Skyscan 1172 microtomograph

Micro-computed tomography (µ-CT) analysis was performed using a SkyScan^® 1172 microtomograph (Bruker^®, Billerica, MA, USA). It was applied to examine the structure of the 3DP tablets with 20%, 35%, and 60% of infill and to verify the repeatability of printing process (the data collected for three tablets with 35% of infill were compared). The image pixel size was 6.9 µm for measurements of all samples. A cone beam reconstruction software program (Nrecon SkyScan^®, Bruker^®, Billerica, MA, USA) based on the Feldkamp algorithm was used for the reconstruction of the projections. A CT-Analyser^® (SkyScan^®, Bruker^®, Billerica, MA, USA) was used for binarization purposes. The procedure was based on density distribution histograms collected for the whole sample volume. A CT-Analyser^® was also used for the characterization of the morphological features of the tablets, their volume, and surface. CTVox^® software (Bruker^®, Billerica, MA, USA) was applied to present the 3D results.

The xenograft (n = 6) and intra‐bone models (n = 6) were established on NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ as before.²⁷, ²⁸ After indicated time, mice were treated with BTZ (i.p., 0.5 mg/kg) and MLN8237 (5 mg/kg, i.g., with β‐cyclodextrin) solo or together every 2 days. For xenograft models, mice were weighed and tumours were measured every 2 days. Micro computed tomography (CT) analysis with Skyscan 1172 microtomograph (BrukermicroCT, Kontich, Belgium), three‐dimensional (3D) models reconstruction with a surface‐rendering program (Ant, release 2.0.5, Skyscan) and 3D measurements with the CtAn software (release 2.5, Skyscan) were performed on the intra‐bone models. The corresponding parameters including trabecular separation, trabecular volume (BV/TV, in %), cortical thickness and trabecular number (Tb.N, in mm‐1) were calculated on the femur body of intra‐bone model.

MicroCT analyses were performed with a Skyscan 1172 microtomograph (Bruker MicroCT, Kontich, Belgium) operated at 70 kV, 100 μA, 340-ms integration time. The isotropic pixel size was fixed at 4 μm, the rotation step at 0.25° and exposure was done with a 0.5-mm aluminum filter. To calibrate for bone mineral density, hydroxyapatite (HA) rods (2-mm diameter, at hydroxyapatite densities of 0.25 and 0.75 gHA/cm³) and volumes of saline and air were also scanned with the same parameters. Each 3D reconstruction image dataset was binarized using global thresholding. Trabecular volume of interest (VOI) was located at the proximal tibia, 0.5 mm below the growth plate and extended 2 mm down. Using the hydroxyapatite calibration scans, the grayscale dataset was transformed into Hounsfield units (HU) by assigning an HU value of 0 to the average grayscale index of saline, and an HU value of −1,000 to black pixels (air). Next, average HU values for the two mineralized phantoms (0.25 and 0.75 gHA/cm³) were used to create a linear relationship between HU and mineral density.The threshold value to discriminate mineralized tissue from bone marrow was set at 0.4 g/cm³ and resulting binarized images were compared visually with original images. All microCT parameters were determined according to guidelines and nomenclature proposed by the American Society for Bone and Mineral Research (32 (link)).

SkyScan 1172 micro-tomograph (Bruker, Kontich, Belgium) was used to create a projection of cells and regenerated tissue growing within scaffolds. This scanner uses a tungsten X-ray source and is equipped with an 11 MP CCD camera (4000 × 2672 pixels). All specimens were scanned at a voltage of 59 kV and 167 μA. Camera was without filter; it was fully rotating 360° at the highest resolution. SkyScan’s NRecon software (NRecon, Bruker, Kontich, Belgium) was used to reconstruct sliced projected images (6 μm/slice) into cross-section images by implementation of a modified Feldkamp’s back-projection algorithm. All scans were loaded into the CTVox software (CTVox, Bruker, Kontich, Belgium) to render the proportion of the area occupied by the cells and the regenerated tissue throughout the scaffold’s architecture. Animated videos and images were created to visually represent the tissue formation inside scaffolds. Scaffolds under all described experimental conditions and at all three time points were studied with micro-CT.