Quanta 200 environmental

For biofilm visualization, hydroxyapatite (HA) discs (5 mm diameter, 2 mm thickness, Clarkson chromatography products inc., PA. USA) were insert into a 48-well plate. The biofilm formation was conducted as described above. After 24 h, the HA discs were taken and washed twice using DDW. All samples were fixed using 4% formaldehyde for 20 min and washed using DDW. The samples were gold-coated and visualized using an analytical Quanta 200 Environmental High-Resolution Scanning Electron Microscope (EHRSEM) (FEI, Eindhoven, The Netherlands) [24 (link)].

To examine the microstructure of as-printed 316L stainless steel, EBSD tests were performed in an FEI Quanta 200 environmental scanning electron microscope equipped with a Hikary backscatter electron detector from EDAX at an accelerating voltage of 30 KeV. A step size of 0.1–2 μm was used depending on the magnification selected. EBSD samples were hand polished with abrasive papers of grits up to 5 µm, followed by polishing with diamond suspensions up to 1 µm and a final vibratory polishing for several hours. EBSD data were analysed with the Orientation Imaging Microscopy Analysis™ software provided by EDAX. To calculate the grain size, each grain was considered as a sphere from which the diameter (grain size) was deducted. Interfaces between grains were considered as low-angle grain boundary when the misorientation angle θ was 2–10° and high-angle grain boundary when θ > 10°.

Tissue culture plastic manufactured from polystyrene was purchased to Corning. Highly-porous (>90%) scaffolds of cross-linked polystyrene (Alvetex^®, Reprocell), referred to as “3D substrates”, were manufactured using an emulsion template technique to control the size of the pores^{36 (link)}. Following manufacturer’s instructions, 3D substrates were immersed in 70% ethanol for 5 min and then thoroughly washed with phosphate buffered saline (PBS) before cell seeding. Polyester cell culture inserts (Corning), referred to as “2D substrates”, were used as flat permeable supports. Topographical characterization of the investigated substrates was assessed by means of scanning electron microscopy (SEM) using an FEI Quanta 200 Environmental scanning electron microscope (Fig. 1a). Pore size distribution in the substrates was analyzed in the course of our previous work^{17 (link)}. 2D substrates present a homogenous pore size distribution with a mean diameter of 0.51 µm while 3D substrates show randomly oriented and interconnected pores which diameters range between 2 to 70 µm (90% pores with diameters lower than 20 μm). The average surface roughness (R_a) of flat supports was 7.2 nm for tissue culture plastic and 4.7 nm for cell culture inserts, as determined using an optoelectronic profilometer (p-6 Stylus Profiler, KLA-Tencor Corporation).