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Joint motion in the absence of normal loading does not maintain normal articular cartilage.

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Articular cartilage from the knees of 4 dogs
whose ipsilateral paws had been transected 6 weeks
earlier (knee,,,,,), and from their contralateral knees
(knee,,), was examined. Knee,,,,, did not bear weight as
a result of the surgical procedure but active motion of
the joint, determined with an angular displacement monitor during walking, was comparable to that of knee,,..
In comparison to knee,, cartilage, knee,,,, samples
showed decreases in thickness, Safranin-0 staining of
the matrix, and uronic acid content (mean, 24.4%), and
increase in water content (mean, 5.9%). Incorporation of
into proteoglycans was 34-67% less in knee,,,,, than
in knee,,, cartilage. Proteoglycan (PC) aggregation in
knee,, cartilage was normal, whereas in knee,,... cartilage the bulk of the proteoglycans, and also those that
had been newly synthesized ('5S-proteoglycans), did not
exist in aggregates. This defect in aggregation was due,
at least in part, to an abnormality in the hyaluronatebinding region of the core protein of the proteoglycans,
since they did not interact in vitro with hyaluronic acid
These changes are essentially identical to those shown
Supported in part by a grant from the National Institute of
Arthritis, Metabolism and Digestive Diseases (#AM 20582) and
awards from the Arthritis Foundation. the Indiana Chapter of the Arthritis Foundation. and the Grace M. Showalter Trust.
From the Kheumatology Division and the Department of
Orthopedic Surgery. Indiana University School of Medicine. Indianapolis, Indiana.
Marshall J. Palmoski, PhD: Assistant Professor of Medicine;
Robert A. Culyer, MI): Assistant Professor of Orthopedic Surgery;
Kenneth D. Brandt, MI): Professor of Medicine and Chief, Rheumatology Division, Indiana University School of Medicine, Indianapolis.
Address reprint requests to Marshall J . Palmoski, PhD,
Rheumatology Division Indiana University School of Medicine, I I 0 0
W. Michigan Street, Indianapolis, Indiana 46223.
Submitted for publication August 22, 1979: accepted in revised form November IS. 1979.
Arthritis and Rheumatism, Vol. 23, No. 3 (March 1980)
to occur in canine knee cartilage after immobilization of
the leg in a cast. Thus, the loading of the joint which occurs from contraction of the muscles that span the knee
and stabilize the limb in stance, and not merely joint
movement, may be required to maintain the integrity of
the articular cartilage.
As part of an ongoing study of the effects of
physical factors on the biochemistry of articular cartilage, we recently described changes in knee cartilage of
normal dogs after immobilization of the hind limb in a
cast for a few weeks (I). The atrophy of knee cartilage
which occurs under this condition was accompanied by
a decrease in the tissue content of uronic acid, suppression of net synthesis of proteoglycans (PGs), and defective macromolecular organization of the cartilage (i.e.
impaired PG aggregation). These biochemical changes
have biomechanical implications and lead to diminished elasticity and compressive stiffness of the cartilage. Although all the abnormalities were fully reversed
2 weeks after removal of the cast, usage of the limb
while they were present might be expected to lead to
chondrocyte injury.
It has been considered that the degenerative
changes in joint cartilage induced by immobilization are
the result of the lack of joint movement (2-5). Since the
nutrition of normal adult articular cartilage is derived
chiefly from the synovial fluid (6), joint motion has been
considered to be important for the pumping of nutrient
molecules from the synovial space into the cartilage,
and for the movement of catabolites from the cartilage
into the synovial space (7). Maroudas, however, has
concluded, on the basis of the relatively high diffusion
coefficients of small ionic and nonionic solutes, that
compressive force will not appreciably increase the rate
of transport of nutrients in articular cartilage above that
which can be achieved by diffusion alone (8).
If diffusion of nutrients and metabolic wastes
through cartilage is indeed adequate without joint motion, what other factors might account for the biochemical, metabolic, and morphologic changes that occur
with immobilization? The immobilization procedure
that we employed ( I ) secured the hip and knee in approximately 90" of flexion. In addition to precluding
joint movement, it prevented the normal loading of the
articular cartilage which occurs with contraction of the
muscles that span the knee and stabilize the limb. The
present study was designed to examine the possibility
that the degenerative changes which arise in articular
cartilage with immobility may be the result of a decrease in loading of the cartilage and may thus occur
even with normal joint movement. Our results strongly
support that contention.
