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Development and Reversal of a Proteoglycan Aggregation Defect In Normal Canine Knee Cartilage After Immobilization.

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508
DEVELOPMENT AND REVERSAL OF A
PROTEOGLYCAN AGGREGATION DEFECT IN
NORMAL CANINE KNEE CARTILAGE AFTER
IMMOBILIZATION
MARSHALL PALMOSKI, ELAINE PERRICONE, and KENNETH D. BRANDT
Healthy adult dogs were studied for a defect in
proteoglycan aggregation by immobilizing one limb for
varying periods of time. Immobilization for 6 days resulted in a 41% reduction in proteoglycan synthesis by
articular cartilage from the restrained knee compared
with the contralateral control knee. After 3 weeks of immobilization, proteoglycan aggregation was no longer
demonstrable in cartilage from the constrained limb.
The aggregation defect was rapidly reversible and aggregates were again normal size 2 weeks after removal
of a cast that had been worn for 6 weeks.
In normal articular cartilage most of the proteoglycans (PG) exist in large aggregates that are noncovalently linked to hyaluronic acid (HA) (1). In osteoarthritis (OA) an aggregation defect exists within the
tissue, since a greater than normal proportion of the
PGs is not aggregated and aggregates which are present tend to be smaller than normal (2-5).
Aggregation defects similar to those of OA in
From the Rheumatology Division, Indiana University
School of Medicine, Indianapolis, Indiana 46202.
Supported in part by grants from the National Institute of
Arthritis, Metabolic and Digestive Diseases (AM-20582), the National
Institutes of Health, Division of Research Resources ( 5 SO7 RR
0537I), and the Grace M. Showalter Trust.
Marshall Palmoski, PhD: Assistant Professor of Medicine;
Elaine Perricone, MS:Research Assistant; Kenneth D. Brandt, MD:
Professor of Medicine and Head, Rheumatology Division, Indiana
University School of Medicine.
Address reprint requests to Marshall J. Palmoski, PhD,
Rheumatology Division, Indiana University School of Medicine, 1 100
West Michigan Street, Indianapolis, Indiana 46202.
Submitted for publication August 23, 1978; accepted in revised form December 26, 1978.
Arthritis and Rheumatism, Vol. 22, No. 5 (May 1979)
morphologically and histochemically normal hip cartilage from aged humans have been recently demonstrated by the authors (6). In other samples from aged
individuals (4), however, and in cartilage from younger
persons (less than 50 years old), PG aggregation has
been normal. Neither the prevalence of aggregation abnormalities in articular cartilage nor their relation to
age is currently known, partly because joint cartilage
from younger individuals is not readily available for
study.
Recently the authors had an opportunity to
study articular cartilage from three patients who were
14, 15, and 30 years old, none of whom had apparent
joint disease. In each case it was anticipated that
marked PG-HA interaction would be demonstrable. In
all three samples, however, profound aggregation defects were found (7). Upon review of the clinical data it
became apparent that in each case the cartilage was obtained from a joint which had not been'used for some
time. In one instance elbow cartilage had been procured
from a limb paralyzed for 9 months by a brachial plexus
injury. In another instance, knee cartilage had been obtained from a patient with a tibia1 sarcoma remote from
the joint who, because of pain, had walked very little for
4 months. In the third case, the cartilage was from the
hip of a patient with spastic diplegia who had never
borne full weight on his legs.
Whether immobilization alone produces changes
in PG aggregation in articular cartilage has not previously been examined. Prompted by the results described above, the present study was undertaken to investigate the effects of immobilization on the
metabolism and macromolecular organization of PGs in
dog knee cartilage.
5 09
PROTEOGLYCAN AGGREGATION DEFECT
MATERIALS AND METHODS
Source of tissue and immobilization procedure. The
right hind limbs of 4 large mature dogs were immobilzed
against the trunk in Lightcast I1 (Merck, Sharpe and Dohme
Co., Oakbrook, Illinois), with ,90" flexion of hip and knee.
The dogs were able to ambulate on three legs but bore no
weight on the immobilized extremity for 6 days, 3, 6, or 8
weeks, after which they were killed with an overdose of sodium pentothal. Three additional dogs were used whose right
hind limbs were immobilized in the same manner for 6 weeks,
following which the cast was removed and the animals were
allowed to ambulate for 1, 2, or 4 weeks prior to killing.
X-rays of both hind limbs were taken before immobilization and at the time of killing. Immediately after death the
knee joints were opened aseptically and the femoral condyles
and patellas were removed. Representative portions of articular cartilage from immobilized and control knees were obtained for histologic study with a Craig biopsy needle. Other
portions (approximately 10 mg) were taken for determination
of dry weight and uronic acid content, while the remainder
was cut into slices less than 0.5 mm thick. Cartilage from the
femoral condyle and patella of each joint was pooled separately.
