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Organization of ground substance proteoglycans in normal and osteoarthritic knee cartilage.

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209
ORGANIZATION OF GROUND
SUBSTANCE PROTEOGLYCANS IN
NORMAL AND OSTEOARTHRITIC
KNEE CARTILAGE
KENNETH D. BRANDT and MARSHALL PALMOSKI
A study of the organization of proteoglycans in
articular cartilage indicates that nonaggregated proteoglycans existed in larger numbers in osteoarthritic than
in normal cartilage and that proteoglycan aggregates in
arthritic cartilage were smaller than normal. After dissociation from hyaluronic acid and tissue glycoproteins,
no difference in hydrodynamic size of disaggregated proteoglycans was noted, but chondroitin sulfate chains of
those from diseased cartilage were shorter than normal.
The data suggest that there is a defect in proteoglycan
aggregation in osteoarthritic cartilage which could be of
pathogenetic significance.
The mechanical properties (eg stiffness on compression) that permit articular cartilage to subserve its
From the Arthritis and Connective Tissue Disease Section,
Evans Department of Clinical Research, University Hospital, and the
Thorndike Memorial Laboratory and Division of Medicine, Boston
City Hospital, Boston, Massachusetts.
Supported by grants from the USPHS National Institute of
Arthritis and Metabolic Diseases (AM-17215. AM-04599. and TIAM-5285) and from the N I H General Clinical Research Centers
Branch of the Division of Research Resources (RR-533).
Kenneth D. Brandt, M.D.: Special Research Fellow, National Institutes of Health, and Associate Professor of Medicine, Boston University School of Medicine; Marshall Palmoski, Ph.D.: Thorndike Memorial Laboratory and Division of Medicine, Boston City
Hospital, and Recipient of a Postdoctoral Fellowship from The Arthritis Foundation.
Address reprint requests t o Kenneth D. Brandt, M.D.,
Indiana University Medical Center, 1100 West Michigan Street,
Indianapolis, Indiana 46202.
Submitted for publication March 29, 1975; accepted June 30,
1975.
Arthritis and Rheumatism, Vol. 19, No. 2 (March-April 1976)
weight-bearing function depend on the presence of proteoglycans (PGs) in the tissue ground substance ( I ) . In
vitro some, but not all, cartilage PGs can be shown to
aggregate after addition of hyaluronic acid (HA) (2),
and it is probable that in vivo the PGs exist as very large
aggregates (3) with many PG molecules noncovalently
linked to HA (4). A tissue glycoprotein (GPL) (3) is also
present in these complexes and appears to stabilize the
PG-HA interaction (5).
I n osteoarthritis (OA) the fundamental defect,
although still unknown, seems to reside in the articular
cartilage, where loss of ground substance occurs. Recent
studies have shown that some PGs of OA cartilage are
more readily extracted by neutral isoosmotic salt than
are those of normal cartilage ( 6 ) , but whether this difference reflects an abnormality in PG aggregation in
OA remains to be determined.
Although few comparisons of intact PGs of normal and OA cartilage have been reported (7-9), their
glycosaminoglycans (GAGs) have been compared more
extensively. The diseased tissue may contain less
chondroitin sulfate and keratan sulfate (7,lO-13) and
shorter chains of chondroitin sulfate than normal
(l0,14). GAGs do not exist free in vivo, however, but
rather as constituents of PGs, so that analyses of total
tissue GAGs reflect the composition of PGs that were
not aggregated as well as of those that were.
Taking into account the possibility that differences existed in the degree of their aggregation in the tissues, the present study compares PGs of normal and OA
cartilage before and after density gradient centrifugation
210
in 4 M guanidinium chloride, a procedure k n o w n t o
dissociate them f r o m complexes with H A and GPL (3,
15). In addition a subpopulation o f PGs extracted f r o m
t h e same tissues merely by brief stirring in neutral isoosmotic sodium acetate, and presumably n o t aggregated in
vivo, was also studied. T h e results indicate t h a t in OA
some PGs are held i n t h e cartilage less firmly t h a n
normal a n d t h a t t h e PG aggregates in OA cartilage tend
t o b e smaller t h a n normal. T h e s e results may be o f
pathogenetic significance.
