The occurrence of chondrocyte apoptosis may be critical
in determining the extent of a lesion and subsequent repair
after mechanical injury to articular cartilage. Agents that prevent apoptosis
and increase chondrocyte survival may prove beneficial in the treatment
of these lesions. This set of experiments was designed
to determine the efficacy of some known apoptosis inhibitors in
preventing chondrocyte apoptosis after mechanical injury.
In the first set of experiments, thirty full-thickness cartilage explants,
5 mm in diameter, were harvested from the weight-bearing regions
of the medial femoral condyle of three fresh bovine joints with
use of a dermal punch. Explants were cultured for forty-eight hours
in Dulbecco modified Eagle medium supplemented with 10% fetal
bovine serum. Explants were divided into three groups: control,
loaded, and loaded + CI. The loaded group was subjected
to a radially unconfined single static load of 23 MPa for 500 msec
and recultured in fresh media for forty-eight hours postinjury.
The loaded + CI group underwent the same loading, but the
specimens were cultured in fresh media containing 100 mM of the
caspase inhibitor z-VAD.fmk. The control group did not
undergo loading. In a second set of experiments, forty-eight bovine
cartilage explants were divided into four groups: control, loaded, loaded + D,
and loaded + I. The loaded, loaded + D, and loaded + I
groups were loaded as described above. The loaded + D explants
were cultured in media containing 0.1 mM dexamethasone,
and the loaded + I explants were cultured in media containing
50 ng/mL IGF-1 (insulin-like growth factor-1). In a third
set of experiments, thirty explants were harvested from macroscopically
normal fresh human cadaver tibial articular cartilage
and were divided into control, loaded, and loaded + CI
groups. Both loaded groups were loaded at 23 MPa, and the loaded + CI
group was cultured in media containing z-VAD.fmk. In a
fourth set of experiments, explants were similarly harvested from
macroscopically normal fresh human cadaver tibial articular cartilage
and were divided into control, loaded, and loaded + I groups.
Both loaded groups were subjected to 30% strain applied
for 500 msec. Loaded + I explants were cultured in media
containing 50 ng/mL IGF-1. Forty-eight hours after injury,
the number of apoptotic cells in each explant was counted with use
of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end
labeling). Apoptosis was confirmed by electron microscopy in selected
samples. For the first experiment, glycosaminoglycan levels in media
were also measured, with use of dimethylene blue assay.
The levels of apoptotic cells in all of the loaded groups were significantly
higher than those in the control groups (p < 0.001).
Caspase inhibition: Culturing loaded explants
in z-VAD.fmk significantly reduced both the percentage of cells
demonstrating apoptosis and the glycosaminoglycan levels in bovine
and human explants (p < 0.01) (Figs. 1 and 2).
IGF-1: Both bovine and human explants demonstrated
a decrease in apoptosis rates when cultured in media containing
50 ng/mL of IGF-1 (p < 0.05) (Fig. 1).
Dexamethasone: Loaded bovine explants demonstrated
a decrease in apoptosis rates when cultured in media containing
0.1 mM dexamethasone (p < 0.05) (Fig. 1).
Glycosaminoglycan release: Compared with control
bovine explants, loaded bovine explants demonstrated an increase
in glycosaminoglycan levels in the media. This increase in glycosaminoglycan
levels with loading was reduced by the addition of z-VAD.fmk
to the media after loading (p < 0.01) (Fig. 2).
In the present and previous studies1,2,
we characterized cartilage response to acute mechanical injury.
It appears that chondrocytes undergo apoptosis when subjected to
various forms of injury. Both apoptosis and proteoglycan release
correlated with load intensity and with each other, suggesting a
possible link between apoptosis and matrix loss after mechanical
injury. This finding supports the notion that cell death, even in
the form of apoptosis, could be linked to matrix loss in cartilage.
Since cartilage does not contain tissue macrophages, there is no
apparent mechanism for removing dead cells or apoptotic bodies.
This raises the possibility that chondrocyte apoptosis could cause
further tissue damage and affect subsequent repair. The induction
of apoptosis is time-dependent, which offers a therapeutic window during
which apoptosis may be inhibited.
The contribution of cell death to cartilage degradation has been
previously suggested for human and experimentally induced osteoarthritis,
in which a close correlation between the frequency of chondrocyte
apoptosis and the severity of osteoarthritic changes was seen. The
inhibition of apoptosis as a therapeutic modality may therefore
have an even more far-reaching impact on osteoarthritis than the
initial preservation of cell viability.