Life Sciences 86 (2010) 538–543

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Life Sciences
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Evaluation of the effect of glucosamine on an experimental rat osteoarthritis model

Kiyohito Naito a,b, Taiji Watari a,b, Atsushi Furuhata c, Shin Yomogida d, Koji Sakamoto e, Hisashi Kurosawa f,
Kazuo Kaneko a, Isao Nagaoka d,⁎
a
Department of Orthopaedic Surgery, Juntendo University Shizuoka Hospital, 1129 Nagaoka, Izunokuni, Shizuoka 410-2295, Japan
b
Sportology Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
c
Division of Biomedical Imaging Research, Biomedical Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
d
Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
e
Koyo Chemical Co., Ltd., 3-11-15 Iidabashi, Chiyoda-ku, Tokyo, 112-0072 Japan
f
Department of Orthopaedic Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Aims: To investigate the in vivo effect of glucosamine on articular cartilage in osteoarthritis (OA), we
Received 14 October 2009 evaluated serum biomarkers such as CTX-II (type II collagen degradation) and CPII (type II collagen
Accepted 7 February 2010 synthesis) as well as histopathological changes (Mankin score, toluidine blue staining of proteoglycans in an
experimental OA model using rats.
Keywords:
Main methods: OA was surgically induced in the knee joint by anterior cruciate ligament transection (ACLT)
Glucosamine
in rats. Animals were divided into three groups: sham-operated group (Sham), ACLT group without GlcN
Osteoarthritis
Type II collagen
administration (− GlcN) and ACLT group with oral administration of glucosamine hydrochloride (+ GlcN;
Biomarkers 1000 mg/kg/day for 56 days).
Key findings: ACLT induced macroscopic erosive changes on the surfaces of articular cartilage and histological
damages such as increase of Mankin score. Of note, glucosamine administration substantially suppressed the
macroscopic changes, although the effect on Mankin score was not significant. In addition, serum CTX-II
levels were elevated in −GlcN group compared to that in Sham group after the operation. Of importance, the
increase of CTX-II was significantly suppressed by GlcN administration. Moreover, serum CP-II levels were
substantially increased in + GlcN group compared to those in Sham and − GlcN groups after the operation.
Significance: GlcN has a potential to exert a chondroprotective action on OA by inhibiting type II collagen
degradation and enhancing type II collagen synthesis in the articular cartilage.
© 2010 Elsevier Inc. All rights reserved.

Introduction
Ostaoarthritis (OA) is a joint disease characterized by pain, cartilage
degeneration and joint stiffness. The pathogenesis of OA involves a
complex multi-factorial diseased process with cartilage catabolism and
anabolism as well as changes in other tissues such as synovium,
subchondral bone and tendons (Lohmander 2000). Currently several
treatments are available for OA ranging from the most conservative to
more surgical extremes. Conservative measures involve lifestyle
modifications, physical therapy and pharmacologic treatment with
nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular
injection of hyaluronan (Puhl et al. 1993). These treatments are not
disease modifying, but only reduce symptoms. In contrast, irreversible
joint disability in advanced OA usually requires surgical intervention to
relieve pain and improve joint function (Buckwalter and Mankin 1998).
⁎ Corresponding author. Tel.: +81 3 5802 1032; fax: +81 2 3813 3157.
E-mail address: nagaokai@juntendo.ac.jp (I. Nagaoka).
Thus, current treatments are mostly targeting the symptoms but not
addressing the destructed structure of articular cartilage in OA.
Glucosamine, a naturally-occurring amino monosaccharide, is
present in the connective and cartilage tissues, and contributes to
maintaining the strength, flexibility and elasticity of these tissues.
