Materials Science and Engineering C 39 (2014) 134–142

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Nanocrystalline hydroxyapatite doped with selenium oxyanions: A new

material for potential biomedical applications
Joanna Kolmas a,⁎, Ewa Oledzka a, Marcin Sobczak a, Grzegorz Nałęcz-Jawecki b
a
Medical University of Warsaw, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, ul. Banacha 1, 02-097 Warsaw, Poland
b
Medical University of Warsaw, Faculty of Pharmacy, Department of Environmental Health Sciences, ul. Banacha 1, 02-097 Warsaw, Poland

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

Article history: Selenium-substituted hydroxyapatites containing selenate SeO2− 2−

4 or selenite SeO3 ions were synthesized using
Received 8 June 2013 a wet precipitation method. The selenium content was determined by atomic absorbance spectrometry. The raw,
Received in revised form 23 January 2014 unsintered powders were also characterized using powder X-ray diffraction, middle-range FT-IR spectroscopy
Accepted 16 February 2014
and transmission electron microscopy with energy-dispersive X-ray spectroscopic microanalysis. The synthe-
Available online 22 February 2014
sized apatites were found to be pure and nanocrystalline with a crystal size similar to that in bone mineral.
Keywords:
The incorporation of selenium oxyanions into the crystal lattice was confirmed.
Selenium The toxicity of hydroxyapatites containing selenite or selenate ions was evaluated with a protozoan assay and
Nanocrystalline hydroxyapatite bacterial luminescence test.
Biomaterials © 2014 Elsevier B.V. All rights reserved.
FT-IR
XRD
TEM

1. Introduction in diverse metabolic processes and that it is a constituent of seleno-

Calcium phosphates including apatites are commonly used for
manufacturing bioceramic materials with high biological activity and
osteoconductive properties in a living organism. The best known repre-
sentative of apatites is stoichiometric calcium hydroxyapatite, described
by the molecular formula Ca10(PO4)6(OH)2 [1–3]. An exceptional fea-
ture of apatites as biomaterials is their chemical resemblance to biolog-
ical apatite, which is the major inorganic constituent of mineralized
tissues, especially of bone, dentin, cement and tooth enamel. Biological
apatite, except that in tooth enamel, is nanocrystalline and non-
stoichiometric. It is characterized as carbonated hydroxyapatite con-
taining various substituted ions (i.e., Mg2 +, Na+, K+, Zn2 +, F−, Cl−,
HPO24 −) that determine its physicochemical and biological proper-
ties [4]. The facility of various ionic substitutions and the resulting capa-
bility of apatites to be doped with various ions have recently attracted
much attention. Those ions could enhance the biological, physicochem-
ical and/or mechanical properties of apatites [5–13].
One of the most important microelements is selenium, which is one
of the essential constituents of the human diet. Until recently, selenium
was considered a highly toxic element, harmful to human health [14].
However, it has been shown that this element plays a significant role
⁎ Corresponding author. Tel.: +48 22 5720755; fax: +48 22 5720784.
E-mail address: joanna.kolmas@wum.edu.pl (J. Kolmas).
proteins, as well as of glutathione peroxidase, the enzyme that
protects cellular membranes against harmful agents [15,16]. Nu-
merous studies indicate that a selenium deficiency may cause an
inhibition of bone growth in rats as well as a severe reduction of
bone strength [17–20]. Selenium may prevent carcinogenesis and
inhibit the growth of tumor cells [21–23]. A beneficial impact of se-
lenium on the inflammatory response of osteoblasts in the metas-
tasis of certain bone tumors has been recently evidenced [24].
Other studies indicated that inorganic selenium compounds (sele-
nates and selenites) 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, which lack oxi-
dation capability [22].
Therefore, it seems reasonable to prepare the apatite doped with
inorganic selenium. Such an apatite could serve as a constituent of
bone augmentation and implant materials that, besides providing a
scaffold for the newly grown bone tissue, would also inhibit the growth
and proliferation of tumor cells.
Until now, a number of methods for the preparation of hydroxy-
apatite in a powder form have been developed. They include wet pre-
cipitation [25], molten salt [26] (with the addition of fluxing agents),
mechanochemical [27], hydrothermal and sol–gel synthetic methods
[28]. There are also alternative procedures for obtaining apatites from
natural sources, such as animal [29] and fish bones [30] or coral skele-
tons [31]. It should be emphasized that the most commonly used are

