Journnl q f Orthopnedir Resecir(.

h
4~379-392, Raven Press, New York
0 1986 Orthopaedic Research Society

Tensile Properties of Human Knee Joint Cartilage:

I. Influence of Ionic Conditions, Weight Bearing, and
Fibrillation on the Tensile Modulus

Shaw Akizuki, *Van C. Mow, ?Francisco Muller, $Julio C. Pita, §David S . Howell, and
?Daniel H. Manicourt
Biomechanics Research Laboratory and *Department of Mechanical Engineering, Aeronairtical Engineering and
Mechanics, Rensselaer Polytechnic Institute, Troy, New York; and +Internal Medicine Department, $Department of
Medicine, and §Arthritis Division, University of Miami, Miami, Florida, U.S.A.

Summary: The flow-independent (intrinsic) tensile modulus of the extracel-

Mar matrix of human knee joint cartilage has been measured for normal, fi-
brillated, and osteoarthritic (removed from total knee joint replacements) car-
tilage. The modulus was determined in our isometric tensile apparatus and
measured at equilibrium. We found a linear equilibrium stress-strain behavior
up to -15% strain. The modulus was measured for tissues from the high and
low weight-bearing areas of the joint surfaces, the medial femoral condyle and
lateral patello femoral groove, and from different zones (surface, subsurface,
middle, and middle-deep) within the tissue. For all specimens, the intrinsic
tensile modulus was always less than 30 MPa. Tissues from low weight-bearing
areas (LWA) are stiffer than those from high weight-bearing areas (HWA). The
tensile modulus of the ECM correlates strongly with the collagen/proteoglycan
ratio; it is higher for LWA than for HWA. Osteoarthritic cartilage from total
knee replacement procedures has a tensile stiffness less than 2 MPa. Key
Words: Intrinsic tensile modulus-High and low weight bearing areas-
Normal, fibrillated, and osteoarthritic cartilage- Ion concentration effects-
Correlation with biochemical composition.

Articular cartilage exhibits both viscoelastic and
biphasic responses under tension, compression,
and shear (18,31,32,Sl). Even under simple uniaxial
tension, it exhibits mechanical behaviors that de-
pend on strain rate, ionic conditions in the bathing
fluid, biochemical composition, and the structural
organization of the dominant components of the
tissue (collagen fibrils and proteoglycans)
Address correspondence and reprint requests to Dr. V. C .
Mow at Department of Orthopedic Surgery, Columbia-Presby-
terian Medical Center, 622 W. 168th St., PH5-129, New York,
NY 10032, U.S.A.
Dr. Akizuki’s present address is Department of Orthopaedic
Surgery, Shinshu University, Medical School, Matsumoto,
Japan.
(15,23,25,36,40,.50). Thus, to understand the influ-
ence of composition and structure on the mechan-
ical behavior of normal and degenerating human ar-
ticular cartilage, all these factors must be consid-
ered and carefully controlled. In this investigation
we assessed the influence of all these factors on the
equilibrium or intrinsic tensile modulus of normal,
fibrillated, and osteoarthritic knee joint cartilage.
To explain the motivation for our experimental
protocol, a brief description of the structural organ-
ization of the extracellular matrix (ECM) of artic-
ular cartilage is required. The main components of
the ECM are collagen fibrils, proteoglycans, and
water. The collagen fibrils are organized into a fine
meshwork of definite architecture, with interfi-

3 79
380 S. AKIZUKI ET AL.
brillar spaces filled with proteoglycans and water.
It is generally believed that the elaborate structural
features of proteoglycan aggregates are essential
for the organization of the ECM by promoting
physical, chemical, and mechanical interactions
and/or entanglement sites with the surrounding col-
lagen network (17,34). Together, the collagen net-
work and the proteoglycans contained within the
network form a cohesive fiber-reinforced com-
posite matrix with all the essential characteristics
of a porous-permeable solid material filled with
water (32). This porous-permeable solid matrix pro-
vides the ability for the tissue to withstand the
enormous stresses of joint articulation (1,9,10,20)in
situ under conditions of large deformation (3).
Each of the structural components of the ECM is
endowed with specific characteristics that provide
the ECM with some outstanding mechanical prop-
erties. Native collagen fibrils are strong in tension
(6,21,40,50), and proteoglycans are highly hydro-
phylic (41). In addition, proteoglycans exhibit an
elastic behavior when compressed (16). In the
ECM, negatively charged proteoglycans are con-
fined to about 20% of their free-solution volume
(17). Thus, a large Donnan osmotic swelling pres-
sure is exerted by the trapped proteoglycans onto
the surrounding collagen network. This swelling
pressure is balanced within the ECM by the tension
developed in the constraining collagen network
(28).
The internal equilibration of forces may be dis-
turbed chemically by changing the counter-ion con-
centration in the external bathing solution. An in-
crease of the Na+ concentration in the bathing so-
lution will cause a decrease of the Donnan osmotic
swelling pressure of the proteoglycans (27), thus
decreasing the tensile stress acting in the collagen
network. Myers and co-workers (37) have shown
that for isometrically stretched cartilage specimens,
the measured change of tension associated with a
change of ionic conditions in the bathing solution
may be theoretically modeled and predicted. Thus,
one objective of this investigation was to use this
method to assess the modulation of the tensile stiff-
ness of the ECM of human knee joint cartilage by
changing N a + concentration.
COMPOSITIONAL AND
STRUCTURAL HETEROGENEITIES
The composition of cartilage varies significantly
over the joint surface (46). These variations appear
J Orthop Res, Vol. 4 , N o. 4 , 1986
to be related to joint loading (7,11,39,45). In gen-
eral, regions that are habitually highly loaded have
more proteoglycan content than regions that are
habitually lightly loaded, and regions that are habi-
tually lightly loaded have more collagen content
than regions that are habitually highly loaded. This
variation provides a convenient way to study the
effect of natural variation of biochemical composi-
tion on the tensile modulus of the ECM. In addi-
tion, layerwise variation of collagen ultrastructure,
composition, and structural anisotropies for this
tissue a r e well known (5,12,33). Thus, tensile
samples must be carefully controlled in terms of the
depth from within the tissue. In this investigation,
specimens were taken from four distinctively dif-
ferent ultrastructural zones (surface, subsurface,
middle, and middle-deep). All specimens were
taken from an orientation, as close as practically
possible, “parallel” to the local split-line direction
(23,4030). Where possible, tissues from fibrillated
joint surfaces and some osteoarthritic surfaces
were tested to assess the effect of these degenera-
tive processes.
LOAD CARRIAGE IN CARTILAGE
Load carriage within articular cartilage is shared
between the interstitial fluid and the intrinsic mate-
rial properties of the ECM. For example, during
rapid compressions, the frictional drag associated
with interstitial fluid flow may account for, de-
pending on the rate of compression, more than 90%
of the compressive stiffness; the stiffness of the
ECM contributes to the remainder of the measured
compressive stress (24). This is a consequence of
the nonlinear biphasic behavior of the tissue (32).
Thus, in order to determine the “intrinsic” flow-
independent properties of the ECM in compres-
sion, equilibrium measurements must be made or
measurements must be made under infinitesimal
shearing conditions (18) to avoid a false high value
for the intrinsic moduli of the ECM resulting from
this flow-generated stiffening effect.
The same situation applies for determining the
intrinsic tensile properties of the ECM. Constant
strain-rate tensile experiments (23,40,50) yield ten-
sile stiffnesses that a r e not flow-independent
(25,26). For tensile specimens 1-2 cm long, an im-
posed displacement rate of 4-5 mm/min will cause
a significant flow-generated stiffening effect (25).
For example, for human medial femoral condyle
cartilage surface zone, Kempson (22) determined a
 TENSILE MODULUS OF HUMAN KNEE CARTILAGE 381

