Nrecon program

The extracted skulls were studied using a μCT machine (SKYSCAN1173, Ver. 1.6, BRUKER-CT, KONTICH, Belgium) for imaging. The pre-set imaging conditions were 60 μA tube current, 130 kVp tube voltage, 500 ms exposure time, 1 mm aluminum filter, and 0.3° rotation angle. The pixel size was 13.85 μm, and the number of pixels was 2240 × 2240. With the help of a μCT scan, a total of 800 raw high-resolution images were obtained. The NRecon program (Ver 1.7.0.5, BRUKER-CT, KONTICH, Belgium) was used for the cross-sectional reconfiguration. The Dataviewer program (Ver. 1.5.1.3, BRUKER-CT, KONTICH, Belgium) and the Ct-VOX program (Ver. 1.14.4.2, BRUKER-CT, KONTICH, Belgium) were used for 3D reconstruction. The volume of newly formed bone inside the scaffolds was calculated using the difference in grayscale level. In the program, since the HU (Housefield unit) of the scaffold was 400, the HU of the cortical bone was 600 or more, and that of the soft tissue was 100 or less, the grayscale threshold of the new bone was set to 150–350 HU. Newly formed bone volume in the scaffolds was calculated using these programs, according to the following calculation: $$\text{Percent}\text{bone}\text{volume}\left(\%\right)=\text{New}\text{bone}\text{volume}/\text{Total}\text{volume}\text{in}\text{scaffold}\times 100$$

After Microfil perfusion, animals were scanned using a Skyscan 1276 micro-CT (Bruker), and images were acquired with a pixel size of 20 μm at 2,016 × 1,344 resolution. CT scans were reconstructed using the NRecon program (Bruker) to adjust for beam hardening and ring artifacts. Image sets were saved as DICOM or BMP files (~1,200–1,500 images/animal). Reconstruction in 3D was performed using the 3D Slicer program. All bone and vasculatures not of interest were removed using the scissors tool within the program to display the aorta and its major branches. To visualize SMAs, all the other vasculatures were removed using the scissors tool.