DOI: 10.1021/cg101437h
The Role of the Air-Liquid Interface in Protein-Mediated Biomineralization of Calcium Carbonate
2011, Vol. 11 803–810
David C. Bassett,*,† Marc D. McKee,†,‡ and Jake E. Barralet† †
Faculty of Dentistry and ‡Department of Anatomy and Cell Biology, McGill University, Montr eal, Qu ebec H3A 2B2, Canada Received October 28, 2010; Revised Manuscript Received January 5, 2011
ABSTRACT: Crystals formed by natural biomineralization processes often display exquisite morphologies not normally seen in synthetic laboratory preparations. This may be attributable to the influence of biomolecules that act to modify or direct crystal growth and assembly. Many attempts have been made to mimic these processes in synthetic systems to render novel crystal shapes and to form otherwise unstable crystal polymorphs. To determine protein influenced crystallization mechanisms, polymers, synthetic peptides, and polypeptides are often used. An established biomineralization model is the use of carbon dioxide gas diffusion into a calcium ion solution since it yields a slow precipitation and so lends itself to observation. However, reported observations are often assumed to be purely solution-phase phenomena,1 despite it being well-known that macromolecules adopt particular conformations at the air-liquid interface.2 In this study, we demonstrate that novel nanostructured cones of calcium carbonate (calcite) formed in the presence of osteopontin are created at the air-liquid interface. On one hand, this suggests that caution should be exercised in interpreting data from this clearly nonbiomimetic model. On the other, we demonstrate that we could physically manipulate air-liquid interface crystallization to align anisotropic nanostructures using 1D microscale guidance.
1. Introduction Precise control of crystallization, mediated by proteins, is essential for the unique structural and mechanical properties of hard tissues formed by living organisms.3 Calcium carbonate is an abundant mineral that may be biogenically formed to provide the basis for skeletal support in marine organisms such as mollusks and crustaceans and also for protection in the shell of avian eggs.4 This biomineral is often formed with a complex yet well-defined microstructure made possible by protein-mediated biomineralization processes. A protein of particular significance to calcium carbonate biomineralization is osteopontin (OPN), an acidic glycoprotein found in avian eggshells, whose mineral phase is calcite, and also in many vertebrate mineralized tissues and pathologically in soft tissue ectopic mineralization.5 OPN is a highly phosphorylated protein, which is thought to be a key property in its mineral binding ability.6 OPN has been shown to limit the growth of calcite crystals7 and other calcium biominerals such as calcium oxalate5b,8 and hydroxyapatite.5a,9 Protein(s) controlling biomineralization processes likely fulfill two roles: (i) nucleation of the desired mineral phase and (ii) control of the growth of the biomineral.3 The process may proceed by the aggregation and fusion of protein nanoparticulate biominerals, which self-assemble to create the final mesocrystal; this process has recently been termed “nonclassical crystallization”.10 Indeed, the influence of templating molecules, particularly on prenucleation structures in synthetic systems, is currently an active and fertile area of research.11 Many attempts have been made to synthetically mimic in vivo calcium carbonate biomineralization phenomena, and the reader is directed to recent reviews on the subject.12 A key aspect of biomineralization is that it often occurs in confined spaces such as that offered by collagen during the formation of *Corresponding author. E-mail:
[email protected]. r 2011 American Chemical Society
