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Mechanical and biomechanical changes in

the superficial zone of articular cartilage in a
canine model of osteoarthritis

Article in Journal of Orthopaedic Research · July 1994

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Journal of Orthopaedic Research
12474484The Journal of Bone and Joint Surgery, Inc
0 1994 Orthopaedic Research Society

Mechanical and Biochemical Changes in the Superficial Zone

of Articular Cartilage in Canine Experimental Osteoarthritis

Farshid Guilak, Anthony Ratcliffe, *Nancy Lane, Melvin P. Rosenwasser, and Van C. Mow

Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Columbia University,New York, New York,
and *Division ofRheumatology, University of California at San Francisco, San Francisco, California, U.S.A.

Summary: The changes in the tensile mechanical properties and biochemical composition
of the superficial zone of articular cartilage were examined in a canine model of early
osteoarthritis generated by transection of the anterior cruciate ligament. Sixteen weeks
following ligament transection, the tensile stiffness of the articular cartilage was decreased
by 44% and the ion-induced stress relaxation of the tissue was increased by 57% compared
with the contralateral control. Biochemical analyses indicated that the water content of
the experimental tissue was increased by 13%, which was reflected as an apparent 37%
decrease in the proteoglycan content and a 36% decrease in the collagen content (ex-
pressed per wet weight). The hydroxypyridinium crosslink density was decreased in the
experimental tissue by 11%. A significant negative correlation was found between the
ion-induced stress relaxation and the hydroxypyridinium crosslink density in both control
tissue (R = -0.56) and experimental tissue (R = -0.70). No correlation was noted between
the tensile stiffness and the biochemical composition of the tissue. These results suggest
that, in the superficial zone of articular cartilage, the structure of the tissue may play a
more important role than the composition in the determination of its mechanical proper-
ties. A major event observed in the model of early osteoarthritis appears to be the
disruption and remodeling of the collagen network in the superficial zone of the articular
cartilage.

The normal mechanical behavior and healthy
function of diarthrodial joints are strongly depend-
ent on the composition and ultrastructural organiza-
tion of the articular cartilage (29). Articular cartilage
is composed primarily of water (60-85%), and the
remaining solid matrix is composed of a crosslinked
network of type-I1 collagen (1522% of the wet
weight), aggregating proteoglycan (4-7% of the wet
weight), and lesser amounts of other collagen types
(including types IX and XI) and noncollagenous pro-
teins and glycoproteins (19). An important modifica-
Received May 5,1993; accepted August 31,1993.
Address all correspondence and reprint requests to Dr. F.
Guilak at Musculo-Skeletal Research Laboratory, Health Sci-
ences Center T18-030, State University of New York at Stony
Brook, Stony Brook, NY 11794-8181,U.S.A.
tion of the collagen in cartilage is the formation of
covalent intrafibrillar and interfibrillar trifunctional
hydroxypyridinium crosslinks, which are the ma-
ture products derived via borohydride-reducible hy-
droxylysine aldehydes (14J5). Both the composition
and macromolecular organization of articular carti-
lage are heterogeneous with depth from the tissue at
the surface and vary with age (10,24,45). In particu-
lar, the superficial zone contains collagen fibrils ori-
ented primarily parallel to the tissue surface. The
proteoglycans are present at a relatively low concen-
tration in this zone and increase in concentration
with depth.
The ability of cartilage to withstand applied loads
depends on mechanical, biochemical, and biophysi-
cal factors, such as the composition and structural
integrity of the solid matrix and the hydration and

