Structural and dielectric properties of yttrium ...

Materials Science and Engineering C 47 (2015) 333–338

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Structural and dielectric properties of yttrium-substituted hydroxyapatites Omer Kaygili a,⁎, Sergey V. Dorozhkin b, Tankut Ates a, N. Canan Gursoy c, Serhat Keser d, Fahrettin Yakuphanoglu a, A. Birkan Selçuk e a

Department of Physics, Faculty of Science, Firat University, 23119 Elazig, Turkey Kudrinskaja sq. 1-155, 123242 Moscow, Russia Department of Microbiology and Clinic Microbiology, Inonu University, 44280 Malatya, Turkey d Department of Chemistry, Faculty of Science, Firat University, 23119 Elazig, Turkey e Technology Department, Saraykoy Nuclear Research and Training Centre, 06983 Ankara, Turkey b c

a r t i c l e

i n f o

Article history: Received 18 June 2014 Received in revised form 2 October 2014 Accepted 10 November 2014 Available online 11 November 2014 Keywords: Hydroxyapatite Yttrium (Y) X-ray diffraction (XRD) Antimicrobial activity Dielectric properties

a b s t r a c t Hydroxyapatite (HAp) samples doped with 0, 2 and 4 at.% of yttrium (Y) were characterized using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy attached with energy dispersive X-ray (EDX) spectroscopy, antimicrobial activity tests and dielectric studies. The hydroxyl groups observed in FTIR spectra confirmed the formation of HAp phase in the studied samples. The crystallite size, crystallinity degree and lattice parameters of the samples were changed with Y content. The volume of the unit cell was gradually decreased with the addition of Y. Undoped and Y-containing HAp samples were screened to determine their in vitro antimicrobial activities against the standard strains. It was found that no samples have any antimicrobial effect. The relative dielectric permittivity and dielectric loss are affected by Y content. While the alternating current conductivity increases with increasing frequency, it decreases with increasing Y content. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Due to the great similarity with the inorganic components of human bones and teeth, calcium orthophosphates and calcium orthophosphate-based materials have a great interest for biomedical applications. Among them, hydroxyapatite (HAp, Ca10(PO4)6(OH)2) is one of the most known implant materials used in several clinical applications (i.e., orthopedics, dentistry, neurosurgery and plastic surgery) due to its superior biological responses (e.g., non-toxicity, high biocompatibility and osteoconductivity) in the physiological environments [1–6]. The stoichiometric HAp has hexagonal crystal structure with the lattice parameters a = b = 0.9418 nm, c = 0.6884 nm and the unit cell volume of V = 0.5288 nm3 [7–9]. However, both the composition and properties of the chemically pure HAp do not fully correspond to those of bones and teeth. Therefore, doping of HAp with various ions has been used to improve its properties. Metal ions such as Ag+, Mg2 +, Zn2 +, Sr2 +, Al3 +, Ce3 +, La3 +, Bi3 +, Y3 + and Eu3 + can substitute Ca2 + ions in the HAp structure [10–22]. These ionic substitutions affect the crystallinity, lattice parameters and morphology of HAp. Since the physical, chemical and biological properties of HAp directly linked with its crystal structure ⁎ Corresponding author. E-mail address: okaygili@firat.edu.tr (O. Kaygili).

http://dx.doi.org/10.1016/j.msec.2014.11.039 0928-4931/© 2014 Elsevier B.V. All rights reserved.

and composition, the ionic substitutions provide the possibilities to control the characteristic properties of HAp [22–26]. Yttrium (Y) has been extremely used in medical applications. Some applications given in the literature can be summarized as follows: Y3+ is used in the treatment of hepatocellular carcinoma [27,28]. Thomas et al. [29] reported that 90Y-containing HAp could serve as an alternative therapy for chronic synovitis because of bleeding disorders. Zhang et al. [30] reported that Y3+ promoted the adipocyte transdifferentiation of primary mouse osteoblasts. Liu et al. [31] reported that Y-containing HAp synthesized by hydrothermal method accelerates the human periodontal fibroblast growth and restricts slightly the oral bacterial growth. The work of Nathanael et al. [32] revealed that the mechanical performance of the Y-doped HAp nanorod reinforced high molecular weight polyethylene (HMWPE) composites was higher than those of the pure HAp nanorod reinforced HMWPE composites. The dielectric and electric properties of a biomaterial synthesized for bone substituting or bone repairing applications have a great importance because bone is a dielectric material. Additionally, the electromagnetic fields have been used for bone healing applications, and the effects of the electrical stimulation have been reported [33–39]. Many authors have investigated and determined these properties for HAps and calcium phosphate based samples [40–44]. Even though, in the literature, there are some investigations related to Y-containing HAps, the investigation of their antimicrobial and

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dielectric properties is new in comparison to the earlier studies. In present work, we synthesized the pure- and Y-doped HAp samples using the precipitation method and investigated the effects of the addition of Y on the crystal structure, phase composition, crystallinity, chemical composition, morphology, dielectric properties and antimicrobial activity of HAp.

by the following relation [46]: X C ≈1−

V 112=300 I300

ð5Þ

where V112/300 is the intensity of the hollow between (112) and (300) crystal planes, and I300 is the intensity of the (300) plane.

