Microstructural Analysis of Bone Tissue

To assess the bone mass and microarchitecture among the five groups,
microcomputed tomography (micro-CT) was performed using the Inveon MM system
(Siemens, Munich, Germany) as previously described.^{21 (link)} In brief, the specimens were scanned at an effective pixel size of
8.89 μm, a voltage of 60 kV, a current of 220 μA, and an exposure time of 1500
ms in each of the 360 rotational steps, in vivo and ex
vivo. The images consisted of 1536 slices and had a voxel size of
8.89 μm in all three axes. Three-dimensional (3D) visualization images were
reconstructed by two-dimensional images and the parameters were calculated using
Inveon Research Workplace (Siemens) as follows: BMD, bone mineral content (BMC),
cortical bone area/total bone area (%Ct.Ar), bone volume/total volume (BV/TV),
trabecular number (Tb.N), bone surface area/bone volume (BS/BV), trabecular
separation (Tb.Sp), and trabecular thickness (Tb.Th) in the region of interest
of the femur (1–2 mm below the distal growth plate; Figure S1), L4 vertebra (trabecular bone of the centrum),
mandible (septa interradicularia mandibulaeta and mandibular angle), and growth
plates, as described previously^{22 (link)–24 (link, link)} according to guidelines
set by the American Society for Bone and Mineral Research.^{25 (link)}