The key role of the surface membrane in why gastropod nacre grows in towers

Antonio G. Checaa,1, Julyan H. E. Cartwrightb, and Marc-Georg Willingerc

aDepartamento de Estratigrafía y Paleontología, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain; bInstituto Andaluz de Ciencias de la Tierra, Consejo Superior de Investigaciones Cientificas–Universidad de Granada, Campus Fuentenueva, E-18071 Granada, Spain; and cDepartamento de Química, Centro de Investigac¸a˜ o em Materiais Ceraˆmicos e Compositos, Campus Universitario de Santiago, Universidade de Aveiro, 3810- 193 Aveiro, Portugal

Edited by Steven M. Stanley, University of Hawaii, Honolulu, HI, and approved November 25, 2008 (received for review September 4, 2008) The nacre of gastropod molluscs is intriguingly stacked in towers. in a step-like manner (7, 9). The nacre thus produced is said to It is covered by a surface membrane, which protects the growing have a terraced arrangement (Fig. 1A). nacre surface from damage when the withdraws into its In gastropods, however, the biomineralization compartment shell. The surface membrane is supplied by vesicles that adhere to of nacre is enclosed by a surface membrane first reported by it on its mantle side and secretes interlamellar membranes from the Nakahara (8) in Monodonta and Haliotis. Since its discovery, its nacre side. Nacre tablets rapidly grow in height and later expand existence went unremarked, until Cartwright and Checa (10) sideways; the part of the tablet formed during this initial growth realized that it is widespread in nacre-secreting gastropods and phase is here called the core. During initial growth, the tips of the that the interlamellar membranes must necessarily detach from cores remain permanently submerged within the surface mem- it. The surface membrane acts as a protective seal, which brane. The interlamellar membranes, which otherwise separate the prevents the organic compounds and minerals involved in nacre nacre tablet lamellae, do not extend across cores, which are aligned growth from being lost to the external environment when the soft in stacked tablets forming the tower axis, and thus towers of nacre body of the gastropod withdraws into its shell, something evi- tablets are continuous along the central axis. We hypothesize that dently not necessary with bivalves. Below the surface membrane, in gastropod nacre growth core formation precedes that of the many parallel interlamellar membranes with tablets growing interlamellar membrane. Once the core is complete, a new inter- between them can be found. These tablets are typically stacked lamellar membrane, which covers the area of the tablet outside the in towers (Fig. 1B), with the smaller, more recently begun tablets core, detaches from the surface membrane. In this way, the found at the top. Although the nacre of gastropods, in particular tower-like growth of gastropod nacre becomes comprehensible. that of the abalone, i.e., the genus Haliotis, has been intensively studied, there are still many pieces to be assembled in the puzzle. biomineralization ͉ molluscs ͉ organic membranes ͉ epitaxy One, perhaps key piece, is the surface membrane, key both because it is intimately related to the other components of nacre acre is by far the most intensively studied non-human and because the mineral ions and organic molecules for nacre Norgano–mineral biocomposite. It has a high proportion, growth are necessarily introduced into the biomineralization Ϸ 5%, of organic matter (proteins and polysaccharides; ref 1), the compartment through it. Its ultrastructure, growth, and secre- mineral fraction being exclusively in the form of aragonite. tional activity have never been elucidated. Jackson et al. (2) estimated that its work of fracture is 3,000 times This work is dedicated to determining the relationship of the higher than that of inorganic aragonite, although later estimates surface membrane to the interlamellar membranes and mineral reduce this figure considerably (see the review in ref. 3). Its tablets. Our conclusions shed light not only on the dynamics of superior biomechanical properties, together with its interest to gastropod nacre growth but also bear on the present debate the pearl industry and its possible biomedical uses (see e.g., ref. about whether superimposed nacre tablets nucleate and grow 4), make nacre the subject of many biomimetic studies. An onto the organic interlamellar matrix or, alternatively, whether ultimate aim of such work is to mimic nacre in the laboratory, there is crystallographic continuity between them across the following the biological principles used by molluscs to produce interlamellar membranes. such a biomaterial (5). It is sine qua non for this objective to have a complete understanding of the mechanisms involved in nacre Results growth. Surface Membrane. The surface membrane extends between the Nacre is secreted only by the molluscan classes , adoral and apical boundaries of the nacreous layer, usually Bivalvia, Cephalopoda, and, to a minor extent, Tryblidiida. It has bounded by the external spherulithic layer and an internal a lamellar structure consisting of alternating tablets of aragonite aragonitic lamellar layer of uncertain microstructure (Fig. 2A). 300–500 nm thick and 5–15 ␮m wide and organic interlamellar In Gibbula and Monodonta, at least, its mantle-side surface is membranes Ϸ30 nm thick, which have a core of ␤-chitin sur- dotted with bodies adhering to it (Fig. 2 A and F). In transmission rounded by acidic proteins (6). It is now clear that the sequence electron microscopy (TEM) sections these structures are seen to of nacre formation involves the secretion of interlamellar mem- be hollow (Figs. 2 B and C and 3C) and thus may be called branes (7) separated by a liquid rich in silk fibroin (5); only subsequently is the liquid replaced with mineral (7–9). This Author contributions: A.G.C. and J.H.E.C. designed research; A.G.C., J.H.E.C., and M.-G.W. pattern is the same for the bivalves and gastropods, and it is likely performed research; A.G.C. and M.-G.W. analyzed data; and A.G.C. wrote the paper. so too for the other nacre-secreting molluscs, although this is yet The authors declare no conflict of interest. to be determined. There are, however, structural differences This article is a PNAS Direct Submission. between bivalve and gastropod nacre. In the former group, the 1To whom correspondence should be addressed. E-mail: [email protected]. interlamellar membranes are secreted with just the liquid-filled This article contains supporting information online at www.pnas.org/cgi/content/full/ extrapallial space between them and the cells of the mantle 0808796106/DCSupplemental. epithelium, and mineralization within the membranes proceeds © 2008 by The National Academy of Sciences of the USA

