Dual roles of brushite crystals in calcium oxalate crystallization provide physicochemical mechanisms underlying renal stone formation.

Calcium oxalate monohydrate (COM) crystals are the major mineral component of most kidney stones, and thus have an important role in chronic human disease. However, the physicochemical mechanisms leading to calcium oxalate (CaOx) stone disease are only partially defined. As spontaneous precipitation of CaOx is rare under renal conditions, an alternative pathway for CaOx crystallization seems necessary to resolve this central issue. We performed kinetic studies using the dual constant composition method to simultaneously analyze the crystallization of COM and brushite, the form of calcium phosphate that is most readily formed in the typical slightly acidic urinary milieu. These studies were supported by parallel analysis by scanning electron and atomic force microscopy. In these studies, mineralization of a thermodynamically stable phase (COM) was induced by the presence of brushite, a more readily precipitated inorganic phase. Furthermore, once formed, the COM crystals grew at the expense of brushite crystals causing the dissolution of the brushite crystals. These studies show that brushite may play crucial roles in the formation of COM crystals. The definition of these two roles for brushite thereby provides physicochemical explanations for the initiation of COM crystallization and also for the relative paucity of calcium phosphate detected in the majority of CaOx renal stones.

Enamel demineralization in primary and permanent teeth.

Although enamel demineralization is important for our understanding of caries formation, no consensus has been reached regarding the possible differences in susceptibility of primary and permanent enamel. We used the constant composition (CC) technique to investigate the acid-induced demineralization of these tissues at a relative undersaturation with respect to hydroxyapatite (HAP) of 0.902, pH = 4.5, and ionic strength = 0.15 mol L(-1). The demineralization rates showed significant differences, primary enamel having the greater susceptibility to dissolution during an initial linear stage: 1.5 +/- 0.5 x 10(-10) mol mm(-2) min(-1) compared with 2.6 +/- 0.5 x 10(-11) mol mm(-2) min(-1) for permanent enamel. During the reactions, we observed nanosized crystallites which attached to the enamel surfaces or escaped into the bulk solution. These nanosized crystallites were kinetically protected against further dissolution, even though the solutions remained undersaturated. It is hypothesized that they may contribute to the remarkable mechanical and dynamic characteristics of enamel.

Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite.

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to bone mineral also contributes to their potency and duration of action. The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension. Affinity constants (K(L)) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant composition method at 37 degrees C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone, there were significant differences in K(L) among the bisphosphonates for HAP growth (pH 7.4) with a rank order of zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their side-chain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate and zoledronate compared with the more readily reversible effects of etidronate and risedronate.

Influence of laser irradiation on the constant composition kinetics of enamel dissolution.

The influence of 9.6 microm CO2 laser irradiation on enamel dissolution kinetics was investigated using a constant composition method designed for rate measurements of enamel dissolution as a function of depth, on a micrometer scale. In contrast to lower irradiation intensities (< or =1.0 J cm(-2)), which consistently showed reduced dissolution rates, higher fluences (energy per surface area) resulted in initially increased dissolution rates, which rapidly decreased, after dissolution times corresponding to removal of a few micrometers, to rates similar to those acquired using lower fluences. It was also demonstrated that surface damage during laser irradiation could be limited to the first 1-2 microm by lowering the number of pulses per spot during the irradiation procedure. The constant composition method can be used to measure detailed kinetics of inhibition of acid dissolution of dental enamel that has been treated by low fluence 9.6-microm CO2 laser irradiation.

Molecular modulation of calcium oxalate crystallization by osteopontin and citrate.

Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is a source of chronic human disease, forming the major inorganic component of kidney stones. Understanding molecular mechanisms of biological control over COM crystallization is central to development of effective stone disease therapies and can help define general strategies for synthesizing biologically inspired materials. To date, research on COM modification by proteins and small molecules has not resolved the molecular-scale control mechanisms. Moreover, because proteins directing COM inhibition have been identified and sequenced, they provide a basis for general physiochemical investigations of biomineralization. Here, we report molecular-scale views of COM modulation by two urinary constituents, the protein osteopontin and citrate, a common therapeutic agent. Combining force microscopy with molecular modeling, we show that each controls growth habit and kinetics by pinning step motion on different faces through specific interactions in which both size and structure determine the effectiveness. Moreover, the results suggest potential for additive effects of simultaneous action by both modifiers to inhibit the overall growth of the crystal and demonstrate the utility of combining molecular imaging and modeling tools for understanding events underlying aberrant crystallization in disease.

