ARTHRITIS & RHEUMATISM
Vol. 62, No. 2, February 2010, pp 463–471
© 2010, American College of Rheumatology
A Longitudinal Analysis of Urinary Biochemical Markers and
Bone Mineral Density in STR/Ort Mice as a Model of
Junichiro Sarukawa, Masaaki Takahashi, Mitsuhito Doi, Daisuke Suzuki, and Akira Nagano
Objective. To investigate the longitudinal changes
both in the urinary concentrations of biochemical mark-
ers and in bone mineral density (BMD) during disease
progression in the STR/Ort mouse model of osteoarthri-
Methods. Male STR/Ort mice were studied, with
CBA mice used as nonarthritic controls. Radiographic
evaluation and grading of the knee and measurements
of urinary C-terminal crosslinking telopeptide of type II
collagen (CTX-II), pyridinoline (Pyr), and deoxypyr-
idinoline were performed between 8 weeks and 40 weeks
of age. The BMD of the femoral shaft was measured
from 20 weeks to 40 weeks of age and adjusted for body
weight. Histologic evaluation and grading were per-
formed at 40 weeks of age. STR/Ort mice were divided
into 2 subgroups (STR OA and STR non-OA) based on
Results. No significant differences between STR/
Ort and CBA mice were observed for any biochemical
marker or BMD at any time point. Urinary CTX-II
levels and BMD in the STR OA subgroup were higher
than those in the STR non-OA subgroup before radio-
graphic changes of OA were apparent. Higher urinary
Pyr levels in the STR OA subgroup were observed at the
advanced stage of OA.
Conclusion. Urinary CTX-II could be a useful
marker in the early diagnosis and predicting the pro-
gression of OA, and urinary Pyr may be a potential
marker to assess the severity of OA at an advanced
stage. An increase in BMD prior to the establishment of
radiographic OA may be related to the induction of OA.
Osteoarthritis (OA) is one of the most common
and disabling disorders in the general population, and its
pathology is characterized by the degeneration of artic-
ular cartilage, sclerosis of subchondral bone, and osteo-
phyte formation. To date, radiographs are generally
used to define these features of OA in clinical practice
and in the research setting (1,2). Osteoarthritic changes
on radiographs, however, are detected in fairly advanced
stages of the disease, and relatively long examination
periods are needed to demonstrate significant changes
on successive radiographs (3). Therefore, it is often
difficult to diagnose OA at an earlier stage and to
monitor the disease status of OA using radiographs
To improve the diagnosis and monitoring of
arthritis, biochemical markers have been developed with
the aim of detecting changes in OA. Two major catego-
ries of molecules are used as biochemical markers of
arthritis: molecules of the extracellular matrix and pro-
teolytic enzymes or cytokines. Most of the molecules of
the extracellular matrix are related to cartilage and bone
metabolism. In particular, proteoglycans and type II
collagen are the major constituents of cartilage, and type
II collagen is localized almost exclusively in cartilage.
Therefore, urinary C-terminal crosslinking telopeptide
of type II collagen (CTX-II) may potentially represent a
specific marker for cartilage turnover (4,5). Pyridinoline
(Pyr) and its minor analog, deoxypyridinoline (D-Pyr),
are major crosslinks of mature collagen (6,7). Urinary
Pyr and D-Pyr have been used as markers of bone
metabolism. Because the tissue distribution of Pyr favors
cartilage in addition to its presence in bone, urinary Pyr
content may serve as a marker of both cartilage and
bone metabolism. D-Pyr is distributed almost exclusively
in the matrix of mature bone (8). Therefore, urinary
Junichiro Sarukawa, MD, Masaaki Takahashi, MD, Mitsuhito
Doi, MD, Daisuke Suzuki, MD, Akira Nagano, MD: Hamamatsu
University School of Medicine, Hamamatsu, Japan.
Address correspondence and reprint requests to Junichiro
Sarukawa, MD, Department of Orthopaedic Surgery, Hamamatsu
University School of Medicine, 1-20-1, Handayama, Higashi, Hama-
matsu, Shizuoka, Postal Code 431-3192, Japan. E-mail: firstname.lastname@example.org-
Submitted for publication September 25, 2008; accepted in
revised form October 9, 2009.
concentrations of Pyr and D-Pyr in patients with OA
may be affected by cartilage degeneration, synthesis of
osteophytes, and sclerosis of subchondral bone (7).
However, the relationship between changes in the levels
of ?1 biochemical marker and the progression of OA
over long-term periods is not yet fully clarified. This
problem stems from 2 reasons; one of these reasons is
the difficulty in diagnosing OA at an early stage, partic-
ularly before the radiographically detectable stage (9),
and the other reason is that changes in the progression
of OA are usually small and relatively long-term in
Over several decades, many animal models have
been developed to investigate the pathology of OA. For
example, degenerative changes can be induced by the
injection of chemicals or by surgical intervention (11–
13). In contrast to these models, STR/Ort mice experi-
ence spontaneous histologic degeneration in the medial
tibial plateau, which closely resembles that in human
OA. Approximately 85% of male STR/Ort mice show
signs of cartilage degeneration by 35 weeks of age (14).
Because the time course for the progression of OA in
STR/Ort mice is much shorter than that in humans, the
use of this model enables us to longitudinally investigate
the relationship between changes in biochemical mark-
ers and the onset and progression of OA in an acceler-
ated time frame.
