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Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of miceAn in vivo microfocal computed tomography study.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 9, September 2011, pp 2690–2699
DOI 10.1002/art.30307
© 2011, American College of Rheumatology
Osteoarthritis Induction Leads to Early and Temporal
Subchondral Plate Porosity in the Tibial Plateau of Mice
An In Vivo Microfocal Computed Tomography Study
Sander M. Botter, Gerjo J. V. M. van Osch, Stefan Clockaerts, Jan H. Waarsing,
Harrie Weinans, and Johannes P. T. M. van Leeuwen
(mean ⴞ SEM cumulative porosity volume 0.05 ⴞ 0.04
ⴛ 10ⴚ3 mm3 in control joints versus 2.52 ⴞ 0.69 ⴛ 10ⴚ3
mm3 in collagenase-injected joints). Four weeks after
injection, the previously formed perforations disappeared, coinciding with a significant rise in osteoblast
activity in the subchondral trabecular bone in collagenase-injected joints compared to control joints (mean ⴞ
SEM bone formation rate/bone surface 0.62 ⴞ 0.13
␮m3/␮m2 per day versus 0.30 ⴞ 0.03 ␮m3/␮m2 per day).
Conclusion. The current study is the first to
provide quantitative longitudinal data on the dynamic
changes in the subchondral bone plate after OA induction. The development of plate perforations may enhance
mutual interaction between subchondral trabeculae,
bone marrow cells, and the articular cartilage in OA.
Objective. In osteoarthritis (OA), changes occur
in both cartilage and subchondral bone. The subchondral bone plate facilitates normal cross-talk between
articular cartilage and trabecular subchondral bone,
and adaptive changes in the plate due to OA may
therefore disturb cross-talk homeostasis. To investigate
these changes over time, we examined the cartilage–
subchondral bone interface using a combined approach
of histologic analysis and in vivo microfocal computed
tomography.
Methods. Sixteen-week-old male C57BL/6 mice
(n ⴝ 32) received intraarticular injections of collagenase in 1 joint to induce instability-related OA and
received saline injections in the contralateral knee joint
(control joint). At 2, 4, 6, 10, and 14 weeks after
injection, changes in the tibial subchondral bone plate
and subchondral trabeculae were analyzed.
Results. Two weeks after injection, collagenaseinjected joints had significantly more cartilage damage
and osteophytosis than did control joints. Osteoclast
activity directly underneath the subchondral bone plate
was significantly elevated in collagenase-injected joints
compared to control joints (mean ⴞ SEM osteoclast
surface/bone surface 11.07 ⴞ 0.79% versus 7.60 ⴞ
0.81%), causing the plate to become thinner and creating a large increase in subchondral bone plate porosity
In osteoarthritis (OA), changes in bone are
thought to accompany cartilage deterioration, although
it remains unclear which process is responsible for the
initial homeostatic disturbance. Located directly underneath the articular cartilage, the subchondral bone plate
provides structural support and acts as a portal for
biochemical interactions between the cartilage layer and
bone marrow and/or subchondral trabecular bone (1–5).
Therefore, any changes in its structure may well have an
effect on the overlying cartilage as well as on the
underlying subchondral bone. In OA, subchondral bone
is hypomineralized and of inferior quality as a consequence of abnormally high turnover (6–10). The exact
cause of this increased bone turnover is as yet unknown,
but the involvement of changes in the overlying articular
cartilage is proposed as the principal cause (11–13). In
contrast, other studies have shown that the OA process
starts with an increase in subchondral bone turnover,
which in turn, initiates cartilage damage (12–15). Whichever theory is true, extensive interaction between carti-
Supported by Erasmus Medical Center, Rotterdam, The
Netherlands.
Sander M. Botter, MSc, Gerjo J. V. M. van Osch, PhD, Stefan
Clockaerts, MD, Jan H. Waarsing, PhD, Harrie Weinans, PhD,
Johannes P. T. M. van Leeuwen, PhD: Erasmus Medical Center,
Rotterdam, The Netherlands.
Address correspondence to Johannes P. T. M. van Leeuwen,
PhD, Erasmus Medical Center, Department of Internal Medicine,
EE585, PO Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail:
j.vanleeuwen@erasmusmc.nl.
Submitted for publication June 22, 2010; accepted in revised
form February 10, 2011.
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IN VIVO TRACKING OF SUBCHONDRAL BONE PLATE POROSITY
lage and subchondral bone is likely to occur. In 2003,
Burr and Radin proposed that microcracks in the subchondral bone plate or calcified cartilage may initiate
targeted remodeling of the subchondral bone, accounting for the increased turnover and reduced material
density of the subchondral plate (16). In addition, an
increase in subchondral bone plate porosity has been
described in OA patients as well as in animal models of OA
(17,18), but it is unclear if this increase in porosity also
leads to increased interaction between cartilage and subchondral bone.
