Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis.

Abstract

Oestrogen depletion may influence onset and/or progression of osteoarthritis. We investigated in an ovariectomized mouse model the impact of oestrogen loss and oestrogen supplementation on articular cartilage and subchondral bone in tibia and patella, and assessed bone changes in osteoarthritis development.
C3H/HeJ mice were divided into four groups: sham-operated, oestrogen depletion by ovariectomy (OVX), OVX with estradiol supplementation (OVX+E) and OVX with bisphosphonate (OVX+BP). Each mouse had one knee injected with low-dose iodoacetate (IA), and the contralateral knee was injected with saline. Cartilage was analysed histologically 12 weeks postsurgery; bone changes were monitored over time using in vivo micro-computed tomography.
In tibiae, OVX alone failed to induce cartilage damage, but OVX and IA combination significantly induced cartilage damage. In patellae, OVX alone induced significant cartilage damage, which was enhanced by IA. In both tibiae and patellae, OVX in combination with IA significantly decreased subchondral cortical thickness in an additive manner. OVX+E and OVX+BP inhibited tibial and patellar subchondral cortical thinning, inhibited patellar and tended to diminish tibial cartilage damage. In patellae, IA interacted with BP, leading to increased subchondral cortical and trabecular bone.
This study demonstrates the significance of oestrogen for articular cartilage and subchondral bone and maintenance of healthy joints, supporting an etiological role for altered oestrogen signaling in osteoarthritis either by directly affecting cartilage or increasing susceptibility for an osteoarthritis trigger. The data strongly support the concept of involvement of subchondral bone plate in osteoarthritis.

Full text

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RESEARCH ARTICLEOpen Access
Oestrogen is important for maintenance of
cartilage and subchondral bone in a murine
model of knee osteoarthritis
Yvonne H Sniekers1,2, Harrie Weinans1, Gerjo JVM van Osch1,3, Johannes PTM van Leeuwen2*
Abstract
Introduction: Oestrogen depletion may influence onset and/or progression of osteoarthritis. We investigated in an
ovariectomized mouse model the impact of oestrogen loss and oestrogen supplementation on articular cartilage
and subchondral bone in tibia and patella, and assessed bone changes in osteoarthritis development.
Methods: C3H/HeJ mice were divided into four groups: sham-operated, oestrogen depletion by ovariectomy
(OVX), OVX with estradiol supplementation (OVX+E) and OVX with bisphosphonate (OVX+BP). Each mouse had one
knee injected with low-dose iodoacetate (IA), and the contralateral knee was injected with saline. Cartilage was
analysed histologically 12 weeks postsurgery; bone changes were monitored over time using in vivo micro-
computed tomography.
Results: In tibiae, OVX alone failed to induce cartilage damage, but OVX and IA combination significantly induced
cartilage damage. In patellae, OVX alone induced significant cartilage damage, which was enhanced by IA. In both
tibiae and patellae, OVX in combination with IA significantly decreased subchondral cortical thickness in an
additive manner. OVX+E and OVX+BP inhibited tibial and patellar subchondral cortical thinning, inhibited patellar
and tended to diminish tibial cartilage damage. In patellae, IA interacted with BP, leading to increased subchondral
cortical and trabecular bone.
Conclusions: This study demonstrates the significance of oestrogen for articular cartilage and subchondral bone
and maintenance of healthy joints, supporting an etiological role for altered oestrogen signaling in osteoarthritis
either by directly affecting cartilage or increasing susceptibility for an osteoarthritis trigger. The data strongly
support the concept of involvement of subchondral bone plate in osteoarthritis.
Introduction
Osteoarthritis (OA) of the knee is a common, disabling
and expensive disease [1,2]. The knee is a complex joint
consisting of three compartments: medial and lateral
tibiofemoral joint (TFJ) compartments and the patellofe-
moral joint (PFJ). Although clinical studies of osteoarthri-
tis of the knee have tended to focus on the tibiofemoral
compartment alone, the patellofemoral compartment is
often affected as well. An investigation in people with
knee pain revealed that the most common radiographic
pattern is combined TFJ and PFJ disease (40%), followed
by isolated PFJ OA (24%) [3]. Isolated TFJ OA occurred
in only 4% of subjects [3]. The patellofemoral joint is an
important source of symptoms associated with knee OA,
such as pain, stiffness and disability [4].
It has been suggested in the literature that oestrogen
depletion plays a role in the onset or progression of OA.
