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Running Inhibits the Reversal of Atrophic Changes in Canine Knee Cartilage After Removal of a Leg Cast.

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arthritis
and
rheumatism
Official Journal of t h e American Rheumatism Association Section of t h e Arthritis Foundation
-
__ - -
.
-___
~
-
RUNNING INHIBITS THE REVERSAL OF ATROPHIC
CHANGES IN CANINE KNEE CARTILAGE AFTER
REMOVAL OF A LEG CAST
MARSHALL J. PALMOSKI and KENNETH D. BRANDT
The effect of vigorous exercise on the reversibility
of canine knee cartilage atrophy produced by immobilization OF the leg was studied. In comparison to cartilage
from the contralateral control knees, cartilage from
knees which had been immobilized in a cast For 6 weeks
showed an increase in water content and decreases in
thickness, Safranin 0 staining of the matrix, uronic acid
content, and net proteoglycan synthesis. In addition, the
ability OF both newly synthesized (3’S) and total tissue
proteoglycans to interact with hyaluronic acid to Form
aggregates was diminished; this was apparently due to
an abnormality in the hyaluronate-binding region of the
core proteins. If the casts were removed and the animals
were then allowed to ambulate ad libitum for 3 weeks,
a11 of these changes were reversed. However, knee
cartilage from 3 dogs which had been run daily on a
treadmill (6 miledday) for 3 weeks after removal of the
casts exhibited continuing decreases in thickness, Safranin 0 staining, and uronic acid content (mean 31%),
even though net proteoglycan synthesis was increased
(mean 16%) in comparison to that in control cartilage
from the contralateral (nonimmobilized) knee. FurtherFrom the Rheumatology Division, Indiana University
School of Medicine. Indianapolis, Indiana.
Supported in part by grants from the National Institute of
Arthritis, Metabolism and Digestive 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. Rheumatology Division, Indiana University School of
Medicine.
Address reprint requests to Marshall J . Palmoski, PhD,
Rheumatology Division, Indiana University School of Medicine,
1100 W. Michigan Street, Indianapolis. IN 46223.
Submitted for publication October 14. 1980; accepted in
revised form April 17, 1981.
Arthritis and Rheumatism, Vol. 24, No. 11 (November 1981)
1329
more, the abnormality in both 35!3- and total tissue
proteoglycans which precluded their interaction with
high molecular weight hyaluronic acid persisted. In this
respect, the proteoglycans were indistinguishable from
those obtained from knee cartilage immediately following 6 weeks in a cast.
A number of reports have detailed the morphologic features of the degeneration that occurs in articular cartilage with immobilization of a limb (1-5). Few
studies, on the other hand, have focused on the
alterations in articular cartilage proteoglycans (PG)
that result from immobility (6-7). In normal joint
cartilage, most PG exist in large aggregates, in which a
number of PG are noncovalently associated with a
single filamentous molecule of hyaluronic acid (HA) in
a linkage stabilized by tissue glycoproteins (8). PG
account for most of the elasticity ofjoint cartilage and
for its ability to resist compression (9). The biologic
function of the aggregate has yet to be defined, but by
ddtons) (lo),
virtue of its enormous size (over50 x
it presumably serves to contain the PG within the
collagen meshwork of the cartilage.
We have recently shown that the atrophy of
knee cartilage which develops when the hind limb of a
normal adult dog is immobilized in a cast for 6 weeks is
accompanied by decreases in uronic acid content and
net PG synthesis (7). Furthermore, newly synthesized
PG from cartilage of the immobilized limb did not
aggregate in vitro, due to an apparent abnormality in
the HA-binding region of the PG core protein (7). All
of these effects of immobilization were rapidly reversible if the casts were removed and the dogs were then
allowed to ambulate ad libitum “on all fours” for 3
1330
PALMOSKI AND BRANDT
weeks. The present study was designed t o extend that
observation b y examination of the effect of immobilization on total tissue proteoglycans and t o answer an
important question: Do chondrocyte injury and articular cartilage damage result if a limb is vigorously
exercised immediately following a period of immobility-at a stage when the cartilage is atrophic and the
macromolecular organization of the matrix is defective?
MATERIALS AND METHODS
Source of tissue and procedure for immobilization and
exercise. The right hind limbs of 7 adult mongrel dogs (25-30
kg) were immobilized against the trunk in a plaster cast with
90" Aexion of hip and knee. The dogs were thus able to
ambulate on three legs but bore no weight on the immobilized limb for 6 weeks, after which the casts were removed.
Two of the animals were killed immediately with an overdose of sodium pentothal, while the remaining 5 dogs were
killed 3 weeks after removal of the casts. During this
interval, 2 of the dogs were allowed to ambulate ad libitum
while 3 were placed on a vigorous treadmill exercise program (6 miledday at 3 mph).
