Hydroxyapatite: Hexagonal or Monoclinic? - Crystal Growth & Design


Department of Physics, Faculty of Science, National University of Singapore, 2 Science Drive 3, Singapore 117542, and National Laboratory of Solid Sta...
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CRYSTAL GROWTH & DESIGN

Hydroxyapatite: Hexagonal or Monoclinic? Guobin Ma†,‡ and Xiang Yang Liu*,† Department of Physics, Faculty of Science, National UniVersity of Singapore, 2 Science DriVe 3, Singapore 117542, and National Laboratory of Solid State Microstructures, and Department of Physics, Nanjing UniVersity, Nanjing 210093, China

2009 VOL. 9, NO. 7 2991–2994

ReceiVed February 10, 2009; ReVised Manuscript ReceiVed May 29, 2009

ABSTRACT: Bioapatite, the major constituent of mineralized tissues in mammalian bones and teeth, has been modeled to the hexagonal hydroxyapatite phase. Monoclinic hydroxyapatite, synthesized before only at very high temperature, is the thermodynamically most stable phase and is expected to exist also in hard tissues. In this work, hydroxyapatite nanobelts are produced by hydrolysis of brushite crystals and are identified to be the monoclinic phase based on electron microscopy and electron diffraction techniques. This is the first report of fabricating monoclinic hydroxyapatite crystals at low temperature. As the structural differences between hexagonal or monoclinic hydroxyapatite are very subtle, the success of this characterization also shows the great potential of electron microscopy and electron diffraction techniques for precise phase identification. The calcium phosphates comprise the largest group of biominerals in vertebrate animals. The structure of the calcium phosphate biominerals can generally be one of several model compounds. Three of the most common model compounds include hydroxyapatite (Ca5(PO4)3OH; HAP), octacalcium phosphate (Ca8H2(PO4)6 · 5H2O; OCP), and brushite (CaHPO4 · 2H2O). The biologically most important compound is HAP, as it is the primary mineral in hard tissues such as bones and teeth. The structural synergy between HAP and organic substrates determines the unique and amazing elastic and mechanical properties of hard tissues. The form of HAP most frequently encountered is hexagonal, having the P63/m space group symmetry, with the lattice parameters of a ) b ) 9.432 Å, c ) 6.881 Å, and γ ) 120°. The structure consists of an array of PO4 tetrahedra held together by Ca ions interspersed among them. The Ca ions occur in two markedly different sites, in accurately aligned columns (Ca(I)) and in equilateral triangles (Ca(II)) centered on the screw axis. The OHs occur in columns on the screw axes, and the adjacent OHs point in opposite directions. Note that this configuration implies that there would be steric interference between adjacent OHs. Therefore, a reversal of the OH direction within a column requires that some reversal points be provided by omission of an OH-. This can be accomplished by replacement of an OH- with a vacancy, or F-, Cl-, etc.1 HAP can also exist in another form, that is, the monoclinic form.2,3 The monoclinic form of HAP is the most ordered and the thermodynamically most stable form, even at room temperature.1,4 The discovery of the monoclinic form occurred much later than the hexagonal form. Fractional millimeter-sized single crystals of chlorapatite were converted to single crystals of monoclinic HAP by being exposed to steam at 1200 °C.2 The space group for stoichiometric HAP is P21/b, and the monoclinic unit cell has parameters a ) 9.421 Å, b ) 2a, c ) 6.881 Å, and γ ) 120°.2 The major difference between the monoclinic HAP and the hexagonal HAP is the orientations of the hydroxyl groups. In the monoclinic HAP, all of the OHs in a given column are pointed in the same direction, and the direction reverses in the next column. On the other hand, in the hexagonal HAP, the adjacent OHs point in the opposite directions as mentioned above. Although the structural differences between monoclinic and hexagonal HAP are very small, they are sufficient to exert a strong impact on some of its physicochemical properties.1,5 Because the * Corresponding author. E-mail: [email protected] Phone: +65-6516 2812. Fax: +65-6777 6126. † National University of Singapore. ‡ Nanjing University.

