Biomineralogy is an ancient discipline, based initially on techniques that were those of classical crystallography and histology, in light or in electron microscopy. Recently, the research landscape has been modified by new methodologies coming from molecular genetics and also from inorganic chemistry, driven by hopes to create a genuine ‘chimie douce’, close to physiological conditions, in which biomineralisations would provide useful models. Historically, belief preceded basic understanding. Today, mineralised tissues appear to be highly structured composites at nanoscales and their morphogenesis might be a model system for genetics.
Most advances in biomineralogy have come from the emergence of new chemical and physical techniques. However, the main concepts were based on geometric considerations, as in crystallography, but differently, since biological shapes are curved in general, whereas straight lines and planes dominate in crystals. This aspect was developed in several chapters of a famous book written by D'Arcy Thompson
These differences between chemistry in factories and chemistry in biological systems were considered very early, but the dream is now the possibility of a genuine ‘chimie douce’ as suggested by Livage
Organizing this meeting on biomineralisation was a splendid initiative of two professors at the ‘Collège de France’, from the very different disciplines of inorganic chemistry and palaeontology. Their long-term views were not limited to these nanofactories for the future, since the topic covers large domains in biological sciences and life history at the surface of the Earth. The fossil record is based mainly on mineralised tissues. Recognizable remains of cells and molecules of biological interest were found in various rocks, including the most ancient ones. Purely inorganic nanofactories were possibly at work for long periods before the very beginning of biosphere and biomineralisations probably began with life itself, but were certainly preceded by innumerable mineral-organic associations.
Simple but difficult questions have appeared regularly about the mineralised parts of living beings but, even today, in many cases, answers are not on the horizon (see Marin in this issue). Some words suffice to explain this situation, say ‘the paradox of the sculptor and the mollusc’ for instance. To create a statue, an artist uses hard materials such as marble or wood, and hard tools to carve them, whilst soft living bodies, as molluscs, do not need such instruments and nevertheless produce solid shells comparable to sculptures, with precise geometries and decorations; there are also soft matters such as clay plus water, shaped by fingers, but hardening requires high temperatures in an oven, whereas the mollusc ‘works’ at the water temperature, in normal environmental conditions.
So, we return to Livage's hope of a ‘chimie douce’ with morphogenetic capabilities, but the route will be long, as suggested by the state of the art related in some optimistic articles, which however cannot hide the distances separating the genuine shells or other elaborated biological materials from the products of some beautiful experiments
Many good ideas were debated by people for centuries, but did not survive simply, because various wrong conceptions could provide easy answers to innumerable questions, and spread among people and even scholars, by a kind of natural selection. Examples of such convenient beliefs were reported in a book by Metzger
Ancient observations and ideas on biominerals were presented in general in books and articles about pearls
The first studies on biominerals were conducted by zoologists who described skeletons, either
Most animals were considered as machines producing sculptured stones, with an excellent efficiency, whereas plants were at the origin of woods and related fibrous materials. However, corals represented a major exception, since they were first classified in the plant kingdom by Tournefort, for instance, as recalled in
Some seaweed as
Amorphous silica forms hard deposits in spicules of most sponges and also in the skeleton of unicellular organisms as diatoms and radiolaria, often at the origin of rocks as diatomites and radiolarites (see
Diverse bacteria also produce inorganic precipitates and their nature depends on their anabolism. Stromatolites, for instance, have been present on diverse seashores since the earliest times of life, and superpose calcareous layers, produced by intercalated cyanobacteria, first confused with blue–green algae. Some bacteria produce a hard calcite patina at the surface of calcareous stones, the biocalcin, which is a natural protection against erosion
At the Renaissance and later, important collections were created by zoologists, botanists and also in hospitals. Conservation methods in appropriate liquids were improved to prepare long-term samples in tight flasks. Shells and skeletons were washed and ordered in boxes or in specialized pieces of furniture. Comparative observations and dissections led to reasoned descriptions, drawings, and paintings. Among ideas from these past times, several still prevail.
Metres of calcareous deposits in the bulk of coral reefs are formed by a thin external layer of living matter (some millimetres thick), associating polyps and tissues in between. We know today that ions in the sea, Ca2+ and
Measurements of lengths, weights, and growth rates began mainly in the 18th century and also physiological investigations. Two distinct types of growth were considered by Buffon and contemporaries:
In the 19th century, considerable advances on biominerals came from collaborations with chemists, affording their new chemicals, often well purified, and their analytical methods. The organic matrix of bone, the ‘ossein’, was discovered by a simple demineralisation in solutions of hydrochloric acid, and this lead also to recognize the mineral itself, a hydrated calcium phosphate. Shells, carapaces, spicules, and most mineralised tissues, pathological or not, presented an organic matrix.
