Faces Of Calcium Oxalate Monohydrate Research Articles

The effects of different types of additives on growth of biomineral phases investigated by in situ atomic force microscopy

Interactions between different types of additives with specific crystal surfaces of mineral phases found in biominerals were investigated at nanometer and micrometer scales by in situ atomic force microscopy (AFM). Firstly, the inhibitory effect of magnesium ion (Mg2+) and that of a peptide molecule, L-aspartic acid 6mer (L-Asp6) on the growth of a fast growing [−1 0 −1] step on the (−1 0 1) face of calcium oxalate monohydrate (COM) crystal are quantitatively compared by the step speed data. Differences in the inhibitory effect are explained based on a De Yoreo model of step kinetics in detail. The step speed data show that Mg2+ had a little inhibitory effect on the growth of the [−1 0 −1] step, approaching a limiting value of about 15% reduction in the step speed, whereas L-Asp6 was substantially more effective in inhibiting the step growth with an ability to stop the growth completely. Secondly, the effects of a protein molecule, bovine serum albumin (BSA) and a pseudo protein-like organic particle PSPMA30-PDPA47 micelle on the growth of calcite on its {1 0 4} faces are compared. Both BSA and PSPMA30-PDPA47 micelle temporarily pinned the passing steps without or negligibly inhibiting the step growth overall. The BSA molecule remained at the crystal surface without incorporation into the crystal because the steps passed through it, pushing it up and out. However, in contrast to the BSA, the micelle was incorporated into the crystal by the passing steps. These results together with other relevant studies suggest that the decisive factor in determining for macromolecules to be incorporated into growing minerals or to remain intact on the mineral surface with no incorporation is the magnitude of binding strength of the macromolecules to the growing step against the step’s propagation force.

Reversible Inhibition of Calcium Oxalate Monohydrate Growth by an Osteopontin Phosphopeptide

Calcium oxalate, primarily as calcium oxalate monohydrate (COM), is the primary constituent of most kidney stones. Certain proteins, such as osteopontin (OPN), inhibit stone formation. The complexity of stone formation and the effects of urinary proteins at various stages of the process make it hard to predict the exact physiological roles of these proteins in growth inhibition. The inhibition of crystallization due to adsorbed impurities is usually explained in terms of a model proposed in 1958 by Cabrera and Vermilyea. In this model, impurities adsorb to growth faces and pin growth steps, forcing them to curve, thus impeding their progress via the Gibbs-Thomson effect. To determine the role of OPN in the biomineralization of kidney stones, crystal growth on the {010} face of COM was examined in real time with atomic force microscopy in the presence of a synthetic peptide corresponding to amino acids 65-80 (hereafter referred to as pOPAR) of rat bone OPN. We observed clear changes in the morphology of the growth-step structure and a decrease in step velocity upon addition of pOPAR, which suggest adsorption of inhibitors on the {010} growth hillocks. Experiments in which pOPAR was replaced in the growth cell by a supersaturated solution showed that COM hillocks are able to fully recover to their preinhibited state. Our results suggest that recovery occurs through incorporation of the peptide into the growing crystal, rather than by, e.g., desorption from the growth face. This work provides new insights into the mechanism by which crystal growth is inhibited by adsorbants, with important implications for the design of therapeutic agents for kidney stone disease and other forms of pathological calcification.

Mimicking the Biomolecular Control of Calcium Oxalate Monohydrate Crystal Growth: Effect of Contiguous Glutamic Acids

