Rey 2017

1.
11 Bioactive Calcium Phosphate Compounds: Physical Chemistry☆

C Rey, C Combes, C Drouet, D Grossin, G Bertrand, and J Soulié, CIRIMAT, University of Toulouse, CNRS, INPT, UPS, ENSIACET,
Toulouse, France
r 2017 Elsevier Ltd. All rights reserved.
1.11.1 Introduction 246

1.11.2 Calcium Orthophosphates Used as Biomaterials: Composition, Occurrence, and Brief Synthesis
Description 246
1.11.2.1 Calcium Phosphates Obtained by Wet Synthesis Methods 246
1.11.2.1.1 Monocalcium phosphates 246
1.11.2.1.2 Dicalcium phosphates 247
1.11.2.1.3 Tricalcium phosphate, whitlockite, and amorphous tricalcium phosphate 248
1.11.2.1.4 Octacalcium phosphate 248
1.11.2.2 Calcium Phosphate Obtained by Dry Synthesis Methods 249
1.11.2.2.1 Crystalline tricalcium phosphates: a- and b-TCP 249
1.11.2.2.2 Tetracalcium phosphate 249
1.11.2.3 Apatites 250
1.11.2.3.1 Well-crystallized apatites 250
1.11.2.3.2 Nanocrystalline and biomimetic apatites 251
1.11.2.3.2.1 Nanocrystalline apatites 251
1.11.2.3.2.2 Biomimetic nanocrystalline apatites 251
1.11.2.3.3 Synthesis and formation of apatites 252
1.11.2.3.3.1 Wet preparations 252
1.11.2.3.3.2 Dry preparations 253
1.11.2.4 Other Phosphate Compounds 253
1.11.3 Characterization Methods of Calcium Phosphates 254
1.11.3.1 Chemical Analysis 254
1.11.3.1.1 Major ions 254
1.11.3.1.1.1 Calcium 254
1.11.3.1.1.2 Phosphorus 254
1.11.3.1.1.3 Carbonate 255
1.11.3.1.1.4 Hydroxide ions 255
1.11.3.1.2 Minor and trace elements 256
1.11.3.1.3 Meaning of the Ca/P ratio 256
1.11.3.2 Diffraction Methods 256
1.11.3.3 Electron Microscopy Techniques 258
1.11.3.4 Vibrational Spectroscopies 259
1.11.3.5 Solid-State Nuclear Magnetic Resonance 260
1.11.4 Properties of Calcium Phosphates 261
1.11.4.1 Thermal Properties of Calcium Phosphates 261
1.11.4.1.1 Reversible decomposition of stoichiometric apatites 262
1.11.4.1.2 Irreversible decomposition of nonstoichiometric apatites 263
1.11.4.2 Thermodynamic Considerations 264
1.11.4.3 Solubility 266
1.11.4.4 Hydrolysis and Aqueous Conversion 268
1.11.4.5 Surface Charge and Surface Energy 268
1.11.4.5.1 Surface charge 268
1.11.4.5.2 Interfacial energy 270
1.11.4.6 Chemical Alterations of the Surface 270
1.11.4.6.1 Ion exchange 270
1.11.4.6.2 Adsorption 271
1.11.4.7 Nucleation Properties 273
☆
Change History: November 2016. C. Rey, C. Combes, C. Drouet, D. Grossin, G. Bertrand, and J. Soulié changed the title of the chapter, added Figs. 6, 8 (the
old Fig. 8 was removed); Sections 1.11.2.3.2, 1.11.4.2, 1.11.6.1, 1.11.6.3, 1.11.7; and Table 8. Updated Section 1.11.4.7; Tables 3–7, 9–11; and text
throughout the chapter and also References section.
This is an update of C. Rey, C. Combes, C. Drouet and D. Grossin, 1.111 – Bioactive Ceramics: Physical Chemistry. Comprehensive Biomaterials, edited by
Paul Ducheyne, Elsevier, Oxford, 2011, pp. 187–221.
244 Comprehensive Biomaterials II, Volume 1 doi:10.1016/B978-0-12-803581-8.10171-7

Bioactive Calcium Phosphate Compounds: Physical Chemistry 245
1.11.5 Biological Response and Physical–Chemical Characteristics 273

1.11.5.1 Active and Passive Biomaterials With Regard to Ca-P Nucleation 274
1.11.5.2 Simulated Body Fluid Testing 275
1.11.5.3 Biodegradation 276
1.11.6 Examples of Applications and Processing 277
1.11.6.1 Apatite Nanocrystals in Suspension, Interactions With Cells 277
1.11.6.2 Porous Ceramics 277
1.11.6.3 Coatings, Example of HA-Plasma Spraying 279
1.11.6.4 Coatings and Bulks, Low Temperature Processing 279
1.11.6.5 Cements 280
1.11.7 Associations of Calcium Phosphates With Other Compounds 282
1.11.7.1 Organic-Based Composites 282
1.11.7.1.1 Proteins 282
1.11.7.1.2 Non-protein natural polymers 282
1.11.7.1.3 Synthetic polymers 283
1.11.7.2 Association of Ca-Ps With Other Bioactive Inorganic Compounds 283
1.11.7.2.1 Calcium carbonates 283
1.11.7.2.2 Calcium sulfate hemihydrate 283
1.11.7.2.3 Silicate-based or phosphate-based bioactive glasses 284
1.11.8 Conclusion and Future Perspectives 284
References 285
Relevant Websites 290
Abbreviations ICP Inductively coupled plasma

AAS Atomic absorption spectroscopy ISO International organization for standardization
ACP Amorphous calcium phosphate JCPDS Joint committee for powder diffraction standards
ATCP Amorphous tricalcium phosphate lzc Line of zero charge
BMP Bone morphogenetic protein MCPA Monocalcium phosphate anhydrous
Ca-P Calcium phosphate MCPM Monocalcium phosphate monohydrate
CC Calcium carbonate MES Metastable equilibrium solubility
CHN Carbon, hydrogen, nitrogen analyzer NMR Nuclear magnetic resonance
ClA Chlorapatite nsHA Nonstoichiometric hydroxyapatite
CS Calcium sulfate OCP Octacalcium phosphate
CSD Calcium sulfate dihydrate (or gypsum) OCPt Octacalcium phosphate triclinic phase
CSH Calcium sulfate hemihydrate OHA Oxy-hydroxyapatite
DC Direct current PLLA poly-L-lactic acid
DCP Dicalcium phosphate PMMA Polymethylmethacrylate
DCPA Dicalcium phosphate anhydrous pzc Point of zero charge
DCPD Dicalcium phosphate dihydrate SAED Selected area electron diffraction
DSC Differential scanning calorimetry SBF Simulated body fluid
DTA Differential thermal analysis SEM Scanning electron microscopy
EDS Energy dispersive spectroscopy TCP Tricalcium phosphate
EDTA Ethylene diamine tetracetic acid TEM Transmission electron microscopy
EXAFS Extended X-ray absorption fine structure TRIS Trishydroxymethyl aminomethane
FA Fluorapatite TTCP Tetracalcium phosphate
FGF Fibroblast growth factor VEGF Vascular endothelial growth factor
FTIR Fourier transform infrared spectroscopy WDS Wavelength dispersive spectroscopy
HA Hydroxyapatite XRD X-ray diffraction
ICDD International centre for diffraction data
Symbols mn (n integer) domain of vibration of a molecule

a, b, c crystallographic unit-cell angles a, b, c crystallographic unit-cell angles
D represents a difference dhkl inter-reticular distance for the (hkl) crystallographic
h X-ray diffraction angle planes
k wavelength of a radiation G Gibbs free energy
246 Bioactive Calcium Phosphate Compounds: Physical Chemistry
H enthalpy pKsp –logKsp

Ksp solubility product S entropy
Glossary Pyrophosphate (also called diphosphate) Salts of

Isomorph Compounds with identical crystalline structure. the pyrophosphoric (or diphosphoric) acid H4P2O7.
Metaphosphate Salt of the hypothetic metaphosphoric Rietveld analysis Series of computing techniques
acid HPO3, often corresponding to chain or cyclic permitting to extract from X-ray diffraction patterns of
polyphosphates Pn O3nþ1 ðnþ2Þ and Pn O3n n ions . powders of crystalline substances information relative to
Nonstoichiometry Always relative to a given the size, strains, preferential orientations of crystals, phases
stoichiometric composition (see stoichiometry). ratios, and, in favorable cases, the atoms locations and
Compound exhibiting ions or elements ratios different from site occupancy relative to a model structure (these last
that of the given composition. points were in fact at the origin of Rietveld refinement
Orthophosphate Salts of the orthophosphoric acid: H3PO4. techniques).
Polymorph Compounds with identical composition but Stoichiometry Proportion of ions or elements in a given
different crystalline structures. compound.
1.11.1 Introduction
Bioactive ceramics based on calcium orthophosphates (Ca-P) have considerably developed in the last decades; their applications
have progressively spread, worldwide, and they have become the most implanted bioactive ceramics. The initial use of Ca-P as
bioceramics was based on their analogy of composition with bone mineral and the need for versatile and risk-free bone substitute
materials immediately available without the constraint of bone graft. One of the major properties of most Ca-P materials is their
osteoconductivity, their ability to favor bone healing and to bind firmly to bone tissue. In addition, some Ca-P materials have been
shown to be able to initiate bone formation de novo in nonosseous sites. Today, Ca-P compounds are mainly used as bone
substitute materials in the form of porous ceramics, coatings on metallic prostheses, composite materials with organic macro-
molecules, injectable calcium phosphate cements and scaffolds for tissue engineering. They are also used in nonosseous appli-
cations such as ocular implants, allowing eyes movements. Many other applications have been proposed: drug carriers, tubing for
nerve reconstruction, transfection support for cell therapy, adsorption substrates in some types of cancer treatments triggering the
immune system. This chapter presents the different Ca-P compounds and their main physical–chemical properties of importance
for biomedical applications. This chapter is primarily intended to describe the physical chemistry of these materials. In its last part,
this chapter briefly presents associations of calcium phosphates with other, non-Ca-P materials.
1.11.2 Calcium Orthophosphates Used as Biomaterials: Composition, Occurrence, and Brief Synthesis
Description
Calcium orthophosphates result from the neutralization of the different acidities of the orthophosphoric acid (H3PO4). Different
calcium salts are formed corresponding to the progressive replacement of acidic protons by calcium ions. The chemical compo-
sition of these calcium orthophosphates with indications of composition and structures are reported in Table 1. The presentation is
divided into two parts: calcium orthophosphates obtained by low-temperature wet synthesis methods and those obtained by high-
temperature dry synthesis methods. Apatites, among the most important calcium phosphate compounds, which can be obtained
by these two routes will be treated separately. All of these calcium phosphates do not give bioactive ceramics, but they are involved
as intermediary phases or compounds in different types of biomaterials and/or they may appear as secondary phases or impurities.
1.11.2.1 Calcium Phosphates Obtained by Wet Synthesis Methods

1.11.2.1.1 Monocalcium phosphates
The first acidic proton of H3PO4 corresponds to a strong acid. The associated calcium salts, monocalcium phosphates, exist as
hydrated and anhydrous crystalline phases. The soluble monohydrated monocalcium phosphate (MCPM) can be prepared by
partial neutralization of the phosphoric acid with calcium hydroxide, followed by evaporation of water at low temperature, in
acidic conditions.13 It crystallizes as elongated platelets.
The anhydrous phase can be obtained with the same method but at a higher phosphoric acid concentration. Some commercial
products appear, in fact, as a mixture of the two monocalcium phosphates. Monocalcium phosphate anhydrous (MCPA) also
forms by slow thermal dehydration of the hydrated phase above 1001C. This reaction is reversible and, in moist air MCPA, can
be converted back into MCPM. Monocalcium phosphates can incorporate many different mineral ions in their crystal lattice
þ 14
(NHþ 4 , K , Cl , Br ).
Table 1 Main calcium phosphate compounds
Name and chemical Most used abbreviations Structural characteristics (unit-cell Density (g.cm3) References
formulae and mineralogical name dimensions in Angstrom)
Monocalcium phos- MCPM Triclinic P1̄ 2.23 1

phate monohydrate: a ¼5.6261; b¼11.889; c¼6.4731
Ca (H2PO4)2, H2O a¼ 98.633; b¼ 118.262; g¼ 83.344
Monocalcium phos- MCPA Triclinic P1̄ 2.57 2
phate anhydrous: a ¼7.5577; b¼8.2531; c¼5.5504
Ca (H2PO4)2 a¼ 109.87; b¼93.68; g¼109.15
Dicalcium phosphate DCPD brushite Monoclinic Ia 2.3 3,4
dihydrate: a ¼5.812; b¼15.180; c¼6.239
CaHPO4, 2H2O b ¼116.25
Dicalcium phosphate DCPA monetite Triclinic P1̄ 2.93 5
anhydrous: a ¼6.916; b¼6.619; c¼6.946
CaHPO4 a¼ 96.18; b¼103.82; g¼88.34
Amorphous tricalcium ATCP (ACP) Ca9(PO4)6 clusters, S6 symmetry – 6
Phosphate:
Ca3 (PO4)2, n H2O
Octacalcium phosphate OCP (OCPt) Triclinic P1̄ 2.67 7
(triclinic structure): a ¼19.692; b¼9.523; c¼6.835
Ca8 (PO4)4(HPO4)2, a¼ 90.15; b¼92,54; g¼108.65
5H2O
b-Tricalcium phosphate b-TCP Trigonal R3ca 3.07 8,9b
Ca3(PO4)2 a ¼10.439; b¼37.375
a-Tricalcium phosphate a-TCP Monoclinic P21/a 2.86 10
Ca3(PO4)2 a ¼12.887; b¼27.280; c¼15.219
b ¼126.20
Tetracalcium phosphate TTCP (TeTCP) Monoclinic P21 3.05 11
Ca4(PO4)2O a ¼7.023; b¼11.986; c¼9.473
b ¼90,90
Phospho-calcium HA Hydroxyapatite Hexagonal P63/mc 3.16 12
hydroxyapatite: a ¼9.418; c¼ 6.881
Ca10(PO4)6(OH)2
a
Hexagonal settings.
b
Neutron diffraction on powder, Rietveld refinement.
c
The structure of pure HA is monoclinic, however in biomaterials it is essentially the hexagonal form which is encountered.
The monocalcium phosphates are very soluble Ca-P salts and they do not form crystals in living organisms, but these salts are
used in biomedical mineral cement compositions.
1.11.2.1.2 Dicalcium phosphates

The second acidity of phosphoric acid corresponds to a weak acid. The neutralization of two acidities of phosphoric acid with
calcium hydroxide leads to dicalcium phosphates. Two crystalline forms exist: dicalcium phosphate dihydrate (DCPD), also called
brushite by mineralogists, and dicalcium phosphate anhydrous (DCPA), also called monetite. These sparingly soluble calcium
phosphates have some biological importance. Brushite may form as an intermediary phase in ectopic mineralization occurring in
slightly acidic media, for example, in dental or urinary calculi.15 DCPD and DCPA are largely used as biomaterials in mineral
cements and coatings and also in composite materials, but these compounds have other applications also, in tooth paste, for
instance. These compounds crystallize easily and they can be obtained industrially with a very high purity. They are often used for
the preparation of other calcium phosphates.
DCPD crystals can be prepared simply by neutralization of phosphoric acid with calcium hydroxide at pH between 3 and 4 at
room temperature. Generally, DCPD is obtained by double decomposition between calcium and phosphate-containing solutions
in slightly acidic media. It can also be formed by conversion of calcium phosphate salts, in acidic media, or by reaction of calcium
salts such as calcium carbonate in acidic orthophosphate solutions.
DCPA is most conveniently obtained by dehydration of DCPD at 1801C. However, it may also be directly precipitated in
aqueous acidic solutions (pH 3–4) at mild temperature (601C).
The dicalcium phosphates can accommodate different biologically relevant ionic substitutes. Calcium, for example, can be
replaced by Sr2 þ ions and HPO2 2
4 ions by SO4 ions. These phases appear generally stoichiometric and no report has mentioned
the existence of cationic or anionic vacancies in these compounds, to our knowledge.
Dicalcium phosphate also exists as an amorphous solid, with interesting properties. This phase can be obtained in nonaqueous
media and by mechanosynthesis and some reports treat of the transitory formation of an amorphous DCP phase on heating
DCPD.16–18
1.11.2.1.3 Tricalcium phosphate, whitlockite, and amorphous tricalcium phosphate

The third acidity of orthophosphoric acid is very weak and, although the PO3 4 exists only in a very small amount at pH lower than
11, tricalcium phosphate salts (TCP) can precipitate due to their very low solubility. The term TCP is used here in its strict chemical
meaning to designate phases with a chemical composition represented by Ca3(PO4)2 with a Ca/P ratio close to 1.5. Several
different phases with a composition close to TCP exist (Table 1): crystalline TCP (a- and b-TCP) form only at high temperature and
it has been generally considered that crystalline, pure, TCP salt cannot be obtained by direct precipitation from aqueous media.19
Recently however several publications report the formation of crystalline b-TCP in aqueous or non-aqueous media,20–23 these
results have to be considered with caution, as very often, minor impurities such as magnesium, iron in calcium salts can lead to the
formation of whitlockite, a native calcium-magnesium (or iron) phosphate mineral, which is isostructural to b-TCP. Although very
often the term whitlockite is used to designate the b-TCP, this confusion should be avoided, as significant differences exist between
these two phases in terms of composition and properties despite a structural analogy.24 The precipitated phases recorded as b-TCP,
unfortunately are not always carefully analyzed for trace elements and chemical composition. Some of these precipitated b-TCP
have been shown to contain also HPO2 4 ions, like whitlockite, and they appear, thus, chemically different from the real high
temperature b-TCP, which shows only PO3 4 ions. These phases exhibit probably different solubility (and other) properties than
b-TCP, and they might present some biological interest.
Whitlockite forms very easily in biological media and it contains an appreciable amount of Mg2 þ ions and HPO2 4 ions.
The composition can be represented by the following chemical formula:
Ca18 Mg2 ðHPO4 Þ2 ðPO4 Þ12
but other compositions exist with Mn2 þ or Fe2 þ .24 The crystal structure of whitlockite can be accessed on a number of miner-
alogical Web sites.
In mammals, Mg-whitlockite generally appears in ectopic calcifications, for example, in joints and in calculi.
Several preparation methods of whitlockite, generally involving coprecipitation of a calcium and magnesium solution with a
phosphate solution, have been described.25 The structure of whitlockite has been determined, but its solubility is not clearly
known. The conversion of Mg-whitlockite into apatite in aqueous solution has never been described and this compound might be
possibly considered as one of the most insoluble calcium phosphate-based compounds.24
Aqueous precipitation of TCP generally leads to an amorphous phase often designated as amorphous calcium phosphate
(ACP) or more accurately as amorphous tricalcium phosphate (ATCP). It is obtained most conveniently by double decomposition
between calcium and phosphate solutions in alkaline media.26 ACP is considered as one of the most important calcium phos-
phates in living organisms. It has been found in calcifications occurring in primitive organisms27 and it has also been described as
an intermediary phase in the formation of bone mineral in vertebrates.28 Although it has, since then, been shown that in most
cases no amorphous phase could be detected in bone, due to its rapid conversion into apatite,29 the existence of this phase as a
precursor of bone mineral formation is still debated.30–32 In relation with this fast ability to convert into apatite ATCP is being
used as a major or minor phase in calcium phosphate self-setting injectable cements for bone substitution.33 ACP also occurs, as a
high temperature phase in calcium phosphate coatings obtained by plasma-spray.34,35 Owing to its reactivity, it has also been
proposed as a remineralizing agent in toothpaste and in several composite materials for dental restoration.36
ATCP exhibits a short-range organization of mineral ions, very similar to that occurring in apatite and other calcium phos-
phates, often referred as Posner’s clusters.37 It has been shown that these clusters correspond to the most stable association of
calcium and phosphate ions.6 ATCP accepts different mineral ion substitutes and often impurities, which are not always con-
sidered and taken into account. Many different ions can replace calcium or phosphate ions in ATCP. For example, cations of
biological importance such as Mg2 þ , Sr2 þ , Zn2 þ , or anions like CO2 2
3 , HPO4 can be incorporated.
It is often difficult to prepare very pure ATCP, thus, the presence of HPO2 4 leads to a Ca/P atomic ratio under 1.5. Due to
stoichiometric ratio limitations, ATCP which contain only Ca2 þ and PO3 4 can only show a Ca/P ratio equal to 1.5. Therefore
2
Ca/P over 1.5 can only be obtained when foreign anions, most often CO2 3 (or O in high temperature ACP phases) are present.
The best method to test the composition of an ACP is probably calcination and transformation into crystalline phases as explained
in the section relative to high-temperature TCPs.
In addition to ATCP, crystalline TCPs with an apatitic structure exist and have been named “apatitic TCP.” Some industrial TCP
products correspond, in fact, to apatitic TCP. Such compounds are described in the apatite section.
Other ACP phases may exist with lower Ca/P ratio than TCP (and thus containing both HPO2 3
4 and PO4 ions) like amor-
phous octacalcium phosphate,38 however little is known on their physical-chemical properties. Several articles on ACP have been
recently provided.39–42
1.11.2.1.4 Octacalcium phosphate

In addition to amorphous phases, several crystalline calcium phosphate phases associate HPO2 3
4 and PO4 ions such as whi-
tlockite, octacalcium phosphate, and apatite. Octacalcium phosphate, OCP, has been the subject of a book edited by Chow and
Eanes.40 The term “OCP,” refers to a chemical composition, but it is also considered to designate most generally the triclinic
crystalline phase associated with this composition (noted here as OCPt). Very often this confusion makes some publication or
patents rather imprecise. Like for TCP, OCP represents stricto sensus, a chemical composition, and this term has been used to name
any compound with a close chemical composition; thus, “apatitic OCP” or “amorphous OCP” have been described.38,43
The original triclinic phase, OCPt, has initially been obtained by hydrolysis of DCPD in slightly acidic media at 401C,44 but
other preparation methods have been developed, especially the double decomposition between a calcium and a phosphate
solution in slightly acidic medium at mild temperatures.45 The structure of OCPt is characterized by the association of alternated
apatite layers and hydrated layers containing Ca2 þ and HPO2 7
4 ions. OCP has been proposed as a precursor of apatite in several
biological mineralization processes, for example, in bone tissue and tooth enamel but also in dental calculi, although some of
these identifications appear rather incomplete and uncertain and have not always been reproduced. The use of OCP in bioma-
terials has been proposed, in injectable bone mineral cements, or for the preparation of thin coatings, due to its high reactivity.46
OCP coatings have been found to exhibit an osteoinductive behavior.47 However due to difficulties of preparation and con-
servation OCP does not seem to have yet found yet any large-scale industrial use.
The hydration degree of OCP depends on the preparation and storage conditions, and two types of OCP differing by the
amount of attached water molecules have been described by Fowler et al.48
The incorporation of mineral ions within the OCPt structure has been studied by a few authors, and OCP with variable
amounts of Sr, Mg, Zn or silicate ions have been described.49,50 Like ATCP, OCP hydrolyzes easily into apatite.51 In fact, most OCP

preparations lead to a partly hydrolyzed phase that contains an excess of HPO2 4 and OH ions observable by Fourier transform
infrared spectroscopy (FTIR)48 and solid-state nuclear magnetic resonance (NMR).52 OCP has the ability to incorporate
dicarboxylic acids and also citric acid in the hydrated layer.53–55 These interactions between OCP and organic molecules have
found applications in the biomaterial field, it has also been recently suggested that the model of citrate-OCP could explain the
junction between bone nanocrystals.55
A last but important type of calcium phosphate compound can be precipitated in aqueous media: apatite, which will be
described in a specific section.
All these phases obtained from aqueous preparations exhibit a thermal decomposition at moderate temperature and cannot be
transformed into bioceramics by conventional high-temperature treatments (see the thermal properties Section 1.11.4.1). This is
not the case of calcium phosphates obtained at high temperature (dry synthesis methods), which can easily be processed and
sintered by traditional methods used by ceramists.
1.11.2.2 Calcium Phosphate Obtained by Dry Synthesis Methods

