Synthesis and physical chemical characterizations of octacalcium phosphate–based biomaterials for hard-tissue regeneration

Synthesis and physical chemical characterizations of octacalcium phosphate
based biomaterials for hard-tissue regeneration

8

Christian C. Rey, Christe`le Combes and Christophe Drouet National Polytechnique Institute of Toulouse, Materials Engineering Department, CIRIMAT, University of Toulouse, CNRS, Toulouse INP
ENSIACET, Toulouse, France

Abbreviations ACP am-OCP ap-OCP ATCP BNA c-BNA Ca-P CDA CDL DCPA DCPD FTIR HA ICP nc-BNA NMR nt-OCP OCP OCPC t-OCP SEM TCP TEM TTCP XRD

Amorphous calcium phosphate Amorphous octacalcium phosphate Apatitic octacalcium phosphate Amorphous tricalcium phosphate Biomimetic nanocrystalline apatite Carbonated BNA Calcium phosphate Calcium-deficient apatite Central dark line Dicalcium phosphate anhydrous Dicalcium phosphate dihydrate Fourier-transform infrared spectroscopy Hydroxyapatite Inductively coupled plasma Non-carbonated BNA Nuclear magnetic resonance Nanocrystalline triclinic octacalcium phosphate Octacalcium phosphate Octacalcium phosphate carbonate Triclinic octacalcium phosphate pentahydrate Scanning electron microscopy Tricalcium phosphate Transmission electron microscopy tetracalcium phosphate X-ray diffraction

Octacalcium Phosphate Biomaterials. DOI: https://doi.org/10.1016/B978-0-08-102511-6.00008-X Copyright © 2020 Elsevier Ltd. All rights reserved.

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Octacalcium Phosphate Biomaterials

Introduction

Octacalcium phosphate (OCP) is the abbreviated name for “octacalcium bis(hydrogenphosphate) tetrakis(phosphate),” which refers to a chemical composition Ca8 ðHPO4 Þ2 ðPO4 Þ4

(8.1)

most generally associated with the triclinic crystallographic structure of the pentahydrate salt [triclinic OCP (t-OCP)] [1]. However, other OCP phases have been described with a similar composition but a different structure. Apatitic OCP (ap-OCP), corresponding to the lower limit of calcium-deficient apatite compositions reported by several authors [1
4], is probably the earliest pseudopolymorph, although it has been the object of a very few dedicated studies. Precipitations in water
ethanol mixed solutions allowed the preparations of amorphous OCP (amOCP) [5] and of the related ap-OCP, which has been the object of detailed studies in relation with its interesting compressive properties and its possible applications as a support for drug-delivery systems [6]. The t-OCP has often been considered as one of the precursors of bone mineral and tooth enamel formation. Another OCP phase, carbonated OCP, reported by Hayek [7] and more recently by Shen et al. [8], could be seen as a transition member in this chain of precursors leading to biological apatites. The study of bone mineral has led to the synthesis of a peculiar kind of nanocrystalline, calcium-deficient apatites (CDA), very close to the biological model, called biomimetic nanocrystalline apatites (BNAs). These minerals are characterized by a surface ionic hydrated layer, considered as amorphous by some authors [9], structured by others, and showing some analogies with t-OCP [10]. It seems interesting to see where we stand and how this layer could be related to the t-OCP pentahydrate Ca8 ðHPO4 Þ2 ðPO4 Þ4 5H2 O

(8.2)

and finally how it behaves on aging and in different environments.

8.2

Different types of octacalcium phosphates and related compounds

8.2.1 Triclinic octacalcium phosphate pentahydrate The t-OCP is one of the most studied phases of the OCP family. Its crystal structure is well established [11]. The water content in the lattice may decrease under its nominal value, depending on water partial pressure, heating, and handling conditions, but except minor alterations of the Fourier-transform infrared spectroscopy (FTIR) and Raman spectra [12], and of the X-ray diffraction (XRD) pattern, this partial water loss is reversible [13]. Nonstoichiometric t-OCPs have been described based on their Ca/P ratio and HPO22 4 content. Calcium-deficient OCPs with a higher hydrogen phosphate content

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than in the theoretical chemical formulae have been described [14]. The excess of HPO22 4 in these nonstoichiometric OCP samples has been shown to be partly but reversibly removed or incorporated by dipping in alkaline or acidic solutions, respectively; this behavior has been related to surface and bulk protonation with some alterations of the crystal structure [15]. Among other alterations, hydroxylated OCP, containing OH2 ions, with a normal Ca/P ratio but an excess of HPO22 4 ions, corresponding possibly to an internal hydrolysis involving intrastructural water and PO32 4 groups, has been identified: 22 2 H2 O 1 PO32 4 ! HPO4 1 OH

(8.3)

A last nonstoichiometric t-OCP, with OH2 ions and a Ca/P ratio above 1.33, sometimes called “hydrolyzed OCP,” could correspond to possible interlayering/ intergrowth with a stoichiometric apatite [hydroxyapatite (HA): Ca10(PO4)6(OH)2] [16
18] (Fig. 8.1). In the two last cases, the OH2 ions show similar characteristics

Figure 8.1 Structure of t-OCP (projection on the 0 0 1 plane) showing the hydrated layer and the apatitic domains with the corresponding unit cell of the apatite (Redraw from Elliott JC. Structure and chemistry of the apatites and other calcium orthophosphates. Amsterdam: Elsevier Science BV; 1994). On the right is given an example of OCP
HA interlayering, showing HA and OCP arrangements in the same “crystal.” (According to Brown WE, Mathew M, Chow LC. Roles of octocalcium phosphate in surface chemistry of hydroxyapatite. In: Misra DN, editor. Adsorption on and surface chemistry of hydroxyapatite. New York: Plenum Press; 1984. pp. 13
28 [19]). HA, Hydroxyapatite; OCP, octacalcium phosphate; t-OCP, triclinic octacalcium phosphate pentahydrate.

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to those present in stoichiometric HA, by FTIR and Raman spectroscopies, or solidstate nuclear magnetic resonance (NMR). This possibility of interlayers of HA and t-OCP domains results in a theoretical continuity between the OCP and the HA composition expressed in the following equation: yCa8 ðHPO4 Þ2 ðPO4 Þ4 1ð12yÞCa10 ðPO4 Þ6 ðOHÞ2 !Ca1022y ðPO4 Þ622y ðHPO4 Þ2y ðOHÞ222y (8.4) where y is the molar fraction of OCP in the interlayered crystal. According to Brown, the surface layer is always a t-OCP hydrated layer [18]. This conception has received some confirmation in the study of well-crystallized as well as poorly crystalline precipitated HA, where HPO22 4 ions have been detected on the surface of the crystals by solid-state NMR [9,20,21]. An interesting property of the t-OCP structure is to trap linear organic dicarboxylate ions (but also other carboxylates like citrate ions) as substitutes of HPO22 ions in the hydrated layer [22,23]. These t-OCP-carboxylate compounds 4 exhibit a larger unit cell than that of regular t-OCP, in relation with the carbon chain length of the di-carboxylate and a larger amount of water expanding the hydrated layer [24]. The structure of the t-OCP-adipate and -succinate has recently been analyzed by solid-state NMR [25]. The case of t-OCP-citrate has received special attention as citrate ions reach a relatively high concentration in bones (around 2%). Considering its role in OCP-citrate crystals, the citrate ion has been proposed as a bridging molecule between bone nanocrystals [26].

8.2.2 Amorphous octacalcium phosphate Although amorphous calcium phosphate (ACP) is often mentioned as a precursor of t-OCP [27,28], references to am-OCP are scarce. In fact, ACP phases rich in HPO22 4 and obtained at physiological pH are very unstable and transform within a few minutes into poorly crystalline apatite. The ACP1 and ACP2 amorphous phases described by Christoffersen [29,30], for example, which are obtained at a rather acidic pH (6.5
5.5) with a Ca/P ratio of 1.35, very close to that of OCP compounds, behave according to these observations. ACP1 has a lifetime less than 5 min and transforms into ACP2, which begins to transform into t-OCP after about 10 min. ACP obtained at alkaline pH are more stable but their composition is closer to that of tricalcium phosphate (TCP) (for which Ca/P 5 1.5), and they contain much lower amounts of hydrogen phosphate ions than OCP [31]. Spray drying of precipitates formed in situ with a two-nozzle system, has led to an amorphous phase with traces of dicalcium phosphate anhydrous (DCPA). As discussed later in this chapter, the use of water
ethanol solutions allows the preparation of an am-OCP [5].

