Micro-CT Analysis of Bone Mineralization

After explantation, constructs were fixed in 4% paraformaldehyde overnight and stored in PBS until micro-computed tomography (μ-CT) analysis. Data were acquired by using a phoenix nanotom m scanner (General Electric, Fairfield, CT) with 0.5-mm aluminum filtered x-rays (applied voltage 70 kV; current 260 μA). Transmission images were acquired during a 360° scan rotation with an incremental rotation step size of 0.25°. Reconstruction was made using a modified Feldkamp algorithm at an isotropic voxel size of 2.5 μm. Threshold-based segmentation and 3D measurement analyses (bone mineral density and volume) were performed using ImageJ software (Schneider et al., 2012 (link)) with the BoneJ (Doube et al., 2010 (link)) and 3D Shape (Sheets et al., 2013 (link)) extensions. The threshold employed for the segmentation was set at 325 mg/cm³, as previously determined to clearly distinguish between ceramic and newly formed mineralized tissue (Scheufler et al., 2008 (link)). Three-dimensional rendering of the structures was performed using VGStudio MAX 2.2 software (Volume Graphics, Heidelberg, Germany).