Men are known to have a higher prevalence of OA than
women before the age of 50 [5], but after this age the pre-
valence is higher in women [6,7]. The prevalence increases
with age in both men and women, but in women, it
increases dramatically around the age of 50 [5,8,9], which
coincides with menopause.
Also, in animal models, a link between oestrogen and
OA has been found. A number of animal studies have
been performed to investigate the effect of oestrogen
depletion and oestrogen replacement on articular carti-
lage (reviewed in [10]). In several animal models,
* Correspondence: j.vanleeuwen@erasmusmc.nl
2Department of Internal Medicine, Erasmus MC, University Medical Center, ‘s
Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands
Full list of author information is available at the end of the article
Sniekers et al. Arthritis Research & Therapy 2010, 12:R182
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© 2010 Sniekers et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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ovariectomy leads to osteoarthritic changes, and oestro-
gen replacement therapy reduces cartilage degradation
[11-13]. Oestrogen acts via oestrogen receptors (ERs),
which have been found in articular cartilage, bone, syno-
vial tissue and ligaments [14-17], which may all be
involved in OA. Several studies have reported associa-
tions between OA and polymorphisms in ERa and ERb
[18-20]. Also, low serum estradiol levels have been
found to be associated with OA [21]. Taken together,
these findings argue for a role of oestrogen in the devel-
opment of osteoarthritis. However, patella OA has not
yet been included in studies on oestrogen and OA.
The exact mechanism by which oestrogen affects OA
is not known. Apart from a direct effect of oestrogen on
cartilage, bone may also be involved. Oestrogen is
known to affect bone metabolism and to regulate the
balance between bone formation and resorption [22].
Subchondral bone changes have been reported in OA
patients [23-25] and in animal models for OA [26-28].
It has been suggested that subchondral bone changes
are important in the aetiology of OA [29]. Alteration in
subchondral bone remodelling, and subsequently in
bone structure, may lead to changes in load distribution.
This may in turn cause or accelerate cartilage damage.
Therefore, bone changes induced by oestrogen depletion
may play a role in OA development.
Although the prevalence increases after the age of 50,
not all postmenopausal women get osteoarthritis [30],
indicating that hormonal changes alone are not enough
to cause OA. We hypothesize that oestrogen depletion
increases the susceptibility of tissues in the joint for
changes, but an additional trigger is needed to develop
osteoarthritic changes. This concurs with the idea that
OA is a multifactorial disease. We addressed this
hypothesis by investigating bone and cartilage changes
in the proximal tibia and patella of knee joints of ovar-
iectomized mice and ovariectomized mice receiving oes-
trogen replacement, combined with a mild osteoarthritis
trigger induced by iodoacetate, an inhibitor of glycolysis
that is an accepted model for osteoarthritis [31-34]. The
impact of bone changes was investigated by using
bisphosphonates to inhibit bone resorption after ovar-
iectomy and iodoacetate.
Materials and methods
Animals
Female C3H/HeJ mice (Jackson Laboratories, Bar
Harbor, ME, USA) were chosen because of their sub-
stantial bone loss after OVX [35]. Four animals were
housed per cage and fed ad libitum. At 12 weeks of age,
mice were randomly allocated to a treatment group (as
explained below). At 24 weeks of age, the experiment
was finished, and serum, knee joints and uteri were
collected. The experiment was approved by the animal
ethics committee DEC consult.
Ovariectomy, oestrogen supplementation and
bisphophonate treatment
All animals received a subcutaneous injection of bup-
renorphine (Temgesic; 0.05 mg/kg body weight) as an
analgesic before the operation. Eight animals underwent
sham ovariectomy (Sham), and 32 animals were bilaterally
ovariectomized (OVX) to induce oestrogen depletion. Of
the 32 OVX animals, 8 animals received estradiol supple-
mentation and 8 animals were treated with the bispho-
sphonate alendronate. Estradiol was supplemented by
subcutaneous implantation of an oestrogen pellet (Innova-
tive Research of America, Sarasota, FL, USA) (OVX+E).
This pellet continuously released 17b-estradiol at a rate of
12 μg/day. Bisphosphonate treatment was performed by
weekly intraperitoneal injections of alendronate dissolved
in saline (2 mg/kg body weight; donated by Merck, White-
house Station, NJ, USA) (OVX+BP). After OVX or sham,
all mice received an intra-articular injection with 6 μl of
0.5% iodoacetate (IA, Sigma-Aldrich, St. Louis, MO, USA)
in one knee, and 6 μl of saline (Sal) in the contralateral
knee by an experienced researcher according to the proto-
col described by van der Kraan et al. [31]. This gave us the
following experimental groups: Sham+Sal, Sham+IA, OVX
+Sal, OVX+IA, OVX+E+Sal, OVX+E+IA, OVX+BP+Sal
and OVX+BP+IA. In the OVX+E group, four mice died
before the end of the experiment. The cause of this is
unclear, but it has been shown that long-term estradiol
supplementation causes death in an ovarian atrophy
model in APPswe transgenic mice [36] as well as in a myo-
cardial infarction model in C57BL/6J mice [37].
