Effects of Heat Treatment of Calcium Hydroxyapatite Particles on the

Jul 15, 2009 - The effects of heat treatment of calcium hydroxyapatite (Hap) on the protein adsorption behavior were examined using typical proteins o...
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J. Phys. Chem. B 2009, 113, 11016–11022

Effects of Heat Treatment of Calcium Hydroxyapatite Particles on the Protein Adsorption Behavior Kazuhiko Kandori,*,† Saki Mizumoto,† Satoko Toshima,† Masao Fukusumi,‡ and Yoshiaki Morisada‡ School of Chemistry, Osaka UniVersity of Education, Asahigaoka 4-698-1, Kashiwara-shi, Osaka, 582-8582, Japan, and Department of Processing Technology, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan ReceiVed: May 13, 2009; ReVised Manuscript ReceiVed: June 16, 2009

The effects of heat treatment of calcium hydroxyapatite (Hap) on the protein adsorption behavior were examined using typical proteins of bovine serum albumin (BSA: isoelectric point (iep) ) 4.7, molecular mass (Ms) ) 67 200 Da, acidic protein), myoglobin (MGB: iep ) 7.0, Ms ) 17 800 Da, neutral protein), and lysozyme (LSZ: iep ) 11.1, Ms ) 14 600 Da, basic protein). The TEM, XRD, and gas adsorption measurements ascertained that all of the Hap particles examined were highly crystallized and nonporous. The Hap single phase was continued up to the heat treatment temperature of 600 °C. However, after treatment above 800 °C in air, the β-Ca3(PO4)2 (β-TCP) phase slightly appeared. TG and ICP-AES measurements suggested that all of the Hap particles are Ca2+-deficient. Also, it was indicated from FTIR and XPS measurements that a partially dehydrated oxyhydroxyapatite (pd-OHap) was formed after treatment at high temperature. The saturated amounts of adsorbed BSA (nsBSA) did not vary on the Hap particles after heat treatment at 200 and 400 °C. However, nBSA values were increased by raising the heat treatment temperature above 600 °C. The adsorption s coverage of BSA was increased up to ca. 1.4. This adsorption coverage of BSA (θBSA) over unity suggests that the BSA molecules densely adsorbed and a part of BSA molecules adsorbed as end-on type on the Hap particle surface or BSA molecules became contracted. Similar adsorption behavior was observed on the LSZ system, but the adsorption coverage of LSZ (θLSZ) values are much less than θBSA. On the other hand, no effect of the heat treatment of Hap particles was observed on the adsorption of MGB. The increases of nsBSA and nLSZ were explained by the increase of calcium and phosphate ions in the solutions dissolved from β-TCP s formed after heat treatment of Hap, especially treated at high temperature. The dissolved Ca2+ and PO43ions may act as binders between proteins and Hap surfaces; the adsorption of Ca2+ ions on the Hap surface offers an adsorption site for BSA owing to its positive charge. In the case of adsorption of positively charged LSZ molecules, PO43- ions act as a binder in an opposite way. Since the MGB molecules are neutral, no binding effect of either ion was observed. Introduction It is well-known that calcium hydroxyapatite [Ca10(PO4)6(OH)2, Hap] is a bioactive or biocompatible material mainly because of its calcium-to-phosphorus (Ca/P) atomic ratio being similar to that of natural bone and teeth, and it possesses a high affinity to the proteins. Hap is in the space group P63/m; its unit cell parameters are a ) b ) 0.943 nm and c ) 0.688 nm, and it possesses two different binding sites (C and P sites) on the particle surface. Thus, it contains a multiple-site binding character for proteins.1-3 After dispersing Hap particles in aqueous media, calcium atoms (C sites) are exposed on the Hap surface by dissolution of OH- ions at the particle surface. Therefore, the C sites, rich in calcium ions or positive charge to bind to acidic groups of proteins, arranged on the ac or bc particle face in a rectangular manner with interdistances of 0.943 and 0.344 nm (c/2) for the a (or b) and c directions, respectively. Indeed, Chen et al. reported that the -COO- claw of protein grasps the calcium atoms of the Hap surface with its two oxygen atoms in a triangle form.4 The solid state NMR study also * Author to whom all correspondence should be addressed. E-mail: [email protected]. † Osaka University of Education. ‡ Osaka Municipal Technical Research Institute.

