Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts

Osteoblasts are bone-forming cells that secrete small vesicles into their extracellular matrix, ECM. These vesicles, called matrix vesicles, are equipped with membrane transporters and enzymes that enable calcium and phosphate ion entry, forming mineral crystals within. As crystals accumulate, they break through the vesicle membrane to form mineralized nodules in the ECM, making up the bone mineral density.

To visualize mineralization, begin with osteoblast cells on a biocompatible scaffold in a suitable growth medium. The osteoblasts attach to the scaffold and use growth factors from the medium to secrete matrix vesicles that subsequently form mineralized nodules in the ECM.

Aspirate the culture medium and rinse the cells to remove any media traces. Treat the cells with a formalin solution, which induces ECM crosslinking, fixing, and preserving the tissue structure. Wash the cells to remove any remaining formalin.

Next, add Alizarin Red staining solution and incubate the culture. The dye molecules bind to the mineralized nodule and associate with calcium, a key bone mineral, forming large stained complexes.

Aspirate the staining solution and wash thoroughly to remove any unbound dye. When observed under a microscope, the calcium-rich mineral deposits appear as bright-red nodules in the ECM, indicating mineralization by the osteoblasts.