Immediately after killing, the right and left knees of
each animal were opened aseptically and the distal femurs
removed with a bone saw. Representative samples of cartilage from the experimental (right) knee and contralateral
control knee were taken with a Craig biopsy needle (internal
diameter, 3 mm) from the central portion of each medial
condyle for histologic study. The cartilage was then removed
with a scalpel from the weight-bearing portions of both
femoral condyles of each joint (250-300 mg wet weight) and
cut into slices less than 0.5-mm thick. All the cartilage from
each joint was pooled separately. Approximately 20 mg (wet
weight) of the sliced cartilage was taken for analysis of dry
weight and uronic acid content and approximately 50 mg
(wet weight) for tissue culture experiments, while the remainder was used for sequential extraction of PG (see
below).
Tissue culture. Cartilage slices from each joint 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 Na235S04 (10 pCi/ml) (New England
Nuclear Corporation, Boston, MA), and were incubated for
18 hours at 37" C in a mixture of air-C02 (19: I). Following
incubation, the medium was decanted and the tissue was
washed twice with 3-ml portions of cold Ham's F-12 nutrient
mixture. The spent medium and washes were combined and
dialyzed against 200 volumes of O.05M 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 PG were extracted and isolated.
Sequential extraction of PG. PG were extracted in
sequential fashion from the labeled and unlabeled cartilage
slices with 0.4M guanidinium chloride (GuHCI) and then
with 4.OM GuHCI, as previously described (7). Both solvents contained the protease inhibitors ethylenediamenetetraacetic acid (O.OlM), 6-aminohexanoic acid (0. lM), and
benzamidine hydrochloride (0.005M).
After dialysis against distilled water, the uronic acid
content of aliquots of the 0.4M and 4.OM GuHCl extracts
was determined (12). The tissue which remained after the
two sequential extractions was digested with pronase and
the uronic acid content of the residual glycosaminolycans
(GAG) in the pronase digest was analyzed following precipitation with 9-aminoacridine hydrochloride, as reported previously (7).
In the tissue culture experiments, samples of the
spent culture medium, of the sequential 0.4 and 4.OM GuHCl
extracts, and of the pronase digest were dialyzed against
distilled water. Then 0.1 ml aliquots were added to ReadySolv HP (Beckman Instruments, Inc., Fullerton, CA) and
counted in a Beckman liquid scintillation spectrometer.
Results were adjusted for differences in the wet weight of the
tissues. Net PG synthesis was determined from the sum of
the nondialyzable radioactivities in the medium, the sequential GuHCl extracts, and the pronase digest.
Isolation and purification of aggregated and disaggregated PG. PG in the medium and 4.OM GuHCl extract were
isolated by equilibrium density gradient centrifugation in
cesium chloride under associative conditions favoring forMtion of PG aggregates (14). The bottom 215 of the gradient
(d 2 1.76 g d m l ) was recovered and dialyzed against several
changes of 0.05M sodium acetate, pH 6.9, to yield fractions
from the medium and from the GuHCl extract which were
designated
and AG,, respectively.
To obtain purified disaggregated PG, aliquots of
fraction AG,, were made 4.OM with respect to GuHCl and
subjected to a second cesium chloride density gradient
centrifugation (di = 1.50 gdm l ). The bottom 215 of this
gradient (d 2 1.59 g d m l ) , which contains disaggregated PG
(141, was dialyzed exhaustively at 4°C against O.05M sodium
acetate, pH 6.9, and designated fraction AcuDGu.
Digestion of fraction Acu with HA p1+3 hydrolase.
One-milliliter aliquots of fraction AG,, were digested with HA
p1-3 hydrolase (Biotrics, Inc., Arlington, MA) as previously described (7), and Sepharose 2B chromatographs of the
PG before and after incubation were compared. The lack of
activity of the enzyme against purified disaggregated PG was
confirmed by its failure to alter the Sepharose 2B elution
profile or the viscometric properties of fraction AG,,DG" (nsp
= 2.58) after the latter had been treated with the hyaluronidase as above.
Interaction of fraction AG,,DG,,with HA. To assess the
ability of "S-PG to undergo aggregation in vitro, fraction
AG~DG,,
was incubated with high molecular weight HA from
human umbilical cord (Sigma Chemical Co., St. Louis, MO)
as previously described (7). Elution profiles (35S)of the PG
in the presence and absence of HA were compared.
Gel chromatography. Samples (0.5 ml) of PG in 0.5M
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
mlhour. Radioactivities (35S)or uronic acid concentrations
of 1-ml effluent fractions were determined and the partition
coefficient (kav)of the PG sample was calculated from the
RUNNING AND CANINE KNEE CARTILAGE ATROPHY
1331
formula: K,, = (V, - Vo)/(Vt - Vo), in which V, was taken
as the peak fraction in the elution diagram, Vo as the void
volume, and Vt as the total column volume.
