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Effect of oral glucosamine on cartilage degradation in a rabbit model of osteoarthritis.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 4, April 2005, pp 1118–1128
DOI 10.1002/art.20951
© 2005, American College of Rheumatology
Effect of Oral Glucosamine on Cartilage Degradation in
a Rabbit Model of Osteoarthritis
Gabrielle Tiraloche,1 Christiane Girard,1 Luc Chouinard,2 John Sampalis,3 Luc Moquin,1
Mirela Ionescu,4 Agnes Reiner,4 A. Robin Poole,4 and Sheila Laverty1
Objective. To determine whether oral glucosamine alleviates cartilage degradation in an animal
model of osteoarthritis (OA).
Methods. The effect of 8 weeks of daily oral
glucosamine hydrochloride on degeneration of articular
cartilage was evaluated in rabbits in which anterior
cruciate ligament transection (ACLT) was performed to
induce OA. Animals were treated with glucosamine (n ⴝ
16) or a placebo (n ⴝ 16) and necropsied at 11 weeks.
Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and
glycosaminoglycan (GAG) content.
Results. Histologic analysis revealed that loss of
proteoglycan, based on Safranin O–fast green staining,
was significantly reduced in the lateral tibial plateau
cartilage of ACL-transected limbs in the glucosamine
group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for
the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial
plateau alone had a significantly lower rate of disease in
the glucosamine group, which was consistent with the
results of the independent histologic assessment. However, no significant treatment effect was detected when
composite histologic scores were analyzed. A significant
reduction in GAG content was observed in the femoral
condyles of placebo-treated ACL-transected joints, but
not in the same region of glucosamine-treated ACLtransected joints, compared with their respective contralateral unoperated joints.
Conclusion. Oral administration of glucosamine
had a detectable, site-specific, partial disease-modifying
effect in this model of OA. From a clinical perspective,
the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all
of the treated animals, and no effects were detected in
the medial joint compartments.
Ms Tiraloche’s work was supported by the Canadian Arthritis
Network and Clintrials Bioresearch. Dr. Poole’s work was supported
by the Shriners Hospitals for Children, the NIH (National Institute on
Aging), the Canadian Institute for Health Research, and the Canadian
Arthritis Network. Dr. Laverty’s work was supported by the Groupe de
Recherche en Médecine Équine du Québec, the Fond du Centenaire,
the Faculté de Médecine Vétérinaire of the Université de Montréal,
and the Canadian Arthritis Network.
1
Gabrielle Tiraloche, Christiane Girard, DMV, MSc, Dipl
ACVP, Luc Moquin, Sheila Laverty, MVB, Dipl ACVS, Dipl ECVS:
Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Quebec, Canada; 2Luc Chouinard, DMV, Dipl ACVP: Clintrials Bioresearch, Senneville, Quebec, Canada; 3John Sampalis, MSc,
PhD: JSS Medical Research, Montreal, Quebec, Canada; 4Mirela
Ionescu, MSc, Agnes Reiner, MSc, A. Robin Poole, PhD: Shriners
Hospitals for Children, McGill University, Montreal, Quebec, Canada.
Dr. Poole has received consulting fees of more than $10,000
per year from Ibex Technologies, Montreal, Quebec, Canada.
Address correspondence and reprint requests to Sheila
Laverty, MVB, Dipl ACVS, Dipl ECVS, Département des Sciences
Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal,
C. P. 5000, St. Hyacinthe, Quebec J2S 7C6, Canada. E-mail: sheila.
laverty@umontreal.ca.
Submitted for publication May 13, 2004; accepted in revised
form December 21, 2004.
In addition to decreasing pain, an ideal therapeutic agent for osteoarthritis (OA) would also prevent or
retard the progression of established OA by reducing or,
preferably, reversing the underlying pathologic processes (structure modifying), resulting in the retention or
restoration of more normal articular cartilage function.
The most common pharmacologic therapeutic agents
currently used for OA are primarily palliative and
include acetaminophen, nonsteroidal antiinflammatory
drugs, corticosteroids, and hyaluronic acid (1,2). None of
these drugs for OA are disease modifying.