Surgical procedure. The right hind limbs of 4 adult
mongrel dogs (approximately 30 kg) were transected immediately above the ankle with aseptic technique under sodium
pentothal anesthesia. Upon recovery from anesthesia the dogs
ambulated freely on 3 legs in pens large enough to allow
walking (12 feet X 5 feet). The incisions healed promptly.
None of the animals developed a wound infection.
After 6 weeks the dogs were killed with an overdose of
sodium pentothal. The knees were opened aseptically and
both distal femurs were removed with a bone saw. Representative samples of cartilage were taken for histologic study with
a Craig biopsy needle (internal diameter, 3 mm) from the central portion of the medial condyle. After other portions (approximately 15 mg) were obtained for determinations of dry
weight and uronic acid content, the remainder of the cartilage
from the weight-bearing portions of the medial and lateral
femoral condyles (approximately 250 mg per joint) was
shaved with a scalpel into slices less than 0.5 mm thick. The
shavings from each joint were pooled and analyzed separately.
Tissue culture. All of the cartilage slices from the
knees of the limbs whose ipsilateral paws had been transected
(knee,,,,,) and from the contralateral knees (knee,,,) of dogs 1,
2, and 3, and 50 mg of the cartilage from each knee of dog 4
were placed in flasks containing culture medium consisting of
Ham's F-12 nutrient mixture, pH 7.4, and fetal calf serum
(8: 2, v/v), streptomycin (50 pg/ml), penicillin (50 Units/ml),
and Na, 3sS0, (10 pCi/ml) (New England Nuclear COT.,
Boston, Massachusetts). The cultures were maintained at
37OC for 20 hours under 5% C02: 95% air. The medium was
then 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 Spectrapor No. 3 dialysis tubing (Spectrum Medical Industries, Inc., Los Angeles, California), which
has an approximate molecular weight cutoff of 3,500 daltons.
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 from the cartilage. After
removal of the culture medium the washed cartilage slices
were suspended in 10 ml of 0.4M guanidinium chloride
(GuHCI) in O.OSM sodium acetate, pH 5.8, containing the
protease inhibitors EDTA (O.OlM), 6-aminohexanoic acid
(0. IM), and benzamidine hydrochloride (0.05M) (9). After
stirring for 48 hours at 4°C the suspension was filtered, the
residue was washed with 5 ml of 0.4M GuHCI, and the filtrate and washings were combined and dialyzed as above. The
cartilage residue was stirred for 48 hours at 4°C in 10 ml of
4.OM GuHCl in 0.05M sodium acetate, pH 5.8, containing the
above protease inhibitors. The suspension was then filtered,
the residue was washed with 5 ml of fresh 4.OM GuHCl, and
filtrate and washings were combined and dialyzed to 0.4M
with respect to guanidinium. Finally. the cartilage residue was
suspended in 3 ml of O.lM borate, pH 8.0, containing 0.2M
calcium chloride. Pronase (Calbiochem, San Diego, California) was added (1 mg enzyme/SO mg cartilage) 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 and the supernatant was dialyzed overnight
against distilled water. The sacs were rinsed with a small
amount of distilled water, the retentate and rinses were combined, and the glycosaminoglycans (GAGS)in the pronase digest were isolated by precipitation with 9-aminoacridine hydrochloride and converted to their sodium salts with Bio Rad
AG 50 (Na') (10).
Aliquots (0.1 ml) of the dialyzed samples of medium,
0.4M and 4.OM GuHCl extracts and of the pronase digest
were separately added to 10 ml of Ready-Soh HP (Beckman
Instruments Inc., Fullerton, California) and counted in a
Beckman liquid scintillation spectrometer. Results were adjusted for differences in wet weight of the tissues.
Although the above procedures provided information
about newly synthesized '3-PGs, they did not permit conclusions concerning the status of the bulk of the PGs in the
tissue. Therefore, approximately 200 mg of the sliced cartilage
from each knee of dog 4 were not placed in tissue culture, but
were suspended immediately in 0.4M GuHCl in 0.05M sodium acetate, pH 5.8, containing the protease inhibitors. The
PGs were then sequentially extracted from this tissue by procedures identical to those described above for the "S-PGs.
