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Acta Biomaterialia 6 (2010) 4457–4475

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review

Amorphous calcium (ortho)phosphates


Sergey V. Dorozhkin *
Kudrinskaja sq. 1-155, Moscow 123242, Russian Federation

a r t i c l e i n f o a b s t r a c t

Article history: Amorphous calcium phosphates (ACPs) represent a unique class of biomedically relevant calcium
Received 4 May 2010 orthophosphate salts, having variable chemical but essentially identical glass-like physical properties,
Received in revised form 22 June 2010 in which there is neither translational nor orientational long-range ordering of the atomic positions. Nor-
Accepted 23 June 2010
mally, ACPs are the first solid phases, precipitated after a rapid mixing of aqueous solutions containing
Available online 4 July 2010
ions of Ca2+ and PO34 ; however, other production techniques are known. Interestingly, ACPs prepared
by wet-chemical techniques were found to have a relatively constant chemical composition over a rela-
Keywords:
tively wide range of preparation conditions, which suggests the presence of a well-defined local struc-
Amorphous
Calcium orthophosphate
tural unit, presumably with the structure of Ca9(PO4)6 – so-called Posner cluster. However, the
Apatite presence of similar clusters in ACPs produced by other techniques remains uncertain. All ACPs are ther-
modynamically unstable compounds and, unless stored in dry conditions or doped by stabilizers, spon-
taneously tend to transform to crystalline calcium orthophosphates, mainly to calcium apatites. This
solution instability of ACPs and their easy transformation to crystalline phases are of a great biological
relevance. Specifically, the initiating role ACPs play in matrix vesicle biomineralization raises the impor-
tance of ACPs from a mere laboratory curiosity to that of a key intermediate in skeletal calcification. In
addition, due to significant chemical and structural similarities with calcified mammalian tissues, as well
as excellent biocompatibility and bioresorbability, all types of ACPs are very promising candidates for the
manufacture of artificial bone grafts. This review summarizes the current knowledge on the occurrence,
preparation, composition, structure, major properties and biomedical applications of ACPs. To assist read-
ers in looking for the specific details on ACPs, a great number of references have been collected and
systematized.
Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction enamel [9], as well as in the mitochondria [1] and sarcoplasmic


reticulum [10] of some cells. Despite intensive efforts, the accumu-
In nature, amorphous phases exist extensively with readily lated evidence for ACP as an integral mineral component in major
moldable isotropic properties, and serve as structural materials. hard tissues, such as bones and teeth, is equivocal and has for
For example, amorphous structures represent about 20% of approx- many years been the subject of considerable debate [11–24]. Fur-
imately 60 different inorganic compounds or minerals formed by thermore, recent studies on bone and teeth formation have sug-
living organisms. These biologically formed minerals are often gested the presence of transient amorphous mineral precursors
called biominerals, while the process of their formation is called and a universal strategy for calcium carbonate-based [21,25] and
biomineralization [1]. A recent review on the subject indicates that calcium orthophosphate-based [21,22,25–28] biomineralization
many biominerals are formed by amorphous precursors and, fur- in both vertebrates and invertebrates.
thermore, the amorphous phases may possess fluidic properties An interesting study on a potential role of ACP in facilitating
that impart new processing capabilities to the system [2]. Among assembly of hydroxyapatite (HA) nanoparticles into highly ordered
existing biogenic amorphous minerals, those composed of calcium structures has been published recently [29]. Higher-order HA
orthophosphates are most abundant in the teeth and exoskeletal architectures were detected only when the starting particles were
structures of marine invertebrates [2–6]. On the other hand, the aggregates of nanospheres with HA cores and ACP shells. Surface
existence of similar amorphous calcium phosphate (ACP) minerals ACP initially linked HA nanoparticles in a way that allowed a par-
in vertebrate organisms has not been well established experimen- allel orientation of the HA nanoparticles and then was incorpo-
tally except in highly specialized locations such as the inner ear rated into HA by phase transformation to produce more ordered
structures of embryonic sharks [1], mammalian milk [7,8], dental architectures with the characteristic features of apatites in biolog-
ical structures. Further, it was demonstrated that enamel- and
* Tel.: +7 499 255 4460. bone-like apatites could be prepared by using nanodimensional
E-mail address: sedorozhkin@yandex.ru HA and ACP under the control of various biological additives

1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2010.06.031
4458 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

[29]. This study points to an important role ACP might play in bone as amorphous or crystalline materials based on the macroscopic
formation. However, due to a lack of absolute proof, the question as properties such as their external shapes, fracture mechanisms
to the occurrence of ACP phases in newly mineralized tissues of and optical properties long before X-ray diffraction techniques
vertebrates remains unanswered [30]. and other methods became available to reveal the atomic struc-
This review is devoted to amorphous calcium orthophosphates, tures of these materials. Only in the past century has an under-
which are of a great biomedical importance due to their chemical standing of the microscopic nature of amorphous materials
and structural similarities to calcified mammalian tissues. A com- become possible [79]. However, there is still much debate concern-
plete list of available calcium orthophosphates, together with their ing the exact nature of these materials. For example, in a recent
standard abbreviations, chemical formulae and major properties, is article, Sheng et al. [81] state: ‘‘the atomic arrangements in amor-
given in Table 1 [31,32]. Since all of these compounds are calcium phous alloys remain mysterious at present”.
orthophosphates, strictly speaking, all abbreviations in Table 1 are An amorphous structure is distinctly different from a densely
incorrect; however, they are extensively used in the literature. As packed assembly of microcrystals and is closely related to the struc-
the majority of calcium orthophosphates are crystalline, this re- ture of a liquid phase. Ideally, an amorphous solid should be de-
view is devoted to the detailed description of ACP (emphasized scribed by the model of a perfectly random structure [82];
by bold font in Table 1). Furthermore, with a few important excep- however, this is the boundary condition. As such, the structure of
tions, neither ion-substituted ACPs [33–45], nor ACP-containing amorphous solids is normally described in terms of statistical distri-
composites [46–72] are considered and discussed. The readers butions. Nevertheless, prior to a further description, one must spec-
interested in either these topics and/or other types of amorphous ify the existing atomic length scales. The shortest length scale
calcium phosphates (e.g. amorphous calcium polyphosphate [73– usually used to describe the structure of a material consists of an
77] and amorphous calcium metaphosphate [78]) are referred to atom and its nearest neighbors, out to perhaps a distance of two or
the original publications. three atoms. All solids and liquids have some structure on this scale,
which is called a short-range order (SRO). For crystalline solids,
2. Basic definitions and knowledge on amorphous solids structural order persists over much longer distances (at least tens
or hundreds of atomic distances), such that the atoms occupy sites
According to the laws of thermodynamic, a perfect infinite crys- in a periodic three-dimensional array. Such materials are said to
tal cannot exist in the real world. Various disorders in the forms of have a LRO and include most metals and many covalently bonded
vacancies, interstitial atoms, impurities, dislocations, grain bound- solids. Non-crystalline solids, including glasses, lack a LRO and are
aries, surfaces and other interfaces disrupt the periodicity of other- said to be amorphous even though they can have a SRO that is quite
wise ‘‘perfect” crystals and in many cases determine their physical well defined [83].
properties. By contrast, highly disordered solids are those solids According to the available literature, for each particular atom of
that are so irregular that the concept of a reference crystal lattice any solid there exists a SRO of 2–5 Å, a medium-range order
must be abandoned. Such highly disordered materials are called (MRO) of 5–20 Å and a LRO at distances exceeding 20 Å [84,85].
amorphous materials [79]. As wikipedia, the free encyclopedia, In the case of covalent materials, in which a directed chemical bond-
has it: an amorphous (from the Greek term aloquo1, which ing is dominant, a SRO can be characterized in terms of the well-de-
means ‘‘shapeless” or ‘‘without form”) solid is a solid, in which fined coordination polyhedra, which, in many cases, appear to
there is no translational and orientational long-range order (LRO) concur with the unit cells. The definition of a MRO is more conten-
of the atomic positions [80]. Early researchers categorized solids tious and it is helpful to subdivide MRO into three subcategories.

Table 1
Existing calcium orthophosphates and their major properties [31,32].

Ca/P molar Compound Formula Solubility at 25 °C, Solubility at pH stability range in aqueous
ratio log (Ks) 25 °C, g l1 solutions at 25 °C
0.5 Monocalcium phosphate monohydrate Ca(H2PO4)2H2O 1.14 18 0.0–2.0
(MCPM)
c
0.5 Monocalcium phosphate anhydrous Ca(H2PO4)2 1.14 17
(MCPA)
1.0 Dicalcium phosphate dihydrate (DCPD), CaHPO42H2O 6.59 0.088 2.0–6.0
mineral brushite
c
1.0 Dicalcium phosphate anhydrous (DCPA), CaHPO4 6.90 0.048
mineral monetite
1.33 Octacalcium phosphate (OCP) Ca8(HPO4)2(PO4)45H2O 96.6 0.0081 5.5–7.0
a
1.5 a-Tricalcium phosphate (a-TCP) a-Ca3(PO4)2 25.5 0.0025
a
1.5 b-Tricalcium phosphate (b-TCP) b-Ca3(PO4)2 28.9 0.0005
b b d
1.0–2.2 Amorphous calcium phosphate (ACP) CaxHy(PO4)znH2O, n = 3–4.5; 5–12
15–20%H2O
1.5–1.67 Calcium-deficient hydroxyapatite (CDHA)e Ca10x(HPO4)x(PO4)6x(OH)2xf 85.1 0.0094 6.5–9.5
(0 < x < 1)
1.67 Hydroxyapatite (HA, HAp or OHAp) Ca10(PO4)6(OH)2 116.8 0.0003 9.5–12
1.67 Fluorapatite (FA or FAp) Ca10(PO4)6F2 120.0 0.0002 7–12
a
1.67 Oxyapatite (OA or OAp) Ca10(PO4)6O 69 0.087
a
2.0 Tetracalcium phosphate (TTCP or TetCP), Ca4(PO4)2O 38–44 0.0007
mineral hilgenstockite
a
These compounds cannot be precipitated from aqueous solutions.
b
Cannot be measured precisely. However, the following values were found: 25.7 ± 0.1 (pH 7.40), 29.9 ± 0.1 (pH 6.00), 32.7 ± 0.1 (pH 5.28). The comparative extent of
dissolution in acidic buffer is: ACP  a-TCP  b-TCP > CDHA  HA > FA.
c
Stable at temperatures above 100 °C.
d
Always metastable.
e
Occasionally, CDHA is named precipitated HA.
f
In the case x = 1 (the boundary condition with Ca/P = 1.5), the chemical formula of CDHA looks as follows: Ca9(HPO4)(PO4)5(OH).
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4459

At the shortest length scale (5 Å), a near-MRO describes the material changes from a supercooled liquid, with properties one
connections among the coordination polyhedra. At the next length would expect from a liquid state material, to a solid. The tempera-
scale (5–8 Å), an intermediate MRO can be associated with corre- ture at which this transition occurs is called the glass transition
lations between pairs of preferred dihedral angles for neighboring temperature. If a cooling rate is faster than the rate at which atoms
bonds. Finally, on a yet larger length scale (8–20 Å), a far-MRO and/or molecules can be organized into a more thermodynamically
can be associated with the total dimensionality of the covalently favorable crystalline state, then an amorphous solid will be formed.
bonded amorphous network. Fig. 1 [86] represents an excellent In contrast, if atoms and/or molecules have a sufficient time to be
visual demonstration of the differences between a SRO and a MRO. organized into structures with two- or three-dimensional order,
Further details on this topic are available in the literature [84,85]. then a crystalline (at least, a semicrystalline) solid will be formed.
In covalent solids, bond angles and bond lengths, as well as a Furthermore, in many cases amorphous materials can be produced
number of the nearest neighbors, are all part of the appropriate by additives, which interfere with the ability of the primary con-
bonding scheme. Thus, due the nature of chemical bonding, even stituent to crystallize. For example, addition of soda to melted sil-
truly amorphous materials have some structural SRO and, perhaps, icon dioxide results in amorphous window glass, and addition of
some MRO. For example, MRO regions of 15 Å in dimensions and glycols to water results in a vitrified solid [80]. More to the point,
comprising about 100 atoms have been directly observed in amor- amorphization of many solids can achieved by applying mechani-
phous carbon [87]. Some order in two-dimensional projections of cal forces, e.g. by intensive milling [90].
thin amorphous three-dimensional structures was found [88]. In From a thermodynamic point of view, amorphous materials are
addition, covalent amorphous solids were found to exhibit a at best metastable. Given sufficient time, they tend to transform to
MRO at length scales up to 20 Å or so [85]. Such MRO clusters crystalline phases that are thermodynamically more stable. Inter-
are called paracrystals [83]. These paracrystals have a crystalline estingly, when an unstable crystalline solid is transformed to an
topology but the atomic positions are highly distorted from those amorphous phase, this transformation frequently exhibits features
of a perfect crystal. However, in solids there is a serious problem that are associated with ordinary melting, i.e. amorphization fre-
of very small particles. Specifically, if the crystal sizes are extre- quently begins at grain boundaries, surfaces or other defect sites,
mely small, it is difficult to make a distinction between the truly as does ordinary melting. Further, as the transformation proceeds,
amorphous and crystalline solids. Namely, if a powder consists of a sharp interface that separates the amorphous materials from the
tiny perfect crystals with dimensions of 2  2  2 nm (8 nm3) or untransformed crystalline material is always observed [79].
less, both this powder and any bulk materials prepared from this
powder (e.g. by compaction) will be amorphous, simply due to 3. Amorphous calcium phosphates (ACPs)
the fact their sizes are below the minimum value of LRO. Addition-
ally, in very small crystals a large fraction of the atoms are located 3.1. History
at/or near surface. Relaxation of the surface and various interfacial
effects distort the atomic positions, decreasing the structural order. According to Eanes [20], the history of ACPs looks as this: ‘‘In
Thus, even the most advanced structural characterization tech- 1955, Robinson and Watson [91] were the first to suggest that a
niques, such as X-ray, neutron and electron diffraction, as well as substantial portion of newly formed mineral in young bone was
transmission electron microscopy (TEM), have difficulties in distin- not crystalline. Instead, they described this early mineral as being
guishing between the amorphous and crystalline structures on more similar in character to an amorphous-like precipitate they
these length scales [80,89]. had prepared in a study on synthetic HA [92]. This precipitate,
Many studies have revealed that the majority of solids can be which appeared initially in their synthesis when sufficiently con-
found or prepared in an amorphous state. For example, cooling centrated solutions of CaCl2 and Na2HPO4 were mixed at room
strongly reduces atomic and/or molecular mobility. Thus, in princi- temperature and neutral pH, had as its most distinctive features
ple, given a sufficiently high cooling rate, any liquid can be trans- an extremely fine, non-crystalline texture when examined by
formed into an amorphous solid. As cooling is performed, the TEM and no discernible electron diffraction pattern. This latter fea-
ture led them to infer that the considerably more diffuse electron
diffraction pattern of newly formed bone mineral as compared to
more mature bone mineral, although still apatitic in character,
indicated the presence also of an amorphous component” [20].
However, Boskey [19] has reported another story: ‘‘In 1964, Dr.
Paul Tannenbaum, a graduate student in periodontics at Columbia,
and a research assistant in Dr. Posner’s laboratory at hospital for
special surgery, was studying the effect of fluoride on apatite crys-
tal size. He prepared a synthetic apatite by mixing high concentra-
tions (30 mM) of calcium chloride and (20 mM) sodium acid
phosphate in buffer, and, being anxious to confirm that the precip-
itate which formed was apatite, pelleted it by centrifugation, dried
it with acetone and placed it on a holder for analysis by wide-angle
X-ray diffraction (XRD). The pattern obtained (Fig. 2.1, bottom) was
broad and diffuse, with a maximum at 30° 2h had no features,
and was clearly not apatite. Dr. Posner suggested that Dr. Tannen-
baum did not have the settings correct on the X-ray diffractometer,
but since it was late on Friday, decided to correct the settings on
Monday. On Monday, the sample, which had been left on the
diffractometer over the weekend, was again subjected to XRD
analysis, but now the pattern had the appearance of a poorly
Fig. 1. A formation of skydivers illustrates disorder on an intermediate length scale.
Each skydiver has a simple set of rules for bonding to the next skydiver (SRO) but
crystalline apatite (Fig. 2.1, middle). Dr. Tannenbaum was certain
there is a sufficient flexibility for different patterns of ordering to be created on the that the settings on the diffractometer were not different from
scale of a few body lengths (MRO). Reprinted from Ref. [86] with permission. those he had used previously. Instructed by Dr. Posner to repeat
4460 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

Fig. 2. Fragments of: (2.1) XRD patterns (Cu Ka radiation, 0.154 nm) of synthetic ACP (bottom), poorly crystalline CDHA (middle) and highly crystalline HA (top); (2.2)
infrared spectra of ACP (upper), CDHA (middle) and HA (lower). Fig 2.2 is reprinted from Ref. [123] with permission.