Analyses of many randomized clinical trials of
oral glucosamine in OA patients support the contention
that glucosamine is a symptom-modifying agent in OA
(3–5), while investigators in many other trials have
1118
ORAL GLUCOSAMINE IN RABBIT OA
detected no effect (6–8). It remains a subject of controversy whether glucosamine modifies cartilage structure
in OA. Reports of recent clinical trials and metaanalyses concluded that, based on joint space narrowing,
oral glucosamine slowed the radiographic progression
of OA. This suggests that glucosamine has a structuremodifying effect (5,9,10). Although it remains the
current method of reference (11), the validity of the
use of radiographic assessment of joint space width
as a measure of disease modification has been questioned (12).
The apparent discrepancy between the various
clinical trial results could, in part, be explained by the
use of different formulations of glucosamine. The majority of the clinical trials have evaluated glucosamine
sulfate, which is a prescription medicine in Europe.
However, in the US and Canada, glucosamine is considered a dietary supplement (13), and content and
purity have been shown to vary markedly between
preparations (14,15).
Animal models of OA offer an important opportunity to resolve the current controversy as to whether
oral glucosamine administration has a structuremodifying effect in OA at the currently recommended
standard dose. To date, there have been few reports of
studies in animal models of OA examining this crucial
question. In an instability model in rabbits, using a
modification of the technique of Hulth et al (16),
glucosamine administration (at 20 times the equivalent
standard dose) had no identifiable effects on histologic
parameters used to evaluate the severity of OA (17).
This model of OA was severe, in that both cruciate
ligaments were sectioned and the medial meniscus was
removed. It is possible that the model was too severe
(18) for a drug effect to be identified. A more recent
study of a model of articular insult in the rabbit using
chymopapain, which induces proteoglycan loss, revealed
that glucosamine increased the glycosaminoglycan
(GAG) content in the injured knees compared with that
in controls (19).
One of the most widely used experimental models of OA is the anterior cruciate ligament transection
(ACLT) model. The use of experimental ACLT models
is of particular clinical relevance, since rupture of the
ACL occurs in humans and also leads to the development of OA (20). Rabbit ACLT is increasingly being
used in OA studies (21–23) because disease onset is
rapid. Furthermore, cartilage-specific analyses that measure proteoglycan content and the degradation of type II
collagen, an important structural macromolecule of the
matrix, are now available (24) and provide important
1119
additional quantitative information about matrix degradative events in animal models (25).
The objective of our study was to determine
whether oral administration of a clinically relevant dose
of glucosamine had a detectable structure-modifying or
metabolic effect on articular cartilage in a standard
rabbit ACLT model of OA. To this end, we comprehensively evaluated combined macroscopic, histologic, and
compositional parameters relating to cartilage damage,
GAG content, and type II collagen degradation.
MATERIALS AND METHODS
Animals. We used 39 skeletally mature (9-month-old)
male NZW rabbits (Charles River, St. Constant, Quebec,
Canada) weighing a mean ⫾ SD of 3.69 ⫾ 1.4 kg. Radiographs
of both femorotibial joints were taken to exclude animals with
joint pathology. Thirty-two rabbits underwent unilateral ACLT
under general anesthesia. The experimental animals were
randomly assigned to 3 groups. Rabbits in the glucosamine
group (n ⫽ 16) were administered 100 mg glucosamine hydrochloride (Sigma, St. Louis, MO) in a 5-gm wafer (Bio-serv,
Frenchtown, NJ) daily for a period of 8 weeks starting 3 weeks
after surgery. These doses are close to the recommended daily
dose for humans with OA (⬃20 mg/kg). The wafers were given
in the morning before the rabbits were fed. Rabbits in the
placebo group (n ⫽ 16) were administered the same wafer
without glucosamine. There were 7 unoperated and untreated
control rabbits (control group). All experiments were conducted with the approval of the Institutional Animal Care and
Use Committee.
ACLT surgery. An analgesic (buprenorphine hydrochloride, 0.03 mg/kg injected intramuscularly every 6 hours)
was administered preoperatively and for 3 days following
surgery. Antibiotics (sulfadiazine and trimethoprim [24%], 15
mg/kg injected subcutaneously twice a day) were also administered preoperatively and for 2 days postoperatively. Anesthesia for the surgical procedure was maintained with a mixture of
isoflurane (Technilab, Mirabel, Quebec, Canada) in oxygen.