2. Materials and method 2.1. Synthesis of the samples The yttrium doped HAp samples were synthesized for the various molar ratios of Y/(Ca + Y):0, 0.02 and 0.04, and were named as Y1, Y2 and Y3, respectively. The (Ca + Y)/P molar ratios were adjusted to 1.67. Diammonium hydrogen phosphate ((NH4)2HPO4, Merck) was dissolved in the distilled water using a magnetic stirrer and heated to temperature of 90 °C. Meanwhile, calcium nitrate tetrahydrate (Ca(NO3) 2·4H2O, Merck) and yttrium(III) nitrate hexahydrate (Y(NO3)3·6H2O, Sigma-Aldrich) were also dissolved separately in the distilled water and poured in one flask and then as-prepared solution was added drop by drop to (NH4)2HPO4 solution. During the synthesis, the pH of the mixture was adjusted and kept at ~10 with ammonium hydroxide (NH4OH, Sigma-Aldrich). The reaction was performed at 90 °C for 6 h. Afterwards, the suspension was filtrated and the precipitates were washed out by distilled water and dried in an oven at 110 °C for 22 h. The obtained powders were calcined at 700 °C for 2 h. 2.2. Characterization of the samples 2.2.1. X-ray diffraction (XRD) measurements X-ray diffraction (XRD) analyses were performed on a Bruker D8 Advance diffractometer using a CuKα radiation with wavelength of λ0.15406 nm produced at 40 kV and 40 mA, and the XRD data were collected over the 2θ range of 20°–55° at every 0.02° for the scan speed of 2° min− 1. The crystalline phases were identified by reference to the Joint Committee on Powder Diffraction Standards (JCPDS) files. The percentage of the formation of the hydroxyapatite phase was estimated in the 2θ range of 20°–55° using the following relation: %HAp phase ¼

Area under peaks belonging to HAp phase 100%: Total Area under all peaks

ð1Þ

Similarly, the percentage of the beta tricalcium phosphate (β-TCP) was computed. The lattice parameters (a and c) were calculated with the relation belonging to hexagonal structure [45]: 1 4 h2 þ hk þ k2 ¼ 2 3 a2 d

! þ

l2 c2

ð2Þ

where d is the distance for two adjacent planes, and h, k and l are the Miller indices. The volume of the hexagonal unit cell was calculated by the following relation [45]: 2

V ¼ 0:866a c

ð3Þ

and Scherrer equation can be used to determine the crystallite size (D) [45]: D¼

0:9λ β cosθ

ð4Þ

where β is the full width at half maximum (FWHM) in radian and θ is the diffraction angle in degree. The crystallite size of the samples was evaluated for the perpendicular crystal planes of (002) and (300) as D002 and D300, respectively. The crystallinity degree (XC) was calculated

