Mineralization Mechanism of Calcium Phosphates under Three Kinds

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Mineralization Mechanism of Calcium Phosphates under Three Kinds of Langmuir Monolayers Li-Juan Zhang, Hong-Guo Liu, Xu-Sheng Feng,* Ren-Jie Zhang, Li Zhang, Ying-Di Mu, Jing-Cheng Hao, Dong-Jin Qian, and Yu-Feng Lou Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, Jinan 250100, People’s Republic of China Received July 29, 2003. In Final Form: December 20, 2003

Three kinds of Langmuir monolayers formed by dipalmitoylphosphatidylcholine (DPPC), arachidic acid (AA), and octadecylamine (ODA) were used as templates to study the initial stage of nucleation and crystallization of calcium phosphates. It was demonstrated that the combination of calcium ions (or phosphates) to the monolayer/subphase interface is a prerequisite for subsequent nucleation. It was found that calcium phosphate dihydrate (DPCD) formed at 25.0 °C for 12 h has a biphasic structure containing both amorphous and crystalline phases. These results showed that calcium phosphates were formed through a multistage assembly process, during which an initial amorphous phase DPCD was followed by a phase transformation into a crystalline phase and then the most stable hydroxyapatite (HAp). This provided new insights into the template-biomineral interaction and a mechanism for biomineralization.

Introduction Biomineralization is an important phenomenon in nature. Studies on the biomineralization mechanism have been aimed at developing a detailed understanding of interfacial interactions associated with biomineralization and template-directed crystallization. To research natural phenomena such as bone, tooth, and shell formation, as well as to assist scientists and engineers to apply biomimetic strategies to develop novel materials such as advanced composites and coatings for medical, chemical, optical, and electronic applications,1-3 one approach focuses on the in vitro4-7 or vivo8-11 functions of the biological matrix, but a major problem with this method is due to the formidable structural complexity. Much work has been done to illustrate the nature of these biological systems. A recent development in this area involved the use of Langmuir monolayers of surfactant molecules as tem* To whom correspondence should be addressed. Telephone: +86531-8564750.Fax: +86-531-8565167.E-mailaddress: [email protected]. (1) Aksay, I. A.; Tran, M.; Mann, S. Science 1996, 273, 892-898. (2) Bunker, B. C.; Rieke, P. C.; Tarasevich, B. J.; Campbell, A. A.; Fryxell, G. E.; Graff, G. L.; Song, L.; Liu, J.; Virden, J. W.; Mcvay, G. L. Science 1994, 264, 48-55. (3) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495-498. (4) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977-11985. (5) Albeck, S.; Aizenberg, J.; Addadi, L.; Weiner, S. J. Am. Chem. Soc. 1993, 115, 11691-11697. (6) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Sicence 1996, 271, 67-69. (7) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56-58. (8) Shenton, W.; Pum, D.; Sleytr, U. B.; Mann, S. Nature 1997, 389, 585-587. (9) Vaucher, S.; Dujardin, E.; Lebeau, B.; Hall, S. R.; Mann, S. Chem. Mater. 2001, 13, 4408-4410. (10) Zaremba, C. M.; Belcher, A. M.; Fritz, M.; Li, Y.; Mann, S.; Hansma, P. K.; Morse, D. E.; Speck, J. S.; Stucky, G. D. Chem. Mater. 1996, 8, 679-690. (11) Schaffer, T. E.; Ionescu-Zanetti, C.; Proksck, R.; Fritz, M.; Walters, D. A.; Almqvist, N.; Zaremba, C. M.; Belcher, A. M.; Smith, B. L.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Chem. Mater. 1997, 9, 1731-1740.

