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Contents lists available at ScienceDirect Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys (BaeCa)TiO 3 nanopowder: Synthesis and their electrical and biological characteristics Narges Ahmadi, Mahshid Kharaziha , Sheyda Labbaf Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran HIGHLIGHTS (Ba x Ca 1-x )TiO 3 were synthesized by two-step sol-gel and mechanical acti- vation process. Substitution of Ca in (Ba x Ca 1-x )TiO 3 changed the cubic structure to tetra- gonal mode. The substitution of Ca for barium in BT nanopowder reduced dielectric constant. Bioactivity of (Ba x Ca 1-x )TiO 3 en- hanced with increased calcium con- tent. Substitution of Ca with Ba sig- nicantly enhanced MG63 cell pro- liferation. GRAPHICAL ABSTRACT ARTICLE INFO Keywords: Electroactive ceramics Barium-calcium titanate Sol-gel process Bone tissue engineering ABSTRACT Despite the applicable piezoelectric property of BaTiO 3 (BT), its biomedical applications have been limited due to its weak bioactivity and osteoconductivity. To overcome these issues, in the present study, (Ba x Ca 1-x )TiO 3 (x = 1, 0.9, 0.8, 0.6, and 0) nanopowders were synthesized using a combination of sol-gel and mechanical activation processes. The changes in the chemical, electrical, structural and biological properties of BaTiO 3 , as a result of the substitution of Ba 2+ with Ca +2 ions, were thoroughly studied. Substitution of Ba 2+ with Ca 2+ in the host (Ba x Ca 1-x )TiO 3 lattice changed the cubic structure of pure BT to tetragonal mode. Moreover, 40 at. % Ca 2+ substitution in BT nanopowder ((Ba 0.6 Ca 0.4 )TiO 3 ) resulted in decreased crystallite and particle size and reduced dielectric constant. In addition, bioactivity of (Ba x Ca 1-x )TiO 3 powder enhanced with increasing calcium content. MTT assay showed that 20 at.% Ca 2+ substitution in the BT nanopowder resulted in signicant increase in MG63 cell proliferation. Overall, calcium-substituted BT nanopowder could be a suitable candidate for bone repair. 1. Introduction Piezoelectric materials are electroactive components which can produce electrical activity in response to mechanical stress [1]. Dif- ferent natural or synthetic piezoelectric ceramics (e.g. calcium titanate, barium titanate and lead zirconate titanate (PZT)) [2] and polymers (e.g. PVDF) [1,3,4] have been introduced for various applications in- cluding actuators, piezoelectric motors, piezoelectric transformers, piezoelectric sensors, and piezoelectric benders [5]. In the last two decades, electroceramics, especially piezoelectric ones, have shown great potential for engineered scaolds [6,7]. Previous studies have conrmed that specic cellular activities such as attachment, https://doi.org/10.1016/j.matchemphys.2018.12.081 Received 19 January 2018; Received in revised form 11 September 2018; Accepted 27 December 2018 Corresponding author. E-mail address: [email protected] (M. Kharaziha). Materials Chemistry and Physics 226 (2019) 263–271 Available online 28 December 2018 0254-0584/ © 2018 Elsevier B.V. All rights reserved. T

Materials Chemistry and Physics · 2020. 3. 5. · ferent natural or synthetic piezoelectric ceramics (e.g. calcium titanate, barium titanate and lead zirconate titanate (PZT)) [2]

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  • Contents lists available at ScienceDirect

    Materials Chemistry and Physics

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

    (BaeCa)TiO3 nanopowder: Synthesis and their electrical and biologicalcharacteristics

    Narges Ahmadi, Mahshid Kharaziha∗, Sheyda LabbafDepartment of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran

    H I G H L I G H T S

    • (BaxCa1-x)TiO3 were synthesized bytwo-step sol-gel and mechanical acti-vation process.

    • Substitution of Ca in (BaxCa1-x)TiO3changed the cubic structure to tetra-gonal mode.

    • The substitution of Ca for barium inBT nanopowder reduced dielectricconstant.

    • Bioactivity of (BaxCa1-x)TiO3 en-hanced with increased calcium con-tent.

    • Substitution of Ca with Ba sig-nificantly enhanced MG63 cell pro-liferation.

    G R A P H I C A L A B S T R A C T

    A R T I C L E I N F O

    Keywords:Electroactive ceramicsBarium-calcium titanateSol-gel processBone tissue engineering

    A B S T R A C T

    Despite the applicable piezoelectric property of BaTiO3 (BT), its biomedical applications have been limited dueto its weak bioactivity and osteoconductivity. To overcome these issues, in the present study, (BaxCa1-x)TiO3(x= 1, 0.9, 0.8, 0.6, and 0) nanopowders were synthesized using a combination of sol-gel and mechanicalactivation processes. The changes in the chemical, electrical, structural and biological properties of BaTiO3, as aresult of the substitution of Ba2+ with Ca+2 ions, were thoroughly studied. Substitution of Ba2+ with Ca2+ in thehost (BaxCa1-x)TiO3 lattice changed the cubic structure of pure BT to tetragonal mode. Moreover, 40 at. % Ca2+

    substitution in BT nanopowder ((Ba0.6Ca0.4)TiO3) resulted in decreased crystallite and particle size and reduceddielectric constant. In addition, bioactivity of (BaxCa1-x)TiO3 powder enhanced with increasing calcium content.MTT assay showed that 20 at.% Ca2+ substitution in the BT nanopowder resulted in significant increase inMG63 cell proliferation. Overall, calcium-substituted BT nanopowder could be a suitable candidate for bonerepair.