Many factors, including obesity, previous injury,
and bone mineral density (BMD), have been related to
the pathogenesis of OA. Numerous cross-sectional stud-
ies have examined the relationship between BMD and
OA (15,16). In general, patients with radiographic OA
have higher BMD than individuals without OA; how-
ever, results differ somewhat according to the site of
BMD measurement and the specific joint groups af-
fected by OA. Few data are available on the longitudinal
changes in BMD in patients with radiographic OA.
In this study, we investigated the longitudinal
relationship between changes in the concentrations of
several biochemical markers and the onset and progres-
sion of OA in STR/Ort mice, as well as the relationship
of OA and BMD. We hypothesized 1) that the levels of
some potential biochemical markers change during dif-
ferent stages of OA development and establishment, and
that such changes elucidate cartilage and bone metabo-
lism during OA development, and 2) that high or low
BMD is associated with the development of OA.
MATERIALS AND METHODS
Animals. In this study, 8-week-old male STR/Ort mice
(n ? 22) and 8-week-old male CBA mice (n ? 12; used as
controls) were purchased from Charles River Japan (Yoko-
hama, Japan). The mice were housed individually in standard
mouse cages (room temperature 24°C; 12-hour light/12-hour
dark cycle) with free access to standard mouse food and tap
water. All experiments were performed according to the
protocol approved by the Animal Care and Use Committee of
Hamamatsu University School of Medicine.
Radiographic analysis. Prior to imaging and after
measuring their body weight, the mice were anesthetized by an
intraperitoneal injection of sodium pentobarbital. Radio-
graphs of the left and right knee joints were obtained in lateral
projections longitudinally at 8, 12, 16, 20, 24, 28, 32, 36, and 40
weeks, using a soft x-ray apparatus (M-60; Softex, Tokyo,
Japan). Exposure was for 20 seconds at 30 kV and 5 mA, and
the film was processed using an automatic film developer.
Radiographs were graded according to our original radio-
graphic grading scale for OA progression, as follows: grade 0 ?
no apparent change; grade 1 ? identification of sclerosis of
subchondral bone, or osteophyte formation, or joint space
narrowing (JSN), or calcification of the patellar tendon; grade
2 ? identification of any 2 of the grade 1 features; grade 3 ?
identification of any 3 of the grade 1 features; grade 4 ?
identification of all 4 grade 1 features (Figure 1A). These
features were characteristic of the osteoarthritic changes in
STR/Ort mice observed in previous studies (17–19). The values
for radiographic grading of the left and right knee joints were
averaged for each mouse and used in the statistical analysis.
A single observer who was blinded with regard to the
experimental group evaluated the radiographic grading.
Cohen’s kappa statistics were used to assess the intraob-
server and interobserver agreement of radiographic grading
by 2 readers. The kappa values for intraobserver and
interobserver agreement were 0.73 and 0.71, respectively.
These values are regarded as reliable enough for the method
used in the present study.
Bone densitometry. The BMD of the left and right
femoral shaft was measured longitudinally in all mice at 20, 24,
28, 32, 36, and 40 weeks of age, by dual x-ray absorptiometry
(DXA) with a Hologic QDR-1000 Plus system (Hologic,
Waltham, MA). Because a relatively long period of time is
required to measure BMD, and the mice need to be anesthe-
tized, which sometimes risks death, we avoided measuring
BMD before 20 weeks of age, because it has been reported that
OA developed in STR/Ort mice during the period from 20
weeks to 35 weeks of age (14,20). While under general
anesthesia, the mice were placed in a supine position with the
bilateral hind limb over the scanning surface and the hip, knee,
and ankle articulations in 90° flexion. The foot and proximal
tibia were secured to the machine with tape just outside the
scanning area; scans included the entire femur. In the resulting
scans, the region of interest (ROI) was defined as the femoral
shaft (middle 60% of the total femoral length). The values for
left and right femoral shaft BMD were averaged for each
mouse and used in the statistical analysis. To evaluate the
reproducibility of the DXA technique, the left femoral shaft
BMD of a mouse at 20 weeks of age was repositioned and
measured 7 times on the same day. The coefficient of variation
(CV) for scanning was 1.1%.
Histologic grading of OA. At 40 weeks of age, all mice
were killed, and the whole knee joints of all mice were
dissected and fixed in 10% neutral buffered formalin for 24
464SARUKAWA ET AL
hours. The specimens were decalcified in 10% EDTA for 2
weeks. After dehydration and embedding in paraffin, 10 serial
sections were prepared from the central region of the medial
tibial plateau in the sagittal plane, because it has been reported
that changes in OA occur more pronouncedly in the medial
tibiofemoral compartment than in other compartments of the
knee joints (21). Sections were stained with hematoxylin and
eosin and Safranin O. Grading of OA progression in the
medial tibiofemoral compartment was performed according to
the procedure described by Walton (14) and Schu ¨nke et al
(22), as follows: grade 0 ? no apparent changes, grade 1 ?