plates to induce the oriented nucleation of organic and inorganic crystals. Mann, Heywood, and co-workers have investigated the growth of CaCO312-18 and BaSO419 under Langmuir monolayers of fatty acids, aliphatic phosphates, and aliphatic sulfates. Cooper et al. 20,21 have studied the nucleation of the amino acids, aspartic acid, and asparagines using the films formed by amphiphilic molecules with different headgroups. Calcium phosphates and calcium oxalates are investigated extensively under the Langmuir films because they are the main inorganic components of hard tissue.22-29 The well-ordered twodimensional structure of the Langmuir monolayer at the air/water interface acting as the nucleating template can (12) Heywood, B. R.; Mann, S. Chem. Mater. 1994, 6, 311-318. (13) Mann, S.; Didymus, J. M.; Sanderson, N. P.; Heywood, B. R.; Samper, E. J. A. J. Chem. Soc., Faraday Trans. 1990, 86, 1873-1880. (14) Heywood, B. R.; Rajam, S.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 735-743. (15) Rajam, S.; Heywood, B. R.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 727-734. (16) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692-695. (17) Mann, S. Nature 1988, 332, 119-124. (18) Buijnsters, P. J. J. A.; Donners, J. J. J. M.; Hill, S. J.; Heywood, B. R.; Nolte, R. J. M.; Zwanenburg, B.; Sommerdijk, N. A. J. M. Langmuir 2001, 17, 3623-3628. (19) Heywood, B. R.; Mann, S. J. Am. Chem. Soc. 1992, 114, 46814686. (20) Cooper, S. J.; Sessions, R. B.; Lubetkin, S. D. Langmuir 1997, 13, 7165-7172. (21) Cooper, S. J.; Sessions, R. B.; Lubetkin, S. D. J. Am. Chem. Soc. 1998, 120, 2090-2098. (22) Backov, R.; Lee, C. M.; Kahn, S. R.; Mingotaud, C.; Fanucci, G. E.; Talham, D. R. Langmuir 2000, 16, 6013-6019. (23) Whipps, S.; Khan, S. R.; O’Palko, F. J.; Backov, R.; Talham, D. R. J. Cryst. Growth 1998, 192, 243-249. (24) Tunik, L.; Addadi, L.; Garti, N. J. Cryst. Growth 1996, 167, 748-755. (25) Lu, H. B.; Ma, C. L.; Cui, H.; Zhou, L. F.; Wang, R. Z.; Cui, F. Z. J. Cryst. Growth 1995, 155, 120-125. (26) Ma, C. L.; Lu, H. B.; Wang, R. Z.; Zhou, L. F.; Cui, F. Z.; Qian, F. J. Cryst. Growth 1997, 173, 141-149. (27) Onuma, K.; Oyane, A.; Kokubo, T. J. Phys. Chem. B 2000, 104, 11950-11956. (28) Xu, G. F.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 2001,123, 2196-2203. (29) Ngankam, P. A.; Lavalle, P.; Voegel, J. C. J. Am. Chem. Soc. 2000, 122, 8998-9005.