38–43 ͉ PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808796106 Downloaded by guest on October 1, 2021 membrane and the surface membrane and partly encased within A it (Fig. 2 D and E). Where tablets have been torn off upon contraction of the membranes during sample preparation, the scars remaining can also be seen (Fig. 2D). The width of these cores is estimated to be between 100 and 200 nm (Figs. 2 E and F, and 3 A, C, and D). The topographical relationship observed in SEM samples is so recurrent that the possibility that this is artifactual can be excluded. The relationship is further demon- strated by TEM, which reveals that the tip of a growing tablet (i.e., the last 50–70 nm) is directly embedded within the surface membrane (Fig. 3 A, C, and D). In the few instances when towers are fortuitously sectioned exactly through their central axis, the interlamellar membrane possesses a fuzzy appearance or is totally absent. The disappear- ance of the interlamellar membrane at the very axes of the towers is evident in some exceptional TEM views (Fig. 3). The partial dissolution sometimes produced during sample preparation (Fig. 3 D and E) does not affect the tower axis area. TEM views of decalcified towers of Gibbula umbilicalis show too that the interlamellar membranes are missing at the very axes of the B towers, across a maximal width of Ϸ100 nm, or are replaced by a fuzzy band of organic matter with a different orientation (Fig. 4A). SEM observation in back-scattered electron (BSE) mode of polished axial sections of nacre towers of the same species, in which we can safely assume that membranes have not been disturbed during sample preparation, manifests that the same effect may take place across tens of tablets in a tower (Fig. 4B). The fuzzy band, when present, usually curves slightly toward the top of the tower. When the same samples are decalcified with methanolic solution, the axes of the towers are marked by a succession of holes (Ϸ150 nm wide) with coarsened rims, sometimes traversed by organic threads (Fig. 4 C and D). The regularity and persistence of such structures exclude the possi- bility that they are artifacts caused by dissolution. Treatment with 2% EDTA preferentially removes the calcified organic-rich components: the interlamellar organic membranes, the lateral boundaries between tablets and, interestingly, the parts of the Fig. 1. Bivalve and gastropod nacre growth compared. (A) Oblique view of tablets coinciding with the axes of towers [supporting informa- the terraced nacre of the bivalve P. margaritifera.(B) Oblique view of the tion (SI) Fig. S1]. towered nacre of the gastropod Perotrochus caledonicus. High-resolution TEM observations show that the interface between two superimposed tablets is fully crystalline at the axis (Fig. 5A). Observation of lattice fringes and fast Fourier trans- vesicles. They vary in shape from spherical, when they are just form (FFT) analysis of small areas provide additional evidence touching the surface membrane, to strongly compressed, when of this crystalline character. The patterns obtained, although they are partially or wholly integrated into the surface membrane indicative of nonuniform orientation (Fig. 5B), are comparable (Fig. 2 B and C). Their walls are electron-dense and have a mean with those obtained within the interior of nacre tablets, which B Inset thickness of 10–15 nm (Fig. 2 ). are composed of nanodomains with variable orientations (X. Li, In section, the surface membrane has a mean thickness of 100 personal communication). nm, markedly thicker than the underlying interlamellar mem- branes (Ϸ30 nm) (Figs. 2 B, C, E, and F,3C and D,4A). In Discussion fracture and TEM sections, it has a homogeneous appearance. Our results, together with those of Nakahara (8, 9, 11), dem- onstrate that the surface membrane is present in representatives Relationship Between the Surface Membrane and Other Nacre Com- of at least three (Haliotidae, Trochidae, and Turbinidae) of the ponents. Examination of the nacre side of the surface membrane six nacre-secreting families of gastropods (12), all grouped within