Tissue culture. The cartilage slices were placed in medium consisting of Ham's F-12 nutrient mixture, pH 7.4 (8
ml), and fetal calf serum (2 ml). Streptomycin (50 pg/ml),
penicillin (50 units/ml), and Na,?30, (10 pCi/ml, 825 mCi/
mM; New England Nuclear Corp., Boston, Massachusetts)
were added and the cultures were maintained at 37°C for 5 or
20 hours under 5% C0,:95% air. In pulse-chase experiments,
slices incubated with 35S for 20 hours were washed three times
with 5 ml portions of Hank's balanced salt solution and transferred to flasks containing 10 ml of fresh medium for an additional 4 or 20 hours.
Following incubation the medium was decanted and
the tissue washed twice with 3 ml portions of cold saline. Medium and washes were combined and dialyzed against 200
volumes of 0.05M sodium acetate, pH 6.8, for 48 hours at 4°C
in tubing previously heated to reduce its pore size (8). The
sacs were rinsed with 5 ml of distilled water, the retentate and
rinses were combined, and the PGs were isolated as described
below.
Sequential extraction of PGs. The washed cartilage
slices were frozen in liquid N, and pulverized in a steel die
cooled with liquid Nz (9). PGs were sequentially extracted
from the cartilage powder as follows:
The pulverized cartilage was suspended in 10 ml of
0.4M guanidinium chloride (GuHCl) in 0.05M sodium acetate, pH 5.8, containing the protease inhibitors EDTA
(O.OlM), 6-aminohexanoic acid (0. IM), and benzamidine hydrochloride (0.05M) (10). After stirring for 48 hours at 4"C,
the suspension was filtered, the residue washed with 5 ml of
0.4M GuHCl, and filtrate and washings were combined. The
residue was then stirred for 48 hours at 4°C in 10 ml of 4.OM
GuHCl in 0.05M sodium acetate, pH 5.8. The suspension was
filtered, the residue was washed with 5 ml of fresh 4.OM
GuHCl, and filtrate and washings were combined and dialyzed to 0.4M guanidinium. Finally, the cartilage residue was
suspended in 3 ml of 0.1M borate, pH 8.0, containing 0.02M
calcium chloride. Pronase (Calbiochem, San Diego, California) was added (1 mg/50 mg cartilage wet weight; 45 PUK/
mg of enzyme) and the sample was digested under toluene for
24 hours at 55°C. The small amount of tissue remaining after
digestion was removed by centrifugation, then the supernatant was dialyzed overnight against distilled water.
Aliquots (0.1 ml) of the dialyzed samples of medium,
0.4M and 4.OM GuHCl extracts, and of the pronase digest
were added to Aquasol I1 (New England Nuclear Corp.) and
counted in a Beckman liquid scintillation spectrometer. Results were adjusted for differences in the wet weight of the tissues.
Purification of PGs under associative and dissociative
conditions. PGs in the medium, 0.4M and 4.OM GuHCl extracts were isolated and purified by sequential cesium chloride
density gradient centrifugations in 0.4M guanidinium (i.e.,
under associative conditions favoring formation of aggregates)
and then in 4.OM guanidinium (i.e., under conditions favoring
dissociation of aggregates) (1 1). Gradient fractions normally
containing either aggregated or purified disaggregated PGs
(11,12) were combined and dialyzed exhaustively at 4°C
against distilled water in the presence of the above protease
inhibitors, yielding Fractions A and D, respectively.*
Gel chromatography. Samples (0.5 ml) of PGs 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, New Jersey) and eluted with the acetate buffer at
a rate of 2 ml/hour. The radioactivity (?3) of 1.0 ml effluent
fractions was determined. Partition coefficients (Ka.,)
were calculated from the formula: K,, = (V, - V,)/(V, - VJ. V, represents the peak fraction in the elution diagram, V, the void
volume, and V, the total column volume.
Interaction of PGs with HA. To assess their ability to
undergo aggregation in vitro, purified disaggregated PGs
(Fraction D) were incubated with HA from human umbilical
cord (Sigma Chemical Corp., St. Louis, Missouri) (3). The
PGs were made 0.5M in sodium acetate, pH 6.9, HA was
added, and the solution was allowed to stand for 18 hours at
20"C, following which an aliquot was chromatographed on
the Sepharose 2B column. Elution diagrams of the PGs before
and after incubation with HA were compared. The proportion
of HA to PG (l:72, uronic acid) (13) was similar to that effecting maximal aggregation in vitro (14) and provided a ratio of
the two moieties comparable to that found in purified PG aggregates (1).
Digestion of PG aggregates with HA fll + 3 hydrolase. Aliquots (2 ml) of AM,, or AGu containing approximately 5 X 10' cpm " S were made O.IM in citric acid and
0.2M in Na,HPO, 7 H,O. The pH was adjusted to 5.6, and
1.5 p g of HA fll + 3 hydrolase (Biotrics, Inc., Arlington,
Massachusetts) was added over a 5-hour incubation period at
37°C (3). The activity of the enzyme was determined viscosimetrically; 0.3 p g of the hyaluronidase decreased the specific viscosity of a 0. I% solution of HA from 4.10 to 0.24 in 75
-
The following system of abbreviations is used: A and D refer to the PG fractions obtained by equilibrium density gradient centrifugation in 0.4M and 4 M GuHCI, respectively. The subscripts
and G~ refer to the source of the sample, i.e., culture medium or 4.OM
GuHCl extract, respectively.