The monohydrogen phosphate and dihydrogen phosphate groups are unstable and condense respectively into metaphosphate and
pyrophosphate ions on heating. Thus, all pure calcium orthophosphates obtained by dry synthesis methods contain the PO3
4 ion.
Two main compositions: TCP and tetracalcium phosphate (TTCP), in addition to apatites, are obtained. None of these compo-
sitions is naturally present in biological systems.
1.11.2.2.1 Crystalline tricalcium phosphates: a- and b-TCP

Two main crystalline phases belong to this category: a- and b-TCP, in addition to a very high temperature and unstable a TCP
phase. A review on TCP has recently been published.56
The TCP forms easily by heating precipitated TCP (amorphous TCP or apatitic TCP) at high temperatures, or by dry
preparation, heating mixtures of any calcium phosphate/carbonate with a 1.5 Ca/P ratio such as, for example,
CaCO3 þ Ca2 P2 O7 -Ca3 ðPO4 Þ2 þ CO2
but many other combinations with Ca/P ¼ 1.5 are possible such as
Ca10 ðPO4 Þ6 ðOHÞ2 þ Ca2 P2 O7 -4Ca3 ðPO4 Þ2 þ H2 O
b-TCP is obtained at temperatures between 8501C and the transition temperature to the a-TCP, 11251C. The metastable a-TCP
is most conveniently obtained by quenching TCP heated above the transition temperature. However, heating ATCP between
650 and 7001C results, also, in the formation of a-TCP. The a-TCP exists only at high temperature (above 14301C) and, unlike
a-TCP, it does not seem to have been quenched at room temperature.
Several ion substitutions can occur in each of these phases and may modify the transition temperature. Mg2 þ and other
bivalents ions substituting for calcium, such as Zn2 þ , Mn2 þ , and Fe2 þ ions, for example, stabilize the b-TCP structure,57,58
whereas silicate ions substituting for phosphate stabilize the a-TCP structure.59 Alkali metals-containing b-TCP with a higher
solubility than regular b-TCP have also been evidenced and studied.60,61
The main impurities associated with TCP can be apatite or calcium pyrophosphate corresponding to a Ca/P ratio respectively
higher or lower than that of regular TCP. Apatite can be easily detected by X-ray diffraction (XRD); however, this is not the case of
pyrophosphate which is best evidenced by FTIR spectroscopy.
TCP phases are, with apatites, among the most widely used Ca-P biomaterials, mainly as bioresorbable ceramics.56
1.11.2.2.2 Tetracalcium phosphate

A specific review on TTCP has been recently published.62 This phase is easily obtained by heating at high temperature a mixture of
calcium and phosphate salts with a Ca/P ratio close to 2, for example,
2CaCO3 þ Ca2 P2 O7 -Ca4 ðPO4 Þ2 O þ 2CO2
The reaction has, however, to be carried out under dry atmosphere in order to avoid, in the presence of water vapor, the
conversion of TTCP into hydroxyapatite (HA) according to the reversible reaction:
3Ca4 ðPO4 Þ2 O þ H2 O3 Ca10 ðPO4 Þ6 ðOHÞ2 þ 2CaO
The TTCP formed at high temperature is generally quenched to prevent any evolution. It is used as an alkaline, Ca-rich phase in
multicomponent mineral cements. Owing to the alkaline pH generated by dissolution of TTCP in water, this phase transforms very
easily into apatite. Ion substitutions in this phase do not seem to have been much studied although the incorporation of Sr has
been mentionned.63
1.11.2.3 Apatites
The apatites are the most frequently encountered crystalline biological calcium phosphates, and they are also among the most used
Ca-P compounds as biomaterials. Apatite is also one of the most extensively studied phases.24 Apatites are found in all mineralized
tissues of mammals and more generally in mineralized tissues produced by vertebrates (bones, tooth enamel fish scales, antlers,
defenses, rostrum of whales) except for egg shells and otoliths. The presentation is divided into two parts: well-crystallized apatites
and nanocrystalline apatites including biomimetic apatites for which surface structure and composition have to be considered.
1.11.2.3.1 Well-crystallized apatites

The chemical composition of apatites is given by the generic formula:
Me10 ðXO4 Þ6 Y 2
where Me represents a bivalent metallic ion such as Ca2 þ , Sr2 þ , Ba2 þ , Cd2 þ , Pb2 þ ; XO4 a trivalent anion such as PO3 3
4 , AsO4 ,
3
VO4 ; and Y a monovalent anion such as OH , F , Cl , Br . The composition of apatites may include many foreign ions
and several solid solutions are possible. The bivalent cations may be replaced partly by trivalent cations (eg, La3 þ , Eu3 þ ) or
monovalent ones (eg, Na þ ) or by cationic vacancies. The trivalent anions can be partly replaced by bivalent or tetravalent ones such
4
as CO2 2
3 (type B carbonate apatite), HPO4 , or SiO4 . The monovalent anions can be replaced partly by bivalent anions such
2 2
as CO3 (type A carbonate apatite), O , or anionic vacancies. No large amounts of vacancies have been reported in trivalent anionic
sites, probably because of the instability of the lattice resulting from the creation of bulky defects. The maximum amount of cationic
and/or monovalent anionic vacancies can be rather large and it may reach 2 per unit formula. This means that apatite with all
monovalent anionic sites empty can exist. It seems, however, that such sites in apatites obtained in aqueous media are not empty and
can be occupied by water molecules as suggested by Rietveld analyzes of calcium-deficient apatites64 or solid-state NMR studies.65
Apatites containing vacancies or ionic substituents can be easily prepared in aqueous media. In order to preserve the elec-
troneutrality of the solid, several charge compensation mechanisms are observed. Only a few examples related to calcium
phosphate apatites and biological applications will be given.
For example, the replacement in part of bivalent cations by trivalent ones can be compensated for by the increase of the charge
of monovalent anions or by that of trivalent anions:
Ca8 La2 ðPO4 Þ6 O2
Ca8 La2 ðPO4 Þ4 ðSiO4 Þ2 ðOHÞ2
The replacement of trivalent PO3

4 anions by bivalent ones results in the formation of both a cationic vacancy (represented by
squares in the chemical formulas) and an anionic vacancy in monovalent site, for HPO2 4 – and CO3 -containing apatites:
2
Bone mineral composition has been approximated with some success using such chemical formulas.66
However, other phenomena can disturb this rather simple charge compensation mechanism. For example, additional Ca2 þ
and OH ions can be incorporated in the lattice at alkaline pH in carbonate apatites:

An internal hydrolysis of PO3
4 groups (giving rise to hydrogen phosphate ions and additional OH ions) may also occur with
residual water molecules associated with the solid:
The precise composition of the apatite unit-cell can be obtained traditionally by determining the density of the solid and the
unit-cell dimensions. Although Rietveld refinements can be performed to determine the site occupancy, the simulation becomes
rapidly rather complex when several possibilities of charge compensation have to be considered. A homogeneous distribution of
foreign atoms is generally assumed, although heterogeneous distribution and atoms clustering can occur in the structure. This is
especially the case for nanocrystalline apatites.
The term apatite corresponds also to a structure, or more precisely, to a series of structures. Most apatites crystallize in a
hexagonal structure and belong to the P63/m group.67 In this structure, the calcium ions can occupy two sites labeled as I and II.
Calcium I sites are on the trigonal axis of the structure at 1/4, 3/4, 1/2 and 3/4, 1/4, 1/2 positions. The Ca II ions form equilateral
triangles at z¼ 1/4 and z ¼ 3/4, on the 63 axis of the structure. These ions constitute part of the walls of “channels” where the
monovalent sites are located. They correspond to the narrowest part of the channels with a diameter of 0.27 nm for Ca-P apatites.
At z ¼1/2, the channels appear slightly larger (0.29 nm) and they are limited by a distorted hexagon of oxygens belonging to PO3 4
ions. Owing to the existence of these channels, apatites have sometimes been compared to zeolites; the channels appear, however,
smaller than those generally found in zeolites, especially in the case of calcium phosphate apatites, and they are monodimensional
and obstructed by ions, which limit considerably the exchanges at low temperature and the trapping of molecules. Nevertheless, it
has been shown that small molecules such as O2 and possibly, CO2 or small organic molecules like glycine,68 formate ion,69 and
even acetate ions can be trapped in these channels. In addition to the hexagonal structure, several Ca-P apatites, always stoi-
chiometric, such as HA, chlorapatite, and type A carbonate apatite crystallize in the monoclinic structure, P21/b group, due to an
ordering of the monovalent anions (or bivalent anions and vacancies) in the channels of the structure.24
1.11.2.3.2 Nanocrystalline and biomimetic apatites

Except for enamel and a few other mineralized tissues, most biological apatites are nanocrystalline. However all nanocrystalline
apatites are not similar to biological ones: in addition to their size, biomimetic nanocrystalline apatites exhibit several other
characteristics.
1.11.2.3.2.1 Nanocrystalline apatites

Several data support the concept that hydroxyapatite nanocrystals synthesized or in contact with an aqueous media exhibit a
surface composition very different from that of the bulk. One of the first model of an hydroxyapatite surface in water was provided
by Brown who suggested that the surface was close to the hydrated layer found in OCPt structure.70 Later a publication involving
different teams and reporting data obtained using different characterization techniques established that hydroxyapatite synthe-
71
sized in aqueous media showed distinctive HPO2 4 ions on their surfaces. Another alteration occurring on hydroxyapatite surface
is their carbonation often observed in carbonate containing solutions or even in CO2 rich atmospheres.72 These surface reactions
are amplified in nanocrystalline apatites. Another characteristic of nanocrystals, depending on their preparation conditions, is a
broadening of lines of XRD patterns and different types of spectra (FTIR, Raman, Solid-state NMR, EXAFS) assigned to size effects
and a high level of crystal strains.
1.11.2.3.2.2 Biomimetic nanocrystalline apatites

In addition to their nanocrystalline size, these apatites exhibit three main characteristics73:
1. Nonstoichiometric chemical composition mainly related to the replacement of PO3 2 2
4 ions by HPO4 and CO3 ions leading to
a Ca/(P þ C) ratio close to 1.3 (see Section 1.11.3.1.3)
2. Plate-like habit of crystals, with the hexagonal c axis along the length of the plates, a morphology rather unusual for apatites.
3. A developed hydrated layer on the surface of plate-like crystals containing mainly bivalent mineral ions (Ca2 þ , Mg2 þ , CO23 ,
HPO24 ), in non apatitic environments (Fig. 1).
Specific surface reactions involving the hydrated layer have been studied in the early 1950s by Neuman,74 and these con-
ceptions have been developed and refined since then to explain spectroscopic data and chemical behavior.75
Spectroscopic data suggest that the core of biomimetic apatite nanocrystals is formed by a nonstoichiometric apatite. However
it is at this date difficult to discriminate the chemical composition of the core and that of the surface.
The proportion of hydrated layer has been found to be particularly important in freshly precipitated apatites. Upon aging in
solution, the more stable apatite domains develop, progressively, incorporating ions from the hydrated layer, if they can be
inserted in the apatite lattice. This process determines the composition of the relatively stable apatite domains. The extent of the
hydrated layer has been evaluated in synthetic biomimetic apatites, through the determination of labile HPO2 4 species in close
contact with water molecules. The solid state NMR data show generally a very high labile HPO2 4 content in freshly prepared
biomimetic apatites representing about 50% of the total P content.76,77 FTIR data allow the discrimination of different HPO2 4
78
ions (apatitic and non-apatitic or labile) with lower amounts close to about 30% of the total P content for the labile HPO2 4 . The
discrepancy between these two techniques could be related to different preparation and sampling methods although alteration of
these very fragile entities could also occur during the analysis.
The ions in the hydrated layer appear to be relatively mobile and they can be very easily exchanged by other mineral ions or charged
organic groups in adsorption reactions (see Sections 1.11.4.5.1 and 1.11.4.5.2 on surface properties). In addition, the possibilities of
ionic substitutions appear much more numerous in the hydrated layer than in the apatite lattice; thus, ions like magnesium, for
Fig. 1 Model of the surface of biological and synthetic biomimetic apatite. The core of the nanocrystals is made of a nonstoichiometric apatite
domain constituting a rigid and stable crystalline lattice. The surface is formed by a fragile structured hydrated layer which is destroyed by drying.
This layer contains mainly bivalent mineral ions. On aging (maturation) in aqueous media, the very stable apatite domain grow (arrows)
incorporating the mineral ions of the hydrated layer which can be inserted in the apatite lattice. The ions of the hydrated layer can be rapidly and
reversibly exchanged by ions of the solutions (double arrows) and such reactions alter strongly the structure of the hydrated layer. Charged groups
of macromolecules and proteins (labeled Pr) can also participate to such ion exchanges leading to adsorption of molecules on the crystals. The
adsorption (or ion exchange) affinity and the amount of molecules (or ions) which can be adsorbed (or exchanged) vary with the extent of the
hydrated surface domains. Adapted from Ref. [197].
example, can easily replace calcium ions in the hydrated layer but they are only incorporated in minute quantities in the apatite lattice;
they are preferably located on the hydrated surface of the crystals and then always remain, for a major part, exchangeable.
The surface hydrated layer has been found to be structured in aqueous media, using wet spectroscopic characterization methods
with ambivalent features: FTIR spectroscopy suggests the existence of OCPt-like environments, whereas NMR indicates the pre-
sence of mineral ions in an environment close to that in DCPD structure.79 Upon drying, these surface structures are strongly
altered and the surface environments appear amorphous-like, which induces some mis-interpretations regarding the nature of
biological mineralizations.31,80 The structure of the hydrated layer depends also on its composition and appears to be reversibly
altered in ion exchange reactions.81 These observations point out the necessity to use various techniques to accurately characterize
nanocrystalline apatites. It also appears that conclusions regarding the presence of foreign phases in bone, or materials involving
nanocrystalline apatites cannot be drawn from spectroscopic data alone. Wet characterization methods should be performed to
prevent any alterations of these fragile entities on drying.
1.11.2.3.3 Synthesis and formation of apatites

Apatites can be prepared by wet and dry methods.
1.11.2.3.3.1 Wet preparations

Wet preparation methods of apatites include direct neutralization reaction between Ca(OH)2 and H3PO4 solutions, double
decomposition between soluble calcium and phosphate salts, hydrolysis reactions of nonapatitic calcium phosphate phases, or else
the conversion of other calcium or phosphate salts; in addition, several sol–gel and nonaqueous methods have been proposed.
The neutralization method does not leave any counter-ion residue and any by-product to discard. It thus appears as a good
method for industrial preparations; however, it generally gives precipitated apatites with carbonate ions impurities. Double
decomposition seems to be the preferred method at the laboratory level and it is often used, also, for industrial production.
Continuous precipitation processes using double decomposition have been studied, giving apatites with different chosen com-
positions (from TCP to stoichiometric HA).82 Calcium nitrate and ammonium phosphate are the preferred starting salts. Although
tiny proportions of nitrate or ammonium ions can be trapped in the precipitate during synthesis, they are easily eliminated by
heating. Hydrolysis reactions are also relatively clean methods: they are generally based on the hydrolysis of DCPD or DCPA.83
However, they require a large amount of water and part of the phosphate is lost in the waste water. Conversion methods are
mostly based on calcium carbonate treatment in phosphate-containing solution.84
The important parameters for wet methods are the pH, temperature, maturation time in solution, and the way of addition of
the reactants, in case of double decomposition. It has been shown that the rate of growth of apatite crystals is rather low, and in
many cases, apatite formation proceeds through the hydrolysis of precursor phases, essentially ACP and OCP, having a higher rate
of formation but a lower stability.
Apatites are generally obtained at slightly acidic or alkaline pH. The solubility isotherms indicate that HA is the most stable
Ca-P compound in aqueous solution at pH greater than 4 at 371C.24 The nonstoichiometry of apatites is directly related to the pH
85
value, and, at neutral or slightly acidic pH, the solids generally contain a large amount of HPO2
4 ions and are nonstoichiometric.
The synthesis temperature also affects the chemical composition, degree of nonstoichiometry, and crystallinity of apatites. The
crystallinity is, in general, greatly improved when the apatites are synthesized at rather high temperature, although the rate of
formation and the supersaturation ratio are also important. At, or under, room temperature, the first precipitate formed is, most of
the time, an amorphous phase, which is then transformed into more stable apatites at a rate strongly related to temperature and
pH. At constant pH, the elevation of temperature leads to apatites closer to stoichiometry.
The importance of the maturation time or aging time in solution is related to the process of formation of apatites involving
intermediate nonapatitic phases and/or nonstoichiometric apatite precursors. Aging is associated with an evolution of the apatite
toward more stable less soluble ones. Different processes seem, however, to be involved depending on the type of apatites. Three
types of aging mechanisms can be distinguished: (1) the development of the apatite domains at the expense of the less
stable surface hydrated domains, especially active in nanocrystals, (2) the chemical evolution leading to more stable apatites closer
to stoichiometry, and (3) the Ostwald ripening process, based on the decrease of interfacial energy of crystals, which favors the
growth of large crystals at the expense of smaller ones. The fastest process, especially active in biological systems, seems to be the
first one.
The order of addition of the reactants and relative proportions also seem to be important parameters in apatite preparation:
when a calcium excess is present in the solution, the apatites obtained are generally closer to stoichiometric ones, whereas such a
stoichiometric composition seems more difficult to obtain in the presence of an excess of phosphate ions.
Some impurities are often present in apatites synthesized by wet methods: magnesium, carbonate, and HPO2 4 ions are among
the most important ones.
Magnesium is always the major cation impurity, found in variable concentrations, in most commercial calcium salts. Very
disappointingly, some providers of such salts do not think it useful to signal the presence of this impurity. We encourage the
different actors in the biomaterial field to determine and control the magnesium impurities in their products. This element has
some chemical and biological effects, which may modify the properties of biomaterials.86 It has an inhibitory effect on apatite
nucleation and crystal growth and it alters the apatite crystal morphology. It can be located on the surface of the crystals and be
readily available, modifying the biological behavior of implants and scaffolds for tissue engineering.
Carbonate impurities are related to reactions with atmospheric CO2 during preparation and washing of precipitated apatites.
Surface carbonation may also occur by simple contact with carbonate-containing solutions. Like magnesium, carbonate is an
inhibitor of apatite crystals nucleation and growth. Carbonate can be eliminated by heating at 900–10001C with a possible partial
decomposition of the apatite phase (see Section 1.11.4.1).
Hydrogen phosphate ions result from the precipitation reaction or the internal hydrolysis reaction of PO3 4 ions by water
molecules. They are eliminated on heating (see Section 1.11.4.1 on thermal stability of apatites), but as in the case of carbonate,
decomposition of the apatite may occur depending on the Ca/P ratio.
1.11.2.3.3.2 Dry preparations

Several dry preparation methods of apatites have been published, essentially for stoichiometric apatites. Basically, they consist in
calcination between 900 and 11001C of solid reactants in the stoichiometric ratio (Ca/P ¼1.67). HA, for example, can be obtained
by heating a mixture of calcium carbonate and TCP at 10001C under water vapor:
CaCO3 þ 3Ca3 ðPO4 Þ2 þ H2 O-Ca10 ðPO4 Þ6 ðOHÞ2 þ CO2
Fluorapatite (FA) or Chlorapatite (ClA) can be prepared with similar reactions:
CaF2 ðor CaCl2 Þ þ 3Ca3 ðPO4 Þ2 -Ca10 ðPO4 Þ6 Fðor ClÞ2
But many other reactants can also be chosen such as pyrophosphate in preparation of hydroxy- or fluorapatite:
4CaCO3 þ 3Ca2 P2 O7 þ H2 O-Ca10 ðPO4 Þ6 ðOHÞ2 þ 4CO2
or a more complex reaction87:
14CaF2 þ 18Ca2 P2 O7 -5Ca10 ðPO4 Þ6 F2 þ 6POF3
releasing POF3 as a gas.

Other interesting processes, concerning apatites, are ion substitutions at high temperature (900–10001C). It is thus possible to
convert a HA into carbonated (type A), chlorinated or fluoridated apatites, for example,
2HCl þ Ca10 ðPO4 Þ6 ðOHÞ2 ⇄Ca10 ðPO4 Þ6 Cl2 þ H2 O
Substances like NH4Cl can be more conveniently used to generate HCl directly in the furnace.
NH4Cl is sublimated and decomposes into HCl and NH3 above 3501C in the oven. If the reaction is made in dry atmosphere, a
pure stoichiometric, monoclinic ClA is obtained.88 These rather fast reactions do not change the crystal or ceramic characteristics;
however, in bulk thick ceramics, only the surface might be modified due to the limited mobility and slow diffusion of the ions.
1.11.2.4 Other Phosphate Compounds

Several other phosphates are of potential interest for biological applications either because they may appear as impurities in Ca-P
compounds or because of their properties.
Calcium pyrophosphates contain the P2 O4 7 anion. Dicalcium pyrophosphates Ca2P2O7 may appear as an impurity in TCP.
Calcium pyrophosphates may also form in ectopic calcifications as calcium pyrophosphate dihydrates.89 Several polymorphs of
these pyrophosphates exist. Pyrophosphates have been tested as biomaterials; they are bioresorbable and seem well tolerated.90
Polyphosphates can exist as linear chains of phosphate groups: [P2O7(PO3)n](n þ 4) or cyclic anions ðPO3 Þn
n . Cyclic poly-
phosphates give soluble complexes with calcium and they are mainly used in washing powders. Linear polyphosphates may give
amorphous glasses or crystalline substances and some of them have been proposed as biomaterials.91
Several other calcium-containing orthophosphates are of potential interest:
Rhenanite (or Buchwaldite), NaCaPO4 or Chlor-spodiosite, Ca2PO4Cl which could possibly be used in cements; Merillite:
Ca18Na2Mg2(PO4)14, Brianite Na2CaMg(PO4)2.
1.11.3 Characterization Methods of Calcium Phosphates
Characterization of calcium phosphates can be the easiest or the most difficult task depending on the type of compound and the
extent of information required. The characterization of DCPD or DCPA phases, for example, is rather straightforward and can be
obtained by different techniques. Systems such as biomimetic nanocrystalline apatites prepared in close-to-physiologic conditions
have been shown to exhibit a rather complex composition and structure as shown in Fig. 1, involving an apatitic core (generally
nonstoichiometric) and a hydrated surface layer-containing anions and cations in positions which do not correspond to the regular
apatite structure (see Section 1.11.2.3.2). In this case, no single characterization technique can provide sufficient data, as it only
enables to determine one characteristic of the system. Therefore, the conjoint use of several techniques along with chemical
analyzes is of foremost importance in the quest for precise characterization of complex Ca-P materials. Chemical composition is
the first step, followed by XRD, electron microscopy, and spectroscopic techniques such as FTIR, Raman and solid-state NMR
spectrometries.
1.11.3.1 Chemical Analysis

The thorough physicochemical characterization of calcium phosphate-based ceramics (as for any other advanced materials)
requires, as one of the main steps, to determine precisely the nature and relative amounts of the chemical elements composing the
system. Such data then allow deriving the overall chemical formula of the compound studied. This consideration appears
especially essential for apatitic materials, which can accommodate a large number of ionic vacancies and which can also tolerate
many ionic substitutions.
It is possible to distinguish three types of ions in calcium phosphates of biological origin and in many synthetic apatites:
• major ions like Ca2 þ , orthophosphates, including PO3 2 2

4 and HPO4 , CO3 , and OH ;
• minor ions like Na þ , Mg2 þ ;
• trace elements such as Sr2 þ , Zn2 þ , F.
The techniques which are possible to use depend on the abundance of the elements.
1.11.3.1.1 Major ions

