Direct Visualization of Calcium Oxalate Monohydrate Crystallization

Of the two calcium oxalate crystalline forms found in kidney stones, clinical ...... Michael M. Mbogoro , Massimo Peruffo , Maria Adobes-Vidal , Emma ...
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Direct Visualization of Calcium Oxalate Monohydrate Crystallization and Dissolution with Atomic Force Microscopy and the Role of Polymeric Additives Shouwu Guo,† Michael D. Ward,*,† and Jeffrey A. Wesson*,‡ Department of Chemical Engineering and Materials Science, University of Minnesota, Amundson Hall, 421 Washington Avenue S.E., Minneapolis, Minnesota 55455-0132, and Nephrology Division, Department of Veterans Affairs Medical Center and the Medical College of Wisconsin, 5000 West National Avenue, Milwaukee, Wisconsin 53295 Received December 5, 2001. In Final Form: March 21, 2002 The growth and dissolution of calcium oxalate monohydrate (COM) were investigated by real-time in situ atomic force microscopy (AFM). The (100) surfaces of COM crystals were sufficiently rough that direct AFM imaging of terrace growth and step motion was not feasible. In undersaturated aqueous solutions, however, COM crystals dissolved, developing elongated hexagonal pits oriented along the [001] direction and having perimeters defined by {010} and {021} planes, which mimics the habit of the macroscopic crystals. Increasing the concentration of calcium oxalate to supersaturated levels reversed etching, resulting in gradual filling of the pits, which is tantamount to crystal growth. The confinement of growth within the pits permitted observation of crystal growth events, in real time and at the microscopic level, which could not be deduced by inspection of the rough surfaces of the COM crystals. This approach allowed determination of growth and dissolution rates along specific crystallographic directions, as well as the influence of solute concentration and additives on those rates. The polymeric additives tested were effective inhibitors of both COM growth and dissolution (pit filling and etching, respectively). Small amounts of poly(aspartate) (polyD) altered the aspect ratio of the (100) pits during growth and dissolution when compared to the behavior observed in the absence of polymer. The observed effects were consistent with preferential binding of the polymer to the {001} or {021} apical planes relative to the {010} planes. In contrast, poly(glutamate) (polyE), which was approximately 16 times less effective than polyD with respect to suppressing growth or dissolution, altered the aspect ratio in a manner consistent with preferred binding of polyE at the {010} surface. These observations provide a basis for understanding, and potentially regulating, calcium oxalate crystallization in important biomineralization processes, such as kidney stone formation.

Introduction Characterization of calcium oxalate crystal formation remains an important area of scientific and medical research, largely because of the importance of these crystals in kidney stones. Of the two calcium oxalate crystalline forms found in kidney stones, clinical studies suggest that adverse physiological effects are correlated with the formation of calcium oxalate monohydrate (COM) crystals (also known as whewellite), which are observed more frequently in stones than calcium oxalate dihydrate (COD or weddellite) crystals.1-4 Furthermore, COD-rich stones contain small amounts of COM on microscopic examination, often in the central portion of the stone, presumably the nidus, where stone growth is initiated.4 Crystals in the urine of asymptomatic individuals or those grown ex vivo in urine are usually COD,5,6 whereas the precipitation of calcium oxalate from water and simple * To whom correspondence should be addressed. E-mail: [email protected] (M. D. Ward) and [email protected] (J. A. Wesson). † University of Minnesota. ‡ Medical College of Wisconsin. (1) Mandel, N.; Mandel, G. Urolithiasis 1996; Pak, C. Y., Resnick, M. I., Preminger, G. M., Eds.; VIII International Symposium on Urolithiasis; Millet the Printer: Dallas, 1996; pp 454-455. (2) Schubert, G. Urolithiasis 1996; Pak, C. Y., Resnick, M. I., Preminger, G. M., Eds.; VIII International Symposium on Urolithiasis; Millet the Printer: Dallas, 1996; pp 452-453. (3) Mandel, N. S.; Mandel, G. S. J. Urol. 1989, 142, 1516. (4) Prein, E. L. J. Urol. 1963, 89, 917. (5) Elliot, J. S.; Rabinowitz, I. N. J. Urol. 1980, 123, 324. (6) Dyer, R.; Nordin, B. E. Nature 1967, 215, 751.

salt solutions exhibits a strong preference for COM.7 The importance of COM in kidney stone formation is further supported by several laboratory studies. In crystal binding studies, more COM crystals were found adherent to renal tubule cells grown in culture than COD crystals of comparable size.8 In animal models of stone disease, COM was the principal crystalline constituent adhered to the tubule cell surfaces in kidney sections.9,10 Macromolecules isolated from human urine were found to inhibit COM crystal growth in vitro and favor the formation of COD over COM, which suggests the possibility that the presence of these macromolecules in urine plays a role in the natural defense against stone formation.8,11,12 Collectively, the evidence suggests that the nucleation and growth of COM in urine and subsequent trapping of the crystal within the kidney lead to stone formation, while forming COD may be protective. Consequently, further characterization of COM crystallization and the factors that suppress its formation, to the extent that growth is redirected toward COD, may provide important clues to a cure for this disease. (7) Martin, X.; Smith, L. H.; Werness, P. G. Kidney Int. 1984, 25, 948. (8) Wesson, J. A.; Worcester, E. M.; Wiessner, J. H.; Mandel, N. S.; Kleinman, J. G. Kidney Int. 1998, 53, 952. (9) Wesson, J. A. Unpublished results. (10) Khan, S. R.; Finlayson, B.; Hackett, R. L. Lab. Invest. 1979, 41, 504. (11) Wesson, J. A.; Worcester, E. Scanning Microsc. 1996, 10, 415. (12) Wesson, J. A.; Worcester, E. M.; Kleinmann, J. G. J. Urol. 2000, 163, 1343.

