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α-Tricalcium Phosphate: Synthesis, Properties and Biomedical

Applications

Raúl G. Carrodeguas and Salvador De Aza

Instituto de Cerámica y Vidrio, CSIC

C/ Kelsen, 5 - 28049 Madrid, Spain

Corresponding author:

Raúl G. Carrodeguas; Instituto de Cerámica y Vidrio, CSIC, C/ Kelsen, 5 -

28049 Madrid, Spain;

E-Mail: rgc@icv.csic.es; Phone: +34 917 355 840; Fax: +34 917 355 843

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Abstract:

Nowadays α-tricalcium phosphate (α-TCP, α- Ca3(PO4)2) is receiving growing

attention as raw material for several injectable hydraulic bone cements,

biodegradable bioceramics and composites for bone repair. In the phase

equilibrium diagram of the system CaO-P2O5, three polymorphs corresponding

to the composition Ca3(PO4)2 are recognized; they are β-TCP, α-TCP, and α’-

TCP. α-TCP is formed by heating the low temperature polymorph β-TCP or by

thermal crystallisation of amorphous precursors with the proper composition

above the transformation temperature. α-TCP phase may be retained at room

temperature in metastable state, and its range of stability is strongly influenced

by ionic substitutions. It is as biocompatible as β-TCP but more soluble than this

and hydrolyses rapidly to calcium deficient hydroxyapatite, which make α-TCP a

useful component for preparing self-setting osteotransductive bone cements

and biodegradable bioceramics and composites for bone repairing. The

literature published on the synthesis and properties of α-TCP is sometimes

contradictory, therefore, this article is focused to review and critically discuss

the synthetic methods and physicochemical and biological properties of α-TCP-

based biomaterials (excluding α-TCP-based bone cements).

Keywords: α-tricalcium phosphate, β-tricalcium phosphate, synthesis, thermal

stability, phase transition

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SUMMARY

1 Introduction .................................................................................................. 4

2 Structural characteristics of α-TCP and its polymorphs .......................... 5

3 Identification and characterization techniques ......................................... 7

4 Solubility and biodegradability ................................................................... 8

5 Commercially available α-TCP .................................................................. 10

6 Synthesis of α-TCP .................................................................................... 11

6.1 Thermal transformation of a precursor with Ca/P≈1.50 ........................ 12

6.2 Solid state reaction of precursors .......................................................... 14

6.3 Self-Propagating High Temperature- and Combustion Synthesis....... 15

6.4 Effects of ionic substitutions on the synthesis of α-TCP ..................... 15

6.5 Thermodynamic considerations relevant to the synthesis of α-TCP .. 17

7 Biological behaviour .................................................................................. 18

8 Clinical applications .................................................................................. 23

9 Concluding remarks .................................................................................. 24

Acknowledgments .......................................................................................... 25

References ...................................................................................................... 26

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1 Introduction

There are three polymorphs of TCP the low temperature, β-TCP, and the high

temperature forms, α- and α´-TCP. The last one lacks of practical interest

because only exists at temperatures above ~1430ºC and reverts almost

instantaneously to α-TCP by cooling below the transition temperature. On the

other hand, β-TCP is stable at room temperature and transforms

reconstructively [1, 2] at ~1125ºC to α-TCP, which can be retained during

cooling to room temperature [3]. Several phase equilibrium diagrams have been

proposed to describe the phase relationships in the system CaO-P2O5 [4-8].

The main difference between them is to consider [4, 7] or not consider [5, 6, 8]

the existence of a TCP solid solution field at the high P2O5 content side of the

TCP composition.

The more recent study is that by Kreidler and Hummel [8] where TCP solid

solution was not found (Figure 1). They considered that loss of P2O5 at

temperature over 1600 ºC was the cause for a mixture of Ca2P2O7 and

Ca3(PO4)2 to shift to the composition of Ca3(PO4)2 and subsequent finding of

this phase by X-ray analysis in the work by Welch et al. [7], leading them to

misinterpret their experimental results [8].

α- and β-TCP are currently used in several clinical applications in dentistry,

maxillo-facial surgery, and orthopedics; β-TCP is the component of several

commercial mono- or biphasic bioceramics and composites and α-TCP is the

major constituent of powder component of various hydraulic bone cements [9,

10].

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In spite of having the same chemical composition, α- and β-TCP considerably

differ in their structure, density and solubility, which in turn determine their

biological properties and clinical applications.

β-TCP is mainly used for preparing biodegradable bioceramics shaped as

dense and macro-porous granules and blocks, whereas the more soluble and

reactive α-TCP is mainly used as a fine powder to prepare calcium phosphate

cements, although some commercial bioceramic granules and blocks made of

α-TCP may be found in the market. Both β- and α-TCP materials are used in

clinics for bone repairing and remodelling applications.

In Table 1 some commercial calcium phosphate bone cements using α-TCP in

their formulae are listed. Detailed description and discussion of the physico-

chemical and biological properties of α-TCP-based bone cements can be found

in previous reviews by other authors [9-20]. Additional information on the

properties and applications of α-TCP may be found in references [3, 9, 21-25].

2 Structural characteristics of α-TCP and its polymorphs

α-TCP crystalline structure was related to that of the mineral glaserite

(K3Na(SO4)2) by Dickens and Brown in 1972 [26] and later studied in detail by

Mathew et al in 1977 [27] and more recently by Yashima et al. [28]. α-TCP

crystallizes in the monoclinic crystal system and belongs to the space group

P21/a. Cell parameters (a, b, c, α, β and γ), cell volume (V), number of formula

units per cell (Z), volume per formula unit (V0) and theoretical density (Dth); and

the projections of the unit cells along the [001] direction are displayed in Table 2

and Fig. 2, respectively, for α-TCP [27, 28] and its polymorphs, β-TCP [29, 30]

and α’-TCP [28, 31].

