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Organic-Inorganic Hybrid Structure of Calcite Crystalline Assemblies Grown in a Gelatin Hydrogel Matrix: Relevance to Biomineralization Olaf Grassmann, Gerd Mu¨ller, and Peer Lo¨bmann* Lehrstuhl fu¨ r Silicatchemie, Universita¨ t Wu¨ rzburg, Ro¨ ntgenring 11, D-97070 Wu¨ rzburg, Germany Received May 27, 2002. Revised Manuscript Received August 21, 2002
Calcite particles are grown in a collagenous matrix using a counter-diffusion arrangement. Although microstructural analysis revealed a heterogeneous intergrowth of organic and inorganic phases within the particles, the composite growth is rather a consequence of local chemical environment that is not specific to the gelatin gel. However, using an artificial poly-acrylamide hydrogel results in a very different growth morphology. The addition of poly-aspartate to the pore solution in either gelatin or poly-acrylamide hydrogel appears to overcompensate the physical properties of the organic matrix, leading to morphologies independent of which hydrogel is used. Our results stress the importance of noncollageneous proteins within a physical growth environment.
Introduction The formation of mineral phases in organic matrices is a key feature of natural biomineralization processes. For example, in multicellular organisms enamel and bone1 are formed in gellike extracellular networks. In nacre, the mineral phase as well as the microstructural assembly of the crystalline building blocks are governed by complex interaction of the organic matrix and acidic macromolecules.2 Much work has been done to understand the influence of key factors such as the microstructure of the growth medium, functional groups within the matrix, and the presence of proteins either in solution or adsorbed to the network surface.3 Because certain types of collagen are known to be involved in the biomineralization of bone, gelatin is widely used to study the general role of protein networks on the formation of mineral phases in vitro. For instance, Kniep and co-workers4,5 have observed the fractal growth of fluorapatite-gelatin composites in a double-diffusion arrangement. Nevertheless, the role of the organic matrix remains ambiguous: a unique coreshell assembly of the mineral could be obtained only in gelatin from porcine skin, on the other hand the morphogenesis largely seems to be determined by the intrinsic electric fields of the crystals. The same type of gelatin has been used to prepare cross-linked xerogel substrates for the nucleation of CaCO3, poly-L-aspartate (poly-L-Asp) was applied as a * Corresponding author: phone +49 (0)931 31 26 31; fax +49 (0)931 31 21 09; e-mail
[email protected]. (1) Lowenstam, H.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (2) Weiner, S.; Traub, W. Philos. Trans. R. Soc. London B 1984, 304, 425. (3) Fallini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem. Eur. J. 1997, 3, 1807. (4) Kniep, R.; Busch, S. Angew. Chem. 1996, 108, 2788. (5) Busch, S.; Dolhaine, H.; DuChesne, A.; Heinz, S.; Hochrein, O.; Laeri, F.; Podebrad, O.; Vietze, U.; Weiland, T.; Kniep, R. Eur. J. Inorg. Chem. 1999, 1643, 3.
model substance for noncollagenous proteins.6 The mineralization of calcite, aragonite, and vaterite could be induced as a function of the polypeptide concentration and the mechanical elongation of the gelatin substrate. A model for the relationship between the deformation of the collagen matrix, adsorption of poly-L-Asp, and the alignment of crystal faces was derived. Because of the substrate geometry it was rather difficult to obtain additional information about the involvement of the organic matrix in the further morphogenesis. The authors suggest that the structural evolution during the growth of acicular crystal aggregates in the gels is mainly related to the presence of gel released by the substrate film. In this paper we describe the nucleation and growth of CaCO3 in a continuous gelatin matrix. Significant amounts of this matrix are incorporated into the inorganic phase, and its distribution within the composite is studied. The microstructure and growth mechanism are compared with our previous work on mineralization in artificial poly-acrylamide (p-AAm) hydrogels.7 New results indicate that the addition of polypeptides to the growth media overcompensates the influence of both different types of networks. Experimental Section Gelatin gels were prepared according to Kniep and Busch,4 consisting of 10 mass% gelatin (300 Bloom, Aldrich). To prevent bacterial growth 0.1 mass% sodium azide (Aldrich) was added to the gels. After preparation the gelatin gels were kept for 24 h at ambient temperature. The gels were then extracted for 4 days in a 0.05 M solution of tris(hydroxymethyl)-aminomethane (Aldrich) which was adjusted with a 2 M solution of HCl to a pH of 8.35 (Tris-HCl). As confirmed by pH measurement with an inserted pH electrode, the pH of (6) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. J. Chem. Soc., Dalton Trans. 2000, 3983. (7) Grassmann, O.; Neder, R.; Putnis, A.; Lo¨bmann, P. Am. Mineral. Submitted for publication.
