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CERAMICS
INTERNATIONAL
Ceramics International 41 (2015) 9203–9231
www.elsevier.com/locate/ceramint

Review paper

Substituted hydroxyapatites for biomedical applications: A review

Monika Šupován
Department of Composites and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, V Holešovičkách 41,
Prague 182 09, Czech Republic
Received 11 February 2015; received in revised form 27 March 2015; accepted 30 March 2015
Available online 9 April 2015

Abstract

This review summarizes recent and very recent work on preparing substituted hydroxyapatites. Ease of atomic doping or substitution in apatite
opens this mineral up for a wide range of biomedical applications. It can be used for repairing and replacing diseased and damaged parts of
musculoskeletal systems, and also as a drug or gene delivery agent, as a bioactive coating on metallic osseous implants, biomagnetic particles and
fluorescent markers. First, the physicochemical properties of bioapatites are described and discussed. Then a general summary on substitution
reaction for hydroxyapatite is made. Special attention is paid to describing anionic, cationic and multisubstituted hydroxyapatites used for various
biomedical applications. Finally, conclusions are drawn and future perspectives are discussed.
& 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Keywords: Bioapatite; Calcium phosphate; Hydroxyapatite; Substitution

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9204
2. Bioapatites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9204
3. Substitution in bioapatite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9205
4. Substitution in synthetic hydroxyapatite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9207
4.1. Anionic substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9208
4.1.1. Carbonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9208
4.1.2. Fluorides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9210
4.1.3. Chlorides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9211
4.1.4. Silicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9212
4.1.5. Sulfur and selenium oxyanions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9212
4.2. Cationic substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9213
4.2.1. Magnesium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9213
4.2.2. Lithium, potassium and sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9214
4.2.3. Strontium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9215
4.2.4. Zinc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9216
4.2.5. Other cationic substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9217
4.3. Multi-ionic substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9219
4.3.1. Cationic co-substitution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9219

n
Tel.: þ42 266 009 221; fax: þ 42 284 680 105.
E-mail address: supova@irsm.cas.cz

http://dx.doi.org/10.1016/j.ceramint.2015.03.316
0272-8842/& 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
9204 M. Šupová / Ceramics International 41 (2015) 9203–9231

4.3.2. Anionic co-substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9220

4.3.3. Anionic-cationic co-substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9221
5. Summary and concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9223
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9223
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9224

1. Introduction micron-sized or synthetic HA. The release of calcium ions from

The class of ceramics used for repairing and replacing diseased
and damaged parts of musculoskeletal systems are termed
bioceramics. They range in biocompatibility from ceramic oxides,
which are inert in the body, to the other extreme of bioresorbable
materials. The most widely-used bioresorbable ceramics include
calcium orthophosphates (CaPs). They are present in bones, teeth,
deer antlers and the tendons of mammals, giving these organs
hardness and stability. There are eleven [1] known non-ion-
substituted calcium orthophosphates with a Ca/P molar ratio
between 0.5 and 2.0. The most widely used member of the family
of CaPs is hydroxyapatite (HA).
Other well-known biomedical applications of CaPs are as a drug
delivery agent [2,3] and as a gene delivery agent [4], and as a filler
for biocomposites, as a scaffold for seeding stem cells and
bioactive coatings on metallic osseous implants [5]. In addition,
recent studies have also shown that HA particles inhibit the growth
of many kinds of cancer cells [6].
Generally, nano CaP powders can be synthesized by a variety of
methods, including solid state and wet chemical methods, hydro-
thermal processes, mechanochemical techniques, pH shock waves,
microwave processing, and hydrothermal microemulsion and
nanoemulsion techniques. In past decades, many different methods
have been introduced and presented for preparing CaP nanoparti-
cles with precise control for the microstructure, particle shape
and size.
A number of previous reviews have reported the synthesis and
properties of CaP materials, especially for HA. The most updated
review paper about synthesizing HA, written by Sadat-Shojai et al.
[7] and published in April 2013 in Acta Biomaterialia, presents
procedures for preparing HA particles reported in recent years
(1999–2011). Brilliantly and in detail, it summarizes all available
information on preparation methodologies, and highlights the
inherent advantages and disadvantages involved in each method.
The most updated review that summarizes the current state of the
art for the synthesis of CaP crystals with controlled sizes from
nanoscale to macroscale, and with diverse shapes ranging from
zero-dimensional to three-dimensional, was published by Lin et al.
[8] in June 2014. In addition, Lin et al. summarized studies on the
controlled formation of nano-/microstructures on the surface of
bulk ceramics, and the preparation of macroscopical bone grafts
with 3-D architecture nano-/microstructured surfaces.
The mineral phase can also be obtained from a wide range of
natural sources, referred to as bioapatite (BAP), as an alternative to
processes for preparing synthetic apatite. It is believed that
nanosized BAP isolated or prepared from biogenic sources is the
best material for tissue replacement and regeneration, because it
exhibits enhanced resorbability and much higher bioactivity than
nanosized BAP is also similar to the behavior of the apatitic phase
in real tissue. In recent decades, a number of isolation and
preparation routes for producing BAP have been developed and
published [9]. Since bioapatites have better biocompatibility than
hydroxyapatite, many scientific studies and research projects have
dealt with “synthetic bioapatites” (SHAs) prepared by substitutions,
which chemically and structurally mimic and imitate real, natural
bioapatite. The substitution can affect the biological and mechan-
ical properties. An ionocovalent structural model of the apatite
family shows a structure that can accept both cationic and anionic
substituents. These substitutions induce modifications in the lattice
parameters and in crystallinity, which substantially influence the
solubility of SHA under physiological conditions, without generat-
ing significant changes in the hexagonal apatitic system. Ease of
atomic doping or substitution in apatite has opened this mineral up
for a wide range of compositions. Metal doped or substituted
hydroxyapatites can be used not only as a bone-forming agent [10],
but also as a microbial agent [11,12], as a drug delivery system
[13], as a coating [14] and as a biomarker for nanomedical
applications [15,16].
A number of papers have been published on the production
of SHAs. The effects of various ionic substitutions in HA have
been discussed in numerous articles, in an excellent overview
chapters by LeGeros [17], by Pan and Fleet [18], and in a
review paper of Boanini et al. [19] provided an overview of
studies until 2010 on ion-substituted calcium phosphates
prepared at low temperature, i.e. by direct synthesis in an
aqueous medium or through hydrolysis of more soluble
calcium phosphates. However, no well-arranged and well-
summarized updated review paper on the preparation of SHAs
for biomedical applications has been published in recent times.
This review is organized into a number of sections. After the
introduction (the current section), general knowledge on
bioapatites is presented in the second section. The third section
provides a general summary of substitution in native bioapa-
tites. The fourth section offers a detailed description from the
literature on anionic and cationic substitutions in hydroxyapa-
tite for obtaining SHAs. Finally, the summary presents
concluding remarks on this active research area and discusses
future perspectives.
2. Bioapatites
The major components of apatite-mineralized tissue (bone,
dentin, enamel) are the mineral phase (calcium phosphate), the
organic phase (collagen) and water. Bone and dentin consist of
about 45–70 wt% mineral, 10 wt% water, and the rest is collagen
 M. Šupová / Ceramics International 41 (2015) 9203–9231 9205

with a small proportion of non-collagenous proteins [20]. Enamel 2–3 nm in thickness and tens of nanometers in length and
is strikingly different in its total lack of collagen and its 96 wt% width. The c-axes of apatite crystallites lie parallel to the long
mineral content [21]. axis of the microfibril [25,26] (Fig. 1).
The bone mineral is idealized as hydroxyapatite The degree of crystallinity of biological apatites is always
(Ca10(PO4)6(OH)2), but it is associated with minor groups poor. Handschin and Stern [24] determined the average
and elements (e.g. CO3 2  , HPO4 2  , Na þ , Mg2 þ ) and trace crystallite size of human bone in the direction of the c-axis
elements (e.g. Sr2 þ , K þ , Cl  and F  ), some of them at as 28 nm within the 0–25 age group, reaching a constant
ppm level [20], which play a vital role in the biochemical average domain size of 34 nm within the 30–80 age group.
reactions associated with bone metabolism. Several crystal- There are four different types of crystallographic positions
lochemical formulas describing the chemistry of bioapatite in the apatitic unit cell [21,27]. Four calcium ions per unit cell,
were proposed by Skinner [20] as (Ca, Na, Mg, K, Sr, Pb,. )10 called Ca(I) (Fig. 2), are in columns parallel to the c-axis and
(PO4, CO3, SO4,. )6 (OH, F, Cl, CO3)2, whereas Cazalbou are surrounded by nine oxygen atoms. Other calcium ions, six
et al. [22] used Ca8.3▯1.7(PO4)4.3 (HPO4 and CO3)1.7 (OH and per unit cell, called Ca(II), form two equilateral triangles along
0.5CO3)0.3▯1.7 (where ▯ is a vacancy). The chemical composi- the c-axis at z¼ 1/4 and 3/4, a so-called anion channel, which
tion of apatite in bone (Table 1) [23] can be varied, but not is typically occupied by two monovalent anions (most com-
with the same randomness and flexibility, because apatite has monly OH  , F  , Cl  ) but in some cases by bivalent CO3 2 
several different crystallographic sites where atomic exchanges per unit cell. The large size of the Ca(II) triangle allows motion
can occur, and many different elements with different ionic of the anion along the column axis. The structure is completed
charges can be accommodated or substituted in those positions. by six PO34  anions, where each P atom is coordinated to four
Handschin and Stern [24] analyzed human bone specimens and oxygen atoms. HA has a hexagonal structure with the space
determined the ion concentrations. They found a significant group P63/m.
average variation in concentrations of CO3 2  , Na þ , Mg2 þ , Bone apatite does not have a high concentration of OH 
K þ and Cl  ions within the 0–30 age group; i.e. concentra- groups [29] (Fig. 3(A)), and some bone apatite may not
tions of Na þ and CO3 2  ions increased with age, whereas contain any OH  groups.
concentrations of Mg2 þ , K þ , Cl  ions decreased with age. Another very striking feature of biological nanocrystalline
Apatite crystallites nucleate on and grow within the collagen apatites is the presence of “non-apatitic” environments [30,31].
network in the form of plate-shaped nanocrystals, which are They can be observed by several spectroscopic methods, e.g.
by infrared spectrometry (FTIR)—see Fig. 3(A). These non-
Table 1
apatitic environments correspond to hydrated domains of the
Composition and lattice parameters of the mineral phase of bone compared
with stoichiometric HA, as reported by Dorozhkin and Epple [23]. nanocrystals distinct from apatite domains containing rela-
tively mobile ions (mainly bivalent cations and anions: Ca2 þ ,
Composition Concentration level Bone Stoichiometric HA HPO24  and CO3 2  ). These hydrated surface layers confer
Calcium (Ca) (wt%) 34.80–36.60 39.6
unexpected properties and offer a wide range of reaction
Phosphorus (P) 15.20–17.10 18.5 possibilities, such as crystal–crystal, crystal–substrate, crystal-
Carbonates (CO3) 4.80–7.40 – molecules (e.g. proteins) and crystal–cell interactions, which
Sodium (Na) 0.90 - 1.00 – living organisms can benefit from (Fig. 3(B)) [28,30,32,33].
Magnesium (Mg) 0.60–0.72 –
Chlorine (Cl) 0.10–0.13 –
Fluorine (F) 0.03–0.10 –
3. Substitution in bioapatite
Potassium (K) 0.03–0.07 –
Strontium (Sr) 0–0.05 – The number of ionic substitutions in bioapatites is smaller
Silicon (Si) (ppm) 0–500 –
than for geological apatites, owing to the limited number of
Zinc (Zn) 0–39 – elements present in body fluids. Foreign ions known and/or
Chromium (Cr) 0–0.33 – reported in bone and tooth apatites are F  , Cl  , Na þ , K þ ,
Cobalt (Co) 0–0.025 – Fe2 þ , Zn2 þ , Sr2 þ , Mg2 þ , citrate and carbonate. However,
Manganese (Mn) 0–0.17 – there are structural limits that determine how much of a given
Lattice parameters Bone Stoichiometric HA ion can be incorporated.
a-axis (nm) 0.9410 0.9430 The major substituent in biological apatite consists of carbo-
c-axis 0.6890 0.6891 nates (CO3 2  ), which occur in bone mineral at levels typically of
5–8 wt%. Highly CO3 2  bone crystallites also express a platelet
morphology that interfaces very effectively with collagen fibrils
(Fig. 1) [34]. The carbonate ion can substitute at two sites in the
bioapatite
apatite structure, namely the hydroxyl position (A-type) and the
platelets c-axis phosphate ion position, giving B-type carbonate-apatite. How-
ever, CO3 2  has a planar structure differing from the linear
collagen structure of OH  and the tetragonal structure of PO4 3  .
microfibrils
Numerous geometric configurations of carbonate substitution
Fig. 1. Arrangement of bioapatite nanocrystals along the collagen microfibrils. are therefore possible. The A/B ratio in bone is influenced by
9206 M. Šupová / Ceramics International 41 (2015) 9203–9231

(I) Ca
(II) Ca, Na, Mg
A- P, C
- O
A anion – channel
A- OH¯, F¯, Cl¯, CO

a A- c

b b

Fig. 2. View of the atomic structure of hydroxyapatite as a stand-in for bioapatite. Viewed (A) down the c-axis, and (B) perpendicular to the c-axis.

