carbonated hydroxyapatite nanobiocomposites

Journal of Molecular Structure 1150 (2017) 188e195

Contents lists available at ScienceDirect

Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Effect of sintering temperatures on the in vitro bioactivity, molecular structure and mechanical properties of titanium/carbonated hydroxyapatite nanobiocomposites Rasha A. Youness a, Mohammed A. Taha b, Medhat A. Ibrahim a, * a b

Spectroscopy Department, National Research Centre, El Buhouth St., 12622, Dokki, Giza, Egypt Solid-State Physics Department, National Research Centre, El Buhouth St., 12622, Dokki, Giza, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 July 2017 Received in revised form 17 August 2017 Accepted 20 August 2017 Available online 20 August 2017

Titanium-containing carbonated hydroxyapatite (Ti-CHA) nanocomposite powders, with different CHA contents, have been prepared using high-energy ball milling method. The effect of sintering temperatures, 900, 1100 and 1300
C on molecular structure and microstructure of these samples were examined by XRD; Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM), respectively. Furthermore, their mechanical properties including hardness, longitudinal modulus, Young's modulus, shear modulus, bulk modulus and Poisson's ratio were measured by ultrasonic nondestructive technique. Moreover, bioactivity of sintered samples at different firing temperatures was assessed by immersing them in simulated body fluid at 37 ± 0.5
C for 7 days and then, analyzed by FTIR spectroscopy. The results pointed out that increasing sintering temperature up to 1100
C caused significant increases in densities and mechanical properties of these nanocomposite samples. However, further increase of firing temperature to 1300
C was responsible for complete CHA decomposition and the resultant a-tricalcium (a-TCP) phase greatly affected these properties. On the contrary, better bioactivity was observed for sintered samples at 900
C only. However, increase of sintering temperature of these samples up to 1300
C led to severe decrease in their bioactivity due to the formation of highly soluble a-TCP phase. © 2017 Elsevier B.V. All rights reserved.

Keywords: In vitro bioactivity Mechanical properties FTIR spectroscopy Nanobiocomposites

1. Introduction Owing to the ageing populations worldwide, there is a strong need for developing biomaterials for tissue engineering applications [1]. Accordingly, hydroxyapatite (HA) is considered as the most common bioceramic which can be used extensively for repairing as well as reconstructing diseased or damaged hard tissues as a result of its excellent biological characteristics over other biomaterials [2]. Furthermore, carbonated hydroxyapatite (CHA) exhibits better biological properties than stoichiometric one due to a considerable decrease in its crystallinity character and a marked increase in surface area. Thus, it presents higher bioactivity and consequently; CHA is more applicable in biomedical applications [3e6]. Additionally, nano-sized CHA achieves better bioactivity over micron-sized one due to its large surface to volume ratio and

* Corresponding author. E-mail address: [email protected] (M.A. Ibrahim). http://dx.doi.org/10.1016/j.molstruc.2017.08.070 0022-2860/© 2017 Elsevier B.V. All rights reserved.

unusual chemical/electronic synergistic effects. Unfortunately; low toughness and weak bending strength are the main drawbacks of its uses in load-bearing sites applications [7]. Therefore, these mechanical limitations can be avoided by reinforcing CHA with another phase to achieve the desirable mechanical strength [8e11]. Considering the excellent properties of titanium (Ti) and titania (TiO2) such as superior photocatalysis, high corrosion resistance, appropriate mechanical properties, biocompatibility, non-toxicity and excellent antibacterial characters [12], they can be widely used in many biomedical applications such as joint replacement parts for hip, dental implants, knee, elbow, spine, shoulder etc. Furthermore, TiO2 promotes cell growth, exhibits good osteoconductive property and enhances osteoblast adhesion [13e15]. It is worth to note that TiO2 have been used as excellent photocatalyst for efficient decay of organic compounds and several environmental contaminants [16e21]. Consequently; CHA/Ti nanocomposites can achieve these basic requirements for using in orthopaedic field. Mechanical alloying (MA) method is considered as the most