10.1021/cm0212156 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/15/2002
Hybrid Structure of Calcite Particles in Gelatin the pore solution is set to a value of 8.35. Artificial p-AAm hydrogels were obtained by a procedure described elsewhere.7 Hydrogels containing poly-L-aspartate (poly-L-Asp) were obtained by addition of 0.1 g poly-L-Asp (sodium salt; Sigma Co.) to 100 mL of aqueous gelatin respectively acrylamidmethylenbisacrylamide solution. The monomer/poly-L-Asp massratio amounts to 100:1, corresponding to a poly-L-Asp concentration of 0.11 mmol/L within the hydrogels. Mineralization experiments were conducted in a doublediffusion arrangement. Hydrogels of 25-mm diameter and approximately 35-mm thickness were placed into a U tube between two aqueous solutions. The different sides contained solutions of 0.1 M CaCl2 and 0.1 M NaHCO3, respectively. To avoid an influence of a nonuniform pH throughout the gel column the solutions were buffered with Tris-HCl to a constant pH of 8.35. The pH of the solutions remained unaltered during double diffusion, as confirmed by pH measurement after terminating the experiments. For cathodoluminescence microscopy investigations 50 ppm Mn2+ (MnCl2 solution) was added to the 0.1 M CaCl2 solution. To prevent leaching from the gels by diffusion, identical concentrations of polyamino acid were added to the mineral stock solutions for the respective experiments. In the course of diffusion the components met in the hydrogel, and precipitation of CaCO3 occurred. After one week the double-diffusion experiments were terminated, and the gels were removed from the tube and cut into slices of 5-mm thickness. To isolate the precipitates, the gelatin slices were dissolved by immersion in hot water, and the p-AAm samples were incubated for 24 h in sodium hypochlorite solution. The remaining inorganic material was separated by centrifugation in both cases. Thermal analysis was performed on samples that were picked manually out of the organic matrix and shortly exposed to leaching conditions. The complete removal of possible surface contamination was shown by electron microscopy. The thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were performed using a Setaram TAG24 with a heating rate of 10 K/min under synthetic air atmosphere. To gain insight into the composite nature of the precipitates, particles isolated mechanically were immersed in 0.25 M EDTA solution. The solvent was adjusted with a 0.1 M NaOH solution to a pH of 7.0 (25 °C). The dissolution process of the inorganic component was monitored after 3, 7, and 28 days. The solid phases were analyzed by powder X-ray diffraction (XRD, Stoe Stadi P), polarization microscopy (Leica DMRM), and scanning electron microscopy (SEM, Hitachi S800). Investigations of thin-cut samples with an analytical SEM (LEO 1450VP) and an optical hot-cathode-luminescence microscope (voltage 14 kV, current density 10 µA/mm2) revealed information on the microstructure of the precipitates. The cathodoluminescence microscopy was performed with Mn2+ doped crystals to enhance the fluorescence of the particles.
Results and Discussion Mineralization of Calcite in a Gelatin Hydrogel Matrix. In gelatin gels first mineralization can be visually observed after approximately 60 h at the Ca2+rich side of the experimental setup. Subsequently, typical Liesegang-rings are formed indicating mineralization under oscillating concentration conditions of the respective components.8 X-ray powder diffraction experiments (XRD) of ground samples (data not shown) proved that the crystalline material consists of calcite. In Figure 1 a typical SEM-image of a precipitate isolated from the center of the gelatin gel after 7 days is shown: barrel-shaped particles composed of two (8) Henisch, H. Crystals in Gels and Liesegang Rings; Cambridge University Press: Cambridge, U.K., 1988.