the experiment, he observed the same phenomenon. Immediately 3.2.1. Wet chemistry
after being mixed, the precipitate formed was amorphous, while By the early 1970s researchers had already established that the
after several hours, it converted to poorly crystalline apatite. It final and stable product of a reaction between calcium and ortho-
seemed plausible to Dr. Posner that were such an ‘‘amorphous” phosphate salts in neutral or basic aqueous solutions was crystalline
material (i.e. one that did not give a crystalline diffraction pattern) stoichiometric HA. However, stoichiometric, highly crystalline HA
present in bone, along with the apatite, it might account for the could only be prepared at elevated temperatures; thus, in aqueous
broad diffraction pattern of bone mineral” [19] (It should be solutions CDHA is formed instead [31,32]. During CDHA precipita-
emphasized that both Chow et al. [93] and Eanes [94] published tion, over a broad range of solution conditions, an ACP precursor
corrigenda to this story by Boskey). phase is formed [107–117], in some cases, via a short intermediate
In the 1960s, both XRD and infrared spectroscopic techniques stage of octacalcium phosphate (OCP) formation [118,119]. Data
were used to obtain a quantitative estimate of the amorphous con- are available that CDHA crystallization from ACP simply involves
tent of bone mineral, and then, based on the methods used in poly- an increase in LRO in the structure [110]. It should be emphasized
mer chemistry, an algorithm to estimate the amount of ACP in that by the mid-1970s ACP had been found not to be a mandatory
bones was developed [95–97]. Early XRD estimates indicated the precursor to CDHA: in dilute aqueous solutions CDHA was found
presence of 30% or more of a non-crystalline mineral in bones to precipitate without going through an ACP precursor [120]. A mod-
of several animal species. Later estimates by X-ray radial distribu- el was subsequently developed to illustrate factors influencing the
tion analysis placed the upper limit of 10% ACP in bones and nature of non-stoichiometric amorphous precursor phases precipi-
brought into question whether all X-ray amorphous bone minerals tating in highly supersaturated solutions [121].
were truly non-crystalline [17,98–101]. However, further studies The basic approach for synthesizing ACP still consists of a spon-
by higher-resolution techniques have shown that 99% of the min- taneous precipitation by mixing concentrated aqueous solutions of
eral in bone is a poorly crystalline ion-substituted calcium-defi- calcium and orthophosphate ions, first developed in 1953 by Wat-
cient hydroxyapatite (CDHA) of biological origin [19]. son and Robinson [92]. Another commonly used method is to pre-
Morphological evidence establishing the extent of ACP in skeletal pare an acidic (pH 4–5) subsaturated aqueous solution of a calcium
tissues is equally ambiguous. Although some studies [91,102–106] orthophosphate salt (e.g. dicalcium phosphate dihydrate (DCPD))
report a presence of small spheroidal particles atypical of crystalline and afterwards to induce precipitation by rapid addition of a strong
material, primarily in actively metabolizing regions, most TEM stud- base (e.g. NaOH, KOH, NH4OH) to reach the desired solution pH
ies of bones do not even mention finding such possibly amorphous [122]. Vigorous mixing is highly desirable. By means of both ap-
structures. Furthermore, during aging, the amount of ACP in bones proaches, various types of ACPs have been prepared from solutions
and teeth decreases while the crystalline forms of biologically encompassing a wide range of pH (from 6.5 to 13), Ca/P ionic ra-
formed ion-substituted CDHA increase during early stages of forma- tios (from 0.1 to 10), calcium and orthophosphate concentrations
tion [3,11]. Since physical and morphological evidences for ACP in (from 0.002 to 1 mol l1), as well as at temperatures from 0 to
skeletal tissue have been difficult to establish directly, much of our 50 °C. However, the Ca/P ratio of the mixing reagents (classically,
progress in clarifying the possible roles of ACP in biogenic calcifica- Ca(NO3)24H2O and (NH4)2HPO4) is typically kept within 1.50–
tion has come from both synthetic and in vitro studies [20]. 1.67, while the mixing solutions are frequently rendered basic by
addition of NH4OH [46,110,122–126]. At acidic pH, crystalline
3.2. Preparation calcium orthophosphates are normally precipitated. However, in
the presence of stabilizers (Mg and/or citrates), ACP could be
There are various methods for producing ACPs. We will look precipitated at solution pH of 6.0–6.5 [127]. No information on
first at wet chemistry methods, then at mechanical and thermal ACP precipitation from even more acidic aqueous solutions has
techniques. been found in the literature. The obtained precipitates should be
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4461

Fig. 3. XRD patterns of: (3.1) freeze-dried precipitates prepared from aqueous solutions containing different amounts of polyethylene glycol (note a shift of the center of the
broadened peak from 32° toward 31° with increasing polyethylene glycol/Ca molar ratio, which implies some structural differences in the resulting ACPs); (3.2) heat-
treated (800 °C) ACPs prepared from aqueous solutions containing different amounts of polyethylene glycol. Reprinted from Ref. [142] with permission.

collected shortly after the preparation (the sooner, the better), be- example, an ACP was prepared using a mechanochemical method
cause in aqueous media ACP is spontaneously converted to other involving a dry mixture of DCPD and Ca(OH)2 reactants with a
calcium orthophosphates, mainly to CDHA [110,128]. Furthermore, Ca/P ratio of 1.67 [147]. Other authors have shown that a pro-
it was shown that the final calcium orthophosphate (a dry powder) longed high-energy ball milling of either a-TCP, b-TCP powder in
would be amorphous if, beside the appropriate key factors of the ethanol or a dry mixture of ACP and DCPD powders lead to ACP for-
synthesis (a high concentration of reagents, a basic solution pH mation after 24 h [148–150]. However, there is a non-negligible
and a low temperature), both a high addition rate and a mandatory risk of powder contamination (ball wear) when using this process-
freeze-drying of the precipitates were employed [39,110,126,129]. ing route over extended periods to obtain an ACP [30]. Further-
The precipitated ACP phases appear to be spherical (Fig. 4a and b) more, a crystalline to amorphous transition has been detected for
in an electron microscope (diameter 30–100 nm), unlike the nee- various calcium orthophosphates at very high (up to 10 GPa) pres-
dle-like crystals of CDHA (Fig. 4c and d). The solution pH, the con- sures (Fig. 5) [166,167].
centration of the mixing reagents and the preparation temperature
all affect the ACP particle sizes: a higher supersaturation produces 3.2.4. Thermal
smaller ACP particles [123]. Although ACP can be prepared with a Finally, ACPs might be prepared using high-energy processing
Ca/P molar ratio as low as 1.2 (at low pH: see Fig. 6) or as high as at elevated temperatures. This method is based on a rapid quench-
1.7 (at high supersaturation), departure from a Ca/P of 1.5 has ing of melted calcium orthophosphates occurring, for example,
been shown to be due to surface-adsorbed soluble phases, which during plasma spraying of HA [151–163]. A plasma jet, which pos-
can be washed away, or to occluded Ca, respectively [98]. sesses very high temperatures (5000–20000 °C), partly decom-
poses HA. Furthermore, it has been suggested that thermal
3.2.2. Non-aqueous solutions and solvents (sol–gel) spraying produces the amorphous phase, not only due to the high
In addition, ACPs can be easily prepared in either non-aqueous cooling rate, but also because removal of hydroxyl ions makes it
or solvent + water media [125,130–144]. The presence of organic more difficult for the crystalline phase to form [156]. This generally
compounds and/or solvents results in a decrease in the dielectric leads to calcium orthophosphate phases with variable composi-
constant. Therefore, all ions in solutions appear to be less solvated tions, often containing impurities, which is not convenient for
than those in water. The consequence of this is a strong decrease of preparation of pure ACPs. Interestingly, the amorphization degree
solubility and an increase in precipitation kinetics, which simpli- of plasma-sprayed HA coatings appeared to correlate with the
fies amorphization [145]. Furthermore, in such systems, complexes presence of vacancies of hydroxyl ions in the structure of HA: the
of calcium with organic agents can be formed. This favors ACP for- more vacancies there were in the apatite structure due to missing
mation, which is attributed to the coordinated complexing agents hydroxyl sites, the more ACP was present in the resultant coatings
remaining in the structure of ACP [136]. The influence of the pres- [155]. This conclusion was drawn based on the fact that particles
ence of organic solvents to an amorphization degree of precipitated resident in plasma for a longer period of time will lose more struc-
calcium orthophosphates is well illustrated in Fig. 3.1. In some tural water. Other studies have shown more amorphous phase lo-
cases, incorporation of organic compounds into ACPs has been de- cated adjacent to the substrate and a gradient tending towards a
tected [125,142]. Interestingly, replacement of a freeze-drying lower ACP content at the top of the coating [30]. Therefore, in plas-
stage of a wet ACP precipitate by oven drying at 80 °C resulted in ma-sprayed HA coatings, amorphous phases are in intimate mix-
its transformation to CDHA [131]. This process was ascribed to tures with both crystalline calcium orthophosphates and other
an internal hydrolysis of a part of orthophosphate ions of ACP to compounds, such as CaO [9], and so far nobody has succeeded in
those of CDHA according to the schemes [131,146]: separating ACPs from the coatings. However, the amorphous re-
PO3 2  gions in plasma-sprayed HA coatings might be mapped using a
4 þ H2 O ! HPO4 þ OH ð1Þ
scanning cathodoluminescence microscopy technique [159]. Fur-
Ca9 ðPO4 Þ6 þ H2 O ! Ca9 ðHPO4 ÞðPO4 Þ5 OH ð2Þ thermore, due to a number of uncertainties, the reproducibility
of such experiments is poor; thus, the plasma-spaying technique
is not considered as a valuable method to produce ACPs. A flame
3.2.3. Mechanical spray synthesis, in which a liquid precursor solution is fed through
In addition to the aforementioned solution-based methods, var- a capillary into a burning methane/oxygen supporting flame,
ious types of ACPs can be prepared by dry chemical techniques. For seems to be more preferable to produce ACPs at high temperatures
4462 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

Fig. 4. Bright-field transmission electron micrographs of ACP ? CDHA transformation at reaction times of (a) 5 min, (b) 3 h, (c) 9 h, (d) 48 h. Reprinted from Ref. [168] with
permission.

Fig. 6. Ca/P molar ratios of washed (dashed line) and unwashed (solid line) ACP
Fig. 5. Effect of pressure on XRD patterns of HA and hydrated TCP at 1 bar and high precipitates as a function of their formation pH. Reprinted from Ref. [169] with
pressures. Reprinted from Ref. [167] with permission. permission.
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4463

[164,165]. Unfortunately, it remains unclear to what extent the remain unknown and, in the available literature, the various ACPs
high-temperature ACPs are similar to the dry-heated dehydrated are distinguished only by Ca/P ratio. Since most ACPs have a Ca/P
ACPs prepared by wet-chemistry. Further details on and additional ratio close to 1.5 (see below), a term ‘‘amorphous TCP” (ATCP) is
examples of ACP preparation can be found in a recent review [30]. standard in the literature [30,132,146,150,164,175–181]. Other
To conclude this part, one should mention an interesting at- terms such as ‘‘amorphous carbonated apatite” [39,182,183],
tempt to precipitate separately hydroxyapatites of Mg, Ca, Sr and ‘‘amorphous CaHPO4” [132], which is equal to ‘‘amorphous DCPA”
Ba from basic supersaturated orthophosphate solutions containing (Table 1), ‘‘amorphous Ca8(HPO4)2(PO4)4” [132], which is equal to
a 10:6 divalent cation/PO4 molar ratio [170]. The first phase which ‘‘amorphous OCP” (Table 1), ‘‘amorphous OCP” [134], ‘‘amorphous
precipitated in the case of Mg, Ca and Sr had a 3:2 ratio (i.e. that of Ca10(PO4)6(OH)2” [184], which is equal to ‘‘amorphous HA” (Table
a TCP), while only Ba went directly to the stable 10:6 HA phase. 1) and ‘‘amorphous dicalcium phosphate” [185] are rare but have
Precipitated magnesium orthophosphate was amorphous and did been already mentioned. It should be noted that ACPs with the
not convert to a magnesium-deficient HA. Precipitated calcium Ca/P ratio <1.0 currently remain unknown.
orthophosphate formed ACP, which converted to CDHA by a solu-
tion-mediated autocatalytic mechanism [124]. Precipitated stron- 3.4.1. Precipitated ACPs
tium orthophosphate was not amorphous but poorly crystallized Although first described in 1953 [92], quantitative chemical
and readily converted in solution to a strontium HA. Thus, the studies on precipitated ACPs were not reported until 1965, when
smaller alkaline earth cation systems tended to form the more sta- methods were devised to isolate large amounts of unstable solids
ble amorphous 3:2 compounds [170]. for analysis. To minimize changing during sample drying, those
methods utilized filtration and/or centrifugation to wash excess ions
3.3. Morphology of precipitated ACPs from ACP slurries, then freezing wet ACPs under high vacuum to re-
move any remaining entrapped solvent by sublimation [122]. Early
When viewed by TEM, ACP solids precipitated from aqueous chemical studies [128,186] on ACPs prepared at pH 10.5, filtered,
solutions usually have a curvilinear appearance rather than the washed and lyophilized, showed that the Ca/PO4 molar ratio was
faceted and angular shape of crystalline calcium orthophosphates very close to 1.5, suggesting a TCP composition (as Ca3(PO4)2nH2O
[171,172]. However, this curvilinear aspect has only been clearly [97,187]). No OH ions were found in this compound. Furthermore,
established for dried ACP. The morphological form of highly hy- the electron spin resonance spectra of vanadyl (VO2+) ions adsorbed
drated flocculent solids that appear initially in freshly precipitated on ACP formed under varying conditions also revealed that ACP is
ACP suspensions is not known. What is observed when drops of either a non-crystalline form of hydrated TCP or a solid solution with
these suspensions are placed on carbon-coated grids, excess solu- the composition Ca3(PO4)l.87(HPO4)0.2 [188]. Other researchers re-
tion removed and air-dried are irregularly shaped, anastomosing ported a stoichiometry of the precipitated ACP more akin to OCP
aggregates of low-contrast, disk-shaped particles varying widely [130,132,134,189–194], DCPA [132] and DCPD [195]; however, with
in lateral dimensions (from 0.01 lm to >5 lm) [172,173]. These a few exceptions [132,134] the terms ‘‘amorphous OCP” and ‘‘amor-
highly flattened particles represent collapsed, desolvated residues phous DCPD” were not introduced: although the Ca/P ratio of early
of the initial wet ACP flocculates. formed ACP phases was varied between 1.35 and 1.38, which was
As ACP suspensions age, high-contrast particles with a more close to that of OCP (Table 1), the authors of Refs. [192–194] used
spherical aspect begin to appear, initially evolving as bud-like exten- the term ACP2 to explain initial variations in solution pH during
sions from the disks [172,173]. With time, these spherical forms the transformation of ACP into more crystalline phases of OCP
become the dominant shape for ACP. Although generally smaller and/or CDHA. Further, based on the results of TEM analysis, ACP2
(20–300 nm in diameter) than the disks they supplant, the spherical was identified as a separate amorphous phase with a floccular mor-
forms, like the disks, frequently aggregate into irregularly shaped phology and no electron diffraction pattern, if compared with a
and branching clusters. The progression from disk-shaped to ball- spherular morphology of the initially precipitated amorphous phase
like particles most probably represents a spontaneous desolvation (ACP1) [192–194]. The same terms ACP1 and ACP2 were used in an-
in situ or the initial gel-like flocculates into smaller, denser, less-hy- other study [137], in which the authors studied aging of calcium
drated structures [174]. That the spherules are formed in suspension orthophosphate precipitates in methanol at room temperature, fi-
and are not a drying artifact is supported by the crystallization nally leading to formation of a nanosized b-TCP. Furthermore, in pre-
behavior of ACP preparations. Although the evolution of a spherical cipitation experiments from aqueous solutions containing
morphology would be favored during consolidation as this shape polyethylene glycol, two types of ACPs were detected [142]: one
minimizes interfacial tension with the surrounding solution, it also with a broadened peak centered at 31° and another with a broad-
requires that the contracting surface be isotropic. This is possible for ened peak centered at 32° (Fig. 3.1, spectra 16:1 and 4:1, respec-
uniformly curved surfaces only when the enclosed structure re- tively). According to the authors, the first one was similar to the
mains non-crystalline while desolvating [20]. basic structure of b-TCP, while the second one was similar to the ba-
sic structure of HA [142]. A similar shift in the position of the amor-
3.4. Chemical composition phous maximum but obtained at a different aging time of ACP
precipitates (Table 2) has been detected in another study [126].
ACPs should be recognized as a special class of biomedically rel- Additional analyzes of ACPs prepared from aqueous solutions at
evant calcium orthophosphate salts having variable chemical but pH 7.4 with wide variations (from 5:1 to 1:5) in starting Ca/PO4
essentially identical glass-like physical properties. Presumably, molar ratios showed that the compositional Ca/P ratio decreased
all compounds mentioned in Table 1 might somehow be fabricated only slightly from 1.5 due to the presence of small amounts
in an amorphous state. Therefore, perhaps, some time in the future (<15%) of HPO2 4 ions [169,196–198]. This is an indirect confirma-
people will deal with an amorphous phase corresponding to the tion of the existence of an amorphous TCP. The presence of
chemical composition of MCPM (‘‘amorphous MCPM”), an amor- HPO24 in ACP is easy to detect by infrared spectroscopy, most nota-
phous phase corresponding to the chemical composition of DCPA bly by an absorption peak at about 890 cm1, due to a P–O(H)
(‘‘amorphous DCPA”), an amorphous phase corresponding to the stretching mode of protonated orthophosphate species (Fig. 7)
chemical composition of TTCP (‘‘amorphous TTCP”), etc. (in most [127,132]. Furthermore, in infrared spectra of HPO2 4 -containing
cases, stabilization procedures will become necessary), as well as ACPs, there is a large band of a weak intensity near 2300 cm1,
with various mixtures thereof. Currently most such compounds which corresponds to H–O(P) stretching in HPO2 4 ions [132]. More
4464 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

