Effect of Phosvitin on the Nucleation and Growth of Calcium

Phosphoric acid esters cannot replace polyvinylphosphonic acid as phosphoprotein analogs in biomimetic remineralization of resin-bonded dentin. Sui Ma...
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J. Phys. Chem. B 2005, 109, 8257-8262

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Effect of Phosvitin on the Nucleation and Growth of Calcium Phosphates in Physiological Solutions Kazuo Onuma* Institute for Human Science & Biomedical Engineering, National Institute of AdVanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ReceiVed: NoVember 30, 2004; In Final Form: January 27, 2005

The promoting effect of phosvitin on the nucleation of hydroxyapatite (HAP) and the inhibitory effect of phosvitin on the transformation from amorphous calcium phosphate (ACP) to HAP were investigated. Atomic force microscopy observations showed that the nucleation of HAP on collagen substrate was greatly enhanced when the phosvitin was bound on the collagen surface. Nucleated crystals were uniformly distributed with a high nucleation rate on the collagen surface in the presence of phosvitin, while, in the absence of phosvitin, crystals nucleated slowly and were observed only at some particular area. Time-resolved static light scattering measurements revealed that the transformation from ACP to HAP was inhibited when free phosvitin was present in the calcium phosphate solutions. The transformation kinetics in the absence of phosvitin, which is a direct reconstruction of the inner ACP structure to HAP, was changed to heterogeneous growth of HAP on ACP with time.

Introduction The formation kinetics of hydroxyapatite (Ca10(PO4)6(OH)2, HAP) is an important issue from the viewpoints of the development of functional biomaterials that show similar physicochemical properties in vivo condition and insight into the biological mineralization in human hard tissues.1-8 The difficulties of understanding the formation mechanism of HAP are related to the complex conditions in which HAP nucleates and grows and to the presence of several kinds of metastable calcium phosphate phases. Especially under in vitro pseudophysiological conditions, metastable phases such as octacalcium phosphate (OCP), amorphous calcium phosphate (ACP), and dicalcium phosphate dihydrate (DCPD) are formed depending on a slight change in the solution concentration, pH, and presence of buffer. These metastable materials transform to HAP under physiological conditions when they coexist with HAP. Many kinds of inorganic and organic materials can affect the nucleation and growth of HAP; they can either promote or inhibit growth. For example, magnesium and zinc, which are essential elements in a human body, strongly inhibit HAP growth.9,10 Organic materials such as lipids and proteins affect the growth kinetics of HAP.11-14 Because such organic materials, especially proteins, are easily adsorbed on the faces of the HAP crystals, advancement of the growth steps on the surface is severely retarded. Immobilized proteins on substrates can sometimes promote HAP nucleation because such proteins possess radicals that act as favorite sites for heterogeneous nucleation of HAP. Wellknown radicals are carboxylic and phosphate;15,16 phosphoproteins especially should enhance HAP nucleation.17 Phosphoproteins are also thought to affect the transformation process from metastable calcium phosphates to HAP. I previously showed that ACP transforms into HAP with direct reconstruction of the loose inner structure to a rigid one with * To whom correspondence should be addressed. Phone: +81-29-8614832. Fax: +81-29-861-2565. E-mail: [email protected].

