Volume 227, Issue 11 -

Heinz A. Lowenstam’s discovery in 1967, together with Ken Towe that the magnetite mineral in mature chiton teeth forms from a disordered transient precursor phase, ferrihydrite, remained an isolated curiosity for 30 years. During the last 15 years, many more examples were found in both invertebrates and vertebrates, where the mature crystalline mineral phase is formed through a transient amorphous precursor phase. Here we review this widespread phenomenon, and also describe the details of the transformation process in the formation of the calcitic spicules of the sea urchin larva. We identify many open questions.

It is demonstrated that magnesium and carbonate ions can form pre-nucleation clusters in analogy to calcium carbonate. If a mixed calcium and magnesium solution is brought in contact with carbonate ions, mixed pre-nucleation clusters form. The equilibrium constants for their formation are reported revealing that over the entire range of possible cation mixing ratios, calcium gets enriched over magnesium in the pre-nucleation clusters. This can explain high magnesium contents in amorphous calcium carbonate. However, this enrichment alone is not sufficient to explain the magnesium content of less than 41 mol% which is found in magnesium calcite biominerals nucleated from seawater.

The size, morphology and species-specific texture of mollusc shell biominerals is one of the unresolved questions in nature. In search of molecular control principles, chitin has been identified by Weiner and Traub (FEBS Lett. 1980, 111:311–316) as one of the organic compounds with a defined co-organization with mineral phases. Chitin fibers can be aligned with certain mineralogical axes of crystalline calcium carbonate in a species-specific manner. These original observations motivated the functional characterization of chitin forming enzymes in molluscs. The full-length cDNA cloning of mollusc chitin synthases identified unique myosin domains as part of the biological control system. The potential impact of molecular motors and other conserved domains of these complex transmembrane enzymes on the evolution of shell biomineralization is investigated and discussed in this article.

The synthesis of a new class of hybrid materials with two differently oriented layers of calcite at adjacent sides of an organic template is demonstrated. A Langmuir monolayer of the amphiphilic block copolymer poly(butyl acrylate)- b -poly(hydroxypropyl acrylate) directs the formation of a first CaCO 3 phase through interaction with the hydrophilic poly(hydroxypropyl acrylate) blocks. After partial hydrolysis of the hydrophobic poly(butyl acrylate) segments a second CaCO 3 phase is formed on the monolayer associated to the first mineral phase. Thus, bilayered CaCO 3 -based hybrid materials are obtained with two differently oriented calcite phases at opposite sides of the polymer film. By using DNA as an additive the crystal orientation in the second layer can be modified.

Proteins have found their way into many of Nature’s structures due to their structural stability, diversity in function and composition, and ability to be regulated as well as be regulators themselves. In this study, we investigate the constitutive amino acids that make up some of these proteins which are involved in CaCO 3 mineralization – either in nucleation, crystal growth, or inhibition processes. By assaying all 20 amino acids with vapor diffusion and in situ potentiometric titration, we have found specific amino acids having multiple effects on the early stages of CaCO 3 crystallization. These same amino acids have been independently implicated as constituents in liquid-like precursors that form mineralized tissues, processes believed to be key effects of biomineralization proteins in several biological model systems.

Sea urchin spines of Holopneustes porossisimus are porous single crystals, with the pores being filled with a material rich in carbon, silicon, fluorine and sodium. The magnesian calcite constituting the spine is highly strained. Even though the spines appear to be single crystalline on a macroscopic scale, the calcitic material exhibits an extended defect network. We find dislocations as well as rotational and other, not yet identified boundaries. We also observe within spine calcite a patterned distribution of sulphur. Both distributions, that of the defect network and that of sulphur resemble in their pattern to each other and have a similar mesh size of 50 nm. We conclude from these observations that they arise from the growth process of the spine and account for the mosaicity within the spine single crystals.

