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In vivo effect of aspirin on canine osteoarthritic cartilage.

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994
IN VIVO EFFECT O F ASPIRIN ON CANINE
OSTEOARTHRITIC CARTILAGE
MARSHALL J. I’ALMOSKI and KENNETH D. BRANDT
The in vivo effect of aspirin on degeneration of
knee cartilage in a canine model of osteoarthritis was
examined. When dogs were fed aspirin daily after
anterior cruciate ligament transection, the degeneration
of articular cartilage in the unstable knee was more
marked 9 weeks later than that in the operated knee of
dogs which did not receive aspirin. Compared with
samples from the contralateral knees, the thickness of
articular cartilage in the operated knees of aspirin-fed
dogs was reduced, while it was increased in the operated
knees of dogs not fed aspirin. In addition, the proteoglycan (uronic acid) content and the augmentation of
proteoglycan synthesis in cartilage from the unstable
knee were significantly lower when the dogs were fed
aspirin than when they were not, and Safranin-0 staining of the matrix was less intense. However, cartilage
from the contralateral knees of aspirin-fed dogs was
histochemically and biochemically normal in every respect. When metatarsal bones, with their overlying
articular cartilage intact, were cultured in the presence
of 10-4M and lO-’M salicylate, net glycosaminoglycan
synthesis was suppressed by 25% and 15%, respectively. These concentrations of salicylate had previously
From the Rheumatology Division. Indiana University
School of Medicine. Indianapolis. Indiana.
Supported in part by grants from the National Institute of
Arthritis. Diabetes. Digestive and Kidney Diseases (AM 20582 and
AM 27075) and awards from the Arthritis Foundation and the Grace
M. Showalter Trust.
Marshall J. Palmoski, PhD: Associate Professor of Medicine and Anatomy; Kenneth D. Brandt. MD: Professor of Medicine
and Chief. Rheurnatology Division. Indiana University School of
Medicine.
Address reprint requests to Marshall J. Palmoski. PhD,
Rheurnatology Division, Indiana University School of Medicine, 541
Clinical Dnvc, Room 492. Indianapolis. I N 46223.
Submitted for publication October I I , 1982; accepted in
revised form March 22. 1983.
Arthritis and Rheumatism, Vol. 26, No. 8 (August 1983)
been shown to have no effect on glycosaminoglycan
metabolism in normal cartilage from the weightbearing
region of the femoral condyle. Since the uronic acid
content of metatarsal cartilage is lower than that of
femoral cartilage, and that of osteoarthritic femoral
cartilage is lower than that of normal femoral cartilage,
the present results are consistent with the concept that
cartilage is more permeable to aspirin when its matrix is
depleted of proteaglycans.
When 10-3M salicylate is added to the culture
medium, glycosaminoglycan (GAG) synthesis in slices
of normal articular cartilage from habitually loaded
regions of canine femoral condyles is reduced by about
30%, while lower concentrations of the drug have no
effcct (1,2). Notably, this in vitro inhibition of GAG
synthesis by salicylate is much greater in osteoarthritic
(OA) than in normal cartilage (3). Furthermore, salicylate may affect articular cartilage in vivo as well as in
vitro; when aspirin was fed to dogs in quantities
sufficient to achieve a serum salicylate concentration
of 20-25 mg/dl (i.e., approximately lO-’M), the degeneration of femoral articular cartilage caused by immobilization of the leg was aggravated (4). Thus, knee
cartilage from the constrained legs of the aspirin-fed
dogs showed decreases in uronic acid content and net
GAG synthesis and an increase in extractability of
proteoglycans (PG),which were significantly greater
than those seen in atrophic cartilage from the immobilized knees of dogs which did not reccive aspirin.
However, aspirin had no apparent in vivo effect on
articular cartilage of the contralateral (nonconstrained)
knees.
