Aniline blue solution

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.

We cultured c643 overexpressing OPN-SV and TPC-1 cell lines in 24-well plates (1.5 × 10⁵ cells/well) for 31 days. Following 10, 17, 24, or 31 days in culture, cells were fixed with 4% paraformaldehyde (PFA) in PBS and evaluated for collagen fibers formation by Masson’s trichrome staining. Briefly, cells were immersed in Richard-Allan Scientific^® Bouin’s fluid (Thermo Fisher Scientific, Waltham, MA, USA) solution for 5 min at room temperature, washed in deionized water, followed by incubation in Celestin Blue (Thermo Scientific) for 6 min. Then, cells were incubated in Gil’s hematoxylin staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Cells were washed with 1% acid alcohol and then underwent three sequential washes in deionized water. Cells were then immersed in Biebrich scarlet-acid fuchsin (Thermo Scientific) for 5 min, placed in phosphotungstic and phosphomolybdic acid solution (Thermo Scientific) for 5 min, moved to Aniline Blue solution (Thermo Scientific) for 5 min, and then placed in 1% acetic acid solution for 2 min. Finally, cells were rinsed in deionized water. Representative images for each culture condition were captured with a NIKON microscope and NIKON Digital DLS Camera.