Evaluating Cell Orientation in Bilayer Scaffold

Cell orientation was assessed at 1, 7, and 14 days using SEM and confocal microscopy. For SEM (Hitachi S3000N VPSEM, Berkshire, UK), samples (n = 2) were washed in PBS (Sigma-Aldrich, UK) and fixed in 2.5 %v/v glutaraldehyde in PBS at 4°C for 2 h. As previously described (Shamsah et al., 2019 (link)), samples were dehydrated through increasing concentrations of ethanol in distilled water (50–100 %v/v), chemically dried in hexamethyldisilazane (Sigma-Aldrich, UK), mounted on carbon-tabbed stubs, and gold-sputter coated. In order to image the cells positioned in between the bilayer scaffold, several small cuts were made on the top layer using a scalpel, which allowed the upper surface to be carefully removed using forceps to reveal the cell layer below. This allowed representative images of the cells present on the bottom layer of the bilayer scaffold to be captured. For confocal microscopy (Leica SP8, Leica Biosystem UK Ltd, UK), samples with live AF cells were washed with PBS, stained in the dark with calcein solution (1 μl calcein solution to 2.5 ml PBS) (Fluorescence-based Molecular Probes, ThermoFisher, Waltham, USA), and incubated at 37°C for 20 min. Samples were subsequently washed with PBS (Sigma-Aldrich, UK) and placed on glass coverslips for imaging.