Characterization of nano-biphasic calcium phosphates synthesized

Journal of Nano Research Vol. 3 (2008) pp 67-87 online at http://www.scientific.net © (2008) Trans Tech Publications, Switzerland Online available since 2008/Oct/01

Characterization of nano-biphasic calcium phosphates synthesized under microwave curing Wafa I. Abdel-Fattah 1a, Fikry M. Reicha 2b, Tarek A. Elkhooly1c 1

Biomaterials Department National Research Center, Cairo, Egypt. 2

Physics department Faculty of Science, Mansoura University [email protected], [email protected], [email protected]

a

Received Feb. 27, 2008; received in revised form May. 4, 2008; accepted July. 30, 2008

Keywords: Nanoparticles, calcium deficient hydroxyapatite, chemical precipitation, microwave curing, Biphasic calcium phosphate

Abstract

Two biphasic BCP ceramic samples were synthesized by chemical precipitation and microwave curing of calcium deficient hydroxyapatite CDHA under the same pH value and temperature but varied in their initial Ca/P molar ratio. Precipitates were characterization after thermogravimetric analysis, fourier transform infrared spectroscopy, X-ray diffraction, atomic absorption spectroscopy and TEM. Hydroxyapatite (HA) contents were measured for the two biphasic calcium phosphate (BCP) ceramics by sintering the calcium-deficient apatites (CDHA). The results reveal two condensation mechanisms of HPO42- affecting the Ca/P molar ratio after calcination. The X-ray diffraction patterns of BCP powders show the in situ formation of -TCP in the BCP powder. The amount of -TCP phase increases as the initial Ca/P molar ratio decreases due to more calcium deficiency in CDHA structure. The influence of HPO42- incorporation on increasing -TCP phase content after calcination is evaluated. TEM micrographs proved the effect of microwave curing during the preparation process on reducing of particle size to nanoscale range and the destruction of CDHA to finer HA and -TCP particles upon calcination.

Introduction Hydroxyapatite (HA) and tricalcium phosphate (TCP) are widely used in the field of orthopaedic and dentistry. Recently, biphasic calcium phosphate (BCP) ceramics has drawn much attention as an ideal bone substitute due to its controlled degradability. The material is gradually dissolves in the body, seeding new bone formation as it releases calcium and phosphate ions into All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, www.ttp.net. (ID: 41.237.146.96-28/03/09,11:05:33)

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Journal of Nano Research Vol. 3

the biological medium. An ideal biomaterial for hard tissue repair should be biocompatible, osteoconductive, resorbable, and osteoinductive. Attention is directed recently to the development of resorbable and osteoinductive biomaterials from calcium phosphates [1]. Tricalcium phosphate (TCP) degrades in an unpredictable way, i.e., it dissolves 12.3 times faster than HA in acidic medium and 22.3 times faster in basic medium and so it may not provide a scaffold for new bone to grow [2]. The biphasic calcium phosphate (BCP) ceramic has controlled biodegradability, thus producing calcium ions in the microenvironment inducing the bone formation and gradual remodeling of the new bone [1]. Due to the preferential dissolution of -TCP component, bioreactivity of BCP bioceramics can be controlled by manipulating the composition (HA/-TCP ratio) and/or its crystallinity [3]. Therefore, it is necessary to produce the biphasic ceramics with various HA/TCP ratios in a simple and systematic way. Biphasic calcium phosphates of varying HA/-TCP ratios can be prepared by mechanical mixing of -TCP and HA powders in desired quantities [4] or by chemical methods through calcium-deficient apatites [CDA; Ca10x(HPO4)x(PO4)6-x(OH)2-x],

