4.5. Visualization of Mineralized Cell Matrix by Von Kossa Staining

Cell-scaffold constructs, i.e., PCL and PCL/CaP scaffolds cultured with OB or OB+PBMC, were harvested, washed twice with PBS, and fixed with ice-cold methanol for 15 min. Cell-scaffold constructs were then incubated sequentially in the following solutions at room temperature: (i) a 3% silver nitrate solution for 30 min, (ii) a 1% pyrogallol solution for 3 min, (iii) 5% sodium thiosulphate for 5 min, (iv) a Kernechtred solution for 5 min, and (v) 96% ethanol for 1 min. In between the incubation steps, cell–scaffold constructs were washed with distilled water at least three times. Stained samples were kept in PBS at 4 °C and imaged with a digital microscope (VHX-1000, Keyence, Neu-Isenburg, Germany). The assay was performed in duplicate.