    1. Introduction

    Piezoelectric materials are electroactive components which canproduce electrical activity in response to mechanical stress [1]. Dif-ferent natural or synthetic piezoelectric ceramics (e.g. calcium titanate,barium titanate and lead zirconate titanate (PZT)) [2] and polymers

    (e.g. PVDF) [1,3,4] have been introduced for various applications in-cluding actuators, piezoelectric motors, piezoelectric transformers,piezoelectric sensors, and piezoelectric benders [5]. In the last twodecades, electroceramics, especially piezoelectric ones, have showngreat potential for engineered scaffolds [6,7]. Previous studies haveconfirmed that specific cellular activities such as attachment,

    https://doi.org/10.1016/j.matchemphys.2018.12.081Received 19 January 2018; Received in revised form 11 September 2018; Accepted 27 December 2018

    ∗ Corresponding author.E-mail address: [email protected] (M. Kharaziha).

    Materials Chemistry and Physics 226 (2019) 263–271

    Available online 28 December 20180254-0584/ © 2018 Elsevier B.V. All rights reserved.

    T

    http://www.sciencedirect.com/science/journal/02540584https://www.elsevier.com/locate/matchemphyshttps://doi.org/10.1016/j.matchemphys.2018.12.081https://doi.org/10.1016/j.matchemphys.2018.12.081mailto:[email protected]://doi.org/10.1016/j.matchemphys.2018.12.081http://crossmark.crossref.org/dialog/?doi=10.1016/j.matchemphys.2018.12.081&domain=pdf

  • migration, proliferation and differentiation of cells could be improvedby electric pulses generated using piezoelectric scaffolds [8,9]. There-fore, piezoelectric based scaffolds could provide electrical stimulationwithout external electrical sources or implanting batteries [10].

    In addition to the synthetic materials, many body tissues like bone,cartilage and tendons are naturally piezoelectric [10]. Bone is a dy-namic tissue with a very low piezoelectric coefficient (0.7 pC/N) [11].Therefore, in response to micromechanical stresses such as bodymovement, bone produces electrical dipoles and signals which canimprove bone growth and remodeling [10,12]. Indeed, calcium andphosphate ions in the body fluid move towards dipoles with oppositecharges leading to better bone regeneration [12]. Therefore, utilizationof the piezoelectric materials as charge provider agents could be apromising approach for stimulating bone healing process and re-construction of bone defects [6,12].

    Barium titanate is the most frequently used lead free piezo-ceramicin bone tissue engineering owing to its remarkable piezoelectric andmechanical characteristics [11]. Barium titanate is the first perovskite(ABO3) applied for bone repair with 200 pc/N piezoelectric coefficientand curie point of 130 °C [11–15]. Moreover, compared to other com-monly applied bioceramics such as bioactive glass (compressivestrenght:800–1200MPa), hydroxyapatite (compressive strength:300–900MPa) and tricalcium calcium phosphate (compressivestrength: 450–650MPa), barium titanate shows better compressivestrength (compressive strength: 411–561MPa) for bone tissue en-gineering applications [16,17]. This ceramic displays appropriate bio-compatibility and could provide a strong interfacial bond with bone[12]. Moreover, in vivo studies revealed bone formation at a greater rateon the barium titanate/hydroxyapatite than pure hydroxyapatiteceramic [7]. However, despite these favorable properties, in vitro stu-dies demonstrated weak bioactivity of barium titanate compared toother bioactive materials like bioactive glasses and hydroxyapatite[18]. On the other hand, calcium titanate is another electroceramicwhich has been applied for biomedical applications in various forms(powder, coating) [18,19]. Recent results indicated that calcium tita-nate coatings on titanium promoted osteoblast adhesion compared totitanium implants [19]. More recently, Bagchi et al. [18] developednanocomposite of poly (ε-caprolactone) (PCL) with different perovskitenanoparticles including calcium titanate, barium titanate and strontiumtitanate. In vitro studies revealed enhanced osteogenic gene expressionon PCL/calcium titanate nanocomposites compared to other composites[18]. However, the piezoelectric property of calcium titanate is weakerthan barium titanate [18]. Therefore, it is expected that the in-corporation of calcium ions within barium titanate ceramic and theformation of (Ba,Ca)TiO3 could stimulatory promote both piezoelectricand biological properties. Doping of external elements is an effectiveway to improve the electrical behavior of electroceramics [20]. Zhuanget al. [21] reported the possibility of Ca2+ occupation in BaTiO3structure, for the first time. Following this report, different approachesare applied for Ca2+ substitution in BaTiO3 structure and synthesis of(Ba, Ca)TiO3 powder [21]. Conventionally, (Ba, Ca)TiO3 powder issynthesized by solid-state reaction between a mixture of TiO2, BaCO3and CaCO3. However, obtained powders often consist of coarse grainswith non-uniform particle size and shape [22]. In addition, (Ba,Ca)TiO3 powder synthesized via solid-state reaction consists of lowsolubility limit of Ca2+ [22]. Nowadays, wet chemical processes arepreferred ways of synthesis due to high solubility limit, stoichiometrycontrol, reproducibility, purity and small particle size of products [22].These wet chemical techniques consist of carbonate-oxalate [21], gel-carbonate [21], gel-to-crystallite conversion [21], hydrothermal [23],sol-gel [24] and polymeric precursor [25] approaches. Amongst them,sol-gel process enables a better control of size and morphology withleast impurity [26]. Li et al. [24] synthesized (BaxCa(1-x))TiO3 powder(x= 0.9) with grain size of 2 μm using sol-gel process. However, to thebest of our knowledge, the substitution of Ca ions in the host BaTiO3lattice, using sol-gel method, has been performed in a limited