8.2.3 Apatitic octacalcium phosphate ap-OCP is often considered as the lower limit of calcium-deficient apatites (CDA), based on compilations of various chemical compositions of carbonate-free CDA

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obtained in a wide range of synthesis conditions [1]. These are usually represented by the following chemical formula: Ca102x ðPO4 Þ62x ðHPO4 Þx ðOHÞ22x with 0 # x # 2

(8.5)

22 showing that the replacement of one trivalent PO32 4 ion by a bivalent HPO4 ion in the stoichiometric HA (for x 5 0), inducing the loss of a negative charge, is most generally associated with the creation of one Ca21 vacancy and another one in an OH2 site to restore the charge balance. According to this conception, the limit number of calcium vacancies is thus reached when x 5 2 (empty OH2 sites), corresponding to an apatite phase with the composition of OCP (8.1). In these CDA, the calcium deficiency (x) is related to the atomic Ca/P ratio often used to characterize phosphate-CDA: x 5 10-6(Ca/P). The CDA composition represented by the formula (8.5) and that of OCP
HA interlayers (8.4) are equivalent (with x 5 2y). CDAs with the OCP composition are however difficult to obtain: generally, the lowest Ca/P values that were reached at physiological pH are close to 1.38 [10], and the samples obtained evolve rather rapidly toward more stoichiometric apatites in solution. Although it is possible to reach lower Ca/P ratios at pH under the 7.4 physiological value, the synthesis becomes erratic and the formation of dicalcium phosphate dihydrate (DCPD) or DCPA and t-OCP impurities is frequent. In this case as well the use of hydroalcoholic media can facilitate the formation of an ap-OCP.

8.2.4 What about carbonated octacalcium phosphate? The similarity between type B
carbonated and HPO4-containing apatites has been established several decades ago [2,3], and these can be represented by a unique, although simplified, chemical formula derived from (8.5): Ca102x ðPO4 Þ62x ðHPO4 =CO3 Þx ðOHÞ22x with 0 # x # 2

(8.6)

22 HPO22 4 and CO3 , two divalent anions, play the same role and can replace each other with identical electrical charge compensation mechanism (see Section 8.2.3). Such carbonate species are called type B carbonates to distinguish them from type and a A carbonates corresponding to the replacement of 2OH2 by one CO22 3 vacancy in the monovalent anionic sites of HA [1,32]. In these mixed phosphatecarbonate CDA, the calcium deficiency (x) is related to the atomic Ca/(P 1 C) ratio, where C represents the carbonate content: x 5 10-6(Ca/(P 1 C)). The fully carbonated type B carbonate apatite would thus correspond to

Ca8 ðPO4 Þ4 ðCO3 Þ2

(8.7)

which, according to its chemical composition, can be considered as an octacalcium bis(carbonate) tetrakis(phosphate).

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8.2.4.1 Triclinic octacalcium phosphate carbonate The chemical composition of the octacalcium bis(carbonate) tetrakis(phosphate), named octacalcium phosphate carbonate (OCPC), has been introduced by Hayek [7]. However, this OCPC was more likely a paracrystalline form of apatite contain22 ing both HPO22 4 and CO3 and not a carbonate-containing t-OCP structure [33]. More recently, Shen et al. [8] claimed the successful synthesis of a triclinic octacalcium phosphate carbonate (t-OCPC); however, no quantitative data regarding the chemical composition of this compound have been given, and the presence of HPO22 4 species on the FTIR spectra demonstrates that the fully carbonated OCPC has not been obtained. In addition, the carbonate ions in these syntheses are reported to occupy type B carbonate position found in carbonated apatite, which raises some questions, considering the possible existence of interlayered domains including carbonate apatite and t-OCP. To this day, fully carbonated t-OCPC has not been obtained, but these first results deserve more thorough investigations.

8.2.4.2 Other phases with the octacalcium phosphate carbonate chemical compositions Several attempts have been made to prepare OCPC chemical compositions in the form of amorphous or apatite solids.

8.2.4.3 Amorphous octacalcium phosphate carbonate The amorphous OCPC (am-OCPC) can be easily obtained by direct co-precipitation; pouring a phosphate and carbonate solution (with a C/P atomic ratio of 0.5) into an alkaline calcium solution (with an excess of Ca21). It shall be noticed, however, that mixed amorphous calcium phosphate and carbonate (ACPC) can be prepared in a very wide range of compositions by changing the C/P ratio of the phosphate
carbonate solution, and that OCPC is simply one of those. Interestingly, the OCPC Ca/P ratio corresponds to that of a high-temperature phase, tetracalcium phosphate (TTCP): Ca4(PO4)2O and the am-OCPC could thus be an interesting intermediate to prepare TTCP by a thermal decomposition of the carbonate anion leading to oxide ions [34].

8.2.4.4 Apatitic octacalcium phosphate carbonate Apatitic octacalcium phosphate carbonate compounds, sometimes also involving sodium incorporation, have been prepared by various ways [1]. They also appear, however, as one composition in a range of solid solutions, with no specific properties compared to the rest of the series. It shall be noticed that the composition of carbonated apatites can go beyond the limit of two carbonates per four phosphates corresponding to formula (8.6) [35].

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8.2.4.5 Octacalcium phosphate carbonate formation in surface reactions Although, to our knowledge, t-OCPC has not yet been prepared, recent investigations of the reactivity of nanocrystalline t-OCP (nt-OCP) reveal interesting information regarding the interaction of carbonate ions with t-OCP. These results were obtained by ion-exchange experiments on nt-OCP at room temperature. These experiments affect only the surface layers of the crystals and, although they do occur on regular t-OCP crystals, their effects remain difficult to observe and to be studied in relation with the very low specific surface area of regular t-OCP crystals and the very limited surfaceexchange reactions. This relative inertia of OCP in carbonate solutions, at room temperature, was already noticed by Chickerur et al. [33]. These surface exchanges are only clearly observable on nt-OCP with specific surface area above 100 m2/g. Reversible ion-exchange reactions can occur in solution at room temperature between ions in solution and those of the solid sample in suspension. Cationic and anionic exchanges have been performed and described on BNA [36
38]. They appear as very fast reactions inducing alterations of the ion environments in the surface layer but no visible change in the crystalline domains. All these reactions are reversible, and the initial ionic environments of the original sample can be restored after a reverse exchange. The exchange of surface HPO22 ions by carbonate ions performed on well4 crystallized t-OCP results only in a very low carbonate uptake, barely observable by FTIR spectroscopy. The preparation of nt-OCP (see Section 8.2.5) offers however samples with high specific surface areas (above 100 m2/g) allowing a convenient follow-up 22 of the CO22 3 ! HPO4 ion-exchange reactions by FTIR. The data obtained (Fig. 8.2) show a clear carbonate uptake with lines at 1420, 1480 cm21 and a weaker, relatively narrow one at 870 cm21, which seems mainly correlated with the specific displacement of one of the HPO22 4 ions of the t-OCP structure, HPO4(6) (see Figure 8.1). The FTIR spectra show that the lines assigned to this particular HPO22 4 ion are reduced in the exchanged sample. The XRD pattern of the CO3 exchanged nt-OCP (not shown) exhibits the characteristic peaks of the initial nt-OCP sample without major alterations, indicating that the exchange reaction remained a surface phenomenon. The reverseexchange reaction (spectrum 3 in Fig. 8.3) restores the original nt-OCP FTIR spectrum with the initial intensity of the HPO4(6) lines and with faint residual carbonate species. These data suggest the reversible replacement of HPO4(6) of the surface-hydrated layer of t-OCP by CO22 3 ions from the solution. Such insertions in the hydrated layer of t-OCP have been suggested by Chickerur et al. as possibilities [33]. The carbonate-ion FTIR vibrational lines in the carbonate-exchanged t-OCP reveals spectral characteristics analogous to those of the nonapatitic labile carbonate species present in bone mineral apatites and synthetic analogues.

8.2.5 Biological apatites and octacalcium phosphate Chemical composition and crystal habits of bone mineral crystals present some analogies with OCP; however, despite a few isolated reports [41], the t-OCP structure has never been reproducibly identified in bone samples of mammals.

