Formation of Aragonite Crystals in the Crossed Lamellar

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Formation of Aragonite Crystals in the Crossed Lamellar Microstructure of Limpet Shells Michio Suzuki,†,‡ Toshihiro Kogure,‡ Steve Weiner,† and Lia Addadi*,† † ‡

Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan ABSTRACT: The crossed lamellar microstructure is the most commonly formed shell structure in mollusks. Although the mechanical properties and the architecture of the crossed lamellar microstructure have been extensively studied, its formation mechanisms are largely unknown. In this study, we obtained information on the formation processes of the aragonite crystals in the crossed lamellar microstructure using two converging approaches: cryo-scanning electron microscopy performed on the forming shell layer and in vitro crystallization experiments with extracted matrix macromolecules. We show that deposition of the first crystalline particles occurs in a thin granular layer on the growing surface of the mature crossed lamellar microstructure. The granules subsequently grow to form the lamellar prisms. In vitro, the ensemble of organic macromolecules extracted from the crossed lamellar layers induced the formation of only aragonite crystals from solutions containing Ca/Mg ion ratio of 1/2.5 within 6 h, and that the morphology and the crystallographic orientation of the synthetic crystals are the same as those of the biogenic crystals. In the absence of matrix macromolecules, both calcite and aragonite crystals formed. These observations provide insights into the early crystal nucleation and growth events of aragonite crystals in the formation of the crossed lamellar microstructure.

’ INTRODUCTION Mollusk shells are composed of calcium carbonate, in the form of calcite or aragonite, within a matrix framework of organic macromolecules. The mineral constitutes 95 99% of the shell weight and the organic matrix material 0.01 5 wt %.1 The organic material is composed of proteins and polysaccharides, which are thought to provide the microenvironment where mineral deposition occurs. These macromolecules, some of which are unusually acidic, are also occluded inside the mineral phase, where they presumably exert direct control over crystal nucleation, crystal growth, polymorph type, and morphology.2 4 The crossed lamellar microstructure originally identified by Bøggild5 7 is formed by bivalves, gastropods, scaphopods, and polyplacophorans and is the most common type of mollusk shell microstructure. The crossed lamellar microstructure has a complicated hierarchical structure with first-, second-, and third-order lamellae (Figures 1a and b).6 8 The third order lamellae are composed of aragonite crystals up to several micrometers in length and about 100 200 nm in cross-section. In contrast to nonbiogenic aragonite, which typically grows as needles elongated along the fast-growing c axis, and to the large aragonite plates of nacre that develop in the ab plane, the crystals in the crossed lamellar microstructure have a variable orientation in different species but are almost always elongated in a direction approximately perpendicular to the crystallographic c-axis.9 12 It r 2011 American Chemical Society

has been proposed that the much smaller building blocks within the crystal (100 nm thick, 200 300 nm wide and 100 200 nm long), possibly created by frequent (110) twinning, are a further subdivision of the third lamellae crystals into fourth-order lamellae.13,14 The thin prisms of the third-order lamellae are aligned parallel to each other to form plates (second-order lamellae) inclined to the shell surface. The inclined plates are in turn stacked to form thick blocks, which may be hundreds of micrometers long (firstorder lamellae). These blocks are arranged in layers where the planes of the lamellae are rotated in orientation (Figures 1a and b). Previous studies of the organic matrix of the crossed lamellar microstructure show that it contains the smallest amounts of organic molecules compared to other shell microstructures, such as nacre.13,15,16 The third-order lamellae are surrounded by thin layers of organic matrix, and thicker layers of organic matrix are also present between the layers of second-order lamellae.15 Peak shape analysis using infrared spectroscopy17,18 shows that the aragonite crystals forming the third-order lamellae of the crossed lamellar microstructure are well-ordered compared to the aragonite crystals of nacre, prismatic microstructure, or the aragonite crystals in bivalve hinge ligaments.19 The complex architecture of Received: May 24, 2011 Revised: September 5, 2011 Published: September 07, 2011 4850

dx.doi.org/10.1021/cg2010997 | Cryst. Growth Des. 2011, 11, 4850–4859

Crystal Growth & Design the crossed lamellar microstructure endows the material with exceptional mechanical properties.20,21 All these intriguing properties make the understanding of the formation mechanism of crossed lamellar crystals of much interest. This understanding may also lead to the design of tough, lightweight structures in materials engineering. We adopted two different approaches to better understand the formation mechanisms of aragonite crystals in the crossed