1.11.3.1.1.1 Calcium
The amount of calcium contained in a given specimen can be drawn by way of many techniques such as atomic absorption
spectroscopy (AAS), inductively coupled plasma (ICP) emission spectroscopy, ionometry using a specific electrode, ion chro-
matography, old but still competitive chemical methods like complexometry (eg, with a complexing agent such as ethylene
diamine tetracetic acid (EDTA)), or even X-ray fluorescence or electron microscopy-based techniques (microprobe analyzes,
energy-dispersive X-ray analyzes). Although the latter offer the possibility to observe the spatial distribution of the elements(s)
across the sample, they require a precise preliminary calibration step and do not appear generally accurate enough to distinguish
phases with close composition or to determine a departure from stoichiometry. The other techniques cited above are thus more
frequently used for the routine determination of the amount of calcium contained in a specimen. Determinations based on AAS or
ICP seem to provide relatively accurate data, and these techniques are more specific than complexometry where the complexation
of other cations may simultaneously occur during the titration, thereby leading to nonspecific cationic content measurements.
Direct determinations of calcium ions in solution can also be made by ion chromatography or by ionometry. These methods are
not as sensitive as AAS or ICP-related methods, but they can be useful for a follow-up of calcium concentration in solutions in
contact with Ca-Ps.
1.11.3.1.1.2 Phosphorus
Phosphorus contained in calcium phosphates is generally present either under the form of orthophosphate ions (PO3 2
4 , HPO4 ,
H2 PO 4 ) or as pyrophosphate ions P O4
2 7 . It is then such oxygenated phosphorus species that are generally followed. The
total phosphorus content can be determined by some of the methods, which can also be used for calcium such as ICP, or
electron microprobe analysis. However, these methods do not allow discrimination between orthophosphate and other
phosphorus-containing molecules or ions. Ion chromatography can be used in such a situation, regarding the orthophosphate and
pyro- or polyphosphate content, but the distinction between the different orthophosphate ions is impossible due to fast equi-
librium reactions between these species. This distinction can only be made directly on the solid samples using vibrational
spectroscopies or solid state NMR or indirectly using the method developed hereafter.
Orthophosphate ions can usually be satisfactorily titrated by spectrophotometry of the phospho-vanado-molybdenum com-
plex either directly (yellow coloration) or after reduction (blue coloration).92 The first one is more accurate but less sensitive. All
orthophosphate ions present in the system, under any form, are analyzed. Other phosphate groups such as pyrophosphate ions or
metaphosphates are not analyzed. When such ions are present, however, a slow hydrolysis may occur in the aqueous medium
needed for the analysis, and it is advised to carry out the determination using these spectrophotometric methods just after
dissolution. Such hydrolysis reactions could also occur with esters of phosphoric acid.
The HPO2 4 content appears as an important characteristic in several calcium phosphates, especially in dicalcium phosphates,
ACP, or octacalcium phosphates. In apatites, the amount of HPO2 4 ions is related to nonstoichiometry, and in nanocrystalline
apatites, it may also appear as a characteristic of the surface composition. HPO24 in calcium orthophosphates can be determined
after thermal conversion into pyrophosphate ions as shown by Gee and Dietz.93,94 Samples are heated at 5001C for 3 h (or
preferably at 6001C for 1 h):
4 -P2 O7 þ H2 O
2HPO2 4
The amount of pyrophosphate ions is then determined either after separation on an ion exchange resin or simply by difference
in orthophosphate analyzes before and after pyrophosphate formation or, more accurately, before and after hydrolysis of the
pyrophosphate ions in solution, in identical aliquots of the sample. A limitation to this determination of the HPO2 4 content,
however, exists in the simultaneous presence of carbonate ions, as side reactions between HPO2
4 and carbonate ions may interfere
(see Section 1.11.4.1).78 Another difficulty is the possible internal hydrolysis of PO3
4 with water molecules attached to the solid
during heating.85 An alternative to the rather time-consuming analytical method proposed by Gee and Dietz exists for nano-
crystalline apatite where most of the HPO2 2
4 ions belong to a surface hydrated layer. The HPO4 content can be evaluated from the
mathematical decomposition of the n4PO4 band of the FTIR spectrum.78 Indeed, a rather good correlation could be obtained
between the amount of HPO2 4 drawn from chemical analyzes and by this FTIR-based method. However, the curve-fitting
parameters need probably to be refined for obtaining more precise determinations.
1.11.3.1.1.3 Carbonate
Several methods can be used to determine the carbonate content of calcium phosphate samples. One of the best methods is
probably coulometry. The carbonate content of Ca-P samples can be determined using a CO2 coulometer measuring the CO2
released upon sample dissolution in acidic media or heating of the sample. The CO2 released is transferred into a photometric cell
in a nonaqueous medium and titrated through an acid–base reaction.95 A second method is the CO2 analysis through gas
chromatography or a carbon-hydrogen-nitrogen (CHN) classical analysis system, releasing the CO2 by heating, although the
heating parameters have to be adjusted to be sure to decompose all carbonate ions.96 Thermogravimetric determination of
the CO2 evolved can be carried on; however, generally, CO2 and water loss cannot be easily separated, and in this case also, gas
chromatography has to be used. Another method for total carbonate content is FTIR spectroscopy. The spectral lines integrated
intensity of carbonate is determined relatively to that of phosphate bands.97 The accuracy seems lower than that of chemical
methods; however, FTIR spectroscopy allows the type of carbonate (type A and/or B, see Section 1.11.2.3.1) involved to be
determined on very limited amount of sample.
1.11.3.1.1.4 Hydroxide ions

For HAs, the evaluation of the amount of OH ions is also important, although this determination is rarely made. The chemical
method proposed by Trombe and Montel98 consists in a substitution of OH ions by F ions at 10001C; when HA or oxy-HAs are
heated in the presence of an excess of CaF2 in a dry atmosphere, FA is formed and H2O is released:
CaF2 þ Ca10 ðPO4 Þ6 ðOHÞ2 -Ca10 ðPO4 Þ6 F2 þ CaO þ H2 O
The mass of the released water corresponds to the amount of OH ions initially present in the solid. An excess of CaF2 is used
(up to 20%) to be sure that the reaction is completed. The application of this method supposes, however, that there are no other
possible reactions. The decomposition of HA at high temperature under vacuum appears also as a possible method, although it
does not seem to have been utilized much:
Ca10 ðPO4 Þ6 ðOHÞ2 3 Ca10 ðPO4 Þ6 O þ H2 O
Other reactions like that involving pyrophosphate and HA (around 800–11001C):

Ca2 P2 O7 þ Ca10 ðPO4 Þ6 ðOHÞ2 -4Ca3 ðPO4 Þ2 þ H2 O
are being studied with interesting results and could give information on the possible nonstoichiometry of well-crystallized HA.
Among other methods, the relative intensity of FTIR OH lines at 3570 or 633 cm1 can also be used and applications have
been developed for the determination of OH in oxy-HA coatings,99 which can be extended to bone and nanocrystalline apatites.
An alternative method is to use the Raman OH stretching band at 3570 cm1. These necessitate, however, calibration curves
obtained with well-characterized reference samples (or mixtures of samples) in a preliminary step.
1.11.3.1.2 Minor and trace elements

The determination of minor and trace elements is usually performed by spectroscopic methods such as AAS or ICP-related
methods using emission spectroscopy or mass spectroscopy. In some cases, neutron activation analyzes have also been used to
determine trace elements in biological samples. Fluoride is found in apatites of biological origin at low concentration (less than
0.1%) and with higher amounts in the case of a bone disease, fluorosis.100 This ion is generally determined quite accurately by
ionometry using a specific electrode.
1.11.3.1.3 Meaning of the Ca/P ratio

The atomic Ca/P ratio of calcium phosphates is generally considered as one of their major characteristics. The Ca/P ratio of most
calcium phosphate phases is generally restrained to a narrow domain of variations. In the case of apatites, however, the Ca/P ratio
may vary in a wide domain and it is frequently used to characterize the apatite composition. Two cases shall be considered:
The first one concerns nonstoichiometric apatites with Ca/P ratios lower than that of the stoichiometric composition
(Ca/P ¼ 10/6¼ 1.667), which occur when the apatite contains HPO2 3
4 ions in place of PO4 ions, as discussed in the section on
apatite composition. Considering that phosphate sites do not accommodate large amounts of vacancies, the Ca/P ratio represents
then the amount of cationic vacancies.
The second one concerns nonstoichiometric apatites with carbonate ions replacing phosphate groups (type B carbonate
apatites). In this case, the Ca/P ratio does not represent the amount of cationic vacancies and it has no simple meaning. However,
considering that there are only a few vacancies in the trivalent anionic sites of the apatite structure (all six sites are occupied either
by phosphate or by carbonate groups), it is the Ca/(P þ C) ratio (where C represents the amount of carbonate ions replacing
phosphate groups) which represents the departure from stoichiometry and the cationic vacancies.
In more complex compositions when other cations than calcium, for example, Sr2 þ , are present in carbonated apatite, the
atomic ratio, related to cation vacancies, to consider is (Ca þ Sr)/(P þ C). Similarly when silicate or other ions are incorporated in
place of phosphate the cationic vacancies possibly present are given by Me/(P þ Si), where Me represents all the cations in the
apatite structure.
For nanocrystalline biomimetic apatites, the situation is even more complex due to the surface composition, which cannot be
neglected, and which is different from the core of the crystals. An important proportion of bivalent ions is on the surface, but this
amount is difficult to estimate, and there is no adequate model describing these compounds (see Section 1.11.2.3.2.2). In this case,
the Me/(P þ C) ratio does not have a meaning and it has no correlation with the amount of cationic vacancies in the apatite domains.
Special techniques for the determination of Ca/P ratio using XRD have also been developed and standardized in the case of HA
ceramics and coatings, which also imply several assumptions (see Section 1.11.3.2).
1.11.3.2 Diffraction Methods

XRD is an essential method for the characterization of calcium phosphate materials. XRD experiments have been performed on
monocrystals, polycrystalline bulk ceramics, coatings, or powder samples. The first point is not discussed here, although it has
been involved in the determination of most Ca-P structures (see Table 1 for references). The peak position of XRD diffraction
patterns allows the identification of all crystalline calcium phosphate compounds as recorded in the databases of diffraction
patterns such as International centre for diffraction data (ICDD), Joint committee for powder diffraction standards (JCPDS) or
other. In everyday uses, some specific intense peaks are used to recognize the main Ca-P phases (Table 2).
The identification of phases has been standardized for several biological applications (ISO Standard 13779-3), mainly related
to high-temperature processing of apatites as coatings or ceramics. The proposed method is based on the use of reference samples
and single peaks integrated intensity ratios and needs the establishment of a standardization curve using known mixtures of phases
in presence. However, other methods have now been developed using the integrated intensity of all the peaks of phases in presence
and involving Rietveld analysis. These methods suppose a knowledge of the crystallographic structure, including atomic positions
of each crystallized phase. In addition it is assumed that the phases are “pure,” and contain only specified atoms.
Table 2 Useful lines for the identification of main Ca-P phases in ceramics and thermal coatings by diffraction
techniques (ISO Standard 13779-3)
Phase JCPDS folder hkl dhkl (A )̊ 2y (lCu ¼1.54 A )̊
a-Tricalcium phosphate 09-0348 4 4 1 2.905 30.753

b-Tricalcium phosphate 09-0169 02 1 0 2.88 31.027
Tetracalcium phosphate 25-1137 0 4 0 2.995 29.807
Calcium oxide 82-1690 2 0 0 2.405 37.361
Apatite 09-0432 2 1 0 3.08 28.966
Apatite 72-1243 2 1 1 2.81 31.820
The determination of the ACP, or oxyhydroxyapatite (OHA) content existing in plasma-sprayed coatings, however, remains an
open question. The standards define only a crystallinity ratio not necessarily related to the amount of amorphous phase as it has
been shown in the case of bone mineral 30 years ago.101 The methods using standardization curves ACP/HA appear rather
inaccurate and one of the best direct method at present is probably the determination of the heat of crystallization of the
amorphous phase by differential scanning calorimetry (DSC) or differential thermal analysis (DTA).102,103 Rietveld techniques can
also be used in this case, however they do not detect really amorphous phases but more likely diffuse diffraction phenomena
which might not be necessarily associated with a distinct amorphous phase with clear boundaries, but could be associated with
defects and disorders in the crystals.101 For OHA, the XRD pattern is very close to that of HA and these phases are rather difficult to
distinguish based on peak positions. Recently however, the quantitative identification of foreign Ca-P phases in HA and ClA
coatings, including ACP and OHA, has been proposed using Raman spectroscopy.35
Among the difficulties related to phase identification is the distinction of poorly crystalline apatites and OCPt, due to XRD
patterns analogies and peak broadening. The most intense peak of OCPt, (100), corresponding to a 1.868 nm d spacing, has been
shown to be characteristic of this phase. The observation of this peak might need special settings of the diffractometer to reach low
angle. Another difficulty arises from the preferential orientation of the crystals due to several causes, such as the preparation of
the samples for XRD, a preexisting crystal orientation like in biological tissues (bone, dental enamel), or the processing of the
Ca-P-based biomaterial. Such preferential orientations can considerably change the intensity of the different peaks and can make
phase identification and quantification difficult.
In addition to phase recognition, XRD can be used to determine the unit-cell dimensions of the different crystallized Ca-P
compounds. These crystallographic parameters can also be used to estimate the effect of ion substitutions on the structure. Such
effects have been particularly well documented for the apatite structure. It is well established that the substitution of cation by a
bigger one induces an increase of both dimensions of the hexagonal unit-cell. The replacement of a trivalent anion by a bigger one
has the same influence. However, when a monovalent anion is replaced by a larger one, it induces an increase of the hexagonal a
unit-cell dimension and a decrease of the c dimension. Generally, substitutions in apatite follow the Vegard’s law, and the increase
or decrease of the unit-cell dimensions is linearly proportional to the amount of ions introduced. Thus, increments can be
determined for different ions of biological interest as shown by McConnel (Table 3).104
Important information brought by XRD concerns the crystal size and strains, which induce a peak broadening. Basic appli-
cation in the biological field for the crystal size determination uses Scherrer’s method neglecting the contribution of micro-
strains.105 More elaborated treatments can be performed considering the effect of microstrains and crystal sizes, which are included
in Rietveld analysis of the XRD diagrams available from different Web sites. Such analysis allows the determination of crystal size
in different crystallographic directions, microstrains, unit-cell dimensions, phase composition but also structural information such
as the atomic position of substitutes or vacancies in favorable cases or the site occupancy. For example, the orientation of
carbonate species in carbonate apatites (type A and type B),106,107 the location of vacancies in nonstoichiometric apatites64 or that
of Sr in Sr-containing apatites have been determined using Rietveld analysis.108 Although these very powerful methods have
brought valuable information on calcium phosphates, they may sometimes lead to unrealistic solutions, and a critical examination
of the data is advised. One of the difficulties, especially annoying in the case of apatites, is that these treatments do not consider
the possibilities of a distribution of solid solutions with variable compositions, which is known to exist in bone and synthetic
preparations (in fact in all Rietveld analyzes, the phases are considered as homogeneous). Such samples would exhibit peaks
broadening falsely interpreted as strains and/or size effects.
A special, very accurate technique, for the determination of the Ca/P ratio by XRD has been developed and standardized for
Ca/P ratios between 1.50 and 2 (ISO standard 13779). Nonstoichiometric apatites are not stable and they decompose on heating
at 900–11001C depending on their Ca/P ratio. If the Ca/P ratio is lower than 1.67, the heated sample is constituted by stoi-
chiometric b-TCP and HA provided the samples are chemically pure (only Ca2 þ , orthophosphate and hydroxide or oxide ions).
If the Ca/P ratio is higher than 1.67, a mixture of stoichiometric HA and calcium oxide is observed. For a Ca/P ratio equal to 1.67,
no decomposition is observed.109,110 The foreign phases (b-TCP and calcium oxide) can be detected with high accuracy by XRD
Table 3 Increments of crystallographic parameters of the apatite unit cell related to cationic and monovalent anionic substitutions in phosphate
apatites for biological relevant ions. The increments are determined for 1 atom substituted (D) (values in Angstrom)
a Parameter FA D (OH- F) HA D (Cl-OH) ClA D (Cl-F)
Ca 9.370 0.025 9.420 0.105 9.630 0.130

D (Sr- Ca) 0.034 0.034 0.024
Sr 9.710 0.025 9.760 0.055 9.870 0.080
c Parameter FA D (OH - F) HA D (Cl-OH) ClA D (Cl-F)
Ca 6.880 0.000 6.880 0.050 6.780 0.050

D (Sr- Ca) 0.040 0.040 0.024
Sr 7.280 0.000 7.280 0.045 7.190 0.045
Source: According to McConnel Ref. [104].

and quantified. The accuracy of the method lies on the observation that when the chemical Ca/P atomic ratio varies by about 10%
between 1.50 (pure TCP) and 1.67 (pure HA), the phase composition varies by 100%.
A similar technique can be used for the determination of the purity of TCP. Heating at 9001C shall give only b-TCP phase.
Samples with a Ca/P lower than 1.5 give an additional Ca-pyrophosphate phase and samples with a Ca/P ratio higher than 1.5
contain stoichiometric apatite as explained above. However, in this case, the detection of pyrophosphate by FTIR spectroscopy
appears much more sensitive than by XRD.
1.11.3.3 Electron Microscopy Techniques

Electron microscopy techniques are widely used by materials scientists with several objectives:
• determination of the crystal morphology, their size and organization, including pores distribution in ceramics;
• identification of phases at the microscopic level using electron diffraction;
• composition determination using Energy Dispersive Spectrometry (EDS) or Wavelength Dispersive Spectrometry (WDS); and
• analyzes of crystal defects through lattice imaging at high resolution.
Most Ca-Ps exhibit specific crystal morphologies, which can be used for their identification. Mathew and Takagi111 proposed a
classification of the most used Ca-Ps into three major structural types: (1) the apatite-type structure also including apatite-related
structures such as OCP, (2) the glaserite-type structure in which all the TCP polymorphs can be included, and (3) the Ca-PO4
sheet-containing compounds including dicalcium and monocalcium phosphate phases. However, this classification cannot be
used to determine the crystal morphology of these compounds, as other parameters can control nucleation and crystal growth of
these Ca-P phases leading, for example, in the cases of apatite or DCPD, either to needle-like or platelet-like morphologies. Some
of the typical and most common morphologies are shown in Fig. 2.
Some crystals can show a variable morphology depending on their formation conditions; however, some morphologies can be
considered as recurrent: this is the case, for example, of OCPt crystals appearing as platelet crystals forming imbricated leaflet with
irregular edges often compared to “sand-roses.” However, OCPt can hydrolyze into apatite, without change of crystal habit, and
these morphologies may also be associated with apatites. DCPD also forms platelet crystals with, generally, larger dimensions than
OCP crystals. Precipitated ATCP appears most generally as spherical units (diameter between 20 and 300 nm). Well-crystallized
apatites generally exhibit a needle-like morphology with crystals elongated along the c axis of the hexagonal structure. Platelets
hexagonal crystals with a short dimension along c have also been reported in special preparation conditions.112 Upon heating, the
morphology of apatite crystals changes considerably: the length is reduced and the width-thickness is increased, resulting in a
lower crystal surface energy. The hexagonal shape, however, remains recognizable. Some preparations such as hydrothermal
synthesis, slow hydrolysis of more soluble Ca-P salts (DCPD, TCPs), and reactions in fused salts may give apatite crystals a very
Fig. 2 Example of typical morphologies of Ca-P compounds. (a) Scanning electron microscopy (SEM) image of DCPD crystals; (b) transmission
electron microscopy (TEM) image of ACP (reproduced from Kim, S.; Ryu, H.-S.; Shin, H.; Jung, H. S.; Hong, K. S. Mater. Chem. Phys. 2005, 91,
500–506, with permission); (c) SEM image of OCP crystals (reproduced from Combes, C.; Rey, C.; Frec̀ he, M. J. Mater. Sci. Mater. Med. 1999,
10, 153–160, with permission); (d) TEM image of apatite crystals (reproduced from Ref. [115], with permission); (e) SEM image of b-TCP crystals
(reproduced from Choi, D.; Kumta, P. N. Mater. Sci. Eng. C 2007, 27 (3), 377–381, with permission); (f) SEM image of TTCP crystals.
high aspect ratio (length/width ratio) that can be considered as fibers.113 In most preparations, the needle-like crystals have the
tendency to form dendritic spherical units. Tooth enamel crystals show the typical morphology of apatite with very elongated
crystals. Bone crystals, however, appear essentially as platelet crystals with a rather irregular shape, elongated along the c axis of the
hexagonal structure, but with different perpendicular dimensions along a and b axis directions. This very unlikely morphology for
hexagonal crystals (supposed to possess equivalent a and b directions) has been considered as an indication of the formation of
OCP precursor phases.24 This morphology is also observed in nanocrystalline synthetic apatites precipitated at physiologic pH and
it is not confined to the growth of apatite crystals in bone matrix.114
Identification of phases at the microscopic level using electron diffraction, selected area electron diffraction (SAED), can be
performed on Ca-P samples. All these observations can be disturbed by beam damage especially when fragile hydrated phases or
nanocrystalline compounds are present.115
Composition determinations using EDS or WDS are often performed. These techniques are used for the determination of main
constituents of biomaterials and biological Ca-P compounds: essentially Ca, Mg, P, F. Their accuracy is, however, linked to the
stability of the Ca-P compound and the sample characteristics (surface roughness, conditions of analyzes), and even with the use
of adequate standards, the determination of the apatite nonstoichiometry, using these methods, remains difficult.
High-resolution transmission electron microscopy (TEM) is also a common technique often difficult to perform on very small
unstable crystals sensitive to beam damage. The lattice fringes allow an identification of the crystallographic planes, the deter-
mination of the d-spacings, the orientations of crystals, and the observation of crystal defects. Amorphous domains have also been
observed, at high resolution, at the surface of apatite nanocrystals corresponding probably to the dehydration of the structured
layer existing in wet media (see spectroscopic characterizations).
One of the problems of these techniques is the beam damage, which is frequently observed especially with hydrated Ca-Ps and
may lead to the decomposition of hydrated phases or even to the volatilization of phosphorus.116–118 As calcium phosphates are
nonconductive, it is necessary to limit the charge effect. In scanning electron microscopy (SEM), several techniques can be used:
(1) coating with an electrically conductive layer, (2) working at low accelerating voltage at short working distance, and (3) working
under alterated vacuum. The solution (1) is preferred for morphology examination. The high vacuum may also alter the hydrated
Ca-P phases; although environmental microscopes can be used.
1.11.3.4 Vibrational Spectroscopies