Thus, glucosamine has been widely used to treat osteoarthritis for
more than two decades in humans (Crolle and D'Este 1980). Several
short- and long-term clinical trials in osteoarthritis have shown the
significant symptom-modifying effect of glucosamine (McAlindon
et al. 2000; Reginster et al. 2001; Pavelká et al. 2002). Moreover, the
updated Osteoarthritis Research Society International (OARSI) recom-
mendations for management of hip and knee OA have recently
suggested that glucosamine has symptom-relieving and structure-
modifying effects in knee OA (Zhang et al. 2008). Importantly, it has
been previously revealed in vitro that glucosamine can inhibit the
degradation and stimulate the synthesis of glycosaminoglycans
(proteoglycans), thereby possibly exhibiting chondroprotective ac-
tion (Fenton et al. 2000; Gouze et al. 2001). Moreover, glucosamine
has been shown to reduce radiographic progression of joint space
narrowing in knee OA (Reginster et al. 2001). On the other hand, GAIT

0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2010.02.015
 K. Naito et al. / Life Sciences 86 (2010) 538–543
study reported that glucosamine did not reduce pain effectively in
patients with OA (Clegg et al. 2006). Thus, the effect of glucosamine
on OA is still controversial.
A number of OA models, using aging animals, genetically modified
mice as well as animals with surgically, enzymatically or chemically
induced OA (Bendele 2001; van den Berg 2001), have been developed
to investigate the pathogenesis of OA and evaluate the potentials of
new disease/structure-modifying drugs (Oegema et al. 2002; Høegh-
Andersen et al. 2004; Jean et al. 2006; Tang et al. 2008). Among these,
the anterior cruciate ligament transaction (ACLT) model has been
widely used to analyze the histological and biochemical changes
observed during the progression of OA (Ameye and Young 2006).
Furthermore, ACLT results in joint instability and induce cartilage
degeneration, subchondral bone sclerosis and osteophyte formation,
which mimics the pathological changes detected in human OA
(Hayami et al. 2006). Thus, this model is considered to be suitable
for evaluating therapeutic agents for OA.
In the present study, to investigate the in vivo mechanism how
glucosamine affects the articular cartilage in OA, we evaluated serum
biomarkers such as CTX-II (type II collagen degradation) and CPII (type II
collagen synthesis) as well as histopathological changes (Mankin score
and toluidine blue staining of proteoglycans) in an ACLT-induced
experimental rat OA model. Usually, 1.5 g/day of glucosamine sulfate
or hydrochloride (approximately 25 mg/kg) is administered to humans
for treatment of osteoarthritis (McAlindon et al. 2000; Reginster et al.
2001; Pavelká et al. 2002), and serum glucosamine level reaches 0.020 ±
0.006 mM, as measured by a high performance liquid chromatography
(Hua et al. 2004). In contrast, because of a poor bioavailability due to a
low absorption and/or substantial loss in the gastrointestinal tract, a high
dose of glucosamine hydrochloride (350 mg/kg) was orally administered
to rats to obtain the serum level of glucosamine (0.05 mM), which was
almost the same as that in humans (Aghazadeh-Habashi et al. 2002).
Thus, in this study, to attain an enough effect of glucosamine, rats orally
received an average of 1000 mg glucosamine hydrochloride/kg/day, as
described in Materials and methods.
Materials and methods
Animal model and glucosamine administration
All procedures were carried out according to the Institutional
Animal Care and Committee Guide of Juntendo University School of
Medicine. Male Sprague-Dawley rats (10-week-old; Charles River
Breeding Laboratories, Tokyo, Japan) were used in the present study.
Two and three rats per cage were housed under a specific pathogen-
free condition (controlled temperature of 24 ± 3 °C and humidity of
55 ± 15%) and fed standard laboratory food ad libitum.
OA was surgically induced in the knee joint. Each rat was anesthe-
tized with halothane, and after being shaved and disinfected, the right
knee joint was exposed through a medial parapatellar approach. The
patella was dislocated laterally and the knee placed in full flexion,
followed by anterior cruciate transection (ACLT) with micro-scissors
(Hayami et al. 2006). After the surgery, the joint surface was washed
with sterile saline, and both capsule and skin were sutured using
Vicryl 4-0 absorbable suture and monofilament 4-0 Nylon threads,
respectively. In sham-operated animals, the right knee joint was
exposed and incisions were closed after subluxation of the patella and
washing the joint surface with saline.
