Biomineralization: crystal engineering

a novel approach to

Stephen Mann

This article, describes the main types and functions of biominerals, the biological strategies used in their synthesis, and recent developments in modelling these processes in the laboratory. Emphasis is placed on the understanding of molecular interactions involved in the recognition of mineral surfaces by organic molecules present in the mineralization environment.

Oyster shells, coral, ivory, pearls, sea urchin spines, cuttlefish bone, limpet teeth, magnetic’ crystals in bacteria:- these are just a few of the vast variety of biomineralized materials engineered by living creatures. Many of these biologic- al building materials consist of inorganic minerals intricately combined with organic polymers. Together, these are fashioned into a fascinating variety of shapes and forms which serve many different functions. Not only are biominerals highly refined but the ex- tent of their production is enormous. For example, geological deposits such as the white cliffs of Dover contain billions of exquisite calcium carbonate scales formed within the cells of marine algae. Chemists are interested in how biominerals are made because the abil- ity to produce well-defined inorganic materials is of great value in fields such as ‘catalysis, electronics, magnetism, and ceramics. For materials scientists, biomineralization provides a unique opportunity to study solutions to key problems in mechanical design. Mate- rials such as bones, teeth, and shells are synthesized as complex composites and the organization and interfacial chemis- try of the components are optimized for functional use. Mimicking such struc- tures would be a significant step towards so-called ‘smart’ materials [l, 21. For instance, the fabrication of high strength, ‘macro-defect-free’ cements [3] was much inspired by the high strength and toughness of the nacre (mother-of-pearl) layer of sea shells and a similar perspective could be applied to

S Mann, B.Sc., MSc., D.Phil.

Is Professor of Chemistry in the School of Chemistry, University of Bath, U.K. His research interests are in new aspects of inorganic che- mistry involving crystalline or amorphous materials synthesized in the presence of orga- nic supramolecular assemblies. Much of his work has been inspired by structural studies of biominerals formed within living organisms.

Endowour. NW Beries, Volume 15, No. 3,lBBl. OlBo-9327/w $3.00 + 0.00. @ 1501. Petgamon Press pk. Printed in Great Britain.

the recent production of cheap, tough skeletons of vertebrates comprise cal- ceramics [4]. cium phosphate is unclear. Other, more

esoteric, functions are known. For ex- Biominerals: types and functions ample, the mammalian inner ear con- Crystalline and amorphous biominerals tains hundreds of small calcite crystals are synthesized by a wide range of which act as an inertial mass for the organisms (Table 1) [5, 61. The main detection of changes in linear accelera- minerals are insoluble calcium salts such tion. Single crystals of calcite were also as carbonates and phosphates which are used in the compound eyes of trilobites. utilized in structural support or special- Interestingly, single crystals of calcite ized hard parts (figure 1). Why the are renown for their ability to doubly garden snail uses calcium carbonate to refract white light, suggesting that the support soft tissue whereas the trilobites suffered a life of continual

TABLE 1 THE TYPES AND FUNCTIONS OF THE MAIN INORGANIC SOLIDS FOUND IN BIOLOGICAL SYSTEMS

Mineral Formula Organism/function

Calcium carbonate: Calcite CaCO,* Algae/exoskeletons

Trilobites/eye lens Aragonite CaCO, Fish/gravity device

Molluscs/exoskeleton Vaterite CaCO, Ascidians/spicules Amorphous CaCOBnH20 PlantsKa store

Ca phosphate: Hydroxyapatite Calo(PO&OH12 Vertebrates/

endoskeletons teeth, Ca store

Octa-calcium %J-MPOJs Vertebrates/precursor phosphate phase in bone? Amorphous ? MusselsKa store

Vertebrates/precursor phases in bone?

Calcium oxalate: Whewellite CaC204.H20 PlantsKa store Weddellite CaC2042H20 PlantsKa store

Group IIA metal sulphates: Gypsum CaSO, Jellyfish larvae/gravity

device Barite BaSO., Algae/gravity device Celestite SrSO, Acantharialcellular

support Silicon dioxide: . Silica Si0,nH20 Algae/exoskeletons

Iron oxides: Magnetite WA BacteriaImagnetotaxis

Chitons/teeth Goethite aFeOOH Limpets/teeth Lepidocrocite yFeOOH Chitons (Mollusca) teeth Ferrihydrite 5Fe203.9H20 Animals and plants/Fe

storage proteins

*A range of magnesium-substituted calcites are also formed.

