E. D. Eanes

A thermodynamic analysis of the amorphous to crystalline calcium phosphate transformation.

SummaryA thermodynamic analysis of the precipitation of amorphous calcium phosphate (ACP) and its transformation to crystalline apatite has been made. A nearly constant ion product, over a wide variety of conditions, was obtained for a tricalcium phosphate (TCP)-like phase suggesting that the molecular unit which governs the solubility of ACP may be similar in composition to TCP. The introduction of 10% acid phosphate into the formula for the TCP ion product improves the fit of experimental data and results in an invariant ion product. The stability of ACP in solution was found to be dependent upon its thermodynamic instability with respect to an octacalcium phosphate (OCP)-like phase. The dependence of the induction period for the amorphous to crystalline transformation upon the pH and the Ca/P ratio of the solution is best explained by the assumption that an OCP-like phase is initially nucleated on the surfaces of the ACP particles. The events that occur in the immediate post-transition period suggest the hydrolysis of this OCP-like material to an apatitic phase.

The maturation of crystalline calcium phosphates in aqueous suspensions at physiologic pH

SummaryThe maturation of calcium phosphate crystals formed by the conversion of spontaneously precipitated amorphous calcium phosphate (ACP) was studied in aqueous media at temperatures ranging from 20° to 37°. Reaction pH was kept at 7.4 with either Hepes buffer or by the pH-stat addition of base. Reaction kinetics were followed by monitoring solution calcium and total phosphate, and, in the pH-stat controlled reaction, by recording the amount of KOH needed to maintain pH. Reaction products were examined chemically and by X-ray diffraction and transmission electron microcopy. The first crystals to form deviated markedly from apatite in morphology, composition, structure, and solubility. They were extremely thin and flaky in appearance, had a low Ca/P molar ratio (1.4), contained an appreciable amount of acid phosphate (16%), and had an exceptionally largea-axis (10.5 Å vs. 9.4 Å for apatite). With maturation, the crystals became thicker but smaller in lateral dimensions, more apatitelike in structure and composition, and less soluble. However, this ripening of the crystals was accompanied by unusual inflections in the solution Ca and total PO4 curves, and, in the case of the pH-stat experiments, in the OH consumption profiles as well. These anomalous post-ACP solution changes suggest that a phase change had taken place during crystal maturation. Although the observed structural and compositional changes are not inconsistent with the perfection of an initially defective apatite, the changes in crystal morphology and the anomalous behavior of the reaction solution may more accurately reflect a conversion of the ACP first into an OCP-like crystalline phase which subsequently hydrolyzes into apatite.

Quantitative Assessment of the Efficacy of Amorphous Calcium Phosphate/Methacrylate Composites in Remineralizing Caries-like Lesions Artificially Produced in Bovine Enamel

Recent studies show that methacrylate-based composites with amorphous calcium phosphate (ACP) as a filler can release supersaturating levels of calcium and phosphate ions in proportions favorable for apatite formation. These findings suggest that such composites could be effectively used as coatings for remineralizing teeth damaged by tooth decay. To examine this hypothesis, we tested composites in vitro for their efficacy to remineralize artificially formed caries-like lesions in extracted bovine incisors. Single 120-μm-thick sagittal tooth sections were placed in holders that exposed only the carious enamel surface. The exposed surfaces were coated with a 1-mm- to 1.5-mm-thick layer of the composite containing, by mass, 40% apatite, silica, or P2O7 4--stabilized ACP and 60% photoactivated resin comprised of Bis-GMA, TEGDMA, HEMA, and ZrM. The photocured composite-coated sections were immersed either in a remineralizing solution for 4 weeks at 37°C (static model) or cyclically immersed in demineralizing (0.5 h) and remineralizing solutions (11.5 h) for 2 weeks (dynamic model). Quantitative digital image analysis of matched 102 μm x 220 um areas from contact microradiographs taken of the sections before and after immersion showed that lesions coated with ACP-filled composites fractionally recovered 71% ± 33% of their lost mineral compared with 14% ± 13% for apatite controls in the static model and 38% ± 16% compared with -6% ± 24% in the dynamic model. The results suggest that sealants based on ACP-filled methacrylate composites have the potential to remineralize carious enamel lesions.