The uronic acid contents of the GuHCl extracts and of the residual GAGS, after isolation and purification of the latter by
precipitation with 9-aminoacridine as described above, were
determined ( I I).
Isolation of aggregated and disaggregated PGs. Following dialysis of the medium and 4.OM GuHCl extracts,
these fractions were subjected to cesium chloride equilibrium
density gradient centrifugation in 0.4M GuHC I according to
the method of Hascall and Sajdera (12), i.e., under associative
conditions which allow recovery of PG aggregates. Material
from the bottom 2/5 of the gradient (average density = 1.76
gm/ml) was dialyzed exhaustively at 4°C against 0.05M sodium acetate, pH 6.9, to yield Fractions AHcdand A,, from
the medium and GuHCl extract, respectively. These fractions
normally contain PG aggregates, i.e., PGs noncovalently
bound to hyaluronic acid (HA) in a linkage stabilized by link
Figure 1. Angular displaccment monitor. See text for details.
glycoproteins (13). The fractions also contain some nonaggregated PGs ( 13).
Purified disaggregated PGs were obtained by centrifugation of Fraction Ao. in a second cesium chloride gradient in
the presence of 4M GuHCI, i.e. under dissociative conditions
(12). Material from the lower 2/5 of this gradient (average
density = 1.51 gm/ml) was dialyzed exhaustively at 4°C
against 0.05M sodium acetate, pH 6.9, and designated Fraction DGu.
Gel chromatography. Samples (0.5 ml) of PGs in
0.05M sodium acetate, pH 6.9, were applied to a column (90.0
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 "SO4 radioactivity (dogs 1 4 ) or uronic
acid concentration (dog 4) of 1 ml emuent fractions was determined as above. Partition coefficients (Kav) were calculated
from the formula: K,, = (Ve-Vo)/(V,-Vo), where V, represents the peak fraction in the elution diagram, V, the void volume, and V, the total column volume.
Interaction of PGs with hyaluronic acid (HA). To assess their ability to undergo aggregation in vitro, purified disaggregated PGs (Fraction DG.)were incubated with HA from
human umbilical cord (Sigma Chemical Corp., St. Louis, Missouri). This HA preparation was sufficiently large in hydrodynamic size to elute completely in the void volume on Sepharose 2B chromatography. The PGs were made 0.5M with
respect to sodium acetate, pH 6.9, HA was added, and the solution was allowed to stand for 18 hours at 2 0 ° C after 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 (1 :72,
uronic acid) was similar to that effecting maximal aggregation
in vitro (14) and provided a ratio of the 2 moieties comparable
to that found in puriEed PG aggregates (13).
Digestion of PG aggregates with HA b1-3 hydrolase.
Aliquots (2 ml) of AM&or AGuwere made O.IM with respect
to citric acid and 0.2M with respec! to Na,HPO, x 7 H,O.
The pH was adjusted to 5.6, and 1.5 pg of leech HA /31+3 hydrolase (Biotrics, Inc., Arlington, Massachusetts) were added
over a 5 hour incubation period at 37°C; Sepharose 2B chromatographs of the PG samples before and after incubation
were then compared. The activity of the hyaluronidase was
determined viscosimetrically; 0.3 pg of the enzyme decreased
the specific viscosity of a 0.1% solution of HA from 4.10 to
0.24 in 75 minutes at 37°C. The lack of activity of the enzyme
against purified PGs was confirmed by its failure to alter the
Sepharose 2B elution profile of Fraction DGU.
Analytical methods. For determinations of wet weight
of the tissue, approximately 15 mg of cartilage were blotted
gently with Whatman #2 filter paper (CLiRon, New Jersey)
and weighed immediately. Water content of the cartilage was
determined after the tissue had been placed in two changes of
acetone for 24 hours and dried to constant weight in yacuo at
80°C. The dried cartilage was digested with pronase as above;
the GAGS then were isolated by precipitation with %aminocridine hydrochloride and converted to their sodium salts with
Bio Rad AG 50(Na') (10). After the resin was removed by 61tration, the uronic acid content of the filtrate was determined
( 1 1).
Histologic examination of the cartilage. Full thickness
samples of the cartilage and a portion of the underlying subchondral bone were obtained from the central portion of the
medial condyle with a Craig biopsy needle (3 mm diameter).