superficial fibrillation of articular cartilage, grade 2 ? defects
limited to uncalcified cartilage, grade 3 ? defects extending
into calcified cartilage, and grade 4 ? exposure of subchondral
bone at the articular surface (see Figure 2). The final grade was
determined as the highest grade in all sections, by a single
observer who was blinded with regard to the experimental
group. A mouse was defined as having histologic OA if the
histologic grade for at least 1 knee joint was ?2. Based on
these criteria for histologic OA, STR/Ort mice were further
categorized into 2 subgroups: STR OA (bilateral OA and
unilateral OA) and STR non-OA (no signs of OA). The kappa
Figure 1. A, Typical radiographs of the knees of mice in the lateral projection, representing the grades of radiographic
knee osteoarthritis (OA). The 4 features used in radiographic grading were osteophyte formation (OF), sclerosis of
subchondral bone (SS), calcification of the patellar tendon (CP), and joint space narrowing (JSN). The grades were
defined as follows: grade 0 ? no signs of OA, grade 1 ? identification of 1 of the 4 features, grade 2 ? identification
of any 2 of the 4 features, grade 3 ? identification of any 3 of the 4 features, and grade 4 ? identification of all 4 features.
Bar ? 1 mm. B and C, Time course of the progression of OA by radiographic grading in STR/Ort and CBA mice (B)
and mice in the STR OA and STR non-OA subgroups (C). The values for the radiographic grading of the left and right
knee joints were averaged for each mouse. Bars show the mean and SD. ? ? P ? 0.001 versus CBA; † ? P ? 0.01 versus
STR non-OA; ‡ ? P ? 0.001 versus STR non-OA.
Figure 2. Histologic sections representing the grades of degenerative lesions of articular cartilage. Degeneration of articular
cartilage was evaluated by systematic analysis of knee joints sectioned in a sagittal plane. Tissue sections were stained with
Safranin O and graded as follows: grade 0 ? no apparent changes, grade 1 ? superficial fibrillation of articular cartilage, grade
2 ? defects limited to uncalcified cartilage, grade 3 ? defects extending into calcified cartilage, and grade 4 ? exposure of
subchondral bone. Bar ? 200 ?m.
URINARY BIOCHEMICAL MARKERS AND BMD IN A MOUSE MODEL OF OA465
values for intraobserver and interobserver agreement of histo-
logic grading were 0.91 and 0.83, respectively. These values are
regarded as reliable enough for this method.
Urine samples. At 8, 12, 16, 20, 24, 28, 32, 36, and 40
weeks of age, urine samples were collected from the mice by
keeping them overnight in metabolic cages for 24 hours. The
samples were centrifuged at 3,000 revolutions per minute for
10 minutes to remove debris, stored in aliquots, and frozen at
?30°C until assayed.
Measurement of urinary CTX-II. Urinary CTX-II lev-
els were measured by enzyme-linked immunosorbent assay
(Urine Pre-Clinical CartiLaps; Nordic Bioscience, Herlev,
Denmark) (5). The standard for CTX-II was a synthetic
peptide (EKGPDP) that was included in the kit. The levels of
CTX-II were corrected for urinary creatinine, measured enzy-
matically on an autoanalyzer (Hitachi, Tokyo, Japan) accord-
ing to the manufacturer’s protocol. The formula for calculating
the concentration was as follows: corrected CTX-II value
(?g/mmole creatinine) ? Urine Pre-Clinical CartiLaps (?g/
liter)/creatinine (mmole/liter). The intraassay and interassay
CVs for CTX-II were 5.7% and 9.4%, respectively.
Measurement of urinary Pyr and D-Pyr. Aliquots of
urine samples were hydrolyzed with an equal volume of 6N
HCl at 110°C for 20 hours. The levels of urinary Pyr and D-Pyr
were measured by high-performance liquid chromatography
directly linked to an automated sample preparation with
extraction columns (ASPEC) system (23). The external stan-
dard for Pyr and D-Pyr was isolated from human cortical bone
(24). In several studies, standards isolated from animal species
different from mice have been used to measure the levels of
urinary Pyr and D-Pyr in mice (25,26). The levels of urinary
Pyr and D-Pyr were corrected for creatinine. The formula for
calculating the concentration was as follows: corrected Pyr or
D-Pyr (nmoles/mmole creatinine) ? Pyr or D-Pyr (nmoles/
liter)/creatinine (mmoles/liter). The intraassay and interassay
CVs for Pyr were 6.4% and 8.3%, respectively, and those for
D-Pyr were 5.9% and 9.5%, respectively.
Statistical analysis. The Mann-Whitney U test was
used to assess differences in radiographic grading between
groups at each time point. Differences in BMD between the 2
groups, adjusted for body weight at each time point, were
assessed by analysis of covariance. A repeated-measures two-
way analysis of variance for urinary biochemical markers
determined whether there was an age effect, a group effect,
and a group-by-age interaction. When a group-by-age interac-
tion was significant, post hoc pairwise comparisons with Bon-
ferroni adjustment were conducted. Spearman’s correlation
coefficients were used to examine the correlation between the
2 measurements. P values less than 0.05 were considered
significant. SPSS version 11.0 statistical software was used for
all analyses (SPSS, Chicago, IL).
