CRYSTAL GROWTH & DESIGN
Molecular Tectonics: Abiotic Control of Hydroxyapatite Crystals Morphology
2002 VOL. 2, NO. 6 489-492
Vincent Ball,*,† Jean-Marc Planeix,‡ Olivier Fe´lix,† Joseph Hemmerle´,§ Pierre Schaaf,† Mir Wais Hosseini,*,‡ and Jean Claude Voegel§ Institut Charles Sadron, Unite´ propre 22 CNRS, 6 Rue Boussingault, Strasbourg 67083 Cedex, France, Laboratoire de Chimie de Coordination Organique, 4 rue Blaise Pascal, Strasbourg 67000, France, and Institut National de la Sante´ et de la Recherche Me´ dicale Unite´ 424, 11 Rue Humann, Strasbourg, France Received July 2, 2002
ABSTRACT: Ultra flat hydroxyapatite platelets with characteristic lateral dimensions in the micrometer range and a thickness of a few nanometers were synthesized at ambient temperature from supersaturated calcium and phosphate solutions and in the presence of a bis-amidinium cation. Hydroxyapatite Ca5(PO4)3OH (OHAP), the most stable form of calcium phosphates,1 is the main inorganic constituent of bone and teeth where it appears often in a calcium deficient form.2 Both the nucleation and the morphology of OHAP crystals are under control of biomolecules produced by specialized cells such as osteoblasts and ameloblasts for bone and enamel crystals, respectively.3 In the case of bone, the platelet-shaped crystals are embodied between the holes of collagen bundles associated with acidic proteins controlling the nucleation and the growth of the inorganic phase.4 Enamel crystals, on the other hand, have a needlelike shape, and specific proteins such as amelogenins control their growth along the c-axis.5 Although the body fluids of mammals are supersaturated with respect to OHAP, the crystal nucleation is inhibited not only by specific proteins but also by small anionic entities such as citrate or pyrophosphate.6 In vitro, largesized platelet-shaped crystals of OHAP may only be obtained under high pressure, high temperature, or by overgrowth on octacalciumphosphate (OCP).7 The understanding, at different hierarchical levels, of molecular mechanisms and supramolecular events controlling biomineralization is important and of current interest. In particular, the control of the growth and the morphology of OHAP crystals by abiotic molecular components remains a challenge. Pursuing our effort in the area of molecular tectonics,8 in the present paper, we report an investigation dealing with the role played by the specific dicationic phosphate receptor 12+ (Scheme 1) on both the induction time and the morphology of calcium phosphate crystal formation under physiological conditions. Cyclic bisamidinium derivatives are interesting H-bond donor tectons9 for the crystal engineering of H-bonded molecular networks in the solid state. Indeed, because of the conjugated nature of the amidine group, upon double protonation, such a compound leads to an amidinium dication possessing four acidic N-H protons pointing outwardly and capable of interacting with anionic species through the formation of H-bonds. The dication 12+ presents several interesting features such as its ability to form H-bonded molecular networks in the presence of dicarboxylates,10 phosphate, pyrophosphate,11 and metal cyanide * To whom correspondence should be addressed. V.B.: E-mail: ball@ ics.u-stasbg.fr. M.W.H.: E-mail:
[email protected]. † Unite´ propre 22 CNRS. ‡ Laboratoire de Chimie de Coordination Organique. § Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 424.
Scheme 1
anions.12 Because of structural features of 12+, it appeared interesting to study its role in the formation of OHAP crystals. Here, we describe the structure and morphology of OHAP particles obtained in the presence of 12+. As a preliminary study, the formation of H-bonded molecular networks engaging the dication 12+ and phosphate anion was carried out. After a solution of the free base 1 (0.1 mmol) and H3PO4 (0.1 mmol) in a EtOH (16 mL)/H2O (3.6 mL) mixture was refluxed, high quality platelet type crystals were obtained upon cooling to room temperature. The single crystal thus obtained was analyzed by X-ray diffraction, which revealed the following features13 (Figure 1): the crystal (monoclinic, P21/n as the space group) is exclusively composed of 12+ and H2PO4- anion in a 1/2 stoichiometry. For the organic tecton, the two NCN fragments (NCN angle of 122.2°) are parallel (dihedral angle of 180°) but not coplanar as expected due to the presence of the ethylene chain connecting the two amidinium moieties. For the amidinium group, both CN bonds are identical (dCN ) 1.30 Å) indicating, as expected from the difference in the pKa of the amidine group and the phosphoric acid, that the compound 1 is indeed diprotonated (Figure 1a). Dealing with the anionic component H2PO4-, because of the amphoteric nature of dihydrogenophosphate (both donor and acceptor of H-bond), a one-dimensional (1D) H-bonded network (Figure 1b) through a monohapto mode is obtained (dO-O ) 2.55 Å). The dicationic and monoanionic units are interconnected through strong H-bonding (dN-O ) 2.78 Å) forming a neutral two-dimensional (2D) network (Figure 1c). The bidimensional network may be described as the interconnection of anionic 1D H-bonded networks by dications 12+. Indeed, each dication 12+ is surrounded by four H2PO4anions (a set of two on each side), which as stated above, are interconnected. On the basis of the results mentioned above on the formation of 2D networks between the bisamidinium 12+ and the phosphate anion, we thought that it would be of
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Communications
Figure 2. Example of a turbidity curve displaying the particle growth kinetics from a solution containing 12+ (5.3 × 10-4 M). The tangent of the experimental curve at its inflection point (solid line) is used to determine the lag time τ characterizing the formation of stable nuclei.
