1.1 Hard-Tissue Biomineralisation: How Nature Works
The bones and teeth of all vertebrates are natural composite materials (Figure 1.1), where one of the components is an inorganic nanocrystalline solid with apatite structure and the chemical composition of a carbonated, basic calcium phosphate, hence it can be termed a carbonate-hydroxy-apatite. It amounts to 65% of the total bone mass, with the remaining mass formed by organic matter and water.1
Most of the biominerals are inorganic/organic composite materials.2 In this sense, the bones of vertebrates are also formed by the combination of an inorganic calcium phase – carbonate-hydroxyl-apatite – and an organic matrix.3 The benefits that the inorganic part brings to this combination are toughness and the ability to withstand pressure.
On the other hand, the organic matrix formed by collagen fibres, glycoproteins and mucopolysaccharides, provides elasticity and resistance to stress, bending and fracture. Such symbiosis of two very different compounds, with markedly different properties, confers to the final product, i.e. the biomineral, some properties that would be unattainable for each of its individual components per se. This is a fine example in Nature of the advantages that a composite material can exhibit, in order to reach new properties with added value. In fact due to this evidence, a large portion of the modern materials science field is currently focused on the development of composite materials.
1.1.1 Bone Formation
The bone exhibits some physical and mechanical properties that are rather unusual. It is able to bear heavy loads, to withstand large forces and to flex without fracture within certain limits. Besides, the bone also acts as an ion buffer both for cations and anions. From the material point of view, the bone could be simplified as a three-phase material formed by organic fibres, an inorganic nanocrystalline phase, and a bone matrix. Its unique physical and mechanical properties are the direct consequence of intrinsic atomic and molecular interactions within this very particular natural composite material.
Bone is not uniformly dense. It has a hierarchical structure. Due to its true organic-inorganic composite nature, it is able to adopt different structural arrangements with singular architectures, determined by the properties required from it depending on its specific location in the skeleton. Generally speaking, most bones exhibit a relatively dense outer layer, known as cortical or compact bone, which surrounds a less dense and porous, termed trabecular or spongy bone, which is in turn filled with a jelly tissue: the bone marrow.4 This complex tissue is the body deposit of nondifferentiated trunk cells, precursors of most repairing and regenerating cells produced after formation of the embryonic subject.5,6 The bone fulfils critical functions in terms of a structural material and an ion reservoir. Both functions strongly depend on the size, shape, chemical composition and crystalline structure of the mineral phase, and also on the mineral distribution within the organic matrix.
The main constituents of bone are: water; a mineral phase, calcium phosphate in the form of carbonated apatite with low crystallinity and nanometric dimensions, which accounts for roughly two thirds of the bone's dry weight; and an organic fraction, formed of several proteins, among which type-I collagen is the main component, which represents approximately the remaining one third of bone dry weight. The other intervening proteins, such as proteoglicans and glycoproteins, total more than two hundred different proteins, known as noncollagen proteins; their total contribution to the organic constituent, however, falls below 10% of the said organic fraction. These bone constituents are hierarchically arranged with, at least, five levels of organisation. At the molecular level, the polarised triple helix of tropocollagen molecules are grouped in microfibres, with small cavities between their edges, where small apatite crystals – approximately 5 nm × 30 nm sized – nucleate and grow. These microfibres unite to form larger fibres that constitute the microscopic units of bone tissue. Then, these fibres are arranged according to different structural distributions to form the full bone.7
It was traditionally believed that the inorganic phase was mainly amorphous calcium phosphate that, in the ageing process, evolved towards nanocrystalline hydroxyapatite. Results of solid-state 31P NMR spectroscopy, however, showed that the amorphous phase is never present in large amounts during the bone development process.6 Besides, this technique did detect acid phosphate groups. Phosphate functions correspond to proteins with O-phosphoserine and O-phosphotreonine groups, which are probably used to link the inorganic mineral component and the organic matrix. Phosphoproteins are arranged in the collagen fibres so that Ca2+ can be bonded at regular intervals, in agreement with the inorganic crystal structure, hence providing a repeating condition that leads to an ordered sequence of the same unit, i.e. the crystallinity of the inorganic phase. The cells responsible for most of the assembling process are termed osteoblasts. When the main assembling process is completed, the osteoblasts keep differentiating in order to form osteocytes, which are responsible for the bone maintenance process. The controlled nucleation and growth of the mineral take place at the microscopic voids formed in the collagen matrix. The type-I collagen molecules, segregated by the osteoblasts, are grouped in microfibres with a specific tertiary structure, exhibiting a periodicity of 67 nm and 40 nm cavities or orifices between the edges of the molecules.7 These orifices constitute microscopic environments with free Ca2+ and PO ions, as well as groups of side chains eligible for bonding, with a molecular periodicity that allows the nucleation of the mineral phase in a heterogeneous fashion. Ca2+ ions deposited and stored in the skeleton are constantly renewed with dissolved calcium ions. The bone growth process can only be produced under a relative excess of Ca2+ and its corresponding anions, such as phosphates and carbonates, at the bone matrix. This situation is achieved due to the action of efficient ATP-powered ionic pumps, such as Ca2+ ATPases for active transportation of calcium.8–10 In terms of physiology, carbonate and phosphate are present in the form of HCO, HPO and H2PO anions. When incorporated to the bone, the released protons can move throughout the bone tissue and leave the nucleation and mineralisation area. The nucleation of thin, platelet-shaped apatite crystals, takes place at the bone within discrete spaces inside the collagen fibres, hence restricting a potential primary growth of these mineral crystals, and imposing their discrete and discontinuous quality (Figure 1.2).
