*2.3. Evaluation of Osteogenic Di*ff*erentiation Using Alkaline Phosphatase Activity Assays*

Cell spheroids were grown in osteogenic medium and were collected on Days 1, 3, 5, and 7. A commercially available alkaline phosphatase assay kit (K412-500, BioVision, Inc., Milpitas, CA, USA) was used for the evaluation of osteogenic differentiation. In short, the resultant supernatant was mixed and incubated with p-nitrophenyl phosphate substrate (BioVision, Inc.) for 40 min at room temperature. Absorbance was measured using a microplate reader at 405 nm (BioTek, Winooski, VT, USA).