The limb was clipped and prepared for surgery in a standard
manner. A medial arthrotomy was performed on the left
femoropatellar joint to permit transection of the ACL. Routine skin incision closure was performed. Tissues were harvested following euthanasia, 11 weeks after surgery.
Macroscopic cartilage assessment. All femorotibial
joint compartments (medial femoral condyle [MFC], lateral
femoral condyle [LFC], medial tibial plateau [MTP], and
lateral tibial plateau [LTP]) in all animals were examined for
gross morphologic changes of the articular cartilage and
immediately graded, following application of India ink, as
described previously (26). Briefly, compartments were graded
from 1 to 4 (grade 1 ⫽ no uptake of India ink, indicating an
intact surface; grade 2 ⫽ minimal focal uptake of India ink,
indicating mild surface irregularity; grade 3 ⫽ evident large
focal dark patches of ink uptake, showing overt fibrillation;
grade 4a ⫽ erosion of cartilage [whiter area demonstrating
visible bone] ⱕ2 mm; grade 4b ⫽ erosion of cartilage ⬎2 mm
but ⱕ5 mm; and grade 4c ⫽ erosion of cartilage ⬎5 mm). The
1120
examination was performed by 2 independent observers
(SL and GT) who were blinded to the treatment groups. The
lesions were documented digitally using a D1 camera (Nikon,
Tokyo, Japan).
Histologic assessment of cartilage. Histologic assessment was performed on the femorotibial joints of rabbits in the
glucosamine (n ⫽ 8), placebo (n ⫽ 8), and control (n ⫽ 7)
groups. The femoral and tibial articular surfaces were fixed in
10% neutral buffered formalin. Tissue blocks were decalcified with 14% EDTA (Fisher Scientific, Nepean, Ontario,
Canada) in 10 mM phosphate buffer (pH 7.4), dehydrated
through graded alcohols, cleared with toluol, and embedded
in paraffin. Six-micrometer sections were cut at a standard
site centrally in the MFC, LFC, MTP, and LTP. The sections
were stained with Safranin O–fast green and hematoxylin–
eosin–saffron. Two histologic sections from each site were
evaluated using a modified Mankin grading system (27–29)
(Table 1) by 2 independent, board-certified veterinary
pathologists (CG and LC) who were blinded to the treatment
groups.
Cartilage markers of type II collagen degradation
(Col2-3/4C and Col2-3/4m neoepitopes) and type II collagen
content. Full-depth articular cartilage was removed by careful
dissection with a scalpel from the tibial plateau and femoral
condyles (either operated or unoperated), avoiding the periarticular margins and osteophytes, and was analyzed for each
of the rabbits in the glucosamine (n ⫽ 8), placebo (n ⫽ 8), and
control (n ⫽ 7) groups. Wet weights were determined and
recorded for each region after blotting to remove any excess
moisture.
For solubilization, cartilage was incubated overnight
at 37°C, first with ␣-chymotrypsin and then with proteinase K
as described previously (30,31). The samples were assayed for
both the Col2-3/4C neoepitope (using the ␣-chymotrypsin
extract) (30) and the Col2-3/4m hidden epitope (using the
␣-chymotrypsin and proteinase K extracts) (31) to determine
denatured and total type II collagen content. The Col2-3/4C
neoepitope assay was a modification of the reported assay (30).
The peptide CGPP(OH)GPQG (the Col2-3/4C neoepitope
generated by collagenase) was conjugated to ovalbumin, and a
polyclonal antibody was prepared as described (30). Binding of
the biotinylated antibody was detected using dissociationenhanced lanthanide fluoroimmunoassay (DELFIA)
europium-labeled streptavidin (EG&G Wallac, Turku, Finland) diluted 1:1,000 in DELFIA assay buffer (EG&G Wallac). Fluorescence at an excitation of 340 nm and with an
emission at 615 nm was measured by a 1412 plate reader
(EG&G Wallac). A commercial assay for the Col2-3/4C neoepitope, called the C1,2C assay, is available from Ibex Technologies (Montreal, Quebec, Canada). Total type II collagen
content of each sample was determined from the total amount
of Col2-3/4m epitope in the ␣-chymotrypsin extract and in the
proteinase K digest (31). The amount of denatured collagen
(Col2-3/4m epitope) found in the ␣-chymotrypsin digest was
recorded.