http://dx.doi.org/10.1016/j.msec.2014.02.018
0928-4931/© 2014 Elsevier B.V. All rights reserved.
 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142
the wet synthetic processes, especially those carried out in aqueous so-
lutions [32–35].
So far, there have been only a few literature reports on the applica-
tion of selenium in biomaterials, taking advantage of anticancer and
antimicrobial properties of this element. Tran et al. [36] examined a tita-
nium surface (implant material) covered with a layer formed by seleni-
um nanoclusters and demonstrated an inhibitory effect of selenium on
the proliferation of cancer cells. Then, Tran et al. [37] showed that sele-
nium can be covalently bound to the surface of a wound dressing,
prolonging its effectiveness. They found that an organoselenium coating
of cellulose prevented the formation of a Pseudomonas aeruginosa and
Staphylococcus aureus biofilm. Osteoblast adhesion on selenium nano-
particles was examined by Perla and Webster [38]. Selenite ions were
incorporated into apatite coatings produced by Pulsed Laser Deposition
in order to endow them with antibacterial properties [39]. Likewise,
they were introduced to hydroxyapatite nanoparticles to enhance
their ability to induce apoptosis of osteosarcoma cells [40]. Ma et al.
have synthesized hydroxyapatites containing sodium and selenite ions
with a different Se/P ratio and have proved their thermal stability in
sintering tests [41].
Inspired by previous research on selenium-doped apatites, we de-
cided to explore new routes in this research by directing attention to
the oxidation state of the incorporated selenium species. Therefore,
our study is aimed at the wet chemical synthesis of hydroxyapatites
doped with selenite (SeO23 −) or selenate (SeO24 −) ions containing
Se(IV) and Se(VI), respectively and the subsequent comparison of the
physicochemical properties of those materials. The obtained apatites
are characterized using powder X-ray diffractometry (PXRD), middle-
range Fourier transformed infrared (FT-IR) spectroscopy, transmission
electron microscopy (TEM) equipped with an energy-dispersive X-ray
analysis (EDS spectrometer) accessory and atomic absorption spectros-
copy (AAS). The toxicity of selenite or selenate doped hydroxyapatites is
evaluated with a bacterial luminescence test (Vibrio fisheri) and a proto-
zoan assay (Spirotox test with the Spirostomum ambiguum).
2. Experimental
2.1. Preparation
Samples of hydroxyapatite (HA), undoped and doped with selenite
(SeO23 −) or selenate (SeO24 −) ions, were obtained by precipitation
from aqueous solutions (wet method). Ca(NO3)2·4H2O (Sigma-Aldrich,
purity 99%) and (NH4)2HPO4 (Sigma-Aldrich, purity 99%) were used as
sources of calcium and phosphorus, respectively. Na2SeO3·5H2O
(Sigma-Aldrich, purity 99%) and Na2SeO4 (Sigma-Aldrich, purity 98%)
were used as sources of selenium for HA, doped with selenite and sele-
nate ions, respectively.
There are two locations in the HA crystal unit cell capable of accom-
modating either SeO2− 3 or SeO2−
4 ions: the channels running along the
c-axis, normally occupied by columns of structural hydroxyl groups
and the orthophosphate sites. The SeO2− 4 ion has a tetrahedral structure
as the PO3−
4 ion [42]. However, the selenate is slightly larger: 249 pm vs.
238 pm in diameter [43]. On the other hand, the SeO2− 3 ion has a very
similar diameter (239 pm) to that of the PO3− 4 ion, but it has the quite
different geometry of a flat trigonal pyramid [42,44]. Considering the
sizes of the SeO23 − and SeO24 − ions, the incorporation of those ions
into the HA crystal lattice is possible, but they can easily replace only
the phosphate ions (see the schematic model in Fig. 1S in Supplementa-
ry materials). Furthermore, as the planned substitution was of a doubly
charged anion for a triply charged one, an electric charge balancing
mechanism similar to that proposed by Barralet et al. [45] for introduc-
ing B-type carbonates into the HA crystal lattice was adopted. Thus, for
selenium-doped HA the following molecular formula was proposed:
Ca10−x ðPO4 Þ6−x ðSeOn Þx ðOHÞ2−x ;
135
where 0 b x b 2, x refers to the amount of selenium in the form of sele-
nium oxyanions and n is 3 or 4, for the selenite or selenate ions, respec-
tively. The obtained materials are denoted by HA-xSeO3 or HA-xSeO4,
respectively (see Table 1).
The substitution of a bivalent selenate or selenite anion (charge −2)
for an orthophosphate anion (charge −3) creates a positively charged
vacancy (+ 1). According to the proposed formula, this charge defect
is then compensated by a joint release of one calcium cation and one hy-
droxyl anion. As a consequence, we can observe a simultaneous decalci-
fication and dehydroxylation in the apatite sample. It can be described
as follows:
Ca10 ðPO4 Þ6 ðOHÞ2 þ x SeOn
2−
→Ca10−x ðPO4 Þ6−x ðSeOn Þx ðOHÞ2−x
3− 2þ –
þ x PO4 þ x Ca þ x OH :
In order to comply with the aforementioned Formula (1), the
amounts of reagents with the assumption that one orthophosphate
ion is to be replaced by one selenite or selenate ion were calculated.
In this study, it was intended to synthesize materials with x up to 1.6.
First, adequate amounts of aqueous solutions of (NH4)2HPO4 and of
the selenium salt were added dropwise under stirring to an aqueous so-
lution of Ca(NO3)2. The pH of the reaction mixture was maintained
within the range of 8–9 by adding aqueous NH3. Next, the reaction mix-
ture was kept at about 60 °C under stirring for 4 h. After that, the resul-
tant suspension was left for 48 h at room temperature without stirring.
The obtained precipitate was filtered out under reduced pressure. After
the filtering, the precipitate was intensively washed with distilled water
in order to remove any excess of ammonia (until the pH of the filtrate
became neutral). Finally, the powder was dried at about 80 °C for 12 h.
2.2. Analytical methods
The phase composition and crystallinity of our samples were exam-
ined by X-ray diffraction (XRD; Bruker D8 Discover diffractometer)
using Cu Kα radiation (λ = 1.54 Å). The PXRD patterns were also
used to calculate crystal dimensions (Scherrer's method) and the di-
mensions of crystallographic unit cells (the Rietveld method). Unit cell
parameters were calculated using TOPAS software (version 3, Bruker).
This program uses Rietveld refinement based on analytical profile func-
tions and least squares algorithms to fit a theoretical to a measured XRD
pattern. However, we have not performed a full refinement due to the
poor resolution of the patterns. An example of Rietveld fitting is pre-
sented in Fig. 2S in Supplementary materials. The error of the measure-
ments did not exceed 0.3%.
The morphology of apatite crystals was observed using transmission
electron microscopy (TEM). The measurements were performed with
an FEI Titan 80–300 Cubed microscope operated at an acceleration volt-
age of 300 kV and equipped with an energy dispersive X-ray spectrom-
eter (EDS spectrometer). For TEM inspection a droplet of ultrasonically
treated suspension of the powder in methanol was deposited onto a po-
rous carbon film and dried.
The selenium content was determined (after dissolving the powder
in the concentrated HNO3) using the atomic absorption spectrometer
from GBC Scientific Equipment, equipped with an electrothermal atom-
izer (ET-AAS). Background correction was performed using the longitu-
dinal Zeeman's effect, measuring ET-AAS signals in the peak height
mode.
In order to study the structure of the obtained materials, mid-
infrared spectroscopy (MIR) and Raman spectroscopy were used. MIR
spectra in the 400–4000 cm−1 range were recorded from KBr pellets
using a Spectrum 1000 spectrometer (Perkin Elmer, Cambridge, UK)
with a spectral resolution of 2 cm− 1 and 30 scans. The spectra were
processed using GRAMS/AI 8.0 software (Thermo Scientific 2008).
Raman spectra (shown in Supplementary materials) were per-
ð1Þ formed at room temperature and in ambient air under a laser excitation
136 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142

Table 1
Various parameters of the obtained samples.