time under pseudo-physiological conditions.18 Because the essential unit that constructs the ACP aggregate is the same as that of HAP, that is, the calcium phosphate cluster, the transformation can proceed without passing through the normal dissolution and growth route. If the phosphoproteins affect the nucleation and growth of HAP, the transformation kinetics from ACP to HAP should also change. I have investigated the effect of phosvitin, a representative phosphoprotein, on HAP nucleation on a collagen substrate. Nucleation was observed both in the presence and absence of immobilized phosvitin on the collagen surface. I also investigated the effect of free phosvitin in the calcium phosphate solution on the transformation process from ACP to HAP. The results provide useful insight into the formation of bone structure, which is the composite of collagen and HAP. Experimental Section 1. Preparation of the Phosvitin-Bound Collagen Plate. A type I collagen deposited glass plate (Matsunami Glass Ind., Ltd. Osaka, Japan) was soaked in a solution containing egg yolk phosvitin (Sigma Aldrich Japan KK, Mw ) 34 kDa) and dimethyl suberimidate (Nakalai Tesque, Inc., Tokyo, Japan; hereafter DMS), a cross-linker between proteins, for a few days to prepare the phosvitin-bound collagen (PBC) plate. Phosvitin (12 µM) and 1.5-11 mM DMS were separately dissolved in 200 mM tris(hydroxymethyl)aminomethane (Tris) at a pH of 8.5. Both solutions were stirred for 12 h at 4 °C and then mixed with the same volume. The collagen deposited glass plates were soaked in this solution for 1-5 days to prepare the PBC plates. The prepared plates were finally washed with ultrapure water for 24 h to completely remove the unbound phosvitin, which would affect the nucleation and growth of HAP. 2. Physiological Solution and Simplified Calcium Phosphate Solution. A pseudo-physiological solution containing 140 mM NaCl, 1 mM K2HPO4‚3H2O, 2.5 mM CaCl2, and 50 mM Tris, buffered at a pH of 7.4 at 25 °C, was prepared. PBC and normal collagen plates were soaked in this physiological solution

10.1021/jp044550l CCC: $30.25 © 2005 American Chemical Society Published on Web 03/25/2005

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Figure 1. XPS spectrum for P2p of PBC plates prepared by soaking collagen plates in constant concentration of phosvitin and various concentrations of DMS solutions. The time dependence of the phosphate spectrum is shown for each DMS concentration: (a) 1.5 mM; (b) 7.5 mM; (c) 11 mM.

(PS), and the nucleation behaviors of HAP were compared using atomic force microscopy. The calcium phosphate solution used in the transformation experiment contained 2.5 mM CaCl2 and 1 mM H3PO4 and was buffered at a pH of 7.4 by KOH. This solution was used because concentrated NaCl and Tris greatly decrease the transformation rate. The effect of free phosvitin, 0.1 µM concentration, on the transformation kinetics was observed using time-resolved static light scattering. 3. Observation and Characterization of HAP Nucleation. Atomic force microscopy (AFM) (Veeco Japan Inc., Tokyo, Japan) was used to observe the surfaces of the PBC plates after soaking in the PS. All observations were done in air by tapping mode using silicon single-crystal cantilevers. Characterizations of the nucleated materials on the PBC plates were performed using thin-film X-ray diffraction (TF-XRD) (Rigaku Corporation, Japan) and X-ray photoelectron spectroscopy (XPS) (Quantum-2000, ULVAC-PHI, Inc., Japan). 4. Dynamic Light Scattering and Time-Resolved Static Light Scattering Measurements. Dynamic light scattering (DLS) measurements to estimate the dispersion of phosvitin in the presence of calcium or phosphate ions were performed using a DLS-7000 optical system (Otsuka Electronics Co., Ltd., Osaka, Japan) with an Ar+ laser (Spectra-Physics Lasers, Mountain View, CA) at a wavelength of 488 nm and using an ALV5000/E correlation system (ALV-Laser Vetriebsgesellschaft). All sample solutions were filtered using a 0.22 µm pore filter before measurement. Exponential function fitting and CONTIN analysis of autocorrelation data determined the apparent hydrodynamic radius of the phosvitin. The instrument used for the time-resolved static light scattering (TR-SLS) is described elsewhere.18,19 An ellipsoidal mirror and a high-speed CCD camera enabled simultaneous correction of the scattered light from a wide range of scattering angles. TR-SLS has superior time and scattering angle resolutions compared to conventional SLS. The angular dependence on the scattering intensity can be obtained at angles ranging from 10 to 170° simultaneously within a time interval of 1 s with a maximum angular resolution of 0.6°. TR-SLS was used to observe the changes in the molecular weight, radius of gyration, and fractal dimension of the calcium phosphate aggregate during transformation from ACP to HAP in the presence of phosvitin. A concentrated phosvitin solution containing 1 mM phosphate buffered at a pH of 7.4 was added to adjust its concentration at 0.1 µM in the calcium phosphate solution immediately after the pH of the calcium phosphate solution reached 7.4. Characterization of the initial (ACP) and final (HAP) products in the solution was performed using XRD

