μct50 system

Femora (one per mouse) were examined as reported previously30 (link), using the μCT50 system (Scanco Medical AG, Switzerland). Briefly, scans were performed at a 10-μm resolution. The mineralized tissues were segmented by a global thresholding procedure. Trabecular bone parameters were measured in the secondary spongiosa of the distal femoral metaphysis and cortical parameters were determined in the mid-diaphyseal region according to the standardized nomenclature.

At autopsy, the left tibiae were harvested and fixed in 10% formalin (Thermo Fisher Scientific) for 48 hours at 4°C. A high-resolution microcomputed tomography (μCT) 50 system (Scanco Medical) was used to analyze the mouse tibiae bone volume and microarchitecture. Tomographic images were acquired of hindlimbs in 70% ethanol (70 kVp, 12 μm voxel size, 300 ms integration time). μCT images were reconstructed, filtered (σ = 0.2, support = 1.0), and thresholded at 230. Tibiae were contoured starting 10 slices below the growth plate and continued 100 slices in the distal direction using the Scanco software algorithm. Images of individual tibiae were analyzed using the Scanco Medical Imaging software to determine the morphometric parameters.

Femora (one per mouse) were examined using the μCT50 system (Scanco Medical AG, Switzerland) (27 (link), 28 (link)). Briefly, scans were performed at a 10 μm resolution. The mineralized tissues were segmented by a global thresholding procedure (29 (link)). Trabecular bone parameters were measured in the secondary spongiosa of the distal femoral metaphysis, which was further divided into proximal and distal halves. Cortical parameters were determined in a 1 mm height ring in the mid-diaphyseal region. Volumetric bone mineral density (vBMD) was calculated by the proprietary Scanco software with reference to a calibrated phantom, and expressed as mg hydroxyapatite per cm³ of tissue (mgHA/cm³).

μCT analysis was performed using μCT50 system (Scanco Medical AG, Brüttisellen, Switzerland) as described in (Raygorodskaya et al., 2016 (link)). In the entire intact femur we measured volumetric bone mineral density (vBMD) and axial length using the calibrated μCT software (Scanco, uct_evaluation v6.5-3). In the femoral cortical bone, we used a 1-mm-height diaphyseal segment extending distally from the mid-shaft. The femoral trabecular traits were examined in the distal half (1.5 mm height) of the distal metaphysis (dVOI). The following cortical and trabecular parameters were measured in femora (Bouxsein et al., 2010 (link); Manolagas & Kronenberg, 2014 (link)): diaphyseal (Dia.Dia) and medullary cavity (Med.Dia) diameters, cortical thickness (Ct.Th), tissue mineral density (TMD), trabecular bone volume fraction (bone volume/tissue volume ratio, BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). In the 3rd lumbar vertebrae we measured the following trabecular parameters: BV/TV, Tb.N, Tb.Th and Tb.Sp.

Femurs (one per mouse) were examined using the μCT50 system (Scanco Medical AG, Bruttisellen, Switzerland) [73 (link),74 (link)]. Briefly, scans were performed with a 10 μm resolution, 90 kV energy, 114 mA intensity, and 1100 ms integration time. The mineralized tissues were segmented by a global thresholding procedure following the Gaussian filtration of the stacked tomographic images [75 (link)]. Trabecular bone parameters were measured in the secondary spongiosa of the distal femoral metaphysis.

The skeletons were scanned and analyzed using a μCT 50 system (Scanco, Brüttisellen, Switzerland) with a spatial resolution of 10 μm. For trabecular bone morphometric analysis of distal femur, the starting slice was taken where the growth plates were fully merged and extended for 100 slices proximally. The morphometric analysis was performed for bone mineral density (BMD), bone volume per tissue volume (BV/TV; %), and cortical bone thickness by μCT 50 system. The 3D reconstruction of the skeletons and cross-section images were performed by Dataviewer and CTvox (Skyscan, Belgium).