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Proteoglycan-degrading acid metalloprotease activity in human osteoarthritic cartilage and the effect of intraarticular steroid injections.

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Cartilage samples from both the immediate and
remote lesion areas were obtained from the tibia1 plateaus of 21 patients with osteoarthritis, and were subjected to histologic and enzymatic study. There was a
frequent loss of pericellular metachromatic staining in
the OA cartilage. Seven patients had received
intraarticular injections of steroids, and in 21% of those
cartilage samples, a pericellular halo was seen. This halo
was seen in 71% of patients who had not received steroid
injections. The total acid metalloprotease activity was
increased more than twofold in specimens from OA
lesions and in those samples graded moderate, as compared with age-matched control cartilages. These differences were greater when the specimens from patients
who had received steroid therapy were excluded from
the data. The cartilage specimens from steroid-treated
patients were not significantly different from those of
controls with respect to the enzyme activity in the lesions
or in cartilage with moderate disease. The active form of
the protease was suppressed by steroids. In samples
from patients who did not receive steroid injections and
who had a moderate grade of OA, a significantly elevated level of the active protease was present, as compared with control samples. Those samples graded
moderate which came from patients who received steroid treatments showed no difference in the active
protease level versus that of controls. Our results are
consistent with the hypothesis that acid metalloprotease
activity is involved in the degradation of the cartilage
matrix in OA. Since the protease retains a significant
fraction (40%) of its activity at neutral pH, its physiologic role might occur either at acid pH or at neutral pH.
The halo surrounding the chondrocytes (the result of a
local loss of proteoglycan) might be related to the action
of this enzyme. Preliminary evidence has suggested that
steroids reduce cartilage enzyme activity.
From the Department of Medicine, University of Montreal,
School of Medicine, Centre de Recherche, HGpital Notre-Dame,
Montreal, Quebec, Canada, and the Department of Biochemistry,
University of Miami, Miami, Florida.
Supported by the Medical Research Council of Canada, the
Canadian Arthritis Societv. and NIH erant AM-16940.
Osteoarthritis (OA) is a slowly progressive disease that is characterized mainly by degradation and,
eventually, by complete disappearance of the articular
cartilage from the joint surfaces. Histologic studies of
University of Montreal, School of Medicine, and Head, Department
of Rheumatology, Centre de Recherche, HGpital Notre-Dame; J.
Martel-Pelletier, PhD: Assistant Professor of Medicine, University
of Montreal, School of Medicine, Centre de Recherche, HBpital
Notre-Dame; J. M. Cloutier, MD: Professor of Surgery, University
of Montreal, School of Medicine, and Head, Department of
Orthopaedic Surgery, HGpital St-Luc, Montreal, Quebec, Canada;
J. F. Woessner, Jr., PhD: Professor of Biochemistry, University of
Miami, School of Medicine.
Address reprint requests to J. p. Pelletier, MD, Unit6 des
Maladies Rhumatismales, HGpital Notre-Dame, 1560 rue Sherbrooke Est, Montreal, Quebec, Canada H2L 4K8.
Submitted for publication April 28, 1986; accepted in revised form November 11, 1986.
changes is a progressive decrease in metachromatic
Staining, which indicates a loss of proteoglycan content (1). Recent reports by Sachs et a1 (2) and Goldberg
et (3) describe types Of changes in metachromatic
staining. Type A is characterized by a lack of perkellular staining, so that a halo appearance is present
around the lacunae. The remainder Of the intertenitorial matrix has norma] metachromatic staining. The
second pattern, type
represents diminished Or
sent staining in the interterritorial areas. Type C'is
Arthritis and Rheumatism, Vol. 30, No. 5 (May 1987)
found mainly in osteophytic tissues and consists of an
irregular staining of the matrix.
Because a physiologic pH is found in the
extracellular matrix, the changes described as type B
are consistent with the enzymatic digestion of proteoglycans by neutral proteases. Recently, it has been
demonstrated in our laboratory (4) that neutral metalloproteases are the enzymes most likely to be involved
in this process. Proteoglycan degradation at the
pericellular level (type A) suggests the possible involvement of acid proteases, since it has been postulated that cells might be able to produce an acid pH
environment close to the cell surface (5). Cathepsins B
and D, active at pH 6 and at pH 5, respectively, have
been found in increased concentrations within OA
cartilage (6,7). These cathepsins could be responsible
for pericellular degradation. However, Sapolsky et a1
(8) reported the presence of another protease that acts
at acid pH in human articular cartilage. This enzyme
belongs to the metalloprotease class.