The cartilage was fixed in 1Wo formic acid for 5 days and imbedded in paraffin. Sections (6p) perpendicular to the surface
were cut on a microtome and stained with Safranin-0 fast
Measurement of knee joint motion. Movement in
knee,,., as well as in kneeconwas obvious upon inspection of
each animal during walking. Joint motion was recorded by
means of an angular displacement monitor (Figure 1). The
unit, which was enclosed in a pouch, was strapped externally
over the knee joint (Figure 2) while the animal was led on a
leash through a 60-step walk. Recordings were taken in quadruplicate from knee,. and knee,,,,, of dogs 3 and 4 four weeks
after surgery.
The instrument records movement of the joint
through a programmed angle referenced to the vertical and
operates independently of its vertical axis orientation. It contains a memory register that records the number of times the
unit is moved through n X 5", where n can be set by the researcher to range from 1 to 11. The transducer in this activity
monitor is a curved bubble chamber filled with methanol and
containing 12 platinum electrodes spaced 5" apart. As the
moving bubble comes into contact with an electrode, the resistance between the fluid and electrode increases. The contact is
recorded and the information is stored and compared with the
subsequent positions of the migrating bubble as the monitor is
pivoted. A count is displayed digitally if the bubble has traveled through the preset angle. Additional counts are generated
with each change in direction of the bubble if the distance
(number of electrodes) traversed is equal to or greater than
the preset value of n.
Although the device employed would detect angular
displacement of the extremity produced by hip motion as well
as that produced by knee motion, when the monitor was preset at any angle between 10"-20", the counts recorded with
the instrument strapped to the leg below the knee (and hence
recording principally tibiofemoral movement) were essentially
identical to those obtained when it was strapped to the leg at
the level of the knee joint.
At the time of killing, none of the knee joints
contained a synovial effusion. Femoral articular cartilage from knee,,,,, , as well as from knee,, , appeared
normal and was white and smooth with no softening,
pitting, or osteophytes. The cartilage from knee,,,,, of
dog 1 was considerably thinner than that of the knee,,,
of this animal, but there was no obvious difference between the thickness of the cartilage from knee,,, and
knee,,,,, of the other animals.
Histology and histochemistry. The surface of the
cartilage was intact in every case, and the number and
distribution of chondrocytes appeared normal, with no
evidence of brood capsules. No abnormalities of the
tidemark were seen. In all samples of knee,,,, intensity
and distribution of staining of the extracellular matrix
with Safranin-0 were judged normal. Invariably, cartilage from knee,,,,, stained less intensely than cartilage
from knee,,, of the same animal (Table 1). The thickness of the knee,,,,, cartilage from dogs 2,3, and 4, measured from surface tidemark in triplicate sections, was
1 4 2 9 % less than that of the cartilage from the corresponding knee,,,, while that from dog 1, which was
noted to be thin on gross inspection (see above) was
55% less upon measurement of the histologic sections
(Figure 3). The knee,,,,, cartilage from dog 1 also exhibited the most marked decrease in Safranin-0 staining of
all the samples examined, and some horizontal fraying
Figure 2. The angular displacement monitor. The unit is enclosed in, a
pouch which is sewn onto an orthopedic stocking covering the dog’s
leg. The stocking is secured by pinning it to a coat strapped onto the
dog’s back.
of the subsurface layer was observed in this dog but not
seen in the other specimens.
Water and uronic acid contents. In every case
cartilage from knee,,,,, had a higher water content and
lower uronic acid content than that from knee,,, (Table
1). The increases in water content ranged from 3.8 to
9.8% (mean 5.9%) of the tissue wet weight while the decreases in uronic acid content were variable. A maxi-
Table 1. Characterization of canine knee cartilage from limbs in which the ipsilateral paw was
transected and from the contralateral control limbs
Dog I
Dog 4
Source of
Severe reduction
Slight reduction
Slight reduction
Moderate reduction
I .o
8 of tissue
wet weight
acid content,
8 of tissue
dry weight
Knee,,. = control knee; Knee,,,,, = knee from limb whose ipsilateral paw had been transected 6
weeks earlier.
t Mean thickness, from surface to tidemark, of 3 histologic sections from medial femoral condyle. The
thickness of the 3 sections examined from each joint varied by less than 0. I mm.
