Ct analyser 1

The porosity of the biomaterials was studied by micro-computed tomography—micro-CT (Skyscan 1174, Bruker microCT, Kontich, Belgium) with 12 µm voxel resolution. Reconstruction of the 360 cross-section images representing the middle of the samples was conducted using the NRecon 1.7.1.6 software (Bruker microCT, Kontich, Belgium), whereas porosity (closed, open, total) and pore diameter assessment was performed using the CTAnalyser 1.17.7.2 software (Bruker microCT, Kontich, Belgium) which determines porosity in the whole selected 3D volume of interest (VOI).

Micro-CT scans of the maxillas of mice in each group were performed (SCANCO Medical AG, Switzerland) with a voxel resolution of 10 μm. Sagittal images of the middle tooth were selected, and the distance between the alveolar crest and enamel junction was measured to quantify the loss of the distal aspect of the first molar and the mesial aspect of the second molar. Bone volume fraction (BV/TV) analysis per tissue volume in the region of interest was performed using CT-Analyser 1.13 software (Bruker). The area of interest was the rectangular area of approximately 5.2 mm² of the alveolar bone at the mesial and distal root bifurcation of the maxillar first molar^{57 (link)}.

Spine sections of the 3 experimental groups (naïve, saline, and BoNT/A), containing the damaged area, were soaked in a potassium iodine contrast agent, 0.1N (v/v) Lugol solution (Sigma), at room temperature, in the dark, for 3 months, replacing the solution several times during the exposure. The acquisition of tomographic datasets was performed through a high-resolution 3D Micro-CT Imaging System (Skyscan 1172G Bruker, Kontich, Belgium), at 12 µm pixel/size (2 × 2) and the reconstructions, using built-in NRecon Skyscan Software (Version 1.6.6.0, Bruker).
Tridimensional volumes were analyzed by using 3D Visualization Software CTvox v. 2.5 (Bruker) for creating images and movies in grayscale and reversed B/W. Size measurements were calculated as the average of 5 manual evaluations, performed using Bruker CT-Analyser 1.13 Software, at the level of the same transverse slide for each sample, selected from the samples with the bone fracture. The entire transverse dataset was aligned based on the orientation of the fractured vertebra, and therefore the same vertebra for each specimen, using the Atlas of the Mouse Brain and Spinal Cord (Sidman–Angevine–Pierce, 1971) for guidance to accurately identify the correct orientation of the virtual sections.

Maxillae were dissected from WT and Gnat3^−/− mice. The maxillae samples were soaked in a 10% sodium hypochlorite solution for 30 minutes. The soft tissues were removed, and the maxillae were stained in a 2% methylene blue solution for 1 minute. After repeated cleaning with deionized water, images were collected under a stereomicroscope (Olympus, SZX10). Additionally, fixed maxillae were randomly selected and scanned by microCT (μCT 50, Scanco). The X-ray beam was set at 70 kVp and 200 μA. All samples were scanned in the sagittal position at a voxel resolution of 8 μm. The alveolar bone loss (mm) was measured from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) of the distal area of M1 and the mesial area of M2 by Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD). After 3D reconstruction, the region of interest (ROI) was defined as a rectangular region from the CEJ to the alveolar bone base between M1 and M2. Histomorphometry of trabecular bone at the ROI was assessed by CT-Analyser 1.13 software (Bruker), including bone volume per tissue volume (BV/TV) and trabecular number (Tb.N).