Recently, Woessner and Selzer (9) and Azzo
and Woessner (10) have extracted and purified this
acid metalloprotease from human articular cartilage. It
occurs in a latent form of M , 55,000, and can be
activated by p-aminophenylmercuric acetate (APMA)
to constitute an active form of M , 35,000. The optimum digestion of the proteoglycan monomer occurs at
pH 5.3. However, at pH 7.5, the digestion is still 40%
of the maximum value, so that action at physiologic
pH is also possible. The proteoglycan is degraded to
fragments of about 140,000 daltons.
We studied the activity of this acid metalloprotease on the endogenous proteoglycans within
the cartilage. The role of the enzyme in the development of OA and its possible regulation by steroids was
also explored.
Chemicals. The chemicals used were either certified
by the American Chemical Society or were the best commercially available grade. Chondroitin sulfate and pepstatin
were purchased from Sigma (St. Louis, MO). APMA,
cetylpyridinium chloride, and carbazole were obtained from
Eastman-Kodak (Rochester, NY). Ortho-phenanthroline,
trichloroacetic acid (TCA), and guanidine hydrochloride
were purchased from Fisher Scientific (Fairlawn, NJ). We
obtained 1,9-dimethyImethylene blue from Poly Science,
Inc. (Warrington, PA). Sepharose CL-2B was purchased
from Pharmacia (Montreal, Quebec, Canada).
Selection of specimens. Twenty-one tibial plateaus
were obtained from 10 men and 1 1 women who had primary
OA. Their mean age was 68 years (range 54-81). Specimens
Figure 1. Sites of specimen selection from human osteoarthritic
knee cartilage. Tissues were excised from 2 sites on the tibial
plateau: the fibrillated or lesion zone (L) and an area remote from
that site (R).
were taken during total knee replacement surgery. The
patients were from Notre-Dame and St-Luc Hospitals in
Montreal. The diagnosis of OA was determined from the
results of clinical and radiologic evaluations (1 1).
These patients had been referred to our orthopedic
reconstructive surgery unit from different hospitals and
clinics throughout the province, and in many cases, the
original medical records were not available. We therefore
asked all patients which medications they had received,
whether they had received intraarticular injections of steroids into the affected knee, and if so, how often and the
approximate dates. At the time of hospitalization, all 21
patients were taking nonsteroidal antiinflammatory drugs.
Seven patients had received at least 1 intraarticular injection
of steroid within the year prior to surgery.
The tibial plateaus were excised, swathed in sterile
pads, soaked with cold physiologic saline, placed in a sealed
container, and carried on ice to the laboratory. The plateaus
were further washed in physiologic saline and then dissected. Tissue blocks, including the subchondral bone, were
sectioned from each of the 21 tibial plateaus at fibrillated or
lesional zones, and at sites remote from these zones (Figure
1). A portion of each block was selected for histologic
examination, and the remainder was used for the enzyme
Control cartilages were obtained from 13 subjects at
autopsy, within 12 hours of death. The controls were age
matched. Only macroscopically normal cartilage was used
for this study.
Histologic procedures. OA cartilage specimens were
fixed in 10% formaldehyde and embedded in paraffin. Sections were cut at a thickness of 6 p and double-stained with
Safranin 0 and hematoxylin and eosin. The specimens were
graded histologically, according to the scale described by
Mankin et al ( I ) (grades 1-5 = mild, grades 6-9 = moderate,
grades 1@-14 = severe). The changes in the pattern of
Safranin 0 staining were also examined, with particular
attention to the pericellular staining.
Acid metalloprotease assay. We used a modification
of our previously described assay method for neutral
proteoglycan-degrading proteases (4). Briefly, cartilage samples (150 mg wet weight) from tibia1 plateaus were homogenized in 8 volumes of acetate buffer (200 mM sodium acetate,
200 mM acetic acid, 10 mM CaCI2, 150 mM NaCI, 40 pg of
tobramycin, and 40 pg of gentamycin), pH 5.5, containing 1
p M pepstatin. Aliquots (100 pl) were put in test tubes.