2.2.2. Fourier transform infrared (FTIR) analysis Fourier transform infrared (FTIR) spectra were collected by a PerkinElmer Spectrum One spectrometer in the region 450–4000 cm−1 using KBr pellets with a spectral resolution of 4 cm−1. 2.2.3. Microstructural observations The microstructure and elemental compositions of the samples were investigated using a scanning electron microscope (SEM, ZEISS EVO 50) equipped with an energy dispersive X-ray (EDX, Oxford Instruments Inca Energy 350) spectrometer operated at 10 kV. The as-synthesized HAp samples were uniaxially compacted into disks, with a diameter of 13 mm and a thickness of 2 mm, using a MTI 24T Desktop Hydraulic Pressing Machine under pressure of 10 MPa. All the samples were coated with a conductive layer of gold for 20 s using a Denton Desk V coater, and then the microstructures were observed. 2.2.4. Antimicrobial activity tests Antimicrobial activities of the pure and Y-containing HAp samples were determined by using agar dilution procedure recommended by the Clinical and Laboratory Standards Institute [47,48]. Minimal inhibitory concentrations for each compound were investigated against standard bacterial strains; Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were obtained from American Type Culture Collection (Rockville, MD.) and the fungal strains Candida albicans and Candida tropicalis were obtained from the Department of Microbiology, Faculty of Medicine, Ege University (Turkey). The inoculum was prepared by making a direct broth of isolated colonies selected from an 18- to 24-hour blood agar plate. Bacterial strains were subcultured on Muller Hinton Broth (HiMedia Laboratories Pvt. Ltd., Mumbai—India) and fungal strains were also on RPMI 1640 Broth (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Their turbidities matched that of a McFarland no. 0.5 turbidity standard, and the absorbance should be 0.08 to 0.13 at 625 nm for this standard [49]. Since the pure and Y-containing HAp samples have limited solubility, the stock solution of all compounds was prepared in dimethyl sulfoxide (DMSO) at 800 μg/ml concentration. All of the dilutions were done with distilled water. 1 ml of this stock solution was mixed with 9 ml of Muller Hinton Agar (MHA), sterilized using an autoclave and cooled until 50 °C. Then MHAs containing the HAp samples were waited until they solidify. By making serial twofold dilutions, the concentrations of the tested compounds were 800, 400, 200, 100, 50, 25, 12.5 and 6.25 μg/ml. Ampicillin and ciprofloxacin were used as antibacterial standard drugs, while fluconazole were used as antifungal standard drugs whose minimum inhibitory concentration (MIC) values are provided. A loopful (0.01 ml) of the standardized inoculum of the bacteria and yeasts (106 CFU/ml) was spread over the surface of agar plates. All the inoculated plates were incubated at 35 °C and results were evaluated after 16–20 h of incubation for bacteria and 48 h for yeasts. The lowest concentration of the compounds that prevented visible growth was considered as the minimal inhibitory concentration (MIC). 2.2.5. Dielectric studies To make the dielectric measurements, all the samples were grinded and uniaxially compacted into disks, with a diameter of 13 mm and a thickness of 2 mm under pressure of 10 MPa. The dielectric measurements were performed using a HIOKI 3532-50 LCR HiTESTER at room temperature. Using Eqs. (6), (7) and (8), the relative permittivity (ε'),


dielectric loss (ε″) and alternating current conductivity (σac) were determined by the following relations [50] ε′ ¼

Cl εo A

ð6Þ

ε″ ¼ tanδ ε0

σ ac ¼

ð7Þ

l ZA

ð8Þ

where εo is the permittivity of free space, A is the area of the electrode, tan δ is the loss tangent and C, l and Z are the capacitance, thickness and impedance of the sample, respectively. The areas of the sample and gold electrode used in the dielectric measurements are 1.327 × 10− 4 and 1.130 × 10− 4 m2, respectively. To determine the conductivity mechanism of the as-prepared HAp samples, the wellknown Jonscher relation [51] was used σ ac ¼ σ dc þ Bω

s

ð9Þ

where σdc is the direct current conductivity, B is a constant, ω is the angular frequency and s is an exponent. 3. Results and discussion 3.1. Phase and crystal structure analyses of the samples The powder XRD patterns of the prepared samples are shown in Fig. 1. The patterns showed that all the peaks are matched perfectly with the standard HAp (PDF no: 09-432) except for a peak belonging to the β-tricalcium phosphate (β-TCP, PDF no: 09-169) phase detected at around 2θ = 31.22°. The formation percents of HAp phase are estimated to be 98.0, 97.4 and 95.7% for Y1, Y2 and Y3, respectively. The percents belonging to the β-TCP phase are found to be 2.0, 2.6 and 4.3% for Y1, Y2 and Y3, respectively. The formation of HAp and β-TCP phases is affected by Y content. With increasing amount of Y, the Hydroxyapatite Y3 -TCP

(300)

(004)

(213)

(321) (410) (402)

(222) (203)

(312)

(310)

(311)

(202) (301)

(210)

(002) (102)

(111)

(200)

3.2. Detection of the functional groups of the samples Fig. 2 shows the FTIR spectra of the pure and Y-containing HAp. The observed bands and their assignments are given as follows. The bands detected at 1037, 601 and 569 cm−1 were assigned to the vibrational modes of the phosphate groups. The sharp band at 3571 cm−1 and a weak one at 631 cm−1 were related with the characteristic vibrational modes of the hydroxyl group. With addition of Y, while the intensity of the band at 3571 cm−1 increased, the intensities of the bands at 1037, 601 and 569 cm−1 decreased. A wide band centered at ~3642 cm−1 and a weak band observed at 1635 cm−1 were associated with the water, which was absorbed in the samples and/or in the KBr pellet [55]. The doublet at 1458 and 1415 cm−1 was attributed to the vibrational modes of the carbonate groups [56], which incorporated at the precipitation stage.