10.1021/la011754+ CCC: $22.00 © 2002 American Chemical Society Published on Web 05/01/2002

Calcium Oxalate Monohydrate Crystallization Chart 1

A number of studies have relied on characterization of macroscopic COM crystals formed in the presence of various natural and synthetic macromolecular inhibitors to gain insight into the molecular mechanisms of the important crystal interactions. Inhibition of COM crystal growth has been demonstrated for poly(aspartic acid) (polyD, Chart 1),13 glycosaminoglycans (GAGs),14 citrate,15,16 uropontin,17 sodium diisooctyl sulfosuccinate (AOT),18 sodium dodecyl sulfate,19 nephrocalcin,20 and poly(glutamic acid) (polyE, Chart 1).13 In contrast, phospholipids21,22 have been used as promoters for COM nucleation. The selectivity between the three common crystalline forms of calcium oxalate, COM, COD, and calcium oxalate trihydrate (COT), can be controlled by varying the concentration of molecular additives such as dodecylammonium chloride,23 sodium cholate,24 sodium, potassium, ammonium, or magnesium salts of mono- and multicarboxylic acids,25 and various anionic polyelectrolytes26 introduced to supersaturated CaOx solutions (this latter report suggests that COD is increasingly favored as the concentration of anionic polyelectrolytes is increased). The evidence overwhelmingly supports a role for anionic functional groups in the growth modification of calcium oxalate. Moreover, COM growth suppression is commonly accompanied by the crystallization of COD. Additionally, investigations of COM growth in the presence of polyD and polyE have demonstrated that the crystal habit and growth characteristics of COM can be sensitive to slight differences in the structure of macromolecular inhibitors, beyond the chemical functional group in the anionic side chains.12 Though the aforementioned investigations have demonstrated the ability of inhibitors to alter calcium oxalate growth, morphology, and composition, the molecular level interactions leading to these influences remain unexplored. During the past decade, advances in real-time in (13) Ito, H.; Coe, F. Am. J. Physiol. 1977, 233, F455. (14) Shirane, Y.; Kurokawa, Y.; Sumiyoshi, Y.; Kagawa, S. Scanning Microsc. 1995, 9 (4), 1081. (15) Millan, A.; Sohnel, O.; Grases, F. J. Cryst. Growth 1997, 179, 231. (16) Antinozzi, P. A.; Brown, C. M.; Purich, D. L. J. Cryst. Growth 1992, 125, 215. (17) Worcester, E. M.; Blumenthal, S. S.; Beshensky, A. M.; Lewand, D. L. J. Bone Miner. Res. 1992, 7, 1029. (18) Tunik, L.; Addadi, L.; Garti, N.; Furedi-Milhofer, H. J. Cryst. Growth 1996, 167, 748. (19) Skrtic, D.; Filipovic-Vincekovic, N. J. Cryst. Growth 1988, 88, 313. (20) Deganello, S. Calcif. Tissue Int. 1991, 48, 421. (21) Letellier, S. R.; Lochhead, M. J.; Campbell, A. A.; Vogel, V. Biochim. Biophys. Acta 1998, 1380, 31. (22) Backov, R.; Khan, S. R.; Mingotaud, C.; Byer, K.; Lee, C. M.; Talham, D. R. J. Am. Soc. Nephrol. 1999, 10, S359. (23) Skrtic, D.; Filipovic-Vincekovic, N.; Fu¨redi-Milhofer, H. J. Cryst. Growth 1991, 114, 118. (24) Skrtic, D.; Filipovic-Vincekovic, N.; Babic-Ivancic, V.; TusekBozic, L.; Fu¨redi-Milhofer, H. Mol. Cryst. Liq. Cryst. 1994, 248, 149. (25) Cody, A. M.; Cody, R. D. J. Cryst. Growth 1994, 135, 235. (26) Manne, J. S.; Biala, N.; Smith, A. D.; Gryte, C. C. J. Cryst. Growth 1990, 100, 627.