bone.13 Mimicking this effect by spatially restricting crystal growth in synthetic systems through the use of templated surfaces,14 gels,15 and collagen matrices16 has emerged as a technique to guide inorganic crystal morphology into remarkable forms. In this study, we examined the effect of supraphysiological levels (0.1-10 mg mL-1) of OPN on calcite formation in an established biomineralization model, namely, the ammonium carbonate sublimation technique. Here the diffusion of carbon dioxide into aqueous CaCl2 solution yields a slow precipitation, which is convenient to monitor. We found that OPN caused the formation of vateritic plates, calcitic nanofibers, and self-assembled nanofibers in the form of cones up to 150 μm long. The nanostructured cones of calcite were formed from the edges of floating vateritic plates at the airliquid interface. Cone formation appeared be a result of point nucleation of nanofibers, and cones were orientated toward the bottom of the reaction vessel. Additionally we were able to physically manipulate the air-liquid interface crystallization to affect 2D control of nanofiber growth using 1D microscale guidance. 2. Results In protein-free negative control reactions, regular rhombohedral crystals of calcite typically around 50-100 μm in size were formed after 72 h reaction (Figure 1A). However, when the reaction was repeated in the presence of 1 mg mL-1 OPN, radically different crystal morphologies were obtained (Figure 1B-D). There were three morphologies: hollow conical structures ranging in length from about 30 to 150 μm that were found in the reaction liquid (Figure 1B shows this structure after drying), nanofibers, around 40 nm in diameter and several hundred micrometers long, observed to form on the base of the reaction vessel (Figure 1C), and large (ca. 150-200 μm diameter) microplates found at the air-liquid interface (Figure 1D Published on Web 01/26/2011
pubs.acs.org/crystal
804
Crystal Growth & Design, Vol. 11, No. 3, 2011
Figure 1. SEM micrographs of protein-free control (A), conical calcium carbonate formed in the presence of OPN (B), and small, 50 ( 18 nm, fibers formed on the base of the OPN crystallization dish (C) and environmental SEM of vateritic plates formed on the surface of the reaction liquid taken at an angle of 30° (D).
Figure 2. X-ray diffraction patterns of the different calcium carbonate structures obtained in the presence of osteopontin as shown in Figure 1. Plates formed at the air-liquid interface (A), a mixture of plates and cones from the reaction liquor (B), and the nanofibers formed on the base of the dish (C). Peaks labeled 9 = calcite and O = vaterite.
shows a side view of these structures after careful removal of the liquid phase). In order to determine the crystalline phase of the different morphologies, samples were separated, and powder X-ray diffraction (XRD) scans were taken. It was possible to separate the microplates from the cones by carefully scraping the top layer of the reaction liquid; however, it was extremely challenging to separate the cones from the microplates in sufficient quantities for XRD analysis since some of the microplates sank and were mixed with the cones. It was found that the microplates were predominantly composed of vaterite, the cones and microplates contained a mixture of vaterite and calcite, and the nanofibers formed on the base of the dish were purely calcitic (Figure 2). Considerable peak broadening was evident for all the samples indicating small crystallite size; this was estimated to be 18 nm using the Scherrer relation, (fwhm = 0.46° at 2θ = 29.40°). Microscale precipitates also formed at the air-liquid interface under protein-free conditions and were found to be poorly formed calcite.
Bassett et al.
Figure 3. SEM micrographs depicting the cone surface morphology in detail (A, insert shows texture of the surface at high magnification) and interior structure (B) and high (C) and low (D) magnification images of the plates found at the air-liquid interface revealing a distinctly different morphology of bundled fibers.