To study changes in the subchondral bone, the
use of microfocal computed tomography (micro-CT)
imaging has become an invaluable tool (19,20). In
contrast to histologic assessment, micro-CT enables
3-dimensional (3-D) monitoring and quantification of
changes in dense tissues such as bone. Over the last few
years, the use of in vivo micro-CT has become the new
standard (21). This technique allows longitudinal followup within 1 animal and enables better discrimination
of cause and consequence. Nevertheless, relatively few
studies have looked at in vivo changes in the subchondral
bone plate (22,23). Therefore, in this study, we examined
changes in the subchondral bone plate with the development of OA, using a combined approach of histologic
analysis and in vivo micro-CT.
MATERIALS AND METHODS
OA induction. Thirty-two 16-week-old male C57BL/6
mice (Harlan) were used. They were maintained on a 12-hour
light/12-hour dark cycle, housed in individually ventilated
cages with 4 littermates per cage, and fed ad libitum. Body
weight of all animals was measured at weekly intervals
throughout the experiment. For induction of OA, we used the
collagenase OA model as described previously (24–26). On day
0 of the experiment, mice were anesthetized using a 5%
isoflurane/N2O/O2 mixture, and intraarticular injections of
either 1 unit of purified bacterial type VII collagenase (Sigma)
dissolved in 6 ␮l saline (right knee joints) or 6 ␮l saline only
(left knee joints; hereinafter called control left knee joints)
were given using a syringe with a 33-gauge needle. The animals
received pain medication subcutaneously while anesthetized
(buprenorphine hydrochloride; 0.01 mg/kg body weight). All
injections were repeated on day 1.
Animals were divided into 3 cross-sectional (cross)
groups, termed cross/0–2, cross/0–4, and cross/0–14, with
numbers referring to followup time (in weeks), and 1 longitudinal (lt) group (lt/0–14), with 8 mice per group. Sixteen mice
(cross/0–2, n ⫽ 4; cross/0–4, n ⫽ 4; and cross/0–14, n ⫽ 8)
received an intraperitoneal calcein injection (0.01 mg/gm body
weight; Sigma) 8 days and 1 day before they were killed. After
the mice were killed, their hind legs were excised and fixed in
4% buffered formalin. All procedures were performed in
accordance with applicable federal guidelines and institutional
policies.
2691
Figure 1. Schematic overview of the scanning procedure. A, Left,
Animals were anesthetized, and a foam block was secured between
their hind legs with soft adhesive tape to prevent any soft tissue from
entering the scanned area. The tail of the animal was bent out of view
dorsocranially. After a fast scout scan using a low-radiation dose for
orientation, the x-ray beam was focused on the proximal tibia for the
actual scan, leaving all areas outside shielded from radiation. Right, A
3-dimensional model of the proximal tibia is shown, with 2 volumes
of interest (VOIs; 1 on the medial side and 1 on the lateral side),
which were chosen for analysis. These VOIs included the subchondral
bone plate (black rectangles; dark blue in color figure shown online)
and subchondral trabeculae (VOI; turquoise in color figure shown
online). B, Animals were divided into 3 cross-sectional (cross) groups,
termed cross/0–2, cross/0–4, and cross/0–14, with numbers referring
to followup time (in weeks after injection), and 1 longitudinal (lt)
group (lt/0–14). S indicates time points at which animals of each group
were scanned. The first scan (at time zero) was performed just before
the first of the 2 intraarticular injections. The last scans (at 2, 4, and
14 weeks after injection) were performed immediately after mice were
killed (†). Their hind limbs were then harvested and processed for
ex vivo scanning and histologic analysis (H). Color figure can be viewed
in the online issue, which is available at http://onlinelibrary.wiley.
com/journal/10.1002/(ISSN)1529-0131.
Micro-CT. To monitor bone changes, we used a SkyScan 1076 in vivo x-ray microtomograph. Prior to scanning,
each animal was anesthetized, and a small synthetic foam block
was mounted between its hind legs to clear the scan area of
surrounding soft tissues (Figure 1A). The mouse was placed
supine in a polystyrene bed. During scanning, the x-ray beam
was directed at the proximal tibia, scanning an area 17 mm in
length and 35 mm in width, while leaving the rest of the animal
shielded from radiation. The following settings were used:
voxel size 9 ␮m, voltage 40 keV, current 250 ␮A, exposure time
2,356 msec, frame averaging 2, beam filtration filter 1 mm
2692
aluminum. Data were recorded every 1.5-degree rotation step
through 180 degrees. Left and right legs were scanned in the
same scan session. The total time per scan was 21 minutes; the
hind legs were irradiated for a total of 11 of these minutes
due to the use of a shutter that shielded the beam between
recordings.