Cartilage GAG analysis. The ␣-chymotrypsin and proteinase K cartilage digests were assayed to determine total
GAG content (primarily a measure of the proteoglycan aggrecan content) using a modification of the colorimetric dimethylmethylene blue dye assay (32). All results were expressed per mg wet weight of cartilage.
TIRALOCHE ET AL
Table 1.
Criteria (grading) for histologic evaluation
Safranin O–fast green staining
0 ⫽ uniform staining throughout articular cartilage
1 ⫽ loss of staining in the superficial zone for less than one-half
of the length of the condyle or plateau
2 ⫽ loss of staining in the superficial zone for one-half or more of
the length of the condyle or plateau
3 ⫽ loss of staining in the superficial and middle zones for less
than one-half of the length of the condyle or plateau
4 ⫽ loss of staining in the superficial and middle zones for onehalf or more of the length of the condyle or plateau
5 ⫽ loss of staining in all 3 zones for less than one-half of the
length of the condyle or plateau
6 ⫽ loss of staining in all 3 zones for one-half or more of the
length of the condyle or plateau
Chondrocyte loss
0 ⫽ no decrease in cells
1 ⫽ minimal decrease in cells
2 ⫽ moderate decrease in cells
3 ⫽ marked decrease in cells
4 ⫽ very extensive decrease in cells
Structure
0 ⫽ normal
1 ⫽ surface irregularities
2 ⫽ 1–3 superficial clefts
3 ⫽ ⬎3 superficial clefts
4 ⫽ 1–3 clefts extending into the middle zone
5 ⫽ ⬎3 clefts extending into the middle zone
6 ⫽ 1–3 clefts extending into the deep zone
7 ⫽ ⬎3 clefts extending into the deep zone
8 ⫽ clefts extending to calcified cartilage
Clone formation
0 ⫽ normal
1 ⫽ minimal (ⱕ4)
2 ⫽ moderate (⬎4 but ⱕ8)
3 ⫽ marked (⬎8)
Tidemark integrity
0 ⫽ intact
1 ⫽ crossed by blood vessels
Statistical analysis. Macroscopic grades. Since the
macroscopic grades were not continuous, the chi-square test
was used to compare the glucosamine and placebo groups. In
addition, the 6 grades were divided into 2 categories for
statistical analysis (diseased joints [evident fibrillation or erosion] with grades 3 or 4 and nondiseased joints with grades 1 or
2), since the 6 grades of classification defined disease and
normal states. The odds ratio was used to estimate the relative
risk for having a diseased joint in the placebo group compared
with the glucosamine group.
Histologic scores. Student’s t-test was used to assess
differences between the glucosamine and placebo groups with
respect to histologic parameters (Safranin O–fast green staining, structure, chondrocyte loss, chondrocyte clusters, tidemark
integrity) of the operated limbs. These analyses were done for
the total joint and for each individual compartment (MFC,
LFC, MTP, LTP) separately, for each of the histologic parameters. When a statistically significant difference between the
2 groups was detected, analysis of variance (ANOVA) was
used to adjust for the confounding effects of animal and
pathologist. Spearman’s rank correlation coefficient was used
to evaluate the association between macroscopic grades and
ORAL GLUCOSAMINE IN RABBIT OA
1121
Figure 1. Macroscopic grades of the articular surfaces of the femoral condyles and tibial plateaus. Four compartments were graded (medial femoral
condyle [MFC], lateral femoral condyle [LFC], medial tibial plateau [MTP], and lateral tibial plateau [LTP]). Data are presented as the mean
percentage distribution of grades. Grades 1 and 2 represent normal cartilage with no or minor focal uptake, respectively, of India ink; grades 3 and
4 represent overt fibrillation and erosion, respectively (see Materials and Methods). Control ⫽ joints from unoperated untreated animals; placebo
ACLT ⫽ anterior cruciate ligament–transected joints from placebo-treated animals; placebo unop ⫽ contralateral unoperated control joints from
placebo-treated animals; GlcN ACLT ⫽ ACL-transected joints from glucosamine-treated animals; GlcN unop ⫽ contralateral unoperated control
joints from glucosamine-treated animals.
histologic scores for each of the different joint compartments.