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Ca and PO4 ions constituting the unit cells of α-TCP and its polymorphs are

packed in columns along the [001] direction. Two kinds of columns exist in α-

TCP; one kind, named C-C in Figs. 2 and 3, contains only Ca cations and the

other, named C-A, contains both Ca cations and PO4 anions. Each C-C column

is surrounded by six C-A columns, and in turn, alternating C-C and C-A columns

to the total number of six surround each C-A column. C-C columns are quite

distorted from the straight line as shown in Fig. 3 [27].

Thin solid-line rhombus inscribed within the unit cell of α-TCP in Fig. 2 outlines

a cell related to that of hydroxypatite, in which the OH columns could replace

the C-C columns at the cell corners. By analogy, the Ca-PO4 columns in

hydroxyapatite may be considered as very distorted C-A “columns” and each

“column” is surrounded by three C-A “columns” as in α-TCP, and by two C-C

and one OH column [27].

A significant structural difference between α-TCP and β-TCP is that there are

no C-C columns in the latter. Instead, there are two kinds of C-A columns in β-

TCP; A columns with the sequence …-P-Ca-Ca-P-…, and B columns with the

order …-P-Ca-Ca-Ca-P-P-…, and each A column is surrounded by six B

columns, while each B column by two A and four B columns [30].

On the other hand, α’-TCP consists of alternating C-C and C-A columns

similarly to α-TCP (See Fig. 2) [28, 31].

The structure of α-TCP is less densely packed than β-TCP, and more densely

than α´-TCP, as shows the volumes per formula unit (V0) and the calculated

theoretical density (Dth) of the three substances listed in Table 2. Difference in

packing densities of the three polymorphs is consistent with thermodynamic

considerations and with their stability temperature ranges. Besides, it should be

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expected that in a physiological environment the dissolution and degradation of

the “looser” α-TCP structure proceeds faster than β-TCP as has been

experimentally observed [11, 23, 32].

3 Identification and characterization techniques

Crystalline α-TCP is usually identified, and sometimes quantified when mixed

with other calcium phosphates by means of x-ray or neutron diffraction. The

calculated X-ray diffraction pattern for CuKα is displayed in Fig. 4 for α-TCP and

its polymorphs. X-ray diffraction is a useful tool to differentiate α- and β-

polymorphs.

X-ray absorption near edge structure (XANES) spectroscopy has also been

proposed as a useful technique to identify and differentiate calcium phosphate-

based biomedical materials. In contrast to traditional techniques such as X-ray

diffraction, neutron diffraction, and energy dispersive spectroscopy, XANES

spectroscopy provides information on the valence, oxidation state, coordination

number of individual elements as well as the chemical structure of compounds.

In Fig. 5 distinctive characteristics are observed for the P L2,3- (a) and P K-

edge (b) XANES spectra of α-and β-TCP [33].

Other instrumental tools often employed in identifying and characterising α-TCP

are Fourier-Transform Infrared (FTIR) [34], Raman [34] and 31P MAS-NMR [35]

spectroscopies. Typical infrared and Raman spectra of α-TCP are shown in Fig.

6, and the main absorption bands and their characteristics are listed in Table 3

for α- and β-TCP [34, 36].

High-resolution 31P solid-state nuclear magnetic resonance spectroscopy has

been employed to analyze bioceramics composition and to estimate

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osteoformation during implantation [37]. 31P MAS-NMR spectra of α-TCP, as

reported by Bohner et al. [35], and β-TCP [38] are presented in Fig. 7.

Differently from hydroxyapatite [39, 40] and β-TCP [41], there are no standards

establishing the requirements of chemical composition for α-TCP-based

materials intended for surgical implants. Nevertheless, chemical analysis is

almost customary for an exact characterization of α-TCP and the other calcium

orthophosphates used as bone repairing materials. Particularly, Ca and P

contents, their molar ratio, and minor and trace elements exert marked

influence on thermal stability, phase purity, and solubility of α-TCP, as will be

discussed ahead. Quantitative X-ray fluorescence methods have been

successfully employed to determine Ca and P contents in pure and substituted

α-TCP as well as minor and trace amounts of Mg and Si among other elements

[42, 43]. Besides, keeping in mind that β-TCP and HAp are practically identical

to α-TCP from the point of view of chemical constitution, analytical methods

standardized [44] and developed [45-49] for the formers may be used for

chemical characterization of α-TCP.

4 Solubility and biodegradability

The structural differences between β- and α- polymorphs of TCP are

responsible for their different chemical and biological properties, among them,

solubility and biodegradability.

Solubility products of α-TCP and other calcium orthophosphates are listed in

Table 4 [23, 32], and the calculated isotherms of solubility at 37ºC are

represented in Fig. 8 as the log of the concentration of Ca (a) and P (b) vs. pH

[11]. The analysis of Fig. 8 reveals that at physiological pH (7.2 - 7.4) the

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concentration of Ca and P dissolved from calcium orthophosphates decreases

in the order TTCP > α-TCP > DCPD > DCPA > OCP > β-TCP > HAp. On the

other hand, in that conditions HAp is the most stable of all calcium

orthophosphates, this way it should precipitate as α-TCP dissolution progress.

This is the thermodynamic base of calcium phosphate cements derived from α-

TCP, which set as result of the Equation 1 [10, 16, 17].

3 α-Ca3(PO4)2(s) + H2O(g) → Ca9(HPO4)(PO4)5(OH)(s) Eq. 1

However, empirical systems not always fulfil thermodynamic predictions and

other calcium phosphates different from HAp may be obtained as result of the

dissolution of α-TCP [9, 10].

On the other hand, the increase in solubility of α-TCP is considerable at pH < 5

and other calcium phosphates different from HAp may exist in thermodynamic

equilibrium with the saturated solution (see Fig. 8) [11]. The increase in

solubility of α-TCP by diminishing pH from 7.4 to 5.4 is illustrated in Fig. 9,

where kinetic curves of dissolution at both pH are plotted for α- and β-TCP [50].