as we did in the previous study.18 The result was the same as that in the solution without phosvitin. Results and Discussion 1. Determination of the Appropriate Concentration of DMS. The appropriate concentration of DMS to prepare the PBC plate was estimated using XPS as described below. The collagen plates were soaked in a solution containing a constant concentration of phosvitin, 12 µM, and various concentrations of DMS, 1.5-11 mM. The plates were rinsed in pure water after removal from the solution, and then, the amount of phosphate on the surface, which resulted from the phosphate radicals in the phosvitin, was measured by XPS. Figure 1 shows the XPS narrow scan spectrum of phosphate, P2p, for the five soaking times. When the concentration of DMS was 1.5 mM (Figure 1a), the P2p peak showed an irregular change with time. The peak was broad after 3 days of soaking and then became sharper after 5 days of soaking. Figure 2a shows the XPS analysis of calcium, Ca2p, after soaking each sample for which the P2p spectrum is shown in Figure 1a in PS for 3 days. The peaks corresponding to Ca2p were all weak, independent of the reaction time between phosvitin and collagen via DMS. This indicates that HAP nucleation on the PBC plates was insufficient, probably due to less phosvitin bound by the small concentration of DMS. When the concentration of DMS was 11 mM, as shown in Figure 1c, the P2p peak reached a maximum after 3 days of soaking and then decreased. This suggests that the major portion of DMS was used to bind the phosvitin and collagen, while the excess free DMS in the solution simultaneously adsorbed on the formed PBC surface over time. When the plates for which the P2p spectrum is shown in Figure 1c were soaked in PS for 3 days, the Ca2p peak (Figure 2c) showed the same trend as that shown in Figure 1c; it reached a maximum after 3 days of soaking and then decreased. The 11 mM DMS was excessive and affected the nucleation of HAP in the PS. The DMS readsorbed DMS on the PBC plate, which polluted the surface and reduced the number of active nucleation sites for HAP. When the concentration of DMS was 7.5 mM, as shown in Figure 1b, the amount of phosphate continued to increase up to 5 days of soaking. After 5 days, it showed almost the same spectrum, indicating the reaction had reached equilibrium and that no excess DMS adsorbed on the PBC surface. Figure 2b shows the Ca2p peaks after each sample for which the P2p spectrum is shown in Figure 1b was soaked in PS for 3 days. The peak intensity increased consistent with the change in the

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Figure 2. XPS spectrum for Ca2p of PBC plates soaked in PS for 3 days. Time dependences of the calcium spectrum corresponding to (a) Figure 1a, (b) Figure 1b, and (c) Figure 1c.

Figure 3. Time-lapse AFM images of PBC plates soaked in PS: (a) original PBC surface; (b) surface after 3 h of soaking; (c) surface after 12 h of soaking; (d) surface after 3 days of soaking with nucleated HAP stacked three-dimensionally evident; (e) magnified image of part d; (f) lowmagnification image of part d. White dots are all HAP.

P2p spectrum observed in Figure 1b. The amount of calcium stayed constant for the PBC plate soaked more than 5 days. XPS measurements with various kinds of DMS demonstrated that 7.5-9.0 mM DMS is appropriate for preparing PBC plates. PBC plates soaked for 5 days in a solution containing 12 µM phosvitin and 7.5 mM DMS were therefore used in the experiments described below. 2. Nucleation of HAP on PBC and Collagen Plates. Figure 3 shows AFM images of the surface of a PBC plate soaked in PS for 3 h to 3 days. The solution was not stirred to prevent three-dimensional nucleation caused by high supersaturation of the PS with respect of the HAP. Figure 3a shows the surface before soaking; the fiber structure of the collagen can be seen. After 3 h of soaking (Figure 3b), small particles with diameters of 10-20 nm started to become evident on the surface. The precipitation of these particles obscured the fiber structure of collagen. Nucleated particles covered the surface after 12 h of soaking, as shown in Figure 3c. The fiber structure was not evident, and the roughness of the surface was temporarily reduced compared with that shown in part b. The black spots