Mice in groups cross/0–2, cross/0–4, and cross/0–14
were scanned at time zero (right before OA induction) and
immediately after they were killed 2 weeks (cross/0–2), 4 weeks
(cross/0–4), and 14 weeks (cross/0–14) after OA induction.
Mice in group lt/0–14 were scanned at time zero and at weeks
2, 4, 6, 10, and 14 after OA induction (Figure 1B). After mice
were killed, all excised hind limbs were scanned again using an
extended ex vivo scanning protocol with improved signal-tonoise ratio, where the rotation angle was lowered to 0.8
degrees, and frame averaging was increased to 3. All other
settings remained unchanged. Reconstruction was performed
using SkyScan NRecon software, version 1.4.3. Resulting grayscale cross sections were composed of isotropic voxels measuring exactly 8.88 ␮m in all dimensions.
Radiation dose. Radiation dose per scan was measured
with a Solidose 400 dosimeter equipped with a DCT16-77
ionization chamber probe (RTI Electronics). The received
radiation dose was 0.08 Gy per minute using the in vivo scan
settings. This dose rate needs to be corrected for tissue depth
since part of the radiation dose will be absorbed by the skin
and overlying tissues. SkyScan recommends a tissue correction
factor of 0.656 for the mouse hind leg, which means that the
hind legs of each animal received 11 minutes ⫻ 0.08 ⫻ 0.656 ⫽
0.58 Gy per scan.
Quantification of bone morphometric parameters.
Each knee joint data set, consisting of reconstructed cross
sections, was aligned to the same spatial orientation. Scans
were segmented with a local threshold algorithm (27). Using
3-D data analysis software (CTAnalyzer; SkyScan), the proximal tibia was selected for further analysis (Figure 1A). The
cortex (including the subchondral bone plate) and trabeculae
of the proximal tibia were separated using software developed
in house (available upon request from the corresponding
author). By applying subsequent steps of image dilation and
erosion on the binary data sets, the software extracts different
anatomic compartments of the proximal tibia: subchondral
bone plate, subchondral and metaphyseal trabeculae, bone
marrow, and perforations inside the subchondral bone plate.
Perforations were defined as tunnels through the subchondral
bone plate, enabling contact between the articular cartilage
and the subchondral bone marrow. The different compartments were analyzed with the freely available software package
3D-Calculator (http://www.erasmusmc.nl/47460/386156/
Downloads) using a binary selection feature.
To measure subchondral bone plate thickness (in ␮m),
we selected medial and lateral subsections of the weightbearing region of the subchondral bone plate measuring 0.50
mm mediolateral in width and 1.15 mm ventrodorsal in length
(Figure 1A). For reasons of clarity, medial and lateral thickness values were averaged after measuring. Total perforation
volume inside the subchondral bone plate was measured in
subsections expanded to 1.00 mm mediolateral width, and
medial and lateral volumes of each cross section were summed.
For subchondral trabeculae (Figure 1A), the following 3-D morphometric parameters were calculated: trabecular bone volume fraction (BV/TV), representing the ratio
of trabecular bone volume (BV; in mm3) to endocortical
BOTTER ET AL
tissue volume (TV; in mm3); trabecular thickness (TbTh; in
␮m); trabecular separation (TbSp; in ␮m); and connectivity
density (in mm⫺3), indicating the number of trabecular
connections per unit volume (28). Medial and lateral values
were averaged. In order to evaluate whether bone changes
also occurred at distal sites in the mouse hind leg due to the
collagenase injection, BV/TV of the calcaneus (scanned ex
vivo) was measured in mice killed 4 and 14 weeks after
saline or collagenase injection.
To visualize the subchondral bone changes that took
place within 1 animal, scans derived from 1 animal at time zero
and at 4 and 14 weeks were spatially matched by rotation and
translation using the registration software M.I.R.I.T. (29). This
software uses an optimization criterion based on maximizing
mutual information. The resulting rotated data sets were used
to create overlaps between different time points after injection,
and differences were highlighted by color coding. All 3-D
images were made using the software program ANT (SkyScan).
Histologic analysis. Left and right hind limbs of groups
cross/0–2 (n ⫽ 4), cross/0–4 (n ⫽ 4), and lt/0–14 (n ⫽ 8) were
decalcified in 10% EDTA/5% paraformaldehyde for 14 days
and embedded in paraffin. Hind limbs of animals that received
calcein injections remained undecalcified and were embedded
in methylmethacrylate.
