After the pilot experiments involving multiple appendages (see above), tissue samples from definitive experiments were obtained from distal femur only. Bone samples were fixed in 10% neutral buffered formalin overnight, then decalcified in TBD-2 (Thermo Fisher Scientific, Waltham, MA). The specimens were processed routinely and embedded in paraffin. Tissue sections were cut at 4 microns, stained with hematoxylin and evaluated by light microscopy.
Tissue samples were analyzed in a blinded fashion by an experienced veterinary pathologist (KB). Osteonecrosis was defined as the presence of all three of the following criteria: empty lacunae, pyknotic nuclei of ghost osteocytes in the bone trabeculae, and necrosis of the adjacent marrow and stromal elements.8 (link),11 (link),34 (link) All mice that had at least one osteonecrotic lesion in a stifle joint were considered positive for osteonecrosis, whereas those with no osteonecrotic lesions were considered negative for osteonecrosis.
Tissue samples were analyzed in a blinded fashion by an experienced veterinary pathologist (KB). Osteonecrosis was defined as the presence of all three of the following criteria: empty lacunae, pyknotic nuclei of ghost osteocytes in the bone trabeculae, and necrosis of the adjacent marrow and stromal elements.8 (link),11 (link),34 (link) All mice that had at least one osteonecrotic lesion in a stifle joint were considered positive for osteonecrosis, whereas those with no osteonecrotic lesions were considered negative for osteonecrosis.
