Effects of density of BMSCs on osteogenic differentiation and mineralization of 3D bio-printed hydrogel scaffolds. (a) Mineralization of hydrogel scaffolds with different densities of BMSCs in vitro for day 7, 14, and 21 as shown by alizarin red staining. (b) Quantitation of mineralization levels of hydrogels. Scale bar: 200 μm αp<0.05 vs T0; βp<0.05 vs T5; γp<0.05 vs T6; δp<0.05 vs T7. (c) Elemental composition of mineralized nodules as determined by Energy Dispersive X-ray Spectroscopy (EDS). Scanning electron micrograph of mineralized nodules, elemental distribution of P and Ca elements, and the spectrum of various elemental compositions are shown. (d) Quantitative assays of Alkaline phosphatase (ALP) activity of encapsulated BMSCs in hydrogel scaffolds after culturing for 7, 14, and 21 days. αp<0.05 vs T5; βp<0.05 vs T6; γp<0.05 vs T7. Relative gene expression levels of osteogenic markers (e) Runt-related transcription factor 2 (Runx2), (f) Osterix, (g) ALP, (h) osteocalcin (OCN) and (i) type I collagen (Col-1) of BMSCs after day 7 and day14 culture in the hydrogels. αp<0.05 vs control; βp<0.05 vs T5; γp<0.05 vs T6; δp<0.05 vs T7. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)