FTIR and Raman spectroscopies allow the identification of most calcium phosphate phases. Generally, these methods focus
on the use of internal vibrational levels of molecules and ions which may vary according to the short-range environments of these
molecules or ions. Phosphate, carbonate, hydroxide ions, and water molecules are the main species detected by FTIR and Raman
spectroscopies.
1 1 1
The PO34 group has four main vibrational domains: n1 (at about 950 cm ), n2 (400–470 cm ), n3 (1000–1150 cm ), n4
1
(500–620 cm ). A detailed presentation of vibrational spectroscopy of calcium phosphates has been proposed recently.119
The main characteristic bands of the FTIR and Raman spectra of different calcium phosphates are shown in Tables 4 and 5.
The phases that cannot be easily distinguished by XRD can be discriminated using FTIR or Raman spectroscopies. This
is the case, for example, of OCP and apatite or HA, OHA, and amorphous phases present in plasma-sprayed coatings.126 In
addition to phase identification, these vibrational spectroscopies can be used for phase quantification using standard mixtures and
standardization curves based on line intensity ratios. Raman spectroscopy allows also a mapping of the different phases in
coatings, for example.35 However, the principal advantage of vibrational spectroscopies over XRD is the identification of fine
structural details, especially concerning apatites, such as the location of carbonate species on the different sites of the apatite

structure, the presence of HPO2 4 species, the quantification of the amount of OH ions, or the fine characterization of nano-
crystalline apatites.119
Carbonate ions, like phosphate, have four main vibrational domains: n1 (at about 1050 cm1), n2 (820–900 cm1),
n3 (1400–1550 cm1), and n4 (650–750 cm1). Carbonate ions are always present in biological apatites and they may occupy
different types of sites historically labeled as A and B: site A corresponds to monovalent anionic sites of the apatite structure
and site B corresponds to trivalent anionic sites (Table 6). Several additional carbonate sites have been described127 and several
deviations have also been distinguished corresponding to different combinations with vacancies, ions substitutions, water
molecules or simply the combination of the two carbonate substitutions.127,128 It has been shown that carbonate may also be
incorporated in the surface hydrated layer of nanocrystalline apatite.119
One of the most powerful uses of vibrational spectroscopies concerns apatite nanocrystals and poorly crystalline apatites for
which XRD lacks accuracy. These methods are able to distinguish surface species and allow their quantification in simple cases.
1
Surface HPO24 have been shown to exhibit a specific band, at 534 cm in the n4 domain of the ion.131 In addition, FTIR and Raman
spectroscopies can be used on wet samples revealing specific structural environments, which are lost upon drying, as shown in Fig. 3.
Concerning OH ion, there is only one vibrational domain corresponding to the stretching of the O H bond (3400–
3650 cm1). However, a libration line, corresponding to a rotational energy level of the ion in the lattice, is also observed
(630–750 cm1), within the usual wavenumber window of the technique. OH bands are essentially detected in apatites and related
compounds like partly hydrolyzed OCP. The OH stretching and libration movements are very sensitive to hydrogen bonding, and it
has been shown in fluoride- and chloride-containing apatites that the OH bands can be considerably shifted away from their
position in HA.132,133 The OH vibration and libration modes are also very sensitive to the nature of the surrounding cations.119
Table 4 FTIR spectral characteristics of phosphate ions in different calcium phosphates salts (wavenumbers in cm–1)
DCPD DCPA OCP ATCP b-TCP a-TCP HA TTCP
n3PO4 1400 w
1350 w
1215 m 1295 w
1175 m,sh 1193 w
1132 s 1128 s 1137 vw
1121 s 1119 s
1103 s 1105 w
1094 w,sh 1092 s 1093 w
1070 s 1077 s 1080 w,sh 1073 w
1060 s 1064 s 1055 s 1055 s 1062 s
1041 vs 1039 s 1040 vs 1046 s
1037 s 1025 s 1033 m
1023 s 1013 s
1000 w,sh 1000 vvw 1010 w,sh 997 s 1010 s
984 s
n1PO4 984 s 992 m 972 s 989 s
962 w 962 w 962 w
954 m 956 w
945 m 946 w
941 w
P-OH st. of HPO2
4 917 w
872 m 892 m 861 w
n4PO4 627 vw 613 m 620 w
601 m 602 m 597 m 601 m 594 w
589 w 585 m
577 m 576 s 575 w 563 m 575 m,sh 571 s
563 s 560 m 550 m 551 m 561 m
526 s 525 m 524 w 541 m 501 w
n2PO4 480 471 w 472 vw 471 m
466 vw 463 w
454 w
418 sh 428 vw 449 vw 432 vw 430 w 450 w
400 m 405 m 415 w 429 w
398 sh 399 m
References 120 121 121 121 122 121 123
Water molecules exhibit three vibrational modes: the symmetric stretching mode (ns) and the antisymmetric stretching mode
(nas) have very close energy and they are difficult to distinguish. They generally give a broad absorption band on FTIR spectra
(3000–3700 cm1). The bending movement around 1640 cm1 gives a narrower band. Like OH, the water molecules are very
sensitive to hydrogen bonding. In some calcium phosphates such as DCPD, the lines due to water molecules appear very specific and
allow a clear identification. In other calcium phosphates (OCP, apatite, ACP), however, the water bands appear rather unspecific.
Recently, studies of water molecules in nanocrystalline apatites using near infrared spectroscopy have been published.134,135
1.11.3.5 Solid-State Nuclear Magnetic Resonance

Solid-state NMR allows the identification of the short-range environment of different nuclei possessing a magnetic moment, such
as 1H, 13C, 31P, 19F, which can be found in calcium phosphates.136
This very powerful technique also gives information on magnetic interactions between neighbor magnetic species. The main
lines detected in calcium phosphates and the corresponding assignments are reported in Table 7.
Like FTIR, NMR spectrometry is particularly suitable for the study of nanocrystalline and poorly crystalline apatites. Solid state
76,77,138
NMR data have thus confirmed the concept of the hydrated layer rich in HPO2 4 ions. In bone, several types of water
139,140
species have been distinguished.
The study of carbonate species by NMR spectrometry has confirmed the existence of the two main types of carbonate sites in
apatite named A and B, respectively at 166.5 and 170.2 ppm (TMS reference).141 In addition, a third type of carbonate species has
been detected in apatites, at 168.2 ppm probably close to water molecules confirming the FTIR results. Two-dimensional NMR
analysis of carbonated apatites has also confirmed the location of carbonate species in a hydrated domain.142
19
F solid-state NMR studies of fluoride-containing carbonated apatites have clearly indicated the existence of two crystal-
lographic sites corresponding to fluoride ions in monovalent anionic sites, (chemical shift at 68 ppm) and close to type B
carbonate ions (chemical shift at 78 ppm) (reference hexafluorobenzene).142
Table 5 Raman spectral characteristics of phosphate ions in different calcium phosphates salts (Raman shifts in cm1)
DCPD DCPA OCP ATCP b-TCP a-TCP HA TTCP
n3PO4 1132 vw 1131 m

1119 vw 1118 w 1119 vw
1112 w 1101 w
1079 w 1094 m 1079 vw 1090 w 1077 w 1077 w 1091 sh
1061 m 1064 w 1076 vw
1052 w 1058 w 1057 w
1048 w 1050 w 1048 w 1045 vw
1041 w
1036 vw 1034 w
1027 vw 1027 w 1029 w 1026 w
1011 m 1015 w 1012 w 1008 w
1005 w,sh 998 w
n1PO4 976 s 983 vw
986 s 988 s 966 s 970 s 964 s 964 vs 961 vs
959 vs 948 s 954 sh 956 vs
951 s 946 s
940 s
P-OH st. of HPO2
4 900 m 916 w
878 m 874 w
n4PO4 619 vw 620 w 614 w 615 vw
609 mw 612 w 610 w 607 w 608 vw
588 w 588 m 591 m 594 m 593 w 591 w 597 w
563 m 577 m 577 w 580 w 576 vw
563 w 566 sh
549 w 556 vw
525 w 523 w, b 495 sh
n2PO4 480 w 481 sh
451 m 451 m 451 w 448 w 463 sh
439 w 433 w 449 w
420 w 427 m 419 m 421 w 414 sh
411 w 409 m 408 w 407 w
381 w 394 w 353 w 389 w
References 124 124 54 – 124 122 124 123
Table 6 FTIR and Raman spectral characteristics of carbonate ions in different calcium phosphates (wavenumbers in cm–1)
Type A carbonate Type B carbonate (in HA) Type B carbonate (in FA) Carbonate in ACP
FTIR Raman FTIR Raman FTIR FTIR
n3CO3 1542 s 1490 s

1465 s 1462 s 1452 s
1412 s 1425 s 1425 s
n1CO3 1107 m 1070 m
n2CO3 883 m 872 m 862 m 868 m
n4CO3 757 vw 765 w 718 vw 716 vw
670 vw 675 w 692 vw 695 vw
References 129,130 24,130 129,130 125 129,130 129
Solid-state NMR spectroscopy also proved to be particularly useful in the study of the evolution of amorphous phases and the
formation of apatite nuclei undetectable by other techniques.
1.11.4 Properties of Calcium Phosphates
1.11.4.1 Thermal Properties of Calcium Phosphates

The thermal properties of Ca-Ps are important parameters to consider when processing calcium phosphate materials, or simply
when preparing biological samples for different types of analyzes. They are schematized in Fig. 4.
Fig. 3 Example of FTIR PO3 4 lines in the n3 domain of wet nanocrystalline apatite samples progressively dried in the spectrometer. The fresh wet
samples (bottom) show rather narrow well-resolved lines which can be associated to phosphate groups belonging to the hydrated layer. On drying
(in minutes), the lines become broadened and poorly resolved indicating the destruction of the surface structure and resulting in amorphous-like
environments of the phosphate ions.
The monocalcium phosphate monohydrate loses water at 1081C and the MCPA obtained decomposes into calcium dihydrogen
pyrophosphate, and then, due to continuous water loss, into calcium metaphosphate above 1861C.
The DCPD loses water at 1801C and transforms into DCPA. At higher temperature, another water loss is observed corre-
sponding to the formation of calcium pyrophosphate associated with the condensation of HPO2 4 ions already mentioned in the
characterization in Section 1.11.3.1.1.2.
The ATCP loses water up to its crystallization into a-TCP first and then b-TCP above 8001C.143 The formation of the
metastable a-TCP before the stable b-TCP might appear surprising. In fact, this phenomenon is rather common and it is
observed also, for example, with amorphous zirconium dioxide or in the crystallization of polymeric substances. It is known
as “Ostwald’s step rule” or law of successive reactions, stating that in any crystallization process, the state that is generally
observed is not the most stable state but the least stable state that is closest in terms of free-energy change to the original
state.144
The decomposition of OCP is more complex and involves the formation of DCPA and HA, and at higher temperature calcium
pyrophosphate, which then partly reacts on HA to give b-TCP.
Considering apatites, two different types of decomposition reactions can be distinguished: reversible decomposition
affecting stoichiometric apatites at very high temperature and irreversible decomposition related to nonstoichiometric apatites or
apatites-containing thermally unstable ions such as carbonate and HPO2 4 , for example.
1.11.4.1.1 Reversible decomposition of stoichiometric apatites

The stoichiometric HA is thermally very stable and is generally sintered at 1200–12501C without strong alteration. However, HA is
considered to begin to lose water at 8501C at a very low H2O partial pressure leading to the formation of oxyapatite.98 This loss is
strongly dependent on the water partial pressure. Then, oxyapatite decomposes into TCP and TTCP at about 10501C and, at higher
temperature (17201C), CaO and a liquid phase form.
All these reactions are theoretically reversible and, on cooling under an adequate water partial pressure, the HA structure is
reformed, provided there is no fast quenching which prevents the completion of the reverse reaction. A problem which has been
raised is the volatilization of P at very high temperatures, for example, in plasma spraying processes. This type of reaction is well
31
Table 7 Solid state NMR isotropic chemical shifts of P and 1H in calcium phosphates (in ppm relative to H3PO4 and TMS (tetramethylsilane),
respectively)136,137
Nuclei MCPM MCPA DCPD DCPA OCP ATCP b-TCP a-TCP TTCP HA
31
P 4.6
1.5
0.1 0.5 0.0 0.3 0.23
0.5 0.6 0.04
0.35
0.66
0.95
1.14
1.39
1.7 1.7 1.7
2 1.91
2.53
3.2 3.0 2.99 2.8
3.5 3.44
3.92 3.8
4.22
4.67 4.7
5.1
1
H 16.2
13.6 13.0
10.4 9.6
6.5
5.0 5.5 5.6
0.2 0.18
known in the presence of reducing agent (carbon essentially or hydrogen in plasma gas) and SiO2 and corresponds to the
preparation of pure P from phosphate ores:
Ca10 ðPO4 Þ6 F2 þ 5SiO2 þ 7C-5Ca2 SiO4 þ 7CO2 þ 6P
1.11.4.1.2 Irreversible decomposition of nonstoichiometric apatites

Nonstoichiometric apatites exhibit a complex decomposition behavior depending on their initial composition, as shown in
Fig. 4(b). All these reactions are related to the instability of calcium-deficient apatites and they are irreversible. The decomposition
involves the formation of pyrophosphate in the case of HPO2 4 -rich apatites and the formation of oxide ions in the case of
carbonate-containing apatites:
4 -P2 O7 þ H2 O
2HPO2 4
3 -O
CO2 þ CO2
2
Curiously, the pyrophosphate ions formation is not easily detected by FTIR spectroscopy but it can be evidenced by chemical
methods. It is only at above 7501C that calcium pyrophosphate crystallizes as a separate phase when the Ca/P ratio of the apatite is
under 1.5. The formation of oxide ions, related to the decomposition of carbonate ions leads to the formation of OH in the
apatite structure, or, in highly carbonated type B apatites with a Ca/P ratio above 1.67, to stoichiometric HA with a CaO phase.
When HPO2 2
4 and CO3 are both present, cross-reactions can be observed such as
4 þ CO3 -2PO4 þ CO2 þ H2 O

2HPO2 2 3
or
4 -P2 O7 þ H2 O
2HPO2 4
followed by:
7 þ CO3 -2PO4 þ CO2

P2 O4 2 3
In the case of nanocrystalline apatite, the first decomposition reactions can occur at very low temperature 100–2001C.
The end products at 900–10001C of the irreversible decomposition of nonstoichiometric Ca-P apatites are only related to the
initial Ca/P ratio:
• for Ca/Po1.5, b-TCP and b-calcium pyrophosphate are obtained;

Fig. 4 (a) Thermal decomposition of DCPD, DCPA, ATCP, and OCPt. (b) Thermal decomposition of apatites. Depending on their composition
(HPO2 2
4 or CO3 or both) vacancies-containing apatites decompose differently, these reactions are always irreversible. At high temperature (above
8501C) stoichiometric hydroxyapatite decomposes reversibly into different Ca-P compounds depending on the temperature and water partial pressure.
• for 1.5oCa/Po1.67, a mixture of b-TCP and stoichiometric HA is obtained;

• for Ca/P41.67, a mixture of stoichiometric HA and CaO is obtained.
These decomposition reactions can be used to determine the Ca/P ratio of an unknown Ca-P phase or of the global Ca/P ratio
of mixture of phases (as implicitly exposed in the dry preparation methods of Ca-P phases) and some of them have been
standardized (see Section 1.11.3.2).
The irreversible decomposition of calcium-deficient apatites can be used to prepare biphasic ceramics made of TCP and HA.83
1.11.4.2 Thermodynamic Considerations

For better understanding the formation and evolution in solution or in vivo of calcium phosphate phases, whether of natural or
synthetic origin, knowledge of their thermodynamic properties is a prerequisite.
A review of the thermodynamic properties of phosphate apatite compounds Me10(PO4)6Y2 with varying Me2 þ and Y ions has
been recently reported.145 In all cases, it may be noted that the standard Gibbs free energy of formation DG0f :
DG0f ¼ DH0f T DS0f
which is directly linked to the stability of the phase, is dominantly enthalpy-driven: the DH0f contribution being significantly larger,
in absolute value, than the T.DS0f entropy part (the latter representing only a few percent of the total DG0f value), which may often
allow drawing stability statements on the basis of enthalpy data from calorimetry measurements. Ion substitutions in the
phosphate apatite array are shown to modify significantly the thermodynamic properties of the obtained apatite compounds: for a
given cation Me2 þ , the phase is all the more stable (more negative DG0f ) that the electronegativity of the element Y is high and
its ionic radius is small. Thus the stability of apatite end-members ranges in the order F-apatites4OH-apatites4Cl-apatites4Br-
apatites, with oxyapatite Ca10(PO4)6O remaining the phase (within calcium apatite end-members) with the lowest DG0f value, in
link with vacancies in OH sites. For a given anion Y, the impact of the cationic radius is less direct and the phase is all the more
stable that the electronegativity of the element Me is low, generating then a more ionic character of the M–O bond. Considering
some divalent cations of biomedical interest such as calcium, strontium, magnesium, zinc and copper, the experimentally-based
DG0f stability order for end-members is as follows: Ca-apatites4Sr-apatites4Mg-apatites4Zn-apatites4Cu-apatites, whether for
hydroxide- or fluoride-bearing apatites.145 Trivalent cations can also be incorporated, such as lanthanides (Ln3 þ ions such as
Eu3 þ , Tb3 þ ) which can, for example, convey luminescence features to ceramic-based nanoparticles for use in nanomedicine.146
The thermodynamic properties of lanthanide-bearing apatites have been explored experimentally by calorimetry.147 For such ions
of similar valence electron configurations and differing mainly by the filling of internal 4f orbitals, the role of ionic radius was
found to be noticeable: although enthalpies of formation are exothermic in all cases, Ln-doping has a destabilizing effect on the
apatite phase, and the destabilization increases with the Ln doping content and with the size of the incorporated Ln3 þ ion.
In biological apatites as well as in synthetic analogs, end-member compositions are however rarely observed: the compounds
most generally correspond to solid solutions, often nonstoichiometric, and composition is bound to have a direct implication on
thermodynamic properties. On the basis of the exploration of 33 different apatite end-members for which thermodynamic
properties had been reported, an additive model (ThermAP) was recently established to determine the thermodynamic con-
tribution of each ion contained in an apatite phase,145 much in the same way as was done previously for zeolites, simple
phosphates or silicates.148–153 Indeed, the energetic contribution of a given ion, for example, Ca2 þ , in an apatitic phosphate
environment is likely to be rather similar for all apatites involving this ion. This model allows estimating (typically with a mean
relative error of less than 1% and often less than 0.5%) the enthalpy of formation, Gibbs free energy of formation and entropy of
not only end-members but also solid solutions or nonstoichiometric apatite compounds.
In such ionic compounds, the three-dimensional cohesion of the system is ensured by strong electrostatic interactions between
anions and cations. Therefore, for nonstoichiometric apatite compounds, the existence of vacancies in calcium and hydroxide sites
is expected to lower the overall cohesion of the system, leading to less stable crystals. The difference in thermodynamic properties,
directly linked to the chemical content can again be evaluated using the ThermAP model. Even if nanocrystalline apatites do not
correspond to actual solid solutions but involve an apatitic core (nonstoichiometric) surrounded by a hydrated ionic layer, the
application of the ThermAP model was shown to remain applicable in practice for evaluating their standard thermodynamic
properties.145 It may also be noted that application of the ThermAP method to the case of another compound involving hydrated
and apatitic layers (in a stacked manner), namely OCP, leads again to very close values to experimental ones (eg, estimated
DG0f ¼ 12282 kJ/mol to be compared to 12263 kJ/mol for the reported experimental value).24 From an experimental view-
point, the thermodynamics of synthetic biomimetic apatites has been explored, using drop solution calorimetry.154 In that work,
the enthalpy of formation DH0f of nanocrystalline apatites exhibiting an increasing state of maturation was determined from the
direct measurement of drop solution enthalpies. At 298 K, DH0f varied from 12058.9712.2 down to 12771.0721.4 kJ/mol
for apatites (noncarbonated) precipitated with maturation times increasing from 20 min to 3 weeks, thus tending toward the value
for stoichiometric hydroxyapatite. Through entropy considerations, the Gibbs free energies of formation of such apatites were also
evaluated. As expected, the solid phases were quantitatively found to be all the more stable that the composition approached
stoichiometry. The variation in free energy of maturation DGmaturation(i-f) from an initial maturation stage “i” to a more
advanced stage “f” was estimated from these data154: the negative values of DGmaturation obtained (typically ranging from 0 to
117723 kJ/mol as maturation increases between 20 min and 3 weeks) allowed quantifying the thermodynamic driving force
along which, in this particular case of HPO2 4 -containing apatites, immature apatites evolve in solution toward stoichiometry,
concomitantly with a decrease of the surface hydrated layer. These data represent a quantitative background for nanocrystalline
calcium phosphate apatite aging in solution.
As mentioned previously, the flexibility of the apatite structure, allowing departure from stoichiometry and ionic substitutions,
is remarkable as it allows apatitic systems to adapt to different functions in the organism. In particular, while tooth enamel
is expected to last all life long, in many species, and to endure systematic bacterial acidic attacks, bone apatite has to remain
reactive so as to take part in bone remodeling/microfracture auto-healing and for playing an active role in homeostasis. Based
on thermodynamics considerations, this is made possible by way of an adapted composition and stoichiometry: enamel
corresponds to a chemical composition close to stoichiometric hydroxyapatite (low solubility, greater stability under acidic
conditions), in contrast bone apatite is composed of nanocrystals of nonstoichiometric apatite (larger solubility facilitating
remodeling, see following subsection on solubility). Other factors than thermodynamic ones are however involved in biological
adaptation.80
Apart from apatites, other calcium phosphate phases of biomedical relevance may also be encountered, for example, as
intermediates in the formation of bone in vivo or in reactive settings such as those involving inorganic bone cements formulations.
Table 8 summarizes the main standard thermodynamic properties of several calcium phosphate phases of interest (at 298 K
and 100 KPa).
Table 8 Main standard thermodynamic properties of calcium phosphate phases, at 298 K and 1 bar (formation energetics refer to elements
taken in their standard state)
Compound (298 K, 1 bar) Name DG0f (kJ.mol1) DH0f Z (kJ.mol1) S0 (J.mol1.K1) References
Ca10(PO4)6(OH)2 Hydroxyapatite (HA) 12 634 13 432 780 145

Ca10(PO4)6F2 Fluorapatite (FA) 12 899 13 658 778 145
Ca10(PO4)6Cl2 Chlorapatite (ClA) 12 434 13 185 835 145
Ca10(PO4)6O Oxyapatite 12 290 13 066 634 145
b Ca3(PO4)2 b-Tricalcium phosphate (b TCP) 3 884.7 4 120.8 236 155
a Ca3(PO4)2 a-Tricalcium phosphate (a TCP) 3 875.5 4 109.9 240.91 155
CaHPO4 Monetite (DCPA) 1 681.18 1 814.39 111.38 155
CaHPO4 2H2O Brushite (DCPD) 2 154.58 2 403.58 189.45 155
Ca8(PO4)4(HPO4)2 5H2O Octacalcium phosphate (OCP) 12 263 155
Table 9 Solubility products of different calcium phosphates
Ca-P compound Common abbreviation pKsp at 251C References
DCPD, CaHPO4.2H2O DCPD (monoclinic) 6.59 158

