Effects of aluminum(III), chromium(III), and iron(III) on the rate of dissolution of calcium hydroxyapatite crystals in the absence and presence of the chelating agent desferrioxamine

Summary Aluminum ions (Al) and chromium (III) ions (Cr), as they exist in aqueous solution at neutral pH, adsorb onto calcium hydroxyapatite crystals (HAP) and severely inhibit their dissolution process, when present in concentrations less than 1 μM. Iron (III) ions (Fe), at concentrations up to 10 μM, have no effect on the dissolution process of HAP. The Fe-chelating agent desferrioxamine also forms strong complexes with Al but not with Cr. Desferrioxamine prevents the adsorption of Al to HAP and removes pre-adsorbed Al from the HAP surface, although not instantaneously, but has no significant effect on the adsorption of Cr to HAP. Desferrioxamine is also found to be capable of removing Al preadsorbed to bone powder.     

The effect of aluminum on the rate of dissolution of calcium hydroxyapatite—A contribution to the understanding of aluminum-induced bone diseases

Summary Aluminum ions, Al³⁺, as these ions exist in aqueous solutions at pH≊7, adsorb onto calcium hydroxyapatite (HAP) crystals and severely inhibit the dissolution process of these crystals at 0.1 μM concentrations of aluminum. Human bone crystals have also been shown to adsorb these ions or molecules. A mechanism explaining why aluminum, in connection with bone resorption, causes less demineralization in the osteoid/calcified bone region than deeper in the calcified bone, is suggested.     

The seeded growth of calcium phosphates. The kinetics of growth of dicalcium phosphate dihydrate on hydroxyapatite

The kinetics of growth of calcium phosphate on synthetic HAP seed crystals has been studied at 37°C and at pH values of 4.5 and 5.0 by the pH-stat controlled addition of base. Following an induction, DCPD crystal growth takes place with growth kinetics characteristic of the crystallization of pure DCPD seed. The effect of stirring rate, HAP seed concentration and initial degree of supersaturation with respect to DCPD on the kinetics of the growth process have been determined. The crystallization appears to be controlled by a surface reaction and nucleation of DCPD on the HAP substrate is completed in the initial induction period. Formation of DCPD under these conditions closely simulates plaque and calculus production in the mouth. Thus calculus that develops from the initial plaque is known to contain an appreciable amount of DCPD.     

The seeded growth of calcium phosphates. The kinetics of growth of dicalcium phosphate dihydrate on hydroxyapatite

The kinetics of growth of calcium phosphate on synthetic HAP seed crystals has been studied at 37°C and at pH values of 4.5 and 5.0 by the pH-stat controlled addition of base. Following an induction, DCPD crystal growth takes place with growth kinetics characteristic of the crystallization of pure DCPD seed. The effect of stirring rate, HAP seed concentration and initial degree of supersaturation with respect to DCPD on the kinetics of the growth process have been determined. The crystallization appears to be controlled by a surface reaction and nucleation of DCPD on the HAP substrate is completed in the initial induction period. Formation of DCPD under these conditions closely simulates plaque and calculus production in the mouth. Thus calculus that develops from the initial plaque is known to contain an appreciable amount of DCPD.     

A comparison of some effects of fluoride on apatite formationin vitro andin vivo

Summary In a liquid (22°C) saturated with and in contact with powdered bone apatite, the fluoride ion activity was adjusted to 1–10 parts/10⁶. Due to the fluorapatite (FAP) supersaturation produced hereby, a rapid formation of this salt occurred leading to a decrease of the concentration of the ions involved. When no more fluoride was available in the liquid, the concentrations of calcium and phosphate increased again, due to dissolution of presumeably hydroxyapatite (HAP). Fifty four rats were given either 10, 20, or 40 mg NaF per kg body weight intraperitoneally. The animals were sacrificed from 5 min to 96 h after the injection. The fluoride concentration in plasma increased to a peak, after which it decreased. Plasma calcium decreased and remained low until the fluoride had attained normal levels. The intensity of mineralization of the growing dental hard tissue was monitored on microradiographs. Corresponding to the plasma fluoride peak and the decrease of plasma calcium, a hypermineralized layer was formed while a hypomineralized zone was formed during plasma calcium increase after disappearance of fluoride. Similarities and dissimilarities between thein vitro and thein vivo experiments are discussed.     

