Fib sem crossbeam 550 field emission microscope

Chondrocyte attachment onto CS/Gel/PVA hydrogels was visualized using scanning electron microscopy (SEM) (16538, Electron Microscopy Science, Hatfield, PA, USA). Constructs (5 × 10⁵ cells) were fixed with 2.5% v/v glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate (Sigma-Aldrich) at pH 7.2 for 24 h. Samples were washed for 5 min in 0.1 M PBS at pH 7.4 and further dehydrated for 10 min in a serially diluted ethanol solution, ranging from 30% to 99.99%, for 30 min each wash (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany). All specimens were dried with a critical point dryer CO₂ chamber (K850, Quorum Technologies, Kent, UK). Images were acquired with a FIB-SEM Crossbeam 550 field emission microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) at 5 kV.

The microarchitecture of the native samples and dECM-BD was observed by SEM. Briefly, all samples were fixed in 3% (v/v) glutaraldehyde in a buffering solution of 0.1M sodium cacodylate (pH 7.2) for 48 h, (16538, Electron Microscopy Science, Hatfield, PA, USA). Subsequently, all samples were dehydrated at various ethanol concentrations, ranging from 30% to 99.99%, 30 min each, (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany) Finally, they were dried using a critical point dryer CO₂ chamber (K850, Quorum Technologies, Kent, UK), as previously reported [18 (link),19 (link)]. The images were acquired with a FIB-SEM Crossbeam 550 field emission microscope (Zeiss Oberkochen, Germany) at 5 kV.

The microarchitecture and topology of the Gel/CS/PVA hydrogel; as well as the cell adherence and morphology in chondrogenic constructs, were observed by SEM. Briefly, all samples were fixed in 3% (v/v) glutaraldehyde in a buffering solution of 0.1 M sodium cacodylate (pH 7.2) for 48 h (16538, Electron Microscopy Science, Hatfield, PA, USA). Subsequently, the samples were dehydrated in ethanol ranging from 30% to 99.99% for 30 min each (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany). Then, they were dried using a critical point dryer CO₂ chamber (K850, Quorum Technologies, Lewes, UK), as previously reported. Finally, all dried samples were mounted in 25 mm diameter aluminum pin stubs and mounted on them using 25-mm-diameter carbon-based electrically conductive carbon double-sided adhesive discs (Agar Scientific, Stansted, UK). The samples were coated with gold in two thin layers in two cycles of 20 sec each by plasma-assisted deposition using a manual sputter coater (AGB7340, Agar Scientific, Stansted, UK). The images were acquired with a FIB-SEM Crossbeam 550 field emission microscope Zeiss (Oberkochen, Baden-Württemberg, Germany) at 4 kV.