Aggregation of Calcium Phosphate and Oxalate Phases in the Formation of Renal Stones.

The majority of human kidney stones are comprised of multiple calcium oxalate monohydrate (COM) crystals encasing a calcium phosphate nucleus. The physiochemical mechanism of nephrolithiasis has not been well determined on the molecular level; this is crucial to the control and prevention of renal stone formation. This work investigates the role of phosphate ions on the formation of calcium oxalate stones; recent work has identified amorphous calcium phosphate (ACP) as a rapidly forming initial precursor to the formation of calcium phosphate minerals in vivo. The effect of phosphate on the nucleation of COM has been investigated using the constant composition (CC) method in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings indicate COM nucleation is strongly promoted by the presence of phosphate; this occurs at relatively low phosphate concentrations, undersaturated with respect to brushite (dicalcium phosphate dehydrate, DCPD) formation. The results show that ACP plays a crucial role in the nucleation of calcium oxalate stones by promoting the aggregation of amorphous calcium oxalate (ACO) precursors at early induction times. The coaggregations of ACP and ACO precursors induce the multiple-point nucleation of COM. These novel findings expand our knowledge of urinary stone development, providing potential targets for treating the condition at the molecular level.

Tracking Amorphous Precursor Formation and Transformation during Induction Stages of Nucleation.

Hydroxyapatite (HAP) participates in vertebral bone and tooth formation by a nonclassical hitherto unknown nucleation mechanism, in which amorphous precursors form and transform during long induction periods. Elucidation of the mechanism by which amorphous precursors assemble and transform is essential to understanding how hard tissues form in vivo and will advance the design and fabrication of new biomaterials. The combination of conductance and potentiometric techniques to monitor Ca-P mineral formation has given new insight into the mechanism of nucleation. Differences detected in the dehydration rates of calcium and phosphate ions indicate the formation of nonequilibrium calcium-deficient clusters. The aggregation of these clusters forms a calcium-deficient amorphous phase I [Ca-(HPO4)1+x ·nH2O]2x-) early in the induction period, which slowly transforms to amorphous phase II [Ca-(HPO4)·mH2O] by dehydration. Precritical nuclei form within amorphous phase II later in the induction period, leading to mineral formation.

Kinetics of canine dental calculus crystallization: an in vitro study on the influence of inorganic components of canine saliva.

This work identifies carbonated hydroxyapatite (CAP) as the primary component of canine dental calculus, and corrects the long held belief that canine dental calculus is primarily CaCO3 (calcite). CAP is known to be the principal crystalline component of human dental calculus, suggesting that there are previously unknown similarities in the calcification that occurs in these two unique oral environments. In vitro kinetic experiments mimicking the inorganic components of canine saliva have examined the mechanisms of dental calculus formation. The solutions were prepared so as to mimic the inorganic components of canine saliva; phosphate, carbonate, and magnesium ion concentrations were varied individually to investigate the roll of these ions in controlling the nature of the phases that is nucleated. To date, the inorganic components of the canine oral systems have not been investigated at concentrations that mimic those in vivo. The mineral composition of the synthetic calculi grown under these conditions closely resembled samples excised from canines. This finding adds new information about calculus formation in humans and canines, and their sensitivity to chemicals used to treat these conditions.

Dynamics of Biomineralization and Biodemineralization.

In order to understand the fundamental processes leading to biomineralization, this chapter focuses on the earliest events of homo/heterogeneous nucleation from an initial supersaturated solution phase and subsequent growth involving various possible precursor phases (amorphous or crystalline) to the final mineral phase by specific template and other influences. We also discuss how the combination of macroscopic constant composition and microscopic atomic force microscopy provides insights into the physical mechanisms of crystal growth and phase stability and the influences of proteins, peptides or other small molecules.Biodemineralization reactions of tooth enamel and bone may be inhibited or even suppressed when particle sizes fall into certain critical nanoscale levels. This phenomenon actually involves particle-size-dependent critical conditions of energetic control at the molecular level. Clearly, this dissolution termination is a kinetic phenomenon and cannot be attributed to reaction retardation as a result of surface modification by additives. Almost all biomineralized structures are highly hierarchical at many different length scales. At the lowest level they often consist of tiny crystals, typically tens to hundreds of nanometers. This size is not arbitrary; rather, it seems to give biominerals such as bone and tooth remarkable physical characteristics.