The technical advances in microscopy and microtomy in the 19th century led to the discovery of most cell types and their assembly in diverse tissues. An extraordinary set of techniques was invented to see the organization of living matter through series of thin sections, where the order was remarkably preserved. This was the origin of two associated disciplines, named histology and cytology, since they considered tissues and cells. Their methods were adapted to mineralised organs by devising procedures of demineralisation, which did not alter strongly the matrix and the cells themselves, as can do hydrochloric acid solutions, replaced by trichloracetic acid for instance. Calcium chelators and acid salts were used later, as diluted ammonium citrate, preserving some enzymes and most proteins. The polarizing microscope was essential in the study of minerals. X-ray radiographs and diffraction appeared during the following century.
Some stains were recognized to bind to precise components, and specific chemical reactions were observed at the level of cellular details. These histochemistry and cytochemistry have now their immunological and molecular prolongations in light or in electron microscopy. All these techniques are long and difficult, but often remain the sole way to define biomaterials, their nature, structure, shape, localization in cells, tissues and organisms, and to ask clear questions, a prerequisite to any further studies in X-ray and electron diffraction, or to apply microanalytical methods.
The concept of epithelium was established in the 19th century, from microscopical studies, and corresponds to single or multilayered sheets of densely packed cells. These epithelia delimit compartments, with distinct physiological properties, and are able of considerable works, for instance building carapaces in crustaceans and shells in molluscs. The carapace of crabs, or ‘cuticle’, can be one or two millimetres thick and is secreted by a single layered epidermis, which can be forty times thinner
It appeared that matrices and mineral concretions were absent from cytoplasm in epithelial cells at the origin of shells in molluscs or cuticles in crustaceans, so that it was concluded that cell membranes separating these cells from their external productions represented an essential part of the machinery elaborating the matrices. In bones, the collagen molecules assemble into fibrils at the close periphery of cells, the osteoblasts, and mineralisation occurs later, at a distance from membranes. The matrix assembly always precedes mineralisation. The synthesized matrix proteins are first packaged and exported in the reticulum and in Golgi vesicles, separated by a membrane from hyaloplasm, and they assemble into the matrix outside of the cell. This was made clear also from studies on mineralised skeletons of Protozoa, coccoliths for instance by Pienaar
The demineralisation methods even by hydrochloric solutions do not alter the bone shape but, after some days and washings, the bone becomes flexible and is easily cut by a razor blade, so that histological sections can be prepared. The mineral salts were first considered to correspond to hydrated Ca3(PO4)2, with a small amount of carbonates, but more precisely it is an hydroxyapatite (see Rey in this issue). This demineralised bone is organic, with some mineral traces. This organic matter was called ossein, its main component being collagen, a fibrous protein also present in connective tissues. Heating of ossein or connective tissues in water leads to the production of a well-known glue, which is gelatine, and corresponds to denatured collagen. Organic matrices are present in most mineralised tissues and intracellular stones or spicules, but the mass ratio is highly variable between mineral, organic matter, and water. This ratio is not easily estimated, because a part of the organic matter is not the matrix, but comes from diverse associated cells and tissues, as nerves, vessels, etc.
Important matrices were found in vertebrate bones, in mineralised carapaces of crustaceans and in shells of molluscs, but are often less developed in spicules. Fibrous proteins and polysaccharides form these matrices, mainly proteins with collagen in bones and mainly chitin, a polysaccharide, in crustacean carapaces. In both cases, the fibrils form a highly structured matrix, with a plywood-like arrangement, the fibril direction differing in successive layers, either by an angle close to 90°, in compact bone, or by a series of small angles leading to a continuously twisted packing, found in cholesteric liquid crystals, but stabilized as in crab carapaces, for instance, by the assembly of chitin chains into fibrils (see
Composites are materials made of distinct parts, differing by their physical and chemical properties, these parts being distributed according to a definite geometry. This term was introduced by Maxwell in very general terms. There is the example of materials associating transparent media of different refractive indices, for instance, a stack of layers of refractive index
Skeletal tissues are ‘fibre-reinforced composites’, a kind of system where ‘the whole is more than the sum of its parts’, a conception often discussed by Plato himself. Such composites are realized in bones, carapaces, and shells. Separately, the mineral or the fibrils do not show ‘useful’ mechanical properties, the mineral being made of brittle crystals and the polymers being supple, but the composite structure resists strong constraints, since crystal fractures stop where they meet fibrils, and these latter do not bend, because the inter-distances are fixed in the mineral.