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provide the microspace for the biomineral growth. This method has provided important insight into the relationships between the structure of the substrate and the overgrowing crystals. Nucleation beneath monolayer films relies upon the degree of molecular recognition between the film and the nucleating species. This recognition may be manifested by an exact match of the headgroup of the film formation molecules to the nucleating species, usually called “template matching”, whereby the film mimics a particular plane in the nucleating crystal and so leads to nucleation bounded by this plane.30-33 Alternatively, electrostatic attraction and either geometrical matching or stereochemical complementarity may play a main role in the nucleation and crystallization of minerals34-36 in biomineralization. It is commonly accepted that the protein matrix plays a very important role in regulating biomineral formation. However, there remain many unknown problems as to how the matrix affects the crystallization process, especially the initial nucleation. In general, the formation of certain mineral phases can proceed through two pathways. A crystal could precipitate via epitaxy directly from the liquid solution, so that the nuclei bear the same structure as the final crystal. The traditional view of epitaxy emphasizes the structural similarity between the matrix and the assumed nucleating crystal face, implying a direct crystallization process. Consequently, this leads to the belief that the matrix controls the crystallization from the very beginning. Although there are some observations that could be explained by this mechanism, strong and direct evidence for such a process is lacking. In this paper, we employed Langmuir films that formed from amphiphilic molecules with different headgroups at the air/water interface as templates to biomimetic calcium phosphate formation. The template inducing and controlling, molecular recognition, and, especially, electrostatic attraction and lattice matching were investigated. More interestingly, we observed a phase transformation process from an initially deposited amorphous phase to a crystalline phase during the initial stage of calcium phosphate formation. The thermodynamically unstable calcium phosphate dihydrate (DPCD) and the most stable hydroxyapatite (HAp) were studied, and they provide direct evidence for a multistep crystallization process in this biomimetic model system. This sequence of events stands in contrast to the prevailing view that nuclei form as crystalline particles directly, which is coincident to that of calcium phosphate formed by many organisms.37,38 Experimental Section Materials. Dipalmitoylphosphatidylcholine (DPPC), arachidic acid (AA), and octadecylamine (ODA) were purchased from Fluka and used without further purification. The molecular structures are shown in Chart 1. HAp and tris(hydroxymethyl)aminomethane were purchased from Beijing Yili Fine Chemical Co. Ltd. Hydrochloric acid was obtained from Tianjin Chemical Plant. (30) Landau, E. M.; Levanon, M.; Leiserowitz, L.; Lahav, M.; Sagiv, J. Nature 1985, 318, 353-356. (31) Landau, E. M.; Grayer, W. S.; Levanon, M.; Leiserowitz, L.; Lahav, M.; Sagiv, J. J. Am. Chem. Soc. 1989, 111, 1436-1445. (32) Weissbuch, I.; Berkovic, G.; Yam, R.; Als-Nielson, J.; Kjaer, L.; Lahav, M.; Leiserowitz, L. J. Phys. Chem. 1995, 99, 6036-6045. (33) Popovitz-Biro, R.; Wang, J. L.; Majewski, J.; Shavit, W.; Leiserowitz, L.; Lahav, M. J. Am. Chem. Soc. 1994, 116, 1179-1191. (34) Tang, R.; Tai, Z.; Chao, Y. Chem. Lett. 1996, 7, 535-536. (35) Mann, S.; Heywood, B. R.; Rajam, S.; Walker, J. B. A.; Davey, R. J.; Birchall, J. D. Adv. Mater. 1990, 2, 257-261. (36) Heywood, B. R.; Mann, S. Adv. Mater. 1994, 6, 9-20. (37) Bermen, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. Science 1995, 269, 515-518. (38) Mann, S.; Ozin, G. A. Nature 1996, 382, 313-318.

Zhang et al. Chart 1. Molecular Structures of DPPC, AA, and ODA

Scheme 1. Experimental Process of Calcium Phosphates Induced by Langmuir Monolayers

Triple-distilled water (resistivity of 18 MΩ‚cm) was used in the experiments. Disposal of Silicon Slides. To eliminate the organic compounds on their surfaces, silicon slides were first cleaned with chloroform and acetone, respectively. Then, they were soaked in sulfuric acid at 100 °C for 12 h and cleaned with triple-distilled water. Finally, they were put in triple-distilled water for reservation. Preparation of HAp Supersaturated Solution. The HAp supersaturated solution subphase was prepared as follows: HAp’s were dissolved in 0.01 mmol‚L-1 HCl solution. The concentration of calcium ion was 2.5 mmol‚L-1, which is simlar to that of human body fluid.39 The pH of the solution was adjusted to 6.8 by adding the appropriate volume of 0.01 mol‚L-1 tris(hydroxymethyl)aminomethane and 0.01 mol‚L-1 HCl solution at the temperature 25 °C. Experimental Procedures. Chloroform solutions of DPPC, AA, and ODA were spread on the fresh surface of the HAp supersaturated solution that was poured into a glass container to form a condensed monolayer, respectively. The amount of the three chloroform solutions added was calculated according to their mean molecular areas and the open area of the glass container so that the resulting monolayer corresponded to a liquid-condensed state. The whole setup was sealed and left unstirred for different times during the period of crystal growth from the subphase. Meanwhile, a solution without a monolayer was prepared for the sake of comparison under the same conditions. The calcium phosphates formed were transferred to clean silicon slides by carefully touching the monolayer and were cleaned with triple-distilled water three times and dried in air. The experimental process is shown in Scheme 1. Samples for TEM were collected on Formvar-coated, carbon-reinforced 230 mesh copper grids that had been placed in the subphase and positioned at an angle of approximately 15° with respect to the bottom of the container to allow for better drainage before spreading the monolayer by slowly lowering the subphase surface through siphoning the solution out of the container. (39) Rhee, S. H.; Lee, J. D.; Tanaka, J. J. Am. Ceram. Soc. 2000, 83, 2890-2892.