Since the PG content of OA cartilage and
atrophic cartilage is lower than that of normal carti-
ASPIRIN AND CANINE OA CARTILAGE
lage. these data suggest that a reduction in PG content
may increase the susceptibility of articular cartilage to
salicylate, perhaps by increasing the permeability of
the drug. This possibility is strengthened by our recent
data indicating that the suppressive effects of salicylate in vitro are increased if the proteoglycan content
of normal cartilage has been reduced by hyaluronidase
digestion prior t o incubation with the drug (5). In view
of the potential clinical significance of the question, we
considered it important t o ascertain whether the
changes produced by salicylate in OA cartilage in vitro
(3) occurred also in vivo. The present study examines
this question and provides some new information
concerning the permeability of intact articular cartilage t o salicylate.
MATERIALS AND METHODS
Animals and surgical procedure. Eight adult mongrel
dogs (25-30 kg) were anesthetized by intravenous administration of sodium pentothal. The joint capsule of the right
knee was opened through a short oblique anteromedial
incision permitting visualization of the anterior cruciate
ligament, which was cut with a scalpel. Care was taken to
avoid damage to the articular cartilage. The joint capsule
was closed with 2-0 chromic sutures and the skin with
interrupted 4-0 nylon. Postoperatively the animals were
maintained in pens large enough to allow walkingm(8'x 10').
All dogs bore weight on the operated limb by the third day
after surgery. None developed a wound infection. Four of
the dogs were fed acetylsalicylic acid (120 mg/kg/day in 3
evenly divided doses) from the day after surgery until they
were killed. Serum salicylate concentrations were measured
weekly. Blood samples were obtained in the morning before
the first aspirin capsule of the day was administered, and
averaged 20-25 rngidl in each dog that received aspirin.
Nine weeks postoperatively all dogs were killed with
an overdose of sodium pentothal and both distal femurs were
removed aseptically with a bone saw. Representative fullthickness samples of articular cartilage from the central
weightbearing portion of the medial femoral condyles were
obtained with a Craig biopsy needle for histologic study,
while other full-thickness portions (approximately 10 mg wet
weight) of cartilage from the same region were taken for
determination of dry weight and uronic acid content. The
remainder of the cartilage from the habitually loaded portions of both femoral condyles was shaved into slices less
than 0.5 mm thick and used for tissue culture studies.
Cartilage from the operated (Cru-X) knee and the contralatera1 control knee of each dog was pooled separately.
Tissue culture studies. Cartilage slices from each joint
(approximately 60 mg) were placed in Ham's F-12 nutrient
mixture, pH 7.4, containing 10% newborn calf serum, streptomycin (50 mg/ml), penicillin (50 unitslml), and Naz "SO4
(10 pCi/ml) (New England Nuclear Corp., Boston, MA), and
were incubated with gentle rocking for 18 hours at 37°C in a
mixture of air-C02 (19: 1). After incubation the medium was
decanted and the tissue was washed twice with 3-ml portions
995
of cold Ham's F-12 nutrient mixture. The spent medium and
washes were combined and dialyzed against 200 volumes of
0 . 0 9 4 sodium acetate, pH 6.8, for 48 hours at 4°C in
Spectrapor No. 3 dialysis tubing (Spectrum Medical Industries, Inc., Los Angeles, CA). which has an approximate
molecular weight cutoff of 3,500 daltons. The sacs were
rinsed with 3 ml of distilled water and the retentate and
rinses were combined, following which the PGs were isolated as described below.
Sequential extraction of PGs from the cartilage and
measurement of net PG synthesis. PGs were extracted sequentially from the slices of labeled cartilage with 0.4M
guanidinium chloride (GuHCI) and then with 4.OM GuHCI.
as previously described (6). Both solvents contained the
protease inhibitors EDTA (O.OlM), 6-aminohexanoic acid
(0. IM), benzamidine hydrochloride (0.00SM). and phenylmethylsulfonyl fluoride (0.5 mM) (7). Following extraction
with 4.OM GuHCI, the cartilage residue was digested with
pronase as described previously (6).