CaHPO4 DCPA (triclinic) 6.90 159
Ca8(PO4)4(HPO4)2 5H2O OCP (triclinic) 96.6 160
a-Ca3(PO4)2 a-TCP (monoclinic) 25.5 161
b-Ca3(PO4)2 b-TCP (trigonal) 28.9 162
ATCP non-crystalline 24.8–25.5 39
Ca10(PO4)6(OH)2 HA (stoichiometric) 116.8a 163
Ca10(PO4)6F2 FA (stoichiometric) 121a 164
Ca4(PO4)2O TTCP (monoclinic) 38 165
a
Expressed for the chemical formula “Ca10(PO4)6Y2.”
1.11.4.3 Solubility
Per definition, the solubility of a solid (in our case an ionic solid) in an aqueous solution is characterized by an equilibrium
between the solid surface and the species in solution, which exhibit a constant ion activity product corresponding to the solubility
product (Ksp). The Ksp is characteristic of a solid compound and is related to its ionic composition; it is not directly representative
of the amount of solid dissolved, which may change as the species in solution vary, due, for example, to protonation or
deprotonation of orthophosphate groups, the formation of ion pairs in solution, or the change of the activity coefficients of the
different ions due to alterations of the ionic strength. The speciation of different entities present in the solution is an important
factor. Such alterations are considered in computer programs available on the web. In several cases, however, authors have not
really evidenced an equilibrium and they report solubility data, which might correspond to a dissolution rate rather than to real
solubility equilibrium. In the case of phases that cannot form in the conditions of the dissolution experiments, for example, in
physiologic conditions (371C, pH¼ 7.4) such as a- and b-TCP, DCPA, and TTCP, an apparent dissolution equilibrium is reached;
however, the risk exists to precipitate non detectable amounts of more stable Ca-P phases. The determination of Ksp of
unstable calcium phosphates, which can be easily hydrolyzed into more stable phases in aqueous media, is particularly delicate
(eg, OCP, ACP) and, in fact, several different solubility products have been reported in these cases.24,39 Another confusion arises
from apparent solubilities often determined in conditions where an equilibrium is not really reached, for example, because of
uncontrolled release of CO2 in dissolution studies of carbonated phases. Other problems of reliability include uncontrolled
impurities levels, the consideration of different ion pair formations or ion activities corrections, which are responsible for
discrepancies of Ksp of sparingly soluble solid phases. Tang and Nancollas156 have raised a new concern regarding the dissolution
process of crystals. A stepwise dissolution behavior has been shown in the case of well-crystallized OCPt assigned to the need
of additional energy for the formation of dissolution kinks on a flat crystal surface and resulting in difficulties to determine a
solubility equilibrium. In the case of solid solutions and nanocrystalline apatites, other difficulties have been raised, as discussed
later in this paragraph. Nevertheless, the solubility products of most stoichiometric well-crystallized calcium phosphates have been
determined (Table 9), although divergences and discussion are still existing. A recent direct method, especially based on the total
dissolution of increasing amounts of crystals until the detection of residual crystals in suspension,157 has given solubility products
with much lower values than those generally admitted, but this rather unconventional procedure could be biased due to the
stabilization of crystals in undersaturated solutions as shown by Tang and Nancollas.156
The solubility isotherms of stoichiometric Ca-P compounds have been studied in detail by several authors.166,167 At physio-
logic pH, the solubility, expressed as the amount of Ca2 þ and orthophosphate ions in solution at the equilibrium, is found to
decrease in the order TTCP4a-TCP4DCPD4DCPABOCP4b-TCP4HA, while FA is itself less soluble than HA. Moreover,
among the nonfluorinated Ca-P compounds, stoichiometric HA is the least soluble phase in a wide range of pH values down to
pH close to 4, at room temperature. Below this pH value, DCPA becomes the least soluble phase, as shown by the dissolution
isotherms.24 Although DCPA does not form generally spontaneously in these conditions, and DCPD appears often as the
stable phase in solution at moderate temperatures.
Most solubility products generally increase as the equilibrium temperature increases, an interesting finding is that several
calcium phosphate salts exhibit a retrograde solubility, ie, a solubility which decreases as the temperature increases, among which
several calcium phosphates such as DCPD, DCPA, OCP, b-TCP, HA.24
These data permit some general observations. At room temperature, for the same composition, phases with lower density show
a higher solubility, for example, considering the dicalcium phosphates or the crystalline TCP, in relation with the hydration or
simply the structure. This observation can be related to the degree of cohesion of the structure, assuming that the main factor
involved in the cohesion of Ca-P crystals is the electrostatic attraction between anions and cations and if we neglect the events
occurring in solution (protonation, ions pairs), which, for the same or similar compositions, should not be crucial. Such a
reasoning can be extended to apatites; thus, Ca-P apatites with the smaller unit-cells and, therefore, the higher the cohesion forces,
the lesser the solubility, and FA is less soluble than HA. Similarly, Ca-P apatites are less soluble than strontium-phosphate apatites,
with larger unit-cell dimensions. This behavior can probably be generalized to apatites-containing vacancies: apatites with a high
vacancies content should be more soluble than those with a low vacancies content. Considering carbonate and HPO2 4 -containing
apatites with similar Ca vacancies content, type B carbonated apatites, with a smaller unit-cell, should be less soluble and they
have been found, in fact, to form preferentially during maturation.168 The cohesion forces can also be increased by raising the
electric charge of the constitutive ions, thus, silicate- and lanthanide-containing apatites are less soluble than regular Ca-P apatites.
These considerations neglect, however, the existence of partial covalent bondings between ions in the apatite crystals and the ions
interactions with the solution and, although they are indicative, no definitive conclusion should be made based only on elec-
trostatic cohesion forces. More precise solubility product determinations can be determined based on Gibbs free energy of the
dissolution chemical equilibrium equation and inversely.24,155
It is also worth mentioning that the presence of foreign ions in the composition of a given Ca-P compound or even in the
surrounding solution may noticeably modify the dissolution behavior. For example, in the case of HA, the incorporation of
fluoride ions was found to decrease the solubility, while carbonate or else magnesium ions induced an increase in the apparent
solubility.169,170 In the presence of fluoride ions in solution, a decrease in solubility is also observed probably related to a surface
alteration of the HA. Such phenomena involving a very low amount of surface ions impurities affecting considerably the solubility
of apatites are used in the biomedical field, such as in fluorinated tooth paste, for example, or in apatite coatings of prosthetic
devices, for decreasing the solubility of HA.
Besides the above considerations, dealing with stoichiometric Ca-P compounds, the solubility behavior of nonstoichiometric
biomimetic nanocrystalline apatites is also interesting to address due to their resemblance with bone mineral and their high
potential in the bioceramic field. However, the physicochemical characteristics of apatite nanocrystals highly depend on their
conditions of formation. Several factors can influence the solubility, such as the size, composition/nonstoichiometry of the
nanocrystals and the surface structure and composition. A detailed analysis of the solubility behavior of such biomimetic apatites
indicates that, unlike stoichiometric HA, they do not reach a constant solubility product. In fact, the apparent equilibrium which is
attained depends on the amount of solid dissolved (Fig. 5); the ionic product at the apparent equilibrium state shifts progressively
toward that of stoichiometric HA as the amount dissolved increases. This observation has led to the concept of “Metastable
Equilibrium Solubility” (MES). This behavior has been evidenced and studied in various works171–174 and has been related to the
Fig. 5 Illustration of the Metastable Equilibrium Solubility (MES) of biomimetic apatites.174 For a stoichiometric hydroxyapatite, the ion activity
product in solution is independent of the amount dissolved. For biomimetic nanocrystalline apatites (from biological or synthetic origin), the ion
activity product in solution decreases as the amount of dissolved apatite increases leading to a MES. The shape and position of the MES curves
depend on the maturation time.
level of microstrains in the crystals.174 However, other explanations are possible such as a variable surface composition and
structure or the existence of mixed crystals population with different size, composition, and solubility.
Noncongruent dissolution (ie, dissolution leading to a mineral ion composition of the solution different from the solid) has
often been reported in the case of calcium phosphates. This phenomenon can have several causes, for example, a surface hydrolysis
or the preferential dissolution of undetected impurities possibly on the surface of the crystals. In the case of high solid/solution
ratios with solids of high specific surface area, noncongruent dissolution can also be related to surface reactions such as adsorption
of constitutive ions, as discussed in Section 1.11.4.4.1, or surface hydrolysis or precipitation; but these experimental conditions,
favoring the detection of surface reactions, and depending on solid/solution ratios, do not necessarily correspond to an anomaly
of dissolution. It shall be emphasized that a dissolution of a pure homogeneous phase leads to a congruent dissolution as it is
most commonly found, provided the surface of the crystals in equilibrium with the solution is small enough compared to the ions
in solution. In case of solid solutions however, even if they are homogeneous, subsequent surface equilibrations between the ions
dissolved and the residual mineral surface can alter both the composition of the solution and that of the solid surface and these
alterations dependent on the solid/solution ratio. It shall be kept in mind that it is the surface of the solid which is in equilibrium
with the solution. In the case of phase with a heterogeneous distribution of atoms, ie, when a solid surface is altered compared to
the bulk, a “noncongruent” dissolution may be observed.
1.11.4.4 Hydrolysis and Aqueous Conversion

The most soluble Ca-P compounds (ACP, DCPD, DCPA, OCP, TCP, TTCP, nonstoichiometric HA) can be transformed into more
stable phases in aqueous media. The important parameters are pH, calcium and phosphate concentrations, and temperature. Most
generally from slightly acidic to alkaline pH, the end term of these conversion reactions is apatite, in agreement with the
dissolution isotherms.24 However, these reactions can lead to apatites with very different compositions depending on the mineral
ions in the solution. Thus, in the presence of carbonate and/or fluoride, carbonated and/or fluorinated apatites can be obtained.
Several reaction mechanisms have been proposed for the hydrolysis reactions. Dissolution–reprecipitation seems to be the
dominant process for hydrolysis involving phase transformations. Although at physiologic pH, the most stable, least soluble,
phase is apatite, even stoichiometric apatite, this phase is rarely obtained in solution, in these conditions, and generally,
metastable nonstoichiometric apatites are formed especially at human body temperature. These conversion reactions may involve
intermediary phases according to the Ostwald step rule (see Section 1.11.4.1); thus, the transformation of ACP into apatite at
neutral pH is considered to occur through an OCP precursor at pH close to neutrality. In the case of OCP hydrolysis into apatite, a
topotactic transformation which could give rise to interlayered crystals of OCP and apatite has been suggested.175 The HA crystals
formed can appear in this case as pseudomorphs of OCP crystals. In the case of apatite nanocrystals with a surface hydrated layer,
the evolution in aqueous media appears more complex and is often called “maturation.” In this case, the hydrolysis is associated
with an alteration of crystals characteristics. At physiologic pH, maturation has been found to be related to the hydrated layer. The
apatite domains develop at the expense of the ions in the hydrated layer and when the hydrated layer has practically vanished, the
process stops.168 Even when maturation has stopped, slower evolution processes remain active like Ostwald ripening. However, if
they play a role at geological time scale, their implication in biological minerals is questionable.
Aqueous conversions of Ca-P are mainly used in self-setting Ca-P cements. One may distinguish rapidly hydrolyzable Ca-Ps
such as ACP, a-TCP, which can lead to apatites in less than 1 h at 371C and can be used in neutral monocomponents mineral Ca-P
cements, and slowly hydrolyzable Ca-Ps such as DCPD, DCPA, and OCPt, which need generally longer reaction times, higher
temperature, or an alkaline pH to react rapidly. These are used in “acid–base” cements in combination with TTCP, which
hydrolyzes relatively rapidly and generates an alkaline pH. This distinction between rapidly and slowly hydrolyzable Ca-Ps is,
however, rather artificial and depends essentially on the characteristics of the crystals, especially the crystal dimensions.
1.11.4.5 Surface Charge and Surface Energy

Interfacial phenomena play a major role regarding the biological properties of Ca-Ps, especially in adsorption and cell adhesion,
and surface charge and surface energy have often been considered as the most important physical characteristics of the surface.
1.11.4.5.1 Surface charge

The surface charge of solids, at the origin of “zeta potential,” arises from different phenomena: the preferential adsorption/
desorption of the constituting ions of this solid in solution, the protonation of surface species like PO3
4 ions, for example, or the
surface adsorption of foreign ions or molecules from the solution. For ionic solids such as calcium phosphates, the surface charge
is closely related to the concentration, in the surrounding fluid, of specific ions that can interact with the surface of the solid. Such
ions are often referred to as “potential-determining.” A difficulty is the time-dependent variation of the zeta potential, which may
result in discrepancies.176 The point of zero charge (pzc) defines the conditions of the solution (in particular, the pH value) for
which the surface density of positive charges (contribution of cations) equals that of negative charges (anions). It is often regarded
as a characteristic parameter for a given surface in a given aqueous solution.
When considering calcium phosphates, most studies have been dedicated to stoichiometric HA, which involves at least three
types of potential-determining ions (calcium, phosphate, and hydroxide or proton). A variety of concentration conditions exist for
conducing theoretically to a condition of zero surface charge,177 which therefore leads to define a line of zero charge (lzc) in the
calcium–phosphate–hydroxyl ternary diagram. Chander and Fuerstenau177 calculated the position of this lzc for HA and com-
pared it to experimental reports: discrepancies were unveiled. An isoelectric point in the range of pH 6.4–8.5 has been repor-
ted178,179 for stoichiometric HA. However the conditions of preparation, washing, and storage of the apatite material as well as the
presence of impurities and structural defects are bound to play a key role in the electrochemical properties179,180 of the surface.
Also, even under constant pH and ionic strength, a change in the calcium or phosphate concentrations can affect the surface charge
of HA significantly: an increase of calcium concentration will lead to a surface charge shifted toward more positive values
(traducing the adsorption of calcium ions on the surface of the solid), while an increase of phosphate concentration would render
the surface more negative. These effects were, for example, observed experimentally by Somasundaran and Wang.180 Ionic
substitutions and apatite exact composition play a role in the modification of apatite surface charge. The pzc of FA was reported to
lie in the range of pH 4.5–6.9,179 and the following order was found for the pH values of the pzc of the four apatite samples:
FAoHAostrontium-HAobarium-HA.181 This order also corresponds to the order of increasing solubility of the four apatites.
Other types of ionic substitutions were also investigated; for example, the surface charge of silicated-HA182 was found to be
noticeably more negative than regular HA in similar conditions, which was assigned to the presence of tetravalent silicate ions
exposed on the surface of the solid rather than phosphate ions. Nonstoichiometry may also affect the surface charge of apatites,
and Ca-deficient apatites, more soluble than stoichiometric ones, have been reported to exhibit zeta potential greater than
stoichiometric HA.
However, recent observations (Drouet et al. data to be published) on both stoichiometric and nonstoichiometric apatites run
on well-characterized synthetic samples (stoichiometric HA and nonstoichiometric apatite as well as on the well-known Durango
fluorapatite standard suggest that the domain where such phospho-calcic apatites particles exhibit a net negative surface charge can
in fact be significantly wider than what may be expected from previous literature reports, even in acidic conditions (eg, covering
the wide pH range 4–14 in some instances, see Fig. 6). These observations could lead to change the paradigm along which apatites
such as HA expose positively-charged surfaces at acidic pH values.
The above discussion has pointed out the possible artefactual role of parameters such as nonstoichiometry, impurities/ion
substitutions, structural defects, etc. on the determination of pzc or lzc. It then appears that “universal” isoelectric points should
not be considered a priori for a given type of apatite sample placed in any medium, but that a direct measurement should be done
on each compound to check its actual surface charge when contacted to a selected medium.
It is also worth noting that the adsorption of organic molecules on the surface of solids can also significantly modify the surface
charge of the particles, especially when the molecules exhibit charged functional end-groups (or at least strongly electropositive or
electronegative elements). An example of this effect was reported by Mangood et al.183 referring to the adsorption of acet-
aminophen on HA, leading to a surface noticeably more negatively charged. In contrast, the adsorption of amino acids184 or an
amine-terminated phospholipid moiety185 on calcium-deficient apatites were found to lead to surface charges shifted toward more
positive values. It must, however, be stated that, in the case of nonstoichiometric nanocrystalline apatites prepared in biomimetic
conditions (low temperature, physiologic pH, etc.), the existence of a nonapatitic surface layer on the apatite nanocrystals (see
previous section on the “preparation of Ca-P compounds”) leads to a more complex system that cannot anymore be adequately
defined by using the regular HA surface model, which thus makes it difficult to appreciate the amounts of phosphate or calcium
ions actually exposed on the surface (as they are bound to vary depending on the conditions of formation and storage).
Apart from these data regarding apatitic compounds, the surface charge data relative to other calcium phosphate compounds
are scarce. Zeta potential measurements have been reported on octacalcium phosphate (OCP) in solutions equilibrated with
Fig. 6 Direct zeta potential measurements versus pH (at 251C in KNO3 102 M) on well-characterized synthetic hydroxyapatite, stoichiometric and
nonstoichiometric (biomimetic apatite matured 1 day in precipitation medium at room temperature and pH 7.2, Ca/PD1.45) as well as on Durango
fluorapatite standard apatite.
respect to that phase.186 In these conditions, a positively charged surface was noticed beyond pH 5 but, as mentioned above for
apatites, further measurements would be needed in other media for unveiling a possible modification of behavior, and
approaching the net surface charge actually in place in applicative conditions. As discussed in the solubility section, the solid/
solutions ratio play certainly a role in the final surface equilibration: as zeta potential is the result of an equilibrium between ions
on a solid surface and ions in the solution. The amount of ions on the surface (related to the solid/solution ratio) has certainly to
be considered.
1.11.4.5.2 Interfacial energy

Interfacial energy is also an important parameter to consider when dealing with interactions between a solid surface and the
surrounding solution. Yet, only few data are available in the literature on this matter in the case of calcium phosphates. Generally,
two main components of the interfacial tension are distinguished, determined by the type of interactions with the surface polar
(dielectric bondings) and apolar (van der Waals bonding) components, sometimes completed by a hydrophilic parameter related
to hydrogen bonding. Such interfacial energies (with respect to water) have been reported for HA, FA, OCPt, and DCPD, with
respective values: 9.0, 18.5, 4.3, and 0.4 mJ m2.187,188 Interestingly, among those four compounds, the lowest interfacial tensions
were found for the hydrated phases (OCP and DCPD). A value for the interfacial tension in water was also reported for b-TCP:
3.8 mJ m2,189 also pointing out a lower value than for the apatites HA or FA. The surface tension of a carbonated apatite sample
(well-crystallized, 3 wt.% CO3) was also determined,190 with a value (9.0 mJ m2) found similar to that of HA. In all cases, the
surface tension of these calcium phosphate compounds was found to correlate well with their solubility in water, the lowest
surface tensions being observed for the most soluble phases.
1.11.4.6 Chemical Alterations of the Surface

Two main chemical alterations of the surface of Ca-P have been described in addition to preferential adsorption of constitutive
ions discussed in Section 1.11.4.4.1: ion exchange and the adsorption of molecules.
1.11.4.6.1 Ion exchange

The term “ion exchange” can be misused and may sometimes be used to describe ion substitutions inside an apatite lattice in
synthesis reactions. This is not, however, what is intended here, and ion exchange corresponds to a reaction specifically affecting
the accessible ions on the surface of given crystals.
The surface ion exchanges are of utmost relevance in the case of nanocrystalline calcium phosphate apatites, which were found
to exhibit a structured, metastable, nonapatitic surface layer that was shown to be highly reactive (see Section 1.11.2.3.2). These
phenomena were first studied by Neuman and Mulryan191 who suggested the possibility of carbonate-HPO2 4 exchanges in
biological nanocrystalline apatites. Several studies have explored surface ion exchange capabilities of biomimetic apatites, as
illustrated recently in the case of Ca/Mg and Ca/Sr exchanges,134,192 where Mg2 þ and Sr2 þ are biologically active ions. Other ion
sorptions were also investigated for HA. For instance, exchanges of part of the Ca2 þ ions with metal ions Cu2 þ , Cd2 þ , Pb2 þ , or
else Zn2 þ were studied.193,194
These reactions generally comply with the Langmuir isotherm equation linking the activity of ions in solution (C) to the
amount taken up on the solid surface (Q):
Q ¼ KNC=ð1 þ KCÞ
and introducing two main constants K and N representing respectively the affinity constant and the maximum exchangeable
amount (surface saturation). However, the use of Langmuir, or other adsorption equation, considers only one part of the ion
exchange reactions: the uptake of a mineral ion from the solution onto the solid surface, and forgets the possible release of calcium
(or phosphate) ions, from the Ca-P mineral surface to the solution. To fully explain all the characteristics of these reactions, such as
an apparent lack of reversibility on dilution, or release reactions, a more complete and accurate treatment based on the real
chemical equation of the ion exchange has to be considered. For a cationic ion exchange, for example, the chemical equation195:
ðsolutionÞ þ CaðsolidÞ 3 MeðsolidÞ þ CaðsolutionÞ

Me2þ 2þ 2þ 2þ
leads to an expression very close to the Langmuir equation:

Q ¼ ðKeNC=CaÞ=ð1 þ KeC=CaÞ
where the activity of the ion in solution in the original equation is replaced by the ratio of this activity on that of the calcium ions
released in solution: C/Ca; and the affinity constant becomes the equilibrium constant of the ion exchange equation (Ke).196
The ion exchange capabilities of nanocrystalline apatites have been related to the development of the surface hydrated layer.
Thus, higher amount of ions are exchanged in freshly precipitated nonmatured apatites, where the surface hydrated layer may
contain up to 20% of the exchangeable ions; in matured apatites where the apatite domains have developed at the expense of the
hydrated layer, the ion exchange capabilities can be considerably reduced especially for carbonate–hydrogen-phosphate or anion
exchanges. Curiously, cation exchanges appear to always involve higher amounts than anion exchanges. All these exchange
reactions are very fast (the equilibrium is reached in a few minutes) and they do not alter the main characteristics of the apatite
domains (crystal size and nonstoichiometry).197 The ion substitution capabilities in the hydrated layer are considerably larger than
those of a Ca-P apatite lattice. Thus, a considerable amount of surface Ca2 þ can be replaced by Mg2 þ , whereas Mg2 þ ions can
only substitute for a very small amount of Ca2 þ ions in the apatite lattice. The reversibility of ion exchange reactions is always very
high when the reverse reaction immediately follows the direct exchange reaction. However, when a maturation period occurs after
the exchange, two kinds of behavior have been distinguished. When the mineral ion introduced in the hydrated layer can
substitute calcium ions in the apatite domains, the reverse exchange reaction can only exchange back decreasing amounts of
foreign ions still present in the decreasing hydrated layer. This is the case of Sr–Ca exchanges. When the mineral ion introduced
cannot substitute in appreciable proportion the calcium ion in the apatite domain, they stay in the decreasing hydrated layer and
remain almost totally exchangeable. This is the case of Mg–Ca exchanges.198
It is often difficult to determine if the ion exchange reaction corresponds really to a surface ion exchange involving a
re-equilibration of the surface or to a dissolution–reprecipitation process with the possible formation of a new phase. This
distinction is especially difficult in the case of apatites where continuous solid solutions exist and where surface exchange can
modify the surface composition to reach an equilibrium with the solution by modifying only the first surface atomic layers. The
formation of a distinct phase by dissolution of the original apatite and reprecipitation of a more stable phase incorporating
the exchanging ion in solution, is not generally compatible with a Langmuir-type isotherms with a typical saturation domain. On
the contrary, the increase of the amount of exchanging ion in solution results in more (and eventually total) destruction of the
original apatite. In some cases, however, this process could result in a pseudo Langmuir-like adsorption, with an apparent
saturation domain if there is a coating of the original apatite surface by the new phase, preventing or hampering further reaction.
Sometimes both real surface exchange reaction and dissolution–reprecipitation reactions can be observed depending on
exchanging ion concentration and other parameters (pH, temperature, etc.). These phenomena can generally be identified by XRD,
SEM or TEM examinations.
1.11.4.6.2 Adsorption
Adsorption phenomena on calcium phosphates are also of major interest, as molecules interacting with these compounds can play
a key role in the recruitment and expression of cells, and they are strongly involved in the biointegration of implants. This is
especially true for apatites due to their biomimetic nature and their numerous potentialities in the biomedical field.
The adsorption of proteins or other active molecules on apatites is also considered to play a determining role in mineralized
tissues as it may control apatite crystal growth and dissolution in vivo, and it also appears as a clever way to associate drugs (eg,
antibiotics, antiosteoporotic agents, etc.) and other active agents (growth factors, etc.) to control their range of action. Such
adsorption phenomena have thus been the object of a great number of studies. Proteins, like osteocalcin, osteopontin, or else,
albumin, and other biomolecules (phospholipid, growth factors such as fibroblast growth factor (FGF), vascular endothelial
growth factor (VEGF), bone morphogenetic protein (BMP)) have, for example, been shown to adsorb on apatite and interact
strongly with its surface.199–201 Several macromolecules have been considered to act either as promoters or inhibitors of crystal
precipitation.202 It has been shown, however, that such classifications can be misleading.203 Of course, an adsorbed molecule on a
given calcium phosphate surface crystal appears to delay in most cases the growth of the corresponding crystalline face; however,
adsorption also stabilizes crystal nuclei with a subcritical size, and paradoxically low concentrations of adsorbing molecules in
solution were shown to increase the nucleation rate and accelerate precipitation, whereas high concentrations inhibit precipita-
tion.203 All these adsorption reactions exhibit some common characteristics: they are generally well described by a Langmuir
adsorption isotherm and they appear irreversible by dilution, which is often considered as an anomaly with regard to the original
Langmuir model (Fig. 7).
Several factors have been shown to be involved in the adsorption properties of molecules on calcium phosphates either related
to the solid surface or to the molecules themselves: surface energy, surface charge, functional chemical groups of the molecules,
conformation, competition with other potential adsorbents, etc.
Historically, interfacial energy has been one of the first parameters to be considered in adsorption and, according to Gibbs,
adsorption may be considered as determined by a decrease of the interface energy. This global physical view, of interest when weak
interactions are involved with the adsorbate, neglects the possibility of strong chemical interactions at the atomic level which very
often occur with the surface of Ca-P and determine the major adsorption processes of importance in biology.
Indeed, due to the ionic nature of Ca-P compounds, two main types of interactions have been distinguished corresponding to
electrostatic attraction between negatively charged end-groups and calcium ions on the one side, and/or positively charged end-
groups and phosphate ions on the other side. The zeta potential of apatites has thus been, and is still considered to play a major
role in the adsorption process and most adsorption behaviors have been related to this surface parameter. Once again, however,
the correlation between this global physical parameter describing the total surface charge of apatite particles and chemical
interactions with specific ions or charged groups able to interact an atomic level is not straightforward. It appears that the global
charge of a particle does not give any information on the local distribution of cations and anions on a mineral surface, and as a
matter of fact, the zeta potential does not appear to be a decisive parameter: for example, the adsorption of positive molecules on a
globally positive apatite surface and, inversely, negative molecules on a globally negative apatite surface have been descri-
bed.204,205 Adsorption is also observed on neutral Ca-P surfaces near their pzc. Similarly, the isoelectric point of proteins, by itself,
does not give specific information on the ionization state of the different functions of a molecule and should be considered with
caution when predicting the adsorption behavior most often related to a chemical interaction and not on global (molecular, or
particle) electrostatic attractions.
Fig. 7 Example of an adsorption isotherm of citrate molecular ions, on apatite from aqueous solutions (221C). The isotherms (curve A)
correspond to a Langmuir adsorption equation: Q ¼KNC/(1 þ KC) where Q is the amount adsorbed (ordinate), and C is the equilibrium
concentration (abscise). Two parameters characterize the adsorption reaction: K, the affinity constant, and N, the maximum amount adsorbed at
saturation. Generally, the Langmuir equation is linearized to extract more conveniently the adsorption parameters (curve B): C/Q ¼ C/N þ 1/KN.
Reproduced from Ref. [210], with permission from Elsevier.
Interestingly, the nature of the functional chemical groups exposed by the adsorbent is one of the key parameters to take into
account when considering the interaction with a calcium phosphate surface. Phosphate esters and similar groups especially interact
strongly with apatite surface, and phosphorylated compounds are considered to play an important role in biomineralization.
Among the other functional groups considered to interact strongly with apatite surfaces are silicate and sulfate. In general,
carboxylate groups interact weakly, except when they are associated in polyacids.206,207 Positively charged groups can also adsorb
on apatites but generally less strongly than negatively charged ones.208
In numerous examples, the adsorption of negative molecular ions has been related to the release of phosphate ions,
demonstrating a chemical ion exchange reaction with the apatite surface. This process has been described in different instances, as,
for example, in the adsorption of polyacids,209 citrate ions,210 bisphosphonates,114,211 or serum proteins on apatites. This che-
mical process seems rather general and supports a chemical description of the adsorption reaction rather than a physical one based
on global electrostatic interactions or pure interfacial energy considerations. Adsorption could thus be represented, for example, as
a chemical equilibrium as follows (in the case of a one-to-one exchange with surface phosphate groups):
A sol þ Pap 3 A ap þ Psol
with A being the adsorbate in a given ionic state either in solution (sol) or on the apatite surface (ap), and P being the phosphate
group in a given ionic state either in solution or on the apatite surface.
As for the ionic exchange process described in the preceding section this treatment is compatible with a Langmuir-like isotherm
and explains specific characteristics of this type of “adsorption” reaction like the irreversibility of dilution.114,212 Other ion
exchange possibilities have been discussed by Misra210 in the case of citrate adsorption. These descriptions seem consistent with
the use of apatites in chromatography where negatively charged molecules are desorbed using phosphate solutions and positively
charged ones using calcium solutions.
Here again, like for ion exchanges, surface reaction and dissolution–reprecipitation, with the formation of a new phase
incorporating the adsorbate, can be considered as possible competitive processes. However in this case, unlike for ion exchange on
apatites, no solid solutions are generally possible between the calcium phosphate and the calcium salt of the molecules (if we
except the case of OCP-carboxylates associations).53 Thus, in the case of insoluble calcium salts formation and precipitation with
the adsorbate molecules, the apatite phase is progressively destroyed as the new phase forms, leading to the release of phosphate
into the solution until a new equilibrium is reached. Analyzes of the solid, after the adsorption reaction, by XRD or electron
microscopy generally allows the formation of new phases to be detected.
1.11.4.7 Nucleation Properties