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Figure 8.2 FTIR spectra in the ν4
ν1 PO4 domain illustrating the carbonate
hydrogen phosphate exchanges on nt-OCP samples: (A) initial nt-OCP sample; (B) carbonateexchanged nt-OCP in a 1 M NH4HCO3 solution during 10 min at room temperature (pH 7.75); (C) reverse exchange experiment on the carbonate-exchanged sample 2, in a 1 M phosphate solution in the same conditions. The carbonate uptake on the nt-OCP sample appears related to a decrease of the lines assigned to HPO4(6) (see Figure 8.1) of the t-OCP structure. This substitution is reversible as seen on spectrum 3 except for a small residual amount of carbonate. FTIR, Fourier-transform infrared spectroscopy; nt-OCP, nanocrystalline t-OCP; t-OCP, triclinic OCP.

8.2.5.1 Relationships between bone mineral and octacalcium phosphate Chemical composition The composition of bone mineral of several mammal species can be represented by the following chemical formula proposed by Legros et al. [42]: Ca8:3 ðPO4 Þ4:3 ðHPO4 =CO3 Þ1:7 ðOHÞ0:3

(8.8)

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Figure 8.3 TEM images of isolated bone crystals and of synthetic BNA. (A) Bovine bone treated with NaOCl to remove the organic matrix. (B) Carbonated BNA aged for 1 month (5.6% CO22 3 ) [39]. In both pictures, flake-like crystals with irregular shapes are shown (white arrow); in the bone, it has been shown that they lie on a 1 0 0 face. Although vertical crystals (dashed white arrow) allow the estimation of thickness, it has been shown that these crystals have the tendency to pile up in bone. BNA, Biomimetic nanocrystalline apatite; TEM, transmission electron microscopy. Source: (A) Reproduced with permission from Schwarcz HP, Mcnally EA, Botton GA. Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals. J Struct Biol 2014;188:240
48 [40].

was derived from (8.6) with x 5 1.7, a value very close to the maximum one. In this 22 formula, as already noticed (8.2.4), the HPO22 4 and CO3 bivalent ions play a similar role, and, on aging, in most vertebrate species studied so far, the carbonate con2 tent increases at the expense of the HPO22 4 content [42]. The OH content in bone is always very low as reported in several studies [43
45], and this ion is barely observed by vibrational spectroscopies [46]. In addition, the identification, in bones of low amounts of carbonate ions replacing OH2 (type A carbonate) [47], contributes to the reduction of even more the amount of hydroxide ions, which reaches at most 3%
4% of that found in stoichiometric HA, according to Raman and FTIR evaluations [46], and about 20% according to solid-state NMR determinations [44,48]. Such discrepancies are not uncommon; they can be related, in part, to the reactivity and relative instability of the bone mineral and possibly to technical bias. This bone mineral formula could correspond to partly carbonated and slightly hydrolyzed ap-OCP. Some misunderstandings are common regarding bone mineral composition. It is generally believed, based on the increase of the Ca/P ratio with age, that bone mineral composition evolves toward that of a stoichiometric HA on aging. In fact, if the Ca/P ratio is an important marker of calcium deficiency of purely calcium phosphate HAs, it has strictly no meaning in carbonated apatites such as those of bone mineral. If we admit that most carbonate ions in bone mineral

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replace phosphate groups in the apatite structure (type B substitution) and that no significant amount of vacancies exist on these sites, the calcium deficiency in type B
carbonated apatites is measured by the value of the Ca/(P 1 C) ratio, which remains almost unchanged during aging and is very close to that of an apatitic, partly carbonated OCP. It shall be remembered, however, that the bone composition is rather heterogeneous at the tissue level due to remodeling, growth, and repair, but also at the bone crystal level due to the existence of nonapatitic environments of mineral ions [47,49,50] detected by spectroscopic methods and assigned to ions in a hydrated layer at the surface of the tiny, nanometric bone crystals [51]. Thus the chemical composition reported earlier corresponds to median values and does not take into account subtle variations at micrometric or nanometric scales. This point will be developed in the following Section 8.2.5 relative to biomimetic apatites.

Crystal habit Bone mineral is constituted by plate-like crystals with irregular shapes [39,52] (Fig. 8.3) of nanometric dimensions about 50 nm long, 25 nm wide, and 10 nm thick [53]. XRD patterns reveal that the plate-like crystals are elongated along the c-axis of the hexagonal apatite structure and thus present along the two orthogonal, in principle equivalent, a and b directions, very different dimensions, which is rather unusual in geological or synthetic apatites. The XRD patterns only allow a median value for the thickness
width of the crystals to be evaluated. Although dimensions of isolated bone crystals have been obtained by transmission electron microscopy (TEM) or atomic force microscopy (AFM), the problem of possible alterations of these very fragile crystals in the different isolation procedures, in vacuum or under electron beams, has not yet been solved and the determination of their thickness remains delicate. AFM, especially, has given very low values (less than 2 nm), equivalent to a unit cell of HA (or OCP) for calf bones [54]. The very unusual crystal habit of bone mineral has been suggested to originate from the hydrolysis of a nanocrystalline precursor [55,56], possibly t-OCP into apatite, considering that such phase conversion, often described as topotactic, can preserve the original crystal morphology.

8.2.5.2 Relationship between tooth enamel and octacalcium phosphate In accordance with the biological adaptation of the tissue to their function [57], bone and tooth-enamel crystals present very different characteristics. Unlike bone, the enamel tissue cannot be renewed, and it does not participate in homeostasis. It has to resist, for a lifetime, strong compressive strains, mechanical abrasion, and chemical dissolution related to the acidic fluids of extracellular matrix of oral bacteria, food, and beverage. To fulfill this objective the composition of enamel apatite crystals appears closer to that of the less soluble stoichiometric HA than to bone mineral; hydroxide ions are clearly detected and the amount of calcium vacancies 22 22 and of bivalent ions substituted for PO32 4 , essentially HPO4 and CO3 , are much less important than in bone apatite; this increases the crystals’ cohesion and lowers

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their solubility. The effect of Ca21 and OH2 vacancies in apatites has been quantified recently from drop-solution calorimetry measurements, which clearly pointed to the role of nonstoichiometry on apatite stability [58]. A particularity of enamel crystals is to show a “central dark line” (CDL) observable by electron microscopy [59], which is also evidenced in synthetic apatites [60] and has been suggested to be a remnant of a t-OCP precursor [61] corresponding to the first-formed thin, elongated ribbon-like mineral crystals appearing at early stages of enamel formation [62]. However, this assignment has been questioned as the CDL exhibits a calcium concentration that is higher than expected for t-OCP [63]. High-resolution TEM studies at the dentine
enamel junction have also revealed the possible existence of OCP as a transient phase [64].

8.2.5.3 Hypothesis on the formation of bone crystals Several models have been proposed for the formation of bone mineral mainly based on the study of synthetic calcium phosphate formation and evolution. Among the proposed precursors, ACP, t-OCP, and DCPD have received a special attention [46]. The first suggestion of the involvement of t-OCP resulted from its structural affiliation with HA [11], and its ability to be converted into carbonated apatite in aqueous media [33]. A transient role of t-OCP was also reported based on the studies of the hydrolysis of ACP. The t-OCP phase was not formally identified but indirectly deduced from the behavior of supersaturated aqueous solution [65,66]. More recently, t-OCP was identified by XRD as a transient phase in the chain of reactions occurring in simulated body fluid (SBF)-like supersaturated solutions and leading to apatite formation [67]. A model, largely diffused, has been proposed by Gower [68] regarding the order of potent calcium carbonate phases formed during biomineralization processes in invertebrates. This model is based on the Ostwald step rule or law of successive reactions, considering that in a crystallization process the state that is generally observed is not the most stable one, but the less stable state that is closest in terms of Gibbs free energy change to the original state. The application of this empirical law to calcium phosphate biomineralization has been proposed [57,69,70] in which t-OCP appears as a transient phase in apatite formation. However, the application of the Gower’s model to calcium phosphate system does not take into account the large range of nonstoichiometry and solid solutions in biomineral apatites compared to biomineral calcium carbonates and the correlated wide ranges of free energies of formation [58]. We will show in the following sections that, in fact, t-OCP may form by hydrolysis of a nanocrystalline, sodium-containing CDA.

8.2.6 Biomimetic nanocrystalline apatites and octacalcium phosphate The adjective “biomimetic,” regarding bone mineral, is rather imprecise. Most often, this term designates apatites obtained in conditions supposed to be those of mineral formation in the body, especially in simulated body fluids (SBF), largely used to evaluate the osteogenic potential of biomaterials for bone substitution [71]. For us, “BNAs” refer to apatites with physical
chemical properties similar to those

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of bone mineral (crystal habit and dimensions, chemical composition, regarding the main constituents and characterized by a surface-hydrated layer with mineral ions in labile nonapatitic environments). In addition, these apatites have been shown to behave like bone minerals on aging [72].