tensile modulus ranging from 40 MPa (for a spec- TABLE 1. Characteristics of the human knee joint
imen obtained from a donor more than 85 years old) specimens
to 125 MPa (for a 20-year-old specimen) when the Joint no. Age Sex Side Condition Criteria
applied stress was 5 MPa. In similar constant
1 24 Male Left Whole Normal
strain-rate experiments on 2-year-old bovine carti- 2 31 Male Right Whole Normal
lage, both Woo and co-workers (50) and Roth and 3 35 Male Left Whole Normal
Mow (40) found tensile moduli to be less than 30 4 42 Male Right Whole Normal
5 51 Male Right Whole Fibrillated
MPa when the applied stress was 5 MPa. This dis- 6 60 Male Right Whole Fibrillated
crepancy may be due to differences in the composi- 7 65 Female Left TKR parts Osteoarthritic
tion and structure between bovine and human car- 8 68 Female Right TKR parts Osteoarthritic
9 74 Female Right TKR parts Osteoarthritic
tilage, ages of the specimens, and fibrillation, or it
could be due to the mechanical testing protocol. In TKR, total knee replacement.
this investigation, to avoid ambiguous interpreta-
tions and the possibility of the flow-generated stiff- of Miami. These joints were divided into two
ening effect, all tensile moduli of the ECM were groups: group 1-joints with visually normal carti-
measured at equilibrium. lage with no India staining over the surface (ages:
In addition, Woo and co-workers (51) showed 24, 32, 35, 42 years) and group 2-joints with par-
that similar cartilage specimens exhibit pronounced tial fibrillation of the cartilage surface visible to the
stress-relaxation effects in tension and that this be- naked eye and stained with India ink (age: 52 and
havior may be described by the quasi-linear visco- 60 years). In this group, India ink stain grades of
elastic theory proposed by Fung (14). Hence, it is two and three, according to the scheme developed
difficult to interpret correctly the results of con- by Emery and Meachim (13,29), were noted over
stant strain-rate tensile experiments where both the some aspects of the joint surface. However, most
flow-generated stiffening and stress-relaxation ef- of the specimen surfaces did not exhibit any India
fects are occurring simultaneously. Once again, ink uptake. Parts from three osteoarthritic joints
these difficulties may be circumvented if equilib- were obtained from total knee replacements (TKR)
rium measurements are made. (ages: 65, 68, and 74 years)2. In this study, we de-
Thus, in this investigation the intrinsic tensile fine group 1 specimens as normal, group 2 spec-
modulus of the ECM were determined at equilib- imens as fibrillated, and TKR specimens as OA.
rium. The variation of this property with the natu- Following the protocol established at the Tissue
rally occurring differences of tissue composition Bank and Surgical Research Laboratories, knees
and surface fibrillation were assessed. Specimens harvested from fresh cadavers were stripped of all
were categorized as being from high and low soft tissues and flash-frozen in liquid nitrogen. The
weight-bearing areas (HWA and LWA) and from specimens were then placed in heavy-duty plastic
different zones throughout the depth of the tissue bags, sealed, packed with dry ice, and shipped in
(surface, subsurface or transition, middle, and an insulated box by overnight delivery to Rensse-
middle-deep), and whether they were normal, fi- laer. All specimens were received frozen and were
brillated, or osteroarthritic (OA). These intrinsic placed immediately in a freezer and stored at
tensile moduli of the ECM were measured in deion- -20°C until ready for dissection. Parts from the
ized water and 0.15 M NaCl solution. TKRs were received from the operating room after
sufficient amounts were taken for ordinary patho-
logical examinations. They were placed in plastic
MATERIALS AND METHODS
bags, frozen, and sent; all specimens arrived frozen
Specimens and Tissues and were subsequently processed in the same
manner.
A total of nine knee joints were tested (Table 1).
Preparation of Tensile Specimens
Six knees were obtained from the Tissue Bank and
Surgical Research Laboratories' at the University In this study, cartilage from the distal femur was

I Courtesy of Dr. Theodore I. Malinin, Director, Tissue Bank These joints were obtained from Queen's University Hos-
and Surgical Research Laboratories, Department of Surgery, pital, courtesy of Dr. Derek T. V. Cooke, and Women's and
University of Miami. Brigham Hospital, courtesy of Dr. Clement B. Sledge.

