Bone evaluation software

BMAT content was analyzed at the distal femur using osmium staining and subsequent µCT analysis (vivaCT40, Scanco Medical, Brüttisellen, Switzerland). Femurs from mice and rats were fixed with 10% PBS-buffered formalin for 24 h and decalcified in Osteosoft (Merck, Darmstadt, Germany). After decalcification, bones were scanned using the vivaCT40 using a standard protocol to ensure complete demineralization. Afterward, bones were incubated with 2% osmium tetroxide (Sigma-Aldrich, Mannheim, Germany) dissolved in 0.1 M sodium cacodylate buffer (Sigma-Aldrich, Mannheim, Germany) for 1 h at room temperature. Specimens were washed and immediately scanned using the µCT with an X-ray energy of 55 keV, 300 ms integration time, and an isotropic voxel size of 10.5 µm. The fat volume (FV) in the rats was calculated from 700 slices using manual contouring and the Scanco bone evaluation software. The threshold was set to 448 mg HA/cm³. In mice, 400 slices were used with a threshold of 180 mg HA/cm³.

Left hind paws and tibiae were fixed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 2 days and then stored in 70% ethanol until micro-CT analysis. The PFA-fixed hind paw and tibia were separately placed in a microcentrifuge tube with 70% ethanol and scanned with viva CT40 (Scanco Medical AG, Bassersdorf, Switzerland) with an X-ray energy of 55 kVp (145 µA), a voxel resolution of 15 µm, and an integration time of 200 ms. The threshold was set to 300 (equivalent to 471.1 mg hydroxyapatite (HA)/cm³) for the hind paw, 260 (384.7 mg HA/cm³) for the cortical bone of the tibia, and 220 (298.2 mg HA/cm³) for the trabecular bone of the tibia to distinguish mineralized tissues from non-mineralized tissues. The talar bone volume was evaluated for a quantitative measurement of bone erosion [32] (link). The region of cortical bone comprised 30 slices of midshaft (1 mm proximal to the tibio-fibular junction); the region of trabecular bone comprised 70 slices of secondary spongiosa beginning just beneath primary spongiosa. Bone properties were analyzed using Scanco bone evaluation software. All micro-CT analysis procedures were performed according to the international guideline [33] (link). After the analysis, bone samples were stored in 70% ethanol at 4°C until histological analysis.