Glucosamine hydrocholoride was supplied by Koyo Chemical Co.,
Ltd., (Tokyo, Japan). Glucosamine hydrochloride solution dissolved in
tap water (1%, 10 mg/ml) was orally administered to rat ad libitum for
8 weeks after surgery. The intake volume of glucosamine solution was
measured twice a week, and it was found that the animals received an
average of 50 ml glucosamine solution (1000 mg glucosamine hydro-
choloride/kg/day).
539
Rats were divided into three groups: 1. Sham-operated group
(Sham; n = 6), 2. ACLT group without glucosamine (−GlcN; n = 6), 3.
ACLT group with glucosamine (+GlcN; n = 6). All groups (Sham,
−GlcN and + GlcN) of rats were allowed to move freely in plastic
cages. Animals were sacrificed at 56 days after the surgery.
Gross morphology
After disarticulation, both femurs and tibias were cleaned and fixed
for 24 h in phosphate-buffered formalin (10%). The gross appearance of
distal femur and proximal tibia were recorded by a digital camera (D2X;
Nikon, Tokyo, Japan). To quantitatively evaluate the gross morphology,
erosive area was measured using Adobe Photoshop 7.0 (Adobe Systems,
San Jose, CA) and expressed in mm2.
Tissue preparation and histopathological evaluation
Histopathological evaluation was performed on the sagittal sections
of cartilage in the weight-bearing area of the medial tibia plateau. Knee
joint samples were dissected, fixed in 10% formalin for 24 h,
decalcificated by Gooding and Stewart's fluid (equal volume of 10%
formalin and 10% formic acid solution), and embedded in paraffin.
Sections (5 µm) were stained with 0.05% toluidine blue (pH 4.1). The
severity of OA lesion was graded on a scale of 0–13, using the modified
Mankin scoring system (Oegema et al. 2002; Tang et al. 2008) with a
combined score of structure (0–6 points), cellular abnormalities (0–3
points) and matrix staining (0–4 points) (Table 1). Histopathological
evaluation was performed by two independent blinded observers, and
the inter-observer average variation was 0.76 in the highest Mankin
score of 13 (a combined score of structure, cells and staining). The
averaged data of the two observers were used for calculating Mankin
score.
Enzyme linked immunoassay (ELISA) of serum biomarkers
Blood was obtained from tail vein immediately before and at 7, 14,
28 and 56 days after the surgery, and sera were stored in aliquots at
−80 °C.
Assays for serum CTX-II (Rousseau and Delmas 2007) were performed
with a serum Pre-Clinical CartiLaps ELISA kit (Nordic Bioscience
Diagnostic A/S, Herlev, Denmark), which detects C-telopeptide degrada-
tion products of type II collagen (CTX-II) in sera.
Table 1
Mankin score for the histological grading of cartilage degeneration.
Grade Structure
0 Normal
1 Surface irregularities
2 Pannus and surface irregularities
3 Clefts to transitional zone
4 Clefts to radial zone
5 Clefts to calcified zone
6 Complete disorganization
Grade Cells
0 Normal
1 Diffuse hypercellularity
2 Cloning
3 Hypocellularity
Grade Staining
0 Normal
1 Slight reduction
2 Moderate reduction
3 Severe reduction
4 No dye noted
540 K. Naito et al. / Life Sciences 86 (2010) 538–543

Assays for serum CPII (Rousseau and Delmas 2007) were
performed with a Procollagen type II C-propeptide ELISA kit (IBEX
Technologies Inc., Montreal, Canada), which detects carboxy propep-
tide of type II collagen (C-propeptide, also referred as CPII) in sera.
CPII is cleaved from type II procollagen during processing of newly
synthesized procollagen, and thus can be used as a type II collagen-
synthesis marker.
Assays for serum CTX-I (Rousseau and Delmas 2007) were
performed with a RatLaps ELISA kit (Nordic Bioscience Diagnostic A/S),
which detects C-telopeptide degradation products of type I collagen
(CTX-I) in both serum and urine.