Micro-CT analysis
Mice were scanned using an in vivo micro-computed
tomography (micro-CT) [38,39] scanner (Skyscan 1076;
Skyscan, Kontich, Belgium) at 9-μm voxel size. The
mice were anaesthetized using a 5% isoflurane/oxygen
mixture. Hindlimbs were stretched, taped to a polystyr-
ene foam block and placed in a perspex holder to image
both knee joints simultaneously without interference of
abdominal tissues or tail and without needlessly radiat-
ing the abdomen. Mice were scanned every 3 weeks
starting just prior to OVX or Sham operation (t = 0).
Reconstructed grey-scale images were aligned visually
using anatomical landmarks. The scans were segmented
using a local thresholding algorithm [40], and the proxi-
mal tibia was isolated (Figure 1a). Using 3-D data analy-
sis software (CTAnalyzer; Skyscan) the tibial epiphysis
was selected as a region of interest for further analysis.
Care was taken not to include any osteophytes. The epi-
physis was further divided into a cortical and trabecular
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part [27], which were analyzed separately using a 3-D
calculator (freely available [41]).
In the cortical compartment of the epiphysis, regions
of interest (0.5 mm in mediolateral direction, 0.7 mm in
anteroposterior direction) were selected at the middle of
both the medial and lateral plateaus, representing the
subchondral plate [28] (Figure 1b) to calculate 3-D
thickness. To describe the bone structure of the epiphy-
seal trabecular compartment (Figure 1c), we calculated
bone volume fraction, describing the ratio of bone
volume over tissue volume (BV/TV). In the metaphysis,
a region of interest (1 mm high), containing only trabe-
cular bone (Figure 1d) was selected to calculate BV/TV.
To follow bone changes over time within one mouse,
data sets of t = 0 and t = 12 were matched by rotating
and translating one data set with respect to the other
[38,39]. Registration (matching) software was used,
which automatically matches two data sets using an
optimization criterion based on maximizing mutual
information [42].
In addition to the proximal tibia, the entire patella was
selected as a region of interest (Figure 2). The patella was
further divided into a cortical part and a trabecular part
(Figure 2d), which were analyzed separately. In the cortical
compartment of the patella, a region of interest was
selected containing cortical bone in contact with the
articular cartilage of the patella (subchondral cortical
bone; Figure 2e). The size of this region was 0.50 mm in
the mediolateral direction and 0.97 mm in the proximal-
distal direction. For this region, 3-D thickness was calcu-
lated. For the trabecular compartment, bone volume frac-
tion, which describes the ratio of bone volume over tissue
volume (BV/TV), was calculated.
Histological analyses
Knee joints were fixed in 4% formalin, decalcified with
ethylenediaminetetraacetic acid and embedded in paraf-
fin. Frontal sections (6 μm thick) were stained with
safranin O. Glycosaminoglycan depletion was assessed
by loss of safranin O staining. The severity and extent
of cartilage erosion in medial and lateral tibia plateaus
and in patella were scored by a blinded observer using
the grading and staging scoring system described by
Pritzker et al. [43]. Per area, the average of three sec-
tions (100 μm apart) was determined, with a maximum
score of 24 for each area.
The presence or absence of osteophytes was scored
both on histology and on micro-CT scans. Osteophytes
visible on histology can be either purely cartilaginous or
(partially) calcified. Osteophytes visible on micro-CT are
(partially) calcified; otherwise, they would not be visible.
Joints were evaluated for the presence of exudate or
infiltrate in the synovium, presence of hyperplasia of the
synovium or fibrosis of synovium and joint capsule.
Statistical analysis
Results are expressed as means ± SEM. Data from multi-
ple groups were compared using one-way analysis of
Figure 1 Regions that were analyzed using micro-CT. (a) Cross-sectional image of proximal tibia, including epiphysis (epi) and metaphysis
(meta). (b) Epiphysis with medial (M) and lateral (L) subchondral plate depicted in light grey. (c) Epiphysis with trabecular bone depicted in light
grey. (d) Metaphysis with trabecular bone region depicted in light grey.