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Figure 1 Scanning electron micrograph of a fractured section of a cuttlefish bone. The cuttlebone is a modified shell that is used as a buoyancy device. It is constructed of calcium carbonate (aragonite) and the polysaccharide, Bchitin. The biomineral is deposited in two distinct but interdependent structures; viz. thin sheets and S-shaped pillars. The mineral lameflae are propped by the crystalline pillars such that a network of chambers are formed throughout the cuttlebone. The number of chambers filled with liquid or gas dictates the density of the material and enables the cuttlefish to maintain a constant depth without the expenditure of metabolic energy. Scale bar is 100 pm. (Photograph courtesy of Mr J. B. A. Walker, Bath University.)

double vision! However, studies of well- preserved fossilized material shows that each crystal is aligned in the eye such that the unique (non-refracting) c axis is perpendicular to the surface of each lens [7]. In this orientation, the calcite lens behaves isotropically like glass and a single well-defined image is formed.

Perhaps bone, more than any other biomineral, reflects the greatest distinc- tion between an inorganic and a bioinorganic solid. For many purposes the calcium phosphate of bone is best thought of as a ‘living mineral’ since it is undergoes continual growth, dissolu- tion, and remodelling. The structure and mechanical properties of bone are derived from the organized mineraliza- tion of hydroxyapatite within a matrix of collagen fibrils, proteoglycans, and many other proteins. The initial sites of mineralization are located within holes between adjacent collagen molecules and the plate-like crystals are crystallog- raphically oriented in parallel arrays across the fibrils. Recent observations have also shown that this level of orga- nization is often coherent across neigh- bouring fibrils and that this long range order may be responsible for much of the unusual fracture properties of bone PI.

Whilst the Ca-containing biominerals are common artefacts of most

seashores, iron-containing biominerals are less well known. However, it turns out that these biominerals are in fact almost as widespread as their Ca- containing counterparts. In particular, the formation of magnetite (FeRO,) in magnetotactic bacteria is a feature of most water/sediment environments [9]. These organisms synthesize a chain of intracellular magnetic particles which they use to navigate in the earth’s magnetic field [lo]. Very recently, new types of magnetotactic bacteria have been discovered [ll-131. These organ- isms live in sulphide-rich environments and synthesize discrete crystals of the magnetic mineral, greigite (Fe&) (figure 2). These bacteria are interesting because they represent the first known examples of controlled iron sulphide biomineralization. Moreover, as sul- phide rather than oxide chemistry is likely to have been dominant in the early stages of the Earth’s history, the formation of intracellular bacterial iron sulphides could be representative of an ancient process in which inorganic materials were adapted to specific biolo- gical functions.

Other iron oxides, such as goethite (@eOOH) and lepidocrocite (/3- FeOOH), are deposited in the teeth of certain molluscs. For example, the com- mon limpet is armed with sabre-like

rust-coloured goethite teeth. During feeding, these hardened structures are rasped across rocks encrusted with algae. In certain species, the chitons, the teeth contain both lepidocrocite and magnetite, and are therefore magnetic! Another important and widespread iron oxide is the hydrated mineral called ferrihydrite. This is the brown gelati- nous precipitate that is readily formed in a test tube by the addition of sodium hydroxide to an iron (III) solution. The iron storage protein, ferritin, contains a 5 nm central core of this mineral wrap- ped up in a protein coat. Encapsulation of the mineral in this way protects the organism from the all-familiar problem of rusting.

The main amorphous biomineral is silica (Table 1). Many unicellular organ- isms synthesize elaborate exoskeletons from this material. In plants, the pre- sence of a large number of silica spines and nodules presents an unpalatable meal to a discerning predator. In some plants such as the horsetail, the level of silica is extremely high, being of the order of 20-25 per cent of the dry weight. The dried plant is essentially akin to sandpaper and was used (so the story goes) by the early American pioneers as an effective means of clean- ing teeth.

