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Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 8, August 2005, pp 2386–2395
DOI 10.1002/art.21215
© 2005, American College of Rheumatology
Mechanical Injury of Cartilage Explants Causes Specific
Time-Dependent Changes in Chondrocyte Gene Expression
Jennifer H. Lee, Jonathan B. Fitzgerald, Michael A. DiMicco, and Alan J. Grodzinsky
Objective. Joint injury in young adults leads to an
increased risk of developing osteoarthritis (OA) later in
life. This study was undertaken to determine if injurious
mechanical compression of cartilage explants results in
changes at the level of gene transcription that may lead
to subsequent degradation of the cartilage.
Methods. Cartilage was explanted from the femoropatellar groove of newborn calves. Levels of messenger RNA encoding matrix molecules, proteases, their
natural inhibitors, transcription factors, and cytokines
were assessed in free swelling control cultures as compared with cartilage cultures at 1, 2, 4, 6, 12, and 24 hours
after application of a single injurious compression.
Results. Gene-expression levels measured in noninjured, free swelling cartilage varied over 5 orders of
magnitude. Matrix molecules were the most highly
expressed of the genes tested, while cytokines, matrix
metalloproteinases (MMPs), aggrecanases (ADAMTS5), and transcription factors showed lower expression
levels. Matrix molecules showed little change in expression after injurious compression, whereas MMP-3 increased ⬃250-fold, ADAMTS-5 increased ⬃40-fold, and
tissue inhibitor of metalloproteinases 1 increased ⬃12fold above the levels in free swelling cultures. Genes
typically used as internal controls, GAPDH and ␤-actin,
increased expression levels ⬃4-fold after injury, making
them unsuitable for use as normalization genes in this
study. The expression levels of tumor necrosis factor ␣
and interleukin-1␤, cytokines known to be involved in
the progression of OA, did not change in the chondrocytes after injury.
Conclusion. Changes in the level of gene expression after mechanical injury are gene specific and time
dependent. The quantity of specific proteins may be
altered as a result of these changes in gene expression,
which may eventually lead to degradation at the tissue
level and cause a compromise in cartilage structure and
function.
Acute traumatic joint injury in young adults leads
to an increased risk of developing osteoarthritis (OA)
later in life (1–3), despite efforts to intervene in this
process by surgically stabilizing injured joints (4). Although the mechanism by which injury leads to tissue
degeneration remains to be elucidated, several injuryrelated factors may contribute to the development of
OA. These factors include, but are not limited to,
instability in the joint due to ligament, tendon, or
meniscal tear, and/or initiation of a cellular response in
cartilage or other joint tissues at the time of the injury.
Previous clinical studies have shown an increase
in the protein levels of matrix metalloproteinase 3
(MMP-3) and tissue inhibitor of metalloproteinases 1
(TIMP-1) as well as an increase in proteoglycan and type
II collagen fragments in the synovial fluid of patients
following a tear in the anterior cruciate ligament (ACL)
or meniscus from 1 day to 20 years after the injury (5,6).
During the first week after ACL injury, a significant
increase in synovial fluid levels of tumor necrosis factor
␣ (TNF␣) and interleukin-1␤ (IL-1␤) can also occur. By
3 weeks after injury, the levels of these cytokines decreased to the levels observed in samples from patients
with chronic arthritis (7). Studies of gene expression in
normal and OA cartilage have shown up-regulation of
MMP-13 in late-stage OA, whereas MMP-3 is downregulated (8). In addition, bone morphogenetic protein
2 is increased in OA cartilage and colocalizes with newly
synthesized type II procollagen, which suggests that
anabolic remodeling of the tissue is taking place (9).
With the use of the lapine ACL transection
Supported by the NIH (grant AR-45779). Dr. Lee’s work was
supported by a National Science Foundation predoctoral fellowship.
Jennifer H. Lee, PhD, Jonathan B. Fitzgerald, BSE, Michael
A. DiMicco, PhD, Alan J. Grodzinsky, ScD: Massachusetts Institute of
Technology, Cambridge.
Address correspondence and reprint requests to Alan J.
Grodzinsky, MD, Massachusetts Institute of Technology NE47-377, 77
Massachusetts Avenue, Cambridge, MA 02139. E-mail: alg@mit.edu.
Submitted for publication December 16, 2004; accepted in
revised form May 3, 2005.
2386
GENE EXPRESSION AFTER CARTILAGE INJURY
model to study the pathogenesis of OA in vivo, investigators have observed an increase in MMP-3 expression
after 9 weeks (10) and location-specific changes in
messenger RNA (mRNA) levels of several genes after 3
weeks and 8 weeks (11). Expression of type II collagen,
aggrecan, biglycan, MMPs 1, 3, and 13, and TIMP-1
increased during the development of OA in this animal
model, whereas decorin and fibromodulin showed decreased expression (11).
