Figure 1.

Experimental design and methods. (A) Cartilage explants were harvested from the medial femoral condyle (MFC) for the first set of experiments, and from two sites for the second set of experiments; the MFC and the distal patellofemoral groove (PFG). One explant from each region was impacted at a higher impact magnitude, one was impacted at a lower magnitude, and one served as an un-impacted control. Explants were then divided for use in several assays; chondrocyte viability was quantified using live/dead staining, mitochondrial (MT) membrane polarity was determined as red to green fluorescent intensity (R:G) ratio on confocal imaging and MT respiratory function was assessed via microrespirometry. Cell membrane damage was assessed by measuring lactate dehydrogenase (LDH) activity in cartilage conditioned media. (B) MT respiratory function was quantified by measuring oxygen consumption rate (OCR) over time. After basal OCR was determined, a MT stress test was performed by sequential addition of (i) oligomycin, an ATP synthase inhibitor; (ii) FCCP, a proton circuit uncoupler; and (iii) rotenone (Rot) and antimycin A (Ant A), inhibitors of MT complexes I and III. Parameters of respiratory control were calculated as depicted by the shaded areas under the curve.