MATERIALS AND METHODS
Tissues
Knee joints from 6 steers 6-9 years of age were obtained at the abattoir and stored at -20°C. After thawing to
4°C the articular cartilage was removed from the distal femurs
with a scalpel. Small samples were taken for histologic study
and the cartilage from a ) normal and b) softened, fibrillated
areas was pooled separately and then diced. Portions of
approximately 150 mg were weighed and placed in acetone for
24 hours and then dried to constant weight in vacuo at 80°C.
Samples of the acetone-dried cartilage were taken for determination of hydroxyproline, while the remainder was digested
with pronase at 55°C for 24 hours in 0.1 M borate buffer, pH
8.2, containing 0.2 M CaCI,. For each gram of tissue, 10 mg of
enzyme in 50 ml of buffer were employed. The small amount of
undigested material remaining after incubation was removed
by centrifugation, following which the G A G S in the supernatant were isolated by precipitation with 9-aminoacridine
hydrochloride, as described below.
The remainder of the pooled cartilage was divided into
two portions: one was frozen in liquid nitrogen, pulverized in a
steel die cooled in liquid nitrogen, and extracted with sodium
acetate. The other, unpulverized portion was extracted directly
with guanidinium chloride, as follows.
Extraction and Isolation of Proteoglycans
Sodium Acetate Extract. The pulverized cartilage was
suspended (15 g/lOO ml) in cold 0.15 M sodium acetate, pH
6.8, and agitated for 7 minutes at the lowest speed of a Virtis
homogenizer, following which the suspension was filtered
through lint. The residue was washed with sodium acetate, the
clear filtrate and washings were combined, and the PGs were
isolated by precipitation with 9-aminoacridine hydrochloride
(16) and converted to their sodium salts by ion exchange with
Bio-Rad-AG-50 (Na+). The resin was removed by filtration,
the filtrate was concentrated by rotary evaporation, and the
PGs were further purified by a second precipitation with 9aminoacridine. After removal of the acridine as above, the
PGs were precipitated with 80% (v/v) ethanol, washed with
80% (v/v) ethanol, absolute ethanol, and acetone, and dried in
vacuo over P,O,. I n each case the amount of uronic acid
remaining in the supernatant after precipitation with 9-aminoacridine was determined after the excess acridine had been
removed with additional resin.
BRANDT AND PALMOSKI
Guanidinium Chloride Extract. A standard procedure
(3) was used to prepare a ) aggregated PGs and b) purified
disaggregated PGs, dissociated from HA and GPL, from the
unpulverized cartilage:
The tissue was stirred for 48 hours at 20°C in 4 M
guanidinium chloride in 0.05 M sodium acetate, pH 5.8, after
which the suspension was centrifuged and the supernatant
removed by decantation. The cartilage residue was washed
twice with fresh guanidinium chloride and supernatant and
washings were combined and made 0.5 M in guanidinium
chloride by dialysis, after which the retentate was removed and
the sacs were rinsed with additional guanidinium. Cesium
chloride was added to a density of 1.69 g/ml, and the solution
was centrifuged at 4 ° C for 48 hours in the No. 40 angle rotor
of a Beckman ultracentrifuge (average speed: 95,000 X g).
Following centrifugation the gradient was fractionated
into five equal parts and the fractions corresponding to the
bottom two-fifths (density L 1.73 g/ml) were pooled. After
exhaustive dialysis against distilled water the retentate was
removed and the sacs were rinsed with water. Retentate and
rinses were combined and lyophilized to yield a fraction containing the aggregated PGs.
To obtain dissociated PGs, free of HA and GPL,
portions of the lyophilized material were dissolved in 4 M
guanidinium chloride, pH 5.8. The density was adjusted with
cesium chloride to I .SO g/ml and the samples were centrifuged
for 42 hours as described above. The bottom 2/5 of the
gradient (density L 1.53 g/ml) was then recovered and, after
dialysis as above, the PGs were lyophilized.
Gel Chromatography. Columns of Sepharose 6B* (40
X 0.8 cm) and Sepharose 2B* (45 X 0.8 cm) were employed;
their void volumes had been previously determined with Blue
Dextran*. With both, samples of 1.5 mg were applied in 0.5 ml
of 0.5 M sodium acetate, pH 6.5, and eluted with the acetate
solution at a flow rate of 2 ml/hour. Fractions of 1 ml were
collected and the uronic acid content of each fraction was
determined.