Scanning confocal interference microscopy (SCIM) and molecular dynamics (MD) simulations were used to investigate the adsorption of the synthetic polypeptide poly(l-glutamic acid) (poly-glu) to calcium oxalate monohydrate (COM) crystals and its effect on COM formation. At low concentrations (1 μg/mL), poly-glu inhibits growth most effectively in ⟨001⟩ directions, indicating strong interactions of the polypeptide with {121} crystal faces. Growth in <010> directions was inhibited only marginally by 1 μg/mL poly-glu, while growth in <100> directions did not appear to be affected. This suggests that, at low concentrations, poly-glu inhibits lattice-ion addition to the faces of COM in the order {121} > {010} ≥ {100}. At high concentrations (6 μg/mL), poly-glu resulted in the formation of dumbbell-shaped crystals featuring concave troughs on the {100} faces. The effects on crystal growth indicate that, at high concentrations, poly-glu interacts with the faces of COM in the order {100} > {121} > {010}. This mirrors MD simulations, which predicted that poly-glu will adsorb to a {100} terrace plane (most calcium-rich) in preference to a {121} (oblique) riser plane but will adsorb to {121} riser plane in preference to an {010} terrace plane (least calcium-rich). The effects of different poly-glu concentration on COM growth (1-6 μg/mL) may be due to variations between the faces in terms of growth mechanism and/or (nano)roughness, which can affect surface energy. In addition, 1 μg/mL might not be adequate to reach the critical concentration for poly-glu to significantly pin step movement on {100} and {010} faces. Understanding the mechanisms involved in these processes is essential for the development of agents to reduce recurrence of kidney stone disease.

Citrate Modulates Calcium Oxalate Crystal Growth by Face-Specific Interactions

Because of its ability to inhibit the growth of calcium oxalate monohydrate (COM) crystals, citrate plays an important role in preventing the formation of kidney stones. To determine the mechanism of inhibition, we studied the citrate-COM interaction using a combination of microscopic and simulation techniques. Using scanning confocal interference microscopy, we found that addition of citrate preferentially inhibits crystal growth in <100> and, to a lesser extent, <001> directions, suggesting that citrate adsorbs to the faces of COM in the order {100} > {121} > {010}. Scanning electron microscopy showed that the resulting crystals are plate shaped, with large {100} faces and rounded ends. Molecular-dynamics simulations predicted, however, that citrate interacts with the faces of COM in a different order, i.e. {100} > {010} > {121}. Our simulations showed that citrate molecules align with the rows of Ca²⁺ ions on the {010} face but do not form close contacts, presumably because of electrostatic repulsion by the carboxylate groups that project from the Ca²⁺-rich plane. We propose that this weak interaction is responsible for citrate’s limited inhibition of COM growth in <010> directions. Overall, these findings indicate that electrostatic interactions with the Ca²⁺-rich faces of COM crystals are responsible for the growth-modulating properties of citrate.

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)6DDD and (DDDG)6DDD (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 left( {bar{1}01} right) 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.

Kinetics of Calcium Oxalate Crystal Growth in the Presence of Osteopontin Isoforms: An Analysis by Scanning Confocal Interference Microcopy

Proteins that inhibit the growth and aggregation of calcium oxalate crystals play important roles in the prevention of kidney stone disease. One such protein is osteopontin (OPN), which inhibits the formation of calcium oxalate monohydrate (COM) in a phosphorylation-dependent manner. To determine the role of phosphate groups in the inhibition of COM growth by OPN, we used scanning confocal interference microscopy to compare the effects of highly phosphorylated OPN from cow milk, less phosphorylated OPN from rat bone, and nonphosphorylated recombinant OPN. COM growth was measured in the principal crystallographic directions <001>, <010>, and <100>, representing lattice-ion addition to {121}, {010}, and {100} faces, respectively. While the shapes of growth curves were very consistent from crystal to crystal, absolute growth rates varied widely. To control for this, results were expressed as changes in the aspect ratios <010>/<001> and <100>/<001>. Compared to control, bone OPN increased <010>/<001> and had no effect on <100>/<001>; milk OPN had no effect on <010>/<001>and decreased <100>/<001>; recombinant OPN had no significant effect on either aspect ratio. These findings indicate that milk OPN interacts with COM crystal faces in order of preference {100} > {121} approximately {010}, whereas bone OPN interacts in order of preference {100} approximately {121} > {010}. As {100} is the most Ca(2+)-rich face of COM, while {010} is the least Ca(2+)-rich, it appears that the OPN-mediated inhibition of COM growth occurs through a nonspecific electrostatic interaction between Ca(2+) ions of the crystal and phosphate groups of the protein.