474
 COMPOSITION A N D TENSILE PROPERTIES OF OSTEOARTHRITIC CARTILAGE
ionic environment of the tissue (23,29). Proteogly-
cans, due to their negative charge, are responsible for
the ion-induced swelling behavior of the tissue and
are believed to provide compressive stiffness to the
tissue (23,24). Collagen, due to its fibrillar structure,
provides tensile stiffness and strength to cartilage
(21,40). The heterogeneities in the structure and
composition of cartilage are believed to be responsi-
ble for the observed variations in mechanical prop-
erties of the tissue with depth (47). Similarly, the
mechanical properties of cartilage vary with age
and disease. The interrelationships among the struc-
ture, composition, and tensile properties of articu-
lar cartilage have been the subject of many recent
investigations on both healthy and diseased tissue
(2,3,21,31,39,43).
Osteoarthritis is characterized by progressive
changes in the ultrastructure and biochemical com-
position of articular cartilage and the surrounding
tissues (7). A canine model of experimental osteoar-
thritis generated by transection of the anterior cru-
ciate ligament (17,35) yields reproducible changes in
the morphology, composition, and metabolic activity
of articular cartilage. These changes are represent-
ative of early human osteoarthritis and are believed
to be initiated by the instability of the joint following
transection of the anterior cruciate ligament. Im-
portantly, these changes progress with time to ero-
sion and degeneration of the articular cartilage and
subchondral bone (5,27). Significant early changes
include increased tissue hydration (26) and disorga-
nization of the collagen network (46). Metabolic
changes include increased rates of proteoglycan and
collagen synthesis (13,38), increased proteoglycan
breakdown (8,36), and changes in the structure of
the glycosaminoglycan chains being synthesized on
the aggrecan protein core (9,37). Reported changes
in the biomechanical properties of articular carti-
lage include a decrease in the tensile stiffness and
an increase in the swelling behavior (4,31,43). These
structural and biochemical changes are responsible
for significant loss of the mechanical function of
the joint (7,20,34). However, it still is unclear how
the normal relationships between structure, compo-
sition, and mechanical properties of cartilage may be
changed with degeneration.
The objectives of this study were to examine the
role of tissue composition in the determination of
the tensile mechanical properties of the superficial
zone of articular cartilage and to investigate how the
interrelationships may be altered in a canine model
of early osteoarthritis. To minimize joint trauma and
475
any lacerative damage to the joint tissues, transec-
tion of the anterior cruciate ligament was performed
with a newly developed arthroscopic technique. The
isometric tensile swelling test was used to determine
the changes in the tensile mechanical properties and
swelling behavior of the articular cartilage. Impor-
tant compositional analyses included determination
of the hydroxypyridinium crosslink density and the
changes in the collagen, proteoglycan. and water
content caused by the experimental procedure.
MATERIALS AND METHODS
Canine Model
Skeletally mature female beagles, weighing 10- 12
kg, were kept in the animal facilities at Columbia-
Presbyterian Medical Center. After a 1-week quar-
antine and acclimation period, the animals were
anesthetized and the anterior cruciate ligament of
the right knee (stifle joint) was transected with use
of a newly developed arthroscopic procedure. In
brief, the animals were placed under general anes-
thesia, and the right leg was shaved and prepared
with antibiotic soap. The joint capsule was inflated
with Ringer's lactate solution so that medial and
lateral parapatellar stab incisions could be made. A
2.7 mm, 30" arthroscope was inserted through the
lateral portal, proximal to the menisci, and a right-
angle blunt probe was inserted in the medial portal.
This probe was used to palpate the intra-articular
structures atraumatically. Following a thorough ex-
amination to ensure the absence of previous liga-
ment or cartilage damage, a retrograde knife with a
blunt leading edge was used to transect the ante-
rior cruciate ligament under direct observation. An
anterior-posterior subluxation drawer test was per-
formed immediately to ensure full transection. The
incisions then were closed with a single interrupted
