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

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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 b

Medical University of Warsaw, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, ul. Banacha 1, 02-097 Warsaw, Poland 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

Article history: Received 8 June 2013 Received in revised form 23 January 2014 Accepted 16 February 2014 Available online 22 February 2014 Keywords: Selenium Nanocrystalline hydroxyapatite Biomaterials FT-IR XRD TEM

a b s t r a c t 2− Selenium-substituted hydroxyapatites containing selenate SeO2− 4 or selenite SeO3 ions were synthesized using a wet precipitation method. The selenium content was determined by atomic absorbance spectrometry. The raw, unsintered powders were also characterized using powder X-ray diffraction, middle-range FT-IR spectroscopy and transmission electron microscopy with energy-dispersive X-ray spectroscopic microanalysis. The synthesized apatites were found to be pure and nanocrystalline with a crystal size similar to that in bone mineral. The incorporation of selenium oxyanions into the crystal lattice was confirmed. The toxicity of hydroxyapatites containing selenite or selenate ions was evaluated with a protozoan assay and bacterial luminescence test. © 2014 Elsevier B.V. All rights reserved.

1. Introduction 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 representative of apatites is stoichiometric calcium hydroxyapatite, described by the molecular formula Ca10(PO4)6(OH)2 [1–3]. An exceptional feature of apatites as biomaterials is their chemical resemblance to biological 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 nonstoichiometric. It is characterized as carbonated hydroxyapatite containing various substituted ions (i.e., Mg2 +, Na+, K+, Zn2 +, F−, Cl−, HPO24 −) that determine its physicochemical and biological properties [4]. The facility of various ionic substitutions and the resulting capability of apatites to be doped with various ions have recently attracted much attention. Those ions could enhance the biological, physicochemical 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: [email protected] (J. Kolmas).

http://dx.doi.org/10.1016/j.msec.2014.02.018 0928-4931/© 2014 Elsevier B.V. All rights reserved.

in diverse metabolic processes and that it is a constituent of selenoproteins, as well as of glutathione peroxidase, the enzyme that protects cellular membranes against harmful agents [15,16]. Numerous 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 selenium on the inflammatory response of osteoblasts in the metastasis of certain bone tumors has been recently evidenced [24]. Other studies indicated that inorganic selenium compounds (selenates 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 oxidation 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 hydroxyapatite in a powder form have been developed. They include wet precipitation [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 skeletons [31]. It should be emphasized that the most commonly used are

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

the wet synthetic processes, especially those carried out in aqueous solutions [32–35]. So far, there have been only a few literature reports on the application of selenium in biomaterials, taking advantage of anticancer and antimicrobial properties of this element. Tran et al. [36] examined a titanium surface (implant material) covered with a layer formed by selenium nanoclusters and demonstrated an inhibitory effect of selenium on the proliferation of cancer cells. Then, Tran et al. [37] showed that selenium 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 nanoparticles 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 decided 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), middlerange Fourier transformed infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM) equipped with an energy-dispersive X-ray analysis (EDS spectrometer) accessory and atomic absorption spectroscopy (AAS). The toxicity of selenite or selenate doped hydroxyapatites is evaluated with a bacterial luminescence test (Vibrio fisheri) and a protozoan 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 selenate ions, respectively. There are two locations in the HA crystal unit cell capable of accomor SeO2− ions: the channels running along the modating either SeO2− 3 4 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. ion has a very 238 pm in diameter [43]. On the other hand, the SeO2− 3 ion, but it has the quite similar diameter (239 pm) to that of the PO3− 4 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 Supplementary 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 introducing 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 ;

ð1Þ

135

where 0 b x b 2, x refers to the amount of selenium in the form of selenium oxyanions and n is 3 or 4, for the selenite or selenate ions, respectively. 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 hydroxyl anion. As a consequence, we can observe a simultaneous decalcification and dehydroxylation in the apatite sample. It can be described as follows: Ca10 ðPO4 Þ6 ðOHÞ2 þ x SeOn þ x PO4