8.2.6.1 Physical
chemical characteristics of biomimetic nanocrystalline apatites The crystals of BNA appear close to isolated bone crystals (Fig. 8.3); they do show an elongated plate-like morphology with XRD-measured apparent dimensions close to those of bone nanocrystals. Their composition is also similar to that of bone mineral as shown in Table 8.1 with a Ca/(P 1 C) ratio close to 1.3
1.4 and the amount Table 8.1 Evolution of the composition of bone samples and of maturated, carbonated biomimetic nanocrystalline apatite (BNA). Maturation time or animal’s age

Atomic ratios Ca/P ( 6 0.02)

Ca/(P 1 C) ( 6 0.02)

C/P ( 6 0.01)

1.44 1.51 1.51 1.54 1.62 1.62

1.41 1.42 1.40 1.39 1.40 1.38

0.03 0.06 0.09 0.15 0.16 0.20

1.57 1.62 1.65 1.69 1.77

1.33 1.31 1.35 1.37 1.39

0.18 0.23 0.22 0.24 0.27

1.51 1.60 1.65

1.38 1.39 1.38

0.10 0.15 0.20

1.65 1.69

1.40 1.39

0.18 0.21

Carbonated BNA 0 6h 24 h 3 Days 10 Days 4 Months

Chicken bone Embryonic (14 days) 4 Weeks 14 Weeks 32 Weeks 50 Weeks

Rat bone At birth 30 Days 1 Year

Cow bone 2 Months 7 Years

Source: The values of the rat and cow bones were reported from Legros R, Balmain N, Bonel G. Age-related changes in mineral of rat and bovine cortical bone. Calcif Tissue Int 1987;41:137
44.

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of carbonate, which increases on aging in solution after precipitation such as during bone aging [72]. In fact, a few minutes after precipitation, even in the presence of carbonate ions, obtained BNA samples show a large amount of HPO22 4 ions in nonapatitic environments (hydrated layer) and a very low amount of carbonate. However, we never could structurally identify a t-OCP phase in these conditions.

8.2.6.2 The nonapatitic environments and the hydrated layer Mineral ions in the nonapatitic environments of the hydrated surface layer have been evidenced and confirmed using spectroscopic methods in bone as well as in 22 BNA. The most documented data concern HPO22 4 and CO3 , the two main mineral ions involved in the hydrated layer [47,73,74]. They have been shown to be close to water molecules by solid-state NMR [9,50,74,75]. Regarding cations of the hydrated layer, only a few data are available [36,38]. The composition of the hydrated layer is difficult to determine at this stage. Spectroscopic methods associated with ion-exchange experiments have established that this layer contains biva22 lent ions (mainly Ca21 ; Mg21 ; CO22 in bone) in equilibrium with the 3 ; HPO4 surrounding solution and participating in the growth of apatite domains (see Section 8.2.5.4). The role of water in this layer appears crucial, and it has been recently discussed [76]. Paradoxically, most of the studies concerning the hydrated layer have been realized on dry, often lyophilized samples; however, as it could have been guessed, drying considerably alters the environment of these ions.

8.2.6.3 The hydrated layer in situ and triclinic octacalcium phosphate Curiously, when the first transmission FTIR spectrum of humid gel-like BNA was recorded, just after precipitation and washing, thinner and better defined lines than those of the lyophilized sample were observed [77] (Fig. 8.4). However, with time, as the sample was drying in the spectrometer, a considerable broadening occurred and spectra with broad bands analogous to that of the dry lyophilized sample were eventually recorded. Similar data were observed by solid-state NMR [77] (see Fig. 8.9), and these experiments revealed that drying strongly alters these nanocrystalline samples and blurs the data. This evolution on drying appears only partly reversible. This is probably one of the reasons for the divergences reported regarding the structure of the hydrated layer considered as amorphous when analyzed in dry samples but which appears structured when analyzed wet. These structures are so fragile (the amorphization on drying takes only a few minutes) that the physical
chemical characterizations of such nanocrystalline calcium phosphates should be made, whenever possible, in their wet original state for synthetic compounds as well as for biological samples despite additional experimental difficulties. Other causes for amorphization of the hydrated layer are the maturation process and alteration of its structure and composition by foreign ions, such as 21 CO22 3 , Mg , or molecular ions such as citrates.

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Figure 8.4 FTIR spectra (ν1-ν4 PO4 domain) illustrating the effect of drying on a wet noncarbonated BNA. Series of absorbance spectra obtained on a thin layer of BNA gel suspension deposited on a polyethylene film during the time of drying (a few minutes) in the spectrometer. The loss of resolution is attributed to the broadening of lines related to the alteration of the structured hydrated layer and its “amorphization” on drying. BNA, Biomimetic nanocrystalline apatite; FTIR, Fourier-transform infrared spectroscopy. Source: Reproduced with permission from Rey C, Combes C, Drouet D, Bertrand G, Soulie´ J. Bioactive calcium phosphates compounds: physical chemistry. In: Ducheyne P, Grainger DW, Healy KE, Hutmacher DW, Kirkpatrick CJ, editors. Comprehensive biomaterials II, vol. 1. Oxford: Elsevier; 2017. p. 244
90.

The data obtained on wet, carbonate-free, freshly precipitated BNA reveal interesting analogies with t-OCP (Fig. 8.5); however, some very specific lines of this compound are missing, such as the very specific HPO4(6) lines of t-OCP at about 1298 and 913 cm21, and that assigned at HPO4(5) at 1207 cm21. Similarly (see also Section 8.4.4), the 31P solid-state NMR spectra of wet samples appear somewhat different from that of t-OCP (see Fig. 8.9). Although distinct lines assigned to 22 PO32 4 and HPO4 were identified [77], their positions are different from those in t-OCP and the chemical shift of the line assigned to HPO22 4 ions in BNA seems closer to that of DCPD [78]. These discrepancies could possibly be due to alterations of these fragile entities in the spectrometer or to real differences in the structures. However, the similarity of FTIR spectra of t-OCP and freshly precipitated wet BNA appears striking. Besides, one should not expect an exact coincidence between the hydrated layer of t-OCP within a tridimensional crystal and that of the nanocrystalline apatite-hydrated layer on a free surface. The reactivity of this hydrated layer is also much higher and diverse than that of the intracrystalline layer of t-OCP. This peculiar feature seems to correspond, however, to a transitory stage

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Figure 8.5 Comparison of wet t-OCP and wet noncarbonated BNA FTIR spectra obtained on diamond ATR. 1—wet noncarbonated BNA; 1 —resolution-enhanced spectrum 1 by selfdeconvolution (Omnic software, Thermos Fisher Scientific 2016); 2—wet t-OCP. The dashed lines illustrate the major differences: bands assigned to HPO22 4 ions in t-OCP at 917, 861, and 1193 cm21 are missing. The band at 1077 cm21 also assigned to HPO22 4 ions is considerably broadened and loses most of its height, such as other very small bands, also attributable HPO22 4 , at 905 and 1297 cm21. ATR, attenuated total reflectance; BNA, Biomimetic nanocrystalline apatite; FTIR, Fourier-transform infrared spectroscopy; t-OCP, triclinic OCP.

and on aging, the analogy with t-OCP is fading out; in addition, if this structured hydrated layer is altered, when carbonate ions or Mg21 ions are incorporated by ion exchange, this phenomenon is reversible and the original features are retrieved by a reverse ion exchange [79]. Recent studies on a fresh sheep’s cortical bone (2-year-old animal) by solid-state NMR have revealed that the HPO22 4 ions were located in the surface-hydrated layer and none of them were detected in the apatite core. The analogy with t-OCP was not observed in this mature, carbonated bone sample [48].

