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 o rganic phosphates [beta-glycerophosphate (beta-GP), Ser-P, or Thr-P], inorganic phosphate (P-i), or calcium. In a dose-dependent manner, the se agents (2-10 mM) stimulated confluent cultures to deposit mineral i n the cell layer (ED(50) of similar to 4.6 mM for beta-GP (30 +/- 2 nm ol Ca2+/mu g DNA) and similar to 3.8 mM (29 +/- 2 nmol Ca2+/mu g DNA) for P-i) with a plateau in mineral formation by 20 h (ET(50) approxima te to 12-15 h). beta-GP or P-i treatment yielded mineral crystals havi ng an x-ray diffraction pattern similar to normal human bone, Alizarin red-S histology demonstrated calcium mineral deposition in the extrac ellular matrix and what appeared to be intracellular paranuclear stain ing. Electron microscopy revealed small, needle-like crystals associat ed with fibrillar, extracellular matrix deposits and intracellular sph erical structures. Mineral formation was inhibited by levamisole (ED(5 0) approximate to 250 mu M), pyrophosphate (ED(50) approximate to 1-10 mu M), actinomycin C-1 (500 ng/ml), cycloheximide (50 mu g/ml), or br efeldin A (1 mu g/ml). These results indicate that UMR 106-01 BSP cell s form a bio apatitic mineralized matrix upon addition of supplemental phosphate. This process involves alkaline phosphatase activity, ongoi ng RNA and protein synthesis, as well as Golgi-mediated processing and secretion.