Interobserver agreement between the 2 pathologists (CG and
LC) was measured with an interclass correlation coefficient.
Cartilage type II collagen and GAG analyses. A KruskalWallis test was used to evaluate differences in cartilage wet
weight, in results of type II collagen analyses, and in GAG
levels among control unoperated, glucosamine-treated ACLtransected, placebo-treated ACL-transected, and contralateral
unoperated TP and FC regions. When significant differences
occurred among treatments, post hoc tests with adjusted P values
were used to determine which pairs of treatments actually differed. Differences between glucosamine- and placebo-treated
ACL-transected regions and their respective contralateral unoperated control regions were examined with the Wilcoxon signed
rank test. The 5% significance level was used.
RESULTS
Experimental animals. The rabbits rapidly ingested the wafers, usually within 5 minutes of administration, thus effectively mimicking a bolus dose of glucosamine. Postmortem inspection revealed that ACLT
was complete in all rabbits included in the study. Two
animals with some persisting ACL fibers were excluded
from the study. These had been assigned to the biochemical analysis section of the glucosamine group. Therefore, 6 animals remained for biochemical analysis of
articular cartilage.
Effect of glucosamine on macroscopic parameters. In the unoperated right femorotibial joints (n ⫽
32) and control rabbit joints (n ⫽ 7), the articular
Table 2. Proportion of animals with disease (cartilage fibrillation or
erosion) on macroscopic evaluation using India ink*
Group
Compartment
Glucosamine
(n ⫽ 14)
Placebo
(n ⫽ 16)
OR
P†
MFC
LFC
MTP
LTP
43
43
21
14
56
56
19
44
0.42
0.42
1.18
0.21
0.450
0.450
0.780
0.046
* Values are percentages of animals with disease in a given compartment. OR ⫽ odds ratio; MFC ⫽ medial femoral condyle; LFC ⫽
lateral femoral condyle; MTP ⫽ medial tibial plateau; LTP ⫽ lateral
tibial plateau.
† By chi-square test.
1122
TIRALOCHE ET AL
Figure 2. Representative Safranin O–fast green–stained histologic sections illustrating cartilage lesions. a, Normal articular cartilage with intact
surface stained with Safranin O–fast green. b, Superficial diffuse loss of Safranin O–fast green staining, with clusters of cells (arrowhead) present
in the superficial layer and surface irregularity and fibrillation. c, Severe loss of Safranin O–fast green staining and loss of cartilage, with cleft
extending to the middle zone (arrowhead). d, Erosion of cartilage matrix (solid arrowhead) to calcified cartilage (open arrowheads). (Original
magnification ⫻ 40.)
cartilage macroscopic grades were 1 (normal) or 2
(scarcely perceptible ink uptake), with the exception of 1
animal in each group that had grade 3 lesions (overt
fibrillation with distinct focal patches of ink uptake). No
cartilage erosions (grade 4) were observed in any of the
joint compartments (results not shown). The unoperated
control group was included for comparative purposes to
demonstrate that the ACLT surgery performed by us
induced articular cartilage changes.
The induced OA lesions (fibrillation and erosion
of articular cartilage) were focal and occurred at the
same sites in all ACL-transected joints. The glucosamine
group (n ⫽ 14) tended to have a lower severity grade of
macroscopic articular cartilage lesions in all 4 joint
compartments compared with the placebo group (n ⫽
16) (Figure 1), but this difference was not significant.
The macroscopic grade data, categorized as disease
(overt fibrillation and cartilage erosion) or nondisease,
ORAL GLUCOSAMINE IN RABBIT OA
1123
Figure 3. Histologic scores at 11 weeks. Shown are scores for the histologic parameters with comparisons between groups. Values are the mean and
SD. Histologic analysis revealed that loss of proteoglycan, based on Safranin O–fast green staining, was significantly reduced in the LTP cartilage
of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant,
trend for the LFC cartilage. See Figure 1 for definitions.
showed 44% of the placebo group with disease in the
LTP compartment, compared with 14% in the glucosamine group (P ⫽ 0.046). This indicated a statistically
significantly lower rate of disease for the glucosamine
group compared with the placebo group at this site
(Table 2).