J Orthop Res, Vol. 4 , N o , 4 , 1986

382 S . AKIZUKI ET AL.
used. Care was taken to harvest and categorize
specimens from what is generally regarded as the
HWA and LWA regions over the femoral condyles
(9,10,43). The lateral patellofemoral groove (LPG)
and the medial femoral condyle (MFC) sites were
chosen because their broad surfaces make it easy to
obtain tensile specimens (Fig. 1).
Our methods and procedures for preparation of
tensile specimens have been reported (37,40), but
to briefly summarize, rectangular osteochondral
blocks were cut from the LPG and MFC. These
were then divided into several blocks, some from
the HWA region and others in the LWA region. The
exact locations of the HWA and LWA regions for
each joint, of course, are not known. Thus, the
HWA and LWA classifications should be regarded
only as a qualitative guide. Cartilage was then re-
moved from the subchondral bone at the tidemark
and subsequently sectioned using a freezing stage
Leitz sledge microtome. The superficial 50 km of
the tissue was microtomed from each block of car-
tilage to yield a flat surface. Subsequently, seven to
ten slices, -250 krn thick, were removed serially
from each block (Fig. 2). In some blocks, due to the
thinness of the tissue o r fibrillation, specimens
from the middle-deep and deep zones could not be
obtained, which explains the unequal number of
samples in our data pool (Table 2). From each slice
of tissue, two to three 1.8 x 15 mm tensile strips
may be obtained. The strips were taken, as close as
practically possible, “parallel” to the local split-
line direction (21,40). However, in some cases, par-
ticularly with the LPGiHWA specimens, a slight
angle may exist between the axis of the specimen
and the split-line direction.
In total, we successfully tested 130 strips of
normal cartilage from joints showing no signs of
India ink staining, 47 strips of “fibrillated carti-
lage’’ (obtained immediately adjacent to regions
stained with India ink), and 23 strips of “os-
Lateral Patello Femoral Groove ( L P G )
ow Weight Bearing
H,qhWe,ght
Lateral Femoral Medial Femoral
Condyle (LFC) Condyle ( M F C I
J Orthop Res, Vol. 4 , No. 4 , 1986
teoarthritic cartilage” (obtained adjacent to se-
verely osteoarthritic lesions or regions with no car-
tilage). In severely osteoarthritic TKR materials,
only LWA specimens were obtained because no
HWA cartilage existed. The number of specimens
in each anatomical category are indicated in Ta-
ble 2.
Biochemical Analyses
Initially, we prepared -100 mg of tissue for each
category of specimen for the complete set of bio-
chemical assays. To obtain this amount of mass, the
remaining material in each slice and the immedi-
ately adjacent slices (above and below) were
grouped together to yield the required mass. The
mechanically tested strip was not included in the
-100 mg of tissue for biochemical assay. In fibril-
lated and osteoarthritic cartilage, some of the mate-
rials pooled in these samples necessarily included
tissues from regions of frank fibrillation, which un-
doubtedly must influence the biochemical results.
With all the samples, we measured hydroxyproline,
hexosamine, glucosamine, galactosamine, uronic
acid, collagen, proteoglycan, and water contents.
Water content was determined and expressed as
the ratio of the wet weight minus the dry weight
relative to the wet weight of the specimen. Prior to
weighing, each sample harvested for biochemical
analyses was equilibrated in 0.15 M NaCl solution
for 1 h. Upon removal from the solution, extra fluid
covering the surfaces of the specimen was gently
removed using soft absorbent tissue paper, and the
specimen was weighed immediately in a micro-
balance (with an accuracy of 0.01 mg) to determine
its wet weight. The specimen was then dried in a
vacuum oven at 60°C for 48 h. The dry weight was
then determined and the water content calculated.
Dried cartilage specimens were subjected to pa-
pain digestion with an enzyme-to-substrate ratio of
FIG. 1. Schematic diagram of location of
high weight-bearing area (HWA) and low
weight-bearing area (LWA) on the distal fem-
oral condyle (right). Five sample locations
were chosen to be in areas of relative high
and low weight-bearing regions over the
joint surface (left). The exact nature of
loading over a specific joint is, of course, not
known
 TENSILE MODULUS OF H U M A N KNEE CARTILAGE 383

Depth prn
Surface Zone ( S )
Sub Surface Zone ( S S )

-Middle Zone ( M 1

2000 -Middle Deep Zone ( M D )

Subchondral Bone
I Split Line Direction

FIG. 2. Osteochondral block of material removed from the

femoral condyle was sliced into 250 p m thick slices. Usually,
eight to ten slices could be harvested from each block of
material. The top 50 p m was removed to obtain a flat planar
surface. Each slice was subsequently cut into strips such
that the long axis of the strip was aligned parallel to the local
split-line direction. The length and width, L and w, of the
strip equal 15 and 1.8 mm, respectively.