Analytic methods. For determination of tissue dry
weight, portions of cartilage (approximately 20 mg wet
weight) were placed in two changes of acetone for 48 hours
and then dried to constant weight in vacuo at 80°C. The dried
cartilage was digested with pronase as above, after which the
GAG were isolated by precipitation with 9-aminoacridine
and converted to their sodium salts with Bio-Rad AG-SO
(Na+) (13). After the resin was removed by filtration, the
uronic acid content of the filtrate was determined (12).
Histologic examination. A full-thickness sample of the
cartilage, including the underlying bone, was obtained with a
Craig biopsy from the medial femoral condyle of each joint.
Microscopic sections 6p in thickness were examined after
staining with Safranin 0-fast green. The cartilage depth from
surface to tidemark was measured with a reticule eyepiece
attachment.
RESULTS
Gross observations. Articular cartilage from
both knees of each animal appeared grossly normal
and was white and smooth with no softening, pitting,
or osteophytes. None of the knee joints contained a
synovial effusion, and the synovial membranes were
normal with no intracapsular adhesions. No difference
was apparent between the cartilage from the immobilized knee (IK) of dogs which had been killed immediately after 6 weeks of cast wear, dogs allowed to
ambulate ad libitum for 3 weeks after removal of the
cast (IK-AL), or dogs exercised on a treadmill for 3
weeks after removal of the cast (IK-TM), and cartilage
from the contralateral (nonimmobilized) knee of the
same animals (CK, CK-AL, and CK-TM, respectively).
Histology and histochemistry. In all samples, the
cartilage surface and tidemark were intact. Fibrillation
was uniformly absent. After 6 weeks of immobilization. a marked reduction in Safranin 0 staining was
evident in IK cartilage accompanied by a decrease in
depth (Figure 1, Table 1). Thus, IK cartilage from
Dogs 1 and 2 was only 75% and 67%. respectively, as
thick as the cartilage from the contralateral knee,
(Table 1). Three weeks after removal of the casts,
however, in the dogs that had been allowed to ambulate ad libitum, Safranin 0 staining was normal, and
the thickness of IK-AL cartilage was comparable to
that of the control cartilage (Dogs 3 and 4) (Figure I ,
Table 1 ) . In marked contrast, IK-TM cartilage from
Dogs 5, 6, and 7, which had been subjected to treadmill exercise after cast removal, showed moderate to
marked reduction in Safranin 0 staining and was about
Figure 1. Cartilage from A. Dog 1 , control (left) knee; B Dog I ,
right knee. immobilized in a cast for 6 weeks; C, Dog 3, control (left)
knee; D, Dog 3, right knee, immobilized in a cast for 6 weeks,
followed by 3 weeks of ambulation ad libitum; E, Dog 5 , control
(left) knee; F. Dog 5 , right knee immobilized in a cast for 6 weeks,
followed by 3 weeks of treadmill exercise.
20% thinner than that of the corresponding CK-TM
cartilage (Figure 1 , Table 1 ) .
Water and uronic acid contents. The water content of IK cartilage from Dogs 1 and 2 killed immediately following the period of casting was 9% and 6%
greater, respectively, than that of cartilage from the
contralateral knee, while the uronic acid content was
about 20% less than that of the controls (Table 1). In
1332
PALMOSKI AND BRANDT
Table 1. Characterization of knee cartilage from dogs which had borne casts for 6 weeks
Interval,
cast removal
t o killing
Treadmill
exercise
Cartilage
thickness
(mm)
Source of
cartilage*
Animal
Safranin 0
staining
Water
content
(% of tissue
wet weight)
Uronic acid
content
(% of tissue
dry weight)
Normal
Marked decrease
Normal
Marked decrease
Normal
Normal
Normal
Normal
Normal
Marked decrease
Normal
Marked decrease
Normal
Moderate decrease
72.1
78.5
71.9
76.5
71.3
72.5
72.0
73.2
72.0
75. I
73.3
76.2
71.1
77.6
5.1
4.0
4.2
3.3
3.6
3.7
3.9
4.0
4.4
2.5
3.8
2.6
3.9
3.2
________I__
Dog 1
None
No
3 weeks
No
(
Dog 3
Dog4
Dog 5
3 weeks
0.80
0.60
0.90
0.60
0.95
I .OO
0.70
0.70
0.90
0.75
0.80
0.65
0.85
0.70
CK
IK
CK
1K
CK-A I,
I K-A L
CK-AI,
IK-AL
CK-TM
IK-TM
CK-TM
IK-TM
CK-TM
IK-TM
Yes
* IK = immobilized knee of dog killed immediately after removal of cast: CK = contralateral control knee of same animal. IK-AL =
immobilized knee of dog permitted to ambulate ad libitiim “on all fours” for 3 weeks after cast removal but before killing; CK-AL =
contralateral control knee of same animal. IK-TM = immobilized knee of dog which was wbjected to daily treadmill exercise (see text) for 3
weeks after cast removal and before killing; CK-TM - contralateral control knee of zame animal.
contrast, water and uronic acid contents of the IK-AL
cartilage from Dogs 3 and 4 were essentially the same
as those of the CK-AL cartilage. However, the water
content of IK-TM cartilage of Dogs 5 , 6 , and 7, which
had been subjected to treadmill exercise, remained
greater (4%, 3%, and 9%) than that of the control
cartilage, and the uronic acid content was decreased to
57%, 68%, and 82%, respectively, of that of the CK-
TM cartilage from these dogs (Table 1). Thus, the
decreases in uronic acid content of IK-TM cartilage
tended to be greater than those in IK cartilage.