Table 2 presence of 20% of HPO2 4 in two ACP samples [199]. Furthermore,


Position of the XRD amorphous maximum at different aging times of ACP precipitates orthophosphate units close to water and a third (as yet unknown)
[126].
type of orthophosphate group were found in the NMR spectra of
Aging time 2 min 1h 2h 3h 4h 5h 6h the ACPs. Furthermore, substantial differences were noticed be-
2h (°) 29.5 30.1 30.4 30.3 30.5 30.8 29.0 tween the NMR spectra of two ACP samples, donated by two differ-
ent research groups [199]. In other studies, two ACP samples of
Note: The 2h values were derived from the profile analysis of the scattering curves.
Negligible changes were found for longer aged ACPs. The experimental
different chemical composition (since they had been prepared at
error ± 0.5°2h [126]. solution pH 6.5 and 10.0, respectively) were found to give very sim-
ilar extended X-ray absorption fine structure (EXAFS) spectra
[127,200]. The latter indicated that the SRO around calcium ions of
both ACPs is very similar; however, the reasons why the pH differ-
ences were not reflected in the calcium environment remained un-
clear. These experiments confirm the fact that ACPs are not a
single chemical compound but represent a special class of amor-
phous calcium orthophosphate salts. Thus, ACPs cannot be de-
scribed by a single chemical formula; a sketch CaxHy(PO4)znH2O
(Table 1) seems to be the most reasonable element demonstration
of this class of calcium orthophosphate salts. Presumably, ACPs with
the Ca/P ratio exceeding 1.6 should also contain some hydroxide
anions; however, no information on this point has been found in
the literature. However, the presence of CaO is frequently detected
in ACP-containing calcium orthophosphate coatings prepared by
plasma spraying.
Nevertheless, in slightly alkaline aqueous solutions, ACP might
have a well-defined chemical composition. For example, ACP slur-
ries in the pH range 7.4–9.25 appeared to have a nearly constant
solution ion activity product of 1.6  1025 when the solid-phase
composition is postulated to be Ca3(PO4)l.87(HPO4)0.2, i.e. when the
local chemical unit in ACP is postulated to have a Ca/P molar ratio
of 1.45, with 10% HPO2 -
4 , but without OH ions [197]. Thus, in
spite of the absence of LRO, this relative constancy in the composi-
tion over such a wide pH range indirectly suggests that ACP should
have some well-defined local chemical units. However, at solution
pH exceeding 9.25, ACP does not appear to have a nearly constant
solution ion activity product. This breakdown in the solution con-
Fig. 7. Infrared spectra after baseline subtraction for ACPs prepared at: (A) pH 10.0, stancy suggests that the solubility-controlling ions of ACP are sub-
(B) pH 7.0, (C) pH 6.5 and (D) pH 6.0. Reprinted from Ref. [127] with permission. The tly dependent compositionally on the preparation conditions. It is
complete (from 400 to 4000 cm1) infrared spectrum of ACP is available in Ref. [30].
possible that at solution pH > 9.25 the content of HPO2 4 ions no
longer remains constant and gradually decreases with increasing
to the point, the amount of HPO2 pH [122]. Furthermore, at more acidic pH 6.9, ACP precipitates
4 in ACP strongly depends on the
solution pH: the lower the Ca/P ratio for any given ACP precipitate, with Ca/P molar ratios as low as 1.15 have been reported [201].
the greater is its HPO2 These latter precipitates are extremely unstable and rapidly
4 content (Fig. 8).
The presence of HPO2 change into crystalline DCPD. Again, the term ‘‘amorphous DCPD”
4 ions in ACP has been confirmed by solid-
state nuclear magnetic resonance (NMR): the results revealed the was not been used in that study.
Even after lyophilization, solution-matured, spheroidal ACP sol-
ids still retain 15% water by weight [187,202]. A temperature-
programmed description analysis by Sedlak and Beebe indicated
that most (75%) of this retained water was tightly bound inside
the solid, the rest was a more loosely held surface water, with acti-
vation energies of 20.0 and 10.5 kcal mol1, respectively [203].
These results suggest that ACPs do not completely desolvate in
solutions but remain partially hydrated with about three water
molecules per formula unit. Other researchers found that water oc-
curred in regions that were only loosely associated with calcium
cations in ACP [5]. Furthermore, when prepared from carbonate-
containing solutions, ACPs can readily incorporate carbonate an-
ions [34–37,39,204,205]. The amount of carbonate incorporated
at any given pH increases with solution carbonate concentration.
At a given concentration, carbonate uptake also increases with
pH. Incorporating carbonate into ACP does not affect the HPO2 4
content but increases the Ca/P molar ratio. At physiological pH,
the carbonate content of ACP precipitated from solutions contain-
ing 30 mmol l1 carbonates is 3 wt.% [173]. These data suggest
Fig. 8. Acid phosphate content (per cent of total P as (HPO2
that ACP, if present in skeletal tissues, would contain appreciable
4 ) as a function of pH for
washed (dashed line) and unwashed (solid line) ACP precipitates. Reprinted from amounts of carbonates, although less than those present in the apatitic
Ref. [169] with permission. phases of bones [205]. Two other ions that readily incorporate into
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4465

the ACP structure are Mg2+ [36,43,127,170,206–208] and P2 O4 7 characteristic diffuseness resulted from a breakdown in atomic
[205,209,210]. Ions such as P2 O4 7 , carbonate and Mg
2+
increase LRO within the interior of the synthetic ACP [213–215]. The major
the solution stability of ACP and, in the case of the latter two ions, peak positions were found to occur at distances of 2.55, 3.75 and
could possibly play an important role in maintaining the presence 6.40 Å with evidence of a peak at 2.9 Å [213]. Furthermore, any or-
of ACP in skeletal tissue. In addition, other ionic substitutions are derly atomic arrangements did not extend beyond 0.95 nm in
possible; however, such inorganic additives alter the ACP composi- diameter, which corresponds to the smallest values of a far-MRO.
tion, which would enhance the negative effects in the biomedical A contiguous periodic regularity in the distribution of these do-
application of ACP. Besides, with a few important exceptions, mains typical of crystalline materials was absent. EXAFS spectros-
ion-substituted forms of ACP [33–45] are not discussed in this copy [110,216] indicated that possible regularities in the local
review. environment around individual Ca2+ ions were even more circum-
scribed, not extending beyond distances of 0.3 nm (Fig. 9). This is
3.4.2. Other types of ACPs a SRO scale. In addition, an infrared analysis showed a similar lack
Little is known on the chemical composition of ACPs prepared of crystalline order for orthophosphate anions in the ACP structure
by other amorphization techniques. For example, various ACP sam- [30,123,132,218]. The method is based on the observation that a
ples prepared by compressing of several calcium orthophosphates splitting of the P–O antisymmetric bending mode at 550–
at very high pressures revealed collapse of their initial crystal 600 cm1 (Fig. 2.2) increases as crystallinity increases. This appar-
structures but possible changes in their chemical compositions ent lack of crystalline regularity is one of the striking features of
were not investigated [166,167]. Interestingly, the authors found ACP that distinguishes it from other calcium orthophosphates
that in the region below 550 cm1 the infrared spectra of DCPD and provides the structural basis for its name [20].
in amorphous phase resembled that of HA in the crystalline phase, The apparent absence of observable crystalline features in ACPs
and conversely the spectra of DCPD in the crystalline phase resem- does not exclude the possibility that ACPs might have a well-de-
bled that of HA in the amorphous phase [167]. In the case of fined local structural unit. The compositional constancy of ACPs
milling, calcium orthophosphates were found to become amor- over a wide range of preparative solution conditions suggests an
phous; however, no additional phases were detected [149,150]. existence of such a core structure [98]. Several lines of evidence,
Presumably, this means that during amorphization their chemical however, indicate that the local unit of ACPs is not a cryptostruc-
composition remained unchanged. tural variant of one of the crystalline calcium orthophosphates.
Concerning the ACPs formed in plasma-sprayed HA coatings, For example, although in the majority of cases precipitated ACPs
the authors of one study have reported that ‘‘the amorphous phase appear to be closest in composition to TCP, in aqueous solutions
mostly consists of a dehydroxylated calcium phosphate” [163], ACPs transform into either OCP or CDHA but not into TCP. If ACP
which, presumably, meant dehydroxylated HA. If so, the chemical were a cryptocrystalline TCP, direct growth into observably crys-
composition of that particular ACP should be close to amorphous talline TCP by crystal ripening would be expected to occur. The
OA. The authors of another study considered ‘‘that the amorphous aforementioned possibility that ACP may be a cryptocrystalline
phase substance consists of HA molecules” [152]. However, in a CDHA or HA cannot be as easily excluded. Only below pH 9 can
subsequent study, the same authors mentioned that ‘‘the plasma- CDHA be ruled out for the same reason as TCP. However, even
sprayed amorphous phase is an oxyapatite” [153]. No further clar- above this pH, the finding that CDHA forms primarily as an out-
ification has been provided; however, all these authors reached the growth from the surface of ACP suggests that an in situ ripening
same conclusion regarding the apatitic chemical composition of process does not occur. Dissimilarity in composition rules out
the plasma-sprayed amorphous phases. Furthermore, these ACPs ACP as being made up of highly disordered arrays of OCP unit cells.
are definitively anhydrous, contrary to the precipitated ACPs. It is equally unlikely that ACP is an orthophosphate-deficient OCP
To conclude the chemical part, the solubility of ACPs should be as the structural integrity of the latter depends on a full comple-
briefly mentioned. Due to chemical variations, this value cannot be ment of orthophosphate groups in the unit cell. The finding that
measured precisely (Table 1). Several different solubility products the Ca/P molar ratio remains relatively constant at 1.5 over
have been proposed for ACPs and interested readers are referred a wide range of pH (7.4–9.25) appears to exclude ACP being a
to Table 1 of Ref. [30] for details.

3.5. Structure

The atomic arrangement within ACPs depends on the chemis-


try. The first quantitative studies on a synthetic ACP were done
in the mid-1960s on a material precipitated at pH 10 [128,186].
As Watson and Robinson found at neutral pH, the initial phase that
spontaneously formed immediately upon mixing concentrated
alkaline Ca- and PO4-containing solutions was structurally non-
crystalline [92]. The XRD pattern of this rapidly precipitated phase
showed only two very broad and diffuse peaks, typical for sub-
stances that lack periodic LRO [186].
Initially, there were suggestions that a synthetic ACP was, in
fact, HA of such small crystal dimensions that its XRD pattern
was widely broadened to appear amorphous in character. How-
ever, calculated XRD patterns assuming that ACP consisted of small
groups of HA unit cells, or even a single HA unit cell, did not match
the observed ACP diffraction data [211]. The NMR spectra of ACP
are also sufficiently different from HA to suggest that they do not
Fig. 9. Diagrammatic representation of the SRO structure of four phosphate
have the same structural motif [212]. The probability that ACP tetrahedra and two water molecules about calcium ions in an acidic ACP, calculated
was structurally distinct from HA then led to a study of this mate- using the DCPD shell model. The positions of hydrogen atoms are not determined.
rial by the X-ray radial distribution method, which showed that its Reprinted from Ref. [217] with permission.
4466 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

cryptocrystalline mixture of OCP and CDHA. Instead of remaining


constant, an increase in pH would be expected to increase the overall
Ca/P molar ratio of such a mixture as the relative proportion of the
OCP and CDHA components would shift in favor of the latter [20].
Based on the relatively constant Ca/P ratio of ACP formed under
varying conditions, such as different concentrations of calcium and
orthophosphate ions, solution pH and different temperatures, as
well as spectroscopic and structural analyzes, Posner and Betts
hypothesized that the initial solid phase of ACP (more precisely,
of amorphous TCP) precipitated in vitro consisted of spherical par-
ticles ranging from 300 to 1000 Å in diameter with water mole-
cules in the interstices [98,214,215]. In other words, the defining
structural unit of ACP is a spatial subset of the HA unit cell consist-
ing of a central Ca2+ ion coordinated by the oxygens of six sur-
rounding orthophosphate groups, which, in turn, are stabilized
by another eight Ca2+ ions spherically distributed around the outer
boundary of the subset (Fig. 10). This is a neutral ion cluster 9.5 Å Fig. 11. A model of Posner’s cluster (in a circle) showing its relationship with the
in diameter, whose composition is expressed by the formula HA crystal structure. Black lines forming a rhomb delimiting the borders of one unit
cell of HA. Reprinted from Ref. [30] with permission. A similar relationship is also
Ca9(PO4)6. Since then this structure has been defined in the litera- available in Ref. [242]. This correlation between Posner’s clusters and HA structure
ture as a Posner’s cluster. It was later suggested that these clusters was first published in Ref. [98]. One should note that in Ref. [220] another set of
in fact possess a S6 symmetry [219,220]. This subset, which has a atoms of the HA crystal structure has been chosen to represent a Posner’s cluster.
composition close to that of TCP, is linearly expanded by 3% and
its radial distribution function is similar to that calculated from
the diffuse XRD profile of ACP. The authors postulated that at the faces associated with [Ca3(PO4)2]n clusters were analyzed in detail
far-MRO level these slightly enlarged spherical subsets are ran- using ab initio calculations for n = 1–4 [223]. The energy criteria
domly clustered in ACP particles (Fig. 10) with water filling the were found to favor the Posner’s cluster, which is the core of the
intervening spaces [98,214,215]. No data have been found in the actual structural model of ACP (more precisely, of amorphous
literature to suggest that plasma-spayed ACPs cannot contain Pos- TCP). Moreover, the calculations showed that an aggregation of
ner’s clusters as the structural units; however, undoubtedly, they the clusters corresponded to a large energy stabilization irrespec-
cannot contain water molecules in the intervening spaces. Interest- tive of the cluster considered [223], which is in agreement with
ingly, an atomic arrangement of Posner’s clusters appears to be Posner’s hypothesis in which clusters are proposed to be closely
analogous to that existing in several other calcium orthophos- packed in ACP structure. Furthermore, the vibrational spectra of
phates, such as HA, OCP and b-TCP. For example, the relationship ACP have been modeled as well [224]. In spite of this, it is not quite
between the original Posner’s cluster and the atomic structure of clear from what experimental spectroscopic or structural data the
HA is represented in Fig. 11. However, it should be stressed that cluster model (Fig. 10) has been derived, except the TCP composi-
this is only a model because the arrangements of clusters in larger tion. Further, it is not entirely clear whether there are volumetric
structures are not known. regions within the large 300–1000 Å particles that are free of the
In general, an electrostatic interaction, a hydrophobic interac- 9.5 Å clusters. In other words, are there some volumetric regions
tion and a ‘‘cementing” effect of water molecules are among the within the large 300–1000 Å particles where the atoms are com-
main factors holding ions of a cluster together [221]. Again, a pletely random with no SRO? Moreover, the results of EXAFS anal-
‘‘cementing” effect of water molecules is applied for the ACPs pre- ysis indicated that the range of orderly Ca–Ca and Ca–P
cipitated from aqueous solutions only. Theoretical investigations interactions in ACP were much shorter than would be predicted
on the stability of different calcium orthophosphate clusters with from the Betts and Posner model [110,216]. Another weakness in
an increasing number of ions have confirmed that Posner’s clusters the model is that water only serves to fill the interstices between
are the most stable arrangement [222]. The potential energy sur- the HA subsets and is not an essential part of the ACP structure,
contrary to the assumptions by Sedlak and Beebe [203]. Both of
these weaknesses could possibly be remedied if a smaller spatial
domain is carved from a portion of the OCP unit cell that contains
part of the hydration layer. However, this suggestion has not been
examined in detail. It is also possible, however, that the defining
structural unit for ACP is a truly unique entity with no satisfactory
crystallographic model. In fact, there is no a priori need for such a
model, as the defining unit for ACP would not be constrained struc-
turally by the symmetry requirements for crystalline arrange-
ments. Therefore, ACP could have a structural motif not found, or
even permissible, in any of the crystalline calcium orthophos-
phates [20].
More to the point, mechanisms of calcium and orthophosphate
ion association in aqueous solutions have been elucidated by
means of quantum and classical molecular mechanics simulations
[225]. A special focus was dedicated to the role of the protonation
state of orthophosphate ions and depronation of the hydrogen
orthophosphate ions appeared to be necessary during crystal
growth. According to the simulation results, a triple ion [Ca2+(H-
Fig. 10. A model of an ACP particle, showing one Posner’s cluster. Reprinted from PO4)2Ca2+]2+ could form in aqueous solutions and subsequently
Refs. [19,99] with permission. yield another triple ion [Ca2+(PO4)3Ca2+]+ by releasing a proton.
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4467