Phosphorylation of Osteopontin Peptides Mediates Adsorption to and Incorporation into Calcium Oxalate Crystals

Phosphorylated peptides of osteopontin (OPN) have been shown to inhibit the growth of the {100} face of calcium oxalate monohydrate (COM). The inhibitory potency has been shown to be dependent on the phosphate content of the peptide. The purpose of this study is to better understand the means by which phosphate groups promote crystal growth inhibition by OPN peptides. Peptides of rat bone OPN 220–235 peptides have been synthesized with zero (P0), 1 (P1) or 3 (P3) phosphate modifications. COM crystals were grown in the presence of 0.1–10 μg of P0, P1 or P3. P0 incorporation into COM crystals was evident at 10 μg/ml of peptide, whereas the phosphorylated peptides P1 and P3 were incorporated at all tested concentrations. At 5 μg/ml of P3, COM crystals exhibited a ‘dumbbell’ morphology. To study the peptide-mineral interaction, surface frequency plots were constructed from molecular dynamics simulations of OPN peptide adsorption. Carboxylate and phosphate groups were found to adsorb in specific orientations to the COM {100} surface. In conclusion, it appears that the phosphate groups on OPN peptides are capable of interacting with the COM {100} surface. This interaction appears to increase the adsorption energy of the peptide to the surface, thus enhancing its inhibitory potency.

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.

Control of Calcium Oxalate Crystal Growth by Face-Specific Adsorption of an Osteopontin Phosphopeptide

Mineral-associated proteins have been proposed to regulate many aspects of biomineralization, including the location, type, orientation, shape, and texture of crystals. To understand how proteins achieve this exquisite level of control, we are studying the interaction between the phosphoprotein osteopontin (OPN) and the biomineral calcium oxalate monohydrate (COM). In the present study, we have synthesized peptides corresponding to amino acids 220-235 of rat bone OPN (pSHEpSTEQSDAIDpSAEK), one of several highly phosphorylated, aspartic-, and glutamic acid-rich sequences found in the protein. To investigate the role of phosphorylation in interaction with crystals, peptides containing no (P0), one (P1), or all three (P3) phosphates were prepared. Using a novel combination of confocal microscopy and scanning electron microscopy, we show that these peptides adsorb preferentially to {100} faces of COM and inhibit growth of these faces in a phosphorylation-dependent manner. To characterize the mechanism of adsorption of OPN peptides to COM, we have performed the first atomic-scale molecular-dynamics simulation of a protein-crystal interaction. P3 adsorbs to the {100} face much more rapidly than P1, which in turn adsorbs more rapidly than P0. In all cases, aspartic and glutamic acid, not phosphoserine, are the amino acids in closest contact with the crystal surface. These studies have identified a COM face-specific adsorption motif in OPN and delineated separate roles for carboxylate and phosphate groups in inhibition of crystal growth by mineral-associated phosphoproteins. We propose that the formation of close-range, stable, and face-specific interactions is a key factor in the ability of phosphoproteins to regulate biomineralization processes.

Specific Adsorption of Osteopontin and Synthetic Polypeptides to Calcium Oxalate Monohydrate Crystals

Protein-crystal interactions are known to be important in biomineralization. To study the physicochemical basis of such interactions, we have developed a technique that combines confocal microscopy of crystals with fluorescence imaging of proteins. In this study, osteopontin (OPN), a protein abundant in urine, was labeled with the fluorescent dye AlexaFluor-488 and added to crystals of calcium oxalate monohydrate (COM), the major constituent of kidney stones. In five to seven optical sections along the z axis, scanning confocal microscopy was used to visualize COM crystals and fluorescence imaging to map OPN adsorbed to the crystals. To quantify the relative adsorption to different crystal faces, fluorescence intensity was measured around the perimeter of the crystal in several sections. Using this method, it was shown that OPN adsorbs with high specificity to the edges between {100} and {121} faces of COM and much less so to {100}, {121}, or {010} faces. By contrast, poly-L-aspartic acid adsorbs preferentially to {121} faces, whereas poly-L-glutamic acid adsorbs to all faces approximately equally. Growth of COM in the presence of rat bone OPN results in dumbbell-shaped crystals. We hypothesize that the edge-specific adsorption of OPN may be responsible for the dumbbell morphology of COM crystals found in human urine.