revealed that the -COO- terminus of amelogenin is orientated to the Hap surface.5 The P sites, lacking calcium ions or positive charge to attach to basic groups of proteins, are arranged hexagonally on the ab particle face with a minimal distance of 0.943 nm. In addition, Hap is the most stable calcium phosphate under physiological conditions. Hence, Hap is widely applied for separating various proteins, used as a column for a highperformance liquid chromatograph (HPLC) apparatus. Many essential studies therefore have been reported.2,6,7 The Hap ceramics used for the HPLC column are also occasionally reproduced by baking at high temperature for eliminating organic substrates. Furthermore, Hap is an ideal candidate for clinical applications either in the form of a fully dense sintered material or as a coating material on a bioinert metallic implant. After sintering, the bioceramics have been shown to favor protein adsorption and cellular attachment and growth.8 However, the mechanism of this fact has not been fully elucidated. The adsorption characteristics of BSA and LSZ on the Hap surfaces were compared by changing the heating temperature of the particles at 250-800 °C in air by Akazawa et al.9,10 They explained the characteristic changes of protein adsorption from a change in a proportion of C to P sites on Hap particles. However, since the morphology of Hap particles they treated

10.1021/jp904481z CCC: $40.75  2009 American Chemical Society Published on Web 07/15/2009

Effects of Heat Treatment of Calcium Hydroxyapatite

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TABLE 1: Properties of Hap Particles after Treatment at Various Temperatures Ca/P atomic ratio heat treatment temperature size (nm) particlea surfaceb SN (m2/g) SW (m2/g) SW/SN nontreated 200 °C 400 °C 600 °C 800 °C 1000 °C a

12 × 80 13 × 85 14 × 94 18 × 73 109 g1000

1.58 1.59 1.61 1.59 1.59 1.61

1.83 1.76 1.69 1.73 1.78 1.69

84.2 81.6 85.0 52.2 11.8 7.2

77.9 66.2 62.1 45.2 11.4 8.4

0.92 0.81 0.73 0.86 0.97 1.17

From ICP-AES. b From XPS.

TABLE 2: Properties of Proteins no. of functional groups/molecule proteins

isoelectric point

molecular weight (Da)

size (nm)

-NH2

-COOH

BSA LSZ MGB

4.7 11.1 7.0

67 200 14 600 17 800

4 × 14 3 × 3.5 3.5 × 4.5

680 155 34

680 32 36

was drastically changed by fusion, the result may not only be explicable by the proportion of C to P sites. Rouahi et al. also investigated the effect of sintering temperature on the protein adsorption capacity.8 Since the Hap ceramic disks they examined were prepared by pressing Hap powders, the effect of heat treatment was strongly correlated to the formation of intergranular microporosity. A study to explore the effects of heat treatment temperature on the protein adsorption behavior onto Hap particles without micropore, therefore, has been desired. The aim of this paper is to explore this subject. The results obtained in the present study will contribute to the development of a way to reproduce the high-quality HPLC column, and may be useful to the researchers in the fields of biomaterials, biomineralization, and biosensors.

Experimental Section Materials and Methods. The colloidal Hap particles were prepared by the following wet method:11-15 0.405 mol of Ca(OH)2 was dissolved into 20 dm3 of deionized-distilled water free of CO2 in a sealed Teflon vessel. After being stirred for 24 h at room temperature, 0.226 mol of H3PO4 was added into the solution and the suspension was stirred for a further 24 h at room temperature. This suspension was aged in an air oven at 100 °C for 48 h. The Hap particles generated were filtered off, thoroughly washed with distilled water, and finally dried at 70 °C in an air oven for 24 h. All chemicals were reagent grade, supplied from Wako Chemical Co., and used without further purification. The Hap particles employed were further treated between room temperature and 1000 °C in 200 °C intervals for 6 h in a conventional air oven. The shape, specific surface area, crystal phase, and Ca2+ and PO43- contents of Hap particles were determined by a transmission electron microscope (TEM), N2 and H2O adsorption measurements, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The adsorption isotherm of N2 was measured at the boiling point of liquid nitrogen with the use of a computerized automatic volumetric apparatus built in-house. Adsorption isotherms of H2O were also determined by a gravimetric technique at 25 °C. Specific surface areas were obtained by fitting the BET equation to these N2 and H2O adsorption isotherms and were abbreviated as SN and SW, respectively. Prior to these gas adsorption measurements, the samples were evacuated at 300 °C for 2 h. The XRD patterns were taken with Ni-filtered Cu KR radiation (40 kV, 120 A). The zeta potential (zp) of the particles was also measured by an electrophoresis apparatus. The characteristics of Hap particles examined are listed in Table 1. Protein Adsorption Measurement. The amounts of proteins adsorbed on Hap particles were measured by a batch method as following the method employed in our previous papers.16-19

Figure 1. TEM pictures of Hap particles after treatment at various temperatures.