8.2.6.4 Interactions and dynamics within the biomimetic nanocrystalline apatite crystal structure in solution The surface-hydrated layer is a determining feature of bone mineral and BNA. It is involved in fast ion-exchange reactions and participates most probably in

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homeostasis [80]. It is also involved in the adsorption of bioactive molecules, such as bisphosphonates, through an ion exchange process [81
83] and probably many other biomolecules, for example, albumin, but also growth factors such as BMP-2 [84]. These surface reactions can be seen as analogous to those involved in di-carboxylates’ t-OCP, already mentioned (Section 8.2.1), corresponding to the replacement of HPO22 ions of the hydrated layer by the di-carboxylate 4 anions. However, on a surface layer, there are lesser constrains than in an intracrystalline layer. This reactivity of the surface-hydrated layer is mainly related to its ability in hosting and interacting with a large range of mineral ions and active functional groups of various molecules due to its adaptability in a much easier way than solid surfaces with rigid ion positions, which only offer limited epitaxial interactions. Such a versatility also exists in t-OCP where the hydrated layers present in the stacked structure may accept many substituents and adapt itself to them [13,24]. The model of BNA crystal organization and reactivity is schematized in Fig. 8.6, showing a two-interface system. The hydrated surface layer is in constant equilibrium with the surrounding solution. It is subjected to dissolution equilibrium but

Figure 8.6 Representation of the hydrated layer at the surface of bone mineral crystals and synthetic BNA crystals. The arrows show the dynamics of the different domains. The thermodynamic more stable apatite domains develop continuously using some of the ions in the hydrated layer. The hydrated layer is in continuous equilibrium with the surrounding solution and participates directly in the homeostasis. This layer is also involved in the interaction of the crystals with organic molecules of the extracellular fluid such as citrate ions, peptides, drugs, such as bisphosphonates, and functional groups of larger biological molecules (symbolized by Org). The reconstruction of the hydrated layer during circadian variations of calcium, phosphate, and carbonate ions in blood cannot be excluded. BNA, Biomimetic nanocrystalline apatite. Source: Reproduced with permission from Rey C, Combes C, Drouet C, Sfihi H, Barroug A. Physico-chemical properties of nanocrystalline apatites: implications for biominerals and biomaterials. Mater Sci Eng, C 2007;27:198
205 [85].

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also to ion exchanges with cations or anions from the solution, which are able to replace ions of the hydrated layer: this includes cations, such as Mg21, Sr21, Cu21, 22 Zn21, and anions such as CO22 3 and HPO4 [86]. This flexibility of the surfacehydrated layer also allows interactions with molecular charged functional groups, which may displace the relatively mobile, more or less hydrated mineral ions [82]. Another property of the hydrated layer is to offer a lower interfacial energy than nonhydrated mineral surfaces [19,87]. This surface layer could then determine the equilibrium face of the nanocrystal complex and the growth of the underlying apatite domains. The second interface, hydrated layer/apatite core, is also active, although less easy to study. The apatite is the thermodynamic stable phase and its development utilizes the ions present in the hydrated layer. Thus the apatite domains grow at the expense of the hydrated domains. Not all the ions of the hydrated layer can be incorporated in the growing Ca-P apatite crystals; CO22 3 ions, for example, delay the progression of the apatite front, but they are incorporated in the lattice on apatite sites (type A and B carbonates). This is not the case of Mg21 ions, which can be exchanged at more than 80% in coprecipitated Mg-containing BNA samples, and which thus remain in the hydrated layer even in matured BNA [36], despite the possibility of magnesium entering in Ca-P apatites [88]. The existence of solid solutions with Ca-P apatites is a prerequisite for an ion of the hydrated layer to enter the apatite domains; however, the case of Mg21 shows that it is not a sufficient condition. Ions, once they have entered the apatite domains, cannot be exchanged anymore. This is, for example, the case of Sr21, which can be exchanged in large proportions with calcium ions in freshly Sr-loaded hydrated layer but which become less and less exchangeable as the samples are aging [36].

8.3

Synthesis routes of octacalcium phosphates

Several reliable preparation methods of OCP and related compounds have been published; however, new syntheses are still being studied and unveiled. The main difficulties in t-OCP synthesis are the spurious formation of DCPD (or DCPA) and calcium-deficient apatite (CDA). Among the important parameters to control are the reaction solution pH and temperature. Several synthesis routes have been explored, and the hydrolysis of precursors of OCP is one of the oldest methods that is still frequently used [89].

8.3.1 Hydrolysis Several hydrolysis methods have been proposed essentially for the synthesis of t-OCP. One of the most popular is the hydrolysis of DCPD [89], but the hydrolysis of α-TCP is also possible [90] and has been used to associate t-OCP with organic acids [91]. More recently, the hydrolysis of precipitated nanocrystalline, sodiumcontaining CDA into t-OCP has been shown to occur.

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8.3.1.1 Hydrolysis of dicalcium phosphate dihydrate The hydrolysis of DCPD into t-OCP, schematized by the following equation 8 CaHPO4 ; 2H2 O ! Ca8 ðHPO4 Þ2 ðPO4 Þ4 ; 5H2 O 1 2 H3 PO4 1 11 H2 O

(8.9)

releases a considerable amount of protons (artificially represented above as phosphoric acid), which needs to be neutralized. This was obtained in the original synthesis [89] by buffering the hydrolysis media with sodium acetate (0.5 M). The solution was renewed when its pH reached 6.1. One risk related to this buffer is sodium incorporation in OCP [92]; in addition, we could detect traces of acetate in t-OCP prepared in the presence of this anion. Other buffer solutions can be chosen such as phosphate buffer. Hydrolysis studies of DCPD performed in mixed Na2HPO4:(NH4)2HPO4 solutions have established the incorporation of Na in t-OCP and more interestingly the fact that this ion favors t-OCP formation [92]. An alternative to buffer solutions is to use a pH-stat or a constant composition crystal-growth method as presented in Section 8.3.3. A second parameter of importance is the hydrolysis temperature; historically, the temperature was 40 C. The tendency, in order to improve the rate of hydrolysis and the crystallinity of the obtained t-OCP, is to shift to a higher temperature of 60 C [92,93] or even 70 C
80 C [94]. The hydrolysis time depends on the starting DCPD features (crystal size especially); grinding can increase the hydrolysis rate and facilitates the reaction. The advancement of the reaction has to be controlled in order to stop the reaction at the end of total DCPD hydrolysis and before the beginning of the hydrolysis of the t-OCP formed into apatite. Sometimes, these reactions can be superimposed and no pure t-OCP phase can be obtained.

8.3.1.2 Hydrolysis of α-tricalcium phosphate This original method was promoted by Monma [90]. The reaction 3 Ca3 ðPO4 Þ2 1 7 H2 O ! Ca8 ðHPO4 Þ2 ðPO4 Þ4 ; 5H2 O 1 CaðOHÞ2

(8.10)

releases OH2 ions [represented by Ca(OH)2] and, here as well, buffering is advised at a slightly acidic pH. The recommendations for the hydrolysis of 1 g of α-TCP in 50 mL are [90] 70 C with pH 4.5
5 or 60 C at pH 5. The hydrolysis is completed in 3 h for α-TCP powder-ground and sieved through a 200 in. mesh.

8.3.1.3 Hydrolysis of CDA This reaction seems rather improbable as thermodynamically the reverse reaction is supposed to occur. It allows the preparation of nt-OCP from a first instable calcium-deficient nanocrystalline apatite. Three steps are distinguished. The first one is the precipitation of ACP obtained by pouring, at room temperature, a CaCl2 solution (CaCl2, 2H2O 21.8 g in 250 mL) in a phosphate solution (Na2HPO4, 43.2 g in 750 mL). The pH of the mixture decreases rapidly, due to the precipitation, and then more slowly to 6.5 in a few minutes. After about 10 min,

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a nanocrystalline Na-containing CDA has crystallized. At 6 h after precipitation, t-OCP can be detected. The crystallinity of this phase increases progressively with the aging of the suspension. In the long run, this nanocrystalline OCP progressively hydrolyzes into CDA. The presence of Na is crucial in this reaction probably because of the suggested role of sodium in OCP formation [92]. The second point is that the first crystallized phase in this system, sodium-doped CDA, shows some instability related in part to the sharp pH decrease due to the precipitation of the ACP. The details of the growth of t-OCP crystals at the expense of CDA and the origin of their limited crystal growth remain however unknown.

8.3.2 Precipitations 8.3.2.1 Aqueous solutions Triclinic octacalcium phosphate The direct precipitation of t-OCP, or other OCP phases in aqueous solutions, raises some difficulties. The first one is that there is always a lag time between the formation of the first precipitate and the beginning of solution addition, which can strongly alter the conditions of precipitation and favor the formation of stable phases at given local pH and concentrations (DCPA/DCPD or CDA) instead of the metastable t-OCP. The second point is that a precipitation reaction from supersaturated solutions, in the pH domain of formation of t-OCP (about 4
7), always induces a release of protons, which has to be managed. A last point is that the pH affording the formation of t-OCP is around neutrality, at 7.2, and in a very narrow range ( 6 0.1) at room temperature but acidic and tolerant to larger pH variations at higher temperature. The first important direct synthesis of t-OCP was reported by LeGeros [95]. It was obtained by double decomposition between equimolar (0.02 or 0.04 M) calcium acetate solutions and sodium hydrogen phosphate solutions (250 mL for both of them) at different sets of temperatures and pH (60 C and pH 5
6; 70 C and pH 4.5; 80 C and pH 4). The partial removal of protons from the precipitation reaction is obtained by the buffering effect of the acetate and the excess of phosphate in solution. The use of relatively high temperatures allows the formation of wellcrystallized t-OCP, containing possibly traces of sodium and acetate. Several variants of this reaction have been proposed [96
100].

