Hexamethyldisilane

Bacterial colonization on MAP-PLA and commercial membranes was besides CFU semi quantitatively evaluated using scanning electron microscopy (SEM). After incubation in a humified atmosphere of 37 °C at 5% CO₂, the culture medium was removed and cells were fixed with glutaraldehyde (Sigma, St. Louis, USA) 3% in PBS at a pH value of 7.4 for 24 h. After removal of the glutaraldehyde solution, the samples were dehydrated in an ascending alcohol dilution for 300 s for each series. After that, drying with hexamethyldisilane for 1 min (Sigma-Aldrich, St. Louis, USA) and a gold vapor deposition with a thickness of 15 nm (SCD 500, CAL-Tec, Ashford, UK) were performed and SEM analysis (Jeol, Freising, Germany) was conducted at a voltage between 10–15 kV.

Osteoclast progenitors (1 × 10⁶) were seeded onto the 3D-printed BG-/Mo-scaffolds and incubated in culture medium for 24 h. For cell adhesion and morphology, each sample was fixed in 2.5% glutaraldehyde (Sigma–Aldrich) overnight at 4 °C and then gradually dehydrated in ethanol solutions with gradient concentrations (10%, 20%, 40%, 60%, 80%, 90%, and 100%). Subsequently, the samples were critical-point dried using hexamethyldisilane (Sigma–Aldrich) at room temperature before sputter-coating with Pb/Au. Cell adhesion and morphology on both scaffolds were finally observed and compared using field-emission SEM (Hitachi S-4800).To assess cell viability, a Calcein-AM/PI Double-Stain Kit (Yeasen Biotechnology, Shanghai, China) was used according to the manufacturer’s instructions. Each sample was stained with 5 mL of PBS containing 15 μL of 200 calcein-AM and 5 μL of PI for 15 min in the dark. Then, each sample was rinsed and observed with a confocal laser microscope (A1 PLUS, Nikon, Tokyo, Japan).

12 mm coverslips were coated with FN and put in a 24-well plate. 100.000 HUVECs were seeded in each well and grown to confluency in 2 days. TNFα was added 24 hours before the experiment. Cells were fixated in a 4% PFA and 2% glutaraldehyde in PBS++ solution for 1 hour. Afterwards, cells were dehydrated with increasing (50%, 60%, 70%, 80%, 90% to 96%) ethanol concentrations of 20 minutes each and submerged in hexamethyldisilane (Sigma-Aldrich) for 30 min. Next, samples were mounted on aluminum SEM stubs with Leit-C carbon cement (PLANO GmbH). A Leica EM ACE600 (Leica Microsystems) was used for sputter-coating with a 4 nm-thick platinum-palladium layer. Imaging was done on a Zeiss Sigma 300 SEM (Zeiss) at 2.00 or 3.00 kV, with magnifications ranging from 2.000x to 10.000x.