The spent culture medium, the sequential 0.4M and
4.OM GuHCl extracts, and the pronase digest were dialyzed
against distilled water, following which 0. I-ml aliquots were
added to Ready-Solv HP (Beckman Instruments, Inc., Fullerton, CA) and counted in a Beckman liquid scintillation
spectrometer.
Net PG synthesis was determined from the sum of
the nondialyzable counts per minute in the medium, the
sequential GuHCl extracts, and the pronase digest.
Isolation and purification of PGs. After dialysis of the
medium and 4.OM GuHCl extract, PGs were isolated and
purified by cesium chloride equilibrium density gradient
centrifugation under associative conditions in the presence
of 0.4M GuHCI, according to the method of Hascall and
Sajdera (8). Fractions from the bottom two-fifths of the
density gradient (density = 1.76 g d m l ) were combined and
dialyzed exhaustively at 4°C against O.05M sodium acetate.
pH 6.9, to yield fractions designated AMedand AG", respectively.
Gel chromatography. Samples (0.5 ml) of Fraction
kU
in 0.05M sodium acetate, pH 6.9, were applied to a
column (95 x 1.0 cm) of Sepharose 2B (Pharmacia Fine
Chemicals, Piscataway, NJ) and eluted with the same buffer
at a rate of 2 ml/hour. The "SO4 radioactivity in I-ml effluent
fractions was determined as above, following which the
partition coefficient (Kav) was calculated from the formula:
Ka,< = We - V,)/(V, - V,)
in which V, represents the peak fraction in the elution
diagram, V, the void volume, and V, the total column
volume.
Studies with normal metatarsal cartilage. In an initial
experiment, cartilage was shaved from the proximal and
distal ends of metatarsals 11-V of both paws of a normal dog
and pooled separately for determination of dry weight,
uronic acid content, and for histologic and histochemical
examination. To ascertain whether the levels of GAG synthesis in cartilage from proximal and distal ends were
similar, the metatarsal bones from another dog, with their
caps of cartilage intact, were each incubated individually as
above for 24 hours in 7 ml of Ham's F-12 medium containing
20 pCi/ml of Naz ' 3 0 4 . The medium was decanted, the
PALMOSKI AND BRANDT
placed in 2 changes of acetone for 48 hours and dried to
constant weight in vacuo at 80°C. The dried cartilage was
digested with pronase as above, after which the GAGs were
isolated by precipitation with 9-aminoacridine hydrochloride
and converted to their sodium salts with Bio-Rad AG-50
(Na+) (9). After the resin was removed by filtration, the
uronic acid content of the filtrate was determined (10).
Serum salicylate concentrations were determined by the
method of Trinder (1 1).
Histologic examination. Histologic sections (6p) of
the full thickness samples of articular cartilage including
underlying bone were examined microscopically after staining with Safranin-0 and counterstaining with fast green. The
depth of the cartilage from surface to tidemark was measured with a reticule eyepiece.
bones were washed with cold medium, and the spent medium and wash were combined, dialyzed overnight against
water at 4"C, and counted. The articular cartilage was then
shaved from each bone, and that from the proximal and
distal end was digested separately with pronase: The digests
were dialyzed overnight against water at 4"C, after which the
radioactivity in the retentates was counted as above.ahe
tissue content of newly synthesized GAGs was determined
from the nondialyzable "S cpm in the pronase digest.
To assess the effect of sodium salicylate on GAG
synthesis in the intact metatarsal cartilage, metatarsals 111-V
were obtained from both paws of 2 additional normal adult
dogs. The metatarsals from the right paw were incubated
separately as above for 24 hours in medium containing
or lO-'M sodium salicylate; those from the left paw
were cultured in identical fashion but with no salicylate in
the medium. The addition of sodium salicylate did not
change the pH of the culture medium. Naz "SO4 (20 pCilml)
was added for the final 4 hours of culture, after which the
tissue content of newly synthesized GAGs was determined
as above.
