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Hierarchical Texture of Calcium Carbonate Crystals Grown on a Polymerized Langmuir-Blodgett Film Kimiyasu Sato,*,† Yuri Kumagai,‡ Koji Watari,† and Junzo Tanaka§ Advanced Sintering Technology Group, Ceramic Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Nagoya, Japan, CREST, Japan Science and Technology Agency (JST), Japan, and Biomaterials Center, National Institute for Materials Science (NIMS), Japan Received October 28, 2003. In Final Form: January 12, 2004
Introduction The textures that are found in shells, bones, and teeth are utilized for the design of biomaterials with desired functions. Recently, there has been a growing interest in the principles governing the architectures in living bodies. Much of this interest stems from the desire to create new synthetic highly functional materials, consuming only a small amount of energy in their production. The process of biomineralization is characterized by the close association of inorganic and organic substances throughout the process. The molecular interactions at the inorganic/ organic interfaces are an important aspect of biomineralization because the nucleation, growth, and organization of biominerals are mediated by an organic supramolecular system.1 In our previous works,2 organic monolayer films prepared by the Langmuir-Blodgett (LB) method were used to investigate the nucleation mechanism of calcium phosphate crystals in a body environment. It was shown that the crystallization of hydroxyapatite was induced by the chemical interaction between the carboxyl groups and inorganic ions (Ca2+) in a body fluid. Recently, it was reported that a template with regularly spaced functional groups induces inorganic crystals with specific crystallographic orientation.3 On the other hand, the surrounding environment also influences the phase, shape, and size of * Author to whom correspondence should be addressed: Anagahora 2266-98, Shimoshidami, Moriyama-ku, Nagoya 4638560, Japan. Tel: +81-52-7367154. Fax: +81-52-7367164. Email:
[email protected]. † AIST. ‡ JST. § NIMS. (1) For an example, see: (a) Mann, S.; Archibald, D. D.; Didymus, J. M.; Douglas, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286-1292. (b) Mann, S. Nature 1993, 365, 499-505. (c) Mann, S. Biomimetic Materials Chemistry; VCH Publishers: New York, 1996. (d) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, U.K., 2001. (2) (a) Sato, K.; Kumagai, Y.; Tanaka, J. J. Biomed. Mater. Res. 2000, 50, 16-20. (b) Sato, K.; Kogure, T.; Kumagai, Y.; Tanaka, J. J. Colloid Interface Sci. 2001, 240, 133-138. (3) (a) Berman, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. Science 1995, 269, 515-518. (b) Berman, A.; Charych, D. Adv. Mater. 1999, 11, 296-300. (c) Berman, A.; Charych, D. J. Cryst. Growth 1999, 198/199, 796-801. (d) Champ, S.; Dickinson, J. A.; Fallon, P. S.; Heywood, B. R.; Mascal, M. Angew. Chem., Int. Ed. 2000, 39, 2716-2719. (e) 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. (f) Travaille, A. M.; Donners, J. J. J. M.; Gerritsen, J. W.; Sommerdijk, N. A. J. M.; Nolte, R. J. M.; Kempen, H. Adv. Mater. 2002, 14, 492-495.
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the resultant inorganic crystals. Because of the calcium carbonate crystals, many studies have shown that divalent cations or polyelectrolytes control the crystallographic features.4 In the present paper, an attempt to control the nucleation and growth of the calcium carbonate crystals by using an organic template and additives and to form the assemblages of the crystals into a hierarchical texture is reported. Materials and Experiments The polymerized LB film of 10,12-pentacosadiynoic acid (PDA ) CH3(CH2)11CtCCtC(CH2)8COOH), a 25-C chain with a diacetylene functionality, was prepared according to a method reported in a preceding work.5 Monomeric PDA was dissolved in chloroform with a concentration of 1 mM, and 300 µL of the obtained solution was spread on a subphase of ion-exchanged distilled water. After chloroform was evaporated for 5 min, the residual organic film was slowly compressed up to a surface pressure needed for the ordering of the organic molecules, typically 20 mN‚m-1, and allowed to equilibrate for 30 min. The organic thin film was polymerized in situ with a 254-nm UV light. The ordered and packed structure of the organic molecules on the water surface results in a linear-conjugated polymer. Fused-silica glass slides (hydrophobized with 1,1,1,3,3,3-hexamethyldisilazane beforehand) of 13 × 38 × 1 mm3 in size were used as substrates. The polymerized PDA film was transferred to the substrate by horizontally touching the subphase surface (Langmuir-Schaefer