Simulating calcium salt precipitation in the nephron using chemical speciation

Theoretical modeling of urinary crystallization processes affords opportunities to create and investigate scenarios which would be extremely difficult or impossible to achieve in in vivo experiments. Researchers have previously hypothesized that calcium renal stone formation commences in the nephron. In the present study, concentrations of urinary components and pH ranges in different regions of the nephron were estimated from concentrations in blood combined with a knowledge of the renal handling of individual ions. These were used in the chemical speciation program JESS to determine the nature of the solution complexes in the different regions of the nephron and the saturation index (SI) of the stone-forming salts calcium oxalate (CaOx), brushite (Bru), hydroxyapatite (HAP) and octacalcium phosphate (OCP). The effect of independent precipitation of each of the latter on the SI values of other salts was also investigated. HAP was the only salt which was supersaturated throughout the nephron. All of the other salts were supersaturated only in the middle and distal regions of the collecting duct. Supersaturations were pH sensitive. When precipitation of CaOx, Bru and OCP was simulated in the distal part of the collecting duct, little or no effect on the SI values of the other stone forming salts was observed. However, simulation of HAP precipitation caused all other salts to become unsaturated. This suggests that if HAP precipitates, a pure stone comprising this component will ensue while if any of the other salts precipitates, a mixed CaOx/CaP stone will be formed. Application of Ostwald’s Rule of Stages predicts that the mixed stone is likely to be CaOx and Bru. Our modelling demonstrates that precipitation of stone-forming salts in the nephron is highly dependent on the delicate nature of the chemical equilibria which prevail and which are themselves highly dependent on pH and component concentrations.     

Effects of fluoride on human enamel and selachian enameloid in vitro: A high-resolution TEM and electron diffraction study

Summary The effects of fluoride on human enamel and selachian enameloid in vitro were visualized in TEM and analyzed with electron diffraction. It is demonstrated that under precise pH conditions, inducing concentration balance between F⁻ ions and apatite, calcium fluoride is no longer formed, and crystalline changes occur instead. A secondary growing process, inducing a twofold increase in crystal size, involves all crystal faces, altering the hexagonal symmetry. It is suggested that the mechanism involved is not a dissolution/precipitation process but rather a secondary growth of residual crystallites induced by apatite dissolution.     

Influence of preparation conditions on the composition of type B carbonated hydroxyapatite and on the localization of the carbonate ions

Summary It is shown how certain aspects of the composition and structure of carbonated apatites depend strictly on preparation conditions, for example, excess of phosphate or calcium ions in the reaction medium, CO₃ ²⁻ concentration, pH, ammonia added or not. Depending on those conditions, either one or the other of the two proposed mechanisms of introduction of carbonate ions into the B sites is dominant. The mechanisms are (1) replacement of a phosphate ion by a carbonate ion with the formation of three vacancies, one in a phosphate oxygen site and one each in the neigh-boring Ca²⁺ and OH⁻ sites; and (2) replacement of a phosphate ion by a carbonate accompanied by a hydroxyl ion. Whether mechanism (1) is observed to dominate over mechanism (2), or vice versa, is accounted for by the relative concentrations of the various ions in the reaction medium. The number of vacancies is decreased by the presence of either, or both, excess calcium ions or ammonia in the reaction medium. A structural-chemical mechanism is advanced for the view that, with the smallest CO₃ ²⁻ content, the A sites are favored but with increasing carbonate content the B sites become favored and the A-site content becomes less than it is when the total carbonate content is less.     

In vitro studies of composite bone filler based on poly(propylene fumarate) and biphasic α-tricalcium phosphate/hydroxyapatite ceramic powder

While many different filler materials have been applied in vertebral augmentation procedures, none is perfect in all biomechanical and biological characteristics. To minimize possible shortages, we synthesized a new biodegradable, injectable, and premixed composite made from poly(propylene fumarate) (PPF) and biphasic α-tricalcium phosphate (α-TCP)/hydroxyapatite (HAP) ceramics powder and evaluated the material properties of the compound in vitro. We mixed the PPF cross-linked by N-vinyl pyrrolidinone and biphasic α-TCP/HAP powder in different ratios with benzoyl peroxide as an initiator. The setting time and temperature were recorded, although they could be manipulated by modulating the concentrations of hydroquinone and N,N-dimethyl-p-toluidine. Degradation, cytocompatibility, mechanical properties, and radiopacity were analyzed after the composites were cured by a cylindrical shape. We also compared the study materials with poly(methyl methacrylate) (PMMA) and PPF with pure HAP particles. Results showed that lower temperature during curing process (38-44°C), sufficient initial mechanical compressive fracture strength (61.1±3.7MPa), and gradual degradation were observed in the newly developed bone filler. Radiopacity in Hounsfield units was similar to PMMA as determined by computed tomography scan. Both pH value variation and cytotoxicity were within biological tolerable limits based on the biocompatibility tests. Mixtures with 70% α-TCP/HAP powder were superior to other groups. This study indicated that a composite of PPF and biphasic α-TCP/HAP powder is a promising, premixed, injectable biodegradable filler and that a mixture containing 70% α-TCP/HAP exhibits the best properties.     