Coronal (frontal) histologic sections (6-␮m thick) were
taken through the joint at 100-␮m intervals. Paraffin sections
were stained with Safranin O–fast green. Cartilage damage was
scored at the medial and lateral tibial plateau in 3 histologic
sections per joint using the semiquantitative grading and
staging system devised by the Osteoarthritis Research Society
International Working Group (30). Medial and lateral damage
values were summed and subsequently averaged for the 3
sections, yielding a maximum obtainable cartilage damage
score of 48. Loss of proteoglycans (stained by Safranin O) was
scored using a 0–3-point scale, where 0 represents no loss of
proteoglycans and 3 indicates complete loss of staining for
proteoglycans in more than half of the cartilage layer. Osteophyte presence was scored according to the 4-point scoring
system described by Kamekura et al (31), where 0 ⫽ no
osteophytes, 1 ⫽ formation of cartilage-like tissues, 2 ⫽
increase in cartilaginous matrix, and 3 ⫽ endochondral ossification. Finally, osteoclasts were visualized using histochemical
staining for tartrate-resistant acid phosphatase activity as
described previously (32), using Gill’s hematoxylin as counterstain. Osteoclast quantification included osteoclast surface/
bone surface (OcS/BS) and osteoclast number/bone surface
(OcN/BS) using Bioquant Osteo software, version 7.20.
Methylmethacrylate-embedded sections were photographed in brightfield and fluorescent light to visualize the
outline of the bone and calcein labels, respectively. These
photomicrographs were merged in Paint Shop Pro software,
version 7.02 (Jasc Software) (i.e., the brightfield photomicrograph was made 70% transparent and added as layer to the
fluorescent photomicrograph). The whole surface area of
epiphyseal trabecular bone was analyzed, and single and
double labels were manually traced using Bioquant Osteo
software, version 7.20. Mineralized surface/bone surface (MS/
BS; in %), mineral apposition rate (MAR; in ␮m/day), and
bone formation rate/bone surface (BFR/BS; in ␮m3/␮m2 per
day) were then calculated.
Statistical analysis. Results were statistically analyzed
with GraphPad Prism software, version 5.02. For the cartilage
IN VIVO TRACKING OF SUBCHONDRAL BONE PLATE POROSITY
2693
damage, osteophytosis, osteoblast, and osteoclast data, a
repeated-measures two-way analysis of variance (ANOVA)
was applied with treatment (intraarticular injection with saline
or collagenase) and time as factors and Bonferroni posttests.
For the micro-CT–derived morphometric data, a two-way
ANOVA was used with treatment (intraarticular injection with
saline or collagenase) and time as factors and Bonferroni
posttests. When studying effects in time within a single group
or treatment, a one-way ANOVA with Tukey’s posttest was
chosen. In all cases, P values less than 0.05 were considered
significant. In all graphs, data are presented as the mean ⫾ SEM.
RESULTS
Figure 2. A, Cartilage damage and osteophyte presence in collagenaseinjected knee joints and contralateral control knee joints. B, Changes in
subchondral bone plate thickness (SbPlTh) over time. Thickness curves
from saline-injected and collagenase-injected knee joints differed significantly as indicated by analysis of variance (P curves). ⴱ ⫽ P ⬍ 0.05 versus
collagenase-injected knee joints; ⫹ ⫽ P ⬍ 0.05 and ⫹⫹ ⫽ P ⬍ 0.01 versus
2 weeks after injection (within treatment); ## ⫽ P ⬍ 0.01 versus time
zero (within treatment). C, Photomicrograph of tibial subchondral bone
plate area examined (dotted area) (top). Inset shows a tartrate-resistant
acid phosphatase–positive osteoclast (arrowhead) at higher magnification. M ⫽ meniscus; BM ⫽ bone marrow; GP ⫽ growth plate; AC ⫽
articular cartilage; SBP ⫽ subchondral bone plate. Osteoclast activity in
the region of interest was evaluated after collagenase or saline injection
(bottom). Osteoclast surface/bone surface (OcS/BS) and osteoclast
number/bone surface (NOc/BS) were determined. Values are the mean ⫾
SEM (at 2 and 4 weeks after injection, n ⫽ 4 mice for each bar; at 14
weeks after injection, n ⫽ 8 mice from the longitudinal group for each
bar). See Figure 1 for explanation of groups. Color figure can be viewed
in the online issue, which is available at http://onlinelibrary.wiley.
com/journal/10.1002/(ISSN)1529-0131.