m m - 0

1: 100 (wtlwt). After digestion, the material was

centrifuged at 10,000 rpm for 10 min, the superna-
tant was removed and filtered, and known volumes
of sample solutions were made. Total hexuronate
was determined in an aliquot of the solution using
the carbazole method as modified by Heinegard
(19). The remaining solution was divided into two
parts that were independently hydrolyzed in HCl
6 M at 100°C for 17 h, and then evaporated to dry-
ness at 60°C under a carefully controlled circulation
of dry air. One of the residues was dissolved in 1 .O
ml of buffer solution containing 0.25 M citric acid,
0.60 M sodium acetate, 0.80 M NaOH, and 0.20 M
acetic acid. Hydroxyproline was determined in this
solution by the method of Woessner (49) using hy-
droxyproline gold labeled standards from Aldrich.
The second residue was dissolved in the alkaline
sodium acetate buffer a s described by Blu-
menkrantz and Asboe-Hansen (8) and modified by
Wagner (47). This method was used to determine
the total hexosamine and the galactosamine to glu-
cosamine ratio. In these determinations, galactos-
amine HC1 (Sigma G-0500) was used as standard.
Collagen was calculated by assuming hydroxy-

J Orthop R P S ,Vol. 4 , N O , 4 , 1986

384 S . AKIZUKI ET A L .
proline residues in 100 FW, and proteoglycan con-
tent was calculated by assuming 22% hexuronate in
the monomer. Initially, we restricted the sample
size to 100 mg for the biochemical analyses for each
category of tissue, e.g., LPG/HWA, because we
wished to correlate the site-specific biomechanical
properties with the site-specific biochemical com-
position. However, we found this to yield samples
that were too small for accurate biochemical
assays. Thus, the same zones of different catego-
ries, e.g., surface zones of the LPG/HWA and
MFClHWA, were grouped together for these bio-
chemical assays. Consequently, in our statistical
correlations, mean values of biomechanical proper-
ties of each zone were correlated with the corre-
sponding biochemical data.
Dimensional Measurements of Cartilage Strips
In order to calculate the engineering stress
(force/original cross-sectional area) acting on the
specimens, an accurate cross-sectional area must
be determined. Further, as both normal and os-
teoarthritic tissues swell upon a change of salt con-
centration in the external bathing solution (fibril-
lated and osteoarthritic cartilage swelling much
more than normal cartilage), the dimensions of the
tensile specimens were determined after complete
equilibration in deionized water and again after
complete equilibration in 0.15 M NaCl solution.
The thickness was measured in a new apparatus
designed to sense the electrical conductivity of the
tissue. The tensile strips were gently placed on a
precision-ground stainless-steel plate attached to a
Leitz electro-optical micrometer. The tip of the mi-
crometer, also electrically connected to the circuit,
was aligned perpendicular to the ground steel plate
and at a known distance above the plate. The ver-
tical traverse of the tip was controlled by a preci-
sion electric motor. The downward traverse of the
tip was stopped when the tip touched the electri-
cally conducting tissue. The distance traveled by
the tip yielded a reading for the specimen thick-
ness. The accuracy of the devise has been verified
to be within 23.0 pm against an optical noncon-
tacting (but much more tedious) method. Thickness
measurements of the specimens were made at ten
points along the length of the strip and averaged.
All measurements were completed within 1 min to
minimize evaporation effects.
The lengths and widths were measured at ten and
five points along the respective edges, using a
J Orlhop Res, Vol. 4, N o . 4 , 1986
Bausch and Lomb stereo-zoom microscope
adapted with an Olympus precision X-Y translation
stage coupled with a Microcode digital microm-
eter, and averaged. The difference in the length of
the strip equilibrated in deionized water and 0.15 M
NaCl solution must be taken into account in the
calculation of the true tensile strain for the 0.15 M
NaCl equilibrated strip (37).
Tensile Experiment
Figure 3A shows our isometric tensile apparatus
(ITA) used to determine the equilibrium tensile
properties of the ECM. [This apparatus is also
especially effective in studying the kinetic swelling
behavior of cartilage strips held in isometric tension
(37). This phenomenon, for human articular carti-
lage, was measured and is discussed in a com-
panion paper, see Akizuki et al. (2).] The principle
of the apparatus is very simple: elongation of the
specimen is determined by the movement of an air-
activated piston (Fig. 3A). The displacement of the
piston, and hence the right jaw, is controlled by a
micrometer stop. The micrometer provides a
simple method for determining the exact jaw-to-jaw
length of the specimen. In this way, the precise
amount of stretch experienced by the specimen
may be i re determined.^ The load is measured by
the load cell attached to the stationary left jaw.
Hence, the entire load history, including the equi-
librium load, is easily monitored. In addition, pro-
visions for a stepwise change of the bathing solu-
tion are provided by the continuous stream of fresh
solutions from two separate sources attached to
two rows of shower nozzles (Fig. 3B). Thus, the
tensile properties of the specimen equilibrated in
various solutions may be determined in a single ex-
periment.
Figure 4 provides the complete details of the ex-
perimental protocol for the determination of the
equilibrium tensile properties of cartilage strips as
well as the kinetic swelling properties (2). Figure
4A shows the sequences for displacement and sa-
Recent results have indicated inaccuracies in using jaw-to-
jaw strain measurements (40,51) and inhomogeneities in the
strain field in other soft tissues in similar tensile experiments
(52). We have nevertheless used this measure of strain and as-
