Inhibition of Pathological Mineralization of Calcium Phosphate by

Sep 7, 2014 - We present an in situ study of the interaction of osteopontin (OPN) peptide-bearing solutions with brushite (DCPD), CaHPO4·2H2O, (010) ...
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Inhibition of Pathological Mineralization of Calcium Phosphate by Phosphorylated Osteopontin Peptides through Step-Specific Interactions Shiyan Li,† Shanshan Wu,† Defeng Nan, Wenjun Zhang, and Lijun Wang* College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China S Supporting Information *

ABSTRACT: We present an in situ study of the interaction of osteopontin (OPN) peptide-bearing solutions with brushite (DCPD), CaHPO4·2H2O, (010) surfaces using atomic force microscopy. We show that in situ observations of the [1̅00]Cc step kinetics are consistent with classic Cabrera-Vermilyea model of step pinning combined with adsorption dynamics of phosphorylated OPN peptides, highlighting the effects of supersaturation and peptide concentration on step movement and pinning as a mechanism of inhibitor action. In addition to a kinetic effect, the presence of phosphorylated OPN, preferentially binding to the [1̅00]Cc steps, may alter mineral interfacial energies, thus delaying the formation of active steps during growth. This is consistent with the bulk nucleation observations. Furthermore, the phosphorylation-deficient form of this segment fails to inhibit DCPD crystallization. These in vitro results may reveal that the dual control of step kinetics and interfacial energy by phosphorylated OPN peptides may have much broader utility for improving our understanding of the mechanisms through which pathological mineralization of calcium phosphate is inhibited.

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INTRODUCTION In terms of chemical diversity, all pathological calcifications mainly contain calcium oxalate (CaOx) and calcium phosphates (Ca−Ps). However, secreting organs such as biliary tract and kidneys show a more complex constituent consisting of calcium salts (oxalates, phosphates, sulfates, and carbonates) or organic compounds (purines, amino acids, lipids, and proteins).1 More than 50% of CaOx stones contain variable amounts of brushite (CaHPO 4 ·2H 2 O, DCPD) and hydroxyapatite (Ca10(PO4)6(OH)2, HAP).2,3 For example, the Randall’s plaques (RP) in the kidney are subepithelial deposits of calcium phosphate, and it is thought that these nucleate CaOx formation and evolve into kidney stones.4 In addition, for a Ca−P stone, a radial distribution of acicular brushite crystallites with a center of apatites is the most common mineral phases.1 It has long been known that certain natural proteins, for example, osteopontin (OPN) present in urine,5 inhibit crystallization of CaOx6,7 and HAP.8,9 Moreover, expression of OPN in a variety of tissues indicates a multiplicity of functions. For example, one species, a highly phosphorylated sialoprotein, is a prominent component of the mineralized extracellular matrices of bones and teeth.10 Native OPN © 2014 American Chemical Society

undergoes extensive post-translational modifications such as phosphorylation and glycosylation.11 OPN is characterized by the presence of a polyaspartic acid sequence, and sites of Ser/ Thr phosphorylation that can mediate HAP binding.12 Much evidence has suggested that the interaction of OPN with Ca− Ps is determined by the extent of protein phosphorylation and that phosphorylation enhances OPN’s ability to adsorb and to inhibit the growth of Ca−P crystals, although other anionic groups also contribute to these properties.13−16 A recent study shows that recombinant OPN (rOPN), which lacks phosphorylation, treated with protein kinase to phosphorylate the molecule (p-rOPN) produced an effect similar to that of native OPN, suggesting that phosphorylations are critical to OPN’s role to inhibit nucleation, whereas the growth of HAP crystals is effectively controlled by the highly acidic OPN polypeptide.17 Combining in situ atomic force microscopy (AFM) with molecular modeling, Qiu et al. have shown that OPN or citrate controls calcium oxalate monohydrate (COM) growth habit Received: June 10, 2014 Revised: September 2, 2014 Published: September 7, 2014 5605