Calcium phosphate nanocrystals in bone, formed at the mentioned spaces left between the collagen fibres, exhibit the particular feature of being monodispersed and nanometre-sized platelets of carbonate-hydroxyl-apatite. There is no other mineral phase present, and the crystallographic axis c of these crystals is arranged parallel to the collagen fibres and to the largest dimension of the platelet. In the mineral world, the thermodynamically stable form of calcium phosphate under standard conditions is the hydroxyapatite (HA).11 Generally speaking, this phase grows in needle-like forms, with the c-axis parallel to the needle axis. Figure 1.3 shows the crystalline structure of hydroxyapatite, Ca10(PO4)6(OH)2, which belongs to the hexagonal system, space group P63/m and lattice parameters a = 9.423 Å and c = 6.875 Å.
Besides the main ions Ca2+, PO and OH−, the composition of biological apatites always includes CO at approximately 4.5%, and also a series of minority ions, usually including Mg2+, Na+, K+, Cl−, F−.12 These substitutions modify the lattice parameters of the structure as a consequence of the different size of the substituting ions, as depicted in Figure 1.3. This is an important difference between minerals grown in an inorganic or biological environment.
The continuous formation of bone tissue is performed at a peripheral region, formed by an external crust and an internal layer with connective tissue and osteoblast cells. These osteoblasts are phosphate-rich and exude a jelly-like substance, the osteoid. Due to the gradual deposit of inorganic material, this osteoid becomes stiffer and the osteoblasts are finally confined and transformed in bone cells, the osteocytes. The bone-transformation mechanism, and the ability to avoid an excessive bone growth, are both catered for by certain degradation processes that are performed simultaneously to the bone formation. The osteoclasts, which are giant multinucleated cells, are able to catabolyse the bone purportedly using citrates as chelating agent. The control of the osteoclast activity is verified through the action of the parathyroid hormone, a driver for demineralisation, and its antagonist, tireocalcitonin.
The collagen distribution with the orifices previously described is necessary for the controlled nucleation and growth of the mineral, but it might not suffice. There are conceptual postulations of various additional organic components, such as the phosphoproteins, as an integral part of the nucleation core and hence directly involved in the nucleation mechanism. Several immuno-cyto-chemical studies of bone, using techniques such as optical microscopy and high-resolution electron microscopy, have clearly shown that the phosphoproteins are restricted or, at least, largely concentrated at the initial mineralisation location, intimately related to the collagen fibres. It seems that the phosphoproteins are enzymatically phosphored previously to the mineralisation.13
The crystallisation of the complex and hardly soluble apatite structures evolves favourably through the kinetically controlled formation of metastable intermediate products. Under in vitro conditions, amorphous calcium phosphate is transformed into octacalcium phosphate (OCP) that, in turn, evolves to carbonate hydroxyapatite; at lower pH values, the intermediate phase seems to be dehydrated dicalcium phosphate (DCPD).14,15
The mechanisms of bone formation are highly regulated processes,7 which seem to verify the following statements:
Mineralisation is restricted to those specific locations where crystals are constrained in size by a compartmental strategy.
The mineral formed exhibits specific chemical composition, crystalline structure, crystallographic orientation and shape. The chemical phase obtained is controlled during the stages of bone formation. In vertebrates, said chemical phase is a hydroxyl-carbonate-apatite, even though the thermodynamically stable form of calcium phosphate in the world of minerals, under standard conditions, is hydroxyapatite.
Since the mineral deposits onto a biodegradable organic support, complex macroscopic forms are generated with pores and cavities. The assembling and remodelling of the structure are achieved by cell activity, which builds or erodes the structure layer by layer.
Without a careful integration of the whole process, bone formation would be an impossible task. The slightest planning mistake by the body, for instance in its genetic coding or cell messengers, is enough to provoke building errors that would weaken the osseous structure.
The hard tissues in vertebrates are bones and teeth. The differences between them reside in the amounts and types of organic phases present, the water content, the size and shape of the inorganic phase nanocrystals and the concentration of minor elements present in the inorganic phase, such as CO32−, Mg2+, Na+, etc.12 The definitive set of teeth in higher-order vertebrates has an outer shell of dental enamel that, in an adult subject, does not contain any living cells.16 Up to 90% of said enamel can be inorganic material, mainly carbonate-hydroxyl-apatite. Enamel is the material that undergoes more changes during the tooth development process. At the initial stage, it is deposited with a mineral content of only 10–20%, with the remaining 80–90% of proteins and special matrix fluids. In the subsequent development stages, the organic components of the enamel are almost fully replaced by inorganic material. The special features of dental enamel when compared with bone material are its much larger crystal domains, with prismatic shapes and strongly oriented, made of carbonate-hydroxyl-apatite (Figure 1.4). There is no biological material that could be compared to enamel in terms of hardness and long life. However, it cannot be regenerated.