method). Crystals were grown from a supersaturated solution with respect to calcium carbonate. The supersaturated solution was prepared using the method of Kitano.6 CaCO3 was suspended in distilled water, and carbon dioxide gas was bubbled into the stirred suspension for 3 h at room temperature. The remaining CaCO3 solids were removed by filtration. In the present study, we added Mg2+ ion and a polyelectrolyte, poly(L-aspartate), to the solution after the filtration. Poly(L-aspartate) is considered to be an analogue of the macromolecules in mollusk shells. Mg2+ ion and poly(L-aspartate) were added as chloride and sodium salt, respectively. The Mg2+/Ca2+ molar ratio was 6.0, which is close to the value for seawater, and the concentration of poly(L-aspartate) was adjusted to 4.4 × 10-4 wt % according to work by Sugawara and Kato.7 The molecular weights of poly(L-aspartate) determined by viscosity and low-angle light scattering were 10 300 and 8900, respectively. The polymerized LB film supported by a fused-silica substrate was soaked in the supersaturated solution for 3 days to deposit the calcium carbonate crystals onto the film surface. After being gently washed with ion-exchanged distilled water and dried at room temperature, the specimens were evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface topography of the crystals was investigated using an SEM (S-5000; Hitachi, Japan), operated at 10 kV after tungsten coating. Samples for TEM were sonicated in an appropriate amount of ethanol, and the precipitates and polymerized films were suspended in the ethanol. A droplet of the suspension was transferred onto a carbon microgrid supported by a Cu mesh, and ethanol was removed by a filter paper. TEM observations were performed using a JEM-2010 microscope (JEOL, Japan) equipped with an energy-dispersive X-ray spectrometer (EDS), operated at 200 kV.
Results and Discussion Figure 1 shows SEM images of the polymerized film soaked in the calcium carbonate supersaturated solution (4) For an example, see: (a) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67-69. (b) Gower, L. A.; Tirrell, D. A. J. Cryst. Growth 1998, 191, 153-160. (c) Zhang, Y.; Dawe, R. A. Chem. Geol. 2000, 163, 129-138. (5) Lio, A.; Reichert, A.; Nagy, J. O.; Salmeron, M.; Charych, D. H. J. Vac. Sci. Technol., B 1996, 14, 1481-1485. (6) Kitano, Y. Bull. Chem. Soc. Jpn. 1962, 32, 1980-1985. (7) Sugawara, A.; Kato, T. Chem. Commun. 2000, 487 and 488.
10.1021/la0360198 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/21/2004
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Figure 1. SEM images of the precipitates formed on the polymerized LB films. White arrows indicate the directions of polymerization. (a) Aligned precipitates on a LB film. (b) A domain boundary (indicated by a broken line).
for 3 days. The precipitates of ∼1 µm in size formed on the film showed a characteristic spindlelike shape. Furthermore, thin-film domains, in which many parallel cracks are formed, appeared on the substrate. The orientation of the spindlelike precipitates was very regular; their longitudinal axes were at right angles to the cracks of the film and almost uniformly inclined to the substrate. It is known that the polymerized PDA film breaks parallel to the polymer backbone.8 The thin film with parallel cracks corresponds to the polymerized LB film,9 and the cracks in the LB film are along the direction of the conjugated polymer backbone. Part b of Figure 1 shows an SEM image of the boundary of the LB film domains, where the directions of the cracks in each domain were different. Note that the geometrical relationships between the cracks of the LB films and precipitates were identical in each domain. Part a of Figure 2 shows a TEM image of a spindlelike precipitate and the selected-area electron diffraction (SAED) pattern from the precipitate (the inlet). The obtained SAED pattern, showing a characteristic fiber diffraction pattern, indicates that the spindlelike precipitate was an aggregate of the calcite crystals, whose caxes were parallel to the longitudinal direction of the aggregate. Detailed analyses of the dark-field TEM observations indicate that the spindlelike aggregate was composed of calcite crystals much smaller than 100 nm (8) Putman, C. A. J.; Hansma, H. G.; Gaub, H. E.; Hansma, P. K. Langmuir 1992, 8, 3014-3019. (9) We investigated the polymerized PDA film by atomic force microscopy before soaking and found the parallel cracks to be identical with those found in the SEM images.
Figure 2. (a) Bright-field TEM image of a precipitate and the corresponding electron diffraction pattern. (b) Dark-field image observed using a 110 diffraction spot.
