Nanostructure of mouse otoconia.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Microstructure and crystallography of the wall plates of the giant barnacle Austromegabalanus psittacus: a material organized by crystal growth.

In biomineralization, it is essential to know the microstructural and crystallographic organization of natural hard tissues. This knowledge is virtually absent in the case of barnacles. Here, we have examined the crystal morphology and orientation of the wall plates of the giant barnacle Austromegabalanus psittacus by means of optical and electron microscopy, and electron backscatter diffraction. The wall plates are made of calcite grains, which change in morphology from irregular to rhombohedral, except for the radii and alae, where fibrous calcite is produced. Both the grains and fibres arrange into bundles made of crystallographically co-oriented units, which grow onto each other epitaxially. We call these areas crystallographically coherent regions (CCRs). Each CCR elongates and disposes its c-axis perpendicularly or at a high angle to the growth surfaces, whereas the a-axes of adjacent CCRs differ in orientation. In the absence of obvious organic matrices, this pattern of organization is interpreted to be produced by purely crystallographic processes. In particular, due to crystal competition, CCRs orient their fastest growth axes perpendicular to the growth surface. Since each CCR is an aggregate of grains, the fastest growth axis is that along which crystals stack up more rapidly, that is, the crystallographic c-axis in granular calcite. In summary, the material forming the wall plates of the studied barnacles is under very little biological control and the main role of the mantle cells is to provide the construction materials to the growth front.

Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation.

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.

Structure and crystallography of foliated and chalk shell microstructures of the oyster Magallana: the same materials grown under different conditions.

Oyster shells are mainly composed of layers of foliated microstructure and lenses of chalk, a highly porous, apparently poorly organized and mechanically weak material. We performed a structural and crystallographic study of both materials, paying attention to the transitions between them. The morphology and crystallography of the laths comprising both microstructures are similar. The main differences were, in general, crystallographic orientation and texture. Whereas the foliated microstructure has a moderate sheet texture, with a defined 001 maximum, the chalk has a much weaker sheet texture, with a defined 011 maximum. This is striking because of the much more disorganized aspect of the chalk. We hypothesize that part of the unanticipated order is inherited from the foliated microstructure by means of, possibly, [Formula: see text] twinning. Growth line distribution suggests that during chalk formation, the mantle separates from the previous shell several times faster than for the foliated material. A shortage of structural material causes the chalk to become highly porous and allows crystals to reorient at a high angle to the mantle surface, with which they continue to keep contact. In conclusion, both materials are structurally similar and the differences in orientation and aspect simply result from differences in growth conditions.

Crystallographic orientation inhomogeneity and crystal splitting in biogenic calcite.

The calcitic prismatic units forming the outer shell of the bivalve Pinctada margaritifera have been analysed using scanning electron microscopy-electron back-scatter diffraction, transmission electron microscopy and atomic force microscopy. In the initial stages of growth, the individual prismatic units are single crystals. Their crystalline orientation is not consistent but rather changes gradually during growth. The gradients in crystallographic orientation occur mainly in a direction parallel to the long axis of the prism, i.e. perpendicular to the shell surface and do not show preferential tilting along any of the calcite lattice axes. At a certain growth stage, gradients begin to spread and diverge, implying that the prismatic units split into several crystalline domains. In this way, a branched crystal, in which the ends of the branches are independent crystalline domains, is formed. At the nanometre scale, the material is composed of slightly misoriented domains, which are separated by planes approximately perpendicular to the c-axis. Orientational gradients and splitting processes are described in biocrystals for the first time and are undoubtedly related to the high content of intracrystalline organic molecules, although the way in which these act to induce the observed crystalline patterns is a matter of future research.

Nacre and false nacre (foliated aragonite) in extant monoplacophorans (=Tryblidiida: Mollusca).

Extant monoplacophorans (Tryblidiida, Mollusca) have traditionally been reported as having an internal nacreous layer, thus representing the ancestral molluscan condition. The examination of this layer in three species of Neopilinidae (Rokopella euglypta, Veleropilina zografi, and Micropilina arntzi) reveals that only V. zografi secretes an internal layer of true nacre, which occupies only part of the internal shell surface. The rest of the internal surface of V. zografi and the whole internal surfaces of the other two species examined are covered by a material consisting of lath-like, instead of brick-like, crystals, which are arranged into lamellae. In all cases examined, the crystallographic c-axis in this lamellar material is perpendicular to the surface of laths and the a-axis is parallel to their long dimension. The differences between taxa relate to the frequency of twins, which is much higher in Micropilina. In general, the material is well ordered, particularly towards the margin, where lamellae pile up at a small step size, which is most likely due to processes of crystal competition. Given its morphological resemblance to the foliated calcite of bivalves, we propose the name foliated aragonite for this previously undescribed biomaterial secreted by monoplacophorans. We conclude that the foliated aragonite probably lacks preformed interlamellar membranes and is therefore not a variant of nacre. A review of the existing literature reveals that previous reports of nacre in the group were instead of the aragonitic foliated layer and that our report of nacre in V. zografi is the first undisputed evidence of nacre in monoplacophorans. From the evolutionary viewpoint, the foliated aragonite could easily have been derived from nacre. Assuming that nacre represents the ancestral condition, as in other molluscan classes, it has been replaced by foliated aragonite along the tryblidiidan lineage, although the fossil record does not presently provide evidence as to when this replacement took place.