Histologic evaluation and categorization into
subgroups. Among the 24 joints of 12 control CBA mice
at 40 weeks of age, 21 joints (87.5%) were histologic
grade 0, and 3 joints (12.5%) were grade 1. Histologic
grades of ?2 were not identified in CBA mice. Among
the 44 joints of 22 STR/Ort mice at 40 weeks of age, 10
(22.7%) were histologic grade 0, 10 (22.7%) were grade
1, 4 (9.1%) were grade 2, 16 (36.4%) were grade 3, and
4 (9.1%) were grade 4. All 22 STR/Ort mice were
further categorized into 2 subgroups, with 16 in the STR
OA subgroup (histologic grade of ?2 for at least 1 knee
joint) and 6 in the STR non-OA subgroup (histologic
grade of 0 or 1 for both knee joints). In the STR OA
group, 9 mice had unilateral OA, and 7 mice had
bilateral OA (Table 1).
Changes in body weight. At the beginning of the
study, the body weights of the STR/Ort mice were
almost equal to those of the CBA mice. Although the
body weights of the STR/Ort and CBA mice increased
throughout the study period, those of the STR/Ort mice
were ?15% heavier than those of CBA mice older than
age 16 weeks. There were no differences in body weight
between mice in the STR OA and STR non-OA sub-
groups throughout the study period (data not shown).
Changes in radiographic grading. The radio-
graphic grades for the CBA mice did not increase
throughout the study period, while those for the STR/
Ort mice increased after 24 weeks of age. The radio-
graphic grades for the STR/Ort mice at 28, 32, 36, and 40
weeks of age were significantly higher (16.4-, 46.8-, 18.3-,
and 7.6-fold, respectively) than those for the CBA mice
(P ? 0.001 for all) (Figure 1B). The radiographic grades
for mice in both the STR OA and STR non-OA
subgroups increased gradually after 24 weeks of age. The
radiographic grades for mice in the STR OA subgroup at
28, 32, 36, and 40 weeks of age were significantly higher
in CBA and STR/Ort mice*
Histologic evaluation and grading of cartilage degeneration
(no. of joints/no. of mice)
no. of joints
STR OA unilateral, no.
STR OA bilateral, no.
* All mice in the STR osteoarthritis (OA) subgroup had OA of grade
?2, based on a histologic grading scale of 0–4 (see Materials and
Methods). STR/Ort mice were further categorized into 2 subgroups:
STR OA (unilateral and bilateral) and STR non-OA (no sign of OA).
466 SARUKAWA ET AL
(2.7-, 2.1-, 2.2-, and 2.3-fold, respectively) than those for
mice in the STR non-OA subgroup (P ? 0.004, P ?
0.004, P ? 0.002, and P ? 0.001, respectively) (Figure
1C). The radiographic grading at 40 weeks of age
correlated with the histologic grading at 40 weeks of age
(r ? 0.788, P ? 0.001).
Changes in femoral shaft BMD adjusted for body
weight. In STR/Ort mice, femoral shaft BMD adjusted
for body weight increased gradually from 20 weeks to 28
weeks of age and reached a plateau thereafter. The
adjusted BMD in CBA mice increased gradually
throughout the study period. There were no significant
differences in the adjusted BMD between STR/Ort and
CBA mice at each time point (Figure 3A). In the STR
OA subgroup, the adjusted BMD increased gradually
from 20 weeks to 28 weeks of age and reached a plateau
thereafter. Meanwhile, the adjusted BMD in the STR
non-OA subgroup increased gradually from 20 weeks to
Figure 4. Time course of urinary C-terminal crosslinking telopeptide of type II collagen (CTX-II) levels (A and B), pyridinoline (Pyr) levels (C and
D), and deoxypyridinoline (D-Pyr) levels (E and F) in STR/Ort and CBA mice and mice in the STR osteoarthritis (OA) and STR non-OA subgroups.
All urinary biochemical markers were corrected for creatinine (creat). Bars show the mean ? SD. ? ? P ? 0.001 versus mice younger than age 4
weeks in the STR OA and STR non-OA subgroups; † ? P ? 0.05; ‡ ? P ? 0.01; § ? P ? 0.001, versus STR non-OA.
Figure 3. Time course of femoral shaft bone mineral density (BMD) adjusted for body weight in STR/Ort and
CBA mice (A), and mice in the STR osteoarthritis (OA) and STR non-OA subgroups (B). The values for the left
and right femoral shaft BMD were averaged for each mouse. Bars show the mean ? SD. † ? P ? 0.05; ‡ ? P
? 0.01, versus STR non-OA.
URINARY BIOCHEMICAL MARKERS AND BMD IN A MOUSE MODEL OF OA 467
28 weeks of age, decreased after 28 weeks of age, and
then increased again. The adjusted BMD was signifi-
cantly greater (5%) in the STR OA subgroup compared
with the STR non-OA subgroup at 28, 32, 36, and 40
weeks of age (P ? 0.033, P ? 0.007, P ? 0.041, and P ?
0.047, respectively) (Figure 3B).
Changes in urinary CTX-II. In STR/Ort and
CBA mice, there was a significant age effect (F[3,106] ?
45.76, P ? 0.001). However, a group effect and group-
by-age interaction were shown not to be significant (P ?
0.439 and P ? 0.180, respectively) (Figure 4A). In the
STR OA and STR non-OA subgroups, there was a
significant age effect (F[8,160] ? 50.56, P ? 0.001), a
significant group effect (F[1,20] ? 5.28, P ? 0.033), and
a significant group-by-age interaction (F[8,160] ? 2.02,
P ? 0.047). Urinary CTX-II levels in the STR OA and
non-OA subgroups decreased significantly (55% [P ?