Samples Crystal size Crystal size Ca/(P + Se) Expected Se content Measured Se Measured type Calculated formula
along c axis along a axis molar ratiob wt. % (mol %) contentc B CO2−
3 content
d
Ca10 − x − y(PO4)6 − x − y(SeOn)x(CO3)y(OH)2 − x − ye
(nm)a (nm)a wt. % (mol %) (wt. %)

HA 25 7 1.63 0.0 (0.0) 0.0 1.4 Ca9.71(PO4)5.71(CO3)0.29(OH)1.71

HA-0.1SeO3 20 8 1.65 0.79 (0.10) 0.78 (0.1) 2.1 Ca9.56(PO4)5.56(SeO3)0.10(CO3)0.34(OH)1.56
HA-0.4SeO3 15 6 1.59 3.24 (0.40) 2.96 (0.36) 2.4 Ca9.26(PO4)5.26(SeO3)0.36(CO3)0.38(OH)1.26
HA-0.8SeO3 16 7 1.62 6.42 (0.80) 6.11 (0.73) 2.5 Ca8.88(PO4)4.88(SeO3)0.73(CO3)0.39(OH)0.88
HA-1.2SeO3 15 5 1.64 9.70 (1.20) 9.65 (1.15) 2.3 Ca8.49(PO4)4.49(SeO3)1.15(CO3)0.36(OH)0.49
HA-1.6SeO3 16 5 1.68 13.10 (1.60) 9.85 (1.17) 2.4 Ca8.46(PO4)4.46(SeO3)1.17(CO3)0.37(OH)0.46
HA-0.1SeO4 25 9 1.62 0.79 (0.10) 0.72 (0.09) 0.9 Ca9.76(PO4)5.76(SeO4)0.09(CO3)0.15(OH)1.76
HA-0.4SeO4 22 7 1.58 3.22 (0.40) 2.89 (0.35) 2.4 Ca9.26(PO4)5.26(SeO4)0.35(CO3)0.39(OH)1.26
HA-0.8SeO4 20 6 1.64 6.34 (0.80) 5.83 (0.72) 1.2 Ca9.08(PO4)5.08(SeO4)0.72(CO3)0.20(OH)1.08
HA-1.2SeO4 20 8 1.62 9.52 (1.20) 9.36 (1.14) 1.9 Ca8.55(PO4)4.55(SeO4)1.14(CO3)0.31(OH)0.55
HA-1.6SeO4 25 10 1.65 12.80 (1.60) 9.76 (1.21) 0.9 Ca8.64(PO4)4.64(SeO4)1.21(CO3)0.15(OH)0.64
a
From XRD diffractograms (Scherrer's formula).
b
Calculated from EDS data.
c
Measured by atomic absorption spectrometry AAS.
d
From FTIR spectra (using method described by Clasen and Ruyter [55]).
e
n = 3 for selenite, n = 4 for selenate; x and y — selenium oxyanion and carbonate content, respectively.

wavelength of 1064 nm using the Nicolet NXR spectrometer (Thermo Sci- responses were studied with the use of a dissection microscope (magni-
entific). The spectra were recorded with spectral resolution of 1 cm−1 and fication 10×), deformation of the protozoan cell (i.e., bending, shorten-
2000 scans. ing) or lethal response.

2.3. Toxicity assays 3. Results and discussion

For the toxicity test, the hydroxyapatite powders with the highest
concentration of selenite or selenate ions were used. Two different sus-
pensions were prepared: 1 mg/ml and 2 mg/ml.
For the toxicity evaluation, we used the Microtox® test with the lu-
minescent bacteria V. fisheri. The test was performed in special glass dis-
posable cuvettes with the lyophilized bacteria purchased from SDI
(USA). Samples were incubated for 15 min and then the light output
of the samples was measured in the Microtox® M500 Analyzer. As a dil-
uent and a control 2% NaCl was used.
A Spirotox test with the protozoan S. ambiguum was performed
according to the standard protocol [46]. The test was performed in dis-
posable, polystyrene multiwell plates. Ten organisms of S. ambiguum
were introduced to each well of the multiwell. The samples were
incubated in darkness at room temperature for 24 h and 48 h. As a dil-
uent and a control, a Tyrode solution was used. After incubation, test
The synthesized materials were subjected to comprehensive analyt-
ical and structural analysis.
Figs. 1 and 2 show PXRD patterns of pure HA, regarded as the refer-
ence material, and patterns of synthesized HA doped with selenite or
selenate ions. No peak, other than from the HA crystalline phases, was
detected. As the materials were found to be nanocrystalline, the HA
peaks were wide, poorly resolved and some of them were too broad
to be detected. Furthermore, the materials were well hydrated because
they were dried at a low temperature (80 °C). For this research, consid-
ering the lack of the literature data on selenium-containing HA, we were
mostly interested in materials with a hydrated surface layer. The effect
of thermal treatment on the physicochemical properties of selenium-
doped HA will be discussed in our future work.
The HA crystallinity (crystal size and crystal perfection) can be
assessed visually by looking at the width and resolution of the PXRD

Fig. 1. The PXRD patterns of the selenite-doped HA (HA-xSeO3) and HA undoped.