Effect of glucosamine on histologic parameters.
Representative histologic sections are shown in Figure 2.
We observed a normal integrity of the articular surface.
This was lost with early mild fibrillation, and a loss of
Safranin O–fast green staining was observed along with
chondrocyte clustering. With increased degeneration,
there was a loss of Safranin O–fast green staining
accompanying cleft formation that extended into the
middle and deep zones. In severe cases, calcified cartilage sometimes remained, but often, there was eburnation of bone.
Unoperated limbs (including contralateral unoperated joints in glucosamine and placebo groups [n ⫽ 8
in each group] and joints of control unoperated rabbits
[n ⫽ 8]) exhibited some mild degenerative histologic
changes, such as changes in Safranin O–fast green
staining, changes in structure, or chondrocyte loss, in the
different joint compartments. These were most evident
in the MTP compartment (Figure 3). Interestingly,
ACLT did not change the histologic score for these
parameters at the MTP site to the extent seen in the
other compartments.
There was a significantly lower mean score for
Safranin O–fast green staining in the LTP compartment
of the ACL-transected limbs in the glucosamine group
compared with the ACL-transected limbs in the placebo
group (P ⫽ 0.049). There was a trend (P ⫽ 0.11) toward
lower Safranin O–fast green staining scores in the LFC
1124
TIRALOCHE ET AL
Figure 4. Biochemical analysis of cartilage from the femoral condyles (FC) and tibial plateaus (TP). Shown are concentrations of glycosaminoglycan
(GAG), type II collagen, collagenase-cleaved type II collagen, and denatured type II collagen. Values are the mean and SD. A significant loss of
GAG occurred in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected
joints, compared with their respective contralateral unoperated joints. No other significant changes were detected. See Figure 1 for other definitions.
compartment of the ACL-transected limbs in the glucosamine group. There were no statistically significant
differences in the Safranin O–fast green staining scores
in the MTP and MFC compartments between ACLtransected limbs in the glucosamine group and those in
the placebo group. No significant differences in structural scores, chondrocyte loss, or amount of chondrocyte
clusters were detected between the glucosamine and
placebo groups in any of the ACL-transected joint
compartments (Figure 3). Chondrocyte cluster formation (cloning) was only seen in ACL-transected joints,
never in unoperated joints. Alterations in the tidemark
integrity were not observed in this study. When all
histologic parameters were combined from each site to
obtain a total joint score, no statistically significant
differences were detected between ACL-transected
joints in the glucosamine group and those in the placebo
group.
Results of the multivariate ANOVA with treatment and pathologist entered as covariates indicated
that the largest component of observed variation in
histologic measurements was due to treatment, followed
by variation due to the pathologist, indicating a high
interrater agreement. There was no significant variation
attributed to animal effect.
Statistical correlations. In ACL-transected joints
in both the glucosamine and placebo groups, a high
correlation was observed between the macroscopic
grades and the histologic scores in the MFC (rs ⫽ 0.9,
P ⫽ 0.006 for the glucosamine group; rs ⫽ 0.9, P ⫽
0.0001 for the placebo group), LFC (rs ⫽ 0.9, P ⫽ 0.001
for the glucosamine group; rs ⫽ 0.9, P ⫽ 0.007 for the
ORAL GLUCOSAMINE IN RABBIT OA
placebo group), and LTP (rs ⫽ 0.7, P ⫽ 0.04 for the
glucosamine group; rs ⫽ 0.8, P ⫽ 0.03 for the placebo
group) compartments. In the MTP compartment, the
values were rs ⫽ 0.5, P ⫽ 0.19 for the glucosamine group
and rs ⫽ ⫺0.1, P ⫽ 0.8 for the placebo group.
Effect of glucosamine on cartilage type II collagen and proteoglycan. There were significant effects of
experimental group on the amount of cartilage harvested from the femoral condyles (P ⫽ 0.003) and tibial
plateaus (P ⫽ 0.044). Post hoc tests revealed that
significantly more cartilage was harvested from the
femoral condyles of ACL-transected joints in both the
glucosamine and placebo groups than from the femoral
condyles of control unoperated joints (data not shown).