The latter ion was suggested to be the smallest stable aggregate, of hydroxyl-depleted areas of the amorphous phase to OA and
and to contain an entirely deprotonated orthophosphate ion 230 kJ mol1 for crystallization of hydroxyl-rich areas of the amor-
[225]. Some indirect experimental evidence in support of this phous phase to HA [163]. Therefore, hydroxylated ACP regions were
hypothesis has been obtained recently [226]. Nevertheless, verifi- found to crystallize more readily compared to the dehydroxylated
cation of the aforementioned structural models of ACPs will ones. A heat of recrystallization of ACP to HA was calculated to be
require details of the chemistry and the processing conditions to- 43 kJ mol1 in yet another study [161].
gether with viewing techniques at the atomic scale. Unfortunately, To conclude the thermal part, the results mentioned in this sec-
no additional information on the structure of high-temperature tion appear to be additional indirect confirmation of the fact that
ACP phases has been found in the literature. Thus, for want of any- ACP is not a single chemical compound but represents a special
thing better, one is forced to assume, that except for water mole- class of amorphous calcium orthophosphate salts.
cules and the possible presence of HPO2 4 ions, all the
aforementioned is valid for ACPs produced using plasma-sprayed 3.7. Amorphous-to-crystalline transformations in aqueous solutions
coatings. This assumption should clearly be verified.
To conclude the structural part, determination of the specific As previously stated, in the vast majority of cases, ACPs are the first
surface area of ACPs generally leads to surprisingly low numbers. solid phases to appear upon mixing of calcium- and orthophosphate-
This might be related to the larger spherical associations of Pos- containing aqueous solutions at pH > 7 and concentrations
ner’s clusters and the apparent hindrance to nitrogen adsorption sufficiently high to produce an immediate precipitation. The sponta-
of these inner surfaces [180,202]. neous formation of ACPs is a kinetically driven process. A rapid mixing
of highly concentrated solutions creates sufficiently strong stochastic
3.6. Thermal properties interactions between the ions and they quickly coalesce into irregu-
larly coordinated, highly hydrated clusters large enough to separate
All types of ACP are thermally unstable and sustain neither cal- from solution in a gel-like state before they have a chance to rearrange
cining nor sintering. If precipitated ACPs are heated, they first of into orderly nuclei capable of growth as crystals. This structural
all lose water. Two types of water loss occur, corresponding to arrangement, however, is inherently unstable. In addition to desol-
loosely bound water molecules adsorbed on the surface of ACP vating, ACPs kept in solution eventually disappear, being supplanted
agglomerates and more strongly bound internal water molecules, by more stable crystalline phases such as OCP or CDHA [20].
respectively. The first loss is essentially reversible, whereas the sec- Watson and Robinson in their pioneering study on ACP [92] were
ond is mostly irreversible [203,227]. Furthermore, in experiments the first to observe the transient nature of ACP when kept in contact
where water interactions were minimized by allowing for the with its preparative medium. They found that electron diffraction
escape of volatile components, crystallization of ACP was found to patterns of ACP taken later in the precipitation reaction were no
begin at about 530 °C [187]. Below this temperature, the non- longer diffuse but resembled patterns of a poorly crystalline CDHA.
crystalline features of ACPs seemed thermally stable. The first crys- Further investigations revealed that this amorphous-to-crystalline
talline phase to appear was invariably b-TCP. However, between 600 transition was not gradual but occurred rather precipitously. Ini-
and 800 °C, depending upon the preparation, a-TCP was found to tially, there is a period of a relative stability, where surfaces of the
become generally the favored ignition product, even though b-TCP high-contrast spherules generally remain smooth and regular
is normally the stable phase up to 1200 °C. Neither washing/drying [172]. However, as seen from the data of Table 2 [126], some changes
procedures employed to isolate the amorphous material, nor the occur with solid ACP during this time. Afterwards, the transition fol-
choice of soluble orthophosphate salt used in its preparation, were lows a sigmoid evolution with the solid phase rapidly progressing
found to have any significant effect on the thermocrystallization from being barely crystalline to where the amorphous features dis-
properties of ACP [187]. However, in other studies, both a-TCP appear. Once the first crystals appear on the surface of the spherules,
[139,164,165,180] and carbonated CDHA [133] appeared to be the the transition proceeds rapidly to completion. Simultaneously, dra-
first detectable crystalline phase obtained from heating various matic declines in ionic concentrations of calcium and orthophos-
ACPs to 550–600 °C. Interestingly, the presence of organic solvents phate ions occur in the mother solution. The time it takes to reach
(in that case, polyethylene glycol) at the ACP preparation stage this amorphous-to-crystalline boundary varies considerably with
was found to influence the products formed at elevated tempera- the preparation conditions, being particularly sensitive to tempera-
tures [142]. Namely, when the amount of polyethylene glycol was ture and pH [66,124]. For example, at pH 7.4, ACP converts five
small, a-TCP was formed on heating; when the amount of polyeth- times faster at 37 °C than at 20 °C [229]. The pH dependency is some-
ylene glycol was large, b-TCP was formed on heating; biphasic what more complex than that for temperature. Namely, at 25 °C the
(a-TCP + b-TCP) formulations were formed when the amount of aqueous lifetime of freshly precipitated ACP is less than 0.3 h at pH
polyethylene glycol was average (Fig. 3.2). Similar effects of both 7.4. It increases to a maximum lifetime of over 9 h between pH
aging time and the solution pH were also detected [143]. 10.0 and 10.5, then rapidly decreases until at pH 12.8 the lifetime
ACPs with Ca/P ratios of 1.00 (‘‘amorphous DCPA”) and 1.34 is nearly as short as that at pH 7.4 [197,230]. The solution lifetime
(‘‘amorphous OCP”) were found to remain amorphous after heating of ACP can be greatly extended by inclusion of simple inorganic ions
to 600 °C, while crystalline compounds (b-Ca2P2O7 in the case of Ca/ such as Mg2+, Zr2+, silicates, carbonates and pyrophosphates
P = 1.00 and a-TCP + b-Ca2P2O7 in the case of Ca/P = 1.34) started to [39,40,42,127,170,194,206–209,231,232]. As an extreme example,
appear at 620 °C [132]. In the same study, ACP with Ca/P ratio of 1.51 ACP prepared from Mg2+-containing solutions at pH 10.0 and
(‘‘amorphous TCP”) was found to remain amorphous at heating up to 32.5 °C remained in a gel-like amorphous state for up to 20 weeks
550 °C, while crystalline compounds (b-TCP) started to appear at when the reactant Mg/Ca molar ratio was set at 0.2 [170]. Other sub-
600 °C. Interestingly, heating of a crystalline DCPA (monetite) stances can increase the stability of ACP in aqueous solutions include
leads to c-Ca2P2O7 and this phase is then transformed at 750 °C F [229,230], various polyelectrolytes [65,66], polyalcohols and
to b-Ca2P2O7 [228]. Thus, amorphous DCPA showed a thermal polyglycols [125,136,138], phospholipids [233], dentin phospho-
behavior different from that of crystalline DCPA [132]. protein [234], phosvitin [210,234], glycochenodeoxycholic acid
Furthermore, the crystallization of ACPs is an exothermic pro- [235], biomacromolecules such as casein phosphopeptide [67], as
cess. The heat produced was found to be 21 kJ mol1, while the well as adenosine di- and triphosphates (but not the monophos-
activation energy was 450 kJ mol1 [157]. Other researchers phate) [236,237]. On the other hand, an excess of Ca2+ ions in the
reported activation energy values of 440 kJ mol1 for crystallization solution accelerates the transformation of ACP into a crystalline
4468 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

CDHA [168]. Interestingly, collagen, which is the principal matrix To conclude this part, as ACPs represent a special class of cal-
protein in skeletal tissues, has no effect on ACP stability [229,234], cium orthophosphates, it is reasonably to assume that several
while the presence of gelatin promotes the transformation of ACP amorphous-to-crystalline transformation mechanisms might exist
into crystalline phases [47]. It should be noted that in a wet atmo- due to the initial differences in the investigated ACPs. Furthermore,
sphere solid ACP with a Ca/P ratio of 1.33 (amorphous OCP) was various competitive processes might occur simultaneously and
found to recrystallize into a mixture of CDHA + DCPA [134]. their importance might depend on the conversion conditions. Fur-
Conversion of ACPs to solid phases with XRD patterns similar to ther details on this topic are available in the literature [30,117].
that of CDHA has been studied largely in aqueous suspensions
[65,66,110,113,115,124,126,146,168,176,181,186,208,238–241]. 4. ACP in vivo
Transmission electron micrographs of this transformation are
shown in Fig. 4. The kinetics of this process can be described by As stated in the Introduction, both physical and morphological
the following empirical equation: dC/dt = k1 + k2C, where C is the evidence for the presence of ACPs in skeletal tissue has been diffi-
fraction of ACP converted into the crystalline phase by time t, k1 cult to establish directly, and the validity of inferential evidence for
is a rate constant associated with the nucleation of the first crystals the presence and amounts of ACPs has been the subject of consid-
and k2 is an autocatalytic rate constant indicative of the observa- erable debate. Indirect assessments, such as by XRD methods, have
tion that the transition rate is proportional to the mass fraction al- produced widely varying estimates of the amorphous content of
ready crystallized and not to the fraction of remaining ACP bone mineral, placing it at less than 1% [19] to more than 30%
[128,186]. Numerical values for k2 appear to be much larger than of the total mineral mass, the rest presumably being poorly crystal-
those for k1, reflecting the exponential rapidity of the transition line ion-substituted CDHA (biological apatite). Even if ACPs only
once this has started [186]. occur at a lower percentage, one would expect TEM to reveal some
The amorphous-to-crystalline transformation mechanisms of evidence [9], but, as stated earlier, most TEM studies of bones and
ACPs have not been well elucidated. Chemically, this process is de- teeth do not even mention the existence of amorphous-like struc-
scribed by Eqs. (1) and (2), while in the reality it might proceed tures that could be ascribed to ACPs. However, the absence of such
along several pathways. For example, it might occur via dissolution structures could have been a negative artifact caused by aqueous
of ACP and reprecipitation of crystalline phases (e.g. CDHA) dissolution of more labile ACPs during the sample preparation for
[20,112,115,122,124,146,181,238], internal structural rearrange- examination. A few early TEM studies [102,103] that avoided aque-
ments [168,181,208,219,242,243], development of a LRO without ous processing of bone specimens by directly embedding and
changing the immediate environment of Ca [110], formation of sectioning freeze-dried material revealed a zone of electron dense,
crystalline phases directly within the ACP phases [141,226], or 6–20 lm diameter spheroidal bodies adjacent to crystal-rich areas
self-aggregation and surface-mediated transformations [30]. For of bone tissue. Electron diffraction of these sites revealed a hazy,
example: ‘‘When the density reached a critical value, the random diffuse pattern similar to that observed by Watson and Robinson
arrangement of growth units became disadvantageous in terms [92] in their synthetic, amorphous-like precipitates. Whether or
of total free energy, resulting in a sudden regularization of the not these spheroidal particles represented ACPs has been much de-
structure, which was deposited as HA” [219]. Furthermore, Yin bated, but the labile nature of ACPs suggests the need to maintain
and Stott suggested that, in the transformation from ACP to CDHA, carefully the anhydrous conditions in order to preserve this phase
ACP needed only to dissociate into clusters rather than undergo in calcified material for examination [20].
complete ionic solvatation [244]. Another possibility that could account for the inability to estab-
Having summarized findings of their own and previously pub- lish with certainty the existence of ACPs in bone is that the ACP
lished results by other researchers, Wang et al. [226] depicted does not exist as separate particles but, instead, as an amorphous
the following picture of the main events that take place during layer on crystals of the poorly crystalline phase of biological apa-
the induction period and finally trigger the rapid precipitation of tite. Although there has been no direct evidence for this possibility,
calcium orthophosphates from supersaturated aqueous solutions: similar coatings appear to form in physiological-like solutions
‘‘Calcium and orthophosphate ions form pairs and clusters succes- seeded with HA [245,246]. Under these in vitro conditions, the
sively in the first few seconds. These ions, pairs and clusters then initial accretions appeared to form an amorphous calcium carbon-
compose the initial solid phase that is heavily hydrated and con- ate-ACP coating on the seed crystals. The initial phase also incorpo-
tains hydrogenorthophosphates. Growing and aggregating, the so- rated a small amount of Mg2+ ions from solution that was
lid increases in size and quantity without affecting the solution pH. subsequently released upon the ancillary formation of CDHA crys-
During the induction period, the solid particles exhibit a steady tals. This Mg2+ behavior, consistent with the amorphous-to-crys-
size distribution, which is around 300 nm at the early stage and talline transition, is the most compelling compositional evidence
shifts toward 1000 nm with time. These particles are agglomerates from these studies for the initial coat being an amorphous layer
of primary particles of 60–100 nm in diameter and are originally [245].
amorphous in structure. At multiple sites inside a particle, crystal- A proper assessment of the possibility that some of the mineral
line domains develop from ion pairs and/or clusters by taking up in skeletal tissues is in a free-standing amorphous state is further
calcium and releasing hydrated proton, possibly through a stage complicated by the fact that the minimum ion activity product
at which the more compact cluster Ca9(PO4)6 presents. Since the needed to form ACP de novo in physiological-like synthetic solu-
expansion of crystalline domains consumes surrounding calcium tions at pH 7.4 is considerably greater than that calculated for ser-
and orthophosphate ions (or their pairs and clusters) and releases um [247,120]. If extracellular skeletal fluids were in electrolyte
hydrated protons, the mechanic strength decreases in the inter-do- equilibrium with serum, it would appear unlikely that ACPs could
main regions. Finally, under the action of the shearing strength of form in vivo except possibly as a coating on crystals of biological
the fluid, these primary particles collapse and the liberated crystal- apatite of bones. Without stronger evidence than that described
lites induce the rapid precipitation of calcium orthophosphates, to- above, even this possibility is highly speculative. However, there
gether with the previously trapped hydrated protons in primary is some evidence that suggests that calcifying bone matrix may
particles, resulting in the abrupt pH drop. Indeed, it is the crystal- be compartmentalized with the establishment of an interior milieu
lization at multiple sites inside amorphous particles that finally different from that of serum [248]. Unfortunately, it is not known
triggered the rapid precipitation of calcium phosphate from the whether such compartmentalization results in an extracellular
supersaturated solution” [226]. fluid space capable of initiating de novo ACP formation [20].
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4469