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(St. Louis, Missouri, USA)) was incorporated into the gel and 0.5 M CaCl2 (Fluka (St. Louis, Missouri, USA)) in the top solution of a tube, which remained at room temperature for 30 days.22 The crystals were harvested and subjected to Bruker D8 X-ray diffraction (Billerica, Massachusetts, USA) to identify crystal phase(s). Synthetic brushite crystals were used for in situ AFM surface growth experiments. Bulk DCPD Nucleation. The basis for the possible differences in inhibitory effects of 3P-OPN and NPP on bulk DCPD crystallization was investigated in nucleation experiments to determine the effects of these peptides on induction times for nucleation. The relative supersaturation (σ) and supersaturation ratio (S) for DCPD nucleation is given by

and kinetics by pinning step motion on different faces through specific interactions.18 Nene et al. observed a decrease in step velocity and changes in the morphology of the growth hillocks upon addition of a synthetic OPN phosphopeptide adsorbed on the COM (010) faces using AFM.19 It appears likely that OPN inhibition of Ca−P growth occurs by a similar mechanism. The present study tests the hypotheses that the interaction of OPN peptides with DCPD crystal faces is determined by the extent of protein phosphorylation and that the surface growth dynamics is modulated by step-specific interactions. In this study, we investigate the DCPD (010) surface growth in the absence and presence of OPN peptides using in situ AFM coupled with a fluid reaction cell through which solutions were flowed with varying supersaturations (σ) under nearphysiological ionic strength (0.15 mol L−1). We chose DCPD because Randall’s plaques are calcium oxalate crystals on a nidus of calcium phosphate,4 and it has long been recognized as one of the more important mineral Ca−P phases that has frequently been invoked as a precursor phase in the biological/ geological formation of apatite.20−22 It is also a good model system for monitoring Ca−P crystal growth and dissolution by AFM because the platelike DCPD crystals with smooth (010) surfaces are large enough to be employed as suitable substrates for dynamic AFM imaging.23 Moreover, we chose this OPN species based on refs 5 and 6, and this protein had been isolated from human urine and the amino acid sequences of osteopontin proteins from different tissues are identical. The isoforms from bone, plasma, breast milk, urine, and cells differ only in post-translational modification.10 We selected from the full OPN sequence a 14 amino acid segment with highly conserved aspartic acid residues (DDVDDTDDSHQSDE) (Figure 1) (sequence of amino acids from 93 to 106 of OPN

⎡ IAP ⎤1/2 ⎥ −1 σ=S−1=⎢ ⎢⎣ K sp ⎥⎦

(1)

where IAP is the ionic activity product and Ksp is the solubility activity product of brushite (2.36 × 10−5 mol2 L−2,23 25 °C). Solution speciation calculations were made by using the extended Debye−Hückel equation proposed by Davies from mass balance expressions for total calcium and phosphate with appropriate equilibrium constants by successive approximation for the ionic strength (IS).26 Supersaturated solutions were prepared by the slow mixing (dropwise) calcium chloride (Fluka (St. Louis, Missouri, USA)) and potassium dihydrogen phosphate (Sigma-Aldrich (St. Louis, Missouri, USA)) with sodium chloride to maintain the physiological ionic strength, IS = 0.15 mol L−1. The pH value was finally adjusted to 5.6 with 0.1 mol L−1 KOH solution using Metrohm 888 Dosimat Plus (Herisau, Switzerland) to carefully make the reaction solution supersaturated (the final volume of 60 mL) in a double-walled Pyrex vessel (250 mL) by magnetic stirring. The final solutions were mixed to prevent local supersaturation monitored by a calcium ion selective electrode and a reference electrode. All supersaturated solutions were prepared using distilled deionized water (>18 MΩ·cm). The experimental conditions are summarized in Table 1. For nucleation experiments in the Table 1. Conditions in DCPD Nucleation and Growth Experiments

Figure 1. Phosphorylated (3P-OPN) and nonphosphorylated 14-mer peptide (NPP) segment 93−106 of osteopontin (OPN) used for dicalcium phospate dihydrate (brushite, CaHPO4·2H2O, DCPD) nucleation and growth studies has three phosphorylation sites (two serine (S) and one threonine (T)).

final solution concentration (mmol L−1)a

with three phosphorylation sites including two serine (S) and one threonine (T)).24 We expect that the general trends found for this model DCPD−OPN system will also hold for the other Ca−P pathological minerals such as HAP.

