Adhesive carbon tabs

Cells cultured for 48 h in 2D culture plates, or for 14 d in 3DPS, were gently washed once in cell medium and fixated for 1 h at room temperature in 2.5% glutaraldehyde (Product ref G7651-10ML, Sigma-Aldrich Sweden AB, Stockholm, Sweden) diluted in cell medium. Scaffolds were washed once in TBS (50 mM Trizma-HCl, Sigma-Aldrich, Stockholm, Sweden; 150 mM NaCl, Merck, Stockholm, Sweden; pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), fixated for 1 h at room temperature in 1% osmium tetroxide (Sigma-Aldrich, Stockholm, Sweden) in TBS (50 mM Trizma-HCl (Sigma-Aldrich, Stockholm, Sweden); 150 mM NaCl, (Merck, Stockholm, Sweden); pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), rinsed in deionized water, plunge-frozen in liquid propane (EMS-002 Rapid Immersion Freezer, Electron Microscopy Sciences) and freeze-dried overnight (VirTis Sentry 2.0 Benchtop Freeze Dryer, SP Scientific, Warminster, PA, USA). Samples were mounted on aluminum stubs by adhesive carbon tabs (Agar Scientific) and coated with a 10 nm Au/Pd conducting thin film by sputter-coater (PECS Mod 682, Gatan Inc., Pleasanton, CA, USA). Samples were imaged by Zeiss SUPRA 40VP scanning electron microscope operated in secondary electron mode at 3.0–4.1 kV acceleration voltage, 9–17 mm working distance and 100–250,000× magnification range.

Tissue samples were fixed with 2.5% vol/vol buffered glutaraldehyde (Sigma-Aldrich), for 2 h at room temperature. Fixed tissue was washed in DPBS (three times: 5–10 min each) and dehydrated with a series of ethanol solutions, starting with 50% vol/vol and progressing through 70%, 80%, 95% and 100% absolute ethanol. Samples were dried in adhesive carbon tabs (12 mm, Agar Scientific), sputter-coated with a 15 nm Au/Pd film and visualized in a Hitachi TM3030Plus tabletop scanning electron microscope operated in BSE mode at an accelerating voltage of 15 keV. The alignment of ECM fibers was measured with the FibrilTool Fiji plugin. In brief, the mean fiber orientation direction and anisotropy index (0—random alignment/isotropic array, 1—perfect alignment/purely anisotropic array) were computed for each ROI and across several photomicrographs. Density histograms were generated in R (v.4.0.3) using the ggplot2 package (v.3.3.3) with a bin of 15°.

Samples were fixed with 1.5% glutaraldehyde solution at 4°C for 30 min, followed by dehydrating through ascending grades of ethanol (from 50% to 100%). Dehydrated samples were further dried by evaporation of the HMDS. Next, samples were mounted onto aluminum pin stubs (Agar Scientific) with Adhesive Carbon Tabs (Agar Scientific). Samples were sputter‐coated with Au/Pd before imaging with Phenom Pro desktop SEM (Phenom‐World) at approximately 500× magnification.