decision to submit the
manuscript for publication. Publication of this article was contingent
upon approval by Amgen or UCB Pharma.
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