E5100

SEM images were obtained by means of Inspect Microscope (FEI, Hillsboro, OR, USA) working with W (tungsten) filament. Regarding sample preparation, when necessary, PZT-5%Fe₃O₄ samples were sputtered with a thin layer (20–50 nm) of Au under medium vacuum (10⁻¹ Torr) by means of a typical sputtering unit (E5100, Quorum Technologies Ltd, Eats Sussex, UK). Then the sample was placed onto conventional pin stubs. During SEM we employed typical values for the interfering parameters (i.e. acceleration voltage within 15–30 kV, working distance within 8–15 mm and spot size 3–8). Information on the topography for the evaluation of the microstructure was obtained with secondary electron imaging (SEI), while elemental analysis to obtain both energy-dispersive x-ray spectroscopy (EDS) information and compositional mapping images was recorded with backscattered electron imaging (BSE). Au-coated samples were used for SEI, while both Au-coated and non-coated samples were used for BSE to cross-check the results due to the overlapping of the M-spectral line of Au (2.123 keV) with the Lα-spectral line of Zr (2.044 keV) and the M-spectral line of Pb (2.342 keV).

The nanofibers and cell morphology were examined by SEM. Scaffold samples containing cells were fixed with 4% PFA for 15 min, washed once with PBS, and dried by immersion in graded concentrations of ethanol solutions in water (25, 50, 75, and 100% vol/vol). The samples were kept in an aseptic environment until complete drying. Prior to SEM visualization, the samples were coated with a 45-nm gold/palladium layer by a sputter coater (model E5100, ex-Polaron; Quorum Technologies, ON, Canada) and observed under a conventional SEM (model S2400; Hitachi, Japan) with an electron beam with 20 kV of accelerating voltage. SEM images were analyzed with ImageJ (National Institutes of Health, United States) to estimate both orientation and fiber diameter profiles. At least 50 samples were individually measured for each condition.

The casted brain, left hepatic lobe, spleen and kidney specimens were individually placed into a macerating solution of 5–7.5% potassium hydroxide solution (Sigma-Aldrich Company Ltd, Dorset, UK). The macerating solution was renewed daily until all of the tissue that surrounded the casted vasculature had been removed. Each resin-casted specimen was carefully washed using dd.H₂O and placed into a dust-protected container, lined with fine filter paper. Each specimen air-dried for 72 h [24 ] prior to being secured onto an aluminium stub and homogeneously coated with a thin layer of palladium and gold (E5100; Quorum Technologies Ltd, East Sussex, UK) for SEM examination.