Sequential extraction of PG from the cartilage.
No difference was seen between control cartilage and
cartilage from knees which had been immobilized
(regardless of usage following immobilization) with
respect to the distribution of newly synthesized (35S)
Table 2. Distribution of total tissue proteoglycans between sequential 0.4M and 4.OM guanidinium chloride extracts and pronase digest of the
cartilage residue
% of total tissue uronic acid contained
in sequential extracts of the tissue
Interval, cast
removal to killing
Treadmill
exercise
None
No
3 weeks
No
3 weeks
Yes
.
-
Animal
(
{
1
_
_
.
_
I
_
Dog 1
Dog2
Dog 3
Dog4
Dog 5
Dog 6
Dog 7
Source of
Cartilage*
0.4M GuHCl
extract
4.0M GuHCl
ex tract
Cartilage
residue
CK
IK
CK
IK
CK-AL
IK-AL
C K-AL
IK-AL
CK-TM
IK-TM
CK-TM
IK-TM
C K-TM
IK-’I’M
________
1
1
2
3
7
9
3
4
I
1
5
4
7
13
71
65
65
70
78
71
67
70
68
62
69
64
73
71
28
34
32
27
15
20
30
26
31
37
26
32
20
16
__
* IK = immobilized knee of dog killed immediately after removal of cast which had been borne for 6 weeks; CK = contralateral control knee
of same animal. I L A L = immobilized knee of dog which had borne a cast for 6 weeks, then was permitted to ambulate ad libitum “on all
fours” for 3 weeks before killing; CK-AL = contralateral control knee of same animal. IK-TM = immobilized knee of dog which had borne a
cast for 6 weeks, then was subjected to daily treadmill exercise (see text) for 3 weeks before killing: CK-TM = contralateral control knee of
same animal.
1333
RUNNING AND CANINE KNEE CARTILAGE ATROPHY
or total tissue (uronic acid) PG between the various
sequential extracts of the cartilage. Thus, in each
tissue culture experiment. the medium contained
about 10-20% of the total nondialyzable 35S material
and the 0.4M GuHCl extract held about 1-6%, while
the greatest proportion (45-60%) of the total 3'S-PG
was accounted for in the 4.0M GuHCl extract. Similarly, in every case. about 70% of the total tissue PG
(uronic acid) was extracted with 4.0M GuHCI, while
only 1-1396 was present in the 0.4M GuHCl extract
(Table 2).
Net proteoglycan synthesis. Rased on the sum of
the nondialyzable "S radioactivities in the culture
medium, sequential GuHCl extracts, and pronase digest of the cartilage residue immediately following 6
weeks of casting, net PG synthesis in IK cartilage of
Dogs I and 2 was suppressed to levels of 69% and
5696, respectively. of the control values. On the other
hand, net PG synthesis in IK-AL cartilage from Dogs
3 and 4, which were permitted 3 weeks of ad libitum
exercise following removal of the cast, returned to
control levels. Notably, PG synthesis in IK-TM cartilage from Dogs 5 and 7, subjected to treadmill running.
was 125% and 117% greater, respectively, than that in
control cartilage, while that in IK-TM cartilage of Dog
6 increased to a lesser extent (107% of control).
PG aggregation. In each experiment fraction
AMedaccounted for about 40% of the total nondialyzable 35Sradioactivity in the culture medium. Since the
medium contained about 10-2095 of the total nondialyzable "S material, AMedrepresented only about 48% of the total "S-PG and it was not analyzed further.
Similarly, since the 0.4M GuHCI extracts represented
only 1-13% of the total PG ("S and uronic acid). no
attempt was made to characterize them.
Fraction A(;,, represented about 80-90% of the
total nondialyzable 3sS counts per minute (cpm) or
uronic acid in all 4.0M GuHCl extracts and thus
contained over 65% of the total labeled and unlabeled
PG extracted from the cartilage. About 24-43% of the
3sS-AGusamples from the control tissues and 30-32'3
from IK-AL cartilage were large enough in average
hydrodynamic size to be excluded from Sepharose 2R,
while the K,, of the PG which were retarded by the gel
ranged from 0.15 to 0.42 (Figure 2. Table 3). I n
contrast, none of the 35S-AG11samples from I K and
IK-TM cartilage contained any PG which eluted in the
Sepharose 2B void volume. The K,, of the retarded
PG in these samples. however, were not substantially
different from those of the corresponding CK and CKTM cartilage.
