Aaron S. Posner

Infrared analysis of rat bone: age dependency of amorphous and crystalline mineral fractions.

Quantitative infrared spectrophotometric analysis of whole femurs from male rats demonstrates that anorphous calcium phosphate is a major component of bone mineral. The amount of amorphous calcium phosphate in whole bone decreases while the crystalline bone apatite increases during early stages of bone formation. Mature rat bone contains constant levels of both amorphous and crystalline calcium phosphate.

Calcium phosphate formation in vitro: II. Effects of environment on amorphous-crystalline transformation☆

The amount of time that actually elapsed during the solution mediated transformation of amorphous calcium phosphate into crystalline apatite was short and invariable. On the other hand, the time required to reach this period of rapid precipitate conversion was strongly dependent upon the exact solution environment. Amorphous calcium phosphate was more stable when formed from solutions high in pH, ionic strength, and viscosity, rich in Ca2+ relative to HPO42−, and low in initial supersaturation and temperature. The suspension lifetime of amorphous calcium phosphate was also sensitive to changes in dielectric constant. Amorphous calcium phosphate was also more stable in the presence of poly-l-glutamate, polyacrylate, phosvitin, casein, and large amounts of the protein-polysaccharides, or by the addition of small amounts of Mg2+, P2O74−, CO32− and F−. Macromolecule-calcium phosphate flotation reactions (inhibition of ready sedimentation) were found for the casein, poly-l-glutamate, polyacrylate, and poly-l-lysine molecules as well as for the protein-polysaccharide complex.

Calcium phosphate formation in vitro. I. Factors affecting initial phase separation.

Abstract The major events comprising the de novo formation of calcium phosphate salts from buffered solutions were: (1) the initial deposition of amorphous calcium phosphate; (2) its subsequent transformation into small crystals of apatite; and (3) the ultimate growth or ripening of those crystals. The degree of metastability for any given calcifying solution was shown to be an exact function of pH, ionic strength, temperature, sol Ca P molar mixing ratio, and initial Ca × P millimolar product. Initial mineral-phase formation lag time was also sensitive to changes in solution viscosity and dielectric constant. The presence of pyrophosphate, fluoride, magnesium, carbonate, collagen, or gelatin always enhanced amorphous calcium phosphate formation, while addition of lysozyme, casein, phosvitin, poly- l -lysine, or protein-polysaccharides enhanced initial mineral-phase formation only at relatively low initial Ca × P millimolar products. Citrate, poly- l -glutamate, and polyacrylate always inhibited amorphous calcium phosphate formation, whereas chondroitin sulfate and protein-polysaccharides were inhibitory only at relatively high initial Ca × P millimolar products.

Magnesium stabilization of amorphous calcium phosphate: A kinetic study

The kinetics of the conversion of amorphous calcium phosphate (ACP) to hydroxyapatite (HA) at pH 8 at 26.0°, 37.5 and 48.0 C, in the presence of Mg has been studied in two sets of experiments in which (a) Mg-free ACP was added to solutions containing different amounts of Mg, or, (b) ACP precipitated in the presence of Mg was left in contact with its mother liquor. If the Mg/Ca ratio in the system exceeded 0.2 no conversion was observed. In the range of Mg/Ca ratios found in bones and teeth (mole ratios from 0.004 to 0.04), the induction period of the transformation (time before first HA crystals observed) increased with increasing Mg concentration, but the rate of the first order transformation was independent of Mg content. It is shown that the Mg effects the transformation by reducing the ACP solubility.

Effect of proteoglycans on in vitro hydroxyapatite formation

SummaryWell-characterized bovine nasal proteoglycan A1 fraction (aggregate) and proteoglycan D1 fraction (subunit) have been shown to be effective inhibitors of hydroxyapatite (HA) formation in two in vitro test systems: (a) the transformation of amorphous calcium phosphate (ACP) to crystalline HA, and, (b) the direct precipitation of HA from low-concentration calcium phosphate solutions. A1 or D1 in solution slowed the transformation kinetics in system (a) without affecting the time to the onset of conversion. In system (b), A1 or D1 in solution increased the time to the onset of HA formation without affecting the HA formation kinetics. In both test systems A1 was a more effective inhibitor than D1, although the difference was not great. In both systems the inhibitory effect was proportional to the A1 or D1 solution concentration. The action of solutions of low and high molecular weight neutral dextrans on both test systems showed that high molecular weight and/or extended spatial molecular conformation has a much stronger correlation with inhibitory ability than solution viscosity. Proteoglycans have been implicated as playing a role in regulating biological mineralization particularly in the epiphyseal growth plate. Our study suggests that just enzymatic cleavage of aggregate into subunit is not sufficient to allow mineralization to occur, since we find that D1 itself is a potent inhibitor of HA formation. Further degradation and/or removal of D1 appears to be necessary for calcification to take place.