which should be subsequently sintered above 700°C to give both HA and

-TCP whose ratio reflects the former CDA Ca/P [5]. Solid state reaction between two commercially available calcium-based precursors namely, tricalcium phosphate (TCP) and calcium hydroxide (Ca(OH)2) is another alternative [6]. It was reported that an optimal composition of HA/TCP as a scaffold for stem-cell-induced bone formation was 20/80 HA/TCP as, it have the greatest effect on bone formation and acquire an osteoinductive properties [7]. Preparation of BCP from sintered calcium deficient hydroxyapatite (CDHA) would appear to be an effective process over mechanical mixing in conservation of bioactivity and biodegradability, increased the purity of the ceramic and displayed interesting mechanical properties. The -TCP ceramics which have a good ability of osteointegration show poor ability of apatite layer formation both in vitro as well as in vivo [8]. Nanomaterial is considered as a new class of material since it possesses superior properties over its microscale counterpart. Nanocrystalline HA promotes osteoblast cells adhesion, differentiation, and proliferation, osteointegration and deposition of calcium containing minerals on its surface better than its microcrystalline; thus enhancing the formation of new bone tissue within a short period [9]. Additionally, it was reported to improve sinterability and densification due to its greater surface area, which could improve the fracture toughness and other mechanical properties. Nano-HAp is also expected to have better bioactivity than coarser crystals. Nanocrystalline calcium phosphate has the potential to revolutionize the field of hard tissue engineering from bone repair and augmentation to controlled drug delivery devices [10].


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The microwave has an advantage over previous methods of very short time, small particle size, narrow particle size distribution and high purity [11]. It is also believed that microwave irradiation could improve the microstructures of products, thus if the microwave irradiation is introduced in HAp preparation process, the reaction does not occur via crystallograsphic transformation consequently HAp should be precipitated in very small nanosized crystallites [12]. In this study, biphasic HA/TCP compound powders were synthesized using a coprecipitation method and microwave curing in mother solution. The influence of initial Ca/P ratio on the degree of calcium deficiency in the prepared dried CDHA is investigated. The microwave technique for preparing biphasic calcium phosphate nanoscaled powders was applied. Also, the role of HPO42- on the ratio of BCP in calcined powders is followed.

Experimental

Biphasic HA/TCP powders were synthesized by using co-precipitation method with Ca(OH)2 (ANALAR, UK) and (NH4)2HPO4 (NORMAPUR, CE-EMB). Two initial Ca/P ratios were chosen to be 1.56 and 1.61 coded B1D and B2D respectively. Calcium hydroxide is a strong base of pH equal to 12. Therefore in order to decrease and maintain its pH value around 8, both NH4OH (ADWIC, Egypt) or HCl (POCH, Poland) were added during precipitation method.

The

temperature of the calcium hydroxide solution was maintained at 50oC during the addition of (NH4)2HPO4 in dropwise manner at rate of 5 ml/min. The precipitates formed were left in the mother liquor to mature overnight about 16 hrs. The obtained precursor solutions were transferred to a domestic microwave oven (2450 Hz, moulinex, china) and irradiated with microwave for 1 hrs [13]. The operating power of the oven was adjusted to be 350 W to avoid the overflow of the solution from the reaction vessel during the process inside the microwave oven. Care was taken to release the ammonia gas every 15 min evolved during the reaction [14]. The prepared powders were dried overnight at 100oC and then calcined in an oven (JLabJech, Korea) at 900oC for 3 h and left to cool overnight to obtain BCP ceramic phases.

Characterization Ca2+ ions concentrations were determined using an atomic absorption spectrophotometer (spectraAA Varian, Australian). The structure of dried and calcined samples was assessed using an X-ray powder diffractometer (Bruker Axs D8 Advance, Germany) with Cu Kα target (Ni filter), wavelength (λ) of 1.54 Å was used. The XRD runs were carried out at a scanning speed of 2θ= 2o/min. The functional groups present in dried and calcined samples were ascertained by Fourier transform infrared spectroscopy (FT-IR) (Nicolet Spectrometer model 670 with a FT-Raman

