2.12. Alkaline phosphatase assay

The colorimetric alkaline phosphatase (ALP) assay kit (Abcam) was used to assess ALP activity in ADSCs seeded on the scaffolds in osteogenic differentiation medium after 14 and 21 days. Staining was performed according to the manufacturer’s instructions (n = 6). In brief, 80 μl of supernatant was collected and then 50 μl of the p-nitrophenyl phosphate (pNPP) was added to the samples as a substrate followed by the addition of ALP enzyme solution for 60 min at 25 °C. The reaction was then stopped by adding 20 μl of Stop Solution and the absorbance was read at 450 nm (n = 6).