All samples were measured in duplicates on the same microtiter
plate. The detection limits of CTX-II, CPII and CTX-I were b6.9 pg/ml,
b50 ng/ml and b7.7 ng/ml, respectively.
Statistical analyses
Data are presented as mean± SD. Statistical significance was de-
termined by one-way ANOVA analysis using a StatView 5.0 pro-
gram (SAS Institute Inc., NC, USA). p b 0.05 was considered statistically
significant.

Fig. 1. Gross appearance of femoral condyles and tibial plateau at 56 days after the ACLT operation. Sham-operated joints showed no detectable changes on both femoral condyle (A)
and tibial plateau (E). In −GlcN group, ACLT induced the erosive change on the joint surface (B and F, indicated by dotted circles). Of note, glucosamine administration (+GlcN)
noticeably suppressed the degenerative changes (C and G). The erosive area quantitated was significantly decreased in + GlcN compared to − GlcN (D and H). **p b 0.01, ***p b 0.001.
Scale bar = 5.0 mm.
 K. Naito et al. / Life Sciences 86 (2010) 538–543 541

Results Evaluation of biomarkers

Effect of glucosamine administration on the gross morphology of
articular cartilage
In the joints of sham-operated rats, no macroscopic changes were
detected on the articular surfaces of femoral condyles and tibial plateau
(Fig. 1A and E). In contrast, ACLT obviously induced the erosive change
on the surfaces of articular cartilage (−GlcN; Fig. 1B and F). Of interest,
the degenerative changes were substantially suppressed by glucos-
amine administration (+GlcN; Fig. 1C and G). Moreover, quantitative
analysis indicated that the erosive area was significantly decreased in
+GlcN compared to −GlcN (Fig. 1D and H).
Effect of glucosamine administration on the histopathological changes in
articular cartilage
Next, we evaluated the effect of glucosamine administration on the
histopathological changes in articular cartilage. ACLT apparently
induced histopathological changes such as surface irregularity of
medial tibia plateau, surface depletion and reduced toluidine blue-
staining in the cartilage (− GlcN vs. Sham; Fig. 2A and B), as
previously reported (Hayami et al. 2006). These changes were further
evaluated using Mankin score. As expected, the score was significantly
increased by ACLT (− GlcN vs. Sham, p b 0.01) (Fig. 2D). Of interest,
surface irregularity of medial tibia plateau, surface depletion and
reduced toluidine blue-staining were suppressed by glucosamine
administration (+ GlcN; Fig. 2C). Consistent with this, Mankin score
was reduced in + GlcN compared with − GlcN, although the
difference was statistically not significant (Fig. 2D).
Furthermore, we evaluated the effect of glucosamine administra-
tion on articular cartilage by using biomarkers for collagen degradation
and synthesis (Rousseau and Delmas 2007). Preoperational levels of
serum CTX-II (a marker for type II collagen degradation) were 59.45±
13.73 pg/ml in Sham group, 60.89±17.06 in −GlcN group and 65.97±
19.12 in+GlcN group; preoperational levels of serum CPII (a marker for
type II collagen synthesis) were 1247.47±417.60 ng/ml in Sham group,
1301.33±421.49 in −GlcN group and 1221.66±344.85 in +GlcN group;
preoperational levels of serum CTX-I (a marker for type I collagen
degradation) were 17.98±3.67 ng/ml in Sham group, 22.97±6.11 in
−GlcN group and 20.46±5.12 in +GlcN group. Thus, there were no
significant differences in the preoperational levels of CTX-II, CPII and CTX-I
among three groups.
Serum CTX-II levels were elevated in −GlcN group compared to that
in Sham group at 7 (p b 0.01) and 14 (p b 0.001) days after the operation
(Fig. 3A). Of note, the increase of CTX-II levels was significantly
suppressed by glucosamine administration, and CTX-II levels were not
different between +GlcN and Sham groups. Moreover, serum CPII levels
were appreciably increased in +GlcN compared to those in Sham and
−GlcN groups at 7, 14 and 28 days after the operation (+GlcN vs Sham
and −GlcN: p b 0.001)(Fig. 3B). In contrast, CTX-I levels were not
essentially changed during 56 days after the operation in three groups
(Sham, −GlcN and +GlcN) (Fig. 3C).
