Materials Science and Engineering C 33 (2013) 440–445
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation and characterization of selenite substituted hydroxyapatite Jun Ma a, b, Yanhua Wang a, b, Lei Zhou a, b, Shengmin Zhang a, b, c,⁎ a b c
Advance Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074, PR China Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China China–Korea Center for Biomaterials and Nano-biotechnology, Life Science Building, 1037 Luoyu Road, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2012 Received in revised form 16 August 2012 Accepted 17 September 2012 Available online 23 September 2012 Keywords: Hydroxyapatite Apatite structure Selenium FTIR XRD (X-ray diffraction)
a b s t r a c t Selenite-substituted hydroxyapatite (Se-HA) with different Se/P ratios was synthesized by a co-precipitation method, using sodium selenite (Na2SeO3) as a Se source. Selenium has been incorporated into the hydroxyapatite lattice by partially replacing phosphate (PO43−) groups with selenite (SeO32−) groups. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) techniques reveal that substitutions of phosphate groups by selenite groups cause lower carbonate groups occupying at phosphate sites and change the lattice parameters of hydroxyapatite. The powders obtained are nano-crystalline hydroxyapatite when the Se/P ratios are not more than 0.1. The particle shape of Se-HA has not been altered compared with selenite-free hydroxyapatite but Se-incorporation reduces the crystallite size. The crystallinity was reduced as the Se/P ratios increased until amorphous phase (Se/P =0.3) appeared in the Se-HA powder obtained, and then another crystal phase presented as calcium selenite hydrate (Se/P=10). In addition, the sintering tests show that the Se-HA powders with the Se/P ratio of 0.1 have thermal stability at 900 °C for 2 h; hence they have great potential in the fabrication of bone repair scaffolds. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (HA) has been intensively studied as biomaterials for bone and tooth repairs. Ion-substituted, calcium deficient HA is well known as the main inorganic component of hard tissues in vertebrates [1–3]. Electron microscopy studies have shown that the mineral in bone and dentin consists of tiny and plate-like HA crystals with a size of 25–35 nm, whose c-axes are aligned with the collagen fibril axis [4,5]. HA, tricalcium phosphate (TCP) and biphasic calcium phosphate (BCP) appear to be excellent bioactive implantation materials with different rates of resorption in vivo. The synthesis of HA and these apatite-like materials has attracted great interest in recent decades. Nowadays, the composites of synthetic HA and apatite-like materials with biodegradable polymer are considered to be promising candidates as grafts and scaffolds for hard tissue engineering [6–8]. The crystal structure of natural HA is modified by ionic substitutions. For instance, CO32− substitutes for PO43− (B-type) and OH − (A-type), Na + for Ca 2+, Zn 2+ for Ca 2+, and F − for OH −. The influence of ionic substitutions on the physical and chemical properties of apatites has been investigated. The foreign ion incorporation affects the crystal growth, solubility and morphologies [9–11]. Furthermore, the biocompatibility of HA, such as mitochondrial activity, membrane integrity, cell density and adhesion, is also influenced by ⁎ Corresponding author at: Life Science Building, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, PR China. Tel.: +86 27 87792216; fax: +86 27 87792205. E-mail addresses: [email protected], [email protected] (S. Zhang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.011
the incorporation of ionic substitutions [12]. It is considered that the alterations in the physiochemical properties and biological responses correspond to the substitution mechanisms. The structural change and the related substitution mechanisms have been explored in the past few years [9,13,14]. Both experimental and theoretical studies have been conducted to analyze the substitutions in HA and other apatites [15]. Substitutions of the carbonate groups in HA are observed in human bone mineral and the carbonate content reaches 7.4 wt.% in natural bone. The carbonate substitutions are divided into A- and B-types, depending on whether they occupy the OH − or PO43− sites in HA lattice, respectively [16]. When substituted with Na + and SiO44− together, the charge compensation mechanisms become difficult to analyze [17]. In this condition, more than one charge compensation mechanism may exist at the same time. Selenium plays an important role in the protein functions and it has a significant effect on the induction of cancer cell apoptosis [18]. The incorporation of selenium into apatites seems like a hopeful idea for treating bone cancers to reduce the recurrence probability. Recently nano-crystalline selenite-substituted HA (Se-HA) has been studied for the inhibiting effect on the growth of osteosarcoma cells in our group [19]. Therefore, the role of selenite substitutions in the HA lattice is important with respect to the physicochemical properties and bioactivities. However, the structure of the synthetic Se-HA nanoparticles and the exact mechanisms of charge compensation are not well known, especially when the selenium content is high. The aim of this study is the preparation and characterization of several Se-HA powders with different Se/P ratios. The structure modification and charge compensation mechanisms for the selenium incorporation
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into HA were investigated according to the spectroscopy studies and phase analysis. 2. Materials and methods The HA and Se-HA powders were prepared by an aqueous co-precipitation method [20]. Briefly, the precipitation was carried out in a three-neck bottle at open atmosphere and in an oil-bath of 80 °C. The precipitates were obtained by dropwise addition of 200 ml solution of (NH4)2HPO4 and Na2SeO3 into 200 ml solution of Ca(NO3)2 (50 mM). Carbon dioxide from the air was involved in the reaction. The pH value was kept at 10 by adding 25% ammonium solution during synthesis. All of the chemical reagents used were purchased in chemical purity grade. HA and six grades of Se-HA powders were synthesized by maintaining Ca/(P + Se) ratios fixed at 1.67, as represented in Table 1. Stirring was continued for 16 h, then the precipitates were collected by centrifugation and washed three times using distilled-water. All samples were dried at 60 °C overnight and stored in vacuum for further studies. X-ray diffraction (XRD) analysis was performed using an automated diffractometer (X'Pert PRO, PANalytical, B.V., Netherlands), at a step size of 0.02°, scanning rate of 1.2° in 2θ/min, and a 2θ range from 10 to 70°. The value of full width at half-maximum (FWHM) of the peak of (002) plane, representative of the crystallite along the c-axis, was used in the calculation according to Scherrer's formula. D ¼ Kλ=β cos Θ Where D is the crystallite size; K is the shape factor (K = 0.9); λ is the wavelength (λ = 0.154 nm); Θ is the diffraction angle and β is the line width (FWHM) in radians. The particle morphology was investigated using transmission electron microscopy (TEM, Tecnai G2 20, FEI, Holland). The powders were dispersed in ethanol and a drop of this suspension was applied on a copper grid coated with carbon film. The copper grid was dried in air and then readied for TEM observation. Fourier-transform infrared spectroscopy (FTIR) was carried out using a spectrometer (Vertex 70, Bruker, German). The powder samples were mixed with KBr and compressed into pellets for measurements. The wavenumber range of 4000–400 cm −1 was recorded. The Se/P ratios in the dried powders were measured by X-ray fluorescence (XRF) spectroscopy. In order to study the sintering properties, the synthetic powders were sintered at 900 °C and 1100 °C for 2 h, respectively. The thermal stability and phase transition behaviors were investigated by XRD. 3. Results 3.1. XRD analysis The XRD patterns of HA and Se-HA powders, as shown in Fig. 1, reveal no secondary phases other than HA (JCPDS no. 09‐0432) when the Se/P ratios are under 10. However, the reflections are broadened and only the peaks of (211) and (002) planes are clearly observed. The broadening and loss of reflection peaks in the XRD patterns
Fig 1. X-ray diffraction patterns of different hydroxyapatite powders dried at 60 °C overnight.
