Rapidly Forming Apatitic Mineral in an Osteoblastic Cell Line (UMR 106—01 BSP)

Abstract

This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [β-glycerophosphate (β-GP), Ser-P, or Thr-P], inorganic phosphate (Pi), or calcium. In a dose-dependent manner, these agents (2-10 m M) stimulated confluent cultures to deposit mineral in the cell layer (ED50of ∼ 4.6 m M for β-GP (30 ± 2 nmol Ca2+/μg DNA) and ∼3.8 m M (29 ± 2 nmol Ca2+/μg DNA) for Pi) with a plateau in mineral formation by 20 h (ET50≈ 12-15 h). β-GP or Pitreatment 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≈ 250 μM), pyrophosphate (ED50≈ 1-10 μM), actinomycin C1(500 ng/ml), cycloheximide (50 μg/ml), or brefeldin A (1 μg/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, on-going RNA and protein synthesis, as well as Golgi-mediated processing and secretion. This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [β-glycerophosphate (β-GP), Ser-P, or Thr-P], inorganic phosphate (Pi), or calcium. In a dose-dependent manner, these agents (2-10 m M) stimulated confluent cultures to deposit mineral in the cell layer (ED50of ∼ 4.6 m M for β-GP (30 ± 2 nmol Ca2+/μg DNA) and ∼3.8 m M (29 ± 2 nmol Ca2+/μg DNA) for Pi) with a plateau in mineral formation by 20 h (ET50≈ 12-15 h). β-GP or Pitreatment 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≈ 250 μM), pyrophosphate (ED50≈ 1-10 μM), actinomycin C1(500 ng/ml), cycloheximide (50 μg/ml), or brefeldin A (1 μg/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, on-going RNA and protein synthesis, as well as Golgi-mediated processing and secretion.