N. C. Blumenthal

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).

Gallium increases bone calcium and crystallite perfection of hydroxyapatite

SummaryGallium, a group IIIa metal, is known to interact with hydroxyapatite as well as the cellular components of bone. In recent studies we have found gallium to be a potent inhibitor of bone resorption that is clinically effective in controlling cancer-related hypercalcemia as well as the accelerated bone resorption associated with bone metastases. To begin to elucidate galliums mechanism of action we have examined its effects on bone mineral properties. After short-term (14 days) administration to rats, gallium nitrate produced measurable changes in bone mineral properties. Using atomic absorption spectroscopy, low levels of gallium were noted to preferentially accumulate in regions of active bone formation, 0.54±.07 μg/mg bone in the metaphyses versus 0.21±.03 μg/mg bone in the diaphyses,P<0.001. The bones of treated animals had increased calcium content measured spectrophotometrically. Rats injected with radiolabeled calcium during gallium treatment had greater 45-calcium content compared to control animals. By wide-angle X-ray analyses, larger and/or more perfect hydroxyapatite was observed. The combined effects of gallium on bone cell function and bone mineral may explain its clinical efficacy in blocking accelerated bone resorption.

Amorphous calcium phosphate in casein micelles of bovine milk

SummaryThe calcium phosphate remaining after hydrazine deproteination of casein micelles isolated from bulk skim milk exhibits under the electron microscope a very fine and uniform granularity being formed by small subunits with a true diameter of approximately 2.5 nm. This material, which is about 10 percent by weight citrate, termed calcium phosphate citrate (CPC) complex, also contains Mg and Zn at molar ratios of 0.03 and 0.003 respectively. Radial distribution function (RDF) and infrared analyses show that CPC is a Mg-containing amorphous calcium phosphate (ACP) similar to synthetic and cytoplasmic ACP. Presence of CPC in casein micelles as an amorphous colloid bonded with phosphoproteins provides the means for storing in milk large amounts of Ca (16 mM) and Pi (10 mM) in a readily utilizable form but at a higher ion concentration than found in biological solutions.

Formation and structure of Ca-deficient hydroxyapatite

SummaryWhen amorphous calcium phosphate (ACP) was transformed to crystalline hydroxyapatite (HA) in a series of aqueous slurry concentrations ranging from low to high, the higher slurry concentrations produced more Ca-deficient HA as measured by Ca/P ratio and heat-produced pyrophosphate. We feel that the excess solution phosphate produced in the higher slurry transformations results in lower Ca/P ratio HA. It has been suggested that an ACP is the precursor to bone apatite. Regulation of the in vivo ACP slurry concentration could then control the stoichiometry and, therefore, the metabolic activity of bone apatite. X-ray radial distribution function (RDF) analyses showed that CO32− substitution in HA creates far greater structural distortions than do Ca deficiencies. The latter, however, do produce small, but observable, structural distortions when compared to stoichiometric HA. It now seems clear that the RDF of bone apatite can be modeled by a synthetic, Ca-deficient, CO32−-containing HA.

Nucleotide stabilization of amorphous calcium phosphate

Abstract Amorphous calcium phosphate granules, containing substantial amounts of magnesium and adenosine triphosphate (ATP) have been found in the hepatopancreas of the blue crab. These stable amorphous deposits are similar in local atomic order to synthetic amorphous calcium phosphate (ACP), which, in contrast, converts to hydroxyapatite in an aqueous environment. Magnesium is known to be a stabilizing agent, and the present study shows that ATP, at levels comparable to those in the granules, also inhibits the conversion to hydroxyapatite (HA). Increasing amounts of ATP in solution, increased the time before ACP began to convert to HA, without greatly altering the rate once conversion began, an effect similar to that of Mg. Conversion was also inhibited by ADP, although less effectively, and AMP was found to be ineffective as a stabilizer. These nucleotides did not decrease the solubility of ACP, indicating that they act in solution to suppress the nucleation and/or growth of hydroxyapatite crystals. Possible stabilization mechanisms are discussed and the relationship to the action of other condensed phosphates is considered.

Comparison of bone apatite in osteoporotic and normal Eskimos

SummaryAn infrared and x-ray diffraction study of osteoporotic and normal, archaeological Eskimo bones. Osteoporotic bone apatite is greater in crystal size and/or perfection and lower in CO3 than normal bone apatite.

Properties of nucleating systems

Abstract A brief review of the various theories of bone mineralization is presented. It is not clear at present whether the many mineralization events which have been proposed are redundant or cooperative. A short description is given of homogeneous and heterogeneous nucleation of solids from solution and how these mechanisms affect hydroxyapatite formation, in vitro and in vivo . The remainder of the paper deals with specific experiments by the authors on (a) the stabilization of amorphous calcium phosphate and (b) the inhibitory role of proteoglycans in preventing cartilage mineralization. Stable amorphous calcium phosphate is found in the mitochondria of cells involved in tissue mineralization. In addition, it is believed that in bone mineral deposition an unstable amorphous calcium phosphate is formed first before transforming to bone apatite. A number of chemical species have been shown to act as stabilizers of amorphous calcium phosphate. The example given here in detail is the stabilization of the amorphous granules found in mitochondria by a mixture of Mg and adenosine triphosphate. The effect of proteoglycan aggregates and subunits from calcifying and non-calcifying cartilage was observed on the following two hydroxyapatite formation systems: (a) transformation of amorphous calcium phosphate to hydroxyapatite; (b) direct precipitation of hydroxyapatite from supersaturated calcium phosphate solutions. The aggregates were more effective inhibitors of apatite formation in both test systems, than equal weight solutions of subunits, but the subunit also delayed apatite formation. This suggests that in the epipryseal growth plate even the proteoglycan subunit must be removed by diffusion or enzyme cleavage for cartilage calcification to take place.