Biomimetic nanocrystalline apatites Carbonated or noncarbonated BNA are obtained by double decomposition between a solution of a soluble calcium salt (generally calcium nitrate or chloride) and a solution of a soluble phosphate (and carbonate) salt(s), generally di-ammonium hydrogen phosphate or di-sodium hydrogen phosphate (Table 8.2). The calcium solution is rapidly poured into the phosphate solution. After a chosen maturation time, the precipitate is filtered on a Buchner funnel, washed, and lyophilized. The impact of the synthesis parameters on the physical
chemical characteristics of the obtained nanocrystalline apatites have been studied, pointing in particular to the role of maturation time and conditions, temperature, and pH [101].

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Table 8.2 Composition of solutions for biomimetic nanocrystalline apatite (BNA) synthesis. Preferred conditions in bold. Calcium solution (250 mL)

Noncarbonated BNA Carbonated BNA

Phosphate solution (500 mL) Phosphate and carbonate solution (500 mL)

Calcium nitrate (chloride or acetate) 0.3 M Magnesium, strontium, or other cations may be added to this solution after adequate adjustments of the calcium content Di-ammonium (or di-sodium) phosphate 0.6 M Di-ammonium (or di-sodium) phosphate 0.45
0.6 M Ammonium or sodium bicarbonate 0.36
0.70 M

The pH adjustment to 7.4 is made just after precipitation (when needed). Maturation (aging) in the mother solution (10 min to several months in sealed flasks).

8.3.2.2 Synthesis in water
ethanol solutions Amorphous octacalcium phosphate The precipitation of phases with OCP composition even more reactive and metastable than t-OCP, such as am-OCP and ap-OCP, necessitates the use of water
ethanol solutions. These solutions, exhibiting a lower dielectric constant than pure water, are considered to favor the less dissociated species and to slow down crystallization reactions [102]. The am-OCP can be obtained in ethanol
water solutions by a classical doubledecomposition reaction between a calcium nitrate solution [30 mmol Ca(NO3)2, 4H2O dissolved in a solution made of equal volumes (100 mL) of water and ethanol; solution A] and a di-ammonium hydrogen phosphate solution [30 mmol (NH4)2HPO4 dissolved in a solution containing 250 mL of water, 45 mL of an 11 M aqueous ammonia solution and 295 mL of ethanol; solution B] [5]. Both solutions were heated to 37 C and solution A was rapidly mixed with solution B; the precipitate formed was immediately filtered, washed with a solution made of water: 210 mL and ethanol: 210 mL; and freeze-dried [5].

Apatitic octacalcium phosphate ap-OCP, although predicted theoretically, is not so easy to obtain in aqueous media. Very close compositions can be reached, with Ca/P ratios between 1.38 and 1.40 corresponding to BNA, but these are not really identical to an OCP composition. The gel-like amorphous precipitate resulting from the am-OCP preparation in water
ethanol solutions depicted earlier, when dried at 80 C, crystallizes as an apatite with the composition Ca8 ðPO4 Þ3:5 ðHPO4 Þ2:5 ðOHÞ0:5

(8.11)

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197

2 containing an excess of HPO22 4 and OH ions compared to the ideal chemical formula (8.1) but still with a Ca/P ratio of 1.33 [103]. This compound has been the object of several studies due to its good compaction ability and its possible use as drug-delivery substrate [104].

8.3.3 Constant composition crystal growth Another interesting synthesis route of t-OCP is that proposed by Nancollas’ group using the elaborated technique of constant composition crystal growth, which involves nucleating seeds and where the characteristics of the precipitation solution, including pH, supersaturation, ionic strength, and all ions’ concentrations, are maintained at a constant level [105]. This technique revealed, for example, that the rate of crystal growth of t-OCP at constant supersaturation was considerably larger than that of HA in the same conditions (about 3000 times) [106]. It also showed that tOCP could nucleate and grow on HA and vice versa [17]. Similarly, OCP can grow on TiO2 anatase [107] or calcium carbonates such as calcite and aragonite [108]. A development of this technique, the dual constant-composition method, allows mixed, dissolution, and crystal growth reactions to be followed, for example, DCPD dissolution and t-OCP crystal growth [109] and also helps analyze the inference of additives such as Mg21 and Zn21. The study of the effect of biological molecules, such as bovine serum albumin (BSA), on t-OCP nucleation and growth on a collagen matrix has also been evaluated [110]. An interesting phenomenon is that, in the lower range of BSA concentration studied, at a given supersaturation, a considerable increase of the growth rate of t-OCP has been observed, explained by the increase of nucleation rate of t-OCP related to BSA adsorption followed by a burst growth of these multiple nuclei (Fig. 8.7). This phenomenon could be involved in the seeding of organic matrix in biomineralization.

8.4

Evolution of the different octacalcium phosphate phases and related compounds in aqueous media

All OCPs and related compounds are metastable and they evolve more or less rapidly in aqueous media.

8.4.1 Triclinic octacalcium phosphate t-OCP is converted into calcium-deficient apatite as shown in several works [17,111
115]. This phenomenon is associated with an increase of the Ca/P ratio of the solid phase and a correlated acidification and lower Ca/P ratio in the aqueous phase. In the presence of carbonate the conversion of well-crystallized OCP into type B carbonate apatite is very slow at room temperature, and it has been first studied at elevated temperatures (85 C
95 C) and at 37 C [33] and in different

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Figure 8.7 Illustration of the effect of adsorption of BSA on OCP crystal growth. By decreasing the interfacial energy, the adsorbed substance, here BSA, facilitates the crystals’ nuclei formation at a critical radii inferior to that regularly observed for a solution with the same supersaturation but without adsorbate molecules. This surface reaction inhibits, partly, the growth of these nuclei; a multiplication of nuclei during a latency period is thus observed, which progressively depletes the solution in adsorbate; when the conditions for an efficient coverage of the surface by adsorbate molecules are no more satisfied, a burst crystal growth occurs with a crystal growth rate much higher than that observed in the regular adsorbent-free solution. BSA, Bovine serum albumin; OCP, octacalcium phosphate. Source: Published in Combes C, Cazalbou S, Rey C. Apatite biominerals. Minerals 2016;6:1
25.

conditions [116]. In a wet atmosphere (saturated water vapor) the t-OCP can decompose into DCPA and apatite at 40 C and above [104].

8.4.2 Amorphous octacalcium phosphate Just after precipitation and washing, at room temperature, when in gel-like form, am-OCP is very rapidly converted (in about 1 h) into CDA. This reaction occurs even on lyophilized am-OCP in an atmosphere saturated with water vapor, with a transitory formation of t-OCP [5]. In these conditions the formation of the CDA is associated with that of DCPD and then of the more stable DCPA.

8.4.3 Apatitic octacalcium phosphate The ap-OCP hydrolyzes in aqueous solutions toward a more stoichiometric apatite associated with an acidification of the media. This reaction is faster when the temperature increases. In wet atmosphere, this evolution leads to decomposition with the formation of an hydroxylated apatite closer to stoichiometry and DCPD/DCPA depending on the temperature and time of hydrolysis [104].

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8.4.4 Biomimetic nanocrystalline apatites Two different evolution processes have been found in BNA, without carbonate and with carbonate.