Average molecular size of chondroitin sulfate chains
was assessed by chromatography on Sephadex G-200* (17).
Approximately 10 mg of PGs were dissolved in 2 ml of 0.1 M
sodium acetate buffer, pH 5.5, containing 2 mg EDTA and 0.6
mg cysteine hydrochloride, to which crude papain? was added.
After digestion under toluene for 24 hours at 65"C, GAGS
were precipitated with 80% (v/v) ethanol, washed with 80%
(v/v) ethanol, absolute ethanol, and acetone, and dried in
vacuo. Samples (approximately 3 mg) were applied to a column ( 1 15 X 0.8 cm) and eluted with 0.2 M .sodium acetate, pH
6.5. One-milliliter fractions were collected at a rate of 4
ml/hour and their uronic acid contents determined.
Analytical Methods. Hexuronic acid, protein, hexose,
and xylose were all determined as described previously (16).
Hexosamine was determined by the method of Kraan and
Muir (18), with glucosamine hydrochloride employed as
standard. Samples were hydrolyzed for 3 hours in 8 M HCI
under N2 at 95°C (19), following which the acid was rapidly
removed by boiling under N,. Molar ratios of glucosamine:galactosamine were determined on a JEOL-SAH
automated analyzer with samples hydrolyzed as they would be
* Pharmacia Fine Chemicals, Piscataway, New Jersey.
t Sigma Chemical Company, St. Louis, Missouri.
G R O U N D SUBSTANCE PROTEOGLYCANS IN O A CARTILAGE
21 1
Table 1. Composition of Normal and Osteoarthritic Bovine Knee Cartilage
~
~~
~~
Composition (percent o f tissue dry weight)
Tissue
Dry Weight
(percent of wet weight)
Hydroxyproline
Uronic Acid
Hexosaniine
Glucosamine: Galac(osaniine
(molar ratio of total
glycosaminoplycans)
24.5
28.0
7.9
8.3
2.67
2.3 I
2.38
2.10
I : 1.5
I : 1.3
Normal
OA
for total hexosamine. Amino acid analyses were performed on
the same instrument with samples hydrolyzed in 6 M HCI
under N, for 24 hours at 105"C, following which the acid was
removed by boiling under N,. Hydroxyproline contents of
acetone-dried cartilage were determined on the amino acid
analyzer after hydrolysis of the tissue for 24 hours at 105°C in
6 M HCI and removal of the acid as above.
Histochemistry. Samples of cartilage from each joint
were stained with Safranin-0 and examined histologically. The
severity of osteoarthritis was graded in each specimen according to the criteria of Mankin et a1 (20).
RESULTS
Cartilage that was grossly normal was also histologically normal and, after examination of Safranin-0
stained sections, was graded 0, whereas the grossly osteoarthritic cartilage ranged between Grades 5 a n d 8
(20). Whole normal a n d OA cartilage were similar with
respect to dry weight per gram of fresh tissue (24.5% and
28.0% respectively) and hydroxyproline content (approximately 8% of dry weight). Consistent with the observed loss of Safranin-0 staining, the diseased cartilage
contained less uronic acid a n d hexosamine than normal,
as indicated by analysis of the G A G s isolated after
pronase digestion of the tissue. T h e proportion of glucosamine relative to galactosamine in the total G A G s ,
however, was essentially the same (Table I ) . Uronic acid
was not found in any of the supernatants after
precipitation with 9-aminoacridine, a fact indicating
that all t h e compounds containing uronic acid were
precipitated.
PGs were not quantitatively extracted by either
of the procedures employed. Those in normal cartilage
were less readily extracted by sodium acetate than those
in OA cartilage, although there was no appreciable difference in yields of the guanidinium extracts, which
contained much more of the tissue PGs (Table 2). Thus
the sodium acetate extract of the OA cartilage contained
seven times as much of the total tissue uronic acid (9%)
as the sodium acetate extract of the normal cartilage
(1.3%), whereas the guanidinium extracts of normal and
O A cartilage contained 60% a n d 62% respectively of the
tissue uronic acid (Table 2).