Stabilization of Amorphous Calcium Phosphate by Mg and ATP

SummaryA synergistic effect has been demonstrated when magnesium and adenosine triphosphate (ATP) are used together in solution to delay the conversion of a slurry of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA). Conversion is delayed in some instances more than 10 times as long as with either ATP or Mg alone. In all experiments conversion did not begin until ATP in solution had decreased through hydrolysis to an undetectable level. The effect of Mg is to decrease substantially the rate at which ATP hydrolysis occurs. Once conversion began it proceeded more slowly in the presence of both Mg and ATP than with Mg or ATP alone. ATP was also found to prevent the formation of HA from metastable solutions of calcium and phosphate which did not contain any solid phase. Over the time period of these experiments, ATP hydrolyzed to a negligible extent in Tris-HCl buffer and in solutions containing Ca, PO4, and Ca plus PO4 ions. Hydrolysis of ATP does occur in the presence of ACP or HA, presumably by transphosphorylation on the surface of the solid calcium phosphate phase. It was concluded that ATP stabilized ACP, not by affecting its dissolution, but either by poisoning heteronuclear growth sites, or by poisoning the growth of embryonic HA nuclei (formed heterogeneously or homogeneously) before their critical size is reached, or by poisoning both. In the case of embryonic HA nuclei, the poisoned nuclei would go back into solution preventing HA crystal formation. In addition, it was found that the neutral Ca9(PO4)6 clusters, which are believed to be the basic structural unit of ACP, break down into individual Ca and PO4 ions when ACP dissolves in aqueous medium.

Effect of carbonate and biological macromolecules on formation and properties of hydroxyapatite

Amorphous calcium phosphate (ACP) was transformed at 25° to hydroxyapatite (HA) in horse and bovine serum; solutions of serum-protein fractions in tris-HCl buffer (pH 7.4), and pH 7.4 buffers containing from 0.1 to 10 times physiological CO32− concentration. The ACP-to-HA transformation was slower in whole serum and serum fractions than in control buffer solution. The observed adsorption of serum proteins on ACP and HA probably inhibits both the dissolution of the ACP particles and the growth of HA crystals. After 72 h all transformations were complete as determined by X-ray diffraction. The HA crystal dimensions decreased with increasing CO32− but the shape, as shown by X-ray linewidths, was relatively constant up to about 4% CO32−. At 15% CO32− the crystals were more equiaxial and less needle-like in habit. The radial distribution function (RDF) of HA with 3.7% CO32− is less well resolved than the RDF of HA with ambient CO32− (1.1%). The peaks are less sharp and their amplitude falls more rapidly with increasing atomic separation than for low CO32−-HA. These effects show that CO32− decreases the regularity of the atomic arrangement when incorporated in HA. The rapid decrease, with increasing CO32− content, of the IR splitting of the P−O bending mode of CO32−-HA is attributed to reduced crystal size and possibly to a perturbation of the crystal field due to CO32−-induced lattice distortion. Finally, for bone mineral, it is probable that the poor resolution of the X-ray and IR patterns is due, in large part, to small crystal size and internal disorder caused by CO32−.

In vitro model of aluminum-induced osteomalacia: Inhibition of hydroxyapatite formation and growth

Introduction: 0steomalacia renal osteodystrophy in patients on long term hemodialysis has often been associated with aluminum accumulation in bone (i). Aluminum has been shown to be localized at the mineralization front in dialysis osteomalacia, suggesting it as a possible etiologic agent in this disorder. Animal models of aluminum-induced osteomalacia have been defined which accurately reflect the clinical disease (2,3).

The role of synthetic and bone extracted Ca-phospholipid-PO4 complexes in hydroxyapatite formation.

SummaryThe calcium-phospholipid-phosphate (Ca-PL-PO4) complex isolated from young bone has been shown to initiate hydroxyapatite formation from a metastable calcium phosphate solution. The action of the complex was compared to that of the acidic phospholipids: phosphatidyl serine, phosphatidyl inositol and phosphatidic acid. These phospholipids first remove calcium, and a small amount of phosphate from the metastable solution forming a material similar to the complex isolated from bone, and then form hydroxyapatite. The rate of hydroxyapatite proliferation, once phosphatidyl serine and phosphatidyl inositol are converted to Ca-PL-PO4 complexes, is the same as the rate observed for comparable weights of the complex isolated from bone. It is suggested that the complex isolated from bone was formed in a manner similar to the complexes in our in vitro experiments. Finally, our evidence supports the possiblity that a similar complex is responsible for the initial mineralization in matrix vesicles.

Effect of preparation conditions on the properties and transformation of amorphous calcium phosphate

Amorphous tricalcium phosphate (ACP) is the invitro and invivo precursor of hydroxyapatite (HA) and bone apatite, respectively. ACP prepared under conditions of high supersaturation and/or high pH is small in particle size and shows a high Ca/P ratio due to occluded CaCl2. The smaller ACP particles transform more slowly to HA than larger particles in bovine blood serum.