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Figure 2. XRD patterns of Hap particles after treatment at various temperatures.

This measurement was conducted at 15 °C employing a 1 × 10-4 mol dm-3 KCl solution of the protein in 10 cm3 Nalgen polypropylene centrifugation tubes. The centrifugation tubes were gently rotated end-over-end at 15 °C for 48 h in a thermostat. The concentrations of proteins were measured by the microbiuret method using an UV absorption band at 310 nm after centrifuging the dispersions. Most of the UV experiments were triplicated and reproducible within 2%, indicating an uncertainty of 2 × 10-2 mg m-2 for the amounts of protein adsorbed. All proteins were purchased from Sigma Co. (BSA, A-7030; MGB, M-0630; LSZ, L-6876). The properties of proteins used in this study were listed in Table 2. Results and Discussion Properties of Hap Particles. The TEM pictures of Hap particles used in this study are shown in Figure 1. The particles as precipitated (called nontreated hereafter) are rod-like and 12 × 80 nm2 in size. The particle size is not remarkably changed up to 600 °C. However, the heat treatment above 800 °C induced the agglomeration of the particles to ca. 109 nm (at 800 °C) and to over 1000 nm (at 1000 °C). The X-ray diffraction spectra of the Hap particles are shown in Figure 2. From these results, it can be inferred that nontreated Hap particles were highly crystalline and composed of a pure Hap single phase. The Hap single phase was continued up to the heat treatment temperature of 600 °C. However, after treatment above 800 °C, the characteristic weak peaks of β-Ca3(PO4)2 (β-TCP) appeared. The formation of the CaO crystal phase was not detected in this study. From the N2 adsorption experiment, it was revealed that all of the particles employed are nonporous (data not shown). The Ca/P molar ratios of Hap particles thus prepared and assayed by the ICP-AES measurement were less than 1.67, suggesting that all of the particles are Ca2+-deficient. Figure 3 shows TG curves of the Hap particles. The weight loss in all of the materials occurs in a temperature range of 25-1000 °C, though no change in TG curves is detected for the particles after treatment at 800 and 1000 °C. The weight loss accompanies a small step at ∼800 °C. Berry20 and Monma et al.21 have reported for nonstoichiometric HAP that the large weight loss up to ca. 700 °C is caused by the release of adsorbed and bound H2O molecules and evolves a small amount of H2O molecules by dehydration of HPO42- ions, while the smaller weight loss above 700 °C resulted from a reaction of the P2O74ions formed by the reaction up to 700 °C with OH- ions. Applying the proposed mechanisms to the present materials with

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Figure 3. TG curves of Hap particles after treatment at various temperatures.

H2O molecules in the lattice, the corresponding reactions can be expressed by eqs 1 and 2.

Ca10-x(HPO4)x(PO4)6-x(OH)2-x(H2O)x · nH2O f Ca10-x(P2O7)x(PO4)6-2x(OH)2(H2O)x + nH2O (1) Ca10-x(P2O7)x(PO4)6-2x(OH)2(H2O)x f (1 - x)Ca10(PO4)6(OH)2 + 3xCa3(PO4)2 + 2xH2O (2) This result strongly suggests that Hap particles are Ca2+deficient. The appearance of β-TCP is due to a proceeding of reaction 2. It is well-known that nonstoichiometry occurs not only in biological Hap but also in synthetic Hap (both precipitated and high temperature preparation) by formation of vacant lattice sites, and complicates their crystal chemistry. Yamashita et al. revealed that a partially dehydrated oxyhydroxyapatite (pd-OHap),22,23 which has the apatite structure, is obtained after sintering Hap particles according to the following reaction:

Ca10(PO4)6(OH)2 f Ca10(PO4)6[(OH)2-2xOx0x] + xH2O (3) where the notation 0 expresses a vacancy. In the occurrence of a considerable amount of OH- vacancies, the apatite structure will be retained no longer. Figure 4 shows the FTIR spectra between 3000 and 4000 cm-1 of the Hap particles. The large and broad absorption band with a peak at 3400 cm-1 can be recognized. This band is ascribed to the OH stretching band of water molecules adsorbed. Therefore, the absorption band is weakened by raising the heat treatment temperature along with the reduction of their specific surface area. Another finding in Figure 4 is the strength of a sharp band at 3572 cm-1, identified as the stretching band of OH- ions in the Hap crystal, is diminished by raising the heat treatment temperature. This diminution of the 3572 cm-1 band substantiated the formation of pd-OHap. XPS spectra of the Hap particles were taken in order to characterize the surface layer of the particles. The spectra of the samples treated at different heat temperatures are shown in Figure 5. The shift of the ordinary peaks is calibrated with respect to the C1s peak position set at 284.6 eV. The C1s spectra were partially taken before and after the measurements of Ca and O spectra, and the peak positions were averaged. The dotted

Effects of Heat Treatment of Calcium Hydroxyapatite

Figure 4. FTIR spectra of Hap particles after treatment at various temperatures.

lines in each figure represent the literature values of Hap monocrystalline reported elsewhere.24 Clearly, the binding energies of XPS spectra peaks of the nontreated and treated particles at 200 °C are lower than the literature values, but they shifted to higher binding energy and agreed with the literature after treatment at 400 °C. Finally, they shifted to much higher binding energy after treatment above 600 °C, though all of the peaks possess a nearly equal full width at half-maximum (fwhm) of 1.8-2.0 eV. The peak strength is diminished after treatment over 800 °C because of a reduction of their specific surface area. It has been found that the increase in the binding energy of a core-electron is correlated not only to the change in the ionic valency induced by chemical environment25,26 but also to the change in the polarization and lattice energies for dielectric materials.27 Since the fwhm’s are almost equal among all of the samples, the increase in the binding energy cannot be ascribed to the increase in the lattice energy with the heat treatment. Also, β-TCP is hexagonal in crystal structure as well as Hap; this fact cannot be attributed to the phase transformation from Hap to β-TCP. Therefore, the reason for the increase in the binding energy by lattice energy should be excluded. As already described before, Yamashita et al.22 reported that the polarization of Hap takes place more effectively at higher temperatures due to the ionic diffusion mechanism of pd-OHap; polarization is attributed to the proton rotation of OH- ions in the apatite column channels. Hence, the shift of binding energy can be ascribed to the polarization of Hap particles by formation of pd-OHap. Of course, it is difficult to neglect completely electrostatic changes by a neutralizer attached to the XPS apparatus, because Hap is not an electronic but ionic conductor. The atomic ratios of Ca/P in the surface layer of the particles, estimated from the area intensity of the XPS peaks, are shown in Table 1 along with the total atomic ratios of the particles obtained by the ICP-AES method. Note that the surface Ca/P ratios are larger than the theoretical atomic ratio of 1.67, opposite to the result of the total atomic ones. This result reveals that the surface composition of Hap differs from the bulk ones in spite of the heat treatment, indicating that the surface phase is more Ca2+ rich than the bulk one, though the reason of this fact is obscure at the moment. Adsorption Behavior of Proteins on Heat Treated Hap Particles. BSA. Adsorption isotherms of BSA onto the Hap particles were shown in Figure 6 along with zp. All of the adsorption isotherms of BSA from 1 × 10-4 mol dm-3 KCl solution are the Langmuirian type. The saturated amounts of adsorbed BSA (nsBSA) for the nontreated sample were 0.6 mg/