evident in part c are areas where the particles did not precipitate. After 3 days of soaking, the surface was completely covered with the particles (Figure 3d). The magnified image in Figure 3e shows three-dimensional stacking of particles with 10-20 nm in diameter, the same as with 3 h of soaking. The larger scanning area image (Figure 3f) shows that the particles uniformly nucleated all over the surface. The white dots in part f are nucleated particles. This remained the case with 5 days of soaking, although the three-dimensional stacking of particles slowly continued. Examination of the nucleation feature not only at 25 °C but also at 37 °C, the human body temperature, revealed no remarkable differences. Figure 4 shows AFM images of the surface of the collagen plate (a) and the surface after 5 days of soaking in PS (b and c). When the phosvitin was not bound to the collagen, nucleation was drastically changed; the nucleation was not observed for up to 4 days, and the nucleation took place at some particular area, not over the whole surface, a stark contrast to the case using a PBC plate. Figure 4b shows the nucleated particles, and the low-magnification image in part c shows that the nucleation

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Figure 4. AFM images of a collagen plate soaked in PS for 5 days: (a) original surface; (b) surface after 5 days showing HAP nucleated within a limited area; (c) low-magnification image of part b.

Figure 5. Thin-film XRD patterns of PBC and collagen plates soaked in PS for 5 days.

was limited. Because the height ranges in parts b and c are more than 10 times that in part a, the detailed structure of the collagen is not visible. The nucleated particles grew into larger grains than those observed in the plate shown in Figure 3d, and the stacking was more in the vertical direction. The height range in Figure 3d is 10 nm, while that in Figure 4b is 15 times larger, 150 nm. Figure 5 shows the TF-XRD patterns for nucleated materials on both PBC and collagen plates. Peaks at 2θ ) 10.8, 21.8, and 32.9° were observed in the PBC sample. These peaks coincide with those of (100), (200), and (300) reflections of HAP.20 Theoretically, the (300) peak of HAP should be more intense than the (100) peak, contrary to the present case. This is probably an artifact caused by the setup of the sample. Because the nucleated layer on the PBC plate is very thin, a slight change in the incident angle of the X-ray caused by an increment in the sample plate might affect the relative intensity of each peak. The small size of each particle (less than 15 nm) might have some effect. The reflections seen in the figure show that the nucleated materials were HAP, arranged with their c-axis parallel to the PBC surface. A similar HAP arrangement was previously observed in a study using a thiol self-assembled monolayer (SAM).16 When a carboxylic thiol SAM substrate was immersed in PS, HAP nucleated with its c-axis parallel to the substrate. This is because the growth unit of HAP is Ca9(PO4)6 clusters with a slightly positive surface charge.21 The two calcium ions in the cluster were bridged by neighboring negative carboxylic radicals and took a similar to HAP orientating its c-axis parallel to the substrate. A similar mechanism apparently operated in the present study. Because the phosphate radicals have a greater ability to bind calcium than carboxylic radicals, the nucleation of the calcium phosphate proceeded more easily, forming a HAP (100) structure with the help of a relatively flexible distance between the phosphate radicals in the phosvitin as compared to the fixed distance between the carboxylic radicals in a SAM.