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EXPERIMENTAL SECTION OPN Peptide Synthesis. Phosphorylated (3P-OPN) and nonphosphorylated 14-mer peptide (NPP) fragments were synthesized according to standard procedures of solid-phase peptide synthesis from Niusiter Biotech (Wuhan, China). These two peptide segments were purified, and the molecular weight was verified by mass spectrometry. Details of the synthesis procedure are provided elsewhere.9,25 DCPD Crystal Synthesis. DCPD single crystals were synthesized using 4.3% solution of sodium metasilicate (Aldrich) and the pH was adjusted to 5.6−6.0 to form the gel. Potassium dihydrogen phosphate (1.0 M; Sigma-Aldrich

σDCPD

NaCl

CaCl2

KH2PO4

0.397 0.450 0.503 0.555 0.607 0.736 1.09

112.0 111.0 109.0 108.0 106.0 103.0 93.0

9.6 10.0 10.4 10.8 11.2 12.2 15.0

9.6 10.0 10.4 10.8 11.2 12.2 15.0

a

The solutions were freshly prepared using the stock solutions (1.0 M NaCl, 0.04 M CaCl2, and 0.04 M KH2PO4).

presence of peptides, 3P-OPN or NPP (30.0, 50.0, or 70.0 nM) solutions were added prior to pH adjustment. Nitrogen presaturated with water vapor was passed through the reaction solutions to exclude carbon dioxide. DCPD nucleation experiments were performed at a high supersaturation (σDCPD = 1.09, IS = 0.15 mol L−1, pH 5.6, 25 °C) (Table 1). The lowering of the pH in all crystallization experiments was monitored by a glass pH electrode coupled with a singlejunction Ag/AgCl reference electrode. All data (induction 5606

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times) with their mean values ± standard deviation (SD) of three independent sets of experiments are presented. In Situ Atomic Force Microscopy (AFM). All in situ DCPD surface growth experiments were performed in contact mode (Nanoscope V-Multimode 8, Bruker (Santa Barbara, California, USA)) on the (010) surfaces of synthetic DCPD crystals (about 2.0 × 2.0 mm in size) anchored inside the enclosed fluid cell. DCPD surface growth experiments using AFM were performed at a range of supersaturation (σDCPD = 0.397−0.736, IS = 0.15 mol L−1, pH 5.6) (Table 1) in the absence and presence of peptides. AFM images were collected using Si3N4 tips (Bruker, tip model NP-S10, spring constants 0.12−0.35 N/m) with scan rates of 2−4 Hz, while minimizing tip−surface interactions when solutions were passed through crystal substrate. All experiments were performed under ambient conditions (25 ± 1 °C). The chosen flow rate (about 1 mL/min) was enough to ensure surface-controlled rather than diffusion-controlled reactions,23 and this flow rate does not influence the adsorption of the peptides (10−100 nM) on the crystal faces. Measurements were made on more than three crystals per solution composition to ensure reproducibility of the results, and each was imaged in different locations on the crystal (>3 points). The images were analyzed using the NanoScope analysis software. Circular Dichroism (CD). CD spectra of 50 μM peptides in solutions (0.5 mM 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) (Fluka (St. Louis, Missouri, USA)), 150 mM NaCl in the absence and presence of 200 mM CaCl2 at pH 5.6) were collected on a Jasco Model 810 Spectropolarimeter (Hachioji, Tokyo, Japan) using 1 mm quartz sample cells. The mean residue ellipticity ([θ]) was calculated using the following equation [θ ] =

θobs 1 × 10lc r

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RESULTS In Situ AFM Growth of the DCPD (010) Face. Figure 2A shows a representative growth hillock on the (010) face of

Figure 2. Direct imaging of a growing (010)-DCPD face at a fixed supersaturation of σ = 0.736 using in situ AFM. (A) AFM deflection and (B) height images showing development of a new spiral segment generated at a complex dislocation hillock, which is triangular in shape with crystallographically distinct steps along the [101]Cc, [1̅00]Cc, and [101]̅ Cc directions. Steps advance only if the length of the step, L, exceeds a critical value, Lc. Arrows indicate advancing step segments. (C) Cross-sectional analysis of the step height along a dotted line in (B). (D) Cross-sectional view of the [1̅00]Cc step. Oxygen atoms are red, phosphorus atoms are purple, calcium atoms are blue, and hydrogen atoms are black. The step height is as marked at 7.6 Å. Figure 2D was reproduced from ref 22.