sumed it to be uniform in our calculations for the tensile modulus
because of the extreme difficulties encountered in measuring
gage-length strains with our Optron electro-optical noncon-
tacting extensometer when a continuous stream of fluid bathes
the specimen.
 TENSILE MODULUS OF HUMAN KNEE CARTILAGE
3A
PISTON ROD
//
MOVING J A W
38
F
linity changes imposed on the specimen. After
mounting the specimen in the ITA, preconditioning,
and equilibrating in deionized water, a very small
amount of stretch was imposed onto the specimen
at time T,. This stretch caused an immediate load
response, followed by stress relaxation occurring
during T,-T, (Fig. 4 B ) . Complete relaxation
usually occurred within 15 min (37,51). The equilib-
rium stress attained during the TI-T, period was
adjusted to be below 1 gf. We used this protocol to
define the tare loads and tare lengths of the spec-
imens. This tare load was chosen because it is the
load required to maintain an initially “straight”
configuration for the naturally curled strips. The
tare load was subtracted from all subsequent values
385
TER
OMETER STOP
FIG. 3A and B. Schematic diagram of the isometric tensile
apparatus (ITA) used in the experiments. See text for a de-
scription of its function. Figure 3B is reproduced with per-
mission from Myers et al. Journal of Biomechanical Engi-
neering, ASME, 1984.
of force in our calculations for the stress, and the
tare length was used as the initial length in our cal-
culations for strain when the specimen was equili-
brated in deionized water. Calculations of strains
for specimens equilibrated in 0.15 M NaCl were ad-
justed by the amount of free contraction between
deionized water and 0.15 M NaCl determined under
free swelling conditions.
The kinetic isometric swelling (2) and the tensile
experiments began at time T, (Fig. 4). At this time,
the bathing solution was changed stepwise to 0.15
M NaCl from deionized water (Fig. 4A), and the
resulting time variation of stress change, i.e., the
nonequilibrium swelling kinetics, was monitored
during T,-T, (Fig. 4B). Upon equilibration in 0.15
J Orthop Res, Vol. 4, N o . 4, 1986
386 S. AKIZUKI ET AL.
PROTOCOL OF EXPERIMENT
- Displacement Chonge 0
C known that articular cartilage does fully recover
.... NaCl Cancentrotion Change t 5G
Time
(b)
FIG. 4. Experimental protocol used to determine intrinsic
tensile moduli in different bathing solutions. The solid line
shows the displacement history applied and the dashed line
shows the history of change of the bathing solution imposed
on the specimen (a). Typical changes in stress due to the
applied strain and imposed change of salinity are shown (b).
See text for an explanation of the stress history.
M NaCl and at T,, deionized water was reimposed
on the specimen in a stepwise manner; the resulting
kinetics of stress change was monitored during
T,-T,. Following reequilibration in deionized water
and at T,, an additional increment of strain was
added onto the specimen by the ITA in a stepwise
manner (Fig. 4A); the resulting stress relaxation was
monitored during T,-TS. Upon equilibration (at T,)
the NaCl solution was applied and the resulting ki-
netics of stress change again monitored. This pro-
cedure of cyclical changes of bathing solution and
increments of strain was repeated eight times, until
15% strain was reached. No attempts have been
made by us, and none have ever been reported in
the literature, to determine whether articular carti-
lage, when stretched up to 15%, will return to the
tare length upon removal of the tensile stress. We
believe, however, that at the 15% strain level, col-
lagen alignment within the ECM might be irrevers-
ible. Further investigations are required to quantita-
tively assess the residual tensile strain upon re-
J Orthop Res, Vol. 4, No. 4 , 1986
moval of tensile stress. In compression, it is well
upon removal of the compressive stress (44).
Shown in Fig. 4B is the typical mechanical
stress-relaxation pattern after each increment of
strain and the typical kinetic swelling behaviors of
the cartilage specimens at each level of strain (37).
Note that the nature of stress change associated
with the imposition of the 0.15 M NaCl solution de-
pends on the tensile strain; at low strains, T,-T,, a
contraction effect occurred, while at high strains,
T,-T,, an expansion effect occurred. At interme-
diate strain levels, T,-T,, a transition occurred be-
tween the contraction effect and the expansion ef-
fect. This pattern existed for both bovine and
human articular cartilage. For this investigation,
the equilibrium tensile moduli of the ECM in deion-
ized water and in 0.15 M NaCl were determined by
simply calculating the ratios of the increments of
the equilibrium stress, i.e., points D, E, F and
points A, B , C of Fig. 4B, to the increments of
strain (Fig. 4A), respectively. In the range of strains
chosen (- 15%) the equilibrium stress-strain re-
sponse of the ECM in tension was remarkably
linear (Fig. 5). Figure 5 shows some typical results
for a subsurface zone specimen bathed in deionized
water and 0.15 M NaCl. We note that, over the
years, we have repeatedly studied, for control pur-
poses, the effect of freezing on the biomechanical
properties of bovine, porcine, and dog articular
cartilage. For storage at -20°C up to as long as 1
year, no consistent pattern of biomechanical prop-
erty changes were noted.
Tensile Modulus in Delanued water
I p'I Tensde modulus in 0 15M Nacl
/
1'
0
a
E
v, /
v, 0.50 /
W
[L
t-
v,
0.25 ,I//
0
0
I
0.05
I I
0.10
I I
0.15
-
STRAIN
FIG. 5. Equilibrium tensile stress-strain relationship of the
extracellular matrix for a subsurface specimen. The relation-
ship, under the present experimental protocol, is linear up to
-15% tensile strain.
 TENSILE MODULUS OF HUMAN KNEE CARTILAGE
Statistical Analyses
To evaluate the differences in the mean values of
each category, analysis of variance was used to
