CHAPTER 1: Biological Apatites in Bone and Teeth

Most biominerals are inorganic/organic composite materials.¹  This is also the case for the bones and teeth of all vertebrates, which are formed by the combination of an inorganic calcium phosphate phase and an organic matrix²  (Figure 1.1). The inorganic component is a 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 comprises 65% of the total bone mass, with the remaining mass formed by organic matter and water.³  The benefits that the inorganic component brings to this combination are toughness and the ability to withstand pressure.

In contrast, 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, reaching new properties with added value. In fact due to this evidence, a large portion of the field of modern materials science is currently focused on the development of composite materials.

The bone exhibits some physical and mechanical properties which are rather unusual. It is able to bear heavy loads, to withstand large forces and to flex without fracture within certain limits. Besides this, the bone also acts as an ion buffer both for cations and anions. From the material point of view, bone could be simplified as a three-phase material formed by organic fibers, 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 layer, 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 non-differentiated trunk cells, the precursors of most repairing and regenerating cells produced after formation of the embryonic subject.^5,6  The bone fulfils critical functions in terms of structural material and 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 proteoglycans and glycoproteins, total more than 200 different proteins, known as non-collagen proteins; however, their total contribution to the organic constituent falls below 10% of the organic fraction. These bone constituents are hierarchically arranged, with at least five levels of organization. At the molecular level, the polarized triple helix of tropocollagen molecules are grouped in microfibers, with small cavities between their edges, where small apatite crystals – ∼5 nm × 30 nm sized – nucleate and grow. These microfibers unite to form larger fibers which constitute the microscopic units of bone tissue. These fibers 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 which, in the aging process, evolved towards nanocrystalline hydroxyapatite. However, results of solid-state ³¹P nuclear magnetic resonance spectroscopy evidenced 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-phosphothreonine groups, which are probably used to link the inorganic mineral component and the organic matrix. Phosphoproteins are arranged in the collagen fibers so that Ca²⁺ can be bonded at regular intervals, in agreement with the inorganic crystal structure, hence providing a repeating condition which 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 microfibers 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 PO₄³⁻ 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 forms of HCO₃⁻, HPO₄²⁻ and H₂PO₄⁻ anions. When incorporated to the bone, the released protons can move throughout the bone tissue and leave the nucleation and mineralization area. The nucleation of thin, platelet-shaped apatite crystals takes place at the bone within discrete spaces inside the collagen fibers, 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 as mentioned at the spaces left between the collagen fibers exhibit the particular feature of being monodispersed and nanometer-sized platelets of carbonate-hydroxyl-apatite. There is no other mineral phase present, and the crystallographic axis c of these crystals is arranged in parallel to the collagen fibers 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.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⁺, PO₄³⁻ and OH⁻, the composition of biological apatites always includes CO₃²⁻ at ∼4.5%, and also a series of minority ions, usually including Mg²⁺, Na⁺, K⁺, Cl⁻ and 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 which are performed simultaneously to the bone formation. The osteoclasts, which are giant multinucleated cells, are able to catabolyze 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 demineralization, 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 mineralization location, intimately related to the collagen fibers. It seems that the phosphoproteins are enzymatically phosphored prior to the mineralization.¹³ 

The crystallization 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 which, in turn, evolves to carbonate hydroxyapatite; at lower pH values, the intermediate phase seems to be dehydrated dicalcium phosphate.^14,15 

The mechanisms of bone formation are highly regulated processes,⁷  which seem to verify the following statements:

• Mineralization 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% proteins and special matrix fluids. In 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 synthesize bone replacement materials for clinical applications featuring physiological tolerance, biocompatibility and long-term stability have, up to now, had only relative success; the superiority and complexity of the natural structure shows where, for instance, the 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. Bone 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 stating that the bone is a highly structured porous matrix, made of nanocrystalline and non-stoichiometric apatite, calcium deficient and carbonated, intertwined with collagen fibers 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 macro scale point of view, to the ‘invisible’ nano scale.

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 which 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 fiber-forming aggregates. These fibers 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 4–8% in 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 Ca_8.3(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 fibers where they are deposited. Electron microscopy techniques enabled this information to be obtained.²⁰ 

The bones are characterized by their composition, crystalline structure, morphology, particle size and orientation. The apatite structure hosts carbonate in two positions: the OH⁻ sub-lattice producing so-called type A carbonate apatites or the [PO₄]³⁻ sub-lattice (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 which, combined with their non-stoichiometric composition, inner crystalline disorder and presence of carbonate ions in the crystal lattice, explain their special behaviour.