510
PALMOSKI ET AL
minutes at 37°C. Sepharose 2B chromatographs of AModand
AGubefore and after incubation were compared. The lack of
specificity of the enzyme for PGs was confirmed by its failure
to alter the elution profile of DGU.
Analytical methods. Portions (approximately 20 mg)
of cartilage were placed in two changes of acetone for 24
hours and dried to constant weight in vacuo at 80°C. The
dried cartilage was digested with pronase as described previously, then the glycosaminoglycans (GAGS) were isolated
by precipitation with 9-aminoacridine hydrochloride and converted to their sodium salts with Bio-Rad AG-50 (Na') (15).
After resin was removed by filtration the uronic acid content
of the filtrate was determined (13).
Histologic examination. Full-thickness samples of the
cartilage and a portion of the underlying subchondral bone
were obtained with a Craig biopsy needle from femoral condyles and patellas. Histologic sections 6p in diameter were examined after staining with Safranin-0-fast green. Cartilage
depth from surface to tidemark was measured with a reticule
eyepiece attachment. Cell density/unit area was derived from
photomicrographs (X40) of the full-thickness histologic sections. The average number of cells contained in 3 one-inch
square areas of the photomicrograph was determined.
RESULTS
Articular cartilage from immobilized (IK) and
control knees (CK) of every animal was white and
smooth with an intact surface. Synovial membranes
were grossly normal with no intracapsular adhesions.
No synovial effusions were present. X-rays were normal
in every case, except for slight bone demineralization of
the immobilized leg of the dog which had borne a cast
for 8 weeks.
Histology and histochemistry. In all samples the
cartilage surface and tidemark were intact. Safranin-0
staining and cell distribution indicated that CK cartilage was normal in every case. The earliest change
noted in IK cartilage was a slight reduction in Safranin0 staining after 6 days of immobilization (Table 1).
Casting for periods of 3-8 weeks resulted in progressively greater reductions in staining of IK cartilage, accompanied by decreases (30-50%) in cartilage thickness.
In addition, after 8 weeks IK cartilage contained approximately 30% fewer cells/unit area than that from
CK (Figure 1).
A few cell clusters, brood capsules (16), were
noted in IK cartilage 1 week after cast removal, but 2
and 4 weeks after removal of the restraints cell distribution was normal. The loss of cartilage thickness which
developed in IK during immobilization persisted for up
to 4 weeks following removal of the cast, but at that
point the thickness of IK cartilage was only 15% less
than that of CK cartilage. The reduction in Safranin-0
staining of IK cartilage of dogs killed immediately after
immobilization was apparent also 1 week after cast removal. By 2 weeks after removal of the restraint, however, staining of IK cartilage was normal (Table l).
Water and uronic acid content. IK and CK cartilage of the animal that was immobilized for 6 days had
Table 1. Characterization of canine knee cartilage from immobilized and contralateral control limbs
Experimental group
Dogs killed immediately
following removal of cast
Dogs killed at varying intervals
following removal of cast
* CK
Duration
of immobilization
Interval
of cast
removal to
killing
Source
of
cartilage*
Cartilage
thickness,
mmt
Water content,
% of tissue
wet weight
Uronic acid
content,
% of tissue
dry weight
Safranin-0
staining
6 days
-
CK
IK
I .o
1.o
69.6
68.2
3.2
3.1
Normal
Slight decrease
3 weeks
-
CK
IK
1.5
0.8
71.6
84.2
4.8
2.5
Normal
Moderate decrease
6 weeks
-
CK
IK
0.9
0.6
72.9
71.5
Normal
Marked decrease
8 weeks
-
CK
IK
1.4
0.7
80.8
3.8
3.O
1.4
6 weeks
1 week
CK
IK
0.8
0.5
61.0
75.0
2.4
3.4
Normal
Slight decrease
6 weeks
2 weeks
CK
IK
0.7
0.6
72.0
73.5
2.2
2.6
Normal
Normal
6 weeks
4 weeks
CK
IK
0.7
0.6
71.5
73.0
2.6
2.5
Normal
Normal
= control knee; IK = immobilized knee.
t Measured from surface to tidemark on histologic sections.