Mechanism of dissolution of sparingly soluble electrolytes.

Recent constant composition dissolution studies of sparingly soluble calcium phosphates have revealed an interesting and unusual behavior in that the rates decreased, eventually resulting in effective suppression, even though the solutions remained undersaturated. Contrary to traditional theories of dissolution, these experimental results indicated the importance of not only the particle size on the dissolution rate but also the participation of critical phenomena. In these theories, it is assumed that when the dissolution reactions are initiated, they continue spontaneously until all solid phase has disappeared. In terms of these mechanisms, there are no critical phenomena in the dissolution mechanism. Although the crystal size decreases during dissolution, when the reaction is controlled by polypitting (formation and growth of pits), the edge free energy increases at the very first stage due to the creation of pits and dissolution steps. The constant composition experimental results demonstrate the development of surface roughness as the dissolution steps are formed, implying an increase of the total edge length during the reactions. In an exactly analogous mechanism to crystal growth, the participation of critical conditions involving dissolution steps is a possibility. In contrast to crystal growth, dissolution is a process of size reduction and, when the particle size is sufficiently reduced, critical phenomena become important so that the influence of size must be taken into consideration. This paper proposes such a model for dissolution reactions, and although these unusual phenomena probably apply to all mineral phases, they are more evident for sparingly soluble electrolytes in which the critical conditions are attained much more readily.

Nucleation at surfaces: the importance of interfacial energy.

The nucleation and growth of stone-forming minerals on the surfaces of other crystalline phases, cellular material, and immobilized macromolecules must be important in the formation of stones in the urinary tract. The nucleation and growth of calcium oxalate monohydrate (COM) crystals were studied using the constant composition kinetics technique, in solution supersaturated with respect to COM (sigmaCOM = 1.44). The solid phases during the reaction were examined by x-ray diffraction, scanning electron microscopy, and diffuse reflectance Fourier transform infrared spectroscopy. Human serum albumin was found to nucleate COM crystals when immobilized on hydroxyapatite (HAP) surfaces. The induction period for nucleation of COM on HAP surfaces preadsorbed with albumin significantly decreased to about 65 min from about 230 min for pure HAP particles. The initial growth rate of COM on pure HAP particles, Rm approximately/= 0.56 X 10(-7) mol/min per m2, was slower than that for HAP surfaces preadsorbed with albumin, 2.14 x 10(-7) mol/min per m2. The surface properties were characterized using contact angle measurements by sessile drop and thin layer wicking. The thermodynamic results suggested that surfaces with high Lewis base parameter values (gamma-) and low interfacial tension with water (gammaSL) are more effective in the nucleation and growth of crystal phases.

Determination of interfacial tension from crystallization and dissolution data: a comparison with other methods.

Methods for the determination of interfacial tension between a solid and a liquid are reviewed including solubility/particle size, crystallization and dissolution kinetics. The use of solubility as a function of particle size, originally put forward by Ostwald and later corrected by Freundlich, may be unjustified for determining interfacial tension at solid-liquid interfaces. The interfacial tension values between solutions and sparingly soluble minerals such as hydroxyapatite, fluorapatite, brushite, octacalcium phosphate, calcium oxalate monohydrate, barium sulfate, calcium sulfate, calcite, and divalent metal fluorides are discussed. A comparison of these results is made with contact angle or wetting measurements. The interfacial tension values obtained from constant composition reaction kinetics are of the same order of magnitude as those determined using a contact angle method involving thin layer wicking techniques.

Heterogeneous nucleation of calcium phosphates on solid surfaces in aqueous solution.

