Skyscan 1276

The microstructure of mouse tibiae was monitored by weekly in vivo μCT scanning using SkyScan^® 1276 (Bruker, Kontich, Belgium). Baseline scanning prior to intratibial injection (termed Week 0) was performed on mice in the 4.5 N loading and treadmill running experiments, while the first scanning for the 8 N loading group was on Week 1. For each scan, mice were anesthetized with 3% (v/v) isoflurane and held in the built-in holder, which was rotated 180-degree with a step of 0.8 degrees. One frame was taken per step with the following settings: 900 ms exposure time, X-ray of 200 mA current and 50kVp, and a 0.5mm Al filter. The in vivo scanning lasted only 4-5 min per animal and the calculated radiation exposure (<600 mGy) was low, which did not affect bone property and tumor growth [29 (link)]. The 3D reconstruction of the murine hind limbs was obtained using the NRecon^® software (Bruker) with a voxel size of 7 μm. The left and right tibiae were separated into individual volumes using Python codes (available upon request). The first scan of each tibia was aligned, where its long axis was positioned vertically and the anterior-posterior and medial-lateral axes were arranged orthogonally. This baseline scan served as the image registration reference for subsequent weekly scans using the SimpleITK package in Python [30 (link)]. Our volume of interest (VOI) consisted of 300 slices (2.1 mm) of bone metaphysis below the growth plate at the proximal tibia (Fig. 1E), which was generated using 3D Slicer^® [31 (link)]. This VOI was chosen because it contained nearly all bone perforation found during the study period. Cortical and trabecular bones were segmented using CT Analyzer 1.17^© (Bruker). A global threshold value (73/256) was applied and the processed images were found to agree well with the gray-scaled images. Cortical polar moment inertia (Ct.pMOI), bone mineral density (Ct.BMD), and tissue mineral density (Ct.TMD) as well as trabecular bone volume fraction (Tb.BV/TV), thickness (Tb.Th), separation (Tb.Sp), bone mineral density (Tb.BMD), and tissue mineral density (Tb.TMD) were reported using CTan^® 3D analysis software. By overlaying the subsequent scans with the first available scan (Week 0 or Week 1) of the same tibia, which was assumed to be intact, we could identify the osteolytic lesions (Fig. 1E), where bone tissue in the initial scan was replaced with non-bone tissues (low x-ray attenuation), and quantify the volume of the osteolytic lesions as Ct. VoL. The tibia was also classified into one of two classes (“intact” vs. “perforated”) based on the following criteria: (1) there was one or more “penetrating holes” (i.e., osteolytic lesion through the full thickness of the cortex), and (2) the linear dimension of the lesion was greater than 35 μm/5 pixels (i.e., the size of nutrient foramen, Fig. 1E). Using the cortex perforation as a general read-out of bone integrity, the Kaplan-Meier survival curve was calculated for the probability of tumor-injected tibiae free of perforation with or without exposure of exercise using the free software R (ggplot2-package, version 3.5.2, R Foundation) [32 ].

After clearing had taken place, the samples were scanned in a SkyScan 1276 micro CT (Bruker Corporation, Billerica, MA, USA) at 40 kV voltage, 200 μA current and 10 μm resolution to calculate vascular volume. Three samples were scanned at a time, taking approximately 26 minutes per scan with frame averaging set at three in order to reduce noise. Three-dimensional images of the samples were reconstructed using Hierarchical InstaRecon software (NRecon, 1.7.4.2., InstaRecon, 2.0.4.0. InstaRecon). This software was used to adjust the following parameters while reconstructing the images; Beam Hardening Correction (%) was set at 51, Ring Artifact Correction at 9, Smoothing at 1, Post alignment compensation and Histogram windows were manually adjusted for each scan. After obtaining reconstruction of the images, AnalyzePro software (AnalyzeDirect, Inc., Overland Park, KS, USA) was used to measure the volume of the vasculature and the volume of the total nerve. A vessel/nerve area ratio was calculated and expressed in percentages (vessel%).