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variance (ANOVA) followed by paired or unpaired t-test
as appropriate. A two-way ANOVA was performed to
evaluate whether there was interaction between the sys-
temic treatment (Sham, OVX, OVX+E or OVX+BP) and
IA injection. The presence or absence of osteophytes was
compared using a c2test. P < 0.05 was considered
significant.
Results
Effect of oestrogen depletion and oestrogen replacement
OVX mice weighed more than Sham mice at the end of
the experiment (Sham: 21.9 ± 0.6 g, OVX: 25.1 ± 0.3 g;
P < 0.05), but no difference was found between OVX
and OVX+E mice (OVX+E, 25.0 ± 1.0 g). The strong
reduction in uterine weight in the untreated OVX group
proved successful ovariectomy (Sham: 91.4 ± 8.4 mg,
OVX: 21.8 ± 1.5 mg; P < 0.05). Uterus weight of OVX
+E mice (OVX+E: 189.6 ± 33.5 mg) was increased com-
pared to OVX and Sham mice.
Bone changes were followed over time by 3-weekly
micro-CT scans. In the patella, subchondral cortical
thickness in OVX+IA knees was decreased at week 3,
resulting from an additive effect of OVX and IA
(Figure 3a). The decrease in OVX+IA was significantly
different from the change in Sham+Sal and OVX+Sal
mice. Subchondral cortical thickness of OVX+IA knees
progressively increased from week 3 onwards, resulting
in values similar to Sham+Sal, Sham+IA and OVX+Sal
at week 12. Oestrogen supplementation resulted in a
significantly increased subchondral cortical thickness in
OVX+E+IA compared to OVX+IA knees (Figure 3b).
In the tibia, OVX+IA caused a 10% decrease in sub-
chondral plate thickness already at week 3, but this was
not significantly different from the other conditions (Fig-
ure 3c). The change in medial subchondral plate thick-
ness in OVX+IA knees was significantly different from
Sham+Sal at week 9 and from Sham+Sal, Sham+IA and
OVX+Sal at week 12. This decrease in OVX+IA knees
resulted from an additive effect of OVX and IA. At the
lateral side, the decrease in subchondral plate thickness
in OVX+IA was significantly different from Sham+Sal
from week 6 onwards. At week 12, lateral subchondral
plate thickness was also more decreased in OVX+IA than
in Sham+IA and OVX+Sal knees (Figure 3e). Oestrogen
supplementation significantly increased medial subchon-
dral plate thickness in OVX+E+IA mice from week 6
onwards and prevented the loss in lateral subchondral
plate thickness induced by OVX+IA at weeks 6 and 12
(Figures 3d and 3f).
Registration software was used for the proximal tibia
to compare bone images at the start (week 0) and at the
end of the experiment (week 12) (Figure 4). Thinning of
medial and lateral subchondral plate and osteophyte for-
mation in the OVX+IA mouse at week 12 is clearly visi-
ble. These changes were not observed in OVX+Sal
knees. In OVX+E+IA knees, medial plate thickness was
increased due to endocortical apposition and osteo-
phytes had formed.
Patellar BV/TV was not affected in Sham+IA, OVX+Sal
and OVX+IA knees (Figure 5a). Oestrogen supplementa-
tion strongly increased BV/TV in OVX+E+Sal and OVX
+E+IA knees (Figure 5b). The increase in BV/TV resulted
from increased trabecular bone volume as well as
decreased endocortical volume as a consequence of
increased subchondral cortical thickness.
Epiphyseal BV/TV decreased over time. In OVX+IA
knees, the effect of IA and OVX was additive at week 3 and
6, resulting in a significantly stronger decrease than in
Sham+Sal and Sham+IA. At week 12, the change in BV/TV
of Sham and OVX groups was similar (-12%) (Figure 5c). In
both OVX+E+Sal and OVX+E+IA, oestrogen supplementa-
tion strongly increased BV/TV compared to the OVX
groups (Figure 5d). The increased BV/TV is also visible in
the registered images of week 0 and 12 (Figure 4).
In the metaphysis, OVX caused a very strong decrease
(-64%) in BV/TV (Figure 5e) in both OVX+Sal and
Figure 2 Processing micro-CT data sets illustrated by one cross section. (a) Reconstruction and alignment. P indicates patella, F indicates
femur. (b) Segmentation. P indicates patella, F indicates femur. (c) Isolation of patella. (d) Separation of trabecular bone (grey) and cortical bone
(black). (e) Region of interest of subchondral cortical bone (in light grey).