 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142 137

Fig. 2. The PXRD patterns of the selenate-doped HA (HA-xSeO4) and HA undoped.

peaks. Thus, the crystallinity of HA has no obvious trend with an in-
crease in the selenite or selenate content (Figs. 1 and 2 respectively).
Moreover, the absence of additional crystalline phases, such as CaO
or tricalcium phosphate (TCP) may be proof that the selenium ions
entered into the crystalline structure of hydroxyapatite. If the selenite
or selenate had not been introduced to the HA lattice according to
Eq. (1), the material obtained during the synthesis would have had a
Ca/P molar ration greater than the stoichiometric value of 1.67. It
would be calcium rich and therefore a secondary phase would be pro-
duced and observed in the diffraction pattern, such as CaO or TCP [47].
The dimensions of the crystals along the c and a axes were estimated
from the (002) and (130/310) reflexes, respectively. Scherrer's equation
has been used:
0:94  λ
D¼ ;
β1=2  cos θ
where:
selenites, causes shortening of the a axis [49,50]. It is important to
note that it can be a simple matter of the different ion sizes: carbonates
are significantly smaller (178 nm) than phosphates and selenites. On
the other hand, both the a and c parameters are only insignificantly af-
fected by introducing a slightly larger, but tetrahedral selenate ion
(249 pm), structurally resembling the phosphate ion. It is also impor-
tant to note that the adsorption of selenite and selenate on the hydroxy-
apatite crystal surface has been studied in some papers [51–53]. The
results presented in these papers show that selenium oxyanions were
sorbed on hydroxyapatite mainly by an anionic exchange with phos-
phate ions. It has been proven that selenate and selenite were not exclu-
sively located at the surface of the apatite, but diffused slightly in a
thickness of a few nanometers [51]. It is also very interesting that sele-
nate anions were much less sorbed than selenite [52].
Representative TEM images with electron diffraction patterns of se-
lected samples containing SeO2− 2−
3 and SeO4 ions are presented in Fig. 4.
Fig. 4 a and d, showing pure hydroxyapatite, has been attached as a

D is a crystallite size,
λ is the wavelength of the radiation (in nm),
β1/2 is the peak full-width at half maximum (in radians) and
θ is the diffraction angle of the corresponding reflex.

The results of this estimation are presented in Table 1. The obtained

HA-based materials containing SeO2− 3 and SeO2−
4 ions are nanocrystal-
line, and their crystal sizes are similar to the size of biological apatite
crystals in bone tissue [48].
The effect of introducing selenium into the HA unit cell can be
assessed from its c and a parameters calculated by the Rietveld method.
In this study, it was found that the substitution of a selenite ion for a
phosphate ion resulted in a significant extension of the a axis, while
the c axis remained practically unchanged (Fig. 3).
On the other hand, the introduction of a selenate Se(VI) ion into the
hydroxyapatite crystal cell resulted in a slight increase of the parameter
a up to x = 0.8 without causing any significant changes beyond that
level. As in the case of SeO2− 3 ions, the change of the c parameter was
very small. In this place, it seems reasonable to refer to the spatial struc-
ture of selenium oxyanions. Due to its completely different spatial struc-
ture (a flat trigonal pyramid), the selenite ion significantly affects the
parameter a of the cell, despite its dimensions, which are very close to Fig. 3. The unit cell parameters of the studied samples (a and c — lattice constants (nm);
those of the phosphate ion (239 versus 238 nm). It is very interesting x — the amount of selenium in the form of selenium oxyanions; n — 3 or 4, for the selenite
that a similar substitution of carbonates, which are isotypical with or selenate ions, respectively).
138 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142

Fig. 4. Electron diffraction patterns and bright field images of reference undoped HA (a and d), the HA-1.2SeO3 sample (b and e), the HA-1.2SeO4 (c and f).

reference. It seems noteworthy that the presence of selenium ions af-
fects the shape of the crystals. The crystals of pure HA are elongated
platelet-shaped, whereas selenium-doped HA crystals are more plate-
like in shape. Analysis of TEM images confirms that the obtained mate-
rials are nanocrystalline; the crystals do not exceed 30 nm in length and
10 nm in width (see Table 1). The electron diffractogram patterns pre-
sented in Fig. 4 a–c show that the pure hydroxyapatite (the HA sample)
is more crystalline than the selenium-doped samples. Moreover, diffuse
rings in the case of the sample HA-1.2SeO3 prove to have the smallest
crystallite size and/or the worst quality of crystal structure. HRTEM
images of selected samples are presented in Figs. 3S and 4S in Supple-
mentary materials. They show the nanocrystalline nature of studied
materials. In addition they prove that the apatitic structure of HA-
xSeO3 samples is more disordered than that of HA-xSeO4 samples. This
observation is in accordance with the previously presented conclusions
of the PXRD data.
The Ca/P + Se ratio was determined using EDS analysis (see Table 1).
In all samples, the estimated Ca/P + Se ratio was slightly below 1.67
(the Ca/P ratio for stoichiometric hydroxyapatite).
Concerning selenium content in the tested samples, the data from
AAS are presented in Table 1. The obtained values are slightly lower
than those that were expected. In most cases, they exceeded 90% of
the level assumed before the reaction. This means that even after inten-
sive rinsing in distilled water, the obtained materials contain almost all
selenium taken to the synthesis. If the selenite/selenite ions were
adsorbed on the crystal surface, the selenium content would be signifi-
cantly reduced (soluble ammonium selenite/selenate would be re-
moved during the rinsing process). The content of selenium obtained
 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142 139

Fig. 5. The FT-IR transmission spectra of the studied selenite-doped HA (HA-xSeO3) and HA undoped.