0.001] and 58% [P ? 0.001], respectively) from 8 weeks
to 12 weeks of age. Urinary CTX-II levels in the STR
OA subgroup at 20 weeks and 24 weeks of age were
significantly higher (1.6-fold [P ? 0.017] and 1.5-fold
[P ? 0.001], respectively) than in the STR non-OA
subgroup (Figure 4B). In STR/Ort mice, there were no
significant differences in urinary CTX-II levels between
mice with unilateral OA and those with bilateral OA, at
each time point (data not shown).
Changes in urinary pyridinoline. In STR/Ort and
CBA mice, there was a significant age effect (F[8,256] ?
22.60, P ? 0.001) and a significant group effect (F[1,32]
? 7.57, P ? 0.010). However, a group-by-age interaction
was shown not to be significant (P ? 0.426) (Figure 4C).
In the STR OA and STR non-OA subgroups, there was
a significant age effect (F[8,160] ? 15.39, P ? 0.001), a
significant group effect (F[1,20] ? 10.04, P ? 0.005), and
a significant group-by-age interaction (F[8,160] ? 2.46,
P ? 0.015). Urinary Pyr levels in the STR OA and STR
non-OA subgroups decreased significantly (25% [P ?
0.001] and 30% [P ? 0.001], respectively) from 8 weeks
to 12 weeks of age. Urinary Pyr levels in the STR OA
subgroup were significantly higher (1.5-fold) than those
in the STR non-OA subgroup at 32, 36, and 40 weeks of
age (P ? 0.009, P ? 0.004, and P ? 0.024, respectively)
(Figure 4D). In STR/Ort mice, there were no significant
differences in urinary Pyr levels between mice with
unilateral OA and those with bilateral OA, at each time
point (data not shown).
Changes in urinary D-Pyr. In STR/Ort and CBA
mice, there was a significant age effect (F[8,256] ?
22.60, P ? 0.001). However, a group effect and group-
by-age interaction were shown not to be significant (P ?
0.550 and P ? 0.972, respectively) (Figure 4E). In the
STR OA and STR non-OA subgroups, there was a
significant age effect (F[5,102] ? 4.97, P ? 0.001).
However, a group effect and group-by-age interaction
were shown not to be significant (P ? 0.113 and P ?
0.484, respectively) (Figure 4F).
Correlations between urinary biochemical mark-
ers and radiographic grading. In STR/Ort mice, corre-
lations between the levels of urinary CTX-II and Pyr and
the radiographic grade at each time point from 12 weeks
of age were examined. The radiographic grades were
correlated with urinary CTX-II levels at 24 weeks of age
(r ? 0.390 [P ? 0.048]) and with urinary Pyr levels at 36
weeks and 40 weeks of age (r ? 0.444 [P ? 0.046] and
r ? 0.421 [P ? 0.03], respectively).
In this study, we investigated the longitudinal
relationship between changes in the concentration of
several biochemical markers reflecting cartilage and
bone turnover and the onset and progression of OA in
STR/Ort mice in which knee OA developed spontane-
ously, as well as the relationship between OA and BMD.
To evaluate the onset and progression of knee
OA, we performed longitudinal radiographic assess-
ments of the knee joints of mice and histologically
analyzed the severity of knee OA at the end of the study
period. In this study, the radiographic grades for STR/
Ort mice were higher than those for CBA mice after 28
weeks of age, and histologically evident OA developed
in ?80% of male STR/Ort mice at 40 weeks of age.
Walton also showed that OA lesions developed in ?85%
of male STR/Ort mice by 35 weeks of age (14). Walford
defined the term “old” in mice by using a 50% survival
point (27). For STR/Ort mice, the 50% survival point is
?70 weeks of age (Charles River Japan: unpublished
data). Therefore, it is assumed that age 35 weeks in
STR/Ort mice corresponds to age ?50 years in humans.
Unlike other animal models of OA using chemi-
cal induction or surgical intervention, it is difficult to
unify the onset and severity of disease in STR/Ort mice
in which OA develops spontaneously. We further com-
pared the STR OA and STR non-OA subgroups. Mod-
els of surgically induced OA can use nonsurgically
treated animals as controls; however, no “true” control
group has been available for STR/Ort mice. CBA mice
have been used most frequently as controls for STR/Ort
mice, because knee OA has rarely developed in these
mice (28–30). In our study, there were no significant
differences in any of the biochemical markers used or
the BMD between STR/Ort and sex- and age-matched
468 SARUKAWA ET AL
CBA mice. These results may be attributable to strain
differences in body weight, growth speed (31), and the
baseline level of each biochemical marker and baseline
BMD. Therefore, we attempted to categorize STR/Ort
mice into STR OA and STR non-OA subgroups.
In the present study, urinary CTX-II levels in
mice in the STR OA subgroup were higher than those in
mice in the STR non-OA subgroup at 20 and 24 weeks
of age, before any radiographic changes of OA were
apparent. This indicates that urinary CTX-II may be
useful as a potential marker to predict disease progres-
sion at an earlier stage of OA. Because type II collagen
is the predominant collagen in cartilage and is restricted
in localization to this tissue, urinary CTX-II is regarded
as a sensitive marker that reflects cartilage degeneration.