reveals that the interlamellar membranes develop in close con- the (13). We may hence consider the surface SCIENCES tact with the surface membrane. The difference between them membrane a basic element of gastropod nacre. Although its APPLIED PHYSICAL is evident from their fibrous nature compared with the smooth- growth dynamics is not yet totally elucidated, the surface mem- ness of the surface membrane (Fig. 2D). In section, it is notable brane seems to form by addition of organic vesicles to its that the surface membrane generally intercepts the last-formed mantle-side surface (Fig. 2B), which gradually integrate into it interlamellar membranes at a shallow angle in the apical direc- (Fig. 2C). We hypothesize that this is a means by which com- tion (Fig. 2E; see also ref. 10, Fig. 4 g and h). ponents necessary for the production of organic and crystalline

Scanning electron microscopy (SEM) observations of the structures within the nacre compartment are transported. These SCIENCES nacre side of the surface membrane also reveal the existence of observations are compatible with others that show that the

tablet cores growing between the last-formed interlamellar surface layers of the tablets are of amorphous calcium carbonate APPLIED BIOLOGICAL

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Fig. 2. SEM views of the nacre of G. pennanti.(A) View of the internal surface of the shell, close to the . The nacre compartment is overlain by the surface membrane, visible as the darker area, which has cracked and curled upon contraction during preparation. (Inset) View of the mantle side of the surface membrane with vesicles. (B) TEM section of decalcified nacre with vesicles adhering to the mantle side of the surface layer. (Inset) Bilayered appearance of the wall of one such vesicle. (C)AsinB, with some vesicles apparently in the process of being incorporated into the surface membrane. (D) Nacre-side view of the surface membrane, with the last-formed interlamellar membrane adhering. The surface membrane can be differentiated by its smooth aspect. Arrows indicate two tablet cores semidetached from the surface membrane. (E and F) Transverse views of the surface membrane and of the underlying interlamellar membrane and tablets in formation. (E) The last-formed interlamellar membrane and the surface membrane meet in the apical direction (Right of photograph). (F) The surface membrane (with vesicles) has partly been torn off, which has exposed the last core. il, internal lamellar layer; ilm, interlamellar membranes; n, nacre; os, outer spherulithic layer; sm, surface membrane.