The exoskeleton of crustaceans is formed by the cuticle, a chitin-protein-based nano-composite with hierarchical organization over at least eight levels. On the molecular level, it consists of chitin associated with proteins forming fibres, which are organized in the form of twisted plywood. On the higher levels, the twisted plywood organization is modified and forms skeletal elements with elaborate functions. The load-bearing parts of crustacean cuticle are reinforced with both crystalline and amorphous biominerals. During evolution, all parts of the exoskeleton were optimized to fulfill different functions according to different ecophysiological strains faced by the animals. This is achieved by modifications in microstructure and chemical composition. In order to understand the relationship between structure, composition, mechanical properties and function we structurally characterized cuticle from the dorsal carapace of the edible crab Cancer pagurus using light and scanning electron microscopy (SEM). The local chemical composition was investigated using energy dispersive X-ray spectroscopy (EDX) and confocal m-Raman spectroscopy. Nanoindentation tests were performed to study the resulting local mechanical properties. The results show local differences in structure on several levels of the structural hierarchy in combination with a very heterogeneous mineralization. The distal exocuticle is mineralized with calcite, followed by a layer containing a magnesium, phosphate and carbonate rich phase and ACC in the proximal part. The endocuticle contains magnesian calcite and ACC in special regions below the exocuticle. Structure and mineral phase are reflected in the local stiffness and hardness of the respective cuticle regions. The heterogeneity of structural organization and mechanical properties suggests remarkable consequences for the mechanical behaviour of the bulk material.

The crustacean cuticle is a bio-composite consisting of hierarchically organized chitin-protein fibres, reinforced with calcite, amorphous calcium carbonate and phosphates. Comparative studies revealed that the structure and composition of tergite cuticle of terrestrial isopods is adapted to the habitat of the animals, and to their behavioural patterns to avoid predation. In this contribution we use FE-SEM, polarized SC μ -RSI and EBSD to investigate micro- and nano-patterns of mineral phase distribution and crystal orientation within the tergite cuticle of the two terrestrial isopod species Armadillidium vulgare and Porcellio scaber . The results show that the proximal regions of the exocuticle contain both calcite and ACC, with ACC located within the pore canals. Calcite forms hierarchically organised mesocrystalline aggregates of similar crystallographic orientation. Surprisingly, c -axis orientation preference is horizontal in regard to the local cuticle surface for both species, in contrast to mollusc and brachiopod shell structures in which the c -axis is always perpendicular to the shell surface. The overall sharpness of calcite crystal orientation is weak compared to that of mollusc shells. However, there are considerable differences in texture sharpness between the two isopod species. In the thick cuticle of the slow-walking A. vulgare calcite is more randomly oriented resulting in more isotropic mechanical properties of the cuticle. In contrast, the rather thin and more flexible cuticle of the fast- running P. scaber texture sharpness is stronger with a preference of c -axis orientation being parallel to the bilateral symmetry-plane of the animal, leading to more anisotropic mechanical properties of the cuticle. These differences may represent adaptations to different external and/or internal mechanical loads the cuticle has to resist during predatory attempts.

Biologic structural materials for skeletons or teeth show a hierarchical architecture, where organic macromolecules and mineral substance form a hybrid composite material with its components inter-weaved on many length scales. On the nanostructure level brachiopods form hybrid composite mesocrystals of calcite with occluded organic molecules. On the microstructure level several types of materials are produced, on which the electron back-scatter diffraction (EBSD) technique gives insight in texture and architecture. We describe the calcite single-crystal fiber composite architecture of the secondary layer involving organic matrix membranes, the competitive-growth texture of the columnar layer and the nano-structuring of the primary layer. In the overall skeleton the organic biopolymers provide flexibility and tensile strength while the mineral provides a high elastic modulus, compressive strength, hardness and resistance to abrasion. The hierarchical composite architecture, from the nanostructure to the macroscopic level provides fracture toughness. The morphogenesis of the biomaterial as a whole and of the mineral crystals is guided by the organic matrix and most probably involves amorphous calcium carbonate (ACC) precursors. In this paper we review the hierarchical architecture of rhynchonelliform brachiopod shells, which is very distinct from mollusk nacre.