Losses of uronic acid after dialysis of the guanidinium extracts of the normal a n d arthritic cartilage
were minimal (5% a n d 6% respectively). Sixty-three percent of the uronic acid extracted from normal and 77%
of that extracted from the diseased tissue were recovered
Table 2 . Comparison of Proteoglycans Extracted from Normal and Osteoarthritic Bovine Knee Cartilage by 0.15 M Sodium Acetate,
pH 6.8, and by 4 M Guanidinium Chloride. pH 5.8*
Composition of Purified Proteoglycans*
Percent Dry Weight
Tissue
Normal
OA
Norm al
OA
Extract
Sodium
acetate
Sodium
acetate
Gu an id i n i urn
chloride
Guanidiniurn
chloride
Percent of
Total Tissue
Uronic Acid
Uronic
Acid Hexosamine
Molar Ratio
Protein
Hexose
Xylose
Percent
Glucosamine: Xylose:
Retarded by
Galactosaniine Uronic Acid S e p h a r o e 6 8
1.3
271
32.5
7.9
10.8
0.14
I :3.3
I :29
51
9.0
25.3
32.0
8. I
11.3
0.85
I :2.9
1:23
47
60
23.5
34.0
13.4
14. I
0.61
I : 1.8
I :30
8
62
16.8
33.1
13.0
15.5
0.62
I : 1.4
I :20
II
* Proteoglycans extracted by 4 M guanidiniurn chloride were purified by equilibrium density gradient centrifugation in cesium chloride. then
in cesium chloride in 4 M guanidinium chloride (see text).
B R A N D T A N D PALMOSKI
212
from the bottom two-fifths of the initial cesium chloride gradient.
Based on elution profiles of uronic acid after
Sepharose 2B chromatography of the material in the
bottom two-fifths of the initial cesium chloride gradient, PG aggregates of normal cartilage tended to be
larger than those of OA cartilage. Thus 70% of the
sample from normal, but only 40% of that from OA
cartilage, eluted with a K,, 5 0.5 (21). In contrast the
disaggregated PGs of normal and OA cartilage, which
represented about 40% of the total uronic acid in each
case, were essentially the same in average hydrodynamic
size. Both eluted with a rather broad unimodal peak,
somewhat after the void volume of the Sepharose 2B
column.
Over 9070 of the uronic acid in the aggregates of
both normal and diseased cartilage was recovered as
disaggregated proteoglycans after the second density
gradient centrifugation. Whereas over 90% of the disaggregated PGs in the guanidinium extracts were excluded from Sepharose 6B, 50% of the PGs extracted by
sodium acetate from normal as well as from OA cartilage were small enough to be retarded. In addition to
their differences in hydrodynamic size, there were apparent differences in composition between the PGs exTable 3. Amino Acid Composition, Residues/1000 Residues, of
Purified Proteoglycans+ of Normal and Osteoarthritic Bovine
Knee Cartilage and Bovine Nasal Cartilage
Normal Knee
HYPro
GlY
Pro
Leu
Ala
Val
I le
Phe
ASP
Arg
LYS
Glu
Ser
Thr
TY r
His
CYS
M el
0
1
on
I12
ni
79
65
35
29
74
37
23
I33
I12
65
in
20
5
4
OA Knee
Nasal Cartilage?
0
I14
I17
78
69
68
36
32
70
34
25
I 38
99
60
25
25
7
3
0
I00
99
86
79
64
33
40
67
38
23
141
I12
62
21
23
9
3
* Proteoglycans were extracted in 4 M guanidinium chloride and
purified by equilibrium density gradient centrifugation in cesium
chloride, then in cesium chloride in 4 M guanidinium chloride (see
text).
t Reference (3).
tracted in sodium acetate and the purified disaggregated
PGs prepared from the guanidinium extracts (Table 2).
Thus PGs obtained by sodium acetate extraction contained more uronic acid and xylose, less protein and
hexose, and approximately twice as much galactosamine, relative to glucosamine, as the disaggregated
PGs in the guanidinium extracts.
