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Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase

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Science  12 Nov 2004:
Vol. 306, Issue 5699, pp. 1161-1164
DOI: 10.1126/science.1102289

Abstract

The skeletons of adult echinoderms comprise large single crystals of calcite with smooth convoluted fenestrated morphologies, raising many questions about how they form. By using water etching, infrared spectroscopy, electron diffraction, and environmental scanning electron microscopy, we show that sea urchin spine regeneration proceeds via the initial deposition of amorphous calcium carbonate. Because most echinoderms produce the same type of skeletal material, they probably all use this same mechanism. Deposition of transient amorphous phases as a strategy for producing single crystals with complex morphology may have interesting implications for the development of sophisticated materials.

Many organisms—including mollusks, echinoderms, calcisponges, corals, certain algae, and others—form their hard parts out of calcium carbonate minerals (1, 2). The echinoderms, which include sea urchins, sea stars, and brittle stars, among others, are among the few groups that form skeletal parts consisting of large single crystals of Mg-bearing calcite, some of which are several centimeters long (3, 4). These single crystals usually have smooth, continuously curved surfaces that form a three-dimensional fenestrated mineral network surrounding micrometer-sized spaces that are occupied by living cellular tissue. This so-called stereom appears to be an “all purpose material,”used by members of the whole phylum (5). It is molded into different shapes and sizes and is thus adapted to fulfill different functions.

The strategy evolved by echinoderms to build their spines, test plates, and ossicles has been the subject of study for more than a hundred years (6, 7). The crystals form inside a syncytium (8), a membrane envelope produced by many cells. The cells provide the raw materials necessary for constructing the growing single crystals (9, 10). A key question is how a single crystal of calcite can be molded into such convoluted shapes, when inorganically formed calcite crystals are rhombohedra with flat crystal faces. Another perplexing issue is how the cells can efficiently provide the ions and, at the same time, efficiently remove water, during and after crystal deposition. This question becomes critical if it is assumed that the crystal grows out of an aqueous solution saturated with calcium carbonate (11).

Insights into these questions have been gained from studies of calcitic spicule formation in sea urchin larvae (12). The larval spicules grow on a single calcite crystal seed by transformation of a transient amorphous calcium carbonate (ACC) phase (13). ACC is apparently fed into the syncytium by cells in the form of ACC-containing vesicles (14). Thus, packages of ACC are delivered to the crystal deposition site and then transform in a controlled manner into calcite single crystals. There is also no discernible aqueous phase around the growing spicule (14). Mollusk larvae also form their shells via an ACC precursor phase, which then transforms into aragonite (15). Because this strategy may be a unique feature of larvae adapted to a swimming mode of life in the oceans, it was important to determine whether or not adult echinoderms or mollusks also use transient ACC for forming their hard parts. We show that adult sea urchins do build their regenerating spines via the deposition of an amorphous ACC precursor phase.

Sea urchins are able to regenerate spines that break. Spine regeneration first involves a wound-healing phase during which the epidermis is reconstituted around the broken spine (16, 17). Within this space, the skeleton-forming sklerocytes build a new syncytium, which is in contact with the stump of the old spine. The new crystal is presumably nucleated epitaxially on the old spine (18), because the regenerated and old spines together diffract x-rays as one single crystal. The regeneration process is thus considered to be very similar to the original spine growth. Regeneration starts in the form of small projections emerging from the broken stereom surface. These microspines initially grow parallel to the mature spine long axis, which is also the c crystallographic axis of calcite, and then form the complex three-dimensional fenestrated structure (19) (Fig. 1, A and B). As the process is initiated with the break, a real-time investigation of rapid skeleton deposition is possible. We used etching in water, infrared spectroscopy, electron diffraction, and environmental scanning electron microscopy (ESEM) to investigate the possible involvement of ACC in spine regeneration of the sea urchin Paracentrotus lividus.

Fig. 1.

Scanning electron micrographs of regenerating spines. (A) Five-day-old regenerated spine growing on the original broken spine. (B) Higher magnification view of the tip of the new growth, showing the typical stereom structure and the protruding newly formed microspines. (C) One microspine formed after 4 days of regeneration, observed fresh. (D) Four-day-old microspine, etched in water while fresh. (E) Four-day-old microspine, etched in water 1 month after regeneration. All the spines were observed after removal of the exposed organic material by a 3% NaOCl solution. Etching was performed by immersing cleaned spines in double distilled water for 12 hours.

