Crustaceans protect themselves from predators by secreting at their body surface an organic matrix, called carapace or cuticle, reaching several millimetres in thickness. Calcite crystals then appear and grow within the fibrillar organic network ending in a rigid acellular composite material forming an exoskeleton.

Continuous growth of the animal implies a periodic set aside of this rigid cuticle during moult. The different steps of matrix deposition and mineralisation are identical throughout the successive inter-moult cycles [13]. The organic matrix is formed by four superimposed layers (Fig. 1A). The epicuticle (ep) and pigmented (pi) layers are deposited before moult; the principal (pr) and membranous (mb) layers are deposited after moult. The matrix, composed of chitin associated with proteins, is produced by an underlying monolayer of epidermal cells (Fig. 1B). In a lower ratio the cells also secrete lipids mostly in the epicuticle and pigments in the pigmented layer. Enzymes, for example alkaline phosphatase and carbonic anhydrase, are also synthesized in relation with the control of calcification, which occurs in the two middle layers (pi and pr).

Hierarchical structures of the organic matrix are evidenced in sections analysed in optical microscopy or in transmission electron microscopy (TEM). At low magnifications, sections transverse to the body surface show regular laminae in all the chitin-containing layers (pi, pr, mb) (Fig. 1A). At higher magnifications, the matrix appears formed of regularly disposed fibrils seen in longitudinal, oblique, or transverse views (Fig. 1C); each lamina corresponds to a 180° rotation of the fibrillar directions (Fig. 1D). At high resolution in TEM, in domains where the section plan is perfectly transverse to the fibril axis, a honeycomb pattern appears which reveals the elementary chitin protein units. The central clear rods, a few nanometres in diameter, correspond to chitin crystallites; the dark sheath corresponds to the surrounding proteins (Fig. 1E) [6] and [27]. Each chitin crystallite is composed of about 20 linear chains of N-acetyl-glucosamine, based upon the rod diameter and crystalline lattice dimensions (Fig. 1F). Keeping the same basic structure, different modes of aggregation exist, giving fibrils of various diameters, or reticulated morphologies, precisely disposed from top to bottom of the cuticle [18].

The new cuticle calcifies after each moult by nucleation and growth of calcium carbonate in the form of calcite crystals [9], [21] and [22]. The first crystalline germs appear in the most external part of the pigmented layer (Fig. 2A) and grow in the form of flat discs or spherulites that fuse laterally to form a crystalline mosaic (Fig. 2B). The mineral further grows along the interprismatic septae that correspond to imprints left in the cuticle by the underlying epidermal cells (Fig. 2C and D). Calcification then progresses radially from these vertical axes in the pigmented and principal layers, with various speeds as a function of the animal body curvature and antero-posterior axis [13]. When the space in between the organic fibrils is lower than 300 Å, as in the membranous layer, mineralisation does not occur [9].

Crab cuticles are complex structures where initiation, growth, and orientation of calcite domains are controlled by organic intra- and extracellular elements. Pore canals, which are thin cytoplasmic expansions of epidermal cells, play a role in the transport of calcium ions [32]. Carbonic anhydrase activity, producing carbonate ions, is demonstrated in the crab cuticle during the inter-moult cycle and shows a maximum during the first stages of calcification just after the moult. The enzyme enhances the precipitation of calcium carbonate; it was shown that diamox, its inhibitor, decreases by a factor 2 the growth rate of calcite crystals in the cuticle (Fig. 2D) [16]. Alkaline phosphatase is present in calcified tissues, namely in crustacean carapaces, as it removes phosphate ions, which are considered as inhibitors of calcification [31]. The presence of cation-binding glycoproteins in the sites where calcification is initiated, the interprismatic septae, plays a role in calcium carbonate nucleation [18]. The twisted fibrillar matrix of chitin generates screw dislocations, which are detected in scanning electron microscopy by the presence of spiral steps on the surface of the crystals. These defects favour crystal growth by introducing privilege sites for further crystalline units to grow [22]. All these actors of mineralisation show a precise distribution both in space, over the cuticle structure, and time, during the inter-moult cycle.