indicate the decrease of crystallinity with increasing Se/P ratios, which may be attributed to the substitution of PO43− by SeO44−. However, the broadening of the peaks could result from an effective decrease in the crystallite size. The crystallite size as determined by Scherrer's formula along the c axis (in Table 2) decreases with the increasing Se/P ratios. Cell refinements suggest no obvious trends in the a-axis, but a decrease in the c-axis when the Se/P ratios increase. The volumes of the HA unit cell also decrease when the Se/P ratios increase. The crystallite size and changes in lattice parameters of the Se-HA samples compared with HA are considered to be caused by the selenite substitutions. According to the XRD patterns, Se-HA samples with high selenium content (Se/P = 10 and 100) show many peaks other than calcium phosphate due to the low concentrations of phosphate ions during synthesis. The crystals newly formed are assigned to calcium selenite hydrate (JCPDS no. 35‐0883). In this paper, we focused on the formation of selenite substituted HA, so the following characterizations were mainly conducted using the powder samples containing HA phase.
3.2. TEM analysis TEM micrographs of the different HA particles are given in Fig. 2. The morphological features of HA, Se-HA-0.03, and Se-HA-0.1 have not shown obvious differences between selenite-substituted and unsubstituted HA. All particles are needle-like bundles, which are composed of fine crystallites. The diffraction patterns shown in Fig. 2(a) and (b) suggest that the particles are polycrystalline hydroxyapatite. In Fig. 2(d), the amorphous phase has been observed in the sample of Se-HA-0.3. Although the XRD patterns are almost the same, there is still difference in the crystallinity and phase composition according to the TEM results. From the above XRD and TEM results, the Se-HA-0.1 sample provides the pure nano-crystalline HA with the highest Se/P ratios in all samples obtained.
Table 1 Quantities of reactants and expected molar ratios of selenite-substituted hydroxyapatite. Sample
Ca(NO3)2 (mmol)
(NH4)2HPO4 (mmol)
Na2SeO3 (mmol)
Predicted Se/P
HA HA-Se-0.03 HA-Se-0.1 HA-Se-0.3 HA-Se-1.0 HA-Se-10 HA-Se-100
10.0 10.0 10.0 10.0 10.0 10.0 10.0
6.00 5.83 5.50 4.60 3.00 0.55 0.06
0 0.17 0.55 1.38 3.00 5.50 6.00
0 0.03 0.10 0.30 1.0 10 100
Table 2 Crystallite size, lattice parameters and unit cell volume of different hydroxyapatite powders. Samples
D(002) (nm)
a (Å)
c (Å)
Ω (Å3)
HA HA-Se-0.03 HA-Se-0.1 HA-Se-0.3 HA-Se-1.0
21.1 20.1 17.0 15.7 15.5
9.4089 9.4081 9.4124 9.3895 9.3968
6.8748 6.8689 6.8611 6.8509 6.8497
527.07 526.53 526.41 523.08 523.79
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Fig. 2. Transmission electron micrographs of (a) HA, (b) Se-HA with Se/P = 0.03, (c) Se-HA with Se/P = 0.1 and (d) Se-HA with Se/P = 0.3. The insets in (a) and (b) are diffraction patterns of the crystals.
3.3. FTIR spectra The FTIR spectra of different HA powders contain some systematic changes in phosphate, hydroxyl, carbonate, and selenite bands with increasing Se/P ratios. Fig. 3 represents the FTIR spectra of different HA powders dried at 60 °C overnight. Because all the spectra have an identical intensity scale, the band areas can be compared directly. The spectra of all samples have a broad band at 3800–3000 cm −1, due to the adsorbed H2O. The bending vibration band of molecular H2O appears at 1630 cm −1. It is well known that as hydrogen bonding strength increases, the stretching vibration frequency of the OH group decreases. The hydroxyl stretching and bending bands at 3570 and 630 cm −1 have not been seen for all samples. The strong bands in the range 900–1200 cm−1 correspond to P\O stretching vibration modes of the phosphate groups. The infrared symmetric and asymmetric P\O stretching bands decrease as the selenium contents increase. Similar changes are also observed in the doublet at 604 and 567 cm−1 which correspond to the O\P\O anti-symmetric bending mode. However, the position of both stretching and bending bands is obviously unaffected by the selenite substitutions. The group of bands around 1500 cm −1 belongs to CO32−. The carbonate bands appear in all samples at 1456 and 1413 cm −1 which correspond to the carbonate groups occupying at the phosphate
sites in HA (B-type). Fig. 3 shows that the intensities of the carbonate bands at 1456 and 1413 cm −1 decrease as the Se/P ratios increase. The FTIR spectra also display a band at 1564 cm −1, assignable to the A-type carbonate substitutions. For Se-HA samples, the intensity of this band is larger than that belonging to B-type substitution. The most intense spectral features of selenite groups appear in the range of 900–800 cm −1, which are attributed to asymmetrical stretching vibrations of SeO4 tetrahedron. The band at 864 cm −1 was overlapped with the carbonate group and its intensities decrease as the Se/P ratios increase. The band at 766 cm −1 caused by O\Se\O bending vibration (v3) becomes stronger when the Se/P ratios increase. The band at 1384 cm −1 appears for all Se-HA samples which correspond to the interaction between carbonate groups and selenite groups.
3.4. XRF results As shown in Fig. 4, the Se/P ratios in the dried powders measured by XRF increased with the Se/P ratios in the reactants. When the Se/P ratio was 0.3 in the reactants, the incorporation of Se into HA became much less than expected. It was accordant to the TEM results that the amorphous phase appeared when Se/P ratio was 0.3.