(2)

where θobs is the measured ellipticity in millidegrees, l is the length of the cell, c is the concentration (M), and r is the number of residues per peptide molecule (14 residues in both phosphorylated and nonphosphorylated peptides). Wavelength scans were recorded using a 1 nm step size, 50 nm/min scan speed, and 4 s response time. X-ray Photoelectron Spectroscopy (XPS). After bulk DCPD nucleation experiments (σDCPD = 1.09, IS = 0.15 mol L−1, pH 5.6, 25 °C), nucleated crystallites were mixed and incubated with 1.0 mM 3P-OPN or NPP for 7 h, and then crystallites were separated from the slurry solutions by centrifugation at about 3800g. After vigorous washing in deionized water, peptide-bound DCPD samples were dried under vacuum for at least 8 h and were placed on an aluminum (Al) platform for XPS measurements (VG multilab 2000 equipment ThermoVG scientific, East Grinstead, West Sussex, UK) using the Al Kα X-ray line of 1486.6 eV excitation energy at 300 W. To correct for sample charging, high-resolution spectra were used as a reference by setting the C 1s hydrocarbon peak to 284.6 eV. The background was linearly subtracted. Data analysis was performed with the Thermal Advantage software (http://www.tainstruments.com). The ratios of atomic concentrations were calculated using the peak areas normalized on the basis of acquisition parameters and sensitivity factors proposed by the manufacturer.

DCPD crystals grown in a pure supersaturated solution at σ = 0.736, exhibiting a triangular-shaped morphology with crystallographically distinct steps along the [101]Cc, [1̅00]Cc, and [101̅]Cc directions.27 DCPD atomic steps measure about 0.77 nm tall (Figure 2B), reflecting that it is close to 0.76 nm (Figure 2C), half the size of the lattice constant of the b axis. According to its atomic structure (as space group Ia with lattice parameters a = 5.812 Å, b = 15.18 Å, c = 6.239 Å, and β = 116.25°),27 DCPD is a noncentrosymmetric monoclinic crystal which is composed of corrugated rows of calcium cations with HPO42− anions (Figure 2C). Water molecules bound to the calcium cations result in layers of water between the Ca2+- and HPO42−-containing sheets.27 For this reason, the fully hydrated {010} faces have a relatively low interfacial energy of 4.5 mJ m−2 compared with apatite (8 mJ m−2).28 In pure supersaturated solutions at σ ranging from 0.397 to 0.607, 5607