Before incubation (at 37°C for 42 hours), one of the following
was added to each tube: (a) APMA (1 mM), to activate latent
enzyme; (b) APMA (1 mM) plus o-phenanthroline (5 mM), to
serve as a blank; (c) no addition, to measure the activity of
the proteases already in their active form; or (d) ophenanthroline (5 mM), to be used as a blank for group c.
After incubation, the charges were equilibrated, and
the tubes were centrifuged at 12,OOOg for 30 minutes at 4°C.
This yielded the first pellet, PI. Each supernatant (S,) was
then treated with cetylpyridinium chloride to a final concentration of 1.7%, until a flocculent precipitate formed (5
minutes at 20°C). TCA was added to a final concentration of
9%, for 30 minutes at 4°C. The suspension was then centrifuged at 12,OOOg for 10 minutes. This yielded the TCAinsoluble substrate, pellet P2, and the supernatant, Sz.
Uronic acid measurements (12) were made of the last supernatant, S2 (TCA-soluble, proteoglycan-digested products).
These measurements were also made of P I , which was
resuspended in a 1: 1 volume of concentrated H2SO4 and
distilled water, and Pz, which was solubilized in 2M KCI.
Enzymatic activity was expressed as the percentage
of total uronic acid released in supernatant S?. Total metalloprotease activity was determined according to the digestion found in tube a minus that found in tube b. Active
metalloprotease activity was determined according to the
tube c value minus that of tube d. The use of ophenanthroline blanks ensured that only metal-dependent
activity was measured.
Chromatographic procedure. Acid metalloprotease
effects on the proteoglycan macromolecule were examined.
For this experiment, we analyzed 2 separate OA cartilages.
The specimens were selected from the lesion zones of 2
patients who were not treated with steroid injections. Each
specimen was homogenized in the acetate buffer, pH 5.5,
which contained 1 p M pepstatin. The homogenate was
divided into 2 aliquots: 1 was treated with APMA (1 mM);
the other was treated with o-phenanthroline ( 5 mM). These
were incubated for 42 hours at 37°C.
After incubation, the proteoglycans were extracted
in 4M guanidine hydrochloride in a 50 mM sodium acetate
buffer, pH 5.8, which contained protease inhibitors. The
extraction continued for 24 hours at 4°C. The samples were
then centrifuged at 12,OOOg for 20 minutes at 4"C, dialyzed
against distilled water, and lyophilized. The resultant precipitate was dissolved in a 500 mM sodium acetate buffer,
pH 6.8, containing 2M guanidine hydrochloride, and the
solution was applied to a Sepharose CL-2B column (1.5 cm
x 90 cm) and eluted with the same buffer. Fractions (2 ml)
were collected. The proteoglycans were determined
spectrophotometrically by the 1,9-dimethyImethylene blue
method (13), which uses chondroitin sulfate as a standard.
Statistical analysis. Values for the mean and standard
errors of the mean were calculated and were compared by
Student's 2-tailed t-test. P values <0.05 were considered
significant. The regression line was fitted to the equation
y = mx f b, using the least squares method and assuming
Table 1. Proteoglycan-degrading acid metalloprotease activity in cartilage specimens*
Normal controls
OA patients
Histologic grade
No. of
staining, 7%
pericellular halo
Enzyme activity, 9% uronic
acid released
(mean t SEM)
3.21 t 0.57
1.20 t 0.30
7.13 -c 1.43t
5.10 t 1.02
2.94 t 0.98
3.02 t 0.81
8.14 f 2.06f
4.14 f 0.06
3.23 ? 1.40
2.48 t 0.57
* Four of the osteoarthritis (OA) specimens were not processed adequately for histologic analysis;
these were from patients who had not received intraarticular injections of steroids (I-). I + = patients
who had received at least 1 intraarticular injection of steroids within 1 year prior to surgery.
t P < 0.05 versus controls, by Student's 2-tailed r-test.
f P < 0.04 versus controls and versus OA cartilage graded mild, by Student's 2-tailed r-test.
Figure 2. Photomicrograph of the surface and middle zone of a
human osteoarthritic cartilage specimen. Cell clones of 2 or more
stained nuclei are abundant. Absence of Safranin 0 staining is noted
at the superficial layer. In the middle zone, there is minimum
pericellular staining, with a halo around the lacunae. The interterritorial matrix staining appears normal (original magnification
x 220).
errors in both x and y. The P value for the correlation
coefficient was calculated using the r test.