A
m
0
r
X
E
2
6
W
E F F L U E N T VOLUME ( m l )
Figure 2. Chromatography on a column (95 x 1.0 crn) of Sepharose
2B of "S-proteoglycans prepared under associative conditions
(fraction A(;,,) (--)
and of the same fraction after incubation with
hyaluronic acid P I 4 3 hydrolase (------). The "S radioactivity of I -
ml fractions was determined. A , cartilage from right knee of Dog I
which had been casted for 6 weeks: B, cartilage from right knee of
Dog 4 which had been casted for 6 weeks and permitted to ambulate
ad libitum for 3 weeks after removal of the cast; C , cartilage from
right knee of Dog 7 which had been casted for h weeks and subjected
to a treadmill exercise regimen for 3 weeks after removal of the cast.
The proportion of the total tissue AGu fraction
from IK and IK-TM cartilage which was excluded
from Sepharose 2B was substantially less than that of
the corresponding controls (Table 4). Thus, only 10%
and 25% of AGu from IK cartilage of Dogs I and 2,
respectively, but 35% and 4l%, respectively. of AG,,
from CK cartilage of these animals eluted in the
Sepharose 2B void volume. Similarly, the proportion
of fraction Acil, from IK-TM cartilage (Dogs 5. 6, and
7) present in the void volume peak was only about onehalf that from the CK-TM samples (Table 4). In
contrast, elution profiles of
from IK-AL cartilage
of Ilogs 3 and 4 were quite similar to those of CK-AL
cartilage ('Table 4).
PALMOSKI AND BRANDT
1334
Table 3. Sepharose 2B chromatography of 'S-proteoglycans extracted from canine knee cartilage of immobilized and contralateral limbs with
4.0M guanidinium chloride and purified under associative and dissociative conditions
Interval.
cast
removal
to killing
Treadmill
exercise
None
No
3 weeks
No
Animal
(
(
Dog I
Dog2
Dog 3
Dog4
Dog 5
3 weeks
Dog 7
Source of
cartilage*
CK
IK
CK
IK
CK-AL
IK-AL
CK-AL
IK-AL
CK-TM
IK-TM
CK-TM
IK-TM
C K-TM
1K-'TM
Fraction Aciu t
HA p l+3 hydrolase
Fraction A<;,,
Fraction AG,,Dc,,,
Fraction AtiuDtiu +
hyaluronic acid -
% Vo
Ka,,*V,
76 V,,
Kzlv.V,
%I V,,
Kav- Vr
7% Vo
K,,, V,
25
0
43
0
25
30
35
32
24
0
30
0
40
0
0.42
0.38
0.20
0.35
0.3 I
0.30
0.35
0.34
0
0
0.42
0.40
0.38
0.35
0.32
0.35
0.35
0.32
0.40
0.42
0.41
0.40
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.43
0.42
0.40
0.37
0.35
0.35
0.38
0.35
0.45
0.42
0.38
0.35
0.40
0.40
35
0
50
0
30
25
38
35
25
0
35
0
44
0
0.42
0.42
0.44
0.40
0.35
0.38
0.3s
0.34
0.40
0.45
0.36
0.38
0.40
0.42
0.15
0.38
0.32
0. 28
0.40
0.38
0
0
0
0
0
0
0
0
0
0
* IK = immobilized knee of dog killed immediately after removal of cast which had been borne for 6 weeks; CK = contralateral control knee
of same animal. IK-AL = immobilized knee of dog which had borne a cast for 6 weeks. then was permitted to ambulate ad libitum "on all
fours" for 3 weeks before killing; CK-AL = contralateral control knee of same animal. IK-TM = immobilized knee of dog which had borne a
cast for 6 weeks, then was subjected to daily treadmill exercise (see text) for 3 weeks before killing: CK-TM = contralateral control knee of
same animal
Incubation with H A P I 4 3 hydrolase. an enzyme specific for H A . eliminated all 35S and uronic
acid-containing material in the Sepharose ZB void
volume (Figure 2. Tables 3 and 4) and strongly suggested that this material represented PG associated
with HA. In most cases, the elution profiles of PG
which were retarded by the gel were unaltered by
incubation with the enzyme; this indicated that they
were not aggregated. However, in 2 cases (CK from
Dog 2, and CK-TM from Dog 5), treatment of 35S-Ac;U
with the hyaluronidase resulted in a slight increase in
the K,, of those PG retarded by the gel; this suggested
that some relatively small aggregates were present.
