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Interleukin-1╨Ю┬▒ induction of tensile weakening associated with collagen degradation in bovine articular cartilage.

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Vol. 54, No. 10, October 2006, pp 3267–3276
DOI 10.1002/art.22145
© 2006, American College of Rheumatology
Interleukin-1␣ Induction of Tensile Weakening
Associated With Collagen Degradation in
Bovine Articular Cartilage
Michele M. Temple, Yang Xue, Michael Q. Chen, and Robert L. Sah
Objective. To determine whether interleukin-1␣
(IL-1␣) induces tensile weakening of articular cartilage
that is concomitant with the loss of glycosaminoglycans
(GAGs) or the subsequent degradation of the collagen
Methods. Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured
with or without IL-1␣ for 1 week or 3 weeks. Then,
portions of the explants were analyzed for their tensile
properties (ramp modulus, strength, and failure strain);
other portions of explants and spent culture medium
were analyzed for the amount of GAG and the amount of
cleaved, denatured, and total collagen.
Results. The effect of IL-1␣ treatment on cartilage
tensile properties and content was dependent on the
duration of culture and the depth of the explant from
the articular surface. The tensile strength and failure
strain of IL-1␣–treated samples from the superficial
and middle layers were lower after 3 weeks of culture,
but not after 1 week of culture. However, by 1 week of
culture, IL-1␣ had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3
weeks of culture, IL-1␣ induced significant collagen
degradation, as indicated by the amount of total,
cleaved, or denatured collagen in the medium or in
explants from the superficial and middle layers.
Conclusion. IL-1␣–induced degradation of carti-
lage results in tensile weakening that occurs subsequent
to the depletion of GAG and concomitant with the
degradation of the collagen network.
The tensile integrity of articular cartilage decreases with age and in the presence of osteoarthritis
(OA). In macroscopically normal adult human articular
cartilage from the femoral condyles, both the tensile
strength and stiffness of the superficial layer decrease
⬃10% per decade of age after reaching peak values at
the age of 24 years (1). In cartilage that is mildly
fibrillated and osteoarthritic, tensile equilibrium moduli
are reduced ⬃70% and ⬃85%, respectively, compared
with that in young normal cartilage from the human
knee joint (2). Such tensile deterioration of cartilage is
detrimental to its load-bearing properties, with a resultant increase in transverse deformation in response to
applied compressive load (3). Cartilage tensile properties are dependent primarily on the integrity of the
collagen network (1), damage to which may result in
an impairment of the normal counterbalance to
proteoglycan-associated swelling (4) and lead to the
cartilage swelling that occurs in OA (5,6). Thus, the
diminution of cartilage tensile integrity may facilitate
progressive deterioration and development of end-stage
The mechanism by which articular cartilage undergoes tensile weakening in arthritic degeneration remains to be established. In degenerate and osteoarthritic
human articular cartilage from the femoral condyle, the
percentage of degraded collagen (6) is higher than that
in normal cartilage and shows specific alterations of the
collagen network, including cleavage (7) and denaturation (8) of collagen molecules. Whether tensile weakening occurs concomitantly with collagen network degradation or with other preceding degradative processes
remains to be established. Tensile softening and weakening of cartilage have been analyzed in samples sub-
Supported by the NIH. Dr. Temple is recipient of a Predoctoral Fellowship from the NSF.
Michele M. Temple, PhD, Yang Xue, BS, Michael Q. Chen,
BS, Robert L. Sah, MD, ScD: University of California, San Diego.
Address correspondence and reprint requests to Robert L.
Sah, MD, ScD, Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, Mail Code 0412, La Jolla, CA
92093-0412. E-mail:
Submitted for publication January 27, 2006; accepted in
revised form June 30, 2006.
jected to extensive degradation of extracellular matrix
components, including both proteoglycan and collagen
(9,10). In vitro degradation of the collagen network by
the application of elastase or collagenase has been
shown to result in an ⬃45% (9) or ⬃90% (10) decrease
in the tensile strength of the superficial layer of macroscopically normal human articular knee cartilage. In
naturally occurring arthritis, degradation of proteoglycan and collagen may be instigated by chemical stimuli
and underlies the biomechanical weakening of the cartilage tissue.