The bones, the body-supporting scaffold, can exhibit different types of integration between organic and inorganic materials, leading to significant variations in their mechanic properties. The ratio of both components reflects the compromise between toughness (high inorganic content) and resiliency or fracture strength (low inorganic content). All attempts to synthesise bone replacement materials for clinical applications featuring physiological tolerance, biocompatibility and long-term stability have, up to now, had only relative success; which shows the superiority and complexity of the natural structure where, for instance, a human femur can withstand loads of up to 1650 kg.17
The bones of vertebrates, as opposed to the shells of molluscs, can be considered as “living biominerals” since there are cells inside them under permanent activity. It also constitutes a storage and hauling mechanism for two essential elements, phosphorus and calcium, which are mainly stored in the bones. Most of what has been described up to this point, regarding the nature of bone tissue, could be summed up by stating that the bone is a highly structured porous matrix, made of nanocrystalline and nonstoichiometric apatite, calcium deficient and carbonated, intertwined with collagen fibres and blood vessels.
Bone functions are controlled by a series of hormones and bone-growth factors. Figure 1.5 attempts to depict these phenomena in a projection from our macroscale point of view, to the “invisible” nanoscale.
Bone's rigidity, resistance and toughness are directly related to its mineral content.18 Although resistance and rigidity increase linearly with the mineral content, toughness does not exhibit the same trend, hence there is an optimum mineral concentration that leads to a maximum in bone toughness. This tendency is clearly the reason why the bone exhibits a restricted amount of mineral within the organic matrix. But there are other issues affecting the mechanical properties of bone, derived from the microstructural arrangement of its components. In this sense, the three main components of bone exhibit radically different properties. From this point of view, the biomineral is clearly a composite.19 The organic scaffold exhibits a fibrous structure with three levels: the individual triple helix molecules, the small fibrils, and its fibre-forming aggregates. These fibres can be packed in many different ways; they host the platelet-shaped hydroxyl-carbonate-apatite crystals. In this sense, the bone could be described as a composite reinforced with platelets, but the order–disorder balance determines the microstructure and, as a consequence, the mechanical properties of each bone. In fact, bones from different parts of the body show different arrangements, depending on their specific purpose.
Bone crystals are extremely small, with an average length of 50 nm (in the 20–150 nm range), 25 nm in average width (10–80 nm range) and thickness of just 2–5 nm. As a remarkable consequence, a large part of each crystal is surface; hence their ability to interact with the environment is outstanding.
Apatite phase contains between 4 and 8% by weight of carbonate, properly described as dahllite. Mineral composition varies with age and it is always calcium deficient, with phosphate and carbonate ions in the crystal lattice. The formula Ca8.3(PO4)4.3(CO)3x(HPO4)y(OH)0.3 represents the average composition of bone, where y decreases and x increases with age, while the sum x + y remains constant and equal to 1.7.12 Mineral crystals grow under a specific orientation, with the c-axes of the crystals approximately parallel to the long axes of the collagen fibres where they are deposited. Electron microscopy techniques were used to obtain this information.20
The bones are characterised by their composition, crystalline structure, morphology, particle size and orientation. The apatite structure hosts carbonate in two positions: the OH− sublattice producing so-called type A carbonate apatites or the [PO4]3− sublattice (type B apatites) (Figure 1.6).
The small apatite crystal size is a very important factor related to the solubility of biological apatites when compared with mineral apatites. Small dimensions and low crystallinity are two distinct features of biological apatites that, combined with their nonstoichiometric composition, inner crystalline disorder and presence of carbonate ions in the crystal lattice, allow their special behaviour to be explained.
Apatite structure allows for wide compositional variations, with the ability to accept many different ions in its three sublattices (Figure 1.7).
Biological apatites are calcium deficient; hence their Ca/P ratio is always lower than 1.67, which corresponds to a stoichiometric apatite. No biological hydroxyapatite shows a stoichiometric Ca/P ratio, but they all move towards this value as the organism ages, which are linked to an increase in crystallinity. These trends have a remarkable physiological meaning, since the younger, less-crystalline tissue can develop and grow faster, while storing other elements that the body needs during its growth; this is due to the highly nonstoichiometric quality of HA, which caters for the substitutional inclusion of different amounts of several ions, such as Na+, K+, Mg2+, Sr2+, Cl−, F−, HPO, etc.21 (Figure 1.8).
Two frequent substitutions are the inclusion of sodium and magnesium ions in calcium lattice positions. When a magnesium ion replaces a calcium ion, the charge and position balance is unaffected. If a sodium ion replaces a calcium ion, however, this balance is lost and the electrical neutrality of the lattice can only be restored through the creation of vacancies, therefore increasing the internal disorder.
The more crystalline the HA becomes, the more difficult interchanges and growth are. In this sense, it is worth stressing that the bone is probably a very important detoxicating system for heavy metals due to the ease of substitution in apatites; heavy metals, in the form of insoluble phosphates, can be retained in the hard tissues without significant alterations of their structural properties.