PG-HA interaction in vitro. Since insufficient
amounts of unlabeled AG,, were available, A C ~ D ~ ; "
fractions were prepared only from the "SAG,, fractions. In every case, exposure of the control 35S-AGu
fraction to the dissociative conditions of the second
cesium chloride gradient eliminated the Sepharose 2B
void volume peak. The K,, of the retarded PG in the
AGUD~,,
fractions were virtually identical to those of
the retarded PG seen after treatment of the corresponding AG,, with H A P1+3 hydrolase. AouDc,, from
all samples of control cartilage and from IK-AL
cartilage interacted extensively with HA in vitro since
25-50% of the sample was excluded from Sepharose
2B after incubation with H A (Table 3). In contrast.
incubation with H A had no effect on the elution
profiles of & j U D G U from either IK or IK-TM cartilage.
DISCUSSION
The present results confirm our previous observations (7) that immobilization of the knee of a normal
dog by casting results in rapid degeneration of articular
cartilage. This degradation is characterized by decreases in both the thickness of the tissue and its
uronic acid PG content, an increase in the water
content, reduction in net PG synthesis, and defective
aggregation of newly synthesized "S-PG.
As noted previously (7), when the period of
immobilization was confined to 6 weeks, the defects
were promptly reversible with ad libitum ambulation.
In other studies (15), we described changes in knee
cartilage after amputation of the ipsilateral paw; these
changes were identical to those produced by immobilization. Notably, the cartilage degeneration in the paw
transection model developed in the presence of a
normal arc of knee joint movement. This strongly
suggested that the changes arising from immobilization
were not due simply to a lack of joint motion but to
reduction in the loading of the cartilage that results
from contraction of the muscles spanning the joint.
The reduced load stabilizes the limb in the stance
phase of gait.
The present study indicates that after 6 weeks
of immobilization, the proportion of newly synthesized PG as well as the total tissue proteoglycans
(uronic acid) capable of interacting with HA to form
1335
RUNNING AND CANINE KNEE CARTILAGE ATROPHY
Table 4. Effect of hyaluronic acid pl+3 hydrolase on Sepharose 2B chromatographs of total tissue
proteoglycans extracted with 4.0M guanidinium chloride
Interval,
Fraction .AGu t
cast
Fraction A<;”
HA PI-33 hydrolase
removal
Treadmill
Source of
to killing
exercise
Animal
cartilage*
% VO
KUv,V,
$6 Vo
K,,,, V,
None
No
3 weeks
No
(
[
Dog 1
Dog 2
Dog3
Dog4
Dog5
3 weeks
Dog6
Dog 7
CK
35
IK
10
CK
1K
CK-AL
IK-AL
CK-AL
IK-AL
CK-TM
I K-TM
CK-TM
IK-TM
CK-TM
IK-TM
41
25
32
29
37
40
36
20
35
20
33
18
0.48
0.44
0.42
0.45
0.40
0.43
0.41
0.39
0.21
0.29
0.32
0.3 I
0.32
0.35
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.46
0.47
0.42
0.46
0.43
0.47
0.41
0.42
0.35
0.32
0.35
0.33
0.34
0.35
* IK = immobilized knee of dog killed immediately after removal of cast which had been borne for 6
weeks; CK = contralateral control knee of same animal. IK-AL = immobilized knee of dog which had
borne a cast for 6 weeks then was permitted to ambulate ad libitum “on all fours” for 3 weeks before
killing; CK-AL = contralateral control knee of same animal. IK-TM = immobilized knee of dog which
had borne a cast for 6 weeks, then was subjected to daily treadmill exercise (see text) for 3 weeks
before killing; CK-TM = contralateral control knee of same animal.
aggregates are diminished. Furthermore, when the
joint was subjected to vigorous exercise, the chondrocytes in the IK-TM cartilage of Dogs 5 , 6, and 7 were
unable to repair the atrophic matrix despite an increase in net PG synthesis.
Immobilization of rabbit knees in extension is
reported to cause morphologic changes similar to
those in osteoarthritis (OA) (16). It should be emphasized that, in the present study, the morphologic
changes which developed as a result of immobility and
those which developed following remobilization were
not the changes of OA. Neither osteophytes, fibrillation, nor pitting of the cartilage was seen. On the other
hand, the increase in cartilage water content which
was encountered in the immobilized joints is a feature
of OA and occurs in the earliest stages of the disease
(17,18). It implies a failure in the elastic restraint of the
collagen network of the cartilage which enables the PG
to swell to a higher degree of hydration than normal
(19). In OA, the increase in water content is accompanied by supernormal extractability of the PG, as
reflected by the fact that an abnormally high proportion of the total PG may be solubilized with low ionic
strength solvents (20-22), and the proportion of 35SPG released into the medium of OA cartilage cultures
is greater than that of controls (23). Notably, despite
the increase in water content of IK and IK-TM
cartilage in the present study, the extractability of the
PG was normal (Table 2).
