Scanning electron microscopy was used for cell observations on samples as well as for the surface morphology of modified films. For this purpose, a Zeiss Sigma 500 VP Scanning Electron Microscope (Zeiss, Oberkochen, Germany) in the BSE (Backscattered-Electron Imaging) detector mode was used. The microscope operated at 20 kV in a vacuum below 10−5 mbar, and the magnification ranged from 100× to 2000×. Samples were covered with Au (sputter current: 40 mA, sputter time: 50 s) using a QUORUM machine and dried before measurements at the critical point in a Leica EM CPD300 dryer.
The SEM analysis was performed to visualize the cell colonies. i.e., macrophages (RAW 264.7) and fibroblasts (3T3 Swiss Albino) on the modified surfaces. Samples with macrophages and fibroblasts were fixed using a 3% glutarate (POCH, Gliwice, Poland) for 15 min at room temperature. Next, the samples were rinsed twice with phosphate buffer (Sigma-Aldrich, Saint Louis, Missouri, USA) for the purpose of fixative elimination. Lastly, dehydration in increasing concentrations of ethanol (25, 60, 95, 100%) for 5 min in each solution was carried out. After that, the samples were further prepared according to the procedure for surfaces without biological material (described above).
The SEM analysis was performed to visualize the cell colonies. i.e., macrophages (RAW 264.7) and fibroblasts (3T3 Swiss Albino) on the modified surfaces. Samples with macrophages and fibroblasts were fixed using a 3% glutarate (POCH, Gliwice, Poland) for 15 min at room temperature. Next, the samples were rinsed twice with phosphate buffer (Sigma-Aldrich, Saint Louis, Missouri, USA) for the purpose of fixative elimination. Lastly, dehydration in increasing concentrations of ethanol (25, 60, 95, 100%) for 5 min in each solution was carried out. After that, the samples were further prepared according to the procedure for surfaces without biological material (described above).