(14) and that mineral may be precipitated intracellularly before growth within the surface membrane, the tablets should incor- being transported to the mineralization site where it is remod- porate organic material from the surface membrane. eled (15), but the exact sequence of events in mineralization is (iii) The interlamellar membrane disappears completely or a matter for further study. continues only as a diffuse band at the very core of the crystalline Based on our results, we reach four main conclusions. (i) tablets (Fig. 3 and Fig. 4 A–D). Interlamellar membranes are Interlamellar membranes detach from the nacre side of the electron-dense and sometimes appear bi- or trilayered (see also surface membrane. From the topographic relationships between refs. 7, 8, and 11 and Figs. 2C and 3D); their aspect as a fuzzy structures, Cartwright and Checa (10) concluded that interla- band in samples cut exactly through the tower axis (Figs. 3 and mellar membranes form at the nacre side of the surface mem- 4) is thus quite distinct (Fig. 4A Inset). brane and detach from it in an apical direction. Our observations (iv) At the location of the core, the interface between growing support this view (Fig. 2 D and E). In this way, the balance tablets is fully crystalline. This observation (Fig. 5) does not between the components acquired via vesicle addition and those totally preclude the existence of the interlamellar membrane at this position because Rousseau et al. (18) showed that the lost because of the formation of interlamellar membranes re- mineralized interlamellar membranes of the pearl oyster mains steady so that the surface membrane maintains a constant Pinctada margaritifera are partly nanocrystalline. In our case, thickness. FFT patterns (Fig. 5B) are identical to those obtained within the (ii) Nacre tablets begin growing within the surface membrane. interior of nacre tablets, being characterized by a nanodomain- It is known that tablets first acquire their maximal height and like ultrastructure. subsequently expand sideways until they impinge on each other These four findings above can be understood coherently with (16). Their initial growth in height keeps pace with the separa- the following model. A new interlamellar membrane does not tion of an interlamellar membrane from the surface membrane penetrate through the very core of the tablet because core so that they stay in contact with the rest of the tower below them, formation precedes that of the interlamellar membrane; when it at the same time that their tips remain permanently submerged detaches from the surface membrane, the tip of the tablet core within the surface membrane (Fig. 3 A, C, and D). Nakahara (8) is already present. The fuzzy band sometimes observed may be and Mutvei (17) concluded, based on TEM and etching tech- explained by competition between the mineral and the organic niques, respectively, that nacre tablets have an organic-rich core. molecules leading to cocrystallization. The sequence of events is In our EDTA-treated samples the cores of tablets etch prefer- thus as follows: (i) formation of an organic-rich tablet core, entially (Fig. S1), which is also indicative of their organic-rich which grows rapidly in height with its tip embedded in the surface composition. This is comprehensible because during their initial membrane (Fig. 6A); (ii) cessation of tablet growth in height and

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Fig. 3. TEM sections that have partly penetrated the axes of nacre towers of M. labio in formation show crystal continuity and/or the absence of interlamellar membranes (arrows). (C and E Insets) Details. (B and F) Details of A and E, respectively. (D and E) White areas are where tablets have partly disappeared during sample preparation. ilm, interlamellar membranes; sm, surface membrane; v, vesicles.

simultaneous detachment of another interlamellar membrane; partments they produce are formed in advance of the nacre organic compounds may adhere to the tip of the tablet within the tablets, thus has to be modified insofar as each interlamellar surface membrane (Fig. 6B); (iii) formation of a new tablet core membrane is formed after the core of the tablet, which thus (Fig. 6C). becomes a distinct part within the tablet, but before the tablet The basic model for gastropod nacre growth of Nakahara (8, 9), which states that interlamellar membranes and the com- A A B

B C D SCIENCES Fig. 4. Nacre of G. umbilicalis.(A) TEM section approximately through the axis of a decalcified sample. The interlamellar membranes are seen as diffuse APPLIED PHYSICAL organic membranes or disappear entirely at the axial zone. (Inset) Detail of two such interlamellar membranes. (B) Polished section through the axis of a nacre tower (BSE mode). (Inset) The organic membranes become diffuse and tend to be convex upward. (C and D) Fixed and decalcified polished section of nacre. The axis of the tower is marked by aligned holes with coarsened rims Fig. 5. High-resolution TEM study of the axial zones of nacre tablets of M. within the interlamellar membranes (arrows). (C) Succession of Ϸ10 such holes labio.(A) The contact between the last two tablets is fully crystalline and

that are perfectly aligned. Their mean diameter is Ϸ150 nm. (D) Similar case in consists of a single crystal domain. (B) The different areas at and around the SCIENCES which a hole has been sectioned. ilm, interlamellar membranes; sm, surface contact between the last two tablets are crystalline, as shown by the FFT