Control of biomineralization The formation of a complex material such as a sea urchin spine involves many features of control at the site of miner- alization. The site must be delineated from the surrounding biological en- vironments, activated at specific times in the life of the organism, constrained in size and shape, and highly regulated with respect to the chemistry of the mineralization process. The principal processes to be controlled are nuclea- tion and crystal growth, both of which are critically dependent on the level of supersaturation of the medium and the nature of the molecular interactions be- tween the biomineral and associated organic macromolecules.

There are three main sites of mineral deposition: on the surface of the cell, within the cell, and outside the cell. In bacteria, minerals are generally formed on or within the polysaccharide sheath that surrounds the cell membrane. Metabolic products (for example NHs, OH-, S*-) pass through the cell wall and combine with extraneous cations (Cu*+, Zn*+, Pb*+ etc.) to give supersaturated solutions from which the minerals are precipitated. Under these conditions, the biominerals are disorganized and have particle shapes and sizes similar to those observed under conditions of sim- ple inorganic precipitation. In contrast, intracellular biomineralization shows a high level of biological control. Crystals are sculpted into elaborate shapes and are organised with respect to their

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Figure 2 Transmission electron micrograph of a magnetotactic bacterium containing magnetic crystals of greigite (Fe&). The inclusions are discrete single crystals of narrow size distribution. They have species-specific morphologies - for example, cube-octahedral or rectangular prismatic - and appear to be crystallographically aligned within chains. In one species, pyrite (FeS,) crystals are associated with the greigite particles. Clearly, these processes are under strict biological control. Scale bar = 0.5 pm. (Photograph courtesy of Dr B. R. Heywood. Bath University.)

crystallographic axes. The controlling factor is the presence of an organic substrate. Often this is a surrounding membrane which controls the direction of crystal growth by limiting the space made available to the development of the mineral. Internal stresses generated by micro-filaments within the cell can induce shaping of the membrane, which then

inorganic and organic components re- spectively. In particular, the close che- mical association of the components gives rise to a structure which is virtually free of voids. This produces a material of much higher tensile strength compared with the pure inorganic mineral.

The molecular details of how organ-

isms achieve this level of control over mineralization are still to be worked out. It is clear that one of the most important facts is the nature of the molecular interactions which take place at the interface between organic macro- molecules and ions in the crystal sur- faces of biominerals. One important role of these organic molecules lies in their ability to control nucleation. Orga- nized surfaces of these macromolecules can act as catalysts and molecular tem- plates for oriented crystallization. For example, there may be a close match between the distances of adjacent func- tional groups arranged on a ppleated sheet protein and those separating Ca ions in a specific face of calcium carbon- ate (figure 3). A similar relationship can be considered for biominerals formed at the surfaces of close-packed phospholi- pid membranes.

Mimicking biomineralization: molecular recognition and crystal engineering From the above discussion it should be clear that the fundamental distinction between inorganic and bioinorganic minerals lies with the enhanced crystal chemical specificity of the biogenic pro- ducts. Although biominerals have re- latively simple inorganic structures compared with those frequently en- countered in synthetic solid state che- mistry, the level of engineering is often much higher. Crystal structure, size, shape, orientation, and assembly are under precise control. How do we mimic these processes? Three approaches are currently under inves- tigation: (i) the use of phospholipid vesicles and micelles to constrain the particle size by membrane encapsula- tion; (ii) the application of growth mod- ifiers to modify the crystal morphology:

acts as an organic mould for the casting of the crystal morphology. In this way, the organic patterning overides the conven- X

tional crystallographic symmetry of the inorganic mineral and many exquisite R R R R R

shapes can be engineered. Extracellular mineralization occurs

primarily in higher animals and plants. //

In these organisms, specialised cells are 1’ involved in assembling macroscopic structures such as shells, bones, and teeth. Again, the controlling factor lies in the ability of an organic matrix to organize and regulate mineral deposi- tion. Constructing a shell is very much like building a wall. Bricks of the in- -y- organic material, calcium carbonate, are precisely assembled into regular stacks across the entire shell. A mortar of organic polymers consisting of pro- Figure 3 Representation of geometric matching at a mineral-protein interface.

teins and polysaccharides glue each Cation-cation distances in the unit cell (v) are commensurate with the spacing of

mineral particle into position. The result- negatively charged binding sites on the ppleated sheet protein (x1. Matching can be in one or two dimensions. In mollusc shells, there is a close match between the

ingcomposite material has acombination Ca-Ca distance along the a axis (4.7 A) and the periodicity of ne atively charged of the strength and elasticity of the aspartic acid residues in the /?-pleated sheet protein sheet (4.96 1 1.