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Figure 1. Morphology of a calcite particle isolated from the middle section of the gelatin growth medium after 7 days as imaged by SEM. A magnified view of terrace planes is shown in the insert (a, top). SEM micrograph of a bisected calcite particle with radial structure (b, bottom).
similar parts, separated by a slight groove, have grown in the gel. Each half is composed of three homogeneous crystals limited by terrace planes (insert in Figure 1a). The two halves of the barrel appear to be twisted 60° about the barrel axis. Upon closer inspection of the particle surface the terraces seem to be the {10-14} planes of the calcite cleavage rhombohedron. A SEM micrograph of a particle bisected along the indention plane is shown in Figure 1b: the concentric structure of the particles is the result of a radial growth from a central nucleation site. This type of morphology is not highly specific to crystals grown in gelatin gel, as McCauley and Roy9 and Fernandez-Diaz et al.10 observed similar calcite aggregates grown in silica hydrogels. Crystals were separated from the gel medium, the thermogravimetric analysis (TGA) and differential thermal analysis of ground samples are shown in Figure 2a. Thermal decomposition of organic material incorporated in the calcite particles is identified by an exothermal reaction and a corresponding weight loss. Because of the disperse distribution of the gelatin gel within the ground particles the decomposition is completed after heating to 400 °C. In contrast to this, thermal degradation of the pure gelatin reference (Figure 2b) is characterized by a two-step mechanism indicating a different decomposition pathway of the organic material. (9) McCauley, J.; Roy, R. Am. Mineral. 1974, 59, 947. (10) Ferna´ndez-Diaz, L.; Putnis, A.; Prieto, M.; Putnis, C. J. Sediment. Res. 1996, 66, 482.
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Figure 3. SEM image of a partly dissolved calcite-gelatin composite after a 3-day period of EDTA treatment. The outer parts of CaCO3 are dissolved.
During heating 3.9 mass% of incorporated organic material is removed by exothermal decomposition. The corresponding volume content is calculated using the theoretical density of calcite (2.72 g/cm3 11) and a density of 1.3 g/cm3 for the incorporated gelatin gel. Under these assumptions it can be estimated that the precipitates contain a volume fraction of about 8% organic material. Isolated particles were incubated for 3, 7, and 28 days in an EDTA solution to dissolve the inorganic phase via complexation of Ca2+. After 28 days a pure calcite reference crystal of comparable size (Iceland spar) is dissolved completely. Under these conditions the particles grown in the gelatin gel are still visible within the EDTA solution, and the morphology of the particles is only slightly altered. Upon drying, the original morphology is lost because of shrinkage of the gelatin hydrogel. Using SEM-EDX analysis no calcium is detectable within the shrunken particles, confirming the complete dissolution of the inorganic CaCO3 phase. After only a 3 day period of dissolution, the initial particle morphology of dried samples is still visible (Figure 3), although the outer parts do not contain any calcium. Obviously, a nondissolved calcite core maintains the particle integrity. In contrast to that of fluorapatite5 the dissolution of the inorganic phase of the examined calcite composites started from the particle surface. Some samples were embedded in epoxy resin and prepared as thin-cuts to facilitate optical transmission microscopy (Figure 4a). Although the radial structure
of the particles resembles spherolites, the sectional extinction observed under crossed polarizers indicates a nonspherolitic crystal growth. Thin-cuts perpendicular to the particle mean axis were characterized by three sections of homogeneous crystallographic orientation, corresponding to the terraces shown in the SEM (Figure 1a). The angles between the optical axes of these crystal individuals were estimated using a universal rotary stage attached to the microscope. It was shown that the crystallographic c-axes of the calcite individuals are slightly tilted relative to the particle mean axis. In a longitudinal cut, pairwise homogeneous extinction throughout the whole particle is observed (arrows in Figure 4a). Consequently, the crystallographic orientation of the respective pairs is identical, indicating a triplet intergrowth of three crystal individuals. The origin of the intergrowth remains ambiguous. It is remarkable, however, that the observed radial structure, together with the flat crystal faces (Figure 1) and the 3-fold symmetry of the barrel-shaped particles, resemble mammalian otoconia.12 To investigate the distribution of the gelatin incorporated in the particles, their cathodoluminescence was studied. The electron beam generates luminescence within the particles. The luminescence properties of the material are determined by the distribution of luminescence centers such as the Mn2+ dopant (extrinsic defect). Because of the substitution of Mn2+ for Ca2+ in the calcite lattice, the luminescence centers are preferentially enriched in the CaCO3 phase of the composites. For this reason the inorganic component of the particles is characterized by a brighter luminescence than the incorporated organic gelatin gel. Obviously, the organic material is heterogeneously distributed at a µm scale (Figure 4b). This does not necessarily rule out a submicroscopic organic-inorganic composite character of the whole particle. Nevertheless, the bulk of the gelatin appears to be preferably incorporated at the core of the respective halves of the particles. Because of the lower resolution of the optical microscope, it is not possible to draw further conclusions about the spatial arrangement of organic and inorganic phases within the particles by this method. Therefore, polished surfaces were imaged by SEM in backscattered
(11) Deer, W.; Howie, R.; Zussman, J. Rock-Forming Minerals; Longman: Harlow, U.K., 1992.