Mineralised compact bone is composed of specialised cells, a dense organic matrix, and inorganic phosphate ions. Skeletal tissues have three functions, a mechanic one supporting the body weight, a protective one of essential body organs, a metabolic one as reservoir of mineral ions, mostly calcium and phosphate. The major part of the organic fraction consists in type-I collagen, a structural protein to which are associated non-collageneous proteins (osteonectin, osteopontine, osteocalcin, sialoproteins…). The organic fraction is first synthesized by cells called osteoblasts, whose cycle of development and interaction with the environment is under genetic control, actively studied by gene manipulation and genetic mutations [25]. In a second step, the organic matrix calcifies by the deposit of calcium phosphate in the form of small crystals. Skeletal tissues are remodelled through lifetime by highly polarised cells called osteoclasts. The physiological process of remodelling alternates synthesis and resorption phases and is controlled by mechanical and hormonal stimuli. The action of osteoclasts through complex biochemical events, will both dissolve the mineral fraction by creating very low pH domains through the action of Na⁺/H⁺ pumps, and disrupt the organic matrix by secreting specialised enzymes, namely metalloproteases [2].

The general structure of compact bone consists of adjacent cylindrical structures of 0.2 mm in diameter, called osteons, laying parallel to the bone long axis. Each osteon is formed by coaxial layers of collagen fibrils running helically around a haversian canal containing nerve and blood vessels. It is generally admitted that collagen fibrils are organised in distinct lamellae, fibrils all running parallel in one lamella and showing a strong change of orientation from one lamella to the next [16]. Three osteon types are described in the literature, depending on their aspect in polarized light microscopy respectively described as bright, intermediate or dark type (Figs. 3A′, A′′, 3B′) [1]. The very dense collagen network in decalcified sections of compact bone shows collagen fibrils arranged either in orthogonal sheets (Fig. 3C), or in nested arcs (Fig. 3D) [15]. Interpreting the origin of the arced patterns has introduced the hypothesis of an analogy between, notably, the fibrillar collagen distribution in bone matrix and the macromolecular arrangements in cholesteric liquid crystals (cf. § 4). From these observations, it has been proposed that two main osteon models coexist in a compact bone; the first obeys the classical description of osteons following an orthogonal plywood model (Fig. 3A), the second obeys the twisted plywood model, where the collagen fibrils follow small and regular changes in their orientation (Fig. 3B). In this last case, the structure is continuous, but stratification, i.e. a lamellar aspect, appears in microscopy sections at each 180° rotation of the fibrillar directions [19].

The bone mineral fraction is made of apatite with the following general composition Ca₁₀(PO₄)₆(OH)₂. However, the mineral phase is heterogeneous, lacunae exist, as well as substitutions of Ca²⁺ by other divalent ions (Mg²⁺, Ba²⁺, Sr²⁺), substitutions of PO 4 3 – $ {{\mathrm{PO}}_{4}}^{3–}$ by HPO 4 2 – $ {{\mathrm{HPO}}_{4}}^{2–}$ , or of OH by H₂O [27]. The mineral composition in vertebrate bones has been shown to vary in function of species, age, and maturation stage. X-ray diffraction diagrams of bone mineral samples appear different from those taken with standard hydroxyapatite crystals. Data correspond to rather amorphous phases for young bones, with a transition toward more crystallized samples as a function of age [23].

Electron micrographs of bones at the beginning stages of calcification show the initial locus of mineralisation at the ultrastructural level. The very first calcium phosphate crystals appear in the gap zones of the collagen fibrils, following a regular 67-nm periodicity [24]. Mineralisation then continues within intra- and inter-fibrillar spaces, more or less completely along the fibrils. The crystals appear as flat platelets in the size range of nanometres with their long axis making a small angle with the direction of the collagen fibrils [33]. The same observation was made in fish scales by small-angle X-ray scattering; the information that collagen fibrils display preferential orientations introduces a finite number of collagen orientations [5].

When observed in transmission electron microscopy, ultra-thin sections of the organic matrix, both in crab carapaces and in compact bone osteons, reveal typical series of arced patterns [7] and [19]. Such figures frequently evidenced in sections of extracellular tissues do not result from authentic curved filaments. In an ideal representation, the molecular directions are drawn as parallel and equidistant straight lines on a series of rectangles and from one card to the next, the lines turn by a small and constant angle (Fig. 4A). Series of nested arcs appear on oblique sides of the model, just as they appear in microscopy after sectioning of the material (Fig. 4B). Another consequence of the twisted plywood arrangement is the presence of periodic extinctions when the sections are viewed in polarized light microscopy, with a planar disposition observed in the crab cuticle (Fig. 4C) and coaxial circles in intermediate type osteons (Fig. 3B′).