    concentration range. Moreover, the role of various concentrations ofsubstituted Ca on the properties of (BaxCa(1-x))TiO3, specifically elec-trical and biological properties, was not investigated.

    The aim of the current study was to synthesize (BaxCa1-x)TiO3(x= 1, 0.9, 0.8, 0.6, and 0) nanopowders by a simple combination ofsol-gel and mechanical activation processes. Moreover, the effects ofCa2+ substitution on chemical, electrical and biological properties ofbarium titanate were investigated. X-ray diffraction (XRD), Ramanspectroscopy, Field emission-scanning electron microscopy (FE-SEM)and energy-dispersive X-ray(EDX) are applied to characterize of(BaxCa1-x)TiO3 powders. Moreover, the dielectric constant of (BaxCa1-x)TiO3 powders was calculated using LCR meter. In vitro bioactivity ofthe barium-calcium titanate powders was considered by immersing thesamples in stimulated body fluid (SBF) solution. Eventually, the cyto-toxic activity of the samples against MG63 cell lines was investigatedusing MTT assay.

    2. Materials and methods

    2.1. Materials

    Glacial acetic acid (100% anhydrous), calcium nitrate tetrahydrate(Ca(NO3)2·4H2O, 99%), barium acetate (Ba(OOCCH3)2), titanium tet-raisopropyl alkoxide (TTIP, 98%) and 2-propanol (> 99%) were pur-chased from Merck company.

    2.2. Synthesis of barium-calcium titanate powder

    Barium-calcium titanate powder was synthesized using a combina-tion of mechanical activation and sol-gel process. In order to synthesize(BaxCa1-x)TiO3 with various Ba:Ca atomic ratios (1:0, 0.9:0.1, 0.8:0.2,0.6:0.4, and 0:1), barium acetate and calcium nitrate were used asbarium and calcium precursors, respectively. Primarily, calcium nitratetetrahydrate and barium acetate were dissolved in pure acetic acid at65 °C, separately. The mole ratio of (calcium nitrate tetra-hydrate + barium acetate): acetic acid was adjusted to 1:1. Moreover,according to Ba:Ca atomic ratios in various samples, pure acetic acidsolution was divided between them. In the next step, both solutionswere cooled down to room temperature (cooling rate = 5 °C/min) and2-propanol and TTIP were added to them, respectively. After mixingtwo solutions, the temperature was reached to 2–3 °C (coolingrate = 10 °C/min) and deionized water was incorporated to the solu-tion to form a transparent sol. The above precursors were selected toprovide acetic acid: TTIP:2-propanol: deionized water molar ratio of6:1:1:150. The sol was transformed to get by heating it upon 60 °C. Thegel was dried was at 100 °C for 24 h. In order to enhance the surfaceenergy of powders and reduce the calcification temperature of powders,as prepared dried gel was milled through high-energy ball milling at250 rpm for 1 h. Finally, calcification was perforated at 1000 °C for 2 hto obtain white powders (the rate of heating and cooling of powderswas adjusted to 10 °C/min). The powder was cooled down in the fur-nace. It needs to mention that, according to Ba:Ca ratio (1:0, 0.9:0.1,0.8:0.2, 0.6:0.4, and 0:1) in the final composition, the samples werenamed as BT, BT1, BT2, BT3 and CT, respectively.