Nucleation and crystal growth are processes involved in normal and pathological biological mineralizations; these processes are
also of primary importance when calcium phosphate-based bioceramics are in contact with a calcium and phosphate super-
saturated solution such as human blood plasma (ceramics implanted in vivo) or the considered equivalent synthetic solutions
such as simulated body fluid (SBF) or other similar solutions used to test ceramics in vitro.
Crystallization at surfaces, that is, heterogeneous crystallization, may be induced at supersaturations lower than those required
for spontaneous precipitation (homogeneous crystallization). This phenomenon is generally considered to be related to the lower
overall free energy in the formation of nuclei of critical size at the surface of a substrate213 and it has been related to the interfacial
energies. Crystallization on foreign substrates is generally preceded by an induction period which is, for a given system, markedly
related to the supersaturation, the temperature, and the presence of additives or impurities. This period has been considered by
many authors to be the time needed for the formation of nuclei of critical size, that is, stable nuclei able to grow.
Other than the classical crystallization theory, several authors have also proposed that biomineralization could take place
following a non-classical theory of crystallization, proceeding via an intermediate self-assembly or ion clustering, prior to crystal
formation or transformation to a metastable or amorphous precursor phase/particle.214–217
The growth of one crystalline phase on the surface of another may be important especially in physiologic mineralization
processes. The epitaxial relationships and kinetics of growth of HA on calcium phosphate crystalline phases or on other com-
pounds such as the ones involved in bioactive ceramics (especially CaCO3) have been investigated by Koutsoukos and Nancol-
las.218 Epitaxy or oriented crystal growth is a special case of heterogeneous crystallization in which the crystal formation on the
substrate is determined by the atoms mapping that offers a good crystal lattice match facilitating the nucleation of a foreign phase.
The authors showed that HA can be grown on different calcium salts as substrates (DCPD, calcium fluoride, and calcite) from
calcium and phosphate solutions with a low supersaturation; moreover, they showed that the crystallization kinetics are similar to
that of HA on HA seed crystals whatever the substrate. b-TCP on the contrary appears as a poor nucleation substrate for HA,219
although it can be used in heteronucleation of OCPt.
The heterogeneous nucleation of apatite crystals on organic substances has been related to functional groups. Thus, phosphate
groups have been found to favor the nucleation of apatite on collagen matrix.220 More recently, Toworfe et al.221 have shown that
apatites form preferentially on surfaces rich in -OH terminal groups, whereas surfaces with -NH2 groups exhibited a low induction
capability. Such findings have been associated with the polarization of the surfaces and especially, the acceleration of crystal growth
on a negative surface evidenced by Yamashita et al.222 and discussed by Calvert and Mann.223 In addition, the existence of an early
precursor step in biomineralization processes has been suggested by several authors, first in the case of the formation of calcium
carbonate exoskeleton, which involves a polymer-induced liquid-precursor (PILP) phase before the amorphous phase formation
step in the presence of anionic polymer.214 Such a phenomenon may be also related to adsorption phenomena discussed in the
previous Section 1.11.4.5.2.80 This PILP phase is considered to be a highly hydrated phase which is more labile than the amorphous
phase and could be formed of stabilized nuclei with subcritical radii. The mechanism proposed by Gower for collagen miner-
alization during bone formation involves a PILP phase,217 that is, liquid-like amorphous precursor micelles that are drawn into the
gap zones of the collagen fibrils through capillary forces. Then, once these micelles have infiltrated the gap zones and interfibrillar
space of collagen, the PILP phase is transformed into the amorphous (solid) phase (ACP), which in turn crystallizes into apatite
nanocrystals. These models have however to demonstrate their compatibility with those of collagen fibrils arrangements in bone.224
In vitro and in vivo crystallization processes can differ substantially. Indeed, two main differences can be noted between in vitro
and in vivo crystallization: (1) the rate of precipitation which is usually faster in vitro compared to in vivo and (2) the presence of
many proteins and ions in vivo that can promote or inhibit mineralization and can possibly alter crystal morphology.
1.11.5 Biological Response and Physical–Chemical Characteristics
Physical–chemical characteristics of materials are directly related to some of their biological properties and determine in part
biointegration, biological activity, and biodegradation of materials. The biointegration of an implant is a crucial property,
especially in the case of bone substitutes, and a poor biointegration is responsible for most implant failures. The biointegration of
implants is determined on the one hand by mechanical factors: the interlocking of implant surface and biological tissue and on the
other hand by chemical factors: the chemical bonding to the tissue constituents. The mechanical integration is mostly determined
by the roughness and porosity of biomaterial surfaces initially created on the medical device and it may also be the result
of biodegradation irregularities. The chemical bonding to the tissue seems a determining factor and it appears mainly related, in
actual bone substitutes, to the ability of the surface to bind directly to the mineral fraction of bone tissue.
Even if bioactivity has different meanings depending on the authors, it is generally recognized that bioactive materials have
the property to bind directly to bone tissue without any fibrous interface. Bioactivity is also used to name materials favoring an
accelerated tissue repair. These definitions are related to different biological phenomena, namely implant–tissue interactions and
implant–cell interactions that can possibly be or not be combined in the same material. It seems important to insist on these two
properties. Cell culture dishes, for example, which have been specially treated to favor cell adhesion, allow osteoblast cells to
proliferate and produce bone tissue. However, their bone-bonding ability is limited and the materials they are made of will never
be a good bone substitute. In fact, once osteoblast cells have reconstructed bone, the interface is essentially implant/tissue and it
is mainly the strength of this interfacial interaction which insures bone bonding. Most calcium phosphate compounds involved
in bioceramics are not in equilibrium with biological fluids; several reactions will thus occur on the Ca-P ceramic surface. As
biological fluids are supersaturated with respect to apatite, the Ca-P ceramics may also serve as templates for the neoformation of
carbonated apatite crystals in vivo.
1.11.5.1 Active and Passive Biomaterials With Regard to Ca-P Nucleation

It has been shown that all bioactive materials that bind chemically to bone tissue and promote the formation of a stable interface
(“bone-bonding materials”) also have the ability to initiate nucleation of Ca-P from biological fluids at their surface. This layer of
Ca-P shows characteristics analogous to bone mineral and appears to be composed of nanocrystalline carbonated apatite with a
high reactivity. The association of the layer with specific bone proteins enables osteoblast cells (responsible for bone tissue
formation) adhesion and activity.225 Their activity is, however, related to nutritive fluid supply. For some authors, this process is
similar to that involved in bone remodeling and the neoformed layer would present a composition analogous to the cement line
at the edge of an osteon.226 However, one can note that the formation of this layer is desirable at the biomaterial surface only for
applications as bone substitutes and not for other applications of biomaterials, like, for example, ball-cup friction materials in
artificial hip joints for which any calcium phosphate nucleation and deposit must be avoided.
The ability to form this layer is generally considered as a measurement of the biological activity of bioceramics. Two main classes
of biomaterials can be distinguished depending on the processes inducing the formation of apatite nanocrystals (see Table 10 and
Fig. 8): in one case, the implant is only a substrate favoring the heterogeneous nucleation (“passive” biomaterials) and in the other
case, it releases mineral ions that locally increase the supersaturation of biological fluids and thus contribute to the fast formation of
the apatitic layer (“active” biomaterials) adding mineral ions from the material to the ions of the body fluids (Fig. 8).
Assuming that the formation of an apatite layer determines the biological activity, “active” biomaterials could be considered
more efficient than “passive” ones and in fact such a conclusion has been drawn by Hench comparing bioactive glass and HA.227
Table 10 Classification of biomaterials based on their ability to promote the formation of an apatitic layer analogous to bone mineral on their
surface
Biomaterial Active ions Induction process
“Active” biomaterials Bioglasses Ca2 þ Diffusion outside the bioglass

Coral Ca2 þ , CO2
3
Dissolution
Plaster of Paris Ca2 þ Dissolution
Ca-P cements Ca2 þ , PO3
4 , OH

Dissolution
Alkaline hydrogels OH Hydrolysis
Biomaterial Nucleation ability Induction process
“Passive” biomaterials Sintered apatites Excellent Epitaxy

2þ
Titanium Average Hydrolysis, PO3
4 and Ca uptake
Neutral hydrogels Good PO3
4 uptake
Collagen Good Phosphorylated entities
Polyactiv Good Ca2 þ uptake
Source: From Ref. [198].
Fig. 8 Illustration of “active” and “passive” bioactive materials with regard to the nucleation of Ca-P apatites. In active biomaterials, the mineral
ions needed for the precipitation of apatite crystals at the surface of the biomaterial can come in part from the material, either by dissolution or by
diffusion leading to a surface depletion. In passive biomaterials, the formation of the apatite crystals involves only ions from the supersaturated
body fluids, and nucleation ability is exclusively an inherent surface property.
However, bone is a living tissue with a remodeling process and one may wonder what may happen at the interface of the “active”
biomaterials and bone after successive remodeling processes. Is the release of mineral ions from the biomaterials still active or is it
no more existing after the first contact resulting in surface depletion of active mineral ions? “Active” biomaterials are probably
superior for bioabsorbable implants, whereas “passive” ones, which will essentially keep their intrinsic nucleation properties even
after multiple remodeling events, could be preferred for permanent implants.
1.11.5.2 Simulated Body Fluid Testing

Considering the importance of nucleation and formation of an apatite layer on bioactive materials, an international standard (ISO
standard 23317, 2007) has been published to evaluate the apatite-forming ability on the surface of biomaterials in a model
solution: simulated body fluids (SBF). The SBF solution, proposed by Kokubo et al. in the 1990s, contains mineral ions at
concentrations nearly equal to those of human blood plasma (see Table 11).228 Indeed it has been shown that this apatite layer can
be reproduced on the surfaces of material in SBF solutions and that nanocrystalline apatite thus formed is quite analogous to bone
mineral. This inexpensive and fast test has been largely used to estimate the potential biological activity of a material and it is
presented as useful for evaluating in a quantitative manner bone-bonding ability of a biomaterial preliminary to animal
experiments, although it shall be acknowledged that there are no quantitative studies correlating SBF tests and the in vivo
biological activity and performance.
The test is quite simple and consists in the immersion of the material to be tested in a SBF solution at 371C up to 4 weeks. The
formation of the biomimetic carbonated apatite on the surface of the biomaterial can be detected by thin film XRD and/or SEM
and the rate of formation can be followed by chemical analyzes of the solution assuming that there is no homogeneous nucleation
and precipitation during the test. This test is commonly considered as a measurement of the bioactivity of a biomaterial; when
apatite forms on the material surface in a shorter time, bioactivity is supposed to increase.
The protocol for the use of this solution to test bioceramics or more generally, biomaterials, is detailed in the ISO standard,
although several errors exist in the available version at this date: the amount of trishydroxymethyl aminomethane (TRIS), used to
buffer the solution, for example, is incorrect and the way of obtaining the SBF solution appears rather awkward, leading to the
elimination of carbonate ions and risks of precipitation. A safer and more correct procedure would consist in preparing two
buffered solutions (500 mL each for 1 L of SBF), a cations solutions (half of the sodium chloride, potassium chloride, magnesium
chloride, calcium chloride, buffered with half the amount of HCl recommended and TRIS) and an anion solution (half of the
sodium chloride, dipotassium hydrogen phosphate, sodium sulfate, buffering the other half of HCl and TRIS. These two solutions
are mixed just before use and the sodium bicarbonate is added, last, in the buffered anion solution without alteration of the pH.
SBF solutions differ in several points from the composition of blood plasma: the carbonate content is not adequate and
proteins are absent. These constituents of blood plasma are important and they have been shown to alter strongly the nucleation
and crystal growth process of apatites.229 The composition of SBF solution has been revised with a view to getting closer to mineral
ion concentrations in human blood plasma by decreasing Cl concentration and increasing HCO 3 concentration compared to
concentration in conventional SBF solution.230 However, reaching the carbonate composition of real blood plasma would need to
work under CO2 partial pressure to avoid a decrease of carbonate content related to its dissociation at physiologic pH in solution:
þ
3 þ 2H 3 2H2 CO3 3 2H2 O þ 2CO2
2HCO2
Whatever the type of SBF solution (conventional or revised) considered, there is still a major difference between this model
solution and human blood plasma: many proteins are present in plasma and the ionic concentrations in SBF solution have been
established considering the ionic content of plasma, which includes compounds formed between mineral ions and organic
molecules (soluble complexes, ion pairs) of serum altering the amount of free ions in solution and the activities of the mineral
ions. It has been demonstrated that ultrafiltrated fractions of blood plasma (excluding mineral ions linked to proteins) had in fact,
an ionic product close to that of OCP,231 whereas the actual SBF solutions exhibit a much larger one and is supersaturated with
most Ca-Ps (DCPD, DCPA, ACP, OCP and HA) and calcium carbonates (with revised SBF). More elaborated analyzes on bone
extracellular fluid and plasma blood ionic content, especially free calcium and phosphate ions, have been published by
Neuman.31,232
Another criticism made to SBF testing is that the mineral ions concentrations is not constant in the batch as it would be in vivo,
and circulating SBF testings have been proposed. However, the test is supposed to measure the apatite nucleation ability of an
implant surface and not really a crystal growth kinetic. In this sense, the batch procedure seems adequate. SBF testing is the only
chemical model presented as a measure of the biological activity of an implant surface based on the assumption that the apatite
Table 11 Ionic composition of blood plasma and SBF solutions (Kokubo et al.228; Kim et al.230)
Concentration 103 mol/L
Na þ Kþ Ca2 þ Mg2 þ HCO3 Cl HPO2

4
SO2
4
SBF 142.0 5.0 2.5 1.5 4.2 148.8 1.0 0.5

Blood plasma 142.0 5.0 2.5 1.5 27.0 103.0 1.0 0.5
nucleation ability of a surface is related to its bioactivity. As it is an inexpensive and rather rapid testing method, it will probably
continue to be extensively used, although it cannot replace animal implantation. The SBF test has been recently criticized by
Bohner and Lemaitre who have proposed some improvements.233 Some anomalies have been reported regarding the minerals
formed during the test: in some case amorphous Ca-P or calcium carbonate have been shown to precipitate. These phenomena
occur frequently with “active” biomaterials where the release of calcium ions and/or OH, and/or phosphate creates an elevated
supersaturation close to the implant and the precipitation of any Ca-P or calcium carbonate susceptible to precipitate depending
on the local supersaturation, nucleation abilities of the surface and dynamic variations in the solution. For example, the formation
of calcium carbonate234,235 in SBF, could be related to the release of excessive calcium ions from the implant, leading, locally, to a
precipitation of all P as an ACP or poorly crystalline apatite and then the precipitation of carbonate in the P-depleted local
solution.236 The control of the solid/solution ratio appears as a crucial parameter in the testing of “active” biomaterials in SBF.
The chemical reproduction of the reactions occurring on an implant surface placed in the body seems difficult to achieve: the
body response to an implantation is a complex phenomenon which varies as time elapses. From a physical-chemical point of view,
the composition and pH of the body fluids at the vicinity of an implant surface after the surgical operation is not really known. It is
believed that the surgical trauma induces acidic pH, which could be responsible for accelerated degradation of Ca-P materials, and
strong alteration of the events supposed to occur from SBF test results. The intensity and duration of the pH alteration related to
surgical trauma is not known; without this information events are difficult to predict, making a modeling of the behavior of an
implant surface from SBF, or other in vitro test results, unrealistic and raising some doubts on the sketches explaining biological
activity based solely on such observations.
1.11.5.3 Biodegradation
Biodeterioration and biodegradation of implants determine their durability. The first term is generally used when the alteration of the
implant is not necessarily wanted and the second when it is one of its advantages. Several processes contribute to biodeterioration
and biodegradation: mechanical wear, spontaneous reactions of the implant materials with body fluids, and cell-mediated altera-
tions. Mechanical wear is of course occurring at the interface between mobile parts of a medical device but it is also observed at the
tissue–implant interface, and appears mainly related to micromovements (fretting) and results in abrasion wear residues and possible
inflammatory reaction. This phenomenon is limited when there is a strong interfacial bonding between the implant and the tissue.
In addition to mechanical wear and surface abrasion, two main processes can be distinguished: one is purely physicochemical
(ionic crystal dissolution) and the other one is linked to cellular (and enzymatic) activity at the implantation site (activated
resorption).237 In fact, both aspects are bound to the intrinsic properties of the ceramic, including its grain shape and size,
porosity, solubility, structure and microstructure, and also the degradation residues of various constituents in combined materials
such as composites or heterogeneous materials.
The overall biodegradation rate in vivo has often been directly linked to the solubility behavior of the compound(s) con-
stituting the bioceramic.238,239 The different Ca-P compounds exhibit solubility products that greatly differ from one another
(see Section 1.11.4.2). Compared with blood plasma, with an ionic activity product generally considered to be very close to
OCP,231 the calcium phosphate phases, that should spontaneously dissolve are TTCP, DCPD, DCPA, ACP, and a-TCP, in addition
to the two very soluble monocalcium phosphates. The other calcium phosphates should not spontaneously dissolve and on the
contrary could exhibit a crystal growth. However, the most stable phase remains apatite, and an evolution of OCP and b-TCP
toward apatite by surface or topotactic transformation cannot be excluded.
Spontaneous dissolution does not mean, however, that the implant, made of the most soluble Ca-P, necessarily disappears. The
supersaturations that are reached locally can be so high when relatively soluble Ca-P are immersed in body fluids that spontaneous
precipitation occurs most of the time and the formation of apatites is observed. On experimental grounds it has been found that
the implants containing the most soluble Ca-P (excluding the very soluble calcium monophosphates) are effectively transformed
rather rapidly after implantation into poorly crystalline apatites.
The cell activity has also to be considered in biodegradation. Multinucleated giant cells involved in the defense response of the
organism and osteoclast-like cells are frequently found in the vicinity of Ca-P bioceramics especially at early stages after
implantation. These cells have the property to generate very acidic pH, and all Ca-Ps, including apatites, become rather soluble
below pH 4, which explains their progressive dissolution, for example, within osteoclastic resorption pits. In this case, the rate of
dissolution becomes the main parameter determining the biological degradation. It is related not only to the solubility product of
the Ca-P and more precisely to the undersaturation achieved locally, but also to the crystals characteristics (size, strains) and the
implant characteristics (surface exposed, porosity). The undersaturation appears as a crucial factor. The rate of dissolution of
stoichiometric HA, for example, is very low and this Ca-P is often considered as a nonresorbable or at least a very slowly resorbable
compound. This is only valid, however, for stoichiometric highly crystalline HA and it is not an inherent property of the structure
itself. In the case of calcium-deficient HA, the biodegradation can be very high, like in some biomimetic cements, for example.
b-TCP is considered also to give rapidly resorbing ceramics.
Generally, bioceramics made of Ca-P are made of a multitude of crystals bound to one another, and they are sometimes
multiphasic compounds. Several additional physical–chemical phenomena affecting biodegradation can then be involved. The
most obvious is probably the decohesion of the material related to the preferential dissolution of the most rapidly soluble
constituent, leading to the release of crystals or particulates of the least soluble, more stable, phase. These phenomena have been
found, for example, in the case of HA plasma-sprayed coatings, and they can also manifest in biphasic HA/b-TCP ceramics or in
composite materials.240 Depending on their size these particles can induce different body responses. When the released particles
are small enough, they can be phagocytized and they are eventually eliminated. However, when the elimination is not possible or
when too numerous particles are released, they may initiate an inflammatory response sometimes devastating. This can occur,
for example, with poorly sintered materials241 or when a Ca-P ceramic is associated with a rapidly absorbed matrix. When the
fragments are big enough and cannot be further fragmented, they might remain in place, encysted in the tissue. Biphasic
compounds, especially, have considerably developed in the last years and their resorption can be regulated by the phase ratio.242
This is the case, for example, of biphasic HA/b-TCP ceramics where the degradation rate can be tailored by modifying the
proportion of the more soluble phase, namely b-TCP, which seems to be degraded preferentially upon cellular activity.
The degradation behavior of nonstoichiometric nanocrystalline apatites is also interesting to consider, although apatite nanocrystals
may exhibit varying physicochemical characteristics depending on their conditions of formation, which then account for differences in
resorption rates in vivo. Various parameters can indeed influence the dissolution and biodegradation processes: in particular, the
nonstoichiometry of the nanocrystals is a major factor to consider, as the dissolution of such ionic compounds depends on the
amount of vacancies. A second effect could be related to the important interfacial energy and the evolution of the dissolution rate with
the crystal size. As already mentioned, the presence of foreign ions in the composition of a given Ca-P compound may greatly modify
its dissolution behavior. For instance, the solubility and in vivo degradation rates of zinc-enriched TCP were found to be significantly
lowered as compared to pure TCP.243 Also, the dissolution of calcium pyrophosphates was found to increase in the presence of ions
such as Mg2 þ or albumin,244 although the in vivo degradation of those compounds is mostly due to enzymatic activity.
Other physical–chemical parameters have been shown to determine the degradation rate of implants in vivo such as the
porosity and the crystallinity. Considering the porosity, macroporosity (pore diameter in the range 150–500 mm) seems to play a
major role especially in the case of ceramics made of “insoluble” Ca-P in body fluids. The macropores allow cell invasion and
increase the number of dissolution sites. The effect of microporosity is more difficult to apprehend: a first effect seems primarily
related to a lesser content in material; a second effect is related to the increase of the surface area and of the related dissolution rate.
Thus, for the same composition, highly microporous mineral cements are faster absorbed than less porous ones. Crystallinity has
been shown to play also a role probably related to the crystals surface area and dissolution rate but also to crystalline defects and
possibly amorphous phase content, as these different characteristics are often considered in the global “crystallinity” parameters.
A last parameter of importance in biodegradation is cell adhesion. In order to be active, the osteoclast-like cells involved in the
biodegradation of Ca-P materials have to adhere to the materials and find favorable conditions. Alkaline pHs especially have been
found to strongly inhibit osteoclast activities.245 Thus, Ca-P generating alkaline pH on hydrolysis, like TTCP, for example, found in
some mineral cements, may inhibit osteoclasts activity, as long as they are present.
1.11.6 Examples of Applications and Processing
1.11.6.1 Apatite Nanocrystals in Suspension, Interactions With Cells