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Fig. 3. Fourier-transform infrared spectra of different hydroxyapatite powders dried at 60 °C overnight. Fig. 5. X-ray diffraction patterns of hydroxyapatite powders sintered at 900 °C for 2 h.
3.5. Sintering properties
4. Discussion
The thermal stabilities of different HA powders were examined at 900 and 1100 °C. Fig. 5 shows the XRD patterns of samples sintered at 900 °C for 2 h. After sintering at this temperature, the crystallinity of all samples increases significantly. When Se/P is 0.3, a sharp peak at 37.5° (indicated by asterisk) is observed which suggests the impurity phase formation. This new phase may be assigned to calcium phosphate (JCPDS no. 49‐0496). The crystal phase newly formed may be transformed from the amorphous phase observed by TEM as shown in Fig. 2(d). Fig. 6 shows the XRD patterns of samples sintered at 1100 °C. The Se-HA samples become more unstable at this temperature. There are more impurity phases in the samples after sintering. For Se-HA-0.03, the phase of tricalcium phosphate (TCP) has been found (indicated by asterisk). For Se-HA-0.1 and Se-HA-0.3, the peak at 37.5° suggests the formation of calcium phosphate phase. According to the above results, the synthetic HA with selenite substitution is stable at 900 °C when Se/P = 0.03 and 0.1. However, after sintering at 900 °C, the crystallinity of Se-HA-0.1 is lower than that of Se-HA-0.03, which is related to the amount of selenite substitutions.
Tissue engineering techniques have provided great hopes for the regeneration of deficient tissues such as bones; nano-crystalline HA and apatite-like minerals are considered to be the most promising biological mimetic materials for bone repair [6,21]. The incorporation of trace elements into HA has brought new biological properties to the synthetic HA materials. Silicate substituted HA is found to improve the biocompatibility of the HA grafts and accelerate the bone healing process [22,23]. Carbonate substituted HA is also a good choice for bone tissue engineering [24]. Besides fabrications into bone grafts and scaffolds, the HA nanoparticles also have good performance in the applications of drug and gene delivery [3,25]. The controlled delivery of proteins has also been modified by the incorporation of divalent ions [26]. It is well-known that selenium plays a very important role in the human body and the up-take of selenium will induce the apoptosis in many cancer cells. The incorporation of selenium into HA may help us to treat bone cancers by lowering the chance of tumor recurrence. In this paper, the nano-crystalline HA powders with selenite substitutions were prepared at open atmosphere. FTIR results show
Fig. 4. The Se/P ratios measured by XRF versus the Se/P ratios in the reactants.
Fig. 6. X-ray diffraction patterns of hydroxyapatite powders sintered at 1100 °C for 2 h.
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that the Se-HA samples prepared have selenite and carbonate co-substitutions. Sodium ions may also be involved into the final products due to the usage of sodium selenite. The characterization by XRD, TEM, and FTIR techniques in this work has revealed the structural evolutions caused by the selenite substitutions. The hydroxyl groups have not been seen in the FTIR spectra. Meanwhile, the carbonate ions were found in the HA powder obtained without addition of sodium selenite. The present FTIR studies provide detailed information about the type of carbonate substitutions and their alteration after the addition of selenite substitutions. The number of the observed FTIR bands and their intensities show mixed A- and B-type carbonate substitutions. The intensities of the bands belonging to B-type carbonate substitutions decrease after the selenite groups are involved in the HA lattice. The incorporation of selenite groups has decreased the possibility of carbonate ions occupying the phosphate sites. According to the FTIR results, the most probable process for charge compensation is the replacement of two hydroxyl groups by one carbonate group [27]. At the same time, some phosphate sites are also occupied by the carbonate groups. The charge compensation mechanism can be proposed as the replacement of one phosphate group and one hydroxyl group by two carbonate groups [27]. Previous studies have clearly demonstrated these substitution mechanisms [16,27,28]. Due to the size limitation, the selenite ions occupy the phosphate sites. The selenite groups can replace phosphate groups inside the HA lattice with the ratio of 1:1 by ion exchange process [29]. Because the selenite groups have the same charges to the carbonate groups, the charge compensations for the carbonate substitutions and selenite substitutions may have no difference. The charge compensations of B-type carbonate hydroxyapatite might be the same as the selenite substitutions. Based on this assumption, the substitution mechanisms for the low Se/P ratios are proposed as the following. 3−
−
2−
2−
2−
2−
PO4 þ OH → CO3 ðAÞ þ CO3 ðBÞ→ SeO3 þ CO3 ðAÞ
ð1Þ
For HA with carbon dioxide involved, the carbonate ions occupy the sites of hydroxyl ions (A-type) and phosphate ions (B-type). When selenite ions are added, the carbonate ions have fewer chances to occupy the phosphate sites. At this time, the carbonate ions prefer hydroxyl column (A-type). In this condition, lower intensity of B-type FTIR spectra has been observed for the Se-HA samples. When the Se/P ratios are high, other charge mechanisms should be considered. In the condition of one HA unit cell having more than two Se atoms, an oxygen void (VO) is generated nearby the Se2O76− structure. This charge compensation mechanism is very similar to that of silicate substituted HA [20,23]. 3−
6−
2PO4 → Vo þ Se2 O7
ð2Þ