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situ atomic force microscopy (AFM) have made possible direct imaging of nucleation and crystal growth events at the microscopic, and even near-molecular, level. These studies have enabled elucidation of crystal growth mechanisms at the microscopic level, including examination of the role of molecular additives on growth rates and crystal surface topography.27-32 We describe herein real-time AFM studies of CaOx dissolution and growth of COM crystals and the effects of sodium polyD, polyE, and their corresponding monomers on these processes. These additives were chosen for our investigations because all natural proteins known to affect calcium oxalate crystallization have been anionic, with 10-40% of their residues identified as aspartate and glutamate.33 Synthetic polymeric forms of these two amino acids, however, clearly affect calcium oxalate crystallization differently.12 The results described herein illustrate that polyD and polyE inhibit both growth and dissolution at the microscopic level, with polyD being substantially more effective in this regard. The direct visualization possible with AFM enables microscopic examination of the preferential growth modes and the influence of additives in real time so that direct comparisons of additives and the effects on the rates of crystal growth along specific crystallographic directions can be examined. This observed suppression of COM growth by these polymers may be crucial to shifting the selectivity toward COD, which in turn may be the natural defense mechanism against kidney stone formation. Experimental Section Materials. Sodium oxalate, sodium chloride, sodium hydroxide, calcium chloride (Aldrich), sodium poly-L-aspartate (molecular weight: 12 kDa), sodium poly-L-glutamate (molecular weight: 13 kDa), sodium aspartate, sodium glutamate, and HEPES [N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)] (Sigma) were used as received. Deionized water with 18 MΩ resistance was obtained with a Barnstead E-Pure purification system. COM crystals were obtained using an established procedure developed by one of our laboratories.11 Briefly, 0.4 mL of 10 mM CaCl2 stock solution was added to a 20 mL glass scintillation vial containing 9.2 mL of a NaCl-buffered solution at pH ) 7.5. After vigorous shaking for several seconds, 0.4 mL of 10 mM Na2Ox stock solution was added, producing a CaOx concentration of 0.4 mM, a NaCl concentration of 150 mM, and a HEPES concentration of 10 mM. This solution was shaken vigorously for 10-15 s and allowed to stand at room temperature (∼25 °C) for 2 weeks, during which crystals of COM grew. The COM crystals formed as elongated hexagonal plates, which were up to 2 microns thick and 10-50 microns in length. Crystals were separated from the solution by filtration through track-etched polycarbonate membranes with 5-micron pores (PCTE, 13 mm diameter, Poretics Corp.) mounted in a Swinney filter holder. The polycarbonate membrane was allowed to dry at room temperature, and the platelike COM crystals on the membrane surface were transferred to an AFM specimen holder that was coated with a partially cured (approximately 2 h) UV-curable optical cement (Type SK9, EMS Acquisition Corp.). Contacting the side of the polycarbonate membrane containing the COM crystals to the partially cured cement coating effectively transferred the crystals to the AFM specimen holder, while avoiding deleterious flow of the cement over the crystals. The cure cycle was then completed. (27) Teng, H. H.; Dove, P. M.; Orme, C. A.; De Yoreo, J. J. Science 1998, 282, 724. (28) Hillier, A. C.; Ward, M. D. Science 1994, 263, 1261. (29) Yip, C. M.; Ward, M. D. Biophys. J. 1996, 71, 1071. (30) Yip, C. M.; Brader, M. L.; DeFelippis, M. R.; Ward, M. D. Biophys. J. 1998, 74, 2199. (31) Davis, K. J.; Dove, P. M.; De Yoreo, J. J. Science 2000, 290, 1134. (32) Mao, G.; Lobo, L.; Scaringe, R.; Ward, M. D. Chem. Mater. 1997, 9, 773. (33) Shiraga, H.; Min, W.; VanDusen, W. J.; Clayman, M. D.; Miner, D.; Terrell, C. H.; Sherbotie, J. R.; Foreman, J. W.; Przysiecki, C.; Neilson, E. G.; Hoyer, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 426.

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Figure 1. (A) Optical microscopy image of a COM single crystal. (B) Schematic of the COM crystal habit with plane assignments drawn with the program SHAPE. (C) Top view of the (100) plane of a COM crystal in the P21/c representation, illustrating the molecular packing and the crystal habit. Calcium atoms are green, carbon atoms gray, oxygen atoms red, and hydrogen atoms off-white. This view is perpendicular to the (100) plane (i.e., along a*). (D) AFM image of the (100) plane of a COM crystal acquired after etching in H2O for 1 h, revealing several etch pits with shapes and aspect ratios similar to the habit of the bulk crystals. Methods. Real-time in situ atomic force microscopy was performed in contact mode with a Digital Instruments Nanoscope IIIa Multimode system using a glass liquid cell (Digital Instruments), 200 µm Si3N4 cantilever tips with a force constant of 0.06 N/m, and a scanner with a maximum lateral scan range of 15 µm × 15 µm. All data were acquired in height and deflection modes using the lowest tip forces possible, scan rates ranging from 2.5 to 3 Hz, and look ahead gain of 0.00. All solutions were introduced to the AFM cell via a Teflon tube inserted through a feed-through of the AFM cell and attached to a reservoir of the working solution. In every experiment, the AFM cell was refreshed with the working solution at 5-min intervals to maintain a relatively constant solution condition (either supersaturated or undersaturated). X-ray diffraction was performed with a Bruker-AXS Microdiffractometer that provides collimation of the X-ray beam (Cu KR, λ ) 1.54 Å) to a 50-micron spot size. Optical microscopy images were obtained with an Olympus-BX60 microscope.