However, the ability to exchange ions in this structure is not a coincidence. Nature designed it, and the materials scientist can use it as a blueprint to design and characterise new and better calcium phosphates for certain specific applications. It is known that the bone regeneration rate depends on several factors such as porosity, composition, solubility and presence of certain elements that, released during the resorption of the ceramic component, facilitate the bone regeneration carried out by the osteoblasts. Thus, for instance, small amounts of strontium, zinc or silicates stimulate the action of these osteoblasts and, in consequence, the new bone formation. Carbonate and strontium favour the dissolution, and therefore the resorption of the implant.12 Silicates increase the mechanical strength, a very important factor in particular for porous ceramics, and also accelerate the bioactivity of apatite.22 The current trend is, therefore, to obtain calcium phosphate bioceramics partially substituted by these elements. In fact, bone and enamel are some of the most complex biomineralised structures. The attempts to synthesise bone in the laboratory are devoted at obtaining biocompatible prosthetic implants, with the ability to leverage natural bone regeneration when inserted in the human body. Its formation might imply certain temporary structural changes on its components, which demand in turn the presence, at trace levels, of additional ions and molecules in order to enable the mineralisation process. This is the case, for instance, with bone growth processes, where the localised concentration of silicon-rich materials coincides precisely with areas of active bone growth. The reason is yet unknown, although the evidence is clear; the possible explanation of this phenomenon would also justify the great activity observed in certain silicon-substituted apatite phases and in some glasses obtained by sol-gel method, regarding cell proliferation and new bone growth.
1.1.2 A Discussion on Biomineralisation
Biomineralisation is the controlled formation of inorganic minerals in a living body; said minerals might be crystalline or amorphous, and their shape, symmetry and ultrastructure can reach high levels of complexity. Bioinorganic solids have been replicated with high precision throughout the evolution process, i.e. they have been reproduced identically to the primitive original. As a consequence, they have been systematically studied in the fields of biology and palaeontology. However, the chemical and biochemical processes of biomineralisation were not studied until quite recently. Such studies are currently providing new concepts in materials science and engineering.17
Biomineralisation studies the mineral formation processes in living entities. It encompasses the whole animal kingdom, from single-cell species to humans. Biogenic minerals are produced in large scale at the biosphere, their impact in the chemistry of oceans is remarkable and they are an important component in sea sediments and in many sedimentary rocks.
It is important to distinguish between mineralisation processes under strict biological – genetic – control, and those induced by a given biological activity that triggers a fortuitous precipitation. In the first case, these are crystal-chemical processes aimed at fulfilling specific biological functions, such as structural support (bones and shells), mechanical rigidity (teeth), iron storage (ferritin) and magnetic and gravitational navigation, while in the second case there are minerals produced with heterogeneous shapes and dimensions, which may play different roles in the increase of cell density or as means of protection against predators.23
At the nanometre scale, biomineralisation implies the molecular building of specific and self-assembled supramolecular organic systems (micelles, vesicles, etc.) which act as an environment, previously arranged, to control the formation of inorganic materials finely divided, of approximately 1 to 100 nm in size (Figure 1.9). The production of consolidated biominerals, such as bones and teeth, also requires the presence of previously arranged organic structures, at a higher length scale (micrometre).
The production of discrete or expanded architectures in biomineralisation frequently includes a hierarchical process: the building of organic assemblies made of molecules confers structure to the synthesis of arranged biominerals, which act in turn as preassembled units in the generation of higher-order complex microstructures. Although different in complexity, bone formation in vertebrates (support function) and shell formation in molluscs (protection function) bear in common the crystallisation of inorganic phases within an organic matrix, which can be considered as a bonding agent arranging the crystals in certain positions in the case of bones, and as a bonding and grouping agent in shells (Figure 1.10).
Our knowledge of the most primitive forms of life is largely based upon the biominerals, more precisely in fossils, which accumulated in large amounts. Several mountain chains, islands and coral reefs are formed by biogenic materials, such as limestone. This vast bioinorganic production during hundreds of millions of years has critically determined the development conditions of life.24 CO2, for instance, is combined in carbonate form, decreasing initially the greenhouse effect of the earth's crust. Leaving aside the shells, teeth and bones, there are many other systems that can be classified as biominerals: aragonite pellets generated by molluscs, the outer shells and spears of diatomea, radiolarian and certain plants, crystals with calcium, barium and iron content in gravity and magnetic field sensors formed by certain species, and the stones formed in the kidney and urinary system, although the latter are pathological biominerals. The protein ferritin, responsible for iron storage, can also be considered a biomineral, taking into account its structure and inorganic content.
Bones, horns and teeth perform very different biological functions and their external shapes are highly dissimilar. But all of them are formed by many calcium phosphate crystals, small and isolated, with nonstoichiometric carbonate-hydroxyl-apatite composition and structure, grouped together by an organic component. Nucleation and growth of the mineral crystals is regulated by the organic component, the matrix, segregated in turn by the cells located near the growing crystals (Figure 1.11).
This matrix defines the space where the mineralisation shall take place. The main components of the organic matrix are cellulose, in plants, pectin in diatomea, chitin and proteins in molluscs and arthropods, and collagen and proteoglycans in vertebrates.
1.1.3 Biomineralisation Processes
Different levels of biomineralisation can be distinguished, according to the type and complexity of the control mechanisms. The most primitive form corresponds to biologically induced biomineralisation, which is mainly present in bacteria and algae.23 In these cases, biominerals are formed by spontaneous crystallisation, due to supersaturation provoked by ion pumps, and then polycrystalline aggregates are formed in the extracellular space. Gases generated in the biological processes, by bacteria for instance, can (and often do) react with metal ions from the environment to form biomineral deposits.
More complex mechanisms involve processes with higher biological control. The obtained, well-defined bioinorganic products are formed by inorganic and organic components. The organic phase is usually made of fibrous proteins, lipids or polysaccharides, and its properties will affect the resulting morphology and the structural integrity of the composite.
