**10.9.2. Preparation of nanocrystalline apatites**

**Typical acronym**

? "Tricalcium phosphate" Ca9(Mg,Fe2+)

**Table 4** Different apatitic and non-apatitic calcium phosphates [108].

**10.9.1. Biological apatite in bone tissue engineering**

CPPD Calcium pyrophosphate dihydrate

DCPD Dicalcium phosphate dihydrate

substrates (scaffolds8

[109],[115].

**Chemical name Chemical formula Mineral name Structure Ca/P ratio**

Geologically occurring

Non-apatitic 1.28

whitlockite

Ca2P2O7·2H2O — Non-apatitic 1.0

Ca(HPO4)·2H2O Brushite Non-apatitic 1.0

(PO4)6(HPO4)

480 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

γ-CCP γ-Calcium pyrophosphate Ca2P2O7 — Non-apatitic 1.0 OCP Octacalcium phosphate Ca8H2(PO4)6·5H2O — Non-apatitic 1.33 MON Dibasic calcium phosphate Ca(HPO4) Monetite Non-apatitic 1.0

Small organic molecules incorporated in apatite crystals act as porogens that controlthe porous structure of apatite single crystal. The presence of amino acid under the apatite synthesis conditions leads to firm bindings and encapsulation of amino acid within apatite single crystals. The amino acid elimination by heating or electron beam irradiation enhances the pore formation in apatite single crystals. Moreover, the incorporation of acidic amino acid into apatite induces the peapod-like nanotubes in apatite single crystals. That suggests the potential of using small organics for nanostructural control of apatite single crystals, which would be valuable for enhancing thedrug loadings orfor modulating the materialdigestion in vivo [111].

Tissue engineering (TE) techniques were developed to recover or enhance lost tissue func‐ tion and structure. In biological hard tissues, for example, lost portions can be effectively reconstructed by the control of environmentalfactors, physical stimulation, addition of growth factors and by the use of degradable materials. These factors strongly facilitate the regenera‐ tion of macroscopic shape of defected hard tissues. Nevertheless, the differences in micro‐ structure, and also in mechanical and physical properties, between regenerated and original

For in vitro engineering of living tissues, cultured cells are grown on bioactive degradable

tion and assembly into three-dimensional structures. One of the most critical issues in TE is the realization of scaffolds with specific physical, mechanical and biological properties. Scaffolds act as substrate for cellular growth, proliferation and the support for new tissue

<sup>8</sup> Scaffolds might be defined as artificial structure capable of supporting the three-dimensional tissue formation, which allows the cell attachment and migration, the delivery and retaining of cells and biochemical factors and enables the diffusion of vital cell nutrients and expressed products. In the case of bone, scaffolds should replicate its architecture and three-dimensional structure with predetermined density, hierarchical pore distribution and interconnected pathways

) that provide the physical and chemical cues to guide their differentia‐

hard tissues must be examined prior to clinical application [112],[113],[114].

Nanocrystalline calcium phosphate apatites play an important role in biomineralization9 and in the field of biomaterials. Biological nanocrystalline apatites are the main inorganic compo‐

<sup>9</sup> The enamel of vertebrate teeth, vertebrate bone and tooth-like microfossils of conodonts.

nents of hard tissues in mammals (bone10 and tooth11 (**Fig. 13**)) with the exception of enamel (which is closer to stoichiometric hydroxylapatite) and are involved in several pathological calcifications such as dental calculi, salivary stones and blood vessel calcification. In compar‐ ison with hydroxylapatite, which is a stoichiometric apatitic phase and is the most stable and the least soluble calcium phosphate at ambient conditions, nanocrystalline apatites are nonstoichiometric and calcium (and OH-) deficient and may incorporate substituted ions in their nanosized crystals. Their calcium and hydroxide deficiencies are responsible for higher solubility than HA. Besides, they have the ability to mature when submitted to humid environments [108],[112],[117],[118].

**Fig. 13.** The cross-section of lower jaw bone (a) and tooth (b) of European roe deer (*Capreolus capreolus*) and human tooth (c, d): enamel (1), dentin (2), pulp chamber (3), cementum (4) and root canal (5).