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lamellar microstructure. We examined the newly formed shell growth layer with cryo-SEM to determine the nature of the first formed crystals and whether or not a precursor mineral phase exists at the shell-tissue interface. In the second approach, we extracted the organic matrix from the crossed lamellar microstructure and tested its function using in vitro crystallization experiments. We studied the crossed lamellar layers formed by three species of limpets: Lottia gigantea, Lottia dorsuosa, and Cellana rota.12,22,23 The limpet shell has five laminated layers.These layers are divided into outer and inner layers that are separated by the myostracum, a thin shell layer formed by the cells responsible for adhering the muscle to the shell.22 The position of each layer is defined by its location relative to the myostracum (abbreviated as M).22 The outer layers are referred to as M + 1, M + 2, M + 3, from the inside to the outside, and likewise the inner layer on the other side of the myostracum is referred to as M 1. Most limpets, and among those the species studied here, have the crossed lamellar microstructure in M + 1 and M 1 (Figure 1c). The orientation of the third-order lamellar crystals in the limpet species Lottia kogamogai, and presumably also in the limpet species studied here, was found to be along the (110) twinning direction, with the c axis almost perpendicular to the plates.12

’ EXPERIMENTAL SECTION Figure 1. (a) Schematic representation of the hierarchical organization of the crossed lamellar microstructure (modified from ref 12). (b) SEM image of the crossed lamellar microstructure from C. rota. White rectangles show the first-, second-, and third-order lamellae. (c) Internal surface of the shell of C. rota. M shows the location of the myostracal layer that is attached to the adductor muscle of the soft body. M + 1 and M 1 layers have the crossed lamellar microstructure.

Sample Preparation. Fresh specimens of Cellana rota were collected along the Mediterranean coast of Israel (Haifa beach). The living individuals were kept in a small amount of seawater at 4 °C to prepare fresh samples for cryo-SEM. Specimens of Lottia dorsuosa were collected in Jogashima Island, Kanagawa-Pref., Japan. Specimens of Lottia gigantea were collected in Santa Barbara, U.S.A. After capture, the soft parts of the latter two species were mechanically removed and the shells were washed with pure water before drying. The dried shells were

Figure 2. Cryo-SEM images of fracture surfaces of the crossed lamellar microstructure of C. rota. Samples were high-pressure frozen and fractured at 120 °C, and the ice was etched for 10 min at 105 °C. (a) The growing surface of the crossed lamellar microstructure. White arrow indicates the granular layer. Inset: high magnification image of the granular layer. Asterisk indicates the 1-hexadecene layer that covers the sample. (b) In-lens backscattered electron images of the same area shown in (a). The bright contrast shows the mineralized crossed lamellar layer and thin overlying granular layer, and the dark contrast shows the 1-hexadecene layer. (c) High magnification image of the granular layer. (d) Fracture along a plane forming a shallow angle to the shell internal surface. White arrow indicates a crystal formed inside the granular layer. 4851

dx.doi.org/10.1021/cg2010997 |Cryst. Growth Des. 2011, 11, 4850–4859

Crystal Growth & Design

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Figure 3. (a, b, c) High magnification images of representative area (white rectangles indicate the areas of 300 300 nm) where particle counts were performed in the granular layer, at the surface (a), middle (b) and at the boundary with the crossed lamellar layer (c). (d) Particle numbers counted in each region (10 areas taken from at least two different individuals in each region). transported to the laboratory at room temperature. The shell of L. dorsuosa and L. gigantea were used for amino acid composition analysis and SDS-PAGE. Cryo-SEM. Before preparation for cryo-SEM, the outer layer of C. rota shells (at least eight individuals) was mechanically cleaned. After carefully removing the soft body parts without damaging the inner surface of the shell, small pieces of shell were cut out from the inner side of the fresh shells and were mounted on an aluminum platelet with a cavity 200 μm deep, filled with 1-hexadecene (Sigma). The samples were then cryo-immobilized in an HPM 10 high pressure freezing device (BalTec). The frozen samples were mounted on a holder and transferred to a BAF 60 FF device (Bal-Tec) using a VCT 100 Vacuum Cryo Transfer device (Bal-Tec). Frozen samples were fractured at a temperature of 120 °C, etched for 5 min at 105 °C and coated with 4 nm Pt/C by double axis rotary shadowing. Samples were transferred to an Ultra 55 SEM using the VCT 100 Cryo transfer device and were observed using secondary electron and backscattered electron in-lens detectors at 120 °C. The backscattered electron in-lens detector was operated at 2 kV with an ESB grid voltage of 300 V. Stronger signals in the backscattered