Na

Non-apatitic Ca
OH Mg
groups HPO CO
Apatite
Ca
core
Mg
HPO
CO Na
Ca
Hydrated
layer Solution

Fig. 3. Infrared spectrum of poorly crystalline bioapatite (A) isolated from bovine bone by alkaline hydrothermal treatment followed by calcination at 500 1C,
showing the non-apatitic environment of phosphate ions after spectral deconvolution. (B) Surface reactions in the hydrated layer that contains loosely bound ions
which can be exchanged by foreign ions and molecules, such as charged groups of proteins [28].

its maturation [35]. B-type carbonate substitution causes changes
in various physical properties, e.g. decreases in a-axial length
together with increases in c-axial length, changes in crystallite
size, and changes in the amount of crystallographic microstrain,
optical birefringence [27] and mechanical strengthening of the
bone [36]. There is an increase in solubility [24,37], because the
Ca–CO3 bonds are weaker than the Ca–PO4 bonds. Smaller
crystallite size and also higher carbonate concentration account
for the much greater solubility of bone apatite than of tooth
enamel. Synthetic A-type carbonate apatite can be produced only
at very high temperature by solid-state reactions at 1000 1C,
whereas synthetic B-type carbonated apatite can be precipitated
from solutions in the temperature range up to 100 1C [17].
However, Rey et al. [38] have demonstrated that at least three
different major sites of carbonate exist in bone mineral. Decon-
volution of the ν2 CO3 2  band in infrared spectra enables the
type of carbonate substitution to be assigned. The major
component at 871 cm  1 is due to B-carbonate, while a band at
878 cm  1 can be assigned to A-carbonate. A band at 866 cm  1
corresponds to labile carbonate ions in an amorphous environ-
ment, in an early state of apatite crystal formation. Carbonates
appear to belong to disordered domains of the apatite crystal, but
their precise locations are unknown. However, it now seems to be
generally accepted that carbonates dominantly replace phosphates
in biological apatite [39]. The deficit in the negative charge
caused by the replacement of PO4 3  by either CO3 2  or
HPO4 2  , can be compensated by the loss of a positive charge,
for example, through removal of Ca2 þ from the lattice. Another
option is to substitute a monovalent ion, typically sodium, for
calcium, because the ionic radii of Ca2 þ and Na þ ions are quite
similar (0.099 and 0.098 nm, respectively) [40]. The essential
characteristic of sodium ions in biological apatites is their
potential role in cell adhesion and also in the bone metabolism
and resorption process [41]. HPO4 2  containing apatites have
traditionally been called Ca-deficient apatites, and they are
defined as those apatitic phases with a Ca/P ratio smaller than
1.67, which is a typical value for stoichiometric HA.
Another influental cationic substitute is magnesium, with
ions typically amounting to as much as to 10 mol% (2 wt%) in
cartilage and bone tissue during the initial phases of osteogen-
esis, while it tends to disappear when the tissue matures [42].
The range of Mg concentrations in the natural dentin and bone
are 1.23 wt% and 0.73 wt%, respectively. Magnesium defi-
ciency adversely affects all stages of skeletal metabolism,
causing cessation of bone growth, a decrease in osteoblastic
and osteoclastic activities, osteopenia and bone fragility [43].
The incorporation of Mg2 þ ions implicates shortening of the
c-axis, owing to the considerably smaller radius of Mg2 þ
(0.069 nm) than of Ca [44]. Moreover, magnesium inhibits
apatite crystallization and destabilizes the apatitic structure.
This consequently favours its thermal conversion into
β-tricalcium phosphate (β-TCP) [42,45–47]. The significance
of potassium in bone composition lies in its tremendous
influence on the biomineralization process [48] and its
important role in the apatite mineral nucleation process [49].
Strontium is one of the metallic elements found in bones,
 M. Šupová / Ceramics International 41 (2015) 9203–9231
where it is present in significant amounts, especially in regions
of high metabolic turnover [50]. It appears to be one of the
most effective substances for the treatment of osteoporosis
[51]. A newly-developed drug, strontium ranelate, has been
shown to have dual effects of stimulating osteoblast differ-
entiation and inhibiting osteoclast activity and bone resorption,
which could reduce the incidence of fractures in osteoporotic
patients [52]. Because calcium and strontium share the proper-
ties of group 2A elements, strontium can replace calcium in
hydroxyapatite, and hence in bone, without much difficulty. It
has been reported that stable strontium is non-toxic, even when
it is administered in large doses in the human body for
prolonged periods [53]. Zinc can be found in all biological
tissues, and bone contains the major part of the total body zinc
[54]. It is an important mineral in the normal growth and
development of the skeletal system, and zinc deficiency is
associated with a decrease in bone density [55]. Zinc inhibits
osteoclast differentiation and promotes osteoblast activity [56].
Moreover, it is well known that it plays an important role in
some biological functions, such as enzymatic activity, nucleic
acid metabolism, protein synthesis and preservation of the
structures and functions of the membranes and hormonal
activity.
Pasteris et al. [27,57] studied the correlation between the degree
of hydroxylation and the degree of atomic ordering in natural and
synthetic apatites. They developed the following mechanistic model:
the smaller the crystallite size and the greater the atomic disorder
within the unit cells of the crystal, the less energetically favorable it
is for apatite to incorporate OH  into its channel sites. “Apatites
with different functions (bone, dentin, enamel) have a different
degree of hydroxylation. The concentration of hydroxyl groups
influences the dynamic equilibrium between bioapatite-collagen, as
they share OH groups. Alternatively, this coupling occurs with non-
collagenous proteins associated with bone mineralization. The
contrast in the degree of hydroxylation between bone and enamel
is reflected in the different solubility of these two bioapatites.
Whereas bone undergoes constant dissolution owing to remodeling,
dissolution of this type is undesirable for enamel. The so-called
anion channel can also be occupied by substituting F  and/or Cl 
anions. Fluoride anions are able to fit best into the channel site at
z=1/4 and 3/4 in the center of the triangles of Ca(II). The OH  ions
in HA are displaced along the c-axis and have split atom positions
with occupancy of 0.5: the hydroxyl oxygen is at z=7(0.198 and
0.302) [58]. A smaller fluoride radius (0.119 nm) than the radius of
a hydroxide anion (0.153 nm) permits the most symmetric position
in the channel, so fluorapatite is the apatite with the highest
symmetry (hexagonal space group P63/m). The incorporation of F 
instead of OH  results in contraction of the unit cell along the a-
axis direction. Substitution of fluoride ions occurs readily, even at
room temperature or at body temperature [59]. This substitution is
desirable, because fluorapatite is less soluble in acidic solutions than
the original hydroxylapatite of the tooth. A positive effect of
fluoride anions was proved in a dental application. Wang et al. [60]
demonstrated that fluoroapatite paste with a neutral pH value
significantly enhanced the hardness of the enamel surface, increased
the surface roughness and also prevented enamel mass loss.
Another positive fact is that fluorine-substituted HA possesses
9207
notable corrosion resistance in the body fluids, and it is an important
therapeutic agent in the treatment of osteoporosis [61]. The structure
of fluorapatite, and partial or complete substitution of the fluoride
ions by hydroxyl ions, is quite well understood [62]. By contrast,
Cl  does not substitute readily for OH  in bone and tooth apatite,
due to its larger ionic size (0.167 nm), although the abundancy of
Cl  in the blood plasma is of the order of tenths of wt%. A large
Cl  anion is displaced along the c-axis at z¼ 7(0.432 and 0.068)
[58] and the coordination of the Cl  anion with Ca(II) is therefore
nearly octahedral in chloroapatite. The significance of chloride ions
lies in their ability to develop an acidic environment on the surface
of bone that activates osteoclasts in the bone resorption process and
digestion of the organic matrix by the acid hydrolases that
osteoclasts secrete. Inorganic sulfate is the fourth most abundant
anion in human plasma, and is required by cells in order to function
properly. Sulfate ions are required by all mammalian cells for cell
growth, for cell matrix synthesis and for maintenance of cell
membranes [63].
4. Substitution in synthetic hydroxyapatite
Ionic substitution in apatites can be well illustrated in
geological apatites. The geological environment provides a wide
range of elements, and temperatures can reach hundreds of
degrees Celsius, which makes the apatite structure more prone
to substitutions, e.g. OH  , Cl  and F  anions can substitute for
each other in the channel sites in almost any proportion. Anionic
complexes can replace the phosphate group and a large number
of bivalent and trivalent metal cations can substitute for calcium.
In fact, apatite is able to incorporate almost half of the elements of
the periodic system in its atomic structure [62]. However,
laboratory conditions related to temperature and pressure do not
enable such a wide range of options as are enabled by a
geological environment, in synthetic hydroxyapatite substitutions.
The incorporation of ions into the HA structure will result in
some structural changes, which can be detected with various
analytical techniques. Ionic substitution can affect the crystal
structure, the crystallinity, the surface charge, the solubility and
other vital properties, leading to major changes in biological
performance upon implantation.
Some previous studies on synthetic hydroxyapatites have
explained the role of substitution of biocompatible cations,
such as Mg2 þ , Zn2 þ , Sr2 þ . Substitutions of bivalent ions can
cause no charge imbalance in the apatite lattice, but substituted
monovalent ions like Na þ and K þ cause a charge imbalance
that can be neutralized by creating supplementary vacancies
[64], or by the occurrence of simultaneous substitutions of
cations and anions without the formation of any vacancy or
loss of charge balance [65]. Replacement of Ca2 þ ions by
other cations can lead to contraction or expansion of the lattice
parameters, depending upon ionic size. It might be assumed
that more voluminous cations can modify the cell volume and
also extend the a-axis parameter. However, this statement is
not valid, mainly in cases when a bivalent cation is substituted
by a monovalent cation. The formation of vacancies in the
channel reduces the diameter of the channel diameter, and
consequently reduces the a parameter [66]. The localization of
9208 M. Šupová / Ceramics International 41 (2015) 9203–9231
substituting cations in the apatitic structure is also generally
correlated to their ionic radius [67]. Site Ca(I) is smaller in
volume than site Ca(II) [68]. Simplistic reasoning might lead
to the conclusion that a cation with a larger radius than radius
of Ca2 þ must prefer and occupy site (II). However, some
experiments have shown the converse. The kind of anion in the
channel is probably of great importance. As an example, in
fluorapatite, the Mn2 þ ion (0.080 nm) substitutes for calcium
(0.099 nm) in site (I); however, in chlorapatite, it substitutes
simultaneously in both sites [67]. The charge of the ions and
the strength of the corresponding bonds affect the distribution
of the cations between these two sites. Another explanation is
that the driving force in the distribution process is optimization
of the Metal–Oxygen interaction: site (I) allows larger cations
to be accommodated because of the longer Metal (I)–O
distances. However, when the number of the bigger ions
increases, the repulsion between atoms in the Metal (I) position
would cause an enlargement of the c-axis, which is partially
restrained by accommodating metal atoms in site (II) [69]. To
maintain the charge balance, trivalent cations would be
incorporated as lower valence hydroxo-ions. The ion-
exchange ability of trivalent ions seems to depend more on
their charge density than on their ionic radii [70].
Monovalent anions (F  , Cl  ) substitute OH  anions in the
anion channel without charge imbalance. Bivalent anion
(HPO4 2  , CO3 2  , SO4 2  , SeO3 2  , SeO4 2  ) substitutions
instead of the trivalent phosphate group are balanced by the
formation of both hydroxide and calcium vacancies. In the
case of tetravalent anion (SiO4 4  ) substitutions, the negative
charge are compensated by a hydroxide vacancies.
Synthetic HA is not ideal for use as a bone substitute in
loadbearing applications, due to its brittleness and lack of
strength. Other disadvantages include a high degree of crystal-
linity, which results in reduced degradability of pure HA when
it is implanted into the organ. In order to overcome this
obstacle, HA is doped or substituted with various metals, such
as magnesium [71–73], manganese [74], zinc [75], titanium
[76] and strontium [53] to improve its mechanical strength.
Moreover, minerals and traces of metal elements accelerate
bone formation [69] and resorption on bone cells or bone
mineral in vivo and in vitro [53].
4.1. Anionic substitution
4.1.1. Carbonates
Carbonates are among the most abundant substituents in
bioapatite. Nanosized carbonate apatites (CHA) have been synthe-
sized by many chemical-processing routes, including wet chemical
precipitation, co-precipitation [36,40,77], emulsion methods
[78,79], sol–gel synthesis [80], mechanochemical methods [81],
mechanochemical–hydrothermal reactions [82], and microwave
precipitation [83]. Sodium hydrogen carbonate (NaHCO3)
[36,40], di-ammonium carbonate ((NH4)2CO3) [68,84], calcium
carbonate (CaCO3) [81] or a carbon dioxide (CO2) saturated
solution [85] can be used as a source for carbonates. Emulsion
processing enables the clustering to be refined and the formation of
hard agglomerates to be restricted. The smaller crystallite size of
HA prepared by the microemulsion method is attributed to the
nanosize discrete microreactors constituting the microemulsion
system that inhibited the growth of crystals. A microemulsion is
a thermodynamically stable isotopic transparent dispersion of two
immiscible liquid phases, such as water and organic (oil), stabilized
by the presence of surfactants. A nanoemulsion is a new class of
emulsions having very fine and uniform droplet sizes up to
500 nm, typically in the range of 20–200 nm. Nanoemulsions are
not thermodynamically stable, but are highly kinetically stable
against sedimentation and creaming. One major disadvantage of
microemulsions is that they require larger amounts of surfactants
(typically over 20 wt%) than conventional emulsions. By contrast,
nanoemulsions offer the possibility to obtain microemulsion-like
dispersions using low surfactant concentrations. Zhou et al. [79]
used the nanoemulsion technique to synthesize nanosized CHA,
even without a surfactant. The nanospheres were in an amorphous
state and became highly crystalline after calcination at 900 1C. The
sol–gel method can reduce the segregation of metal elements, and
can improve the molecular-level mixing of reactants and improve
the chemical homogeneity of the resulting powder, but the major
disadvantages are the high cost of the starting material (metal
alkoxides) and sometimes (or often) the precursor that is formed is
extremely moisture sensitive. The resulting powder exhibits a
stoichiometric structure with a large surface area and a small cluster
size (ranging from 50 nm to about 1 mm). A notable advantage is
that a several hundred degrees lower temperature is required for