Because loading variables are difficult to control
in vivo, a number of investigators have developed in
vitro models to isolate cartilage and study tissue- and
cellular-level effects of mechanical injury. Mechanical
loads applied in vitro range from single compressions of
up to 50% strain (12–17) to large-amplitude cyclic
compression at varying frequencies (⬃0.05–0.3 Hz) for
up to 2 hours (18–21). Injurious mechanical compression of cartilage in vitro can damage the extracellular
matrix (ECM), leading to increased water content
(12,16,18,22), decreased stiffness (12,22), increased hydraulic permeability (20), loss of glycosaminoglycan
(GAG) to the culture medium (12–15,17,20,22,23), loss
of collagen to the medium (20), and temporary denaturation of collagen in the tissue (16,18,20,21). In addition,
injurious mechanical compression can lead to cell death
by both apoptosis and necrosis (14,16,19,21–23), as well
as decreased matrix biosynthesis rates in the remaining
viable cells after injury (12).
Although many studies have focused on the effects of in vitro injurious compression on cartilage tissue,
the resulting modulation of chondrocyte gene transcription has not been fully elucidated. The objective of this
study was to quantify the effects of cartilage injury in
vitro on 24 genes central to cartilage maintenance,
including genes encoding macromolecules of the ECM,
proteases that can cleave ECM proteins and their natural inhibitors, transcription factors, and cytokines known
to affect cartilage metabolism. Using real-time polymerase chain reaction (PCR), we measured the levels of
mRNA of these molecules at 6 time points after acute
mechanical injury. We observed distinct changes in the
pattern and kinetics of expression that may suggest a
role for certain catabolic processes associated with eventual cartilage degradation.
MATERIALS AND METHODS
Tissue harvest. Articular cartilage explant disks were
harvested from the femoropatellar grooves of 1–2-week-old
calves using previously developed methods (24). Briefly,
9-mm–diameter cartilage-bone cylinders were drilled perpen-
2387
Figure 1. Loading device used to induce injurious compression of
bovine cartilage explants, and example of compression waveforms. A,
An incubator-housed loading apparatus was used to apply injurious
compression in displacement control to individual cartilage disks. The
load and displacement were recorded by transducers during loading. B,
Polysulfone chamber used to hold cartilage disks during loading in
unconfined compression. C, Representative data acquired during
compression to 50% strain at a strain rate of 1.0/second. Peak stress
reached a maximum value of 20.7 MPa. Color figure can be viewed in
the online issue, which is available at http://www.arthritisrheum.org.
dicular to the cartilage surface. These cylinders were then
placed in a microtome holder and the most superficial,
⬃200-␮m layer was removed to obtain a level surface. Up to 3
sequential 1-mm slices were cut from each cylinder, and 4 disks
(1-mm thick, 3-mm diameter) were cored from each slice using
a dermal punch, yielding a total of 48 disks from each joint.
These disks were then equilibrated in culture medium for 2
days (low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 10 mM HEPES
buffer, 0.1 mM nonessential amino acids, 0.4 mM proline, 20
␮g/ml ascorbic acid, 100 units/ml penicillin G, 100 ␮g/ml
streptomycin, and 0.25 ␮g/ml amphotericin B) in a 37°C, 5%
CO2 environment.
Injurious compression. After equilibration of the explants, a custom-designed incubator-housed loading apparatus
(25) was used (Figure 1A) to injuriously compress 36 cartilage
disks from each joint, while the remaining 12 disks served as
free swelling controls. Cartilage samples to be injured were
placed individually into a polysulfone chamber (Figure 1B),
which allows radially unconfined compression of the disk by
impermeable platens (12,13,15). The measured thickness of
the cartilage disk just prior to loading was recorded, and the
zero-strain position was identified by the point of first contact
between the loading platen and the cartilage surface. The
injury protocol consisted of a single displacement ramp to a
final strain of 50% at a velocity of 1 mm/second (strain rate
1.0/second in displacement control), followed by immediate
removal of the displacement at the same rate (Figure 1C).
Application of these strain and strain rate parameters resulted
in an average peak stress of ⬃20 MPa; this loading protocol
has previously been shown to produce damage to the ECM, a
significant decrease in cell viability, a decrease in cell biosynthesis by the remaining viable cells, and an increase in GAG
2388
loss to the medium in similar bovine cartilage explants
(12,13,15,22,23).