8.4.4.1 Evolution in carbonate-free solutions In the absence of carbonate ions, in phosphate-buffered solutions, at room temperature, the Ca/P ratio of BNA samples increases progressively [10] (Table 8.3) toward that of stoichiometric apatite but never reaches this supposed end term even after several months of maturation. This evolution corresponds to the formation of less calcium-deficient apatites, and FTIR spectra attest an increase of the OH2 content and a decrease in the amount of HPO22 4 ions with time in agreement with the chemical analysis. These evolutions are also associated with a decrease of ΔGf (more and more negative) with the maturation time [58]. Simultaneously, an increase of the crystallinity is observed. These changes appear rather fast at the beginning of the maturation process and slow down with time. Although HPO22 4 can be detected in the apatite domains, the labile HPO22 environments subsist even after long-term 4 maturation—what might be considered as a preservation of the hydrated layer for maintaining a minimal interfacial energy. This maturation process is associated with a release of protons in the solution, which, when there is no, or only a weak, buffering (such as in cell culture media), may lead to net acidification. When only a Table 8.3 Comparison of the evolution of noncarbonated biomimetic nanocrystalline apatite (BNA) and forming tooth enamel. Maturation time or tissue-formation stage

Atomic ratios Ca/P ( 6 0.02)

Ca/(P 1 C) ( 6 0.02)

C/P ( 6 0.01)

1.38 1.39 1.45 1.47 1.49 1.50

1.38 1.39 1.45 1.47 1.49 1.50









1.40 1.52 1.54 1.57 1.59

1.21 1.33 1.35 1.40 1.47

0.16 0.14 0.14 0.12 0.08

Noncarbonated BNA 0 6h 24 h 3 Days 9 Days 1 Month

Pig enamel Outer layer (first formed) Early secretory Late secretory Early maturing Mature

Source: The data on tooth enamel were reported from Aoba T. Recent observations on enamel crystal formation during mammalian amelogenesis. Anat Rec 1996;245:208
18.

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small amount of water is present (wet samples or gels), this process can even lead to a very acidic medium inducing the formation of DCPD or DCPA. Such alterations could be at the origin of the observation of these phases in bone, which is sparsely reported [41,78].

8.4.4.2 Evolution in carbonate-containing solutions In carbonate and phosphate-buffered solutions the evolution is shown in Table 8.1. During maturation, the carbonate content increases and the HPO22 content 4 decreases, but the calcium vacancies’ content, as determined by the Ca/(P 1 C) ratio, remains high and practically unchanged. In this case as well the crystallinity of the samples improves, mainly due to a reduction of residual strains in the apatite lattice. The labile carbonate and HPO22 4 environments subsist, but these ions are also incorporated in the apatite lattice, such as carbonate ions, essentially as type B carbonate. No apatitic OH2 incorporation is detected in this maturation process. 22 A difference appears here between the roles of HPO22 4 and CO3 in BNA. In the presence of carbonate the formation of a CDA can occur without change in the global composition, and the incorporation of carbonate allows the formation of a relatively 2 stable calcium-deficient apatite incorporating also HPO22 4 but without OH . If we consider t-OCP as a surface model of BNA (Fig. 8.8) with its HA-like layer [Ca6(PO4)4]

Figure 8.8 Schematization of the differences of behaviors of BNA between maturation processes in the presence and in the absence of carbonate ions in solution using a model of t-OCP-like hydrated layer at the interface between the apatite domains and the hydrated layer. In the presence of carbonate the development of the apatite domains can occur without change in the local chemical composition, except for the carbonate content determined by a surface equilibrium with the solution. In the absence of carbonate, additional ions’ incorporation and elimination are activated to allow the growth of the apatite domains. BNA, Biomimetic nanocrystalline apatite; OCP, octacalcium phosphate.

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22 and hydrated layer [Ca2(CO3/HPO4)2] containing bivalent CO22 3 and HPO4 ions, the growth of apatite domains does not need any additive ions, and the rearrangement of mineral ions from the surface and the hydrated layer and the evacuation of some of the water molecules of the hydrated layer suffice theoretically to explain the evolution. This is not the case in the absence of carbonate, when only HPO22 4 ions are available as divalent ions; the unstable ap-OCP-like domains cannot form. Instead, it appears that OH2 ions are needed for the building of the apatite domains in conjunction with additional calcium ions and a deprotonation of HPO22 4 ions. These two processes are associated with a decrease of the amount of nonapatitic mineral-ion environments partly explained by an increase in the apatite domain’s average dimensions. The second maturation process is analogous to that of bone mineral, although the labile species are always found in higher quantities in bone. The difference between these processes is the formation of OH2 ions in the first one, leading to apatites with a higher stability [58], which appears inhibited in the second process when carbonate is present, and allows the formation of a carbonated apatite with a composition close to that of OCPC.

8.5

Physical
chemical characterization

8.5.1 Chemical analyses Chemical analyses are an essential step in the characterization of calcium phosphates. The calcium content can be obtained by different ways with different advantages and drawbacks [32]: the old but still competitive complexometry determination with ethylenediaminetetraacetic acid, inductively coupled plasma (ICP) techniques, atomic absorption spectroscopy, specific calcium electrode, or else ion chromatography. The total orthophosphate content can be determined by spectrophotometry of the phosphovanadomolybdic acid or by ICP-atomic emission spectroscopy (AES). In the case of OCPs a specific determination of the HPO22 4 content is often a necessity. The method proposed by Gee and Dietz [117], although often criticized, is still used. It consists in preliminarily heating the sample to condense 42 HPO22 4 ions into pyrophosphate ions, P2 O7 42 2HPO22 4 ! P2 O7 1 H2 O

(8.12)

followed by the determination of the residual orthophosphate ions (PO32 4 ions) by the spectrophotometric method cited earlier, which is insensitive to pyrophosphate ions. The comparison between total and residual orthophosphate then gives the initial HPO22 4 content. An aqueous hydrolysis of the pyrophosphate ions in the analysis solution into orthophosphate ones, by heating the solution, can be made for double checking. Some artifacts may however occur, such as overvaluations of the HPO22 4 content, due to the internal hydrolysis reaction between hydration water and PO32 4 ion on heating [reaction (8.3)] [4,11]. It is advised to heat the samples rapidly, to 600 C for 20 min, in order to minimize the internal hydrolysis reaction, or

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to heat under vacuum. The occurrence of parasite reactions, in particular between 42 HPO22 4 or P2 O7 ions and carbonate ions, prevents the use of this method in the presence of a carbonate. The presence of organic compounds, susceptible to give carbonate on heating, can also potentially affect the pyrophosphate determination. 2 At higher temperature (at about 700 C), a reaction between P2 O42 7 and OH ions (from apatite) is observed: 2 32 P2 O42 7 1 2OH ! 2PO4 1 H2 O

(8.13)

The determination of the carbonate content, when present, can be made by coulometry or by carbon, hydrogen, nitrogen analyzer measurements. This last method can also be used to detect the presence of ammonium impurities or that of organic molecules.

8.5.2 X-ray diffraction The XRD pattern of t-OCP is very specific, especially with the low angle 1 0 0 peak, at 2θ 5 4.72 degrees (CuKα1 anticathode) most often superimposed to the diffusion background of air when the diffraction is not carried out under vacuum. The peaks doublet 1 2 1 0, 2 0 0 (2θ 5 9.44 degrees), and 0 1 0 (2θ 5 9.77 degrees) is generally preferred for an identification; close to the 1 0 0 peak of HA (2θ 5 10.82 degrees), these peaks can be used to assess the presence of mixtures and their evolution. For poorly crystalline phases, however, the poorly resolved XRD patterns lose accuracy. For amorphous samples, XRD patterns look very similar and amorphous TCP (ATCP) or am-OCP cannot be distinguished. Although pair distribution functions or radial distribution functions allow the characterization of amorphous phases, they are often poorly selective and sensitive to the hydration level.

8.5.3 Vibrational spectroscopies The main lines of the FTIR and Raman spectra of t-OCP have been assigned by Fowler [12] (Table 8.4). The mixtures of t-OCP and HA differ essentially by the OH2 stretching line at 3570 cm21. The OH2 libration band at 630 cm21 is superimposed to a broad composite line assigned by Fowler to PO4 groups (608 cm21) and H2O (627 cm21). For am-OCP (Table 8.4), lines attributed to HPO22 4 ions are observed at 875, 1285, and 2340 cm21 in addition to the broad lines due to PO32 4 vibrations, which are slightly shifted compared to ATCP. The ap-OCP spectrum (Table 8.4) appears similar to that of a noncarbonated aged BNA, with specific lines assigned to HPO22 at 535, 870, 1140, 1220, and 4 2430 cm21 and to OH2 lines at 630 and 3570 cm21, in addition to PO4 lines. The FTIR spectral lines reported in Table 8.4 are given for lyophilized samples. For aged samples, there is no major difference between the spectra of wet and

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Table 8.4 Positions of Fourier-transform infrared spectroscopy lines of octacalcium phosphate (OCP) phases and parent biomimetic nanocrystalline apatite (BNA) phases according to the molecular ionic species (wave number in cm21). Molecular ions