    2.3. Characterization of barium-calcium titanate powders

    The phase composition, lattice parameter as well as crystallite sizeof the synthesized powders were characterized with X-ray diffraction(XRD, Philips X’ Pert) technique. The powders were irradiated using Nifiltered Cu kα (λCukα=0.154 nm) with step size of 0.05° and a scan steptime of 1.25 s. The working voltage and current were 40 kV and 30mA,respectively. The samples were scanned in the diffraction angles (2θ)between 30° and 80°. To determine phase compositions, the patternswere compared with the Joint Committee on Powder DiffractionStandards (JCPDS) reference cards. Moreover, the crystallite size of the

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  • powders was calculated from XRD patterns using the modified Scherrerformula (eq. (1)) [27]:

    = +lnβ ln kλL

    lnθ

    1cos (1)

    where L is the crystallite size (nm), λ is the wavelength of Cu kα ra-diation (λ=1.5404 A), β (radians) is the peak width of the diffractionpeak profile at half maximum height, θ is the diffraction angle (°) and kis the broadening constant. Furthermore, in order to calculate thecrystal parameters, Rietveld analysis was performed using a MAUD(material analysis using diffraction) software package. Moreover, toverify the crystal structure of the synthesized powders, Raman spectrawere recorded through a Teksan spectroscopy (Takram model), usingNd:YAG (532 nm laser excitation) from 200 to 2000 cm−1. The spectralresolution was adjusted to 2 cm−1. Dielectric measurement was alsocarried out on LCR meter (GW Instek LCR-819) at a frequency of20 kHz.

    Finally, the morphology and elemental composition of powderswere investigated by Field emission-scanning electron microscopy (FE-SEM, Philips XL30: Eindhoven) and energy dispersive X-ray (EDX).Before imaging, the powders were suspended in ethanol and sonicatedfor 30min to prevent from the agglomeration of particles.Consequently, the suspensions were drop-casted on a glass slide andallowed to dry. Finally, the samples were gold sputter-coated and im-aged using FE-SEM (Zeiss SUPRA 60). Moreover, the average particlesizes of the synthesized samples were estimated using Image J software.In this regard, three FE-SEM images from various sections of sampleswere taken and the average size of about 50 particles were determined.

    2.4. In vitro bioactivity evolution of barium-calcium titanate powders

    In vitro bioactivity of the barium-calcium titanate powders wasevaluated via immersing the samples in stimulated body fluid (SBF)solution prepared by Bohner et al. method [28]. Primarily, the pellets ofbarium-calcium titanate powders (2 mm in diameter and 0.5 mm inthickness) were uniaxially pressed at 300MPa. Consequently, the pel-lets were soaked in 10ml SBF (pH 7.4) at 37 °C for 14 days. After dryingthe samples in a vacuum desiccator at room temperature, SEM, EDS andelemental map analysis were performed to investigate the formation ofapatite on the surface of samples. In order to compare the bioactivity ofbarium-calcium titanate powders, Inductivity coupled plasma massspectroscopy (ICP) was applied to determine Ca and P ion concentra-tions of SBF solutions after 14 days soaking. In addition, pH values ofSBF solutions were recorded during sample soaking.

    2.5. Cell culture

    In order to investigate the biocompatibility of the samples, cyto-toxicity and spreading of MG63 osteoblast-like cell-line (purchasedfrom the National Cell Bank of Iran at the Pasteur Institute (NCBI code:C555)) were investigated. MG63 cells were cultured in Dulbecco'sModified Eagle Medium (DMEM-low glucose, Bioidea, Iran) containing10 vol% fetal bovine serum (FBS) (Bioidea, Iran) and 1 vol% strepto-mycin/penicillin (Bioidea, Iran) at 37 °C under 5% CO2 condition. Priorto cell culture, the samples were sterilized in 70 vol% ethanol for30min followed by 2 h exposure to ultraviolet (UV) light.

    2.5.1. Cytotoxicity assayThe cytotoxicity assay was evaluated using a dilution of nano-

    powder extracts in contact with MG63 cells according to theInternational Standard Organization (ISO/EN 10993.5, 1999). Briefly,MG63 cell lines were exposed to a series of nanopowder extracts in thefull culture medium. Prior to cell culture, the 200 μg/ml of calcium-barium titanate powders were prepared in complete culture medium.After 24 h incubation at 37 °C, the mixtures were diluted further toprovide powder extracts at concentrations of 50 and 100 μg/ml. MG63

    cells were seeded at density of 104 cells/well in a 96-well plate. After24 h incubation, the medium was removed and replaced with dilutedextracts (50, 100 and 200 μg/ml).

    The relative viability of cells was studied by 3-(4,5-dimethylthia-zolyl-2)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) col-orimetric assay. After one day of culture, the medium was discarded,the wells were washed with PBS and the samples were incubated withMTT solution (0.5 mg/ml MTT reagent in PBS) for 4 h. As formed darkblue formazan crystals were solubilized with dimethyl sulfoxide(DMSO, Sigma) and kept for 30min at 37 °C. Subsequently, the opticaldensity (OD) of each well was measured with a microplate reader (BioRad, Model 680 Instruments) against DMSO (blank) at a wavelength of490 nm. The relative cell survival was calculated based on the followingequation (eq. (2)):

    =

    A AA A

    Relative cell survival (%) sample bcontrol b (2)

    where Asample, Ab and Acontrol are the absorbance of sample, blank(DMSO) and control (TCP), respectively.