suture (Prolene; Ethicon, Somerville, NJ, U.S.A.),
and a sterile dressing was applied. The average oper-
ative time was approximately 30 minutes.
All animals were monitored closely for p,ain post-
operatively and were given analgesics as needed. The
dogs were allowed free activity in their cages postop-
eratively and were killed at 16 weeks. The hindlimbs
were removed immediately post mortenz and frozen
at -80°C for later analysis. As in previous studies, the
contralateral joint was used as a control (1,4,8,35).
Preparation of Specimens
Articular cartilage used for this study was har-
vested from the posterior aspect of the medial fem-
oral condyle (Fig. l ) , which is considered to be a
J Orthop Res. Vol. 12. No. 4, 1994
476
-
Superficial-zonespecimens for
biomechanicaland biochemical testing
-
(1.7 mm x 8 mm x 200 pm)
Lateral Medial
FIG. 1.Specimens for tensile testing were removed fromthe postcrior aspect of the medial femoral condyle. Biochemical analysis was
performed on an identically prepared specimen taken from the superficial zone, adjacent to the site of tensile testing.
weight-bearing area in the canine knee joint (22).
Prior to mechanical testing, the frozen joints were
thawed at 4°C and carefully dissected open. A min-
iature osteotome was used to remove full-thickness
blocks (800-1,000 pm) of articular cartilage from
the femoral condyle, in an orientation parallel to the
local split-line direction (the predominant orienta-
tion of collagen fibers at the articular surface). A
freezing stage microtome was used to remove the
top 180-200 pm (superficial zone) of each block
for testing (Fig. l),and a rectangular strip (approxi-
mately 1.7 x 8 mm) was cut from this sample with a
razor cutting jig (2). An adjacent sample from the
superficial zone was prepared in an identical manner
for biochemical analysis (water, collagen, hydroxy-
pyridinium crosslink, and proteoglycan contents).
Small pieces of waterproof polishing paper were
attached to the ends of the specimen with cyanoac-
rylate adhesive in order to prevent the sample from
slipping from the jaws of the testing instrument. The
dimensions of the specimen were measured as de-
scribed previously (2). The width and length of the
sample were measured with a noncontacting optical
system, and the thickness was measured with an ap-
paratus that senses the conductivity of the tissue.
Each dimensional measurement was made at five
different points on the sample, and the average was
calculated.
Tensile Testing
The test strip was mounted between two steel jaws
in a specially designed tensile testing apparatus (Fig.
J Orthop Res, Vol. 12, No. 4, 1994
E GUILAK ET AL.
t
Depth
0
200
Articular Surface
w
w
400
600
800
f
Full-thickness
1000
cartilage strips
Subchondral Bone
2). This instrument is a computer-controlled version
of an instrument described previously (2). One sta-
tionary jaw was connected to a miniature load cell
(0-20 N; Sensotec, Columbus, OH, U.S.A.), and the
other, to the shaft of a computer-controlled microm-
eter (StepperMike; Oriel, Stratford, CT, U.S.A.).Mo-
tion of the micrometer and acquisition of the force
signal from the load cell were controlled by an Ap-
ple IIgs microcomputer (Apple Computers, Cuper-
tino, CA, U.S.A.) and custom-written software, as
described previously (18). This system allows either
load or deformation-controlled testing of the sample
at a force resolution of approximately 0.001 N and a
displacement resolution of 1.O pm. The specimens
were bathed continuously in one of two fresh solu-
tions, which were regulated by step changes of an
electromagnetic valve under the control of the mi-
crocomputer. The step changes in the bathing solu-
tion provided information on the ion-induced force
response and swelling behavior of the tissue. The
microcomputer was programmed to specify an en-
tire sequence of step displacements (strains) and
changes in bathing solution.
With use of the load-control mode of the instru-
ment, a tare load of 0.02 N was applied to the tissue
for 1,000 seconds to define an initial testing geome-
try. The creep response of the sample during this tare
load was recorded, and the final length of the speci-
men was used to calculate the applied displacements.
To determine the tensile modulus, strain was in-
creased incrementally and the load on the tissue was
allowed to relax to an equilibrium state (1,200 sec-
 COMPOSITION A N D T E N S I L E P R O P E R T I E S OF O S T E O A R T H R I T I C C A R T I L A G E 477