Calcium oxalate: calcium phosphate transformations.

Knowledge of the physical-chemical mechanisms responsible for the crystal growth and dissolution events involved in stone formation might enable the manipulation of thermodynamics in such a way as to increase the solubility of sparingly soluble phases (such as calcium oxalates and phosphates), thereby reducing the driving force for stone formation. This may be accomplished through modification of pH, reduction of supersaturation with respect to nucleating phases, and the presence of key inhibitors. If these modifications are made during the initial stages of crystallite nucleation, they could potentially reduce the participation of phases such as Randall's plaques in stone formation.

Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo.

Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647-labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14(high) bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14(+) cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

Molecular mechanisms of crystallization impacting calcium phosphate cements.

The biomineral calcium hydrogen phosphate dihydrate (CaHPO(4).2H(2)O), known as brushite, is a malleable material that both grows and dissolves faster than most other calcium minerals, including other calcium phosphate phases, calcium carbonates and calcium oxalates. Within the body, this ready formation and dissolution can play a role in certain diseases, such as kidney stone and plaque formation. However, these same properties, along with brushite's excellent biocompatibility, can be used to great benefit in making resorbable biomedical cements. To optimize cements, additives are commonly used to control crystallization kinetics and phase transformation. This paper describes the use of in situ scanning probe microscopy to investigate the role of several solution parameters and additives in brushite atomic step motion. Surprisingly, this work demonstrates that the activation barrier for phosphate (rather than calcium) incorporation limits growth kinetics and that additives such as magnesium, citrate and bisphosphonates each influence step motion in distinctly different ways. Our findings provide details of how, and where, molecules inhibit or accelerate kinetics. These insights have the potential to aid in designing molecules to target specific steps and to guide synergistic combinations of additives.

How amelogenin orchestrates the organization of hierarchical elongated microstructures of apatite.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving a coassembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.

Pathways to biomineralization and biodemineralization of calcium phosphates: the thermodynamic and kinetic controls.

Although extensive investigations of calcium phosphate crystallization have been performed, many have focused only on the final structures and morphologies and have not emphasized the need to consider the molecular contacts between mineral and matrix that drive nucleation nor the thermodynamic and kinetic controls imposed by matrix and soluble proteins during the nucleation stage. This review focuses on the earliest events of homo/heterogeneous nucleation from an initial supersaturated solution phase and subsequent growth. We also discuss how the combination of macroscopic constant composition (CC) and microscopic atomic force microscopy (AFM) provides insights into the physical mechanisms of crystal growth and phase stability and the influences of proteins, peptides or other small molecules. In addition, a new model for nanoscale enamel and bone demineralization suggests biodemineralization reactions may be inhibited or even suppressed when particle sizes fall into certain critical nanoscale levels. This size is not arbitrary; rather, it seems to give biominerals such as bones and teeth remarkable physical characteristics including self-preservation in the fluctuating physiological milieu.

Surface aggregation of urinary proteins and aspartic Acid-rich peptides on the faces of calcium oxalate monohydrate investigated by in situ force microscopy.

The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin, and the 27-residue synthetic peptides (DDDS)(6)DDD and (DDDG)(6)DDD (D = aspartic acid, S = serine, and G = glycine) was investigated via in situ atomic force microscopy. The results show that these four growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition of or an increase in the step speeds (with respect to the impurity-free system), depending on a range of factors that include peptide or protein concentration, supersaturation, and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we propose a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at the crystal surface.

Phosphorylation of osteopontin is required for inhibition of calcium oxalate crystallization.