Interleukin-1 (IL-1) is a mediator of inflammation and articular cartilage destruction in several arthritic diseases and is also a target for therapeutic
intervention. IL-1 is a cytokine produced by activated
synoviocytes, mononuclear cells, and chondrocytes (11–
13), and levels of IL-1 are elevated in the synovial
membrane, synovial fluid, and cartilage of patients with
OA and in patients with rheumatoid arthritis (RA)
(11,14,15). When injected into rabbit knee joints, IL-1
induces joint swelling, inflammation, and degradation of
cartilage proteoglycan (16,17), events that also occur in
arthritis in humans. These effects were shown to be
suppressed by intravenous administration of IL-1 receptor antagonist (18), with a decrease in inflammation and
an inhibition of proteoglycan loss. Damage to articular
cartilage may arise, in part, from direct activation of
chondrocytes by IL-1.
Bovine cartilage explant cultures have been used
to identify biochemical pathways by which chemical
stimuli affect cartilage composition, structure, and biomechanical function and relationships between these
properties. Incubation of adult bovine cartilage explants
with growth factors that maintained proteoglycan content was shown to also maintain the compressive modulus (19). In contrast, incubation of adult or immature
bovine cartilage explants with IL-1 induced a decrease in
the compressive modulus as well as a correlated decrease in proteoglycan content (20). Treatment of adult
human and bovine cartilage explants in vitro with IL-1
was shown to induce aggrecan cleavage and loss (21,22),
as well as subsequent collagen cleavage and denaturation (23,24). This occurred through stimulation of chondrocyte synthesis and secretion of proteases (25–27)
such as aggrecanase 1 (ADAMTS-4) and aggrecanase 2
(ADAMTS-5), which mediate IL-1–induced aggrecan
degradation (28,29) and the matrix metalloproteinases
(MMPs) collagenase and stromelysin, which degrade
proteoglycan subunits (30) and type II collagen (23,24).
While cytokine-induced effects on the composition of the collagen network (23,24,27) have been stud-
ied extensively, the concomitant effect on collagen network function and cartilage tensile properties has not
been studied previously. Thus, the objective of the
present study was to determine whether IL-1 induces
tensile weakening of articular cartilage and whether such
weakening is associated temporally with the initial loss
of proteoglycan or the subsequent degradation of the
collagen network in the proteoglycan-depleted tissue.
Materials and reagents. Materials for cartilage explant
isolation and culture and for extraction of degraded collagen
were obtained as described previously (6,19). Enzyme-linked
immunosorbent assays (ELISAs) to detect cleaved (using the
polyclonal antibody Col2-3/4Cshort) and denatured (using the
monoclonal antibody Col2-3/4m) collagen epitopes were obtained from Ibex (Montreal, Quebec, Canada), and recombinant human IL-1␣ was obtained from R&D Systems (Minneapolis, MN).
Cartilage isolation and culture. Osteochondral fragments were obtained from the medial and lateral aspects of the
patellofemoral groove of young adult (1–2 years old) bovine
knee joints (n ⫽ 4). From each fragment, cartilage was cut
sequentially, parallel to the articular surface, to a thickness of
⬃0.3 mm to yield cartilage slices from the superficial, middle,
and deep layers, with the superficial cartilage explants having
an intact articular surface. From the slices, a total of 192
cartilage explants measuring 10 mm long and 5 mm wide were
prepared. The cartilage explants were weighed wet and were
either tested immediately for tensile properties (week 0) or
were incubated in medium (Dulbecco’s modified Eagle’s medium with 10 mM HEPES, 0.1 mM nonessential amino acids,
0.4 mM L-proline, 2 mM L-glutamine, 100 units/ml of penicillin,
100 ␮g/ml of streptomycin, 0.25 ␮g/ml of amphotericin B, and
25 ␮g/ml of ascorbate) supplemented with 0.01% bovine serum
albumin alone (basal medium) or with an additional 5 ng/ml of
recombinant human IL-1␣.
Cartilage explants were incubated at 37°C in an atmosphere of 5% CO2, with 3 medium changes each week, for 7 or
21 days. The spent medium from each sample was pooled each
week. After culture, each cartilage explant was weighed wet,
measured for thickness, and cut into 2 portions, a tapered
specimen for biomechanical testing and adjacent tissue for
biochemical analysis. Biochemical measures of week 0 explants
were not performed. However, based on a previous study (23),
under basal conditions, tissue contents on day 0 and at day 7
would be expected to be similar for Col2-3/4m and Col2-3/
4Cshort and to be decreased by ⬃25% for glycosaminoglycan
Biomechanical analysis. Tapered specimens were
tested in tension, and data were analyzed to determine tensile
ramp modulus, strength, and strain at failure, as described
previously (31). Each tapered specimen had a gauge area of 4
mm ⫻ 0.8 mm (length by width), and specimens were elongated at a constant rate of extension (5 mm/minute) until
failure, while monitoring load. Throughout mechanical testing,
specimens were kept hydrated by immersion in a phosphate
buffered saline bath or drip at room temperature (⬃22°C) and
Figure 1. Measures of collagen network degradation after explant
culture of young adult bovine cartilage. The components present in
tissue or medium fractions were intact collagen, cleaved collagen,
denatured collagen, and smaller fragments of collagen. Amounts of
cleaved collagen, denatured collagen, and total collagen (encircled
plus signs) were quantified by enzyme-linked immunosorbent assay
(ELISA) for Col2-3/4Cshort epitope, ELISA for Col2-3/4m epitope,
and by colorimetric assay for hydroxyproline content (33), respectively.