The heterogeneous nucleation of calcium phosphates on solid surfaces of poly(methyl methacrylate) (PMMA), poly-(tetrafluoroethylene-co-hexafluoropropylene) (FEP), silicone rubber, mica, and radiofrequency glow discharge (RFGD)-treated PMMA, FEP, and silicone rubber has been studied in solutions supersaturated with respect to hydroxyapatite. The surface properties of the substrates were characterized by contact angle measurements. For the RFGD-treated surfaces, the Lifshitz-Van der Waals surface tension component changes very little, but the Lewis acid-base surface tension parameters vary greatly depending upon the materials. With scanning electron microscopy, nucleation of calcium phosphates was observed only on the surfaces: mica, RFGD-treated PMMA and FEP, with relatively high values of the Lewis base surface tension parameter. The more hydrophobic surfaces having low Lewis acid-base surface tensions, untreated PMMA and FEP, silicone rubber, and even RFGD-treated silicone rubber showed no nucleation.

The relationship between surface free-energy and kinetics in the mineralization and demineralization of dental hard tissue.

The interfacial free-energy is an important factor in the regulation of mineralization and dissolution at the surfaces of dental hard tissues. However, few thermodynamic studies have been aimed at the elucidation of the interfacial terms. Contact angle measurements (sessile drop and thin layer wicking) and kinetic dissolution and growth techniques have been used to study the interfacial properties of root dentin (D), human enamel (E), and hydroxyapatite (HAP). The interfacial tensions between water (w) and each of these phases were calculated from contact angle data according to surface tension components theory. The values gamma wD = 4.5 x 10(-3) J m-2, gamma wE = 8.8 x 10(-3) J m-2, and gamma w,HAP = 10.4 x 10(-3) J m-2 were of the same order of magnitude as those obtained from dissolution kinetic data (pH = 4.5): gamma wD = 1.4 x 10(-3) J m-2, gamma wE = 3.2 x 10(-3) J m-2, and gamma wHAP = 9.3 x 10(-3) J m-2. Kinetics studies of the crystallization of HAP on HAP, dentin, and enamel yielded the interfacial free-energy values, gamma wHAP = 17.1 x 10(-3) J m-2, 17.7 x 10(-3) J m-2, and 9.4 x 10(-3) J m-2, respectively, probably reflecting the interfacial energies of the deposited phases rather than those of the dental hard-tissue substrata. The lower interfacial tension values are consistent with the higher solubilities of these solid phases: logKSO = -52.0, -55 approximately 57, and -58 approximately 59, for root dentin, enamel, and HAP, respectively, expressed as an equivalent HAP ionic product. The higher interfacial free-energy is also consistent with the slower mineralization of HAP on dentin and enamel surfaces.

Constant composition dissolution kinetics studies of human dentin.

The constant composition (CC) method has been used to study the dissolution kinetics of whole powdered human dentin as a function of calcium phosphate concentration at relative undersaturations with respect to hydroxyapatite (sigma HAP), ranging from +0.8 to -2.8, ionic strength from 0.05 to 0.30 mol/L-1 in sodium chloride or potassium nitrate, pH 4.00 to 5.50, and molar calcium/phosphate ratio in the reaction solutions from 0.05 to 11.1. The results suggest that human dentin behaves as a mixture of at least two calcium phosphate phases, HAP-like and octacalcium phosphate-like, OCP-like. Significant dissolution took place in solutions that were even supersaturated with respect to HAP, and the rates exhibited a striking insensitivity to relative undersaturation, while influenced by ionic strength, pH, and molar calcium/phosphate ratio in the reaction solutions. Although the dissolution was retarded in the presence of magnesium ion, the reaction rate showed the same insensitivity to undersaturation with respect to calcium phosphate.

Pre-conditioning and dual constant composition dissolution kinetics of pulsed laser deposited hydroxyapatite thin films on silicon substrates.

The kinetics of dissolution of pulsed laser deposited crystalline and amorphous thin films of hydroxyapatite on silicon substrates were measured at 37 degrees C and a pH value of 6.5 using the dual constant composition method. Solutions in which the pulsed laser deposited films were pre-conditioned (0.15 M NaCl) remained undersaturated or slightly supersaturated with respect to hydroxyapatite after equilibrium was reached, indicating only a very small coating release and the absence of re-precipitation on the surfaces. The amorphous films released more calcium and phosphate during pre-conditioning than the more crystalline films. Dual constant composition dissolution rates decreased as film crystallinity increased. The film with the lowest dissolution rate (approximately one sixth that of a crystalline film deposited using a hydroxyapatite powder target) was fabricated using a human tooth as the laser target. During pre-conditioning of plasma-sprayed coatings, more calcium and phosphate were released than for pulsed laser deposited films, and dual constant composition dissolution rates were much higher.