It has been well documented that the setting reaction of Eq. 1 taking place in α-

TCP-based cements is initially controlled by surface reaction that depends on

the dissolution rate of the powdered reagent. This way, modulation of the

dissolution rate of α-TCP is very important in controlling the overall setting

reaction [18]. There are several approaches to modify the dissolution rate of α-

TCP powders [18]:

a) Change of the contact area between powder and mixing liquid,

b) Change of the powder solubility in the mixing liquid,

c) Change of the saturation of the mixing liquid towards α-TCP,

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d) Use of dissolution inhibitors, or

e) Surface modification (passivation [51, 52] or activation[53] of the surface)

5 Commercially available α-TCP

Whereas commercial β-TCP products with different degrees of quality can be

easily found in the market, suppliers of α-TCP are quite rare and dispersed over

the world. Only a few manufacturers and distributors of α-TCP could be found

during a survey carried out in scientific and commercial data bases in the web.

Taihei Chemical Industrial Co. Ltd. (Osaka, Japan) manufactures Technical

Grade α-TCP [54]. According to the manufacturer the product is supplied as

white granulate of 1.0-2.0 g/cm3 of bulk density with granule size lower than 1.7

mm and specific surface area in the range of 0.1-2.0 m2/g. The pH of an

aqueous suspension of the material is in the range of 7-10. No further

information on chemical and phase purity of the product is provided by the

manufacturer.

Wako Pure Chemical Industries, Ltd. (Osaka, Japan) is another Japanese

manufacturer of α-TCP with subsidiaries in Germany and USA [55]. They offer a

product named “apatite alpha-TCP, monoclinic”, but no further information on

the chemical characteristics is provided online.

Ensail Beijing Co., Ltd. (Beijing, P. R. China), is a manufacturer of calcium-based

biomaterials, α-TCP among others. It is supplied as high purity (> 99 %) single phase

powder with particle size under 50 μm. The Ca/P ratio of the product is 1.5 and the levels of

As, Cd, Hg and Pb, fulfil the requirements of the ASTM standard [56]. The

manufacturer claims that the product is suitable as raw material for calcium

phosphate cements as well as for bioceramics for orthopaedic implants [57].

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A few reagent grade α-TCP’s are also commercially available. Sigma-Aldrich

Co. (St. Louis, USA) distributes a sintered powder consisting of more than 75 %

of α- phase and less than 25 % of β- phase, hydroxyapatite and/or calcium

pyrophosphate. According to the manufacturer, the product is suitable as

standard for analysis of bone cements [58].

Zimmer Dental GmbH (Freiburg, Germany) offers biomedical grade α-TCP

bioceramic granules approved for clinical applications. Biobase® was licensed

by the German Board of Public Health as early as 1991. Biobase® consists of

granules with several size ranges: 0.2-0.5 and 0.5-1.40 mm intended for dental

applications, and 1.40-3.2, 3.2-5.0 and 5.0-8.0 mm for orthopedics [59].

Biobase®, is distributed in Africa by Implant Support Services CC (Pretoria,

South Africa) [60].

InnoTERE GmbH (Dresden, Germany) produces α-TCP 13-1000, which is

described as particulate α-TCP with a phase purity ≥ 95 % and particle size ≤ 1

mm. According to the manufacturer, α-TCP 13-1000 is intended “for laboratory

use” only, even though they might deliver “different bulk sizes, custom packing

and raw materials for the production of medical devices on query” [61].

BrainBase Corporation (Tokyo, Japan) markets biomedical grade α-TCP

granules under the brand name of ArrowBoneTM-α. The product consists of

single phase α-TCP shaped in open porous (75 % porosity) granules of size

250-1000 µm [50].

6 Synthesis of α-TCP

Most of the published researches dealing with α-TCP have been carried out on

lab-made products probably because of the above mentioned shortage of

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commercial products. Basically, the synthesis of α-TCP is accomplished by

thermal transformation of a precursor with molar ratio Ca/P≈1.5 (calcium-

deficient hydroxiapatite, CDHA; amorphous calcium phosphate, ACP; or β-TCP)

previously obtained [62-68], or solid state reaction of a mixture of solid

precursors at high temperatures [12, 15, 27, 28, 31, 35, 64, 69-76]. Self-

propagating high temperature synthesis (SHS) [77] and combustion synthesis

[78-80] have also been employed.

6.1 Thermal transformation of a precursor with Ca/P≈1.50

Thermal decomposition of CDHA into α-TCP is represented in Eq. 2.

Ca9(HPO4)(PO4)5(OH)(s) → 3 α-Ca3(PO4)2(s) + H2O(g) T≥1150ºC Eq. 2

Camiré et al. [64] employed a CDHA precursor obtained by precipitation from a

solution containing Ca(NO3)2 and (NH4)2HPO4 with a molar ratio Ca/P = 1.5,

followed by thermal transformation into α-TCP by heating at 1250 ºC for 2 hr.

Jokic et al. [65] also prepared α-TCP powder by thermal decomposition of

CDHA (Ca/P = 1.56) obtained by hydrothermal method according to Eq. 2.

CDHA decomposed into β-TCP at T≥800 ºC and β-TCP in turn started to

transform into α-TCP at T ≥ 1200 ºC. Maximum transformation was obtained

after heating 2 hr. at 1500 ºC even though a small amount of HAp remained

untransformed, obviously because of the positive deviation from the proper

stoichiometry to get pure TCP, i.e. Ca/P=1.5.

Similarly to Eq. 2, Eq. 3 represents the thermal transformation of ACP into α-

TCP.