As determined by a Student’s I evaluation (paired observations) the increase in water content in
Kneelran,was significant (P< 0.025).
Figure 3. Cartilage stained with Safranin-0, from (A)left knee (knee,,,) of dog I, and (B) from right knee of
same animal, 6 weeks after transection of the ipsilateral paw. The cartilage thickness in (B) is reduced by 56%
and Safranin-0 staining is markedly decreased in comparison with (A)(magnification x 40).
tal '3 PGs/lO mg wet weight of cartilage, the distribution of newly synthesized PGs between the culture medium, the 2 GuHCl extracts, and the pronase digest of
the cartilage fron knee,,,,, was similar to that seen with
cartilage from knee,,,. (Table 2). The medium contained
about 15% of the total "S-PGs, and the 0.4M GuHCl
extracts only about 5%. In every case the greatest proportion (52.5-60%) of the total '3-PGs was extracted
with 4.OM GuHCl. Based on the sum of the non-dialyzable 35S radioactivities in the medium, GuHCl ex-
mum decrease of 53% was seen in the cartilage from
knee,,.,, of dog 1, and decreases of 16, 5, and 21% were
noted in cartilage from knee,,,,, of dogs 2, 3, and 4, respectively. In contrast to the changes in water content,
the decreases in uronic acid content in cartilage from
knee,,,,, were not statistically significant as determined
by a Student's t evaluation using paired observations
Sequential extraction of PCs from the cartilage
and net synthesis. Based upon the proportion of the to-
Table 2. Incorporation of " S into proteoglycans* of femoral cartilage from limbs in which the
ipsilateral paw was transected and from the contralateral control limbs
76 Distribution of total "S-cpm insequential extracts of cartilage
Dog I
Dog 2
Dog 3
Dog 4
Total 3JS$
Non-dialyable ' 5 S radioactivity.
= control knee; Knee,,,,, = knee from limb whose ipsilateral paw had been transected
6 weeks earlier.
Total "S incorporation into proteoglycans/lO mg wet weight of cartilage. Results are shown as percentage of control
t Knee,,,.
Table 3. Sepharose 2B chromatography of 3sS-proteoglycanaggregates (Fraction A,") and nonaggregated proteoglycans (Fraction DGU)
Fraction tL;.
Fraction D,.
+ hyaluronic acid
Fraction Atiu
Dog I
Dog 3
Source of
81 + 3 hydrolase
Fraction D,,
hyaluronic acid
K,,. VrS
K.,. VrS
K.,. VrS
0.5 1
control knee; KneetranJ= knee from limb whose ipsilateral paw had been transected 6 weeks earlier.
t V,, = Proportion of sample eluting in the Sepharose 2B void volume.
+ V, = Material retarded by the gel.
tracts, and the pronase digest of the cartilage residue,
net PG synthesis was invariably lower (3467%) in
knee,,.,, cartilage than in cartilage from the corresponding knee,,. (Table 2).
PG aggregation. Fraction Ahleaaccounted for
about 50% of the total nondialyzable ' 3 radioactivity in
the culture medium, and thus represented only about
7% of the total 3 - P G s . It was not analyzed further.
Similarly, since the 0.4M GuHC 1 extracts represented
only about 5% of the total '3-PGs, no attempt was
made to characterize the PGs.
Fraction AG. represented about 90% of the total
nondialyzable radioactivity in the 4M GuHC 1 extracts
from knee,,, and knee,,,,,. Fifteen to thirty-five percent
of A," from the knee,, samples were sufficiently large
in average hydrodynamic size to elute in the void volume on Sepharose 2B chromatography (Table 3)
whereas the K,, of the PGs that were retarded by the gel
(V,) ranged from 0.35 to 0.44 (Table 3). In contrast, A,,
from knee,,,, of dogs 1, 3, and 4 contained no material
sufficiently large in hydrodynamic size to elute in the
Sepharose 2B void volume whereas the K,,s of the retarded PGs were 0.440.46, indicating that they tended
to be slightly smaller in average hydrodynamic size than
the retarded PGs from the corresponding knee,. cartilage, whose K,,s were somewhat lower (Table 3). However, the differences in the K,,s of the retarded material
in knee,,. and knee,,,,, are not statistically significant as
determined by a Student's t evaluation using paired observations. Although approximately 15% of the "S-PGs
in Atiu from knee,,,,, of dog 2 were recovered in the void
volume peak, more than twice as much (35%) of the corresponding fraction from knee,, of this animal were excluded by Sepharose 2B.