(12) Mann, S.; Parker, S.; Ross, M.; Skarnulis, A.; Williams, R. Proc. R. Soc. London B 1983, 218, 415.
Figure 2. TGA and DTA of calcite particles isolated from the gelatin growth medium (heating rate 10 K/min, synthetic air atmosphere) (a, top). TGA and DTA of dried (110 °C) bulk gelatin gel (b, bottom).
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Figure 4. Optical polarization microscopy image of thin-cut calcite particles obtained from growth in a gelatin gel matrix. Areas of identical brightness within the particles (arrows) indicate uniform crystallographic orientation (a, top). Optical cathodoluminescence micrograph of thin-cut calcite samples, heterogeneous distribution of luminescence centers within the particles (b, bottom).
Figure 5. SEM micrograph of a polished calcite-gelatin particle in backscattered electron mode. Three sections of the particle are highlighted.
electron mode (Figure 5). Because the nonuniform distribution of the gelatin matrix has been proven by
optical cathodoluminescence microscopy, the contrasts within the image can be attributed to differences in the energies of electrons backscattered from mostly inorganic material and regions of high content of organic matrix. Due to shrinkage of the incorporated gelatin gel upon drying, some parts show large porosity. The cut perpendicular to the particle mean axis is characterized by three sections (highlighted in Figure 5) corresponding to the crystal individuals identified in Figure 4a. Obviously, regions of high gelatin content and parts composed of mostly inorganic material are distinguishable. An apparently lamellar assembly of the organic and inorganic material may result from the orientation of the thin-cut relative to the crystallographic orientation of the constituent crystallites. Because the overall morphology is not specific for calcite prepared in gelatin gels9,10 we propose that the organic-inorganic intergrowth is not the reason for, but a consequence of, the respective growth mechanism.
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Figure 6. SEM image of a calcite particle crystallized near the CaCl2 solution of the counterdiffusion setup (a, top). Micrograph of a calcite particle isolated from a part of the gelatin gel matrix near the NaHCO3 solution (b, bottom).
Relevance to Biomineralization. Even though significant amounts of gelatin are incorporated, we find no indication for any influence of the natural protein network on the morphogenesis of the aggregates by means of chemical interaction of, for example, functional groups with the growing crystals. Samples isolated from different zones of the gel show distinct variations in their appearance, although the variations of the morphology are less pronounced than calcium carbonate grown within silica gels using solutions with deviating pH values.10 Upon closer inspection of the particle surfaces with SEM, the terraces of the particles crystallized near the CaCl2 solution are rounded (Figure 6a). Moreover the crystal faces appear rough, indicating a crystal growth process under high supersaturations.10 Particles isolated from zones near the NaHCO3 solution are characterized by smooth faces and sharp edges, corresponding to near-equilibrium crystal growth (Figure 6b). Thus, we postulate that the appearance of the particles is mainly dominated by the local supersaturation within the growth environment. As influences of varying pH throughout the gel column could be ruled out by buffering, we propose an impact of the gelatin matrix on the supersaturation. The asparaginic and glutaminic acids contained in the gelatin gel immobilize Ca2+ via an ionotropic process13 leading to high local supersaturation. Thus, the gelatin gel affects the crystal growth mainly by fine-tuning of the specific chemical environment. Compared to crystal growth in gelatin, calcite aggregates grown in artificial p-AAm hydrogels are highly
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specific for the growth matrix used.7 The hydrogel network used in our previous work provides a growth environment with a specific spatial arrangement. Whereas gelatin is composed of electrostatically coiled polypeptide chains, p-AAm hydrogels consist of covalently cross-linked monomers. As estimated from freeze-drying experiments, the pore size of p-AAm is significantly less compared to that of gelatin.14,15 Because ionic functional groups are not present, the influence on crystal aggregation is predominantly governed by the microstructural properties of the hydrogel. For calcite crystals grown within the p-AAm hydrogels we observed a high degree of order in the assembly of rhombohedral calcite crystallites, which formed pseudooctahedral aggregates.7 The subcrystals are highly aligned within the artificial hydrogel matrix so that the total assembly