In previous studies, the utility of urinary CTX-II for
detecting the earliest stage of OA and predicting OA
progression has been described (10,32). In contrast, no
significant differences in urinary CTX-II levels between
the STR OA and STR non-OA subgroups were ob-
served after 28 weeks of age in the present study. Jordan
et al (33) reported that once a certain level of JSN has
been reached, CTX-II production reaches a plateau and
possibly starts to decline. This indicates a breakdown of
the repair mechanism in the advanced stage of OA. In
the present study, the observation of no apparent differ-
ences in urinary CTX-II levels between mice in the STR
OA and STR non-OA subgroups at an advanced disease
stage is in accordance with the observations by Jordan et
Several previous studies have indicated that uri-
nary Pyr and D-Pyr levels are associated with radio-
graphic progression of knee OA (6,7). Rudolphi et al
(34) reported that urinary levels of hydroxylysylpyridino-
line (i.e., Pyr) in STR/1N mice, a strain very closely
related to STR/Ort mice, predominantly reflected carti-
lage destruction. Graverand et al (35) reported that Pyr
levels were higher in patients with late OA than in those
with early OA, and that increased Pyr levels reflect bone
erosion and/or increased sclerotic bone remodeling of
the joint epiphyses. We previously investigated the con-
centrations of Pyr and D-Pyr in joint tissues, such as
cartilage, bone, and synovium, and observed that Pyr is
more abundant in cartilage and to a lesser extent in
synovium, and that Pyr concentrations reflect not only
cartilage and bone metabolism but also synovial metab-
olism (8). In the present study, elevated levels of Pyr in
the STR OA subgroup at an advanced stage of disease
may be attributable to changes in cartilage, bone, and
synovium and may reflect, to a small degree, systemic
bone turnover, because there were no differences in
urinary D-Pyr levels at different disease stages. Urinary
D-Pyr derived only from the bone matrix and urinary
CTX-II derived only from the cartilage matrix may be
insufficient to reflect the disease status of OA at the
Figure 5. Schematic representation of the course of the relationship between differences in the levels of urinary
biochemical markers and bone mineral density (BMD) and the occurrence of osteoarthritis (OA) in the STR OA
and STR non-OA subgroups. CTX-II ? C-terminal crosslinking telopeptide of type II collagen; Pyr ?
pyridinoline; D-Pyr ? deoxypyridinoline.
URINARY BIOCHEMICAL MARKERS AND BMD IN A MOUSE MODEL OF OA469
In general, arthritic changes among the major
joints of STR/Ort mice were restricted to the knee joints.
Some male STR/Ort mice are known to experience a
spontaneous ankle deformity and calcification of the
Achilles tendon, but it has been reported that there was
no correlation between changes in ankle deformity and
calcification of the Achilles tendon and the presence of
OA (36). In the present study, STR/Ort mice showed no
detectable radiographic changes of OA in the other
major joints and only mild calcification of the Achilles
tendon in 12 hind paws. Therefore, in STR/Ort mice,
other major joints do not contribute to the urinary
concentrations of biochemical markers.
Most previous studies have indicated that the
skeletal sites of higher BMD differ among patients with
OA (37,38). A comparative analysis of bone and carti-
lage metabolism in Hartley guinea pigs with naturally
occurring OA showed that femoral shaft BMD was
higher in guinea pigs with OA than in controls (39).
Meanwhile, Calvo et al (40) showed that lower BMD
increased the severity of cartilage damage in an experi-
mental model of knee OA in rabbits. The results of the
present study suggest that high BMD may be related to
the onset and progression of knee OA in STR/Ort mice,
based on the observation of higher BMD in the STR OA
subgroup. Possible systemic factors that might explain
the relationship between BMD and OA include obesity
(41), bone turnover (37,38), and genetics (42). In our
study, there were no significant differences in body
weights and the urinary D-Pyr level, which is a bone-
specific biochemical marker, between the STR OA and
STR non-OA subgroups. Further studies are needed to
clarify the mechanism of BMD in relation to the patho-
physiology of knee OA.
In our study, we measured femoral shaft BMD in
mice to evaluate the relationship between BMD and
OA. In small animals, the entire femur is more fre-
quently used as the ROI for measuring BMD (43–45).
Fink et al showed that the most precise ROI in small
animals is the entire femur, because larger sites have less
variation than smaller sites (46). We considered that
femoral shaft BMD indicates the most appropriate site
reflecting systemic bone turnover in mice, because the
entire femur may be affected by changes in subchondral
bone in knee OA.
Figure 5 is a schematic summarization of the
relationship between the establishment of radiographic
OA and biochemical markers and BMD in the STR OA
subgroup compared with the STR non-OA subgroup.
Higher levels of urinary CTX-II and an increase in BMD
in the STR OA subgroup were observed prior to the
establishment of radiographic OA. In contrast, higher
levels of urinary Pyr in the STR OA subgroup were
observed at the advanced stage of OA. These results
indicate that the period during which a biochemical
marker contributes to assessing the disease status of OA
differs according to each biochemical marker, and that
high BMD may be related to the induction of OA.
In conclusion, urinary CTX-II could be a useful
marker in the early diagnosis and predicting the progres-
sion of OA, and urinary Pyr may be a potential marker
to assess the severity of OA at an advanced stage. An
increase in BMD prior to the establishment of radio-
graphic OA may be related to the induction of OA.
We wish to thank Ms Ayako Okamoto and Mr. Yuichi
Kaneko for their technical help in this study.