Although the general compartmentalization of bone-producing with considerable amounts of TTCP and trace amounts of a-TCP
conditions favorable for ACP needs to be further established, a con- [264]. Therefore, these cases can hardly count as biomedical appli-
siderable body of evidence suggests that local micro-compart- cations of ACPs.
ments exist in bones that could allow for ACP development. The There is another type of calcium orthophosphate coating, ob-
most thoroughly studied of these micro-spaces are the mem- tained by a biomimetic route. Some of these coatings contain
brane-enclosed aqueous cores of matrix vesicles [249,250]. Most ACP as the major component [265–268]. For example, an ACP coat-
commonly found near osteoblasts in the extracellular regions of ing on polyethersulfone plates was prepared by precipitation from
rapidly mineralizing embryonic bone, these spherical bodies of cel- a simulated body fluid (SBF) at 35 °C. It took the authors 12 days to
lular origin are the sites of initial mineral formation [251]. Preced- obtain a 20 lm thick ACP coating and 28 days for a 50 lm thick
ing appearance of the first crystals at these sites is an accumulation one [265]. Similar results were obtained in other studies
of calcium and orthophosphate ions within the aqueous cores of [266,267]. One should stress that, in the vast majority of cases,
the vesicles to levels that far exceed the threshold level for de novo the biomimetic approach presumes application of SBF, which con-
ACP formation [252,253]. Studies with synthetic liposomes confirm tains ions of sodium, magnesium, potassium, sulfate and hydrog-
that ACP should readily form under such compartmentalized con- encarbonate; thus, ion-substituted ACPs are always formed as a
ditions [254]. However, Raman spectroscopic data indicate that result.
ACP in matrix vesicles is not in a pure chemical state but instead Furthermore, ACP coatings can be deposited on titanium using
calcium and orthophosphate ions are combined in a single-phase an electrochemical technique at 36 °C and solution pH 6.4 [268].
complex with lipid and protein moieties found within the vesicles The obtained ACP coatings appeared to be unstable and trans-
[255]. Also consistent with formation of an amorphous precursor formed into those of CDHA. By choosing the electrochemical
phase are infrared and Raman spectroscopic findings that the first parameters, a homogeneous coating of ACP, CDHA or some inter-
crystals in matrix vesicles are OCP and not CDHA [255,256]. These mediate phases could be achieved, thus allowing formation of
crystals, in turn, penetrate the enclosing membrane and initiate a the coatings with different morphologies and solubilities [268].
chain of crystallization events that appears to trigger the mineral- Among the self-setting calcium orthophosphate cements and
ization of the collagenous matrix. Thus ACP, even when present in concretes [258,259], there is only one ACP-based formulation cur-
small amounts, may be an important initiating factor in the calci- rently on the market, namely BiobonÒ (a-BSMÒ) [269,270]. This ce-
fication of skeletal tissues [20]. ment comprises a mixture of ACP (50 wt.%) and DCPD (50 wt.%)
In addition to the questionable cases of calcified tissue, ACPs are which is mixed with an appropriate amount of aqueous medium
found in mammalian milk [7,8,70]. Clearly, the presence of the (deionized water or saline) with a liquid to solid ratio of 0.8 ml g1
most easily biodegradable calcium orthophosphates in the form at room temperature. An injectable paste is obtained which sets in
of ACPs in milk is necessary to construct skeletons of young less than 20 min at 37 °C. After hardening, the cement is consti-
organisms. tuted of nanocrystalline CDHA with crystal dimensions close to
those in human bones [30]. A similar cement formulation but addi-
5. Biomedical application of ACPs tionally containing mechanoactivated b-cyclodextrins has been
studied as well [147]. Furthermore, an ACP + DCPA cement formu-
Currently, biomaterials and bioceramics of calcium orthophos- lation is known [271]. ACP can also be added as an admixture
phates are available in various physical forms: powders, particles, phase to standard calcium orthophosphate cement formulations
granules, dense blocks, porous scaffolds, injectable formulations, [43,272]. In such cases, addition of ACP resulted in cements exhib-
self-setting cements and concretes, implant coatings, as well as iting shorter setting times, a compressive strength suitable for
composite components of different origin (natural, biological or non-load-bearing applications and full conversion to nanocrystal-
synthetic) often with the specific shapes, such as implants, pros- line CDHA. Moreover, ACP-containing formulations demonstrated
theses or prosthetic devices [31,32,257]. In principle, all these good cell viability, making them suitable candidates for biomedical
physical forms should apply to ACPs; however, not all of them have applications [272]. More to the point, amorphous TCP nanoparti-
yet been realized. It is easy to prepare ACPs in powder form by a cles of 13, 19 and 40 nm diameter were found to be highly reactive
wet-precipitation technique (see Section 3.1); however, manufac- and set to CDHA within minutes, if compared with microcrystalline
turing of other physical forms of ACPs is not so simple. Further- powders of both a-TCP and b-TCP [178,179]. A similar approach
more, for use in surgery, all implantable 3-D constructions must was reported in other studies [148–150].
possess the necessary mechanical properties, which is difficult to Since ACPs do not sustain heating above 600 °C (see Section
achieve in the case of ACPs. For example, both dense blocks and 3.5), the possibility of preparing dense ACP bioceramics was stud-
3-D porous scaffolds made of calcium orthophosphates achieve ied using spark plasma sintering at temperatures ranging from 150
their desired mechanical properties only after sintering at temper- to 200 °C with processing times of <15 min. Unfortunately, the ob-
atures exceeding 1000 °C, which is impossible in the case of ACPs served mechanical strength of the prepared consolidated disks
(see Section 3.5). Furthermore, an ACP powder might be easily (sintered for 6 min at 150 °C) was poor [273]. However, an increase
added as a component to self-setting cements and concretes; how- in the sintering processing time to 13 min indicated a possibility of
ever, no cement formulation is known which results in ACP forma- improving the mechanical properties. The low-temperature condi-
tion as the major end-product [258,259]. More to the point, ACPs tions appeared to be more adaptive to the processing of ACP when
are present as components of various calcium orthophosphate compared with the experimental conditions in conventional sin-
coatings; however, only a few studies are known in which ACP tering. However, the physicochemical characterization of the pre-
coatings have been fabricated [260–263]. pared consolidated bioceramics indicated a crystallization of the
Not many examples of biomedical application of ACPs are cur- initial ACP to an apatitic phase with no other detectable crystalline
rently available. Most cases of plasma-sprayed calcium orthophos- phase [30].
phate coatings should be excluded because the coatings consist of However, the majority of cases of the biomedical applications of
a complex mixture of various phases, of which ACP is just one ACPs comprise various biocomposites and hybrid biomaterials
[9,151–163]. Furthermore, both the amount and the composition containing ACP as one of the phases [47–72,147,274–285]. Several
of amorphous phases cannot be accurately controlled. Even in ACP-containing formulations (e.g. Recaldent™ and Enamelon™)
the case, when ACP was plasma sprayed, the coating was found are now commercially available [285]. As can be seen from these
to be a mixture of a crystalline carbonate-containing apatite phase references, most of the applications are in the field of dentistry.
4470 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

In all cases, addition of calcium orthophosphates, including ACPs, a SRO) matter? A good attempt to discuss this topic is available in
imparts both biocompatibility and bioactivity to the biocomposites the literature [312], and interested readers are referred to this.
[278]. For example, to improve cell adhesion, a hydrophilic array of To conclude, the biomedical applications of ACPs are currently
ACP was fabricated on a surface of hydrophobic polystyrene [286]. limited due to the great difficulties in consolidating these materials
A similar effect was found when coatings composed of ACP and into 3-D structures, which have to possess sufficient mechanical
hyaluronic acid were used [287]. Furthermore, in the acidic oral properties. As ACPs do not sustain heating at temperatures exceed-
environment the ACP-containing biocomposites take advantage ing 600 °C, they must be consolidated by low-temperature tech-
of the ability of ACPs to release calcium and orthophosphate ions, niques only. Spark plasma sintering is one such method [30,273];
which potentially can take part in enamel remineralization [49– another comprises mixing of thermally unstable calcium orthophos-
64,276,277,179,288–307]. Such ACP-containing biocomposites phates with water-soluble porogens, followed by cold isostatic
and hybrid biomaterials might be coatings [279] and cements pressing of the prepared mixture and dissolving the porogens
[147,274,275]. In dentistry, the ACP-containing formulations are [313]. Obviously, other consolidation approaches are possible, and
used mainly as anticariogenic and/or remineralizing agents [288– these need to be developed.
307], e.g. in chewing gums [291–293], sugar confections [71], var-
ious tooth mousses [294–296], bleaching gels [297,298], various Acknowledgements
drinks [299,300] or even in milk [304,305]. Furthermore, ACP-con-
taining formulations are used in orthodontics [280–284]. Many thanks to various contributors for their kind permissions
to reproduce figures. Special thanks to Dr. Marc Bohner for sending
6. Conclusions me a copy of ‘‘Amorphous calcium phosphate”, a chapter from
Eanes [20].
ACPs can be both found in living organisms (especially inverte-
brates) and synthesized in the laboratory. Due to their chemical
variability, they should be recognized as a special class of calcium Appendix A. Figures with essential colour discrimination
orthophosphates offering a wide variety of compositions. Presum-
ably, all known calcium orthophosphates (see Table 1) might be Certain figures in this article, particularly Figures 1 and 3, are dif-
prepared in an amorphous state; however, not all of them (espe- ficult to interpret in black and white. The full colour images can be
cially, those with Ca/P ratio <1.1) have been prepared. Further- found in the on-line version, at doi:10.1016/j.actbio.2010.06.031.
more, in the available literature nothing has been found on the
existence of amorphous TTCP. The vast majority of available publi- References
cations on ACPs in fact describe wet-precipitated compounds with
Ca/P ratio close to 1.5, i.e. amorphous TCP. As a Poster’s cluster [1] Lowenstam HA, Weiner S. On biomineralization. New York: Oxford University
has the chemical composition of TCP, it might be located in the Press; 1989.
[2] Gower LB. Biomimetic model systems for investigating the amorphous
atomic structures of HA (Fig. 11), TCPs and OCP. As TTCP and all precursor pathway and its role in biomineralization. Chem Rev
acidic calcium orthophosphates have Ca/P ionic ratios far away 2008;108:4551–627.
from 1.5, presumably all of these calcium orthophosphates in an [3] Lowenstam HA, Weiner S. Transformation of amorphous calcium phosphate
to crystalline dahillite in the radular teeth of chitons. Science 1985;227:51–3.
amorphous state should contain structural units that are different [4] Stricker SA, Weiner S. Amorphous calcium phosphate in the stylets produced
from Posner’s clusters. Nothing on this point has been found in the by a marine worm (Nemertea). Experientia 1985;41:1557–9.
available literature. Obviously, this topic needs to be investigated [5] Mitchell PCH, Parker SF, Simkiss K, Simmons J, Taylor MG. Hydrated sites in
biogenic amorphous calcium phosphates: an infrared, Raman, and inelastic
in the future. neutron scattering study. J Inorg Biochem 1996;62:183–97.
In aqueous solutions, all currently known ACPs are easily con- [6] Becker A, Ziegler A, Epple M. The mineral phase in the cuticles of two species
verted into crystalline phases of calcium orthophosphates, espe- of Crustacea consists of magnesium calcite, amorphous calcium carbonate,
and amorphous calcium phosphate. Dalton Transactions 2005;10:1814–20.
cially into poorly crystalline CDHA. Therefore, advantage can be [7] McGann TCA, Buchheim W, Kearney RD, Richardson T. Composition and
taken of ACPs’ high reactivity to prepare various bioactive bioma- ultrastructure of calcium phosphate–citrate complexes in bovine milk
terials. Currently, ACPs are involved as transient or constitutive systems. Biochim Biophys Acta 1983;760:415–20.
[8] McGann TCA, Kearney RD, Buckheim W. Amorphous calcium phosphate in
phases in several commercial substitute bone materials, such as
casein micelles of bovine milk. Calcif Tiss Int 1983;35:821–3.
plasma-sprayed coatings on metal prostheses and injectable ce- [9] Brès EF, Moebus G, Kleebe HJ, Pourroy G, Werkmann J, Ehret G. High
ments for orthopedic applications. ACPs are also used for dental resolution electron microscopy study of amorphous calcium phosphate. J
applications as fillers in ionomer cements to fill cavities or as col- Cryst Growth 1993;129:149–62.
[10] Raeymaekers L, Agostini B, Hasselbach W. The formation of intravesicular
loidal suspensions in toothpastes, chewing gums or mouthwashes calcium phosphate deposits in microsomes of smooth muscle: a comparison
to promote remineralization of carious lesions and/or to prevent with sarcoplasmic reticulum of skeletal muscle. Histochemistry
tooth demineralization [30]. Obviously, these examples are just 1981;70:139–50.
[11] Termine JD, Posner AS. Infrared analysis of rat bone: age dependency of
the initial steps of the biomedical applications of ACPs. amorphous and crystalline mineral fractions. Science 1966;153:1523–5.
As noted above, the atomic structure of amorphous compounds is [12] Termine JD, Wuthier RE, Posner AS. Amorphous–crystalline mineral changes
difficult to investigate due to the lack of LRO. Thus, in some of the during endochondral and periosteal bone formation. Proc Soc Exp Biol Med
1967;125:4–9.
aforementioned studies on ACPs, the ‘‘amorphous” character of the [13] Eanes ED, Termine JD, Posner AS. Amorphous calcium phosphate in skeletal
phases might be due to the formation of nanodimensional and/or tissues. Clin Orthop Relat Res 1967;53:223–35.
nanocrystalline calcium orthophosphates [308,309] with crystal [14] Tannenbaum PJ, Schraer H, Posner AS. Crystalline changes in avian bone
related to the reproductive cycle. II. Percent crystallinity changes. Calcif Tiss
dimensions of 2  2  2 nm (8 nm3) or so (see Section 2). Employ- Int 1974;14:83–6.
ment of novel high-resolution techniques raised doubts as to the [15] Glimcher MJ, Bonar LC, Grynpas MD, Landis WJ, Roufosse AH. Recent studies
existence of various calcium orthophosphate samples in an amor- of bone mineral: is the amorphous calcium phosphate theory valid? J Cryst
Growth 1981;53:100–19.
phous state [310,311]. Therefore, there is still an unanswered ques-
[16] Grynpas MD, Bonar LC, Glimcher MJ. On the question of amorphous
tion concerning the structure of large numbers of nanodimensional tricalcium phosphate in bone mineral. Dev Biochem 1981;22:279–83.
and/or nanocrystalline calcium orthophosphates: is their almost [17] Grynpas MD, Bonar LC, Glimcher MJ. Failure to detect an amorphous
amorphous appearance (according to numerous results of XRD stud- calcium–phosphate solid phase in bone mineral: a radial distribution
function study. Calcif Tiss Int 1984;36:291–301.
ies) due to the exceedingly small dimensions of well-crystallized [18] Aoba T, Moreno E. Changes in the nature and composition of enamel mineral
structures or to them actually being amorphous (i.e. retaining only during porcine amelogenesis. Calcif Tiss Int 1990;47:356–64.
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4471