monoclinic HAP is structurally more stoichiometric than the hexagonal HAP, it is predicted that the dissolution kinetics and the kinetics of diffusion along the OH-column region will be quite different.5 In addition, the dielectric properties of HAP are closely related to the structural difference.6,7 Therefore, the knowledge of the occurrence and synthesis of the monoclinic HAP phase is particularly important for our understanding of the biological apatite formation mechanisms and perspective applications in bone remodeling practices. Very unfortunately, in spite of its importance, not much attention has been devoted to the formation of monoclinic HAP under normal conditions until now. It was believed that the monoclinic HAP phase could only occur in the bulk at high temperatures.1-3,8,9 Moreover, as in organisms HAP crystallites contain many “impurity” ions and vacancies that could provide the inversed centers for the OH directions; thus, the occurrence of hexagonal HAP is believed to be the most likely event compared to monoclinic HAP. We notice that the above conclusion is mostly based on X-ray diffraction (XRD) results. However, natural bioapatites and most synthetic HAP crystals are highly defective or poorly crystallized, leading to broad diffraction lines. As a result, we should point out that the slight structural difference between hexagonal HAP and monoclinic HAP makes it difficult to accurately distinguish the structures by XRD techniques. For example, the ribbon-like morphologies existing in developing dental enamel crystals may suggest the monoclinic phase from the viewpoint of crystalline symmetry, although they are always assigned to the hexagonal phase.2 Even though brushite is more soluble than other calcium phosphates, it forms under many physiological, geochemical, and laboratory conditions. Brushite is also an important compound in biomineralization processes. It is known to participate in tooth, bone, calculus, and renal stone formation.1 Brushite will hydrolyze to more basic calcium phosphates, but the hydrolysis process is different from OCP.10 Brushite seems to always dissolve first, then reprecipitate, while OCP usually transforms topotactically. In the view of the biological importance of HAP and brushite, the emphasis should be given to the understanding of the hydrolysis process. In this paper, we present a study of the formation and characterization of the monoclinic HAP nanobelts by hydrolysis of brushite crystals. To overcome the shortcomings of XRD in the HAP phase characterization, selected area electron diffraction (SAED) as well as high resolution transmission electron microscopy (HRTEM) is employed in this study. This work is to clarify the most stable phase of HAP occurring at low temperatures.

10.1021/cg900156w CCC: $40.75  2009 American Chemical Society Published on Web 06/03/2009

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Figure 2. XRD patterns for (a) as-prepared sample and (b) after treatment at 95 °C for 24 h, respectively. The former is indexed to brushite, whereas the latter is denoted with the monoclinic HAP indices.

Figure 1. SEM images for (a, b) as-prepared sample from the reaction of K2HPO4 and CaCl2, and the sample after treatments at (c) 80 °C for 1 h, (d) 80 °C for 24 h, (e) 95 °C for 1 h, and (f) 95 °C for 24 h, respectively.

All of the reagents (analytical grade purity) were purchased from Sigma Aldrich Company and were used directly without any further purification. To prepare brushite crystals, 1 M dipotassium phosphate (K2HPO4) aqueous solution was added slowly to a continuously stirred 1 M calcium chloride (CaCl2) solution. After mixing, white precipitates appeared in the solution. The mixed solution was further stirred for 24 h to ensure a thorough reaction. The reacted solutions were treated at 80 or 95 °C for a period from 1 to 24 h, respectively. Then the solutions were cooled to room temperature and the products were washed out by centrifugation. X-ray diffraction (XRD) measurements for the products were conducted on an X’pert Pro diffractometer with Cu KR radiation at 40 kV and 40 mA. Scanning electron microscopy (SEM) investigation of the morphologies was performed on a JEOL JSM-6700F field emission scanning electron microscope (FESEM). A JEOL JEM-2010F high resolution transmission electron microscope (HRTEM) was employed for microstructural and SAED measurements. The typical morphology of the as-prepared sample is elongated slabs with an average length of 10 µm and width of 5 µm (Figure 1a,b). After heat treatment at 80 °C for 1 h, a small portion of nanobelts appears (Figure 1c). However, as shown in Figure 1d, the slabs will convert entirely to belt-shaped products upon 24 h treatment at 80 °C. The length of the belts reaches several micrometers, whereas the width varies from several hundreds of nanometers to 1 µm. Higher temperature will greatly speed the formation of nanobelts. As one can see from Figure 1e, the entire conversion takes place within 1 h at 95 °C. Although further treatment does not lead to significant morphology change, thinner nanobelts such as that shown in Figure 1f will appear. XRD measurements of the samples show that the as-prepared and the heat-treated samples are of different phases. As presented in Figure 2a, the XRD plot of the as-prepared sample is characteristic of brushite crystals (JCPDS 72-0713). On the other hand, the plot of the sample treated at 95 °C for 24 h can be assigned either to the hexagonal HAP (JCPDS 72-1243) or monoclinic HAP