in HA-1.6SeO3 and HA-1.6SeO4 samples is much lower than the expect-
ed value. Presumably, the substitution of phosphate ions for such a large
amount of selenium oxyanions was too difficult; the excess of selenate
and selenite ions has remained in the solution. Moreover, selenium
oxyanions adsorbed on the surface of hydroxyapatite crystals were un-
doubtedly removed during intensive repeated rinsing in distilled water
and filtering the precipitate.
Figs. 5 and 6 show the FT-IR spectra of the tested hydroxyapatite
samples, HA-xSeO3 and HA-x SeO4, respectively. The bands that are
characteristic for hydroxyapatite are presented in all spectra (a spec-
trum of pure hydroxyapatite is shown at the bottom of Figs. 5 and 6).
The dominant band at 1200–900 cm− 1, as well as the bands at 602
and 563 cm−1, corresponds to the vibrations of the phosphate groups
of hydroxyapatite. The broad band at 3700–2500 cm−1 and the band
at 1635 cm−1 could be assigned to the stretching and bending vibra-
tions of water adsorbed on the surface of the crystals [32,54]. The
weak, narrow band at 3570 cm−1 and the band at 630 cm−1 correspond
to the structural hydroxyl groups (stretching and librational vibrations).
It is clear that the intensity of the band at ca. 3570 cm−1 decreases
with an increase of the selenite content (Fig. 5) as is in accordance
with the proposed mechanism of substitution and with Formula (1).
However, for samples containing selenates, there are no significant
changes in the relative intensity of this band. The interpretation is not
easy and requires a more detailed study.
Consider that the regions of 1550–1400 cm−1 and 873 cm−1 corre-
spond to vibrations of the carbonate groups. Carbonates are present in
water and in air and therefore can be easily incorporated in the hy-
droxyapatite crystals. Due to their small size (178 pm), carbonates not
only substitute phosphate ions (type B) but are also situated in the col-
umns of structural hydroxyl groups. It was possible to estimate carbon-
ates by using a method described by Clasen and Ruyter [55]. All the
studied materials contain a significant amount of carbonates, especially