When Se/P ratio is above 1.0, apatite structure may not be formed and calcium selenite hydrate begins to precipitate in the reaction mixture. From the TEM results, it is obvious that the amorphous phase has been obtained when the Se/P ratio is only 0.3. After sintering at 900 °C for 2 h, other calcium phosphate phases appeared in the products because of the phase transformation. However, the main phase in the products with the Se/P ratio less than 0.3 after sintering is HA. According to the XRD results, the increasing Se/P ratios have led to the shrinkage of HA lattice. The changes in the lattice parameters are mainly caused by the replacement of phosphate ions by selenite ions. However, the radius of Se 4+ (0.50 Å) is much larger than that of P 5+ (0.35 Å); and the length of Se\O bond (0.164 nm) is greater than that of the P\O bond (0.155 nm). The size of the PO4 tetrahedron would be expected to be smaller than that of the SeO4 tetrahedron. The occupation of selenium in phosphorus sites should enlarge the
lattice size which is not accordant to the XRD analysis. It is noted that charge compensations are needed for the selenite substitution. The oxygen void may exist close to the selenite groups or one oxygen atom in the SeO4 tetrahedron is shared with the close phosphate and carbonate groups. The presence of more oxygen voids in HA lattice leads to the smaller unit cell. Another possible reason for the shrinkage of unit cell is the oxidization of selenite group. When the selenite groups (Se 4+) are oxidized to selenate groups (Se 6+), the shortened Se\O bands result in the decrease of HA lattice parameters. The charge compensations associated with the carbonate and hydroxyl groups have obvious effects on the lattice change of HA [30]. Due to the multiple substitutions, the trends of the lattice size with the incorporation of selenium into HA are complicated. In this manuscript, the above two mechanisms of charge compensation have not been approved by the direct experimental data. Further experiments about the detailed structure of Se-HA are needed to analyze the charge compensation mechanisms. Considering the structure stability and phase purity, the synthetic reactants with Se/P = 0.1 may produce the nano-crystalline Se-HA powders containing high Se content. They have stable HA structure after sintering at 900 °C for 2 h and only small amount of TCP phases appear after sintering at 1100 °C for 2 h. Because HA and TCP are both successful bone grafting materials in clinic, Se-HA-0.1 is the most promising candidate for the fabrication of bioactive ceramics to treat bone and osteosarcoma cancers among the samples prepared in this study. 5. Conclusion The facile precipitation technique using sodium selenite as a source of selenite ions has been successfully applied for the preparation of Se-HA. This low cost and facile method allows for the incorporation of carbonate and selenite ions into HA lattice. With the Se/P ratios no more than 0.1, the powders without sintering are nano-crystalline HA. Increasing substitutions of selenite ions for PO43− are accompanied by a decrease of CO32− substitutions. The powder crystallinity is strongly reduced as the extent of substitution increases, and when the Se/P ratios reach 10, the apatite structure changes to the phase of calcium selenite hydrate. The selenite substitutions in PO43− positions import obvious structural changes. The powder of Se-HA-0.1 having the sintering stability at 900 °C for 2 h is considered to be a promising candidate for the fabrication of bone tissue engineering scaffolds. Acknowledgments This work was supported by National Basic Research Program of China (grant no.: 2012CB933601), National Natural Science Foundation of China (grant nos.: 81071263, 30870624), International S&T Cooperation Program of China (grant no.: 0102011DFA31430), National Key Technology Research and Development Program of China (grant no.: 2012BAI17B02), and National High Technology Research and Development Program of China (grant no.: 2011 AA03105). References [1] D. Tadic, M. Epple, Biomaterials 25 (2004) 987–994. [2] N. Huebsch, D.J. Mooney, Nature 462 (2009) 426–432. [3] V. Uskokovic, D.P. Uskokovic, J. Biomed. Mater. Res. B Appl. Biomater. 96 (2011) 152–191. [4] W. Zhang, S.S. Liao, F.Z. Cui, Chem. Mater. 15 (2003) 3221–3226. [5] F.-Z. Cui, Y. Li, J. Ge, Mater. Sci. Eng. R Rep. 57 (2007) 1–27. [6] A. Tampieri, G. Celotti, E. Landi, Anal. Bioanal. Chem. 381 (2005) 568–576. [7] K. Zhao, Y.-F. Tang, Y.-S. Qin, J.-Q. Wei, Ceram. Int. 37 (2011) 635–639. [8] L.C. Wu, J. Yang, J. Kopecek, Biomaterials 32 (2011) 5341–5353. [9] A.M. Pietak, J.W. Reid, M.J. Stott, M. Sayer, Biomaterials 28 (2007) 4023–4032. [10] D.M. Ibrahim, A.A. Mostafa, S.I. Korowash, Chem. Cent. J. 5 (2011) 74. [11] L.T. Bang, K. Ishikawa, R. Othman, Ceram. Int. 37 (2011) 3637–3642. [12] T.J. Webster, E.A. Massa-Schlueter, J.L. Smith, E.B. Slamovich, Biomaterials 25 (2004) 2111–2121.
J. Ma et al. / Materials Science and Engineering C 33 (2013) 440–445 [13] Y. Tang, H.F. Chappell, M.T. Dove, R.J. Reeder, Y.J. Lee, Biomaterials 30 (2009) 2864–2872. [14] D. Laurencin, N. Almora-Barrios, N.H. de Leeuw, C. Gervais, C. Bonhomme, F. Mauri, W. Chrzanowski, J.C. Knowles, R.J. Newport, A. Wong, Z.H. Gan, M.E. Smith, Biomaterials 32 (2011) 1826–1837. [15] D.E. Ellis, J. Terra, O. Warschkow, M. Jiang, G.B. Gonzalez, J.S. Okasinski, M.J. Bedzyk, A.M. Rossi, J.G. Eon, Phys. Chem. Chem. Phys. 8 (2006) 967–976. [16] M.E. Fleet, X. Liu, Biomaterials 28 (2007) 916–926. [17] I.R. Gibson, W. Bonfield, J. Mater. Sci. Mater. Med. 13 (2002) 685–693. [18] J. Brozmanova, L. Letavayova, V. Vlckova, Toxicology 227 (2006) 1–14. [19] Y. Wang, J. Ma, L. Zhou, J. Chen, Y. Liu, Z. Qiu, S. Zhang, Interface Focus 2 (2012) 378–386. [20] N.Y. Mostafa, H.M. Hassan, O.H. Abd Elkader, J. Am. Ceram. Soc. 94 (2011) 1584–1590. [21] A. Tripathi, S. Saravanan, S. Pattnaik, A. Moorthi, N.C. Partridge, N. Selvamurugan, Int. J. Biol. Macromol. 50 (2012) 294–299.