in size (Figure 2b). We then performed EDS analyses of the calcite aggregate. From the aggregate, calcium and magnesium were detected, indicating that the constitutive crystals of the aggregate were magnesian calcite. The common biologically formed calcium carbonate polymorphs are calcite and aragonite, and calcite is thermodynamically more stable than aragonite at ambient temperatures and pressures. Calcite crystals formed in the calcium carbonate supersaturated solution containing neither other cations nor polyelectrolytes could grow up to several tens of micrometer single crystals in an hour. Mg2+ is known to exert a significant effect on the calcium carbonate precipitation and, when present in sufficient concentration, generally precipitates aragonite rather than the thermodynamically favored calcite phase. Mg2+ ions can be easily incorporated into the calcite crystal structure at Ca2+ ion sites, which distort the lattice form.10 The distortion of the calcite lattice results in a rate
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reduction of crystal growth and, relatively, aragonite formation becomes insistent. However, the precipitates obtained in the present experiment were aggregates of magnesian calcite crystallites. Some polyelectrolytes including poly(L-aspartate) also reduce the crystal growth rate of calcium carbonate through binding to its growth sites. The presence of poly(L-aspartate) should inhibit the crystal growth of both calcite and aragonite. The calcite phase is favored when the rate of precipitation is lowered by the polyelectrolytes.11 This probably helped the formation of the tiny magnesian calcite crystals. Nanosized calcite crystals smaller than 100 nm are not stable.12 Therefore, the crystal growth of the tiny metastable calcite must proceed further in the stable precipitation process. If not only the growth but also the dissolution process of the subcritical nuclei is sufficiently hampered by the adsorbed organic molecules, the critical size required for further growth can be reached by the formation of polycrystalline supercritical precipitates composed of subcritical crystals. In the polycrystalline texture, the nanosized crystals are glued to each other via the organic molecules. The “glue” in the present experiment, poly(L-aspartate), consists of carboxylated residues that favor an R-helical structure in solution but is suggested to adopt a β-strand conformation when adsorbed to calcite surfaces.13 Poly(L-aspartate) interacts selectively with {11h 0} facets; the facets lie parallel to the c axis. When poly(L-aspartate) is bound on a (11h 0) face in the direction perpendicular to the c-axis, there is no chain strain because of the mismatch between the surface lattice ions and adsorbate. The binding of poly(L-aspartate) in that direction is most advantageous according to the energyoptimization calculations.14 On the other hand, crystallization of calcite in the presence of poly(L-aspartate) with a β-pleated sheet structure results in the orientation of the c-axis perpendicular to the polypeptide sheet.15 The alliance of the two phenomena, adsorption of poly(L-aspartate) on calcite surfaces and crystallization of calcite upon the poly(L-aspartate) in β-sheet conformation, probably yielded the aggregate of calcite nanosized crystallites, whose c-axes were in a specific direction. As the above crystal maturation progressed, the longitudinal direction of the spindlelike aggregate, corresponding to the calcite c-axes, was likely to elongate along the c-axis of the initial nucleus. On a polymerized PDA film, the calcite single crystals deposit most frequently on (012) basal planes, owing to optimization in the lattice match and stereochemical fit.3a,c In the geometry, their caxes are at right angles to the polymer backbone, and an approximate 60° inclination between the film normal and the c-axes is yielded. Probably the crystal embryos in the (10) Berner, R. A. Geochim. Cosmochim. Acta 1975, 39, 489-504. (11) (a) Falini, G.; Gazzano, M.; Ripamonti, A. J. Cryst. Growth 1994, 137, 577-584. (b) Meldrum, F. C.; Hyde, S. T. J. Cryst. Growth 2001, 231, 544-558. (12) Lin, R.; Zhang, J. Y.; Zhang, P. J. Cryst. Growth 2002, 245, 309-320. (13) Addadi, L.; Berman, A.; Oldak, J. M.; Weiner, S. Connect. Tissue Res. 1989, 21, 127-135. (14) Wierzbicki, A.; Sikes, C. S.; Madura, J. D.; Drake, B. Calcif. Tissue Int. 1994, 54, 133-141. (15) Falini, G. Int. J. Inorg. Mater. 2000, 2, 455-461.
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Figure 3. Schema showing the probable maturation process of the calcite aggregates on the polymerized LB film. The geometric relation between the calcite aggregates and LB film is illustrated, as viewed from the polymerization direction.
present experiment formed in a manner identical with that described above, and the directions of the c-axes of the embryos were retained throughout the successive crystal maturation. This maturation process of the calcite aggregates is illustrated in Figure 3. Conclusion An elaborate integration of the interfacial interactions between the inorganic/organic materials led to assemblages of calcium carbonate crystallites in the hierarchical texture. The geometry in the arrangement of the carboxyl groups obtained by PDA-film polymerization induced uniformly aligned calcite crystal embryos. Incorporation of poly(L-aspartate) into the growing precipitates resulted in spindlelike assemblages of magnesian calcite whose caxes are aligned to a specific direction. Generally, the cellular involvement is imperative for the formation of tissues and organs in the body. However, nanoscaled textures much smaller than cells could be formed only through the self-assemblage processes such as that shown in the present paper. Acknowledgment. The authors thank Dr. T. Kogure, University of Tokyo, for helpful discussions. LA0360198