After injury, the disks were placed into fresh culture
medium. Groups of 6 cartilage disks were removed from
culture at 1, 2, 4, 6, 12, and 24 hours and then flash-frozen in
liquid nitrogen and stored at ⫺80°C. Two groups, each comprising 6 free swelling disks, were frozen at 4 and 24 hours to
serve as controls. Explant disks in each group of 6 specimens
were purposely matched across depth and location along the
joint surface, to prevent bias based on location; as a result, in
each experimental condition, the specimens used are representative of the specimens within the joint surface.
RNA extraction. RNA was extracted from the 6 pooled
cartilage disks by first pulverizing the tissue and then homogenizing in TRIzol reagent (Invitrogen, San Diego, CA) to lyse
the cells. Extracts were then transferred to Phase Gel tubes
(Eppendorf, Hamburg, Germany) with 10% volume/volume
chloroform and spun at 13,000g for 10 minutes. The clear
liquid was removed from the phase gel, and RNA was isolated
from the sample using the RNeasy Mini Kit (Qiagen, Chatsworth, CA). Genomic DNA was removed by a DNase digestion
step (Qiagen) during purification. Absorbance measurements
were read at 260 nm and 280 nm to determine the concentration of RNA extracted from the tissue and the purity of the
extract. The mean ⫾ SD 260 nm:280 nm ratio of absorbencies
was 1.86 ⫾ 0.12. Reverse transcription of equal quantities of
RNA (2.5 ␮g) from each sample was performed using the
AmpliTaq-Gold Reverse Transcription Kit (Applied Biosystems, Foster City, CA).
Real-time PCR. Real-time PCR was performed using
the Applied Biosystems 7700 instrument and SYBR Green
Master Mix (Applied Biosystems). Primers were designed on
the basis of bovine sequences for matrix molecules (type II
collagen, aggrecan, link protein, fibronectin, fibromodulin, and
type I collagen), proteases (MMP-1, MMP-3, MMP-9, MMP13, ADAMTS-4, and ADAMTS-5), protease inhibitors
(TIMP-1 and TIMP-2), cytokines (TNF␣ and IL-1␤), housekeeping genes (␤-actin and GAPDH), transcription factors
(c-fos, c-jun, and SOX9), and growth factors (insulin-like
growth factor 1 [IGF-1], IGF-2, and transforming growth
factor ␤ [TGF␤]), using Primer Express software (Applied
Biosystems). Standard curves for amplification using these
primers were generated; all primers demonstrated approximately equal efficiency, with standard curve slopes of ⬃1,
indicating a doubling in complementary DNA quantity in each
cycle. Expression levels in injured samples were normalized to
those in free swelling control samples for each gene.
Statistical analysis. Changes in gene-expression levels.
In each experiment, expression levels measured in injured
sample groups were normalized to those in free swelling
control groups for each gene; expression data are presented as
the mean (⫾SEM) of 3 replicate experiments. Changes in
gene-expression levels in the injured samples with respect to
the levels in free swelling controls at the 4-hour and 24-hour
time points were examined using a nonparametric t-test (26).
The t-test was made nonparametric by estimating the P values
from permuted data sets (27); the t statistic was calculated
from each of the permuted data sets to create a distribution of
possible values. Using this method, all changes in expression
that were ⱖ5-fold were found to be statistically significant.
Changes between 2-fold and 5-fold were also found to be
LEE ET AL
significant, with 3 exceptions (c-fos at 4 hours and 24 hours,
and c-jun at 24 hours); in certain instances, a lower magnitude
of change was found to be significant.
Gene clustering. To distinguish the main expression
trends, a k-means clustering algorithm was applied to the
injury time-course data (28–31). Each gene was grouped on
the basis of correlations between the time-course expression
profile and a set of randomly chosen starting genes. Group
profiles were then calculated as the mean of the expression
profiles of the genes in each group. The correlation between
each gene and each group profile was calculated and the genes
were then regrouped in an iterative manner until convergence.
To ensure that an optimal clustering solution for the 24 genes
was found, the algorithm was run a sufficient number of times
to cover every possible selection of starting genes. Each set of
randomly chosen starting genes produced a deterministic
grouping of the genes, with each gene paired with the group
profile showing the highest correlation. The optimal solution
was chosen as the grouping that had the highest overall
correlation of genes with group profiles, by averaging over all
of the genes (for details, see ref. 31). The number of groups
was varied from 3 to 6. Ultimately, 5 groups were chosen to
best represent the trends. To determine the distinctiveness of
the main expression trends, the final group profiles were
compared using a comparison-means Student’s t-test. The
Euclidean distance between 2 group profiles represents the
difference of means, and the average squared distance of
the genes within a group to the group profile represents the
variance. The number of genes in each group corresponds to
the degrees of freedom for that group.