-
Normal
Marked decrease
51 1
PROTEOGLYCAN AGGREGATION DEFECT
B
A
Figure 1. Cartilage, stained with Safranin-0, from (B) femoral condyle of knee which had been casted for 8 weeks, and from
contralateral control knee (A). Immobilization resulted in a 40% decrease in thickness of the cartilage of the immobilized
knee, reduction in staining of the extracellular matrix and moderate decrease in cellularity. (Magnification X 35.)
similar water content (68.2 and 69.6%, respectively, of
tissue wet weight) (Table 1). The water content of CK
cartilage did not change appreciably in the other animals in the study, except in one instance where it fell to
61% of the tissue wet weight 1 week after removal of the
cast. Immobilization appeared to produce an increase in
the water content of IK cartilage, which was about 20%
greater than that of the corresponding CK cartilage after 3 weeks of casting and about 10% greater after 6
weeks (Table 1). However, by 2 weeks after removal of
the cast, water content of IK cartilage had fallen to control levels.
The slight decrease in Safranin-0 staining seen in
IK cartilage after 6 days of immobilization was accompanied by a minimal decrease in tissue uronic acid content in comparison with the corresponding CK cartilage
(3.1 and 3.2% of tissue dry weight, respectiveiy) (Table
1). After 3 and 6 weeks of immobilization, however, the
Table 2. Incorporation of 3 5 S 0 4into proteoglycans of canine knee cartilage from immobilized and contralateral control limbs
Nondialyzable %04cpm/l0 mg wet weight
of cartilage, % distribution of total cpm
Duration of
immobilizazation
Experimental group
Dogs killed immediately
following removal of cast
Dogs killed at varying intervals
following removal of cast
* CK= control knee; IK
=
Interval
of cast
removal to
killing
Cartilage
sample*
Residual
Total ' 5 S cpm
glycosamino- incorporation,
glycans
% of control
Medium
0.4M
GuHCl
extract
4.OM
GuHCl
extract
25.6
27.3
7.8
9.4
31.1
38.5
35.5
24.8
6 days
-
CK
IK
3 weeks
-
CK
IK
18.5
21.1
I .5
2.8
35.4
33.7
44.6
42.4
6 weeks
-
CK
1K
4.3
7.4
3.7
5.1
54.9
48.4
37.1
38.5
8 weeks
-
CK
1K
24.8
35.1
10.6
54.9
43.2
4.9
21.4
5. I
6 weeks
I week
CK
IK
30.7
21.8
2.5
8.1
42.4
38.7
24.4
31.4
6 weeks
2 weeks
CK
IK
24. I
25.4
10.5
11.9
24.1
23.5
41.3
39.2
6 weeks
4 weeks
CK
IK
18.6
23.7
4.5
5.9
27.5
19.8
49.4
50.6
immobilized knee.
59.0
45.3
-
40.2
56.5
-
162.0
67.4
73.0
PALMOSKI ET AL
512
0
5
10
15
T I M E IN H O U R S
20
Figure 2. Rates of incorporation of "S into proteoglycans in articular
cartilage from immobilized (04)
and control (A-A)
limbs of
the dog immobilized for 8 weeks. Cpm represents the sum of nondialyzable 35S radioactivities in medium, sequential 0.4M and 4.OM
guanidinium chloride extracts, and pronase digest of tissue residue
(see text).
uronic acid content of IK cartilage was considerably
lower than that of CK cartilage. In contrast, 1 and 2
weeks following removal of the cast the uronic acid content of IK cartilage (3.4% and 2.6%, respectively) was
greater than that of CK (2.4% and 2.2%, respectively),
whereas 4 weeks after removal of the restraint uronic
acid contents of IK and CK cartilage were similar
(Table 1).
PG metabolism. Based on the sum of nondialyzable "S radioactivities in the medium and GuHCI
extracts and GAGS obtained after pronase digestion of
the cartilage residue, a reduction in PG synthesis was
apparent in IK cartilage after only 6 days of immobilization; '5S incorporation into GAGS ranged from 4059% of control values throughout the immobilization
period (Table 2). The cartilage remained metabolically
active in culture, as indicated by the increase in 35Sincorporation into PGs during the 20-hour period of incubation (Figure 2). The decrease in tissue content of
newly synthesized PGs was not due to increased catabolism since pulse-chase experiments on cartilage from the
dog subjected to the longest period of immobilization
showed similar rates of PG degradation in IK and CK
cartilage (loss of radioactivity over 20 hours = 67 and
6076, respectively) (Figure 3).
One week after removal of the cast, PG synthesis
in 1K cartilage appeared greatly increased-162% of
that in the CK cartilage-but at 2 and 4 weeks it was
about 70% of controls (Table 2).
Sequential extraction of PGs from the cartilage. After 6 days and 3 weeks of casting, IK and CK
cartilage both had a similar distribution of PGs between
the culture medium, GuHCl extracts, and pronase digests. The greatest proportion (3 l-38%) of the total
PGs was extracted with 4.OM GuHCl (Table 2). After 6
weeks of casting a relatively smaller proportion of the
total PGs were present in the medium and a relatively
greater proportion in the 4.OM GuHCl extract, but no
major difference was noted between IK and CK samples. However, after 8 weeks of immobilization, the PG
distribution among these fractions was markedly altered: the 0.4M GuHCl extract of the IK contained
54.9% of the total -"S PGs, whereas it accounted for only
10.6%of the PGs extracted from the CK cartilage. Some
4.9% of the total 35S PGs was present in the 4.OM
GuHCl extract of the IK cartilage, whereas nearly 10
times as much, i.e., 43.2%, was contained in the analogous CK extract.