High-resolution SEM analysis confirmed that the cones were comprised of very small particles (10-20 nm diameter) of calcium carbonate arranged in concentric layers of repeating triangles (Figure 3A). The cones appeared to be hollow (Figure 3B) often with a well-defined edge at the base. Microplates appeared to be similarly made of small particles, albeit slightly larger (20-30 nm) and rougher. These particles were arranged in fibrous bundles rather than organized conical shapes (Figures 3C,D). Since the XRD analysis was ambiguous in determining the crystal polymorph of the cones, a thin cross-section of a cone was prepared using ion beam milling for TEM and SAED analysis (Figure 4). Ion beam milling exposed the internal structure of the cone and confirmed it to be hollow (Figure 4B). By use of dark field TEM, it was possible to measure the size of the small crystalline domains making up the large structure to be on the order of 10-20 nm in diameter, which confirmed earlier SEM and XRD observations. SAED confirmed the presence of polycrystalline domains of calcite; there was a small amount of preferred orientation observed for {202} and {110} planes, which appeared to be aligned with and perpendicular to the symmetrical axis of the cone, respectively. To elucidate the cone growth mechanism, light micrographs of the reaction solution were taken at various time points, and example images are depicted in Figure 5. The first microscale precipitates were found at 12 h reaction time and were comprised of microfilaments either singly or branching as shown. By 24 h, conical shapes had formed that continued to grow in size and number up until 72 h, after which there were no further changes observed over an additional 3 weeks. Note that the images shown in Figure 5 are of different cones made in separate reactions and do not follow the growth of one cone. This is because the reaction was sensitive to disturbance such that cone formation would be halted following manipulation. We hypothesized that this was due to the cones forming at the air-liquid interface; however due to inevitable disturbance of the reaction upon examination with either a conventional light microscope or SEM, this process was not directly observable. To image this process with minimal disturbance, we used a confocal scanning microscope to observe the growing crystals in situ. A modified tissue culture dish with a section of the base removed to which a glass coverslip was
Article
Crystal Growth & Design, Vol. 11, No. 3, 2011
805
Figure 4. FIB milling procedure used to extract a section of cone for TEM analysis: (A) The cone was positioned and then welded to the micro manipulator. (B) A section was cut through the cone, exposing the interior hollow structure. (C) View of the extracted section prior to thinning for TEM analysis. TEM in light (D) and dark (E) field of the cross section and accompanying SAED pattern (F). Inset in panel E indicates the position and orientation of the sample taken for TEM analysis in relation to the whole cone for comparison with the SAED pattern.
Figure 6. Confocal image of a growing cone at the air-liquid interface showing a plate-like formation on the liquid surface with attached fibrous processes from which the cones form (arrow) (A). Adjacent light micrographs show the results of experiments performed in normal (B) and inverted orientations (C). Note that cone formation was inhibited in the inverted state.
Figure 5. Light micrographs depicting stages of cone growth at 12, 24, 36, and 72 h.
glued to create a thin base was used for microscopy. On the interior surface a second smaller coverslip was placed such that it could be moved to one side to clearly image the cone and microplate structure in the reaction liquid without interference from the precipitates, which formed in the base of the dish. Reaction liquid was then carefully removed such qthat the structures of interest were within the focal plane of
the microscope. Interestingly, our hypothesis appeared to be correct because it was found that the cones were growing from semispherical microplates floating on the surface of the liquid and form from filaments originating from the microplates protruding into the liquid (Figure 6). In order to further investigate this growth mechanism, the experiment was performed in the inverted state, that is, the crystallization was performed with the gas surface below the liquid, using surface tension to retain the liquid in the reaction vessel. This simple change in conditions prevented cone formation and resulted in spherical particles with small rounded appendages and no fibrous material (Figure 6). The primary particle size and crystal polymorph (calcite) was, however, unchanged. To investigate whether we could spatially control crystallization in our system, we modified the reaction chamber as depicted in Figure 7A. A thin space 150 μm across was created between two glass coverslips by using small strips of coverslip as a spacer and gluing together. The remaining well opening
806
Crystal Growth & Design, Vol. 11, No. 3, 2011
Figure 7. Diagram showing experimental set up of glass plates (depicted in blue) arranged to restrict growth (A) and accompanying SEM micrographs showing resulting aligned fibers formed in the presence of OPN at low (B) and high magnification (C). Aligned fibers were found between the glass plates in the area depicted in yellow. TEM micrographs of thin sections of the aligned fibers taken in light (D) and dark fields (zone axis [010]) (E) show orientation of the constituent calcite nanocrystals. High magnification TEM (F) and accompanying SAED pattern clearly show the morphology and orientation of the prismatic calcite nanocrystals in the [010] zone axis.