appears to be single crystalline in X-ray diffraction experiments. Dominguez Bella and GarciaRuiz describe a comparable aggregation of subunits in silica gels leading to single crystalline fibers of CaCO3.16 In the field of biomineralization an analogous microstructure is known from the nacreous layer of abalone shells, where aragonite tablets are regularly arranged within a protein matrix.17 In both cases of our in vitro experiments (calcite in p-AAm and in gelatin) we suggest that the different networks solely provide a spatial environment in which specific growth mechanisms of the mineral phase dominate. Whereas the gelatin gel provides the chemical microenvironment, crystal growth in p-AAm appears to be dominated by compartimental restriction. A similar argument may hold true for the fractal morphogenesis of fluorapatite in gelatin, which may be ruled mainly by internal electric fields of the inorganic phase.5 Falini and co-workers6 were able to control the polymorphism of CaCO3 nucleated on cross-linked gelatin-xerogels by the addition of poly-L-aspartate and the uniaxial deformation of the collagen network. These results stress the importance of macromolecules (i.e., noncollagenous proteins18) adsorbed on the matrix surface on mineral nucleation. On the other hand, further growth and morphogenesis of the material may be governed by “physical” network parameters. We applied the double-diffusion arrangement to compare CaCO3 grown within gelatin and artifical p-AAm gel matrices in the presence of p-L-Asp (Figure 7c and d) to the respective controls without polypeptide addition (Figure 7a and b). Whereas without polypeptides the particles exhibit distinct, characteristic morphologies, in the presence of p-L-Asp the products obtained from the different growth environments show quite similar morphologies. Powder-XRD (data not shown) identifies the CaCO3 polymorph vaterite, comparable to the observations of Falini et al.6 Under ambient condi(13) Greenfield, E.; Wilson, D.; Crenshaw, M. Am. Zool. 1984, 24, 925. (14) Halberstadt, E.; Henisch, H.; Nickl, J.; White, E. J. Colloid Interface Sci. 1969, 29, 469. (15) Ru¨chel R.; Steere, R.; Erbe, E. J. Chromatogr. 1978, 166, 563. (16) Dominguez Bella, S.; Garcia-Ruiz, J. J. Cryst. Growth 1986, 79, 236. (17) Sarikaya, M.; Liu, J.; Aksay, I. In Biomimetics: Design and Processing of Materials; Sarikaya, M., Aksay, I., Eds.; AIP Press: New York, 1995; p 35. (18) Belcher, A.; Wu, X.; Cristensen, J.; Hansma, P.; Stucky, G.; Morse, D. Nature 1996, 381, 56.
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Figure 7. Morphology of CaCO3 particles obtained from growth in gelatin (a, top left) and artifical p-AAm hydrogels (b, top right) without additives and in the presence of 0.11 mmol/L poly-L-aspartate in gelatin (c, bottom left) and p-AAm matrix (d, bottom right), respectively.
tions vaterite is thermodynamically metastable, thus the polypeptides within the hydrogels inhibit the transformation to calcite by kinetic effects. Considering that for the p-AAm hydrogel network no ordered adsorption of the polypeptide can be assumed, which at least would be possible within the partial fibrous gelatin network, the nucleation and growth in both networks is governed by the polypeptide in solution. Effects of the different matrix structures seem to be overcompensated by the additive. In further studies, the concentration of the additive will be systematically reduced. Below a certain threshold concentration, the polypeptide is expected to be mainly adsorbed on the collagen surface and provide nucleation sites, whereas further growth of the aggregates should be matrix-specific. Conclusions Calcite particles were grown by means of a collagenous matrix. Even though significant amounts of the organic material were incorporated in the particles, the crystal growth is rather a consequence of local chemical environment that is not unique to gelatin gel. No direct
influence of functional groups of the matrix on the morphogenesis of the particles was found. The analogy of the gelatin-grown particles to some biominerals, however, suggests that biological crystallization may take place under comparable conditions. On the basis of our previous work and the results of other groups, it is proposed that the control of crystal assembly in biomineralization is attributed, to a considerable extent, to the physical properties of the growth environment. In contrast to this, the nucleation of specific polymorphs is associated with the presence of macromolecules either in solution or adsorbed on the respective framework surface. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the Schwerpunktprogramm (SPP) “Prinzipien der Biomineralisation”. We thank J. Go¨tze, R. Neder, and A. Putnis for experimental support and helpful discussions. CM0212156