aCT ⫽ ␣-chymotrypsin; ProK ⫽ proteinase K.
an approximately physiologic pH (7.2). From the displacement
and load data, tensile mechanical properties were determined.
Stress (in MPa) was calculated as the load normalized to the
initial width and thickness of the gauge region. Strain (unitless)
was calculated by normalizing the displacement data to the
initial sample length, which was taken as the initial grip-to-grip
length. The ramp modulus was calculated as the slope of the
stress–strain curve between 25% and 75% of the maximum
strain, and the strength and failure strain were recorded as the
stress and strain, respectively, at which the maximum stress was
Biochemical analysis. Previous in vitro studies (23)
have developed methods, which were adopted for the present
study, showing that IL-1␣ treatment of cartilage explants
induces collagen degradation, altering the collagen in both the
culture medium and tissue (Figure 1). Newly synthesized and
intact collagen molecules may be deposited in the tissue or
released into culture medium. Such collagen molecules may
be subsequently catabolized into cleaved, denatured, or fragmented forms, the latter of which diffuse easily within cartilage and out into the culture medium. Treatment of cartilage
with ␣-chymotrypsin selectively releases cleaved and denatured collagen, but not intact collagen, and subsequent treatment of the tissue with proteinase K completely solubilizes
the residual collagen. All of these forms of collagen molecules
can be measured as hydroxyproline (23) in the medium, in
␣-chymotrypsin extracts of tissue, and in proteinase K digests
of tissue. In culture medium and ␣-chymotrypsin extract,
cleaved and denatured collagens can be delineated by competitive ELISAs with the Col2-3/4Cshort and Col2-3/4m antibodies, respectively (7,8).
Based on these findings, the surrounding residual
tissue was analyzed for the quantity of matrix components,
including sulfated GAG as an index of proteoglycan, and
cleaved and denatured collagen. The tissue was weighed wet
and then treated with 1 mg/ml of ␣-chymotrypsin for 18 hours
at 37°C to extract denatured collagen (6). The residual tissue
was solubilized with 0.5 mg/ml of proteinase K for 24 hours at
60°C. The spent medium, ␣-chymotrypsin extracts, and proteinase K digests were analyzed for GAG (32) and hydroxyproline (33) contents. Because the amount of hydroxyproline in
medium was small, medium from multiple blocks from the
same animal were pooled and analyzed. The spent medium
and ␣-chymotrypsin extracts were analyzed for cleaved (Col23/4Cshort) and denatured (Col2-3/4m) collagen by ELISA (7,8).
Controls for each assay were prepared in the appropriate
buffer for the samples being analyzed.
Hydroxyproline content was converted to collagen
content using a mass ratio of collagen to hydroxyproline equal
to 7.25 (34). GAG content was calculated by comparison to
known concentrations of shark chondroitin sulfate. The contents of GAG, collagen, Col2-3/4Cshort, and Col2-3/4m were
scaled up from the amount in the tissue used for this analysis
to the amount in the whole explant (based on postculture
measurements of wet weight), and these quantities were then
normalized to the preculture wet weight. Therefore, both the
medium and tissue contents of each particular component
represent the values relative to the wet weight of the initial
tissue explant. Presentation of the data in this form provides
values that are comparable with those normalized to the wet
weight at the end of culture, since the changes in wet weights
were minimal (ranging on average from –8% to 2% in the
different experimental groups). The amounts of these constituents in media were reported as the amount above that in basal
(day 0) culture media. The percentage of collagen in
␣ -chymotrypsin (degraded) was calculated as that in
␣-chymotrypsin compared with the sum in the ␣-chymotrypsin
and proteinase K solutions.
Statistical analysis. Data are expressed as the mean ⫾
SEM. The effects of culture on tensile properties were assessed
by one-way analysis of variance (ANOVA), followed by Dunnett’s test, with data from week 0 as the control. The effects of
IL-1␣ on mechanical and biochemical parameters were assessed using repeated-measures ANOVA, with tissue depth
(superficial, middle, deep) as a repeated factor and with
1-week and 3-week experiments being performed and analyzed
separately. For analysis of medium, the week in culture (first,
second, or third week) was an additional repeated factor, and
data from day 0 were not included, since the data were
reported as values above amounts in day 0 culture medium.