Incubation with hyaluronic acid p1+3 hydro-
lase, an enzyme specific for hyaluronic acid, eliminated
the Sepharose 2B void volume peak of Fraction A,"
from each of the knee,,,. samples, as did exposure to the
dissociative conditions of the second cesium chloride
density gradient (run in the presence of 4.OM GuHCI)
(Figure 4, Table 3). These results strongly suggest that
the AG. fractions from knee,,, contained PGs in macromolecular association with HA.
Treatment of AG. with hyaluronic acid p1+3
hydrolase in some cases (knee,,, and knee,,,,,, dogs 1
and 2) produced a modest increase in the K,, of PGs retarded by the gel, suggesting that some PG aggregates
that were small enough to elute behind the void volume
peak were present in these samples. Notably, the K,,s of
the PGs in Fraction DG. were virtually identical to
those of the PGs in the V, fraction of A,, after that
sample had been treated with hyaluronic acid p1+3 hydrolase (Figure 4, Table 3).
PG-HA interaction in vitro. When Fraction DGu
from knee,,, of dogs 1 and 3 was incubated with high
Table 4. Movement of control knee and knee of limb with ipsilateral
paw transected, determined with an external angular displacement
monitor during a 60-step walk
Cumulative counts recordedt
Dog 3
Dog 4
Source of
45.3 f 0.4
53.0 0.6
41.3 0.7
44.6 f 0.9
42.5 f 3.3
52.0 f 2.9
39.0 f 0.6
43.3 f 2.3
8.5 f 1.6
9.3 f 1.3
Knee,,. = control knee; Knee,,,,, = knee from limb whose ipsilateral paw had been transected 6 weeks earlier.
t Number of times which knee passed through the preset angle (in
either Bexion or extension). Counts represent mean f 1 SE of quadruplicate determinations.
molecular weight HA in vitro, evidence of PG-HA interaction was readily apparent. Whereas the purified
D<,"fractions did not contain any PGs eluting in the
Sepharose 2B void volume, 14%and 38% of these samples were excluded from the gel after incubation with
hyaluronic acid. In contrast, incubation with HA did
not alter the elution profiles of Dcrufrom knee,,,,, of
these animals (Figure 5, Table 3).
Analyses of nonlabeled PGs. The bulk of the unlabeled PGs in cartilage from knee,,,,, and knee,,,, of
dog 4 (55% and 6296, respectively) was extracted with
4M GuHCI, with only 2-3% contained in the 0.4M
GuHCl extract. Although 24% of the PGs in Fraction
A,,, from knee,,, eluted in the Sepharose 2B volume
(uronic acid), those in the corresponding fraction of
knee,,,,, were completely retarded by the gel (K,,, 0.52).
After digestion with hyaluronic acid PI-3 hydrolase,
the void volume material in Fraction A,, from knee,.
was abolished, while the K,, of the retarded material in
Fraction A,,, from knee,,,. and knee,,,,, was unaffected.
Measurement of knee joint motion. During walking, the range of motion in knee,,, and knee,,,., of dogs
3 and 4 appeared normal on gross inspection. When
these animals were guided through a 60-step walk with
33 1
the angular displacement monitor strapped externally to
either knee,, or knee,,,,,, the cumulative counts obtained with the unit set to record movement at 15" from
the vertical were similar to those obtained when it was
set to record at 10" (approximately 40-50 counts)
(Table 4). At either angle, counts from knee,,,,, of dog 3
were greater (17%, 24%, respectively) than those from
knee,,,,. Minimal difference was observed, however, between the counts from knee,,, and knee,,,, of dog 4 with
the monitor set at 10" or 15". When the unit was set at
25", it registered far fewer counts than when it was set
at the lesser angles (dog 3, knee,, = 8.5, knee,,,,, = 9.3
counts) (Table 4).