RESULTS
Gene-expression levels in noninjured control
cartilage disks. Real-time PCR was used to determine
the expression levels of 24 genes of interest in noninjured control cartilage disks for comparison with mechanically injured disks. Levels of expression of the
tested genes varied over 5 orders of magnitude, as seen
in Figure 2, with data normalized to the level of the
lowest expressed gene, ADAMTS-4 (aggrecanase 1).
ECM molecules, as well as SOX9, a transcription factor
promoting expression of matrix molecules in cartilage,
showed the highest levels of expression. Genes typically
used as internal controls (GAPDH and ␤-actin) showed
intermediate levels of expression, whereas certain cytokines, MMPs, and transcription factors displayed relatively lower levels of expression. ADAMTS-4 and
ADAMTS-5 (aggrecanase 2) showed the lowest levels of
expression of the genes tested.
Effects of injurious compression on gene expression. Levels of gene expression in noninjured free swelling controls (shown in Figure 2) changed selectively in
response to injurious compression. P values comparing
expression levels after injury with expression levels in
noninjured controls were calculated at the 4-hour and
GENE EXPRESSION AFTER CARTILAGE INJURY
Figure 2. Expression levels of 24 genes in free swelling control
cartilage cultures, ranked by relative abundance. Medium was changed
2 days after harvest and samples were obtained at 4 and 24 hours after
medium change for gene-expression quantification. Levels at the 2
time points were averaged to give a single value for each tissue sample.
Expression levels were normalized to expression of ADAMTS-4, the
least abundant gene measured. Data are reported as the mean and
SEM of 3 replicate experiments using tissue from 3 different joints.
IGF-2 ⫽ insulin-like growth factor 2; MMP-3 ⫽ matrix metalloproteinase 3; IL-1␤ ⫽ interleukin-1␤; TIMP-2 ⫽ tissue inhibitor of
metalloproteinases 2; TGF␤ ⫽ transforming growth factor ␤; TNF␣ ⫽
tumor necrosis factor ␣.
24-hour time points (Table 1). Although expression
levels of some genes remained unchanged in response to
injury, others exhibited dramatic differences compared
with their free swelling controls. After compression,
GAPDH and ␤-actin increased in expression ⬃4-fold
above the levels in free swelling controls (Figure 3A).
Because these housekeeping genes showed variations in
expression levels within the 24 hours after loading, they
were not used as internal controls to normalize the data
acquired on the other genes; instead, all expression
levels were normalized by using a fixed quantity of
extracted RNA for reverse transcription. By using a
fixed quantity of RNA from each sample, decreased cell
viability in injuriously compressed cartilage should not
affect the observed levels of gene expression; rather,
changes in expression should represent the changes
occurring within the remaining viable cells in the tissue.
Matrix molecules showed no more than a 2-fold
change in gene-expression levels during the 24 hours
immediately following compression. Levels of type II
collagen and aggrecan (Figure 3A), as well as fibromodulin and link protein (results not shown), did not
fluctuate during the 24 hours immediately following
injury. Fibronectin increased ⬃2-fold in gene expression
at the 12- and 24-hour time points (results not shown).
2389
The most dramatically changing gene in this
study was MMP-3, which, following injurious compression, increased in expression ⬃250-fold above the levels
in free swelling controls (Figure 3B). MMP-3 expression
began to increase within 2 hours after injury, peaked by
12 hours, and then declined to an ⬃50-fold increase
above free swelling levels by 24 hours. MMP-13, in
contrast, showed only an ⬃2-fold increase above the
level in free swelling controls during the 24 hours after
injury (Figure 3B). Moreover, MMP-1 and MMP-9 increased by ⬃6-fold and ⬃4-fold, respectively, above the
levels in their free swelling controls (results not shown).