compare data from three or more groups. The Stu-
dent's t test was used to compare data from two
groups when the population variances of each
group could be safely assumed to be equal. When
the variances of the two populations were different,
we used the method of Welch (48). The F test was
used for the evaluation of the equality of the popu-
lation variances. For comparing the differences in
the tensile moduli of the specimens in the two
bathing solutions, we used the correlated t test.
Most of the statistical and linear regression anal-
yses used in this study were performed with
MIDAS, a statistical software package.
RESULTS
Zonal Differences of the Tensile Modulus
In general, from our previous studies, we found
that the tensile modulus did not vary in a statisti-
cally significant manner from strip to strip; varia-
tion from slice to slice was significant. Random
variations occurred from joint to joint for each cate-
gory. The representation of our data reflects this
general trend.
Figure 6 shows a typical variation of the intrinsic
tensile modulus of the ECM for the four different
zones (surface, subsurface, middle, and middle-
deep) of the LPGIHWA cartilage samples. The ten-
sile moduli of all the human knee joint cartilage
tested were less than 30 MPa; most fell in the range
between 1.0 MPa and 15 MPa. For these adult
human cartilage, the tensile modulus generally de-
creased with increasing distance from the surface.
The tensile moduli of the surface zone samples
from fibrillated cartilage were significantly dimin-
ished as compared with normal cartilage; for LPGI
HWA regions p < 0.05, and for LPGiLWA p <
0.01. However, the moduli of the lower zones were
very similar to those of normal cartilage. In some
fibrillated specimens, particularly with the LPGI
LWA specimens, the subsurface zone had signifi-
cantly greater tensile moduli than those of the sur-
face zone (p < 0.01) (Table 2). For the osteoarthritic
specimens, only LWA tissue existed, and the ten-
sile moduli for these specimens were very low, less
than 3.0 MPa, and no zonal differences were de-
tected (Table 2 ) . In addition, Fig. 6 shows that the
387
H2O H20
A 0.15M NaCl A 0.15M NaCl
Nr8***
lo-
T
f I k
S SS M MD S SS M M D
FIG. 6. Zonal differences of the mean value of the intrinsic
tensile moduli of normal and fibrillated adult human knee
joint cartilage from the high weight-bearing areas (HWA) of
the lateral patellofernoral groove (LPG). The bars represent
one standard deviation. Differences of mean intrinsic tensile
moduli between deionized H,O and 0.15 M NaCl solution are
also shown. Significance of differences between deionized
H,O and 0.15 M NaCl are shown as: *I*, p < 0.001; **, p <
0.01; *, p < 0.05; S, surface zone; SS, subsurface zone; M,
middle zone; MD,middle-deep zone.
intrinsic tensile moduli of the ECM are greater in
deionized water than in 0.15 M NaCl. These differ-
ences were statistically significant for cartilage
from every zone for both normal and fibrillated
joints.
There were no statistical differences or ten-
dencies in the variation of the tensile moduli with
age in any of the categories of tissues from normal
knee joints. However, as the average ages of the
fibrillated knee joints and osteoarthritic (TKR)
specimens were successively higher, when all the
material was taken together there was a trend for a
decrease of the tensile modulus with age.
Topographical Differences (HWA vs. LWA)
Table 2 gives the mean values and standard de-
viations of the intrinsic tensile moduli of all the
specimens tested. In normal cartilage, the LWA
specimens for both MFC and LPG locations had
higher tensile moduli than the corresponding HWA
specimens. This was true for every zone. There
J Orthop Res, Vol. 4, No. 4 , 1986
388 S . AKIZUKI ET AL.
was no consistent pattern of differences between
LPG and MFC, though the LPG/LWA specimens
were twice as stiff as the MFCILWA specimens (p
< 0.001). However, for fibrillated joint surfaces, no
significant differences of the intrinsic tensile moduli
were found for cartilage specimens from the surface
zone of the HWA and LWA regions. The difference
between the tensile moduli of the lower zones of
the HWA and LWA became less well defined.
Biochemical Data and Tensile Modulus
Table 3 summarizes all our statistical correlations
between the tensile modulus and our biochemical
measurements. For normal knee joint cartilage,
strong correlations existed, positive and negative,
between the tensile modulus and all biochemical
measures except water and hydroxyproline/wet
weight. All the correlations disappeared, except the
collagen/proteoglycan ratio, for cartilage specimens
from fibrillated surfaces and no correlations existed
for specimens from the osteoarthritic (TKR) carti-
lage.
Figure 7 shows the correlation between the col-
lagen/proteoglycan ratio and mean values of the
tensile modulus for the different zones of normal
cartilage, r = 0.714 and p < 0.0001.4Note that the
collagen/proteoglycan ratio was higher for LWA-
specimens than for HWA specimens. As expected,
the surface zones also had higher collageniproteo-
glycan ratios that corresponded to their higher ten-
sile moduli. Interestingly, for normal knee joint car-
TABLE 3 . Summary of statistical significance of
correlations between intrinsic tensile modulus and
biochemical parameters
Normal Fibrillated Osteoarthritic
Water content
Collagen/proteogl ycan C A
Hexosamine/uronic acid A tended to also be on the low side (Fig. 8).
Hydroxyproline/dry wt . A
Hydroxyprolineiwet wt.
Hexosamine/dry wt. B DISCUSSION
Hexosamine/wet wt. B
Uronic acidldry wt. A Our equilibrium or flow-independent tensile ex-
Uronic acid/wet wt. B periments on human knee joint cartilage showed
A, p < 0.05; B, p < 0.01; C, p < 0.001. Collagen as calculated
assuming 100 hydroxyproline residue in 100,OOOformula weight.
Proteoglycan content was calculated assuming 22% uronic acid
in the monomer.