The scanning electron microscope (SEM) micrographs and energy dispersive X-ray (EDX) results of the synthesized samples are shown in Fig. 3. The fine-grained morphology was observed and the particle size did not exceed 1 μm. The Ca/P molar ratio of Y1 was found to be 1.68, which is almost equal to that of the stoichiometric HAp (1.67). For the Y-containing HAp samples, the (Ca + Y)/P molar ratios were found to be 1.66 and 1.69 for Y2 and Y3, and these values also are very close to the stoichiometric value of 1.67. The Y/(Ca + Y) molar ratios

(112)

Y1

Table 1 The calculated values of the crystallite size, crystallinity degree, lattice parameters and volume of the unit cell of the as-synthesized samples.

Standard HAp (JCPDS 09-432)

20

formation of HAp phase decreases, whereas the amount of β-TCP phase increases. No other peaks, nor additional phases were detected with Y addition ranging from 0 to 4 at.%. The relative intensity of the peak at ~ 40.12° belonging to (221) crystal plane was gradually decreased with the addition of Y, and the calculated values were found to be 0.303, 0.288 and 0.257, respectively. The relative intensity of the peak at ~ 32.38° belonging to (112) crystal plane was gradually increased with adding of Y, and the calculated values were found to be 0.464, 0.506 and 0.680 for Y1, Y2 and Y3, respectively. The calculated values of the crystallite size, crystallinity degree, lattice parameters and the unit cell volume are given in Table 1. All the mentioned parameters were significantly affected by the amount of Y. For each sample, the calculated values of D002 and D300 appeared to be close to each other. The estimated values of the crystallite size for (221) crystal plane were found to be 37.59 nm for Y1, 34.39 nm for Y2 and 32.41 nm for Y3. With the addition of Y, the crystallite size was gradually decreased for (221) crystal plane, whereas the change in this parameter did not significantly increase or decrease for (002) and (300) crystal planes. The lattice parameter c, volume of the unit cell and crystallinity degree were gradually decreased by the addition of Y ranging from 0 to 4 at.%, and this result is in good agreement with the reported works in the literature [20,32,52,53]. Since Y3+ ions (ionic radius 0.09 nm) are smaller than Ca2+ ions (ionic radius 0.10 nm), it is expected that Y incorporation might cause the size reduction (shrinkage) of the apatitic structure [20,21,32]. The decrease in the crystallinity degree can be related with the presence of β-TCP phase, namely, the relative intensity of the peak at around 2θ = 31.22° was found to increase with the addition of Y (0.188 for Y1, 0.202 for Y2 and 0.386 for Y3). As known, the amount of the β-TCP content in the HAp can affect the crystallinity [54].

3.3. Microstructure and composition

(211)

Intensity (a.u.)

Y2

335

25

30

35

2

40

45

( )

Fig. 1. XRD patterns of the as-synthesized samples.

50

55

Sample

D002 (nm)

D300 (nm)

XC

a (nm)

c (nm)

V (nm3)

Y1 Y2 Y3

34.13 31.99 32.77

34.84 30.49 33.71

0.906 0.897 0.880

0.9368 0.9351 0.9356

0.6838 0.6828 0.6818

0.5197 0.5170 0.5168

336

O. Kaygili et al. / Materials Science and Engineering C 47 (2015) 333–338 Table 2 Minimum inhibitory concentrations (μg/ml) of the tested compounds.

Transmittance (a.u.)

Y3

Y2

Y1 OH

H2O

H2O 2

CO3

OH 3

PO4 PO4

4000

3600

3200

2800

2400

2000

Wavenumber (cm

1600 —1

1200

800

)

Fig. 2. FTIR spectra for the pure- and yttrium-containing HAp samples.

400

Microorganism

Y1

Y2

Y3

Ampicillin

Ciprofloxacin

Fluconazole

E. coli S. aureus E. faecalis P. aeruginosa C. albicans C. tropicalis

N800 N800 N800 N800 N800 N800

N800 N800 N800 N800 N800 N800

N800 N800 N800 N800 N800 N800

3.12 3.12 1.56 – – –

1.56 0.39 0.78 3.12 – –

– – – – 3.12 3.12

for Y2 and Y3 samples were found to be 0.003 and 0.010, respectively. One can see that both values were smaller than the expected values (i.e., 0.02 for Y2 and 0.04 for Y3). Thus, not all amounts of Y were incorporated into the crystal structure of HAp. Probably, this was due to the charge imbalance. Moreover, this result is in a very good agreement with the results reported by Capuccini et al. [57]. Namely, despite the incorporation of Y rises with increasing amount of Y, this increase is smaller than that of the as-expected or theoretical value.