Effect of the operation and scanning. All animals
were closely monitored up to 12 hours after the operation, corresponding to the therapeutic effective timespan of the applied analgesic, and since cage activity was
normal, no further analgesia was applied. Although
some weight loss was measured in the first week after
surgery (2–6% of the initial body weight at time zero),
this change was not significant, and all animals had
normal weight gain thereafter. From 10 weeks after
injection onward, the average body weights of both the
cross/0–14 group (scanned 2 times, including once after
mice were killed) and the lt/0–14 group (scanned 6
times, including once after mice were killed) were
significantly higher than the body weights measured at
time zero (data not shown). The bone volume fraction in
the proximal tibia of control left knee joints did not
differ significantly between cross/0–14 (mean ⫾ SEM
12.6 ⫾ 0.71%) and lt/0–14 (mean ⫾ SEM 10.17 ⫾
1.04%) at 14 weeks, indicating that repeated scanning
did not have a measurable deleterious effect on normal
bone metabolism.
To assess whether the animals would preferentially unload one of their hind legs due to the operation,
we analyzed the amount of trabecular bone in the calcaneus of collagenase-injected and contralateral control
legs. In collagenase-injected legs, the mean ⫾ SEM
BV/TV was 104.9 ⫾ 6.3%, 98.6 ⫾ 5.2%, and 96.0 ⫾
3.8% of that in contralateral control legs and the
mean ⫾ SEM TbTh was 95.7 ⫾ 2.7%, 99.5 ⫾ 1.9%, and
96.9 ⫾ 4.3% of that in contralateral control legs at 2, 4,
and 14 weeks, respectively, after collagenase injection.
No significant differences were found between collagenase-injected and contralateral control joints at any time
point.
OA induction and subchondral bone changes.
Two weeks after OA induction, cartilage damage was
significantly higher in collagenase-injected knee joints
than in contralateral control joints (Figure 2A). Mineralized osteophytes were also observed after 2 weeks in
collagenase-injected joints (Figure 2A). Reduction of
2694
Figure 3. A, Temporal changes in the subchondral trabecular bone of
collagenase-injected knee joints and contralateral control knee joints.
Shown are trabecular (Trab) bone volume fraction (BV/TV) and trabecular separation (TbSp). # ⫽ P ⬍ 0.05; ## ⫽ P ⬍ 0.01; ### ⫽ P ⬍ 0.001
versus time zero (within treatment). Although no differences were found
at each individual time point, the analysis of variance (P curves) indicated
that the curves were significantly different for both morphometric parameters. B, Merged brightfield and fluorescent photomicrographs of the
proximal tibia of a control knee joint and a collagenase-injected knee joint
at 4 weeks after injection, with calcein labeling evident (light gray; green
in color figure shown online) and the epiphysis delineated by the dashed
line. In the collagenase-injected joint, increased osteoblast activity is
evidenced by the larger distance between the 2 calcein labels given 8 days
and 1 day before killing (arrowheads). GP ⫽ growth plate; M ⫽
metaphysis. C, Quantification of osteoblast activity, shown by mineralized
surface/bone surface (MS/BS) and bone formation rate/bone surface
(BFR/BS). Values in A and C are the mean ⫾ SEM (at 4 weeks after
injection, n ⫽ 4 mice for each bar; at 14 weeks after injection, n ⫽ 8 mice
from cross-sectional group/0–14 for each bar). See Figure 1 for explanation of groups. Color figure can be viewed in the online issue, which is
available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.
BOTTER ET AL
Safranin O staining, indicative of proteoglycan loss, was
significant at 4 weeks after OA induction, but not at 2 or
14 weeks (data not shown).
In the subchondral volumes of interest of collagenase-injected knee joints, bone loss was observed.
Compared to contralateral control knee joints, the thickness of the subchondral bone plate in the collagenaseinjected knee joints decreased from 2 weeks after injection
onward (Figure 2B), indicating increased osteoclastic activity. This initial thickness decrease was temporary, since
at 10 weeks after injection, the bone plate returned to its
initial thickness, although the average plate thickness measured in the contralateral control joints (which also increased in time) could not be equaled.
When we quantified the presence of osteoclasts
located directly underneath the subchondral bone plate
(Figure 2C), we found similar time-dependent dynamics
in OcS/BS and OcN/BS in saline- and collagenaseinjected knees. A significant increase in OcS/BS was
detected in collagenase-injected knee joints only during
the first period after injection (at 2 weeks) (Figure 2C).
The OcN/BS was elevated as well, although this difference did not reach significance (P ⫽ 0.09) (Figure 2C).