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Ca9(PO4)6.nH2O(s) → α-Ca3(PO4)2(s) + nH2O(g) Eq. 3

α-TCP has been synthesised at temperatures much lower than the transition

temperature of ≈ 1130 ºC predicted by the phases diagram of Fig.1. Somrani et

al. [62] found that ACP (Ca/P=1.52) prepared by double decomposition from

aqueous solutions of Ca(NO3)2.4H2O and (NH4)2HPO4 is almost totally

converted into α-TCP (4-7 % β-TCP) by heating to 600-800 ºC. However, by

heating to 900 ºC, pure β-TCP was obtained. The apparent contradiction had

been previously reported by Eanes [81] and was explained in the light of the

Ostwald step rule.

Bohner et al. [66] prepared nanosized ACP powders by flame synthesis which

rendered α-TCP (β-TCP ≤ 6 %, HAp traces) by thermal treatment at

temperatures as low as 600 ºC. Increasing the temperature to 700 ºC favoured

the formation of β-TCP (40-60 %) and pure β-TCP was obtained at 800 ºC. A

more detailed study on the evolution of crystalline phases during the thermal

treatment of the ACP performed by Dobelin et al. [67, 68] showed similar

results.

Thermal transformation of crystalline β-TCP is the most direct and perhaps the

simplest and cheapest approach to the synthesis of α-TCP. According to the

phase equilibrium diagram of Fig. 1 a temperature above 1130 ºC should be

enough to accomplish the β- to α-TCP transformation. Kitamura et al. [63] have

employed commercial β-TCP powder with a mean particle diameter under 45

µm to prepare porous blocks of sintered α-TCP. The starting powder was

suspended in an aqueous starch solution and polyurethane sponges were

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impregnated in the slurry by dipping. After drying, the polyurethane sponge was

burned out by gradually heating to 1000 ºC. Total conversion of the resulting

porous blocks into α-TCP was achieved by heating to 1400 ºC for 12 hr. and

cooling to room temperature in the furnace.

6.2 Solid state reaction of precursors

Solid state reaction between solid precursors is the preferred synthetic route in

the literature reports. The solid state reactions more employed are listed below:

CaCO3(s) + 2CaHPO4(s) → α-Ca3(PO4)2(s) + CO2(g) + H2O(g)

Eq. 4 [12, 15, 27, 28, 31, 64, 69-73, 76]

3CaCO3(s) + 2NH4H2PO4(s) → α-Ca3(PO4)2(s) + 3 CO2(g) + 3 H2O(g) + 2 NH3(g)

Eq. 5 [71, 75]

CaCO3(s) + Ca2P2O7(s) → α-Ca3(PO4)2(s) + CO2(g)

Eq. 6 [64, 71, 74, 82]

Ca10(PO4)6(OH)2(s) + 2CaHPO4(s) → 4α-Ca3(PO4)2(s) + 2H2O(g)

Eq. 7 [35, 69]

The synthesis is carried out obeying the general rules for solid state reactions,

i.e., solid precursors are milled together to reduce particle size, increase the

contact area and mix them intimately. Wet milling is generally preferred. After

milling the mixture of powders may be directly heated above the transformation

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temperature or previously pressed to improve contact among particles. The

recommended reaction temperatures varied between 1250-1500 ºC, and the

dwell time was between 2-48 hr. Most authors recommend quenching to avoid

the reversion of the α-phase in spite of the well-recognised reconstructive

character of the first order α→β transformation [42, 83] and the significant

measured value of the apparent activation energy for the β↔α transformation of

1.0467 MJ/mol [84].

On the other hand, the resulting α-TCP often presented variable amounts of

other crystalline phases, mainly β-TCP ([12, 15, 35, 64, 69, 70, 74, 82] or HAp

[74, 82].

6.3 Self-Propagating High Temperature- and Combustion Synthesis

Ayers et al. [78, 79] prepared pellets of a mixture of powders of CaO and P2O5

in 3:1 molar ratio. After reacting under argon by heating a tungsten filament to

the point of igniting the bottom of the reactant pellet, α-TCP containing

significant amounts of HAp and β-TCP was obtained.

Auto-combustion of aqueous dissolutions of calcium and phosphate salts with

the required Ca/P molar rate and containing an organic fuel like urea was

employed for Burkes et al. [77] and Volkmer et al. [80] to synthesise α-TCP, as

well.

6.4 Effects of ionic substitutions on the synthesis of α-TCP

It is well documented that some ionic substitutions may exert drastic effect on

the thermodynamic relationships between α- and β-TCP [85, 86]. For example,

partial substitution of Mg for Ca in TCP increases the thermal stability of β

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phase [42, 69, 83, 87, 88] and gives rise to a binary phase field where β+α-

Ca3(PO4)2 solid solutions coexist. This binary field lies between the single-

phase fields of β- and α-Ca3(PO4)2 solid solution in the Ca3(PO4)2-rich zone of

the system Ca3(PO4)2- Mg3(PO4)2 [42, 88]. In the same way, partial substitutions

of Zn and Sr ions for Ca have similar effect to Mg [89, 90]. Taking this into

account, it becomes evident the need for excluding Mg, Sr, Zn, and any other

impurity able to stabilize β phase from the raw materials employed in the

synthesis of α-TCP [42, 91].

Alternatively, when PO4 is partially substituted for SiO4 α phase is stabilized and

the temperature of the polymorphic transformation decreases [86, 92]. Thus,

doping with silicate has been employed to synthesise pure phase α-TCP [43,

91, 93-95]. Silicate-doped α-TCP presents faster initial reaction in aqueous

solution of Na2HPO4 and a slower secondary reaction than pure α-TCP [96].

The benefits of silicate-doping from the biological point of view are matter of

discussion. Several in vitro and in vivo studies have found enhanced

osteogenesis for silicon-substituted calcium phosphates in comparison with

pure materials. The effect has been attributed to silicon species released from

the materials which are able to stimulate bone regeneration and remodeling,

among other factors [97]. However, at present no experimental evidence

supports the statement that silicon ionic species from Si-substituted calcium

phosphates are at therapeutic levels, and the linkage between the improved

biological performance of silicon-substituted calcium phosphates and the

release of silicon species to the physiological medium have not been proven yet

[98].