The changes in knee,,,,, cartilage in the present
study-decrease in thickness, reduction in Safranin-0
staining, loss of uronic acid (PG) content, increase in
water content, defective PG aggregation, and reduction
of net PG synthesis-are identical to those that develop
in canine knee cartilage after complete immobilization
of the hind limb for a few weeks (1). The significance of
the present results lies in the fact that they occurred not
Figure 4. Results of chromatography on a Sepharose 28 column (90 X
1.0 cm) of proteoglycans from knee,,,,, and kneecon of dog 3. A =
Fraction A,;" from knee,,,,; B = Fraction Atiu from knee,,,,,. Pro-
teoglycans are shown before (-) and after (---) incubation with
hyaluronic acid B1+3 hydrolase. C = Fraction Dcjufrom knee,,.; D
= Fraction Dcjufrom knee,,,.,.
r H A C T I 0 3 ImLJ
Figure 5. Results of chromatography on a Sepharose 28 column (90 X
1.0 cm) of proteoglycans from cartilage of dog 3 prepared under dissociative conditions (Fraction Dcj,). Elution profiles of Fraction Dciv
are shown before (-) and after (---) incubation with hyaluronic acid.
A = knee,,,,; B = knee,,,,,.
with immobilization, but while movement of knee,,,,,
was comparable to, or greater than, that of knee,,,,
(Table 4).
An increase in water content has been noted also
in cartilage from osteoarthritic joints (16,17) and may
imply a failure in the elastic restraint of the collagen
meshwork of the matrix (18), enabling the PGs to become more highly hydrated than normal (19). The decrease in net PG synthesis in the knee,,,., cartilage, however, contrasts with the findings in osteoarthritic
cartilage in which net PG synthesis appears to be increased in the early stages (20,21).
Notably, the defect that permitted knee,,,,, cartilage to take up excessive water was not reflected in an
abnormality in extractability of the PGs. Thus, the proportion of the total “S PGs present in the medium (1020%) was not greater in cultures of knee,,,,, than of
knee,,,., and none of the 0.4M GuHCl extracts contained over 8% of the total PGs (Table 2). Low molarity
salt solvents (e.g., 0.4M GuHCl) do not affect the interaction between the constituents of PG aggregates (PGs,
HA, link glycoproteins) (12) and solubilize only a small
proportion of the total PGs of normal cartilage (22,23).
The PGs that can be extracted with low molarity salt do
not interact with HA in vitro (23), suggesting that they
are not aggregated in vivo. On the other hand, in some
cases PG aggregates are present in 0.4M GuHCl extracts of cartilage (Palmoski M, Stack M, and Brandt K,
unpublished observations), so that solubilization of an
abnormally high proportion of the total tissue PGs by
this solvent may not be due to defective PG-HA-link
protein interaction, but to defective stabilization of the
matrix, perhaps caused by an abnormality in PG-collagen interaction (24).
T h e present d a t a emphasize that all nonaggregated PGs are not extracted by 0.4M GuHCI.
Thus, even though no evidence of (’3)PG aggregation
was present in knee,,,,, of dogs 1, 3, and 4, and aggregation in knee,,,,, cartilage from dog 2 was diminished by
hdlf in comparison with that in knee,,, (based on the
amount of PGs excluded from Sepharose 2B), the proportion of PGs in the 0.4M extracts of knee,,,,, was no
greater than that in the corresponding extract of knee,,,,
and the bulk of the PGs in the tissue required a strong
dissociating solvent-4.0M GuHC1-for extraction.
Evidence of the PG aggregation defect in knee,,,,,
was clear-cut and included 1) the absence of molecules
large enough to be excluded from Sepharose 2B, 2) the
lack of effect of hyaluronic acid B1+3 hydrolase on the
elution profile of Fraction AG,, and 3) the observation
that the average hydrodynamic size of Fraction Aciuwas
only slightly larger than that of the PGs in Fraction
Dcju.Notably, the aggregation defect in knee,,., was
present not only in newly synthesized (3)
PGs but in
the bulk of the PGs in the tissue, as indicated by analysis of the unlabeled cartilage.
The aggregation defect may have resulted from
an abnormality in the HA-binding region of the PG
core protein, since the PGs did not interact with HA invitro (Table 3). Although link glycoproteins serve to stabilize the PG-HA interaction in aggregates and are essential t o t h e integrity of th e aggregate in a n
ultracentrifugal field (25), their absence does not interfere with the demonstration of PG-HA complexes by
gel chromatography. The present data, therefore, do not
preclude defects in the link glycoproteins or in the cartilage HA, in addition to those in the PGs themselves.