Apple I I gs

Signal
Conditioner

I I
TEST CONTROL &
DATA ACQUISITION

SOLENOID
COMPUTERVALVE FOROF
CONTROL
BATHING SOLUTION
J
FIG. 2. Schematic diagram of the automated isometric tcnsile testing instrument. A dedicated microcomputer (Apple IIgs; Apple
Computers) was used for feedback control of load or deformation of the cartilage sample. Computer control of the bathing solution
for the tissue was achieved through a solenoid valve.

onds). Because the equilibrium tensile behavior of
the tissue was found to be linear at strains of less
than approximately 15%, the tensile modulus, which
is defined as the slope of the stress-strain curve,
was detcrmined from this region of the curve. Ten-
sile strains of 0.0, 0.5, 1.0, 2.5, 3.5, 5.0, and 10.0%
were applied to the sample, and a least-squares lin-
ear regression between the equilibrium stress and
the applied strain between 0 and 5% was used to
determine the slope of the stress-strain curve. The
total ion-induced stress relaxation (bR) between
distilled/deionized water and 0.15 M NaCl was meas-
ured at a strain of 5.0%. The rate of ion-induced
stress relaxation was used to determine the Na' dif-
fusion coefficient according to a previously devel-
oped technique (3,30). The normalized transient
stress-relaxation curve, cr(t), is measured following
complete mechanical stress relaxation and may be
described by an exponential function with single
characteristic decay time z:
o(t) = 0 ~+ GR
~ exp(-t/.r)
- methylene blue dye (16) in a microtiter platc assay
where oNd+ is the equilibrium stress in the tissue
bathed in 0.15 M NaC1. An exponential curve-fitting
routinc (RS/1 statistical package; BBN, Cambridge,
M A , U.S.A.) was used to determine oRand z from
the stress-relaxation curve. The Na- diffusion coeffi-
cient is given by: D = h'/(4n2z), where h is the thick-
ness of the specimen.
Biochemical Analysis
Biochemical analysis was performed on samples of
tissue taken from locations adjacent to the biome-
chanical test site from 15 of the 10 animals. To deter-
mine the water content, the samplcs were weighed
before and after a 48-hour period of freeze-drying.
The tissue then was digested with papain (1.25
mg/100 mg of tissue) for 16 hours at 60°C, and ali-
quots of this digest were taken for separate analyses
to determine total sulfated glycosaminoglycan con-
tent, hydroxyproline content, and hydroxypyridinium
crosslink content and density. The amount of sulfated
glycosaminoglycan was determined by 1,9-dimethyl-
(41). as described previously. Shark chondroitin sul-

I Orrhop Rrs. Vol 12, N I L 4, 1YY4

4 78 E GUILAK E T A L .

25
25 m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 rneanisd.
p<O.OOOl vs. control
Animal Number

C 1.5 I 15 I

0.5

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 rneanfs.d.

.
Animal Number p<0.0005vs. control

4 . 4 4 .

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 mean*s.d.
Animal Number p=0.09 vs. control

FIG. 3. Tensile mechanical properties of articular cartilage from experimental joints (transection of the anterior cruciate ligament) and
contralateral control joints.

fate was used as standard. To determine the hydroxy- Statistical Analysis

proline content of the tissue, the papain digest was Statistical analyses were performed with the BLSS
hydrolyzed with HC1 at 107°C overnight, dried, and statistical package (University of California, Berke-
then analyzed by a colorimetric procedure (44). ley, CA, U.S.A.) and a NeXTstation microcomputer
The collagen hydroxypyridinium crosslink con- (NeXT Computer, Redwood City, CA, U.S.A.). Com-
tent was determined with a previously described parisons between samples from the experimental
procedure (14,15), by reverse-phase ion-pair high joint and the contralateral joint were performed with
performance liquid chromatography. Levels were the paired two-tailed t test. A linear correlation
quantified with use of a standard of hydroxypyridin- matrix was formed between the tensile mechanical
ium crosslink from bovine bone (kindly provided by properties (tensile modulus, peak G ~and , Na' diffu-
Dr. Simon Robins, Rowett Research Institute, Aber- sion coefficient) and the biochemical composition
deen, Scotland). The hydroxypyridinium crosslink (hydroxypyridinium crosslink density and content,
density was calculated as moles per mole of collagen, hydroxyproline and sulfated glycosaminoglycan con-
with the assumptions that hydroxyproline contrib- tents [wet and dry weight basis], collagen/proteo-
utes 13.3% of the weight of collagen and collagen glycan ratio, and water content). Significance was
has a molecular weight of 300 kDa. reported at the 95% confidence level.