3−

2þ

þ x Ca

2−

→Ca10−x ðPO4 Þ6−x ðSeOn Þx ðOHÞ2−x –

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 solution 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 mixture was kept at about 60 °C under stirring for 4 h. After that, the resultant 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 examined 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 dimensions 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 functions 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 presented in Fig. 2S in Supplementary materials. The error of the measurements 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 voltage of 300 kV and equipped with an energy dispersive X-ray spectrometer (EDS spectrometer). For TEM inspection a droplet of ultrasonically treated suspension of the powder in methanol was deposited onto a porous 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 atomizer (ET-AAS). Background correction was performed using the longitudinal Zeeman's effect, measuring ET-AAS signals in the peak height mode. In order to study the structure of the obtained materials, midinfrared 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 performed at room temperature and in ambient air under a laser excitation

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Table 1 Various parameters of the obtained samples. Samples

Crystal size along c axis (nm)a

Crystal size along a axis (nm)a

Ca/(P + Se) molar ratiob

Expected Se content wt. % (mol %)

Measured Se contentc wt. % (mol %)

Measured type d B CO2− 3 content (wt. %)

Calculated formula Ca10 − x − y(PO4)6 − x − y(SeOn)x(CO3)y(OH)2 − x − ye

HA HA-0.1SeO3 HA-0.4SeO3 HA-0.8SeO3 HA-1.2SeO3 HA-1.6SeO3 HA-0.1SeO4 HA-0.4SeO4 HA-0.8SeO4 HA-1.2SeO4 HA-1.6SeO4

25 20 15 16 15 16 25 22 20 20 25

7 8 6 7 5 5 9 7 6 8 10

1.63 1.65 1.59 1.62 1.64 1.68 1.62 1.58 1.64 1.62 1.65

0.0 (0.0) 0.79 (0.10) 3.24 (0.40) 6.42 (0.80) 9.70 (1.20) 13.10 (1.60) 0.79 (0.10) 3.22 (0.40) 6.34 (0.80) 9.52 (1.20) 12.80 (1.60)

0.0 0.78 (0.1) 2.96 (0.36) 6.11 (0.73) 9.65 (1.15) 9.85 (1.17) 0.72 (0.09) 2.89 (0.35) 5.83 (0.72) 9.36 (1.14) 9.76 (1.21)

1.4 2.1 2.4 2.5 2.3 2.4 0.9 2.4 1.2 1.9 0.9

Ca9.71(PO4)5.71(CO3)0.29(OH)1.71 Ca9.56(PO4)5.56(SeO3)0.10(CO3)0.34(OH)1.56 Ca9.26(PO4)5.26(SeO3)0.36(CO3)0.38(OH)1.26 Ca8.88(PO4)4.88(SeO3)0.73(CO3)0.39(OH)0.88 Ca8.49(PO4)4.49(SeO3)1.15(CO3)0.36(OH)0.49 Ca8.46(PO4)4.46(SeO3)1.17(CO3)0.37(OH)0.46 Ca9.76(PO4)5.76(SeO4)0.09(CO3)0.15(OH)1.76 Ca9.26(PO4)5.26(SeO4)0.35(CO3)0.39(OH)1.26 Ca9.08(PO4)5.08(SeO4)0.72(CO3)0.20(OH)1.08 Ca8.55(PO4)4.55(SeO4)1.14(CO3)0.31(OH)0.55 Ca8.64(PO4)4.64(SeO4)1.21(CO3)0.15(OH)0.64

a b c d e

From XRD diffractograms (Scherrer's formula). Calculated from EDS data. Measured by atomic absorption spectrometry AAS. From FTIR spectra (using method described by Clasen and Ruyter [55]). 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 Scientific). The spectra were recorded with spectral resolution of 1 cm−1 and 2000 scans.

responses were studied with the use of a dissection microscope (magnification 10×), deformation of the protozoan cell (i.e., bending, shortening) 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 suspensions were prepared: 1 mg/ml and 2 mg/ml. For the toxicity evaluation, we used the Microtox® test with the luminescent bacteria V. fisheri. The test was performed in special glass disposable 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 diluent 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 disposable, 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 diluent and a control, a Tyrode solution was used. After incubation, test

The synthesized materials were subjected to comprehensive analytical and structural analysis. Figs. 1 and 2 show PXRD patterns of pure HA, regarded as the reference 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, considering 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 seleniumdoped 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.