Characterization of native and recombinant bone sialoprotein : Delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain

Bone sialoprotein is a small, sulfated, and phosphorylated integrin‐binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106–01 BSP) culture media is shown to have a hydroxyapatite Kd ≈ 2.6 × 10−9 M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR‐BSP (Fragment 1 ∼133A–∼265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post‐translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral‐binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a15N‐enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR‐BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non‐RGD cell adhesion sites are localized within conserved amino‐ and carboxy‐terminal tyrosine‐rich domains of recombinant human BSP. Given the proximity of the latter non‐RGD cell adhesion site to the RGD tripeptide, a model of BSP‐receptor interactions is presented.

A thermodynamic analysis of the secondary transition in the spontaneous precipitation of calcium phosphate

SummaryA thermodynamic analysis has been made of the secondary transition stage in the spontaneous precipitation of calcium phosphate following the amorphous-crystalline transformation. The first formed crystalline material has a solubility similar to that of octacalcium phosphate (OCP) and the computed thermodynamic solubility product remains invariant in the pH range 7.00–8.60. The duration of the secondary stage is sensitive to pH and the transition appears to occur by hydrolysis of the first formed OCP-like phase to a more basic apatitic phase with a tricalcium phosphate (TCP) stoichiometry. The crystalline material at the end of this transition has an invariant solubility product, in the pH range 7.00 to 8.60, when the TCP-like molecular formula is assumed. Changes in the solution chemistry which accompany the solid-tosolid transitions are consistent with the above conclusions. The results of this study are also consistent with those of a previous study which suggest that the stability of the amorphous calcium phosphate phase is dependent upon the instability of the solution phase with respect to OCP formation.

Phosphoprotein modulation of apatite crystallization.

SummarySeveral phosphoprotein preparations (phosvitin, rat incisor and fetal calf molar dentin phosphoproteins) all inhibit apatite growth/replication from pre-existing crystal seeds in metastable solutions. Two stages of the crystal growth process were inhibited by these phosphoproteins. First, an initial lag period was induced, probably associated with seed surface phenomena. This period was prolonged indefinitely when a combination of phosphoprotein precoated seeds was used together with soluble phosphoproteins in the crystal growth reaction. Second, the phosphoproteins prolonged that stage of the reaction where octacalcium phosphate is the predominant mineral phase present prior to its conversion to the final apatite product. Pretreatment of the phosphoproteins with calcium diminished their inhibitory activity to seeded crystal growth as well as towards de novo apatite formation in synthetic extracellular fluids. The presence of collagen diminished the inhibitory activity of the phosphoproteins towards de novo precipitation but had no effect on phosphoprotein-modulated apatite crystal growth in the seeded systems. These results suggest a potential regulatory role for phosphoproteins in dentin mineralization.

Improved properties of amorphous calcium phosphate fillers in remineralizing resin composites