The nucleation and growth of calcium phosphate crystals at protein and phosphatidylserine liposome surfaces.

The kinetics of calcium phosphate crystal growth at the surfaces of proteins and phospholipids has been investigated using free drift and constant composition methods in supersaturated calcium phosphate solutions (relative supersaturations: with respect to hydroxyapatite, HAP, sigma HAP = 15.0, and with respect to octacalcium phosphate, OCP, sigma OCP = 1.9). Fibrinogen and collagen molecules adsorbed at hydrophobic surfaces as well as uncross-linked collagen fibrils induce ion binding and subsequent nucleation of calcium phosphate. The formation of OCP on phosphatidylserine vesicles introduced to highly supersaturated calcium phosphate solutions probably involves the interaction of the calcium ions with the ionized carboxylic groups of the phospholipid.

Degradation potential of plasma-sprayed hydroxyapatite-coated titanium implants.

The purpose of this study was to develop an analytical method for evaluating the dissolution behavior of plasma-sprayed hydroxyapatite (HAP) coatings. Six commercially available and clinically used coatings applied to nonporous titanium coupons according to their respective specifications for orthopedic devices were used in this study. Dissolution behavior was monitored by first preconditioning the implant in 0.15 mol L-1 sodium chloride solution at 37 degrees C until either equilibrium or the desired change in solution hydrogen and calcium ion concentrations had been reached. In the second step, the implants were subjected to dual constant composition (DCC) dissolution under conditions of controlled undersaturation. Results indicate that the dissolution rates of the HAP coatings may differ by as much as a factor of 5 despite the fact that analytical techniques, including X-ray diffraction and FTIR, indicate no change in crystallinity or composition of the coatings before and after the solution treatment. These results indicate that HAP coatings from different sources react very differently when placed in the same local aqueous environment. However, more work will be necessary before these in vitro results can be used to predict in vivo behavior.

Dual constant composition kinetics characterization of apatitic surfaces.

The use of calcium phosphate phases as implant materials in forms such as ceramic hydroxyapatite (CHAP) and hydroxyapatite plasma-coated implants (HPCTI) as well as the synthetic phases is now quite well established. Although conventional physical chemical characterization methods such as X-ray diffraction may indicate the exclusive presence of hydroxyapatite (HAP), there is little doubt that other calcium-containing surface impurities play important roles in the initial reactions that take place when such materials are brought into contact with aqueous phases. These surface impurities usually dissolve rapidly with the release of excess calcium and hydroxide ions. Based on the constant composition (CC) method, the dual constant composition (DCC) approach has been developed for investigating the dissolution kinetics behavior of these apatitic materials in an attempt to characterize and modify the surfaces, and possibly to predict their behavior when placed in vivo. The results of this study confirm the presence of highly reactive heterogeneous calcium-containing phases on the surface of materials such as CHAP and HPCTI, as evidenced by the release of appreciable amounts of calcium and hydroxyl ions upon introduction of these surfaces in saline solutions. Furthermore, these calcium phosphate phases exhibited a unique dissolution behavior when compared with synthetically prepared phases such as HAP and beta-tricalcium phosphate (beta-TCP), as suggested by the observation that they dissolve in solutions supersaturated with respect to HAP.

Hydroxyapatite mineralization and demineralization in the presence of synthetic phosphorylated pentapeptides.

A constant composition (CC) method was used to compare the influence of statherin-like N-terminal 5-residue fragments having different amino acids in the terminal position on hydroxyapatite (HAP) growth and dissolution. The CC experiments were done in solutions containing 4.00 x 10(-4) mol/l calcium and 2.40 x 10(-4) mol/l phosphate. The solutions used in the crystallization studies were supersaturated only with respect to HAP (pH = 7.40, sigma HAP = 3.60). The CC dissolution studies were done in solutions undersaturated with respect to HAP (pH = 6.00; sigma HAP = -0.39). The HAP mineralization and demineralization processes were markedly inhibited by the negatively charged pentapeptides. Those containing a phosphorylated terminal residue inhibited dissolution to a greater extent than the native statherin fragment having aspartate as the N-terminal residue. Strong dependencies of the degree of inhibition of growth and dissolution reaction rates on the extents of reaction were noted. As the reactions proceeded, the rate inhibition decreased in the case of crystal growth and increased for dissolution.