Apatite nanocrystals have been proposed as transfection vectors, cells markers and therapeutic carriers for different diseases.246
Biomimetic nanocrystalline apatites, especially, exhibit a high specific surface area and a plate-like morphology, both favoring a
strong agglomeration effect between adjacent nanocrystals. In applications as in nanomedicine, particle size is a critical parameter
as particle aggregation does not allow optimized interactions with cells and fundamentally limits the list of possible adminis-
tration routes. To circumvent this drawback, a control of the agglomeration state of apatitic particles is desirable, which can be
achieved by adding a stabilizing agent in the precipitating medium during apatite formation. The organic corona composed
of stabilizing molecules such as 2-aminoethylphosphate (AEP) or phosphonated polyethyleneglycol (PEG)P adsorbed on the
obtained colloidal-like particles has indeed been shown146 to enable tailoring apatite particle size in the nanoscale, typically lower
than 200 nm and down to 30 nm. Other attempts to modulate particle size had also been tested using macromolecules like DNA
247
or ions exposing multiple carboxylate groups like citrates.248 The amount of stabilizing agent can then modify features such as
surface charge and mean particle size, and the colloidal status of the nanoparticles in suspension enables to envision applications
with direct interaction with cells, whether for diagnostic purpose (eg, using luminescent lanthanide-doped apatite nano-
particles),247,249,250 therapy, or even theranostic applications.
The possibility to prepare colloidal apatite nanoparticles capable of addressing more specifically diseased cells via the
adsorption of a cell targeting agent was also considered. For illustration, surface functionalization with folic acid (B9 vitamin)
allowed apatite nanoparticles to interact more specifically with breast cancer cells overexpressing folate receptors, and antibody
surface grafting was also shown to be feasible on apatite particles.251
The biocompatibility of such colloidal apatite nanoparticles has been evaluated by way of direct cytotoxicity assays (on normal
as well as tumoral cells), pro-inflammatory response, and hemocompatibility after contact with blood components like red blood
cells and plasma proteins. In all cases, low toxicity and high biocompatibility have been evidenced,146,252 thus opening the way to
increasing applications in the field of nanomedicine.
1.11.6.2 Porous Ceramics

Porous Ca-P ceramics are among the most used synthetic bone substitute materials. They are generally obtained by natural
sintering at a high temperature of HA, b-TCP, or a mixture of them and stabilized a-TCP. Several types of porosity are found in
such ceramics. The definition of porosity in bioceramics appears historically different from the IUPAC recommendations used
in other fields like zeolites. In bioceramics, macroporosity is generally between 200 and 400 mm and allows cell rehabilitation. In
addition, microporosity (1–10 mm) is frequently present depending on the sintering conditions: longer sintering time and higher
temperatures generally decrease the microporosity. The macroporosity of most industrial ceramics is obtained by replication from
a porous polymer, the use of calibrated porosity agents or with foaming agents.253 Other types of porous Ca-P ceramics have been
obtained from natural compounds like the sintering of cow bone, for example, or the conversion of corals (calcium carbonate)
skeleton.254 Several shapes exist for different surgical uses: granules, cylinders, parallelepipeds, edges. Other techniques have been
proposed to obtain a more regular porosity including additive manufacturing process, however, they do not seem to have been
widely commercialized yet. The total porosity; of porous Ca-P ceramics is generally comprised between 30 and 80%. As usually
observed, the compressive strength decreases when the porosity increases for similar compounds.
HA ceramics are considered as nonbioresorbable and, although osteoclasts adhere on such surfaces effectively, they have
difficulties to spread and create resorption pits.255 HA ceramics may contain two impurities: CaO and TCP, which can be
determined by XRD according to ISO standards. CaO rehydrates as Ca(OH)2 in aqueous media with a 40% increase in volume.
This phenomenon may result in the fragilization and cracking of the ceramic. TCP is a bioresorbable Ca-P, which might dissolve
in vivo and facilitate the bioresorption as in biphasic ceramics, as discussed earlier. HA ceramics can be associated with very low
levels of SiO2 that have been shown to improve their biological activity.256 However, the reasons for these effects remain obscure.
The improvements obtained with silicate-substituted apatite at higher ratios have been assigned to the effect of released silicate.257
It seems interesting to mention that the doping of HA with foreign elements cannot be considered as straightforward as for glasses
or alloys. For example, if a mixture of HA with SiO2 is heated, it will result in a mixture of HA and b-TCP due to the fact that it is
SiO4 3
4 anions which will be incorporated and that these anions will substitute for PO4 anions in the apatite structure. For
example:
5Ca10 ðPO4 Þ6 ðOHÞ2 þ 2SiO2 -10Ca3 ðPO4 Þ2 þ 2Ca10 ðPO4 Þ5 ðSiO4 ÞOH þ 4H2 O
If one wants to get Zn doped HA by heating HA and ZnO, one has to consider that Zn2 þ will substitute for Ca2 þ in the apatite
structure and the result will be HA doped with Zn, and CaO:
Ca10 ðPO4 Þ6 ðOHÞ2 þ xZnO-Ca9x Znx ðPO4 Þ6 ðOHÞ2 þ xCaO
Heating with different oxides may lead to different results and impurities, depending on substitution abilities in the apatite
structure.
b-TCP ceramics have been especially developed by Jarcho et al.258 They are bioresorbable. However, unlike it is sometimes
written, they cannot spontaneously dissolve in body fluids at physiologic pH. b-TCP can hydrolyze and be converted into apatite
in aqueous solution, but this reaction already rather slow at 1001C (at least 48–72 h are needed) is inappreciably slow at 371C.
It is only cell activity after implantation, producing acidic pH, which can dissolve b-TCP. As its biodegradation is determined
by osteoclast cells activity, like in regular bone, it appears as a ceramic, which adapts to the remodeling rate of bone at the loci of
the implantation. Unlike some other Ca-Ps, like DCPD, for example, b-TCP is unable to favor apatite nucleation by epitaxy.
Although the reported data are not consistent, it is considered as a fast resorbing implant due to its rate of dissolution in acidic
media linked to its solubility product; this resorption rate could, however, depend on its composition and especially the Mg
content, a major impurity in calcium salt. The main impurities in b-TCP are calcium pyrophosphate and apatite. They can be
determined with high accuracy by FTIR spectroscopy and XRD respectively. b-TCP has been associated with active ions such as
Zn2 þ . In some cases, behavior akin to osteoinduction has been suggested for b-TCP259; however, the reason of this behavior is not
really known.
Biphasic HA-b-TCP ceramics have been proposed by Daculsi and LeGeros.260 They are bioresorbable and it has been shown
that the bioresorption rate was related to the b-TCP content. Generally, this content varies between 20 and 60% in industrial
products. All these ceramics are osteoconductive and some of them, generally sintered at low temperature, have been shown to be
osteoinductive.261–264 Unlike in pure b-TCP, the presence of HA facilitates the nucleation of apatite in body fluids, and these
ceramics, like pure HA ceramics, can bind firmly to bone tissue. The biodegradation has been reported to involve a faster
dissolution of the b-TCP phase, followed by HA crystals elimination. Curiously, these biphasic Ca-Ps are unlikely to contain any
other crystalline phase: excess of CaO in the HA fraction would lead to additional HA by reaction on b-TCP at sintering
temperature and excess of pyrophosphate in b-TCP would lead to additional b-TCP by reaction with HA. In both cases only the
b-TCP/HA ratio would be affected.
The high-temperature ceramics can be associated with drugs and growth factors; however, because of their very low specific
surface area there is no strong surface interaction and the release is mainly controlled by diffusion through the pores and physical
parameters. This release process can be interesting for some substances like antibiotics, for example, but it might appear as a
drawback for very active substances like growth factors, compared to other Ca-P materials where adsorption occurs and where the
release remains localized and under the control of the organism. A thin layers (a few micrometers) of nanocrystalline biomimetic
apatite can be used to coat the macropores of sintered ceramics without significant alteration of the porosity. This layer increases
the specific surface area of the ceramics and improves their adsorption ability.265
1.11.6.3 Coatings, Example of HA-Plasma Spraying

One of the most efficient solutions to favor biological integration and bioactivity of prostheses and orthopedic implants consists in
coatings with Ca-P ceramics. Many different techniques have been proposed and used to deposit such coatings, but plasma
spraying of HA appears as one of the most applied and most developed at an industrial scale.34
This is a technique in which a DC electric arc is struck between two electrodes, while a stream of mixed gases (generally H2 and Ar)
passes through this arc. The arc turns these gases into an ionized mixture (plasma) of high temperature and with a high speed of up
to 400 m s1. The temperature of the plasma rapidly decreases as a function of distance from 20,000 K close to the arc to
2000–3000K at 6 cm outside the electrodes. A ceramic powder suspended in a carried gas can be incorporated into the plasma and
impinged in a partially molten state toward a surface. Many surfaces of metals or ceramics can be coated with a relatively thick layer
(50–300 mm) of composition close to that of the powder. Ideally, only a thin outer layer of powder particles gets into the molten
state which is necessary to ensure dense and adhesive coatings. The bonding of the coating is mainly due to mechanical interlocking.
HA appears certainly as the major plasma-sprayed Ca-P. One of the main difficulties in HA plasma spraying is the thermal
decomposition of HA at high temperature and the mixture of phases, which constitute the coating (Table 12).266 These phases are
heterogeneously distributed in the particle reaching the surface and in the coating. At contact with the metal and on the surface, a
rapid quenching of the molten fraction leads mainly to an amorphous phase, a slower quenching inside the particle may allow the
crystallization of high-temperature phases in their order of stability: CaO (with a very high melting point: above 25001C), TTCP
and a-TCP, and then b-TCP and oxy-HA. However, when a second pass is made, the surface is reheated and a recrystallization may
proceed again. In addition to this decomposition, volatilization of phosphorus has also been evidenced, especially in H2-rich
flames, resulting in a change in stoichiometry; thus, even when using pure HA in the plasma spraying, the resulting coatings may
contain an excess of calcium appearing as CaO or TTCP. Depending on processing parameters, many different coatings can be
obtained and ISO standards have been published regarding some important physical–chemical characteristics of the coating: the
Ca/P ratio, the content in crystalline impurities, and a crystallinity ratio (ISO 13779-2: 208 and ISO 13779-3: 2008), but curiously
not really the amorphous phase content. Although several post-treatments have been proposed as remedial and have been shown
to restore the apatite structure, they all have some drawbacks and weaken at some point the coating cohesion and adhesion to the
metal surface.
The decomposition of HA and the formation of different Ca-P phases, especially the amorphous phase, can have a positive
effect, especially on biological activity, as most of these phases will spontaneously release mineral ions and will thus “activate” the
coating (see Section 1.11.4.1). However, these phases weaken the coating which may degrade and lead to a deterioration of the
implant–bone interface. In addition, the metal-coating interface appears as the weak point of the process and delamination of
the coating may occur.267 In fact, the adhesion of the coating on the metallic substrate is mainly due to mechanical interlocking on
the porous implant surface, although chemical bonding cannot be totally excluded in the case of Ca-P. High levels of amorphous
phase in the coating lead to improved interfacial adhesion on the metal and biological activity but poor durability. Raising the
crystalline HA content, on the contrary, lowers the biological reactivity and weakens the metal-coating interface but the coating can
last longer. The solution to this dilemma could be a coating made of several layers with different characteristics as proposed by
different authors.268
1.11.6.4 Coatings and Bulks, Low Temperature Processing

Other calcium phosphate compounds than hydroxyapatite are interesting to be produced as coatings. But, as already mentioned
(see Section 1.11.6.2), plasma spraying, which is the most developed process at industrial scale, is using high temperature and high
velocity gases to transfer the feedstock material toward the substrate. Therefore the temperature sensitive calcium phosphates (see
Fig. 4) cannot be processed by plasma spraying without degrading the material and alternative low temperature processing
techniques have to be implemented. Wet-chemical methods have faced this challenge. The most frequently used are the biomi-
metic processes, which consist of immersing a material in a supersaturated Ca-P solution under soft conditions, and was first
proposed by Kokubo et al.228 not for coating samples, but as a convenient way to evaluate surface reactions of an implant in
simulated body fluid (SBF) reproducing the mineral ion composition of blood plasma. Since then, the method has been extended
and often revised by several research groups229,269 to produce at low temperature the so-called biomimetic coatings. The main
Table 12 Phases found in plasma-sprayed coating of HA
Phases Formation
Calcium oxide Decomposition of HA and TTCP; Volatilization of P

Calcium hydroxide and Calcium carbonate Reaction of CaO with humidity and CO2 (on contact with air, or body fluids)
Amorphous calcium phosphate Melting of HA and quenching
a-Tricalcium phosphate Thermal decomposition of HA
b-Tricalcium phosphate Phase transition on cooling from a-TCP
Tetracalcium phosphate Thermal decomposition of HA
Oxy-hydroxyapatite Dehydration of HA
Hydroxyapatite –
problem using the supersaturated SBF solution is that the Ca and P concentrations are very low and that these processes are not
adapted for an industrial production. Several alterations of the initial SBF process have been proposed, like the use of solutions
more concentrated in Ca and P than SBF, and unstable at physiologic pH. Such solutions are prepared in acidic media and the
heterogeneous nucleation is obtained by a pH increase. Several processes have been studied from slow ammonia incorporation
(either directly270 or using urea hydrolysis271) to removal of acidifying agents like CO2.272 A direct use of unstable concentrated
solutions has also been studied.273 A interesting variant is the use of moderate complexing Ca agents like lactate, citrate ions which
allow a stabilization of concentrated pseudo-SBF solutions while keeping their supersaturation properties at physiologic pH.274,275
Such processes exhibit the advantage of allowing the immobilization of organic agents (such as bisphosphonates, proteins, bone
morphogenetic protein, vitamins, and antibacterial agents) within the Ca-P coating due to the mild operating conditions.275–277
Various shapes and materials (metallic coupons, porous polymer matrix, bovine bone granules) could be biomimetically covered
with a calcium phosphate layer. Other low temperature wet-chemical techniques have also been evaluated, among which four
methods should be enlightened:
• Alternate soaking278,279 which forms apatite (mainly, DCPD and HA) by incubating alternately the substrate into Ca2 þ and
278
PO3
4 solutions, was developed by Taguchi et al. in 1998 to increase the efficiency and coating time of the biomimetic
technique. It is able to easily treat polymer and hydrogel scaffolds.
• Micro-arc oxidation280,281 (also called plasma electrolytic oxidation or anodic spark deposition) can produce porous, rough
and adherent ceramic coating by electrochemical oxidation with micro-discharges created on the surface of components
immersed in an electrolyte at room temperature. On a titanium substrate, a composite coating composed of rutile, CaTiO3,
Ca2Ti5O12, b-Ca2P2O7, a-Ca3(PO4)2, and apatite is grown by varying the applied voltage (at fixed pulse frequency and duty
cycle), time, and electrolyte composition.
• Electrophoresis282 that consists in the migration of colloidal ceramic particles under the influence of an electric field enables the
deposition on complex shapes of inorganic compounds modified by organic or biological charged agents.
• Electrochemical deposition283–286 is a very versatile technique allowing deposition of calcium phosphates substituted with
ionic antibacterial agents (Cu, Ag, Zn,…) or other biologically active ions (Sr, F, Si, Mg,…), and of apatite/organic (chitosan,
collagen,…) composite coatings by varying the electrolyte composition as well as the electrochemical method (pulsed,
potentiostatic,…).
Aerosol deposition,287 and also blast coating288 and cold spray289 techniques in which powder particles are accelerated and
have a ballistic impingement on the substrates, have emerged as low temperature alternative physical processing methods for
sensitive substrates like PLLA or PEEK. The process is not only safe, rapid, and simple but also cost-effective and suitable for use in
mass production. Another advantage relies on the fact that the deposited material exhibits the same physical, chemical and
crystallographic characteristics as the feedstock material. Adhesion strength (around 30 MPa) of 1 mm thick aerosol deposited HA
coating on Ti substrate is higher than the values reported for biomimetic coatings that are around 15–20 MPa.
The abovementioned techniques are dedicated only to coat a surface not to produce bulks. Conventional manufacturing
processes including isostatic pressing, slip- or gel-casting, extrusion, and injection molding are flexible methods but they require
machining and firing treatments on green parts, which prevents from using sensitive calcium phosphates. Additive manufacturing
also known as free form fabrication is a method during which components are built layer by layer from a numerical image. A
curing stage or sintering one is also required to achieve final parts with good mechanical properties. However a few attempts have
been made to process apatite powders at very low temperature directly by cold pressing or below 2001C by spark plasma
sintering.290,291 It was demonstrated that pressure conditions can be found to consolidate nanocrystalline biomimetic apatites
while preserving their hydrated layer, nanosized crystals, and nonstoichiometry.
1.11.6.5 Cements
The fast-setting calcium phosphate cements have emerged as an attractive concept essentially as bone filling and reinforcement
biomaterials. Ca-P bone cements have developed considerably in the last 30 years due to their excellent biocompatibility and
bioactivity.292 The applications for the filling of bone defects are very promising, whether in the form of a molded paste placed in
the surgery site or in the form of an injectable paste hardening in situ.
Several types of reactions can be involved in the Ca-P self-setting materials, which lead to the formation of two main classes of
Ca-P cements depending on the end product: apatite cements consisting of more or less crystallized apatite and brushite
cements.292,293 It shall be noticed that brushite is not stable in body fluids and that it shall inevitably be transformed more or less
rapidly, depending on the size of the implant, into nanocrystalline apatite.
The hardening of mineral cements is generally attributed to the growth of interlaced crystals. This has been shown in the
case of some apatite cements where long needle-like crystals can be seen by SEM and in the case of brushite cement,
interlaced large plate-like crystals isomorphous to gypsum crystals responsible for the hardening of plaster can be observed. This
mechanism, however, does not seem to be involved in biomimetic cements resulting in nanocrystalline apatite analogous to bone
mineral. The high specific surface area and strong interactions between nanocrystals could be, in this case, the main factor of
hardening.
Two major types of reaction are involved in phospho-calcium cements formation. The first consists in a reaction between
one or more “basic” calcium compound (ie, rich in calcium) such as TTCP or even Ca(OH)2 and one or more “acidic”
calcium phosphates (ie, rich in phosphate) such as MCPM, H3PO4, DCPD. In aqueous medium, the reaction between these phases
leads to less soluble new phases, whose crystallization brings about the setting. The second type of reaction takes advantage of the
metastability of a Ca-P phase in aqueous medium and its rapid conversion into apatite. Two phases, a-TCP and ACP, can be used.
Various cement formulations have been proposed, sometimes mixing different Ca-P phases and hardening reactions, and several
cements are commercialized: SRS, Bonesource, Cementek, Biobon, a-BSM.
Several properties characterize a biological cement, like construction cements, although most of them have not been
standardized which do not allow comparison between formulations. The workability is the time during which the paste can be
mixed and kept before use; this property is related to the characteristics of the reactions involved in the setting. The ACP
conversion, for example, is extremely slow at room temperature, which is related to a long working time. For some brushite
cements, like for plaster of Paris, mixing the paste for too long may break the growing crystals involved in the setting process and
may result in lower mechanical properties. Additives, which delay the reactions between constituents at room temperature, can be
used to increase the workability. The setting time has been standardized (Gillmore needle test) and corresponds to the time
needed to get a compact block. Generally, the chemical reactions are not achieved at the setting time. Several factors determine this
property related essentially to the rate of reactions. The addition of soluble phosphate salts in the liquid phase, for example, has
been shown to shorten the setting time of “acid–base” cements, probably through the reaction with TTCP or other calcium-rich
phase. The addition of apatite nuclei can also be used like in ACP-based cements, for example.294 Fluoride ions, through their
effect on apatite solubility, may also accelerate the apatite formation and shorten the setting time. The hardening time is related to
the evolution of the cement after setting: in construction cements, it corresponds to the time needed to get the nominal mechanical
properties, for biological cements, this parameter is not always determined and the mechanical properties may possibly degrade
after completion of the chemical reactions. Injectability is an important parameter for biological cements and several techniques
have been proposed to determine this parameter in conditions as close as possible to those of the surgical use. One of the
difficulties in the injection of an aqueous suspension of a solid phase is the filter-press effect responsible for a variation of the
solid/liquid ratio during the extrusion of the paste and eventually the impossibility to continue the injection by applying a
reasonable pressure on the syringe. The injectability is related to the viscosity of the suspension and especially the solid/liquid
ratio, and the stability of the suspension obtained. Several additives have been used to improve the injectability. The wash-out
phenomenon corresponds to the disaggregation or dispersion of the paste during or after its placement; this property is not an
intrinsic characteristic of the cement and is also related to the viscosity of the medium in which the cement paste is introduced.
It has been shown that the viscosity of the paste has to be higher than that of the medium it is injected in to avoid dispersion.
In vivo, wash-out can also be due to the action of blood on the nonset paste. Additives such as gelling agents can be used to
avoid wash-out, provided they do not interfere with the setting reaction. The mechanical properties of hardened Ca-P cements
depend on their microstructure and especially their porosity related to the solid/liquid ratio. They vary in a large domain
in commercial cements (reported compression resistance from 5 to 80 MPa) and they are not suitable for implantation in
locations where mechanical loads are high. In addition to these physical-chemical properties, biological properties of the Ca-P
cements have to be determined and, here also, the absence of standardized methods does not generally allow comparison between
different formulations. Among these properties, the most important is bone reconstruction ability associating bone formation with
cement resorption. Like in the case of bioceramics, the cohesion of the hardened cement is a determining factor for avoiding the
release of too numerous particles and the development of uncontrolled inflammatory reactions. Generally, cements are considered
to behave like bone tissue and their degradation has been defined as “centripetal” resulting in irregular degradation and bone
formation from the surface of the implant to the interior with possible cement residues remaining encysted in the newly formed
bone. The degradation of apatite cements is related to the microstructural and physical–chemical characteristics of the hardened
material. The degradation is faster when the porosity is higher, the crystal size is smaller, and the amount of vacancies in the
structure is higher. The amount of vacancies is not simply related to Ca/P ratio as explained earlier (see Section 1.11.3.1), but to
the substitution ratio of trivalent PO3 2 2
4 ions by bivalent ones (essentially CO3 and HPO4 ions). Biphasic cements have been
295
proposed, in analogy with biphasic ceramics, to vary the bioabsorption properties. Cements may show variability of their
properties depending on their preparation in the operation room, which is often perceived as tedious. To circumvent this
drawback, ready to use cements have been proposed in which the setting reaction is frozen, physically, or chemically, by removing
water from the premixed paste (non-aqueous phase, miscible with water)296 or by using reversible ion exchange processes
inhibiting the setting reaction.297
The main advantages of Ca-P ionic cements are their easiness of use, their biodegradation combined with the progressive
replacement by bone neoformed tissue. Besides, they can easily be associated with therapeutic and/or biologically active com-
ponents (antibiotics, growth factors, platelets, etc.). Radio-opacifier can also be added to the paste provided they are bioab-
sorbable; Sr salts have also been proposed. One of the main drawbacks of actual cements are the small size of pores which does
not favor cell rehabilitation; the poor mechanical properties and the variability in characteristics and procedures for paste
preparation compared to polymeric cements.
As for Ca-P cements, the concept of putty emerged in the 1980s and has also developed in the last decade. Most of the
putties including Ca-P combine a biopolymer (alginate, cellulose derivatives,…) or a protein (collagen, fibrin…) with
Ca-P granules or micro/nanoparticles leading to non-setting hydrogel-Ca-P paste.298 The cohesion of such non-setting
pastes/putties is related to the ability of the biopolymer/protein to form a gel (eg, alginate molecules crosslinking with Ca2 þ
ions). However, such putties may produce negative biological reactions due to particles release in vivo depending on
their size.
1.11.7 Associations of Calcium Phosphates With Other Compounds
Calcium phosphates are often associated with various organic and/or inorganic compounds to combine their respective physico-
chemical and biological properties or even reach a synergistic effect.
1.11.7.1 Organic-Based Composites