Discussion
Glucosamine is currently used as a health supplement to relieve
the pain of OA (Towheed et al. 2005). Several clinical trials have
shown the significant symptom-modifying effect of glucosamine in

Fig. 2. Histopathological evaluation of articular cartilage in a rat OA model. Knee joints were dissected at 56 days after the ACLT operation, and the sagittal sections of cartilage in the
weight-bearing area of the medial tibia plateau were stained with toluidine blue; Sham (A), −GlcN (B) and + GlcN (C). ACLT induced the irregularity of medial tibia plateau, surface
depletion and reduced toluidine blue-staining in − GlcN (B, indicated by arrowheads), which were suppressed by glucosamine administration in + GlcN (C). The severity of OA
lesion was graded on a scale of 0–13 using the modified Mankin scoring system (D). Data represent the mean ± SD of six animals in each group (Sham, − GlcN and + GlcN). Values
were compared between Sham and −GlcN or +GlcN. *p b 0.05, **p b 0.01. Scale bar = 200 µm.
542 K. Naito et al. / Life Sciences 86 (2010) 538–543
Fig. 3. Effects of glucosamine administration on biomarkers in a rat OA model. Blood
was obtained from tail vein before (0 day) and at 7, 14, 28 and 56 days after the ACLT
operation, and sera were used for the assays of CTX-II (A), CPII (B) and CTX-I (C). CTX-II,
CPII and CTX-I levels are expressed as a percentage of baseline levels before the
operation (0 day). Data represent the mean ± SD of six animals in each group (Sham,
closed diamond; −GlcN, closed circle; +GlcN, closed triangle). Values were compared
between − GlcN and Sham or +GlcN. **p b 0.01, ***p b 0.001.
osteoarthritis (McAlindon et al. 2000; Reginster et al. 2001; Pavelká
et al. 2002). In this context, biochemical and pharmacological studies
have suggested in vitro that glucosamine possibly exhibit chondro-
protective action by inhibiting the degradation and stimulating the
synthesis of proteoglycans (Fenton et al. 2000; Gouze et al. 2001). In
addition, glucosamine is expected to exert an anti-inflammatory
action, based on the findings that glucosamine suppresses the
activation of inflammatory cells (such as neutrophils, cytotoxic
T-lymphocytes and dendritic cells), chondrocytes and synoviocytes
in vitro (Hua et al. 2002, 2007; Forchhammer et al. 2003; Nakamura
et al. 2004). In fact, glucosamine has been shown to exhibit preventive
actions on rheumatoid arthritis in humans as well as adjuvant arthritis
in rats (Hua et al. 2005; Nakamura et al. 2007).
Currently, many researchers are trying to evaluate OA using
biomarkers. Markers under study are basically the constituents of
cartilage, such as aggrecan, chondroitin sulfate and collagens
(Rousseau and Delmas 2007). Type II collagen is a major constituent
of articular cartilage, representing 90–95% of a total collagen content
and forming the fibrillar structure that give cartilage its tensile
strength (Garnero et al. 2000). Among several biomarkers reported
(Rousseau and Delmas 2007), components of type II collagen are
recognized as the most important biomarkers for OA (Garnero et al.
2001), since type II collagen is specifically localized in cartilage, and
OA essentially involves catabolism and anabolism of articular type II
collagen.
Usually 1.5 g/day of glucosamine sulfate or hydrochloride (ap-
proximately 25 mg/kg) is administered to humans for treatment of
osteoarthritis (McAlindon et al. 2000; Reginster et al. 2001; Pavelká
et al. 2002.), and serum glucosamine level reaches 0.020 ± 0.006 mM,
as measured by a high performance liquid chromatography (Hua et al.