Mineralization Mechanism of Calcium Phosphates

Figure 1. π-A isotherms of monolayers on pure water and HAp supersaturated solution for different times: (A) DPPC, (B) AA, and (C) ODA at 25 °C. Isotherm Measurement. DPPC, AA, and ODA were dissolved in chloroform at a concentration of 1.0 mmol‚L-1, and the solutions were spread on the subphases, respectively. Two subphases were used in this experiment: pure water (a resistivity of 18 MΩ‚cm) and HAp supersaturated aqueous solution (2.5 mmol‚L-1). The experimental temperature was 25.0 °C. Instruments. The Langmuir minitrough system was a NIMA 600 one (U.K.). The compositions of calcium phosphates formed under the monolayers were analyzed with an Electronprobe microanalyzer (EPM) (JXA-8800R, Japan). Transmission electron microscopy (TEM, JEM-200CX) was used to investigate the morphologies of calcium phosphates at the interface and for electron diffraction analysis.

Results Pressure-Molecular Area (π-A) Isotherms of Three Monolayers. π-A isotherms of three monolayers were obtained on pure water and the HAp supersaturated aqueous solution, and they are shown in Figure 1. The mean areas of DPPC per molecule obtained by extrapolating the slopes of the isotherms to zero pressure are found to be 65 Å2 on pure water and 69, 74, and 92

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Å2 on HAp supersaturated aqueous solution for 30 min, 1 h, and 2 h, respectively. Those of AA are 24 Å2 on a pure water surface and 27, 31, and 36 Å2 on HAp supersaturated aqueous solution for 30 min, 1 h, and 2 h, respectively, and those of ODA are 46 Å2 on pure water and 51 and 66 Å2 on HAp supersaturated aqueous solution for 1 h and 2 h, respectively. All the monolayers are very stable without loss of film after several days under the HAp supersaturated aqueous solution or pure water surface. From these curves, it can be seen that the mean molecular areas of DPPC, AA, and ODA on a HAp supersaturated aqueous solution are larger than those on pure water, indicating that there is a very strong interaction between the monolayers and calcium ions (or phosphate), and calcium ions (or phosphate) could bind into the monolayers. On the other hand, it was found that the collapsed pressures of the last curves were lower than those of the former curves in each group of isotherms. This also suggested that more and more calcium ions (or phosphate) bound to the monolayer/subphase interface, which led to brittle films. This results in a much higher local concentration of calcium phosphate under the monolayers than that in solution and makes it possible for Langmuir films to induce orientated growth of calcium phosphate. The higher the limiting area of forming-films materials in the presence of calcium ions (or phosphate), the more cations (anions) binding to the anionic (cationic) template. TEM Images and the Corresponding Select Area Electron Diffraction (SAED). The shapes of particles formed under three monolayer templates with different structures were different markedly. For DPPC monolayer, particles were dandelion-like (Figure 2a and 2b); regular spherical particles were formed for AA (Figure 3a and 3b); and a cluster of particles was formed for ODA (Figure 4a and 4b). In addition, in Figure 4b, after the sample was kept at 25 °C for 3 days, spherical particles aggregated together along some certain direction and the sizes of the particles became small. The amount of particles formed under a DPPC or AA monolayer for 12 h is more than those of particles formed for 6 h, meaning that more nucleation sites were created at the monolayers/subphase interfaces with time. At the same time, it was found that the diffraction patterns of calcium phosphates formed for 12 h were very obscure (Figures 2c, 3c, and 4c): After 3 days at 25 °C, the spots became clear (Figures 2d, 3d, and 4d), and they exhibited regular hexagons under DPPC on the 7th day (Figure 2d). Under AA, the spots showed a clean quadrangle on the 2nd day (Figure 3d). All the results show that a phase transformation from an amorphous phase to a crystalline phase occurs, especially at the initial stage of nucleation. The part-crystalline particles could be unstable for about 60 h; more crystals were formed when prolonging settlement. From the Lane spots of crystals in the pattern (as shown in Figures 2d, 3d, and 4d), it may be inferred that the (0001) plane of the crystal is parallel to the monolayer plane. This is to say, the crystallographic 〈0001〉 axis of the crystal is perpendicular to the monolayer plane. Compositions of Calcium Phosphates. The compositions of calcium phosphates formed under DPPC, AA, and ODA monolayers at different times were examined with an EPM, respectively. From Figures 5-7, it can be seen that the atomic ratios of Ca/P of calcium phosphate formed under a DPPC monolayer are about 0.85, 0.98, 1.33, and 1.67 for 1, 2, 5, and 7 days, under an AA monolayer they are about 0.90, 1.01, and 1.49 for 1, 2, and 7 days, and under an ODA monolayer they are about 0.91, 0.95, and 1.6 for 1, 2, and 7 days, respectively. The ratios