When IL-1␣ or depth from the articular surface had an effect
(for P ⬍ 0.05), planned comparisons were made between
treatment groups at each depth.
Findings of the biomechanical analysis of bovine
cartilage. IL-1␣ treatment lowered the tensile integrity
of cartilage samples in a manner that was dependent
upon the culture duration and depth of the sample from
the articular surface. In particular, the tensile integrity
Figure 2. Effect of interleukin-1␣ (IL-1␣) on tensile properties of
young adult bovine cartilage explants. Analyses of A–C, tensile ramp
modulus, D–F, strength, and G–I, strain at failure were performed on
explants of cartilage (n ⫽ 7–16) from the superficial (S), middle (M),
and deep (D) layers. Samples were either tested immediately for
tensile properties (week 0) (A, D, and G) or were incubated with or
without 5 ng/ml of IL-1␣ for 1 week (B, E, and H) or for 3 weeks (C,
F, and I). Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍
0.005 by Dunnett’s test for comparisons with week 0 samples and by
planned comparisons between samples cultured with or without IL-1␣.
Asterisks within the bars indicate comparisons with week 0 samples;
asterisks above the bars indicate comparisons between samples cultured with or without IL-1␣.
of IL-1␣–treated samples, as compared with those incubated in basal medium as well as with the week 0
samples, was lower after 3 weeks of culture, but not after
1 week of culture. The tensile ramp modulus, strength,
and failure strain of samples cultured in basal medium
for 1 week (Figures 2B, E, and H) and 3 weeks (Figures
2C, F, and I) of culture were similar to those of week 0
samples (P ⫽ 0.1–0.8) (Figures 2A, D, and G).
After 1 week of culture, the tensile ramp modulus
(Figure 2B), strength (Figure 2E), and failure strain
(Figure 2H) of samples cultured in medium with IL-1␣
were similar to those in samples cultured in basal
medium (P ⫽ 0.3, P ⫽ 0.5, and P ⫽ 0.3, respectively) as
well as those in the week 0 samples (P ⫽ 0.1–0.4, P ⫽
0.1–0.4, and P ⫽ 0.3–0.8, respectively) (Figures 2A, D,
and G). The tensile strength and failure strain were
dependent upon cartilage depth (P ⬍ 0.005 for each
comparison), whereas the ramp modulus was not (P ⫽
0.2), and there was no interaction effect between depth
and IL-1␣ treatment (P ⫽ 0.5–0.8). Planned comparisons of treatment at each depth revealed no significant
effect of IL-1␣ treatment after only 1 week of culture for
ramp modulus (P ⫽ 0.2–0.5), strength (P ⫽ 0.2–0.8), or
failure strain (P ⫽ 0.4–0.9) compared with samples
cultured in basal medium.
In contrast, after 3 weeks of culture, treatment
with IL-1␣ caused a decrease in tensile strength and
failure strain of cartilage explants, with effects most
pronounced in samples from the superficial and middle
layers. Each of the tensile properties was dependent on
depth (P ⬍ 0.05 for each comparison). After 3 weeks of
culture, IL-1␣–treated samples showed a tendency for
an overall decrease in tensile strength, with a decrease
(31%; P ⬍ 0.05) in superficial samples and no effect on
middle (25%; P ⫽ 0.18) or deep (5%; P ⫽ 0.7) samples
as compared with those cultured in basal medium (Figure 2F), and a decrease in superficial (30%; P ⬍ 0.05)
and middle (33%; P ⬍ 0.05) samples, but not deep
samples (23%; P ⫽ 0.6), as compared with week 0
samples (Figure 2D). Failure strain was also affected by
IL-1␣ treatment, being lower in IL-1␣–treated superficial (33%; P ⬍ 0.05) and middle (38%; P ⬍ 0.005)
samples, but not deep samples (16%; P ⫽ 0.2), than in
those cultured in basal medium (Figure 2I) and lower in
IL-1␣–treated middle (47%; P ⬍ 0.005) and deep (30%;
P ⬍ 0.05) samples, but not superficial samples (30%;
P ⫽ 0.1), than in week 0 samples (Figure 2G). Ramp
modulus was not affected by IL-1␣ treatment in superficial, middle, or deep samples as compared with samples cultured in basal medium (P ⫽ 0.2–0.8) (Figure 2C)
as well as with week 0 samples (P ⫽ 0.5–0.9) (Figure
Findings of the biochemical analysis of bovine
cartilage. IL-1␣ treatment had a significant effect on the
release and retention of matrix components by cartilage
explants. The effects were dependent on the culture
duration and the depth of the explant. Release of matrix
components into the medium was stimulated by IL-1␣
treatment, with the release of collagen network components being delayed relative to the release of GAG.
