Osteogenic characterization of the vascularized bone model. (A) Whole mount alizarin red staining imaging and quantification after digestion of the constructs. (B) COL1A1 and BSP immunofluorescence characterization of the cross-section of the constructs. (C) Quantitative RT-qPCR analyses; gene expression is relative to GAPDH levels. ALP = alkaline phosphatase, BSP = bone sialoprotein, OSX = osterix, OPN = osteopontin, COL1A1 = collagen type I, OSC = osteocalcin. (D) Quantitative evaluation of osteogenesis in terms of calcium released in the medium and ALP activity in the culture supernatant. Number of experiments = 3, number of replicates/experiment = 4. ns = non-significant, * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** p < 0.00005.