The decrease in net PG synthesis in IK cartilage
provides a further contrast with the findings in OA,
where net PG synthesis appears to increase until the
disease is far advanced. This presumably reflects an
attempt at repair by the chondrocyte (24). In IK-AL
cartilage, where the atrophic changes in the cartilage
had been reversed, PG synthesis and uronic acid
content were normal. However, in IK-TM cartilage,
in which failure of repair was apparent, the uronic acid
content of the IK-TM cartilage was low despite the
increase in PG synthesis. Thus, the rate of PG degradation in IK-TM cartilage must have been even greater than the rate of synthesis. It is possible that the loss
of PG from LK-TM cartilage, by whatever mechanism
(e.g., increased degradation, diffusion out of the matrix due to defective interaction with HA and/or glycoprotein link, or with collagen), reduced the normal
feedback inhibition on the chondrocyte which controls
the composition of the extracellular matrix (25,261.
The loss of aggregation of newly synthesized
35S-PG in IK and IK-TM cartilage appeared complete
since I ) none of the AGusamples contained PG large
enough to elute in the Sepharose 2B void volume, 2)
no change occurred in the elution profiles of the 35SAGu fraction after incubation with HA (31-3 hydro-
1336
lase, and 3) fractions Acu and AG,,DG"were similar in
average hydrodynamic size. In contrast, not all of the
total tissue PGs were disaggregated after 6 weeks of
immobilization (IK cartilage) , even when followed by
3 weeks of vigorous exercise (IK-TM cartilage), as
indicated by comparison of the Sepharose 2B chromatographs before and after digestion of AGu with
hyaluronic acid p1+3 hydrolase (Table 4).
This observation and the fact that the dissociated 35S-PG and total tissue PG from IK and IK-TM
cartilage were comparable in hydrodynamic size to
those in AGuDGufrom the contralateral control knee
suggest that the aggregation defect was not solely due
to an increase in nonspecific degradative enzyme
activity such as might have resulted from generalized
cell death. The aggregation defect presumably resulted
from limited degradation of the HA binding region at
the N-terminus of the PG core protein (27), since the
PG did not interact with HA in vitro (Table 3) but were
nonetheless comparable in size to PG that were aggregated from control knees. The present results, however, do not preclude additional defects in the link
glycoproteins or cartilage HA.
The relatively small differences among control
A G samples
~
with respect to the proportion excluded
from Sepharose 2B (Tables 3 and 4) presumably reflect
individual variation in PG aggregation in vivo since the
procedures used to extract the PG and prepare A G ~
from the tisues of each dog were identical. They could,
however, reflect differences in the amount of HA
extracted with 4.0M GuHCI, since others have shown
(28) and we have confirmed in unpublished observations that incubation of A G u fractions with HA in vitro
may increase the proportion of PG present in aggregates.
In some experiments with control cartilage, the
proportion of 35S-AGu (Le., newly synthesized PG)
excluded from Sepharose 2B was less than that of the
A G fraction
~
prepared from the whole tissue (uronic
acid) (Tables 3 and 4). Oh the other hand, while all of
the 35S-AGufrom IK or IK-TM joints was retarded by
Sepharose 2B, some of the nonlabeled AG,, prepared
from every IK and IK-TM joint eluted in Vo (Table 4)
although the amount of Vo material was less than that
seen with the corresponding control AGu. These findings are consistent with observations of Sandy et al
(291, who showed that the proportion of newly synthesized PG incapable of interacting with HA in vitro in
normal joint cartilage is greater than that of PG synthesized earlier. Since PG subunits are more susceptible
to proteolytic degradation than PG in aggregates (27),
PALMOSKI AND BRANDT
limited cleavage of the HA binding region of 35S-PG in
IK and IK-TM cartilage by neutral or acid proteases
(30) may have occurred prior to their interaction with
HA.
The present results show that when the integrity of the extracellular matrix of articular cartilage has
been diminished by immobilization, it may be vulnerable to loading during subsequent exercise and that may
damage the chondrocyte and affect its capacity for
repair. These data may have important clinical implications. Studies are currently in progress to determine
whether the experimental conditions employed in this
study or more prolonged periods of treadmill exercise
in previously casted dogs may lead to osteoarthritis.
ACKNOWLEDGMENTS
We would like to thank Jeffrey Wilson for technical
assistance and Margaret Britner for secretarial support.