<sup>10</sup> Bones are rigid organs that form a part of the endoskeleton of vertebrates. Their function is to move, support and protect various organs of the body, produce red and white blood cells and store minerals. Bones appear in a variety of shapes and have a complex internal and external structure described by various hierarchical models [117]. Bone is a complex and hierarchical tissue consisting of nano-hydroxylapatite (70 wt.%) and collagen (30%) as major portions [113].

<sup>11</sup> Teeth consist of a bulk of dentin covered with (inorganic) enamel on the crown and cementum on the root surface. Thick collagen bundles, called periodontal ligaments (PDL), attach to cementum at one end and to the alveolar bone at the other end. The alveolar bone is supported by the jaw [117].

**Fig. 14.** Electron microscopy picture (SEM) of lower jawbone of European roe deer from **Fig. 13** under magnification of 50× (a), 5000× (b), 50,000× (c) and 100,000× (d).

Scanning electron microscopy of deer bone (**Fig. 14**) shows a spongiform texture (*a*, *b*) formed by crystals of carbonated hydroxylapatite with the size below 100 nm (*e*, *f*).

The inorganic portion of bone contains two major mineral phases


nents of hard tissues in mammals (bone10

482 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

environments [108],[112],[117],[118].

and tooth11

(which is closer to stoichiometric hydroxylapatite) and are involved in several pathological calcifications such as dental calculi, salivary stones and blood vessel calcification. In compar‐ ison with hydroxylapatite, which is a stoichiometric apatitic phase and is the most stable and the least soluble calcium phosphate at ambient conditions, nanocrystalline apatites are nonstoichiometric and calcium (and OH-) deficient and may incorporate substituted ions in their nanosized crystals. Their calcium and hydroxide deficiencies are responsible for higher solubility than HA. Besides, they have the ability to mature when submitted to humid

**Fig. 13.** The cross-section of lower jaw bone (a) and tooth (b) of European roe deer (*Capreolus capreolus*) and human

10 Bones are rigid organs that form a part of the endoskeleton of vertebrates. Their function is to move, support and protect various organs of the body, produce red and white blood cells and store minerals. Bones appear in a variety of shapes and have a complex internal and external structure described by various hierarchical models [117]. Bone is a complex and hierarchical tissue consisting of nano-hydroxylapatite (70 wt.%) and collagen (30%) as major portions [113]. 11 Teeth consist of a bulk of dentin covered with (inorganic) enamel on the crown and cementum on the root surface. Thick collagen bundles, called periodontal ligaments (PDL), attach to cementum at one end and to the alveolar bone at

tooth (c, d): enamel (1), dentin (2), pulp chamber (3), cementum (4) and root canal (5).

the other end. The alveolar bone is supported by the jaw [117].

(**Fig. 13**)) with the exception of enamel

In amorphous calcium phosphate, the Ca:P ratio is about 1.33. The non-crystalline or amor‐ phous bone mineral is metastable with respect to bone apatite. Bone apatite crystals are calcium deficient due to the defects in the crystalline lattice and isomorphous substitutions, that is, the replacement of some ions by others in the crystal without disrupting the general symmetry. Young bone tissue was found to be richer in amorphous mineral than crystalline apatite [119].

In the body, only the bone collagen has the property of inducing the mineralization through in vitro; collagens of other tissues do not possess this property. One of the differences between nucleating and non-nucleating collagens might be the presence of a sort of inhibitor bound to the latter. This inhibiting substance was shown to be pyrophosphate. Calcium is accreted in bone tissue in the process of new bone formation or remodeling and resorbed from the bone tissue in the process of bone destruction. The loss of endogenous calcium in urine and feces is compensated for by an equivalent intake of this element (**Fig. 15**) [119].

**Fig. 15.** General scheme of metabolism of calcium [119].

Enamel (**Fig. 17**) is normally the best preserved from hard tissues. It is almost completely a mineral, so the decomposition of organic matter has little effect on it. Archeological enamel nearly always yields good microscope sections, often indistinguishable from fresh enamel [120]. The structure of carbonate dental enamelrefined by WILSON et al [121] is shown in **Fig. 16**.

