Cells morphology and spreading, as well as cells/scaffold interaction, were observed after 1 day (d), 7 day and 14 day of culture by a dual approach: fluorescent labelling (fluorescein isothiocyanate –FITC‐ conjugate phalloidin, Sigma–Aldrich, Steinheim, Germany) and scanning electron microscopy (SEM, Zeiss EVO HD15 Scanning Electron Microscope, Carl Zeiss S.p.A, Italia).
Briefly, for fluorescent labelling the cultures were washed with PBS, fixed with a 4% paraformaldehyde solution in PBS for 15 min at 37°C, permeabilized in 0.5% Triton X‐100 for 15 min, again washed in PBS and labelled with a FITC‐conjugate phalloidin solution 1:100 in PBS for 30 min at 37°C. The cell cytoskeleton, to which phalloidin bounds, was visualized using an inverted microscope equipped with an epifluorescence setup (Eclipse TiU, NIKON Europe BV, NITAL SpA, Milano, Italy): by the excitation/emission setting of 488/530 nm the green fluorescence was appreciated, and the cells attached to the scaffolds, as well that in CTR condition, were easily visualized.
For SEM analysis, the samples were fixed in 2.5% glutaraldehyde, in pH 7.4 phosphate buffer 0.1 M for 1 hr at room temperature and dehydrated in a graded ethanol series. Before SEM observation, samples were air dried after hexamethyldisilazane‐based treatment.
Briefly, for fluorescent labelling the cultures were washed with PBS, fixed with a 4% paraformaldehyde solution in PBS for 15 min at 37°C, permeabilized in 0.5% Triton X‐100 for 15 min, again washed in PBS and labelled with a FITC‐conjugate phalloidin solution 1:100 in PBS for 30 min at 37°C. The cell cytoskeleton, to which phalloidin bounds, was visualized using an inverted microscope equipped with an epifluorescence setup (Eclipse TiU, NIKON Europe BV, NITAL SpA, Milano, Italy): by the excitation/emission setting of 488/530 nm the green fluorescence was appreciated, and the cells attached to the scaffolds, as well that in CTR condition, were easily visualized.
For SEM analysis, the samples were fixed in 2.5% glutaraldehyde, in pH 7.4 phosphate buffer 0.1 M for 1 hr at room temperature and dehydrated in a graded ethanol series. Before SEM observation, samples were air dried after hexamethyldisilazane‐based treatment.