Assignment

t-OCP

am-OCP

ap-OCP

nc-BNA 10 min maturation

nc-BNA 2 months maturation

HPO4

O3PO-H str P-O str (ν3 PO4)

2440 w 6 1295 w 6 1193 w 5 1137 sh 1121 s 6 1103 s 5 1000 sh 917 w 6

2340 w b 1285 sh

2430 w b

2410 w b 1260 sh

2450 w b 1290 sh 1215 sh 1150 sh

O3P-OH str

1215 sh 1140 sh

1009 sh

1007 sh

875 w b

897 sh 871 w b

1140 sh 1125 sh 1110 sh

892 sh 875 w b

900 sh 875 w b 865 sh 550 sh AP

529 sh HL

530 sh HL

1036 s b

1028 s

1030 s

861 w 5 549 sh AP 534 sh HL

Bend (ν4 PO4) 524 w PO4

PO4, HPO4

OH H2O

P-O str (ν3 PO4)

1055 s 1037 s 1023 s

1059 s b

P-O st (ν1 PO4) Bend (ν4 PO4) Bend. (ν2 PO4) P-O str (ν3 PO4) P-O ben (ν4 PO4) str Libration str

962 w

950 sh

961 w

960 sh

961 w

601 m 560 m 466 w

555 m b

603 m 563 m 471 w

600 m 560 m 465 w

603 m 563 m 472 w

Bend Libration

1077 s

575 sh

3600 3525 1642 m 627 sh

1096 s

577 sh

572 sh 3568 w 634 sh

1639

1093 s b 618 sh 575 sh

572 sh 3568 w 633 w

1640

am-OCP, Amorphous OCP; AP, HPO22 4 ions in the apatite domains; ap-OCP, apatitic OCP; nc-BNA, noncarbonated BNA; b, broad; bend, bending; HL, HPO22 4 ions in the hydrated layer; m, medium; s, strong; sh, shoulder; str, stretching; w, weak. 5 and 6 correspond to the two sites for HPO22 in the t-OCP structure (see Fig. 8.1). 4 Source: Assignments for t-OCP from Fowler BO, Markovic M, Brown WE. Octacalcium phosphate. 3. Infrared and Raman vibrational spectra. Chem Mater 1993;5:1417
23.

lyophilized samples. The surface layer structure changes rapidly on aging in the absence of stabilizing ions such as carbonate or magnesium. Surface HPO22 4 ions remain still observable and are identified by a specific line at 530 cm21 distinct 21 from that of apatitic HPO22 4 at 550 cm .

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Octacalcium Phosphate Biomaterials

8.5.4 Solid-state nuclear magnetic resonance Solid-state NMR is another major technique, which brings very valuable information on the OCPs, bone mineral, and related BNA compounds. Only 31P spectra are mentioned here, and chemical shifts were determined using H3PO4 as reference. The 31P spectrum of t-OCP appears very specific with lines, which have received different assignments (Table 8.5), but the data seem constant and robust and allow an unambiguous identification of t-OCP. Some alterations of this spectrum in modified OCP have been reported, for example, during its hydrolysis in apatite [61] or in citrate
OCP combinations [26]. The 31P NMR spectra of am-OCP presents only a broad line at 2.7 ppm assigned 22 to PO32 4 ions and a very broad line at 0.9 ppm assigned to HPO4 ions. No spectrum seems available so far for ap-OCP. For noncarbonated BNA (Fig. 8.9), just after precipitation, the lyophilized samples show a dissymmetric line with a maximum at 2.8 ppm attributed to PO432 ions 31 and a broad shoulder at about 0.5 ppm assigned to HPO22 P 4 ions. However, the spectrum of the wet sample exhibits a much better resolution than the lyophilized one: the dissymmetric line has a maximum at 3.8 ppm, and the line attributed to HPO22 4 is distinct with a maximum at 1.2 ppm. In addition, two weak shoulders at 3.2 and 2.7 ppm can be detected. On aging, the wet sample spectra become closer to that of the lyophilized sample with a maximum of the main line having shifted at about 3 ppm and weaker HPO22 4 line appearing as a shoulder on the main peak at about 0.5 ppm [79]. Some discrepancies have been noticed between FTIR, Raman, and solid-state NMR. Regarding the wet BNA samples, for example, the analogy with OCP appears more obvious in FTIR than in solid-state NMR. Similarly, the solid-state NMR evaluations of the OH2 content of bone are much higher (at least 20% of OH2 compared to stoichiometric apatite) than those obtained by Raman or FTIR (a few percentages). Another anomaly is the very high content of HPO22 ions 4 reported for BNA or bone by solid-state NMR [9,48] (up to 50%), considerably larger than that obtained by chemical analyses. These discrepancies have not yet been explained. Table 8.5 Assignments of the octacalcium phosphate 31P solid-state nuclear magnetic resonance lines (P5 and P6, HPO22 4 ions). Chemical shift (ppm)

Initial assignment [61]

New assignment [118]

3.6 3.2 2.0 2 0.1 2 0.3

P1 P2, P4 P3 P5, P6 P5, P6

P4 P1 P2 P6 P3, P5

Source: According to Davies E, Duer MJ, Ashbrook SE, Griffin JM. Applications of NMR crystallography to problems in biomineralization: refinement of the crystal structure and 31 P solid-state NMR spectral assignment of octacalcium phosphate. J Am Chem Soc 2012;134:12508
15.

Synthesis and physical chemical characterizations of octacalcium phosphate

205

Figure 8.9 31P spectra of t-OCP, DCPD, wet, and lyophilized noncarbonated BNA at a short maturation time. Compared to the lyophilized sample, the improvement of the resolution on the wet BNA sample is clearly apparent, especially on the HPO22 4 line at 0
1 ppm attributed to ions in the hydrated layer. Contrarily to the observations made on similar samples by FTIR (Fig. 8.5), there is no strong resemblance between the wet BNA and the t-OCP 31P solid-state NMR spectra. BNA, Biomimetic nanocrystalline apatite; DCPD, dicalcium phosphate dihydrate; FTIR, Fourier-transform infrared spectroscopy; NMR, nuclear magnetic resonance; t-OCP, triclinic OCP. Source: Figure reorganized from Eichert D, Sfihi H, Combes C, Rey C. Specific characteristics of wet nanocrystalline apatites. Consequences on biomaterials and bone tissue. Key Eng Mater 2004;254
256:927
30.

8.6

Perspectives and conclusion

Among the three OCP polymorphs presented, t-OCP has been the most studied. This phase is often considered as a bone mineral precursor, however, the synthesis of nt-OCP implicitly hypothesized in such an assertion is rarely mentioned [119
122], and its surface properties have been rarely investigated. The two other OCPs have not received much attention. The am-OCP could have been an interesting precursor of t-OCP for self-setting cements or composite materials; however, its fast conversion to CDA has shortcut these possibilities. The ap-OCP with its unusual chemical composition presenting a high level of HPO22 4 deserves probably more attention. The t-OCP structure has revealed a unique association between a rigid and dense partial apatite-like structure and an adaptable hydrated layer containing bivalent ions, which is probably close to the existing interface between

206

Octacalcium Phosphate Biomaterials

BNA crystals and an aqueous solution. The composition of this layer and that of the associated apatite domains in bone or BNA are not totally elucidated and, at this stage, no distribution of the atoms obtained by a bulk analysis can be proposed. From a structural point of view, the characteristics of this very reactive and easily altered surface-hydrated layer containing labile ions are not well known either, but, for sure, an improvement of our knowledge necessitates a careful investigation of samples in their native aqueous medium. The surface-hydrated layer present on bone-apatite crystals or its biomimetic analogues offers more flexibility than that present in the internal structure of t-OCP, although t-OCP surfaces appear to react quite similarly as BNA in carbonate
hydrogen phosphate ion exchanges. The incorporations of nonnative ions or molecular groups of diverse molecules in the hydrated layer involve often a reversible ion-exchange process with native ions (most commonly HPO22 4 ), which determines in fact the behavior of the “adsorbed” substances, especially the nonreversibility on dilution [84] and the release conditions. Another interesting phenomenon concerning BNA is their low-temperature consolidation [123]. This direct contact fusion between nanocrystals, not really studied and understood, could not only be involved in the grouping of nanocrystals in bone or enamel but also in the interactions of BNA with other surfaces or with polymers in composite materials. Such studies, at the scale of isolated nanocrystals, necessitate, however, to get them as individual entities without altering their surface irreversibly. At a larger scale, the role of the nanocrystals on bulk properties of bone tissues or biomaterials has to be clarified.

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