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OVX+IA. As in the epiphysis, oestrogen supplementa-
tion strongly increased BV/TV compared to the OVX
groups (Figure 5f). These changes in metaphyseal BV/
TV are also clearly visible in the registered images of
weeks 0 and 12 (Figure 4). In none of the experimental
conditions was metaphyseal cortical thickness affected
by IA injection (data not shown).
Cartilage damage was assessed on the basis of histol-
ogy (Figure 6) after 12 weeks at the end of the experi-
ment. Overall, 6 μl of 0.5% IA appeared to be a mild
trigger of cartilage damage showing a clear loss of safra-
nin O staining (data not shown). In the patella, both
OVX groups had more severe cartilage damage than the
Sham groups (Figure 6a). Oestrogen supplementation
Figure 3 Time course of subchondral cortical thickness. Mice were scanned every 3 weeks in an in vivo micro-CT scanner. (a and b) Patella.
(b and c) Medial tibia. (d and e) Lateral tibia. (a, c and e) Sham and OVX groups.aP < 0.05 for OVX+IA versus Sham+Sal;bP < 0.05 for OVX+IA
versus Sham+IA, both according to unpaired t-test.cP < 0.05 for OVX+IA versus OVX+Sal according to paired t-test. (b, d and f) OVX and OVX+E
groups.eP < 0.05 for OVX+IA versus OVX+E+IA;fP < 0.05 for OVX+Sal versus OVX+E+Sal, both according to unpaired t-test. Note that in (a),
Sham+IA and OVX+Sal curves are overlapping up to week 9.
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tended to limit the OVX-induced cartilage damage,
albeit not significantly. In the tibia, cartilage damage at
the medial tibia plateau of OVX+IA knees was signifi-
cantly higher than that in the Sham+Sal, Sham+IA and
OVX+Sal knees (Figure 6b), which was also reflected by
significant interaction between OVX and IA injection.
Oestrogen supplementation tended to limit medial carti-
lage damage caused by IA, albeit not significantly. At
the lateral tibia plateau, none of the treatments had a
significant effect (data not shown).
The presence of osteophytes at 12 weeks was evaluated
both on histology (see examples in Figures 6c and 6e) and
in micro-CT scans (see examples in Figures 6f and 6g),
with the latter showing only calcified osteophytes. At the
patella, more osteophytes were present after IA injection
than after Sal injection. In Sham+IA and OVX+IA patel-
lae, almost all (Sham+IA) or all (OVX+IA) osteophytes
visible on histology were also visible on micro-CT, while
in OVX+E+IA patellae only half of the osteophytes were
also visible on micro-CT (Table 1).
At the medial tibia plateau, more osteophytes were
visible on histology in Sham+IA than in Sham+Sal
tibiae. On micro-CT scans, more osteophytes were visi-
ble after IA injection than after Sal injection in both
Sham and OVX mice, but not in OVX+E mice. There
were significantly more osteophytes visible on micro-CT
in OVX+IA tibiae than in OVX+E +IA tibiae. No signif-
icant effects on lateral osteophyte presence were
observed (data not shown).
No signs of exudate or infiltrate were visible after 12
weeks. OVX did not induce changes in the synovium.
All joints injected with IA showed signs of mild hyper-
plasia, and this was not different in OVX and sham ani-
mals. None of the joints had excessive fibrosis.
Effect of bisphosphonate treatment
To study the effect of OVX on cartilage independent of
bone changes, we administered bisphosphonate (BP).
Body and uterus weight of OVX+BP mice were not dif-
ferent from OVX mice (body weight: OVX: 25.1 ± 0.3 g,
Figure 4 μCT overlays visualzing bone changes in proximal tibia from start to end of experiment. Overlaid registered longitudinal cross-
section of proximal tibia scanned at start (week 0) and end of experiment (week 12) for (a) OVX+Sal mouse, (b) OVX+IA mouse, and (c) OVX+E+IA
mouse. M indicates medial, L indicates lateral. Dark grey: present at both time points; black: only present at week 0 (i.e., resorbed in 12 weeks); light
grey: only present at week 12 (i.e., newly formed in 12 weeks). Note the thinning of the subchondral plate (open arrowheads in (b)), loss of
trabeculae in the metaphysis (both (a) and (b)), and the osteophyte formation at the medial epiphysis (closed arrowhead in (b) and (c)).