Fraction A,, represented only about 50% of the
cpm in the culture medium of both
knee,,, and knee,,,,,, whereas the analogous Fraction,
Atiu, represented about 90% of the nondialyzable cpm in
the 4.OM GuHCl extracts. This finding might indicate
that some PGs in the medium were degraded or contained relatively short GAG chains. In previous studies
in which methods identical to those employed here were
used, we were able to recover high molecular weight PG
aggregates from the medium ( I ) . The decrease in average hydrodynamic size of Fraction Acjufrom all samples
of knee,,, and knee,,,,, of dog 2 after incubation with
HA /31+3 hydrolase was due to digestion of the HA in
the aggregates and not to digestion of chondroitin sulfate chains of the PGs. The enzyme had no effect on the
size of purified disaggregated PGs and the K,, of the
PGs in Fraction Acjuafter treatment with the enzyme
was essentially the same as that of the PGs isolated after
dissociation of the aggregates with 4M GuHCl (Table
Whether the knee,,, cartilage should be considered “normal” is arguable; it must have been subjected
to some excessive loading because of the transection of
the paw on the opposite limb. Indeed, we showed that
some decrease in PG aggregation occurred in cartilage
from the left knee of dogs while their right hind limbs
were immobilized in a cast for up to 8 weeks (1). However, in the present study, no defect in PG aggregation
was apparent in knee,,,, cartilage, and the tissue appeared histologically and histochemically normal and
exhibited water (approximately 72%) and uronic acid
contents (approximately 4%), which were similar to
those of numerous other samples of adult canine knee
cartilage which we have examined.
The present results, taken in conjunction with
those of our previous study which showed similar
changes in articular cartilage after immobilization (I),
indicate that joint movement alone may be insufficient
to maintain the integrity of the articular cartilage. What
other physical factors could be involved? Most of the
force across the knee joint is generated by contraction of
the muscles spanning the joints normally used to stabilize the limb in stance. In the normal human quadriceps
muscle this force may be nearly 2000 kg force/cm (26).
It is even greater with isometric contraction (27). In the
animals used in the present study the major muscle
groups spanning knee,,,,,, the quadriceps and hamstring, were intact and functioned in a reasonably normal fashion in joint movement, as indicated by the data
obtained with the angular displacement monitor (Table
4). However, since the limb was no longer in contact
with the ground, its action in stabilizing the leg was
eliminated, thereby reducing the loading of the articular
The present results are consistent with the observation that degenerative changes developed in the lateral tibia1 condyle of rats when contact was eliminated
by excision of the lateral femoral condyle (28). Whether
the degenerative changes that occur in the knee with
femoral condylectomy, immobolization, and ipsilateral
paw amputation are all due to nutritional deprivation of
the chondrocyte or to other factors is unknown. Obviously these changes occur notwithstanding the high diffusion coefficients of small solutes, e.g., glucose, observed in cartilage in vitro (8).
Little is known about the effect of physical forces
on PG metabolism in cartilage. However, the chondroitin sulfate content of weight-bearing areas of articular
cartilage is greater than that of nonweight bearing regions (29). Similarly, GAGSconstitute a higher proportion of the dry weight of weight-bearing than of nonweight bearing sites of human dermis (30). In a more
direct test of the effect of mechanical stress on GAG
metabolism, when cultured smooth muscle cells from
pig aorta were centrifuged GAG production increased
approximately 50%, and heparan sulfate, dermatan sulfate, and chondroitin sulfate showed relatively greater
increases than hyaluronic acid (3 1).
Recently, the prevalence of osteoarthritis was
found to be increased in the contralateral knees of patients with below-the-knee amputation (32), presumably
due to increased load bearing on the normal limb. NO
information was provided concerning load bearing on
the limbs fitted with prostheses, or of their usage. The
present study suggests that these variables might account for some of the degenerative changes in the ipsilateral knee.
We would like to thank Professor John Ryan of the
Department of Electrical Engineering Technology at Indiana
University who designed the angular displacement monitor.
We are grateful to Janet Anderson and Jeffrey Wilson for
technical assistance and to Margaret Britner for secretarial
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motion, absence, maintain, joint, loading, norman, cartilage, articular
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