To determine 14C acetylsalicylic acid uptake by the
intact metatarsal cartilage, metatarsals 11-V were excised
under sterile conditions from both hind limbs of another dog
and freed of soft tissue. With the articular cartilage on its
proximal and distal ends intact, each metatarsal was incubated separately with gentle rocking for 6 hours in a conical
centrifuge tube containing culture medium as described
above, 10-3M acetysalicylic acid, and 0.5 pCi/ml acetylsalicylic acid, ~ar b o x y l - '~(New
C
England Nuclear; 34.3 mCi/
mmole). At the end of the incubation period the medium was
removed and the bones were washed 3 times with 7-ml
aliquots of fresh unlabeled medium.
The proximal and distal ends of each metatarsal were
then transected with a bone saw approximately 0.5 cm below
the articular surface and mounted with a drop of water on a
cryostat chuck at -15°C. Multiple sections (16p) of the
cartilage, which is approximately 300-350p thick, were cut
parallel to the surface down t o the bone and groups of 4-5
adjacent sections were pooled into 5 fractions, with Fraction
1 representing the zone nearest the surface and Fraction 5
the deepest zone. Each fraction was weighed, digested with
pronase, and counted as above.
Analytic methods. For determination of dry weight,
portions of cartilage (approximately 10 mg wet weight) were
RESULTS
Gross observations. Articular cartilage from the
nonoperated knee of every animal was white, glistening, and smooth with a n intact surface. No osteophytes were noted. The articular condyles of Cru-X
knees appeared to be grossly intact. However, the
cartilage was dull and marginal osteophytes were
present on the medial and lateral condyles of each
operated knee. Aspirin administration did not affect
these gross changes. In 1 dog that received aspirin,
pitting of the articular surface was noted on the
femoral condyles of the Cru-X knee.
Histology and histochemistry. Cartilage from the
unoperated knees of dogs which had been fed aspirin
exhibited normal Safranin-0 staining, and its thickness
was comparable with that of cartilage from unoperated
knees of dogs not fed aspirin (Table 1). T h e thickness
of cartilage from Cru-X knees of the aspirin-fed dogs
averaged 17% less than that of the contralateral controls ( P < 0.02), and Safranin-0 staining was moderately-markedly reduced. In addition, frank fibrillation
extending from t he surface through approximately
50% of the cartilage thickness was noted in sections
Table 1. Osteoarthritic changes in knee cartilage of dogs whose anterior cruciate ligament had been
transected 9 weeks Dreviouslv
No. of Aspirin
dogs feeding
4
Source of
cartilage
Cartilage
thickness,
mm 2 1
SEM
Osteophytes
No
Control knee 0.95 ? 0.02
Cru-X knee* 1.12 ? O.OSt
014
414
Yes
Control knee 1.00 ? 0.16
Cru-X knee 0.83 ? 0.10t
014
414
* Cru-X = anterior cruciate ligament transection.
t P < 0.02.
Safranin-0
staining
Normal
Slight-moderate
reduction
Normal
Moderate-marked
reduction
Fibrilla- Chondrocyte
tion
clusters
014
1I4
014
414
014
214
014
414
ASPIRIN AND CANINE OA CARTILAGE
997
Table 2. Composition of canine knee cartilage 9 weeks after transection of anterior cruciate ligament
Uronic acid content
Water content
% of
NO.of
dogs
Aspirin
feeding
4
No
4
Yes
Source of
cartilage
Control knee
Cru-X knee*
Control knee
Cru-X knee
tissue
dry
weight c
1 SEM
% of
control
* 0.2
0.3
* 0.2
4.4
3.5
4.2
2.9 2
-
80
69t
0.2
% of
tissue wet
weight 2
1 SEM
70.9
74.5
71.5
76.8
?
?
2
3.4
3.4
1.4
1.3
% of
control
105
108
* Cru-X
= anterior cruciate ligament transection.