Fig. 6. The FT-IR transmission spectra of the studied selenate-doped HA (HA-xSeO4) and HA undoped.
140 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142
type B: 0.9–2.5 wt.% (see Table 1). Thus, for these samples the Formu-
la (1) was modified:
Ca10−x−y ðPO4 Þ6−x−y ðSeOn Þx ðCO3 Þy ðOHÞ2−x−y ;
where x and y refer to the amount of selenium oxyanions and carbon-
ates, respectively and n is 3 or 4, for the selenite or selenate ions, respec-
tively (see Table 1).
It is important to note that, for this formula, the Ca/P ratios for the
apatite core and in the hydrated layer were assumed the same.
Considering the proposed Formula (2) it can be claimed that the
decrease of structural hydroxyl groups depends on both selenium
oxyanions and carbonate contents. In hydroxyapatites containing
selenite ions, type B carbonate content was almost constant (2.1–
2.4 wt.%). In such cases, the structural hydroxyl groups' concentration
depends only on the selenite content. In hydroxyapatites with SeO2− 4 ,
the amount of carbonates was significantly different: 0.9–2.4 wt.%
(Table 1) and that is why there is no correlation between selenate incor-
poration and the intensity of the band at ca. 3570 cm−1.
The spectra of HA-xSeO3 samples contain two distinct bands at
approximately 767 cm−1 and 840 cm−1 (see Fig. 5). The intensity of
these bands increases with an increased content of selenite in the hy-
droxyapatite. Notably, a weak band at approximately 505 cm−1 appears
in the spectrum of the sample HA-1.6SeO3. According to the literature
data, vibrations of the free SeO23 − ion provide four bands within the
middle IR region: ν1 (symmetrical stretching vibrations) at 807 cm−1,
ν2 (in-plane bending vibrations) at 432 cm− 1, ν3 (asymmetrical
stretching vibrations) at 737 cm−1 and ν4 (out-of-plane bending vibra-
tions) at 374 cm−1 [56–58]. In the spectra, the bands at 767 cm−1 and
840 cm−1 have been assigned to the symmetrical and asymmetrical vi-
brations Se\O of the selenite ion in hydroxyapatite, respectively, while
the band at 505 cm−1 has been interpreted as resulting from bending
vibrations. Due to the limitation of the wave number range, another
band generated by bending (out-of-plane) vibrations is invisible in the
presented spectra.
Moreover, in the presented spectra, the positions of the bands signif-
icantly differ from the bands assigned by Nakamoto [57] for the selenite
ions. Therefore, one can confirm that in the tested samples, selenite ions
are incorporated into hydroxyapatite crystals. Furthermore, it may be
noted that the position of the band at 767 cm−1 is shifted towards the
lower wavenumbers. A detailed analysis of FT-IR spectra, including
this observation, will be performed in our further work.
Vibrations of free selenate ions SeO24 − give two middle-IR active
bands: ν3 (asymmetrical stretching vibrations) at 873 cm− 1 and ν4
(out-of-plane bending Se\O) at approximately 314 cm−1 [58]. Due to
the limited range of the detector, it was impossible to detect the second
band in this study. The weak band at 874 cm−1 is present in both
samples' spectra, doped with selenate SeO2− 4 and selenite SeO2− 3 ions.
In addition, it could be also recognized in the spectrum of pure hydroxy-
apatite. Therefore, this band was assigned to carbonate groups (ν2). The
weak and rather broad band at approximately 910 cm− 1 appears in
the spectra of HA-xSeO4 samples and it is invisible in the spectra of
other materials (Fig. 6). It was assumed that this band comes from
the Se\O stretching vibrations of the SeO24 − group. The shift of this
Table 2
Toxicity of selected samples in Microtox® and Spirotox tests.
Sample Microtox® (15 min-PE)a
1.0 mg/ml 2.0 mg/ml
HA-1.6SeO3 6 12
HA-1.6SeO4 0 0
a
Percent of toxic effect after 15 min of incubation.
b
NT — not toxic; def — deformation of the cells.
band with respect to that coming from the free selenate ion, confirms
the incorporation of the ion into the hydroxyapatite unit cell. As a con-
firmation of selenate substitution for phosphate, Raman spectra of HA,
ð2Þ HA-1.2SeO3 and HA-1.2SeO4 samples were included in Supplementary
materials (see Fig. 5S). Consider that the ν2 bands of carbonates are in-
active in Raman spectroscopy [59]. Only in the HA-1.2SeO4 spectrum,
the bands at ca. 911, 873 and 843 cm−1, which can be assigned to sele-
nates, were detectable.
The luminescent bacteria V. fisheri (Microtox®) and protozoa
S. ambiguum (Spirotox) were used to evaluate the toxicity of obtained
hydroxyapatites doped with selenium. For these tests, the samples
HA-1.6SeO3 and HA-1.6SeO4 with the highest concentration of selenium
were chosen. Although selenium is an essential trace element, it may be
highly toxic at higher concentration [16]. Thus, it was important to
assess the general toxicity of the studied materials. The HA-1.6SeO3
sample containing selenites caused the 6% and 12% inhibition of lumi-
nescence of the bacteria in 1.0 mg/ml and 2.0 mg/ml solutions, respec-
tively. The sample with selenates HA-1.6SeO4 was not toxic to the tested
bacteria in both solutions. In the Spirotox test, the HA-1.6SeO3 sample
was toxic after a 48-hour incubation, causing deformation of the proto-
zoan cells, whereas the HA-1.6SeO4 sample was completely neutral (see
Table 2).
4. Conclusions
New apatite materials, HA-xSeO3 and HA-xSeO4, have been synthe-
sized by the conventional wet method and thoroughly examined by
powder X-ray diffractometry (PXRD), transmission electron microscopy
(TEM), atomic absorption spectrometry (AAS) and FT-IR spectroscopy
in the middle IR range. The most significant conclusions from these ex-
aminations are as follows:
1. The obtained materials are nanocrystalline hydroxyapatites; the ma-
terials do not contain any additional crystalline phases.
2. The examined crystals have the following dimensions: less than
30 nm in length and less than 10 mm in thickness. The values are
close to those of biological apatites of bones, dentin and cement.
3. The introduction of selenite or selenate ions does not significantly af-
fect crystallinity.
4. In all samples, the Ca/P + Se ratio is slightly below 1.67 (the Ca/P
ratio for stoichiometric hydroxyapatite).