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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 increase 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 produced 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: D¼

0:94  λ ; β1=2  cos θ

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 affected by introducing a slightly larger, but tetrahedral selenate ion (249 pm), structurally resembling the phosphate ion. It is also important to note that the adsorption of selenite and selenate on the hydroxyapatite 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 phosphate ions. It has been proven that selenate and selenite were not exclusively located at the surface of the apatite, but diffused slightly in a thickness of a few nanometers [51]. It is also very interesting that selenate anions were much less sorbed than selenite [52]. Representative TEM images with electron diffraction patterns of se2− lected samples containing SeO2− 3 and SeO4 ions are presented in Fig. 4. Fig. 4 a and d, showing pure hydroxyapatite, has been attached as a

where: D λ β1/2 θ

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

The results of this estimation are presented in Table 1. The obtained and SeO2− ions are nanocrystalHA-based materials containing SeO2− 3 4 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 ions, the change of the c parameter was level. As in the case of SeO2− 3 very small. In this place, it seems reasonable to refer to the spatial structure of selenium oxyanions. Due to its completely different spatial structure (a flat trigonal pyramid), the selenite ion significantly affects the parameter a of the cell, despite its dimensions, which are very close to those of the phosphate ion (239 versus 238 nm). It is very interesting that a similar substitution of carbonates, which are isotypical with

Fig. 3. The unit cell parameters of the studied samples (a and c — lattice constants (nm); x — the amount of selenium in the form of selenium oxyanions; n — 3 or 4, for the selenite or selenate ions, respectively).

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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 affects the shape of the crystals. The crystals of pure HA are elongated platelet-shaped, whereas selenium-doped HA crystals are more platelike in shape. Analysis of TEM images confirms that the obtained materials are nanocrystalline; the crystals do not exceed 30 nm in length and 10 nm in width (see Table 1). The electron diffractogram patterns presented 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 Supplementary materials. They show the nanocrystalline nature of studied materials. In addition they prove that the apatitic structure of HAxSeO3 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 intensive 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 significantly reduced (soluble ammonium selenite/selenate would be removed during the rinsing process). The content of selenium obtained

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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 expected 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 undoubtedly 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 spectrum 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 vibrations 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 correspond to vibrations of the carbonate groups. Carbonates are present in water and in air and therefore can be easily incorporated in the hydroxyapatite crystals. Due to their small size (178 pm), carbonates not only substitute phosphate ions (type B) but are also situated in the columns of structural hydroxyl groups. It was possible to estimate carbonates 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.

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type B: 0.9–2.5 wt.% (see Table 1). Thus, for these samples the Formula (1) was modified: Ca10−x−y ðPO4 Þ6−x−y ðSeOn Þx ðCO3 Þy ðOHÞ2−x−y ;

ð2Þ

where x and y refer to the amount of selenium oxyanions and carbonates, respectively and n is 3 or 4, for the selenite or selenate ions, respectively (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 incorporation 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 hydroxyapatite. 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 vibrations) 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 vibrations 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 significantly 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 and selenite SeO2− ions. samples' spectra, doped with selenate SeO2− 4 3 In addition, it could be also recognized in the spectrum of pure hydroxyapatite. 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

band with respect to that coming from the free selenate ion, confirms the incorporation of the ion into the hydroxyapatite unit cell. As a confirmation of selenate substitution for phosphate, Raman spectra of HA, HA-1.2SeO3 and HA-1.2SeO4 samples were included in Supplementary materials (see Fig. 5S). Consider that the ν2 bands of carbonates are inactive 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 selenates, 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 luminescence of the bacteria in 1.0 mg/ml and 2.0 mg/ml solutions, respectively. 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 protozoan 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 synthesized 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 examinations are as follows: 1. The obtained materials are nanocrystalline hydroxyapatites; the materials 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 affect 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 observed 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 hydroxyapatite 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

Table 2 Toxicity of selected samples in Microtox® and Spirotox tests. Sample

HA-1.6SeO3 HA-1.6SeO4 a b

Microtox® (15 min-PE)a

Spirotoxb

1.0 mg/ml

2.0 mg/ml

1.0 mg/ml

2.0 mg/ml

6 0

12 0

NT (24 h) → def (48 h) NT

NT (24 h) → def (48 h) NT

Percent of toxic effect after 15 min of incubation. NT — not toxic; def — deformation of the cells.

J. Kolmas et al. / Materials Science and Engineering C 39 (2014) 134–142 2− hydroxyapatites doped with SeO2− 3 or SeO4 ions can find new applications 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 biocompatibility assays.

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