The structure of apatite allows for wide compositional variations, with the ability to accept many different ions in its three sub-lattices (Figure 1.7).

Biological apatites are calcium-deficient; hence their Ca/P ratio is always <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 is 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 non-stoichiometric quality of HA, which caters for the substitutional inclusion of different amounts of several ions, such as Na⁺, K⁺, Mg²⁺, Sr²⁺, Cl⁻, F⁻, HPO₄²⁻, 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 balance of charge and position is unaffected. However, if a sodium ion replaces a calcium ion, 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 hydroxyapatite, the more difficult interchanges and growth become. 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 their substitution in apatites; heavy metals, in the form of insoluble phosphates, can be retained in the hard tissues without important 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 characterize 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 released during the resorption of the ceramic component that facilitate the bone regeneration performed by the osteoblasts. Thus, for instance, small amounts of strontium, zinc or silicates stimulate the action of these osteoblasts and, in consequence, new bone formation. Carbonate and strontium favour the dissolution and therefore the resorption of the implant.¹²  Silicates increase mechanical strength, a very important factor in particular for porous ceramics, and also accelerate the bioactivity of apatite.²²  Therefore, the current trend is to obtain calcium phosphate bioceramics partially substituted by these elements. In fact, bone and enamel are some of the most complex biomineralized structures. Attempts to synthesize bone in the laboratory are devoted to obtaining biocompatible prosthetic implants, with the ability to leverage natural bone regeneration when inserted into the human body. Its formation might imply certain temporary structural changes of its components, which demand in turn the presence, at trace levels, of additional ions and molecules in order to enable the mineralization process. This is the case, for instance, with bone growth processes, where the localized 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 the sol–gel method, regarding cell proliferation and new bone growth.

Biomineralization is the controlled formation of inorganic minerals in a living body; the 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 biomineralization were not studied until quite recently. Such studies are currently providing new concepts in materials science and engineering.¹⁷ 

Biomineralization 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 mineralization 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 nanometer scale, biomineralization 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 finely divided inorganic materials, of ∼1–100 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 (micrometers).

The production of discrete or expanded architectures in biomineralization 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 crystallization 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 greatly based upon 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 over hundreds of millions of years has critically determined the development conditions of life.²⁴  For instance, CO₂ is combined in carbonate form, decreasing initially the greenhouse effect on the earth’s crust. Leaving aside the shells, teeth and bones, there are many other systems which 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 the 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 non-stoichiometric 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 mineralization will 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.

Different levels of biomineralization can be distinguished according to the type and complexity of control mechanisms. The most primitive form corresponds to biologically induced biomineralization, which is mainly present in bacteria and algae.²³  In these cases, biominerals are formed by spontaneous crystallization, due to supersaturation provoked by ion pumps, and then polycrystalline aggregates are formed in the extracellular space. Gases generated by 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 mineralization process. A living body is able to mineralize 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-catalyzed 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 the creation and maintenance of a supersaturated solution in a biological environment.²³ 

Nucleation is related to the 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 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.

Biomineralization processes can be classified in two large groups: the first includes those phenomena where some kind of control seems to exist over the mineralization process, while the second encompasses those where control seems to be non-existent. According to Mann,²³  biomineralization processes can be described as biologically induced when said biomineralization 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 the case in single-cell species, although some higher order species also verify this behaviour.

However, there are situations where a specific mechanism is acting, which are then described as biologically controlled. An essential element of this process is space localization, whether in a membrane closed compartment, or confined by cell walls, or by a previously formed organic matrix. The biologically controlled process of biomaterials formation 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 which are in turn controlled in terms of composition by the cells in charge; hence the contents of trace elements and stable isotopes in many mineralized areas are not balanced with the concentrations present in the initial medium.

Nucleation in controlled biomineralization 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 mineralization 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 mineralization site, or because the crystal comes in contact with another crystal, or else because the mineral comes into contact with the previously formed organic phase.

Whatever the cause, biomineralization processes are extremely complex, and not yet well known. One of the prevailing issues not yet fully elucidated is the mechanism at the 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 mineralization phenomena, such as in many bacteria. These processes are considered more of an induction than a control of crystallization. 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, but not all, crystal parameters are controlled.²³ 

A basic strategy performed by many organisms to control mineralization is to seal a given space in order to regulate the composition of the culture medium. This is usually done by forming barriers made of lipid bilayers or macromolecular groups. Subsequently, the sealed space can be divided into smaller spaces where individual crystals can be grown, adopting the shape of the 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 specialized cells that regulate each process throughout its 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 biomineralization. These macromolecules are able to control crystal formation processes. It is already known that there is a wide range of biomineralization 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 of 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 those conditions in the biological environment is not as 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 potential general principles improved, new options in material synthesis or modifications of already existing materials could be used in a wide range of applications in materials science.