Significantly greater than the decrease in uronic acid content of cartilage from the Cru-X knees of
dogs not given aspirin (P < 0.02).
t
from the Cru-X knee of 1 of the dogs fed aspirin, and
less extensive fibrillation in sections from a second
aspirin-fed dog (Table 1).
In contrast, when compared with samples from
the unoperated knees, cartilage thickness was increased by 18% (P < 0.02) in OA cartilage from dogs
not given aspirin, and the reduction in Safranin-0
staining of Cru-X cartilage was less marked than that
seen in Cru-X cartilage of dogs which received aspirin.
Surface fibrillation was noted in cartilage of 1 of the 4
dogs which did not receive aspirin (Table 1). Regardless of whether the dogs received aspirin, chondrocyte
clusters containing up to 5 cells per lacuna were seen
near the tidemark in all sections of cartilage from CruX knees, although the tidemark was intact in every
case.
Water and uronic acid content. All samples of
cartilage from the unoperated knees were similar with
respect to their uronic acid and water contents, regardless of whether the dog had received aspirin (Table 2).
The uronic acid content was about 4.3% of the tissue
dry weight and the tissue water content was about 71%
of the total wet weight. While the uronic acid content
of cartilage from the Cru-X knees of dogs not fed
aspirin was 80% of that in the corresponding control
cartilage of the same dogs, the uronic acid content of
cartilage from Cru-X knees of dogs which had received
aspirin was 69% of that in the corresponding cartilage.
Thus, the reduction in uronic acid content of OA
cartilage from dogs which received aspirin was greater
than that in OA cartilage of dogs which did not ( P <
0.02) (Table 2). The water content of cartilage from the
Cru-X limbs was greater than that of control cartilage,
although this increase was not significantly affected by
aspirin feeding (108% in aspirin-fed dogs, 105% in
controls) (Table 2).
Sequential extraction of PGs from cartilage. In
every case the proportion of the total 35S PGs contained in the medium after incubation of cartilage from
the Cm-X knees was greater than that in the medium
of cultures of control cartilage (P < 0.01). This increased release of PGs from Cru-X cartilage was not
influenced by aspirin feeding. Thus, 10-12% of the
total nondialyzable 35Sradioactivity was recovered in
the medium of control cultures and 21-23% in the
medium of cultures of Cru-X cartilage (Table 3). In all
cases the majority of the 35S PG was extracted with
4.OM GuHCl (54-61% of the total), while only a small
percentage was extracted with 0.4M GuHCl (4-7% of
the total) (Table 3).
PG synthesis. Based on the sum of the nondialyzable 35S radioactivity in the culture medium, sequential GuHCl extracts, and pronase digest of the
cartilage residue, net PG synthesis in cartilage from
the Cru-X knees of dogs which did not receive aspirin
averaged 183 ? 9% of that in cartilage from the
contralateral knees (Table 3). The level of PG synthesis in OA cartilage of dogs which had received aspirin
was much lower in every case ( P < 0.01) and averaged
116 4% of that in corresponding control cartilage
(Table 3).
PG aggregation. In all samples AMedaccounted
for 40-50% of the total nondialyzable 35Scpm in the
culture medium which was applied to the associative
cesium chloride gradient, i.e., about 5% of the total
nondialyzable 35S cpm. Since the nondialyzable 3sS
radioactivity in the 0.4M GuHCl extracts also represented only about 5% of the total, neither fraction was
analyzed further.
Regardless of whether the dog was fed aspirin,
Fraction AGu represented 7 0 4 0 % of the total nondialyzable radioactivity in the 4.OM GuHCl extracts of
*
PALMOSKI AND BRANDT
998
Table 3. Effect of aspirin feeding on the distribution of "S proteoglycans between culture medium
and sequential GuHCl extracts of normal and osteoarthritic canine knee cartilage*
% of total 3sS cpm 2 1 SEM
No.
of
dogs
Aspirin
feeding
4
No
4
Yes
Source of
cartilage
Culture
medium
Control knee
Cru-X kneet
Control knee
Cru-X knee
21 + 2f
12 f 1
23 f 3f
10
f
1
0.4M
GuHCl
extract
4.OM
GuHCl
extract
Pronase
digest
Total
"S cpm,
% of
control
5 2 1
72 I
6123
58 2 2
5723
5421
24f3
14 It 3
27f3
17f1
183 f 9
116 2 48
4+1
6 2 1
* Nondialyzable "S counts per minute.
t Cru-X = anterior cruciate ligament transection.
f Significantly greater than the % of the total "S cpm found in the medium from the control knee (P<
0.01).