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accessory, USA). The FT-IR spectra were obtained over the region 400–4000 cm−1 using KBr pellet technique with spectral resolution of 4 cm−1. Two mg of the powder samples were carefully mixed with 198 mg dried KBr powder and pressed into pellets. In order to obtain information about the chemical changes which occur during preparation of calcium phosphate from calcium deficient hydroxyapatite (CDHA), thermo-gravimetric analysis (TGA) was performed. Briefly, 15-25 mg of CDHA were placed into a small platinum crucible and heated from room temperature to 1050 oC in nitrogen atmosphere under 20 ml/min with a heating rate of 10oC.min-1. A TGA system (Perkin Elmer, USA) installation was used. Changes in the morphology of the synthesized and calcined powders were examined via transmission electron microscopy (TEM). 2 mg of powders were dispersed in 20 ml bi-distilled water and then drops of the suspension were placed on carbon-coated grids. The grids were observed with (JEM-1230FX, JEOL Co., Tokyo, Japan) operated at 120 kV. High magnification (80Kx, 150Kx) bright field (BF) images and selected area diffraction (SAD) patterns were recorded.

Results

Chemical analysis revealed that sample B1D prepared with lower Ca/P of 1.56 molar ratio exhibited lower Ca ions (8.26 wt %) concentration proving more deficiency than B2D (12.17 wt %) and consequently higher amount of -TCP phase in the biphasic sample. The Ca ion concentrations increased upon calcination and recorded 12.67 and 17.22 wt% for B1C and B2C samples, respectively.

Table 1 The lattice parameter and x values of synthesized CDHA, the crystallite size of HA and -TCP reflection, the fraction of -TCP formed in the calcined BCP samples, and the HA/-TCP ratio of both calcined BCP samples. Crystal size (nm) HA -TCP

Lattice parameters a=b

c

002

300

214

0 2 10

Fraction of -TCP

B1D B2D

9.4455 9.4404

6.8841 6.8810

57.55 52.52

41.75 40.78

– –

– –

– –

B1C9

–

–

58.78

60.71

79.39

66.92

39.4 %

B2C

–

–

58.98

59

88.44

72.31

31.4 %

BCP samples

D: stands for dried and C: stands for calcined x: degree of deficiency

HA/TCP ratio – – 56% : 44% 66% : 34%

x value of CDH A 0.46 0.34 – –


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Figure 1 X-Ray diffraction patterns of dried BCP samples (a) B2D and (b) B1D… H corresponds to hydroxyapatite maxima XRD patterns of both synthesized biphasic samples (Figure 1) proved poor crystalline HA namely calcium deficient hydroxyapatite CDHA (Ca10-x (HPO4)x(PO4)6-x(OH)2-x .nH2O, x=0-1, n=0-2) with peak positions matching closely those of stoichiometric hydroxyapatite (JCPDS No. 9-432). Negligible difference could be detected between both samples. Crystallographic identification of the phases of synthesized apatites was accomplished by comparing the experimental XRD patterns to

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standards complied by the Joint Committee on Powder Diffraction Standards (JCPDS) using computer program (PCPDFWIN Version 2.2 June 2001 JCPDS-ICDD). The determination of lattice parameters of hexagonal hydroxyapatite using Rietveld refinement (Table 1) revealed that the (a-axis) of sample B1D increased by about 0.005 Å over the other B2D sample, possibly due to more incorporation of HPO42- into the structure synthesized with lower Ca/P molar ratio leading to higher deficiency. Young and Holcomb [15] reported that the (a) lattice parameter is affected much more strongly than is the (c) one by CO32- and many other substitutions. They although claimed that HPO42- expands the a axis at the rate of ~ 0.0015 Å/wt % it is still smaller than the effect of CO32-, which is +0.026 Å/wt % for A type and -0.006 Å/wt % for B type CO32-.Moreover, the replacement of CO32- ions in the OH– site in CDHA structure caused an enlargement in (a-axis) [16]. Using the Debye-Scherrer methods and from the full width at half maximum (FWHM) of the reflection 3 0 0 in the direction of (a-axis), the crystallite sizes for both samples were 41.75 nm and 40.78 nm for B1D and B2D respectively. This is consistent with larger (a-axis) for sample B1D due to its higher deficiency. The (c-axis) of the CDHA hexagonal structure increased for the sample B1D by 0.003 Å over sample B2D due to more CO32- replacement in the phosphate site. Also, the calculated crystallite size in the (c-axis) direction due to reflection (0 0 2) agreed with the increased (c-axis) for sample B1D. The X-ray diffraction patterns of the calcined microwave synthesized BCP powders (Figure 2) were compared with commercial -TCP (Cerasorb®, Germany) coded as R and commercial HA (BDH chemical Ltd Pool, England) coded as HR. The most intense peaks of HA were detected at d values 2.818, 2.781, and 2.725 Å for sample B1C9 while 2.816, 2.778, and 2.721 Å with negligible shift were recorded for sample B2C9 corresponding to reflections 211, 112, and 300, respectively (JCPDS file No. 73-294). Additionally, the most intense peaks of  -TCP were detected at d values 2.883, 2.610, and 3.211 Å for sample B1C9 while 2.880, 2.611, and 3.208 were detected for sample B2C9 corresponding to reflections 0 2 10, 220, and 214 , respectively (JCPDS file No. 9169) (Figure 2). The parent BCP powders synthesized using wet chemical method according to reaction (1) similar to MANJUBALA et al [14] work or according to equation (2) no other phases of calcium phosphate were present in the final product except HA and TCP. 10Ca(OH)2 + (6+x)(NH4)2HPO4 Ca10-x(HPO4)x(PO4)6-x(OH)2-x