Goniometry applied to crystals began on the 18th century, polarizing microscopy on the 19th century and X-ray diffraction on the 20th century, so that there are three phases in the history of crystallography and also in the exploration of biominerals. Chapter 9 in D'Arcy Thompson's book is a reasoned presentation of most concretions and spicules in unicellular organisms and invertebrates
The crystal lattice of a mineral is never perfect and there are defects. Textures correspond to the arrangement of domains of uniform crystalline orientation, often separated by walls, which are narrow zones associating numerous defects. Definite textures were observed in biominerals. They were studied in artificial concretions and in spicules by Prenant
Helical defects were observed in the nacre of molluscs
Mineralisation follows a program of precise events in skeletal structures. The construction of a coccolith for instance begins by the assembly of a thin fibrous matrix in the form of an elliptical disc
Similarly, during their moult, crabs first absorb seawater, so that the body swells, unfolding the new skin, and they can leave their ancient carapace
Experiments by Digby
This electrochemical theory of biomineralisation due to Digby requires some enzymes, the phenol-oxidases involved in protein tanning, which add their effects to those of alkaline phosphatases and carbonic anhydrases to build the calcitic texture. Digby extended his interpretation to mineralisation of mollusc shells
Spicule demineralisation was obtained in living Alcyonaria by adding 0.7 g l–1 of calcium glycero-phosphate to seawater, at pH 7.6 and at 17 °C, in well-aerated conditions
Environment conditions often modify shell growth rates, either at the matrix level, or in the mineral progression, or both. Growth lines are visible in mollusc shells and in some crustacean mineralisations (see Marin and Luquet in this issue). In certain cases, they form age rings, as in otoliths (see Herbomel and Payan in this issue). Their existence is often related to periodic variations of environment, observed daily or yearly or something else. In vertebrates also, aquatic or not, bone mineralisation depends on nutrition and environment. Vitamin D and A are essential to a normal mineralisation of bones, with small doses of ultraviolet light to ‘activate’ sterols. This is a great chapter in mineral physiology with numerous metabolic steps, from absorption
Biominerals are supposed to be deposited directly in a solid state, amorphous or crystalline. Nucleation depends on the existence of interfaces provided mainly by matrices, with diverse structures and defects from place to place, and also epitaxial possibilities
About growth, one has two extreme situations and intermediate ones. Let us first consider the case of well-developed matrices, presenting a uniform distribution, at least locally. Nucleation begins here and there, at points that can correspond to singular structures of the matrix, and then growth is often radial. The main example considered above and studied by Prenant
The situation is radically different in sea urchins, with a skeleton made of large calcite monocrystals (or close to be), and a reduced matrix
Mineral growth in sea-urchin plates is accretive for these calcite monocrystals, as demonstrated by Märkel
Now between these extreme cases, there are intermediate ones. In nacreous layers of molluscs, there are five to seven monocrystalline domains in each nacre plate, and the matrix seems almost absent from them, whereas each plate is completely surrounded by a dense matrix
In bones, mineralisation begins within collagen fibrils, at a distance from osteoblasts, a situation similar to that of crustacean cuticles, but radial growth is absent and there are signs of epitaxy, the first apatite deposit nucleating within the gap zones of collagen fibrils. Later, the free spaces separating fibrils are filled by the mineral, apparently without changing the pre-existent inter-distances between fibrils, as in crustacean cuticle.
The matrix is not much developed in teeth, in the enamel mainly, but also in the dentin, or in the osseous rostrum of certain whales (see Goldberg and Zylberberg in this issue). These tissues with large crystals and reduced matrix are very hard, but fragile. Usual pathologies as caries develop in enamel and dentin and can be stopped at best, but do not repair themselves, in contrast with other tissues, where wound healing is a natural process, and even in bones, fracture reparations are not excluded.
We stressed some distinctive characters between non-biological minerals and skeletal structures (in the first §). Minerals are often limited by planes and straight lines, whereas curved surfaces and lines are found in skeletal shapes. Similar differences separate true crystals from liquid crystals, which also show regularly curved lines and surfaces. Accretive and intussusceptive growth also separates true crystals from liquid crystals, since diffusion is a corollary of fluidity in liquid crystals. It was recalled also (in § 6) that fibrous matrices of mineralised organs presented the symmetries and other geometrical characteristics of liquid crystals, without being liquid, but their assembly was certainly related to that of liquid crystals. This particular state of matter seems today very essential in the study of biomineralisations.
Important mineral deposits are produced by thin layers, and mainly by cell membranes themselves (as indicated in § 4 and 5). It is worth remembering that membranes are fluid and anisotropic, due to the parallel orientation of their components within this bilayer, phospholipids, and proteins mainly. This corresponds to the definition of liquid crystals, which are anisotropic liquids and most biominerals are precipitated in the close vicinity of membranes, in Golgi vesicles or at the periphery of cells or along their narrow projections within carapaces or bones. The liquid crystallinity of biological membranes confers remarkable morphogenetic capacities, from nanoscales to microscales and much larger ones when cells are associated in tissues. One knows also that long-range order arises spontaneously in liquid crystals.