8 Significantly less than the augmented level of glycosaminoglycan synthesis in cartilage from the CruX knee of dogs not fed aspirin (P < 0.01).
control and OA cartilage. Aspirin administration had
no effect on the ability of PGs extracted from control
cartilage to form large aggregates. Thus, 3 6 4 8 % of
the 35S A G samples
~
from all control tissues were
sufficiently large in average hydrodynamic size to
elute in the Sepharose 2B void volume. In contrast,
PGs in fraction AGu from the OA cartilage contained
little or no material which was excluded by the gel.
Elution profiles of AGu prepared from cartilage of the
Cru-X knees of dogs which had received aspirin were
no different from those AGu samples from dogs which
had not.
The K,, of PGs in Fraction A G from
~
all samples of control and Cru-X cartilage small enough to be
retarded by Sepharose 2B were similar (Kav = 0.350.48), and this was not affected by aspirin feeding.
Salicylate uptake by normal metatarsal cartilage.
The above data indicate that aspirin had no in vivo
effect on control cartilage, raising the question of
whether the drug is taken up by intact cartilage. To
examine this point, metatarsal bones from normal
dogs, with the articular cartilage on proximal and
distal ends intact, were incubated in medium containing 14C-acetylsalicylicacid and the distribution of the
radionuclide through the cartilage thickness was determined. 14C-acetylsalicylicacid was present throughout
the depth of the cartilage and its distribution in cartilage from proximal and distal ends was similar (Table
4). In all cases Fraction 3, the middle zone, contained
the highest concentration (mean = 115,860 cpm/mg
cartilage wet weight).
Characterization of metatarsal cartilage. The
thickness of normal metatarsal cartilage, measured
from surface to tidemark, averaged 0.3-0.35 mm. No
difference between proximal and distal cartilage was
apparent with respect to cell density or cartilage
thickness. In comparison with that of normal cartilage
from canine femoral condyles, Safranin-0 staining was
moderately reduced. The uronic acid content of metatarsal cartilage from the proximal and distal ends was
similar (3.2 ? 0.2% and 3.0 ? 0.2% of the dry weight,
respectively) as was the water content (67 2% and 69
2 3% of the wet weight, respectively).
Based on nondialyzable 3sS cpm in pronase
digests of the tissue, the content of newly synthesized
GAGS in cartilage from the proximal ends of each of
the 8 metatarsals from a single dog was similar (Table
5). The mean "S GAG content of cartilage from the
distal ends of each of these 8 metatarsals was also
similar, and was slightly greater than that from the
proximal ends (117%, P < 0.01) (Table 5).
Effect of sodium salicylate on GAG metabolism
in metatarsal cartilage. Various concentrations of sa-
*
Table 4.
cartilage
Uptake of ''C-acetylsalicylic
acid by intact metatarsal
I4C disintegrations per minutelmg wet
weight of cartilaget
Fraction of cartilage
from surface to bone*
Distal end of
metatarsal
43,200 f
85,910 2
110,510 2
78,600 2
45,560 f
3,420
6,520
12,310
3,450
6,217
Proximal end of
metatarsal
48,620 +
88,980 2
121,210 2
92,310 2
53,920 2
5,670
4,680
15,820
6,590
8,231
* Multiple sections ( 1 6 ~ )of the cartilage were cut parallel to the
surface down to bone. Groups of 4-5 adjacent sections were pooled
in each fraction. Fraction 1 = zone closest to joint surface; Fraction
5 = deepest zone of the cartilage.
t Results represent the mean f 1 SEM of results obtained with
metatarsals 11-V from each paw of a single dog.