images are obtained from the higher-atomic-mass elements, that is, in this case the calcium atoms within the mineral phase, providing information regarding the distribution of the mineral relative to the organic material. Extraction of Organic Matrix. The outer layers of limpet shells were polished and removed by drilling leaving only the inner crossed lamellar microstructure. The crossed lamellar part (2 g) was ground in an agate mortar. The mineral was dissolved following the method of Albeck et al.24 and Gotliv et al.25 The fine powder was transferred into a dialysis tube (Spectrapor 3 tubing with a molecular weight cutoff of 16 000 Da, diameter 14 mm). The dialysis bag was placed in a glass cylinder (100 mL) with the two ends of the bag fastened to rubber stoppers. Dowex 50 8 cation-exchange resin (Sigma, H+ form, mesh 50 100; 20 g) prewashed with DDW was placed in the tube and DDW was added to fill the tube. The tube was continuously rotated (30 rpm) in a propeller-like mode at room temperature to keep the resin and mineral in suspension. The DDW was changed daily and the gas, which accumulated inside the dialysis bag was removed daily. The decalcification of 2 g of mineral takes 7 10 days. After complete decalcification, 4852



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Figure 4. Cryo-SEM images of fracture surfaces of the crossed lamellar microstructure of C. rota. (a) The granular layer is not continuous and pinches out (arrowhead). Asterisk indicates the location of the 1-hexadecene layer that covers the sample. (b) Higher magnification of the inner surface of the granular layer and of the juxtaposed crossed lamellar layer. Organic material is visible covering the lamellae (white arrows).

Table 1. Amino Acid Composition Analyses L. dorsuosa residue %

L. gigantea residue %

Asx

16.0

14.7

Ser

7.3

7.6

Glx

9.2

10.1

Gly

15.3

11.4

His

1.0

1.3

Arg

4.6

3.6

Thr Ala

6.8 9.3

12.0 8.7

Pro

4.5

5.1

Tyr

2.4

2.8

Val

5.5

5.3

Met

0.0

1.3

Lys

4.7

4.0

Ile

4.1

3.7

Leu Phe

6.3 3.1

5.5 3.0

the contents of the dialysis bag were extensively dialyzed against DDW and the soluble and insoluble materials were separated by centrifugation

Figure 5. Photograph of an SDS-PAGE gel of soluble organic matrix extracts from the crossed lamellar layer obtained using resin decalcification. The macromolecules were separated by SDS-PAGE and visualized by silver stain. Molecular-mass standards with masses in kDa are indicated on the left. (a) Extract from the crossed lamellar microstructure of L. gigantea. (b) Extract from the crossed lamellar microstructure of L. dorsuosa. at 3000 g for 10 min. The soluble material was concentrated by ultrafiltration (Ultrafree with a molecular weight cutoff of 10 000 Da, Millipore) and desalted using acetone precipitation. After acetone precipitation, the pellet was dried and solubilized in 20 mM Tris-HCl (pH 8.0) buffer. Amino Acid Composition Analysis. Aliquots of the protein solution desalted with acetone precipitation were lyophilized and hydrolyzed under vacuum in 6 N HCl (0.3 mL) at 112 °C for 24 h after flushing twice with nitrogen. Following evaporation of the HCl the hydrolysates were analyzed on a Dionex BIOLC amino acid analyzer with ninhydrin detection.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The soluble extracts from the crossed lamellar microstructure of the shell were subjected to SDS-PAGE (15% gel) under reducing conditions. After electrophoresis, the gel was stained with silver stain reagent (Wako). Calcium Carbonate Crystallization in Vitro. Synthetic crystals were grown in Nunc multiwell dishes (24 wells; 1.5 cm diameter) by diffusion of ammonium carbonate vapor into calcium chloride solutions. Siliconized glass slides (1.2 cm diameter, Hampton Research) were placed on the bottom of each well. A drop (0.05 mL) of a solution containing 20 mM CaCl2 (Merck) and 50 mM MgCl2 (Merck) was deposited on the siliconized glass slide. The sample solutions containing the soluble organic matrix from the crossed lamellar microstructure were added to the drops to reach a final concentration of 0.3 μg/mL. The 4 center wells were used for samples, the 8 wells around the center wells were filled with ammonium carbonate powder (total 2 g), and the 4 corner wells were filled with water (2 mL in each well) to maintain the 4853