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

efficient technique that able to alloying elements those are difficult or even impossible to combine with traditional methods [22]. Additionally, MA causes large dislocation density and produces subgrains fall in nanometer scale. Consequently; the obtained biomaterials have excellent mechanical and surface properties comparable to bone. Interestingly; these materials are considered to be the future generation of biomaterials [23]. Hannora and Ataya [12] prepared HA/TiO2 nanocomposites, with different TiO2 contents up to 25 wt.%, by energy ball milling and they found that compressive strength significantly increased up to 330 MPa in the nanocomposites containing 25% TiO2. Fahami et al. [24] produced HA-20 wt.% Ti nanocomposite by solid state method. The results pointed out that the phase stability as well as structural features of the products was highly influenced by thermal annealing temperature. Many reports [12,25,26] are available on the preparation of Ti/HA nanocomposites and exploring physical and mechanical properties of the prepared nanocomposites. However, at least to the authors' knowledge, the effect of their exposure to elevated sintering temperatures up to 1300
C on the molecular structure and in vitro bioactivity of these nanocomposites has not been studied before. Based upon the above considerations; Ti-CHA nanocomposites were prepared with different CHA contents up to 20 wt.%. The effect of sintering temperatures on molecular structure, density, porosity, mechanical properties and in vitro bioactivity for the prepared nanocomposites were carried out with different characterizing tools. 2. Experimental procedure 2.1. Preparation of Ti-CHA nanocomposites Pure titanium powders (Ti powders; 20e40 mm particle size) was purchased from Aldrich-Sigma. However, CHA has been prepared by mechanochemical synthesis method and well characterized as shown in our recent articles [3,4]. Both Ti and CHA powders, based on their respective wt.% as represented in Table 1, were mechanically blended for 12 h with ball-to-powder ratio (BPR) equals to 1:2 and the diameters of balls were 10 mm. Then, these mixtures were milled for 10 h in a planetary ball mill (type MTI SFM (QM-3SP2)) with rotation speed equals to 400 rpm and BPR ¼ 20:1. The milling was performed using alumina vial and balls. It is worth to mention that the milling was done in a cycle of 2 h and paused for 30 min. 2.2. Ti-CHA nanocomposites characterization X-ray diffraction (XRD) technique, “Philips PW 1373” X ray powder diffractometer with CuK-Ni filtered radiation, was utilized in this work, to investigate milled powders and sintered samples. Fourier transform infrared (FTIR) spectroscopy, (VERTEX 70, Bruker, Germany) was carried out at room temperature using KBr disc technique in the wavenumber range 2000e400 cm1 and 30 scans at 2 cm1 resolution, was employed to investigate the sintered samples for different temperatures and examine the in vitro

Table 1 Scheme of the prepared samples indicating the specimen code and its composition as percentage. Specimen code

Titanium (Ti)

Carbonated hydroxyapatite (CHA)

Ti100 Ti90 Ti80

100 90 80

0 10 20

189

bioactivity of these nanocomposites.

2.3. Density and porosity measurements According to the recent articles of Taha et al. [27,28], the milled powders were consolidated into pellets of 16 mm in diameter and 4 mm in length using hydraulic press at 50 MPa. Archimedes' method (ASTM: B962-13) was employed to determine the bulk density and apparent porosity of sintered samples at different temperatures, i.e. 900, 1100 and 1300
C. Simple mixture role was employed, in this work, to calculate theoretical density of sintered samples, considering the full dense values of Ti and HA are 4.506 and 3.156 g/cm3, respectively. Furthermore, relative density was calculated using the measured bulk density and the calculated theoretical density. The microstructure of the obtained Ti-CHA nanocomposite samples were examined by scanning electron microscopy (SEM) “type Philips XL3000 .