Because the pattern of release of matrix components
into the medium from samples cultured for 1 week was
similar to that of matrix components released after 1
week of culture from samples that were cultured for a
total of 3 weeks, data are shown only for the samples
cultured for 3 weeks.
In particular, IL-1␣ stimulated the release of
GAG from cartilage samples by 1 week of culture (P ⬍
0.005), with an effect on the cumulative release after 2
and 3 weeks of culture (Figure 3A); this release was
dependent on the culture duration (P ⬍ 0.005) and
cartilage depth (P ⬍ 0.05), with an interactive effect of
duration and depth (P ⬍ 0.001). After 1 week of culture,
the amount of GAG released into the medium was
significantly higher (91–116%; P ⬍ 0.005 for each comparison) in IL-1␣–treated samples from the superficial,
middle, and deep layers than in untreated controls
(Figure 3A). Since GAG release from IL-1␣–treated
samples increased little during weeks 2 and 3, whereas
untreated controls still released moderate amounts of
GAG, the difference in the cumulative release of GAG
between IL-1␣–treated and control samples subsequently diminished during week 2 (45–70%; P ⬍ 0.05 for
each comparison) and week 3 (20–45%; P ⫽ 0.082, P ⬍
0.05, and P ⫽ 0.14, for the 3 cartilage layers, respectively).
The effect of IL-1␣ on the release of collagen
into the medium (determined from the hydroxyproline
content in the medium) was delayed relative to the
IL-1␣–enhanced release of GAG into the medium.
IL-1␣ induced the release of collagen from cartilage
samples (P ⬍ 0.05) (Figure 3B), with the release being
dependent on the culture duration (P ⬍ 0.005), but not
the cartilage depth (P ⫽ 0.67), and without an interaction effect (P ⫽ 0.78). The amount of collagen released
from IL-1␣–treated samples during week 1 of culture
was slightly higher (105–177%) than that released from
samples cultured in basal medium. However, IL-1␣
increased the release of collagen dramatically during
subsequent weeks of culture, being higher by 156–368%
at week 2 and higher by 213–788% at week 3.
The effect of IL-1␣ on the release of the collagen
degradation markers Col2-3/4Cshort and Col2-3/4m
epitopes into the medium was also delayed relative to
the IL-1␣–enhanced release of GAG into the medium.
IL-1␣ enhanced the release of Col2-3/4Cshort (Figure
3C) and Col2-3/4m (Figure 3D) epitopes (P ⬍ 0.01 for
each comparison). The cumulative release of Col2-3/
4Cshort was dependent on the culture duration (P ⬍
0.005) and cartilage depth (P ⬍ 0.005), with an interaction effect (P ⬍ 0.005). The cumulative release of
Col2-3/4m was also dependent on the culture duration
(P ⬍ 0.005), but not cartilage depth (P ⫽ 0.30), and
there was no interaction effect (P ⫽ 0.07).
After 1 week of culture, the release of Col2-3/
4Cshort and Col2-3/4m epitopes was not affected by
IL-1␣ treatment in samples from the superficial, middle,
Figure 3. Effect of interleukin-1␣ (IL-1␣) on the cumulative release
of matrix components from young adult bovine cartilage into the
medium. Explants of cartilage (n ⫽ 14–16) from the superficial
(squares), middle (triangles), and deep (circles) layers were cultured
for 3 weeks in the presence (broken lines; open symbols) or absence
(solid lines; solid symbols) of 5 ng/ml of IL-1␣. Samples were then
analyzed for the cumulative release of A, glycosaminoglycan (GAG),
B, collagen (COL), C, cleaved collagen (Col2-3/4Cshort), and D,
denatured collagen (Col2-3/4m) into the medium. Values are the mean
and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.005 by planned
comparisons between samples cultured with or without IL-1␣.
or deep layers (P ⫽ 0.11–0.47). In contrast, IL-1␣
enhanced the cumulative release of the Col2-3/4Cshort
and Col2-3/4m epitopes during week 2 of culture. The
Col2-3/4Cshort epitope release was higher in IL-1␣–
treated superficial layer samples (125%; P ⬍ 0.05) but
not middle (P ⫽ 0.05) or deep (P ⫽ 0.3) layer samples,
whereas the release of Col2-3/4m epitope was higher in
IL-1␣–treated superficial (596%; P ⬍ 0.005) and middle
(517%; P ⬍ 0.05) layer samples, but not deep layer
samples (P ⫽ 0.1). During week 3, the release of
Col2-3/4Cshort and Col2-3/4m epitopes was higher with
IL-1␣ treatment for samples from the superficial (548%
[P ⬍ 0.005] and 2,503% [P ⬍ 0.005], respectively) and
middle (373% [P ⬍ 0.005] and 1,350% [P ⬍ 0.05],
respectively) layers, but not those from the deep (P ⫽
0.08–0.15) layer.