In the trabecular bone underlying the subchondral bone plate, similar temporal bone loss patterns were
found, as assessed by BV/TV (P ⬍ 0.05 between curves)
(Figure 3A) and TbTh (P ⬍ 0.01 between curves) (data
not shown), whereas TbSp was significantly elevated
(P ⫽ 0.001 between curves) (Figure 3A) in collagenaseinjected knee joints compared to contralateral control
joints. As previously found for the subchondral bone
plate, the initial decrease in BV/TV was followed by
an increase, indicating a rise in osteoblastic activity.
This was confirmed by the calcein labeling, which
showed increased osteoblast activity in the subchondral
trabecular bone of collagenase-injected knee joints at
4 weeks (Figure 3B). We observed significant increases
in MS/BS and BFR/BS (Figure 3C) as well as in MAR
(data not shown) in collagenase-injected knee joints
compared to contralateral control joints. At 14 weeks,
these parameters also increased significantly in control knee joints (Figure 3C), indicating normal growth
and providing an explanation for the temporal increase
in subchondral bone plate thickness in controls (see
Figure 2B).
The changes in subchondral bone (i.e., subchondral bone plate and subchondral trabeculae) within the
same anatomic location in 1 animal are shown in Figure
4A. To further visualize the bone remodeling process,
binarized grayscale images derived from time zero, 2
weeks, and 14 weeks were overlapped, and the differences in the amounts of bone between the time points
were visualized (Figure 4B). It became clear that at 2
IN VIVO TRACKING OF SUBCHONDRAL BONE PLATE POROSITY
2695
Figure 4. Visualization of subchondral bone changes within the proximal tibia of a single mouse (from longitudinal group/0–14) at time zero and
at 2, 4, and 14 weeks after injection. A, At the beginning of followup (2 weeks after injection), there was initial thinning of the subchondral bone
plate. This was followed by thickening (at 4 and 14 weeks after injection) at both the lateral (solid arrowheads) and medial (open arrowheads) sides.
Note the development of mineralized osteophytes in this animal (shaded arrowheads) at the medial and lateral sides of the tibial plateau at 4 weeks
after injection and later. In some cases, ectopic calcifications were observed in the collateral ligaments, for example, at 14 weeks after injection
(asterisk). B, Shown are segmented overlaps of the images in A, highlighting differences between time zero and 2 weeks after injection and between
time zero and 14 weeks after injection. Changes in bone architecture are indicated by color coding. See Figure 1 for explanation of groups. Color
figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.
weeks, primarily bone loss was observed at both the
medial and lateral sides of the tibial plateau, whereas at
14 weeks, bone gain was more prominent.
Since we observed that subchondral bone plate
thinning was one of the main outcomes of the experiment, we scrutinized the micro-CT data of the subchondral bone plate. This analysis demonstrated that at
multiple locations, the subchondral bone plate had
become so thin that perforations had started to appear,
forming connecting tunnels between the subchondral
trabecular bone/bone marrow and the articular cartilage
(Figures 5A and B). To exclude the possibility that the
perforations were due to higher noise levels in the in
vivo scans, we checked whether the same perforations
could also be found in scans made with the ex vivo scan
protocol (which had a higher signal-to-noise ratio), and
this proved to be the case (Figure 5A, right). In addition,
we identified the perforations by histologic analysis
(Figure 5C). Subchondral bone plate porosity was observed in both saline-injected and collagenase-injected
knee joints, but the number and size of the perforations
were larger in the collagenase-injected knee joints (Figure 6A). The perforation volume of the tibial plateau
differed significantly between collagenase-injected and
contralateral control joints (0.05 ⫾ 0.04 ⫻ 10⫺3 mm3 in
control joints versus 2.52 ⫾ 0.69 ⫻ 10⫺3 mm3 in
collagenase-injected joints) despite some temporal variation between animals (Figure 6B).
DISCUSSION
This study is the first to show quantitative longitudinal data on the formation of perforations in the
subchondral bone plate in an instability-induced model
of OA appearing within 2 weeks after induction. Increased osteoclast activity acting locally underneath the
subchondral bone plate is the most likely cause of the
formation of these perforations in the initial phase after
inducing instability, followed by repair by increased
osteoblast activity. These data show the high temporal
dynamics in subchondral bone plate metabolism in OA.
Finally, we demonstrated that in vivo micro-CT is sensitive enough to quantify the anatomic changes following
altered osteoblast/osteoclast activities, without disturbing normal bone metabolism. Besides generating statistically powerful data, this technique also helps to reduce
the number of experimental animals needed.