For each OA specimen, the results were first
grouped according to the macroscopic site (lesion or
remote) and the histologic grade. Because of the
possible effect of steroids on the metalloenzyme level
(14-16), we further divided the data according to
whether the patients had received at least 1 intraarticular injection of steroid within 1 year prior to surgery.
Histologic findings. Of the 42 specimens (21
from the center of lesions and 21 from remote sites)
selected from the surgical samples, 38 were graded
using a scale described by Mankin et a1 (1) (Table 1).
Four specimens (from 2 cartilages) were not processed
adequately for histologic analysis; of these, 2 came
from lesion sites and 2 from remote sites. These 4
specimens were from patients who had not received
intraarticular injections of steroids.
The histologic changes observed in the OA
cartilage resembled those previously described (I).
These included fibrillation, clefts, hypercellularity,
clones, tidemark invasion by blood vessels, and a
decrease in Safranin 0 staining. Using Mankin’s scale,
the OA cartilages were graded 2-9. Of the 38 specimens examined, 24 were classified as mild and 14 as
moderate. No specimens were graded as severe.
The 7 patients who had received at least 1
intraarticular steroid injection within 1 year prior to
surgery were designated I + . Of these 14 specimens,
disease severity was graded as mild in 8 and moderate
in 6. Of the 24 specimens (excluding the 4 not processed) from patients who had not received steroid
injections (I-), 16 were graded mild and 8 moderate.
Regarding the changes seen with Safranin 0
staining, 20 specimens showed a disappearance of
pericellular metachromatic staining (Figure 2). This
was more frequently observed in the middle and deep
zones of the cartilage. Three of the 14 I+ specimens,
as compared with 17 of the 24 I- specimens, showed
a consistent pericellular loss of Safranin 0 staining.
Acid metalloprotease activity. Sire selection and
histologic grading. Table 1 shows the levels of enzyme
in OA and normal control cartilages. Compared with
that in the normal controls, total acid metalloprotease
activity in OA cartilages was elevated in the 2 topographic zones selected (lesions and remote sites).
However, there was a significant increase in the total
activity only in those specimens taken from the lesion
zone. Although the mean value of the active form of
enzyme was increased in OA specimens compared
that in with controls, the difference was not statistically significant.
The distribution of total enzyme activity in all
OA specimens, regardless of site, was correlated with
the histologic grade. Linear regression analysis (Figure 3) revealed a direct relationship, with statistical
significance ( P < 0.02, r = 0.38; n = 38). Only those
specimens from patients with disease graded as moderate had increased total enzyme activity (Table 1).
This level was significantly different from that found in
normal controls ( P < 0.04) or in specimens with mild
disease ( P < 0.04), The levels of active enzyme in the
mild and moderate disease groups were about twice
the normal level, but this elevation was not statistically significant.
Steroid effects. We evaluated the effects of
steroids on existing enzyme levels by comparing specimens from I+ and I- patients with those from controls. As shown in Figure 4, specimens from I+
patients had a level of acid metalloprotease activity
that was similar to that of control specimens. The
enzyme activity in specimens from I- patients was
significantly elevated compared with control specimens.
We examined the possibility that the degree of
cartilage degradation could influence steroid action on
enzyme activity. We subclassified each of the OA
cartilage specimens according to its topographic site
and histologic grade. The analysis of individual subgroups (Figure 4) demonstrated that cartilages from I+
patients had much lower levels of total enzyme activity than did the cartilages from I- patients. No I+
subgroup was found to have a level that was different
from that of the control cartilage, However, a significantly increased level of total enzyme activity was
I- I+
I- I+
I- 1'
I- I+
I- I+
Figure 4. Total protepglycan-degrading acid metalloprotease activity in human cartilage. Two osteoarthritis (OA) cartilage groups
were examined: those from patients who had not received
intraarticular injections of steroids (I-) and those from patients who
had received at least 1 such injection within 1 year of surgery (I+).
These cartilage groups were subdivided according to site (lesion or
remote, as selected macroscopicglly) and according to histologic
grade (mild = 1-5; moderate = 6-9) (see Patients and Methods for
details of scoring system and calculations of enzyme activity).