    2.5.2. Cell morphology studyThe morphology of the cells seeded on the pellets were evaluated by

    SEM analysis. Prior to cell seeding, the sterilized samples (n=2) weresoaked in complete culture medium for 8 h, prior to cell seeding. MG63cells were seeded on the samples as well as tissue culture plate (TCP,control) at a density of 104 cells/well in a 96-well plate. MG63 Cellswere incubated for 3 days at 37 °C under 5% CO2 condition. Afterfixation with 2.5%(v/v) glutaraldehyde (sigma) for 3 h, the sampleswere rinsed with PBS, and dehydrated in the graded concentrations ofethanol (30, 70, 90, 96 and 100% v/v) for 10min, respectively. Finally,they were air dried, gold-coated and evaluated via SEM imaging.

    2.6. Statistical analysis

    The cytotoxicity data in this study was considered using one-wayANOVA analyses and reported as mean ± standard deviation (SD). Todetermine a statistically significance difference between groups,Tukey's post-hoc test using GraphPad Prism Software (V.6) with a p-value< 0.05 was applied to be significant.

    3. Results and discussion

    3.1. Characterization of barium-calcium titanate powders

    In order to determine the role of calcium ion, SEM and EDX analysiswere performed. SEM images and EDX analysis of the various nano-powders are presented in Fig. 1(a–e). The average particle sizes esti-mated using Image J software are presented in Fig. 1(f). Results re-vealed that Ca substitution did not significantly alter the sphericalmorphology of the synthesized powders. However, depending on thecalcium ions incorporated into the structure, the particle sizes werechanged. The average particles size of powders reduced from76 ± 4 nm (at BT sample) to 68 ± 2 nm (at BT1) and, consequentlyincreased to 85 ± 3 nm (at BT2), 93 ± 3 nm (at BT3) and finally104 ± 8 nm (at CT). Antonnelli et al. [29] similarly synthesizedBa0.77Ca0.23TiO3 powder by using modified polymeric precursormethod calcinated at 800 °C. As prepared Ba0.77Ca0.23TiO3 powder re-vealed an average particle size of ∼60 nm. It is suggested that the re-duced particle size detected as a result of at least Ca incorporation (BT1sample) could be due to the substitution of smaller Ca 2+ for a largerBa2+ leading to the shrinkage of crystal structure and/or due to the roleof Ca ions in inhabitation of the grain growth during calcification [30].EDX analysis (Fig. 1) also revealed that barium content decreased whilecalcium component enhanced confirming the substitution of barium tocalcium, with increasing the amount of calcium concentration. More-over, according to map analysis (Supplementary Fig. S1), all elements

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  • uniformly distributed in all samples and barium content reduced withincreasing calcium content.

    To confirm the substitution of Ba2+ with Ca2+ in the structure ofBaTiO3, XRD analysis was performed. XRD patterns of the barium ti-tanate (BT), calcium titanate (CT) and barium-calcium titanate nano-powders consisting of various amounts of calcium content (BT1, BT2and BT3) are provided in Fig. 2(a). According to the modified Scherer'sequation (eq. (1)) and Rietveld refinement, the crystallite size (nm),crystalline structure, lattice parameters of a, b and c, lattice volume andc/a ratio were assessed (Table 1). According to JCPDS reference codes(JCPDS 01-079-2263 and JCPDS 01-081-0561), XRD patterns of bariumtitanate and calcium titanate only consisted of the barium titanate and

    calcium titanate peaks, respectively, without any secondary peak con-firming the formation of pure BT and CT powders. Moreover, betweenbarium-calcium titanate nanopowders, BT1 only revealed the char-acteristic peaks of BT (JCPDS 00-008-0725) confirming the formationof solid solution monophasic without any secondary phase. Similarly,previous researches have confirmed that pure barium titanate could beformed when substituted calcium content is less than 23% [31]. Forcalcium ion contents ranging from 20 at.% (BT2) to 40 at.% (BT3), bi-phasic structures of tetragonal BaTiO3 and orthorhombic CaTiO3 wereobserved. Assuming that these structures arose from imperfect homo-geneity due to incomplete diffusion of Ca into BaTiO3 [31]. Therefore,when substituted calcium content reached to 40 t% (BT2 and BT3

    Fig. 1. SEM images and EDX analysis of the (a)BT (b)CT (c) BT1 (d) BT2 (e) BT3 powders. (f) Bar graph of mean particle size of all samples.

    Fig. 2. (a)The XRD patterns and (b) Raman spectra of BT, BT1, BT2 and BT3 samples.

