Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP)

This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [beta-glycerophosphate (beta-GP), Ser-P, or Thr-P], inorganic phosphate (P(i)), or calcium. In a dose-dependent manner, these agents (2-10 mM) stimulated confluent cultures to deposit mineral in the cell layer (ED50 of approximately 4.6 mM for beta-GP (30 +/- 2 nmol Ca2+/microgram DNA) and approximately 3.8 mM (29 +/- 2 nmol Ca2+/microgram DNA) for P(i)) with a plateau in mineral formation by 20 h (ET50 approximately 12-15 h). beta-GP or P(i) treatment yielded mineral crystals having an x-ray diffraction pattern similar to normal human bone. Alizarin red-S histology demonstrated calcium mineral deposition in the extracellular matrix and what appeared to be intracellular paranuclear staining. Electron microscopy revealed small, needle-like crystals associated with fibrillar, extracellular matrix deposits and intracellular spherical structures. Mineral formation was inhibited by levamisole (ED50 approximately 250 microM), pyrophosphate (ED50 approximately 1-10 microM), actinomycin C1 (500 ng/ml), cycloheximide (50 micrograms/ml), or brefeldin A (1 microgram/ml). These results indicate that UMR 106-01 BSP cells form a bio-apatitic mineralized matrix upon addition of supplemental phosphate. This process involves alkaline phosphatase activity, ongoing RNA and protein synthesis, as well as Golgi-mediated processing and secretion.