[19] Boskey AL. Amorphous calcium phosphate: the contention of bone. J Dent Res [50] Skrtic D, Antonucci JM, Eanes ED. Effect of the monomer and filler system on
1997;76:1433–6. the remineralizing potential of bioactive dental composites based on
[20] Eanes ED, Amorphous calcium phosphate. In: Chow LC, Eanes ED, editors. amorphous calcium phosphate. Polym Adv Technol 2001;12:369–79.
Octacalcium Phosphate Monographs Oral Sci, vol 18. Basel: Karger; 2001. pp. [51] Skrtic D, Antonucci JM, Eanes ED. Amorphous calcium phosphate-based
130–147. bioactive polymeric composites for mineralized tissue regeneration. J Res
[21] Weiner S, Sagi I, Addadi L. Choosing the crystallization path less travelled. Natl Inst Stands Technol 2003;108:167–82.
Science 2005;309:1027–8. [52] Skrtic D, Antonucci JM, Eanes ED, Eidelman N. Dental composites based on
[22] Weiner S. Transient precursor strategy in mineral formation of bone. Bone hybrid and surface–modified amorphous calcium phosphates. Biomaterials
2006;39:431–3. 2004;25:1141–50.
[23] Suvorova EI, Petrenko PP, Buffat PA. Scanning and transmission electron [53] Skrtic D, Antonucci JM. Matrix resin effects on selected physicochemical
microscopy for evaluation of order/disorder in bone structure. Scanning properties of amorphous calcium phosphate composites. J Bioactive
2007;29:162–70. Compatible Polym 2005;20:29–49.
[24] Rey C, Combes C, Drouet C, Glimcher MJ. Bone mineral: an update on [54] Skrtic D, Antonucci JM, Eanes ED. Improved properties of amorphous calcium
chemical composition and structure. Osteoporos Int 2009;20:1013–321. phosphate fillers in remineralizing resin composites. Dent Mater
[25] Olszta MJ, Odom DJ, Douglas EP, Gower LB. A new paradigm for biomineral 1996;12:295–301.
formation: mineralization via an amorphous liquid phase precursor. Connect [55] Skrtic D, Antonucci JM. Dental composites based on amorphous calcium
Tiss Res 2003;44:326–34. phosphate–resin composition/physicochemical properties study. J Biomater
[26] Mahamid J, Sharir A, Addadi L, Weiner S. Amorphous calcium phosphate is a Appl 2007;21:375–93.
major component of the forming fin bones of zebrafish: indications for an [56] Skrtic D, Antonucci JM, Liu DW. Ethoxylated bisphenol dimethacrylate-based
amorphous precursor phase. Proc Natl Acad Sci USA 2008;105:12748–53. amorphous calcium phosphate composites. Acta Biomater 2006;2:85–94.
[27] Tsuji T, Onuma K, Yamamoto A, Iijima M, Shiba K. Direct transformation from [57] Skrtic D, Hailer AW, Takagi S, Antonucci JM, Eanes ED. Quantitative
amorphous to crystalline calcium phosphate facilitated by motif- assessment of the efficacy of amorphous calcium phosphate/methacrylate
programmed artificial proteins. Proc Natl Acad Sci USA 2008;105:16866–70. composites in remineralizing caries-like lesions artificially produced in
[28] Beniash A, Metzler RA, Lam RSK, Gilbert PUPA. Transient amorphous calcium bovine enamel. J Dental Res 1996;75:1679–86.
phosphate in forming enamel. J Struct Biol 2009;166:133–43. [58] Park MS, Eanes ED, Antonucci JM, Skrtic D. Mechanical properties of bioactive
[29] Tao J, Pan H, Zeng Y, Xu R, Tang R. Roles of amorphous calcium phosphate and amorphous calcium phosphate/methacrylate composites. Dent Mater
biological additives in the assembly of hydroxyapatite nanoparticles. J Phys 1998;14:137–41.
Chem B 2007;111:13410–8. [59] Lee SY, Regnault WF, Antonucci JM, Skrtic D. Effect of particle size of an
[30] Combes C, Rey C. Amorphous calcium phosphates: synthesis, properties and amorphous calcium phosphate filler on the mechanical strength and ion
uses in biomaterials. Acta Biomater 2010;6:3362–78. release of polymeric composites. J Biomed Mater Res B (Appl Biomater)
[31] Dorozhkin SV. Calcium orthophosphates. J Mater Sci 2007;42:1061–95. 2007;80B:11–7.
[32] Dorozhkin SV. Calcium orthophosphates in nature, biology and medicine. [60] Antonucci JM, Liu DW, Skrtic D. Amorphous calcium phosphate based
Materials 2009;2:399–498. composites: effect of surfactants and poly(ethylene oxide) on filler and
[33] Holt C, van Kemenade MJJM, Harries JE, Nelson Jr LS, Bailey RT, Hukins DWL, composite properties. J Dispersion Sci Technol 2007;28:819–24.
et al. Preparation of amorphous calcium-magnesium phosphates at pH 7 and [61] Skrtic D, Lee SY, Antonucci JM, Liu DW. Amorphous calcium phosphate based
characterization by X-ray absorption and Fourier transform infrared polymeric composites: effects of polymer composition and filler’s particle
spectroscopy. J Cryst Growth 1988;92:239–52. size on composite properties. Key Eng Mater 2005;284–286:737–40.
[34] Bachra BN. Precipitation of calcium carbonates and phosphates from [62] O’Donnell JNR, Schumacher GE, Antonucci JM, Skrtic D. Adhesion of
metastable solutions. Ann NY Acad Sci 1963;109:251–5. amorphous calcium phosphate composites bonded to dentin: a study in
[35] Bachra BN, Trautz OR, Simon SL. Precipitation of calcium carbonates and failure modality. J Biomed Mater Res B (Appl Biomater) 2009;90B:238–49.
phosphates under physiological conditions. Arch Biochem Biophys [63] Antonucci JM, O’Donnell JNR, Schumacher GE, Skrtic D. Amorphous calcium
1963;103:124–38. phosphate composites and their effect on composite–adhesive–dentin
[36] Bachra BN, Trautz OR, Simon SL. Precipitation of calcium carbonates and bonding. J Adhes Sci Technol 2009;23:1133–47.
phosphates. III. The effect of magnesium and fluoride ions on the spontaneous [64] Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV, et al.
precipitation of calcium carbonates and phosphates. Arch Oral Biol Fluoride and casein phosphopeptide–amorphous calcium phosphate. J Dent
1965;10:731–8. Res 2008;87:344–8.
[37] Greenfield DJ, Eanes ED. Formation chemistry of amorphous calcium [65] Amjad Z. Influence of polyelectrolytes on the precipitation of amorphous
phosphates prepared from carbonate containing solutions. Calcif Tiss Res calcium phosphate. Colloids and Surfaces 1990;48:95–106.
1972;9:152–62. [66] Bar-Yosef OP, Govrin-Lippman R, Garti N, Füredi-Milhofer H. The influence of
[38] Olesen PT, Steenberg T, Christensen E, Bjerrum NJ. Electrolytic deposition of polyelectrolytes on the formation and phase transformation of amorphous
amorphous and crystalline zinc–calcium phosphates. J Mater Sci calcium phosphate. Cryst Growth Des 2004;4:177–83.
1998;33:3059–63. [67] Cross KJ, Huq NL, Palamara JE, Perich JW, Reynolds EC. Physicochemical
[39] Tadic D, Peters F, Epple M. Continuous synthesis of amorphous carbonated characterisation of casein phosphopeptide–amorphous calcium phosphate
apatite. Biomaterials 2002;23:2553–9. nanocomplexes. J Biological Chem 2005;280:15362–9.
[40] LeGeros RZ, MijaresD ParkJ, Chang XF, Khairoun I, Kijkowska R, Dias R, et al. [68] Gutiérrez MC, Jobbágy M, Ferrer ML, del Monte F. Enzymatic synthesis of
Amorphous calcium phosphates (ACP): formation and stability. Key Eng amorphous calcium phosphate–chitosan nanocomposites and their
Mater 2005;284–286:7–10. processing into hierarchical structures. Chem Mater 2008;20:11–3.
[41] Aimanova OJ, LeGeros RZ, Sinyayev VA. Antimicrobiologic property hydrated [69] Cushnie EK, Khan YM, Laurencin CT. Amorphous hydroxyapatite–sintered
amorphous calcium phosphates containing silver. Key Eng Mater 2005;284– polymeric scaffolds for bone tissue regeneration: physical characterization
286:439–42. studies. J Biomed Mater Res A 2008;84A:54–62.
[42] Skrtic D, Antonucci JM, Eanes ED, Brunworth RT. Silica- and zirconia- [70] Lin Q, Li Y, Lan X, Lu C, Xu Z. Preparation of amorphous calcium phosphate/
hybridized amorphous calcium phosphate: effect on transformation to tricalcium silicate composite powders. Adv Mater Res 2009;79–82:1643–6.
hydroxyapatite. J Biomed Mater Res 2002;59:597–604. [71] Walker GD, Cai F, Shen P, Adams GG, Reynolds C, Reynolds EC. Casein
[43] Julien M, Khairoun I, LeGeros RZ, Delplace S, Pilet P, Weiss P, et al. Physico- phosphopeptide–amorphous calcium phosphate incorporated into sugar
chemical-mechanical and in vitro biological properties of calcium phosphate confections inhibits the progression of enamel subsurface lesions in situ.
cements with doped amorphous calcium phosphates. Biomaterials Caries Res 2010;44:33–40.
2007;28:956–65. [72] Antonucci JM, Regnault WF, Skrtic D. Polymerization shrinkage and stress
[44] Sinyaev VA, LeGeros RZ, Levchenko LV, Shustikova ES, Karzhaubaeva RA. State development in amorphous calcium phosphate/urethane dimethacrylate
of water in amorphous calcium and calcium–magnesium phosphates. Russ J polymeric composites. J Composite Mater 2010;44:355–67.
General Chem 2008;78:864–7. [73] Sinyaev VA, Shustikova ES, Levchenko LV, Sedunov AA. Synthesis and
[45] Sinyaev VA, Shustikova ES, Levchenko LV, Karzhaubaeva RA, Tokseitova GA. dehydration of amorphous calcium phosphate. Inorg Mater 2001;37:619–22.
Amorphous calcium barium monophosphate and its dehydration in air at [74] Dion A, Berno B, Hall G, Filiaggi MJ. The effect of processing on the structural
room temperature. Russ J Appl Chem 2008;81:1899–903. characteristics of vancomycin-loaded amorphous calcium phosphate
[46] Taylor MG, Simkiss K, Simmons J, Wu LNY, Wuthier RE. Structural studies of a matrices. Biomaterials 2005;26:4486–94.
phosphatidyl serine–amorphous calcium phosphate complex. Cell Mol Life [75] Dion A, Langman M, Hall G, Filiaggi MJ. Vancomycin release behaviour from
Sci 1998;54:196–202. amorphous calcium polyphosphate matrices intended for osteomyelitis
[47] Brečević L, Hlady V, Füredi-Milhofer H. Influence of gelatin on the treatment. Biomaterials 2005;26:7276–85.
precipitation of amorphous calcium phosphate. Colloids Surf [76] Lee B, Kim M, Choi S, Lee YK. Amorphous calcium polyphosphate bone
1987;28:301–13. regenerative materials based on calcium phosphate glass. Key Eng Mater
[48] Ambrosio AMA, Sahota JS, Khan Y, Laurencin CT. A novel amorphous calcium 2009;396–398:209–12.
phosphate polymer ceramic for bone repair: I. Synthesis and characterization. [77] Chen G, Li W, Zhao B, Sun K. A novel biphasic bone scaffold: b-calcium
J Biomed Mater Res 2001;58:295–301. phosphate and amorphous calcium polyphosphate. J Am Ceram Soc
[49] Skrtic D, Antonucci JM, Eanes ED, Eichmiller FC, Schumacher GE. 2009;92:945–8.
Physicochemical evaluation of bioactive polymeric composites based on [78] Chun S, Jeong JH, Kim KM, Kim S. Biodegradation study of amorphous and
hybrid amorphous calcium phosphates. J Biomed Mater Res (Appl Biomater) crystalline calcium metaphosphate in the SBF and tris-buffer solution. Key
2000;53:381–91. Eng Mater 2001;192–195:131–4.
4472 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

[79] Cheng YT, Johnson WL. Disordered materials: a survey of amorphous solids. [118] Brečević LJ, Füredi-Milhofer H. Precipitation of calcium phosphates from
Science 1987;235:997–1002. electrolyte solutions-II The formation and transformation of the precipitates.
[80] <http://enwikipediaorg/wiki/Amorphous>; [accessed in June 2010]. Calcif Tiss Res 1972;10:82–90.
[81] Sheng HW, Luo WK, Alamgir FM, Bai JM, Ma E. Atomic packing and short-to- [119] Liu C, Huang Y, Shen W, Cui J. Kinetics of hydroxyapatite precipitation at pH
medium-range order in metallic glasses. Nature 2006;439:419–25. 10 to 11. Biomaterials 2001;22:301–6.
[82] Lee CY, Stachurski ZH, Welberry TR. The geometry, topology and structure of [120] Boskey AL, Posner AS. Formation of hydroxyapatite at low supersaturation. J
amorphous solids. Acta Mater 2010;58:615–25. Phys Chem 1976;80:40–5.
[83] Hufnagel TC. Amorphous materials: finding order in disorder. Nature Mater [121] Feenstra TP, de Bruyn PL. The Ostwald rule of stages in precipitation from
2004;3:666–7. highly supersaturated solutions: a model and its application to the formation
[84] Elliott SR. Physics of amorphous materials. 2nd ed. London: Longman; 1990. of the nonstoichiometric amorphous calcium phosphate precursor phase. J
[85] Elliott SR. Medium-range structural order in covalent amorphous solids. Coll Interf Sci 1981;84:66–72.
Nature 1991;354:445–52. [122] Eanes ED. Amorphous calcium phosphate: thermodynamic and kinetic
[86] Salmon PS. Amorphous materials: order in disorder. Nature Mater considerations. In: Amjad Z, editor. Calcium phosphates in biological and
2002;1:87–8. industrial systems. Boston, MA: Kluwer; 1998. p. 21–39.
[87] Krivanek OL, Gaskell PH, Howie A. Seeking order in ‘‘amorphous” materials. [123] Blumenthal NC, Posner AS, Holmes JM. Effect of preparation conditions on the
Nature 1976;362:454–7. properties and transformation of amorphous calcium phosphate. Mater Res
[88] Mountjoy G. Order in two-dimensional projections of thin amorphous three- Bull 1972;7:1181–9.
dimensional structures. J Phys Cond Matter 1999;11:2319–36. [124] Boskey AL, Posner AS. Conversion of amorphous calcium phosphate to
[89] Simon V, Lazǎr D, Turcu RVF, Mocuta H, Magyari K, Prinz M, et al. Atomic microcrystalline hydroxyapatite. A pH-dependent, solution-mediated, solid-
environment in sol–gel derived nanocrystalline hydroxyapatite. Mater Sci solid conversion. J Phys Chem 1973;77:2313–7.
Eng B 2009;165:247–51. [125] Li Y, Weng W. In vitro synthesis and characterization of amorphous calcium
[90] Weeber AW, Bakker H. Amorphization by ball milling. A review. Physica B phosphates with various Ca/P atomic ratios. J Mater Sci Mater Med
1988;153:93–135. 2007;18:2303–8.
[91] Robinson RA, Watson ML. Crystal–collagen relationships in bone as observed [126] Zyman ZZ, Rokhmistrov DV, Glushko VI. Structural and compositional
in the electron microscope III crystal and collagen morphology as a function features of amorphous calcium phosphate at the early stage of
of age. Ann NY Acad Sci 1955;60:596–660. precipitation. J Mater Sci Mater Med 2010;21:123–30.
[92] Watson ML, Robinson RA. Collagen–crystal relationships in bone. II. Electron [127] Holt C, van Kemenade MJJM, Nelson Jr LS, Hukins DWL, Bailey RT, Harries JE,
microscope study of basic calcium phosphate crystals. Am J Anat et al. Amorphous calcium phosphates prepared at pH 6.5 and 6.0. Mater Res
1953;93:25–59. Bull 1989;23:55–62.
[93] Chow LC, Takagi S, Vogel GL. Letter to the Editor. J Dent Res 1998;77:6. [128] Eanes ED, Gillessen IH, Posner AS. Intermediate states in the precipitation of
[94] Eanes ED. Letter to the Editor. J Dent Res 1998;77:6. hydroxyapatite. Nature 1965;208:365–7.
[95] Harper RA, Posner AS. Measurement of non-crystalline calcium phosphate in [129] Urch H, Vallet-Regi M, Ruiz L, Gonzalez-Calbet JM, Epple M. Calcium
bone mineral. Proc Soc Exp Biol Med 1966;122:137–42. phosphate nanoparticles with adjustable dispersability and crystallinity. J
[96] Termine JD, Posner AS. Amorphous/crystalline interrelationships in bone Mater Chem 2009;19:2166–71.
mineral. Calcif Tiss Res 1967;1:8–23. [130] Zahidi E, Lebugle A, Bonel G. Sur une nouvelle classe de materiaux pour
[97] Posner AS. Crystal chemistry of bone mineral. Physiol Rev 1969;40:760–92. protheses osseuses ou dentaires. [A new class of materials for bone or dental
[98] Posner AS, Betts F. Synthetic amorphous calcium phosphate and its relation to prostheses]. Bull Sot Chim Fr 1985;4:523–7.
bone mineral structure. Acc Chem Res 1975;8:273–81. [131] Lebugle A, Zahidi E, Bonel G. Effect of structure and composition on the
[99] Betts F, Blumenthal NC, Posner AS, Becker GL, Lehninger AL. Atomic structure thermal decomposition of calcium phosphates (Ca/P = 1.33). React Solids
of intracellular amorphous calcium phosphate deposits. Proc Nat Acad Sci 1986;2:151–61.
USA 1975;72:2088–90. [132] Layrolle P, Lebugle A. Characterization and reactivity of nanosized calcium
[100] Posner AS, Betts F, Blumenthal NC. Formation and structure of synthetic and phosphates prepared in anhydrous ethanol. Chem Mater 1994;6:1996–2004.
bone hydroxyapatite. Progr Cryst Growth Char 1980;3:49–64. [133] Layrolle P, Ito A, Tateishi T. Sol-gel synthesis of amorphous calcium
[101] Bonar LC, Roufousse AH, Sabine WK, Grynpass MD, Glimcher MJ. X-ray phosphate and sintering into microporous hydroxyapatite bioceramics. J
diffraction studies of the crystallinity of bone mineral in newly synthesized Am Ceram Soc 1998;81:1421–8.
and density fractionated bone. Calcif Tiss Int 1983;35:202–9. [134] Rodrigues A, Lebugle A. Behavior in wet atmosphere of an amorphous
[102] Molnar Z. Development of the parietal bone of young mice. I. Crystals of bone calcium phosphate with an atomic Ca/P ratio of 1.33. J Solid State Chem
mineral in frozen-dried preparations. J Ultrastruct Res 1959;3:39–45. 1999;148:308–15.
[103] Molnar Z. Additional observations on bone crystal dimensions. Clin Orthop [135] Ohta M, Honma T, Umesaki M, Nakahira A. Synthesis and evaluation of
1960;17:38–42. amorphous calcium phosphate (ACP) by various synthesis methods. Key Eng
[104] Thyberg J. Electron microscopic studies on the initial phases of calcification in Mater 2006;309–311:175–8.
guinea pig epiphyseal cartilage. J Ultrastruct Res 1974;46:206–18. [136] Li YB, Weng WJ, Cheng K, Du PY, Shen G, Han GR. Complexes of Ca(II) with
[105] Gay CV. The ultrastructure of the extracellular phase of bone as observed in polymers as precursors for preparation of amorphous calcium phosphate.
frozen thin sections. Calcif Tiss Res 1977;23:215–23. Mater Sci Technol 2004;20:1075–8.
[106] Schraer H, Gay CV. Matrix vesicles in newly synthesizing bone observed after [137] Bow JS, Liou SC, Chen SY. Structural characterization of room-temperature
ultracryotomy and ultramicroincineration. Calcif Tiss Res 1977;23:185–8. synthesized nano-sized b-tricalcium phosphate. Biomaterials
[107] Nancollas GH, Mohan MS. The growth of hydroxyapatite crystals. Arch Oral 2004;25:3155–61.
Biol 1970;15:731–45. [138] Li YB, Weng WJ, Cheng K, Du PY, Shen G, Wang J, et al. Preparation of
[108] Blumenthal NC, Betts F, Posner AS. Formation and structure of Ca-deficient amorphous calcium phosphate in the presence of poly(ethylene glycol). J
hydroxyapatite. Calcif Tiss Int 1981;33:111–7. Mater Sci Lett 2003;22:1015–6.
[109] Meyer JL. Phase transformations in the spontaneous precipitation of calcium [139] Wang R, Weng W, Deng X, Cheng K, Liu X, Du P, et al. Dissolution behavior of
phosphate. Croatica Chim Acta 1983;56:753–67. submicron biphasic tricalcium phosphate powders. Key Eng Mater
[110] Harries JE, Hukins DWL, Holt C, Hasnain SS. Conversion of amorphous 2006;309–311:223–6.
calcium phosphate into hydroxyapatite investigated by EXAFS spectroscopy. J [140] Li Y, Wiliana T, Tam KC. Synthesis of amorphous calcium phosphate using
Cryst Growth 1987;84:563–70. various types of cyclodextrins. Mater Res Bull 2007;42:820–7.
[111] Lopez-Valero I, Gomez-Lorente C, Boistelle R. Effects of sodium and [141] Tao J, Pan H, Zhai H, Wang J, Li L, Wu J, et al. Controls of tricalcium phosphate
ammonium ions on occurrence, evolution and crystallinity of calcium single-crystal formation from its amorphous precursor by interfacial energy.
phosphates. J Cryst Growth 1992;121:297–304. Cryst Growth Des 2009;9:3154–60.
[112] Lazić S. Microcrystalline hydroxyapatite formation from alkaline solutions. J [142] Liu S, Weng W, Li Z, Pan L, Cheng K, Song C, et al. Effect of PEG amount in
Cryst Growth 1995;147:147–54. amorphous calcium phosphate on its crystallized products. J Mater Sci Mater
[113] Gadaleta SJ, Paschalis EP, Betts F, Mendelson R, Boskey AL. Fourier transform Med 2009;20:359–63.
infrared spectroscopy of the solution-mediated conversion of amorphous [143] Li Y, Weng W, Tam KC. Novel highly biodegradable biphasic tricalcium
calcium phosphate to hydroxyapatite: new correlations between X-ray phosphates composed of a-tricalcium phosphate and b-tricalcium
diffraction and infrared data. Calcif Tiss Int 1996;58:9–16. phosphate. Acta Biomater 2007;3:251–4.
[114] Tarasevich BJ, Chusuei CC, Allara DL. Nucleation and growth of calcium [144] Li Y, Li D, Weng W. In vitro dissolution behavior of biphasic tricalcium
phosphate from physiological solutions onto self-assembled templates by phosphate composite powders composed of a-tricalcium phosphate and b-
a solution-formed nucleus mechanism. J Phys Chem B 2003;107: tricalcium phosphate. Key Eng Mater 2008;368–372:1206–8.
10367–77. [145] Rodrigues A, Lebugle A. Influence of ethanol in the precipitation medium on
[115] Kim S, Ryu HS, Jung HS, Hong KS. Influence of Ca/P ratios of starting solutions the composition, structure and reactivity of tricalcium phosphate. Coll Surf A
on the crystallization of amorphous calcium phosphate to hydroxyapatite. 1998;145:191–204.
Metals Mater Int 2004;10:171–5. [146] Heughebaert JC, Montel G. Conversion of amorphous tricalcium phosphate
[116] Kim S, Ryu HS, Shin H, Jung HS, Hong KS. Direct observation of into apatitic tricalcium phosphate. Calcif Tiss Int 1982;34:S103–8.
hydroxyapatite nucleation from amorphous phase in a stoichiometric [147] Yu T, Ye J, Wang Y. Synthesis and property of a novel calcium phosphate
calcium/phosphate aqueous solution. Chem Let 2004;33:1292–3. cement. J Biomed Mater Res B (Appl Biomater) 2009;90B:745–51.
[117] Wang L, Nancollas GH. Calcium orthophosphates: crystallization and [148] Tofighi A, Palazzolo R. Calcium phosphate bone cement preparation using
dissolution. Chem Rev 2008;108:4628–69. mechano-chemical process. Key Eng Mater 2005;284–286:101–4.
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4473