The solubility of ACC (200 mg/liter) is 30 times greater than the solubility of calcite (6.7 mg/liter) (20, 21). Etching in water thus reveals the presence of ACC, which is selectively dissolved out of a mixture of ACC and calcite. Four-day-old regenerated microspines were etched in water immediately after severing from the spine (22). Extensive etching occurred in a thin (100 to 200 nm thick) surface layer (Fig. 1D). These etched surfaces were compared to regenerated microspines that were left at room temperature in air for 1 month and then etched. The latter were not affected by the treatment (Fig. 1E) and closely resembled the untreated spines (Fig. 1C).

Fourier transform infrared (FTIR) was used to further investigate the nature of the thin surface layer (Fig. 2, A and C). The characteristic broadening of the in-plane carbonate bending peak at 713 cm–1, relative to the out-of-plane bending peak at 876 cm–1, is well documented for ACC-containing sea urchin larval spicules (13). Regenerated spines, cleaned of organic matrix, were dried in ethanol and sonicated into acetone after rapid freeze shock treatment in liquid nitrogen. Sonication detached small particles from the surface of the microspines. The infrared spectrum of these particles shows a sharp peak at 876 cm–1, whereas the 713 cm–1 peak comprises a sharp peak superimposed on a very broad peak (Fig. 2A, inset). The spectrum of crystalline calcite taken from the mature part of the same spines (Fig. 2B) was then subtracted from the latter. The residual spectrum still shows a sharp out-of-plane bending peak at 876 cm–1, but the 713 cm–1 peak consists only of a broad hump (Fig. 2C), clearly indicative of the presence of ACC. A broad peak at ∼3500 cm–1 is also enhanced in the subtracted spectrum, relative to calcite. This may be due to water molecules that stabilize the amorphous phase and are intimately associated with it. The presence of some water also in the mature crystalline part of the spine indicates, however, that this may be interstitial water or water associated with residual organic matrix.

Fig. 2.

FTIR spectra of particles removed from fresh regenerated spines by freeze shock and sonication. The intensities are normalized to the carbonate stretching peak at 1420 cm–1. (A) IR spectrum of the freshly removed particles. The spectrum corresponds to that of a mixture of ACC and calcite (ratio of intensities I876/I713 = 4.25). (B) IR spectrum of material removed from the mature part of the spines. The spectrum corresponds to that of crystalline calcite (ratio of intensities I876/I713 = 3). (C) IR spectrum of the particles in (A), after subtraction of the spectrum of crystalline calcite from the old part of the spine [taken from (B)]. The spectrum corresponds to that of ACC.

Particles removed from regenerating spines by the same procedure as described above were dispersed on an electron microscope grid and examined by electron imaging and diffraction (Fig. 3) (22). The particles are ∼50 nm in size. Particles obtained from fresh regenerated spines did not produce an electron diffraction pattern (Fig. 3A). The same particles, examined after 3 weeks on the grid and kept in air, were crystalline and produced a diffraction pattern typical of calcite (Fig. 3B).

Fig. 3.

Transmission electron micrographs (TEM) and electron diffraction patterns of particles removed from fresh regenerated spines. (A) Electron diffraction pattern of the particles observed immediately after removal from the spine; the diffuse rings indicate the presence of an amorphous material. (Inset) TEM of the particles. Bar, 50 nm. (B) Electron diffraction pattern of the same particles as in (A) after 3 weeks on the grid. The most prevalent diffraction peaks correspond to a d-spacing of 3.04 Å, characteristic of the calcite plane {104}. They are detected up to fourth order (arrows, enhanced contrast in window). (Inset) TEM of the particles. Bar, 50 nm. Fresh regenerated spines were subjected to freeze shock and sonication. The separated material was mounted and examined on a marked TEM grid, and the position of the particles was recorded. The same particles were examined again after 3 weeks.