The Ca/P atomic ratio of nucleated HAP measured using XPS was 1.57 ( 0.03, indicating a calcium-deficient HAP. The X-ray pattern measured for the materials nucleated on the collagen plate showed a peak at 2θ ) 31.7°, which coincides with the most intense peak of HAP (211). The broad peak at around 2θ ) 20° was from collagen because nucleation occurred within a limited area. The measurement indicated that HAP nucleated with random crystallographic orientations on the collagen surface. The Ca/P atomic ratio could not be determined because of the limited area of the nucleation. 3. DLS Measurement of Phosvitin. In the DLS measurements, the autocorrelation function, g(2)(q,t), and scattering vector, q, were defined as

g(2)(q,t) - 1 ) q)

〈I(q,t) I(q,0)〉 〈I(q,0)〉2

θ 4πn sin λ 2

()

(1) (2)

where I(q,t) is the intensity of the scattered light, t is the time between consecutive photon-counting measurements, n is the refractive index of the solution, λ is the wavelength of the laser light, that is, 488 nm, and θ is the scattering angle. The g(2)(q,t) function was fitted using conventional CONTIN analysis and the following exponential function

g(2)(q,t) - 1 )

{

( )} t

∑i Ki exp - τ

2

(i ) 1, 2, 3, ...)

i

(3)

The decay time, τ, is related to the mutual diffusion coefficient, D, and q2 by 1/τ ) q2D. The translational diffusion coefficient, D0, was converted to the hydrodynamic radius, rH, by using the Einstein-Stokes relationship given by the Boltzmann constant, kB, temperature, T, and solvent viscosity, η.

rH )

kBT 6πηD0

(4)

Figure 6a shows g(2)(q,t) at representative scattering angles of θ ) 30, 60, and 90°. The concentration of phosvitin was 14.5 µM, and 2.5 mM CaCl2 was added to the solution at a pH of 7.4. This solution was stabilized by adding 1 mM Tris, a small enough amount not to affect the dispersion of phosvitin. The autocorrelation functions were fitted at each scattering angle using single-exponential functions assuming one kind of particle in the solutions. This fitting was confirmed by the decay time distribution profile in the CONTIN analysis. Figure 6b shows the relationship between 1/τ and q2 obtained from the CONTIN

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Figure 7. TR-SLS measurement of the transformation from ACP to HAP in the presence of free phosvitin: (a) change in molecular weight (solid circles) and radius of gyration (open circles) with time; (b) change in the fractal dimension of aggregate with time.

Figure 6. DLS measurements of phosvitin solutions containing 2.5 mM CaCl2: (a) autocorrelation functions at different scattering angles. All data can be fitted by a single-exponential function, (b) the relationship between 1/τ and q2, (c) the dependence of the diffusion coefficient of particles on phosvitin concentration.

analysis. The relationship is linear, and the mutual diffusion coefficient of the particle was 20.5 × 10-12 m2/s, as calculated from the slope of the line. As shown in Figure 6c, the dependence of D on the phosvitin concentration showed a linear correlation. The intersection of the line at a zero concentration of phosvitin, which corresponded to D0, was 20.5 × 10-12 m2/ s, which converts to rH ) 11.9 nm. This rH value shows that phosvitin takes an oligomer form in a calcium-containing solution. The small negative slope of the line in Figure 6c indicates a weak attractive interaction between the phosvitin oligomers within the measured concentration range. When 1 mM H3PO4 was added instead of 2.5 mM CaCl2, the absolute scattering intensities drastically decreased to less than 1/40 as compared to the calcium-containing solutions. Although the apparent hydrodynamic radius estimated by CONTIN analysis seemed to be ∼3 nm, conclusive information could not be obtained because of low S/N ratio g(2)(q,t) data and poor linearity on the 1/τ and q2 relationship. However, this means that the phosvitin was dispersed much smaller in the phosphatecontaining solution, probably as a monomer form. 4. TR-SLS Measurements of Calcium Phosphate Solution in the Presence of Phosvitin. TR-SLS measurements of simplified calcium phosphate solutions in the presence of