Similar to MMP-3, ADAMTS-5 showed a dramatic increase in gene expression, to ⬃40-fold above the
levels in free swelling controls by 12 hours after injury,
which, by 24 hours, remained elevated by ⬃10-fold above
Table 1. List of group members with distinct temporal geneexpression profiles induced by injury of cartilage explants, as determined by k-means clustering*
Time point
Group 1
MMP-3
ADAMTS-5
TGF␤
Group 2
c-fos
c-jun
Group 3
MMP-1
MMP-9
MMP-13
Type I collagen
TIMP-1
TIMP-2
Fibronectin
SOX9
GAPDH
␤-actin
TNF␣
Group 4
IGF-1
IGF-2
ADAMTS-4
Group 5
Type II collagen
Aggrecan
Fibromodulin
Link protein
IL-1␤
4 hours
24 hours
0.01
⬍0.01
⬍0.01
0.05
⬍0.01
0.03
0.53
0.03
0.30
0.26
0.07
0.83
0.03
0.02
0.02
0.69
0.49
⬍0.01
0.14
0.08
0.91
0.01
0.04
⬍0.01
0.05
0.03
⬍0.01
0.04
0.92
⬍0.01
0.03
0.33
0.16
0.15
0.03
0.08
0.49
0.02
0.46
0.05
0.09
0.12
0.09
0.09
0.02
0.72
0.18
0.25
* Values are P values in comparison with noninjured control cartilage,
calculated from t-tests performed at the 4-hour and 24-hour time
points after injury. Groups were formed on the basis of gene–group
profile correlations. MMP-3 ⫽ matrix metalloproteinase 3; TGF␤ ⫽
transforming growth factor ␤; TIMP-1 ⫽ tissue inhibitor of metalloproteinases 1; TNF␣ ⫽ tumor necrosis factor ␣; IGF-1 ⫽ insulin-like
growth factor 1; IL-1␤ ⫽ interleukin-1␤.
2390
Figure 3. Changes in expression level of A, matrix molecules, ␤-actin,
and GAPDH genes and B, matrix metalloproteinases and tissue
inhibitor of metalloproteinases 1 after injurious compression. Results
are the fold change (⫻) from free swelling levels, with a value of 1
(broken line) indicating similar expression after injury to the level
measured in free swelling conditions. Six cartilage disks were pooled
for each time point for each experiment. All samples were normalized
to total RNA at the reverse transcription step. Data are reported as the
mean ⫾ SEM of 3 replicate experiments. See Figure 2 for definitions.
control levels (Figure 3B). In contrast, ADAMTS-4 increased only ⬃2–3-fold above the free swelling levels and
showed little variation within the time period up to 24
hours after injury (results not shown). TIMPs, the endog-
LEE ET AL
Figure 4. Changes in expression level of A, transcription factors, B,
growth factors, and C, cytokines after injurious compression. Results are
the fold change (⫻) from free swelling levels, with a value of 1 (broken
line) indicating similar expression after injury to the level measured in
free swelling conditions. Six cartilage disks were pooled for each time
point for each experiment. All samples were normalized to total RNA at
the reverse transcription step. Data are reported as the mean ⫾ SEM of
3 replicate experiments. See Figure 2 for definitions.
GENE EXPRESSION AFTER CARTILAGE INJURY
enous tissue inhibitors of metalloproteinases, were also
affected by injurious compression. TIMP-1 increased to
⬃12-fold over free swelling levels by 12 hours and remained elevated by 24 hours after injury (Figure 3B).
TIMP-2, which was expressed at an overall higher level in
free swelling cartilage (Figure 2), was increased by only
⬃2-fold at 12 hours and 24 hours after injury (results not
shown).
The immediate-response transcription factors
c-fos and c-jun responded to injury with a rapid increase
in gene expression (⬃120-fold for c-fos and ⬃40-fold for
c-jun) within the first hour after injury (Figure 4A). By 4
hours, both genes returned to an ⬃3-fold increase over
free swelling levels and remained moderately elevated
for 24 hours. Another transcription factor, SOX9, which
promotes transcription of matrix molecules, did not
change expression levels significantly during the 24
hours following injurious compression (Figure 4A). This
is consistent with the observed lack of change in geneexpression levels of the matrix molecules shown in
Figure 3A.
Selected growth factors of interest also showed
specific changes in gene-expression levels in response to
injurious compression. TGF␤ increased expression in
the first 4 hours after injury to a peak value ⬃7-fold
above the levels in free swelling controls, which remained elevated through 12 hours and then decreased to
⬃4-fold over the free swelling value by 24 hours (Figure
4B). Insulin-like growth factors IGF-1 and IGF-2 (Figure 4B) and, similarly, the cytokines IL-1␤ and TNF␣
(Figure 4C) showed little variation with time (not exceeding 2-fold increased or decreased levels compared
with noninjured controls) in the 24 hours immediately
following injurious compression.
Clustering analyses of gene-expression profiles.