[149] Gbureck U, Grolms O, Barralet JE, Grover LM, Thull R. Mechanical activation [181] Somrani S, Banu M, Jemal M, Rey C. Physico-chemical and thermochemical
and cement formation of b-tricalcium phosphate. Biomaterials 2003;24: studies of the hydrolytic conversion of amorphous tricalcium phosphate into
4123–31. apatite. J Solid State Chem 2005;178:1337–48.
[150] Gbureck U, Barralet JE, Radu L, Klinger H, Thull R. Amorphous a-tricalcium [182] Dekker RJ, de Bruijn JD, Stigter M, Barrere F, Layrolle P, van Blitterswijk
phosphate: preparation and aqueous setting reaction. J Am Ceram Soc CA. Bone tissue engineering on amorphous carbonated apatite and
2004;87:1126–32. crystalline octacalcium phosphate-coated titanium discs. Biomaterials
[151] Lemons JE. Hydroxyapatite coatings. Clin Orthop 1988;235:220–3. 2005;26:5231–9.
[152] Zyman ZZ, Weng J, Liu X, Zhang X, Ma Z. Amorphous phase and [183] Amin MS, Randeniya LK, Bendavid A, Martin PJ, Preston EW. Amorphous
morphological structure of hydroxyapatite plasma coatings. Biomaterials carbonated apatite formation on diamond-like carbon containing titanium
1993;14:225–8. oxide. Dia Rel Mater 2009;18:1139–44.
[153] Weng J, Liu X, Zhang X, Ma Z, Ji X, Zyman ZZ. Further studies on the plasma- [184] Imai H, Kusunoki M, Hashimoto Y, Nishikawa H, Hontsu S. Evaluation of
sprayed amorphous phase in hydroxyapatite coatings and its deamorphiza- biological molecular adsorption on hydroxyapatite and amorphous
tion. Biomaterials 1993;14:578–82. Ca10(PO4)6(OH)2 thin films using QCM method. IEEJ Trans EIS
[154] Tong W, Chen J, Zhang X. Amorphorization and recrystallization during 2007;127:1839–42.
plasma spraying of hydroxyapatite. Biomaterials 1995;16:829–32. [185] Holt C, Wahlgren NM, Drakenberg T. Ability of a b-casein phosphopeptide to
[155] Weng J, Liu XG, Li XD, Zhang XD. Intrinsic factors of apatite influencing its modulate the precipitation of calcium phosphate by forming amorphous
amorphization during plasma-spray coating. Biomaterials 1995;16:39–44. dicalcium phosphate nanoclusters. Biochem J 1996;314:1035–9.
[156] Gross KA, Berndt CC, Herman H. Amorphous phase formation in plasma- [186] Eanes ED, Posner AS. Kinetics and mechanism of conversion of noncrystalline
sprayed hydroxyapatite coatings. J Biomed Mater Res 1998;39:407–14. calcium phosphate to crystalline hydroxyapatite. Trans NY Acad Sci
[157] Feng CF, Khor KA, Kweh SWK, Cheang P. Thermally induced crystallization of 1965;28:233–41.
amorphous calcium phosphate in plasma-spheroidized hydroxyapatite [187] Eanes ED. Thermochemical studies on amorphous calcium phosphate. Calcif
powders. Mater Let 2000;46:229–33. Tiss Res 1970;5:133–45.
[158] Tong W, Li P. In vitro dissolution of amorphous calcium phosphate (Acp) [188] Oniki T, Oyamada M. Surface structure of amorphous calcium phosphate by
increased the wear particle generation of plasma-sprayed HA coatings. Key ESR of VO2+ adsorbed on it. Calcif Tiss Int 1983;35:477–80.
Eng Mater 2007;330–332:561–4. [189] Boulet M, Marier JR. Precipitation of calcium phosphates from solutions at
[159] Gross KA, Phillips MR. Identification and mapping of the amorphous phase in near physiological concentrations. Arch Biochem 1961;98:157–65.
plasma-sprayed hydroxyapatite coatings using scanning cathodolumines- [190] Newesely H. Changes in crystal types of low solubility calcium phosphates in
cence microscopy. J Mater Sci Mater Med 1998;9:797–802. the presence of accompanying ions. Arch Oral Biol 1961;Spec Suppl
[160] Carayon MT, Lacout JL. Study of the Ca/P atomic ratio of the amorphous phase 6:174–80.
in plasma-sprayed hydroxyapatite coatings. J Solid State Chem [191] VuiIleumier C, Lerch P. Étude de la structure, par la diffraction des rayons X et
2003;172:339–50. la microscopic é1ectronique, de l’hydroxylapatite calcique et des
[161] Kumar R, Cheang P, Khor KA. Phase composition and heat of crystallization of orthophosphates dits tri- et octocalcique. Helv Chim Acta 1966;49:663–70.
amorphous calcium phosphate in ultra-fine radio frequency suspension [192] Christoffersen J, Christoffersen MR, Kibalczyc W, Andersen FA. A contribution
plasma sprayed hydroxyapatite powders. Acta Mater 2004;52:1171–81. to the understanding of the formation of calcium phosphates. J Cryst Growth
[162] Keller L, Dollase WA. X-ray determination of crystalline hydroxyapatite to 1989;94:767–77.
amorphous calcium phosphate ratio in plasma sprayed coatings. J Biomed [193] Christoffersen J, Christoffersen MR, Kibalczyc W. Apparent solubilities of two
Mater Res 2000;49:244–9. amorphous calcium phosphates and of octacalcium phosphate in the
[163] Gross KA, Gross V, Berndt CC. Thermal analysis of amorphous phases in temperature range 30–42 C. J Cryst Growth 1990;106:349–54.
hydroxyapatite coatings. J Am Ceram Soc 1998;81:106–12. [194] Kibalczyc W, Christoffersen J, Christoffersen MR, Zielenkiewicz A,
[164] Döbelin N, Brunner TJ, Stark WJ, Eggimann M, Fisch M, Bohner M. Phase Zielenkiewicz W. The effect of magnesium ions on the precipitation of
evolution of thermally treated amorphous tricalcium phosphate calcium phosphates. J Cryst Growth 1990;106:355–66.
nanoparticles. Key Eng Mater 2009;396–398:595–8. [195] Francis MD, Webb NC. Hydroxyapatite formation from a hydrated calcium
[165] Maciejewski M, Brunner TJ, Loher SF, Stark WJ, Baiker A. Phase transitions in monohydrogen phosphate precursor. Calcif Tiss Res 1971;6:335–42.
amorphous calcium phosphates with different Ca/P ratios. Thermochim Acta [196] Walton AG, Bodin WJ, Furedi H, Sehwartz A. Nucleation of calcium phosphate
2008;468:75–80. from solution. Can J Chem 1967;45:2695–701.
[166] Vaidya SN, Karunakaran C, Pande BM, Gupta NM, Iyer RK, Karweer SB. [197] Meyer JL, Eanes ED. A thermodynamic analysis of the amorphous to
Pressure-induced crystalline to amorphous transition in hydroxylapatite. J crystalline calcium phosphate transformation. Calcif Tiss Res 1978;25:59–68.
Mater Sci 1997;32:3213–7. [198] Meyer JL. Hydroxyl content of solution-precipitated calcium phosphates.
[167] Vaidya SN, Sugandhi V. Pressure induced amorphization in calcium Calcif Tiss Int 1979;27:153–60.
phosphates. J Mater Sci 1999;34:3769–78. [199] Jaeger C, Maltsev S, Karrasch A. Progress of structural elucidation of
[168] Kim S, Ryu HS, Shin H, Jung HS, Hong KS. In situ observation of hydroxyapatite amorphous calcium phosphate (ACP) and hydroxyapatite (HAp): disorder
nanocrystal formation from amorphous calcium phosphate in calcium-rich and surfaces as seen by solid state NMR. Key Eng Mater 2006;309–
solutions. Mater Chem Phys 2005;91:500–6. 311:69–72.
[169] Termine JD, Eanes ED. Comparative chemistry of amorphous and apatitic [200] Nelson Jr LS, Holt C, Harries JE, Hukins DWL. Amorphous calcium phosphates
calcium phosphate preparations. Calcif Tiss Res 1972;10:171–97. of different composition give very similar EXAFS spectra. Physica B
[170] Eanes ED, Posner AS. Intermediate phases in the basic solution preparation of 1989;158:105–6.
alkaline earth phosphates. Calcif Tiss Res 1968;2:38–48. [201] Wuthier RE, Rice GS, Wallace Jr JEB, Weaver RL, LeGeros RZ, Eanes ED. In vitro
[171] Nylen MU, Eanes ED, Termine JD. Molecular and ultrastructural studies of precipitation of calcium phosphate under intracellular conditions: formation
noncrystalline calcium phosphates. Calcif Tiss Res 1972;9:95–108. of brushite from an amorphous precursor in the absence of ATP. Calcif Tiss Int
[172] Eanes ED, Termine JD, Nylen MU. An electron microscopic study of the 1985;37:401–10.
formation of amorphous calcium phosphate and its transformation to [202] Holmes JM, Beebe RA. Surface areas by gas adsorption on amorphous calcium
crystalline apatite. Calcif Tiss Res 1973;12:143–58. phosphate and crystalline hydroxyapatite. Calcif Tiss Res 1971;7:163–74.
[173] Eanes ED. Amorphous intermediates in the formation of biological apatites. [203] Sedlak JM, Beebe RA. Temperature programmed dehydration of amorphous
Physico-chimie et cristallographie des apatites d’intérêt biologique. Coll Int calcium phosphate. J Coll Interf Sci 1974;47:483–9.
CNRS 1975;230:295–301. [204] LeGeros RZ, Shirra WP, Miravite MA, LeGeros JP. Amorphous calcium
[174] Barton SS, Harrison BH. Surface and bulk properties of amorphous calcium phosphates: synthetic and biological. Physico-chimie et cristallographie des
phosphate. In: Kerker M, editor. Colloid and Interface Science, vol 3, 50th apatites d’intérêt biologique. Coll Int CNRS 1975;230:105–15.
proceeding international conference. New York, USA: Academic Press; 1976, [205] LeGeros RZ. Calcium phosphates in oral biology and medicine. Monographs
p. 71. oral science, vol. 15. Basel: Karger; 1991.
[175] Lundager-Madsen HE, Lopez-Valero I, Lopez-Acevedo V. The formation [206] Boskey AL, Posner AS. Magnesium stabilization ob amorphous calcium
product of amorphous tricalcium phosphate at 37 °C. J Cryst Growth phosphate: a kinetic study. Mater Res Bull 1974;9:907–16.
1986;75:429–34. [207] Blumenthal NC, Betts F, Posner AS. Stabilization of amorphous calcium
[176] Roberts JE, Heughebaert M, Heughebaert JC, Bonar LC, Glimcher MJ, Griffin phosphate by Mg and ATP. Calcif Tiss Res 1977;23:245–50.
RG. Solid state 31NMR studies of the conversion of amorphous tricalcium [208] Abbona F, Baronnet AAXRD, EM T. Study on the transformation of amorphous
phosphate to apatitic tricalcium phosphate. Calcif Tiss Int 1991;49:378–82. calcium phosphate in the presence of magnesium. J Cryst Growth
[177] Gbureck U, Barralet JE, Thull R. Thermodynamic study of formation of 1996;165:98–105.
amorphous b-tricalcium phosphate for calcium phosphate cements. Key Eng [209] Fleisch H, Russell RGG, Bisaz S, Termine JD, Posner AS. Influence of
Mater 2004;254–256:249–52. pyrophosphate on the transformation of amorphous to crystalline calcium
[178] Brunner TJ, Bohner M, Dora C, Gerber C, Stark WJ. Comparison of amorphous phosphate. Calcif Tiss Res 1968;2:49–59.
TCP nanoparticles to micron-sized a-TCP as starting materials for calcium [210] Termine JD, Peckauskas RA, Posner AS. Calcium phosphate formation in vitro.
phosphate cements. J Biomed Mater Res B (Appl Biomater) 2007;83B:400–7. II. Effects of environment on amorphous–crystalline transformation. Arch
[179] Brunner TJ, Grass RN, Bohner M, Stark WJ. Effect of particle size, crystal phase Biochem Biophys 1970;140:318–25.
and crystallinity on the reactivity of tricalcium phosphate cements for bone [211] Bienenstock A, Posner AS. Calculation of the X-ray intensities from arrays of
reconstruction. J Mater Chem 2007;17:4072–8. small crystallites of hydroxyapatite. Arch Biochem Biophys 1968;124:604–7.
[180] Somrani S, Rey C, Jemal M. Thermal evolution of amorphous tricalcium [212] Tropp J, Blumenthal NC, Waugh JS, Phosphorus NMR. Study of solid
phosphate. J Mater Chem 2003;13:888–92. amorphous calcium phosphate. J Am Chem Soc 1983;105:22–6.
4474 S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475