2.4. Mechanical properties The microhardness using Vickers in dentor of the sintered samples was measured according to ASTM: B933-09. The ultrasonic wave velocities (longitudinal and shear) propagated in the samples was obtained at room temperature, using pulse-echo technique MATEC Model MBS8000 DSP (ultrasonic digital signal processing) system with 5 MHz resonating. The values of Lame's constants, i.e. l and m were obtained from the longitudinal ðVL2 Þ and shear (V2S ) ultrasonic velocities as follows:



l ¼ r VL2  2V2S



m ¼ rVS2

(1) (2)

where r is the material bulk density. The values of the elastic moduli; longitudinal modulus (L), shear modulus (G),Young's modulus (E), bulk modulus (B) and Poisson's ratio (n) as calculated from the following equations:

L ¼ l þ 2m

(3)

G¼m

(4)

3l þ 2m E¼m lþm

(5)

2 B¼lþ m 3

(6)

ʋ¼

l

2ðl þ mÞ

(7)

2.5. In vitro bioactivity assessment In vitro bioactivity was assessed by soaking the sintered samples in simulated body fluid (SBF) solution prepared according to the recipe described by Kokubo et al. [29,30]. The ratio of sample mass to SBF volume should be equal 0.01 g (mL)1 to achieve the use of excess SBF volume surrounding the samples [31].

190

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

3. Results and discussion 3.1. XRD study The prepared Ti-CHA nanocomposite samples were subjected first to XRD analysis, as the most important tool to assess the structure of the prepared samples. So that, the XRD patterns of Ti and CHA milled powders, with different ratios, for 10 h are examined as illustrated in Fig. 1. Characteristic Ti and CHA peaks are clearly identified according to (JCPDS No.89-5009) and (JCPDS No. 19-0272), respectively. As obviously observed from this figure, the intensity of CHA peaks were gradually increased with increasing of CHA contents. It is worth to mention that the absence of other phases beside Ti and CHA ones indicate that no reaction occurs between them. Furthermore, Al2O3 phases not existed, in this figure, indicating the prepared composite powders are contamination-free. Generally, the exposure of CHA to elevated temperature leads to its partial or complete decomposition causing a considerable effect on the final properties of Ti-CHA nanocomposites [7,22]. Therefore, the effect of different sintering temperatures, i.e. 900, 1100 and 1300
C on the Ti-CHA nanocomposite samples are studied by XRD technique and represented in Fig. 2a-c. Since sintering process is performed in normal atmosphere, most Ti peaks were completely disappeared. Instead, characteristic peaks of rutile TiO2 exhibited (JCPDS No. 89-4920). As clearly observed from Fig. 2a, sintering of these nanocomposite samples at 900
C does not induce CHA decomposition in these samples. The elevation of sintering temperature to 1100
C, as clearly seen from Fig. 2b, lead to the formation of two new phases namely; calcium titanate, i.e. CaTiO3 (JCPDS No. 75-2099) and b-tricalcium phosphate, i.e. b-Ca3(PO4)2 (JCPDS No. 03-0691) beside the characteristic peaks of CHA and TiO2 referring to the occurrence of partial CHA decomposition. Complete CHA decomposition occurs after sintering of these specimens at 1300
C as referred from the absence of its characteristic peaks in Fig. 2c. Furthermore, b-Ca3(PO4)2 is completely transformed to high temperature a-Ca3(PO4)2 (JCPDS No. 29-0359) phase. In order to understand the chemical reactions between HA and TiO2, the following equation can be used: Ca10(PO4)6(OH)2 þ TiO2 / 3 Ca3(PO4)6 þ CaTiO3 þ H2O

(8)

It is well-known that the exposure of CHA to elevated

Fig. 2. XRD patterns of Ti-CHA nanocomposites sintered at a) 900
C, b) 1100
C and c) 1300
C for 2 h.