(1-x) Ca5(PO4)3(OH) + xCa3(PO4)2 +H2O+NH3 (1) (1-x)Ca10(PO4)6(OH)2 + 3xCa3(PO4)2 + xH2O

(2)


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The mass fraction of the HA phase present in the BCP ceramic was calculated by the simple height law equation % mass fraction of HA = I300(HA)/ (I300(HA) + I0 2 10(TCP)) x100

Figure 2. X-Ray diffraction patterns of calcined BCP samples (a) commercial -TCP (b) commercial HA (c) B2C and (d) B1C9 … H, , and P corresponds to hydroxyapatite, beta tricalcium phosphate, and beta pyrophosphate maxima, respectively

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Where I0 2 10(TCP) and I300(HA) are the normalized integrated intensities of the main peaks of TCP and HA, respectively [1] being (d= 2.883 Å, 2θ= 30.996) and (d= 2.8179 Å, 2θ= 31.728) respectively. The ratios of HA/TCP in the BCP was calculated to be 56/44 and 66/34 for B1C9 and B2C samples respectively (Table 1). Since calcium hydroxide and diammonium phosphate were used as precursors, there was no formation of other products in the reaction except for HA and TCP. The second most intense reflection 300 of HA was chosen for % ratio calculation because the most intense reflection of HA 211 is not an isolated peak to calculate its integration under peak. By increasing the x value in both equations 1 and 2 due to the increased deficiency (i.e. more HPO42was incorporated in the structure of (CDHA), and the amount of -TCP phase increased in the calcined BCP samples. Moreover, the amount of -TCP increased as the excess amount of the phosphate solution increased during the preparation procedure i.e. decreasing of the Ca/P molar ratio [14]. For CDHA, the biphasic mixture contains HAP and -TCP. According to reaction (2), the Ca/P ratio of the initial powder could be related to the proportion of HAP or TCP as proposed by S. Raynaud et al [17] wt% HA = 100-wt% -TCP ≈ 600xCa/P-900 Also, the Ca/P molar ratio of the corresponding synthesized apatites could be calculated following the equation proposed by M. Vallet-Regi et al [18] Ca/P = (1.5% TCP + 1.667% HA)/100 Which give the x value in Ca10-x(HPO4)x(PO4)6-x(OH)2-x by: x = 10 – 6Ca/P The later equation was used to calculate the amount of calcium deficiency in the initial precursors. X values of the two samples (Table 1) were 0.46 for the sample B1D for the calculated Ca/P molar ratio 1.59. While, this x value being smaller than the expected value (0.64) for the initial Ca/P molar ratio (1.56) calculated for the sample B1D, indicate the increase in the Ca/P molar ratio during heat treatment of this sample. Sample B2D whose x value (0.34) confirmed its deficiency being prepared with higher initial Ca/P molar ratio (1.61) and final Ca/P ratio was recorded (1.61) from the HA/-TCP wt% ratio. To confirm the increase in the deficiency of the sample B1D, The fraction of -TCP of both samples B1C9 and B2C9 was calculated by comparing the (0 2 10) normalized integrated peak area (2θ = 31.021o) of the sample containing 100% -TCP obtained from Cerasorb®.