0.1 µM phosvitin were done to estimate the effect of phosvitin on the transformation kinetics from ACP to HAP. In preliminary measurements, the transformation was strongly inhibited when the phosvitin concentration exceeded 0.2 µM, and no change in the molecular weight and radius of gyration of the initial aggregate was observed, at least up to 10 h. The decrease in the pH of the solution during the transformation is small, less than 0.2. In the TR-SLS measurements, the apparent molecular weights, Mw, and the radii of gyration of scatterers, 〈S〉, were calculated using a Berry plot as follows.22 The Rayleigh ratio, R, at the scattering angle, θ, was expressed using Mw, the concentration of aggregates or precipitates, c, the particle scattering function of scatterers, P(θ), and the optical constant, K:

1 Kc ) R(θ)c)0 MwP(θ) K)

(5)

4π2n2 dn 2 NAλ4 dc

( )

(6)

where n, dn/dc, NA, and λ denote the refractive index of the solvent, the specific refractive index increment, Avogadro’s number, and the wavelength of the laser light, respectively. Since P(θ) ) 2(e-x - 1 + x)/x2 and x ) q2〈S〉2, eq 1 can be expressed as

(

) ( )(

Kc R(θ)c)0

1/2

)

1 Mw

1/2

1+

)

q2〈S〉2 6

1/2

(7)

Figure 7a shows the change in Mw and in 〈S〉 of the aggregate in the solution with time. Note that this large aggregate was large and did not originate from phosvitin oligomer, as described in the previous section. The aggregate was ACP, the same as

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that in the solution without phosvitin. Mw was constant up to approximately 110 min and then increased 6-fold within 60 min. After 170 min, precipitation of HAP was observed. The change in 〈S〉 showed the same trend. It was initially 200 nm and then started to increase in accordance with increasing Mw at around 110 min, finally reaching 350 nm. This size was approximately half the size of the aggregate that formed in the calcium phosphate solution without phosvitin. The internal structure of the aggregate sometimes showed a similar morphology independent of magnification over a certain limited range of length. This fractal structure is characterized by the fractal dimension, d, which also indicates the compactness of the inner structure. The observed scattered intensity, I(q), can be expressed using q and d as eq 8 because the relationship 1/〈S〉 < q < 1/r holds in the present case. Here, r is the radius of the essential unit of ACP, calcium phosphate cluster, which was approximately 0.4 nm as previously reported.18

I(q) ) Cq-d

(C: constant)

(8)

Figure 7b shows the change in d of the aggregate with time. The fractal nature was confirmed to hold at all stages of transformation by the linear relationship obtained when plotting log I(q) against log q. The initial d value, approximately 1.8, stayed constant up to 110 min and then increased with time, the same behavior as that of Mw and 〈S〉. The final d value was 2.5, which is smaller than that observed in phosvitin-free solution, 2.8. These characteristic features clearly indicate that the transformation process changes from direct reconstruction of the inner structure to heterogeneous nucleation and growth when free phosvitin is present in the solution. As previously reported,18 the ACP aggregate incorporates the growth unit in it with time, which increases the molecular weight. Because its inner structure is very loose, as shown by its low fractal dimension of 1.6, ACP can grow due to an increase in weight but not due to an increase in size, radius of gyration when the phosvitin is absent in the solution. With an increase in the compactness of the aggregate, direct reconstruction of the inner structure into a rigid form proceeds, reducing the total energy, and HAP precipitates. The free phosvitin, at first, inhibits the incorporation of the growth unit into the ACP aggregate, as shown by the delayed transformation. The phosvitin easily adsorbs on the ACP surface, and some of the phosvitin is probably incorporated into the loose ACP. This process completely stops the time-dependent drastic increase in the molecular weight of the ACP; moreover, it keeps the size of the ACP aggregate smaller in the initial stage. The inhibition by phosvitin reaches equilibrium at 110 min, and ACP aggregates in the solution along with immobilization of the phosvitin on their surfaces. The free phosvitin in the solution would be consumed at this stage, which is natural given the low phosvitin concentration. As described in the section on HAP nucleation on collagen, immobilized phosvitin acts as the favored nucleation site for HAP; therefore, the heterogeneous nucleation and growth of HAP on phosvitin-adsorbed ACP aggregate occurs after 110 min. This causes a simultaneous