Figure 1. Portions of the X-ray structure of the H-bonded network formed between 12+ dication and H2PO4- monoanion in the solid state: (a) the conformation adopted by the dication and its surrounding, (b) the formation of a 1D H-bonded network between dihydrogenophosphates, and (c) the 2D network formed upon interconnection of the anionic 1D networks by the dications 12+. Dashed lines represent the H-bonds. For the sake of clarity, H-atoms, except those involved in H-bonding, are not presented. For bond distances and angles, see text.
interest to study the growth and morphology of calcium phosphate crystals in the presence of the dication 12+. The 12+ dication was synthesized as described in ref 14. The results reported here deal mainly with concentration in 12+ of 5 × 10-4 M. All of the other chemicals were purchased from Sigma. Solutions were prepared from Milli-Qplus water (F ) 18.2 MΩ cm). The aqueous calcium and phosphate (12 mM) aliquots were prepared in Tris buffer (10 mM; pH 7.4) and in the presence of NaCl (0.1 M) to maintain the ionic strength constant. The solution was sonicated to remove air bubbles, filtered through Millex GV membranes (Millipore), and mixed in a 1/1 volume ratio.15 Just before the start of the growth experiment (t ) 0), 1 mL of an aqueous solution containing 12+, 2Cl- in various concentrations was added to a fixed volume (20 mL) of the calcium- and phosphatecontaining solution. The calcium and phosphate concentrations were kept constant in all experiments, and only the concentration of 12+ was varied. The turbidity measurements were made in 1.0 cm path length freshly cleaned
Figure 3. Evolution of the induction times preceding fast growth as a function of the concentration in 12+. The concentration of calcium and phosphate ions was constant and equal to 6 mM. The vertical arrow indicates the concentration value in 12+ at which the morphological and crystallographic studies were performed.
quartz cells with a Shimadzu UV-2101 PC spectrophotometer at λ ) 500 nm. A typical nucleation curve followed by means of turbidimetry is shown in Figure 2. The lag time τ preceding crystallization (Figure 3) cannot experimentally be determined for concentration of 12+ lower than 2 × 10-4 M. Indeed, below 2 × 10-4 M in 12+, because one cannot rule out the spontaneous and nonspecific precipitation, the formation of the solid material may not only be attributed to the presence of 12+. Note that in the absence of 12+ but in the presence of calcium and phosphate, nucleation occurred after ca. 180 min and that in the absence of calcium and in the presence of 6 mM phosphate and 12+, no turbidity increase occurred within the investigated concentration range of 12+. This shows that in order to induce precipitation, both calcium and phosphate must be present in the solution containing 12+. Above this concentration of 2 × 10-4 M, the lag time τ first decreases to a minimum value of 15 ( 7 min upon
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Figure 5. Electron micrograph obtained after crystallization from a solution containing calcium and phosphate both at 6 mM but in the absence of 12+.
Figure 4. Electron micrograph of particles obtained with 5.4 × 10-4 M in 12+. The scale bar corresponds to 500 nm.