membrane. patterns. ilm, position of interlamellar membranes. APPLIED BIOLOGICAL

Checa et al. PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 ͉ 41 Downloaded by guest on October 1, 2021 Fig. 6. Scheme for the formation of incipient nacre tablets. (A) The tablet core grows rapidly in height with its tip immersed within the surface membrane. An organic-rich core is formed as the growing tablet absorbs components of the surface membrane. (B) At the same time that vertical crystal growth ceases, a new interlamellar membrane is secreted at the nacre side of the surface membrane. During this time interval, organic material may precipitate on top of the tablet core. (C) Growth of a new tablet commences.

begins to expand laterally. The above model implies that nacre views on how nacre tablets relate to each other (see the review tablets are crystallographically connected at their cores. The in ref. 10). The evidence we have presented here does not rule existence of a fuzzy organic band of material in between should out either theory, but it does show that, for gastropod nacre, not represent an obstacle for crystallographic continuity, as there is a third way: tablets are connected at the tower axes. This shown by our analysis of the lattice fringes. This would explain connection, in turn, explains the tower-like growth of gastropod why nacre tablets stacked along a single tower retain their nacre. overall crystallographic orientation (19 and unpublished data). In more detail, nacre tablets are composed of many Materials and Methods twinned crystals (17), and we present additional TEM evi- SEM. Shells of living specimens of G. umbilicalis, Gibbula pennanti, Mon- odonta sp., and Calliostoma zyzyphinus were fixed with 2.5% glutaraldehyde dence (Fig. S2) that multiple crystal orientations are inherited in a 0.1 M cacodylate buffer. Samples were usually observed intact after CO2 by newborn tablets. critical point drying. Some polished sections were decalcified according to two Cartwright and Checa (10) hypothesized that the different different protocols: (i) 2% EDTA for 2–3 min; (ii) fixation of the organic matrix stacking patterns of gastropods and bivalves could be related to with a mixture of 2.5% glutaraldehyde and 2% formaldehyde and further demineralization with a methanolic solution (3:1:6) in a gel medium (protocol the sizes and densities of the nanopores they observed in the by A´ . Herna´ndez-Herna´ndez, unpublished data). This procedure preserves the interlamellar membranes. The present work shows that the finest details of the organic membranes (note, e.g., the nanopores in the difference is not merely quantitative because the existence of the interlamellar membranes in Fig. 4 C and D). surface membrane and its associated effect on nacre growth TEM. Resin-embedded specimens of G. umbilicalis were completely decalcified strongly promote vertical stacking in gastropods. with 2% EDTA and prepared with an ultramicrotome in the standard way. We In implying the crystallographic continuity of the cores of also had access to original material of Monodonta labio, and Haliotis rufe- tablets, our hypothesis bears implications on the present debate scens of the late H. Nakahara. The samples were prepared in Meikai University of whether tablets communicate across lamellae or not. This by M. Kakei according to the protocol described in ref. 9. Only samples of M. labio rendered significant results. debate began with Weiner and Traub (20), who found that the We used a Leo Gemini 1530 field-emission SEM and a Philips CM20 TEM of fiber axis of the chitin and silk protein forming the interlamellar the Centro de Instrumentacio´n Científica, University of Granada. High- matrix are perpendicular to each other and aligned with the a- resolution TEM analysis was carried out in a Jeol 2200FS at the Centro de and b-axes of the aragonite tablets, respectively. They proposed Investigac¸a˜ o em Materiais Ceraˆmicos e Compositos, University of Aveiro. that the mineral phase grows epitaxially onto the protein chains ACKNOWLEDGMENTS. We wish to express our profoundest appreciation to H. of the organic matrix; this is the heteroepitaxial theory. Scha¨ffer Nakahara (1928–2001). In noticing the existence of the surface membrane and et al. (21) recognized the existence of many pores, several tens of an organic core along the axes of the nacre towers, he largely inspired our of nanometers across, in the intercrystalline matrix of abalone work, which also benefitted from the study of his unique material. We thank M. Kakei (Meikai University) for providing TEM material of H. Nakahara, A´ . nacre, which they showed to be permeable to ions. They sug- Herna´ndez-Herna´ndez (Consejo Superior de Investigaciones Científicas– gested that some of the pores allow tablets to grow from one Universidad de Granada) for sampling preparation with her own fixative and layer to the next, without the need for a new nucleation event; decalcification technique, M. Rousseau (Museum Natural d’Histoire Naturelle, Paris) for Fig. 1B, and E. M. Harper (Cambridge University) for critical revision. this is the mineral-bridge theory. From then on, the heteroepi- This work was supported by Research Project CGL2007-60549 (Ministerio de taxial and mineral-bridge theories have been two conflicting Ciencia e Innovación) and the Research Group RNM190 (Junta de Andalucía).