The dynamic changes in OA subchondral bone
that we found in the present study match previously
reported data. For instance, Hayami et al also found
subchondral bone loss to occur in the rat knee within 2
weeks after surgical destabilization, followed by signifi-
2696
Figure 5. Example of a subchondral bone plate perforation that
pierces the tibial plateau in a collagenase-injected knee joint. All
images were derived from the same anatomic location in the same
animal; open arrowheads indicate the location of the subchondral
bone plate perforation. A, Perforation is visible in the grayscale image
obtained in vivo (left) and in the image obtained with the ex vivo scan
protocol (right), which had a higher signal-to-noise ratio. B, In the
segmented images, each anatomic component shown in the left image
(cortex, trabeculae, plate perforations, bone marrow) was automatically labeled with a different gray tone in the right image. The
indicated plate perforation is shown in very light gray (red in color
figure shown online) for clarity. C, The accompanying histologic
section (dotted area enlarged in inset) confirmed the perforation
(open arrowheads) to be present. Tartrate-resistant acid phosphatase–
stained osteoclasts (solid arrowheads) were observed in the close
vicinity of the perforation. Dashed line within inset represents the
border between the subchondral bone plate (SBP) and the calcified
cartilage (CC). BM ⫽ bone marrow. Color figure can be viewed in
the online issue, which is available at http://onlinelibrary.wiley.com/
journal/10.1002/(ISSN)1529-0131.
cant increases in subchondral bone volume relative to
sham operation up to 10 weeks after surgery (33).
Importantly, the authors observed increased vascular
invasion into the calcified cartilage after 1 week, which
BOTTER ET AL
coincided with the onset of cartilage surface fibrillation.
Two in vivo micro-CT studies also found early bone loss
to occur in the rat knee following destabilization (22) or
after intraarticular injection of monosodium iodoacetate
(23). Similar findings were observed in dogs (18), guinea
pigs (34), rabbits (35), and cats (36).
Subchondral bone loss also occurs in OA patients, especially in progressive OA. Bettica et al found
that levels of urinary markers of type I collagen degradation were significantly higher in patients with progressive knee OA, similar to levels in patients with osteoporosis (10), and Buckland-Wright et al concluded that
risedronate was able to correct the subchondral trabecular bone loss found in progressive knee OA (37). Thus,
although subchondral sclerosis is the final disease outcome, these data indicate that bone loss generally occurs
in OA, which may explain why net bone loss is observed
at early stages of the disease process. To confirm this in
human patients is difficult, since clinical data on changes
in the subchondral bone plate are generally derived from
patients with established OA. This is a pity, since our
current data justify in-depth analysis of the subchondral
bone plate in case of (mild) joint symptoms. This may
identify early stages of OA, which then may be modifiable or treatable.
Our findings that subchondral bone plate perforations are present in normal tibial plateaus and increased after inducing knee joint instability fit well with
previous data. The first reports of holes penetrating the
subchondral bone plate date from the 1980s, when
Duncan et al (38) described numerous perforations
through the subchondral plate in nonarthritic tibial
plateaus, some of which extended to the bone marrow
(38). Similar findings were described by Lyons et al (3)
and Clark and Huber (39). The latter study estimated
the prevalence of these perforations in the human tibial
plateau to be ⬍10/cm2. More recently, imaging studies
showed that small molecules such as gadolinium (2) or
sodium fluorescein (40), initially present in the bone
marrow, are able to pass the subchondral bone plate and
reach the articular cartilage. These studies showed that
next to a mechanical interaction, biochemical interactions between bone and cartilage are also likely to occur.
Finally, Hwang et al found an increasing density of
enlarged subchondral canals penetrating the calcified
cartilage in samples with partially eroded cartilage and
linked this finding to increased hydraulic conductance of
osteochondral tissue samples with progression of OA
(41). Hwang et al also postulated that this increased
hydraulic conductance of the subchondral bone plate
may cause the articular cartilage to lose its high inter-
IN VIVO TRACKING OF SUBCHONDRAL BONE PLATE POROSITY
2697
Figure 6. Formation of subchondral bone plate perforations over time. A, Three-dimensional top views of the tibial plateau at time zero and at 2,
4, and 14 weeks after injection, with subchondral bone perforations (purple) superimposed. Regions of interest from which quantitative data were
derived are indicated on the lateral (L) and medial (M) sides by rectangles superimposed on the tibial plateau. Arrowheads indicate mineralization
of an osteophyte at 4 weeks after injection. B, Quantification of medial perforation volume in individual mice from longitudinal group/0–14 (top).
Some scans that had low quality due to movement artifacts were omitted, but it is clear that collagenase-injected joints show larger and more dynamic
temporal changes in subchondral plate porosity compared to contralateral control joints. The mean ⫾ SEM values for medial perforation volume
are also shown (bottom). # ⫽ P ⬍ 0.05 versus time zero (within treatment); ⴱⴱⴱ ⫽ P ⬍ 0.001 versus contralateral control joints. See Figure 1 for
explanation of groups.
stitial fluid pressurization (important to withstand joint
loads) because of an abnormally high fluid exudation
toward the subchondral bone.