5. The content of selenium in the examined materials is not much lower
than the declared value. A lower content of selenium has been ob-
served only for the samples where x = 1.6.
6. The incorporation of selenium oxyanions into the crystals has been
confirmed by PXRD, FT-IR and Raman spectroscopy data.
7. Carbonate ions (especially type B) have been detected as impurities
in the obtained materials and estimated at less than 2.5 wt.%.
8. Hydroxyapatite containing selenate ions was not toxic, whereas hy-
droxyapatite with the highest concentration of selenites was toxic
in Spirotox and Microtox® tests.
In conclusion, it should be emphasized that partial substitution of
phosphate ions on the selenium oxyanions has been successfully carried
out in this study. We hope that new synthesized nanocrystalline
Spirotoxb
1.0 mg/ml 2.0 mg/ml
NT (24 h) → def (48 h) NT (24 h) → def (48 h)
NT NT
 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142
hydroxyapatites doped with SeO2− 2−
3 or SeO4 ions can find new applica-
tions as material in regenerative medicine. These materials may be bone
substitutes in osteosarcoma or bone metastasis treatment. Selenium
supplementation directly to the bone tissue may prevent the activation
of pro-inflammatory factors and thus contribute to the inhibition of
tumor growth in bone.
Future studies will focus on the evaluation of the in vitro biocompat-
ibility assays.
Acknowledgments
This work was supported by the research program (Project
MNiSzW-2011/03/D/ST5/05793) of the Polish Ministry of Science and
Higher Education and by the European Regional Development Fund
within the Innovative Economy Operational Programme 2007–2013
(No POIG.02.01-00-14-032/08). Special thanks are addressed to Piotr
Dłużewski and Maciej Zieliński for TEM measurement.
The authors are indebted to Andrzej Jaklewicz from the Medical Uni-
versity of Warsaw for the AAS analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.msec.2014.02.018.
References
[1] R.Z. LeGeros, Biological and synthetic apatites, in: P.W. Brown, B. Constanz (Eds.),
Hydroxyapatite and Related Materials, CRC Press, Boca Raton, 1994, pp. 3–28.
[2] S.V. Dorozhkin, M. Epple, Biological and medical significance of calcium phosphates,
Angew. Chem. Int. Ed. 41 (2002) 3130–3146.
[3] S.V. Dorozhkin, Nanosized and nanocrystalline calcium orthophosphates, Acta
Biomater. 3 (2010) 715–734.
[4] B. Wopenka, J.D. Pasteris, A mineralogical perspective on the apatite in bone, Mater.
Sci. Eng. C 25 (2005) 131–143.
[5] Y. Pan, M.E. Fleet, Compositions of the apatite-group minerals: substitution mecha-
nisms and controlling factors, Rev. Mineral. Geochem. 48 (2002) 13–49.
[6] T.J. Webster, E.A. Massa-Schlueter, J.L. Smith, E.B. Slamovich, Osteoblast response to
hydroxyapatite doped with divalent and trivalent cations, Biomaterials 25 (2004)
2111–2121.
[7] S. Kannan, F. Goetz-Neunhoeffer, J. Neubauer, J.M.F. Ferreira, Ionic substitutions in
biphasic hydroxyapatite and β-tricalcium phosphate mixtures: structural analysis
by rietveld refinement, J. Am. Ceram. Soc. 91 (2008) 1–12.
[8] I.R. de Lima, G.G. Alves, C.A. Soriano, A.P. Campaneli, T.H. Gasparoto, E.S. Ramos Jr.,
L.Á. de Sena, A.M. Rossi, J.M. Granjeiro, Understanding the impact of divalent cation
substitution on hydroxyapatite: an in vitro multiparametric study on biocompatibil-
ity, J. Biomed. Mater. Res. A 98 (2001) 351–358.
[9] K. Lin, J. Chang, X. Liu, L. Chen, Y. Zhou, Synthesis of element-substituted hydroxyap-
atite with controllable morphology and chemical composition using calcium silicate
as precursor, Cryst. Eng. Commun. 13 (2011) 4850–4855.
[10] J.H. Shepherd, D.V. Shepherd, S.M. Best, Substituted hydroxyapatites for bone repair,
J. Mater. Sci. Mater. Med. 23 (2012) 2335–2347.
[11] S. Bodhak, S. Bose, A. Bandyopadhyay, Bone cell–material interactions on metal-ion
doped polarized hydroxyapatite, Mater. Sci. Eng. C 31 (2011) 755–761.
[12] H. Eslami, M. Solati-Hashjin, M. Tahriri, The comparison of powder characteristics
and physicochemical, mechanical and biological properties between nanostructure
ceramics of hydroxyapatite and fluoridated hydroxyapatite, Mater. Sci. Eng. C 29
(2009) 1387–1398.
[13] C.S. Ciobanu, S.L. Iconaru, I. Pasuk, B.S. Vasile, A.R. Lupu, A. Hermenean, A. Dinischiotu,
D. Predoi, Structural properties of silver doped hydroxyapatite and their biocompat-
ibility, Mater. Sci. Eng. C 33 (2013) 1395–1402.
[14] A. Fernandez-Martinez, L. Charlet, Selenium environmental cycling and bioavailabil-
ity: a structural chemist point of view, Rev. Environ. Sci. Biotechnol. 8 (2009) 81–110.
[15] D.H. Holben, A.M. Smith, The diverse role of selenium within selenoproteins: a re-
view, J. Am. Diet. Assoc. 99 (1999) 836–843.
[16] M.P. Rayman, The importance of selenium to human health, Lancet 356 (2000)
233–241.
[17] B. Turan, S. Bayari, C. Balcik, F. Severcan, N. Akkas, A biomechanical and spectroscop-
ic study of bone from rats with selenium deficiency and toxicity, BioMetals 13
(2000) 113–121.
[18] R. Moreno-Reyes, D. Egrise, J. Neve, J.-L. Pasteels, A. Schoutens, Selenium deficiency-
induced growth retardation is associated with an impaired bone metabolism and
osteopenia, J. Bone Miner. Res. 16 (2001) 1556–1563.
[19] Y.A. Petrovich, R.P. Podorozhnaya, S.M. Kichenko, M.V. Kozlova, Effects of
selenium-containing compounds and their metabolism in intact rats and in animals
with bone fractures, Bull. Exp. Biol. Med. 137 (2004) 74–77.
141
[20] R. Ebert, F. Jakob, Selenium deficiency as a putative risk factor for osteoporosis, Int.
Congr. Ser. 1297 (2007) 158–164.
[21] G.F. Combs, W.P. Gray, Chemopreventive agents: selenium, Pharmacol. Ther. 79
(1998) 179–192.
[22] E.N. Drake, Cancer chemoprevention: selenium as a prooxidant, not an antioxidant,
Med. Hypotheses 67 (2006) 318–322.
[23] H. Zeng, J.J. Cao, G.F. Combs, Selenium in bone health: roles in antioxidant protection
and cell proliferation, Nutrients 5 (2013) 97–110.
[24] Y.-C. Chen, D.M. Sosnoski, U.H. Gandhi, L.J. Novinger, K.S. Prabhu, A.M. Mastro, Sele-
nium modifies the osteoblast inflammatory stress response to bone metastatic
breast cancer, Carcinogenesis 30 (2009) 1941–1948.