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  • samples), biphasic structures of BT and CT were provided. Moreover,the crystallite size of the powders was calculated from XRD patternswith standard data compiled by the International Center for DiffractionData (ICDD) using the modified Scherer formula (eq. (1)) [27]. Resultspresented in Table 1 confirmed that CT samples revealed the smallestcrystallite size (15.7 nm) between various samples. Moreover, sub-stitution of Ca+2 ions for Ba2+ in BT structure reduced the crystallitesize of powder from 35 nm (at BT powder) to 28 nm (at BT3). Thisresult was similarly reported in previous researches [32,33]. For in-stance, Souza et al. [32] synthesized BCT powder by the microwave-assisted hydrothermal method (MAH) and found that the crystallite sizeof Ba1-xCaxTiO3 powder at x= 0, 0.2 and 0.5 was around 39, 34 and17 nm, respectively [32]. Farzadi et al. [33] also found, similarly, thatincorporation of Mg in the hydroxyapatite lattice reduced crystallitesize. Based on previous researches, substituted Ca ions might act asgrowth inhibitors leading to reduce in the crystallite size of BT powder.

    To calculate the crystal parameters, Rietveld analysis was per-formed using a MAUD (material analysis using diffraction) softwarepackage. In this way, Ba1-xCaxTiO3 (BCT) structure was indexed using

    Table 1The calculated information extracted from XRD patterns.

    Sample name a(A°) b(A°) c(A°) V(A°3) c/a ratio Crystal structure Crystallite size(nm)

    BT (BaTiO3) 4.0094 4.0094 4.0094 64.45 1 Cubic 35BT1((Ba0.9Ca0.1)TiO3)) 4.0087 4.0087 4.0273 64.72 1.004 Tetragonal 35BT2((Ba0.8Ca0.2)TiO3) 4.0016 4.0016 3.9905 63.89 0.997 Tetragonal 34BT3((Ba0.6Ca0.4)TiO3) 3.9880 3.9880 3.9880 63.43 1 Cubic 28CT(CaTiO3) 3.8291 3.8291 3.8288 56.14 – Orthorhombic 16

    Fig. 3. Dielectric constant of BaxCa1-xTiO3 nanopowder as a function of sub-stituted Ca content in BaTiO3.

    Fig. 4. In vitro bioactivity evaluation of BaxCa1-xTiO3 nanopowder: SEM images of (a)BT, (b)CT, (c)BT1, (d)BT2 and (e)BT3 samples after 14 days soaking in SBFsolution. Insets in figures a–c show P ion distribution estimated using map analysis. (f) Bar graph of Ca, Ba and P content deposited on various samples estimatedusing EDX analysis.

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  • tetragonal BaTiO3 (reference code: JCPDS 00-008-0725) for x < 20%(BT1 and BT2 samples) and cubic BaTiO3 (reference code: JCPDS 01-079-2263) for BT and BT3 powders, respectively. Results revealed thatthe lattice volume enhanced in BT1 sample confirming the substitutingTi4+ by Ca2+ ions. Moreover, c/a ratio enhanced at BT1 sample de-monstrating its tetragonal structure. Our results revealed that sub-stituting 10 at.% Ca ions resulted in changing the crystallographicstructure of BaTiO3 from cubic (paraelectric phase) to tetragonal (fer-roelectric phase). At tetragonal form of BaTiO3, due to Ti displacementfrom its centrosymmetric position a permanent electric dipole appears[34]. More Ca substitution for Ba in BT2 and BT3 samples led to reducein its volume structure and tetragonality (c/a). This behavior was si-milarly reported in previous studies [35]. In the case of BT3 sample, thecrystallographic structure changed to cubic one. Besides, our resultsconfirmed that incorporation of calcium atoms prevented the formationof the unwanted hexagonal phase of BaTiO3 which is not ferroelectric.

    The chemical structure of nanopowders was further evaluated usingRaman spectroscopy (Fig. 2(b)). Our results confirmed that all powdersdid not include noticeable traces of intermediate components consistingof BaCO3 due to the absence of any vibrational mode at 1060 cm−1

    [29]. Furthermore, the substitution of Ca in barium titanate powderchanged its crystallographic structure. For instance, owning to the lackof strong vibration modes at ∼470 cm−1, and high intensity of thepeaks at around 254 cm−1 and 517 cm−1, as-prepared BT powder re-vealed cubic-like atomic structure [29]. The vibration modes at around254 cm−1, 517 cm−1 and 715 cm−1 in pure barium titanate (BTpowder) were due to TieO and BaeO bonds, respectively [29]. More-over, Raman spectra confirmed that the predominant phase of both BT1and BT2 powders was tetragonal. Raman spectra of BT1 and BT2powders consisted of sharp and distinct vibration modes at around