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Figure 6. Images obtained through a binocular microscope monitoring the development of calcium carbonate deposition for 24 h in drops containing 20 mM CaCl2 and 50 mM MgCl2 solution. (a) Drop without organic macromolecules from the crossed lamellar microstructure. (b) Drop with organic macromolecules from the crossed lamellar microstructure (0.3 μg/mL). The times from the onset of the experiment are shown on the left-hand side of each image. The glass slides in the field of view have a diameter of 12 mm. humidity in the multiwell dish. Control experiments were performed in parallel in specimen samples containing only the same volume of 20 mM Tris-HCl buffer (pH 8.0) instead of the soluble organic matrix sample. The multiwell dishes were covered with their original lid, sealed with Parafilm and stored at room temperature for 24 h. The crystallization processes in the multiwell dishes were viewed using a stereoscopic microscope. At the end of the experiment, the precipitates formed on the glass slide were washed with water and ethanol, air-dried, and used for TEM, SEM observations, and IR measurements. TEM. Precipitates were suspended in ethanol and poured on a TEM carbon grid. The dried grids were observed using an FEI (Philips) T120Technai TEM operating at 120 kV. The images were recorded in a CCD camera (Gatan ESW-500 W) as digitized images. Focused ion beam (FIB): A Hitachi FB-2100 FIB was used to cut electron-transparent cross sections from synthetic precipitate aggregates. The cross sections were transferred to a JEOL JEM-2010 TEM operating at 200 kV for imaging and electron diffraction. IR. A few hundred μg of powder from the samples were mixed and homogenized with about 40 mg of KBr in a mortar. The mixed samples were pressed into a 7 mm pellet using a manual hydraulic press (Specac). IR spectra were obtained at 4 cm 1 resolution after 32 scan collection using a Nicolet 380 instrument (Thermo).

’ RESULTS Cryo-SEM Observations. We adapted the method of Nudelman et al.26 to examine the forming surface of the limpet crossed lamellar layer, while preserving hydrated physiological conditions. The sample preparation procedure involved sandwiching the sample in a nonpenetrating medium (1-hexadecene) that operates as a filler and cryo-protectant. After high pressure freezing of the embedded sample, fresh fracture surfaces were exposed by freeze fracture. Rapid freezing ensures the preservation of the growth front, while minimizing artifacts due to sample preparation. Figure 2 shows images of cross sections of the crossed lamellar layer in cryo-SEM. In at least five different samples taken from different shells, a granular layer was observed in the central part of the growth surface, overlying the typical prisms of the crossed lamellar microstructure (Figure 2a). This layer was never observed