J. Phys. Chem. B, Vol. 113, No. 31, 2009 11019 m2. The adsorption coverage of BSA (θBSA) on this system, defined as the ratio of the experimental amounts of adsorbed BSA (nsBSA) to the theoretical value, is 0.24. The latter value was estimated as 2.52 mg/m2 by assuming side-on adsorption of globular BSA molecules, which are prolate ellipsoids of 14 × 4 nm2.28 Since the solution pH of the system was ca. 6, BSA molecules were negatively charged. Therefore, the negative values of zp of this system were increased with increase in the amount of adsorbed BSA. The nsBSA values do not vary by heat values are increased treatment at 200 and 400 °C. However, nBSA s by raising the heat treatment temperature up to 1000 °C. The θBSA values were nearly unity for the Hap particles treated at 800 °C, and they were increased up to ca. 1.4 at 1000 °C. This adsorption coverage of BSA over unity suggests that the BSA molecules densely adsorbed and a part of BSA molecules adsorbed as end-on type on the Hap particle surface or BSA molecules became contracted. LSZ. Similar adsorption behavior to the BSA system was observed on the LSZ one, as is displayed in Figure 7. The amount of adsorbed LSZ (nsLSZ) does not vary until treatment of the particles at 600 °C, but it increases above 800 °C. The adsorption coverage of LSZ (θLSZ) was calculated as the ratio of the experimental to the theoretical nsLSZ value as well as the BSA system. The theoretical nsLSZ value was estimated to be 2.02 mg/m2 by assuming a side-on adsorption mode of globular LSZ molecules of which the size is 3.0 × 3.5 nm2.29 The θLSZ values for the Hap particle after treatment at 600 °C were nearly 0.1, though they increase to 0.2 and 0.3 after treatment at 800 and 1000 °C, respectively. However, despite the enhanced θLSZ values, these values are much less than θBSA, indicating that the LSZ molecules adsorbed on the Hap particle surface as the side-on mode. The adsorption of positively charged LSZ at pH 6 results in the polarity change of zp from negative to positive on all Hap particles. MGB. Completely different adsorption behavior to BSA and LSZ systems was observed on the MGB one in Figure 8; all of the adsorption isotherms show similar nsMGB and no significant change was detected among the particles. The adsorption coverage of MGB (θMGB) was ca. 0.3 for all of the systems, when we assume the side-on adsorption mode of the globular MGB molecule with 3.5 × 4.5 nm2 size.30 The low θMGB values also suggest that MGB molecules are adsorbed loosely by the side-on mode. It should be emphasized that there is no effect of the heat treatment of Hap particles on the MGB adsorption. The normalized ratios of saturated amounts of adsorbed proteins are shown in Figure 9, where the saturated amounts of adsorbed protein on nontreated particles in each protein system were taken as unity. It is noted that the normalized ratios of BSA and LSZ systems do not vary up to the heat treatment temperature of 400 °C, while they are increased over that of 600 °C especially for the BSA system. The normalized ratio obtained at 1000 °C is 5.5 times higher than that obtained for the nontreated sample. In the case of LSZ, a 3-fold increase in the normalized ratio can be observed at 1000 °C, though no change appears in the MGB system. Now we should consider these changes in the protein adsorption behavior by the heat treatment. It is well-known that the increase in the protein adsorption is caused by hydrophobic interaction between the hydrophobic part of the protein and adsorbent surface. However, the SW/SN ratio, which represents the surface hydrophilicity of the Hap particles, as shown in Table 1, is around unity. This result suggests that the particle surface hydrophilicity, in turn hydrophobicity, does not vary by the heat treatment. Therefore, a

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Figure 5. XPS spectra of Hap particles after treatment at various temperatures: (a) Ca2p; (b) O1s; (c) P2p.

Figure 6. (a) Adsorption isotherms of BSA and (b) their zeta potential for Hap particles after treatment at various temperatures. Heat treatment temperature: (O) nontreated, (4) 200, (0) 400, (3) 600, (2) 800, and (b) 1000 °C.

possibility of an increase in the nsBSA and nsLSZ values by an increase in the surface hydrophobicity can be excluded. The constant nsMGB value against the heat treatment temperature supports this idea because if the surface hydrophobicity was increased the nsMGB values have shown a similar rise as well as nsBSA and nsLSZ. Furthermore, no correlation can be seen in the surface Ca/P atomic ratio and the heat treatment temperature. Therefore, the change in nsBSA and nsLSZ should be attributed to the solution composition but not to the properties of the Hap particle surface. Now we should consider the production of β-TCP after the heat treatment at high temperature. It has been reported that the presence of R- and β-TCP phases at the adsorbent surface induces considerably the protein adsorption on the ceramic and

influences definitely the cell adhesion compared to cleaned Hap.31 However, the reason of this fact is not clear. Since the solubility of β-TCP is 10 times higher than Hap around pH 6,32 this high solubility of β-TCP can be affected to the protein adsorption behavior. Indeed, β-TCP is a more degradable material, its resorbability being supposed to improve vascularization of the implants.33 Hence, we measured the solubility of heat treated Hap particles by assaying the concentrations of calcium ([Ca2+]) and phosphate ([PO43-]) ions under the same condition of protein adsorption experiment; i.e., 100 mg of Hap particles were dispersed in 8 cm3 10-4 mol/dm3 KCl solution without protein in Nalgen polypropylene centrifugation tubes at 15 °C for 48 h. After the centrifugation, [Ca2+] and [PO43-] were assayed by the ICP-AES method. The results obtained for

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Figure 7. (a) Adsorption isotherms of LSZ and (b) their zeta potential for Hap particles after treatment at various temperatures. Heat treatment temperature: (O) nontreated, (4) 200, (0) 400, (3) 600, (2) 800, and (b) 1000 °C.