increasing the concentration of 12+ to (5 ( 1) × 10-4 M and then reincreases reaching the maximum value of 60 ( 10 min for 12+ concentration of ca. 8 × 10-4 M before decreasing again and reaching the spontaneous precipitation for higher concentration of 12+ dication ((2 ( 1) × 10-3 M). For that reason, the study was conducted at 5 × 10-4 M in 12+ dication. When the turbidity remained constant, the thin platelets thus obtained were stored for 7-10 days before characterization. During this storage period, both the structure and the morphology of particles may change; however, the study reported is based on comparison of morphology in the presence and in the absence of the added organic dication, i.e., the blank experiment. Before characterization by transmission electron microscopy (TEM) (Topcon EM 002B operated at 200 kV), the supernatant was removed after centrifugation (104 rpm during 30 min) and replaced by distilled water, and the pellet was redispersed and centrifuged again (104 rpm during 30 min). This washing step was repeated twice in order to remove the support electrolyte as well as the free calcium and phosphate ions. The concentration of those ions was determined by means of 31P NMR (Brucker at 300 MHz, with glucose-6-phosphate as internal standard) and with a calcium kit (Sigma), respectively. The Ca/P ratio of the particles was determined by means of X-ray dispersive analysis. Before observation, the particles were deposited onto carbon-coated Formvar-covered 300 mesh copper grids. For the thin platelets (Figure 4), the Ca/P ratio of 1.5 ( 0.05, determined by X-ray dispersive analysis, was consistent either with β-tricalcium phosphate or Ca2+ deficient OHAP.1 Within the experimental precision, the analysis of the supernatant gave an almost identical Ca/P ratio. X-ray dispersive analysis also revealed the absence of both Cl- and Na+ or other inorganic salts in the crystal. The formation of ultraflat particles of ca. 1 × 1 µm2 in lateral dimension was due to the presence of 12+ dication. Indeed, for the blank experiment performed under the same conditions but in the absence of the organic additive, the morphology observed was the one corresponding to hydroxyapatite (Figure 5).
Figure 6. Selected area electron diffraction pattern of a single particle displayed diffraction pattern of a single particle displayed in Figure 4. The electron beam is incident with the direct [100] axis. The bright reflections along the direction tilted about 20° with respect to the horizontal direction correspond to the 002, 004, and 006 reflections of OHAP. Table 1. Comparison between Some Measured dhkl Values and the Tabulated Values for OHAPa Miller indices of the reflection
measured dhkl (Å)
tabulated dhkl (Å)
maximum relative difference
002 004 012 022 023
3.68 1.84 3.41 2.97 1.85
3.44 1.72 3.17 2.63 2.00
6.97 6.97 7.57 12.9 8.11
a The maximum relative difference corresponds to the absolute difference between the measured and the tabulated values divided by the minimum of these values.
A crystallographic study based on electron diffraction method performed on a selected area allowed us to determine the pattern parameters for the {100} faces. As an example, the d002 was found equal to 3.68 Å whereas the tabulated one was equal to 3.44 Å. All of the intereticular distances calculated from selected area diffraction are close to those of OHAP within 6-12% as obtained using the Cerius software for OHAP (Figure 6)16. Some typical dhkl values are given in Table 1. This observation indicates that the c-axis is a possible fast growth direction implying growth inhibition along the [001] axes.
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The presence of 12+ in the particles was demonstrated by elemental analysis, which revealed the following (w/w) percent of 0.13 for N, 0.66 for C, and 0.74 for H. These rather low values indicate that the dication although present cannot be incorporated in a periodic fashion in the crystal lattice of OHAP. This is in agreement with the above-mentioned almost identical lattice spacing parameters obtained in the presence and in the absence of the organic dication. Thus, the dication may either be adsorbed on the expressed face or occluded within defects of the crystal, although one may not exclude both situations appearing simultaneously. Assuming that all N atoms present originate only from 12+, it appears that the latter contributes to less than 0.1% of the positive charges incorporated in the crystal lattice. On the other hand, the determined 0.66% for C atom is substantially higher than the expected 0.28% if considering that all carbon originates from the organic dication. The only other carbon source that can be present in the material is carbonate anion. The presence of the latter is certainly due to carbonated water used. Indeed, under our conditions, the preparations were not performed under nitrogen stream. Thus, the 10% calcium deficiency determined from the Ca/P ratio can then be due to both the presence of the dication and the partial substitution of PO43- by CO32- as well as the presence of H2PO4- or HPO42-. On the basis of the above-mentioned formation of a 2D network obtained upon interconnection of phosphate anions and bisamidinium cations (Figure 1c), a possible model for the selective growth of OHAP in the presence of 12+ may be as follows. One may imagine that starting with small domains of 12+/phosphate forming the 2D network, the growth of hydroxyapatite along the c-axis of OHAP may occur upon coordination of Ca2+ cations and further phosphate anions, along the plane of the 2D network. Furthermore, the phosphate-to-phosphate distance of ca. 4.29 Å matches rather well the observed distance for OHAP or related OCP-based materials along the [100] axis.17 Thus, one may propose that the formation of platelets, resulting from the inhibition of growth along (100) face perpendicular to the c-axis of OHAP, may result from lower growth rate perpendicular to the 2D network plane. This kind of mechanism has been demonstrated in the case of calcium carbonate grown in the presence of calcium dicarboxylates.18 In conclusion, it has been demonstrated that upon addition of H-bond donor dicationic tecton 12+, at a concentration of 5 × 10-4 M in a solution containing calcium and phosphate ions at 6 mM concentration, the formation of OHAP platelets may be induced. Although presently no precise description at the molecular level may be given, the observation presented here is of interest since so far in vitro large-sized platelet-shaped crystals of OHAP could only be obtained under high pressure or high temperature. More detailed studies using other concentrations
Communications of 12+ as well as other types of amidinium cations are currently under investigation. Acknowledgment. We thank Pr. C. Rey (Toulouse) for the phosphorus analysis.