Results and Discussion Calcium oxalate (CaOx) is known to crystallize in three forms with different extents of hydration: CaC2O4‚H2O (monohydrate, COM),34 CaC2O4‚(2+x)H2O (x < 0.5), (dihydrate, COD),35 and CaC2O4‚3H2O (trihydrate, COT).36 Both COM and COD are found in kidney stones, but as mentioned above, COM is the primary constituent of clinical stones. In contrast, the evidence suggests that formation of COD crystals may be protective against stone (34) Deganello, S. Acta Crystallogr. 1981, B37, 826. (35) Tazzoli, V.; Domeneghetti, C. Am. Miner. 1980, 65, 327. (36) Deganello, S. Am. Miner. 1981, 66, 859.

formation. Consequently, we have investigated the growth and dissolution characteristics of COM and the influence of macromolecular additives that may suppress COM and favor COD formation. The COM crystals used in these studies were grown in vitro from aqueous buffer solution as elongated hexagonal plates, in agreement with several previous reports. The crystals were up to 2 microns thick and 10-50 microns in length (Figure 1). For the purposes of describing the properties of COM crystals, we rely here on the crystal structure reported by Tazzoli and Domeneghetti35 (P21/c, a ) 6.290 Å, b ) 14.5803 Å, c ) 10.116 Å, β ) 109.46°). This index setting was used recently by Millan in a comparison of theoretical and experimental crystal habits.37 This space group setting and accompanying unit cell choice (Figure 1C) differ from those proposed by Deganello and Piro38 (P21/n, a ) 9.976 Å, b ) 14.588 Å, c ) 6.291 Å, β ) 107.05°), which have been used by others during related investigations of calcium oxalate crystallization. These crystal structures, both collected at room temperature, are identical. The Tazzoli setting was used here for convenience and ease of description (more of the relevant crystal planes and directions are principal ones). Millan has cataloged the indices for both unit cell choices and the equivalent planes for each.39 (37) Millan, A. Cryst. Growth Des. 2001, 1, 245. (38) Deganello, S.; Piro, O. Neues Jahrb. Mineral. Monatsh. 1981, 2, 81.

Calcium Oxalate Monohydrate Crystallization

Figure 2. (A) AFM image of a COM (100) surface acquired in air after retrieval from its crystallization solution. The edges of the (010) and (021) planes and the [010] and [001] directions are indicated. The feature in the upper center appears to be a remnant of a smaller, identically oriented COM crystal on the surface of the larger one. (B) The crystal in (A) after 30 min of immersion in water. (C) The crystal in (B) after growth in 0.25 mM CaOx for 50 min. (D) A different COM crystal after immersion in water for 30 min. The (100) surface has partially dissolved, resulting in numerous pits (see Figure 1 for an enlarged view) oriented along the [001] direction. The inset is the same crystal before immersion.

The crystal plane corresponding to the large upper face of these plates was confirmed to be (100) by X-ray microdiffraction (this plane is equivalent to the (-101) plane in the alternative P21/n setting of Deganello and Piro). Optical microscopy and AFM revealed angles of 143 ( 5° between the planes parallel to the long edges and their adjoining apical planes and 67 ( 5° between the two apical planes themselves (Figure 1). These angles are consistent with {010} planes forming the long edges of the crystals and the {021} apical planes (expected values of 142.6° and 74.7°, respectively). The assignment of these planes agrees with previous reports,20 after accounting for the different unit cell representation.40 The morphology of the COM crystals suggests that growth is fast along the 〈001〉 directions and that the {001} planes have negligible morphological importance, essentially growing out of existence. Therefore, growth along 〈001〉 is expressed by the {021} apical planes. The unambiguous assignment of the crystal faces of COM, coupled with its known crystal structure, can allow characterization of specific crystal growth events at the molecular level with AFM. The thin platelike habit of the COM crystals facilitated direct imaging of the large (100) surfaces (Figure 2). Images of the remaining planes, however, could not be obtained because of their extremely small areas. Unfortunately, the initial (100) crystal surface in air (Figure 2A), as well as during dissolution (Figure (39) COM undergoes a reversible phase transition between 38° and 45° to a high-temperature structure (the so-called “basic” structure), which crystallizes in the I2/m space group. We note that in ref 37 (Millan) the crystal structure depicted in Figure 1 for the (100) plane of the Tazzoli notation is the (-101) plane of the I2/m phase, not the identical room temperature P21/n phase. Consequently, the view of the (100) plane depicted in our Figure 1 differs somewhat from that in ref 37. (40) The most relevant planes for the results described here are the (100), (010), (001), and (021), which correspond to the (-101), (010), (100), and (120), respectively, of the P21/n structure reported by Deganello and Piro in ref 38.