Data points represent mean values of LPG and MFC for
each zone, i.e., subsurface, middle, and middle-deep zones.
J Orthop Res, Vol, 4, N o . 4, 1986
HWA
0 --.
A LWA _I’ ‘,I
0 1 2 3 4 5 6 7
Collagen/Proteoglycan (Ratio1
FIG. 7. Relationship between intrinsic tensile modulus and
collageniproteoglycan ratio for normal human knee joint
cartilage. The variables are related by the linear regression
equation E = 4.31(R) - 9.80( r = 0.714, p < 0.001, n = 25)
where R = collagen/proteoglycan ratio. The mean values
and ?SD of collagen/proteoglycan ratio are 4.00 2 0.20 (n
= 4, HWA surface), 4.85 2 1.00 (n = 3, LWA surface), 3.13 2
0.21 (n = 8,HWA middle), and 3.54 2 0.73 (n = 4, LWA
middle).
tilage, the correlation between the tensile modulus
and hydroxyproline/dry weight was not as strong ( r
= 0.486 and p < 0.05). Negative correlations ex-
isted between the intrinsic tensile modulus and hex-
osamine/wet weight, r = -0.563 and p < 0.01, and
uronic acidiwet weight, Y = -0.491 and p < 0.05.
For specimens obtained from fibrillated knee
joints, the only correlation between the mean value
of the tensile modulus for the different zones of the
ECM and all of our biochemical measurements was
with the collagen/proteoglycan ratio, r = 0.559 and
p < 0.05 (Fig. 8).4 For osteoarthritic specimens
from TKR patients, no correlations existed be-
tween the tensile modulus and any of our biochem-
ical measures. The values of the tensile moduli
were very low and the collagen/proteoglycan ratio
that the ECM behaves linearly in tension, at least
up to 15% strain, and that these intrinsic tensile
moduli of the ECM are generally less than 30 MPa.5
Myers (35) measured the intrinsic tensile modulus of normal
bovine patellofemoral groove cartilage to be of the order of 1.0
MPa.
 TENSILE MODULUS OF HUMAN KNEE CARTILAGE
OA
o/
Normol A of overt fibrillation. No evidence of tissue damage
a o/
O
-i
<
p
12
10
,L
Collogen/Profeoglycon ( A o l i o l
FIG. 8. Relationship between the intrinsic tensile modulus
and collagen/proteoglycan ratio for normal, fibrillated, and-
osteoarthritic (OA) cartilages. For fibrillated tissues, the vari-
ables are related by the regression equation E = 1.18(R) -
0.26 ( r = 0.559, p < 0.05, n = 18) where R = collagenipro-
teoglycan ratio. There is no significant correlation noted for
OA tissues. The mean value and ?SD of collagen/proteog-
lycan (Yo weight) are 4.36 2 0.75 (n = 7, normal surface),
6.66 f 2.66 (n = 3, fibrillated surface), 3.64 2 0.87 (n = 4,
OA surface), 3.26 5 0.46 (n = 12, normal middle), 4.06 f
1.04 (n = 10, fibrillated middle), and 1.36 2 0.15 (n = 4, OA
middle).
These results were significantly lower than those
reported by Kempson (22); for some young adults,
he reported tensile moduli as high as 150 MPa.6
There are at least two reasons for this observed dif-
ference: A flow-generated stiffening effect exists,
associated with the constant strain-rate tensile ex-
periment, which tends to stiffen the specimen in
tension (25) and our use of a low range of tensile
strain in this study (<15%) where the equilibrium
stress-strain relationship is linear (Fig. 5 ) . This
linear tensile equilibrium stress-strain relation of
the ECM is consistent with the linear compressive
equilibrium stress-strain behavior of the ECM and
with the theoretical biphasic formulation for artic-
ular cartilage (3 1). In addition, this biphasic stiffen-
ing effect is very similar to those found in the con-
stant strain-rate, creep, and stress-relaxation com-
pression experiments (24,32). Furthermore, our
results for normal tissues do not show any age de-
pendence. We now believe the age dependence of
the tensile modulus observed by Kempson (22)
might be attributed to surface fibrillation and os-
teoarthritic changes rather than to some intrinsic
aging process causing the decrease of the tensile
Constant strain-rate tensile experiments yield a tensile mod-
ulus for the surface zone of young bovine cartilage of about 30
MPa (40,51).
389
modulus of the ECM. It is to be noted that the “fi-
brillated specimens” tested were removed from vi-
sually normal areas immediately adjacent to areas
exists on these “fibrillated specimens.” Additional
studies need to be pursued to ascertain the reasons
for the discrepancy between our “age-independent”
tensile modulus result for our “normal” cartilage
population and Kempson’s “age-dependent’’ ten-
sile modulus result for his “normal” cartilage pop-
ulation (22).
Normal adult articular cartilage, human and bo-
vine, have very stiff surfaces. This stiffness is prob-
ably required to carry the large tensile stress acting
parallel (hoop stress) to the articulating surface
(30). Stress analysis applicable to the equilibrium
condition (elastic model) shows that the loss of this
stiff protective superficial layer on fibrillated sur-
faces will subject the remaining zones of the ECM
to unusually high stresses (4), thus causing further
damage to the tissue in these lower zones. It is
worth noting that in mildly fibrillated surfaces, the
tensile moduli of tissues from the lower zones were
not diminished; this suggests a progressive degen-
erative process starting from the articulating sur-
face. Apparently, osteoarthritic (TKR) cartilage is
totally disorganized; the tensile moduli of their
ECM are very low and no zonal variations exist.
There are two mechanisms to explain the de-
crease of the tensile modulus of the ECM with in-
creasing Na+ concentration as measured under the
isometric test condition. First, the decrease of the
Donnan osmotic swelling pressure associated with
an increase of the Na+ in the interstitium will result
in an internal release of collagen fibril tension (28).
If, as in the isometric experiment, the collagen net-
work is constrained at constant length, this internal
release is externally manifested in the decrease of
the tensile stress required to maintain the isometric
condition used to stretch the specimen (1S,37).