**Fig. 16.** Crystallographic structure of human dental enamel apatite (perspective view along the c-axis): P63/m, *a* = 9.4081 Å, *b* = 6.8887 Å, *c*:*a* = 0.7322 and *V* = 528.05 Å3 [121].

the latter. This inhibiting substance was shown to be pyrophosphate. Calcium is accreted in bone tissue in the process of new bone formation or remodeling and resorbed from the bone tissue in the process of bone destruction. The loss of endogenous calcium in urine and feces is

> Forming bone

Resorbing bone

Intracellular

Ca

Exchangeable bone Ca

compensated for by an equivalent intake of this element (**Fig. 15**) [119].

484 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

Ca

Extracellular

Enamel (**Fig. 17**) is normally the best preserved from hard tissues. It is almost completely a mineral, so the decomposition of organic matter has little effect on it. Archeological enamel nearly always yields good microscope sections, often indistinguishable from fresh enamel [120]. The structure of carbonate dental enamelrefined by WILSON et al [121] is shown in **Fig. 16**.

**Fig. 16.** Crystallographic structure of human dental enamel apatite (perspective view along the c-axis): P63/m, *a* =

[121].

Ingested

Ca

Faeceal Ca

**Fig. 15.** General scheme of metabolism of calcium [119].

9.4081 Å, *b* = 6.8887 Å, *c*:*a* = 0.7322 and *V* = 528.05 Å3

Gl Tract

Recycling

Urinary Ca

**Fig. 17.** Electron microscopy picture (SEM) of tooth from **Fig. 13**: the crown with enamel-dentin interface (*a*, *b*), nano‐ crystals of carbonated hydroxylapatite in the texture of dentin (*c*), glass-like texture of enamel (*d*) and surface of pulp chamber (*e*, *f*).

Among carbonate apatites, which are formed in mouth cavity, only dental enamel mine belongs to the B-type of carbonate-apatite (**Section 4.6**) and the others (of dentin, salivary and dental stones) belong to the AB-type (B > A). According to the variations in unit-cell parame‐ ters, isomorphic replacements in crystal structures of apatites of pathogenic origin (renal, salivary and dental stones) are more intensive in comparison with physiogenic dental enamel apatites. Among pathogenic apatites, the most considerable compositional variations are observed in renal stone apatites. That indicates strong variability of conditions of their formation. The changes in unit-cell parameters of bone apatites are not completely interpret‐ able, because these apatites consist of nanosized crystals that are smaller than those of other biological apatites [122].

The age variations of the crystal lattice parameters of human enamel apatites are related to complicated processes of de- and remineralization, which result in the increase or reduction of vacancies in Ca positions and in the respective changes of CO3 2−, H2O and HPO4 2− contents in the unit cell:

$$\begin{aligned} \left(\mathrm{n} + \mathrm{m} \,/\, 2\right) &\,\mathrm{Ca}^{2+}\mathrm{n}\,\mathrm{PO}\_{4}^{3-} + \mathrm{m}\,\mathrm{OH}^{-} \rightarrow\\ \left(\mathrm{n} + \mathrm{m} \,/\, 2\right) &\,\left[\begin{array}{c} \mathrm{I} \end{array}\right]\_{\mathrm{Ca}} + \mathrm{n}\,\mathrm{A}^{2-} + \mathrm{m}\,\left(\left[\begin{array}{c} \mathrm{I} \end{array}\right]\_{\mathrm{OH^{-}}},\mathrm{H}\_{2}\mathrm{O} \right) \end{aligned} \tag{21}$$

where A2− = CO3 2− or HPO4 2−, and []Ca, []OH− are the vacancies. Until the age of 50 years, the values of *a* and *c*-parameter of enamel apatites change considerably without any depend‐ ence of particular age that may be explained by essential fluctuations of the content of Ca in human organism. After 50 years of age, significant direct correlation between the age and the *a*-parameter appears [122].

The surface of apatite nanocrystals is possibly doped with foreign elements or functional‐ ized with organic molecules [117],[123],[124]. The course of facile synthesis of B-type carbo‐ nated nanoapatite with tailored microstructure is described by GUALTIERI et al [125].
