Ultrastructural Analysis of ECM Scaffolds

ECM were fixed with 4% paraformaldehyde in phosphate buffer (PB) for 5 h, postfixed in 1% osmium tetroxide in PB for 1 h, dehydrated in ascending degree of ethanol (70–80–95–100%), dried for 30 min in xylene and embedded with paraffin after evaporation of xylene and serially cut as 10 μm thick sections using a Leica microtome. Sections were first deparaffinized in xylene for 2 h and then hydrated in descending degree of ethanol (100–95–80–70%) and H₂O. Ultrathin sections were collected on glass coverslips, mounted on aluminum stubs and sputter-coated 3 min with gold. Morphology and ultrastructure were investigated by SEM on a TM3000 Benchtop SEM (Hitachi, Pleasanton, CA, USA) instrument operating at 15 kV acceleration voltage [30 (link)]. Areas of matrix or empty spaces were calculated with a tool of the ImageJ software (version 2.0, National Institute of Health, Bethesda, MD, USA) and reported as percentage area on the whole image analyzed. Results are also reported as perimeter/area ratio of empty spaces. Repopulated GelMA scaffolds were analyzed by SEM as above, after fixation without embedding.