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2B) and growth (Figure 2C), was rough by AFM standards (root-mean-square roughness of 50-100 Å). Terraces and steps, features that are commonly monitored during dynamic AFM imaging of crystal growth and dissolution, were not evident, although islands of growth without clearly oriented faces or planes could be seen under supersaturated solution conditions, as in Figure 2C. Typically, the observation of well-defined, molecularly flat, large terraces and attainment of high-quality lattice images require an inherent two-dimensional layered structure in the crystal wherein the bonding interactions within a specific set of layers are stronger than those between those layers (e.g., mica,41 zirconium phosphate42). Inspection of the crystal structure of COM suggests that the (100) plane is reasonably flat. However, Ox2- ions alternate orientation along the surface of the (100) plane, such that one-half are nominally in-plane (type I) and the other half project out of the plane (type II). Consequently, the Ca2+ and Ox2- ions essentially form a continuous network of short-range Ca2+ and Ox2- ion contacts both along various directions within the (100) plane, ranging from 2.42 to 2.47 Å, and between the (100) layers, ranging from 2.42 to 2.48 Å. The strong bonding between Ca2+ and Ox2- ions perpendicular to the (100) layers can impart a high surface energy to the (100) plane and frustrate the formation of molecularly flat (100) terraces, which would be preferred for dynamic AFM imaging of crystal growth and dissolution. Also, the roughened appearance seen under our conditions may be exacerbated when crystallization is examined under conditions far from equilibrium, such as those used here, or in growth solutions containing other components, like buffering ions. This limitation was circumvented, however, through direct imaging of pits that formed on the (100) surface under dissolution conditions (water or undersaturated solutions). The pits formed as elongated hexagons, oriented along 〈001〉, which mimicked the morphology of the bulk crystal (depicted under low resolution in Figure 2D and under higher resolution in Figure 1D). Because the pits exhibit an orientation and shape identical to those of the bulk crystal, the base of the pit can be assigned to the (100) plane and the internal edges of the pits can be assigned to the {010} and {021} planes. Specific events related to crystal dissolution can be observed directly by AFM by monitoring further etching of these pits. Conversely, specific events responsible for crystal growth can be observed from pit filling, which was induced through the addition of supersaturated CaOx solutions to the AFM cell. AFM images acquired in real time in pure water revealed etching of the pits at a rate of 26 nm2 s-1, as determined from an average of data acquired for six pits during a given run (Figure 3). Etching (and growth) rates must be expressed in terms of area per unit time because the depth and depth profile of the pits, which would define a volume, could not be established with sufficient accuracy by AFM. The aspect ratio of the pits (defined as the lengthto-width ratio, l/w, based on [001] and [010], respectively) increased with time, indicating that dissolution along the 〈001〉 direction was more rapid than along 〈010〉. Replacement of the pure water solution in the AFM cell with water containing 2 ug/mL of either polyD or polyE caused a very slight, almost immeasurable change in the pit etching rate. However, the addition of these polymers did stop the continual increase in pit aspect ratio with time (41) Baba, M.; Kakitani, S.; Ishii, H.; Okuno, T. Chem. Phys. 1997, 221, 23. (42) Kaschak, D. M.; Johnson, S. A.; Hooks, D. E.; Kim, H.; Ward, M. D.; Mallouk, T. E. J. Am. Chem. Soc. 1998, 120, 10887.

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Figure 3. (A-C) Real-time in situ AFM images acquired during etching of the COM (100) surface in pure H2O. (D-F) Realtime in situ AFM images acquired during etching of the same COM (100) surface after addition of an aqueous solution containing 2 µg/mL of sodium polyD. (G-I) Etching rates and aspect ratios during etching of pits on the COM (100) surface, initially in pure H2O and following addition of an aqueous solution containing 2 µg/mL of sodium polyD and polyE. The data in (G-I) represent averages of measurements for six pits on the COM (100) surface and are reproducible for different COM crystals.

during etching. In fact, the addition of sodium polyD at this concentration actually caused a slight continual decrease in the aspect ratio with time of etching (i.e., the pits become relatively wider with time). In contrast, a low concentration of polyE led to a constant aspect ratio as a function of time. Increasing the concentration of polyD or polyE to 4 and 50 µg/mL, respectively, completely inhibited etching of the measured pits. This was corroborated by bulk studies that revealed significantly slower dissolution of COM in the presence of 4 µg/mL polyD, relative to water alone. Combined, these observations suggest that polymer adsorption on the crystal planes that correspond to the inner surfaces of the pit perimeter ({010}, {021}, and the putative {001}) interferes with dissolution at these surfaces and inhibits enlargement of the pits. Both polymers appear to suppress dissolution of the {021}/{001} planes to a greater extent than {010}, with polyD exhibiting the greater effectiveness and selectivity. When the CaOx concentration in solution was raised to values exceeding 0.1 mM, the etching process was reversed and the pits filled, a process that is tantamount to crystal growth. In 0.25 mM CaOx aqueous solution, the area of the pits decreased monotonically. The initial rate, which corresponds to a growth rate, was approximately 350 nm2 s-1 (Figure 4A), sufficiently fast that AFM data on growth (pit filling) could be collected in a reasonable time frame. A limited number of AFM measurements performed at concentrations between 0.1 and 0.25 mM demonstrated slower rates of pit filling as compared with measurements at 0.25 mM but did not reveal any qualitative changes in crystal growth behaviors or inhibitor interactions. In a 0.25 mM solution, the pits typically filled within 40 min, their aspect ratio increasing with time as the pits filled. This reflects more rapid attachment to the {010} planes and faster growth along 〈010〉, relative to attachment at the {021} planes and growth along 〈001〉 (Figure 1). This observation appears to conflict with the bulk crystal

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Figure 4. Filling (crystal growth) rates and aspect ratios of pits on the COM (100) surface in aqueous solutions containing supersaturated (0.25 mM) CaOx, without polymer and with various concentrations of polyD (A,B) and polyE (C,D). The data represent averages of measurements for six pits on the COM (100) surface and are reproducible for different COM crystals. The rate of pit filling for 0.25 mM CaOx solutions containing 950 µg/mL of aspartate monomer is also indicated. Equimolar concentrations of glutamate monomer (1145 µg/mL) did not have an appreciable affect on the growth rate, though the aspect ratio was affected in a manner similar to that observed for aspartate monomer. Moreover, high concentrations of glutamate resulted in substantial crystallization on the exposed (100) surface. The initial areas of the pits differed for each solution condition tested because a freshly prepared crystal surface was required for each measurement. The initial pit areas for all samples, however, were within 10% of those for the crystal examined at 0 µg/mL. For purposes of comparison in this figure, the data in (A) and (C) were adjusted to the same initial area as the 0 µg/mL value (t ) 0) by vertical translation of the entire data set.