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at high peptide concentrations and low σ, that is, the degree of inhibition increased markedly with decreasing σ and increasing 3P-OPN peptide concentration (Figure 3B). Moreover, there was no obvious change in the spreading rates of both the [101]̅ Cc and [101]Cc steps in the presence of 3P-OPN (Figure S1 in the Supporting Information). In striking contrast, nonphosphorylated 14-mer OPN peptide segment (NPP) has little influence on the (010) surface growth of DCPD at various supersaturations; the velocities of all three steps are not significantly changed in the presence of NPP concentration up to 100 nM (Figure S2 in the Supporting Information). It is noteworthy that although the velocities of the [101]Cc and [101̅]Cc steps were much less influenced by the presence of 3P-OPN than that of the [10̅ 0]Cc step, the step density for both the [101]Cc and [101̅]Cc steps was dramatically decreased with time, especially at relatively low supersaturations, by 3P-OPN (Figure 4A) rather than NPP peptide (Figure S3 in the Supporting Information). The change of the step density and the degree of step pinning with supersaturation (Figure 4B) is consistent with the change of the step velocity along the [1̅00]Cc direction (Figure 3C); that is, as the supersaturation increases, the [10̅ 0]Cc step velocity rises rapidly, and the degree of step pinning by 3P-OPN decreases. Roughening of the [101]Cc and [1̅00]Cc steps was observed due to specific step pinning, and the hillock became irregular at low supersaturations after 20 min of 3P-OPN adsorption, exhibiting highly rounded step corners (intersection of the [101̅]Cc and [101]Cc steps) and a marked decrease of the step density by changing the terrace spacing (width, d) between the growth of [101]Cc or [101̅]Cc steps. To further investigate the differences in the adsorption/ binding of 3P-OPN and NPP to steps and faces of DCPD, in situ AFM was used to image the (010) face of DCPD at increasing levels of these two peptides at 100 nM (9.6 mM CaCl2 + 100 nM 3P-OPN/NPP, pH 5.6 in the absence of phosphate). At this concentration the (010) face is covered similarly with a uniform film of 3P-OPN or NPP peptides at the initial adsorption stages, preventing visualization of the underlying steps and etch pits shown in Figure 5A,B. But following adsorption of NPP for 4 min, desorption behavior of NPP quickly occurred, and just a small amount of NPP remained on the macrosteps (Figure 5B). In contrast, the (010) face was still at high coverage following 12 min of adsorption of the 3P-OPN peptides (Figure 5A), demonstrating that the 3POPN peptides are strongly bound to the (010) face. Induction Time of the Bulk DCPD Nucleation. Figure 6 shows that 3P-OPN dramatically inhibited the bulk nucleation by prolonging the induction time in a concentration-dependent manner at a given supersaturation (σDCPD = 1.09, IS = 0.15 M, pH 5.6, 25 °C). The induction time in pure supersaturated solution was 48 ± 8 min (n = 3), whereas in the presence of 30, 50, or 70 nM 3P-OPN, the induction times increased to 137 ± 25 (n = 3), 232 ± 36 (n = 3), and 440 ± 65 min (n = 3), respectively. In contrast, the induction times (Figure 6) were not significantly changed in the presence of NPP at the same concentration range. To further analyze the difference of adsorption of 3P-OPN and NPP, we collected the DCPD crystallites after pure DCPD bulk nucleation experiments and incubated them with high concentration 3P-OPN or NPP at 1.0 mM for high-resolution XPS analysis. The XPS spectrum displayed a slightly smaller C peak for NPP than for 3P-OPN (Figure 7A), suggesting that the NPP peptide does adsorb to {010} faces of DCPD, but only

anisotropic spreading velocities for a growing spiral segment are present in the order [101]Cc ≈ [101]̅ Cc > [10̅ 0]Cc, that is, [10̅ 0]Cc steps having the lowest growth rate (Figure 3A). Figure 3C shows the [10̅ 0]Cc step velocity in the presence of phosphorylated 14-mer OPN peptide (3P-OPN) (v) relative to peptide-free system (v0) at various supersaturations at σ = 0.397−0.607. The [10̅ 0]Cc step speed was significantly inhibited

Figure 3. (A) Step speed along the [101]Cc, [1̅00]Cc, and [101̅]Cc directions at different supersaturations (σ). Solid lines are linear fits to the data. (B) Dependence of the [1̅00]Cc step velocity on supersaturation in the presence of 3P-OPN. Measurements (solid lines) were made for the {010} face of DCPD. Below a supersaturation given here by σd, the crystal exhibits a dead zone where no growth occurs. As the supersaturation increases beyond σd, the surface begins to grow. Above a sufficiently high supersaturation (denoted here by σ*), the [1̅00]Cc step velocity rises rapidly, approaching the step velocity in pure solutions at the same supersaturation. (C) Relative step velocity (v/v0) as a function of 3P-OPN concentration (Ci) in supersaturated solutions with respect to DCPD. The dashed lines show the trend of the decrease in relative step velocity for experimental data of the [1̅00]Cc step speed. 5608

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Figure 4. AFM time sequence (deflection images) of the spiral growth evolution on a DCPD (010) surface showing effect of 3P-OPN on growth hillock morphology and step density at (A) σ = 0.397 and (B) σ = 0.555 before and after addition of 10 or 100 nM 3P-OPN. In addition to the decrease in the velocity of the [1̅00]Cc step, the terrace widths become larger and the step density following addition of 3P-OPN to growth solutions is decreased on the growing (010) DCPD surfaces by changing the terrace spacing between the growth of [101]Cc or [101]̅ Cc steps. As the supersaturation increases from σ = 0.397 to σ = 0.555, both the change of step density and the degree of step pinning by 3P-OPN decrease.