calcination and sintering of sol–gel HA. However, Rajabi-Zamani
et al. [86] synthesized CHA via a nonalkoxide sol–gel with the use
of ethanol, which led to a stable sol. Recently, there has been
growing interest in synthesizing ceramic materials by self-
propagating combustion synthesis, which provides intimate blend-
ing among the constituents using a suitable fuel, which also acts as
a complexing agent (e.g. citric acid, urea, sucrose, etc.) in an
aqueous medium and a vigorous exothermic redox reaction
between the fuel and an oxidizer (e.g. nitric acid). Sasikumar
et al. [87] proved that by adding citric acid to succinic acid, the
auto-ignition temperature of succinic acid can be lowered. A
mixture of fuels always leads to TCP as a major product, while the
use of a single fuel results in CHA. Iafisco et al. [88] reported a
new methodology, based on the vapor diffusion sitting drop micro-
method, for precipitating CHA nanoparticles. The method was
developed using an innovative device called the “crystallization
mushroom”, a glass device developed for crystallizing protein and
small molecules. This process consists of diffusing vapors of an
aqueous solution of ammonium hydrogen carbonate (NH4HCO3)
through drops containing an aqueous mixture of calcium acetate
(Ca(CH3COO)2) and di-ammonium hydrogen phosphate
((NH4)2HPO4). An advantage is that there is reduced consumption
of reagents during the crystallization process, since the volume of
micro-droplets is around 40 μl. Another advantage is high
reproducibility. Synthesized CHA crystals displayed nanometric
dimensions, a plate-like morphology, and a low degree of crystal-
linity, closely resembling the inorganic phase of bones, teeth, and
many pathological calcifications. Mechanochemical powder
synthesis is a solid-state synthesis method that enables surface-
bonded species to be perturbed by pressure applied via conven-
tional milling equipment ranging from low-energy ball mills to
 M. Šupová / Ceramics International 41 (2015) 9203–9231
high-energy stirred mills. The main advantages are simplicity, low
cost, and easy control of the compositions, and it also provides a
fine homogeneous nanocrystalline powder that can be consolidated
for the purposes of a specific requirement. This method should be
clearly distinguished from mechanochemical–hydrothermal synth-
esis, which incorporates aqueous phases actively participating in
the mechanochemical reaction with processes such as dissolution,
diffusion, adsorption, reaction rate and crystallization. The mechan-
ochemical activation of slurries can generate local zones of high
temperatures (up to 700 1C) and high pressures due to friction
effects and adiabatic heating, although the overall temperature can
reached room temperature. Apatite prepared by a hydrothermal
process can be densified and consolidated by spark plasma
sintering [89]. This technique benefits from simultaneous applica-
tion of high pressure and an efficient heating process.
A significant amount of research work has been devoted to
determining the precise position of CO3 2  ions in HA by various
methods, such as X-ray (XRD) and neutron diffraction, infrared
vibrational spectroscopy, electronspin resonance spectroscopy
(ESR), thermal decomposition, and nuclear magnetic resonance
(NMR). Substitution leads to the preferential formation of B-type
[68,90,91]. Barralet et al. [40] indicated the presence of carbonate in
the A site for low carbonate contents (o 4 wt%), while at higher
contents the carbonate was located predominantly in the B site.
However, there was no agreement among the studies on the exact
CO3 2  location in the channel and its planar orientation with
respect to the c-axis for A-type. For B-type, there was no agreement
among the studies on CO2 3 substitution with respect to the original
CO4 3  configuration. For A-type substitution, Suetsugu et al. [92]
indicated that the CO3 2  ion randomly occupied six equivalent
positions around the c-axis with its triangular plane parallel to the c-
axis and one of the C–O bonds lying along the c-axis, while Fleet
et al. [93] reported that the carbonate ion was ordered along the
apatite channel at z¼ 0.5, with two of the oxygen atoms lying close
to the c-axis and the plane of the ion canted  121. They found a
new space group ðP3Þ and a new orientation for the carbonate ion
in the apatite channel. Further refinement of this structure was
studied by the same authors [94]. With the aid of quantum chemical
ab initio calculations using the supermolecule model, Peeters et al.
[95] calculated that the angle between the planar CO3 2  ion and
the c axis is equal to 71. The carbon atom and two oxygen atoms
(single bonds) were found close to this axis, whereas the bond with
the third oxygen atom (double bond) is almost perpendicular to it.
The dependency of the channel carbonate orientations on pressure
and composition were studied by Fleet et al. [96]. They proved two
separate orientations of carbonate ions: A1 (two oxygen atoms close
to the c-axis) and A2 (only one oxygen atom close to the c-axis),
and re-evaluated the high-pressure configuration (A2) and their
mutual transformation (A1-A2).
For B-type carbonate apatite, Ivanova et al. [97] reported
that carbonate ions randomly occupied the side face of the
phosphate tetrahedron, but Leventouri et al. [98] proposed the
mechanism of carbonate substitution on the mirror plane of the
phosphate tetrahedron. Wilson et al. [90] collected analyses of
several methods, X-ray and time of flight neutron diffraction
data, FTIR, MAS-NMR spectra, inductively coupled plasma-
atomic emission spectrometry (ICP-AES) and carbonate
9209
analyses from a sodium-containing carbonate (12.5 wt%)
apatite. Four structural models from the literature with the
carbonate ion explicitly modeled were fitted to the neutron
diffraction data by the Rietveld method. The best fit was
obtained with the CO3 2  ion in disorder between the mirror
symmetry related faces of a vacant PO4 3  site and with the
normal to the plane of the CO3 2  ion at  301 to the c-axis.
This result contradicts the previous Rietveld studies [97,99].
Substitution configurations of AB-type and corresponding
locations of CO3 2  are more complicated. Fleet and Liu
synthezised AB-type carbonate apatite at high temperature and
pressure (1400 1C, 3 GPa) [100] and at moderate temperature
and pressure (1200 1C, 1 GPa) [101]. Another difference was
the presence of Na þ ions in the final apatite structure [101]. The
crystallographic formula for Na-free AB carbonate hydroxyapa-
tites was established as: (Ca10(PO4)6 y(CO3)x þ (3/2)y(OH)2 2x;
where x 0.7 and y  0.6) [100]. Na-bearing AB carbonate
HA is discused in Section 4.3.3. Ren et al. [102] revealed,
using ab initio quantum mechanical calculations, that the
most energetically stable substitution is AB-type, followed by
A-type and then B-type. The most stable configuration of
A-type substitution has its carbonate triangular plane almost
parallel to the c-axis at z¼ 0.46. The lowest B-type energy
configuration has a calcium ion replaced by a sodium ion,
owing to charge balance, and the carbonate lying almost flat on
the b/c-plane. The lattice parameter changes with carbonate
substitution in the following ways: A-type substitution tends to
increase a and decrease lattice parameter c, whereas B-type
substitution shows the opposite effect. The same trend was
earlier proved by Fleet et al. [103], using XRD and FTIR
methods. Using computer modeling, Peroos et al. [104]
determined that A-type, where two hydroxy groups are
replaced by one carbonate group, is energetically preferred,
followed by the combined AB-type, where both a phosphate
group and a hydroxy group are replaced by two carbonate
groups. B-type substitution, where a phosphate group was
replaced by both a carbonate group and another hydroxy group
was energetically neutral. If the replacement of the phosphate
group by a carbonate group was charge compensated by the
substitution of a sodium or potassium ion for a calcium ion, the
resulting B-type was energetically favorable. It can be stated
that it is always a difficult task to make an experimental
determination of the crystal structure with ionic substitution. This
is particularly true for B-type CHA, because the proposed
carbonate substitution models result indirectly from a best fit of
powder diffraction patterns measured from a limited number of
synthetic specimens, and they do not provide a fully convincing
crystallographic model [105]. Moreover, the final carbonate apatite
products do not contain the same concentrations of carbonates
[90,91,101,106]. However, the carbonate concentration influences
the lattice parameters, as was proved by El Feki et al. [91]. The
lattice parameters of the lower carbonate content apatite (9.75 wt%)
was a¼ 0.93892(4) and c¼ 0.69019(3) nm, compared to the lattice
parameters of the higher carbonate concentration (16.60 wt%),
which are a¼ 0.93249(1) and c¼ 0.69213(1) nm. Regrettably,
a carbonate concentration exceeding 8 wt% does not reflect the real
situation in the apatitic structure of native bioapatites.
9210 M. Šupová / Ceramics International 41 (2015) 9203–9231
For charge balance in B-type apatite, Na þ co-substitution
[68,90,91] was experimentally identified and it was considered
for computer modeling, because it is the most likely case for
apatite formation under physiological conditions. However,
Ivanova et al. [97] prepared synthetic calcium-deficient apatite
using an NH4H2PO4 solution, and the chemical formula was
therefore presented as Ca9.50(NH4)0.10(PO4)5.05(CO3)0.95(OH)2,
and the charge imbalance was primarily compensated by
vacancies in the Ca(I) sites.
Carbonate ions can enhance the sinterability of calcium
phosphate ceramics, but only in the case of B-type CHA [107].
While B-type carbonation decreases the sintering temperature,
A-type carbonation induces a slight shift to higher sintering
temperatures. AB-type HA ceramics with a controlled compo-
sition and fine grains can be fully sintered at temperatures
(T o 900 1C) in mixed flowing atmospheres containing partial
pressures of CO2 and H2O vapor [84,108]. Barralet et al. [40]
studied the effect of reaction temperature and concentration of
bicarbonate ions on particle morphology and phase composi-
tion. These dependencies are summarized in Fig. 4.
4.1.2. Fluorides
Another important anionic substituent present in the bioapa-
tite structure is fluorine. Fluorine-substituted HA (FHA) can
embody the chemical composition of Ca10(PO4)6OH2  xFx
(where x¼ 0–2). The substitution of OH  by F  induces a
shift of (300) XRD reflection to a higher-angle direction,
indicating contraction of the a axis lattice parameter. HA and
fluoroapatite (FA) structures are very similar, but there is a
decrease of approximately 0.00509 nm from HA to FHA due to
the smaller F  ion fitting more easily within the OH/F channels.
By contrast, the c axis parameter increases with fluoride
substitution, however the change in c between HA and FA is
an order of magnitude smaller than the a axis parameter [109].
Rodríguez-Lorenzo et al. [110] found that the introduction of
Fig. 4. Effect of bicarbonate concentration together with temperature of precipitation on the particle morphology and phase composition of carbonate apatite [40].
fluoride promotes hydrogen bonding between the hydroxide and
fluoride within the 63 channel.
Wu et al. [111] prepared FHA by a reaction of HA and CaF2
(0–50 wt%) followed by calcination at 700 1C. They found
that the uncalcined materials were stable in OH vibrational
groups and showed neither OH–F–OH nor OH–F hydrogen
bonds. With increasing CaF2 wt%, OH–F–OH and then OH–F
hydrogen bonds of sintered FHA composites formed. Eslami
et al. [112] proved that thermal stability is substantially
increased with increasing incorporation of F  into the HA
structure. The solubility can be fine-tuned, and correspond-
ingly the biological lifetime, by varying the amount of fluoride
substitutions. Cell culture results revealed that the F  content
affects the behaviors of cultured cells in various ways: a high
F  content produces a low surface potential, which favors cell
attachment. However, a reduction in Ca2 þ release could
inhibit cell proliferation. Fluorine can act as a superior
alternative to coating of titanium implants. Currently, most
HA coatings are produced by a plasma-spraying technique.
The sol–gel technique, which is a thin film method, provides
some benefits over the plasma spraying method, e.g. chemical
homogeneity, a fine grain structure, and a low processing
temperature. It is simple and cost-effective, and also effective
for coating complex-shaped implants. Tredwin et al. [113]
proved that the bond strength increased with increasing F 
content at all calcination temperatures, and this was particu-
larly noticeable at 800 and 1000 1C. FHA has been widely
prepared by multiple wet techniques, e.g. direct precipitation
from Ca2 þ , PO4 3  and F  containing solutions [109,114–
117], sol–gel [118], surfactant-assisted preparation [119], in
the presence of low molecular polyacrylic acid (PAA) [120],
by hydrolysis [121] and by immersion of HA in a sodium
fluoride (NaF) solution [122]. Tredwin et al. [118] synthesized
HA, FHA and FA by the sol–gel method. Calcium nitrate and
triethylphosphite were used as precursors under an ethanol-
water based solution. They found that increasing incorporation
HA + calcite
spheroidal
intermediate
acicular
 M. Šupová / Ceramics International 41 (2015) 9203–9231
of F  into the apatite structure results in a decreased crystal-
lization temperature and a more compact unit cell structure.
Addition of F  results in changes in the rheological properties
of the sol–gel, and may have implications if used to coat
titanium. Zhang and Zhu [119] prepared FHA nanorods by
adding Tween80, a non-ionic surfactant. The nanorods were
elongated along the c-axis owing to the enlarged difference
between the growth rates of the crystal surfaces due to the
adsorption of Tween80. It was found that acidic solutions were
unfavorable for FHA preparation, and the proper pH should be
controlled at  9. The effect of F  on the hydrolysis of
tetracalcium phosphate (TTCP; Ca4(PO4)2O) in 0.1 mol/l
potassium dihydrogen phosphate (KH2PO4) containing 62–
83 mmol/l potassium fluoride (KF) was studied by Kurmaev
et al. [121]. They concluded that hydrolysis of TTCP in the
presence of KF converts into FA, while they observed no
formation of CaF2, which is often found in the hydrolysis of
HA at a high fluoride concentration. The solution combustion
method used by Zhao et al. [123] seems to be an interesting
method for preparing nanostructured FHA. However, most wet
methods need precise controls on the processing conditions,
because the composition and the properties of the final
products are strongly influenced by various parameters. More-
over, the procedure and the apparatus for these processes are
complicated. Post-heat treatment and a long time are needed to
obtain final products with stoichiometric composition and
proper crystallinity. Wet methods are therefore not so suitable
for mass production, especially on an industrial scale. Mechan-
ochemical methods can be used for this purpose [124–126]. A
milling procedure, applied for several hours, mechanically
induced a solid-state reaction between HA and a source of
fluorine (CaF2 [111,124,125] and MgF2 [125]). Fathi and
Zahrani [126] prepared and characterizated FHA nanopowders
with different degrees of fluoridation via a mechanical alloying
method with 0, 25, 50, 75, and 100 % replacement of OH 
group with F  ions. Powders were synthesized using a mixture
of appropriate amounts of calcium hydroxide, phosphorous
pentoxide and CaF2 powders with a high energy planetary ball
mill for 6 h at 300 rpm, using eight balls 20 mm in diameter
and a ball-to-powder weight ratio equal to 35:1. The fabricated
Ca10(PO4)6F2 nanoparticles (35–65 nm) fulfilled the require-
ments of the ASTM 1185-88 standard specification for use as