445
[22] K.A. Hing, P.A. Revell, N. Smith, T. Buckland, Biomaterials 27 (2006) 5014–5026. [23] P. Gillespie, G. Wu, M. Sayer, M.J. Stott, J. Mater. Sci. Mater. Med. 21 (2010) 99–108. [24] E. Sachlos, D. Gotora, J.T. Czernuszka, Tissue Eng. 12 (2006) 2479–2487. [25] M. Motskin, D.M. Wright, K. Muller, N. Kyle, T.G. Gard, A.E. Porter, J.N. Skepper, Biomaterials 30 (2009) 3307–3317. [26] S. Dasgupta, S.S. Banerjee, A. Bandyopadhyay, S. Bose, Langmuir 26 (2010) 4958–4964. [27] S. Peroos, Z. Du, N.H. de Leeuw, Biomaterials 27 (2006) 2150–2161. [28] J. Barralet, S. Best, W. Bonfield, J. Biomed. Mater. Res. 41 (1998) 79–86. [29] F. Monteil-Rivera, M. Fedoroff, J. Jeanjean, L. Minel, M.G. Barthes, J. Dumonceau, J. Colloid Interface Sci. 221 (2000) 291–300. [30] S. Yin, D.E. Ellis, Phys. Chem. Chem. Phys. 12 (2010) 156–163.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation and characterization of selenite substituted hydroxyapatite Jun Ma a, b, Yanhua Wang a, b, Lei Zhou a, b, Shengmin Zhang a, b, c,⁎ a b c
Advance Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074, PR China Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China China–Korea Center for Biomaterials and Nano-biotechnology, Life Science Building, 1037 Luoyu Road, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2012 Received in revised form 16 August 2012 Accepted 17 September 2012 Available online 23 September 2012 Keywords: Hydroxyapatite Apatite structure Selenium FTIR XRD (X-ray diffraction)
a b s t r a c t Selenite-substituted hydroxyapatite (Se-HA) with different Se/P ratios was synthesized by a co-precipitation method, using sodium selenite (Na2SeO3) as a Se source. Selenium has been incorporated into the hydroxyapatite lattice by partially replacing phosphate (PO43−) groups with selenite (SeO32−) groups. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) techniques reveal that substitutions of phosphate groups by selenite groups cause lower carbonate groups occupying at phosphate sites and change the lattice parameters of hydroxyapatite. The powders obtained are nano-crystalline hydroxyapatite when the Se/P ratios are not more than 0.1. The particle shape of Se-HA has not been altered compared with selenite-free hydroxyapatite but Se-incorporation reduces the crystallite size. The crystallinity was reduced as the Se/P ratios increased until amorphous phase (Se/P =0.3) appeared in the Se-HA powder obtained, and then another crystal phase presented as calcium selenite hydrate (Se/P=10). In addition, the sintering tests show that the Se-HA powders with the Se/P ratio of 0.1 have thermal stability at 900 °C for 2 h; hence they have great potential in the fabrication of bone repair scaffolds. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (HA) has been intensively studied as biomaterials for bone and tooth repairs. Ion-substituted, calcium deficient HA is well known as the main inorganic component of hard tissues in vertebrates [1–3]. Electron microscopy studies have shown that the mineral in bone and dentin consists of tiny and plate-like HA crystals with a size of 25–35 nm, whose c-axes are aligned with the collagen fibril axis [4,5]. HA, tricalcium phosphate (TCP) and biphasic calcium phosphate (BCP) appear to be excellent bioactive implantation materials with different rates of resorption in vivo. The synthesis of HA and these apatite-like materials has attracted great interest in recent decades. Nowadays, the composites of synthetic HA and apatite-like materials with biodegradable polymer are considered to be promising candidates as grafts and scaffolds for hard tissue engineering [6–8]. The crystal structure of natural HA is modified by ionic substitutions. For instance, CO32− substitutes for PO43− (B-type) and OH − (A-type), Na + for Ca 2+, Zn 2+ for Ca 2+, and F − for OH −. The influence of ionic substitutions on the physical and chemical properties of apatites has been investigated. The foreign ion incorporation affects the crystal growth, solubility and morphologies [9–11]. Furthermore, the biocompatibility of HA, such as mitochondrial activity, membrane integrity, cell density and adhesion, is also influenced by ⁎ Corresponding author at: Life Science Building, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, PR China. Tel.: +86 27 87792216; fax: +86 27 87792205. E-mail addresses: [email protected], [email protected] (S. Zhang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.011
the incorporation of ionic substitutions [12]. It is considered that the alterations in the physiochemical properties and biological responses correspond to the substitution mechanisms. The structural change and the related substitution mechanisms have been explored in the past few years [9,13,14]. Both experimental and theoretical studies have been conducted to analyze the substitutions in HA and other apatites [15]. Substitutions of the carbonate groups in HA are observed in human bone mineral and the carbonate content reaches 7.4 wt.% in natural bone. The carbonate substitutions are divided into A- and B-types, depending on whether they occupy the OH − or PO43− sites in HA lattice, respectively [16]. When substituted with Na + and SiO44− together, the charge compensation mechanisms become difficult to analyze [17]. In this condition, more than one charge compensation mechanism may exist at the same time. Selenium plays an important role in the protein functions and it has a significant effect on the induction of cancer cell apoptosis [18]. The incorporation of selenium into apatites seems like a hopeful idea for treating bone cancers to reduce the recurrence probability. Recently nano-crystalline selenite-substituted HA (Se-HA) has been studied for the inhibiting effect on the growth of osteosarcoma cells in our group [19]. Therefore, the role of selenite substitutions in the HA lattice is important with respect to the physicochemical properties and bioactivities. However, the structure of the synthetic Se-HA nanoparticles and the exact mechanisms of charge compensation are not well known, especially when the selenium content is high. The aim of this study is the preparation and characterization of several Se-HA powders with different Se/P ratios. The structure modification and charge compensation mechanisms for the selenium incorporation
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into HA were investigated according to the spectroscopy studies and phase analysis. 2. Materials and methods The HA and Se-HA powders were prepared by an aqueous co-precipitation method [20]. Briefly, the precipitation was carried out in a three-neck bottle at open atmosphere and in an oil-bath of 80 °C. The precipitates were obtained by dropwise addition of 200 ml solution of (NH4)2HPO4 and Na2SeO3 into 200 ml solution of Ca(NO3)2 (50 mM). Carbon dioxide from the air was involved in the reaction. The pH value was kept at 10 by adding 25% ammonium solution during synthesis. All of the chemical reagents used were purchased in chemical purity grade. HA and six grades of Se-HA powders were synthesized by maintaining Ca/(P + Se) ratios fixed at 1.67, as represented in Table 1. Stirring was continued for 16 h, then the precipitates were collected by centrifugation and washed three times using distilled-water. All samples were dried at 60 °C overnight and stored in vacuum for further studies. X-ray diffraction (XRD) analysis was performed using an automated diffractometer (X'Pert PRO, PANalytical, B.V., Netherlands), at a step size of 0.02°, scanning rate of 1.2° in 2θ/min, and a 2θ range from 10 to 70°. The value of full width at half-maximum (FWHM) of the peak of (002) plane, representative of the crystallite along the c-axis, was used in the calculation according to Scherrer's formula. D ¼ Kλ=β cos Θ Where D is the crystallite size; K is the shape factor (K = 0.9); λ is the wavelength (λ = 0.154 nm); Θ is the diffraction angle and β is the line width (FWHM) in radians. The particle morphology was investigated using transmission electron microscopy (TEM, Tecnai G2 20, FEI, Holland). The powders were dispersed in ethanol and a drop of this suspension was applied on a copper grid coated with carbon film. The copper grid was dried in air and then readied for TEM observation. Fourier-transform infrared spectroscopy (FTIR) was carried out using a spectrometer (Vertex 70, Bruker, German). The powder samples were mixed with KBr and compressed into pellets for measurements. The wavenumber range of 4000–400 cm −1 was recorded. The Se/P ratios in the dried powders were measured by X-ray fluorescence (XRF) spectroscopy. In order to study the sintering properties, the synthetic powders were sintered at 900 °C and 1100 °C for 2 h, respectively. The thermal stability and phase transition behaviors were investigated by XRD. 3. Results 3.1. XRD analysis The XRD patterns of HA and Se-HA powders, as shown in Fig. 1, reveal no secondary phases other than HA (JCPDS no. 09‐0432) when the Se/P ratios are under 10. However, the reflections are broadened and only the peaks of (211) and (002) planes are clearly observed. The broadening and loss of reflection peaks in the XRD patterns
Fig 1. X-ray diffraction patterns of different hydroxyapatite powders dried at 60 °C overnight.