In all cases, after removal of the restraint only a
small proportion (2.5- 1 1.9%) of the total nondialyzable
35Scpm was extracted with 0.4M GuHCl (Table 2), and
the distribution of newly synthesized PGs among the
I
I
I
I
I
I
16
0 '
I
I
I
0
4
8
12
I
I
1
I
20
T I M E IN HOURS
Figure 3. Pulse-chase experiment showing rate of decrease in tissue content of "S-labeled proteoglycans in articular cartilage from immobilized (M)
and control (A-A)
knees of the dog immobilized
for 8 weeks. Cpm represents the sum of the nondialyzable ' 5 S radioactivities in the medium, sequential 0.4M and 4.OM guanidinium
chloride extracts, and pronase digest of tissue residue (see text).
513
PROTEOGLYCAN AGGREGATION DEFECT
Table 3. Partition coefficient (Kav)and % eluting in void volume (V,) after Sepharose 2B chromatography of 35S proteoglycans in fraction AMcdr
and results of digestion with hyaluronic acid pl += 3 hydrolase
Fraction A,
+
Experimental group
Dogs killed immediately following
removal of cast
Dogs killed at varying intervals
following removal of the cast
* CK
Duration of
immobilization
Duration,
cast removal
to killing
Fraction AMed
H A /31
+3
hydrolase
Source of
cartilage.
9%Vo
6 days
-
CK
1K
17
IS
0.35
0.35
0
0
0.50
0.50
3 weeks
-
CK
IK
19
28
0.79
0.79
0
7
0.79
0.79
6 weeks
-
CK
IK
0
0
0.66
0.69
-
-
-
-
8 weeks
-
CK
IK
0
0
0.69
0.72
0
0
0.69
0.7 I
0.54
0.60
Kaw
v,t
%
vo
Kaw vrt
6 weeks
1 week
CK
IK
23
22
0.23
0.25
0
0
6 weeks
2 weeks
CK
IK
48
43
0.58
0.6 I
-
-
-
-
= control knee; IK = immobilized knee.
t Material eluting from the column after the void volume peak.
various extracts of the IK cartilage was similar to that of
the CK cartilage.
PCs in the culture medium. In CK samples
from all dogs, A,,, accounted for about half of the total
"SO, cpms applied to the associative cesium chloride
gradient. In each case AHedfrom IK represented 6-30%
less of the total "SO, in the gradient than AMedfrom the
corresponding CK, although the difference was not
clearly rated to the duration of immobilization.
After casting for up to 3 weeks, AMCd
from CK
and IK existed as aggregates and eluted partially (1528%) in the Sepharose 2B void volume (Table 3). Digestion with HA pl + 3 hydrolase (Table 3) or exposure to
the conditions of the second cesium chloride gradient
eliminated or markedly reduced the proportion of the
sample which was excluded by the gel and also decreased the hydrodynamic size of the retarded material.
This indicated that the aggregates had undergone dissociation. In marked contrast, PGs in A,=,, of CK and
IK cartilage of the dogs which were casted for 6 and 8
weeks, respectively, were wholly retarded by Sepharose
2B and the elution profiles of AMc,,from the latter animal were unchanged after treatment with HA /?l + 3
hydrolase (Table 3). This indicated that these PGs did
not exist in aggregates.
Removal of the cast was promptly followed by
the reappearance of PG aggregates, excluded from
Sepharose 2B (Table 3). Elution profiles of AMsdfrom
CK and IK cartilage were essentially the same. The hy-
drodynamic size of A,,, from the IK and CK cartilages
of the dog that had been freed of its restraint for only 1
week was reduced by HA pl + 3 hydrolase. Digestion
with the hyaluronidase abolished all material that had
been large enough to be excluded from the gel and
resulted in a decreased average hydrodynamic size of
the PGs retarded by the gel.
PGs in the GuHCl extracts. Fraction AGu
(which normally contains aggregated PGs) represented
70-9576 of the total 35Scpm applied to the associative
gradient, and there was no major difference between the
distribution of radioactivity in the gradient in CK and
IK cartilage.
Some 20-26% of AGufrom CK of dogs immobilized up to 6 weeks was excluded from Sepharose 2B,
while the K,, of the PGs retarded by the gel ranged between 0.21 and 0.40 (Table 4, Figure 4). Incubation
with HA pl + 3 hydrolase or exposure to the conditions of the second density gradient wholly eliminated
the void volume material and resulted in a marked decrease in the hydrodynamic size of each of these samples (Table 4), indicating that this treatment produced
dissociation of the aggregates.