J Orthop Res, Vol. 12, No. 4, 1994

 C O M P O S I T I O N A N D T E N S I L E PROPERTIES OF O S T E O A R T H R I T I C C A R T I L A G E 4 79

1 2 3 4 5 6 7 8 9 101112131415 meartfs.d.
Animal Number p=0.0016 vs. control
0.08
E 0 08
g9 006
0 06
c

2
.- 004 004

2 0.02 0 02

0 n
1 2 3 4 5 6 7 8 9 1011 12131415 meartfs.d.
Animal Number p=O.O18 vs. control

-I 0.04
I I
.m-
alo)
.c
003
0.04

75z002
& S
xal
23 0.01

F 0
1 2 3 4 5 6 7 8 9 101112131415 mean&s.d
Animal Number pc0 001 vs control

25
g.?? 25
E % 2 2
64
YQ 15 15
& a
m a 1 1
i?g
0 %05 05
a
15 0 0
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 15 meartfs d.
Animal Number p=O 025 vs contrr
FIG. 4. Biochemical composition of articular cartilage from experimental joints (transection of the anterior cruciate ligament) and
contralateral control joints S-GAG = sulfated glycosaminoglycan and HP = hydroxypyridinium

RESULTS
Gross Changes
The articular cartilage on the medial femoral con-
dyle from the sites where the strips were taken for
tensile and biochemical studies exhibited a slightly
roughened surface and mild India ink staining in
some animals; however, no gross fibrillation or sur-
face irregularities were present. India ink staining
indicated mild fibrillation of cartilage from the tibia1
plateau of the experimental joints. In many of the
experimental joints, severe meniscal damage was
noted and osteophytes were present at the joint mar-
gins. No lacerative damage was seen in the articular
cartilage from experimental joints. Cartilage from
the contralateral control joints appeared normal,
with a smooth and glistening surface.
Mechanical Properties
The equilibrium stress-strain response of the tis-
sue was found to be linear in the range of 0-10%
strain, resulting in excellent linear-regression fits
(R = 0.92-0.99) for determination of the tensile mod-
ulus. Exponential predictions of oR also were very
consistent. The tensile mechanical properties of the

J Orthop Res, Vol. 12, No. 4, 1994

480 E GUILAK ET AL.

a
C Experimental
-z
0
0
E Control
0 0

0 0.5 1 1.5
Normalized Ion-Induced Stress-Relaxation
FIG. 5. There was a weak but significant correlation between the peak ion-induced stress relaxation and the hydroxypyridinium (HP)
crosslink density for cartilage from control joints (R = -0.56, n = 15), experimental joints (transcction of the antcrior cruciate
ligament) (R = -0.70, n = IS), and the pooled joints (R = -0.61, n = 30).