Results are the mean t SEM of the percent uronic acid released.
The numbers in the lower part of each column are the number of
tissue specimens assayed. Asterisks indicate statistical significance
gs follows: I- OA cartilage versus controls (C), P < 0.05; Ilesional OA cartilage versvs controls, P < 0.02; I- moderate OA
cartilage versus controls, P < 0.01 (by Student's 2-tailed r-test).
Figure 3. Relationship between total acid metalloprotease activity
and histologic grade in osteoarthritic cartilage (see Patients and
Methods for grading system). A positive, direct coFelation was
found (r = 0.38, P < 0.02; n = 38).
found in I- specimens taken from the lesion sites ( P <
0.02) and in specimens which showed advanced degrees of degeneration (moderate), compared with controls (P < 0.01). The less severely diseased cartilages,
i.e., those from remote sites or those in the mild
disease category, appeared to have enzyme activities
that were similar to those of the control specimens.
Figure 5 illustrates the behavior of the active
form of the enzyme. The activity of the active form
paralleled that of the total enzyme. A lower level of the
active form was found in specimens from I+ patients.
When classified according to topographic site, the
cartilages from I- patients consistently showed a
higher level than did the cartilages from both I+
patients and contrals, although these elevations were
not statistically significant. However, I- patient specimens in which degeneration was graded as moderate
had a significantly higher level of activity than that
found in controls ( P < 0.05).
I- I+
I- 1'
I- I+
1- I+
I- I+
Figure 5. Active proteoglycan-degrading acid metalloprotease activity in human cartilage. * = significantly different from control
valutes at P < 0.05 (by Student's 2-tailed t-test). See Figure 4 for
Analysis of proteoglycan macromolecules. Figure
6 depicts the elution profile of proteoglycan from OA
cartilage homogenates which were incubated at pH 5.5
in the presence of either APMA or o-phenanthroline.
Chromatography on Sepharose CL-2B showed that
the extracted proteoglycans eluted as one unimodal,
included peak. The average partition coefficient of the
products generated from the APMA-activated
homogenates was higher (Kav = 0.47) than the partition coefficient from the o-phenanthroline-treated
specimens (Kav = 0.39). These findings are consistent
with the presence of an acid metalloprotease in OA
cartilage that is capable of proteoglycan degradation.
facts that the protease appears in a latent form, can be
activated by APMA, and is inhibited by the tissue
inhibitor of metalloproteases (10) make it likely that
this enzyme functions within the extracellular matrix.
If so, the meaning of the acid pH optimum presents a
The matrix pH is approximately 7.2 (7); thus,
the enzyme would not be at its optimum. However, it
does retain about 40% of its maximum activity at this
pH (10). Alternatively, it may function near the cell
surface, where the pH might be lowered by the secretion of acids ( 5 ) . In this latter case, the halo pattern
developing around the cells is suggestive of, but does
not establish, the validity of the hypothesis, since any
protease secreted by the cell might tend at first to
produce a clearing immediately around the cell. Because neutral metalloproteases have a slightly residual
effect at acid pH, one cannot exclude the possibility
that they are also involved in the pericellular degradation of matrix proteoglycans. It is also possible that
halo formation is a first step, which is then followed by
further diffusion of the proteases into the interterritorial matrix, thereby destroying the proteoglycans in
In previous studies (4,15,17) we have shown
that OA cartilage contains proteases capable of digesting collagen and proteoglycan macromolecules within
the matrix at physiologic pH. These enzymes belong
to the metalloprotease class. They appear to be related
to the development of osteoarthritis, in that their
activities are usually elevated in relation to the degree
of severity of the histologic changes seen in this
In the present study, we explored the activity of
an uiiusual metalloprotease which degrades proteoglycans at acid pH. Normally, such an enzyme might be
considered to have a lysosomal localization and function. The localization is not presently known, but the
Figure 6. Chromatography, on a Sepharose CL-2B column, of
proteoglycan (PG) macromolecules from human osteoarthritic cartilage. The PGs were obtained from the p-aminophenylmercuric
acetate (APMA)-activated homogenates or the o-phenanthrolinetreated homogenates. Sulfated PG content was determined by the
1,9-dimethylmethylene blue method (13). The average partition
coefficient (KaJ of the products from APMA-activated homogenates
was 0.47, while K,, of 0.39 was recorded for the o-phenanthrolinetreated specimens.
that region. The cells may then try to replenish the
matrix by filling in the halo region.