Fluoride uptake and acid resistance of enamel irradiated with Er:YAG laser

This study evaluated the resistance to demineralization and fluoride incorporation of enamel irradiated with Er:YAG. A total of 110 bovine teeth were selected and divided into eight groups: unlased, 37% phosphoric acid, and samples irradiated with the Er:YAG laser at several fluences (31.84 J/cm², 25.47 J/cm², 19.10 J/cm², 2.08 J/cm², 1.8 J/cm², and 0.9 J/cm²). The application of acidulated phosphate fluoride was performed after treatments. All samples were immersed in 2 ml of 2.0 M acetic–acetate acid solution at pH 4.5 for 8 h, and fluoride, calcium, and phosphorus ions dissolved were analyzed by atomic absorption spectrometry and spectrophotometry. The phosphoric acid and 31.84 J/cm² groups presented the lowest dissolution of calcium and phosphorus ions. Higher fluoride incorporation was observed on 1.8 J/cm² and 0.9 J/cm² groups. Based on these results, Er:YAG laser was able to decrease acid dissolution and increase fluoride uptake and can be a promissory alternative for preventive dentistry.     

Kinetic reactions of calcium,phosphate, and fluoride ions at the enamel surface-solution interface

Surfaces of tooth enamel were interfaced with recirculating solutions containing calcium and phosphate. The kinetic interfacial reactions of calcium, phosphate, fluoride, and hydrogen ions were determined in solutions of constant fluoride ion concentrations (0.012, 0.025, 0.050, 0.250, or 0.500 mM/l) and /or constant pH (6.8, 7.0, 7.2, or 7.4). The rates of the calcium, phosphate, fluoride, and hydrogen ion reactions at the solid-solution interface increased under the following conditions: (1) increase of the enamel surface area; (2) increase of the rates of solution flow to the interface; (3) increase of solution pH; and (4) increase of the solution fluoride ion concentration. The nearly proportional rates of the concurrently measured reactions indicated the formation of apatitic minerals on the enamel surfaces throughout the time of solid-solution interface.Support: Veterans Administration. P. H. S. Training Grant No. 5 T01 DE 00007-14.     

Calcium phosphate saturation levels in ultrafiltered serum

Summary Calcifications occurring in arteriosclerotic plaque and other pathological deposits are important health concerns, and the nature of these deposits and their mechanisms of formation warrant investigation. Crystals of the relevant calcium phosphates were equilibrated with the undiluted ultrafiltered human serum (u.f.s.) at 37°C by constant stirring and periodically removing samples for calcium and phosphate analysis and for pH measurement. The solubility measurements were carried out both with and without a 5.5% CO₂ atmosphere, the physiological partial pressure of CO₂. The apparent ion activity products of well-crystallized dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), and hydroxyapatite (OHAp) equilibrated, in u.f.s. were calculated from the calcium and phosphate concentrations and pH in each case for comparison with their known, solubility products. In this way the well-crystallized calcium phosphates serve as fiducial solubility standards, thereby minimizing errors due to complexing of calcium and phosphate ions by u.f.s. constituents. Under 5.5%, CO₂ native u.f.s. was found to be substantially undersaturated with respect to DCPD, slightly supersaturated with respect to OCP, and highly supersaturated with respect to OHAp. The ion activity product of DCPD in DCPD-saturated u.f.s. was 2.4×10⁻⁷, and the ion activity product of OCP in OCP-saturated u.f.s. was 4×10⁻⁴⁹, slightly above their solubility products (K_sp(DCPD)=2.3×10⁻⁷, K_sp(OCP)=2.5×10⁻⁴⁹). The ion activity products of DCPD and OCP in u.f.s. under CO₂ indicate that the concentrations of calcium and phosphate complexing agents (except bicarbonate) are quite low. The u.f.s. remained supersaturated with respect to OHAp even after 2 months of equilibration. This is attributed to the presence of crystal growth inhibitors in u.f.s.     