(JCPDS 76-0694). The XRD database shows that the three strongest diffraction peaks are located at around 31.7, 32.2, and 32.9° 2θ. However, it can be seen from Figure 2b that the peaks at around 25.8 and 28.1° are much more intense than the others. The 25.8° 2θ diffraction corresponds to {0002} planes of hexagonal HAP, or {002} of monoclinic HAP, while the 28.1° one to {202j1} of hexagonal HAP, or {102} and {1j22} of monoclinic HAP. Taking into account of the length of the belts is more than 10 times greater than the width, the much higher intensities of these two peaks can be attributed to the shape effects. Figure 2b also reveals a diffraction peak at 12.6° 2θ position (denoted by an asterisk). The d-spacing of this peak is 6.88 Å, suggesting that it arises from hexagonal {0001} or monoclinic {001}. This diffraction, however, should not appear for ideal hexagonal HAP due to the extinction condition. It will also be too weak to be detectable for perfect monoclinic HAP. The existence of this diffraction peak indicates that the products have a certain amount of defects no matter if they are hexagonal or monoclinic HAP. The defects are likely attributed to Ca deficiency in the products, as it was reported that the Ca to P ratio is always lower than the stoichiometric value of 1.667.1 As mentioned above, it is difficult to assign unambiguously the XRD plot in Figure 2b to the hexagonal or monoclinic HAP phase. However, the occurrence of the belt-shaped morphology suggests the products are more likely of the monoclinic HAP phase. The reason is that the structural symmetry governs the morphology of the crystals. The rod-like morphology with the 6-fold symmetry along the c-axis is energetically more preferable than the belts for the hexagonal HAP, whereas the difference in a and b lattice parameters of the monoclinic structure is consistent with the beltlike shape. Shown in Figure 3a is a TEM image for the 95 °C 24 h sample. The translucent characteristics of the overlapped crystals clearly indicate that the thickness is much smaller than the width; that is, the products are nanobelts. HRTEM image in Figure 3b represents the two-dimensional structural information of the crystal. The image is assigned to monoclinic HAP with the denotation of lattice planes (002) and (2j20) in the figure. Nevertheless, it can also be assigned to hexagonal HAP and the corresponding planes are (0002) and (112j0). Figure 3c is the SAED pattern of a single nanobelt. One can see that besides the strong diffraction spots, there are rows of much weaker spots. If the pattern is denoted to the monoclinic HAP phase as shown, it is a view along the [110] zone-axis. In the figure a characteristic weak spot is indicated by a question mark, which has a d-spacing 2/3 times the 2j20 spot. Taking into account the overall symmetry, the spot can be assigned to 3j30. In fact, another characteristic spot exists in between of the transmission spot and

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Figure 3. (a) TEM image, (b) HRTEM image, and (c) SAED pattern for the sample treated at 95 °C for 24 h. The HRTEM image and SAED pattern are denoted by the indices of monoclinic HAP phase.