articular cartilage from the experimental joints were
significantly different from those of the cartilage
from the contralateral joints. In all 19 animals, the
tensile stiffness was decreased, by an average of
44%, in the articular cartilage from the experimental
joints compared with the contralateral control joint
(p < 0.0001) (Fig. 3a). The isometric swelling test
indicated that oR was increased by an average of
57% in the cartilage from experimental joints (p <
0.0005); this trend was noted in 16 animals (Fig. 3b).
There were no statistically significant changes in the
Na+diffusion coefficient in the tissue from the exper-
imental joints (p = 0.09) (Fig. 3c).
Biochemical Composition
In the samples from the superficial zone of articu-
lar Cartilage in the experimental joints, the average
water content was 86.7%, which was elevated by
13% as compared with tissue from the control joints
(p = 0.0016). This increase was noted in 14 of the 15
animals studied (Fig. 4a). The sulfated glycosamino-
glycan content (Fig. 4b) and hydroxyproline content
(Fig. 4c), expressed per wet weight of the tissue, were
decreased significantly in the cartilage from the ex-
perimental joints; they were 38 and 36% less than
those of the control joints (p = 0.018 and p < 0.001,
respectively). The mean hydroxyproline content (per
dry weight) was 17% higher in the tissue from the
experimental joints than that from the control joints,
but this trend was not statistically significant (p =
0.08). No changes were noted in the sulfated gly-
cosaminoglycan content per dry weight (p = 0.87) or
in the collagen/proteoglycan ratio of the tissue (p =
0.57).
In 11 of 15 animals, the hydroxypyridinium cross-
link density of the samples from the superficial zone
of articular cartilage in the experimental joints was
decreased by an average of 11% compared with the
cartilage from the contralateral joints (p = 0.025)
(Fig. 4d). The hydroxypyridinium crosslink content
per wet weight also was decreased (by 42%) (p <
0.0001). The changes in the crosslink content per dry
weight were not statistically significant (p = 0.33).
Statistical Correlations
The strongest correlations between composition
and mechanical properties were noted with the mag-
nitude O f o R (Fig.5). A negative correlation was found
between hydroxypyridinium crosslink density and
ion-induced stress relaxation in the samples from
control joints (R = -0.56, n = 15), experimental joints
(R = -0.70, n = 15), and pooled control and experi-
mental joints (R = -0.61, n = 30). Similar correlations
were noted between oR and hydroxypyridinium
crosslink content, as normalized to the wet weight
of the sample, for the samples from control joints
(R = -0.70, n = 15), experimental joints (R = -0.52,
n = 15), and pooled joints (R = -0.60, n = 30). In the
tissue from experimental joints, a significant corre-
lation was noted between oKand water content (R =
 COMPOSITION A N D TENSILE PROPERTIES OF OSTEOARTHRITIC C A R T I L A G E
-0.69, n = 1S), sulfated glycosaminoglycan content
per wet weight (R = -0.56. n = 15), and hydroxy-
proline content per wet weight (R = -0.53, n = 15).
No significant correlations were noted between oR
the tensile modulus or the Na' diffusion coefficient
and any of the measured biochemical parameters in
the control. experimental, or pooled values.
DISCUSSION
The canine model of experimental osteoarthritis
(transection of the anterior cruciate ligament) has
been shown to produce changes in articular carti-
lage that are representative of human osteoarthritis
(5,8,9,13,26.27.31.36,38.43).In this study, there were
significant changes in the properties of the speci-
mens taken from the superficial zone of articular
cartilage in experimental joints as compared with
the contralateral controls. The changes in the gross
morphology of the tissue. which included roughen-
ing of the articular surface, mild India ink staining.
and meniscal damage, were consistent with the char-
acteristics of early osteoarthritis and the canine
model of osteoarthritis (7,17.27,28). These morpho-
logic changes also were reflected through significant
changes in both the biochemical composition and
the tensile mechanical properties of the articular
cartilage.
In previous models of canine osteoarthritis, tran-
section of the anterior cruciate ligament generally
has been performed through a blind stab incision
(4.31,35,43). This method involves the risk of iatro-
genic damage to the articular cartilage and other
important joint structures, such as the posterior cru-
ciate ligament or menisci, given their close proximity
within a canine knee joint. Open arthrotomy ( 5 ) pro-
vides visualization of the anterior cruciate ligament.
but it may result in excessive inflammation or he-
marthrosis due to injury of synovial blood vcsscls.
The arthroscopic procedure used in the present
study provided a rapid and reliable technique of
transection of the anterior cruciate ligament under
direct observation while minimizing joint trauma. sy-
novial inflammatory activity, and postoperative heal-
ing time. Although others have found no significant
changes in the cartilage of joints that had a sham
operation (4), these factors are very important in
ensuring that the observed changes were due to tran-
section of the anterior cruciate ligament and were
not hemarthrotic in origin.
From our experience, the isometric swelling test