Effects of pyrophosphate and diphosphonates on the dissolution of hydroxyapatites using a flow system

Summary Pyrophosphate and diphosphonate ions have been said to diminish the dissolution of hydroxyapatite crystals, because they lower the equilibrium concentrations of calcium and phosphate ions in the bulk solution around hydroxyapatite crystals in a closed system. However, in a closed system these effects are not necessarily due to an effect on dissolution alone. In this paper we have used a continuous flow system to study the effects of pyrophosphate and two diphosphonates, ethane-1-hydroxy-1,1-diphosphonate and dichloromethane diphosphonate, on the dissolution of hydroxyapatite. All three compounds decreased markedly the rate of dissolution of hydroxyapatite as well as the exchangeable pools of calcium and phosphate ions around the cystals.     

A study of mineral phase in immobilized rat femur: structure refinements by Rietveld analysis

 The aim of the present work was to examine whether immobilization of a limb influences the structure of bone mineral. The mineral phase in rat femora immobilized for 2 weeks during growth was investigated. Bone mineral was subjected to powder X-ray diffraction, using a scanning method after ashing the bones at 630°C. Occupancy factors of ion positions in bone hydroxyapatite (HAP) were analyzed using the Rietveld refinement method. Occupancy factors of the positions OH⁻, Ca²⁺, and P⁻ were significantly lower in immobilized than in control bones, although the position of ions in the HAP structure did not change. Mineralization of tissue in the immobilized bones was lower than in the controls, but there was no correlation between mineralization and occupancy factors. HAP lattice constants in immobilized bones were slightly but significantly different from those in controls. We conclude that the structure of HAP synthesized in bone during temporary lack of loading differs from that of HAP growing under physiological conditions. The Rietveld refinement method proved to be useful in the estimation of the changes in bone mineral. Received: April 18, 2002 / Accepted: October 17, 2002 Offprint requests to: H. Trebacz     

A comparison of some effects of fluoride on apatite formation in vitro and in vivo

In a liquid (22 degrees C) saturated with and in contact with powdered bone apatite, the fluoride ion activity was adjusted to 1-10 parts/10(6). Due to the fluorapatite (FAP) supersaturation produced hereby, a rapid formation of this salt occurred leading to a decrease of the concentration of the ions involved. When no more fluoride was available in the liquid, the concentrations of calcium and phosphate increased again, due to dissolution of presumably hydroxyapatite (HAP). Fifty four rats were given either 10, 20, or 40 mg NaF per kg body weight intraperitoneally. The animals were sacrificed from 5 min to 96 h after the injection. The fluoride concentration in plasma increased to a peak, after which it decreased. Plasma calcium decreased and remained low until the fluoride had attained normal levels. The intensity of mineralization of the growing dental hard tissue was monitored on microradiographs. Corresponding to the plasma fluoride peak and the decrease of plasma calcium, a hypermineralized layer was formed while a hypomineralized zone was formed during plasma calcium increase after disappearance of fluoride. Similarities and dissimilarities between the in vitro and the in vivo experiments are discussed.     

The influence of multidentate organic phosphonates on the crystal growth of hydroxyapatite

The rate of crystal growth of hydroxyapatite seed crystals in stable supersaturated solutions of calcium phosphate has been studied under reproducible conditions at 25° and at a constant pH of 7.40 in the presence of the organic phosphonates HEDP, 1-hydroxyethylidine 1,1-diphosphonic acid, NTMP, nitrilotri (methylene phosphonic acid); ENTMP, N,N,NN ethylenediamine-tetra (methylene phosphonic acid); TENTMP, triethylenediamine tetra (methylene phosphonic acid). It is suggested that the marked inhibitory influence of the additives upon the rate of crystal growth is due to the formation of strong, substitution inert chelate bonds with the calcium ions present at kinks and dislocations on the crystal surface of HAP. The results of this study show that the potentially hexadentate ligand ENTMP is more effective as a crystal growth inhibitor than the tetradentate NTMP or the tridentate HEDP. The general ineffectiveness of the monophosphonates as crystal growth inhibitors supports the conclusion that the calcium ions are chelated at the surface thereby preventing further deposition of calcium phosphate at that growth site. The relatively low concentration of added phosphonate as compared with the calcium ion concentration rules out calcium chelation in the bulk of the solution as a significant factor in the observed crystal growth inhibition.This investigation was supported in part by grant DE 3223-01 from the National Institute of Health.     

Calcium,phosphate, and fluoride deposition on enamel surfaces

Concurrent calcium, phosphate and fluoride depositions, and the rate of hydrogen ion production, during formation on acid etched enamel surfaces were kinetically measured in a closed system. The deposition rate of calcium and phosphate from solution was dependent on the fluoride concentrations and pH. The proportions of calcium to phosphate and calcium to fluoride indicated fluorapatite or fluorhydroxyapatite formation on the enamel. The hydroxyl ion additions to maintain constant pH, concurrent with mineral deposition, exceeded the total phosphate ions deposited from solution. This indicates the possibility that acid phosphate hydrolysis occurs during mineral deposition.     