the 2j20 spot, which is undetectable due to the very intense transmission signal. This diffraction corresponds to (1j10) planes of monoclinic HAP. If the crystal has the structure of the hexagonal HAP, the indices of these three diffraction spots would have been 1/2 (112j0), (112j0), and 3/2 (112j0). Such fractional indices imply that the observed diffraction spots are neither associated to the ideal hexagonal HAP nor to the hexagonal HAP with statistically distributed defects. In fact, if this assignment is adopted to interpret the diffraction data, a symmetry reduction of the structure from the given hexagonal lattice is inevitable. In Figure 4a,b, we present the schematic drawings of the monoclinic and hexagonal HAP structures projected along the [110] and [1j100] directions, respectively. One can see that the projections of the two structures are very similar. Note that the major difference of the hexagonal and monoclinic structures is reflected by the ordering of the hydroxyl ions. In the hexagonal HAP, the hydroxyl ions are similarly arranged in columns in 2-fold disorder with the O f H direction pointing away from the mirror planes passing through the nearest coordinating calcium ions. The monoclinic HAP, on the other hand, has the P21/b space group, and it has both the intracolumn and intercolumn ordering of hydroxyl ions. HRTEM is a powerful technique for structural study of crystals, especially nanometer-sized crystals, owing to its atomic level resolution capacity. Ideally, an HRTEM image is the potential projection of the crystal and the contrast can be directly correlated to atomic columns of the crystal. However, structural modeling is required for faithful interpretation of experimental HRTEM images, especially for complicated structures or for distinguishing similar phases.11 The background noise in Figure 3b is reduced by the fast Fourier transform (FFT) method, and the result is enlarged and shown in Figure 4c. The insets in Figure 4c are the simulated HRTEM images of the two structures using the same thickness and defocus parameters. It is clear that the simulation of the monoclinic HAP (upper inset) matches very well with the experimental result (Figure 4c). On the other hand, the difference of the experimental image and the simulated hexagonal HAP image (lower inset) is very evident. On the basis of the simulation a thickness of 5-8 nm is also obtained, which agrees with the estimation from Figure 3a. Moreover, the rendering of the symmetry from P63/m to P21/b will lead to additional reflections. On the basis of this fact we assign Figure 3c to monoclinic HAP crystal. This assignment can be confirmed by electron diffraction simulation. Shown in Figure 5 are the experimental SAED pattern and the two simulated diffraction patterns. We index the experimental pattern (Figure 5a) and the simulated pattern of monoclinic HAP single crystal (Figure 5b) using the hkl denotation, with hk values shown on top of the figures and l values at the left. The hkil denotation is used to index the

Figure 4. (a) and (b) Schematic drawings of the atomic structures of monoclinic HAP along [110] and hexagonal HAP along [1j100] view directions, respectively. The hydroxyl ions are emphasized in the drawings to illustrate the primary difference of the two structures, and the symmetry-determined morphologies of the crystals are inserted at the lower right corners. (c) Enlarged HRTEM image of Figure 3b, with the simulated structural images of monoclinic HAP along [110] and hexagonal HAP crystals along [1j100] zone-axis in the upper and lower insets, respectively. A unit cell is also illustrated in (c).

simulated pattern of hexagonal HAP for the purpose to reflect its hexagonal symmetry, and the hki values are given on top of Figure 5c. One can clearly see that the hjkl (where h ) k ) even numbers) diffraction spots of monoclinic HAP match well with the hkil (where

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Figure 5. (a) SAED pattern of a single crystalline HAP nanobelt, and (b, c) simulated electron diffraction patterns of monoclinic HAP and hexagonal HAP structures, respectively. The diffraction spots are indexed by the hk values (hki for hexagonal HAP) on top of the patterns and the l values at the left.

Figure 6. SEM images for HAP produced at 95 °C for 24 h at pH values of (a) 7.6 and (b) 11.0, respectively.