[213] Fawcett RW. A radial distribution function analysis of an amorphous calcium [247] Termine JD, Eanes ED. Calcium phosphate deposition from balanced salt
phosphate with calcium to phosphorus molar ratio of 1.42. Calcif Tiss Res solutions. Calcif Tiss Res 1974;15:81–4.
1973;13:319–25. [248] Soares AMV, Arana-Chavez VE, Reid AR, Katchburian E. Lanthanum tracer and
[214] Betts F, Posner AS. An X-ray radial distribution study of amorphous calcium freeze-fracture studies suggest that compartmentalization of early bone
phosphate. Mater Res Bull 1974;9:353–60. matrix may be related to initial mineralization. J Anat 1992;181:345–56.
[215] Betts F, Posner AS. A structural model for amorphous calcium phosphate. [249] Bonucci E. Fine structure of early cartilage calcification. J Ultrastruct Res
Trans Am Crystal Assoc 1974;10:73–84. 1967;20:33–50.
[216] Eanes ED, Powers L, Costa JL. Extended X-ray absorption fine structure [250] Anderson HC. Vesicles associated with calcification in the matrix of
(EXAFS) studies on calcium in crystalline and amorphous solids of biological epiphyscal cartilage. J Cell Biol 1969;41:59–72.
interest. Cell Calcium 1981;2:251–62. [251] Bernard GW, Pease DC. An electron microscopic study of initial
[217] Holt C, Hukins DWL. Structural analysis of the environment of calcium ions in intramembranous osteogenesis. Am J Anat 1969;125:271–90.
crystalline and amorphous calcium phosphates by X-ray absorption [252] Wuthier RE. Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles,
spectroscopy and a hypothesis concerning the biological function of the and extracellular fluid. Calcif Tiss Res 1977;23:125–33.
casein micelle. Int Dairy J 1991;1:151–65. [253] Wuthier RE, Gore ST. Partition of inorganic ions und phospholipids in isolated
[218] Termine JD, Lundy DR. Vibrational spectra of some phosphate salts cell, membrane and matrix vesicle fractions: evidence for Ca–Pi-acidic
amorphous to X-ray diffraction. Calcif Tiss Res 1974;15:55–70. phospholipid complexes. Calcif Tiss Res 1977;24:163–71.
[219] Onuma K. Recent research on pseudobiological hydroxyapatite crystal [254] Eanes ED, Hailer AW, Costa JL. Calcium phosphate formation in aqueous
growth and phase transition mechanism. Prog Cryst Growth Charact Mater suspensions of multilamellar liposomes. Calcif Tiss Int 1984;36:421–30.
2006;52:223–45. [255] Sauer GR, Zunie WB, Durig JR, Wuthier RE. Fourier-transform Raman-
[220] Treboux G, Layrolle P, Kanzaki N, Onuma K, Ito A. Symmetry of Posner’s spectroscopy of synthetic and biological calcium phosphates. Calcif Tiss Int
cluster. J Am Chem Soc 2000;122:8323–4. 1994;54:414–20.
[221] Georgalis Y, Kierzek AM, Saenger W. Cluster formation in aqueous electrolyte [256] Sauer GR, Wuthier RE. Fourier-transform infrared characterization of mineral
solutions observed by dynamic light scattering. J Phys Chem B 2000;104: phases formed during induction of mineralization by collagenase–released
3405–6. matrix vesicles in vitro. J Biol Chem 1988;263:13718–24.
[222] Treboux G, Layrolle P, Kanzaki N, Onuma K, Ito A. Existence of Posner’s cluster [257] Dorozhkin SV. Bioceramics of calcium orthophosphates. Biomaterials
in vacuum. J Phys Chem A 2000;104:5111–4. 2010;31:1465–85.
[223] Kanzaki N, Treboux G, Onuma K, Tsutsumi S, Ito A. Calcium phosphate [258] Dorozhkin SV. Calcium orthophosphate cements for biomedical application. J
clusters. Biomaterials 2001;22:2921–9. Mater Sci 2008;43:3028–57.
[224] Boldeskul IE, Sukhodub LF, Kalinkevich AN, Khavryutchenko VD. Ab initio [259] Dorozhkin SV. Calcium orthophosphate cements and concretes. Materials
modelling of calcium phosphate clusters and their vibrational spectra. Cond 2009;2:221–91.
Matter Phys 2006;9:669–79. [260] Maxian SH, Zawadsky JP, Dunn MG. In vitro evaluation of amorphous calcium
[225] Zahn D. Mechanisms of calcium and phosphate ion association in aqueous phosphate and poorly crysiallized hydroxyapatite coatings on titanium
solution. Zeitschrift fur Anorganische und Allgemeine Chemie 2004;630: implants. J Biomed Mater Res 1993;27:111–7.
1507–11. [261] Maxian SH, Zawadsky JP, Dunn MG. Mechanical and histological evaluation of
[226] Wang CG, Liao JW, Gou BD, Huang J, Tang RK, Tao JH, et al. Crystallization at amorphous calcium phosphate and poorly crystallized hydroxyapatite
multiple sites inside particles of amorphous calcium phosphate. Cryst coatings on titanium implants. J Biomed Mater Res 1993;27:717–28.
Growth Des 2009;9:2620–6. [262] Garcia F, Arias JL, Mayor B, Pou J, Rehman I, Knowles J, et al. Effect of heat
[227] Kojima Y, Sakama K, Toyama T, Yasue T, Arai Y. Dehydration of water treatment on pulsed laser deposited amorphous calcium phosphate coatings.
molecules in amorphous calcium phosphate. Phosphorus Res Bull J Biomed Mater Res (Appl Biomater) 1998;43:69–76.
1994;4:47–52. [263] Heimann RB, Wirth R. Formation and transformation of amorphous calcium
[228] Wikholm NW, Beebe RA, Kittelberger JS. Kinetics of the conversion of phosphates on titanium alloy surfaces during atmospheric plasma spraying
monetite to calcium pyrophosphate. J Phys Chem 1975;79:853–6. and their subsequent in vitro performance. Biomaterials 2006;27:823–31.
[229] Eanes ED, Meyer JL. The maturation of crystalline calcium phosphates in [264] Liu DM, Chou HM, Wu JD, Tung MS. Hydroxyl apatite coating via amorphous
aqueous suspensions at physiologic pH. Calcif Tiss Res 1977;23:259–69. calcium phosphate. Mater Chem Phys 1994;37:39–44.
[230] Meyer JL, Weatherall CC. Amorphous to crystalline calcium phosphate phase [265] Nagano M, Nakamura T, Kokubo T, Tanahashi M, Ogawa M. Differences of
transformation at elevated pH. J Coll Interf Sci 1982;89:257–67. bone bonding ability and degradation behaviour in vivo between amorphous
[231] Ajibola VO, Thomas SA. Transformation of amorphous calcium phosphate calcium phosphate and highly crystalline hydroxyapatite coating.
hydroxyapatite in the presence of some ions. Bull Chem Soc Ethiopia Biomaterials 1996;17:1771–7.
1997;11:19–24. [266] Leeuwenburgh SCG, Layrolle P, Barrère F, de Bruijn J, Schoonman J, van
[232] Root MJ. Inhibition of the amorphous calcium phosphate phase transition Blitterswijk CA, et al. Osteoclastic resorption of biomimetic calcium
reaction by polyphosphates and metal ions. Calcif Tiss Int phosphate coatings in vitro. J Biomed Mater Res 2001;56:208–15.
1990;47:112–6. [267] Habibovic P, Barrère F, van Blitterswijk CA, de Groot K, Layrolle P. Biomimetic
[233] Wuthier RE, Eanes ED. Effect of phospholipids on the transformation of hydroxyapatite coating on metal implants. J Am Ceram Soc 2002;85:517–22.
amorphous calcium phosphate to hydroxyapatite in vitro. Calcif Tiss Int [268] Rössler S, Sewing A, Stolzel M, Born R, Scharnweber D, Dard M, et al.
1975;19:197–210. Electrochemically assisted deposition of thin calcium phosphate coatings at
[234] Termine JD, Eanes ED, Conn KM. Phosphoprotein modulation of apatite near-physiological pH and temperature. J Biomed Mater Res A
crystallization. Calcif Tiss Int 1980;31:247–51. 2003;64A:655–63.
[235] Qiu SM, Wen G, Hirakawa N, Soloway RD, Hong NK, Crowther RS. [269] Lee DD, Tofighi A, Aiolova M, Chakravarthy P, Catalano A, Majahad A, et al. F-
Glycochenodeoxycholic acid inhibits calcium phosphate precipitation BSMÒ: a biomimetic bone substitute and drug delivery vehicle. Clin Orthop
in vitro by preventing the transformation of amorphous calcium phosphate Relat Res 1999;367(Suppl):S396–405.
to calcium hydroxyapatite. J Clin Investigation 1991;88:1265–71. [270] Tofighi A, Mounic S, Chakravarthy P, Rey C, Lee D. Setting reactions involved
[236] Biumenthal NC, Betts F, Posner AS. Nucleotide stabilization of amorphous in injectable cements based on amorphous calcium phosphate. Key Eng
calcium phosphate. Mater Res Bull 1975;10:1055–60. Mater 2000;192:769–72.
[237] Termine JD, Conn KM. Inhibition of apatite formation by phosphorylated [271] Wang X, Ye J, Wang Y, Wu X, Bai B. Control of crystallinity of hydrated
metabolites and macromolecules. Calcif Tiss Res 1976;22:149–57. products in a calcium phosphate bone cement. J Biomed Mater Res A
[238] Tung MS, Brown WE. An intermediate state in hydrolysis of amorphous 2007;81A:781–90.
calcium phosphate. Calcif Tiss Int 1983;35:783–90. [272] van den Vreken NMF, Pieters IY, Declercq HA, Cornelissen MJ, Verbeeck RMH.
[239] Kazanci M, Fratzl P, Klaushofer K, Paschalis EP. Complementary information Characterization of calcium phosphate cements modified by addition of
on in vitro conversion of amorphous (precursor) calcium phosphate to amorphous calcium phosphate. Acta Biomater 2010;6:617–25.
hydroxyapatite from Raman microspectroscopy and wide-angle X-ray [273] Drouet C, Largeot C, Raimbeaux G, Estournès C, Dechambre G, Combes C, et al.
scattering. Calcif Tiss Int 2006;79:354–9. Bioceramics: spark plasma sintering (SPS) of calcium phosphates. Adv Sci
[240] Pekounov Y, Petrov OE. Bone resembling apatite by amorphous-to-crystalline Technol 2006;49:45–50.
transition driven self-organization. J Mater Sci Mater Med 2008;19:753–9. [274] Mazzaoui SA, Burrow MF, Tyas MJ, Dashper SG, Eakins D, Reynolds EC.
[241] Tao J, Pan H, Wang J, Wu J, Wang B, Xu X, et al. Evolution of amorphous Incorporation of casein phosphopeptide–amorphous calcium phosphate into
calcium phosphate to hydroxyapatite probed by gold nanoparticles. J Phys a glass-ionomer cement. J Dent Res 2003;82:914–8.
Chem C 2008;112:14929–33. [275] Uysal T, Amasyali M, Koyuturk AE, Sagdic D. Efficiency of amorphous calcium
[242] Onuma K, Ito A. Cluster growth model for hydroxyapatite. Chem Mater phosphate-containing orthodontic composite and resin modified glass
1998;10:3346–51. ionomer on demineralization evaluated by a new laser fluorescence device.
[243] Oyane A, Onuma K, Kokubo T, Ito A. Clustering of calcium phosphate in the Eur J Dent 2009;3:127–34.
system CaCl2–H3PO4–KCl–H2O. J Phys Chem B 1999;103:8230–5. [276] Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate
[244] Yin X, Stott MJ. Biological calcium phosphates and Posner’s cluster. J Chem stabilized by casein phosphopeptides: a review. Special Care in Dentistry
Phys 2003;118:3717–23. 1998;18:8–16.
[245] Eanes ED. The interaction of supersaturated calcium phosphate solutions [277] Tung MS, Eichmiller FC. Dental applications of amorphous calcium
with apatitic substrates. Calcif Tiss Res 1976;20:75–89. phosphates. J Clinical Dentistry 1999;10:1–6.
[246] Eanes ED. Crystal growth of mineral phases in skeletal tissues. Progr Cryst [278] Dorozhkin SV. Calcium orthophosphate-based biocomposites and hybrid
Growth Character 1980;3:3–15. biomaterials. J Mater Sci 2009;44:2343–87.
S.V. Dorozhkin / Acta Biomaterialia 6 (2010) 4457–4475 4475

[279] Wei D, Zhou Y. Characteristic and biocompatibility of the TiO2-based coatings [296] Ranjitkar S, Narayana T, Kaidonis JA, Hughes TE, Richards LC, Townsend GC.
containing amorphous calcium phosphate before and after heat treatment. The effect of casein phosphopeptide–amorphous calcium phosphate on
Appl Surf Sci 2009;255:6232–9. erosive dentine wear. Aus Dent J 2009;54:101–7.
[280] Dunn WJ. Shear bond strength of an amorphous calcium-phosphate- [297] Giniger M, MacDonald J, Spaid M, Felix H. A 180-day clinical investigation of
containing orthodontic resin cement. Am J Orthod Dentofac Orthoped the tooth whitening efficacy of a bleaching gel with added amorphous
2007;131:243–7. calcium phosphate. J Clinical Dentistry 2005;16:11–6.
[281] Keçik D, Çehreli SB, Sßar Ç, Ünver B. Effect of acidulated phosphate fluoride and [298] Giniger M, MacDonald J, Ziemba S, Felix H. The clinical performance of
casein phosphopeptide–amorphous calcium phosphate application on shear professionally dispensed bleaching gel with added amorphous calcium
bond strength of orthodontic brackets. Angle Orthod 2008;78:129–33. phosphate. J Am Dent Assoc 2005;136:383–92.
[282] Foster JA, Berzins DW, Bradley TG. Bond strength of an amorphous calcium [299] Ramalingam L, Messer LB, Reynolds EC. Adding casein phosphopeptide–
phosphate–containing orthodontic adhesive. Angle Orthod 2008;78:339–44. amorphous calcium phosphate to sports drinks to eliminate in vitro erosion.
[283] Uysal T, Ulker M, Akdogan G, Ramoglu SI, Yilmaz E. Bond strength of Pediatric Dentistry 2005;27:61–7.
amorphous calcium phosphate-containing orthodontic composite used as a [300] Panich M, Poolthong S. The effect of casein phosphopeptide–amorphous
lingual retainer adhesive. Angle Orthod 2009;79:117–21. calcium phosphate and a cola soft drink on in vitro enamel hardness. J Am
[284] Bröchner A, Christensen C, Kristensen B, Tranæus S, Karlsson L, Sonnesen L, Dental Assoc 2009;140:455–60.
et al. Treatment of post-orthodontic white spot lesions with casein [301] Silva KG, Pedrini D, Delbem ACB, Ferreira L, Cannon M. In situ evaluation of
phosphopeptide-stabilised amorphous calcium phosphate. Clin Oral Invest the remineralizing capacity of pit and fissure sealants containing amorphous
2010. calcium phosphate and/or fluoride. Acta Odontol Scand 2010;68:11–8.
[285] Reynolds EC. Calcium phosphate-based remineralization systems: scientific [302] Bayrak S, Tunc ES, Sonmez IS, Egilmez T, Ozmen B. Effects of casein
evidence? Aus Dent J 2008;53:268–73. phosphopeptide–amorphous calcium phosphate (CPP–ACP) application on
[286] Kim I, Kim HJ, Kim HM. Array of amorphous calcium phosphate particles enamel microhardness after bleaching. Am J Dentistry 2009;22:393–6.
improves cellular activity on a hydrophobic surface. J Biomed Mater Res B [303] Yengopal V, Mickenautsch S. Caries preventive effect of casein phospho-
(Appl Biomater) 2010;93:113–21. peptide–amorphous calcium phosphate (CPP–ACP): a meta–analysis. Acta
[287] Sun W, Zhang F, Guo J, Wu J, Wu W. Effects of amorphous calcium phosphate Odontol Scand 2009;67:321–32.
on periodontal ligament cell adhesion and proliferation in vitro. J Med Biol [304] Walker GD, Cai F, Shen P, Reynolds C, Ward B, Fone C, et al. Increased
Eng 2008;28:107–12. remineralization of tooth enamel by milk containing added casein
[288] Llena C, Forner L, Baca P. Anticariogenicity of casein phosphopeptide– phosphopeptide–amorphous calcium phosphate. J Dairy Res 2006;73:74–8.
amorphous calcium phosphate: a review of the literature. J Contemp Dent [305] Walker GD, Cai F, Shen P, Bailey DL, Yuan Y, Cochrane NJ, et al. Consumption
Pract 2009;10:1–9. of milk with added casein phosphopeptide–amorphous calcium phosphate
[289] Cai F, Shen P, Morgan MV, Reynolds EC. Remineralization of enamel remineralizes enamel subsurface lesions in situ. Aus Dent J 2009;54:245–9.
subsurface lesions in situ by sugar-free lozenges containing casein [306] Willershausen B, Schulz–Dobrick B, Gleissner C. In vitro evaluation of enamel
phosphopeptide–amorphous calcium phosphate. Aus Den J 2003;48:240–3. remineralisation by a casein phosphopeptide–amorphous calcium phosphate
[290] Langhorst SE, O’Donnell JNR, Skrtic D. In vitro remineralization of enamel by paste. Oral Health Prev Dent 2009;7:13–21.
polymeric amorphous calcium phosphate composite: quantitative [307] Mei HL, Chen LY, Zhang D, Zhang PL, Liu B, Zhao W, et al. Effects of casein
microradiographic study. Dent Mater 2009;25:884–91. phosphopeptide–stabilized amorphous calcium phosphate solution on
[291] Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel remineralization. J Clin Rehabil Tiss Eng Res 2009;13:4825–8.
enamel subsurface lesions by sugar-free chewing gum containing casein [308] Dorozhkin SV. Nanodimensional and nanocrystalline apatites and other
phosphopeptide–amorphous calcium phosphate. J Dent Res 2001;80: calcium orthophosphates in biomedical engineering, biology and medicine.
2066–70. Materials 2009;2:1975–2045.
[292] Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Acid resistance of [309] Dorozhkin SV. Nanosized and nanocrystalline calcium orthophosphates. Acta
enamel subsurface lesions remineralized by a sugar-free chewing gum Biomater 2010;6:715–34.
containing casein phosphopeptide–amorphous calcium phosphate. Caries [310] Suvorova EI, Buffat PA. Size effect in X-ray and electron diffraction patterns
Res 2004;38:551–6. from hydroxyapatite particles. Crystallogr Rep 2001;46:722–9.
[293] Cai F, Manton DJ, Shen P, Walker GD, Cross KJ, Yuan Y, et al. Effect of addition [311] Suvorova EI, Buffat PA. Electron diffraction and high resolution transmission
of citric acid and casein phosphopeptide–amorphous calcium phosphate to a electron microscopy in the characterization of calcium phosphate
sugar-free chewing gum on enamel remineralization in situ. Caries Res precipitation from aqueous solutions under biomineralization conditions.
2007;41:377–83. Eur Cells Mater 2001;1:27–42.
[294] Kumar VLN, Itthagarun A, King NM. The effect of casein phosphopeptide– [312] Celotti G, Tampieri A, Sprio S, Landi E, Bertinetti L, Martra G, et al.
amorphous calcium phosphate on remineralization of artificial caries-like Crystallinity in apatites: how can a truly disordered fraction be
lesions: an in vitro study. Aus Dent J 2008;53:34–40. distinguished from nanosize crystalline domains? J Mater Sci Mater Med
[295] Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett 2006;17:1079–87.
DW. The effect of casein phosphopeptide–amorphous calcium phosphate on [313] Tadic D, Beckmann F, Schwarz K, Epple M. A novel method to produce
erosive enamel and dentine wear by toothbrush abrasion. J Dentistry hydroxyapatite objects with interconnecting porosity that avoids sintering.
2009;37:250–4. Biomaterials 2004;25:3335–40.