Whatever the case, the formation of an inorganic solid from an aqueous solution is achieved with the combination of three main physicochemical stages: supersaturation, nucleation and crystal growth.
Nucleation and crystal growth are processes that take place in a supersaturated medium and must be properly controlled in any mineralisation process. A living body is able to mineralise provided that there are well-regulated and active transport mechanisms available. Some examples of transport mechanisms are ion flows through membranes, formation or dissociation of ion complexes, enzyme-catalysed gas exchanges (CO2, O2 or H2S), local changes of redox potential or pH, and variations in the medium's ionic strength. All these factors allow for creating and maintaining a supersaturated solution in a biological environment.23
Nucleation is related to kinetics of surface reactions such as cluster formation, growth of anisotropic crystals and phase transformations. In the biological world, however, there are certain surface structures that specifically avoid an unwanted nucleation, such as those exhibited by some kinds of fish in polar waters to avoid ice formation in body fluids.
The growth of a crystal or amorphous solid from a phase nucleus can be directly produced by the surrounding solution or by a continuous contribution of the required ions or molecules. Besides, diffusion can be drastically altered by any significant change in viscosity of said medium.
The controlled growth of biominerals can be also produced by a sequence of stages, through phase transformations or by intermediate precursors that lead to the solid-state phase.
Biomineralisation processes can be classified in two large groups; the first one includes those phenomena where it seems some kind of control exists over the mineralisation process, while the second one encompasses those where said control seems to be nonexistent. According to Mann,23 biomineralisation processes can be described as biologically induced when said biomineralisation is due to the withdrawal of ion or residual matter from cells, and is verified in an open environment, i.e. not in a region purposely restricted. It is produced as a consequence of a slight chemical or physical disturbance in the system. The crystals formed usually give rise to aggregates of different sizes, with similar morphology to mineral inorganic crystals. Besides, the kind of mineral obtained depends on both the environmental conditions of the living organism and on the biological processes involved in the formation, since the same organism is able to produce different minerals in different environments. This is particularly so in single-cell species, although some higher-order species also verify this behaviour.
There are, however, situations where a specific mechanism is acting, which are then described as biologically controlled. An essential element of this process is the space localisation, whether at a membrane-closed compartment, or confined by cell walls, or by a previously formed organic matrix. The biologically controlled process of formation of biomaterials can be considered as the opposite to a biologically induced process. It is much more complex and implies a strict chemical and structural control.
Most biominerals formed under controlled conditions precipitate from solutions that are in turn controlled in terms of composition by the cells in charge; hence the contents of trace elements and stable isotopes in many mineralised areas are not balanced with the concentrations present in the initial medium.
Nucleation in controlled biomineralisation requires low supersaturation combined with active interfaces. Supersaturation is regulated by ion transport and processes involving reaction inhibitors and/or accelerators. The active interfaces are generated by organic substrates in the mineralisation area. Molecules present in the solution can directly inhibit the formation of nuclei from a specific mineral phase, hence allowing the growth of another phase.
Crystal growth depends on the supply of material to the newly formed interface. Low supersaturation conditions will favour the decrease in number of nuclei and will also restrict secondary nucleation, limiting somehow the disorder in the crystal phase. Under these low supersaturation conditions, growth rate is determined by the rate of ion bonding at the surface. In this scenario, foreign ions and large or small biomolecules can be incorporated to the surface, modifying the crystal growth and altering its morphology.
The final stage in the formation of a biomineral is its growth interruption. This effect may be triggered by a lack of ion supply at the mineralisation site, or because the crystal comes into contact with another crystal, or else because the mineral comes in contact with the previously formed organic phase.
Whatever the cause, biomineralisation processes are extremely complex, and not yet well known. One of the prevailing issues not yet fully elucidated is the mechanism at molecular level that controls the crystal formation process. If we consider the features of many organism-grown minerals, it seems that such control can be exerted at various levels. The lowest level of control would be exemplified by the less specific mineralisation phenomena, such as in many bacteria. These processes are considered more of an induction than a control of crystallisation. The opposite case would be the most sophisticated composites of crystals and organic matter, where apparently there is a total control on crystal orientation during its nucleation, and on its size and shape during the growth stage. This would be the case in the bones of vertebrates. In between these two examples we can find plenty of intermediate situations where some crystal parameters are controlled, but not all of them.23
A basic strategy performed by many organisms to control mineralisation is to seal a given space in order to regulate the composition of the culture medium. This is usually done forming barriers made of lipid bilayers or macromolecular groups. Subsequently, the sealed space can be divided in smaller spaces where individual crystals will be grown, adopting the shape of said compartment. An additional strategy is also to introduce specific acidic glycoproteins in the sealed solution, which interact with the growing crystals and regulate their growth patterns. There are many other routes to exert control, such as introducing ions at very precise intervals, eliminating certain trace elements, introducing specific enzymes, etc. All these phenomena are due to the activity of specialised cells that regulate each process throughout its whole duration.
The stereochemical and structural relationship between macromolecules from the organic matrix and from the crystalline phase is a very important aspect in the complex phenomenon of biomineralisation. These macromolecules are able to control the crystal formation processes. It is already known that there is a wide range of biomineralisation processes in Nature, and that it is not possible to know a priori the specific mechanism of each one. It seems, however, that there are certain basic common rules regarding the control of crystal formation and the interactions involved. The term interaction refers here to the structure and stereochemistry of the phases involved, i.e. nanocrystals and macromolecules.