From our current findings and data from literature discussed above, we would like to propose a concept
of the consecutive events that take place in bone and
cartilage in instability-induced OA in the murine knee
joint. In this concept, we hypothesize that the increased
perforation of the subchondral bone plate found in our
current study leads to an increase in the mutual interaction between subchondral bone and cartilage.
At time zero, directly after inducing instability,
mechanical stress is induced on the articular cartilage
and underlying subchondral bone, causing an increase in
osteoclast activity just below the articular cartilage. This
causes a decrease in subchondral bone plate thickness in
the first 2 weeks. Enlarged plate perforations are formed
within the subchondral bone plate, increasing its permeability and causing a higher than normal fluid exudation
toward the subchondral bone, leading to a net loss of fluid
from the cartilage, which subsequently becomes damaged.
Meanwhile, in an attempt at repair, the damaged
cartilage produces anabolic (growth) factors but also
catabolic factors such as matrix metalloproteinases
(42,43) that reach the subchondral bone via the plate
perforations, while factors derived from the subchondral
bone may also travel toward the articular cartilage
(12,15). This will start a cascade of events in which the
fine balance of bone buildup and breakdown becomes
disturbed. In addition, osteocytes inside the subchondral
bone plate, known to respond to fluid flow, may play a
modulating role as well (44). Then, 4 weeks after the
induction of instability, the functional coupling that
exists between osteoclasts and osteoblasts causes a rise
in osteoblast activity in the subchondral trabecular bone,
decreasing the amount of perforations back to control
levels and increasing the subchondral bone plate thickness. At this stage, osteoclast activity is suppressed. With
more time progressing, osteoblast and osteoclast activity
return to control levels, but the bone turnover is still in
a disordered phase, and at the end of the followup
period (at 14 weeks), the subchondral bone plate remains thinner compared to the contralateral controls.
A limitation of this study is the absence of a
group of healthy animals. Therefore, we cannot rule out
the possibility that some of the parameters measured in
the contralateral control joints may be different compared to intact (i.e., nonoperated) joints (e.g., due to
altered cage activity). However, recurrent monitoring of
the mice did not indicate any changes in daily cage
activities until the end of the followup period (14 weeks),
and we therefore have no reason to believe that this
2698
BOTTER ET AL
might be the case. Also, we did not note progression of
cartilage damage, which is commonly observed in humans. The cause of this is not yet clear, but it may be
related to the OA model we used and/or it may be
explained by the length of the followup period, which
may still be too short. The fact that subchondral bone
osteosclerosis was not observed in our experiment might
be explained by the relatively early phase of the OA
disease process; despite the presence of fully mineralized osteophytes, the amount of cartilage damage at 14
weeks was still mild, and full-thickness erosion of the
articular cartilage layer was only sporadically observed.
Finally, although we acknowledge the fact that anterior
cruciate ligament transection or destabilization of the
medial meniscus is currently more commonly used as a
standard model for instability-induced OA, we believe
that the collagenase model represents instabilityinduced OA as well. Since its establishment by Van der
Kraan et al in 1990 (24), this model has been described
in several reports and in multiple species (45–47). Our
own reported data have demonstrated that the laxity
characteristics of murine knee joints are indeed markedly increased after injection of bacterial collagenase (48).
In conclusion, we used a longitudinal approach in
which subchondral bone changes in instability-induced
OA were quantified using in vivo micro-CT combined
with histologic analysis. We found that, as a result of
increased osteoclast activity, subchondral bone loss occurred within 2 weeks after destabilization, resulting in
increased subchondral bone plate perforation. These
perforations directly connect 2 of the most affected
tissues in OA, articular cartilage and subchondral bone,
providing the possibility of increased cross-talk between
cartilage and bone and contributing to disease progression. Finally, our findings may open up inspection of
subchondral plate perforation as an early indicator of
OA, and it will be a challenge to investigate this concept
in a clinical study.
ACKNOWLEDGMENTS
We kindly acknowledge Yvonne Sniekers for help with
operating the in vivo micro-CT scanner, Nicole Kops for
assistance with histology, and the personnel of the Experimental Animal Facility of the Erasmus Medical Center for taking
care of the animals during the experiment.
AUTHOR CONTRIBUTIONS
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. van Leeuwen 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. Botter, van Osch, Waarsing, Weinans,
van Leeuwen.
Acquisition of data. Botter, Clockaerts, van Leeuwen.
Analysis and interpretation of data. Botter, van Osch, Clockaerts,
Weinans, van Leeuwen.
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