Clustering analysis revealed 5 groups with distinct temporal expression profiles induced by injury. The group
expression profiles are shown in Figure 5, and the
corresponding group members along with their associated level of significance (P values calculated by t-test,
performed at 4 hours and 24 hours after injury) in
comparison with noninjured control cartilage are listed
in Table 1. In general, the group expression profiles are
a reflection of the main traits of the individual genes
within each group, with mean correlation coefficients of
0.90, 1.00, 0.89, 0.77, and 0.88 for groups 1, 2, 3, 4, and
5, respectively. Comparison of means by Student’s t-test
revealed that the group expression profiles were distinct
(Figure 5). The unique profile of group 2 was significantly different from the expression profiles of groups 3,
4, and 5 (P ⬍ 0.05), and the expression profiles of groups
2391
Figure 5. Group expression profiles generated by k-means clustering,
showing the main temporal gene-expression patterns induced by injury
of cartilage explants. Group profiles were calculated by averaging the
expression profiles of genes within each group. Results are the mean
change (⫻) from free swelling levels, with a value of 1 (broken line)
indicating similar expression after injury to the level measured in free
swelling conditions. Color figure can be viewed in the online issue,
which is available at http://www.arthritisrheum.org.
1 and 3 were also significantly different (P ⫽ 0.006). The
expression profiles of groups 1 and 2 were found to be
not significantly different from each other, primarily due
to the low number of genes within each of these groups.
DISCUSSION
A single injurious compression of cartilage has
been shown previously to decrease ECM biosynthesis
rates, compromise mechanical properties, and reduce
chondrocyte viability (12–16,22). We undertook this
study to determine if changes also occur at the level of
gene expression, and to determine whether the changes
are general or are specific to certain genes. Analysis of
samples was performed using real-time PCR, which
allows the measurement of many genes to be achieved in
a high-throughput manner using a relatively small sample volume. We observed significant changes in the
expression of several catabolic and anabolic genes in
response to mechanical injury, and used k-means clustering (31) to further analyze gene-expression patterns
and coregulation of specific genes that may result from
injury. Previous investigators have used similar cluster-
2392
ing techniques to analyze changes in expression caused
by noninjurious static compression (31).
Analysis of the behavior of the gene groups by
clustering resulted in separation of the genes into 5
groups that displayed distinct patterns of behavior after
injury (Table 1 and Figure 5). Group 1 contained
MMP-3, ADAMTS-5, and TGF␤, which all displayed
large changes in expression levels at early time points
(within 4 hours) following injury. In addition to directly
cleaving matrix molecules, MMP-3 has been implicated
as a member of the activation cascades of matrixdegrading enzymes, including other MMPs. Stimulation
of these 3 genes immediately after injury may represent
an attempt to remodel the damaged matrix by removing
some of the matrix molecules or by activating latent
molecules in the matrix. The transcription factors c-fos
and c-jun (group 2) showed an immediate transient
up-regulation followed by a rapid decline within 4 hours;
c-fos and c-jun are members of the activator protein 1
family of genes, which were previously shown to activate
MMPs in a chondrocyte cell line after IL-1␤ treatment
(32). This is consistent with the activation of several
MMPs in groups 1 and 3 of this study, observed at time
points subsequent to the increased expression of c-fos
and c-jun immediately after injury.
Group 3 represents the slowly increasing expression pattern seen for MMPs (other than MMP-3) and
their inhibitors, as well as TNF␣, fibronectin, type I
collagen, GAPDH, and ␤-actin. Although gene expression of SOX9 remained below free swelling levels for all
time points tested (Figure 4A), this gene clustered into
group 3 because its expression increased from 0.6-fold to
1.0-fold the level of free swelling controls during the 24
hours after injury, as was the case for group 3 overall.
Further investigation is required to determine the extent
to which these molecules may affect cartilage behavior
after injury in this system, since their changes in expression were relatively low.
Group 4 (IGF-1, IGF-2, and ADAMTS-4) and
group 5 (type II collagen, aggrecan, fibromodulin, link
protein, and IL-1␤) showed expression patterns that did
not vary significantly with time after injury. Thus, any
immediate alterations in ECM biosynthesis that may
result from mechanical injury are unlikely to be related
to events at the level of matrix gene (group 5) transcription. Any rapid initial repair of the matrix immediately
after acute mechanical injury is not likely to be associated with changes in expression of group 4 genes.