Fig. 3. SEM images and EDX analysis results of the as-prepared samples.

O. Kaygili et al. / Materials Science and Engineering C 47 (2015) 333–338 -2

10

Alternating current conductivity (S m ) ac

Relative permittivity,

'

18

337

16

14 Y1

12 Y2

10

Y3

-3

10

-4

10

-5

10

Y1 Y2 Y3

-6

10

-7

10

-8

10

8

1

0

6

1x10

6

2x10

6

3x10

6

4x10

10

6

5x10

3.4. Antimicrobial activity of the samples The antimicrobial activity of the pure- and yttrium-containing HAp samples was tested against standard strains Gram-negative E. coli, P. aeruginosa as well as Gram-positive E. faecalis, S. aureus and the fungal species C. albicans and C. tropicalis which are common infection-causing organisms in human. As seen in Table 2, irrespective of yttrium content, no samples exhibited any antimicrobial effect at any tested concentrations (800–6.25 μg/ml). In other words, the addition of yttrium in the HAp structure did not cause any positive effect to improve the antimicrobial activity of the HAp. This is not a surprising result for the pureHAp because it does not possess antibacterial properties [58]. 3.5. Dielectric properties of the samples Figs. 4, 5 and 6 show the plots of the relative permittivity, dielectric loss and alternating current conductivity as a function of frequency, respectively. The calculated values of the relative permittivity at 1 kHz frequency were found to be 12.97, 10.65 and 10.10 for Y1, Y2 and Y3, respectively. These values are in a perfect harmony with the reported values given in the literature for the HAp [50,59–61]. The relative permittivity values vary with increasing frequency, as well as Y content (Fig. 4). The dielectric loss gradually decreases with the addition of Y. For all the samples, a relaxation peak was observed at about 4.6 MHz (Fig. 5). The relaxation times were found to be 34.52 ns for Y1, 3.2 2.8

"

4

10

5

10

6

10

7

10

Fig. 6. Alternating current conductivity vs. frequency plots of the as-prepared samples.

Fig. 4. Relative permittivity as a function of frequency plots of the samples.

Dielectric loss,

3

10

Frequency (Hz)

Frequency (Hz)

Y1 Y2 Y3

2.4

2

10

2.0 1.6 1.2 0.8 0.4

34.60 ns for Y2 and 34.67 ns for Y3. As can be seen from Fig. 6, the alternating current conductivity linearly increases with increasing frequency for all the samples and obeys the universal power law behavior. Furthermore, the alternating current conductivity of HAp gradually decreases with the addition of Y. According to Eq. (8), the s values estimated from the slope of logσac vs. logω plot, and these values were found to be 0.99, 1.01 and 0.99 for Y1, Y2, and Y3, respectively. The measured resistance values of all the samples from room temperature to 220 °C were found to be in the range of 1011–1012 Ω, and this result indicates that the as-synthesized samples exhibited the insulator behavior. s values were equal to unity because all the samples lack measurable direct current conductivity. These results explain the conductivity mechanism of the samples. Namely, the hopping motion involved a translational motion with a sudden hopping [62,63]. All the above-mentioned findings indicate that the addition of Y affects the dielectric properties of HAp and the dielectric parameters of the relative permittivity, dielectric loss and alternating current conductivity may be controlled by Y content. As mentioned earlier in the XRD results, the phase composition is affected by Y content. In other words, the dielectric properties are affected by the phase composition changed with the amount of Y [64]. 4. Conclusions High crystalline HAp samples doped by 0, 2 and 4 at.% Y were prepared and investigated. The formation of the HAp phase in all samples was proven by XRD patterns and FTIR spectra. The crystallite size, crystallinity degree, lattice parameters and the unit cell volume were affected by Y content. With the addition of Y into HAp, the crystallinity degree decreased from ~ 91% to ~ 88%, the lattice parameter c was decreased from 0.6838 nm to 0.6818 nm, and the unit cell volume was also decreased from 0.5197 nm3 to 0.5168 nm3. The microstructure and particle size distribution were changed with the addition of yttrium. The numeric values for Ca/P and (Ca + Y)/P ratios for all samples were found to be almost similar and close to the initial values in the beginning of the synthesis. It was found that no samples had any antimicrobial effect. The relative permittivity, dielectric loss and alternating current conductivity change with increasing frequency and the alternating conductivity gradually decreases with the addition of Y. References

0.0 0

6

1x10

6

2x10

6

3x10

6

4x10

6

5x10

Frequency (Hz) Fig. 5. Dielectric loss as a function of frequency plots of the as-synthesized samples.

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