using conventional SEM with or without fixation treatment. When we observed the same areas with backscattered electrons, the granular layers had the same contrast as the cross lamellar microstructure and both had a much higher contrast than the overlying hexadecene, showing that the granular layer is mineralized (Figure 2b). The granular layer has a maximum thickness of about 5 μm and consists of particles ∼10 100 nm in size that appear to gradually increase in size approaching the boundary between the crossed lamellar and granular layers (Figure 2a inset, Figure 2c). To quantify this trend, we counted the number of particles in 5 areas of 300 nm 300 nm each at the surface, in the middle and in the boundary regions close to the mature crossed lamellar layer (Figure 3a c). The particle number/unit area at the surface is twice the number in the boundary region (Figure 3d). From the data in Figure 3d, we deduce that the average size of the particles increases progressively from around 40 nm at the shell surface to 65 nm at the interface with the mature crossed lamellar layer. The larger granules are clearly not spherical, but rather have angular shapes and sharp edges. At the interface, some particles are larger than 100 nm. Furthermore, the interface is not always well-defined, and in several regions the particles appear to merge smoothly into the crossed lamellar layer (Figure 2c). Observations made on a fracture plane intersecting the surface at a shallow angle (Figure 2d) show tightly packed granules, with lamellar plates interleaved among them, suggesting that these nascent lamellar plates grow out of the smaller particles within the granular layer. Significantly the granular layer is not present along the entire inner surface of the shell (Figure 4a). This is consistent with the fact that the mantle cells do not deposit a continuous growth layer at any given time over the whole inner shell surface. The observation of a granular layer in all specimens examined, but not covering the whole surface, indicates that this layer is not a permanent mature feature of the shell, but represents a transition phase to the mature crossed lamellar layer. In the regions where no granular layer is observed, the crossed lamellar layer appears to be fully developed. The hydrated unperturbed conditions of the cryo-SEM enabled the observation of the organic matrix covering the crossed lamellar platelets (Figure 4b) that is impossible to observe using conventional SEM. 4854



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Figure 7. TEM images and electron diffraction patterns of the precipitates from the drops shown in figure 6. (a) Precipitated material after 20 min in the absence of macromolecules (Figure 6a); (b) Electron diffraction pattern of (a): the lack of sharp reflections indicates that the precipitate is amorphous. (c) The precipitate formed after 6 h in the absence of macromolecules; (d) Electron diffraction pattern from (c). (e) The precipitate after 20 min in the sample with macromolecules from the crossed lamellar microstructure (Figure 6b); (f) Electron diffraction pattern from (e). (g) The spindle-shaped crystalline body covered by needles that formed after 6 h in the sample with organic macromolecules; (h) Electron diffraction pattern from (g). The diffraction pattern is compared to the reference diffraction pattern of aragonite crystals (upper right-hand quadrant). 4855



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Figure 8. SEM images of the precipitate extracted from the drops shown in Figure 6. (a) The precipitate after 6 h in the sample without macromolecules. (b) The precipitate after 24 h in the sample without macromolecules. Insert left bottom: some calcite crystals also formed in this sample. (c) The spindlelike crystals that formed after 6 h in the presence organic macromolecules; (d) after 24 h with macromolecules.

Extraction and Analyses of Organic Macromolecules. The total ensemble of intracrystalline and intercrystalline macromolecules was extracted from the crossed lamellar layer of L. gigantea and L. dorsuosa to better understand the function of the organic matrix macromolecules in the crossed lamellar microstructure. Amino acid composition analysis shows that the soluble matrix proteins from L. gigantea contain a relatively high percentage of Asx (14.7%), Ser (7.6%), and Thr (12.0%) residues (Table 1). L. dorsuosa also contains relatively high Asx (16.0%), Ser (7.3%), and Thr (6.8%). The total amounts of water-soluble matrix proteins in L. gigantea and L. dorsuosa are extremely low, namely, approximately 1.6 μg/g (0.0016 wt %) and 1.1 μg/g (0.0011 wt %) of the mineral in dried shells, respectively. When analyzed by SDS-PAGE with silver staining (Figure 5), the extracts showed three major bands (around 100, 60, and 40 kDa) in L. gigantea. Three high molecular weight bands (around 100, 72, and 60 kDa) were also detected after silver staining in L. dorsuosa. In Vitro Calcium Carbonate Crystallization Experiments. The effect of the organic macromolecules extracted from the crossed lamellar microstructure of L. gigantea on aragonite crystallization was investigated by performing in vitro crystallization experiments. 50 μL drops of solutions containing a ratio of Ca/Mg ions of 1/2.5, were deposited on silicon coated glass slides and incubated in sealed well plates with ammonium carbonate powder. In reference solutions, the presence of Mg is known to favor the formation of aragonite at ratios of Ca/Mg ions

Formation of Aragonite Crystals in the Crossed Lamellar

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