morphology, which suggests more rapid growth along 〈001〉. This behavior, however, most likely reflects the uniqueness of growth confined within small pits (vide infra). The observed effects of polyD and polyE on both growth rate and aspect ratio for COM immersed in a 0.25 mM CaOx solution in water are illustrated in Figure 4. The addition of polyD (Figure 4A) to supersaturated 0.25 mM CaOx solutions significantly attenuated the growth rate of COM, the rate decreasing with increasing polyD concentration up to 4 µg/mL, whereupon pit filling was completely inhibited within our experimental time scale. Addition of just 1 µg/mL polyD affords a reduction of the initial filling rate from 350 to 90 nm2 s-1. This level of inhibition compares favorably with the observed 5-fold reduction in bulk COM crystal growth rate we have measured in a seeded constant composition assay (1 µg/ mL of the same polyD in a saline buffered CaOx solution at comparable supersaturation).43 In the presence of polyE (Figure 4C), inhibition of growth was also observed, although the concentrations of polyE required for a given level of COM growth rate inhibition were much greater than for polyD. For example, 64 µg/mL of polyE was required for complete inhibition of growth. Prior studies, using bulk crystallization methods, have demonstrated weaker growth inhibition by polyE compared to polyD.13 In addition, polyD and polyE exhibited qualitative differences with respect to their influence on the aspect ratio during pit filling (Figure 4B and D, respectively). PolyD (43) Wesson, J. A. Unpublished results.

Calcium Oxalate Monohydrate Crystallization

caused an apparent reduction in the rate of increase in aspect ratio with time compared to the 0 µg/mL (control) condition, in a concentration-dependent manner. PolyE caused isomorphic pit filling at all concentrations tested. The aspect ratio data, however, must first be corrected for peculiarities associated with the confinement of growth in small pits. The usual model for step advancement relies on diffusion of “growth units,” in this case “CaOx,” from solution to a terrace plane followed by surface diffusion along the terrace to exposed step edges. Assuming isotropic surface diffusion rates and a pit aspect ratio greater than unity at the initiation of growth, CaOx growth units on the (100) terrace plane at the pit base will always have a greater probability of reaching the {010} steps in a given time increment than of reaching attachment sites located on the apical planes, because of shorter diffusion distances. The logical consequence of this is faster growth along 〈010〉 compared to 〈001〉, resulting in a continual increase in aspect ratio as the pit fills. Of course, any increase in aspect ratio further accelerates the growth rate along 〈010〉, so an accelerating increase in aspect ratio with time is expected from growth in small pits. This underlying anisotropy in pit filling complicates the comparison of changes in aspect ratio with time under the influence of various additives, because the change in aspect ratio with time depends on the extent of pit filling. In other words, the suppression of overall growth rate by polymer additives will reduce the expected aspect ratio at any point in time compared to a control (i.e., in the absence of polymer) because relatively less growth has taken place along the 〈010〉 direction. In the extreme example, 4 µg/ mL of polyD resulted in a constant aspect ratio as a function of time simply because no measurable growth occurred at this concentration. Intermediate concentrations present the same difficulty in interpretation. Therefore, the effect of the polymer additives on growth along the different crystal directions, which reflects their binding affinity for the associated crystal planes, is best deduced from the dependence of the aspect ratio on the extent of pit filling, given as 1 - A/A0, where A and A0 are the time-dependent and initial areas, respectively. This permits comparison of the aspect ratio changes at equivalent extents of pit filling (crystal growth) rather than time, thereby separating the effect of rate differences, pit confinement, and pit anisotropy from the actual binding of the polymer additives. Moreover, data collected in the absence of polymer serve as the benchmark against which the effects of polymers on growth along different crystallographic directions can be compared when analyzed in this manner. The data in Figure 5, which are derived from the data in Figure 4, reveal distinct differences in COM growth in the absence and presence of the polymers. The control condition (no polymer) demonstrated the predicted acceleration in aspect ratio with extent of pit filling. PolyD promoted an increase in the aspect ratio relative to growth in the absence of polymer. This signifies a greater attenuation of the growth along 〈001〉 than 〈010〉 relative to the control, which is tantamount to preferred attachment of the polymer to the {001} or {021} planes relative to the {010} planes. The extent of this effect increases with polyD concentration. PolyE, however, caused a reduction in the aspect ratio relative to growth in the absence of polymer, suggesting preferred attachment to the {010} planes relative to the {001} or {021} planes. In this case, the effect was independent of concentration. The enhanced inhibition afforded by cooperativity associated with binding units attached to a common