The biominerals or organic/inorganic composites used in biology exhibit some unique properties which are not just interesting per se: the study of the formation processes of these minerals can lead 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 materials, as we could consider their role in shells, bones and teeth; they also fulfil many other purposes, for instance, in sensing devices. The biomineralization process is responsible for bone formation, growth of teeth, shells, eggshells, pearls, coral and many other materials which form parts of living species. Biomineralization 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 non-stoichiometric, that is, with frequent variations in their composition, allowing including impurities as interstitial and/or substitutional defects; and (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, which 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 known biominerals 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 generalization is not always true, since there are many biominerals without any calcium content. Therefore, the term biomineralization 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.

Biomineralization 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 the regulation of 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 them 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 evidences deny 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, SiO_n(OH)_4−2n. In an aqueous medium, at pH 1–9, its solubility is ∼100–140 ppm. In the presence of cations such as calcium, aluminium or iron, the solubility decreases markedly, and solubility in sea water is just 5 ppm. In the biosphere, amorphous silicon is dissolved and then easily reabsorbed into the organism; it will then polymerize or connect with other solid structures.

Amorphous silicon biomineral is mainly present in single-cell organisms, in silicon sponges and in many plants, where is located in fitolith form in the 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 humans to single-cell species. Modern molecular biology indicates that single-cell systems may be the best object for research in order to improve our knowledge of biological structure.

At present, a wide range of known inorganic solids are included among the so-called biominerals. The main metal ions deposited in single-cell or multiple-cell species are the divalent alkaline earth cations Mg, Ca, Sr and Ba, the transition metal Fe and the semimetal Si. They usually form solid phases with anions such as carbonate, oxalate, sulphate, 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 the inclusion 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 species, while calcium sulphate 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 recrystallization 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 concentration of elements, 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, SiO_n(OH)_4−2n, where n can be any value in the range 0–2. 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 mineralize 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 polymerized 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 polymerization 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. Several mechanisms have been suggested to try to explain biosilication, but none of them is yet conclusive.

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.

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⁺, SO₄²⁻, HPO₄²⁻ and HCO₃⁻. Understanding biomineral formation depends upon 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 inside the cell itself, as in the case of ferritin, or outside the cell, such as bone collagen. The exact shape of the protein receptacle for ferritin is fixed, and 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 cytoplasmic space, which usually includes crossed fiber structures. As a consequence, when the mould is made of protein or polysaccharides, precipitation must be controlled through the regulation of cytoplasmic 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 macro-pump.

Most of the controlled mineralization processes performed by organisms exhibit associated macromolecules. These macromolecules perform 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 into 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, proteoglycans, 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 variation between species, the opposite can be said of structural macromolecules. They vary from one tissue to another, and there are even some hard mineralized pieces that do not seem to have any kind of acid macromolecule at all. This lack of presence in some tissues allows the inference that their purpose might be to modify the mechanical properties of the final product, not to regulate biomineralization. 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 

Hydroxyapatite, 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  Hydroxyapatite, with hexagonal symmetry S.G. P6₃/m and lattice parameters a = 0.95 nm and c = 0.68 nm, exhibits excellent properties as biomaterial, such as biocompatibility, bioactivity and osteoconductivity. When apatites are synthesized with the aim of mimicking biological ones, 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 strategy is based in the use of chemical synthesis methods to obtain solids with small particle sizes. There are plenty of options among these wet-route processes, which shall 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 into the vertebrate organism, yielding biological-like apatites through a biomimetic process.

Synthetic strategies used to obtain sub-micrometric particles include the aerosol synthesis technique,³¹  methods based on precipitation of aqueous solutions,^32,33  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, variation of the 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 

Conversely, it is difficult to synthesize in the laboratory calcium apatites with carbonate content 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 values in natural bone (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 obtains apatites with carbonate ions in phosphate positions but in lower amounts than in the mineral component of bones,^38,39  although new efforts to prepare B-type carbonated nanoapatites with higher substitution and in an easier manner are still under development.^40,41 

There is currently a large body of knowledge about the role of carbonates in apatites intended for bone grafting. However, research into the development of new carbonated apatites is an ongoing research field. Very recently, the presence of carbonates in nanocrystalline apatites have been demonstrated to enhance the proliferation and differentiation towards osteoblast pathways of human bone marrow cells.⁴² 

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, 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.⁴³ 

Calcium phosphates, glasses and glass ceramics, the three families of ceramic materials from which several bioactive products have been obtained, have given rise to the starting materials used to obtain mixtures of two or more components, in order to improve the bioactive response in a shorter period of time.