Although their protein contents were similar
(Table 2) and the amino acid analyses of normal and
OA PGs in the guanidinium extracts revealed no major
differences between the two (Table 3), PGs from normal
tissue contained more uronic acid and a higher proportion of galactosamine, relative to glucosamine, than did
OA PGs. Based o'n Sephadex (3-200 chromatography of
the GAGS after papain digestion, OA PGs in both extracts had shorter chondroitin sulfate chains than normal (Figure 1). In addition buoyant densities of the
disaggregated PGs from normal cartilage tended to be
greater than those of the disaggregated PGs from OA
cartilage, as indicated by the distribution of uronic acid
within the second cesium chloride gradient. Thus 94%
of the PGs from normal, but only 82%of those from OA
cartilage, had a density L 1.59 g/ml.
DISCUSSION
These data indicate a striking difference i n the
ease with which PGs were extracted from knee cartilage
with isoosmotic sodium acetate and with 4 M
guanidinium chloride. Indeed, unless the surface area is
greatly increased by pulverization, as in the present
study, only negligible amounts of PGs can be extracted
from articular cartilage with sodium acetate (22). In
contrast considerably more PGs can be liberated from
laryngeal cartilage by this solvent even without prior
pulverization. Because the collagen content of articular
cartilage is approximately twice that of laryngeal cartilage, the limited extractability of articular cartilage PGs
may be due to their greater entrapment by collagen.
However, because hydroxyproline contents of the normal and OA cartilage were the same, the several-fold
greater yield of PGs from OA than from normal knee
cartilage by limited sodium acetate extraction (Table 2)
must be due to other factors.
The effectiveness of 4 M guanidinium in extracting PGs from cartilage was presumably related to the
fact that it dissociated PGs that existed in noncovalent
linkage with HA and GPL (3,4). Nonetheless 4 M guanidinium does not extract PGs quantitatively, and PGs
in the guanidinium extracts of both normal and OA
cartilage in the present study represented only 60% of
GROUND SUBSTANCE PROTEOGLYCANS IN OA CARTILAGE
10
+
v~
20
40
30
-Volume,
50
60
213
70
ml+
Fig 1. Gel chromatography of chondroitin suljhte chains on a column ( 1 15 X 0.8 cm) of Sephadex G-200.
eluted with 0.2 M sodium acetate, pH 6.5. Fractions of I ml were collected and their uronic acid contents
determined. The chains were isolated after papain digestion of proteoglycans from normal (0-0 ) and
osteoarthritic (0-0)
cartilage.
the total tissue uronic acid. Thus, although a difference
in the ease of extractability of PGs from normal and OA
cartilage with sodium acetate was readily apparent
(Table 2), this difference was totally obscured by the
much greater efficiency of 4 M guanidinium as an extracting solvent.
The PGs of cartilage are a heterogeneous population and vary among themselves in size and composition (16). It is therefore not surprising that there
were qualitative as well as quantitative differences between the PGs in the sodium acetate and guanidinium
chloride extracts. Because, in general, protein and keratan sulfate contents of PGs are directly related to their
size (22), the analytical data are consistent with the
interpretation that the PGs extracted with sodium acetate tended to be smaller than those extracted with guanidinium. This study, as well as previous work (22),
indicates that sodium acetate extracts of articular cartilage contain significant amounts of smaller PGs, retarded on Sepharose 6B, which contain less protein and
keratan sulfate than the larger PGs (16). These smaller
PGs are also extracted by 4 M guanidine, although they
represent only a small proportion of the total PGs extracted by that solvent (23), and PGs in the sodium
extracts thus may be viewed as a subpopulation of those
in the guanidinium extracts.
The bulk of the PGs extracted by 4 M guanidinium have a buoyant density 2 1.50 g/ml (3) and
are dissociated from HA and GPL, whose buoyant
densities are 5 1.46 g/ml, by equilibrium density
gradient centrifugation in cesium chloride in the presence of 4 M guanidinium (3,15). In the present study a
significant proportion of the PGs isolated after the first
density gradient centrifugation were eluted in the void
volume on Sepharose 2B chromatography whereas
those recovered from the bottom of the second gradient
were essentially wholly retarded; these findings provide
evidence that the PGs in 0.5 M guanidinium were indeed
aggregated and that they were disaggregated by 4 M
guanidinium.