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6.5 Thermodynamic considerations relevant to the synthesis of α-TCP

From the review of published data relating synthesis of α-TCP exposed above

several contradictory results are revealed.

First, most of the synthetic methods reported yield minor amounts of foreign

phases, mainly β-TCP, HAp, and calcium pyrophosphate (CPP).

The presence of β-TCP has been sometimes attributed to partial reversion

during quenching of the already formed α-TCP [12, 15]. However, it should be

noticed that the polymorphic transformation β↔α is reconstructive [42, 83],

according to the Buerger classification, i.e., “involves a major reorganization of

the crystal structure, in which many bonds have to be broken and new bonds

formed” [99] so considerable energy has to be provided to the system to

transform from one to another polymorph. Thus, reverting from α- to β-phase

should be unfeasible, unless either very slow cooling rate or large dwell at

temperature slightly below that of transformation are used. Besides, it has been

shown that it is possible to obtain pure phase α-TCP with cooling rate as

moderate as 10 ºC/min [35, 42].

Thus, the presence of β-TCP in the final product may indicate that:

a) Equilibrium has not been reached and longer dwell is needed above the

temperature of the polymorphic transformation (see diagram in Fig.1).

b) Equilibrium has been reached but the temperature of the polymorphic

transformation is higher than expected from Fig.1 because of the presence of

impurities stabilising β phase in the reaction mixture (see the phase equilibrium

diagram for the system Ca3(PO4)2-Mg(PO4)2 in reference [42]). Then the

temperature required to obtain pure α-TCP has to be increased above the

transformation temperature in the corresponding phase equilibrium diagram.

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c) Partial reversion of the high temperature α- phase has taken place as a result

of too slow cooling rate. In such a case either cooling rate must be increased or

the volume of the product mass must be diminished to improve heat conduction

and removal.

On the other hand, the appearance of small amounts of HAp or CPP together

with the main phase of α-TCP indicates certain deviation of the theoretical Ca/P

molar ratio 1.50. According to the diagram of the Fig. 1 TCP does not form solid

solutions with CPP nor tetracalcium phosphate (Ca4O(PO4)2, TTCP), and there

are binary fields at both sides of the compositional line of TCP. Negative

deviations from the stoichiometric ratio of 1.50 may produce TCP with some

CPP, whereas positive deviations may render TCP with TTCP in anhydrous

conditions. However, atmosphere existing at the lab during synthesis of α-TCP

usually contains some degree of humidity, which makes the diagram of Fig.1

useless. In wet conditions the valid diagram for the system CaO-P2O5-H2O is

displayed in Fig. 11, and explains the presence of HAp as impurity for positive

deviations of the stoichiometric Ca/P molar ratio of 1.50 [100]. Both impurities,

CPP and HAp, may be avoided with accurate adjustment of the Ca/P ratio in the

reaction mixture during the synthesis.

7 Biological behaviour

The biological behaviour of α-TCP based biomaterials has been studied in

several in vitro [101-105] and in vivo studies [106-111].

Mayr-Wohlfart et al. [101] tested three-dimensional porous scaffolds of α-TCP

(BIOBASE, Germany), bioglass, glass-ceramic and solvent dehydrated bone

(SDB) for proliferation and differentiation rates of human osteoblast-like cells

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(SaOS-2) cultured in vitro. The results showed that cells proliferate and

differentiate into osteoproductive osteoblasts better on the α-TCP scaffolds,

than on the other materials tested. Ehara et al. also studied the effects of α-TCP

and TTCP on the proliferation, differentiation and mineralization of cultures of

newborn mouse calvaria-derived MC3T3-E1. It was found that presence of α-

TCP or TTCP increased the pH of the culture media and had not influence on

cell proliferation but did on osteoblast differentiation and mineralization. The

authors concluded that α-TCP and TTCP promote osteogenesis by increasing

collagen synthesis and calcification of the extra-cellular matrix.

Similiarly, surface- and nonsurface-related cell viability of several commercial

bone substitute materials, α- (BioBase, Germany) and β-TCP (Cerasorb,

Germany) among them, was evaluated towards cultures of human primary

osteoblasts, bone marrow mesenchymal cells, and nonadherent

myelomonocitic cells. The report concluded that α- and β-TCP support surface-

and nonsurface related cell viability [104]. In the same sense, Seebach et al.

compared six bone-graft substitutes with regard to cell seeding efficiency,

metabolism and growth behaviour of human mesenchymal stem cells (MSC). α-

(BioBase, Germany) and β-TCP (ChronOs and Vitoss) were among the tested

materials. The number of MSC’s growth in the order Vitoss, BioBase and

ChronOs. The differences in cell adhesion between α- and β-TCP-based

materials was explained by the difference in surface charge density, which is

lower for β-TCP [105].

However, Tamai et al. reported that α-TCP is more cytotoxic than HAp, β-TCP

and TTCP and as cytotoxic as FAp towards in vitro cultured Chinese hamster

V79 lung fibroblasts [103]. The cytotoxic effects of α-TCP and FAp were

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evidenced both in cultures of cells directly seeded onto the materials and in

cultures made in the presence of extracts of the materials. It was proposed that

the cytotoxicity of FAp was due to fluorine ions extracted in the culture medium

whereas the decrease of pH of the medium caused by the phosphoric acid

generated during the hydrolysis of α-TCP was the cause of the cytotoxicity of

this material.

α-TCP in solid and paste forms was neither toxic nor irritant when tested on

intact rabbit skin [111]. In vivo biodegradation of granules of α- (3.2-5.0 mm

diameter) and β-TCP (1.0-2.0 mm diameter) was evaluated in bilateral artificial

bone defects created in the tibial head of Goettingen minipigs. Fluorescent

markers were administered to the experimental subjects during the periods of

observation. Bony regeneration proceeded basally and on the sides of the

defects, according to an angiogenetic reossification pattern. Resorption of the

implanted materials was due to hydrolytic and cellular degradation processes.