As already mentioned, the inorganic and organic components are forced to interact in order to produce a biomineral. They are not two independent elements; the specific extent and method for this interaction can be extremely varied, and the same variability applies to the biomineral's functionality.
The biominerals, natural composite materials, are the result of millions of years of evolution. The mineral phases present in living species can be also obtained in the laboratory or by geochemical routes. The synthesis conditions, however, are very different because the enforcement of said conditions at the biological environment is not so strict. It is worth noting that biogenic minerals usually differ from their inorganic counterparts in two very specific parameters: morphology and order within the biological system. It is quite likely that some general mechanisms exist that govern the formation of these minerals, and if our knowledge of these potentially general principles would improve, new options in material synthesis or modifications of already existing materials could be possible as an answer to a wide range of applications in materials science.
The biominerals or organic/inorganic composites used in biology exhibit some unique properties that are not just interesting per se; the study of the formation processes of these minerals can lead us to reconsider the world of industrial composites, to review their synthesis methods and to try and improve their properties. For instance, comparative studies with biominerals have provided new thinking on improvements of the physical-chemical properties in cements. In fact, the most noticeable property of minerals in biology is to provide physical rigidity to their host. But biological minerals are not just building material, as we could consider their role in shells, bones and teeth; they also fulfil many other purposes such as, for instance, in sensing devices. The biomineralisation process is responsible for bone formation, growth of teeth, shells, eggshells, pearls, coral and many other materials that form part of living species. Biomineralisation is hence responsible for the controlled formation of minerals in living organisms. These biominerals can be either crystalline or amorphous, and they belong in the bioinorganic family of solids. Bioinorganic solids are usually a) remarkably nonstoichiometric, that is, with frequent variations in their composition, allowing impurities to be included as interstitial and/or substitutional defects, b) they can be present in amorphous and/or crystalline form, and in some situations several polymorphs of the same crystalline solid can coexist. Besides, the inorganic component is just a part of the resulting biomineral that actually is a composite material, or more precisely a nanocomposite, formed by an organic matrix which restricts the growth of the inorganic component at perfectly defined and delimited areas in space, determining a strict shape and size control.
The organic component might be a vesicle, perhaps a protein matrix; whatever the case, biominerals are formed by very different chemical systems, since they require the combined participation of mineral components and organic molecules. Vesicles give rise to three-dimensional structures, and are able to fill cavities, while the organic molecules can form linear or layered structures, and also can interact with the inorganic matrix, generating the voids to be filled with minerals.
Almost half of the biominerals known include the element calcium among their constituents. This is the reason why the term calcification is often used to describe the processes where an inorganic material is produced by a living organism. But this generalisation is not always true, since there are many biominerals without any calcium content. Therefore, the term biomineralisation is not only much more generic but also more adequate, encompassing all inorganic phases regardless of their composition; the outcome is the biomineral, that is, a mineral inside a living organism, which is a truly composite material.
Biomineralisation processes give rise to many inorganic phases; the four most abundant are calcite, aragonite, apatite and opal.
In load-bearing biominerals, such as bones, some stress-induced changes may appear and induce in turn certain consequences on their properties, in the crystal growth for instance. The growth of biominerals is related to one of the great unsolved issues in biology: the morphology of its nano- or microcrystals. The skeletons of many species exhibit peculiarities that are clearly a product of their morphogenesis, with direct effects on it, since the gametes of biological systems never or hardly ever produce a biomineral precipitate.
Another question to be considered is the relevance of biominerals from a chemical point of view. Many of these minerals act as deposits that enable to regulate the presence of cations and free anions in cell systems. Concentrations of iron, calcium and phosphates, in particular, are strictly controlled. A biomineral is the best possible regulator of homeostasis. It is important to recall that exocytosis of mineral deposits is a very simple function for cells, enabling to eliminate the excess of certain elements. In fact, some authors believe that calcium metabolism is mainly due to the need to reject or eliminate calcium excess, leading to the development and temporary storage of this element in different biominerals. However, some evidence counters the validity of this point of view: many living species build their skeletons with elements that do not have to be eliminated, such as silicon.25
Mineral deposits such as iron and manganese oxides are used as energy sources by organisms moving from oxic to anoxic areas. Therefore, biominerals are also used by some living species as an energy source to carry out certain biological processes. This fact has been verified in marine bacteria.26
Although silicon – in silicate form – is the second most abundant element in the Earth's crust, it plays a minor part in the biosphere. It may be due in part to the low solubility of silicic acid, H4SiO4, and of amorphous silica, SiOn(OH)4–2n. In an aqueous medium, at pH between 1 and 9, its solubility is approximately 100–140 ppm. In presence of cations such as calcium, aluminium or iron, the solubility markedly decreases, and solubility in sea water is just 5 ppm. At the biosphere, amorphous silicon is dissolved and then easily reabsorbed in the organism; it will then polymerise or connect with other solid structures.
Amorphous silicon biomineral is mainly present in single-cell organisms, in silicon sponges and in many plants, where it is located in fitolith form at cell membranes of grain plants or types of grass, with a clear deterrent purpose. The fragile tips of stings in some plants, such as nettles, are also made of amorphous silicon.