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i.e., fraction f = As/A100%-TCP as suggested in Sz-Chian Liou and San-Yuan Chen work [19] (Table 1). The fraction of -TCP was larger in sample B1C9 (39.4%) due to its lower initial Ca/P molar ratio and while that of sample B2C9 (31.35%), confirm the previous results proving the effect of initial Ca/P molar ratio on the amount of -TCP obtained after calcination. Beta pyrophosphate -Ca2P2O7 most intense peaks are located in positions corresponding to d values 3.22, 3.09, and 3.020Å according to reflections 202, 203, and 008 respectively. The first and third most intense peaks of -Ca2P2O7 were detected in the sample B2C at d values 3.02 and 3.09 Å respectively while, its peak located at 1.997 Å according to reflection 219 was detected in sample B2C. These peaks are indexed according to (JCPDS file No. 9-346). The crystallite size of both HA and -TCP phase (Table 4.5) of reflections 002 and 300 were quite similar for both BCP samples. On the other hand, the reflections 214 and 0210 corresponding to the most intense peaks of -TCP increased in the sample B2C9 may be due to the presence of P2O72- inducing the formation of -TCP phase according to reaction (3). 2 HPO42-

P2O72- + H2O

(3)

Characterization of biphasic calcium phosphates

Dried samples Figure 3 illustrates the FT-IR absorption spectra of the synthesized dried samples. Phosphate bands PO43The bands at 1091 and 1031 cm-1 were assigned to the components of the triply degenerate (3) antisymmetric P–O stretching mode. The 960 cm-1 band was assigned to (1); the nondegenerate P–O symmetric stretching mode. The bands at 603 and 564 cm-1 were assigned to components of the triply degenerate4) O–P–O bending mode and the bands in the range of 434 and 474 cm-1 were assigned to the components of the doubly degenerate 2) O–P–O bending mode [20]. The pure Hydroxyapatite is characterized by the vibration at 472 cm-1 [21] but the appearance of the band at 434 cm-1 could be attributed to the split of 4) vibrational mode which occur in the case of-TCP ceramic phase. Therefore, the increase in the intensity of band 474 cm-1 in sample B2D proved the increased HA amount in B1D sample. On other hand, it’s decreased intensity along with appearance of the band at 434 cm-1 indicate the reduced HA amount. The splitting of the degenerate PO43- 3 and 4 bands gradually appears in the -TCP crystal. This observation could be

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due to site-symmetry splitting of the degenerate modes as the environment of the PO43- groups became more structurally ordered [22]. The 2077 and 1990 cm-1 bands are the overtone and the combination (PO43-) band. [21]

Figure 3 FT-IR spectra of synthesized dried BCP samples (a) B1D and (b) B2D


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HPO42- band The weak peak at 875cm−1 corresponding to P–O–H in-plane and out-of-plane deformation modes, is overlapping with the carbonate band vibrations at 872 cm−1 [20]. This band is exited in nonstoichiometric HA or CDHA.

Water and structural hydroxyl bands Molecular and adsorbed water bands are also discerned at 1646 and 3424 cm-1. The broad band of adsorbed water at 3100–3500 cm−1 remains significant due to the absorption of moisture from environment [20]. A significant concentration of hydroxyl groups remains in the structure as observed from the intensity of the stretching and librational bands at 3570 and 632 cm-1 respectively. Both structural hydroxyl band intensities are reduced in B1C9 sample confirming, therefore, its deficiency.