References (1) Nancollas, G. H. In Vitro Studies of Calcium Phosphate Crystallisation. In Biomineralization. Chemical and Biochemical Perspectives; Mann, S., Webb, J., Williams, R. J. P., Eds.; VCH: Weinheim, 1989; p 157. (2) Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates, Studies in Inorganic Chemistry 1994, 18, 1. (3) Mann, S. Biomineralization; Oxford Chemistry Masters: Oxford, 2001. (4) Weiner, S.; Traub, W. FASEB J. 1992, 6, 879. (5) Fincham, A. G.; Moradian-Oldak, J.; Simmer, J. P.; Sarte, P.; Lau, E. C.; Diekwisch, T.; Slavkin, H. C. J. Struct. Biol. 1994, 112, 103. (6) Robertson, W. G. The solubility concept. In Biological Mineralization and Demineralization; Nancollas, G. H., Ed.; Springer-Verlag: Berlin, 1981; Chapter 1. (7) Falini, G.; Panzavolta, S.; Roveri, N. J. Mater. Chem. 1999, 9, 779. (8) Mann, S. Nature 1993, 365, 499. (9) Simard, M.; Su, D.; West, J. D. J. Am. Chem. Soc. 1991, 113, 4696. (10) (a) Hosseini, M. W.; Ruppert, R.; Schaeffer, P.; De Cian, A.; Kyritsakas, N.; Fischer, J. Chem. Commun. 1994, 2135. (b) Fe´lix, O.; Hosseini, M. W.; De Cian, A.; Fischer, J. Angew. Chem., Int. Ed. Engl. 1997, 36, 102. (c) Fe´lix, O.; Hosseini, M. W.; De Cian, A.; Fischer, J. Chem. Commun. 2000, 281. (d) Braga, D.; Maini, L.; Grepioni, F.; De Cian, A.; Fe´lix, O.; Fischer, J.; Hosseini, M. W. N. J. Chem. 2000, 24, 547. (e) Fe´lix, O.; Hosseini, M. W.; De Cian, A. Solid State Sci. 2001, 3, 789. (11) Hosseini, M. W.; Brand, G.; Schaeffer, P.; Ruppert, R.; De Cian, A.; Fischer, J. Tetrahedron Lett. 1996, 37, 1405. (12) Ferlay, S.; Fe´lix, O.; Hosseini, M. W.; Planeix, J. M.; Kyritsakas, N. Chem. Commun. 2002, 702. (13) Crystallographic data for 12+-H2PO4 (colorless crystals, 173 K): C10H20N4‚2H2PO4, M ) 390.3, monoclinic, a ) 11.089(3) Å, b ) 4.750(1) Å, c ) 15.425(4) Å, β ) 98.25(2), U ) 804.1 Å3, space group P21/n, Z ) 2, Dc ) 1.612 g cm-3, Cu KR graphite monochromated, 819 data with I > 3σ(I), R ) 0.045, Rw ) 0.070. Data were collected on a Philips PW1100/ 16 diffractometer, and structural determination was achieved using the Nonius OpenMolEn package. (14) Oxley, P.; Short, W. F. J. Chem. Soc. 1947, 497. (15) Ngankam, P. A.; Lavalle, Ph.; Voegel, J. C.; Szyk, L.; Decher, G.; Schaaf, P.; Cuisinier, F. J. G. J. Am. Chem. Soc. 2000, 122, 8998. (16) JCPDS (Joint Commitee for Powder Diffraction Spectra), Cambridge Crystallographic Data Center; file number, 9-432. (17) Bertoni, G.; Bigi, A.; Falini, G.; Panzavolta S.; Roveri, N. J. Mater. Chem. 1999, 9, 779. (18) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110.
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