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Figure 5. The trend in aspect ratios during COM growth in the (100) pits in 0.25 mM CaOx aqueous solution, corrected for the extent of pit filling (1 - A/A0) so that the influence of the polymer additives on growth rates along specific crystal directions can be properly compared. The format reveals that polyD inhibited growth and induced an increase in the aspect ratio relative to growth in the absence of polymer. This signifies a greater attenuation of the growth along 〈001〉 than 〈010〉, which is tantamount to preferred attachment to the {001} or {021} planes relative to the {010} planes. The extent of this effect increases with polyD concentration. PolyE, however, inhibited growth and induced a reduction in the aspect ratio relative to growth in the absence of the polymer, reflecting preferred binding to the {010} faces. In this case, the effect was independent of concentration. The influence of polyD and polyE on the change in aspect ratio, relative to the absence of polymer, is schematically represented for each case. The borders represent the perimeter of the pits at different extents of filling. Aspartate and glutamate (not shown here) dramatically reduced the aspect ratio during pit filling but only weakly inhibited growth. The aspartate and glutamate concentration used corresponds to 7 mM, far exceeding the Ox concentration in the sample.

polymer chain deserves special mention. It is apparent from Figure 4A that a nearly 1000-fold greater concentration of aspartate monomer is less effective for growth suppression than the 12 kD polyD. The data in Figure 5 reveal that aspartate monomer causes a decrease in the aspect ratio relative to the no-polymer sample, in contrast to the increase observed for polyD. Measurements of pit filling in the presence of glutamate monomer at a molar concentration equivalent to that of the 950 µg/mL aspartate concentration (corresponding to 1145 µg/mL glutamate monomer) revealed significantly less inhibition of COM growth. At these high concentrations, significant deposition of material on the (100) COM surface was observed. Interpretation of these effects is complicated by the high concentration of aspartate or glutamate (ca. 7 mM), which substantially exceeds that of Ox2- (0.25 mM). At such high concentrations, formation of soluble calciumaspartate or calcium-glutamate complexes, incorporation of aspartate or glutamate into the growing crystal, or changes in crystal phase or composition may occur. Inspection of the data from both growth and dissolution experiments for the two principal directions in the pit (〈010〉 and 〈001〉 in the unit cell) revealed consistent behaviors. The rates for both growth and dissolution were slower in both directions when either polymer was added. Consequently, both polymers appear to inhibit exchange of growth units (CaOx) between the crystal surface and solution phase, and the changes in pit shape during growth or dissolution likely reflect differential adsorption of these

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polymers to specific crystal faces exposed in the pits. The increasing aspect ratios during growth in the presence of polyD suggest that polyD binds preferentially to {021} or putative {001} faces, resulting in slower growth along 〈001〉 relative to 〈010〉. This is completely consistent with the decreasing aspect ratios observed during dissolution (pit etching) in the presence of polyD, which also suggest preferred adsorption of polyD to {021} (or {001}) faces. In this case, slower dissolution from the apical planes than from the {010} planes results in the pits widening as they dissolve. Growth data (pit filling) in the presence of polyE are consistent with greater inhibition of growth along 〈010〉 as compared with 〈001〉, relative to the no-polymer control, suggesting that polyE has a preference for the {010} faces, albeit with lower overall affinity than polyD. In contrast, dissolution data for polyE demonstrated a net suppression along the 〈001〉 directions compared to the control, but to a lesser extent than polyD, which is at least consistent with a stronger interaction with the {010} faces of polyE than polyD. The fact that the aspect ratios achieve constant values in the presence of polyE for both pit etching and filling may reflect a more complicated behavior for polyE than for polyD, but our data do not offer further clues. Regardless, the importance of subtle differences in structural features, of both the polymers and COM crystal planes, on specific binding is still evident. One goal of this study was to gain understanding of behaviors observed in bulk crystallization experiments. In the bulk, comparable concentrations (in the µg/mL range) of polyD shifted the phase selectivity from COM to nearly 100% COD, in a concentration-dependent manner, with relatively little effect on the COM morphology.12 Intermediate concentrations of polyD yielded mixtures of relatively unaltered COM crystals and COD crystals. Under comparable conditions, polyE was not as effective in shifting the selectivity toward COD, with 70% COM/30% COD mixtures achieved at concentrations above 10 µg/mL polyE. This crystal composition was independent of polyE concentration up to the measured maximum of 80 µg/mL. At concentrations of polyE exceeding 1 µg/mL, however, the COM crystals were bunched into aggregates resembling those formed by COM in normal urine samples.44 The more pronounced effect on phase selectivity observed for polyD presumably reflects highly effective inhibition of COM growth along the most rapidly growing 〈001〉 direction, which is entirely consistent with our AFM observations. Conversely, the AFM observations indicate that polyE was not as effective in suppressing growth in the COM pits, and in particular, it may more actively suppress growth along the 〈010〉 directions. This difference in face-specific interactions may explain the relative inability of polyE to direct bulk crystallizations to COD formation. The effect of polyE on the morphology of individual COM crystals formed in bulk crystallization could not be assessed because the COM crystallites were highly aggregated, which prevented direct visualization of the morphology of individual crystals. Regardless of the actual mechanistic details, these observations clearly indicate that polyE is a much less effective inhibitor than polyD and that the additional methylene group affects the crystal face binding selectivity of the protein, thus altering the morphologies observed in bulk crystallization. Some useful data are also available from studies performed with naturally occurring acidic proteins. Among putative inhibitors of kidney stone formation, uropontin (or osteopontin, a glycoprotein that contains about 21% aspartic acid and 14% glutamic acid residues)32 was shown (44) Khan, S. R.; Hackett, R. L. Calcif. Tissue Int. 1987, 41, 157.