biomaterials. Zhang et al. [124] synthesized FHA successfully
from starting materials of CaCO3, CaHPO4  2H2O and CaF2
via a mechanochemical route together with a hydrothermal
process. Dry mechanochemical synthesis involves only a solid-
state reaction. However, the addition of a solvent medium
would generate local zones of high temperatures and high
pressures due to friction and adiabatic heating of gas bubbles.
Effects of this kind are similar to the hydrothermal processes in
autoclaves [82]. This process therefore provides hydrothermal
conditions, but without the need for a pressure vessel and
external heating. A comparison was made [124] between the
formation of FHA using two milling media (H2O, acetone),
and it was found that apatite phases are formed both in
deionized H2O and in acetone, but at varied rates. The kinetics
of reactions in acetone is evidently slower than the kinetics
9211
observed in deionized water. The fast kinetics of reactions in
deionized water therefore resulted from the hydrothermal
process induced by the mechanical collision besides the
solid-state reaction. It was concluded that the starting materials
at first reacted into a poorly crystallized calcium-deficient
apatite, and complete incorporation of the F  ions into the
apatite occurred in the second stage. A very interesting
approach was used by Murugan et al. [127]. They used
bioapatite (BAP) isolated from bovine bone by an alkaline
hydrothermal process, followed by calcination at 700 1C. The
fluoridation of BAP was carried out using two methods: in the
low-temperature method, a stoichiometric amount of hydro-
fluoric acid was allowed to react with BAP in a hydrothermal
apparatus at 250 1C for 30 min, under pressure. In the high-
temperature method, stoichiometric amounts of BAP and
sodium fluoride were ground well in an agate mortar and
heated at 900 1C for 12 h.
Kim et al. [125] evaluated the effect of fluoride ions on the
mechanical properties and on the inhibition of phase decom-
position of HA. They found that the addition of MgF2 (5 vol%)
suppressed the decomposition of HA due to the substitution of
F  for OH  in the apatitic structure. A comparison of two
additives (MgF2 and CaF2), showed that MgF2 was much more
effective for suppressing phase decomposition in the HA/
alumina composites. When MgF2 was added, higher mechan-
ical properties (flexural strength  170 MPa and Vickers
hardness  7 GPa) were obtained than for HA composites. It
has been proved that F  substitution in HA influences the
sintering of FHA compared to pure HA. It is apparently much
more complex to analyze, because it depends on the fluoride
content. Equal amounts of fluoride and hydroxide ions (x 1)
were observed to reduce the sintering greatly [108,128].
4.1.3. Chlorides
Pure chloroapatite cannot be a good biomaterial because total
replacement of the hydroxyl groups by chloride ions will
enhance the acidity of the local environment, leading to rapid
solubilization of alkaline salts. The content of chlorine in natural
bone can be up to 0.13 wt%, whereas in pure chloroapatite the
content is 6.8 wt%. Chloride-substituted HA (ClHA) is therefore
more suitable for biomedical applications. Kannan et al.
[129,130] synthetized ClHA with the crystallochemical formula
(Ca10(PO4)6(OH)2 x(Cl)x, where x = 0.2–2), corresponding to
the weight range (0.64–6.42 wt%), using an aqueous precipita-
tion method with ammonium chloride (NH4Cl) as a source of
chlorine. They studied the effect of incorporated chlorine on the
lattice parameters. Substitution of chlorine in calcium-deficient
apatites tends to form biphasic mixtures of HA and TCP after
calcination, and the proportions in the mixtures were dependent
on the deficiency of calcium in the precursors that were used.
Zhao et al. [123] also used the solution combustion method for
preparing nanostructured ClHA. They proved that the a-axis
parameter of the Cl-substituted samples increased gradually with
the increase in Cl  concentration, unlike in the case of FHA.
All the data indicated a corresponding expansion and contrac-
tion of the a-axis parameter of the substituted Cl  and F  ,
respectively, due to the size mismatch.
9212 M. Šupová / Ceramics International 41 (2015) 9203–9231
Kannan et al. [130] found a significant trend in expansion
also of the c-axis and the unit cell volume parameters. Fahami
et al. [131] produced nanosized ClHA by a mechanochemical
process. Mechanical activation was performed in a high energy
planetary ball mill at a rotational speed of 600 rpm and a ball-
to-powder weight ratio of 20:1, for 5, 30, 60, 180, and
300 min. They found that at the beginning of milling, the
main products were stoichiometrically deficient chlorapatite
and calcium oxide. Highly crystalline ClHA nanopowder was
obtained after 300 min of milling. When the duration of the
milling was increased to 300 min, the lattice strain increased
significantly to around 0.0108 7 0.0005, while the crystallite
size decreased intensely to 28 7 2 nm and the mean particle
size was about 95 nm. The phase transitions and the structural
changes under thermal treatment of ClHA up to 1300 1C were
studied in a further paper [132]. It was proved that during
annealing in the range of 900–1300 1C for 1 h, the residual
elastic strain declined sharply to about 0.01 %, which led to a
considerable increase in crystallite size to around 462 nm. The
same authors used ClHA for producing various bionanocom-
posites with various reinforcing agents based on bioinert
ceramics, such as Al2O3 [133], TiO2 [134,135] and ZnO [136].
4.1.4. Silicates
Many reports have suggested that silicon would be essential
for the metabolic processes associated with growth and skeletal
development and integrity of the extracellular matrix, with
direct effects of Si on the biomineralization process. Si has also
been shown to have effects on the differentiation, proliferation
and collagen synthesis of osteoblasts, and also dose-dependent
effects on the remodeling process and on osteoclast develop-
ment and resorption activities.
Silicate substituted HA (SiHA) powders have been synthetized
by solid state reaction methods [137,138], hydrothermal methods
[139,140], precipitation methods [141–144] and sol–gel methods
[145], with various Si containing precursors, such as calcium
silicates (CaSiO3, Ca2SiO4) [140], tetraethyl orthosilicate Si
(OC2H5)4 [139,145–147], silicon acetate Si(OCOCH3)4 [141–
143,148] and silicon oxide (SiO2) [137]. Silicate ions (SiO4)4
are structurally incorporated into the HA lattice replacing
phosphate ions. The negative charge of the silicate anions
substituting phosphate anions is balanced by the formation of
hydroxide vacancies. The crystallochemical formula for SiHA
can therefore be expressed as Ca10(PO4)6 x(SiO4)x(OH)2 x,
where x 0.05–1.38. The amount of Si that can be incorporated
into HA seems to be limited to a maximum of 5 wt% [149,150],
and amounts around 1 wt% have been suggested to be enough to
elicit major bioactive improvements. However, Palard et al. [143]
used a solution with a molar excess of silicon (x¼ 4) correspond-
ing to  11 wt% of Si. They proved that single phase SiHA, with
0oxo1, could be synthesized after thermal treatment of the raw
powders above 700 1C. The presence of carbonate groups in the
raw apatite played an important role in the incorporation of
silicates during heating. Powders containing more than 1.0 mol of
silicon were always composed of two phases (apatite and α-TCP),
even after calcination appeared simultaneously from 700 1C. For
x¼ 4, the XRD pattern embodied very low crystallinity, even
after thermal treatment. It was hypothesized that a glassy phase
could be formed from polycondensation of the Si(OH)4 second-
ary phase into amorphous silica.
Rietveld refinement method demonstrated [142,144] that the
entry of silicon (0.4 wt%) into the HA lattice results in minor
changes to the parameters of the crystal structure. The a-lattice
parameter contracted by  0.005%, while the c-parameter
expanded by  0.1 %. This substitution has no significant
effect on the interatomic distances in the phosphate tetrahe-
dron. However, the angles between the phosphorus/silicon
atoms and the oxygen atoms of the phosphate tetrahedron are
distorted from the ideal value of 109.471. The angle of
distortion increased by 0.2 % when the slightly larger silicon
atom substitutes for phosphorus in the HA lattice. Arcos et al.
[138] provided an explanation for the Si substitution mechan-
ism, taking into account that HAs obtained by the ceramic
method under an air atmosphere are not pure HA but
oxyhydroxylapatite (Ca10(PO4)6(OH)xOy). They also took into
account the H atoms of the expected HPO24  anions. Theore-
tical studies of Si substitution in CaP are described in detail by
Pietak et al. [151] in their review paper.
SEM observation showed clearly that the substitution of Si
in HA inhibited the grain growth of SiHA [141], and XRD
results confirm a reduction in crystallite size [139,152]. A
differential scanning calorimetry analysis [153] indicated that
silicon substitution does not result in thermal instability of
SiHA. Porter et al. [154] hypothesized that the incorporation of
silicate ions into HA increases its bioreactivity by increasing
the number of defect structures, which are specific sites
vulnerable to dissolution. This explains the mechanism by
which silicate ions increase the in vivo bioactivity of pure HA.
Many other reports have proved that the incorporation of
silicon into a synthetic HA structure improves its bioactivity
[137,139,145,155–160]. Balamurugan et al. [145] evaluated
the biocompatibility effects of SiHA (Si contents of 0, 1, 3 and
5 mol%) in vitro with human osteoblasts. A high Si content
(5 mol%) appears to promote rapid bone mineralization.
However, a high Si content may result in fast dissolution of
the material, owing to a decrease in crystallite size, which may
not be ideal for cell attachment for prolonged time periods. An
optimum level of Si appears to be 3 mol%, which balances
these effects. The general trend is that silicon substitution
decreases the sinterability of HA [108,161]. SiHA powders can
be used for coatings on commercially pure titanium using an
aerosol method [137], magnetron co-sputtering [162] and an
electrostatic method [163].
4.1.5. Sulfur and selenium oxyanions
Only a limited number of studies have been conducted on
the substitution of sulfur and selenium oxyanions in apatite.
Sulfur and sulfates are considered as a biological cement,
which is involved in building and rebuilding skin cells, hair,
nails and cartilage, and protects cartilage from osteoarthritis.
Alshemary et al. [164] substituted sulfate ions into hydroxya-
patite (SHA), with the formula Ca10 x(PO4)6  x(SO4)x(OH)2  x,
where x¼ 0.05–0.5, via a microwave-assisted ion exchange
process. The resulting particles were needle-shaped. It was
 M. Šupová / Ceramics International 41 (2015) 9203–9231
found that doping the HA structure with SO4 2  ion increases
the structural disorder. With increasing SO4 2  ion substitution,
the crystal size of SHA decreased from 92 nm for x¼ 0.05 to
40 nm for x¼ 0.5. The increase in the lattice parameters and in
the cell volume could be attributed to the larger ionic radius of
the sulfate (0.258 nm) than of the phosphate (0.238 nm) which it
replaced.
Selenium oxyanion-substituted hydroxyapatite (SeHA) has
been synthesized as a promising idea for treating bone cancers
to reduce the probability of recurrence, because selenium plays
an important role in protein functions, and it has a significant
effect on the induction of cancer cell apoptosis [165]. Another
study indicated that selenate (SeO4 2  ) or selenite (SeO3 2  )
oxyanions exert their cancer chemopreventive effects by
directly oxidizing critical thiol-containing cellular substrates,
and that they are more efficacious anticarcinogenic agents than
selenomethionine or selenomethylselenocysteine with a lack of
oxidation capability [166]. SeHA materials could therefore
serve as a constituent of implant materials, both providing a
scaffold for the newly grown bone tissue and inhibiting the
growth and proliferation of tumor cells.
SeHA materials have usually been synthesized by a co-
precipitation method [165,167,168] with a Se/P molar ratio up
to 1.1. It should also be noted that the adsorption of selenite
and selenate on the HA crystal surface has been also studied
[166,169,170]. It was proven that selenate and selenite are not
exclusively located at the HA surface, but are diffused slightly
in a thickness of a few nanometers [166]. It is also very
interesting that selenate anions were much less sorbed than
selenite [169].
The SeO4 2  ion has a tetrahedral structure like the PO4 3 
ion, but it is slightly larger—0.249 nm in diameter—than the
phosphate ion, which is 0.238 nm in diameter. By contrast, the
SeO3 2  ion has a very similar diameter (0.239 nm), but it has
a quite different flat trigonal pyramid geometry. The substitu-
tion of bivalent selenium oxyanions forms a positively charged
vacancy compensated by simultaneous decalcification and
dehydroxylation according to the reaction [167]
Ca10 ðPO4 Þ6 ðOHÞ2 þ xðSeOn Þ2  -Ca10
 xðPO4 Þ6  x ðSeOn Þx ðOHÞ2–x
þ xðPO4 Þ3– þ Ca2 þ þ xðOHÞ 
Kolmas et al. [167] found that the introduction of selenate ions
resulted in a slight increase in parameter a up to x¼ 0.8. It did not
cause any significant changes beyond that level, and the change in
parameter c was very small. However, the substitution of selenite
ions resulted in a significant extension of the a axis, while the c
axis remained practically unchanged. Selenite embodies a com-
pletely different spatial structure (a flat trigonal pyramid), and it
therefore has a significant effect on parameter a of cell, despite its
dimensions close to those of the phosphate ion. However, Ma et al.
[168] found no obvious trends in the a-axis, but a decrease in the c-
axis when the Se/P ratios increase. They explained this trend as
follows: “When the Se/P ratios are high, one HA unit cell having
more than two Se atoms, an oxygen void (VO) is generated near to
the Se2 O7 6  structure, which can lead to a smaller unit cell.”
9213
Another possible reason is oxidization of the selenite group (Se4 þ )
to selenate groups (Se6 þ ), which shortens the Se–O bonds,
resulting in a decrease in the parameters of the HA lattice.
However, both the a parameter and the c parameter are only
insignificantly affected by introducing a slightly larger, but
tetrahedral selenate ion, structurally resembling the phosphate ion
[167]. Another disrepancy concerns crystallinity. Kolmas et al.
[167] found that the introduction of selenite or selenate ions does
not significantly affect the crystallinity, while Ma et al. [168]
observed a reduction in crystallinity as the Se/P molar ratios
increased, until an amorphous phase (for an Se/P ratio equal to 0.3,
i.e. x¼ 1.38) appeared in the SeHA powder.
Rodríguez-Valencia et al. [171] proved that there was no
cytotoxicity when an amount of 0.6 at% of Se was added to the
SeHA coatings, and that there was also excellent proliferation
of the MC3T3-E1 preosteoblasts. Antibacterial properties were
also proved with the inhibition of Pseudomonas aeruginosa
and Staphylococcus aureus (S. aureus) from establishing
bacterial biofilms. Kolmas et al. [167] showed that HA-
containing selenate ions were not toxic, whereas HA with
the highest concentration of selenites (x ¼ 1.17) was toxic in
Spirotox and Microtoxs tests.
4.2. Cationic substitution
4.2.1. Magnesium
One of the most widely-used cationic substitutions is the
incorporation of magnesium in concentrations up to 10 mol% in
relation to Ca [172–174]. Syntheses in bulk were realized by a
precipitation method [46,71,172,173,175–186], by sol–gel
[187–189], in the presence of low molecular PAA [120], by
the mechanochemical–hydrothermal route [174], by surfactant-
assisted microwave synthesis [190], by wet mixing [191] or by
melting and extrusion [192]. Wet chemical processes are usually