Under near-physiological pH, temperature, and ionic strength, a kinetics constant composition (CC) method was used to examine the roles of phosphorylation of a 14 amino acid segment (DDVDDTDDSHQSDE) corresponding to potential crystal binding domains within the osteopontin (OPN) sequence. The phosphorylated 14-mer OPN peptide segment significantly inhibits both the nucleation and growth of calcium oxalate monohydrate (COM), inhibiting nucleation by markedly increasing induction times and delaying subsequent growth by at least 50% at concentrations less than 44 nM. Molecular modeling predicts that the doubly phosphorylated peptide binds much more strongly to both (-101) and (010) faces of COM. The estimated binding energies are, in part, consistent with the CC experimental observations. Circular dichroism spectroscopy indicates that phosphorylation does not result in conformational changes in the secondary peptide structure, suggesting that the local binding of negatively charged phosphate side chains to crystal faces controls growth inhibition. These in vitro results reveal that the interactions between phosphorylated peptide and COM crystal faces are predominantly electrostatic, further supporting the importance of macromolecules rich in anionic side chains in the inhibition of kidney stone formation. In addition, the phosphorylation-deficient form of this segment fails to inhibit COM crystal growth up to concentrations of 1450 nM. However, at sufficiently high concentrations, this nonphosphorylated segment promotes COM nucleation. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) results confirm that aggregation of the nonphosphorylated peptide segment takes place in solution above 900 nM when the aggregated peptide particles may exceed a well-defined minimum size to be effective crystallization promoters.

An understanding of renal stone development in a mixed oxalate-phosphate system.

The in vivo formation of calcium oxalate concretions having calcium phosphate nidi is simulated in an in vitro (37 degrees C, pH 6.0) dual constant composition (DCC) system undersaturated (sigma DCPD = -0.330) with respect to brushite (DCPD, CaHPO 4 . 2H 2O) and slightly supersaturated (sigma COM = 0.328) with respect to calcium oxalate monohydrate (COM, CaC2O4 . H2O). The brushite dissolution provides calcium ions that raise the COM supersaturation, which is heterogeneously nucleated either on or near the surface of the dissolving calcium phosphate crystals. The COM crystallites may then aggregate, simulating kidney stone formation. Interestingly, two intermediate phases, anhydrous dicalcium phosphate (monetite, CaHPO4) and calcium oxalate trihydrate (COT), are also detected by X-ray diffraction during this brushite-COM transformation. In support of clinical observations, the results of these studies demonstrate the participation of calcium phosphate phases in COM crystallization providing a possible physical chemical mechanism for kidney stone formation.

Bisphosphonate binding affinity as assessed by inhibition of carbonated apatite dissolution in vitro.

Bisphosphonates (BPs), which display a high affinity for calcium phosphate surfaces, are able to selectively target bone mineral, where they are potent inhibitors of osteoclast-mediated bone resorption. The dissolution of synthetic hydroxyapatite (HAP) has been used previously as a model for BP effects on natural bone mineral. The present work examines the influence of BPs on carbonated apatite (CAP), which mimics natural bone more closely than does HAP. Constant composition dissolution experiments were performed at pH 5.50, physiological ionic strength (0.15M) and temperature (37 degrees C). Selected BPs were added at (0.5 x 10(-6)) to (50.0 x 10(-6))M, and adsorption affinity constants, K(L), were calculated from the kinetics data. The BPs showed concentration-dependent inhibition of CAP dissolution, with significant differences in rank order zoledronate > alendronate > risedronate. In contrast, for HAP dissolution at pH 5.50, the differences between the individual BPs were considerably smaller. The extent of CAP dissolution was also dependent on the relative undersaturation, sigma, and CAP dissolution rates increased with increasing carbonate content. These results demonstrate the importance of the presence of carbonate in mediating the dissolution of CAP, and the possible involvement of bone mineral carbonate in observed differences in bone affinities of BPs in clinical use.

Mimicking the Self-Organized Microstructure of Tooth Enamel.