indicate the decrease of crystallinity with increasing Se/P ratios, which may be attributed to the substitution of PO43− by SeO44−. However, the broadening of the peaks could result from an effective decrease in the crystallite size. The crystallite size as determined by Scherrer's formula along the c axis (in Table 2) decreases with the increasing Se/P ratios. Cell refinements suggest no obvious trends in the a-axis, but a decrease in the c-axis when the Se/P ratios increase. The volumes of the HA unit cell also decrease when the Se/P ratios increase. The crystallite size and changes in lattice parameters of the Se-HA samples compared with HA are considered to be caused by the selenite substitutions. According to the XRD patterns, Se-HA samples with high selenium content (Se/P = 10 and 100) show many peaks other than calcium phosphate due to the low concentrations of phosphate ions during synthesis. The crystals newly formed are assigned to calcium selenite hydrate (JCPDS no. 35‐0883). In this paper, we focused on the formation of selenite substituted HA, so the following characterizations were mainly conducted using the powder samples containing HA phase.
3.2. TEM analysis TEM micrographs of the different HA particles are given in Fig. 2. The morphological features of HA, Se-HA-0.03, and Se-HA-0.1 have not shown obvious differences between selenite-substituted and unsubstituted HA. All particles are needle-like bundles, which are composed of fine crystallites. The diffraction patterns shown in Fig. 2(a) and (b) suggest that the particles are polycrystalline hydroxyapatite. In Fig. 2(d), the amorphous phase has been observed in the sample of Se-HA-0.3. Although the XRD patterns are almost the same, there is still difference in the crystallinity and phase composition according to the TEM results. From the above XRD and TEM results, the Se-HA-0.1 sample provides the pure nano-crystalline HA with the highest Se/P ratios in all samples obtained.
Table 1 Quantities of reactants and expected molar ratios of selenite-substituted hydroxyapatite. Sample
Ca(NO3)2 (mmol)
(NH4)2HPO4 (mmol)
Na2SeO3 (mmol)
Predicted Se/P
HA HA-Se-0.03 HA-Se-0.1 HA-Se-0.3 HA-Se-1.0 HA-Se-10 HA-Se-100
10.0 10.0 10.0 10.0 10.0 10.0 10.0
6.00 5.83 5.50 4.60 3.00 0.55 0.06
0 0.17 0.55 1.38 3.00 5.50 6.00
0 0.03 0.10 0.30 1.0 10 100
Table 2 Crystallite size, lattice parameters and unit cell volume of different hydroxyapatite powders. Samples
D(002) (nm)
a (Å)
c (Å)
Ω (Å3)
HA HA-Se-0.03 HA-Se-0.1 HA-Se-0.3 HA-Se-1.0
21.1 20.1 17.0 15.7 15.5
9.4089 9.4081 9.4124 9.3895 9.3968
6.8748 6.8689 6.8611 6.8509 6.8497
527.07 526.53 526.41 523.08 523.79
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Fig. 2. Transmission electron micrographs of (a) HA, (b) Se-HA with Se/P = 0.03, (c) Se-HA with Se/P = 0.1 and (d) Se-HA with Se/P = 0.3. The insets in (a) and (b) are diffraction patterns of the crystals.
3.3. FTIR spectra The FTIR spectra of different HA powders contain some systematic changes in phosphate, hydroxyl, carbonate, and selenite bands with increasing Se/P ratios. Fig. 3 represents the FTIR spectra of different HA powders dried at 60 °C overnight. Because all the spectra have an identical intensity scale, the band areas can be compared directly. The spectra of all samples have a broad band at 3800–3000 cm −1, due to the adsorbed H2O. The bending vibration band of molecular H2O appears at 1630 cm −1. It is well known that as hydrogen bonding strength increases, the stretching vibration frequency of the OH group decreases. The hydroxyl stretching and bending bands at 3570 and 630 cm −1 have not been seen for all samples. The strong bands in the range 900–1200 cm−1 correspond to P\O stretching vibration modes of the phosphate groups. The infrared symmetric and asymmetric P\O stretching bands decrease as the selenium contents increase. Similar changes are also observed in the doublet at 604 and 567 cm−1 which correspond to the O\P\O anti-symmetric bending mode. However, the position of both stretching and bending bands is obviously unaffected by the selenite substitutions. The group of bands around 1500 cm −1 belongs to CO32−. The carbonate bands appear in all samples at 1456 and 1413 cm −1 which correspond to the carbonate groups occupying at the phosphate
sites in HA (B-type). Fig. 3 shows that the intensities of the carbonate bands at 1456 and 1413 cm −1 decrease as the Se/P ratios increase. The FTIR spectra also display a band at 1564 cm −1, assignable to the A-type carbonate substitutions. For Se-HA samples, the intensity of this band is larger than that belonging to B-type substitution. The most intense spectral features of selenite groups appear in the range of 900–800 cm −1, which are attributed to asymmetrical stretching vibrations of SeO4 tetrahedron. The band at 864 cm −1 was overlapped with the carbonate group and its intensities decrease as the Se/P ratios increase. The band at 766 cm −1 caused by O\Se\O bending vibration (v3) becomes stronger when the Se/P ratios increase. The band at 1384 cm −1 appears for all Se-HA samples which correspond to the interaction between carbonate groups and selenite groups.
3.4. XRF results As shown in Fig. 4, the Se/P ratios in the dried powders measured by XRF increased with the Se/P ratios in the reactants. When the Se/P ratio was 0.3 in the reactants, the incorporation of Se into HA became much less than expected. It was accordant to the TEM results that the amorphous phase appeared when Se/P ratio was 0.3.