Carbonate bands The large levels of CO32- groups in B1D sample prove that they are involved in the lattice of this defective HA, mainly instead of PO43- at this site due to the appearance of band with large intensity at 1403 cm-1. Legeros [3] reported that the BCP composition (HA/-TCP ratio) obtained after sintering depends on the calcium deficiency of the unsintered biologic or synthetic apatite and on the sintering temperature. The presence of other ions during the synthesis of the CDHA can also affect the HA/-TCP ratio after sintering. The incorporation of carbonate or fluoride ions in the synthetic apatite was reported to result in a higher HA/-TCP ratio in the BCP. On the other hand, the incorporation of magnesium (Mg) or zinc (Zn) resulted in a lower HA/-TCP ratio. Therefore, the presence of CO32- ion in the spectrum of sample B1D lead to increase the initial Ca/P molar ratio from 1.56 to 1.59 after calcination at 900oC for 3 h. These carbonate ions are detected in sample B2D with a lower intensity which may not affect Ca/P ratio after sintering. These results complement XRD data. The bands at 2851 and 2927 cm-1 detected in both samples are attributed to 1 C-H symmetric stretch of HCO2- [23]. Calcined samples Phosphate bands PO43Sample B1C9 calcined at 900oC is characterized by intense and well-defined phosphates absorption bands at 1090 and1033 (3 PO43-), 961 and 971 (1 PO43-), 601 and 569 (4 PO43-) cm-1, and 463 and 473 (2 PO43-) cm-1. The other sample B2C9 exhibits phosphate bands at similar positions to sample B1C9 (Figure 4). feature-rich absorption bands between 900–1250 cm–1 and

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450–650 cm–1 are corresponding to 3 and 4 phosphate absorption modes of crystalline -TCP [24]. The structural changes probably involved distortions in the PO43- tetrahedra [25]. Three different types of crystallographically non-equivalent PO43- groups in the -TCP structure were reported [27].

Figure 4 FT-IR spectra of calcined BCP samples (a) B1C9 and (b) B2C9


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Pyrophosphate band A band at 719 cm-1 attributed to pyrophosphate groups (P2O72-) is observed in the spectrum of sample B2C9 (Figure. 4.b) but not in sample B1C9 in agreement with XRD results. The appearance of this band proved the condensation of hydrogenophosphate groups in Ca-dHAP (equation 4) in the range 250–600oC according to a mechanism reported by Hidekazu Tanaka et al. [27]. Above 600oC, the decomposition of the apatite into a mixture of stoichiometric HAP and TCP are described in equation (5). Ca10-x(HPO4)x(PO4)6-x(OH)2-x (0 < x < 1) Ca10-x(P2O7)x(PO4)6-2x(OH)2

Ca10-x(P2O7)x(PO4)6-2x(OH)2

(250-600oC) (4)

(1–x) Ca10(PO4)6(OH)2 + 3x-Ca3(PO4)2 + xH2O(above 600oC)

(5) The presence of pyrophosphate is confirmed by band at 875 cm-1 detected in the dried samples and assigned to the P–(OH) stretch of HPO42- negligible contribution from 2 CO32-. The second most intense CO32- band also absorbs near this frequency. The pyrophosphate groups (P2O72-) is not recorded in the sample B1C9. Another mechanism of HPO42- condensation according to equation (6) is due to the interaction between HPO42- and the large amount of CO32ions, detected in the dried sample, result in PO43- formation [28]. This explains why no pyrophosphate ions are formed in sample B1C9. 2HPO42- + 2CO32-

2PO43- + H2O + 2 CO2

(6)

Water and structural hydroxyl bands The band corresponding to the absorbed water detected in the dried samples records much lower intensity in the calcined samples. The hydroxyl band at 632 cm-1 was reported to be reduced in the samples containing more TCP in BCP mixtures. [14]. Thus this peak area is reduced in sample B1C9 (Figure 4.a) which confirm its lower HA/-TCP ratio as calculated by XRD. Generally, both absorption bands assigned to structural hydroxyl groups located at 633 and 3570 cm-1become sharper with increased intensity after calcination. Thermal Analysis (TGA/DTG) A minute weight loss (1.126%) at 77oC and (1.194%) at 80oC are recorded for B1D and B2D samples respectively (figure 5) due to weakly entrapped water [29]. Major weight loss 7.153% between 135oC and 490oC was recorded in B1D sample. A loss value of only one third of this value (2.459%) between 199oC and 495oC is recorded for B2D sample. This is attributed to the removal of lattice water yielding lager % in the former sample. The area of differential curve at 221oC for sample B1D is larger compared with the sample B2D around 348oC. These results are in good agreement with FT-IR H2O bands. The decomposition of B-type CO32- ions located in