carried out at room temperature [46,175,177,182] or in a
temperature range of 40–60 1C [71,172,173,178,179]. However,
preparation temperatures of  100 1C have also been used
[176]. In the case of wet precipitation, the reactions are
performed in freshly distilled water and under an N2 atmo-
sphere, in order to avoid incorporating carbonates into the Mg-
HA lattice. Khanra et al. [192] prepared magnesium substituted
hydroxyapatite (Mg-HA) by a combination of melting and
extrusion routes with the use of a melted 99.9 % pure Mg
ingot. Lijuan et al. [184] studied the effect of two different
magnesium sources, magnesium nitrate and magnesium stearate.
They proved that Mg-HA synthesized by magnesium stearate
had lower crystallinity and better dispersibility.
Landi et al. [172] proved that ion substitution and synthesis
conditions of 5.7 mol% with respect to Ca in Mg-HA made the
apatite similar to biological apatite in terms of composition,
morphology and crystallinity, and no genotoxicity, carcino-
genicity or cytotoxicity was revealed when in vivo tests were
performed.
Bigi et al. [175] used Rietveld structure refinements, and found
that magnesium incorporated into the HA structure essentially in
the Ca(II) site (Fig. 2). They explained this preference on the
basis of the different Metal–Oxygen interactions and the different
9214 M. Šupová / Ceramics International 41 (2015) 9203–9231
geometries of the two distinct metal sites in the apatite structure.
Partial substitution of Mg2 þ for Ca2 þ causes a reduction in the
dimension of the c-axis [42,44–46,175], owing to the consider-
ably smaller radius of Mg2 þ (0.069 nm) than of Ca2 þ
(0.099 nm) [44]. This difference leads to strong distorsion of
the HA lattice and consequently to a decrease in crystallinity.
However Ren et al. [176] performed a computational study using
ab initio generalized gradient approximation density functional
theory to reveal changes in the lattice parameters. They found that
both lattice constants, a and c, decreased with increasing Mg
substitution, and that the Ca(I) site in the HA lattices provided
energetically favored sites for Mg substitution. Tampieri et al.
[178] proved that the Ca(I) position was preferential for Mg
substitution. Farzadi et al. [177] found that lattice parameter a
increased slightly. These discrepancies can be explained by strong
dependency of the lattice parameters on the preparation method,
as different methods for preparing HA-like materials would
probably result in different levels of hydration and possibly other
defects in the materials.
Changes in lattice parameters due to substitution have a direct
impact on the properties of Mg-HA, compared to their non-
substituted analogs [47,172,174,178]: the changes notably
increase their solubility and biodegradability in physiological
fluids, and favor their thermal conversion into Mg-substituted
β-tricalcium phosphate (β-Ca3 xMgx(PO4)2) [185,186]. Cacciotti
et al. [179] studied the effect of Mg substitution on sinterability,
and they proved that Mg incorporation induced increased residual
porosity. The influence of Mg concentration (up to 1.8 wt%) on
the mechanical properties of Mg-HA was the topic of a detailed
study by Zyman et al. [191]. They found that the compressive
strength and microhardness values for Mg-HA ceramics
decreased sufficiently, but that they were still higher than the
values for bone. However, the fracture toughness, which is a
force characteristic of destruction, showed a tendency to grow
with increasing magnesia concentration up to 0.6 wt% Mg, close
to the magnesium concentration in the natural bone mineral. This
is caused by a decrease in the average grain size, by an increase
in microporosity, by removal of the texture, and by the formation
of a weak intergranular boundary. The same trend was proved by
Fadeev et al. [185].
The increase in the total magnesium content in the solid
phase is always significantly higher than the amount incorpo-
rated into the apatite structure. The excess is probably located
in the amorphous phase and/or on the crystallite surface
[45,47]. Tampieri et al. [178] proved that samples became
completely amorphous between 35 and 50 at% of Mg. Addi-
tional studies found that, according to the synthetic procedure
or the age of the Mg-HA material, higher Mg concentrations at
the surface of the HA crystallites can be observed [181,182].
Bertinetti et al. [181] observed the migration process of Mg2 þ
ions from the bulk towards the surface after 2 months,
indicating that a material of this type stored in air is unstable.
Other studies by Bertinetti et al. [182] proved that HA enriched
with Mg2 þ , particularly in the case of a high Mg content (e.g.
3 wt% Mg), was found to retain more water at its surface than
the Mg-free sample, both at the level of H2O co-ordinated to
cations and adsorbed in the form of multilayers. It has not been
possible so far to unequivocally identify a mineral phase.
However, some similarities have been found with hydrated
magnesium and/or calcium–magnesium phosphates.
Electrolytic deposition Mg-HA coatings with various Mg2 þ
concentrations on titanium were investigated by Jiao et al.
[193]. Smaller crystal size and a morphology with a larger
specific surface at higher Mg concentration (2 wt%) may not
only result in better biocompatibility, but may also be more
suitable for the coatings to carry drugs or proteins such as bis-
phosphonate and bone morphogenetic protein (BMP) for
further improvement of bone induction.
4.2.2. Lithium, potassium and sodium
A limited number of studies have been carried out on the
synthesis of lithium (Li-HA), potassium (K-HA) and sodium
(Na-HA) hydroxyapatites.
It is well known that lithium is present in the human body in
trace amounts. Li, which is an inexpensive drug, has been used
in hematology and psychiatry for the treatment of bipolar
disorder. Moreover, there have been reports of stimulating
effects of Li on the proliferation of human cells, such as
thyroid and pancreatic beta cells, and also an effect on bone
mineral density [194]. Kaygili et al. [195] synthesized lithium
calcium phosphate ceramics by the sol–gel method with two
different atomic ratios of lithium (10 and 20 at%). Their results
indicated that the apatitic structure belonging to HA was
transformed fully to Ca10Li(PO4)7 phase with the addition of
Li, and the crystal structure of HA transformed from hexagonal
to trigonal.
Nordsröm and Karlsson [196] prepared K-HA by soaking
HA in potassium carbonate and potassium chloride solutions for
up to 8 weeks, and they found that after 2 weeks one potassium
ion substituted one calcium ion. Generally, it has been reported
that ionic substitutions in the HA crystal lattice can further alter
the protein adsorption properties. Weissmueller et al. [197] for the
first time described the use of molten salt synthesis for preparing
spheroidal K-HAs, and their bovine serum albumin (BSA)
adsorption propensity. BSA adsorption decreased with higher
potassium content. Highest adsorption was observed for the HA/
K2SO4 ¼ 1/3 weight ratio, which was 7-fold higher than the ratio
of 1/9. Literature reports have revealed that potassium can be
substituted in the apatite lattice without significant changes in the
structural parameters [66,196,198]. However, the cell parameters
for the potassium substituted apatites prepared by Kannan et al.
[198] showed contraction in the a-axis and irregular changes in the
c-axis relative to those of pure HA prepared under the same
experimental conditions. This may be due to the formation of
vacancies in the channel, due to the substitution of a bivalent
cation by a monovalent cation, causing a decrease in the diameter
of the channel, and therefore a decrease in the a-axis parameter,
see an explanation in Section 4. The site occupation preferences of
K þ in the apatitic structure were studied by El Feki et al. [66]. On
the basis of the explanation given in Section 4, it was assumed that
the greater ionic radius of K þ (0.133 nm) must prefer and occupy
site (II). However the experiment performed by El Feki et al. [66]
showed the reverse. Effectively, 83% of the potassium atoms were
located in site (I), and 17% in site (II). Kannan et al. [198] also
 M. Šupová / Ceramics International 41 (2015) 9203–9231
proved that the substitution of potassium into the apatite lattice
improved its thermal stability until 1300 1C. This can be of
immense importance in development for medical applications,
such as porous or granulated materials useful in bone surgery,
since fillers for organic polymers and also coatings, plasma
sprayed or prepared by laser ablation, necessarily need heat
treatments at elevated temperatures.
Some previous studies have dealt with the processing of
sodium hydroxyapatite (Na-HA). However, these studies have
proved that Na þ ions can be substituted in the HA lattice at
calcium sites with supplementary vacancies [64,68,90,91] or,
more frequently, with calcium sites co-substituted by other
anions and cations (this will be described in Section 4.3.).
Many of these studies have focused on low temperature
processing with the formation of single phase apatite. Kannan
et al. [199] prepared sodium substituted biphasic mixtures of
various HA/β-TCP ratios through precipitation at 90 1C. This
biphasic material can be better than a single material in terms
of its biological behavior on implantation. Substitution of Na þ
ions in HA proceeded without any significant modification to
its hexagonal system owing to similarity in the ionic radius.
However, a slight increase in the c-axis parameters leading to
an increase in cell volume was observed in comparison with
pure HA. El Feki et al. [68] found that sodium is located
preferentially in the Ca(II) site of the apatite structure.
Incorporating sodium into the apatite lattice increased the
thermal stability and delayed the allotropic transformation of
β-TCP to α-TCP until 1200 1C [199].
4.2.3. Strontium
Several research works on the synthesis of strontium doped
hydroxyapatite (Sr-HA) using a sol–gel process [189,200,201],
precipitation [53,69,183,202–204] and a hydrothermal method
[205] have been reported. Other methods, e.g. micro-arc
treatment [206], pulsed-laser deposition [207] and laser abla-
tion [208], have been used for producing Sr-HA coatings. The
studied concentrations of strontium (Sr/(Sr þ Ca)) have varied
up to 50 mol%. Precipitation from a supersaturated solution
yields nanocrystalline HA with a degree of Sr substitution
depending on its concentration in the solution [209–211].
With substitution of Sr, the lattice parameters (both the
a-axis and the c-axis) increased steadily [53,69,201,212,213].
The structural refinements of Bigi et al. [69] revealed that the
strontium atoms occupy both (I) and (II) sites of the apatitic
structure. At a very low strontium content (3.5 at%), the
occupancy of site (I) was preferential, but with increasing Sr
content the situation is reversed due to the higher ionic radius
(0.113 nm). Strontium is present only in one cell out of three,
and in site (I) it is present only in one cell out of five, when
apatite contains 3.5 at% of Sr. Terra et al. [214] determined a
preference for the Ca(I) site for Sr concentrations below 1 at%.
The ideal statistical occupancy ratio Sr(II)/Sr(I) ¼ 1.5 was
achieved for  5 at%. Incorporation of Sr2 þ ions preferentially
at site (II) was proved by Li et al. [203] and by Renaudin et al.
[201].
Li et al. [203] revealed that 0.3 mol% and 1.5 mol% Sr-HA
exhibited a single phase spectrum similar to HA. However,
9215
15 mol.% Sr-HA induced the incorporation of HPO24  and
more CO23  [215], and as a consequence the crystallinity
reduced dramatically. Destabilization of the crystal structure by
the larger Sr atom has been invoked to justify the significant
increase in solubility with Sr content [216]. These findings
indicated that the current clinical dosage, which usually results
in Sr incorporation of less than 1.5 mol%, may not change the
chemical composition and the lattice structure of the bone,
while it will broaden the bone crystal size distribution and
strengthen the bone. Studies by Kim et al. [53] have proved
that the addition of Sr over 4 mol% changed the microstructure
from round grains to faceted grains with ambiguous bound-
aries. Powders exhibited highly crystalline characteristics after
calcination at 900 1C, but additional β-TCP was formed and its
intensity increased with increasing Sr addition (up to 8 mol%).
However, Mardziah et al. [200] found that calcined powders
with a doping concentration of 2 mol% of Sr contained HA
and β-TCP as the secondary phase began to appear at a
temperature of 900 1C, whereas in non-Sr-doped material HA
appeared at a temperature below 800 1C. Higher Sr concentra-
tions (up to 15 mol%) helped to stabilize the HA phase, and
thus contributed to the disappearance of the β-TCP phase
together with low crystallinity. This is in agreement with
further reports by Kavitha et al. [212] that the addition of Sr
stabilizes the bioactive HA phase in high temperature proces-
sing and improves its bioactivity by suppressing the formation
of resorbable TCP. The formation of TCP during high
temperature calcination may be promoted by the decomposi-
tion of hydroxyl groups in pure HA. Consequent rearrange-
ment of larger Sr atoms and relieving of the internal strains in
Sr-HA may have retarded the primary particle growth and
phase transformation during calcination. Kavitha et al. [212]
also found that Sr-HA particles embodied nanorods with
multiple grain structures. This morphology is preferred for
drug delivery systems, because of the higher reactivity of the
structures.
It has been proved that the incorporation of Sr influences the
mechanical properties of HA or composites containing Sr-HA.
Landi et al. [204] prepared Sr-HA (with 1.15 mol% of Ca
substituted by Sr) porous materials that mimic the pore size
and the distribution of spongy bone for use as bone substitutes
and drug delivery systems, especially when pathologies con-
nected with Sr deficiency are present. The compressive
strength of the porous samples, 4.527 1.40 MPa, is within
the range of natural bone. Kim et al. [53] proved that there was
a slight increase in Vickers hardness from 5.2 to 5.5 GPa for
Sr-HA with an 8 mol% loading of Sr. Shen et al. [217] proved
that the addition of Sr-HA whiskers (6.6 mol%) significantly
improved the mechanical properties of calcium phosphate
cement (CPC). The 5 wt% Sr-HA whisker reinforced CPC
revealed the highest compressive strength of 2.92 MPa, which
was almost twice higher than the compressive strength of
pure CPC.
The results of an in vitro investigation carried out using
osteoblast-like MG63 cells and human osteoclasts [213] indi-
cated that the bone cell behavior in the presence of Sr2 þ ions is
dose-dependent. Sr concentrations in the range of 3–7 at%
9216 M. Šupová / Ceramics International 41 (2015) 9203–9231
significantly stimulate osteoblast activity and differentiation, and
even 1 at% of Sr substitution is sufficient to affect osteoclast
proliferation, which decreases with increasing Sr content. It has
also been found that ionic extracts of Sr-HA with 3.22 mol% of
Sr substitution showed the best proliferation, osteogenic differ-
entiation and angiogenic factor expression of human osteoblast-
like MG63 cells [205]. However, Chung and Long [206] proved
that coatings with a concentration exceeding 38.9 at% of Sr
significantly inhibit RAW264.7 osteoclast differentiation. Dos
Santos Tavares et al. [218] revealed that hydroxyapatite contain-
ing from 0.5 to 5 mol% of Sr did not affect dehydrogenase
activity, membrane integrity or DNA content.
4.2.4. Zinc
Incorporation of zinc into the HA structure (Zn-HA) has been
abundantly studied, owing to the key effect of Zn2 þ cations in
several metabolic processes that make zinc eligible for use in
many biomedical applications. Its possible antimicrobial aktivity
will be discussed in Section 4.2.5. and in Section 4.3.1. Zn-HA