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Figure 2. TEM images and SAED pattern of calcium phosphates formed under a DPPC monolayer at different times: a, 6 h; b, 12 h; c, the corresponding SAED pattern of part b; d, the SAED pattern after 7 days.

Figure 3. TEM images and SAED pattern of calcium phosphates formed under an AA monolayer at different times: a, 6 h; b, 12 h; c, the corresponding SAED pattern of part b; d, the SAED pattern after 7 days.

of Ca/P increased with the time before 7 days. It could be considered that the particles with the ratios of Ca/P close to the theoretical value of calcium phosphate dihydrate (DPCD) (1.0) were DPCD. In addition, the ratios of Ca/P of calcium phosphate formed under three kinds of monolayers on the 7th day all were close to the theoretical value of HAp (1.67). From these results, we could conclude that,

during the crystallization of calcium phosphate, its unstable precursor (DPCD) was formed first and finally transferred to the stable crystalline phase HAp. Discussion Molecular Recognition. The above results clearly show that not only the DPPC monolayer but also the AA

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Figure 4. TEM images and SAED pattern of calcium phosphates formed under an octadecylamine monolayer at different times: a, 6 h; b, 12 h; c, the corresponding SAED pattern of part b; d, the SAED pattern after 2 days.

Figure 5. EPM spectra of calcium phosphate formed under the DPPC monolayer at different times: a, 1 day; b, 2 days; c, 5 days; d, 7 days.

Figure 6. EPM spectra of calcium phosphate formed under the AA monolayer at different times: a, 1 day; b, 2 days; c, 7 days.

and ODA monolayers could powerfully affect the nucleation and crystallization of calcium phosphates. In addition, the morphologies of calcium phosphate formed were different under Langmuir monolayers with different structures. On one hand, with regard to these three kinds of molecules, they have almost the same length of carbon chains but different headgroups, such as -PO4-, -COO-,

Figure 7. EPM spectra of calcium phosphate formed under the ODA monolayer at different times: a, 1 day; b, 2 days; c, 7 days.