After 1 week of casting, the average hydrodynamic size of AG. from IK was similar to that of A,,
from CK, but after 3 weeks of immobilization no evidence of aggregation could be found in the IK cartilage
(Table 4, Figure 4). Thus, A," from IK of dogs casted
for 3 and 6 weeks was wholly retarded by Sepharose 2B
PALMOSKI ET AL
5 14
Table 4. Sepharose 2B chromatography of proteoglycans extracted with 4M guanidinium chloride from canine articular cartilage of immobilized
and control limbs
Fraction Aou
Experimental
group
Dogs killed
immediately after
removal of cast
Dogs killed at
varying intervals
after removal of cast
Duration
of
immobilization
+
HA pl
Fraction A,
4
3 hydrolase
Fraction D,,
%Vn
K,,, V,t
%Vn
K,,, V,t
%Vn
K,,, V,t
Fraction DGu+ HA
%V,
K,,, V , t
__
-
-
-
6 days
-
CK
IK
26
14
0.2 I
0.25
0
0
0.50
0.49
0
0
0.46
0.48
3 weeks
-
CK
IK
22
0
0.23
0.60
0
0
0.42
0.62
0
0
0.38
0.65
18
0
0.33
0.66
6 weeks
-
CK
IK
20
0
0.40
0.56
0
0
0.52
0.56
0
0
0.60
0.62
29
0
0.62
0.60
8 weeks
-
CK
IK
CK
IK
0
*
0.25
0
0.65
0.67
*
0
+
0.3 I
-
+
0.39
0.33
*
0
15
0
*
-
0
0
0.38
0.40
19
20
CK
IK
43
49
0.23
0.23
0
0
0
0
0.56
0.64
-
0.32
0.33
-
-
-
6 weeks
6weeks
* CK
Duration
of cast
removal Cartilage
to killing sample*
I week
2weeks
4
0.55
0.58
*
= control knee; IK = immobilized knee.
t Material eluting from the column after the void volume peak.
$ Fraction A," contained insufficient material for analysis or for preparation of D,".
(Kav= 0.60 and 0.56, respectively). These Sepharose 2B
elution profiles were unaltered by HA b l -+ 3 hydrolase
and the K,, of Fraction Do" was similar to that of the
corresponding A," (Table 4), indicating that the PGs
were not aggregated.
The PGs in Fraction A," from CK of the dog restrained for 8 weeks were somewhat smaller in average
hydrodynamic size than corresponding PGs from CKs
of animals immobilized for shorter duration. Even
though it did not elute in the Sepharose 2B void volume, A,, from the dog casted for 8 weeks presumably
contained aggregates, since HA 1 * 3 hydrolase caused
a change in its K,, from 0.25 to 0.65 (Table 4), which
was now similar to that of the corresponding D,".
Since the 4.OM GuHCl extract of IK from the
above afforded insufficient material for analysis of aggregation, similar analyses were carried out with Fraction A of the 0.4M GuHCl extract. No evidence of aggregation was found: i.e., the K,, of Fraction A was 0.67
and no material eluted in the Sepharose 2B void volume. In addition, the hydrodynamic size of Fraction A
was unaffected by incubation with HA /I1 + 3 hydrolase or exposure to 4M GuHCl in the dissociative cesium chloride gradient.
As early as I week following removal of the cast,
newly synthesized aggregates were again present in IK
(Table 4, Figure 5). Although none of the material was
excluded from Sepharose 2B, the hydrodynamic size of
Acjufrom the IK was considerably diminished in the
dissociative gradient. Two weeks after removal of the
cast, A,. samples from CK and IK cartilage were similar in hydrodynamic size and evidence of aggregation
was present in each.
PG-HA interaction. Marked aggregation occurred when D,, fractions of CK cartilage from all dogs
killed immediately after immobilization were incubated
with HA, with a consequent increase in hydrodynamic
size of the sample (Figure 6). In contrast, exposure to
HA had no effect on the elution profiles of DGuof IK
cartilage from these animals (Figure 6). DGufrom IK
and CK that was obtained 1 week after removal of the
cast readily formed aggregates following incubation
with HA, as indicated by the fact that 19 and 20%, respectively, now were excluded from Sepharose 2 B
(Table 4).
DISCUSSION
These results indicate that immobilization of a
joint may rapidly result in a defect in PG aggregation
within the articular cartilage. In the dog, after 6 weeks
of immobilization the defect appears promptly reversible after the resumption of normal use of the joint.
During prolonged immobilization intracapsular fibrofatty connective tissue may proliferate and encroach on
the articular cartilage (17), but the changes in macromolecular organization of the cartilage in the present
515
PROTEOGLYCAN AGGREGATION DEFECT
i
0
1
0
1
___-
2 -_---'--I
I
I I
20
V,30
I
40
1
50
loss of uronic acid which is consistent with a decrease in
its GAG content. These changes were progressive and
at 8 weeks IK cartilage was atrophic and hypocellular.