has been prepared predominantly by a precipitation method
[183,219–225], but ion-exchange [226], a hydrothermal techni-
que at 200 1C [75] and the sol–gel process [188,227] have also
been used. Substitution of Zn2 þ into the HA structure through a
hydrothermal method is more difficult than through chemical
precipitation. Precipitation processes have been implemented at
room temperature [219,220,223,225] or in the temperature range
of 60–90 1C [221,222,224,228]. Li et al. [75] compared Zn-HAs
prepared by NaOH and by an ammonium aqueous solution that
adjusts the pH value. The crystallinity and the substitution
content of Zn in Zn-HA prepared with a NaOH solution was
higher than the crystallinity and the substitution content of Zn in
Zn-HA prepared by an ammonium aqueous solution. The
crystallinity and the thermal stability of the apatite decrease
significantly with increasing Zn concentration.
Systematic studies of incorporation and co-precipitation
[224,225] have shown that considerable amounts of Zn can
be incorporated into HA. Miyaji et al. [224] studied progres-
sive Zn substitution in HA, and their findings indicate that with
increasing Zn concentration the final product changes from Zn-
substituted apatite to an amorphous apatite-like phase. They
suggested that the substitution limit of Zn in HA is 15 mol%,
as compared to 25 mol% suggested by Bigi et al. [225]. The
0.2861 nm
0.2245 nm
0.2066 nm 0.1995 nm
v PO4
0.2261 nm
0.2073 nm
Fig. 5. Detailed view of the local coordination of Zn incorporated in the HA structure with interatomic distances. (A) Octahedral coordination, suggested by Terra
et al. [231] and by Ma and Ellis [235]; (B) tetrahedral coordination, suggested by Tang et al. [222].
inhibiting effect of zinc on HA crystallization and its pre-
ference for a β-TCP structure closely resembles the behavior
previously observed for magnesium [225,229]. Chung et al.
[227] found that the sol–gel method is a suitable process for
preparing a Zn-HA coating, and they proved that more than
1000 ppm Zn is needed to show an antimicrobial effect. Zn
addition (100–10,000 ppm) is beneficial for cell attachment.
The lattice parameters of Zn-HA changed owing to the
difference in ionic radius between Zn2 þ (0.074 nm) and Ca2 þ
(0.099 nm). The c lattice parameter of Zn-HA was found by
Miyaji et al. [224] to decrease progressively with increasing Zn
concentration. They also observed an increase in the a-lattice
parameter for a higher Zn concentration (5 mol%). This was
ascribed to increased H2O substitution for OH. Studies
[75,228,230] have observed a decrease in lattice parameter a
with a Zn fraction up to 10 mol% Zn, and then the parameter
begins to increase. Lattice parameter c decreases monoto-
nously as the Zn fraction increases.
Tang et al. [222] systematically examined the local structure
of Zn incorporated into HA at low concentrations by combining
experimental and spectroscopic results with density functional
theory calculations. Their calculations unequivocally showed
that substitution in the Ca(II) position is more energetically
favored. This result agreed with the study by Terra et al. [231],
which employed an embedded cluster LCAO (linear combina-
tion of atomic orbitals) method, and Matsunaga [232], who used
the projector augmented wave method. This fact has also been
proved by other studies [233,234]. Additionally, Ma and Ellis
[235] suggest that the Ca(II) site is to be favored for Zn
substitution at the HA (0001) surface. Both Terra et al. [231]
and Ma and Ellis [235] suggested that Zn is octahedrally
coordinated (Fig. 5(A)), but the calculations of Tang et al.
[222] showed that the most favorable structure for Zn is
tetrahedral coordination, as shown in Fig. 5(B), and this is
consistent with the results of Matsugana [232]. Looking more
closely at tetrahedral coordination (Fig. 5(B)), it can be seen that
among the four oxygen atoms bonded to Zn, one is from the
nearest hydroxyl group and three are from the adjacent
phosphate groups. This arrangement of the atoms causes a
reduction in the a-lattice parameter from 0.9488 to 0.9319 nm.
The hydroxyl group moves off its c-axis position and the Zn
atom moves off the Ca(II) site. Both come close to each other,
OH
0.202 nm
Zn
 M. Šupová / Ceramics International 41 (2015) 9203–9231
and the Zn–O(OH) distance is 0.202 nm compared to 0.269 nm
in ideal distances in HA. Ma and Ellis [235] and Matsugana
[232] have also both observed similar shifts (Fig. 5(A)). The
preference of Zn for the Ca(II) site may facilitate the uptake and
release of Zn by bioapatite because it would not disrupt the
framework of the structure.
4.2.5. Other cationic substitution
There are several limitations to the use of pure HA, including a
lack of antibacterial activity, which decimates its long term stability
and causes implant failures. In recent years, the incorporation of
inorganic antibacterial cations such as silver (Ag þ ), copper
(Cu2 þ ), zinc (Zn2 þ ) and titanium (Ti4 þ ) in biomaterials for the
prevention of microbial infection has been investigated [236].
Among these ions, Ag is believed to have superior chemical and
physical properties, e.g. biocompatibility, high thermal stability,
and also non-toxicity to human cells at low concentration
[237,238]. Kim et al. [239] compared the antimicrobial effect of
metal ion (Ag þ , Cu2 þ and Zn2 þ ) substituted HA against
Escherichia coli (E. coli). An obvious antimicrobial effect was
observed in the case of Ag-HA, but it was difficult to ascertain any
bactericidal effect in the case of Cu-HA and Zn-HA. Moreover,
silver-doped calcium phosphate ceramics has an antimicrobial
activity comparable to that of an antibiotic treatment [240]. In
recent studies reported by Ciobanu et al. [241,242], Ag-HA
nanoparticles showed great promise as antibacterial agents against
both gram-positive and gram-negative bacteria. Ag-HA has been
synthesized using several techniques, e.g. the sol–gel process
[227,238,243], precipitation [241,244–246], ion exchange [247],
combustion synthesis [248], microwave synthesis [237] and a co-
sputtering process [249].
Using chemical analysis, FTIR and XRD methods, a number of
studies [227,237,239–242,245–247,250] have proved the substitution
Ag þ ions for Ca2þ ions in the apatitic structure. Chung et al. [227]
proved the substitution by the presence of a CaO impurity. It was
inferred that the substitution of Ag þ ions in CaP intermediates of an
aged sol resulted in more free calcium ions which, after rapid heating
of the HA precursors, reacted with the surrounding oxygen to form
CaO by-products. Yang et al. [247] found that there were Ag þ ions
substituted in the apatite lattice and also in the form of silver
orthophosphate. However, Sygnatowicz et al. [238] proved the
existence only of silver metal and silver phosphate phases. Metal
silver was also found in a study by Díaz et al. [243], where an Ag-
HA nanocomposite was obtained by a colloidal chemical route and a
subsequent reduction process in an H2/Ar atmosphere at 350 1C that
reduced the silver cations to elemental silver. Other studies
[236,244,249] dealt only with the antibacterial properties of the Ag
doped HA, and they did not investigate the problem of substitution.
Only limited research [237,245,246,250] has been carried out
on changes to lattice parameters due to Ag substitution in the HA
structure. The substitution of Ag þ (0.128 nm) ions takes place
for Ca2 þ (0.099 nm) preferentially in the Ca(I) site of HA, and
this leads to an increase in the lattice parameters linearly with the
amount of silver added in the range of the Ag/(Agþ Ca) atomic
ratio between 0 and 0.06. Yang et al. [247] developed a novel
filter material based on porous Ag-HA. This material demon-
strated excellent bacteria-separating and antibacterial properties as
9217
well as a certain virus-eliminating property. Ag exhibits high
antibacterial activity against E. coli and S. aureus while main-
taining low cytotoxicity. Ciobanu et al. [245] proved that Ag-HA
nanoparticles were effective in protecting macrophages from
lipopolysaccharides from E. coli 055:B5 induced cytotoxicity in
RAW 264.7 macrophage. It is therefore a matter of primary
importance to acquire solid knowledge of the optimal doses that
will provide a balance between the positive effects of Ag-HA and
their possible adverse consequences. Toxic silver concentrations
were in the range of 1–3 mg/ml [244]. In a study by Chung et al.
[227], the addition of 100 ppm Ag þ ions to sol–gel precursor
calcined HA coatings on Ti-6Al-4V substrates resulted in growth
suppression of the pathogen (Streptococcus mutans), and an
apparent inhibition zone was found for an HA film with Ag þ ion
addition up to 10,000 ppm. An in vitro bacterial adhesion study
by Chen et al. [249] indicated a significantly lower number of
Staphylococcus epidermidis and S. aureus on an Ag-HA
(2.0570.55 wt% Ag) surface than on titanium (Ti) and HA
surfaces. In addition, no significant difference in in vitro cyto-
toxicity was observed between HA and Ag-HA surfaces.
Titanium-based metals and alloys have achieved great success as
medical implants. Chemically, titanium is a group IV transition
element, its valence of 4 being the same as the valence of silicon.
However, titanium and its alloys are not osteoconductive. Nano-
composite materials containing titanium and ceramic as a reinforced
phase are therefore promising alternatives to conventional materials,
because they can potentially be designed to match the properties of
bone tissue in order to enhance patients' quality of life. Titanium–
hydroxyapatite nanocomposite materials with various contents of
HA (3–20 vol% HA) were prepared by mechanical alloying and by
a powder metallurgical process [76]. It is important to note that the
mechanical alloying technique enables elements to be alloyed that
are difficult or impossible to combine by conventional melting
methods. Colloidal Ti-HA particles doped with varied amounts of
Ti4 þ ions were prepared by the coprecipitation method [251–253]
and by the sol–gel method [189]. It has been reported that Ti-HA
also has a photocatalytic activity for decomposing acetaldehyde and
albumin under ultra-violet (UV) irradiation [251]. It has also been
proved that these heat-treated Ti-HA particles with a molar ratio
XTi ¼ 0.10–0.15 are suitable for applying this material directly for
decomposing pathogenic proteins in a blood purified therapy under
UV irradiation [252]. Huang et al. [253] studied two concentrations
(0.8 and 1.6 wt%) of Ti incorporated into HA. Rietveld refinement
analysis showed that there was a proportional increase in both the a
axis and the c axis when Ti is incorporated into the HA structure.
However, this increase in both the a and c axes of the Ti-HA unit
cells does not totally support the Ti4þ for Ca2þ substitution. This is
because a decrease in the unit cell parameter was expected as the
ionic radius of Ti4þ (0.0605 nm) is less than the ionic radius of
Ca2þ . Incorporation of Ti into the apatitic structure inhibited grain
growth and, in addition, the grain size decreased with increasing Ti
concentration. Ti-HA with 0.8 wt% of Ti may have great potential
for use in biomedical applications, such as bioactive scaffolds and
coatings on Ti implants. A recent study also found a bactericidal
function of Ti-HA [251].
Manganese (Mn) influences the regulation of bone remodeling,
and a manganese deficit causes a reduction in organic matrix
9218 M. Šupová / Ceramics International 41 (2015) 9203–9231
synthesis and retards endochondral osteogenesis [254]. Bracci et al.
[210] proved that a Mn-HA coating has a remarkably beneficial
effect on bone cells, and that a relatively high content of Mn2þ in
the phosphate has a remarkable beneficial effect on osteocalcin
production, which is even greater than the level of osteocalcin
production observed for Sr2þ . Bigi et al. [255] obtained coatings of
Mn-CHA on Ti substrates by pulsed laser deposition, and proved
their promoting effect on human osteoblast proliferation, activation
of their metabolism, and differentiation. HA containing small
percentages of Mn (up to 2 wt%) can easily be prepared by
precipitation from aqueous solutions, with the Mn concentration in
the solid phase corresponding to as much as 70 % of the Mn
concentration present in solution [256]. Suitch et al. [257]
synthesized fluoroapatite in the presence of Mn2 þ ions followed
by calcination at 900 1C. They found that Mn (ionic radius
0.090 nm) can substitute for Ca up to one atom per unit cell in
the structure (10 at% corresponds to 5.5 wt%), preferentially in the
Ca(I) positions, causing a slight rotation of the phosphate ion [257].
It was proved by Mayer et al. [258] that Mn affects the thermal
stability of apatite, as was proved by partial transformation into
β-TCP at 600 1C. The lattice parameters of β-TCP obtained by
thermal conversion of Mn-HA appear to be reduced with increasing
Mn concentration in the solid phase. However, it has been proved
by several studies that the presence of Mn2þ ions in solution
inhibited HA crystallization and significantly reduced the amount of
precipitate [210,254,256]. Consequently, the smaller and less perfect
HA crystals with a higher Mn content (1.23 wt%) should be more
resorbable, and therefore more biocompatible [259].
Iron substituted HA (Fe-HA) magnetic nanoparticles have
recently been prepared by co-precipitation in an aqueous solution
with the addition of various concentrations of Fe2 þ [260,261], or
with the aid of a microwave process [262]. This Fe-HA magnetic
material can find applications as a heating mediator in hyperther-
mia therapy for cancer [263]. The magnetic properties of these
nanoparticles are influenced by several factors, e.g. microstructure,
anisotropy and orientation. An excess of iron should therefore be
avoided, since it affects the arrangement of the atoms. Zuo et al.
[261] have proved that the presence of Fe2 þ ions with a smaller
radius (0.0835 nm) than Ca2 þ affect the lattice parameters. Doping
of iron ions decreased the crystallinity, inhibited growth of the he
c-axis and promoted growth of the a-axis, with 10 % and 50 %
exhibiting a superparamagnetic character, while Fe-HA particles
with 30 % exhibited a weak ferromagnetic character. Magnetic HA
nanoparticles mixed with phosphate buffer solution (PBS) have
also been tested in vivo by injecting them around a tumor. Tumors
injected with Fe2 þ -HA and treated inside the magnetic field to
achieve hyperthermia showed a dramatic reduction in volume in a
2-week observation period [264]. Pon-On et al. [262] synthesized
Fe3 þ –HA up to 2.23 wt% of Fe. This caused an increase in the
positive charge, together with the appearance of vacancies in the
anion channel, as can easily be seen from the crystallographic
formula: Ca10 xFex(PO4)6(OH)2 x▯x (where ▯ is a vacancy).
Lanthanide-doped HA (Ln-HA) nanoparticles show very
interesting luminescence properties, which make them useful
for biological fluorescence labeling. Their fluorescence is char-
acterized by narrow emission band widths determined by the
lanthanide ions (Ln3 þ ), high photochemical stability and long
fluorescence lifetimes (up to several milliseconds). It is assumed
that ionic substitution of Ln3 þ ions in the apatitic structure is
expressed by luminescence. An absence of luminescence was
observed when the lanthanide ions were only adsorbed on to the
surface, whereas La-HA became luminescent after heating at
800 1C, as the ions diffused into the apatitic lattice [265].
Yasukawa et al. [266] synthesized five types of HA particles
containing light rare earth ions—lanthanides (Ln3 þ : lanthanum
(La3 þ ), cerium (Ce3 þ ), praseodymium (Pr3 þ ), neodymium
(Nd3 þ ) and samarium (Sm3 þ )) with various Ln/(Lnþ Ca) atomic
ratios (XLn ¼ 0–0.15). A mixture of Ln–HA and LnPO4 was
formed in each Ln system at XLn 40.01–0.03. With increasing
XLn, the prepared particles first became longer and then became
smaller. The crystallinity of the particles was altered, in corre-