Studies on the role of calcium phosphate in the process of calcium oxalate crystal formation

Crystals of calcium phosphate (CaP) added to solutions with a composition corresponding to that at different levels of the collecting duct (CD) and with different pH were rapidly dissolved at pH 5.0, 5.25 and 5.5. Only minor or no dissolution was observed at higher pH levels. Despite this effect, CaP crystals induced nucleation or heterogeneous crystallization of CaOx up to a pH of 6.1, whereas CaP was the type of crystalline material that precipitated at higher pH. Accordingly, small crystal volumes were recorded at pH 5.5 and great volumes at pH 6.7 4 h after the addition of CaP crystals to the solutions. Dialyzed urine appeared to counteract the dissolution of CaP and to reduce the rate of secondary crystallization. The CaP induced crystallization of CaOx was confirmed by a reduction of ¹⁴C-labeled oxalate in solution. The AP_CaOx required for a nucleation or heterogeneous crystallization of CaOx in the presence of CaP was around 1.5 × 10⁻⁸ (mol/l)². For CaP crystal formation on CaP, an AP_CaP (^aCa²⁺ × ^aPO₄ ³⁻) of approximately 50 × 10⁻¹⁴ (mol/l)² appeared to be necessary. The CaOx crystals formed were microscopically found in association with the CaP crystalline material and were most frequently of CaOx dihydrate type. Step-wise crystallization experiments comprising supersaturation with CaP (Step A), supersaturation with CaOx (Step B) and subsequently acidification (Step C) showed that CaOx crystal formation occurred when CaP crystals were dissolved and thereby served as a source of calcium. The ensuing formation of CaOx crystals is most likely the result from high local levels of supersaturation with CaOx caused by the increased concentration of calcium. These experimental studies give support to the hypothesis that crystallization of CaOx at lower nephron levels or in caliceal urine might be induced by dissolution of CaP formed at nephron levels above the CD, and that a low pH is prerequisite for the precipitation of CaOx. The observations accordingly provide additional evidence for the important role of calcium phosphate in the crystallization of calcium oxalate, that might occur both at the surface of Randall’s plaques and intratubularly at the papillary tip. Parts of these studies were presented at the Scanning Microscopy Meeting 1996, at the International Symposium on Urolithiasis, Dallas 1996 and at the Eurolithiasis meeting in Istanbul 1998.     

Resolution-enhanced fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigations in thev 3 PO4 domain

Summary Resolution-enhanced Fourier Transform Infrared (FTIR) spectra of early mineral deposits in enamel and bone show bands at 1020, 1100, 1110, 1125, and 1145 cm⁻¹ in thev ₃PO₄ domain which do not belong to well crystallized stoichiometric hydroxyapatite. Bands at 1020 and 1100 cm⁻¹ have been shown to occur in nonstoichiometric apatites containing HPO ₄ ²⁻ ions. Though the bands at 1110 and 1125 cm⁻¹ have not been found in any well crystallized apatite, they are present in newly precipitated apatite. These latter bands disappear progressively during maturation in biological as well as synthetic samples, and partial dissolution of synthetic apatites shows that they belong to species that exhibit an inhomogeneous distribution in the mineral, and that are the first to be solubilized. Comparison of the FTIR spectra of biological apatites with those of synthetic, nonapatitic-containing phosphate minerals shows that the presence of these bands does not arise from nonapatitic, well-defined phases; they are due to the local environment of phosphate ions which may possibly be loosely related or perhaps unrelated to the phosphate groups present in the well-crystallized nonapatitic calcium phosphates. Resolution-enhanced FTIR affords a very precise characterization of the mineral phases which may be very useful in characterizing pathological deposits of Ca−P mineral phases.     

The growth of calcium phosphates on natural enamel

The kinetics of growth of calcium phosphate on seed materials prepared from human tooth enamel has been studied at 25°C and pH 7.40 in stable supersaturated solutions of calcium phosphate. The grown material was characterized chemically, by specific surface area (SSA) measurements and by subsequent dissolution experiments. The marked increase in SSA in the early stages of the precipitation reaction parallel the results obtained with synthetic hydroxyapatite seed crystals and the results of the dissolution experiments indicate a chemical composition close to that of octacalcium phosphate for this intermediate phase. At low supersaturation it is possible to avoid the formation of the precursor phase.