2004). In contrast, because of a poor bioavailability due to a low
absorption and/or substantial loss in the gastrointestinal tract, a high
dose of glucosamine hydrochloride (350 mg/kg) was orally adminis-
tered to rats to obtain the serum level of glucosamine (0.05 mM),
which was almost the same as that in humans (Aghazadeh-Habashi
et al. 2002). In this study, to attain an enough effect of glucosamine, an
average of 1000 mg glucosamine hydrochloride/kg/day was orally
administered to rats, and the effect of glucosamine on the cartilage
was evaluated by using biomarkers for type II collagen (a type II
collagen-degradation marker CTX-II and a type II collagen-synthesis
marker CPII) (Rousseau and Delmas 2007) in an experimental OA
model. Furthermore, we assessed the bone metabolism using CTX-I
(a degradation maker for type I collagen, a major component of bone
collagen)(Rousseau and Delmas 2007), since subchondral bone
remodeling is developed during the progression of OA (Radin and
Rose 1986). Thus, this is the first study to investigate the effects of
glucosamine on OA by evaluating the cartilage changes using
biochemical markers (CTX-II, CPII and CTX-I) as well as histological
criteria (Mankin score and toluidine blue staining).
The present results revealed that CTX-II level was substantially
elevated by ALCT compared to that of Sham group. Notably,
glucosamine administration suppressed the increase of CTX-II after
the operation and lowered the level to that of Sham; CTX-II levels
were not significantly different between + GlcN and Sham groups
(Fig. 3A). These observations suggest that glucosamine administra-
tion suppresses the degradation of type II collagen during the
progression of OA. This could be supported by the findings that
glucosamine inhibits the production of MMP-13, one of the type II
collagen-degrading enzymes, from chondrocytes and synoviocytes in
vitro (Nakamura et al. 2004; Derfoul et al. 2007). Of note, the serum
levels of CPII (a maker for type II collagen synthesis) were
significantly increased in + GlcN group (Fig. 3B), suggesting that
the synthesis of type II collagen in cartilage is enhanced by glucos-
amine administration. Consistent with this, glucosamine has been
shown to augment the expression of type II collagen in chondrocytes
in vitro (Derfoul et al. 2007). In addition to the effects on type II
collagen, the present study revealed that glucosamine administra-
tion restored the ACLT-induced reduction of proteoglycans (assessed
by toluidiine blue staining) and histopathological changes (assessed
by Mankin score) in the articular cartilage (Fig. 2). Of note, glucos-
amine reportedly inhibits the degradation and stimulates the
synthesis of proteoglycans in vitro (Fenton et al. 2000; Gouze et al.
2001). Based on these observations, glucosamine is estimated to
exhibit chondroprotective action on an ACLT-induced OA model by
normalizing proteoglycan metabolism as well as suppressing type II
collagen degradation and possibly enhancing type II collagen syn-
thesis in the cartilage.
In contrast, the levels of CTX-I (a degradation marker for type I
collagen in the bone) were not significantly altered by ACLT in spite the
absence or presence of glucosamine administration (Fig. 3C), although
type I collagen degradation markers such as NTx are reported to be
 K. Naito et al. / Life Sciences 86 (2010) 538–543
increased in human OA (Bettica et al. 2002). Consistent with no
significant changes in the CTX-I levels, alterations in subchondral bone
such as thickness and cell proliferation could not be detected in the
histopathological specimens of our OA model (data not shown).
Conclusion
In summary, the present study performed with an ACLT-induced
rat OA model has suggested that glucosamine has a therapeutic
potential to exert a chondroprotective action on OA by not only
maintaining proteoglycans but also inhibiting type II collagen
degradation and enhancing type II collagen synthesis in the articular
cartilage. However, the detailed mechanism for the protective action
of glucosamine on human OA remains to be elucidated in the future.
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgments
The authors thank Yuko Kojima, Katsumi Miyahara and Yasuko Toi
(Division of Biomedical Imaging Research, Juntendo University
Graduate School of Medicine) for technical expertise. This study was
partially supported by a High Technology Research Center Grant from
the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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