These types of ceramics are also studied to define shaping methods enabling implant pieces in the required shapes and sizes to be obtained, 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 a solid monolith able to fulfil bone replacement functions, but without the regenerative role associated with bioactive ceramics. In order to achieve a chemical reaction throughout the whole material, it is important to design pieces with a 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 a full reaction between the bioactive ceramic and the fluids to be achieved, to yield newly formed bone as reaction product.

From these reasons we can realize that manufacturing bioceramics following a biomimetic strategy involves different factors at different levels, i.e. chemical composition, microstructure and hierarchical macrostructures. Figure 1.12 summarizes these three concepts. First, the apatite intended to mimic the mineral component of the bone must be calcium-deficient, carbonated and non-stoichiometric where phosphate and hydrogenphosphate groups coexist. The microstructural characteristics of the biomimetic apatites should comprise crystallites of 20–50 nm in size with needle-like morphology along the c axis. The X-ray diffraction patterns in Figure 1.12 (right) evidence that the apatites in bone exhibit a degree of crystallinity, which is an intermediate situation between the amorphous structure of a glass and the highly crystalline ordering of hydroxyapatites obtained by high temperature ceramic methods. Finally, the macrostructural characteristic of the bone involves a macroporous architecture, with pores of hundreds of micrometers in size, which allow for the ingrowth of blood vessels and the diffusion of nutrients, as can be seen in Figure 1.12 (bottom). However, this is a very simplified description of the hierarchical porosity of bones. Bones differ from each other depending on anatomical location, sex, age, etc.⁴⁴  Furthermore, we have already seen that for the same bone, cancellous and cortical components posses a very different porosity. Consequently, establishing how or what must be mimicked when designing biomimetic apatites is a very difficult task.

Finally, we must take into account that the goal of biomimetic apatites is commonly to restore the structure and functionality of an impaired bone, not a healthy one. In other words, the bone graft will be in contact with a tissue that, commonly, exhibits an impaired structure. Therefore a new dilemma arises: should whe mimic the properties of a healthy bone or the impaired characteristics of the receptor location, thus matching the implant–tissue interface? This dilemma is especially serious in the case of osteoporotic patients.⁴⁵  Bone weakening due to osteoporosis is far from satisfactory resolution. As a consequence of poor bone quality, surgical procedures performed to implant a device into weakened bone often lead to a clinical result that is worse than if such an intervention was performed on a young and strong bone. The risk of fracture increases exponentially with age, and the recovery process from a fracture is often slow, difficult and may lead to a disability or even to the death of the patient. Figure 1.13 shows micro-computed tomography images obtained of a healthy bone of a young, active woman and for a low-density bone of an older osteoporotic patient. Recent developments in biomaterials converge on the needs of biological enhancement of biomaterials fostering osteoinduction and osteogenesis to support and augment bone healing. Indeed, the reconstruction of osteoporotic bone yields significant difficulties with the solutions available today.

Another alternative is to provide a scaffolding material to the bone, aimed at enhancing its self-healing mechanisms.⁴⁶  The design and development of porous ceramics have attracted much attention in the last years. Pore distribution, as well as pore size can play a fundamental role in bone regeneration, angiogenesis and implant degradation. The incorporation of free-form preparation methods such as three-dimensional printing into the biomaterials field allow the design of hierarchical pore structures to facilitate these processes⁴⁷  (Figure 1.14). An interconnected macropore structure of 150–1000 µm allows cell colonization and enhances the diffusion rates to and from the centre of a scaffold, as well as angiogenesis and bone ingrowth. Small pores allow phagocytic cells to adhere and resorb the scaffolds, whereas larger pores encourage the invasion of new vessels and ingrowth of bone tissue.

Finally, tissue engineering techniques provide excellent tools to mimic apatite biomineralization and bone tissue formation.⁴⁸  In this case, mesenchymal stem cells harvested from patient marrow are seeded onto bioceramic-based scaffolds. By means of adding osteoinductive agents, the osteoblast pathway is activated and osteogenesis is aroused. Finally, the construct inorganic scaffold–bone tissue is implanted to regenerate bone defects (Figure 1.15).