    306 cm−1 [B1, E(TO + LO)] and 185 cm−1 [A1(TO), E(LO)] and broadbands around 260 cm−1 [A1(TO)], 520 cm−1 [A1, E(TO)], and721 cm−1 [A1, E(LO)] [36]. These peaks were the characteristic activemodes of the tetragonal phase of BaTiO3, suggesting the transition fromcubic to pseudotetragonal phase via substitution of Ca ions in the BTstructure [36]. Specifically, the strong contributions at around306 cm−1, relating to asymmetry of TiO6 octahedra was contradictorywith the cubic symmetry of pure BT powder. However, incorporation ofmore Ca ions in the structure of BT powder (BT3) resulted in changingthe structure. In this structure, the band at 185, 305 and 715 cm−1

    disappeared, demonstrating that the phase was changed to the cubicstructure. Previous researches confirmed that there is a critical crys-tallite size for the BT to stabilize the cubic or the tetragonal phase. Inthe case of crystallite size less than the critical value, the phase is cubic[36]. In our study, the transition from tetragonal to cubic structure atBT3 sample might be due to reduced crystallite size of powders to lessthan 27 nm. All these results were in good greement with the results ofXRD analysis.

    The role of Ca substitution on the electrical property of BT wasstudied via evaluation of the dielectric constant values. Fig. 3 presentsthe changes of dielectric constant as a function of Ca substituted contentat ambient temperature. Increasing Ca content resulted in decrease inthe dielectric constant from 288 to 57.65 at 20 kHz. This behavior wassimilarly reported in previous researches [22,35,37]. Various factorsthat influence the dielectric constant include synthesis process, grainsize, temperature of test, frequency and dopants [38]. For instance, Xuel al. [39] showed that increasing the annealing temperature of BT from180 °C to 500 °C reduced hydroxyl defects and enlarged crystallite sizefrom 25 to 33 nm leading to promoted dielectric constant from 80 to205, due to the formation of denser space effective charges at higherannealing temperature. In another study, researches showed that thedielectric constant enhanced as the grain size reduced from 10 μm to1 μm, which is mainly correlated to the grain size and to its distribution[40]. Also, because of the capability to host ions in perovskite structure,various dopants of monovalent, divalent and trivalent can be accom-modated in the BT lattice which could effectively affect the electricalproperties of BT [17]. Our results revealed that incorporation of cal-cium in barium titanate structure reduces the value of dielectric con-stant owning to enhanced crystallographic defects and the motion ofTi4+ which can be affected by the shrinkage of structure by substitutingCa [35].

    3.2. In vitro bioactivity evaluation

    The apatite formation ability of the samples was studied by im-mersing barium-calcium titanate pellets in SBF solution for 14 days at37 ± 0.5 °C. Fig. 4(a–e) represents the SEM images of samples after

    Fig. 5. In vitro bioactivity evaluation of BaxCa1-xTiO3 nanopowders: a) pH value of SBF solution during the immersion of samples as a function of soaking time. b) Theconcentration profiles of Ca, P, ions of SBF solution as a function of substituted Ca content in BaxCa1-xTiO3 nanopowders.

    Fig. 6. Viability of MG63 cells cultured on various samples using MTT assayafter 1 day of culture.

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  • soaking in SBF solution for 14 days. EDX spectra estimated using areamicroanalysis of samples are presented in Supplementary Fig. S2.Moreover, the changes in Ca, P and Ba content on various samples afterimmersion in SBF solution, are presented in Fig. 4(f)). Results showedthat spherical-like particles had deposited on the surface of samples.EDX analysis confirmed that these precipitations consisted of calciumand phosphorus with various ratios owing to Ca content in the samples(Fig. 4(f) and Supplementary Fig. S2). According to bar graph of atomicconcentrations on the surface of all samples (Fig. 4(f)), BT2 and BT3consisted of more P contents than others revealing the formation ofmore bone-like apatite following immersion in SBF solution. SEMimages also demonstrated that increasing the amount of Ca content inthe structure of BCT samples resulted in promoted spherical-like par-ticles deposited on the sample. For instance, most of BT3 surface samplewas covered with a dense layer of CaeP deposition confirming higher

    bioactivity of this sample than others. Element mappings (Fig. 4(a–c))also suggested that compared to BT sample, a dense layer of P elementscovered the surfaces of BT1 and CT nanopowders. This result clearlyconfirmed that substitution of less than 10 at.% Ba for Ca resulted insignificantly enhancement in bioactivity of calcium-barium titanatecompared to barium titanate. The changes of pH value of SBF solutionduring 14 days soaking of pellets are presented in Fig. 5(a). The pHvalue of SBF solution increased in the first 7 days of soaking and thenalmost stable in all samples except CT sample. During soaking in SBFsolution, exchange of Ba2+ and Ca2+with H+ or H3O+ from SBF so-lution and the formation of TieOH bonds in the initial soaking timesmight lead to the increase in the concentration of OH− and, hence pHvalue. Formation of negatively charged surface by TieOH groups re-sulted in the absorption of Ca2+ ions from SBF solution on surface ofthe pellets making these surfaces suitable for deposition of negatively

    Fig. 7. The morphology of MG63 cells cultured for 3 days on the (a)BT, (b)CT, (c)BT1, (d)BT2, (e)BT3 samples.