ASPIRIN AND CANINE OA CARTILAGE
Table 5. Net glycosaminoglycan synthesis in articular cartilage
from proximal and distal ends of metatarsal bones
Nondialyzable "S counts per minutehg
wet weight of cartilage
Right
Left
Proximal
I1
111
IV
V
Mean
Distal
I1
111
IV
V
Mean
349
7,866
6,153
6,68 1
5,804
6,626 t 451
7,984
7,108
7,089
8,245
7,607 f 299
8,247
7,263
7,605
8,016
7,782 f 218
7,506
6,060
6,104
6,184
6.464
f
licylate were added to the culture medium to determine whether under these conditions, a concentration
of drug sufficient to affect GAG metabolism diffused
through the intact cartilage surface. In the presence of
the lowest concentration of sodium salicylate used
(10-5M), the net 35SGAG content of proximal metatarsal cartilage averaged 84 f l % of controls (P <
0.01) and that of distal metatarsal cartilage averaged 87
-t 1% of controls (P < 0.01) (Table 6). At salicylate
concentrations of 10-4M and 10-3M1,GAG synthesis
in cartilage from the proximal and distal metatarsal
cartilage averaged 65 k 3% and 48 ? 3% of controls,
respectively. The proportion of the total 35S GAGS
contained in the culture medium of the metatarsal
cultures was not influenced by the concentration of
salicylate and was similar to that in controls (21 2 5%).
DISCUSSION
The present study illustrates that cartilage degeneration in the Cru-X limbs of dogs which are fed
aspirin is greater than that in Cru-X cartilage from
dogs not fed aspirin. Thus, the decreases in Safranin-0
staining and the fibrillation were more severe in aspirin-fed dogs than in those which did not receive
aspirin. Furthermore, the cartilage thickness was diminished in Cru-X dogs which had received aspirin,
while it was greater than that of the control cartilage in
Cru-X dogs which had not (Table 1).
The biochemical changes in articular cartilage
in this experimental model have been shown in several
studies (3,12-14) to be similar to those of naturally
occurring OA, e.g., an increase in water content and
decrease in PG content, augmented PG synthesis,
increased extractability of PGs, and defective PG
999
aggregation. The PG (uronic acid) content (Table 2)
and augmented PG synthesis (Table 3) were significantly lower in Cru-X cartilage from dogs fed aspirin
than in Cru-X cartilage of dogs which were not, while
the abnormalities in extractability and PG aggregation
were unaffected by the drug (Table 3).
The increased level of net GAG synthesis and
decrease in uronic acid content in OA cartilage suggest
an increase in the rate of PG degradation. The present
data suggest that aspirin inhibits the in vivo synthesis
of GAGS in Cru-X cartilage. It thus interferes with the
repair attempt by the chondrocytes in OA cartilage
(IS). These data are consistent with previous in vivo
data (4)showing that the suppression of PG synthesis
produced in normal canine knee cartilage by application of a leg cast was aggravated if the dog was fed
aspirin during the period of immobilization. Other
studies have shown that salicylate inhibits the enzymes involved in the initial reactions of GAG biosynthesis (16,17).
Although activity of acid and neutral proteases,
which degrade PGs, is greater in OA cartilage than in
normal cartilage (18,19), the present data do not suggest that aspirin increased PG degradation in the CruX cartilage. Thus, the proportions of 35S Pds which
were released into the medium of Cru-X cultures and
the size of the PG monomers in Fraction AGu(Le., the
PGs which were retarded by Sepharose 2B) were
unaffected by aspirin feeding.