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phosphate site in the structure of synthesized CDHA started at 490-650oC and peaked at 575oC with accompanied weight loss of 0.35%. The weight loss in this region for B2D sample record lower value of 0.278% due to the increase in CO32- content in the former sample as proved by the larger the band around 1403 cm-1. The minute weight loss between 650 and 675oC recording 0.38% and 0.278% for B1D and B2D samples respectively are due to decomposition of HPO42- according to equations (6) for sample B1D and equation (7) for the sample B2D. The decomposition of hydrogen phosphate was explained by Hidekazu Tanaka et al.[27] according to equations (4) and (5). P2O74- + 2OH

2PO43- + H2O

(7)

Figure 5 TGA and DTG in nitrogen atmosphere of synthesized and microwave cured biphasic samples (a) B1D and (b)B2D.


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Table 2 TGA data Comparison of synthesized and microwave cured biphasic samples B1D and B2D. Temp. range o C 50-135 135-490 – 490-650 650-835 – 835-960

B1D wt. Peak Loss 0 C % 1.126 77 7.153 221 – – 0.350 575 0.38 754 – – 0.245 907

Total loss %

9.254

B2D Area % -0.185 -4.821 – -0.238 -0.339 – -0.314

Temp. range o C 50-199 – 199-495 495-675 675-820 820-930 –

wt. Loss % 1.194 – 2.459 0.278 0.388 0.163 –

Peak 0 C

Area %

80 – 348 590 755 861 –

-1.045 – -1.63 -0.37 -0.444 -0.219 –

4.482

The final stage of weight loss occurs between 835-960oC with 0.245% and 0.163% loss for samples B1D and B2D respectively. This is assigned to the decomposition of CDHA to the biphasic compound according to equation (2). The increase of weight loss in this region indicates more transformation to -TCP phase. Therefore, the increase in -TCP content in the sample B1D than sample B2D (table 2) is consistent with the results obtained with XRD pattern.

Morphology (TEM) Dried samples The TEM micrographs of the as synthesized dried calcium deficient hydroxyapatite with initial Ca/P molar ratio of 1.56 and 1.61 (Figure 6) reveal agglomerates of nanoparticle with rodlike shape having diameter range of 14-39 nm and length range of 93-159 nm for B1D sample. The diameter range of 25-34 nm and a rod length range of 81-131 nm is detected for B2D sample. The increase in rod diameter for the former sample having lower Ca/P molar ratio is consistent with its increased (a-axis) due to more incorporation of HPO42- in the calcium deficient phosphate site. While, the increase in its rod length is consistent with FT-IR results of dried sample which indicate more incorporation of CO32- (B-Type) leading to an increase in (c-axis) due to the repulsive force between phosphate and hydroxyl in the calcium deficient lattice. The higher incorporation of carbonate as the initial Ca/P molar ratio decreased is inconsistent with Aizawa et al [30] results. They claimed that the carbonate content in calcium deficient hydroxyapatite prepared by chemical precipitation method increased as the initial Ca/P ratio increased. The selected area diffraction (SAD) shows diffuse ring pattern, which characterize the nanocrystallinity of CDHA [31].

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However, more spots are observed in the diffraction pattern of sample B2D indicating more crystallinity as the initial Ca/P ratio increased.

Figure 6 Bright field TEM micrographs and electron diffraction pattern of synthesized dried CDHA powders B1D powders (a) and (b) and B2D powders (c) and (d).

calcined samples The TEM micrograph (Fig. 7) of the calcined biphasic phase has an advantage over SEM image to distinguish between hydroxyapatite rods and -TCP spheres [3]. HA rod-like shape has diameter of


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28 nm and length of 60 nm and b-TCP sphere-like shape has a diameter range from 30-35 nm for both sample. The difference between two samples with different Ca/P molar ratio and different HA/-TCP ratios could not detected by using traditional TEM. It seems that to reveal the effect to HA/-TCP ratios variation on the its morphology, high resolution TEM should be used. The selected area diffraction (SAD) shows sharp diffused ring pattern for B1C sample which indicate its nanocrystalline nature.