Under near-physiological pH, temperature, and ionic strength, amelogenin (Amel) accelerates hydroxyapatite (HAP) nucleation kinetics, decreasing the induction time in a concentration-dependent manner. Hierarchically organized apatite microstructures are achieved by self-assembly involving nucleated nanocrystallites and Amel oligomers and nanospheres at low supersaturations and protein concentrations in a slow and well-controlled constant composition (CC) system. The CC method allows the capture of an intermediate structure, the nanorod, following the formation of the critical nuclei at the earliest nucleation stages of calcium phosphate crystallization. The nanorod building blocks form spontaneously by synergistic interactions between flexible Amel protein assemblies and rigid calcium phosphate nanocrystallites. These intermediate structures further assemble by a self-epitaxial growth mechanism to form the final hierarchically organized microstructures that are compositionally and morphologically similar to natural enamel. This in vitro observation provides direct evidence that Amel promotes apatite crystallization and organization. We interpret our observations to propose that in vivo Amel may maximally exert an influence on the structural control of developing enamel crystals at the earliest nucleation stages.

Amelogenin Promotes the Formation of Elongated Apatite Microstructures in a Controlled Crystallization System.

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that play a pivotal role in controlling the oriented and elongated growth of highly ordered apatitic crystals during enamel biomineralization. However, the mechanisms of amelogenin-mediated mineralization have not yet been fully elucidated. Here we report that amelogenin dramatically accelerates the nucleation kinetics by decreasing the induction time in a dose-dependent manner in a controlled constant composition (CC) in vitro crystallization system. Remarkably, at very low protein concentrations, elongated microstructures which are similar in appearance to apatitic crystals in enamel were formed at relatively low supersaturations, through interfacial structural match/synergy between structured amelogenin assemblies and apatite nanocrystallites. This heterogeneous crystallization study provides experimental evidence to support the concept that templating by amelogenin very early in the crystallization process facilitates the formation of developing enamel crystals.

Modulation of calcium oxalate crystallization by linear aspartic acid-rich peptides.

Calcium oxalate monohydrate (COM) kidney stone formation is prevented in most humans by urinary crystallization inhibitors. Urinary osteopontin (OPN) is a prototype of the aspartic acid-rich proteins (AARP) that modulate biomineralization. Synthetic poly(aspartic acids) that resemble functional domains of AARPs provide surrogate molecules for exploring the role of AARPs in biomineralization. Effects of linear aspartic acid-rich peptides on COM growth kinetics and morphology were evaluated by the combination of constant composition (CC) analysis and atomic force microscopy (AFM). A spacer amino acid (either glycine or serine) was incorporated during synthesis after each group of 3 aspartic acids (DDD) in the 27-mer peptide sequences. Kinetic CC studies revealed that the DDD peptide with serine spacers (DDDS) was more than 30 times more effective in inhibiting COM crystal growth than the DDD peptide with glycine spacers (DDDG). AFM revealed changes in morphology on (010) and (-101) COM faces that were generally similar to those previously described for OPN and citrate, respectively. At comparable peptide levels, the effects of step pinning and reduced growth rate caused by DDDS were remarkably greater. In CC nucleation studies, DDDS caused a greater prolongation of induction periods than DDDG. Thus, nucleation studies link changes in interfacial energy caused by peptide adsorption to COM to the CC growth and AFM results. These studies indicate that, in addition to the number of acidic residues, the contributions of other amino acids to the conformation of DDD peptides are also important determinants of the inhibition of COM nucleation and growth.

Nanosized particles in bone and dissolution insensitivity of bone mineral.

Most of the mineral crystals in bone are platelets of carbonated apatite with thicknesses of a few nanometers embedded in a collagen matrix. We report that spherical to cylindrical shaped nanosized particles are also an integral part of bone structure observed by high resolution scanning electron microscopy. High resolution back scattered electron imaging reveals that the spherical particles have a contrast similar to the crystal platelets, suggesting that they are thus likely to have similar mineral properties. By means of constant composition (CC) dissolution of bone, similar sized nanoparticles are shown to be insensitive to demineralization and are thought to be dynamically stabilized due to the absence of active pits/defects on the crystallite surfaces. Similar reproducible self-inhibited dissolution was observed with these nanoparticles during CC dissolution of synthetic carbonated apatite. This result rules out the possible influence of complicating biological factors such as the possible presence of organic matrix components and other impurities. This phenomenon can be explained by a unique dissolution model involving size considerations at the nanoscale. The unexpected presence of nanoparticles in mature bone may also be due to the stabilization of some nanosized particles during the formation process in a fluctuating biological milieux.