is an accurate and sensitive method to determine
the tensile biornechanical properties of articular car-
481
tilage (2.3,30). The equilibrium-tensile modulus re-
presents the flow-independent stiffness of the colla-
gen matrix of cartilage. This parameter is a function
of the structure (2,47), composition (2,21,39), and
osmotic environment (3,30) of the tissue. The total
ion-induced stress relaxation (oR)represents the
change in the total stress within the sample as caused
by a decrease in the osmotic pressure at 5% strain
(23.30). Due to the highly linear stress-strain rela-
tionship of the tissue, this parameter provides a
measure of the change in tensile modulus between
0.15 M NaCl and distilled water and is thus indicative
of both compositional and structural changes in the
tissue.
The mean tensile modulus of the control cartilage
in distilled water was 15.5 t 4.1 MPa, which is in
general agreement with values previously reported
for samples of cartilage from the superficial zone (2).
A consistent decrease in the tensile stiffness of the
osteoarthritic cartilage was observed in every ani-
mal and was accompanied by an increase in the mag-
nitude of the ion-induced stress relaxation. These
resuits are in strong agreement with previous find-
ings on the tensile properties of cartilage from the
superficial zone in human osteoarthritis (2,3) and
canine experimental osteoarthritis (31.43), where a
decrease in tensile stiffness of 40.60% was observed.
These biomechanical changes are consistent with the
notion of significant "structural" changes such as ma-
trix disruption and disorganization of the collagen
fibrillar network. as observed by electron micros-
COPY (46)-
Significant changes also were observed in the bio-
chemical composition of the superficial zone of the
osteoarthritic articular cartilage. The water content
of the tissue was increased, by an average of 139'0, in
every animal. This change is greater than that found
in other studies of canine osteoarthritic cartilage
(26). The difference between the observations may
reflect the analysis of the superficial zone: previous
studies have analyzed full-thickness cartilage. This
notion is supported further by our finding that com-
positional changes of full-thickness cartilage from
thc tibia1 plateau of the same joints were similar to
compositional changcs that have been reported pre-
viously. This implies that a major change in this stage
of osteoarthritic cartilage occurs specifically in the
superficial zone. This finding is in agreement with
that of previous studies on human osteoarthritic car-
tilage (6) and experimental osteoarthritis (12), in
which an increase in water content and a decrease in
fixed-charge density (proteoglycan content) per wet
J Orthop Xes. Vol. 12, No. 4, 1YY4
482 E G U I L A K ET AL.
weight were observed in the superficial zone. As no
changes were noted in the proteoglycan and collagen
contents on a dry weight basis, it is reasonable to
conclude that the changes per wet weight were due
to an increase in water content and not necessarily
to any overall loss of the matrix components. Again,
these results are consistent with disruption, or “loos-
ening,” of the collagen network in the superficial
zone (1,46) and a resultant expansion of the solid
matrix due to pre-stress of the tissue.
An important new observation in this study is the
decrease in the density of hydroxypyridinium cross-
links in the collagen of the superficial zone. The
cause probably is an increase in collagen turnover
rates (13,33).The hydroxypyridinium crosslink is the
product of a relatively slow maturation event. Thus,
collagen breakdown and loss may be increased, and
replacement of the collagen could occur with in-
creased collagen synthesis, as previously observed
(13). However, the time required for the formation
of the mature hydroxypyridinium crosslinks would
result in a decrease in the detected hydroxypyridin-
ium crosslink density. These results may reflect the
increase in the turnover rate of the collagen and
therefore an increase in the ratio of “immature” to
“mature” collagen. Our findings indicated that the
mean collagen content per dry weight was 17%
higher in tissue from experimental joints, although
the trend was not statistically significant (p = 0.08).
This finding, along with the observation of a signifi-
cant decrease in crosslink density, may explain the
lack of observed changes in crosslink content per dry
weight.
Interestingly, few correlations were noted between
mechanical properties and biochemical composition
in the cartilage from control joints, and no correla-
tions were found between the tensile modulus and
any of the measured biochemical parameters in tis-
sue from either control or experimental joints. In
previous studies, tensile modulus and strength have
been correlated with the level of collagen crosslink-
ing in articular cartilage from middle and deep zones
of skeletally immature animals (39). Akizuki et al.
(2) found a correlation between the intrinsic tensile
modulus and the collagen/proteoglycan ratio in nor-
mal and fibrillated human cartilage pooled through
the depth of the tissue. The results of these investi-
gations, together with those of the present study,
suggest that, in the superficial zone of articular car-
tilage, the highly organized structure of the tissue,