[25] M.R. Saeri, A. Afshar, M. Ghorbani, N. Ehsani, C.C. Sorell, The wet precipitation pro-
cess of hydroxyapatite, Mater. Lett. 57 (2003) 4064–4069.
[26] A. Cüneyt Tas, Molten salt synthesis of calcium hydroxyapatite whiskers, J. Am.
Ceram. Soc. 84 (2001) 295–300.
[27] C. Mochales, R.M. Wilson, S.E.P. Dowker, M.-P. Ginebra, Dry mechanosynthesis
of nanocrystalline calcium-deficient hydroxyapatite: structural characterization,
J. Alloys Compd. 509 (2011) 7389–7394.
[28] A.K. Nayak, Hydroxyapatite synthesis methodologies: an overview, Int. J. Chem.
Technol. Res. 2 (2010) 903–907.
[29] A. Sobczak, Z. Kowalski, Z. Wzorek, Preparation of hydroxyapatite from Animals
bones, Acta Bioeng. Biomech. 11 (2009) 23–28.
[30] M. Boutinguiza, J. Pou, R. Comesana, F. Lusquinos, A. de Carlos, B. Leon, Biological hy-
droxyapatite obtained from fish bones, Mater. Sci. Eng. C 32 (2012) 478–486.
[31] E. Damien, P.A. Revell, Coralline hydroxyapatite bone graft substitute: a review of
experimental studies and biomedical applications, J. Appl. Biomater. Biomech. 2
(2004) 65–73.
[32] S. Koutsopoulos, Synthesis and characterization of hydroxyapatite crystals: a review
study on the analytical methods, J. Biomed. Mater. Res. A 62 (2002) 600–612.
[33] M.P. Ferraz, F.J. Monteiro, C.M. Manuel, Hydroxyapatite nanoparticles: a review of
preparation methodologies, J. Appl. Biomater. Biomech. 2 (2004) 74–80.
[34] A. Banerjee, A. Bandyopadhyay, S. Bose, Hydroxyapatite nanopowders: synthesis,
densification and cell–materials interaction, Mater. Sci. Eng. C 27 (2007) 729–735.
[35] Y.-H. Tseng, C.-S. Kuo, Y.-Y. Li, C.-P. Huang, Polymer-assisted synthesis of hydroxy-
apatite nanoparticle, Mater. Sci. Eng. C 29 (2009) 819–822.
[36] P.A. Tran, L. Sarin, R.H. Hurt, T.J. Webster, Titanium surfaces with adherent selenium
nanoclusters as a novel anticancer orthopedic material, J. Biomed. Mater. Res. A
(2009) 1417–1428.
[37] P.A. Tran, A.A. Hammond, T. Mosley, J. Cortez, T. Gray, J.A. Colmer-Hamood, M.
Shashtri, J.E. Spallholz, A.N. Hamood, T.W. Reid, Organoselenium coating on cellu-
lose inhibits the formation of biofilms by Pseudomonas aeruginosa and Staphylococ-
cus aureus, Appl. Environ. Microbiol. 75 (2009) 3586–3592.
[38] V. Perla, T.J. Webster, Better osteoblast adhesion on nanoparticle selenium — a
promising orthopedic implant material, J. Biomed. Mater. Res. A 5 (2005) 356–364.
[39] C. Rodriguez-Valencia, M. Lopez-Alvarez, B. Cochon-Cores, I. Pereiro, J. Serra, P.
Gonzalez, Novel selenium doped hydroxyapatite coatings for biomedical applica-
tions, J. Biomed. Mater. Res. A 101 (2013) 853–861.
[40] Y. Wang, J. Ma, L. Zhou, J. Chen, Y. Liu, Z. Qiu, S. Zhang, Dual functional selenium
substituted hydroxyapatite, Interface Focus (2012), http://dx.doi.org/10.1098/rsfs.
2012.0002.
[41] J. Ma, Y. Wang, L. Zhou, S. Zhang, Preparation and characterization of selenite
substituted hydroxyapatite, Mater. Sci. Eng. C 33 (2013) 440–445.
[42] F. Renard, G. Montez-Hernandez, E. Ruiz-Agudo, C.V. Putnis, Selenium incorporation
into calcite and its effect on crystal growth: an atomic force microscopy study,
Chem. Geol. 340 (2013) 151–161.
[43] D.S. Tawfik, R.E. Viola, Arsenate replacing phosphate: alternative life chemistries and
ion promiscuity, Biochemistry 50 (2011) 1128–1134.
[44] G. Aurelio, A. Martinez-Fernandez, G.J. Cuello, G. Roman-Ross, I. Alliot, I. Charlet,
Structural study of selenium(IV) substitution of calcite, Chem. Geol. 270 (2010)
249–256.
[45] J. Barralet, S. Best, W. Bonfield, Carbonate substitution in precipitated hydroxyapa-
tite: an investigation into the effects of reaction temperature and bicarbonate ion
concentration, J. Biomed. Mater. Res. 41 (1998) 79–86.
[46] G. Nałęcz-Jawecki, Spirotox test—Spirostomum ambiguum acute toxicity test, in:
C.H. Blaise, J. Ferard (Eds.), Small-scale Freshwater Toxicity Investigations,
vol. 1: Toxicity Test Methods, Springer, 2008, pp. 299–322.
[47] T. Tian, D. Jiang, J. Zhang, Q. Lin, Synthesis of Si-substituted hydroxyapatite by a wet
mechanochemical method, Mater. Sci. Eng. C 28 (2008) 57–63.
[48] S.V. Dorozhkin, Calcium orthophosphates in nature, biology and medicine, Materials
2 (2009) 399–498.
[49] M. Wildner, Isotypism of a selenite with a carbonate: structure of the buetschliite-
type compound K2Co(SeO3)2, Acta Crystallogr. C48 (1992) 410–412.
[50] A. Krajewski, M. Mazzocchi, P.L. Buldini, A. Ravaglioli, A. Tinti, P. Taddei, C. Fagnano,
Synthesis of carbonated hydroxyapatites: efficiency of the substitution and critical
evaluation of analytical methods, J. Mol. Struct. 744–747 (2005) 221–228.
[51] F. Monteil-Rivera, S. Masset, J. Dumonceau, M. Fedoroff, J. Jeanjean, Sorption of sel-
enite ions on hydroxyapatite, J. Mater. Sci. Mater. Med. 18 (1999) 1143–1145.
[52] F. Monteil-Rivera, M. Fedoroff, J. Jeanjean, L. Minel, M.-G. Barthes, J. Dumonceau,
Sorption of selenite SeO32− on hydroxyapatite: an exchange process, J. Colloid Inter-
face Sci. 221 (2000) 291–300.
[53] M. Duc, G. Lefevre, M. Fedoroff, J. Jeanjean, J.C. Rouchaud, F. Monteil-Rivera, J.
Dumonceau, S. Milonjic, Sorption of selenium anionic species on apatites and iron
oxides from aqueous solutions, J. Environ. Radioact. 70 (2003) 61–72.
[54] M. Marcovic, B.O. Fowler, M.S. Tung, Preparation and comprehensive characteriza-
tion of a calcium hydroxyapatite reference material, J. Res. Nat. Inst. Stand. Technol.
109 (2004) 553–568.
142 J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142

[55] A.B.S. Clasen, I.E. Ruyter, Quantitative determination of type A and B carbonate in
human deciduous and permanent enamel by means Fourier transform infrared
spectrometry, Adv. Dent. Res. 11 (1997) 523–527.
[56] S.D. Ross, Inorganic Infrared and Raman Spectra, McGrawHill, London, 1972.
[57] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Com-
pounds, 4th ed. John Wiley & Sons, New York, 1986.
[58] Ch. Su, D.L. Suarez, Selenate and selenite sorption on iron oxides: an infrared and
electrophoretic study, Soil Sci. Soc. Am. J. 64 (2000) 101–111.
[59] A. Awonusi, M.D. Morris, M.M.J. Tecklenburg, Carbonate assignment and calibration
in the Raman spectrum of apatite, Calcif. Tissue Int. 81 (2007) 46–52.