After 86 weeks 95-97 % of both α- and β-TCP were resorbed. Both α- and β-

TCP showed a comparable degradation process and the remains of materials

were completely integrated into newly formed bone trabeculae. In comparison

empty control defects showed only sparse reossification, according to which

both tested materials were classified as bone-rebuilding materials [106].

Kihara et al. performed an in vivo test to observe the biodegradation process of

particles (approximately 300 μm diameter) of pure α-TCP and to determine its

efficacy as space maintainer in bilateral cranial bone defects in rabbits. One

defect in each animal was left empty and used as control. A “reticulate

structure” was developed in the particles after 1 week as a consequence of the

degradation of α-TCP and new bone was observed after 8 weeks. The amount

21

of new bone was not significantly different for experimental and control groups

in any of the observation periods (1, 4 and 8 weeks). However, in the

experimental groups new bone deposited on the surface of α-TCP particles was

observed in the very centre of the defect whereas fibrous connective tissue was

predominant at the centre of control sites. The results suggested that α-TCP is

a degradable osteoconductive material able to act as a space maintainer for

bone regeneration[107].

The osteoconducting ability of α-TCP-based bone filler powder was tested in

comparison with Ti-6Al-4V rods, and polymethylmethacrylate (PMMA) bone

cement. The sites of implantation were holes drilled in the left and right sides of

the femoral condyles of adult New Zealand rabbits. The observation periods

were 1, 3 and 9 weeks. Osteoconduction was evaluated by measuring the

affinity index, bone density, and x-ray diffraction at the implants. More intense

diffraction peaks of HAp that increased with time were observed in sites that

received α-TCP and indicating that HA was formed at the expense of α-TCP

hydrolysis. Furthermore, there was more bone density increase in groups

containing the α-TCP bone filler powder. The results suggested that HA formed

by hydrolysis of the α-TCP bone filler powder could play some role in enhancing

osteoconducting ability [108].

Yamada et al conducted a histological and histomorphometrical study to

compare α-TCP and β-TCP as bone graft material for augmenting alveolar

ridges. The animal model employed porous α- and β-TCP blocks inserted in Ti

cylindrical cages and the set was implanted in 0.5 mm deep circular slits in

Japanese white rabbit calvaria. Animals were sacrificed after 2, 4, and 8 weeks

[109]. No significant difference was observed after 2 weeks but significant

22

differences were observed between both materials after 4 and 8 weeks. The

blocks of α-TCP were notably started degrading after 4 weeks whereas

degradation of β-TCP blocks had just begun at that time and scarcely

progressed after 8 weeks (Fig. 10a). However, α-TCP blocks were severely

degraded after 8 weeks as shown in Fig. 10b. Residual α-TCP particles

surrounded by newly formed bone decreased over time, and both particles and

newly formed bone were simultaneously absorbed by osteoclast-like cells. The

results suggested that α-TCP particles surrounded by newly formed bone may

disappear progressively from bone and could be incorporated into bone

remodelling cycle in combination with newly formed bone [109].

α-TCP particles have also been evaluated as drug carrier of simvastin, which is

a drug able to stimulate BMP-2 and VEGF mRNA expression in osteoblasts and

promote bone growth. Bilateral 5 mm diameter calvarial defects were created in

adult Wistar rats and filled with α-TCP particles containing different doses of

simvastin. Empty defects were used as controls. The observation periods were

2, 4 and 8 weeks [110]. Simvastin doses higher than 0.1 mg caused

imflammation of the surrounding soft tissue, however the microtromography

analysis revealed that the α-TCP with 0.1 mg simvastin yielded significantly

higher volumes of bone than control group at all periods of time. The

percentage of defect closure, bone mineral content and bone mineral density

were also higher in the group of α-TCP with 0.1 mg simvastin than in the others

[110]. Particulate α-TCP is a good carrier for simvastin and probably for other

bone growth stimulating drugs.

The potential of α-TCP powder (particle size 4-10 µm) as capping agent for

exposed dental pulp was tested in anterior teeth of Macaca fuscata with pulp

23

amputation. No inflammation was observed in the residual dental pulp at any

time during the experimental period of 3 month. Bone-like hard tissue was

induced in the pulp capped with α-TCP, and its potential for clinical application

to exposed dental pulp was confirmed [112].

8 Clinical applications

Published scientific literature dealing with clinical applications of α-TCP-based

materials is practically limited to α-TCP bone cements. They are used in

dentistry, craniofacial and maxillofacial surgeries, and orthopaedics,

vertebroplasty and kyphoplasty procedures and as drug carriers; however, more

detailed discussion is beyond the aim of this paper. Recent and excellent

reviews have been already published on the subject [9, 10, 19, 20, 113].

Recommended dental and orthopedics applications for the α-TCP-based

commercial granulates Biobase® [59] and ArrowBoneTM [62] are listed in Table

5.

However the scientific reports dealing with the applications of Table 5 are

extremely scarce and the only data dealing with the clinical performance of the

materials are the technical information at the manufacturers’ web sites.

The effect of a combination of an oily Ca(OH)2 suspension (Osteoinductal®,

Osteoinductal GmbH, Muenchen, Germany) with α-TCP (Biobase® α-pore,

Biovision GmbH, Ilmenau, Germany) vs. α-TCP alone in the treatment of one-

and two-wall intrabony pockets were clinically tested in a six month trial. The

results demonstrated that both treatments may result in significant probe depth

reduction and clinical attachment level gain over a period of 6 months. The

24

combination of Osteoinductal® and α-TCP may, however, additionally improve

the healing process [114, 115].

9 Concluding remarks

Two decades ago α-TCP was proposed as component of several bone

cements. Since then, granules, blocks and composites consisting of α-TCP are

also receiving growing attention as bone repairing materials. However,

commercial α-TCP, reagent or biomedical grade, are very scarce. This forces

researchers and developers to synthesize it on their own.