and NH3+ for DPPC, AA, and ODA, respectively, in the HAp supersaturated solution of pH ) 6.8. From the point of molecular recognition, the arrangement and sizes of different headgroups have affected the magnitude and shapes of calcium phosphates formed. On the other hand, the headgroups of the DPPC and AA molecules are negatively charged while the ODA molecule has positive headgroups. So their electrostatic potentials are also different. The headgroups bound ions in opposite sequences: the headgroups of DPPC and AA attracted calcium ions first, but the headgroups of ODA attracted phosphates. So lattice matching and electrostatic attraction commonly play the main role in the nucleation of calcium phosphates. Template Inducing and Controlling. Template inducing and controlling can be explained as follows: there are the formulas40 ∆GN ) 16π(∆Gl)3/(3∆GB) and ∆GB ) κT ln S, where ∆GN is the activation energy for nucleation, ∆Gl is the energy required to form the new interface as (40) Li, B.; Liu, Y.; Lu, N.; Yu, J. H.; Bai, Y. B.; Pang, W. Q.; Xu, R. R. Langmuir 1999, 15, 4837-4841.

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Figure 8. Two types of critical nuclei: A, crystalline nuclei; B, noncrystalline nuclei.

a new phase grown from the unstable subphase (solution), and ∆GB is the energy released in the formation of bonds in the bulk of the aggregate and is a function of the supersaturated degree S. It is well-known that the surface energy decreases when the monolayer forms. The monolayer itself can also be considered as resulting in heterogeneous nucleation, so that the value of Gl is lowered. On the other hand, the monolayer is an organized organic aggregation. When it is formed on the surface of the solution, the local environment under it also becomes organized, undergoing changes in physical functions such as the electric field, energy, mass transmission, and some other chemical functions that are related to the crystallization. In this paper, it is reasonable thermodynamically to use DPPC, AA, and ODA monolayers as templates to induce the nucleation of calcium phosphate. Otherwise, under given conditions the activation energy for crystallization was further decreased when particles of specific shape (such as dandelion, spherical, or cluster particles) were formed. Therefore, the growth of calcium phosphate becomes much easier. Process of Nucleation and Crystallization. Crystal formation involves two major steps: nucleation and growth. Nucleation is favored when relatively long-lived clusters are formed through the aggregation of ions and/ or molecular species into unstable structural configurations. Growth then proceeds through the addition of further species to nascent crystal faces of variable surface energy. As we known, critical nuclei are of two types,41 which are shown in Figure 8: one is crystalline nuclei that are similar to a piece of crystallization material that is made of dehydrated ions with strong interactions. Its lattice configuration and parameters are consistent with those of the whole crystal at the surface of the matrix, but ions detached to an extent from the normal lattice. The other

Figure 9. Schematic of multistep crystal mechanism.

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one is noncrystalline nuclei that are made of hydrate ions with weak interactions. A highly dispersed hydrated solid phase was formed on the surface of the matrix. The crystal lattice configuration, crystalline parameter, and geometric structure of this solid phase are different from those of the final crystal. The type of critical nuclei decided different pathways of mineralization. If critical nuclei are crystalline nuclei, the approach of mineralization is simple: final crystals were formed from the solution but must overcome higher nucleation energy and growth energy. If critical nuclei are noncrystalline nuclei, a hydrated noncrystalline phase is formed first, then a hydrated crystalline phase, and, at last, an anhydrous crystalline phase. In this process, the nucleation and growth energies are very low. In this article, in the initial stage of nucleation of calcium phosphates, noncrystalline nuclei (DPCDs) were first formed and grew into big particles. It was demonstrated by TEM observation and the halos of SAED patterns that most of these particles are amorphous. Since the amorphous phase formed under conditions of sequential precipitation would be the most soluble phase, a lower energy barrier is expected according to the OstwardLussac law.42,43 So it should be the first solid phase formed during the crystallization and therefore could be prevalent in biomineralization.44,45 In fact, amorphous phase minerals have been identified in many organisms.46 Recently, Addadi and Weiner et al.47,48 have suggested that amorphous calcium phosphate may be much more widespread than the crystalline phase formed from the beginning in biology, but it is often overlooked because of the difficulty of identifying an amorphous phase in the presence of a crystalline phase of the same composition. In our experiment, the transformation of DPCD from the amorphous phase to the crystalline phase and finally to the stable HAp crystal was observed in the process of calcium phosphate formation. On the basis of the above results, the multistep crystal mechanism of the nucleation and growth of calcium phosphates formed under the monolayers is explained in Figure 9. At first, Langmuir monolayers with negative charges bound calcium ions, which caused a local concentration of phosphates, which sequentially attracted more calcium ions, which made the concentration of precursor increase to its supersaturated degree for nucleation (as shown in Figure 9a). The necessity of a significant combination of calcium ions to headgroups of molecules of monolayers that occur at the film/solution interface as a prerequisite for subsequent nucleation is demonstrated in the present studies. The ratio of Ca2+/ PO43- deviated from the stoichiometry that induced the nucleation of calcium phosphates. This interpretation is