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. Sarukawa 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. Sarukawa, Takahashi, Doi, Suzuki,
Acquisition of data. Sarukawa, Suzuki.
Analysis and interpretation of data. Sarukawa, Takahashi, Doi, Su-
1. Buckland-Wright JC, Macfarlane DG, Lynch JA, Jasani MK,
Bradshaw CR. Joint space width measures cartilage thickness in
osteoarthritis of the knee: high resolution plain film and double
contrast macroradiographic investigation. Ann Rheum Dis 1995;
2. Duddy J, Kirwan JR, Szebenyi B, Clarke S, Granell R, Volkov S.
A comparison of the semiflexed (MTP) view with the standing
extended view (SEV) in the radiographic assessment of knee
osteoarthritis in a busy routine X-ray department. Rheumatology
3. Mouritzen U, Christgau S, Lehmann HJ, Tanko LB, Christiansen
C. Cartilage turnover assessed with a newly developed assay
measuring collagen type II degradation products: influence of age,
sex, menopause, hormone replacement therapy, and body mass
index. Ann Rheum Dis 2003;62:332–6.
4. Garnero P, Conrozier T, Christgau S, Mathieu P, Delmas PD,
Vignon E. Urinary type II collagen C-telopeptide levels are
increased in patients with rapidly destructive hip osteoarthritis.
Ann Rheum Dis 2003;62:939–43.
5. Christgau S, Garnero P, Fledelius C, Moniz C, Ensig M, Gineyts E,
et al. Collagen type II C-telopeptide fragments as an index of
cartilage degradation. Bone 2001;29:209–15.
6. Takahashi M, Naito K, Abe M, Sawada T, Nagano A. Relationship
between radiographic grading of osteoarthritis and the biochemi-
cal markers for arthritis in knee osteoarthritis. Arthritis Res Ther
7. Thompson PW, Spector TD, James IT, Henderson E, Hart DJ.
Urinary collagen crosslinks reflect the radiographic severity of
knee osteoarthritis. Br J Rheumatol 1992;31:759–61.
470 SARUKAWA ET AL
8. Takahashi M, Kushida K, Hoshino H, Suzuki M, Sano M, Miy-
amoto S, et al. Concentrations of pyridinoline and deoxypyridino-
line in joint tissues from patients with osteoarthritis or rheumatoid
arthritis. Ann Rheum Dis 1996;55:324–7.
9. Garnero P, Piperno M, Gineyts E, Christgau S, Delmas PD,
Vignon E. Cross sectional evaluation of biochemical markers of
bone, cartilage, and synovial tissue metabolism in patients with
knee osteoarthritis: relations with disease activity and joint dam-
age. Ann Rheum Dis 2001;60:619–26.
10. Reijman M, Hazes JM, Bierma-Zeinstra SM, Koes BW, Christgau
S, Christiansen C, et al. A new marker for osteoarthritis: cross-
sectional and longitudinal approach. Arthritis Rheum 2004;50:
11. Bentley G. Papain-induced degenerative arthritis of the hip in
rabbits. J Bone Joint Surg Br 1971;53:324–37.
12. Williams JM, Brandt KD. Immobilization ameliorates chemically-
induced articular cartilage damage. Arthritis Rheum 1984;27:
13. Hulth A, Lindberg L, Telhag H. Experimental osteoarthritis in
rabbits: preliminary report. Acta Orthop Scand 1970;41:522–30.
14. Walton M. Degenerative joint disease in the mouse knee joint;
histological observations. J Pathol 1977;123:109–122.
15. Star VL, Hochberg MC. Osteoporosis in patients with rheumatic
diseases. Rheum Dis Clin North Am 1994;20:561–76.
16. Sambrook P, Naganathan V. What is the relationship between
osteoarthritis and osteoporosis? Baillieres Clin Rheumatol 1997;
17. Walton M. Degenerative joint disease in the mouse knee; radio-
logical and morphological observations. J Pathol 1977;123:97–107.
18. Evans RG, Collins C, Miller P, Ponsford FM, Elson CJ. Radio-
logical scoring of osteoarthritis progression in STR/ort mice.
Osteoarthritis Cartilage 1994;2:103–9.
19. Munasinghe JP, Tyler JA, Carpenter TA, Hall LD. High resolu-
tion MR imaging of joint degeneration in the knee of the
STR/ORT mouse. Magn Reson Imaging 1995;13:421–8.
20. Gaffen JD, Gleave SJ, Crossman MV, Bayliss MT, Mason RM.
Articular cartilage proteoglycans in osteoarthritic STR/Ort mice.
Osteoarthritis Cartilage 1995;3:95–104.
21. Sokoloff L. Natural history of degenerative joint disease in small
laboratory animals. I. Pathological anatomy of degenerative joint
disease in mice. AMA Arch Pathol 1956;62:118–28.
22. Schunke M, Tillmann B, Bruck M, Muller-Ruchholtz W. Morpho-
logic characteristics of developing osteoarthrotic lesions in the
knee cartilage of STR/IN mice. Arthritis Rheum 1988;31:898–905.
23. Pratt DA, Daniloff Y, Duncan A, Robins SP. Automated analysis
of the pyridinium crosslinks of collagen in tissue and urine using
solid-phase extraction and reversed-phase high-performance liq-
uid chromatography. Anal Biochem 1992;207:168–75.