Similar changes were observed in the tibial plateau, with
the exception that no significant differences were detected between glucosamine-treated ACL-transected
joints and control unoperated joints. In addition, significantly more cartilage was harvested from the femoral
condyles (P ⫽ 0.008) and the tibial plateaus (P ⫽ 0.023)
of placebo-treated ACL-transected joints than from the
corresponding regions of their contralateral unoperated
joints. This was not observed in the glucosamine group.
No significant differences in type II collagen content, denaturation, or cleavage were detected in the biochemical analysis of cartilage in any of the regions (TP or
FC) among the glucosamine (n ⫽ 6), placebo (n ⫽ 8), or
control (n ⫽ 7) groups (Figure 4). In contrast, a significant
reduction (P ⫽ 0.02) in GAG content was observed in the
femoral condyles of placebo-treated ACL-transected
joints, but not in the same region of glucosamine-treated
ACL-transected joints, compared with their respective
contralateral unoperated joints (Figure 4).
The intraassay variation for all of the assays for
cartilage analysis ranged from 5% to 10%. The interassay variation ranged from 7% to 15%.
DISCUSSION
This study provides in vivo experimental evidence
indicating that the oral administration of glucosamine
hydrochloride can alter the degeneration of articular
cartilage in OA. Safranin O–fast green staining revealed
that loss of proteoglycan can be prevented in the lateral
compartment within the operated joint. It is important
to note that the histologic sections were scored by 2
independent, board-certified veterinary pathologists
who were blinded to the treatment groups. The interrater agreement between the 2 pathologists was high,
suggesting that the modified Mankin assessment we
1125
used was reliable and provided consistency in grading
the changes observed on the histologic slides.
Interestingly, when macroscopic findings, reflective of the extent of articular cartilage surface damage
and severity of lesions, were evaluated by 2 other
independent assessors and statistically analyzed as dichotomous variables (disease or nondisease), there was
also significantly less disease in the LTP compartment in
the glucosamine group, which was consistent with the
histologic assessment. However, it is also important to
note from a clinically relevant perspective that, based on
both macroscopic and histologic analysis, the administration of glucosamine did not prevent fibrillation and/or
erosions of the articular cartilage in this model, although
there was an overall trend toward a reduction in severity
of the disease in the operated joints of the glucosamine
group. Furthermore, statistical analysis of the histologic
results revealed that any variation we observed was
primarily due to treatment effects. Taken together, the
results indicate that glucosamine had a positive effect on
proteoglycan retention in the lateral compartment of the
ACL-transected joint (statistically significant in the LTP
compartment and a trend in the LFC compartment).
However, no effects were detected in the MFC and MTP
compartments of the joint.
A separate group of animals was analyzed independently for biochemical changes in cartilage GAG
content and type II collagen degradation. In this case,
for reasons related to tissue requirements for the analyses, we were unable to examine lateral and medial
compartments separately. In spite of this, we found that
glucosamine treatment prevented the loss of proteoglycan observed in the femoral condylar cartilage of the
placebo group compared with unoperated contralateral
joint cartilage from the same region. No significant
changes were detected for the collagen parameters.
Since OA lesions are usually focal, there would have
been less lesional cartilage relative to distant nonlesional
cartilage, and effects of treatment on the lesion could
have been missed. This effect of cartilage sampling has
also been highlighted by others (19). We are confident,
however, that when a significant effect was detected, it
was relevant, since these quantitative analyses involved a
small series of animals in each group.
We unexpectedly harvested a greater amount of
cartilage from the femoral condyles of the ACLtransected limbs (in both the glucosamine and placebo
groups) compared with the unoperated limbs. Intuitively, we would have expected less cartilage in the
ACL-transected limbs because of erosion of the cartilage, particularly at the femoral condyles. It is possible
1126
that this increased yield could reflect the recognized
hypertrophic response of cartilage in OA (33). However,
this finding could also be due to a sampling bias. The
operator may have unknowingly shaved cartilage from
these regions more painstakingly because lesions were
present. Our biochemical results were expressed per mg
wet weight to avoid this confounding issue.
The observed site-specific effects of glucosamine
could be explained by differences in disease development and/or rapidity of progression in the different
joint compartments. Regional differences in the development of OA lesions in the femorotibial joint in the
ACLT rabbit model have already been highlighted by
others (34).