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to cause COD formation in bulk crystallization studies with no alteration in COM morphology, similar to polyD.8 Another potential inhibitor, nephrocalcin (a gla protein that contains about 11% aspartic acid residues and 12% glutamic acid residues, as well as γ-carboxyglutamic acid or gla residues)33 was shown to cause 100% COD formation at high concentrations.8 Furthermore, the nephrocalcin additive caused COM morphology changes at low concentrations, characterized by shortening of the crystal along 〈001〉 and blunting of the {021} faces, suggesting adsorption of the polymers on these faces.8,20 While the COM crystal morphology obtained with nephrocalcin was different than that obtained with polyE, the relatively high glutamic acid content in nephrocalcin compared to uropontin suggests an important link between this microstructural variation and COM morphology. Aspartic acid rich biological proteins are also known to influence the polymorphism of calcium carbonates in mollusk shells,45 which suggests that the interactions may apply more generally to any calcium-containing crystals, presumably through similar molecular level interactions between anionic side chains and the coordination sphere of calcium ions.15 On the other hand, a different COM morphological modification has been described in COM crystals grown in the presence of aspartic acid rich macromolecules (proteins) extracted from COM crystals formed in tobacco and tomato leaves.46 Obviously, complete understanding of the details of protein-crystal interactions at the molecular level requires further investigations. At present, we are unable to comment about possible contributions of protein folding to this interaction, as we have not measured the extent of either R-helix or β-sheet formation for these proteins under these solution conditions. Certainly, specific folded structures could present carboxylate side chains in particular orientations and spacings which might affect interactions with a certain crystal lattice or face, as has been suggested for other systems. The β-sheet conformation was thought to play a major role in interactions between calcium carbonate crystals and either polyD, polyE, or native proteins that led to crystal polymorphism in that system.47-49 The absence of structural characterization of the polymers under our conditions, however, makes such speculations about our results tenuous at best. Viewing the problem from the perspective of the crystal lattice depicted in Figure 1C, the {010} planes of COM expose an equal number of Ox2- ions aligned in two different orientations, one with its carboxylate groups perpendicular to the plane (type I) and the other with its carboxylate groups parallel to the plane (type II). The type I and type II Ox2- anions exhibit two different orientations at other surfaces as well, including the {021} and {001} planes, but they are generally oblique to these surfaces. If site-specific binding by the macromolecules governed adsorption, these structural features would appear to favor adsorption of anionic polymer molecules such as polyD and polyE on the {010} planes, wherein the pendant polymer carboxylates would insert readily into the positions otherwise occupied by the type I oxalates. The type II oxalate orientations on the {010} surfaces, or (45) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110. (46) Bouropoulos, N.; Weiner, S.; Addadi, L. Chem.sEur. J. 2001, 7, 1881. (47) Wierzbicki, A.; Sikes, C. S.; Madura, J. D.; Drake, B. Calcif. Tissue Int. 1994, 54, 133. (48) Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L. Chem.s Eur. J. 1998, 4, 389. (49) Falmi, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem.s Eur. J. 1998, 4, 1048.

Calcium Oxalate Monohydrate Crystallization

the oblique orientations of both types on the {021} and {001} surfaces, would appear to be less favorable adsorption sites owing to steric interactions between the methylene groups of the macromolecules and the crystal surface upon insertion into these sites. The {010} planes are the most calcium rich among these three crystal surfaces, the Ca2+ ion density on these faces decreasing in the order {010} > {100} > {021}. On the basis of these simple arguments, adsorption of the macromolecules on the {021} surfaces would be less favorable than on the {010}, contrary to the trends in the aspect ratio during growth in the presence of polyD but consistent with the behavior in the presence of polyE. Clearly, the binding is governed by subtle macromolecule-surface interactions that are not yet fully characterized with these simple models. Conclusions Face-specific growth and dissolution rates on the COM (100) surface can be measured directly with AFM by observing the evolution of surface pits. We have demonstrated that sodium polyD is an effective inhibitor of COM crystal growth and dissolution. The effectiveness of polyD is roughly 1000 times greater than that of the aspartate monomer, reflecting the cooperative binding of polyD to the crystal surfaces through the aspartate functional groups along the polymer backbone. PolyD macromol-

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ecules exerted greater influence on growth or dissolution in the 〈001〉 directions, presumably due to preferential adsorption to the {021} and putative {001} surfaces of the COM crystal. On the other hand, polyE exerts an overall weaker effect on growth rates but more strongly suppresses growth along the 〈010〉 directions, implying preferential adsorption on the {010} faces. These trends are consistent with prior observations of morphological behavior during bulk calcium oxalate crystallization in the presence of these and other macromolecules and provide hints of the origins of their role in the selectivity for COM and COD. The microscopic events observed by AFM provide a route to understanding the local phenomena that govern calcium oxalate crystallization and the nature of protein-crystal interactions responsible for the regulation of calcium oxalate crystal formation in various biomineral systems, including kidney stones. Acknowledgment. The authors gratefully acknowledge the financial support of the Department of Veterans Affairs (RCD9305), the Medical College of Wisconsin, the NIH (DK48504), and the University of Minnesota Industrial Partnership for Research in Interfacial and Materials Engineering (IPRIME). LA011754+