Figure 7 Bright field TEM micrographs and electron diffraction pattern of calcined BCP samples B1C powders (a) and B2C powders(b).

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Discussion Nanocrystalline biphasic calcium phosphate was successfully prepared via simple chemical precipitation method and microwave curing during aging period. The use of calcium hydroxide as a starting material is useful to eliminate the byproduct. Microwave synthesis of ceramics offers the advantage of efficient transformation of energy and heating throughout the volume efficiently in shorter period leading to finer crystallites [14]. In the present work, the microwave irradiation is considered responsible for reducing biphasic calcium phosphate particle size to the nanometric scale. This reduction should have resulted from the rotation of microwave active dipole H2O molecules which causes repulsion between similar charges in H2O molecules leading to more intensive dispersion during maturation period of the calcium deficient hydroxyapatite. Also, the microwave radiation seems to be effective in the solvation of calcium deficient hydroxyapatite particles during its crystallization with polar water solvent. Each particle surrounded with polar H2O molecule resulted in repulsion between adjacent one which produce well dispersion and prevent its agglomeration. The transformation of calcium deficient hydroxyapatite to finer crystalline BCP with different ratios was proved by Transmission electron microscope (Fig. 7). The difference in the Ca/P ratio during the preparation has lead to the variation in the HA/TCP ratio. The XRD profile of BCP proved the microwave radiation efficiency to produce pure phases as the percentage of pyrophosphate phase is negligible and stabilize -TCP without using stabilizing agent such as Mg2+. The HPO42- resulted from the occupation by H2O molecules at the vacant OH– sites and hydrogen bonding to the nearest PO43− groups [32]. This incorporation of HPO42- resulted in an increase in aaxis of CDHA similar to other work [32]. As its amount calculated from x-ray diffraction (i.e. x value of CDHA) becomes larger the a-axis become longer and the amount of -TCP increased after CDHA calcination. HPO42- group has to mechanisms of condensation or transformation to phosphate group through interaction with B-type carbonate group [28] or through its transformation to pyrophosphate [27] as confirmed by FT-IR results. The amount of HPO42- could be increased by decreasing both pH value during synthesis [33] or by decreasing the synthesis temperature [17]. Also, the increase in ageing time resulted in thermal stable hydroxyapatite by reducing the amount of incorporated HPO42- [34]. In our work, the decrease of HPO42- amount is proved by increasing the initial Ca/P molar ratio. The calculated weight loss from TGA curves recorded double value for lower Ca/P molar ratio sample due to more carbonate and water bands in its FT-IR curve (Fig. 3.a). The calculated number of surface adsorbed and lattice water molecules between 50oC to 495oC collectively in sample B1D is double that of B2D being 0.460 compared to 0.203 coinciding with FTIR spectra (Fig. 3). The increase in weight loss above 650oC for CDHA with lower Ca/P molar


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ratio coincide with the increase in calcium deficiency and -TCP amount after calcination as the starting Ca/P molar ratio decrease.

Conclusion In situ preparation of BCP samples with two initial Ca/P ratio resulted different ratios of HA to -TCP phases, where the HA phase increases as the initial Ca/P ratio increases. The amount of incorporated HPO42- could be varied by controlling the initial Ca/P molar ratio and fix the synthetic temperature, pH value and aging time. The microwave curing of the precipitated solution is efficient method to prepare nanoparticles and increase the particles homogeneity. The destruction of nanorod CDHA to much smaller nano sphere -TCP and HA nanorod is proved in this study to distinguish between the insitu BCP preparation over mechanical mixing.

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Journal of Nano Research Vol. 3 doi:10.4028/www.scientific.net/JNanoR.3 Characterization of Nano-Biphasic Calcium Phosphates Synthesized under Microwave Curing doi:10.4028/www.scientific.net/JNanoR.3.67