Discrepancies between results of our study and
those of Lippiello et al (17), who reported that 20 times
the typical oral dose of glucosamine had no chondroprotective effect in a rabbit model of OA, may be
explained by differences in the models examined. First,
only the cranial cruciate ligament was transected in the
present study, whereas both cruciate ligaments and the
medial meniscus were removed in the study by Lippiello
et al, which would have led to greater instability and
more rapid progression of disease. Second, the femoral
condyles alone were evaluated by Lippiello et al,
whereas the femorotibial sites were also evaluated histologically in our study. Third, we administered glucosamine in a wafer, which was rapidly consumed so as
to simulate bolus dosing, whereas Lippiello et al added
glucosamine to the feed, which the animals eat progressively throughout the day, and this would have resulted
in different doses and different peak serum glucosamine
concentrations. Taken together, it is easy to understand
that divergent results could have been obtained with
these different studies, and this highlights the difficulties
of comparing compound efficacy results in studies using
different animal models of OA conducted with different
dosing regimens.
Oral glucosamine, although at a higher dose, has
been reported to restore GAG in the damaged cartilage
in a chymopapain-injected rabbit model of joint injury,
and the investigators in that study proposed that it may
have been effective because it represented an exogenous
source of glucosamine for GAG synthesis in a tissue
undergoing active repair (19). Interestingly, the data in
the present study revealed no changes in collagen content or turnover following ACLT and no effects of
glucosamine. Similarly, in the chymopapain-injected
rabbit model, Oegema et al (19) reported no significant
changes in collagen content and type II collagen messenger RNA with glucosamine therapy.
TIRALOCHE ET AL
Although an extensive body of in vitro studies to
elucidate the effects of glucosamine on articular cartilage has been published (35–46), the majority of these
studies have identified effects of the monosaccharide at
higher concentrations than those recently reported to be
attainable in vivo in the synovial fluid (0.3–0.7 ␮M)
following the administration of a clinically relevant dose
(20 mg/kg) in an equine model (47). Therefore, to
conclude that these in vitro effects occur in vivo may be
erroneous, since cartilage would not be exposed to these
high levels. It has been proposed that glucosamine may
be concentrated in articular cartilage and that this could
explain its beneficial effects (48), but concrete evidence
for this is currently not available.
Components of the hexosamine pathway (glucosamine or biosynthetic derivatives) have been shown
to be capable of modulating several genes in different
tissues (49–51), and glucosamine effects in articular
tissues could also be induced by the regulation of various
genes. It is also possible that the observed in vivo effects
may be due to an influence of glucosamine on other
tissues that subsequently release factors that modify the
chondrocyte response secondarily (19). Support for the
concept of an indirect effect of glucosamine administration on cartilage is provided by the recent finding that it
affects subchondral bone remodeling in rabbit OA (52).
A weakness of the ACLT model is that the
persistent joint instability may counteract attempts at
cartilage repair, and a drug with less potency may not
have detectable effects. As a result, pathology in the
ACLT model may develop rapidly compared with OA in
humans, the progression of which is usually slow and
may take place over a period of 15–30 years.
A strength of this study is that we elected to use
glucosamine hydrochloride from a chemical company to
avoid any commercial bias, which has been a concern in
previous studies of glucosamine (53). We also based the
oral dose on a typical human dose described in the
literature (53), and we chose to avoid the use of glucosamine sulfate, since sulfation may influence the outcome of therapy, being another building block of proteoglycans (54).
Taken together, these results indicate that glucosamine hydrochloride can produce partial disease
modification at specific sites in a classic model of
articular trauma–induced OA at a dosage relevant to
that used in humans. Ideally, a disease/structuremodifying drug should prevent or alleviate articular
cartilage degeneration generally throughout the joint.
The administration of glucosamine did not prevent
fibrillation and/or erosions of the articular cartilage in all
ORAL GLUCOSAMINE IN RABBIT OA
of the treated animals, and no effects were detected in
the MFC or MTP compartments. It remains to be
determined whether similar changes occur in human
disease and what their clinical relevance may be.
ACKNOWLEDGMENTS
We express our thanks to Helene Richard and Guy
Beauchamp for technical assistance.
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