h ) k ) integer, i ) -2h) spots of hexagonal HAP, but the hjkl (where h ) k ) odd numbers) spots in Figure 5b have no counterparts in Figure 5c. It means that the weaker k-odd diffractions are characteristic of monoclinic HAP. Therefore, from the weak k-odd diffraction spots in SAED pattern of the single belt (Figures 3c and 5a), the structure is further confirmed to be the monoclinic HAP. It has been demonstrated that the hydrolysis of brushite to HAP involves the dissolution of brushite and nucleation and growth of HAP crystals.12,13 To explain why the phase of the products is monoclinic HAP instead of hexagonal HAP, both the energetic and kinetic factors must be taken into account. A study on the structure and energetics of HAP was reported recently by Haverty et al.4 Their calculation using generalized gradient approximation density functional theory showed that the monoclinic model was energetically more favorable than the hexagonal model. Similar results were also reported in other theoretical work.14,15 The impact of kinetics controlling factors including temperature, supersaturation, solvent, impurities, and the period of crystal growth, etc., should be further investigated. The monoclinic HAP has been believed to form in bulk only at elevated temperature.1-3,8,9 Our results, however, show that monoclinic HAP nanobelts can be produced by hydrolysis of brushite crystals at low temperature. We also notice that the size of mono HAP crystallites can be simply modulated by adjusting the pH values. For example, as shown in Figure 6, the size of the nanobelts decreases with increase of pH. The results indicate that these conditions are kinetically in favor of the growth of monoclinic HAP other than hexagonal HAP. The detailed dynamics remains to be examined. However, there might be some possible reasons for no previous reports of the monoclinic growth at low temperature. For example, this work indicates that the precise phase characterization might be an issue in the previous studies. In fact, the characteristic diffraction spots for monoclinic HAP will be too weak to be detectable for smaller or poorly crystallized crystals.

The nanobelts can serve as substrates for epitaxial deposition of HAP particles to mimic bone and enamel formation. Moreover, the monoclinic HAP crystallites are more difficult to dissolve than hexagonal HAP crystallites and the growth kinetics is also different, so the precise identification for the two phases will exert a relevant impact on the control of biomineralization and demineralization.1 Apart from this, the monoclinic phase products reported here may find potential perspective applications in bone remodeling practices. In conclusion, we successfully produced HAP nanobelts by hydrolysis of brushite crystals. On the basis of detailed HRTEM and SAED analysis, the products are assigned to the monoclinic phase instead of the more common hexagonal phase. We show for the first time the ability to grow and identify monoclinic HAP at low temperature. This work shows that HRTEM and SAED are very efficient techniques to distinguish the subtle differences between the two phases. The results are not only helpful for a better understanding of the transformation mechanisms between different calcium phosphates compounds but also are applicable for mimicking bone and enamel formation.

Acknowledgment. The research was supported by Singapore ARC MOE funding (Project No. T206B1114).

References (1) Young, R. A.; Brown, W. E. In Biological Mineralization and Demineralization; Nancollas G. H., Ed.; Dahlem Konferenzen: Berlin, 1982; pp 101-141. (2) Elliott, J. C.; Young, R. A. Nature 1967, 214, 904–906. (3) Elliott, J. C.; Mackie, P. E.; Young, R. A. Science 1973, 180, 1055– 1057. (4) Haverty, D.; Tofail, S. A. M.; Stanton, K. T.; McMonagle, J. B. Phys. ReV. B 2005, 71, 094103. (5) Elliot, J. C. Structure and Chemistry of the Apatites and Other Calcium Phosphates; Elsevier: Amsterdam, 1994. (6) Nakamura, S.; Takeda, H.; Yamashita, K. J. Appl. Phys. 2001, 89, 5386–5392. (7) Kalogeras, I. M.; Vassilikou-Dova, A.; Katerinopoulou, A. J. Appl. Phys. 2002, 92, 406–414. (8) Ikoma, T.; Yamazaki, A. J. Solid State Chem. 1999, 144, 272–276. (9) Suetsugu, Y.; Tanaka, J. J. Mater. Sci.: Mater. Med. 2002, 13, 767– 772. (10) Monma, H.; Kamiya, T. J. Mater. Sci. 1987, 22, 4247–4250. (11) Reimer, L. Transmission Electron Microscopy. Physics of Image Formation and Microanalysis; Springer-Verlag: Berlin, 1984. (12) Prado Da Silva, M. H.; Lima, J. H. C.; Soares, G. A.; Elias, C. N.; de Andrade, M. C.; Best, S. M.; Gibson, I. R. Surf. Coat. Technol. 2001, 137, 270–276. (13) Sˇtulajterova´, R.; Medvecky´, L. Coll. Surf. A 2008, 316, 104–109. (14) de Leeuw, N. H. Phys. Chem. Chem. Phys. 2002, 4, 3865–3871. (15) Treboux, G.; Layrolle, P.; Kanzaki, N.; Onuma, K.; Ito, A. J. Am. Chem. Soc. 2000, 122, 8323–8324.

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