IK cartilage was also consistently thinner than that from
CK (Table 1) although, not unexpectedly, the thickness
of CK cartilage varied widely from animal to animal. In
other studies decreases in metachromasia (20) and hexosamine content (2 I ), also indicating GAG depletion,
were among the earliest changes noted after immobilization. In the present study the diminution in Safranin0 staining was accompanied by a decrease in tissue
content of ' 3 PGs, which indicated a decrease in PG
synthesis since the rate of PG degradation was unaffected. In addition, by 3 weeks the water content of IK
cartilage was increased. In early stages of experimental
osteoarthritis, in which decreased Safranin-0 staining
and defective PG aggregation also occur, a similar increase in water content has been noted (22).
Generally, 20-30% of the total PGs synthesized
in the cultures was present in the medium and there
was no major difference in this respect between CK and
IK cartilage (Table 2). For reasons not apparent and in
contrast to the other samples, the culture medium from
A '
50
.
.
.
I0
V,
80
V, 30
40
I
I
I
1
50
60
I0
Vt
10
8
6
n
II
study occurred when moderate thinning was the only
gross change.
Immobilization of a joint has been shown to produce articular cartilage degeneration and necrosis (1 8).
These changes are more marked and appear earlier in
areas of contact, but they also occur without mechanical
compression. Nutrition of articular cartilage in the adult
is derived from the synovial fluid (19), so that cartilage
degeneration with immobilization may be due to the
lack of pumping action which results from joint movement and is necessary for diffusion of the fluid into the
cartilage. Furthermore, synovial fluid production may
be reduced by immobilization, which causes degenerative changes in synovial cells and atrophy of the synovial membrane (17).
In the present study, after 3 weeks IK cartilage
showed marked reduction in Safranin-0 staining and
I
12
FRACTION (ml)
Figure 4. Results of chromatography on a Sepharose 28 column
(90 X 1.0 cm) of proteoglycans prepared under associative conditions
after extraction of dog knee cartilage with 4M guanidinium chloride
limbs.
from immobilized (----) and contralateral control (-)
Duration of immobilization: A, 6 days; B, 3 weeks; C, 8 weeks.
1 1
x
z
4
2
12
v)
n
10
0
20
FRACTION (ml)
Figure 5. Results of chromatography on a Sepharose 2B column
(90 X 1.0 cm) of proteoglycans prepared under associative conditions
after extraction with 4M guanidinium chloride of dog knee cartilage
following 6 weeks of immobilization. Duration following removal of
the cast: A, 1 week; B, 2 weeks. Proteoglycans from immobilized
(----) and contralateral control (-)
limbs.
516
PALMOSKI ET AL
c
I
0
'
I
I
I
C
A
1
1
aov,
40
1
I
I
1
I
I
I
I
v,
zo v,
40
M,
Vt
rrucnoN (ml)
Figure 6. Results of chromatography on a Sepharose 2B column
(90 X 1.0 cm) of proteoglycans prepared under dissociative conditions
after extraction of dog knee cartilage with 4M guanidiniurn chloride.
Elution profiles of the dissociated proteoglycans are shown before
(-) and after (----) incubation with hyaluronic acid. A, Control
limb from a dog casted for 3 weeks; B, immobilized limb from a dog
casted for 3 weeks; C, control limb from a dog one week after removal
of a cast which had been applied for 6 weeks; D, immobilized limb
from a dog one week after removal of a cast which had been applied
for 6 weeks.
0
eo
the experiments with cartilage from the dog casted for 6
weeks contained only about 4-7% of the 35SPGs. A substantial proportion of AMedfrom the dogs that were immobilized 3 weeks or less eluted in the Sepharose 2B
void volume (Table 4) was digested by HA p l + 3 hydrolase and dissociated by 4M GuHCl, indicating that
PGs in the medium were aggregated. However, there
was no difference between the average hydrodynamic
size of the aggregates in AMc., of CK and IK at any of
the time periods examined. In addition, AMcdrepresented only about half as much of the total nondialyzable
as A,,, suggesting that AMedmay have
contained degraded PGs. In contrast, after 6 or 8 weeks
of casting, PG aggregation was not demonstrable in
AMed and HA pl + 3 hydrolase did not alter the Sepharose 2B elution profile.
Because of the macromolecular organization of
PGs within the collagen meshwork of normal joint cartilage, their quantitative extraction is not possible without degradation of the tissue. Four molar GuHCl dissociates PG aggregates and liberates PGs in relatively
high yields (23). In contrast, 0.4M GuHCl does not affect the interactions between constituents of the aggregate and solubilizes only a small proportion of the total
PGs of normal cartilage. Most PGs extracted from normal cartilage by 4.OM GuHCl will aggregate in vitro
with HA, whereas those obtained with low molarity
salts tend to be smaller, do not interact with HA (12),
and presumably are not aggregated in vivo.
