Quantification of Osteoblast Differentiation

Alkaline Phosphatase (ALP) is one of the most reliable markers for osteogenic differentiation, since it is produced by osteogenic cells such as osteoblasts [34 (link), 35 (link)]. The scaffolds were washed with 1x PBS and then kept at -80°C in dry conditions until further use. The frozen MSC-seeded scaffolds underwent homogenization using a Polytron PT 1200 E benchtop homogenizer (Kinematica, Lucerne, Switzerland) in 1% Triton buffer (Sigma Aldrich, Steinheim, Germany). The homogenate was centrifuged and the supernatant was added to a solution of para-Nitrophenylphosphate (p-NPP) (Sigma Aldrich, Steinheim, Germany) for 90 min. The ALP converts p-NPP to para-Nitrophenol (p-NP) causing a change of color to yellow. The extinction of p-NP which corresponds to the ALP activity was measured photometrically at 405 nm with a reference wavelength of 490 nm using a MRX Microplate Reader (Dynatech Laboratories, Stuttgart, Germany). The ALP activity was normalized to the dsDNA content of each sample, since the amount of dsDNA is directly correlated with the number of cells.