Role of crystal surface adhesion in kidney stone disease

Atomic force microscopy has been used recently to characterize the adhesion force between selected calcium oxalate crystal surfaces and biologically relevant chemical groups attached to the atomic force microscopy probe tip. These measurements have permitted comparisons of the adhesion properties of different, well defined crystal faces, as well as determination of the influence of solution-phase macromolecules on adhesion. These studies have produced new insight into the specific chemical interactions that regulate kidney stone formation. The adhesion force measurements have demonstrated that the large hexagonal (100) face of calcium oxalate monohydrate is the most adhesive. In contrast, the large (101) face of calcium oxalate dihydrate is the least adhesive. Carboxylate and amidinium groups on the atomic force microscopy tip exhibit equivalently large adhesion at a given crystal face, implicating specific binding to crystal surface lattice ions. Solution-phase macromolecules modulate adhesion in a face-selective manner, dependent on their chemical structures. The low adhesion force for calcium oxalate dihydrate predicts a decreased ability of these crystals to aggregate or attach to cells, and correlates with the relative absence of calcium oxalate dihydrate in kidney stones. These measurements provide new understanding of the macromolecular regulation of crystal aggregation and attachment to cells in stone formation.

Effects of amino acids on crystal growth of CaC 2O 4 in reverse microemulsion

Crystal growth of calcium oxalate (CaC 2O 4) in bulk aqueous solution, reverse microemulsion of p-octyl polyethylene glycol phenylether (OP)/ iso-octyl alcohol (IOA)/cydohexane/water and above microemulsions containing different kinds of amino acids, such as aspartic acid (Asp), tyrosine (Tyr) and tryptophan (Trp) were studied. The results indicated that different crystallization types of the crystals, which were calcium oxalate monohydrate (COM), calcium oxalate dihydrate (COD) and calcium oxalate trihydrate (COT), existed in bulk aqueous solution. But CaC 2O 4 growth mainly paralleled with ( 1 ¯ 0 1 ) plane of COM in reverse microemulsion because of the induction of surfactant at water/oil interface. After adding amino acids into microemulsions, the growth of CaC 2O 4 crystals mainly influenced by the varieties of amino acids and the pH values of the amino acid aqueous solution. When pH values of the solutions was higher than isoelectric points of amino acids, CaC 2O 4 crystal paralleled with ( 1 ¯ 0 1 ) plane of COM more easily with the addition of Trp, Tyr, Asp in turn; however, when pH of the solutions was lower than isoelectric points of Trp, CaC 2O 4 crystal growth paralleled with (0 2 0) face of COM. It is obviously that amino acids, pH values of the solutions and surfactant played important roles in the process of crystal growth of CaC 2O 4 in the microemulsions. The formation mechanism of CaC 2O 4 was also discussed in different microemulsions at last.

Crystal Surface Adhesion Explains the Pathological Activity of Calcium Oxalate Hydrates in Kidney Stone Formation

Renal tubular fluid in the distal nephron of the kidney is supersaturated with calcium oxalate (CaOx), which crystallizes in the tubules as either calcium oxalate monohydrate (COM) or calcium oxalate dihydrate (COD). Kidney stones are aggregates, most commonly containing microcrystals of COM as the primary inorganic constituent. Stones also contain small amounts of embedded proteins, which are thought to play an adhesive role in these aggregates, and they often are found attached to the tip of renal papilla, presumably through adhesive contacts. Voided urine, however, often contains COD in the form of single micron-sized crystals. This suggests that COD formation protects against stone disease because of its reduced capacity to form stable aggregates and strong adhesion contacts to renal epithelial cells. Using atomic force microscopy configured with tips modified with biologically relevant functional groups, we have compared the adhesion strengths of the morphologically important faces of COM and COD. These measurements provide direct experimental evidence, at the near molecular level, for poorer adhesion at COD crystal faces, which explains the benign character of COD and has implications for resolving one of the mysteries of kidney stone formation.