The present results indicate that the organizational integrity of IK cartilage was maintained through
6 weeks of immobilization. Until then, less than 10% of
the total "S PGs of IK and CK could be extracted with
0.4M GuHCl. In sharp contrast, after 8 weeks about
55% of the PGs synthesized by IK cartilage were contained in the 0.4M GuHCl extract. These PGs did not
exist as aggregates since their K,, was not altered by
treatment with HA p l + 3 hydrolase, which dissociates
PG-HA complexes (3), and the elution profile of the
PGs from the second (dissociative) cesium chloride gradient was the same as that of the PGs from the initial
(associative) gradient.
Some of these nonaggregated PGs may have
arisen as a result of enzymatic degradation since hydrolytic enzymes, including neutral proteases which will
degrade PGs (24), exist in normal cartilage. In the present study protease inhibitors were used during extraction of the tissues with GuHCl but not during inc u b a t i o n of t h e cartilage, since even in low
concentrations they inhibited "S incorporation into PGs
by up to 90%.
In all but one sample, the majority of the extractable PGs was recovered in A<;".At 6 days of immobilization, PG aggregation in A,. from CK and IK cartilage was similar, but by 3 weeks aggregation in AG.
from IK was absent (Table 4). The Sepharose 2B elution profiles of PG aggregates from CK cartilage varied
from dog to dog. The progressive decrease in aggregation seen with increasing length of the immobilization
period may reflect an effect of supra-normal weight
bearing on the CK cartilage. Alternatively, the results
may simply signify inherent differences in the hydrodynamic size of PG aggregates (Figures 4 and 5).
Impaired aggregation in the present study may
have been due to an abnormality in the PG core protein, since the PGs appeared incapable of interacting
with HA in vitro. The data do not preclude the existence of additional defects in the cartilage HA and/or
517
PROTEOGLYCAN AGGREGATION DEFECT
link glycoproteins. T h e fact that 1 week after cast removal D c j ufrom I K cartilage interacted with HA in
vitro, while Acjufrom the same tissue did not contain
PG aggregates, suggests that the cartilage HA in this
case may have been abnormal, or that inhibitors of PGHA interaction were present.
T h e aggregation defect in the present study is
similar to that described in morphologically normal hip
cartilage from some aged individuals with recent femoral neck fractures (6). It is different, however, from the
aggregation defect n o t e d i n a r t i c u l a r cartilage o f
younger individuals whose joints h a d not been used
normally for some time (7). In the latter cases PGs interacted readily with HA in vitro to form aggregates, a
finding which suggests that the abnormality in vivo was
attributable to a defect in HA, rather than in the PGs.
Immobilization of the joints in those patients was less
complete than that of the canine knees studied here.
Despite the fact that the aggregation defect in the
present study appeared to be reversible, the results emphasize that the effect of disuse must be taken into account in interpreting data relating to PG organization in
articular cartilage. Furthermore, it is likely that the biochemical defect arising from immobility adversely affects the biomechanics of articular cartilage which could
be significant in subsequent use of the joint.
ACKNOWLEDGMENTS
We appreciate the help of Dr. Robert Colyer in apply-
ing the casts. Janet Anderson and Sally Stoehr provided valuable technical support and Lorna Bowman afforded excellent
secretarial assistance. Fritz Lamprey of Merck, Sharpe and
Dohme, Co. generously provided the Lightcast 11.
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Brandt K, Muir H: Characterization of protein-polysaccharides of articular cartilage from mature and immature pigs. Biochem J 114:87 1-876, 1969
Oegema TR, Hascall VC, Dziewiatkowski DD: Isolation
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Hascall VC, Sajdera SW: Protein polysaccharide complex
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in the formation of aggregates. J Biol Chem 244:2384-
2396, 1969
12. Hardingham TE, Muir H: Hyaluronic acid in cartilage
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13. Bitter T, Muir H: A modified uronic acid carbazole reaction. An Biochem 4:330-334, 1962
14. Hardingham TE, Muir H: The specific interaction of
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15. Tsiganos CP, Muir H: Studies on protein-polysaccharides
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16. Sokoloff L: The Biology of Degenerative Joint Disease.
Chicago, The University of Chicago Press, 1969, p 9
17. Enneking WF, Horowitz M: The intra-articular effects of
immobilization on the human knee. J Bone Joint Surg
54Az973-985, 1972
18. Roy S: Ultrastructure of articular cartilage in experimental immobilization. Ann Rheum Dis 29:637-642, 1970
19. Hodge JA, McKibbin B: The nutrition of mature and immature cartilage in rabbits. J Bone Joint Surg 51B:140147, 1969
20. Troyer H: The effect of short-term immobilization in the
rabbit knee joint cartilage. Clin Orthop 107:249-257, 1975
21. 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
22. Mankin HJ, Thrasher AZ: Water content and binding in
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23. Sajdera SW, Hascall VC: Proteinpolysaccharide complex
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development, proteoglycans, immobilization, reversal, knee, norman, cartilage, aggregation, defects, canine
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