spondence with the variations in their size. UV–vis measurements
revealed that Ce-containing HA (Ce-HA) exhibits strong UV
absorption below 400 nm. Cerium can act similarly to calcium in
organisms. It accumulates in small amounts in bones, so Ce-
containing compounds can stimulate the metabolism in organisms.
The electronegativity of metallic Ce and the ionic radius of Ce3 þ
(0.107 nm) are close to those of Ca, so Ca2 þ can be substituted
by Ce3 þ ions. Moreover, it was proved by Yingguang et al. [267]
that Ce3 þ cations possess some antibacterial properties. Kaygili
et al. [268] used the sol–gel method to synthesize Ce-doped HA
with variable amounts of Ce (0.5–2.0 at%). In all samples, the
amount of HA exceeded 92 vol%, while the amount of admixed
β-TCP was always below 8 vol%. The lattice parameter of the c-
axis increased slightly. Feng et al. [269] prepared Ce-HA by a
hydrothermal method at varied XCe (0–10 %). Doping of Ce3 þ
ions resulted in a decrease in the crystallite size, and the particles
grew into long needles with increasing XCe. Huang et al. [270]
prepared a porous Ce-HA coating on commercially pure titanium,
using micro-arc oxidation in an electrolytic solution containing
cerium nitrate, and they proved that this coating has favorable
osteoblast cell affinity and good biocompatibility. Europium-
doped apatite (Eu-HA) can be obtained with a maximum Eu/
(Euþ Ca) ratio of about 2 at% [271–274] in an alcoholic solution
at physiological temperature [272,273,275], or in aqueous solu-
tions [271,276]. Under excitation with 394 nm light, Eu-HA is
red-emitting. Colloidal dispersions stable at neutral pH and
emitting red or green color under excitation at 253 or 365 nm
were obtained for Eu3 þ and terbium (Tb3 þ ) apatites, respectively
[274]. Generally, different colors can be produced by doping with
different lanthanides. The fluorescence efficiency is dependent on
the crystallinity, i.e. amorphous nanoparticles of the same
composition showed much weaker fluorescence [271]. Eu-HA
and Tb-HA ( 2.5 wt%) nanoparticles were stabilized with a shell
of DNA and were taken up by living cells [271]. This shows that
the particles can be used as biocompatible probes for intracellular
labeling, e.g. to follow their way inside the cells during transfec-
tion or antisense experiments. A very interesting project involved
impregnating Eu-HA with Ag þ ions, which were subsequently
reduced. This nanocomposite powder seems to be a promising
antimicrobial agent and fluorescent material for biodetection, due
to its optical and bioactive properties [276].
Wakamura et al. [70] investigated the composition and
structure of HA substituted with metal ions (aluminum
 M. Šupová / Ceramics International 41 (2015) 9203–9231
(Al3 þ ), La3 þ and Fe3 þ ), which are of interest in the clinical
field. It has been reported that Fe3 þ is responsible for black or
brown tooth stains [277], and La3 þ and Al3 þ inhibit dental
caries [278]. Metal3 þ –HAs were prepared by coprecipitation
and immersion methods. Wakamura et al. [70] found that in
coprecipitation these metal ions accelerated the growth of
Metal3 þ –HA particles, markedly for Fe3 þ . Fe–HA prepared
by the coprecipitation method had one kind of surface Fe–OH,
but Fe–HA prepared by the immersion method had no surface
Fe–OH. Al–HA particles prepared by both methods had several
kinds of surface Al–OH in addition to the surface P–OH, while
no surface La–OH was detected on the La–HA. The same
authors [279] further examined the adsorption behavior of Al3 þ ,
La3, þ Fe3 þ hydroxyapatites with BSA. The final HA particles
were elongated (molar metal substitution¼ 0.1), and the extent
of the particle growth was in the order of La3 þ oAl3 þ , Fe3 þ .
The best adsorption results were exhibited by Fe–HA, i.e. a 2.7-
fold increase compared to pure HA. This was explained by
elongation of the mean particle length along with the production
of surface hydroxo ions to induce a hydrogen bond between the
Fe–HA and BSA molecules. It can be summarized that mean
particle length and surface hydrophilicity are determining factors
for the adsorption amounts of BSA.
Other authors have reported advantages of doping HA with
foreign trivalent elements, such as Al [280], gallium (Ga) [281] and
yttrium (Y) [282–284]. It has been proved that yttrium enhances
the mechanical properties of ceramics. Moreover, Webster et al.
[283] demonstrated that there is greater osteoblast adhesion on bulk
HA doped with Y (up to 7 mol%) than on undoped HA. They
provided evidence for a solubility limit of yttrium in HA greater
than 7 mol.% [284], which contrasts with a previous report [285].
Sato et al. [282] investigated the possibility of using nanocrystalline
Y-HA for orthopedic applications. They synthesized nanocrystal-
line powders through a hydrothermal treatment process, and
deposited them on titanium utilizing IonTiteTM multistep proces-
sing, which did not change the chemistry or the crystal size of the
starting HA powders. They found that there was better osteoblast
adhesion on nanocrystalline IonTiteTM HA coatings than on
traditionally-used plasma-sprayed HA coatings. Moreover, IonTi-
teTM Y-doped nanocrystalline HA coatings stimulated osteoblasts
to deposit more calcium. Gallium ions are clinically effective
against bone resorption and for the treatment of osteoporosis and
cancer-related hypercalcemias. They increase the calcium and
phosphorus content of the bone and have direct nontoxic effects
on osteoclasts [286]. A novel gallium-containing orthopedic
biomaterial for local applications stimulating bone growth has
been obtained by introducing gallium ions (up to 11.0 wt%) into
the crystalline lattice of HA [281]. Gallium nitrate and sodium
gallate solutions were used as doping agents. It was found that
gallium does not replace calcium as a result of heterovalent
substitution, and it consequently produces no distortions in the
apatitic lattice. It remains strongly fixed in the form of solid
solution of intercalation.
The effect of cobalt (Co2 þ ), chromium (Cr3 þ ) and nickel
(Ni2 þ ) ions on the in vitro growth of HA has been investigated
in conjunction with orthopedic applications. These metals are
widely used in orthopedics, and some studies have reported
9219
that patients with metal implants have a significant increase in
metal levels in serum and in the synovial fluid, and some
patients with metal-on-metal implants have suffered a femoral
neck fracture for unknown reasons. Bone quality is an
important factor in bone strength, and it is assumed that metal
ions can affect the properties of HA. Mabilleau et al. [287]
demonstrated that these metal ions reduced the quantity of
mineral that was formed, and decreased the Ca/P ratio 1.12-
fold, 1.05-fold and 1.08-fold for Cr2 þ , Cr3 þ and Ni2 þ ,
respectively. Moreover, the presence of metal ions increased
the crystal size as well as the crystallinity of HA, and reduced
the lattice parameter c of the HA lattice. These results
suggested that the quality of the mineral around metallic
implants may be altered.
4.3. Multi-ionic substitution
Several works have dealt with multi-ionic substitutions that
should better imitate the chemical composition and the structural
properties of native bioapatites. Cationic, anionic and mutual
combinations of substitutions in anionic and cationic sites have
been formed. These mutual substitutions of trace elements into
HA have introduced new physico-chemical, mechanical and
biological properties in comparison with pure HA or monoionic
substituted HA materials.
4.3.1. Cationic co-substitution
Sodium and magnesium co-substituted hydroxyapatite (Na/
Mg-HA) and β-TCP mixtures were prepared using an aqueous
precipitation method by Kannan et al. [288]. The results
indicated the influence of the additives (Na and Mg) on the
thermal stability of the apatites. The calculated lattice para-
meters were found to have a more contracted a-axis and a
more expanded c-axis than for pure HA. Kalita et al. [188]
prepared HA substituted with Mg and Zn with the use of sol–
gel synthesis. Prior to calcination, magnesium oxide and zinc
oxide were incorporated separately into the dried amorphous
gel in amounts of 1.0, 2.5 and 4.0 wt%. The presence of
substitutes further improved the compressive strength and the
surface hardness. Biodegradation studies conducted in
dynamic simulated body fluid (SBF) brought out the fact that
presence of these dopants in nanocrystalline HA ceramics
reduces the rate of resorption and surface hardness degrada-
tion, in comparison with pure nanocrystalline HA. Substitution
of Mg2 þ and Zn2 þ in HA and octacalcium phosphate (OCP)
and their possible location in the HA structure were deter-
mined by Matsunaga [232], using first-principles calculations.
OCP can be considered as a structural model of the transition
region between HA and the solution. It is found that
substituted ions with smaller ionic sizes than Ca2 þ cause the
surrounding oxygen ions to undergo considerable inward
relaxation, which results in smaller coordination numbers with
oxygen than the coordination numbers Ca in bulk HA and
OCP. The calculation indicates that the substitutional ions are
quite difficult to dissolve into HA, but can be incorporated
more easily into OCP. Zinc ions can be more favorably
substituted for the specific Ca site of OCP, in comparison
9220 M. Šupová / Ceramics International 41 (2015) 9203–9231
with Mg2 þ . This is attributed to the formation of a covalent
bond between Zn and the surrounding oxygen atoms. It was
therefore considered that zinc may play a role in promoting
bone formation by being incorporated into the transition region
between HA and the surrounding solution. A study by Gopi
et al. [289] reported successful synthesis of Sr, Mg and Zn co-
substituted hydroxyapatite (Sr/Mg/Zn-HA) using glycine as a
template by freezing at 2 1C. They revealed that synthesized
Sr/Mg/Zn-HA not only supports the growth of apatite but also
accelerates growth on to itself.
A very interesting solution for problems connected with
infections after implantation was adopted by Gopi et al.
[290,291]. They prepared strontium/cerium co-substituted
HA (Sr/Ce-HA) [290]. To improve the bioactivity of pure
HA, Sr2 þ ions were substituted and further co-substituted by
various concentrations of Ce3 þ (0.050, 0.075 and 0.100 mol)
to enhance the antibacterial property. In other work [291],
Gopi et al. synthezised antibacterial and bioactive silver/
magnesium co-substituted HA (Ag/Mg-HA) with various
concentrations (0.5, 1.5, 2.5 wt%) of Ag. Magnesium reduces
the cytotoxicity of Ag, as proved by in vitro and antibacterial
activity tests. In the work of Hu et al. [292], Ti4 þ and
antibacterial ions (Ag þ , Cu2 þ and Zn2 þ ) co-substituted
hydroxyapatite (Ti/Ag-HA, Ti/Cu-HA, Ti/Zn-HA) was pre-
pared by a coprecipitation method and by an ion-exchange
method. Ti/Ag-HA, Ti/Cu-HA showed high efficiency for
killing E. coli in the dark and S. aureus under weak UVA
irradiation. They for the first time proved with ESR that O∙2
radicals were formed on Ti/Ag-HA, Ti/Cu-HA in the dark at
ambient temperature. X-ray photoelectron spectroscopy (XPS)
results indicated that the interactions between the redox couple
of Cu0/Cu2 þ or Ag0/Ag þ and O2 possibly resulted in the
formation of O∙2 . The high bactericidal activities were due to
the synergy of the oxidation role of the O∙2 and the
bacteriostatic action of the antibacterial ions.
It is known that heating cells up to 42–45 1C results in the
death of cancer cells [263,264]. This is usually done by
delivering magnetic nanoparticles to the tumor and irradiating
the particles with an ac magnetic field, thus heating up the
particles to the desired temperatures. Since magnetic materials
are usually toxic to living organisms, magnetic nanoparticles
are coated on or incorporated into a material to form a core/
shell structure. These materials are biocompatible with living
organisms. Pon-On et al. [293] prepared core/shell nanoparti-
cles by the coprecipitation method, with the core being formed
by ferrites and the shell by HA. The resulting ceramics had the
empirical formula Ca9.4Fe0.4Mn0.2(PO4)6(OH)2. The ESR
spectrum indicated that the Fe3 þ ions and Mn2 þ ions enter
into the Ca2 þ sites in the HA structure, and it was proved that
the Mn2 þ ions replace the Ca2 þ ions located in the Ca(I) sites
in the HA structure.
4.3.2. Anionic co-substitution
Co-substitution of fluoride and chloride into the apatite
lattice forms a solid solution (Cl/FHA), which will certainly be
a better material for medical applications than the individual
FHA and ClHA phases. Kannan et al. [294] prepared Cl/FHA
through an aqueous precipitation method with concentration
ranges 0.79–3.21 wt% for chlorine and 0.41–1.79 wt% for
fluorine. The cell parameters are expected to be significantly
affected if total replacement of OH  ions by F  or Cl  ions
takes place. When there was combined incorporation of F 
and Cl  ions into the HA lattice, there were indications of
significant differences in the measured lattice values in
comparison with the values for FHA, ClHA and HA [294].
A close examination of the values revealed contraction of the
a-parameter, while a steady increase in the c-axis parameter
was observed. The highest c-axis parameter value is observed
for Cl/FHA, where there was complete replacement of the
OH group.
Co-substitution of fluoride and carbonate ions seemed to be
another possible positive way to combine anions. B-type
carbonate HA (CBHA) can be a suitable material as a host
of F  for slow release. This material can be prepared by
precipitation [295] or by heating a mixture of CHA with CaF2
in dry CO2 at 900 1C [296]. Several kinds of FCBHAs were
prepared and examined, but only for their dissolution property
in terms of Ca2 þ release, e.g. [296]. Sogo et al. [297] studied
the solubility behavior of this material with reference to F 
release. Fluoride was adsorbed on CHA in a NaF solution,
followed by heating at 700 1C in CO2 to substitute F  ions for
the hydroxyl ions. The F  content in the FCBHA powders was
16–22 times greater than the level in normal adult human
bones (0.66 wt%) and the carbonate content (11.03 wt%) was
also much greater. Sogo et al. [297] found that, near the
therapeutic level, the F  concentration increased slowly and
reached 67.20 7 4.81 mg/l, which was 1.5–9.3 times higher
than the level in the body fluid of normal adult humans, but far
lower than the estimated toxic level. A study comparing the
in vivo performance of HA, CHA and FCHA implanted in dog
mandible showed FCHA induced faster bone remodeling, and
a more completely formed Haversian system was obtained than
for HA or CHA [298,299]. The mechanism of this substitution
was studied by Regnier et al. [300]. Their ab initio results
favored the existence of an interstitial position for F  . Their
results showed no evidence for the commonly proposed
hypothetical anion CO3F3  , and no direct bonding between
CO23  and F  was found.
Because the selenite and selenate groups have the same
charges as carbonate groups, the charge compensations during
the precipitaion of SeHA (in the presence of ambient atmosphere
or CO23 ) are very often accompanied by carbonate substitution
[165,167,168]. Kolmas et al. [167] synthesized Se/CHA by a wet
precipitation method. The crystallochemical formula was calcu-
lated as Ca10 x y(PO4)6  x y(SeOn)x(CO3)y(OH)2 x–y, where
n¼ 3 for selenite, n¼ 4 for selenate; x and y are selenium
oxyanions and carbonate content, respectively. For selenite
x¼ 0.1–1.17 and for selenate x¼ 0.09–1.21. They claimed that
the decrease in the hydroxyl groups depends both on the selenium
oxyanions and on the carbonate contents. In apatite-containing
selenite ions, the B-type carbonate content was almost constant
(2.1–2.4 wt%). As a consequence, the concentration of hydroxyl
groups was dependent only on the selenite content. However, in
apatite with selenate ions, the amount of carbonates was
 M. Šupová / Ceramics International 41 (2015) 9203–9231 9221