experimental observations (Figure 5). Collectively, AFM and XPS results suggest that growth inhibition is associated with peptide binding to crystal faces or pinning of the steps by peptides adsorbed on growth hillocks. Circular dichroism (CD) spectra (Figure 7C) of 3P-OPN or NPP in 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) buffer in the absence and presence of CaCl2 revealed that these two peptide segments are present in solution in the same state of unordered structure at about 199 nm (Figure 7C). The presence of 200 mM CaCl2 did not result in a spectrum consistent with any secondary structures such as α-helix or β-sheet, clearly indicating that both peptides are unable to undergo calciumor phosphorylation-dependent conformational changes. The CD results are fully consistent with the previous analysis of this OPN peptide with less ordered structure in solutions.24 Thus, the different AFM surface growth inhibition observed for each peptide further suggests that step-specific binding of negatively charged phosphate side chains to crystal faces controls growth inhibition rather that any secondary peptide structure.

Figure 5. (A1, A2) AFM images showing that the DCPD (010) face becomes covered by 3P-OPN peptide film after addition of solution (9.6 mM CaCl2 + 100 nM 3P-OPN, pH 5.6) for 12 min at 25 °C, preventing visualization of the part of underlying steps or etch pits indicated by arrows. (B1, B2) As shown in Figure 5B1, the image was recorded immediately (0+ min) after injecting NPP-containing solution. After adsorption of NPP (9.6 mM CaCl2 + 100 nM NPP, pH 5.6, 25 °C) for 4 min (the continuous injection of NPP-containing solution for 4 min), desorption of NPP quickly occurred, and just a small amount of NPP remained on the macrosteps.

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DISCUSSION Although DCPD crystals in pure solutions exhibit linear kinetics (Figure 3A) over a range of supersaturation we chose in this study, the linear dependence between step velocity and supersaturation (v = β(a − ae), where a and ae are the actual and equilibrium solute activities and β is called the kinetic coefficient,30) may not be applied in this system due to the presence of wide dead zone (Figure 3B) and/or possible nonlinearity at low supersaturations. Kinetic results (Figures 3B and 4A) show action of Cabrera-Vermilyea (C−V) steppinning mechanism in inhibiting the step velocity by 3P-OPN peptides.31,32 3P-OPN molecules adsorb to the [1̅00]Cc step

weakly (Figure 5), and therefore is not an effective inhibitor of crystal growth. The C 1s peak was fit to four components for aliphatic (CH2 and C−H), phenolic/amine (C−O, C−N), and amide (N−CO) (Figure 7B), exhibiting the multicomponent peaks expected for adsorbed peptide or protein.29 These XPS results (Figure 7 and Figure S4 in the Supporting Information) for peptide adsorption are, in part, consistent with the AFM 5609

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Figure 6. (A) Representative pH curves for DCPD nucleation (free drift experiments) in the absence and presence of phosphorylated (3P-OPN) and nonphosphorylated OPN (NPP) peptides. (B) Plots of induction time against 3P-OPN and NPP peptide concentration for DCPD crystal nucleation.

Figure 7. (A) High-resolution X-ray photoelectron spectra (XPS) of 3P-OPN or NPP adsorbed to DCPD crystallites. (B) C 1s region of 3P-OPN adsorbed to DCPD crystallites was fit with four components: aliphatic (CH2 and C−H), phenolic/amine (C−O, C−N), and amide (N−CO). (C) CD spectra of both peptides (50 μM) at pH 5.6 (25 °C) in 0.5 mM MES buffer with 0.15 M NaCl in the absence and presence of 200 mM CaCl2. (Black square: NPP without CaCl2; red circle: NPP with 200 mM CaCl2; blue triangle: 3P-OPN without CaCl2; green triangle: 3P-OPN with 200 mM CaCl2.)