The effects of mechanical injury on the expression of MMP-3 and MMP-13 (Figure 3B) are similar to
the trends reported by Patwari et al (13) in a study in
LEE ET AL
which Northern analysis was used to determine the
expression levels in similar cartilage explants that were
subjected to the same injury protocol as depicted in
Figure 1C. In that study, injury caused a significant
increase in MMP-3 expression (10-fold) above the level
in controls but no change in MMP-13 in the first 24
hours. In comparison, when our data (shown in Figure
3B) are averaged over the full 24-hour period, MMP-3
appears to be up-regulated 80-fold, while MMP-13
expression increases no more than 2-fold above the
control levels. Taken together, these studies show similar differential changes in the expression of MMPs 3 and
13, obtained using both Northern analysis and real-time
PCR techniques. In vivo studies of OA progression
following joint injury have also demonstrated changes in
MMP gene-expression levels. Le Graverand et al, using
a lapine ACL transection model of OA, found 2–3-fold
increased chondrocyte expression of MMP-3 and 10–30fold increased expression of MMP-13 (11), which differs
in their relative increases as compared with the changes
of MMPs 3 and 13 seen in the present study and in
previous studies (13). This difference may be related, in
part, to the presence of other tissues in the in vivo
model, such as ligaments, tendons, and synovium, that
are not included in the present in vitro model of injury
to cartilage alone.
It is also informative to compare the observed
changes in gene expression reported herein to the
changes in protein levels reported previously in response
to mechanical injury of cartilage in vitro. Immunohistochemical analyses of adult (23-month-old) bovine articular cartilage disks subjected to a rapid ramp displacement to 50% strain and held for 5 minutes revealed an
increase in MMP-1, MMP-3, and MMP-13 as well as a
decrease in TIMP-1 and TIMP-2 (33). A study applying
compressive loading to immature bovine tissue measured increased synthesis and activity of MMP-2 and
MMP-9 after 1–16 hours of loading, while no change was
measured in TIMP-1 or TIMP-2 synthesis (34). Porcine
cartilage disks from 3–6-month-old animals subjected to
a cutting injury showed an increase in synthesis of
MMP-1, MMP-3, and TIMP-1, while collagen synthesis
remained unchanged (35). Interestingly, our study revealed changes in the expression of MMPs and TIMPs,
but no change in type II collagen expression. In addition,
compression injury was found previously to increase
fibronectin protein synthesis (18); the compression injury used in the present study caused an increase in
fibronectin gene expression (Table 1). Specific differences found in these studies may be due to differences in
regulation at the level of translation, as well as to the
GENE EXPRESSION AFTER CARTILAGE INJURY
different injury models used (e.g., cutting versus compression).
Findings from in vivo studies at the protein level
also have certain parallels to the results reported herein.
Lohmander et al analyzed human synovial fluid after
ACL or meniscus injury and found increases in MMP-3
and TIMP-1 protein levels within 1 day after injury,
which persisted for 20 years (5). Similarly, increased
chondrocyte mRNA levels of MMP-3 and TIMP-1 were
found in our study after cartilage injury and in the lapine
ACL transection model (11). Irie et al measured elevated levels of the inflammatory cytokines IL-1␤ and
TNF␣ in human joints within 24 hours after ACL injury
(7). In the current study, cartilage injury did not cause an
increase in chondrocyte expression of IL-1␤ and TNF␣.
Although the major source of the increased levels of
cytokines, MMPs, and TIMPs seen in the synovial fluid
of injured human joints could be the synovium or tissues
other than cartilage, it is informative to be able to
identify specific changes in chondrocyte gene and protein expression for comparison.
Changes in expression of proteases and cytokines
have been found during the progression of OA. Bau et al
(8) compared chondrocytes isolated from patients with
normal articular cartilage with chondrocytes from patients with early and late-stage OA. MMP-13 and
ADAMTS-4 expression increased in late-stage OA,
whereas MMP-3 expression was the highest of the gene
levels tested and was down-regulated in OA (8). Murata
et al (36) measured IL-1␣ and IL-1␤ gene expression (by
reverse transcription–PCR) and protein levels (by
enzyme-linked immunosorbent assay) in OA chondrocytes isolated from cartilage obtained during joint arthroplasty. They reported a decrease in IL-1␣ and IL-1␤
transcript in cells from advanced OA tissue compared
with cells from tissue displaying only moderate degeneration. This decrease in expression was accompanied by
a decrease in protein level in advanced OA (36). In
contrast, we found that the expression of IL-1␤ was not
significantly altered by acute compression injury in vitro
(Table 1).
Other distinct and important differences in gene
expression exhibited by OA tissues versus that in explants subjected to acute mechanical injury have been
observed. For example, types I and II collagen exhibit
increased expression levels with the progression of OA
(37), whereas in our study no significant change in the
expression of type II collagen was observed following
compression injury and type I collagen significantly
increased expression 2.5-fold by 24 hours after injury. It
should be emphasized that the focus of the present study
2393
is on immediate changes after injury (within the first 24
hours), while OA develops over a time span of many
years and involves pathologic processes of the whole
joint (38). It will be important to expand such in vitro
studies to include longer culture periods after injury. In
addition, in vitro models of whole-joint injury that
involve injured cartilage in the presence of exogenous
cytokines (13) or injured cartilage cocultured with other
injured joint tissues (39) may give additional insight into
the cellular pathways underlying chondrocyte response
to injury.
