Quantification of Alkaline Phosphatase Activity

The activity of alkaline phosphatase (ALP), a widely accepted early marker of osteogenesis in bone-forming cells,^{28 (link)} was assessed on cells grown for 14 days on glass or P3HT substrates, under either standard culture conditions or osteogenic stimuli (10 nM dexamethasone, 10 mM glycerol-2-phosphate, 150 mM l-ascorbic acid and 10 nM cholecalciferol, Sigma Aldrich). Briefly, cells were washed in phosphate buffer saline and lysed in 50 μL 0.1% Triton X-100 (Sigma Aldrich). ALP activity was assessed through a colorimetric assay based on the conversion of p-nitrophenyl phosphate into p-nitrophenol, as exhaustively described by Canciani et al.^{29 (link)} The enzymatic activity (U) was inferred based on the amount of produced p-nitrophenol and the reaction time, then normalized with respect to each sample's protein concentration determined by BCA™ Protein Assay (Merck) and expressed as U μg⁻¹.