OBJECTIVES The rationale for this study was based on the hypothesis that the mechanical strength of methacrylate composites containing the bioactive filler, amorphous calcium phosphate, can be enhanced by synthesizing this filler in the presence of glass-forming agents. Specifically, this study was conducted to prepare composites with zirconia- and silica-modified amorphous calcium phosphate fillers, and to determine whether the remineralization potential from the release of calcium and phosphate ions and the mechanical properties of the corresponding methacrylate composites were enhanced. METHODS The modified amorphous calcium phosphates were synthesized at pH 10.5 by mixing 800 mmol/L Ca(NO3)2 solutions and either 250 mmol/L zirconylchloride (ZrOCl2) or 4.4 mol/L tetraethoxysilane (TEOS) solutions with solutions containing 525 mmol/L Na2HPO4 and 11 mmol/L Na4P2O7. After washing and drying, the amorphous calcium phosphates were mixed with visible light-activated resins and photopolymerized to form composite disks that were then examined for their ability to release Ca2+ and total ionic phosphate (PO4(3-) + HPO4(2-) + H2PO4-, hereafter indicated as PO4) by immersion in HEPES-buffered (pH 7.4) saline at 37 degrees C. Solution ion concentrations were compared at regular intervals up to 265 h. Biaxial flexural strengths of the composites before and after immersion were compared, and significant differences were established by Students test (p < 0.05). RESULTS Both ZrOCl2- and TEOS-modified amorphous calcium phosphate composite disks released Ca2+ and PO4 ions at sustained levels requisite for remineralization to occur. The transformation of amorphous calcium phosphate into hydroxyapatite within the composites was also retarded, particularly in the case of amorphous calcium phosphate modified with ZrOCl2. Biaxial flexure strength values of composite disks showed that TEOS- and ZrOCl2-amorphous calcium phosphate-filled composites increased in strength by 33% and 21% before immersion and by 25% and 27% after immersion, respectively, compared to unmodified amorphous calcium phosphate composites (controls). All strength increases except TEOS after immersion were significant (p < 0.05). SIGNIFICANCE Properly modified amorphous calcium phosphate fillers can be used to prepare bioactive composites with enhanced mechanical properties for more demanding dental applications without compromising their remineralizing potential.

Changes in apatite crystal size in bones of patients with osteogenesis imperfecta

SummaryApatite crystal size in compact bone of children (age<11 years) and adolescents (age>12 years) with osteogenesis imperfecta (OI) was analyzed by X-ray diffraction. Eight type I, 4 type II, 11 type III, and 14 type IV OI patients were studied along with 9 controls. The crystal size was most significantly reduced in type II patients, all of whom had died at birth. Crystal size was also diminished in both children and adolescents with types III and IV, whereas with type I OI, crystal size was reduced in children only, returning to normal in adolescence. There was a trend toward increased bone crystal size with age in both OI patients and controls.

Inhibition of Seeded Growth of Enamel Apatite Crystals by Amelogenin and Enamelin Proteins in vitro

The effect of enamel matrix proteins on the seeded growth of enamel apatite crystals was studied in stable supersaturated solutions at pH 7.4 and 37°C. Of the two major protein classes in the enamel matrix, the enamelins were considerably more effective than the amelogenins in retarding seeded growth. However, the amelogenin species that did show significant inhibitory activity are those known to be lost first from the enamel matrix during the rapid mineralization stage of enamel maturation.

Amorphous Calcium Phosphate: Thermodynamic and Kinetic Considerations

Crystalline hydroxyapatite (HAP) is generally considered to be the final, stable product in the precipitation of calcium and phosphate ions from neutral to basic solutions. However, over the broad range of solution conditions in which precipitation occurs spontaneously, unstable amorphous products precede the appearance of the HAP phase. These amorphous calcium phosphates (ACPs) are unique among calcium phosphate salts in that they lack the long-range, periodic atomic scale order of crystalline materials. Although this uniqueness has been the subject of considerable interest, of greater relevance to understanding the dynamics of HAP formation by spontaneous precipitation is the instability of these solids in solution. Above pH 9, ACPs convert directly into HAP. However, in the 7 9 pH range, ACPs are the immediate precursors to octacalcium phosphate (OCP) like phases that, in turn, convert to stable HAP. These transformation processes and the possible role they play in biological calcification, more than anything else, raises the importance of the ACPs above being merely laboratory curiosities and places them in the mainstream of calcium phosphate chemistry. This chapter will cover the preparation and properties of the ACPs, their thermodynamic and kinetic relationship to crystalline calcium phosphates, especially OCP and RAP, the controversy surrounding their true structural nature, and their putative presence and role in vivo.