Calculus formation and inhibition.

The formation, development, and dissolution of hard deposits such as calculus are complex processes that involve numerous calcium phosphate phases as well as the interaction of these ions with organic molecules. Although formation is determined by thermodynamic driving forces, kinetic factors are also important determinants for the precipitation of specific calcium phosphate phases. The overall process, therefore, may involve the formation of metastable intermediates which may subsequently transform into the more stable hard deposits observed in vivo. A knowledge of the kinetics of growth of both individual calcium phosphate phases and their mixtures is important for elucidating the mechanism of calculus formation. Although salivary proteins are effective inhibitors of the mineralization reactions that take place in dental plaque, once adsorbed, their conformation may change to present surfaces that catalyze the nucleation of mineral phases. The variable pH conditions in plaque, expressed in terms of free ionic concentrations, will markedly alter the supersaturations with respect to typical calcium phosphate precursor phases such as dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP). Physical-chemical studies have shown that the mineralization of all the calcium phosphate phases is controlled by reactions at the surface rather than by diffusion of lattice ions through the contacting liquid phase. This makes the rates of reaction very sensitive to ions and molecules in the solution that may absorb at the active growth sites and, while not significantly incorporating into the precipitated crystal phases, markedly influences the rates of mineralization and demineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of histatin-5 fragments on the mineralization of hydroxyapatite.

The adsorption of histatin 5 on hydroxyapatite (HAP) was determined and compared to that of several fragments of histatin 5, such as residues 1-16 (N16), 7-16 (M10), 9-24 (C16), 11-24 (C14), 13-24 (C12), 15-24 (C10). The influence of the adsorbed peptides on the seeded crystal growth of HAP was investigated with the constant composition method. The adsorption affinity of the peptides as well as their ability to inhibit mineralization was influenced by the length of the peptide chain. Histatin 5 showed the highest affinity, as determined by a Langmuir model, whereas the smaller C10 and C12 displayed the lowest equilibrium uptake. The smaller C10 and C12 peptides were, on the other hand, more effective as crystal growth inhibitors, indicating a more efficient coverage of surface active sites. Electrophoretic mobility data indicated an increase in the positive charge at the HAP surface in the presence of these peptides, which were efficient HAP crystallite dispersants.

Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization.

The structural domains of salivary statherin that are partly responsible for the protection and recalcification of tooth enamel were examined with respect to charge, sequence, hydrophobicity, hydrogen bonding potential, and conformation. Several fragments of statherin, 1-15 (SN15), 5-15 (SN11), 15-29 (SM15), 29-43 (SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized. The abilities of these fragments to adsorb at hydroxyapatite (HAP) surfaces and to inhibit its mineralization in supersaturated solutions were determined and compared with those of the whole statherin molecule, reported previously. The conformational preferences of the fragments both in aqueous and nonaqueous solutions were examined by circular dichroism. The highly charged N-terminal SN15 fragment has the greatest adsorption to HAP as compared with statherin and all other fragments. Its mineralization inhibitory activity is significantly greater than those of other fragments and comparable with that of the whole molecule. The dephosphorylated N-terminal fragment SNS15 shows a decreased tendency to adhere to and inhibit the formation of HAP, as compared with SN15. However, the substitution of Asp residues in place of phosphoserines (SNA15), restores the binding affinity and crystal growth inhibition properties, suggesting that the negative charge density at the N-terminal rather than any specific interaction of the phosphate group is important for HAP surface interactions. The C-terminal SC15 and SC25 fragments elicit a much higher affinity for HAP surface than that of the middle sequence (SM15), indicating that hydrogen bonding potential of the C-terminal sequence also contributes to the interaction of statherin with HAP. CD studies provide evidence that the N-terminal SN15 fragment has a strong tendency to adopt an ordered helical conformation, whereas the shorter N-terminal sequence, middle, and C-terminal fragments are structurally flexible and prefer to adopt scattered turn structures or unordered random conformations in organic and aqueous solutions. Collectively, the data indicate that the negative charge density, sequence (1-15), and helical conformation at the N-terminal region of statherin are important for its surface interaction with HAP.