Protocol for Micro Computed Tomography Quantification of Neo-osteogenesis in High Density Additively Manufactured Calcium Phosphate Scaffolds.

This protocol was developed to assess in vivo osteogenesis of 3D-printed, selectively polymerized β-tricalcium phosphate (SP-βTCP) scaffolds placed between the periosteum and native bone of sheep scapulae. The protocol spans the entire development process of scaffold design, infusion, implantation and explant analysis. 3D printed SP-βTCP scaffolds of variable pore size were infused with combinations of gelatin methacryloyl and autologous or allogeneic adipose-derived stem cells (ADSCs), and placed within plasma-treated polyetherketone bioreactor chambers manufactured by laser sintering. These were implanted for 12 weeks on the left sheep scapula and 16 weeks on the right, followed by explantation and scanning using micro computed tomography (μCT). Images were analyzed using Imalytics Preclinical software via six key steps: (i) experimental chamber selection, (ii) chamber isolation, (iii) scaffold thresholding, (iv) bone thresholding, (v) volume generation and (vi) verification. This protocol delineated the border between bone and scaffold material, allowing for reliable quantification of bone formation. ADSCs and scaffolds with a smaller pore size were associated with superior bone formation, regardless of cell origin. These results demonstrate the utility of this protocol in analyzing μCT images in situations where high-density biomaterials degrade at variable rates.