binding of inhibitors to steps extends the time for the first turn of the spiral to create one complete rotation.35,36 In the general case a spiral has N sides, and each corresponding edge (1) grows perpendicular to itself with velocity vi, (2) has some critical length Lci, and (3) forms an angle αi,i+1 with the previous edge of the spiral. Using measured step velocities and assuming that critical lengths for all three orientations are similar and do not change too much in the presence of additive, that is, the terrace width changes if vi change but Lci = constant (i = 1, 2, 3), one can easily show that the terrace width d1 should slightly decrease in the presence of 3P-OPN, whereas two others should significantly increase. In addition to a kinetic effect, increasing of the terrace widths due to the increasing of the critical lengths and step energies (a thermodynamic effect) is a quite possible scenario under our experimental conditions. The structure of the growth hillock depends on the thermodynamics of step advancement through terrace widths and step speeds. Considering the birth of a new spiral segment (Figure 2A), it will advance only if L exceeds a critical value, Lc,37 given by

edges or accumulate on terraces ahead of migrating steps, thereby pinning the [1̅00]Cc step motion and decreasing the velocity of the steps. The classic C−V model of inhibition analyzes the effect of step pinning by comparing the critical curvature of a step to the average impurity spacing.32 Because the critical curvature decreases as the supersaturation is increased, this model predicts32 that (1) at sufficiently high inhibitor concentration, growth ceases because steps can no longer pass between the adsorbed inhibitor molecules, creating a so-called “dead zone” of supersaturation (Figure 3B), and (2) when a threshold supersaturation is met, the steps break through the chain of adsorbed impurities and rapidly achieve the step velocity characteristic of the pure system (Figure 3B). Due to substantial ambiguity in determination of the kinetic coefficient based on the present AFM data, it is insufficient to assume simultaneous action of Bliznakov kink blocking mechanism that exhibits in the COM system33,34 In the frame of the dislocation spiral mechanism terrace widths can change due to (1) change of surface energies and terrace widths and (2) redistribution of step velocities. Both contributions are implemented in eq 3, N

di = vi ∑ i=1

Lc = 2γstepΩ/kBTσ

Lci sin(αi , i + 1) vi + 1

(4)

where Ω is the volume per growth unit in the solid, γstep is the free energy of the step edge, kB is the Boltzmann constant, and T is absolute temperature. Equation 4 implies that step movement does not occur until critical conditions are reached. Rather, a metastable equilibrium condition persists during an induction period τ prior to crystal formation. In this process,

(3)

where for brushite N = 3, i = 1 for direction [10̅ 0]Cc, i = 2 for direction [101̅]Cc, i = 3 for direction [101]Cc and Lci are critical lengths of the step of ith orientation. We can see from eq 3, vi and Lci can affect di. Sizemore and Doherty proposed that the 5610

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differences of these two peptides in the DCPD system. The clusters/aggregates are weakly bound to steps or terraces.43 When an approaching step arrives at a peptide binding site, if the peptide is well-bound, it can block solute access to kinks; but if it is weakly adhered, step propagation can proceed past the site.43 Thus, whether a peptide impedes step motion depends on the characteristic time available to relax into the well-bound state.43,44

surface energetic control may play an important role as it can modify the critical conditions directly,23 as described by eq 4. When the supersaturation is kept constant (since the ratios of calcium to peptide in the reaction solutions are larger than 106 for σ = 0.607 and [peptide] = 100 nM), changes in the calcium concentration as a result of the formation of calcium−peptide complexes can be ruled out; the only variable that can influence the value of Lc is the step energy γstep, which is modified by the presence of peptide in the solution. AFM features in Figure 4 demonstrate that the much larger terrace widths may be associated with these new steps exhibiting higher step edge free energy following addition of 3P-OPN peptide to growth solutions. This in turn may delay the formation of active step sources and increase the time for the formation of critical 1D steps, especially at high peptide concentrations and low σ (Figure 4A), highlighting the effects of supersaturation and peptide concentration. In terms of an isotropic model γstep, the mean value for all crystal-plane-step energies can be considered to be a function of the interfacial energy γSL,23 and according to classical nucleation theory, induction time τ for bulk nucleatioin (Figure 6) depends exponentially on the third power of γSL,38 that is, ln τ ∝ C1 + C2

