Analyzing Pore Structure of Mineralized Scaffolds

The pore size of mineralized collagen and mineralized collagen–amnion scaffolds was analyzed following a JB-4 (Polysciences, Inc., Warrington, PA, USA) embedding procedure (9, 25). Briefly, scaffolds were hydrated in 100% ethanol under vacuum inside a desiccator before embedding. After soaking in JB-4, the scaffolds were placed into wells to harden, with three samples of each type placed flat into the mold and three placed on their side to create transverse and longitudinal sections. These molds were then placed at 4°C overnight to complete polymerization. JB-4-embedded scaffolds were embedded in paraffin to fit in molds for microtome sectioning, and 5-µm sections were cut using an RM2255 microtome (Leica, Wetzlar, Germany) with a tungsten carbide blade. Scaffolds were sectioned and placed onto glass slides, then stained with an aniline blue solution (Thermo Fisher Scientific, Waltham, MA, USA). Slides were then imaged with a NanoZoomer Digital Pathology System (Hamamatsu, Japan). To analyze pore structure, images were captured of each section and images were analyzed by a custom Matlab pore size code [9 (link), 39 (link)] to get an average pore size and aspect ratio for each scaffold.