In the first set of experiments, sixty-four full-thickness cartilage
explants, 5 mm in diameter, were harvested from the weight-bearing
regions of the medial femoral condyles of four fresh bovine knee
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. These explants were divided into four groups: control,
low load, moderate load, and high load. Low-load explants were subjected
to a single static radially unconfined stress of 7 MPa; moderate-load
explants, to 14 MPa; and high-load explants, to 23 MPa. The duration
of loading was 500 msec. The control-group explants were not loaded.
At forty-eight hours after injury, glycosaminoglycan levels were
measured in the media with use of dimethylene blue assay. Apoptotic cells
were counted with use of TUNEL (terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labeling). Apoptosis was confirmed by electron microscopy
in selected samples.
In the second set of experiments, human cartilage explants were
taken from the femoral and tibial condyles and talar domes of macroscopically
normal fresh cadaver joints. These were divided into two groups:
loaded (subjected to 30% strain for 500 msec) and control
(unloaded). Apoptosis levels were measured at forty-eight hours.
In the third set of experiments, tibial cartilage explants were loaded
at 30% strain for 500 msec, and apoptosis levels were measured
at zero, three, six, twenty-four, forty-eight, and ninety-six hours
after loading.