Crystallization of calcium oxalate beneath Langmuir monolayers in the presence of urinary inhibitors

The crystallization of calcium oxalate (CaOxa) beneath Langmuir monolayers of stearic acid (SA) and dipalmitoylphosphatidylcholine (DPPC) was investigated by environmental scanning electron microscopy (ESEM) and X-ray diffraction (XRD) analysis. The headgroups of the monolayer materials, the surface pressure of the monolayers and the biomolecules in subphases affected the formation of CaOxa crystals. More calcium oxalate monohydrate (COM) crystals were observed beneath SA monolayers than that beneath DPPC monolayers. The crystals grown at SA monolayers exhibited unusual morphology, while beneath DPPC monolayers the COM crystals were oriented with the elongated hexagonal face parallel to the plane of the monolayers. More crystals were observed at lower surface pressure of DPPC monolayers. In the presence of l-glutamic acid ( l-glu), irregular polyhedron crystals and three-dimensional hexagonal prismatic crystals were obtained. While urinary macromolecule chondroitin sulfate A (C 4S) made the elongated ( 1 ¯ 0 1 ) face of COM much longer and the crystals became thinner. C 4S can also delay the two-dimensional (2D) growth of the ( 1 ¯ 0 1 ) face of COM crystals.

Crystallization of calcium oxalate monohydrate at dipalmitoylphosphatidylcholine monolayers in the presence of chondroitin sulfate A

The growth and aggregation of calcium oxalate monohydrate (COM) crystals beneath dipalmitoylphosphatidylcholine (DPPC) monolayers in the presence of chondroitin sulfate A (C4S) was systematically examined under different surface pressure. The results indicated that the addition of C4S can inhibit the crystal growth and prevent the aggregation of COM crystals. Under a DPPC monolayer, well-defined three-dimensional hexagonal prisms and three-dimensional rhombus prisms with sharply angled tips were obtained. The DPPC monolayer at a surface pressure of 10mN/m can match the Ca2+ distance of the (1¯01) face of COM better than at 20mN/m. The addition of C4S could cooperatively modulate the interaction strength between the monolayer (or itself) with the specific morphology determining faces such as (1¯01) and (020), and thus results in remarkable stabilization of the (1¯01) faces. The dramatic changes in morphological details were due to the strong electrostatic interactions between the Ca2+-rich (1¯01) crystal faces of COM and the polyanionic polysaccharide C4S together with the negatively charged sites of the zwitterionic DPPC monolayers. The increase of the concentration of C4S can further enhance the stabilization of the (1¯01) face.

Selection of Crystal Face of Calcium Oxalate Monohydrate Beneath Dppc Monolayers in the Presence of Chondroitin Sulfate A

The crystal face selection of calcium oxalate monohydrate (COM) at dipalmitoylphosphatidylcholine (DPPC) monolayers was investigated in the presence of chondroitin sulfate A (C4S). The (1‾01) face was doubly strengthened and the (010) face was doubly weakened in the presence of C4S at DPPC monolayers. This was due to the simultaneous interaction of the Ca2+-rich (1‾01) crystal face of COM with both the polyanionic polysaccharide C4S and the negatively charged sites of phosphate groups of the DPPC monolayers.

Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals by atomic force microscopy.

Atomic force microscopy (AFM) was applied to the (-101) faces of calcium oxalate monohydrate (COM) crystals grown from calcium oxalate (CaOx) solutions. Microstructures of many spiral hillocks with step height of 1 nm were observed on the faces. Then using AFM in situ, we analysed the re-growth process of the spiral steps on the face of COM seed-crystals in CaOx growth solutions that contained growth inhibitors of glycosaminoglycans and studied their inhibition mechanisms on COM crystals. The total morphology of the faces of COM seed crystals re-grown in the CaOx growth solutions was assessed by scanning electron microscopy (SEM). In the growth solution without glycosaminoglycans (control experiment) or with chondroitin sulphate (ChS), AFM images and SEM micrographs of the faces of the re-grown seed crystals showed two-dimensional (2D) nucleation although 2D nucleation was delayed in the presence of ChS. However, the addition of dermatan sulphate (DS) to the growth solution resulted in isotropic growth by a step flow mode and spiral mechanism. With regard to the main inhibition mechanisms of two glycosaminoglycans (ChS and DS) on COM crystals, it can be concluded from these results that ChS delays 2D nucleation and DS inhibits 2D nucleation.