Consistent with the effect on the release of matrix
components into the medium, IL-1␣ had a degradative
effect on the residual collagen network in the cartilage
samples that was delayed relative to the time course of
the decrease in residual GAG. After 1 and 3 weeks of
culture, IL-1␣ caused a marked decrease (P ⬍ 0.005 for
each comparison) in the amount of GAG remaining in
the cartilage samples (Figures 4A and B), with the
amounts being dependent on the cartilage layer after the
3-week (P ⬍ 0.005), but not the 1-week (P ⫽ 0.2),
culture duration. After 1 week of culture, the amount of
residual GAG in IL-1␣–treated samples (Figure 4A) was
53–75% lower in superficial, middle, and deep samples
(P ⬍ 0.05 for each comparison) compared with samples
cultured in basal medium. The amount of GAG remaining in the IL-1␣–treated samples after 3 weeks of culture
(Figure 4B) was considerably lower in superficial (79%;
P ⬍ 0.005), middle (81%; P ⬍ 0.005), and deep (74%;
P ⬍ 0.005) samples. Approximately 94% of the total
GAG from samples cultured in basal medium and 85%
from samples cultured in IL-1␣ were ␣-chymotrypsin–
While it had no detectable effect on the overall
amount of collagen remaining in the cartilage samples,
IL-1␣ caused an increase in the percentage of collagen
in the ␣-chymotrypsin fraction in a manner that was also
delayed relative to the time course of the decrease in
GAG, but that paralleled the time course of the decrease in tensile integrity. The amount of collagen
remaining in the cartilage samples after 1 and 3 weeks of
culture was similar between samples cultured in basal
medium and samples cultured in medium with IL-1␣
(P ⫽ 0.9 and P ⫽ 0.3, respectively) (Figures 4C and D).
However, the percentage of ␣-chymotrypsin–extractable
collagen was significantly higher after 3 weeks (P ⬍
0.005) (Figure 4F), but not after 1 week (P ⫽ 0.1)
(Figure 4E), of IL-1␣ treatment. This was dependent on
cartilage depth for the samples cultured for 1 week and
Figure 4. Effect of interleukin-1␣ (IL-1␣) on the amount of matrix
components remaining in young adult bovine cartilage explants. Analyses of A and B, glycosaminoglycan (GAG), C and D, collagen (COL),
E and F, collagen in ␣-chymotrypsin (COL in aCT), G and H,
Col2-3/4Cshort, and I and J, Col2-3/4m were performed on explants of
cartilage (n ⫽ 7–16) from the superficial (S), middle (M), and deep
(D) layers. Samples were cultured for 1 week (A, C, E, G, and I) or for
3 weeks (B, D, F, H, and J) in the presence or absence of 5 ng/ml of
IL-1␣. Degraded collagen was first extracted with ␣-chymotrypsin
(striped bars), and residual tissue was digested with proteinase K
(solid bars). Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍
0.005 by planned comparisons between samples cultured with or
without IL-1␣.
3 weeks (P ⬍ 0.005 and P ⬍ 0.01, respectively). The
percentage of collagen in the ␣-chymotrypsin fraction
was higher after the 3-week culture duration in samples
from the superficial (102%; P ⬍ 0.005) and middle
(39%; P ⬍ 0.005) layers, but not the deep layer (P ⫽ 0.3)
samples (Figure 4F). The percentage of collagen in
␣-chymotrypsin was not higher after the 1-week culture
duration in superficial, middle, or deep samples (P ⫽
0.6–0.9) (Figure 4E).