We have attempted to relate the degree of halo
formation to the level of acid protease activity, but the
scattering of the data is too great to allow a firm
conclusion to be drawn. However, our findings demonstrate that the cartilages from patients who had been
treated with intraarticular steroid injections showed a
much lower incidence of pericellular proteoglycan
degradation and lower enzyme activity (total and
active) than did those specimens from patients who
had not received steroids. These data strongly suggest
an involvement of acid metalloproteases at the pericellular level. It must be pointed out, however, that
although the level of enzyme activity was lower in
patients who had received steroids, it did not decrease
the severity of OA lesions, when assessed histologically. This can probably be explained by the fact that
the increased level of acid metalloproteases is only one
factor among many that are involved in the occurrence
and progression of osteoarthritis. It seems evident that
the suppression of acid metalloproteases alone may
not be sufficient to stop the progression of the disease.
It should also be noted that all these patients were
considered to have severe OA, and they may have
reached a point at which no treatment would have
been effective in completely stopping the progression
of the disease.
The results of this study corroborate those
obtained by Azzo and Woessner (10) in a preliminary
study. They extracted acid metalloproteases from the
cartilage of human patellas removed at autopsy. In
their study, the levels of extracted enzymes were 3
times higher in OA tissues than in normal tissues. The
present study was more sophisticated, in that it used
fresh surgical tissue, included the histologic grading of
tissue, and provided detailed information about the
patient’s age and previous drug therapy.
The elevation of enzyme activity noted in this
study is substantiated by the results of independent
experiments in which the enzyme was extracted from
the tissue and assayed on an exogenous substrate
(9,10,18). In one of those studies (18), 2 metalloproteoglycan-degrading enzymes were measured in
cartilage extracts of human OA knees. Data on neutral
metallo-enzymes from the extract assay yielded results similar to those obtained from a pellet assay (4).
The same trend was seen for the acid metalloenzymes. Compared with specimens from normal controls, the OA specimens showed a threefold increase
in enzyme activity, which proved to be statistically
The increased levels of latent and active acid
metalloproteases in OA cartilage follow the same
patterns as those observed for neutral metalloproteoglycanase (4) and collagenase (17). This may indicate
that not only are the metalloproteases synthesized
under a similar metabolic pathway, but their synthesis
and the regulation of their activity may be directed
under similar controls. The exact mechanism of the in
vivo activation of the metalloproteases in the cartilage
matrix is still unknown. The in vitro measurement of
the metalloproteases represents the difference between enzyme activation and inhibition. Increased
enzyme activity may represent increased activation,
decreased inhibition, or possibly, both. The greatest
increase in the level of the active form of the enzyme,
which was found in specimens with the most severe
disease, is particularly pertinent. The active form of
the enzyme might be the only one of physiologic
importance that could be responsible for the degradation of the cartilage proteoglycans.
This study further indicates the importance of
knowing the treatment history of the patient, since it
appears that steroids may depress protease levels or,
at least, may prevent them from rising as high as they
do in untreated patients. Although there is no absolute
proof that this decrease in enzyme activity was the
result of steroid therapy, it is possible that the
pharmacologic effects of these agents may last longer
than do their direct effects in the joint. This could vary
from a few hours to a few weeks, depending on their
chemical composition (19,20). It might explain why the
clinical improvement in patients treated with
intraarticular steroids may last for many months (20).
We have found parallel evidence, in both the PondNuki dog model of OA (15) and in rheumatoid arthritis
in humans (14), that low-dose, oral steroids block a
rise in cartilage neutral metalloprotease activity. It
may be that steroids coordinate the regulation of all
metalloproteases in cartilage, regardless of whether
this regulation is mediated by the suppression of
synovial factors (15) o r by direct action o n the
chondrocytes (21). This question awaits further investigation.
We thank FranGois Mineau for technical assistance,
Walentina Nedwetsky for both technical assistance and
revision of the manuscript, and Diane Fournier for secretarial help. We gratefully acknowledge the Pathology Department of Notre-Dame Hospital for their supply of autopsy
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acid, steroid, proteoglycans, effect, injections, activity, osteoarthritis, cartilage, human, degrading, metalloprotease, intraarticular
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