1. Addadi L, Joester D, Nudelman F, Weiner S (2006) Mollusk shell formation: A source of 8. Nakahara H (1983) in Biomineralization and Biological Metal Accumulation, eds new concepts for understanding biomineralization processes. Chem Eur J 12:980–987. Westbroek P, de Jong EW (Reidel, Dordrecht), pp 225–230. 2. Jackson AP, Vincent JFV, Turner RM (1984) The mechanical design of nacre. Proc R Soc 9. Nakahara H (1991) in Mechanisms and Phylogeny of Mineralization in Biological London Ser B 234:415–440. Systems, eds Suga S, Nakahara H (Springer, Berlin), pp 343–350. 3. Lin A, Meyers MA (2005) Growth and structure in abalone shell. Mater Sci Eng 10. Cartwright JHE, Checa AG (2007) The dynamics of nacre self-assembly. J R Soc Interface 390:27–41. 4:491–504. 4. Camprase S, Camprase G, Pouzol M, Lopez E (1990) Artificial dental root made of 11. Nakahara H (1979) An electron microscope study of the growing surface of nacre in two natural calcium carbonate (Bioracine). Clin Mater 5:235–250. gastropod species, Turbo cornutus and pfeifferi. Venus 38:205–211. 5. Heuer AH, et al. (1992) Innovative materials processing strategies: A biomimetic 12. Hedegaard C (1997) Shell structures in the Vetigastropoda. J Molluscan Stud 63:369–378. approach. Science 255:1098–1105. 13. Ponder WF, Lindberg DR (1997) Towards a phylogeny of gastropod molluscs: An 6. Levi-Kalisman Y, Falini G, Addadi L, Weiner S (2001) Structure of the nacreous organic analysis using morphological characters. Zool J Linnean Soc 119:83–265. matrix of a bivalve mollusc shell examined in the hydrated state using cryo-TEM. J 14. Nassif N, et al. (2005) Amorphous layer around aragonite platelets in nacre. Proc Natl Struct Biol 135:8–17. Acad Sci USA 102:12653–12655. 7. Bevelander G, Nakahara H (1969) An electron microscope study of the formation of the 15. Mount AS, Wheeler AP, Paradkar RP, Snider D (2004) Hemocyte-mediated shell min- nacreous layer in the shell of certain bivalve molluscs. Calcif Tissue Res 3:84–92. eralization in the Eastern oyster. Science 304:297–300.

42 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808796106 Checa et al. Downloaded by guest on October 1, 2021 16. Mutvei H (1977) The nacreous layer in Mytilus, Nucula, and Unio (Bivalvia). Calcif Tissue 19. Di Masi E, Sarikaya M (2004) Synchrotron X-ray microbeam diffraction from abalone Res 24:11–18. shell. J Mater Res 19:1471–1476. 17. Mutvei H (1978) Ultrastructural characteristics of the nacre in some gastropods. Zool 20. Weiner S, Traub W (1980) X-ray diffraction study of the insoluble organic matrix of Scripta 7:287–296. mollusk shells. FEBS Lett 111:311–316. 18. Rousseau M, et al. (2005) Multiscale structure of sheet nacre. Biomaterials 26:6254– 21. Scha¨ffer TE, et al. (1997) Does abalone nacre form by heteroepitaxial nucleation or by 6262. growth through mineral bridges? Chem Mater 9:1731–1740. SCIENCES APPLIED PHYSICAL SCIENCES APPLIED BIOLOGICAL

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