24. Ohishi T, Takahashi M, Kawana K, Aoshima H, Hoshino H,
Horiuchi K, et al. Age-related changes of urinary pyridinoline and
deoxypyridinoline in Japanese subjects. Clin Invest Med 1993;16:
25. Mathieu C, Waer M, Casteels K, Laureys J, Bouillon R. Preven-
tion of type I diabetes in NOD mice by nonhypercalcemic doses of
a new structural analog of 1,25-dihydroxyvitamin D3, KH1060.
26. Boregowda R, Paul E, White J, Ritty TM. Bone and soft connec-
tive tissue alterations result from loss of fibrillin-2 expression.
Matrix Biol 2008;27:661–6.
27. Walford RL. When is a mouse “old”? [letter]. J Immunol 1976;
28. Walton M, Elves MW. Bone thickening in osteoarthrosis: obser-
vations of an osteoarthrosis-prone strain of mouse. Acta Orthop
29. Dunham J, Chambers MG, Jasani MK, Bitensky L, Chayen
J. Changes in the orientation of proteoglycans during the early
development of natural murine osteoarthritis. J Orthop Res
30. Gaffen JD, Bayliss MT, Mason RM. Elevated aggrecan mRNA in
early murine osteoarthritis. Osteoarthritis Cartilage 1997;5:
31. Mason RM, Chambers MG, Flannelly J, Gaffen JD, Dudhia J,
Bayliss MT. The STR/ort mouse and its use as a model of
osteoarthritis. Osteoarthritis Cartilage 2001;9:85–91.
32. Garnero P, Peterfy C, Zaim S, Schoenharting M. Bone marrow
abnormalities on magnetic resonance imaging are associated with
type II collagen degradation in knee osteoarthritis: a three-month
longitudinal study. Arthritis Rheum 2005;52:2822–9.
33. Jordan KM, Syddall HE, Garnero P, Gineyts E, Dennison EM,
Sayer AA, et al. Urinary CTX-II and glucosyl-galactosyl-pyridino-
line are associated with the presence and severity of radiographic
knee osteoarthritis in men. Ann Rheum Dis 2006;65:871–7.
34. Rudolphi K. Gerwin N, Verzijl N, van der Kraan P, van der Berg
W. Pralnacasan, an inhibitor of interleukin-1? converting enzyme,
reduces joint damage in two murine models of osteoarthritis.
Osteoarthritis Cartilage 2003;11:738–46.
35. Graverand MP, Tron AM, Ichou M, Dallard MC, Richard M,
Uebelhart D, et al. Assessment of urinary hydroxypyridinium
cross-links measurement in osteoarthritis. Br J Rheumatol 1996;
36. Walton M. A spontaneous ankle deformity in an inbred strain of
mouse. J Pathol 1978;124:189–94.
37. Stewart A, Black A, Robins SP, Reid DM. Bone density and bone
turnover in patients with osteoarthritis and osteoporosis. J Rheu-
38. Sowers M, Lachance L, Jamadar D, Hochberg MC, Hollis B,
Crutchfield M, et al. The associations of bone mineral density and
bone turnover markers with osteoarthritis of the hand and knee in
pre- and perimenopausal women. Arthritis Rheum 1999;42:483–9.
39. Huebner JL, Hanes MA, Beekman B, TeKoppele JM, Kraus VB.
A comparative analysis of bone and cartilage metabolism in two
strains of guinea-pig with varying degrees of naturally occurring
osteoarthritis. Osteoarthritis Cartilage 2002;10:758–67.
40. Calvo E, Castaneda S, Largo R, Fernandez-Valle ME, Rodriguez-
Salvanes F, Herrero-Beaumont G. Osteoporosis increases the
severity of cartilage damage in an experimental model of osteoar-
thritis in rabbits. Osteoarthritis Cartilage 2007;15:69–77.
41. Reijman M, Pols HA, Bergink AP, Hazes JM, Belo JN, Lievense
AM, et al. Body mass index associated with onset and progression
of osteoarthritis of the knee but not of the hip: the Rotterdam
Study. Ann Rheum Dis 2007;66:158–62.
42. Uitterlinden AG, Burger H, Huang Q, Odding E, Duijn CM,
Hofman A, et al. Vitamin D receptor genotype is associated with
radiographic osteoarthritis at the knee. J Clin Invest 1997;100:
43. Cohen SL, Moore AM, Ward WE. Interleukin-10 knockout
mouse: a model for studying bone metabolism during intestinal
inflammation. Inflamm Bowel Dis 2004;10:557–63.
44. Ward WE, Kim S, Chan D, Fonseca D. Serum equol, bone mineral
density and biomechanical bone strength differ among four mouse
strains. J Nutr Biochem 2005;16:743–9.
45. Nordstrom SM, Carleton SM, Carson WL, Eren M, Phillips CL,
Vaughan DE. Transgenic over-expression of plasminogen activa-
tor inhibitor-1 results in age-dependent and gender-specific in-
creases in bone strength and mineralization. Bone 2007;41:
46. Fink C, Cooper HJ, Huebner JL, Guilak F, Kraus VB. Precision
and accuracy of a transportable dual-energy x-ray absorptiometry
unit for bone mineral measurements in guinea pigs. Calcif Tissue
URINARY BIOCHEMICAL MARKERS AND BMD IN A MOUSE MODEL OF OA471