Geometric analysis demonstrates that the three-dimensional organisation of major biological macromolecules is analogous to that of molecules in cholesteric liquid crystals. The formation of connective tissues such as crab cuticles and compact bones is thus suggested, at initial stages of their elaboration, to imply liquid crystalline states of matter [8]. A main difference is that, unlike liquid crystals, the final state exhibits no fluidity. The rigidity of the extracellular matrices is progressively established by molecular cross-links and/or mineralisation.

The demonstration was made in vitro with purified molecules dispersed in aqueous solutions at a proper concentration. Anisotropic distributions of elongated polysaccharide crystallites or protein monomers were evidenced in polarized light microscopy [20] and [29]. The strong birefringence observed between crossed polars arises from spontaneous ordering of the molecules when a critical concentration is achieved. Both systems are intrinsically chiral, thus the nematic order due to parallel alignment of molecules has a long-range helical distortion, termed ‘twist’. This helical arrangement is revealed by the fingerprint patterns typical of cholesteric liquid crystals (Figs. 5A and C). The distance between two dark bands corresponds to a 180° rotation of the molecular orientations and corresponds to the half-cholesteric pitch P/2. The first-order nature of the transition from a disordered isotropic state to a highly ordered cholesteric phase is demonstrated by the existence of a concentration domain in which the two phases coexist. In chitin suspensions, a biphasic domain, i.e. an isotropic phase containing cholesteric droplets, appears at concentrations ranging from 20 to 40 mg ml^–1. At higher volume fractions, the samples are completely chiral nematic (Fig. 5A). The half-cholesteric pitch varies as a function of concentration and ionic strength, and ranges between 10 to 60 μm in our experiments (Fig. 5B) [3]. In collagen solutions, at concentrations around 80 mg ml^–1, cholesteric globules can spontaneously emerge within the isotropic phase. As the concentration is further increased, they merge and fuse, leading to homogeneous cholesteric domains with P/2 values ranging from 0.1 to 4 μm [Fig. 5C]. The general features of the liquid crystal phases obtained with collagen solutions and chitin dispersions are mostly in agreement with theoretical models originally proposed by Onsager to describe the self-assembly of rod-like particles [28].

The passage from the molecular to the fibrillar level in vitro was extensively studied with collagen in dilute solutions, fibrillogenesis being initiated by pH modification [11]. In concentrated systems, collagen liquid crystals are obtained at acidic pH, where repulsions between positively charged residues are large enough to maintain a liquid state.

When the solutions are exposed to ammonia vapours, NH₃ dissolution induces a pH rise without diluting the sample. As the net charge of collagen monomers is strongly decreased, hydrophobic interactions dominate, causing the triple helices to aggregate and form fibrils. This step corresponds to a sol/gel transition and the resulting fibrillar matrices mimic collagen connective tissue organisations [4]. The stabilized samples can be prepared for studies using Transmission Electron Microscopy by classical fixation, dehydration and embedding procedures. The ultrastructure of fibrillar gels obtained in vitro is influenced by the physicochemical parameters of the solution. Regular cholesteric geometries giving superposed series of arced patterns, similar to those observed within compact bones, are now obtained in a well-controlled manner over large domains [Fig. 5D].

Ordered chitin suspensions are obtained in slightly acidic solutions, with electrostatic repulsions that originate from ionised amine groups present on the surface of the rigid crystallites. The suppression of these repulsions by a pH increase results in the precipitation of large aggregates; however, no gelation occurs that would hold the three-dimensional organisation as observed with collagen. Solids have nevertheless been obtained with chitin cholesteric suspensions. Complete evaporation of the solvent results in the formation of rigid films that retain the helical symmetry of the liquid phase, but with much smaller cholesteric pitch values [30]. The macromolecular organisation of the chiral nematic phase was also stabilised by addition of polyacrylamide monomers at concentrations compatible with the liquid crystals and formation of a polyacrilamide gel that blocks the crystallite orientations [3].

Ordered gels and films of collagen and chitin are currently under investigation to form organic-mineral hybrids. An example inspired by biological processes consists of ions that diffuse and precipitate in contact with the organic matrix. Chitin matrices, prepared from decalcified crustacean shells, when soaked in solutions containing appropriate concentrations of calcium and carbonate ions, are covered with rhombohedric calcite crystals that exhibit in Scanning Electron Microscopy chiral features in the form of screw dislocations [22].

Another approach is to co-polymerise silica precursors, namely sodium silicates, with collagen monomers in solution. When mixed in various proportions, the condensed monomers form different morphologies: condensed mineral flakes of silica, polymerised collagen ribbons, or collagen fibrils surrounded by condensed silica (Fig. 5E) [12].