Nrecon software v 1

Scans were performed on a Skyscan 1172 instrument (Bruker, Kontich, Belgium) with a 0.5 mm aluminium filter at a voltage of 50kV, a current of 200μA and a resolution of 5 μm/pixel. 2D images were reconstructed into 3D models using NRecon software v.1.6.10.4 (Bruker) and loaded into CTAn software v.1.16.4.1 (Bruker) for analysis. Quantitative data was recorded for bone and vessel regeneration in rectangular regions of interest (ROI) in the Cortex opposite the defect, measuring 1.5 mm long, 0.9 mm wide and 0.6 mm in depth and in the Defect/Medulla measuring 1.5 mm long, 1.0 mm wide and 1.3 mm in depth. Quantitative data for mineralized tissue includes bone volume/tissue volume (BV/TV %), trabecular thickness (Tb.Th. mm x10-3), trabecular separation (Tb.Sp. mm x10-3), open porosity (%) and open pore volume (Po.V Tot mm3). Quantitative data for vascular channels (Bruker micro CT academy 2016 v5.3) includes vascular channel volume/tissue volume (ChV/TV %), vascular channel number (Ch.N), vascular channel thickness (Ch.Th mm x10-3), and vascular channel connectivity density (Ch.Conn.Dn.).

Rat femora with inserted scaffolds were carefully dissected free of soft tissue, fixed for 24 h in 4% paraformaldehyde at 4°C and rinsed thoroughly with sterile PBS prior to micro-computed tomography (μCT) analysis. Scans were performed at 9 μm/pixel resolution on a Skyscan 1172 instrument (Bruker, Kontich, Belgium) using a 1.0 mm aluminum filter at a voltage of 59 kV, a current of 167 μA. The scan projections were reconstructed into 3D models using NRecon software v.1.6.10.4 (Bruker) and loaded into CTAn software v.1.16.4.1 (Bruker) for analysis. A region of interest (ROI) measuring 4 mm long × 2 mm wide × 2 mm deep was created in the middle of the bone window defect and encompassing the defect, scaffold and medullary cavity of the femur. Quantitative data for mineralized tissue includes bone quantity (BV/TV), number (Tb.N), thickness (Tb.Th), and separation of trabeculae (Tb.Sp), connective density (Conn.Dn), total porosity (Po.Tot), and number of closed pores (Po.N.cl, corresponding to osteocyte lacunae), as described (Ramirez-Garcia-Luna et al., 2019 (link)).