IL-1␣ treatment had a differential effect on the
amount of Col2-3/4Cshort and Col2-3/4m epitopes found
in the ␣-chymotrypsin extracts, with the delay of IL-1␣–
induced collagen network degradation being evident
between 1-week and 3-week cultures. The presence of
the collagen cleavage marker Col2-3/4Cshort was not
higher in samples cultured in IL-1␣ for 1 week (P ⫽ 0.2)
(Figure 4G) or for 3 weeks (P ⫽ 0.06) (Figure 4H). After
the 3-week culture duration, the amount of Col2-3/
4Cshort was higher in IL-1␣–treated superficial layer
samples (95%; P ⬍ 0.05), but not in middle or deep layer
samples (P ⫽ 0.1–0.6). The presence of the Col2-3/4m
epitope, though dependent on cartilage layer (P ⬍ 0.05
for each comparison), was not increased by IL-1␣ treatment after the 1-week (P ⫽ 0.9) (Figure 4I) or 3-week
(P ⫽ 0.6) (Figure 4J) culture duration.
IL-1␣ induced tensile weakening of, and the
release of GAG and collagen components from, adult
bovine cartilage explants, with characteristic time
courses. The IL-1␣–induced loss of GAG was not associated with a loss of tensile integrity, whereas alterations
of the collagen network were. Initially (within the first
week of culture), IL-1␣ reduced the amount of GAG
remaining in the matrix (Figure 4A), without an associated effect on the collagen network (Figures 4C, E, G,
and I) or a detectable decrease in tensile strength or
failure strain compared with either week 0 samples
(Figures 2D and G) or samples cultured in basal medium (Figures 2E and H). In contrast, after 3 weeks of
culture, IL-1␣ treatment caused a decrease in tensile
strength (Figures 2D and F) and failure strain (Figures
2G and I), as well as degradation of the collagen
network. In particular, IL-1␣ induced an increase in the
percentage of degraded collagen (Figure 4F) and
cleaved collagen (Figure 4H), while the amount of GAG
in the cartilage samples remained low (Figure 4B) and
the total amount of collagen was not affected (Figure
4D). These results indicate that IL-1␣ induced degrada-
tion of collagen molecules, but not IL-1␣–triggered
depletion of proteoglycan, results in tensile weakening
of articular cartilage.
Certain factors may limit the interpretation of the
results of this study and the assessment of the relationships between alterations of the extracellular matrix and
the mechanical integrity of articular cartilage. We used
articular cartilage from adult animals, rather than animals undergoing rapid growth (35), because the former
is more similar in tissue organization and composition to
the human adult cartilage that is affected by OA (36),
and the adult bovine knee joint offers a broad, relatively
flat surface, which in contrast to cartilage from older
animals or cartilage from humans, is generally free from
However, certain compositional, structural, and
functional differences between human and bovine articular cartilage do exist. Adult bovine articular cartilage
has lower tensile stiffness and strength (31,35,37) than
adult human cartilage (1,2). The depth-dependent variation in tensile strength and stiffness of young adult
bovine articular cartilage (31) is also somewhat different
from that of adult human cartilage (1). In contrast, the
failure strain (31,37) of adult bovine cartilage is consistent in magnitude and depth-variation with that of young
adult human cartilage (38). Generally, the collagen
content is slightly lower and the GAG content slightly
higher in bovine (35) than in human (38,39) articular
cartilage, and the crosslink composition of the collagen
network can vary, with hydroxypyridinoline crosslinks
being less abundant in adult bovine cartilage (35) than in
adult human cartilage (3). Despite these differences,
bovine cartilage has been used as a model to study
age-related changes in normal human adult articular
cartilage (31).
In addition, the properties of superficial, middle,
and deep layers in this study do not necessarily represent
properties of the superficial, middle, and deep “zones”
as defined classically (36). The thickness of specimens
was chosen to be similar to that used in previous studies
(1,10,31,35) to allow for direct comparisons, and samples
including the articular surface (superficial region) were
used because of its importance to the tensile properties
of articular cartilage (38) and its sensitivity to aging (1),
degeneration (2,40), and the degradative effects of IL-1
Treatment of cartilage with IL-1␣ induced a
number of the changes that are also observed in various
types of arthritis. IL-1 is abnormally elevated in diseases
such as OA and RA, and it is often used in culture
systems to identify cascades of enzymatic activity associated with OA, such as that of the aggrecanases (42,43)
and MMPs (7,24,44). The addition of IL-1␣ to cartilage
explant cultures resulted in a loss of matrix components
similar to that which occurs in arthritis, although in the
absence of mechanical loading and the complicating
effects of surrounding joint fluids and tissues. The
relative amounts of cleaved and denatured collagen as
well as GAG released into the media (Figure 3) and
remaining in the residual cartilage samples (Figure 4)
following culture with IL-1␣ were consistent with those
determined previously, as was the time course of release
(23,24). Indeed, the delay of collagen degradation compared with the loss of GAG may be due to the GAG
shielding of collagen epitopes to cleavage (24,27), as
opposed to the delay in up-regulation of MMPs as
compared with the up-regulation of aggrecanases (45).