Membranes are involved in the production of most macromolecules of the extracellular matrix. For instance, chitin synthetases are present in membranes that produce chitin in extracellular matrices, in mycelia and in arthropod carapaces, for example. Collagen also is produced as other proteins in the endoplasmic reticulum, at the contact point between a ribosome and the reticulum membrane. Glycosylation and triple-helix formation occur later in the Golgi and secretion follows. After cleavage of terminal peptides, collagen molecules assemble into fibrils and form highly ordered matrices, which are stabilized analogues of liquid crystals (as recalled in § 7). A brief passage through a liquid-crystalline phase is not excluded in the first steps of secretion, but the assembling matrix is rapidly gelled by the differentiation of fibrils
Our idea is that liquid crystals could be an essential intermediate state of matter, well devised to accommodate things between the liquid state present in hyaloplasm, say the cytoplasm lying between organelles, and the stabilized state of matrices and even the solid state of minerals in the extrahyaloplasmic compartments, as Golgi vesicles for instance, or in the extracellular spaces. Liquid crystals could be the key of our paradox of the sculptor and the mollusc. The hard tools or the high temperatures used by the sculptor could be replaced by a very special machinery made of a liquid crystal at physiological temperatures, with inserted macromolecules considered as tools in their toolbox. The tools themselves are diverse macromolecules (or macromolecular systems) as receptors, ionic channels pumps, attached ribosomes, or enzymes involved in the production of matrix polymers for instance, or those essential to mineralisation. Most tools are designed to remain attached to their fluid toolbox, since they present amphiphilic properties and are not lost in general, because the liquid crystal is well separated from its aqueous environment, as can be membranes in cells and tissues. Note that certain enzymes are known to be attached to membranes, but are found also in the hyaloplasm (cytosol or cytogel), or in the extracellular matrix, and this can be the case of carbonic anhydrases or alkaline phosphatases, as indicated in § 8. This indicates that some tools, these enzymes, could be released from the fluid toolbox at definite positions in the matrix, possibly with the help of cytoplasmic projections in pore canals, as in the carapace of crabs.
Materials are said to be hybrid when they associate inorganic and organic parts, which strongly interpenetrate at nanoscales, both parts being linked by diverse bond types, covalent, ionic or van der Waals
Another domain of research is essential, that of inorganic liquid crystals discovered by Zocher in 1925 (see
Techniques in molecular genetics were essential in morphogenetic studies and research on (1) defined fibril diameters and inter-distances in the organic matrix; (2) presence of phenoloxidases in the epicuticle and in the twisted layers, just below the epicuticle; (3) very strong twist just below the epicuticle, creating screw dislocations; (4) presence of alkaline phosphatases just below the epicuticle; (5) presence of carbonic anhydrase along interprismatic walls.
These five characters are expressed according to a program defined in the matrix space and in the time of the inter-moult cycle. Points 1 to 5 do not exactly correspond to a chronology. Character 1 is expressed all along the secretion of the fibrous matrix and its expression is modulated according to the level in the cuticle. Character 2 is expressed during secretion of the epicuticle and begins a short time before 1, but stops much before the end of 1. Characters 3 and 4 are expressed at the beginning of the expression of character 1 and character 5 comes after 2, 3 and 4, but stops much before 1. The precise knowledge of the involved molecules and of these steps could provide the best access to a correct interpretation of genetic results.
Mollusc shells also present a subjacent geometry, with a system of coordinates made of intersecting growth lines and conical logarithmic spirals, both visible on the outer periostracum, with its characteristic plywood-like structure. These sharply defined architectures are continued below within the prismatic layer and the nacre, described in clear crystallographic terms. The progressive assembly of these mineralised composites is known for its main aspects and some of the involved proteins and genes are studied today. Similar spatio-temporal programs of mineralisation are expected in most skeletal tissues, as illustrated in this issue by a promising series of results.
There are clear reasons that make biomineralisations very attractive today to geneticists and this can be understood with an example. Great therapeutic perspectives appeared twenty years ago with the progress of molecular techniques. Results accumulate now with large collections of genes, proteins, transcription factors, and some indisputable successes, but not so numerous, and one generally waits for applications. The origin of these difficulties is rather simple: pathologies often lead to slowly destructive effects that remain diffuse, not clearly visible and latent, so that there are neither sharply defined geometries, nor programs. To really grasp the force of these new methods in genetics, we need to apply them to model systems, as morphogenesis, but possibly less complex than the splendid patterns observed in adults of
Thanks to Professor Vic Norris (Rouen University), for his remarks and useful suggestions.