Figure 8. (a) Adsorption isotherms of MGB and (b) their zeta potential for Hap particles after treatment at various temperatures. Heat treatment temperature: (O) nontreated, (4) 200, (0) 400, (3) 600, (2) 800, and (b) 1000 °C.

Figure 9. Plots of normalized saturated amounts of adsorbed proteins as a function of heat treatment temperature.

the heat treated Hap particles are shown in Figure 10. Clearly, [Ca2+] and [PO43-] are increased steeply after treatment of the particles above 600 °C. This fact is completely consistent with the formation of β-TCP, as shown in Figure 2. Assuming that the molar ratio of Ca/P of the heat treated Hap particles is 1.6 (average value of Table 1), the x value in eqs 1 and 2 becomes 0.4. When all of the β-TCPs are dissolved during protein adsorption, the [Ca2+] and [PO43-] can be calculated to 81 and 54 mmol/dm3, respectively. However, the [Ca2+] and [PO43-] dissolved from β-TCP in Figure 10 are much lower than those of calculated values. This result indicated that not all of the

Figure 10. Concentrations of calcium and phosphate ions in the supernatant dissolved from the Hap particles after treatment at various temperatures.

β-TCPs are dissolved. The authors had reported the binding effects of cations such as Ba2+ and Al3+ of those concentrations were less than 1.5 mmol/dm3, as well as the [Ca2+] and [PO43-] values shown in Figure 10; i.e., these ions induce protein adsorption through complexing cations to carboxylic groups of acidic protein BSA.18 Therefore, it can be concluded that the increases of nsBSA and nsLSZ are attributed to the increase of

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calcium and phosphate ions in the solutions. Similar binding effects of cations were represented by Belcourt et al.34 and Rolla et al.35 The adsorption of cations on the Hap surface offers an adsorption site for BSA owing to its positive charge. Moreover, the binding of Ca2+ ions to anionic sites of BSA depresses the inter- and intramolecular electrostatic repulsion between BSA segments, resulting in the dense adsorption of BSA.36 In the case of basic LSZ, it can be postulated that the opposite binding effect of phosphate ions can take place. However, it is hard to originate the binding effects of both ions on the neutral MGB is much molecules with low surface charge. The increase of nBSA s more outstanding than that of nsLSZ, though [Ca2+] is lower than [PO43-]. This fact may be due to the difference in the elasticity between BSA and LSZ molecules. Since BSA is a soft protein, it is susceptible to the inter- and intramolecular electric repulsion with small amounts of [Ca2+], resulting in a remarkable gain in nsBSA in Figure 6. Conclusion The effects of heat treatment of Hap particles on the protein adsorption behavior were examined using BSA, LSZ, and MGB. The Hap single phase was continued up to the heat treatment temperature of 600 °C, but the β-TCP phase appeared after treatment above 800 °C. All of the Hap particles were Ca2+deficient, and it was indicated from FTIR and XPS measurements that a partially dehydrated oxyhydroxyapatite was also values did formed after treatment at high temperature. The nBSA s not vary by heat treatment at 200 and 400 °C, but they were increased by raising the heat treatment temperature up to 1000 °C. Similar adsorption behavior was observed on the LSZ system. On the other hand, no effect of the heat treatment of Hap particles was observed on the adsorption of MGB. The and nLSZ were explained by the binding effects increases of nBSA s s of calcium and phosphate ions dissolved from β-TCP. However, no binding effect of either ion was seen on the neutral MGB system. Acknowledgment. The authors thank Professor Katsumi Kaneko and Professor Hirofumi Kanoh at Chiba University for help with the XPS measurement. References and Notes (1) Kawasaki, T.; Takahashi, S.; Ikeda, K. Eur. J. Biochem. 1985, 152, 361–371. (2) Kawasaki, T.; Niikura, M.; Takahashi, S.; Kobayashi, W. Biochem. Int. 1986, 13, 969–982. (3) Kawasaki, T.; Ikeda, K.; Takahashi, S.; Kuboki, Y. Eur. J. Biochem. 1986, 155, 249–257. (4) Chen, X.; Wang, Q.; Shen, J.; Pan, H.; Wu, T. J. Phys. Chem. C 2007, 207, 1284–1290.

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