Mature bovine stifle joints were obtained after slaughter from a local abattoir (Bud’s Custom Meats, Riverside, IA). Osteochondral explants were prepared by manually sawing a 25 mm by 25 mm square from the lateral tibial plateau, which included the central loaded area of the articular surface that was not covered by menisci. The explants were placed in culture medium containing 45% DMEM, 45% Ham’s F-12, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and incubated at 37°C in an atmosphere of 5% CO2 in air.
Twenty four hours after harvest, osteochondral explants were secured in custom testing fixtures for impact loading and were kept submerged in culture medium at all times. A drop tower was used to impart loads to an indenter resting on the explant surface. The indenter was a flat-faced 5.0 mm diameter brass rod with rounded edges (r = 1 mm). Impact energy was modulated by dropping a 2 kg mass from a height of 7 cm resulting in an impact energy density of 7 J/cm2 and peak stresses in excess of 20 MPa, imposed at a rate of greater than 1000 MPa/sec. The mass was removed from the platen immediately after impact.
To study superoxide production explants were placed in phenol red-free culture medium (10% FBS, DMEM, F12) containing 5μM dihydroethidium (DHE) and 1 mM calcein AM at various time points after impact (1 hour, 3 hours, 6 hours, 24 hours, and 48 hours). The Invitrogen stained explants were imaged on a BioRad 1024 Confocal Microscope equipped with a Krypton/Argon laser (Bio-Rad Laboratories Inc., Hercules, CA). The sites were scanned to a depth of 150 μM at 20 μm intervals using wavelengths of 568 nm and 488 nm and a 10× objective with a field size of ~ 1.0 mm2. Z-axis projections of confocal images were analyzed using Image J (rsb.info.nih.gov/ij), a Java-based public domain image analysis program, to determine the average percentage of DHE-stained cells at each time point. Three sites within the impact site and 3 sites ~0.5 cm away from the impact site were imaged (Figure 1A). Four explants were used for each time point.
To study the effect of rotenone on superoxide production one group of explants was treated with 2.5 μM rotenone (Sigma Aldrich, St. Louis, MO) starting 1 hour before impact and continuing for1 hour post-impact during imaging sessions. Another group of explants was dosed with rotenone 30 minutes post-impact to evaluate the effect of delaying treatment. A third group was impacted but not treated with rotenone and a fourth group was neither impacted nor treated with rotenone (Figure 1B). Impact sites and sites approximately 1 cm away (control) were imaged and analyzed as described above. Three sites within impact sites and three sites outside impact sites were imaged for each explant and 3 explants were analyzed for each group. Impact sites were imaged a final time at 70 minutes post-impact using a 4× objective to record the spatial distribution of staining on the explant surface.
Effects of impact and rotenone on chondrocyte viability were assessed 24 hours after impact, a time when impact-induced chondrocyte death was previously shown to reach a steady state 33 (link). One group of explants was treated with 2.5 μM rotenone for 2 hours before and 2 hours after impact, a second group for 1 hour before and 1 hour after impact, a third group for 1 hour starting immediately after impact. A fourth group went untreated (Figure 1C). Calcein AM (1.0 mM) was used to stain viable cells and ethidium homdimer-2 (1.0 mM) was used to stain dead cells (Invitrogen). Explants were scanned to a depth of 200 μm at 20 μm intervals as described above and the images analyzed using Image J to determine percent viability. Three different projections were recorded within each impact or non-impact control site. Three explants were used for each treatment group.
One-way ANOVA with a post hoc Holm-Sidek correction for multiple comparisons was used to compare treatment groups. A p-value less than 0.05 was considered significant.