Phenom scanning electron microscope

The fibrous PLA-based scaffolds and implants were analyzed with the Phenom scanning electron microscope (SEM) supported with image software (FEI, USA). All SEM samples were incubated in 0.5 % OsO₄ for 1 h at 4 °C, then dehydrated/desiccated with anhydrous ethanol, next automatically critical point dried (Leica EM CPD300, Germany), and finally coated with 15-nm layer of gold (K550 Emitech, USA) prior to SEM analysis.
Live/Dead Cell Staining Kit (Sigma Aldrich, USA) was utilized for simultaneous fluorescence staining and visualization (Nikon Eclipse Ti microscope) of viable and dead cells.
The implants were also analyzed with the Zeiss Axiovert 100 M confocal laser scanning microscope (CLSM) supported with LSM 510 META software (Carl Zeiss Jena GmbH, Germany). CP5 cells were stained (500 ng mL⁻¹) with phalloidin—tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; Sigma, USA) to identify filamentous actin and with DRAQ5™ (Biostatus, UK) intercalating anthraquinone to visualize chromatin. Each imaging analysis was performed at least three times using material collected during three independent experiments.

The SEM analysis was carried out on the optimized microparticles and the MGDDS using a Phenom™ scanning electron microscope (FEI Company, Hillsboro, OR, USA). Prior to the analysis, the samples were gold-sputter coated (SPI Module™ Sputter Coater, SPI Supplies, West Chester, PA, USA) for 90 s (18 mA).