Skyscan 1076

Each calvaria was scanned using a micro-CT scanner (Skyscan 1076, Bruker, Belgium). The resolution was set at 18 µm with the rotational step of 0.40, beam hardening was reduced by the use of 0.025 mm titanium filter, while the frame averaging was set at 2. The obtained images were reconstructed using the NRecon (Bruker, Kontich, Belgium) software and analyzed using the CTAn (Bruker, Belgium) software. For analysis, a 5 mm diameter circular region was delineated along the margins of the initial defect area. To delineate newly formed bone from grafting material, we set specific thresholds for grafting material, while it was kept constant for bone tissue. The threshold for Cerabone^®, Cerabone^® + Al. bone, and Cerabone^® with magnesium was 200–255; for OsteoBiol^® 70–200; and for newly formed bone, it was 50–255 (Figure 5). Due to the difference in material density, threshold range delineated different graft material. After this was done, subtraction was performed to separate the values for newly formed bone versus biomaterial. The calculated parameters included bone volume fraction (BV/TV, %) and trabecular bone parameters such as trabecular thickness (Tb.Th, mm), trabecular number (Tb. N, 1/mm), trabecular separation (Tb.Sp, mm), total porosity (Po (tot), %), and connectivity density (Conn.D, 1/mm³) along with the percentage of residual biomaterial (RB, %).

The skulls of the sacrificed rats were fixed in 4% PFA. We reconstructed the skulls using micro-CT (Skyscan 1076, Bruker micro-CT, Kontich, Belgium) at 70 KV, 114 A, and an isotropic pixel size of 18 μm.

After thawing, each tissue block from L2 to L5 vertebrae was scanned with a voxel spacing of 35 μm (Skyscan 1076; Bruker Micro CT, Belgium). Although computer software is available for subtracting the metallic implant from bone, the scatter caused by the metal screws was too strong to permit the contrast needed to determine the bone-implant contact at the interface with the use of micro-CT. However, the images were useful for a general assessment of osteointegration. Therefore, after scanning, the area of the bone-screw interface of the specimen was quantified by densitometric analysis using videomicroscopy and ImageJ software (National Institutes of Health); the signals were normalized relative to the control group. On the other hand, the CT images were used to measure the bone-volume percentage, which is the percentage of mineralized bone in the total tissue volume [20 (link)]. In addition, the percentage of pedicle screws inside bone tissue was also evaluated by densitometric analysis using CT images.

Radiographic images to detect mineralized tissue in in vivo implanted scaffolds were acquired using a SkyScan 1076 in vivo μCT (Bruker microCT, Kontich, Belgium). Upon sample retrieval and fixation, constructs were scanned using a SkyScan 1172 high‐resolution μCT at a pixel size of 5 μm with a tube voltage of 60 kV and a 0.5 mm aluminum filter. Reconstruction was performed with NRecon v1.6.8 software and mineralized tissue was quantified with CTAn v1.13 software (both from Bruker microCT) using a threshold value of 75 or using custom‐made MeVisLab software. The latter software enabled us to distinguish newly formed bone from scaffold granules based on grayscale values (MeVis Medical Solutions AG, Bremen, Germany).24

After scaffold production (T0) and after explanation (T1), mCT scans were conducted in order to monitor changes in the scaffold structure over the implantation period. T0 and T1 scans were executed with a SkyScan 1076 (Bruker micro-CT, Kontich, Belgium) using the following specifications: tube current 200 µA, exposition time 450 ms, voltage 50 kV, averaging 7, rotation step 0.8°, pixel size 9 µm, 0.5 mm aluminum filter. The resulting duration was 51 min per scan; four scaffolds were scanned in one batch. During scanning, the scaffolds were stored in PBS. The resulting datasets were reconstructed using NRecon (Version 1.6.9.8, Bruker micro-CT, Kontich, Belgium) and were analyzed by Heidelberg-mCT-Analyzer in order to monitor changes in the TIV over the implantation period as a marker for resorption of the scaffold following previously published protocols [62 (link),63 (link)]. For representative 3D visualization, reconstructed datasets were opened in CTVox (Version 3.1, Bruker micro-CT, Kontich, Belgium) and volume rendered after grey-value alignment using the onboard algorithms of the program.

To test the new bone formation, the collected bone blocks were scanned in a micro-CT scanner (SkyScan 1076, Bruker, Kontich, Belgium). The machine was set with the following parameters: images were acquired at 49 kV, 200 μA, through a 0.5 mm thick aluminum filter with a pixel size of 18.27 μm. The reconstructed images were imported into the analysis software (CTAn, Bruker) for calculating bone volume. According to previous studies [18 (link)–20 (link)], the volume of interest (VOI) was defined as the relative changes in bone volume density (BV/TV%), the percentage of bone volume (BV) to the total tissue volume (TV). The new bone growth was evaluated using calculated VOI. In addition, the numbers of HA-βTCP particles found in the defect were counted using the micro-CT images.