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OVX+BP: 23.9 ± 1.0 g; uterus weight: OVX: 21.8 ±
1.5 mg, OVX+BP 22.8 ± 2.1 mg).
BP significantly increased patellar subchondral cortical
bone thickness in OVX+IA but not in OVX+Sal knees
(Figure 7a). There was significant interaction between
OVX+BP and IA at weeks 3 and 9. BP compensated
tibial subchondral plate thinning in OVX+IA knees at
the lateral side (Figure 7b), but not at the medial side
(data not shown).
BP significantly increased patellar BV/TV in OVX+IA
knees (Figure 7c). This is reflected by significant interac-
tion between OVX+BP and IA at weeks 3, 6 and 9. Simi-
lar to observations for subchondral plate thickness, BP
did not affect BV/TV in OVX+Sal patellae (Figure 7c).
Figure 5 Time course of trabecular bone volume fraction. Mice were scanned every 3 weeks in an in vivo micro-CT scanner. (a and b)
Patella, (b and c) epiphysis, (d and e) metaphysis. (a, c and e) Sham and OVX groups.aP < 0.05 for OVX+IA versus Sham+Sal;bP < 0.05 for OVX
+IA versus Sham+IA, both according to unpaired t-test.dP < 0.05 for OVX+Sal versus Sham+Sal according to unpaired t-test. (b, d and f) OVX
and OVX+E groups.eP < 0.05 for OVX+IA versus OVX+E+IA;fP < 0.05 for OVX+Sal versus OVX+E+Sal, both according to unpaired t-test. Note
that OVX+Sal and OVX+IA curves are overlapping in (e) and (f).
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BP increased epiphyseal and metaphyseal BV/TV in both
OVX+IA and OVX+Sal tibiae at all time points
(Figure 7d and data not shown).
BP significantly decreased patellar cartilage damage in
both OVX+Sal and OVX+IA knees (Figure 7e). At both
medial and lateral tibia plateau, BP tended to diminish
cartilage damage caused by IA, albeit not significantly
(Figure 7f).
In five of eight OVX+BP+IA patellae, osteophytes were
detected on histology. Of these five patellae, only one had
a visible osteophyte on micro-CT scans, which is signifi-
cantly less than in OVX+IA patellae, where all patellae
showed osteophytes on micro-CT scans (Table 1). BP did
not affect osteophyte formation at the tibia plateaus.
Discussion
The current study demonstrates that loss of oestrogen
increases cartilage damage in the patella and increases
susceptibility for cartilage damage in the tibia and sub-
chondral bone loss at both sites, while oestrogen supple-
mentation appears to dampen these effects. This
observation corroborates clinical and epidemiological
observations of increased OA incidence after menopause
[5,8,9]. It also illustrates the interplay between hormonal
changes and external triggers in the aetiology of
osteoarthritis.
Figure 6 Cartilage damage and osteophytosis at 12 weeks postsurgery for Sham, OVX, and OVX+E groups. (a and b) Cartilage damage
scored on histology using Pritzker score. (a) Patella cartilage damage. (b) Medial tibia cartilage damage. *P < 0.05 according to unpaired or paired
t-test. (c) Example of patella cartilage damage and osteophytosis (arrows) on histology in an OVX+IA mouse. (d) Example of cartilage damage in
medial tibia plateau. Arrows indicate surface fibrillation. (e) Example of medial knee compartment with osteophyte formation (open arrowheads).
Also note glycosaminoglycan (GAG) depletion (between arrows). (f) Patella without osteophytes on micro-CT image. P = patella, F = femur. (g)
Patella with osteophytes (arrows) on micro-CT image. P = patella, F = femur.
Table 1 Presence of osteophytes on histology and micro-
CT
Patella Medial Tibia
HistologyMicro-CT HistologyMicro-CT
Sham+Sal
Sham+IA
OVX+Sal
OVX+IA
OVX+E+Sal
OVX+E+IA
OVX+BP+Sal
OVX+BP+IA
1/8 a
6/8 a
2/7
7/7
0/4 d
4/4 d
0/8 e
5/8 e
0/8 b
5/8 b
0/7 c
7/7 c,f
0/4
2/4
0/8
1/8 f
2/8 g
8/8 g
6/7
7/7
1/4
4/4
7/8
8/8
0/8 h
5/8 h
0/7 i
7/7 i,j
0/4
0/4 j
0/8 k
6/8 k
Osteophyte presence was scored on histology and in micro-CT data sets of
the patellae and tibiae and are presented as the number of patellae/tibiae in
which an osteophyte was visible on histology or micro-CT. Identical italic
character indicates significant difference between these conditions according
to Fisher’s exact test. CT = computed tomography; Sham = sham ovariectomy;
Sal = saline injection; IA = iodoacetate injection; OVX = ovariectomy; E =
oestrogen supplementation; BP = bisphosphonate treatment.