rather than simply the composition, plays a domi-
nant role in the determination of the tensile stiffness
J Orthop Res, Vol. 12, No. 4, 1994
of the cartilage. In the middle and deep zones of
young cartilage, the organization of the collagen ma-
trix is more random; this suggests that composition
(hydroxypyridinium crosslink density or collagen
content) may play a more important role in the de-
termination of tensile behavior. These changes in the
tensile behavior of superficial zone cartilage thus
seem to be indicative of breakdown of the solid ma-
trix rather than of changes in the collagen content,
proteoglycan content, or collagen/proteoglycan ra-
tio. Because all samples were prepared with the same
geometry, it also may be argued that an increase in
water content (and therefore an apparent decrease
in collagen content) should have been related to the
change in tensile stiffness. The lack of a significant
correlation between these two parameters in tissue
from control, experimental, or pooled joints further
supports the role of the tissue ultrastructure in de-
fining the properties of the superficial zone.
Both ion-induced stress relaxation and hydroxy-
pyridinium crosslink density were decreased in os-
teoarthritic cartilage, and a significant negative
correlation was noted between the two parameters
in tissue from both control and experimental joints.
This finding supports the notion that crosslinking
of the collagen network may be responsible for in-
creased cohesiveness of the solid matrix. In the car-
tilage from experimental joints, increased stress
relaxation was correlated with increased hydration
and decreased proteoglycan and collagen contents.
These relationships, which were not present in the
tissue from control joints, suggest that composition
may not contribute to the tensile behavior of the
tissue unless the structure of collagen matrix some-
how has been compromised. Decreased density of
the mature hydroxypyridinium crosslinks, from the
presence of newly formed collagen or through enzy-
matic degradation, may result in a loss of matrix
cohesiveness and therefore may initiate or promote
an increase in tissue hydration. In turn, the increased
hydration will result in an apparent decrease in pro-
teoglycan and collagen contents.
It has been hypothesized that mechanical factors
play a critical role in the initiation and progression
of osteoarthritis (20,28,34). However, the sequence
of events leading to the observed biomechanical and
biochemical changes is still unclear. Transection of
the anterior cruciate ligament has been shown to
alter the gait pattern and thus the loading history of
the joint (32). It is believed that loss of a major
stabilizing ligament may result in joint laxity, or “in-
stability,” that subjects the cartilage to abnormally
 COMPOSITION A N D TENSILE PROPERTIES OF OSTEOARTHRITIC C A R T I L A G E
high stresses or loading of sites that are not normally
load-bearing. Furthermore, the large decrease in
stiffness observed in this study implies that these
changes may be progressive (i.e., significant changes
in the mechanical properties of the tissue may fur-
ther change the loading environment of the joint).
These factors may be responsible for breakdown
of the collagen network through direct mechanical
damage, which is readily apparent in the superficial
zone. Because the superficial zone plays an impor-
tant role in the compressive viscoelastic behavior of
articular cartilage, any changes in the properties of
this zone may significantly affect the overall mechan-
ical response of the tissue (42).
Alternatively, these biomechanical and biochemi-
cal changes may be due to increased enzymatic
activity of the chondrocytes. An important charac-
teristic of osteoarthritic cartilage is elevated activ-
ity of matrix metalloproteases, including collagenase
(11.25,33). Although the effects of these specific en-
zymes on the mechanical properties of the matrix
or the hydroxypyridinium crosslink density of colla-
gen have not been measured directly, other studies
have indicated that enzymatic degradation of carti-
lage can affect both the transient and equilibrium
tensile properties. For example. degradation of the
nonhelical terminal peptides of collagen molecules
with pancreatic elastase cleaves the principal cross-
linking sites and results in dramatic changes in the
tensile stiffness and strength (39). Furthermore, en-
zymatic digestion of cartilage proteoglycan signifi-
cantly affects the kinetic creep response of the tissue
(40). However, it is not known if the elevated levels
of the matrix-degrading metalloproteases are pres-
ent as part of the initiating degradative process or as
part of a reparative or inflammatory process follow-
ing mechanical damage, or both. The determination
of the sequence of events that result in these changes
will clarify mechanisms that may be important in the
early development of degenerative joint disease.
Acknowledgmenk We are grateful to Dr. Yi Jiang.
Veronica Hlibczuk. Thomas Gardner, and Fatemeh
Saed-Nejad for their excellent assistance. We also would
like to thank Dr. Marcus Seibel and Dr. Susan Shapses
for their assistance in the initial stages of this study. This
study was supported by a grant from Syntex Labora-
tories, Palo Alto. California, and by National Institutes of
Health Grant AR40032.
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