Thermal transformation of β-TCP above the temperature corresponding to the

polymorphic transformation β→α is the rational way to synthesise α-TCP. Thus

all synthetic routes available for the synthesis of β-TCP may be adapted to

obtain the high temperature polymorph α-TCP. However, involuntary or

intentional impurities in the precursors used in the synthesis and the exact

stoichiometry of the reactants affect the phase purity of the α-TCP obtained.

Impurities of Mg, Sr, and Zn substituting Ca stabilise the β phase whereas small

amounts of Si substituting P stabilise α phase. Moreover, even though α-TCP is

thermodynamically metastable at temperatures under ≈1100 ºC, it may be

preserved under this temperature without need of quenching. The reason for

this is that the polymorphic transformation β↔α is reconstructive and a

considerably activation energy is involved. So α-TCP phase may be retained

under ≈1100 ºC, unless quasi-equilibrium conditions (extremely slow cooling

rates) are employed. Cooling rates as low as 10 ºC/min may be used without

risk of reversion to the β phase.

25

From the biological point of view α-TCP is non-toxic, osteoconductive, and

bioactive, both in vitro and in vivo. The main reason of the growing interest in α-

TCP as bone implant material is its biodegradability. It is more bioreabsorbable

than HAp, β-TCP and biphasic (HAp/β-TCP) bioceramics currently used in

clinical practice. This makes α-TCP an ideal implant material able to be

replaced by new bone faster than the other calcium phosphate-based materials

available in the market nowadays. Besides, it may be used as biodegradable

carrier for controlled release of drugs, macromolecules or cells.

As set out above α-TCP seems to be an interesting option for the design of new

biomaterials for emerging bone repairing therapeutic procedures based on

tissue engineering and regenerative medicine.

Acknowledgments

To the “Junta para Ampliación de Estudios (JAE)” of CSIC, Spain, for

supporting R. G. Carrodeguas through the contract JAEDOC087-2009.

To the support from Project CICYT MAT2010-17753.

To the memory of Prof. Salvador De Aza (1933 – 2011) who guided the writing

of this paper and died shortly after it was sent for publishing.

26

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Phosphate (TCP) in Minipigs. Journal of Biomedical Materials Research Part B:

Applied Biomaterials 2002;63:115-21.

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alpha-TCP particles and new bone formation in a rabbit cranial defect model.

Journal of Biomedical Materials Research Part B-Applied Biomaterials

2006;79B (2):284-91.

[108] Yoo JH, Lee SH, Lee JI, Kwon H, Lee K-S. The analysis of

osteoconducting ability of alpha-tricalcium phosphate-based bone filler powder.

Key Eng Mater 2006;309-311:259-62.

[109] Yamada M, Shiota M, Yamashita Y, Kasugai S. Histological and

histomorphometrical comparative study of the degradation and osteoconductive

characteristics of - and -tricalcium phosphate in block grafts. Journal of

Biomedical Materials Research Part B: Applied Biomaterials 2007;82B:139-48.

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combination with -tricalcium phosphate and simvastin on bone regeneration.

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irritative property of -tricalcium phosphate to the rabbit skin. Gen Physiol

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drug delivery systems: A review. J Control Release 2006;113:102-10.

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suspension. A case series. Timisoara Medical Journal 2004;15 (4):410-6.

39

Captions to Figures

Fig. 1. Phase relationships in the system CaO-P2O5 according to Kreidler and

Hummel (this phase equilibrium diagram was published in [8], Copyright

Elsevier)

Fig. 2. Schematic representation of the projections of the α-TCP, β-TCP and α’-

TCP unit cells along the [001] direction [27-31]. Ca2+: green; P5+: magenta, O2-

have not been represented to seek of clarity. C-C: cation-cation column; C-A:

cation-anion column. Thin solid-line rhombus inscribed within the unit cell of α-

TCP outlines a cell related to that of hydroxypatite.

Fig. 3. Fractional projections of the α-TCP, β-TCP and α’-TCP unit cells on the

bc plane showing the disposition of constituent atoms in columns oriented along

the direction [001]. Ca2+: green; P5+: magenta, O2- have not been represented to

seek of clarity. C-C: cation-cation column; C-A: cation-anion column; A: A

column; B: B column.

Fig. 4. Calculated X-ray diffraction pattern of α-TCP and its polymorphs

(2θmin=5º; 2θmax=60; step size=0.05; λ(CuKα1)=0.1540562 nm; Pol=0.5;

Monochrom=None; Ua=Ub=Uc=0.5). (MS Modeling v.4.0.0.0, Accelrys Software,

2005).

40

Fig. 5. Phosphorus L2,3-edge (a) and phosphorus K-edge (b) XANES spectra

of α-and β-TCP (redrawn from [33], copyright 2010, with permission from

Elsevier ).

Fig. 6. Typical Fourier-Transform Infra-Red spectra (a) and Raman spectra (b)

of crystalline α- and β- TCP (redrawn from [34] with permission,

http://www.informaworld.com).

Fig. 7. 31P MAS-NMR spectra of α-TCP (redrawn from [35] with kind permission

from Springer Science+Business Media) and β-TCP (redrawn from [38] with

permission, copyright 2009 Wiley Periodicals, Inc.).

Fig. 8. Solubility phase diagram for the ternary system Ca(OH)2-H3PO4-H2O, at

37 ºC: (a) solubility isotherms showing log [Ca] and pH of solution in equilibrium

with various salts; (b) solubility isotherms showing log [P] and pH of the

solutions (redrawn from [11], with permission from The Ceramic Society of

Japan).

Fig. 9. Kinetics of dissolution in vitro of ArrowBoneTM-α and – β granules

consisting of α- and β-TCP, respectively at pH 5.4 (MES buffer) and 7.4 (Tris

buffer) (redrawn from [44]).