Investigators have studied the effects of static
compression on cartilage explants as well as
chondrocyte-seeded gels to determine if changes occur
at the levels of gene expression and protein synthesis in
these model systems. Cartilage explants were compressed very slowly to 25% and 50% strain and maintained in compression for up to 24 hours. This low strain
rate protocol does not alter cell viability (40), in contrast
to the marked increase in cell death and matrix damage
typically observed after injurious loading. These samples
were compared with unloaded controls. Results by realtime PCR showed a transient increase in mRNA levels
for aggrecan and type II collagen, as well as other matrix
proteins, followed by a down-regulation below control
levels by 24 hours (31). The down-regulation of aggrecan
and type II collagen expression was shown by Northern
analysis to be dose dependent, with 50% compression
causing a greater decrease than that caused by 25%
compression (41). Radiolabel incorporation into proteoglycans and collagen also decreased with increasing
static compression (41). Transcription of many matrix
proteases, including MMP-3, increased with loading
duration, with elevations ranging from 3-fold to 16-fold
by 24 hours of loading (31). Transcription factors c-fos
and c-jun were transiently up-regulated by 6- to 35-fold
after 1 hour of loading (31).
Results similar to those seen for matrix molecule
expression in cartilage explants were obtained in a
cell-seeded construct. Primary chondrocytes were
seeded in type I collagen gels and subjected to static
compression of 0%, 25%, or 50% for up to 24 hours
(42). Results using competitive and real-time PCR
showed inhibition of type I collagen, type II collagen,
and aggrecan mRNA expression. Radiolabel incorporation of proline and sulfate were also inhibited by the
application of static compression (42). In both of these
experiments, changes in mRNA expression levels correlated with changes occurring at the protein level. Notably, changes seen in response to static compression were
markedly different from those observed in injurious
2394
loading scenarios. In the current study, matrix molecules
did not change in expression level, in contrast to that
seen in response to static compression; also, the magnitude of increased expression of degradative enzymes and
transcription factors was higher in response to injurious
compression compared with noninjurious static compression. Thus, the response of chondrocytes to mechanical compression appears to depend on the specific
parameters (rate, amplitude, and duration) of the applied compression.
An unexpected result in the current study was the
relatively high level of type I collagen expression in free
swelling control tissue (Figure 2). Type I collagen molecules can be found in diseased or damaged articular
cartilage; however, it is not abundant in healthy cartilage. Although type I collagen expression was, indeed,
⬃60-fold lower than that of type II collagen, mRNA
levels were higher than most of the other non-ECM
genes studied. Relatively high levels of type I collagen
expression were previously reported in 6-month-old
porcine cartilage and found to be ⬃3-fold lower than the
expression levels of type II collagen (43). The tissue used
in the current study was obtained from newborn bovines,
and type I collagen expression may vary widely with age
and species. In addition, cells from the sparse blood
vessels present in newborn cartilage tissue could contribute to the expression of type I collagen, as seen in the
control data in Figure 2.
One limitation of our study is that samples from
different locations within the femoropatellar groove
were pooled; thus, it was not possible to determine
whether tissue from different depths and locations along
the groove would react differently to injurious compression. Explant disks in each group of 6 specimens were
purposely matched across depth and location along the
joint surface to prevent bias based on location; therefore, the gene-expression results represent an average of
specimens within the joint surface. Another limitation is
the use of newborn tissue. We and other investigators
have previously studied the effects of injury on cell
viability and ECM degradation in the presence and
absence of exogenous cytokines, using both immature
and adult cartilage from bovine and human joint surfaces, with the finding that certain responses vary with
age (12,13,44). It will be important to extend the present
study to identify any age- or disease-dependence of
changes in gene expression caused by mechanical injury.
In summary, injurious compression caused timedependent changes within 24 hours in the expression of
specific catabolic and anabolic genes that can regulate
matrix remodeling and turnover, whereas many ECM
LEE ET AL
molecules were unaffected. Ongoing studies are focused
on determining whether these changes at the level of
gene expression result in changes in protein levels in the
cartilage, and whether the high up-regulation of
ADAMTS-5 and MMP-3 in response to injurious mechanical compression may be associated with cellmediated changes in the proteolytic cleavage of ECM
molecules over extended times after injury.
ACKNOWLEDGMENT
We thank Dr. Moonsoo Jin for design of certain
primers used in this study.
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