Bone cells compose a population of cells of heterogeneous origin but restricted function with respect to matrix formation, mineralization, and resorption. The local, mesenchymal origin of the cells which form the skeleton contrasts with their extraskeletal, hemopoietic relatives under which bone resorption takes place. However, the functions of these two diverse populations are remarkably related and interdependent. Bone cell regulation, presently in its infancy, is a complicated cascade involving a plethora of local and systemic factors, including some components of the skeletal matrices and other organ systems. Thus, any understanding of bone cell regulation is a key ingredient in understanding not only the development, maintenance, and repair of the skeleton but also the prevention and treatment of skeletal disorders.

Bone cell biology: the regulation of development structure, and function in the skeleton

Experiments were performed to determine whether β-glycerophosphate (β-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of β-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither β-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that β-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that β-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM β-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of β-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and β-GP and were deposited into a collagen matrix. We conclude that medium supplementation with β-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.

Mechanism of action of beta-glycerophosphate on bone cell mineralization

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system Mineralization was induced by supplementation of 18 mM of Ca(+2) and 5 mM of beta-GP or Pi Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively Levamisole and beta-GP did not appear to influence DNA synthesis Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism

Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation.

Background
Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO3−)n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

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Control of vertebrate skeletal mineralization by polyphosphates.

Ecto 5′-Nucleotidase and Nonspecific Alkaline Phosphatase TWO AMP-HYDROLYZING ECTOENZYMES WITH DISTINCT ROLES IN HUMAN AIRWAYS

Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PPi. Optimal activities forp-nitrophenyl phosphate, ATP, and PPi are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ and Hg2+ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn2+ ions show no effect. Pi, levamisole, CN−, Zn2+, Ca2+ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. Pi is a linear noncompetitive inhibitor with a Ki of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca2+ ions inhibit noncompetitively with a Ki of 9.3 mM. Zn2+ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be2+ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na2SO4 at 0.5–1.0 M inhibit the enzyme.

Richard Fortuna

Papers

Enzymatic Characterization of the Matrix Vesicle Alkaline Phosphatase Isolated from Bovine Fetal Epiphyseal Cartilage

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

The purification and molecular characterization of alkaline phosphatases from chondrocytes and matrix vesicles of bovine fetal epiphyseal cartilage