significantly different: 0.9–2.4 wt%. It was apparent from the
results of Kolmas et al. [167] that the lattice parameters (discussed
in Section 4.1.5.) are influenced mainly by the incorporation of
selenium oxyanions. Due to their concentration, the influence of
carbonate ions is negligible. It may be a simple matter of the
different ion sizes: carbonates are significantly smaller (0.178 nm)
than phosphates and selenites.
4.3.3. Anionic-cationic co-substitution
Anionic-cationic co-substituted HAs have usually been
prepared as di- and tri-substituted, and multisubstituted HAs
have also been prepared.
Since carbonates are among the most abundant substituents in
bioapatites, co-substitution of Na þ , Mg2 þ and CO3 2  ions is
the most usual and most natural approach [65,68,82,90,91,101].
These co-substitutions also appear in native bone bioapatite.
Handschin and Stern [24] proved that the carbonate content in
human bone increases significantly with maturation. The
positive correlation between Na þ and CO3 2  indicated that
the incorporation of greater amounts of Na þ within the structure
may be facilitated by the incorporation of CO3 2  . By contrast,
the Mg2 þ concentration decreases by 0.14–0.20 wt% within the
0–30 age group.
De Maeyer et al. [65,301] collected at least five fundamental
substitution mechanisms, and studied their contributions to the
incorporation of Na þ and CO3 2  ions into the CBHA lattice.
The contributions of the two predominant mechanisms, i.e.
[Ca2 þ þ PO4 3  þ OH  2VCa þ CO3 2  þ VOH], where V is a
vacancy and [Ca2 þ þ PO4 3  2Na þ þ CO3 2  ] increased with
increasing sodium carbonate (Na2CO3) concentration. In addi-
tion to these two mechanisms, there is a small contribution from
an individual Na þ incorporation [Ca2 þ þ OH  2Na þ þ VOH].
Using the Rietveld method, El Feki et al. [68] and Wilson et al.
[90] determined two faces (O1 O2 O3 and O1 O2 O3´) of the
PO4 empty tetrahedron for the location of the CO3 planar anion
(Fig. 6). The distances of the P–O and C–O bonds are 0.155 and
0.125 nm, respectively. The synthetic Na-bearing AB carbonate
apatite (Na-CHA) with the crystallographic formula
(Ca10 yNay[(PO4)6 y(CO3)y][(OH)2 2x(CO3)x, where x y)]
[101], can serve as a chemical and structural model for
biological apatites with A-type carbonate accounting for up to
50% of the total carbonate. The crystal data for the maximum
content of carbonates (11 wt%) were a¼ 0.93855 nm,
c¼ 0.69142 nm. The authors found that coupling of the A and
B carbonate ion defects and tilting of the B carbonate ion in the
HA structure are required by the close approach (0.114 nm) of
the off-axis oxygen of the channel carbonate ion to an O3 atom
of the phosphate group. Using FTIR, Fleet and Liu [302] also
detected the hydrogen-carbonate ion (HCO3  ) in the c-axis
structural channel of Na-bearing AB carbonate apatite synthe-
sized under conditions of high pressure (up to 1 GPa) and high
temperature (800–1350 1C). The HCO3  ion was only loosely
bound in the apatite channel, and broke down on aging at room
temperature. Volatile decomposition products (CO2 and H2O)
may have a role in mediating acid-base reactions in biological
systems.
Le Geros et al. [303,304] studied biological apatites from
human enamel and dentin from bovine bone and synthetic
apatites with/without Mg2 þ or CO3 2  ions. They proved that
there are synergistic effects of Mg2 þ and CO3 2  on reducing
the crystallinity and increasing the extent of dissolution of
synthetic Mg-CHA, dentin and bone, in comparison with
enamel apatite containing lower levels of Mg2 þ and CO3 2 
ions. A positive effect of Mg-CHA (1 wt% Mg and 6 wt% CO3)
on cell (MSCs and MG63) behavior in terms of adhesion,
proliferation and metabolic activation, in comparison with
stoichiometric HA, was proved by Landi et al. [72]. Barinov
et al. [305] investigated the thermal stability of Mg-CHA
prepared by two different methods: by precipitation from an
aqueous solution, and by a solid–liquid interaction. They proved
that wet synthesized powders are significantly less thermally
stable than powders prepared by a solid–liquid interaction. This
could of course be taken into account when preparing ceramics
for bone substitutes. The effect of Na þ , Mg2 þ and CO3 2 
substitution on the unit cell parameters of HA has been studied
by several authors [73,306,307] (Table 2). Gibson and Bonfield
[73] found that Mg and AB-carbonate co-substitution caused a
significant increase in the a-axis and no change in the c-axis in
comparison with stoichiometric HA, and they proved no direct
effect of the changing Mg content on the unit cell parameters.
However, Kolmas et al. [306] proved an opposite trend. This
can be attributed to a higher carbonate content (than was used in
[73]), which caused an increase in the c-axis parameter. In
addition, if Na2CO3 is used in the precipitation reaction, Na/Mg-
CHA can be prepared [72,307,308]. Sader et al. [307] indicated
O3´
O c 3´
Oc3
P
O3 Oc2
O2
O c1
Na
O1
Fig. 6. Schematic presentation of the arrangement of CO3 plane anions
(dashed line triangles) on two possible sides of the PO4 tetrahedron in Na-
CHA, determined by El Feki et al. [68] and by Wilson et al. [90].
Table 2
Relation between the concentrations (Na, Mg and carbonate) and the lattice
parameters of Mg-CHA and Na/Mg-CHA; * ¼constant concentration.
Maximal concentration Trend in the lattice parameter in Ref.
(wt%) comparison with stoichiometric HA
Na Mg CO3 a-axis c-axis
– 0.330 1.000* ↑ - [73]
– 0.300 5.200 ↓ ↑ [306]
1.977 0.826* 6.830 ↓ ↑ [307]
9222 M. Šupová / Ceramics International 41 (2015) 9203–9231
a decreasing a-axis together with increasing c-axis dimensions
with an increasing Na þ and CO3 2  content with a stable Mg2 þ
content. The synergistic effect of Na þ and CO3 2  ions,
together with their higher content in comparison with Mg, has
a prevalent influence on the change in the lattice parameters.
Larson et al. [308] prepared Na/Mg-CHA by the inexpen-
sive, large-scale and non-stirred crystallization biomimetic
process, using CaCO3 as the Ca, Mg source and phosphate
buffer (non-saline) solutions as the phosphate source. This
method is based on the conversion of CaCO3 into apatite.
Drouet et al. [309] first synthesized carbonated apatite, and
then the powders were contacted with Mg2 þ and Sr2 þ ions in
a second step, in order to mimic the treatment procedures for
post-activation. This study also made an evaluation of the
amount of ions potentially available for interactions with
surrounding fluids in vivo. Inverse exchanges showed that
the proportion of reversibly fixed ions after surface exchange
was close to 85% for Mg and between 75 and 80% for Sr. The
importance of substitution in HA was well established by
individual substitutions. Simultaneous substitution of Zn2 þ /
Sr2 þ and CO3 2  therefore seemed to be a very interesting
approach. Suresh Kumar et al. [228] investigated and com-
pared the lattice parameters of HA, CHA, Zn-HA and Zn-
CHA. The lattice parameters of the Zn-CHA sample showed
an increase in both the a-axis and the c-axis in comparison
with Zn-HA, an increase in the a-axis and a reduction in the
c-axis in comparison with CHA, and a decrease in the a-axis
and an increase in the c-axis in comparison with HA. The
lattice distortion (the ratio of c/a) of HA increased with the
substitutions of Zn2 þ or CO3 2  ions in HA structure.
However, Zn-CHA produced lower lattice distortion than Zn-
HA and CHA, because the calcium vacancy in the HA
structure due to the CO3 2  substitution is compensated by
Zn2 þ substitution. They also proved the regulating effect of
zinc on the apatite forming ability of HA when substituted
along with carbonate. In addition, the presence of carbonates
along with zinc in HA led to thermal stability higher than that
of HA and Zn-HA. Sr-CHA prepared by Landi et al. [215]
demonstrated an increase in the a-axis and in the c-axis in
comparison with HA. This resorbable system is able to release
in situ higher amounts of Sr per day than carbonate-free Sr-
HA. Finally, it can be stated that by adjusting the concentration
of Zn/Sr and carbonate it may be possible to achieve desired
properties for various biomedical applications.
The simple precipitation technique has been successfully
applied for preparing Na-Si/CHA at physiological temperature
[310]. This low-cost and simple method makes it possible to
incorporate Na þ , CO3 2  and SiO4 4  ions in the HA lattice,
with silicate up to 2.23 wt% with the crystallochemical formula
Ca10 xNax(PO4)6  x y 2z(SiO4)y þ z(CO3)x þ z(OH)2 y. Silicate
ion substitutions in phosphate positions import structural
changes around the O–H sites, due to the formation of bent
hydrogen bonding. The powder crystallinity is strongly reduced
as the extent of the substitution increases, but there is no
evidence of a collapse of the apatite structure and the formation
of an amorphous phase, as was stated in other studies [311],
where Mg-Si/CHA was characterized and it was proved that the
simultaneous presence of silicon and magnesium mutually limits
their release.
Mayer et al. [259] studied the morphology of Mn-CHA, and
they found that it depends strongly on the ion concentrations.
Transmission electron microscopy has shown that at relatively low
Mn2 þ content (0.73 wt%) and high CO3 2  content (3.6 wt%),
large platelet and needle-like crystals were present, whereas at a
higher Mn2 þ content (1.23 wt%) and a lower CO3 2  content
(2.1 wt%) only smaller needle-like crystals appeared.
Other beneficial substituents are halogenides, which were
used in co-substitution with carbonates and alkali metals. Yao
et al. [312] prepared Na-F/CHA by coprecipitation, and showed
that increasing CO3 2  and Na þ content with a relatively
constant F  concentration caused a decrease in crystallite size
and consequently an increase in the extent of calcium release.
An increasing F  content had an opposite effect, as was proved
in a further paper by Yao et al. [313]. AB carbonate Na-F/CHA
was synthesized at 1150–1300 1C and 1.0 GPa by Fleet and Liu
[314]. They found that there is a significantly lower uptake of
carbonate in Na-FHA than in Na-CHA for similar crystal
synthesis conditions. The same trend was proved for AB
carbonate Na-Cl/CHA synthesized at the same conditions
[315]. The molar amounts of Na and A-type carbonate main-
tained a near 1:1 ratio, confirming that the Na cation and the A
and B carbonate ion substituents exist as a defect cluster to
facilitate charge compensation and spatial accommodation. The
effects of substituents on lattice parameters are summarized in
Table 3. There is an apparent synergistic effect of CO3 2  and
F  anions on decreasing the a-axis and increasing the c axis
[312,314]. However, in the case of Na-Cl/CHA [315], the
prevalent effect of a relatively high concentration of chloride
showed the opposite trend. Leilei et al. [316] syn-
thesized nanobelt-shaped Na-F/CHA (5–40 nm in thickness,
250–700 nm in width and tens of micrometers in length) using a
combined method of ultrasound-assisted electrochemical deposi-
tion and ion exchange. The nanobelts were interlocked with
each other on the carbon-carbon and they induced the precipita-
tion of apatite layers on its surface in SBF, and exhibited excellent
in vitro biocompatibility.
Mg substituted fluoroapatite (Mg-FHA) has frequently been
prepared in order to integrate the advantages of Mg2 þ with the
low solubility of FHA. Kheradmandfard and Fathi [317]
prepared Mg-FA nanopowders with various Mg contents
(Ca10 xMgx(PO4)6F2, where x¼ 0, 0.25, 0.5, 0.75, and 1) by
Table 3
Relation between the concentrations (Na, Mg, F or Cl and carbonate) and the
lattice parameters of alkali metal and halogenide co-substituted HA;
*¼constant concentration.
Maximal concentration Trend in the lattice parameter in Ref.
(wt%) comparison with stoichiometric HA
Na Cl F CO3 a-axis c-axis
1.70  2.56* 4.68 ↓ ↑ [312]
1.73  3.42 4.68* ↓ - [312]
0.23  3.42 1.80 ↓ ↑ [314]
0.90* 4.90 – 6.48 ↑ ↓ [315]
 M. Šupová / Ceramics International 41 (2015) 9203–9231
the sol–gel method. The incorporation of Mg ions into the FA
resulted in a decrease in both lattice parameters, and only a part
of the Mg entered into the FA lattice, while the remainder
formed MgF2. In their further research [318], Kheradmandfard
and Fathi found that with increasing Mg-substitution, the
solubility of nanopowders and the adsorption of Ca2 þ ions
onto the surfaces of the powders increased simultaneously. It
was concluded that Mg-substitution improves the bioactivity of
FA. Another research group, Cai et al. [319], used Mg and F
incorporation to improve the bioactivity of HA coatings
(Ca10 xMgx(PO4)6F(OH), x¼ 1) on Ti6Al4V alloys. In their
subsequent work [320], Cai et al. determined a significantly
positive effect of Mg ions on cell (MG63) proliferation, and late
differentiation at x¼ 1.5. Finally they studied the interface of the
Mg-FHA coating and the Ti6Al4V substrate, and proved that
Mg incorporation into the FHA coating improves the pull-off
adhesion strength thanks to mutual diffusion of the elements
between the coating and the substrate, and the consequent
formation of chemical bonds [321]. Other cations, Na, Zn and
Sr, were co-substituted with fluoride anions to form Na/Mg-
FHA [322], Zn-FHA [323] and Sr-FHA [324], respectively. It
was proved that the addition of these cations better imitates
natural bone, improves the mechanical properties, and provides
higher corrosion resistance, respectively.
To obtain biomaterials with improved biocompatibility, Mg, Si
co-substituted HA (Mg-SiHA) was prepared by Kim et al. [325],
and it was found that co-substitution of 1 wt% for each ion keeps
their original structures intact for sintering temperatures of up to
1200 1C. Cell proliferation experiments suggested that Mg-SiHA
can be a useful material for implants and bone augmentation. It
was hypothesized that co-substitution of HA nanowires with Si and
Sr elements (Sr-SiHA) can bring about a synergistic effect that will
stimulate the proliferation and differentiation of osteoblasts and
MSCs better than traditional SiHA and Sr-HA materials. Zhang
et al. [326] synthesized Sr-SiHA via hydrothermal treatment in the
absence of any organic surfactants and solvents. An increase in the
substitution amount caused an increase in the lattice constants and
also appeared to improve the degradability of the materials.
Lin et al. [13] prepared biomimetic HA porous microspheres
with co-substituted essential trace ions (Na þ , Mg2 þ , K þ , F  ,
Cl  and CO3 2  ) through a low-temperature hydrothermal
method. Each of the elements mentioned here plays an essential
part in the course of the biological action. The results showed that
the synthetic biomimetic hydroxyapatite porous microspheres were
constructed from two-dimensional nano-sheets about 60 nm in
thickness and 2 μm in width and in length. The novel 3D
architectures resulted in favorable drug loading and release proper-
ties. Co-substitution of essential trace elements provided better
degradability of the porous microspheres than in the case of
traditional pure HA materials.
5. Summary and concluding remarks
Hydroxyapatite and its derivatives have already for several
decades been of general importance in many interdisciplinary fields
of science, including geology, chemistry, ecology, biology and
medicine. The data reviewed in this paper provides evidence of the
9223
growing interest in other ionic substitutions as powerful tools for
improving the biological performance of HAs, either through
modifications to the chemical, structural and morphological
characteristics of the HA or even through utilizing the therapeutic
properties of the substituting ions.
Ease of atomic doping or substitution and the possibility of high
ionic promiscuity in apatite opens this mineral up for a wide range
of biomedical applications. Substituted hydroxyapatites which can
simulate real bioapatite can be used as a filler for a biocomposite,
as a scaffold for newly grown bone tissue, or in the form of a bone
cement for repairing and replacing diseased and damaged parts of
musculoskeletal systems. Drug or gene delivery agents, biomag-
netic particles and fluorescent markers are other biomedical
applications. Last but not least, it is also worth mentioning the
use of substituted hydroxyapatites as a bioactive coating on
metallic osseous implants. These coatings can increase osseointe-
gration, can prevent the release of implants, and can serve as
carriers for antimicrobial drugs.
It has been found that bioapatite ceramic generated partially
or entirely from biogenic sources can be better accepted by
living organs, and it is very inexpensive owing to worldwide
availability. However, this biogenic material can also contain
various impurities and inhomogeneities. By contrast, synthetic
HAs can be synthesized under controlled conditions with
predictable and reproducible properties, together with better
control over impurities. Biogenic apatites have a given
composition, whereas synthetically substituted HAs could be
well regulated by facile tailoring of the substitution level of the
precursors and the reaction ratio of the precursor/solution,
respectively, and a final product with bespoke properties can
be prepared. In addition, they can be substituted by ions which
are not commonly present in bioapatites, e.g. Ag þ , Mn2 þ ,
Ti4 þ , and this can improve their antibacterial and mechanical
properties or their corrosion resistance. Doping or substitution
by some ions, e.g. Ln3 þ , is, at first sight, an almost incredible
option. New alternative ionic substitutions, therefore provide
interesting challenges for future research and for new biome-
dical applications.
However, it must be critically stated that only a limited
number of papers have made comprehensive biological eva-
luations in vitro and/or in vivo. Although many studies of HA
substitutions have already been made, and many papers have
been published, the research results are in many cases very far
from commercial implementation. Much work remains to be
done on the long path from laboratory studies to clinical
practice. There will be much need for effective cooperation
among chemists, materials scientists, biologists, bioengineers
and clinicians to transfer interesting research findings into
valuable materials for use in medical applications.
Acknowledgments
I gratefully acknowledge the financial support provided for
my work by the Long-Term Conceptual Development Research
Organization under Project no. RVO: 67985891. Special thanks
to Mr. R.A. Healey for language correction.
9224 M. Šupová / Ceramics International 41 (2015) 9203–9231
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