Second, an increase of the Na+ in the interstitiurn
can cause the ECM to weaken in tension (37) by
such mechanisms as charge shielding of the electri-
cally interacting groups between the collagen fibrils
and proteoglycans (38,42). Because of this, we be-
lieve that the correlation between the intrinsic ten-
sile modulus of the ECM with the collagen/proteo-
glycan ratio (Fig. 7) is particularly meaningful.
The intrinsic tensile modulus of the ECM for the
LWA and HWA regions of the joint confirms an-
other structure-function hypothesis about the func-
tion of collagen fibrils and proteoglycans in artic-
J Orthop Res, Vol. 4, N o . 4, 1986
390 S . AKIZUKI ET A L .
ular cartilage: proteoglycan is the main structural
component required to bear the compressive loads
for the tissue. Consequently, in lightly loaded re-
gions there should be fewer proteoglycans, thus a
higher collageniproteoglycan ratio. This was con-
firmed biochemically and correlated biomechani-
cally (Fig. 7). In addition, for layered structures
such as the cartilage/bone system at the joint sur-
face, large tensile hoop stresses are most likely to
exist at the periphery of the highly loaded regions.
Thus, a higher tensile stiffness is required at the
LWA regions.
The statistical correlations between our biochem-
ical and biomechanical data show a most inter-
esting result: the intrinsic tensile modulus of the
ECM correlated better with collagen/proteoglycan
ratio than with hydroxyproline or collagen content.
This says that while collagen does contribute to the
stiffness of the ECM, it is not the main parameter in
determining the stiffness. Rather, the tensile stiff-
ness of the ECM is more strongly affected by the
collagen/proteoglycan ratio. This implies that the
interactions responsible for the cohesiveness of the
solid matrix also contribute significantly to the ten-
sile stiffness of the tissue. Disruption of these inter-
actions, by such mechanisms as increasing the
counter-ion concentration in the interstitium of the
ECM, will cause a decline of the intrinsic modulus.
This has also been confirmed.
The tensile specimens from the fibrillated joint
surfaces were taken from areas adjacent to the
areas stained with India ink. Visually, the areas
from which we took our tensile specimen were
normal, i.e., no India ink staining occurred. Yet,
our results show that a biomechanical change has
occurred; this is evidenced by the pronounced de-
crease of the intrinsic tensile moduli of specimens
harvested from the various surface zones. In addi-
tion, there appears to be a stiffening of the imme-
diate subjacent zone associated with this decrease.
This might be a biological response of the tissue to
the disruptions occurring in the surface zones
above by providing another layer of stiff tissue to
withstand the loads of articulation. While the col-
lagen/proteoglycan contents for fibrillated cartilage
remain comparable to normal cartilage (Fig. 8), the
intrinsic tensile moduli of fibrillated cartilage are
definitely lower than those of normal cartilage. This
and the lack of correlation of the tensile moduli of
fibrillated cartilage with any of the other biochem-
ical composition measurements indicate that micro-
scopic structural disorganization may be respon-
J Orthop Re&, Vol. 4 , No. 4 , 1986
sible for the inferior tensile properties of the surface
zone of fibrillated cartilage.
CONCLUSIONS
1 . The equilibrium tensile stress-strain be-
havior of the extracellular matrix is linear up to
- 15% strain. This flow-independent intrinsic
tensile modulus has a value ranging from 1.O MPa
to 30 MPa.
2. The intrinsic tensile moduli of LWA carti-
lage are always greater than those of HWA carti-
lage. This corresponds very well with the higher
collagen/proteoglycan ratio for LWA cartilage
than for HWA cartilage. No consistent pattern of
differences exists for the tensile moduli of carti-
lage from the lateral patellofemoral groove and
those from the medial femoral condyle.
3. The strongest positive correlation exists be-
tween the intrinsic tensile modulus of the extra-
cellular matrix and the collagen/proteoglycan
ratio, followed by a good positive correlation
with hydroxyproline/dry weight and a good nega-
tive correlation with hexosamine/wet weight.
4. Visually normal regions, not stained by
India ink, from fibrillated knee joint surfaces
have much lower tensile moduli. At times, the
subsurface zones adjacent to the fibrillated re-
gions have higher tensile moduli than the visually
normal surface zone.
5 . The interactions between the collagen fi-
brils and proteoglycans appear to be an impor-
tant contributing factor in the tensile stiffness of
the extracellular matrix. Disruption of these in-
teractions by charge shielding of possible electro-
static interaction sites with increased Na+ con-
centration within the interstitium o r os-
teoarthritic processes weakens the extracellular
matrix in tension.
6. The intrinsic tensile modulus of normal ar-
ticular cartilage does not appear to change with
age. Age-related changes in the tensile stiffness
appear to be the result of a fibrillation and/or OA
process rather than some intrinsic aging process
causing the observed decrease of the tensile
modulus.
Acknowledgment: This research was sponsored by the
National Institute of Arthritis, Diabetes, and Digestive
and Kidney Diseases grant AM19094, the Clark and
Crossan Endowment at Rensselaer Polytechnic Institute,
and the Veterans Administration (Miami), University of
Miami. We would like to thank Ms. Rose Boshoff and
 TENSILE MODULUS OF H U MA N KNEE CARTILAGE
Ms. Donna Hutchinson for their help in preparing this
manuscript, and Mr. John M. Schoonbeck for his tech-
nical assistance. The authors with to express special
thanks to Dr. Theodore I. Malinin for providing the
human knee joints, without which this study would not
have been possible.
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