Associations of Ca-P compounds and polymers represent an interesting way to provide advantageous cohesiveness and mechanical
properties to calcium phosphates, in relation with the imitation of the bone tissue composite material. Several composites
for bone substitution, repair or bone tissue engineering involving calcium phosphates and polymers/proteins have been described
in the scientific literature.299 In most of the designed and studied composites the inorganic part is involved as a filler to reinforce
and/or confer bioactive properties to the material. Depending on the way of elaboration, materials with various shapes could
be obtained as scaffolds, monoliths, fibers, microparticles and coatings. A key parameter for these composites is the dispersion
of inorganic Ca-P fillers into polymer matrices. It mainly depends of the size, shape, percentage of particles and the route
of preparation (solid mixing, liquid dispersion, in situ generation…).300 A major difficulty, especially with composites, is the
prediction of their degradation and the in vivo management of the metabolites and particles which can be produced. An ideal
case is when the different components of the composite material are uniformly degraded by cellular activity into soluble man-
ageable metabolites. In many cases however the resorption of the constituents is not homogeneous and the degradation can
be more or less uncontrolled, especially with polymer matrixes which degrade by hydrolysis, like PLA or PGA. These
purely chemical degradation may result in metabolites and particles production which can potentially initiate inflammatory
reactions depending on their number, size and chemical properties, even when each of the constituents, taken alone, are well
tolerated.
Most of calcium phosphates can be associated to a large number of organic components that can be classified into three broad
families: (1) proteins, (2) non-protein natural polymer, and (3) synthetic polymers.
1.11.7.1.1 Proteins
Different associations of Ca-P with proteins have been tested like fibrin301 silk.302 However, apatite/collagen composites have been
particularly studied by many research groups. The possibility for molecules such as collagen to interact with the ions from the
highly reactive surface layer on apatite nanocrystals through ionic functions of proteins (mostly anionic groups) has already been
mentioned in this chapter (see Section 1.11.4.5.2). However, the interactions between the collagen fibrils and the Ca-P (most
generally apatite) are not really known. Solid state NMR studies failed to identify a direct stable chemical bonding between
collagen and the mineral crystals of bone.303 Dos Santos et al.304 have expressed the possibility to study the apatite–collagen
interface by using a FRET technique with luminescent europium-doped apatite. This is an interesting idea for investigating the
interactions between collagen and apatite, although not really at a chemical level.
Many studies have been aimed at producing biomimetic artificial bone-like tissue involving apatite and collagen taken in
varying forms: fiber, gel, or degraded as gelatin.248,305–307 Several protocols have been used to prepare apatite/collagen hybrids.
Testing two methods of preparation of such composites (dispersion of HA in collagen gel or direct nucleation of apatite on
collagen fibers), Tampieri et al.308 have shown that the bioinspired method based on the direct nucleation of apatite leads to
composites analogous to calcified tissue and exhibits intimate associations between apatite and collagen analogous to those
reported in bone. Some researchers have mentioned the preliminary phosphorylation of collagen in order to generate nucleation
sites for mineralization, followed by a subsequent step of apatite nucleation and growth on the collagen by immersion in SBF.309
Other techniques were also tested, such as electrospinning.310 This technique was useful for obtaining composite fibers up to
about 30 wt.% loading with HA, while beyond this amount, a preferred bead-like morphology was observed. Porous scaffolds
associating collagen fibers and poorly crystalline bone-like carbonated apatite nanoparticles have also been generated using freeze-
casting. This way of elaboration allows controlling the shape, direction and size of the porosity of materials.311
In vivo evaluation in weight-bearing sites (in the dog) were performed for testing apatite/collagen composites prepared
by a coprecipitation method.312 The results led to interesting conclusions, especially when associated with BMP-2 growth
factor. The controlled release of BMP-2 from the implant was indeed found to facilitate the early formation of callus and hence
of newly formed bone, enabling early weight bearing. Nishikawa et al.313 have examined the biodegradation of HA/collagen
composites implanted in dogs by a tissue labeling method, and these authors showed that this novel composite allowed bone
augmentation.
These associations have not, however, given materials with the mechanical properties of bone and they have not resulted in
decisive advantages compared to bioceramics bone substitutes.
1.11.7.1.2 Non-protein natural polymers

After collagen, polysaccharides are the main natural polymeric matrix associated with calcium phosphates. Several associations
involving various polysaccharides have been described (cellulose, dextran, hyaluronic acid derivatives) but the most used are
alginate and chitosan. Alginates are linear copolymers of b-D-mannunoric acid and a-L-guluronic acid residues. Sodium alginate
forms stable material through multivalent ionic crosslinking in mild conditions (water, ambient temperature).314 As Ca2 þ is one
of mineral cations necessary to crosslink alginate molecules and precipitate calcium phosphates, various methods including direct
mixing of HA powder with alginate,315 in situ mineralization and 3D plotting technique can be used.316 Despite poorer
mechanical properties compared to synthetic polymer composites, mild processing conditions allow loading of active molecules
or proteins to improve bone regeneration. Chitosan is a linear copolymer of glucosamine and N-acetyl glucosamine. The molar
fraction of N-acetylated units is predominant as higher degrees of acetylation lead to faster enzymatic degradation rates. As for
alginate, several ways of preparation are described like one-step co-precipitation317 or mineralization.318 It is interesting to note
that the chemical modification of chitosan and particularly phosphorylation could improve the nucleation rate of calcium
phosphates.319
1.11.7.1.3 Synthetic polymers

The most utilized synthetic polymers in association with calcium phosphates are saturated poly-a-hydroxy esters, including poly
lactic acid (PLA), polyglycolic acid (PGA) and polylactic-co-glycolide (PLGA) co-polymers.320 Their degradation occurs by uptake
of water followed by hydrolysis of ester bonds. Different intrinsic parameters of the polymer can influence this chemical degra-
dation as molar mass or polydispersity. Unfortunately the fast in vivo degradation of these polyesters can cause abrupt release of
acidic de-esterification products and then a strong inflammatory response.321 Beyond the improvement of mechanical properties
the association of calcium phosphate fillers with PLA, PGA and/or PLGA is expected to modify polymer degradation kinetics, an
autocatalytic process driven by acidic protons. The rapid removal of protons by the dissolution of inorganic particles provides a pH
buffering effect at the polymer surface, modifying the acidic polymer degradation.322 It has been shown that apatite/PLLA
composites are hydrolyzed homogeneously, in vitro.323
1.11.7.2 Association of Ca-Ps With Other Bioactive Inorganic Compounds

Associations of Ca-Ps with other bioactive calcium salts have been investigated to control resorption and/or biological properties
of Ca-P bioceramics. We limit our presentation to examples of three main types of bioactive compounds/materials that have been
associated with Ca-P in bioceramics: calcium carbonate, calcium sulfate and bioactive glasses.
1.11.7.2.1 Calcium carbonates

Calcium carbonates (CaCO3, CC), particularly those produced by marine organisms such as coral and mother of pearl, have been
shown to be biocompatible and bioactive biomaterials, and they have been used for more than 25 years as bone substitutes in the
form of powders, porous ceramics, or gels.324–330 Three crystalline phases of anhydrous calcium carbonates are encountered in nature:
calcite, aragonite, and vaterite and natural calcium carbonates from certain corals and nacre present the aragonite structure. Con-
sidering the mineral ion content, blood plasma seems to be supersaturated with respect to these three calcium carbonate varieties,
although for vaterite, the oversaturation is much smaller than for calcite or aragonite; thus these phases cannot dissolve spontaneously
in vivo. However, in the presence of phosphate ions, the apatite remains the most stable phase and all the three calcium carbonate
varieties should transform, at least superficially, into apatite. The main degradation process of calcium carbonate has been assigned to
a carbonic anhydrase enzyme, which induces a local dissolution and leave place for newly synthesized bone tissue.327
The idea that the interconnected pores structure of coral can replace defective parts of bone331 comes from the similarity in
the structure of some coral skeletons with cancellous bone allowing colonization by cells and blood vessels penetration. Coralline
HA, a carbonated HA, which is prepared by the hydrothermal conversion of calcium carbonate from coral in the presence of
ammonium phosphate is one of the most important example of association of Ca-P with CaCO3.331–333 It has been suggested that
controlling the thickness of HA layer on a CaCO3 matrix could control the rate of resorption of the implant and its replacement by
newly formed bone. Despite a good mechanical strength, this is not sufficient to allow their use in bones subjected to high
mechanical stresses (load-bearing bones). Moreover, their structure is determined by the considered species and their chemical
composition is not well controlled, particularly, with respect to trace elements, and residual organic matrix and implant rejection
have been observed possibly due to the presence of organic matter residue.
Although several attempts have been made to produce synthetic calcium carbonate bioceramics, sintering proves to be difficult,
and the idea of associating calcium carbonate with Ca-P through the cement route (by mixing calcium carbonate and calcium
phosphate phases with an aqueous medium) offers an interesting alternate way to prepare low-temperature Ca-P–CaCO3 bio-
ceramics. Combes et al. demonstrated the feasibility of mixed calcium carbonate-calcium phosphate biomedical cements including
various content of CaCO3 (from 20 to 100%).295,334 Calcium carbonate compounds have a higher solubility compared to that of
apatite and the use of very large proportions (Z20% w/w) of calcium carbonate, especially of metastable amorphous or crystalline
phase (amorphous CC or vaterite), in the dry powder ingredients opens new possibilities which have not yet been exploited to
control the cement resorbability. Later, some other studies also reported the positive effect of the introduction of carbonate on,
in vitro, resorption of calcium phosphate cements.335
1.11.7.2.2 Calcium sulfate hemihydrate

Known as plaster of Paris, calcium sulfate hemihydrate (CaSO4 1/2H2O; CSH), has been used as a building material for at least
5000 years and used by Egyptians to decorate burial tombs of pharaons.336 Dreesman was the first to report a study on the
implantation of plaster of Paris as a bone filler material in eight patients.337 Since this first clinical success, several groups of
research have studied different bioactive ceramics for bone filling and repair involving CSH associated with Ca-P mainly to
improve the handling or the resorbability of Ca-P ceramics (powder, granules, porous blocks).
Nilsson et al. have shown that an association of a-calcium sulfate hemihydrate, the densest form of CS hydrates, and HA
particles gives good clinical outcome in various applications.338 The authors speculated that the good clinical results were due to
fast dissolution of the calcium sulfate phase in combination with the osteoconductive and bioactive nature of HA.
Cortez et al.339 investigated the association of CS with Bonelike granules (glass-reinforced HA with a- and b-tricalcium
phosphate as secondary phases) implanted in non-critical monocortical defects in sheep. Their study showed that CS is an effective
vehicle for Bonelike granules as it facilitates their application and that this composite material functioned as a very satisfactory
scaffold for bone regeneration as it achieved synchronization of the ingrowing bone with biomaterial resorption and subsequent
preservation of the bone graft initial volume. Porous scaffolds have been prepared including a composite coatings based on poly
(D,L-lactide) (PDLLA) polymer solutions, and combination of HA, calcium sulfate and chondroitin sulfate (ChS) powders.340 The
scaffolds with composite coatings resulted in significant improvement in both mechanical and biological properties while
retaining the 3D interconnected porous structure. The authors showed that in vitro mineralizing behaviors were mainly related to
the compositions of CS and ChS powders in the composite coatings.
Finally, three-dimensional (3D) printing of calcium phosphate/calcium sulfate powders blend has been investigated for the
application of tissue engineered bone scaffolds.341 The authors reported the preparation of porous constructs using the 3D
printing technique from optimized Ca-P/CaSO4 powder formulations in terms of particle size, Ca-P/CaSO4 powder ratio and type
of Ca-P powders. High-quality printed scaffolds of HA/CaSO4 powders were manufactured, which exhibited appropriate green
compressive strength and a high level of printing accuracy.
1.11.7.2.3 Silicate-based or phosphate-based bioactive glasses

Initially, phosphate-based glasses (P-glasses), have been used as sintering additives to calcium phosphates (essentially HA and
b-TCP) to improve, with success, the densification and mechanical properties of the resulting composite materials compared to
ceramics obtained by natural sintering.342 Si-based glasses, (Si-glasses) were found globally less efficient in the improvement of the
mechanical properties but exhibited interesting biological properties supported by extensive works.343,344 Both Si- and P-glasses
are absorbable with a degradation rate higher for P-glasses and the most successful associations have involved Si-glasses with the
development of a commercial product.345 These associations, obtained by high temperature treatments, result however in
materials showing a complex physical chemistry not always adequately described. P-based glasses show a rather low Ca/P ratio,
and heating with HA inevitably results in the formation of TCP phases corresponding to a lowering of the global Ca/P ratio, which
is effectively observed (see Section 1.11.4.1.2). These reactions depend on temperature and time of reaction and, as they use the PV
content of the glass, they reduce the glass content of the composite material. Na2O added to P-based glasses can lead to other
phases like rhenanite CaNaPO4 but sodium may also be incorporated in TCP or HA. The formation or modifications of these
phases, also involves a reduction of the glass content. In the case of Si-based glasses, the reactions are even more complex and
depend on the compositions. With the classical 45S5 Bioglass (with a glass initial content of 2.5–5% in HA) a formation TCP is
also observed corresponding to the replacement of phosphate by silicate groups in the apatite structure, as explain above (see
Section 1.11.6.2). The amount of TCP formed was found to increase with the sintering temperature corresponding to the glass-HA
reaction progression.346 In addition to silicate incorporation, sodium can substitute to calcium in the HA and TCP phases as
indicated above. Other reactions may be observed at higher glass content related to the heating temperature.342 Interesting
associations of Si–glasses with Ca-P cements and coatings have also been proposed.347 Such associations trying to benefit of the
properties of different but well defined biomaterials may result in fact in alterations of these constituents, although they may give
good biological results.345,348
1.11.8 Conclusion and Future Perspectives
The evolution in the last decade has shown the emergence of substituted Ca-Ps, and more generally, chemically modified
Ca-Ps, with improved biological properties such as apatite with silicate ions, TCP with Zn ions, and other products. It is probable
that such trials to improve the biological activity will be pursued with possibly other types of ions with a biological action
or combination of ions. Simultaneously, the mechanisms relaying the improved biological activity related to chemically modified
Ca-P are not yet perfectly known in many cases, and the distinction between several effects of doping at the material level,
related to bulk properties of crystals, microstructural characteristics of the ceramics, and surface alterations probably needs to
be clarified. Another point is that the mineral ions, incorporated in such biomaterials, and which are being released are not in
a biologically active form. Most Zn ions of the body, for example, are involved in hundreds of compounds with different
activities. How do the mineral ions which are released interact with the in vivo environment to produce a given researched
effect? When the mineral ions are bone seeking elements found in healthy bone mineral substance, the effects and uses of these
added ions, in a certain limit to be determined, can possibly enter in the global metabolic activity and control. When the
elements are not part of the mineral fraction, it seems legitimate to wonder how they are reacting and how they are
metabolized.
A second route of development concerns nanocrystalline apatites, which exhibit a very high and peculiar reactivity and
constitute the main component of bone substance. Advances in this field will probably also result in a better understanding of
bone biology which in many cases has focused on cell activity and organic macromolecules specificities, forgetting that the mineral
with its high surface area and strong potential interactivity is probably an important player in several bone regulation mechanisms.
Ca-P-based bioceramics with osteoconductive and osteoinductive properties have attracted back the attention of researchers on the
role of Ca-P minerals in living organisms, and advances are expected in this field. In addition to their involvement in nanoma-
terials, Ca-P nanocrystals can also be used in different applications for imaging or cell therapy. The development of the use of
nanocrystals raises the problem of their accurate characterization, evolution, and surface properties, and regulations and standards
will probably flourish.
A third development domain, already active, concerns the associations of bioceramics with active molecules for different
purposes, for example, antiseptic activity or improvement of cell response using growth factors or associations of growth factors
with sequenced controlled release. Additive manufacturing offers in this domain the possibility to elaborate scaffolds with
predetermined functional domains allowing the design of complex architectures withstanding mechanical constrains, but also
favoring bone ingrowth in a controlled manner, and binding to the bone organ.
The Ca-P bioceramics and composite materials are interesting substrates for scaffolding in tissue engineering, and the intense
research activities they generate will certainly develop our knowledge of cell–material and tissue-materials interactions and will
probably allow the distinction of crucial parameters involved in cell colonization and differentiation determined by materials
with, here also, interesting returns in the domain of bone biology.
See also: 1.9 Bioactive Ceramics. 1.13 Bioactive Layer Formation on Metals and Polymers. 1.14 Calcium Phosphates and Bone Induction. 1.16
Calcium Phosphate Coatings. 1.17 Bioactive Ceramics: Cements. 1.20 Silicon-Containing Apatites. 3.14 Molecular Simulation Methods to
Investigate Protein Adsorption Behavior at the Atomic Level. 3.23 Infrared and Raman Microscopy and Imaging of Biomaterials at the Micro and
Nano Scale. 6.1 Bioactive Ceramics and Bioactive Ceramic Composite Based Scaffolds
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Relevant Websites
http://rruff.geo.arizona.edu/AMS/
American Mineralogist Crystal Structure Database.
http://www.ccp14.ac.uk
CCP14 – Collaborative Computational Project Number 14. Series of Open Access Software for Single Crystal and Powder Diffraction. Includes Rietveldt Refinements.
http://www.crystallography.net
Crystallography Open Database.
www.mindat.org
Mindat.org.
http://www.webmineral.com
Mineralogy Database – Description of Mineral Structures With Tri-Dimentional View.
http://rruff.info
RRUFF – Site Collecting the Diffraction Data and Raman and FTIR Spectra of Minerals.
https://vminteq.lwr.kth.se/
Site of “Visual MINTEQ” the Open Access Software for the Speciation of Mineral Ions in Solution.

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1.

11 Bioactive Calcium Phosphate Compounds: Physical Chemistry☆

1.11.1 Introduction 246

244 Comprehensive Biomaterials II, Volume 1 doi:10.1016/B978-0-12-803581-8.10171-7

1.11.5 Biological Response and Physical–Chemical Characteristics 273

Abbreviations ICP Inductively coupled plasma

Symbols mn (n integer) domain of vibration of a molecule

H enthalpy pKsp –logKsp

Glossary Pyrophosphate (also called diphosphate) Salts of

1.11.2.1 Calcium Phosphates Obtained by Wet Synthesis Methods

Table 1 Main calcium phosphate compounds

Monocalcium phos- MCPM Triclinic P1̄ 2.23 1

1.11.2.1.2 Dicalcium phosphates

1.11.2.1.3 Tricalcium phosphate, whitlockite, and amorphous tricalcium phosphate

Ca18 Mg2 ðHPO4 Þ2 ðPO4 Þ12

1.11.2.1.4 Octacalcium phosphate

1.11.2.2 Calcium Phosphate Obtained by Dry Synthesis Methods

1.11.2.2.1 Crystalline tricalcium phosphates: a- and b-TCP

1.11.2.2.2 Tetracalcium phosphate

1.11.2.3.1 Well-crystallized apatites

Ca8 La2 ðPO4 Þ4 ðSiO4 Þ2 ðOHÞ2

The replacement of trivalent PO3

1.11.2.3.2 Nanocrystalline and biomimetic apatites

1.11.2.3.2.1 Nanocrystalline apatites

1.11.2.3.2.2 Biomimetic nanocrystalline apatites

1.11.2.3.3 Synthesis and formation of apatites

1.11.2.3.3.1 Wet preparations

1.11.2.3.3.2 Dry preparations

CaCO3 þ 3Ca3 ðPO4 Þ2 þ H2 O-Ca10 ðPO4 Þ6 ðOHÞ2 þ CO2

Fluorapatite (FA) or Chlorapatite (ClA) can be prepared with similar reactions:

CaF2 ðor CaCl2 Þ þ 3Ca3 ðPO4 Þ2 -Ca10 ðPO4 Þ6 Fðor ClÞ2

4CaCO3 þ 3Ca2 P2 O7 þ H2 O-Ca10 ðPO4 Þ6 ðOHÞ2 þ 4CO2

or a more complex reaction87:

14CaF2 þ 18Ca2 P2 O7 -5Ca10 ðPO4 Þ6 F2 þ 6POF3

releasing POF3 as a gas.

2HCl þ Ca10 ðPO4 Þ6 ðOHÞ2 ⇄Ca10 ðPO4 Þ6 Cl2 þ H2 O

1.11.2.4 Other Phosphate Compounds

1.11.3 Characterization Methods of Calcium Phosphates

1.11.3.1 Chemical Analysis

• major ions like Ca2 þ , orthophosphates, including PO3 2 2

1.11.3.1.1 Major ions

1.11.3.1.1.4 Hydroxide ions

Other reactions like that involving pyrophosphate and HA (around 800–11001C):

1.11.3.1.2 Minor and trace elements

1.11.3.1.3 Meaning of the Ca/P ratio

1.11.3.2 Diffraction Methods

Phase JCPDS folder hkl dhkl (A )̊ 2y (lCu ¼1.54 A )̊

a-Tricalcium phosphate 09-0348 4 4 1 2.905 30.753

a Parameter FA D (OH- F) HA D (Cl-OH) ClA D (Cl-F)

Ca 9.370 0.025 9.420 0.105 9.630 0.130

c Parameter FA D (OH - F) HA D (Cl-OH) ClA D (Cl-F)

Ca 6.880 0.000 6.880 0.050 6.780 0.050

Source: According to McConnel Ref. [104].

1.11.3.3 Electron Microscopy Techniques

1.11.3.4 Vibrational Spectroscopies

DCPD DCPA OCP ATCP b-TCP a-TCP HA TTCP

1.11.3.5 Solid-State Nuclear Magnetic Resonance

DCPD DCPA OCP ATCP b-TCP a-TCP HA TTCP

n3PO4 1132 vw 1131 m

FTIR Raman FTIR Raman FTIR FTIR

n3CO3 1542 s 1490 s

1.11.4 Properties of Calcium Phosphates

1.11.4.1 Thermal Properties of Calcium Phosphates

1.11.4.1.1 Reversible decomposition of stoichiometric apatites