There is a wide range of biological systems with biomineral content, from the human being to single-cell species. Modern molecular biology indicates that single-cell systems may be the best object of research in order to improve our knowledge of a biological structure.
1.1.5 Inorganic Components: Composition and Most Frequent Structures
At present, there is a wide range of known inorganic solids included among the so-called biominerals. The main metal ions deposited in single-cell or multiple-cell species are the divalent alkali-earth cations Mg, Ca, Sr, Ba, the transition metal Fe and the semimetal Si. They usually form solid phases with anions such as carbonate, oxalate, sulfate, phosphate and oxides/hydroxides. The metals Mn, Au, Ag, Pt, Cu, Zn, Cd and Pb are less frequent and generally deposited in bacteria, in sulfide form. More than 60% of known minerals contain hydroxyl groups and/or water bonds, and are easily dissolved releasing ions. The crystal lattice of the mineral group including metal phosphates is particularly prone to inclusions of several additional ions, such as fluorides, carbonates, hydroxyls and magnesium. In some cases, this ability allows for the modification of the material's crystal structure and hence of its properties.
The field of biominerals encompasses a wide range of inorganic salts with many different functions, which are present in several species in Nature. For instance, calcium in carbonate or phosphate form is important for nearly all the species, while calcium sulfate compounds are essential for very few species. All along the evolution of species, there has been a constant development of the control of selective precipitation, that is, of nucleation and growth processes, as well as the shape of the precipitates and their exact location within a living body.27
The minerals in structures aimed at providing support or external protection can be crystalline or amorphous. The generation of amorphous materials in any kind of biological system is undoubtedly a favourable process from an energetic perspective, and is present in several examples such as carbonates and biological phosphates. This amorphous phase usually leads to a series of transformations, either as consequence of recrystallisation processes – which give rise to a crystalline phase, likely to transform itself into other phases due to in-situ structural modifications – or due to redissolution of the amorphous phase, enabling the nucleation of a new phase. If the minerals are crystalline, the biological control can be exerted over several parameters: chemical composition, polymorph formation, and crystal size and shape. Each one of these parameters is in turn closely related to the organic matrix controlling elements concentration, crystal nucleation and growth. If the mineral is amorphous, the chemical composition allows for almost infinite variations, although a certain concentration of the essential elements remains crucial. A typical amorphous biomineral is hydrated silica, SiOn(OH)4–2n, where n can be any value in the range from 0 to 2. Several forms of hydrated silica can be found in living organisms, both in the sea world – such as sponges, diatomea, protozoa and single-cell algae – and in the vegetable kingdom, present in amorphous form. The actions performed by these species to mineralise silicic acids are extremely complex. It seems that this process first involves the transportation of silicic acid towards the inside of the cell, and then to the deposition locations where the monomer will be polymerised to silica. For any silicon structure to be generated, the preliminary essential requirement is the availability of silicic acid, which must also be transported in adequate concentrations. If this stage is verified, the nucleation and polymerisation processes may begin, which will eventually lead to the development of strict and specific morphological features, both at the microscopic and macroscopic scales. Little is known about the early stages, previous to deposition. There are several mechanisms that have been suggested to try to explain biosilication, but none of them is conclusive yet.
Some biominerals perform a very specific function within the biological world; they work as sensors, both for positioning and attitude or orientation. The inorganic minerals generated by some species to carry out this task are calcite, aragonite, barite and magnetite.
1.1.6 Organic Components: Vesicles and Polymer Matrices
The most common cell organ is the vesicle.27 It is an aqueous compartment surrounded by a lipid membrane, impervious to all ions and most organic molecules. The ions required to form the biomineral are accumulated in the vesicle by a pumping action. These ions are, among others, Ca2+, H+, SO, HPO, HCO. In order to understand the biomineral formation, a great deal will depend on the knowledge of cell vesicles and ion pumps.
Proteins or polysaccharides are able to build another kind of receptacle, mould or sealed container, more or less impervious to ions and molecules, depending on the particular system. This receptacle might be into the cell itself, as in the case of ferritin, or outside the cell, such as bone collagen for instance. The exact shape of the protein receptacle for ferritin is fixed, and also the open spaces in collagen where apatite grows always exhibit the same shape. In contrast, the available space in a typical vesicle is not controlled by the organic structure, since vesicles do not have internal crosslinks in their membranes. In fact, vesicle space is very different from cytoplasmatic space, which usually includes crossed-fibre structures. As a consequence, when the mould is made of protein or polysaccharides, precipitation must be controlled through the regulation of cytoplasmatic or extracellular homeostasis. Extracellular fluids have a sustained chemical composition due to the actions of control organs such as the kidney, which actually works as a macropump.
