Fig. 3.

Primary osteoblast isolation and characterization. After calvarial bone fragments were cultured in α-MEM for seven days, osteoblasts migrated from the edge of the bone fragments and proliferated around the fragments (A, B: 1 day; C, D: 4 days; E, F: 7 days; red star: bone fragment; black star: osteoblast). ALP staining indicated violet deposits in the cytoplasm representing intracellular ALP synthesized by primary osteoblasts (G, H). Optical micrograph (I, G) and image of ARS staining (K, L) of mineralized calcium nodules formed after isolated primary osteoblasts were cultured for four weeks (red triangle: unstained calcium nodule; black triangle: ARS-stained mineralized nodule). K and L are the stained versions of I and J, respectively. (The scale bar is 100 ​μm for A – I and K, 50 ​μm for J, 100 ​μm for L). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)