122

t Hz SMaz S

lb)

Figure 4 Schematic representation of the use of the protein, ferritin, in the synthesis of nanometre-size inorganic materials 1161. Ferritin is an iron storage protein that consists of a polypeptide shell and a iron oxide core. The core is formed by specific transport of ions through molecular channels in the protein shell. In the laboratory, the iron oxide core can be converted to a iron sulphide particle by in situ reactions with HzS gas (A). Alternatively, the iron oxide can be removed by reductive dissolution to give apoferritin with an empty cavity. This can then be reconstituted with other inorganic materials such as Mn oxides (B) or uranyl oxides (C).

and (iii) the use of organized organic surfaces to induce oriented nucleation.

Unilamellar vesicles can be readily prepared by ultrasonication of aqueous dispersions of phospholipids. The vesi- cles have a bilayer membrane enclosing an internal aqueous volume often only 20-50 nm in diameter. When formed in the presence of metal ions, the internal space contains encapsulated species which can subsequently undergo in situ precipitation reactions with membrane- permeable species such as OH- and H$. In effect, this is solid state chemis- try in a very small soap bubble. Mate- rials such as metal oxides, sulphides, and phosphates have been prepared as nanometre-size intravesicular particles [ 14, 151. The curved organic membrane has a significant influence on these crys- tallization reactions. For example, changes in crystal structure, size, and morphology are observed compared with analogous reactions undertaken in the absence of the vesicles. Recently, we have shown that a similar approach can be applied to the synthesis of nanophase materials using the protein shell of ferritin [16] (figure 4).

Another approach is to use tailor- made additives that modify the crystal shape through specific interactions of the adsorbed molecules at the crystal faces. Many of the soluble macro- molecules associated with biominerals are considered to act in this manner. For example, acidic glycoproteins ex- tracted from adult sea urchin tests have been shown to bind specifically to the {iio) faces of synthetic calcite [ 171. As many biological molecules con- tain oxyanion functional groups such as carboxylate, phosphate, and sulphate esters, it is interesting to investigate the morphological effect of low molecular weight analogues of these macro- molecules in in vitro crystallization ex- periments. Calcite crystals grown in aqueous solutions containing variable levels of functionalized and non- functionalized a,cu-dicarboxylates have elongated spindle-shaped habits com- pared with the rhombohedral shape of unmodified crystals (figure 5) [18]. A similar effect was observed for crystals grown in the presence of inorganic

phosphate and, to a lesser extent, sul- phate. These morphological changes are analogous to those observed for calcite crystals in solutions containing the sea urchin macromolecules suggest- ing that some of the functional groups in these biological molecules can interact with the calcite lattice in a similar man- ner to the low molecular weight deriva- tives.

A number of different experimental strategies have been developed to eluci- date the mechanisms of inorganic crys- tallization on organized templates. A biological approach has been to isolate the matrix components of mineralized tissue such as bones, teeth and shells, and examine the nucleation of insoluble Ca salts in the presence of these macro- molecules. Studies using extracted mol- lust shell proteins adsorbed onto glass substrates, suggested that stereochemic- al and electrostatic factors were impor- tant [19]. Recent investigations have modelled these processes by using com- pressed films of surfactant molecules formed at the air/water interface as

Figure 5 Scanning electron micrograph of a calcite crystal modified in the presence of the additive, y-carboxyglutamic acid ((HOOC)2CHCH,CH(NH,)COOH). The molecule is highly charged at pH 6 and interacts with one specific set of crystal surfaces. The result is that these surfaces are selectively stabilized and appear in the morphology of the modified crystals. The smooth surfaces in the photograph are the only faces seen in unmodified crystals, whereas the textured faces correspond to those with adsorbed additive molecules. An understanding of the molecular recognition between additives and crystal faces will enable crystals of precise shape to be reproducibly synthesized. Scale bar is 10 pm. (Photograph courtesy of Mr J. M. Didymus, Bath University.)