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  • charged ions such as PO43− and finally bone like apatite formation.According to our results and similar previous researches, calcium ionsrelease from the bioactive samples such as bioactive glass [41,42] andwollastonite [43] could promote spontaneous nucleation and growth ofapatite. Therefore, increasing Ca content in calcium-barium titanatesamples could result in higher precipitation of apatite phase confirmingour results. Fig. 5(b) indicates the concentration of Ca and P ions in SBFsolution after 14 days of soaking. Due to the precipitation of bone-likeapatite on the samples, the concentration of phosphorous and calciumions changed. Our results showed that the concentration of phosphorusion in SBF solution decreased from 5.84 mg/L to 0.03 mg/L when theconcentration of substituted Ca in BT structure increased. However, theconcentration of phosphorus ion in SBF increased when CT was im-mersed in SBF solution. Our result clearly showed barium calcium ti-tanate revealed higher bioactivity than pure BT and CT samples. Itmight be due to role of substituted ion within pure ceramics structurewhich provide high density of point defects leading to higher activity inbiological environment.

    3.3. Cell study

    The viability of MG63 cell line exposed to sample dissolution pro-ducts were studied using MTT assay (Fig. 6). Results demonstrated thatthe proliferation of MG63 cells in contact with BT1, BT2 and CT extractsgradually enhanced from at least concentration (50 μg/ml) to thehighest concentrations (200 μg/ml), while various concentrations ofBT3 did not significantly change it. Noticeably, cell viability was en-hanced from 92.7 ± 12% (control) (at 50 μg/ml of BT1) to218.8 ± 21%(control) (at 200 μl/ml of BT1) (P < 0.05). Moreover,increasing the concentrations of BT sample dramatically reduced theviability of MG63 cells (P < 0.05). Finally, our results confirmed thatincorporation of Ca ions within BT powder upon 20 at.% could sig-nificantly influence the cytotoxicity of BT sample at higher concentra-tions. Xue et al. [44] prepared tricalcium phosphate (TCP) ceramic bydual dopants of magnesium (Mg) and zinc (Zn), and investigated theinfluence of dopants on biological properties of TCP. The highest cellproliferation was found on dual Mg and Zn doped TCP compared topure TCP, TCP-Mg, TCP-Zn and TCP-Mg-Zn samples due to role of thesubstituted ions in control the solubility of TCP and providing a stablecell-material interface. Moreover, Bellucci et al. [45] successfully syn-thesized Mg/Sr bi-doped tricalcium phosphate-bioactive glass compo-site and found that combined stimulatory effect of Mg/Sr substitutioncould significantly promote the differentiation of mouse calvaria-de-rived pre-osteoblastic cells.

    In addition, the morphology of MG63 cells on various samples werestudied. Fig. 7 shows the SEM images of samples after 3 days followingexposure to MG63 cells. Cells appear to have attached and spread onCT, BT1 and BT2 samples with elongated and flattened morphology.Our results clearly revealed that substituting Ca in barium titanatestructure could lead to better cellular behavior which might be due toproducing piezoelectric signals in tetragonal phase of barium titanate[10].

    4. Conclusion

    In summary, combination of sol–gel and mechanical activationtechniques was successfully employed for the synthesis of (BaxCa1-x)TiO3 (x= 1, 0.9, 0.8, 0.6, and 0) nanopowder with high chemicalpurity. Incorporation of Ca in barium titanate structure resulted indecreased average crystallite (from 35 nm to 28 nm) and particle size(from 76± 4 nm to 68± 2 nm) as well as dielectric constant (from288 to 57.65). Moreover, substitution of Ca in barium titanate upto20 at.% resulted in changing the crystallographic structure from cubicsymmetry to tetragonal owning to substitution of Ti4+ by Ca2+.Moreover, substitution of Ca for Ba in the Ba-based perovskite resultedin improved in vitro bioactivity and biocompatibility. Noticeably,

    barium titanate powder upon 10 at.% Ca content significantly promotedMG63 proliferation (around 4 times compared to barium titanate na-nopowder) at higher concentrations. Overall, substituting Ca in bariumtitanate structure can be a promising way for improving barium titanateproperties in bone tissue engineering field.

    Appendix A. Supplementary data

    Supplementary data to this article can be found online at https://doi.org/10.1016/j.matchemphys.2018.12.081.

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    (Ba?Ca)TiO3 nanopowder: Synthesis and their electrical and biological characteristicsIntroductionMaterials and methodsMaterialsSynthesis of barium-calcium titanate powderCharacterization of barium-calcium titanate powdersIn vitro bioactivity evolution of barium-calcium titanate powdersCell cultureCytotoxicity assayCell morphology study

    Statistical analysis

    Results and discussionCharacterization of barium-calcium titanate powdersIn vitro bioactivity evaluationCell study

    ConclusionSupplementary dataReferences