increase in both the molecular weight and radius of gyration of the aggregate. The HAP directly nucleated in physiological calcium phosphate solution is low crystalline. This is in accordance with the small fractal dimension of the HAP that precipitates in a phosvitin-containing solution rather than the HAP that forms due to reconstruction of ACP in a phosvitinfree solution. Conclusion The promoting and inhibitory effects of phosvitin on the nucleation and growth of HAP and on the transformation from ACP to HAP were investigated. Immobilized phosvitin on collagen greatly enhances the nucleation of HAP with uniformly distributed precipitation, showing stark contrast compared to the case in the absence of phosvitin that the nucleation took place only at some particular area. The transformation kinetics from ACP to HAP was completely changed by adding free phosvitin to the solution. The direct reconstruction of the ACP inner structure to HAP changed to heterogeneous nucleation and growth of HAP on ACP. Acknowledgment. I am grateful to Ms. N. Kobayashi for her help in performing the experiments. This study is carried out as a part of “Ground-based Research Announcement for Space Utilization” promoted by Japan Space Forum. References and Notes (1) Brown, W. E.; Smith, J. P.; Lehr, J. R.; Frazier, A. W. Nature 1962, 196, 1050. (2) Bosky, A. L.; Posner, A. S. J. Phys. Chem. 1973, 77, 2313. (3) Barone, J. P.; Nancollas, G. H. J. Dent. Res. 1978, 57, 735. (4) LeGeros, R. Z. Calcif. Tissue Int. 1985, 37, 194. (5) Cheng, P.-T. Calcif. Tissue Int. 1987, 40, 339. (6) LeGeros, R. Z.; Daculsi, G.; Orly, I.; Abergas, T.; Torres, W. Scanning Microsc. 1989, 3, 129. (7) Christoffersen, M. R.; Christoffersen, J.; Kibalczyc, W. J. Cryst. Growth 1990, 106, 349. (8) Iijima, M.; Tohda, H.; Moriwaki, Y. J. Cryst. Growth 1992, 116, 319. (9) Kanzaki, N.; Onuma, K.; Treboux, G.; Tsutsumi, S.; Ito, A. J. Phys. Chem. B 2000, 104, 4189. (10) Kanzaki, N.; Onuma, K.; Treboux, G.; Tsutsumi, S.; Ito, A. J. Phys. Chem. B 2001, 105, 1991. (11) Gliman, H.; Hukins, D. W. L. J. Inorg. Biochem. 1994, 55, 21. (12) Doi, Y.; et al. Calcif. Tissue Int. 1989, 200, 44. (13) Nawrot, C. F.; Cambell, D. J.; Schroeder, J. K.; van Valkenburg, M. Biochemistry 1976, 15, 3445. (14) Hidaka, S.; Liu, S. Y. J. Food Compos. Anal. 2003, 16, 477. (15) Tanahashi, M.; Matsuda, T.; J. Biomed. Mater. Res. 1997, 34, 305. (16) Onuma, K.; Oyane, A.; Kokubo, T.; Treboux, G.; Kanzaki, N.; Ito, A. J. Phys. Chem. B 2000, 104, 11950. (17) Banks, E.; Nakajima, S.; Shapiro, L. C.; Tilevitz, O.; Alonzo, J. R.; Chianelli, R. R. Science 1977, 198, 1164. (18) Onuma, K.; Oyane, A.; Tsutsui, K.; Tanaka, K.; Trevoux, G.; Kanzaki, N.; Ito, A J. Phys. Chem. B 2000, 104, 10563. (19) Onuma, K.; Kanzaki, N.; Kubota, T. J. Phys. Chem. B 2003, 107, 11224. (20) Elliot, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates; Studies in Inorganic Chemistry 18; Elsevier: Amsterdam, The Netherlands, 1994. (21) Oyane, A.; Onuma, K.; Kokubo, T.; Ito, A. J. Phys. Chem. B 1999, 103, 8230. (22) Berry, G. C. J. Phys. Chem. 1966, 70, 4550.