In vitro osteogenic differentiation of hDPSCs after 24 days of culture in osteogenic medium, supplemented with FCS or HS.

A: Alkaline phosphatase assay (AP) and Alizarin Red staining; B: densitometric analysis of AP and Alizarin Red staining. Values are mean ± SD of gray levels (0–255 scale). HS n = 3; FCS n = 3; ** indicate values of paired t-test HS vs. FCS (p<0.01). C: Confocal analysis of osteogenic differentiation of hDPSCs. Double immunofluorescence confocal images showing signals from anti-OPN (green) and anti-Runx2 (red); DAPI (blue) and anti-OCN (green); DAPI (blue) and anti-Osx (red). Bar: 50 µm. D: Western blot (WB) analysis of Coll-I and OCN expression in whole cell lysates of differentiated hDPSCs. Whole cell lysates were collected from three plates of human dental pulp stem cells for each differentiation protocol. Actin bands demonstrate that an equal amount of protein was loaded in each line. Undiff samples show hDPSCs cultured in FCS-medium alone. The same results were obtained in HS-medium culture (data not shown).