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This study was designed to investigate the impact of
oestrogen loss on articular cartilage and subchondral
bone and the role of oestrogen in susceptibility of the
patella and tibia for osteoarthritis, by combining a mild
osteoarthritis trigger with oestrogen depletion. IA has
been widely used in animal models of OA [31-34]. In
both patella and tibia, injection of 6 μl of 0.5% IA did
not cause a significant increase in cartilage damage.
However, OVX together with IA injection led to the
severest cartilage damage in the patella, indicating an
increased susceptibility. At the patella, OVX alone also
increased cartilage damage and supplementation with
estradiol after OVX abolished this effect. Our findings
are in line with previous studies that reported more car-
tilage damage in the tibiofemoral compartments of the
knee after OVX alone [11,13,44] or after OVX in combi-
nation with OA induction [13,45,46].
In contrast to our patella data and data from the litera-
ture, in our study, tibial cartilage damage was not
increased after OVX alone. However, when OVX was
Figure 7 Bone and cartilage parameters for OVX and OVX+BP groups. (a) Time course of patella subchondral cortical thickness. (b) Time
course of lateral subchondral plate thickness. (c) Time course of patella trabecular bone volume fraction. (d) Time course of epiphysis trabecular
bone volume fraction. (e) Patella cartilage damage at 12 weeks postsurgery. (f) Medial tibia cartilage damage at 12 weeks postsurgery.gP < 0.05
for OVX+IA versus OVX+BP+IA;hP < 0.05 for OVX+Sal versus OVX+BP+Sal, both according to unpaired t-test. *P < 0.05 according unpaired t-test.
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combined with IA injection, tibial cartilage damage was
more severe than after either OVX or IA injection alone.
This is reflected in the significant interaction between
OVX and IA injection and further supports the hypothesis
of increased susceptibility for osteoarthritis after oestrogen
loss. Our data are supported by previous studies using
more severe methods of OA induction in combination
with OVX [13,45]. However, the effect of oestrogen sup-
plementation was not investigated in these studies [13,45].
Subchondral cortical thickness in both the patella and
tibia plateaus only decreased in the OVX situation when
the osteoarthritis trigger had been applied. The effects
of OVX and IA treatment were additive, resulting in a
significantly decreased subchondral cortical thickness.
This provides further support for a role of estradiol in
osteoarthritis and adds to the concept of the involve-
ment of bone, specifically the subchondral bone plate, in
osteoarthritis. It remains unknown whether these
changes are directly caused by the osteoarthritis process
triggered by IA or are caused by unloading of the joint
(due to pain); it has been suggested that oestrogen defi-
ciency may exacerbate pain perception [47]. However,
cortical bone in the metaphysis was not affected, sug-
gesting that changes in subchondral cortical bone of the
tibia are not caused by unloading, but are an intrinsic
part of the osteoarthritic pathological process.
The decrease in subchondral plate thickness is in line
with findings from previous OA animal studies evaluating
the early disease stage [26,27,48-50]. In some of these
studies, this was followed by plate thickening [26,49].
This also explains the discrepancy with sclerosis
described in human studies [51-53], which often concern
patients with late osteoarthritis, whereas our present
study reflects an early phase of osteoarthritis. Compari-
son of patella and medial and lateral tibia demonstrated
an as yet unexplained site specificity. At the patella and
tibial medial side after 3 weeks of OVX-IA, the subchon-
dral plate showed a progressive restoration of thickness,
while at the lateral tibia side a progressive thinning was
observed (compare OVX+IA in Figures 3a, c and 3e).
Loss of estradiol did not cause changes in patellar tra-
becular bone, and compared to the metaphysis, caused
only mild changes in epiphyseal trabecular bone. How-
ever, at all three sites, supplementation with estradiol
strongly increased bone volume fraction, suggesting that
these sites are sensitive but that patellar and epiphyseal
bone are less dependent on circulating estradiol than
metaphyseal bone. This may be due to differences in oes-
trogen receptor levels or in local estradiol production.
These as yet unknown differences between epiphyseal
and patellar bone on one side and metaphyseal bone on
the other side could be important for understanding the
functional link between estradiol loss, bone turnover and
development of osteoarthritis.