Not all PGs extracted from cartilage by 4 M
guanidinium will aggregate in vitro in the presence of
HA (2,4). PGs that are extracted by low-speed, brief
homogenization in sodium acetate, as in the present
study, are presumably not aggregated in vivo because
these extraction conditions are unlikely to dissociate
them from H A . Furthermore, when PGs extracted under these conditions were mixed in vitro with HA, no
evidence of aggregation could be found, although aggregation of PGs that had been extracted with 4 M guanidinium chloride was readily demonstrated (24). On the
other hand, after repeated, high speed homogenization of
BRANDT AND PALMOSKI
2 14
articular cartilage in neutral 0.15 M sodium acetate,
during which the tissue was subjected to considerable
shear forces, PGs were eventually liberated whose hydrodynamic size was reversibly diminished by treatment
with 8 M urea, without apparent change in their composition (16). These P G s were similar therefore to those
extracted with 4 M guanidinium.
The present study indicates that nonaggregated
PGs (eg those readily extracted by sodium acetate) exist
in considerably larger numbers in OA than in normal
bovine knee cartilage. Recent studies have shown that
PGs may also be liberated from O A bovine hip cartilage
by isoosmotic neutral sodium acetate in much greater
amounts than normal (6). In agreement with these results is the fact that, when diseased cartilage from dogs
with naturally occurring or experimentally induced O A
was extracted with 2 M calcium chloride, yields of P G s
were approximately twice those obtained from normal
cartilage (8). Comparable results have also been obtained with cartilage from lame pigs that had been
reared intensively with restricted activity but that did
not have OA (25). These data all suggest a n abnormality
of PG aggregation, a possibiiity that is supported by
comparison of the aggregated P G s of normal a n d O A
cartilage in the present study, in which aggregates from
the diseased cartilage tended to be smaller than normal.
After disaggregation, however, P G s of normal and O A
c a r t i l a g e were essentially t h e s a m e in a v e r a g e
hydrodynamic size and had similar protein contents.
Furthermore no appreciable difference was noted between the core proteins of normal a n d O A PGs on
amino acid analysis, or between these and the core proteins of bovine nasal cartilage P G s (3) (Table 3).
Even though they did not differ on Sepharose 2B
gel chromatography, PGs from O A cartilage contained
shorter chondroitin sulfate chains than those from normal. Moreover this difference in chain length was the
same in sodium acetate and guanidinium chloride extracts. Chondroitin sulfate chain lengths of the PGs
of normal cartilage, calculated from molar ratios of
xy1ose:uronic acid under the assumption that all the
xylose is engaged in linking chondroitin sulfate t o protein (26), averaged 30 repeating disaccharide units.
Those o n PGs of O A cartilage averaged approximately
20 repeating units. Elution profiles of t h e GAGs on
Sephadex G-200 chromatography (Figure 1 ) reflect the
difference in chondroitin sulfate chain length indicated
by the analytical data. T h e tendency toward lower buoyant density of O A PGs, in comparison with those of
normal cartilage, may also be attributed to a smaller
amount of polysaccharide, relative to protein, o n O A
PGs. These data therefore agree with studies that reported a decrease in chondroitin sulfate chain length in
O A on the basis of reducing end group analysis (10) or
fractionation of cetylpyridinium chloride complexes of
the G A G s (14). They are consistent also with data showing that PGs of human OA cartilage have a lower uronic
acid:protein ratio than normal (9). T h e present study
did not indicate that the keratan sulfate content of O A
PGs was diminished, although such diminution has been
noted by others (27).
Whether the observed decrease in chondroitin
sulfate chain length represents an abnormality in G A G
synthesis or increased degradation of chondroitin sulfate in O A is not presently established. Hyaluronidase,
which degrades chondroitin sulfate, is present in synovial fluid (28) although it has not been demonstrated in
articular cartilage. Decreased aggregation of PGs in O A
cartilage, as indicated by the present study, could facilitate penetration of synovial fluid hyaluronidase into the
cartilage matrix and thus result in some degradation of
chondroitin sulfate chains a n d possibly also of H A in
the cartilage.
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grounds, proteoglycans, substances, knee, osteoarthritis, norman, cartilage, organization
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