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accordant with that of Crenshaw.49 Then, amorphous calcium phosphates (DPCDs) were formed (as shown in Figure 9b). The phase transformation from amorphous to crystalline calcium phosphates (HAp’s) occurred finally (as shown in Figure 9c). These results were consistent with those found by Mann et al.50 in several cases of biological mineralization. Dissection of the crystallization process into several stages could make the activation energy of each step lower than that of the one-step precipitation. A multistep crystallization process is plausible, especially in a biological environment in which temporal modifications of the crystallization kinetics may be prevalent. The existence of several phases would enable organisms to control mineralization through intervention with the kinetics. By selective interaction with the mineral at different stages during the crystal forming process, the organisms could choose to manipulate both the polymorph and the orientation of the mineral to meet specific biological requirements. Given the great diversity in the phase, morphology, and orientation of biominerals, it may be possible that a (41) Davey, R. J.; Black, S. N.; Bromley, L. A.; Cottier, B.; Dobbs, B.; Rout, J. E. Nature 1991, 353, 549-550. (42) Taylor, M. G.; Simkiss, K.; Greaves, G. N.; Okazaki, M.; Mann, S. Proc. R. Soc. London, B 1993, 252, 75-80. (43) Ziegler, A. J. Struct. Biol. 1994, 112, 110-116. (44) Oliver, S.; Kuperman, A.; Coombs, N.; Lough, A.; Ozin, G. A. Nature 1995, 378, 47-49. (45) Aizenberg, J.; Lambert, L.; Addadi, L.; Weiner, S. Adv. Mater. 1996, 8, 222-227. (46) Ozin, G. A.; Oliver, S. Adv. Mater. 1995, 7, 943-949. (47) Beniash, E.; Aizenberg, J.; Addadi, L.; Weiner, S. Proc. R. Soc. London, B 1997, 264, 461-467.

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complicated multistep crystallization mechanism exits.51 The results of this paper provided direct evidence for a model system for a multistep crystallization process that may be an important pathway for biological mineralization. Conclusion Three kinds of monolayer templates, DPPC, AA, and ODA, have played a critical role in the nucleation and crystallization of calcium phosphates. The binding between calcium ions (or phosphates) and the headgroups of molecules at the monolayer/subphase interface is a prerequisite for subsequent nucleation. Lattice matching and electrostatic attraction between the monolayers and calcium phosphates are the main factors in determing the size and morphology of DPCD formed. The observation of the transformation of DPCD from an amorphous precursor phase to a crystalline phase observed in this paper provided direct evidence for the multistep/stage crystallization process in biomineralization. This work provides a strategy for a synthesis of advanced materials under mild conditions. Acknowledgment. This study was supported by the National Natural Science Foundation of China (20103005). LA035381J (48) Lowenstam, H. A.; Weiner, S. Science 1985, 227, 51-58. (49) Crenshaw, M. A. Biomineralization 1972, 6, 6-11. (50) Mann, S.; Archibald, D. D.; Didymus, J. M.; Douglas, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286-1292. (51) Yuan, C. B.; Zhao, D. Q.; Ni, J. Z. J. Colloid Interface Sci. 1995, 172, 536-541.