To further investigate the relationship of bone changes
with cartilage damage, we studied bone and cartilage
changes in OVX mice in the absence or presence of BP. In
this way, we attempted to assess the impact of OVX on
the IA effect on cartilage while the changes in bone would
be blocked. In the tibia, treatment with BP prevented sub-
chondral plate thinning after IA injection and increased
bone volume fraction in both saline-injected and IA-
injected epiphyses. Both medial and lateral cartilage
damage appeared to be less when OVX-IA mice were trea-
ted with BP. In the patella, the effect of BP treatment was
different. In OVX+BP+Sal patellae, no bone changes
occurred, while in OVX+BP+IA patellae, subchondral cor-
tical thickness and bone volume fraction were strongly
increased (Figures 7a and 7c). Because this occurred only
in IA-injected knees, IA may have activated bone turnover
(increased activity of both osteoblasts and osteoclasts), of
which BP only reduced osteoclastic resorption, resulting in
a net increase. In contrast, estradiol is also directly bone
anabolic, explaining the increase in bone volume in both
OVX+E+Sal and OVX+E+IA groups (compare with Fig-
ures 5b, d and 5f). Interestingly, cartilage damage was less
in both BP groups (OVX+BP+Sal and OVX+BP+IA),
which is in line with reports suggesting that BPs may have
an OA protective effect [54-56]. Since the decrease in
patellar cartilage damage occurred independently of the
observed bone changes in both BP groups, BP may have a
direct effect on cartilage. This precludes drawing definitive
conclusions on the relation between bone changes and
cartilage changes.
It has been suggested in the literature that patella OA is
distinct from tibiofemoral OA [4]. When comparing patel-
lar changes to tibial changes, we notice several differences
between these compartments. First, cartilage damage is
more severe in the patella than in the tibia, which is in line
with previous reports [57,58]. Second, oestrogen depletion
increases cartilage damage in the patella, but increases the
susceptibility for cartilage damage in the tibia. Third, sub-
chondral cortical bone became thinner in the first 3 weeks
in both areas (patella and tibia) after OVX+IA, but in the
tibia the thickness continued to be decreased, while in the
patella the thickness subsequently increased to values
similar to Sham patellae. This may reflect different
dynamics or a different mechanism, which is supported by
differences in biochemical and mechanical properties of
articular cartilage of patella and tibia [59-61] and by differ-
ences in biomechanics between the patellofemoral and
tibiofemoral joint [4].
Conclusions
These data demonstrate the significance of oestrogen for
articular cartilage and subchondral bone and the main-
tenance of healthy joints. Depending on the site in the
joint, oestrogen depletion may directly increase cartilage
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damage and subchondral bone loss or increase suscept-
ibility for an additional trigger. The current data support
an etiological role for altered oestrogen signalling in
osteoarthritis and thereby substantiates the link between
estradiol and the development of osteoarthritis. The cur-
rent data strongly add to the concept of the involvement
of bone, specifically the subchondral bone plate, in
osteoarthritis.
Abbreviations
BP: bisphosphonate; BV/TV: bone volume fraction; Conn D: connectivity
density; CT: computed tomography; E: oestrogen; ER: oestrogen receptor;
GAG: glycosaminoglycan; IA: iodoacetate; OA: osteoarthritis; OVX:
ovariectomy; PFJ: patellofemoral joint; PlTh: subchondral plate thickness; ROI:
region of interest; Sal: saline; SMI: structure model index; TbTh: trabecular
thickness; TFJ: tibiofemoral joint.
Acknowledgements
The authors thank Agnese Ravetto for her help with scoring histological
sections of the patella. All authors are funded by Erasmus MC.
Author details
1Department of Orthopaedics, Erasmus MC, University Medical Center, ‘s
Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands.2Department of
Internal Medicine, Erasmus MC, University Medical Center, ‘s Gravendijkwal
230, 3015 CE Rotterdam, The Netherlands.3Department of
Otorhinolaryngology, Erasmus MC, University Medical Center, ‘s
Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands.
Authors’ contributions
Yvonne Sniekers carried out the experiments and analyses and drafted the
manuscript. All other authors were involved in the design of the study,
interpretation of the data and revision of the manuscript. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 19 January 2010 Revised: 10 June 2010
Accepted: 5 October 2010 Published: 5 October 2010
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doi:10.1186/ar3148
Cite this article as: Sniekers et al.: Oestrogen is important for
maintenance of cartilage and subchondral bone in a murine model of
knee osteoarthritis. Arthritis Research & Therapy 2010 12:R182.
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