Using IL-1␣ in the culture of cartilage tissue explants in
the present study allowed the biologic study of the
relationship between cytokine-induced matrix degradation and alteration of mechanical integrity.
The major result of the present study was that, in
such a biologically triggered system, collagen degradation in a GAG-depleted tissue, as opposed to GAG loss
alone, is associated with tensile weakening of cartilage.
The decrease in tensile strength seen in this study seems
likely to be due to the alterations of the collagen
network and highlights the importance of studies of
collagen degradation in arthritis (7,8). IL-1␣ treatment
for 3 weeks resulted in a 40–100% higher percentage of
␣-chymotrypsin–extractable collagen in superficial and
middle layer samples (Figure 4F). This increase is
similar to the difference in the percentage of
␣-chymotrypsin–extractable collagen between normal
and fibrillated cartilage (6).
The IL-1␣–induced degradation of collagen was
associated with specific alterations to the collagen network. In particular, the amount of Col2-3/4Cshort was
95% higher in samples from the superficial layer and
mildly higher (26–30%) in samples from the middle and
deep layers (Figures 4H and J), differences similar to
that (⬃100%) between normal and OA cartilage (7).
There was, however, no difference in the amount of
denatured collagen (Col2-3/4m) between IL-1␣–treated
and control samples, which is in contrast to the higher
amount (⬃100%) present in OA cartilage compared
with normal tissue (7,8). Indeed, this may be why the
tensile strength was only 28–31% lower in IL-1␣–treated
samples from the superficial and middle layers after 3
weeks of culture, whereas fibrillated cartilage has a
tensile strength that is ⬃50% lower than that of normal
cartilage (40). While additional or other forms of enzymatic or mechanical cartilage degradation may contribute to the tensile weakening observed in OA, IL-1␣–
induced matrix degradation highlighted the role of
collagen degradation in cartilage tensile weakening.
The association between alterations in cartilage
tensile properties and collagen network structure induced by IL-1␣ in this study was consistent with the
findings of previous studies of the tensile properties of
native and degraded cartilage. The values for week 0
samples and samples cultured for 1 or 3 weeks in basal
medium were consistent with the properties of mature
adult bovine articular cartilage from the patellofemoral
groove (31,35). The dramatic decrease (45–90%) in the
tensile strength of the superficial layer of human articular cartilage following treatment with collagendegrading enzymes is consistent with the role of the
collagen network (9,10), although the extent of collagen
cleavage and degradation was not determined in those
studies. In contrast, the tensile strength of articular
cartilage was maintained with enzymatic treatment targeting proteoglycan aggregate constituents (10,46,47),
although such treatments and ionic alterations can affect
viscoelastic tensile behavior (2,10,47).
Sufficiently long and intense exposure of articular
cartilage explants to enzymatic degradation appears to
be needed for tensile weakening. Indeed, the lack of
effect of IL-1␣ on the deep layer tensile properties and
residual collagen network may reflect a protective effect
of proteoglycan on the collagen network, since collagen
network degradation by collagenase is enhanced in an
aggrecan-depleted matrix, and inhibition of aggrecanase
blocks IL-1–stimulated collagen cleavage (27). The
zone-specific effect may also be related to the more
potent effect of IL-1 on chondrocytes from the superficial zone of cartilage than those of the deep zone (41).
Further analysis of the pathway linking biologic
stimuli to biomechanical deterioration of articular cartilage may elucidate the role of specific factors, and their
inhibitors, in the advancement of arthritic disease. Together with other cytokines and mechanical factors, IL-1
may contribute to functional deterioration of the collagen network of cartilage in OA. For example, it has been
found that activation of latent MMPs in immature
cartilage results in compressive softening as well as
hypotonic swelling of the tissue that can be markedly
inhibited by application of tissue inhibitor of metalloproteinases 1 (48). Other interesting targets may be
receptors for advanced glycation end products and their
ligands, which are present in the synovium of OA
patients and increase the expression of MMPs (49).
Cytokine-induced degradation may be the cause of the
alteration in matrix metabolism and content of the
cartilage extracellular matrix, both near and far from
cartilage lesions (50,51) and, thus, trigger weakening of
cartilage throughout the joint. Natural or synthetic mediators or inhibitors of matrix-degrading enzymes may
be tested to elucidate molecular pathways that lead to
tensile biomechanical dysfunction of cartilage and to
identify potential therapies that might prevent functional deterioration of cartilage in arthritis.
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