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l-l WB

ca& + 2HCO& Z CaCC& + C4u + H, 90

--cl PH

Figure 6 Experimental procedure for the growth of CaC03 crystals under compressed Langmuir monolayers. Monolayer films of stearic acid (CHs(CHz),&OOH), octadecylamine (CH3(CH2),,NH2), octadecanol (CH3(CH2),,0H), and cholesterol (C27H450H) have been studied ‘120, 211. Individual surfactants were spread on to the air/water interface of supersaturated calcium bicarbonate solutions by evaporation of chloroform solutions. The resulting monolayer was compressed by a constant perimeter barrier device (CPBD) to specific values of surface pressure as measured by a Wilhelmy balance (WB). Crystals nucleated under the monolayer were studied in situ or removed by dipping glass slides through the air/water interface. The crystals were then studied by a range of physical techniques including optical microscopy (OM); scanning electron microscopy (BEM); high resolution transmission electron microscopy (HRTEM); electron diffraction (ED); and X-ray diffraction (XRD).

organized templates for calcium carbon- ate nucleation (figure 6) [20, 211. With- out the monolayer, a random mass of rhombohedral calcite crystals forms at the solution surface. When a monolayer of stearic acid is present, however, dis- crete oriented crystals of calcite are nucleated under the organic film (figure 7). The crystals are aligned at the mono- layer surface with a specific face, inde- xed as {lTO}, parallel to the plane of the organic film. Changing the surfac- tant headgroup from the negatively charged carboxylate of stearic acid to the positively charged amine group of octadecylamine results in a dramatic change in the crystal structure. Under these conditions, oriented crystals of the metastable mineral, vaterite, are nucleated. Although more work is needed, it seems clear that at least three factors are responsible for the oriented nucleation of calcite: (i) the extent of electrostatic interaction of Ca ions with the carboxylate headgroups; (ii) match- ing of the 58, spacing between adjacent surfactant headgroups with Ca-Ca dis- tances in the { 110) face of calcite; and (iii) complementarity in the stereochemistry of the monolayer carboxylates and car- bonate anions in the { liO} crystal face.

Outlook Perhaps the most important aspect of the above investigations is that they have highlighted the potential for mole- cular recognition at interfaces compris- ing inorganic and organic surfaces. Rec-

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ognition processes are a central feature of biochemical reactions although they have in the past been associated only with molecules in solution. The exten- sion of this concept to molecules in- teracting on the surfaces of inorganic materials is of key importance not only in explaining many biomineralization processes but in the development of new approaches in crystal engineering.

The outlook for biomimicking looks promising [22]. For example, one can envisage the production of tailor-made polymeric surfaces for the organized deposition of oriented films of ultra-fine inorganic materials. Organized three- dimensional inorganic-organic compo- sites could be synthesized by a similar approach. Alternatively, an under- standing of recognition processes could enable precise modifications in crystal shape to be engineered through the use of specifically designed molecular addi- tives.

Finally, whatever the future tech- nological prospects, the integration of biology and solid state science repre- sents a new paradigm in basic scientific research. For example, there are many biological concepts such as self- assembly, dynamics, feedback, and re- modelling that one could foresee as being integral to the study of advanced inorganic materials. Many of the future challenges lie in the unlocking of our imagination from the confines of con- ventional disciplines. Only by this approach can we access the door to new scientific horizons.

Acknowledgments I wish to thank my coworkers Dr B. R. Heywood, J. M. Didymus, F. C. Mel- drum, S. Rajam, V. J. Wade, and J. B. A. Walker for experimental data de- scribed in this article.

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Figure 7 Optical micrograph of oriented calcium carbonate crystals nucleated under a compressed surfactant monolayer of stearic acid. The close packed organic molecules act as a molecular template for the alignment of crystal nuclei forming from supersaturated solution. (Photograph courtesy of Dr B. R. Heywood, Bath University.)

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