The present results differ from other reports in
which aspirin has been shown to inhibit the cartilage
degeneration resulting from superficial scarification
(20) or prolonged joint compression (21) in rabbits and
to reduce the prevalance of degenerative changes in
human cartilage following recurrent dislocation of the
patella (22). The deleterious effects of aspirin in our in
vivo study could be due to the fact that aspirin was
Table 6. Effect of sodium salicylate on glycosaminoglycan
synthesis by intact articular cartilage
Net glycosaminoglycan synthesis,
% of control*
Salicy 1ate
concentration
10-5~
10-4~
10-3~
Dog 9
Dog 10
Proximal
Distal
Proximal
Distal
83
68
54
86
57
45
85
66
45
88
69
56
* Nondialyzable " S counts per minutehg cartilage wet weight in
pronase digest. Controls represent cartilage from the corresponding
proximal or distal end of the metatarsal bones from the opposite foot
cultured in the absence of salicylate.
1000
administered immediately following anterior cruciate
transection, which is different from the clinical situation in which the disease is usually advanced by the
time aspirin is administered. However, in a controlled
prospective study of patients with chondromalacia
patellae in whom arthroscopy was performed before
and after treatment, aspirin administration did not
protect against development of cartilage degeneration
(23). Furthermore, cartilage degeneration in C57 black
mice, which are genetically predisposed to OA, appears to be aggravated by aspirin feeding (24).
The data in this study are consistent with our
previous findings that salicylate suppresses GAG synthesis much more in OA cartilage in vitro (3), and in
atrophic cartilage in vivo (4), than in normal cartilage.
However, the possibility of secondary effects of salicylate in vivo such as analgesia which may allow
increased mechanical trauma, cannot be ruled out.
Serum salicylate concentrations of 20-25 mg/dl, which
are readily achieved therapeutically in humans, represent approximately 10 'M of the drug. Since drugs
reach the articular cartilage chiefly via the synovial
fluid, it is relevant to note that the free salicylate
concentration in synovial fluid is virtually identical to
that in plasma (25).
The data are consistent with the concept that
the effects of aspirin on the chondrocyte are inversely
related to the PG content of the cartilage matrix. This
concept is strengthened, furthermore, by the present
evidence that GAG synthesis in intact metatarsal
cartilage in vitro was suppressed by lo-' and 10 -4M
salicylate (Table 6 ) . Although we have reported these
concentrations of salicylate had no effect on GAG
synthesis in slices of normal cartilage from central
portions of canine femoral condyle (l), the uronic acid
content of metatarsal cartilage is lower than that of the
femoral cartilage (approximately 3.1% and 4.2% of dry
weight, respectively). We have also recently shown
that 10- 3M salicylate suppressed GAG synthesis in
cultures of femoral cartilage from habitually unloaded
regions to a much greater extent than it suppressed
synthesis in condylar cartilage from habitually loaded
regions (whose mean uronic acid contact was about
25% greater than that of the unloaded cartilage) ( 5 ) .
The net negative charge density of the PGs in
the matrix may tend to limit the diffusion of the
salicylate anion through cartilage. The present data
show clearly that salicylate does not diffuse freely into
intact metatarsal cartilage. Based on the ''C-acetylsalicylic acid uptake (Table 4), when the salicylate concentration of the culture medium was lOP3M the
PALMOSKI AND BRANDT
concentration of drug which diffused into the middle
zone of the metatarsal cartilage was calculated to be
0.7 X 10-4M, while that in cartilage near the surface
and adjacent to the subchondral bone was 0.3 x
10-4M. These calculations assume that the water
content in each fraction of cartilage was the same as
that of the full-thickness samples of metatarsal cartilage, i.e., 68% of the total weight (see Results). While
in fact the water content of articular cartilage varies as
a function of depth from the surface (26,27), this
variation does not exceed 10% in normal cartilage, and
differences of this magnitude from zone to zone would
not change the results of these calculations appreciably. Presumably, weightbearing portions of intact knee
cartilage, whose uronic acid concentration is greater,
are more impermeable to salicylate than intact metatarsal cartilage.
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1001
ASPIRIN AND CANINE OA CARTILAGE
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