Etching, IR spectroscopy, and transmission electron microscopy thus provide independent evidence that the regenerated microspines are covered by a thin layer of ACC that, with time, transforms into crystalline calcite. The crystallization process occurs both in situ and in particles removed from the spine.

We were also able to directly observe the crystallization of the amorphous sheath and tip of the microspines into calcite in a wet atmosphere, using ESEM (Fig. 4) (22). The electron beam provides the energy required to induce crystallization. The tip of the microspine first undergoes a reduction in volume (Fig. 4A; compare with Fig. 4, B and C). A single crystal is then observed to form as thin plates with well-defined edges (Fig. 4, D and E). The delimiting faces subtend, between them and with the axis of the microspine, dihedral angles matching those expected for the (001), (102), and (010) faces of a calcite crystal oriented with the c axis parallel to the long axis of the spine. The crystal then grows at the expense of the amorphous material that is consumed in the electron-irradiated region (Fig. 4, F and G), whereas the nonirradiated region remains unmodified (Fig. 4, C and H). When the same procedure is performed on old regenerated spines, or on an old part of the same spine, no such changes are observed, and the spine preserves its dimensions and appearance.

Fig. 4.

Environmental scanning electron micrographs (ESEM) of a fresh, newly formed microspine taken in succession over a period of ∼15 min. (A) Microspine at the beginning of the observation. (B to H) Pictures taken in succession every 2 to 5 min at the same magnification. Between (B) and (C) and between (G) and (H), the viewing frame was moved along the axis of the microspine to compare the condition of a nonirradiated region of the microspine relative to the irradiated region. Lines in (C) and (H) show the boundaries of the previous frames. Arrows in (D) show the growing crystal faces. Regenerated fresh spines were cleaned from epidermal tissue by NaOCl treatment, washed with water, and immediately transferred wet to an ESEM stub, where they were observed under a humid atmosphere (pH2O = 6.6 torr, 5°C, 10 kV).

The reduction in spine volume upon crystallization may indicate expulsion of water, which would be in agreement with the IR data (Fig. 2). This is interesting given that stable ACC contains 15 weight % water, corresponding to the molar composition CaCO3·H2O (23). In contrast, transient ACC in sea urchin larval spicules contains little or no water (24). If the same mechanism is also valid for the growth of adult spines, this would imply that calcium carbonate is first deposited as hydrated ACC, and then dehydrates prior to or concomitant with crystallization. The shrinkage might, however, also be associated with reorganization of the material into a crystalline lattice with or without concomitant expulsion of interstitial water. Irrespective of the mechanism of ACC-to-calcite transition, the introduction and deposition of calcium carbonate as ACC may well provide some answers to the perplexing questions raised above; namely, how the spine is shaped, how the ions are efficiently introduced, and how water is expelled. ACC is introduced into the syncytium as an isotropic noncrystalline solid and can thus be molded into any shape. The solid itself is, of course, a very concentrated source of ions. Water has to be expelled in only small (at most 15%) amounts. This is orders of magnitude lower than if the crystal were deposited directly from solution. The subsequent transformation of the amorphous phase into a composite crystalline solid with much better mechanical properties (25) leads to a functional skeleton. A similar strategy could be used to form synthetic materials with minimal porosity and maximal shape flexibility and has been explored in vitro (2629).

Our investigation of the regeneration of the adult sea urchin spine shows that the skeletal hard part forms through an amorphous precursor phase. Most members of this phylum form the same type of skeletal material (2), the stereom, with similar properties: large single crystals of magnesium-containing calcite, which break with conchoidal fracture and are reinforced by intracrystalline organic matrix. We therefore suggest that all echinoderms probably use this mineral-formation process. It is conceivable that many other animal phyla also use the same strategy. Isolated examples have been demonstrated involving, other than ACC, amorphous ferric oxide and amorphous calcium phosphate [table 3.2 in (2)]. With the discovery that this process is used by adult echinoderms, the possibility that it is a widespread strategy seems likely. Furthermore, this strategy of molding macroscopic elements into any desired morphology and endowing them with the strength and the degree of perfection of a single crystal, but with the reduced brittleness and mechanical properties of a glass, may have interesting implications for the development of sophisticated materials.

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www.sciencemag.org/cgi/content/full/306/5699/1161/DC1

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