was sealed using circular coverslips glued into position. The reaction was then carried out as normal for 72 h in the presence of OPN with the only air-liquid interface being the thin slot created between the glass plates. SEM observation revealed the formation of aligned fibers, the long axes of which were aligned perpendicularly to the air-liquid interface (Figure 7B,C). The fibers were found to be ∼500-600 nm in diameter, in keeping with those that had formed previously in the fibrous microplates and approximately an order of magnitude larger than the tangled nanofibers formed on the base of the reaction vessel. Thin sections of the aligned fibers were made parallel to the fibers long axes for TEM analysis. The fibers were revealed to be composed of aligned prismatic calcite crystals on the order of 18-25 nm wide and 50-100 nm long. SAED and dark field analysis indicated orientation in the [010] zone axis, which is clear evidence that fiber growth occurred via oriented attachment of primary nanocrystals along the c-axis of calcite. The pH of the protein-free reaction rose steeply until reaching a peak value of pH 9.5, which slowly reduced to pH 9.3 over 72 h of measurement. This pH change was closely followed in reactions containing OPN, which initially lagged behind protein-free conditions before reaching a peak at pH 9.9 after which the pH gradually dropped to 8.8 over the same
Bassett et al.
time period (Figure 8A). The rapid increase of pH in both conditions was due to the influx of ammonia gas into the reaction medium resulting in the formation of ammonium hydroxide. Calcium ion concentration rapidly dropped as crystallization began to occur almost immediately after beginning the reaction (Figure 8B). After 2 h, the Ca2þ concentration had dropped to ∼51 mM in both the control and OPNcontaining reactions. Further reduction to 32 mM after 4 h was observed in control experiments; however, this was followed by a gradual rise to ∼45 mM after 24 h, which remained constant. In the presence of OPN, Ca2þ concentration gradually decreased to 42 mM after 12 h after which an increase to a plateau value of 50 mM was observed after 24 h. TGA analysis indicated a protein content of ∼8 wt %, and DSC analysis did not reveal any peaks indicative of phase transformation or crystallization (Figure 8C). The difference in measured pH change dependent on the presence of OPN was visualized by adding a pH indicator to the reaction solution. As shown in Figure 8D, the OPN formed a weak barrier to diffusion because the pH change was slower than in protein-free conditions. At 5 min, small (approximately 0.5 mm) channels of low pH were visible in OPN-containing solutions, which persisted until 20 min (see Figure 8D, black arrows). By 1 h, the pH was uniform and approximately the same for both reaction conditions; however microcrystals were visible in protein-free controls, which continued to grow over several hours (see Figure 8D, white arrows). An alternative method was developed to generate CO2 without changing the pH by feeding HCl into a dish of solid calcium carbonate in the correct amounts to generate the same amount of CO2 as developed by the ammonium carbonate sublimation method. This way reaction pHs were maintained at 4.5, 5.5, 6.5, 7.5, and 8.5 by buffering the system using TrisHCl. In protein-free conditions, precipitation did not occur at pH 4.5 and 5.5; above this no difference in crystal morphology was observed compared with calcite crystals formed by ammonium carbonate sublimation. In the presence of OPN, conical and fibrous structures did not form at pH 4.5. Instead, a small amount of poorly formed calcite rhombohedra 7-9 μm in diameter and long single fibers (up to 500 μm) were found (data not shown). At all other pH values tested, the cones and fibrous structures formed as in the unbuffered system. To directly examine the protein at the air-liquid interface Brewster angle microscopy (BAM), commonly used to investigate thin films, was used to image OPN order and assembly at early stages and at changing pH. At pH 5.5, the OPN appeared to be arranged in a very ordered linear fashion, with slight differences in the thickness of the film giving rise to clear contrast in the BAM images (Figure 9A). The pH was raised to 9.5 by the addition of ammonium hydroxide, and there was a distinct and almost immediate change in the conformation of the protein (Figure 9B). Linear arrangement of the molecule was no longer evident; instead the protein was arranged in multiple dots throughout the film and also tears began to appear. 3. Discussion From electron beam analysis, it appeared that the calcium carbonate crystals formed in the presence of OPN were comprised of discrete nanoparticulate building blocks 10-20 nm in size. The self-assembly of nanoparticles into different morphologies appeared to be interface dependent. Based on calcium ion measurements, the initial formation of these nanoparticles appears to be quite rapid,