Fig. 10. (a) β-TCP and (b) α-TCP porous block inside Ti chambers and

implanted during 8 weeks in 0.5 mm deep circular slits in Japanese white rabbit

calvarias. The α-TCP block is considerably more biodegraded than the β-TCP’s.

41

(reproduced with permission from Yamada et al [109], copyright 2006 Wiley

Periodicals, Inc.).

Fig. 11. System CaO-P2O5-H2O at a fixed P(H2O)=500 mm Hg (reproduced from

[100] with permission).

42

Table 1. Commercial α-TCP-based bone cements.

Brand Name Manufacturer Powder composition

α-TCP+TTCP α-TCP+TTCP

α-TCP+DCP+CC+PHA α-TCP+CC+MCPM

α-TCP+TTCP+DCPD+HAp

Cementek® Mimix®

Calcibon® Norian SRS®

Biopex® MCPC

CallosTM

Teknimed SA, France Biomet, USA

Biomet, Europe Norian, USA

Mitsubishi Materials Co., Japan Biomatlante, France

Skeletal Kinetics, USA α-TCP+ACP+BCP α-TCP+CC+MCPM

TTCP: Tetracalcium phosphate, Ca4(PO4)2O; DCP: Dicalcium phosphate, CaHPO4; DCPD:

Dicalcium phosphate dehydrate; CC: Calcium carbonate, CaCO3; HAp: Hydroxyapatite,

Ca10(PO4)6(OH)2; MCPM: Monocalcium phosphate monohydrate, Ca(H2PO4)2.H2O; PHA:

precipitated HAp, Ca10-x(HPO4)x(PO4)6-x(OH)2-x; ACP: Amorphous calcium phosphate; BCP:

Biphasic calcium phosphate, HAp+β-TCP.

43

Table 2. Structural data of α-TCP and its polymorphs.

Property Ca3(PO4)2 Polymorph

β-Ca3(PO4)2 [30] α-Ca3(PO4)2 [28] α'-Ca3(PO4)2 [28]

Symmetry Rhombohedral Monoclinic Hexagonal Space Group R3C P21/a P63/mmc

a (nm) 1.04352(2) 1.2859(2) 0.53507(8) b (nm) 1.04352(2) 2.7354(2) 0.53507(8) c (nm) 3.74029(5) 1.5222(3) 0.7684(1) α (º) 90 90 90 β (º) 90 126.35(1) 90 γ (º) 120 90 120

Z 21 24 1 V (nm3) 3.5272(2) 4.31(6) 0.19052(8) V0 (nm3) 0.1680(2) 0.180(6) 0.19052(8)

Dth (g/cm3) 3.066 2.866 2.702

44

Table 3. Main bands and their characteristics wavenumbers and intensities in

infrared and Raman spectra of α- and β-TCP [34, 36].

Normal modes Free PO4

3-

(cm-1

)

α-TCP β-TCP

IR (cm

-1)

Raman (cm

-1)

IR (cm

-1)

Raman (cm

-1)

Symmetric P-O stretching, ν1

938 954 (s) 954 (w, sh)

964 (s) 976 (s)

972 (s) 945 (s)

948 (s) 970 (s)

Symmetric P-O bending doubly degenerate, ν2

420

415 (w) 430 (w) 454 (w) 463 (w) 471 (w)

421 (w) 451 (w)

419 (vw) 438 (vw) 458 (vw) 497 (vw)

406 (m) 442 (m) 481 (m)

Anti-symmetric P-O stretching triply degenerate, ν3

1017

984 (s) 997 (s) 1013 (s) 1025 (s) 1039 (s) 1055 (s)

998 (s) 1012 (w) 1027 (w) 1058 (w) 1077 (w)

1025 (s) 1044 (s)

1066 (w, sh) 1083 (w, sh)

1017 (w, br) 1048 (vs)

Anti-symmetric P-O bending triply degenerate,ν4

567

551 (s) 563 (s) 585 (s) 597 (s) 613 (s)

563 (s) 577 (s) 593 (s) 610 (s) 620 (s)

544 (w, sh) 555 (s)

594 (w, sh) 609 (s)

549 (w) 573 (w) 547 (w) 609 (w)

vs: very strong; s: strong; m: medium; w: weak; vw: very weak; sh: shoulder; br: broad

45

Table 4. Solubility of some calcium ortho-phosphates [32].

Ca/P Compound -log Kps Solubility, mg/L

25ºC 37ºC 25ºC 37ºC

1.0 CaHPO4.2H2O 6.59 6.73 87 74 1.0 CaHPO4 6.90 7.04 48 41

1.33 Ca8(HPO4)2(PO4)4.5H2O 96.6 98.6 0.025 0.018 1.5 α-Ca3(PO4)2 25.5 28.5 0.97 0.24 1.5 β-Ca3(PO4)2 28.9 29.6 0.20 0.15

1.67 Ca10(PO4)6(OH)2 116.8 117.2 0.00010 0.000096 2.0 Ca4(PO4)2O 38-44 37-42 0.28-0.038 0.39-0.075

46

Table 5. Recommended dental and orthopedics applications for the α-TCP-based commercial granulates Biobase® [59] and ArrowBoneTM [62].

Dentistry

Periodontology Two and three wall bony defects, can be

used with or without membranes

Implantology

Defect augmentation following extraction tocreate an implant base

Elevation of sinus floor

Gaps between extraction socket andimplant in case of immediate implantplacement

Cysts Defects resulting from cyst removal

Dental and Maxilla-Facial Surgery

Defects resulting from apicectomy

Defects resulting from the removal ofimpacted teeth

Defects resulting from correctiveosteotomies

All other shapes of bony craters and facialbone defects

Orthopedics

Filling of bone defects and cysts

Replenishment of the donor site of autogenous bone

Addition to cancellous bone in spinal fusion, vertebral bodyreplacement and joint replacement

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

http://ees.elsevier.com/actbio/download.aspx?id=195799&guid=c27b8309-0d54-414d-b310-e5ed79d5bd61&scheme=1

Figure 11