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Fig. 3. Fourier-transform infrared spectra of different hydroxyapatite powders dried at 60 °C overnight. Fig. 5. X-ray diffraction patterns of hydroxyapatite powders sintered at 900 °C for 2 h.
3.5. Sintering properties
4. Discussion
The thermal stabilities of different HA powders were examined at 900 and 1100 °C. Fig. 5 shows the XRD patterns of samples sintered at 900 °C for 2 h. After sintering at this temperature, the crystallinity of all samples increases significantly. When Se/P is 0.3, a sharp peak at 37.5° (indicated by asterisk) is observed which suggests the impurity phase formation. This new phase may be assigned to calcium phosphate (JCPDS no. 49‐0496). The crystal phase newly formed may be transformed from the amorphous phase observed by TEM as shown in Fig. 2(d). Fig. 6 shows the XRD patterns of samples sintered at 1100 °C. The Se-HA samples become more unstable at this temperature. There are more impurity phases in the samples after sintering. For Se-HA-0.03, the phase of tricalcium phosphate (TCP) has been found (indicated by asterisk). For Se-HA-0.1 and Se-HA-0.3, the peak at 37.5° suggests the formation of calcium phosphate phase. According to the above results, the synthetic HA with selenite substitution is stable at 900 °C when Se/P = 0.03 and 0.1. However, after sintering at 900 °C, the crystallinity of Se-HA-0.1 is lower than that of Se-HA-0.03, which is related to the amount of selenite substitutions.
Tissue engineering techniques have provided great hopes for the regeneration of deficient tissues such as bones; nano-crystalline HA and apatite-like minerals are considered to be the most promising biological mimetic materials for bone repair [6,21]. The incorporation of trace elements into HA has brought new biological properties to the synthetic HA materials. Silicate substituted HA is found to improve the biocompatibility of the HA grafts and accelerate the bone healing process [22,23]. Carbonate substituted HA is also a good choice for bone tissue engineering [24]. Besides fabrications into bone grafts and scaffolds, the HA nanoparticles also have good performance in the applications of drug and gene delivery [3,25]. The controlled delivery of proteins has also been modified by the incorporation of divalent ions [26]. It is well-known that selenium plays a very important role in the human body and the up-take of selenium will induce the apoptosis in many cancer cells. The incorporation of selenium into HA may help us to treat bone cancers by lowering the chance of tumor recurrence. In this paper, the nano-crystalline HA powders with selenite substitutions were prepared at open atmosphere. FTIR results show
Fig. 4. The Se/P ratios measured by XRF versus the Se/P ratios in the reactants.
Fig. 6. X-ray diffraction patterns of hydroxyapatite powders sintered at 1100 °C for 2 h.
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that the Se-HA samples prepared have selenite and carbonate co-substitutions. Sodium ions may also be involved into the final products due to the usage of sodium selenite. The characterization by XRD, TEM, and FTIR techniques in this work has revealed the structural evolutions caused by the selenite substitutions. The hydroxyl groups have not been seen in the FTIR spectra. Meanwhile, the carbonate ions were found in the HA powder obtained without addition of sodium selenite. The present FTIR studies provide detailed information about the type of carbonate substitutions and their alteration after the addition of selenite substitutions. The number of the observed FTIR bands and their intensities show mixed A- and B-type carbonate substitutions. The intensities of the bands belonging to B-type carbonate substitutions decrease after the selenite groups are involved in the HA lattice. The incorporation of selenite groups has decreased the possibility of carbonate ions occupying the phosphate sites. According to the FTIR results, the most probable process for charge compensation is the replacement of two hydroxyl groups by one carbonate group [27]. At the same time, some phosphate sites are also occupied by the carbonate groups. The charge compensation mechanism can be proposed as the replacement of one phosphate group and one hydroxyl group by two carbonate groups [27]. Previous studies have clearly demonstrated these substitution mechanisms [16,27,28]. Due to the size limitation, the selenite ions occupy the phosphate sites. The selenite groups can replace phosphate groups inside the HA lattice with the ratio of 1:1 by ion exchange process [29]. Because the selenite groups have the same charges to the carbonate groups, the charge compensations for the carbonate substitutions and selenite substitutions may have no difference. The charge compensations of B-type carbonate hydroxyapatite might be the same as the selenite substitutions. Based on this assumption, the substitution mechanisms for the low Se/P ratios are proposed as the following. 3−
−
2−
2−
2−
2−
PO4 þ OH → CO3 ðAÞ þ CO3 ðBÞ→ SeO3 þ CO3 ðAÞ
ð1Þ
For HA with carbon dioxide involved, the carbonate ions occupy the sites of hydroxyl ions (A-type) and phosphate ions (B-type). When selenite ions are added, the carbonate ions have fewer chances to occupy the phosphate sites. At this time, the carbonate ions prefer hydroxyl column (A-type). In this condition, lower intensity of B-type FTIR spectra has been observed for the Se-HA samples. When the Se/P ratios are high, other charge mechanisms should be considered. In the condition of one HA unit cell having more than two Se atoms, an oxygen void (VO) is generated nearby the Se2O76− structure. This charge compensation mechanism is very similar to that of silicate substituted HA [20,23]. 3−
6−
2PO4 → Vo þ Se2 O7
ð2Þ
When Se/P ratio is above 1.0, apatite structure may not be formed and calcium selenite hydrate begins to precipitate in the reaction mixture. From the TEM results, it is obvious that the amorphous phase has been obtained when the Se/P ratio is only 0.3. After sintering at 900 °C for 2 h, other calcium phosphate phases appeared in the products because of the phase transformation. However, the main phase in the products with the Se/P ratio less than 0.3 after sintering is HA. According to the XRD results, the increasing Se/P ratios have led to the shrinkage of HA lattice. The changes in the lattice parameters are mainly caused by the replacement of phosphate ions by selenite ions. However, the radius of Se 4+ (0.50 Å) is much larger than that of P 5+ (0.35 Å); and the length of Se\O bond (0.164 nm) is greater than that of the P\O bond (0.155 nm). The size of the PO4 tetrahedron would be expected to be smaller than that of the SeO4 tetrahedron. The occupation of selenium in phosphorus sites should enlarge the