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CONCLUSIONS Aberrant crystallization within the human body can lead to various diseases including kidney stones, although traditionally the pathogenesis of such diseases has not been studied from a materials science perspective.45 A fundamental understanding of the nucleation and growth, followed by aggregation and adhesion of crystals to cells and tissue, is essential for the elucidation of the mechanisms directing the pathogenesis of these diseases.45 In the present study, we use a combination of surface (in situ AFM) and bulk crystallizaion kinetics methods to investigate nucleation and growth of pathological crystals and to examine the roles of phosphorylation of a 14 amino acid segment (DDVDDTDDSHQSDE) corresponding to potential crystal binding domains within the osteopontin sequence. According to the results of AFM step kinetics (Figures 3 and 4), if a peptide is weakly adhered, step propagation can proceed past the pinning site. It is key to understanding the findings of this study. Morevoer, it appears from the data shown in Figures 5 and 7 that the NPP peptide does adsorb to {010} faces of DCPD, but only weakly, and therefore is not an effective inhibitor of crystal growth. The local binding of negatively charged phosphate side chains to DCPD crystal faces rather than any secondary peptide structure to prevent step movement and increase interfacial/surface energies may be responsible for both the bulk and surface crystallization inhibition. Although the exact role of the whole OPN molecule and its short segments on brushite crystal growth can be substantially different, our present results may be useful for improving understanding of the mechanisms of pathological mineralization of calcium phosphate. Moreover, the Ca density of the faces developed and the interactions of the OPN protein and DCPD crystal faces using molecular simulations deserves a separate study in order to gain further insights.

γSL 3 kB3T 3σ 2

(5)

in which C1 (controlled by entropy changes) and C2 (geometric parameters) are independent constants.38 Equation 5 shows that the increase in the interfacial energy following the introduction of peptide results in the inhibition of DCPD growth through an increase in the induction time of the bulk DCPD crystallization. The only variable relating τ and σ that can be altered by the peptide is the interfacial energy because the supersaturation is kept constant. Although it is difficult to directly correlate nucleation rates of some polygonal nuclei with the growth behavior measured for one single-crystal face by AFM, these collective results may suggest that delayed bulk nucleation and step formation by 3P-OPN is manifested by higher interfacial energies (longer induction times) than by NPP, which are consistent with eqs 4 and 5. The local binding of negatively charged phosphate side chains to DCPD crystal faces rather than any secondary peptide structure to prevent step movement and increase interfacial/surface energies may be responsible for both the bulk and surface crystallization inhibition. The phosphorylated OPN peptide−DCPD crystal face interactions further support an important role for macromolecules rich in anionic side chains in the modulation of stone formation.39,40 Molecular dynamics simulations suggest that the adsorption of OPN peptides to the Ca-rich {100} faces of both HAP41 and COM24,42 is driven by electrostatics and is largely independent of amino acid sequence. Therefore, phosphate groups increase the electronegativity of OPN, resulting in a stronger interaction with cationic crystal faces. As the nonphosphorylated osteopontin peptide is still quite acidic, it is rather surprising that it has little interaction with DCPD crystal faces. Our previous results have demonstrated that the induction times for COM nucleation were not significantly changed in the presence of nonphosphorylated peptide,24 and this nonphosphorylated peptide is more likely to aggregate to form clusters/aggregates than the phosphorylated peptide with increase of concentration, rather than adsorbing on COM crystal faces.24 This may explain the inhibition

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ASSOCIATED CONTENT

S Supporting Information *

(1) The relative step velocity in the presence of 3P-OPN or NPP at various supersaturations (Figures S1 and S2), (3) AFM images of DCPD growth in the presence of NPP peptides (Figure S3), and (4) XPS spectra of 3P-OPN or NPP adsorbed to DCPD crystallites (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 5611

dx.doi.org/10.1021/cm502111v | Chem. Mater. 2014, 26, 5605−5612

Chemistry of Materials

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Article

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ACKNOWLEDGMENTS We thank Dr. Richard Gordon for careful reading of the manuscript. This work was supported by a grant (No. 41071208 to L.J.W.) from the National Natural Science Foundation of China, a Specialized Research Fund for the Doctoral Program of Higher Education (20130146110030 to L.J.W. and 20130146120042 to W.J.Z.), and the Fundamental Research Funds for the Central Universities (2012MBDX014) from the Huazhong Agricultural University (to L.J.W.).

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