Most of the controlled mineralisation processes performed by organisms exhibit associated macromolecules. These macromolecules carry out important tasks in tissue formation and modification of the biomechanical properties of the final product. Although there are thousands of different associated macromolecules, Williams27 stated that they all can be classified in two types: structural macromolecules and acid macromolecules. The main structural macromolecules are collagen, α- and β-quitine, and quitine-protein complexes. The main acid macromolecules are not very well defined in some organisms, but we may include in this group glycoproteins, proteoglicans, Gla-rich proteins, and acid polysaccharides. Little is known about the secondary conformation of acid macromolecules, apart from the fact that all acid glycoproteins with high contents of glutamic and aspartic acids partially adopt in vitro the β layer conformation, in the presence of calcium. Although the composition of these macromolecules shows little variations between species, the opposite can be said of structural macromolecules. They vary from one tissue to another, and there are even some hard mineralised pieces that do not seem to have any kind of acid macromolecule at all. This lack of presence in some tissues allows us to infer that their purpose might be to modify the mechanical properties of the final product, not to regulate biomineralisation. The main means of control over biominerals are the independent areas in the cytoplasmatic space or in the extracellular zones in multiple-cell species, where the organic structures develop well-defined volumes and external shapes.
There are different physical and chemical controls in the development of a mineral phase. Physical controls are determined by the physics of our world and by biological source fields, in the same way that biological chemistry is restricted by the properties of chemical elements in the periodic table.
Mineral and vesicle grow together under the influence of many macroscopic fields. It should be taken into account that the functional values often depend on the interactions with these fields, due to the density, magnetic properties, ion mobility in the crystal lattice, elastic constants and other material properties. These properties do not fall under a strict biological control. Microscopic shape is restricted by the rules of symmetry in crystalline materials, but not in amorphous ones. Any crystal-based biomineral exhibits many restrictions in shape, and the organism adapts itself to them.23
1.2 Alternatives to Obtain Nanosized Calcium-Deficient Carbonate-Hydroxy-Apatites
Hydroxyapatite, (HA), Ca10(PO4)6(OH)2 is the most widely used synthetic calcium phosphate for the implant fabrication because is the most similar material, from the structural and chemical point of view, to the mineral component of bones.28 HA with hexagonal symmetry S.G. P63/m and lattice parameters a = 0.95 nm and c = 0.68 nm, exhibits excellent properties as a biomaterial, such as biocompatibility, bioactivity and osteoconductivity. When apatites aimed to mimic biological ones are synthesised, the main characteristics required are small particle size, calcium deficiency and the presence of [CO3]2− ions in the crystalline network. Two different strategies can be applied with this purpose.
The first one is based in the use of chemical synthesis methods to obtain solids with small particle size. There are plenty of options among these wet-route processes, which will be generally termed as the synthetic route.29
The other strategy implies the collaboration of physiological body fluids.30 In fact, certain ceramic materials react chemically with the surrounding medium when inserted in the organism of a vertebrate, yielding biological-like apatites through a process known as the biomimetic process.
1.2.1 The Synthetic Route
Some synthetic strategies used to obtain submicrometric particles are the aerosol synthesis technique,31 methods based on precipitation of aqueous solutions,32,33 or applications of the sol-gel method, or some of its modifications such as the liquid mix technique, which is based on the Pechini patent.34,35 In these methods, the variation of synthesis parameters yields materials with different properties. Quantum/classical molecular mechanics simulations have been used to understand the mechanisms of calcium and phosphate association in aqueous solution.36
On the other hand, it is difficult to synthesise in the laboratory calcium apatites with carbonate contents analogous to those in bone. Indeed, it is difficult to avoid completely the presence of some carbonate ions in the apatite network, but the amount of these ions is always inferior to that in natural bone values (4–8 wt%) and/or they are located in different lattice positions.12,37 It must be taken into account that biological apatites are always of type B, but if the synthesis of the ceramic material takes place at high temperatures, type-A apatites are obtained. Synthesis at low temperatures allows apatites to be obtained with carbonate ions in phosphate positions but in lower amounts than in the mineral component of bones.38,39
1.2.2 The Biomimetic Process
As in any other chemical reaction, the product obtained when a substance reacts with its environment might be an unexpected or unfavourable result, such as corrosion of an exposed metal, for instance, but it could also lead to a positive reaction product that chemically transforms the starting substance into the desired final outcome. This is the case of bioactive ceramics, which chemically react with body fluids towards the production of newly formed bone. When dealing with the repair of a section of the skeleton, there are two different basic options to consider: replacing the damaged part, or substituting it and regenerating the bone tissue. This is the role played by bioactive ceramics.40
Calcium phosphates, glasses and glass ceramics, the three families of ceramic materials where several bioactive products have been obtained, have given rise to starting materials used to obtain mixtures of two or more components, in order to improve its bioactive response in a shorter period of time.
These types of ceramics are also studied to define shaping methods allowing implant pieces to be obtained in the required shapes and sizes, with a given porosity, according to the specific role of each ceramic implant. Hence, if the main requirement is to verify in the shortest possible time a chemical reaction leading to the formation of nanoapatites as precursors of newly formed bone, it will be necessary to design highly porous pieces, which must also include a certain degree of macropores to ensure bone oxygenation and angiogenesis.
However, these requirements are often discarded when designing the ceramic piece. As a result, the chemical reaction only takes place on the external surface of the piece (if made of bioactive ceramics) or it simply does not occur if the piece is made of an inert material; in both cases, the inside of the piece remains as a solid monolith able to fulfil bone replacement functions, but without the regenerative role associated to bioactive ceramics. In order to achieve a chemical reaction throughout the whole material, it is important to design pieces with bone-like hierarchical structure of pores. In this way, the fluids will be in contact with a much larger specific surface, reaching a higher reactivity phase that allows full reaction between the bioactive ceramic and the fluids to be achieved, thus yielding newly formed bone as reaction product.