lattice size which is not accordant to the XRD analysis. It is noted that charge compensations are needed for the selenite substitution. The oxygen void may exist close to the selenite groups or one oxygen atom in the SeO4 tetrahedron is shared with the close phosphate and carbonate groups. The presence of more oxygen voids in HA lattice leads to the smaller unit cell. Another possible reason for the shrinkage of unit cell is the oxidization of selenite group. When the selenite groups (Se 4+) are oxidized to selenate groups (Se 6+), the shortened Se\O bands result in the decrease of HA lattice parameters. The charge compensations associated with the carbonate and hydroxyl groups have obvious effects on the lattice change of HA [30]. Due to the multiple substitutions, the trends of the lattice size with the incorporation of selenium into HA are complicated. In this manuscript, the above two mechanisms of charge compensation have not been approved by the direct experimental data. Further experiments about the detailed structure of Se-HA are needed to analyze the charge compensation mechanisms. Considering the structure stability and phase purity, the synthetic reactants with Se/P = 0.1 may produce the nano-crystalline Se-HA powders containing high Se content. They have stable HA structure after sintering at 900 °C for 2 h and only small amount of TCP phases appear after sintering at 1100 °C for 2 h. Because HA and TCP are both successful bone grafting materials in clinic, Se-HA-0.1 is the most promising candidate for the fabrication of bioactive ceramics to treat bone and osteosarcoma cancers among the samples prepared in this study. 5. Conclusion The facile precipitation technique using sodium selenite as a source of selenite ions has been successfully applied for the preparation of Se-HA. This low cost and facile method allows for the incorporation of carbonate and selenite ions into HA lattice. With the Se/P ratios no more than 0.1, the powders without sintering are nano-crystalline HA. Increasing substitutions of selenite ions for PO43− are accompanied by a decrease of CO32− substitutions. The powder crystallinity is strongly reduced as the extent of substitution increases, and when the Se/P ratios reach 10, the apatite structure changes to the phase of calcium selenite hydrate. The selenite substitutions in PO43− positions import obvious structural changes. The powder of Se-HA-0.1 having the sintering stability at 900 °C for 2 h is considered to be a promising candidate for the fabrication of bone tissue engineering scaffolds. Acknowledgments This work was supported by National Basic Research Program of China (grant no.: 2012CB933601), National Natural Science Foundation of China (grant nos.: 81071263, 30870624), International S&T Cooperation Program of China (grant no.: 0102011DFA31430), National Key Technology Research and Development Program of China (grant no.: 2012BAI17B02), and National High Technology Research and Development Program of China (grant no.: 2011 AA03105). References [1] D. Tadic, M. Epple, Biomaterials 25 (2004) 987–994. [2] N. Huebsch, D.J. Mooney, Nature 462 (2009) 426–432. [3] V. Uskokovic, D.P. Uskokovic, J. Biomed. Mater. Res. B Appl. Biomater. 96 (2011) 152–191. [4] W. Zhang, S.S. Liao, F.Z. Cui, Chem. Mater. 15 (2003) 3221–3226. [5] F.-Z. Cui, Y. Li, J. Ge, Mater. Sci. Eng. R Rep. 57 (2007) 1–27. [6] A. Tampieri, G. Celotti, E. Landi, Anal. Bioanal. Chem. 381 (2005) 568–576. [7] K. Zhao, Y.-F. Tang, Y.-S. Qin, J.-Q. Wei, Ceram. Int. 37 (2011) 635–639. [8] L.C. Wu, J. Yang, J. Kopecek, Biomaterials 32 (2011) 5341–5353. [9] A.M. Pietak, J.W. Reid, M.J. Stott, M. Sayer, Biomaterials 28 (2007) 4023–4032. [10] D.M. Ibrahim, A.A. Mostafa, S.I. Korowash, Chem. Cent. J. 5 (2011) 74. [11] L.T. Bang, K. Ishikawa, R. Othman, Ceram. Int. 37 (2011) 3637–3642. [12] T.J. Webster, E.A. Massa-Schlueter, J.L. Smith, E.B. Slamovich, Biomaterials 25 (2004) 2111–2121.
J. Ma et al. / Materials Science and Engineering C 33 (2013) 440–445 [13] Y. Tang, H.F. Chappell, M.T. Dove, R.J. Reeder, Y.J. Lee, Biomaterials 30 (2009) 2864–2872. [14] D. Laurencin, N. Almora-Barrios, N.H. de Leeuw, C. Gervais, C. Bonhomme, F. Mauri, W. Chrzanowski, J.C. Knowles, R.J. Newport, A. Wong, Z.H. Gan, M.E. Smith, Biomaterials 32 (2011) 1826–1837. [15] D.E. Ellis, J. Terra, O. Warschkow, M. Jiang, G.B. Gonzalez, J.S. Okasinski, M.J. Bedzyk, A.M. Rossi, J.G. Eon, Phys. Chem. Chem. Phys. 8 (2006) 967–976. [16] M.E. Fleet, X. Liu, Biomaterials 28 (2007) 916–926. [17] I.R. Gibson, W. Bonfield, J. Mater. Sci. Mater. Med. 13 (2002) 685–693. [18] J. Brozmanova, L. Letavayova, V. Vlckova, Toxicology 227 (2006) 1–14. [19] Y. Wang, J. Ma, L. Zhou, J. Chen, Y. Liu, Z. Qiu, S. Zhang, Interface Focus 2 (2012) 378–386. [20] N.Y. Mostafa, H.M. Hassan, O.H. Abd Elkader, J. Am. Ceram. Soc. 94 (2011) 1584–1590. [21] A. Tripathi, S. Saravanan, S. Pattnaik, A. Moorthi, N.C. Partridge, N. Selvamurugan, Int. J. Biol. Macromol. 50 (2012) 294–299.
445
[22] K.A. Hing, P.A. Revell, N. Smith, T. Buckland, Biomaterials 27 (2006) 5014–5026. [23] P. Gillespie, G. Wu, M. Sayer, M.J. Stott, J. Mater. Sci. Mater. Med. 21 (2010) 99–108. [24] E. Sachlos, D. Gotora, J.T. Czernuszka, Tissue Eng. 12 (2006) 2479–2487. [25] M. Motskin, D.M. Wright, K. Muller, N. Kyle, T.G. Gard, A.E. Porter, J.N. Skepper, Biomaterials 30 (2009) 3307–3317. [26] S. Dasgupta, S.S. Banerjee, A. Bandyopadhyay, S. Bose, Langmuir 26 (2010) 4958–4964. [27] S. Peroos, Z. Du, N.H. de Leeuw, Biomaterials 27 (2006) 2150–2161. [28] J. Barralet, S. Best, W. Bonfield, J. Biomed. Mater. Res. 41 (1998) 79–86. [29] F. Monteil-Rivera, M. Fedoroff, J. Jeanjean, L. Minel, M.G. Barthes, J. Dumonceau, J. Colloid Interface Sci. 221 (2000) 291–300. [30] S. Yin, D.E. Ellis, Phys. Chem. Chem. Phys. 12 (2010) 156–163.