REFERENCES
1 . Enneking WF, Horowitz M: The intra-articular effects
of immobilization on the human knee. J Bone Joint Surg
54A:973-985, 1972
2. Roy S: Ultrastructure of articular cartilage in experimental immobilization.Ann Rheum Dis 29:637-643,1970
3. Troyer H: The effect of short-term immobilizationin the
rabbit knee joint cartilage. Clin Orthop 107:249-257,
1975
4. Akeson WH, Woo SLY, Amiel D, Coutts RD, Daniel D:
The connective tissue response to immobility: biochemical changes in periarticular connective tissue of the
immobilized rabbit knee. Clin Orthop 93:356-362, 1973
5. Salter RB, Field P: The effect of continuous compression on living articular cartilage. J Bone Joint Surg
42A:31-49, 1960
6 . Caterson B, Lowther D: Changes in the metabolism of
the proteoglycans from sheep articular cartilage in re-
sponse to mechanical stress. Biochim Biophys Acta
540:4 12-422, 1978
7. Palmoski MJ, Perricone E, Brandt KD: Development
and reversal of a proteoglycan aggregation defect in
normal canine knee cartilage after immobilization. Arthritis Rheum 22508-517, 1979
8. Hascall VC: Interaction of cartilage proteoglycans with
hyaluronic acid. J Supramol Struct 7: 101-120, 1977
9. Kempson GE, Muir H, Swanson SAV, Freeman MAR:
Correlations between the compressive stiffness and
chemical constituents of human articular cartilage. Biochim Biophys Acta 215:70-77, 1970
10. Muir H: The chemistry of the ground substance of joint
cartilage, The Joints and Synovial Fluid. Vol 2. Edited
by L Sokoloff. New York, Academic Press, 1980, pp 2794
RUNNING AND CANINE KNEE CARTILAGE ATROPHY
1 1 . Oegema TR, Hascall VC, Dziewiatkowski DD: Isolation
12.
13.
14.
15.
16.
17.
18.
19.
and characterization of proteoglycans from the swarm
rat chondrosarcoma. J Biol Chem 250:6151-6159, 1975
Bitter T, Muir H: A modified uronic acid carbazole
reaction. Anal Biochem 4:330-340, 1962
Tsiganos CD. Muir H: Studies on protein-polysaccharide from pig laryngeal cartilage: extraction and purification. Biochem J 113:879-884, 1969
Hascall VC, Sajdera SW: Protein polysaccharide complex from bovine nasal cartilage: the function of glycoprotein in the formation of aggregates. J Biol Chem
2242384-23%, 1969
Palmoski MJ, Colyer R , Brandt K: Joint motion in the
absence of normal loading does not maintain normal
articular cartilage. Arthritis Rheum 23:325-334, 1980
Langenskiold A. Michalsson JE, Videman T: Osteoarthritis of the knee in the rabbit produced by immobilization. Acta Orthop Scand 5O:l-14, 1979
Bollet AJ, Nance JL: Biochemical findings in normal
and osteoarthritic articular cartilage: chondroitin sulfate
concentration and chain length, water, and ash contents.
J Clin Invest 45: 1170-1 177, 1966
McDevitt CA, Gilbertson FMM, Muir H: An experimental model of osteoarthritis: early morphological and
biochemical changes. J Bone Joint Surg 59B:24-35, 1977
Maroudas A: Physico-chemical properties of articular
cartilage, Adult Articular Cartilage. Edited by MAR
Freeman. New York, Grune and Stratton, 1973, pp 131I 7n
. I "
20. Brandt KD: Enhanced extractability of articular cartilage proteoglycans in osteoarthrosis. Biochem J
143:475-478, 1974
21. McDevitt CA, Muir H: Biochemical changes in the
cartilage of the knee in experimental and natural osteoarthritis in the dog. J Bone Joint Surg 58B:94-301, 1976
1337
22. Moskowitz RW. Howell DS, Goldberg VM, Muniz 0,
Pita JC: Cartilage proteoglycan alterations in an experimentally induced model of rabbit osteoarthritis. Arthritis Rheum 22: 155-163, 1979
23. Palmoski MJ. Colyer R, Brandt KD: Marked suppression by salicylate of the augmented proteoglycan synthesis in osteoarthritic cartilage. Arthritis Rheum 23:83-91,
I980
24. Mankin HJ: The reaction of articular cartilage to injury
and osteoarthritis. N Engl J Med 291:1335-1340, 1974
25. Wiebkin OW, Hardingham TE, Muir H: Hyaluronic
acid-proteoglycan interaction and the influence of hyaluronic acid on proteoglycan synthesis by chondrocytes
from adult cartilage, Extracellular Matrix Influences on
Gene Expression. Edited by HC Slavkin, PM Johnson.
New York, Academic Press, 1975, pp 209-223
26. Handley CJ, Lowther DA: Extracellular matrix metabolism by chondrocytes. 111. Modulation of proteoglycan
synthesis by extracellular levels of proteoglycan in cartilage cells in culture. Biochim Biophys Acta 500: 132-139,
1977
27. Heinegard D, Hascall VC: Aggregation of cartilage
proteoglycans. 111. Characteristics of the proteins isolated from typsin digests of aggregates. J Biol Chem
249:4250-4256, 1974
28. Oegema TR, Behrens F: Structural alterations in proteoglycans from steroid suppressed articular cartilage.
Trans Orth Res SOC26:125, 1980
29. Sandy JD, Brown HIdG, Lowther DA: Degradation of
proteoglycan in articular cartilage. Biochim Biophys
Acta 543536-544, 1978
30. Howell DS: Degradative enzymes in osteoarthritic human articular cartilage. Arthritis Rheum 18:167-177,
1975
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