Fig. 1. XRD patterns of the investigated Ti-CHA milled powders.

temperatures, i.e. more than 1200
C leads to the formation of tetratricalcium phosphate (Ca4P2O9, TTCP). However, the analysis of XRD patterns of the studied sintered specimens at 1300
C points out that this phase is not present. This observation can be understood by knowing that the existence of TiO2 prevents further phase transformation of HA [9]. These results agree, to a good extent, with

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

those reported in Refs. [12,32,33]. Thermal treatment of the prepared nanocomposites caused observed changes in their crystal sizes as well as lattice strain. Therefore, they are calculated and tabulated in Table 2. As expected, sintering process, up 1100
C, is responsible for the enhancement of crystalline behavior of nanocomposites which leads to subsequent reduction in the width of diffraction peaks and consequently; crystal size increased considerably while, lattice strain decreased. Similar results have been reported elsewhere [34e36]. However, further increase of sintering temperature to 1300
C caused slight decrease in crystal size and weak increase in lattice strain. The explanation of such finding is mainly attributed to that the intensity of TiO2 peaks becomes less intense as a result of the formation of CaTiO3. The mechanisms of nanomaterials crystallization can be interpreted by several theories. The most appropriate models describing this phenomenon are surface energy and diffusion theories. Surface energy theory was proposed by Gibb's and established upon thermodynamic principle. He proposed that total free energy of the crystal, in equilibrium state, greatly affects its growth. Owing to high surface energy of nanomaterials, the crystallites tend to have the lowest surface energy by the growth of nanocrystallite forming larger grains. On the other hand, diffusion theory is closely correlated to Fick's laws for diffusion. Accordingly; recovery process described in surface energy model as well as transfer procedure described in diffusion model could be improved by sintering temperature and lead to significant increase of nanocomposites crystallinity [12]. It is worth to mention that nanostructured materials achieve better interactions with proteins and as a consequence, better biomechanical as well as biological attributes can be obtained [37].

3.2. FTIR spectroscopy The molecular structure of Ti-CHA nanocomposites was evaluated by FTIR spectroscopy as a second important tool to confirm the structure of the prepared samples and investigating their functional groups. Studying stability of Ti-containing CHA nanocomposites after their exposure to elevated temperatures is essential to expect their final characteristics in living tissues [2]. Notably, decomposition temperature is based upon the characteristics as well as preparation method of apatite powder [24]. FTIR spectra of all sintered nanocomposite samples were recorded, displayed in Fig. 3a-c and assigned according to the literature [9,24,26,38e40]. As previously discussed in our recent work [3,4], vibrational modes of phosphate group in HA molecule can be represented by absorption bands at 1040, 972, 605, 560 and 470 cm1. However, the existence of three bands at 875, 1445 and 1470 cm1can be

Table 2 The crystal size and lattice strain for Ti-CHA nanocomposite samples at different firing temperatures. Sample

Temperature (
C)

Crystal size (nm)

Lattice strain (%)

Ti100

0 900 1100 1300 0 900 1100 1300 0 900 1100 1300

31.2 38.2 40.3 41.7 27.7 31.1 34.9 33.5 24.2 27.1 29.2 28.3

0.3007 0.2448 0.2323 0.2247 0.3386 0.3014 0.2684 0.2794 0.3864 0.3456 0.3204 0.3313

Ti90

Ti80

191

Table 3 Theoretical and relative densities of all sintered specimens at different temperatures. Sample code

Ti100 Ti90 Ti80

Theoretical density (g/cm3)

4.5 4.31 4.15

Relative density ((%)) 900
C

1100
C

1300
C

93.23 92.89 92.21

95.31 92.02 91.91

96.11 88.33 86.03

assigned to C-O bending vibrations in the carbonate (CO2 3 ) group that occupies phosphate (PO3 4 ) ion position giving B-type carbonated apatite. Furthermore, band at 1620 cm1 can be assigned to OH associated with HA molecule. It can be seen from Fig. 3a that sintered nanocomposites at 900
C represents the characteristic CHA bands with successive increase in its characteristic bands as apatite contents increase. However, weak bands at 875, 1445 and 1470 cm1 completely disappeared due to sintering process which leads to the releasing of carbonate in gaseous form [24,26,41]. Broad band found at 500-800 cm1 and medium band at 1420 cm1 can be assigned to stretching vibrational modes of Ti-O and Ti-O-Ti, respectively. When sintering temperature increased to 1100
C, as shown in Fig. 3b, there are some noticed changes which can be summarized as the following: a. Medium ill-defined band and new weak band at 1126 and 945 cm1 are clearly observed while, band at 1620 cm1 become less intense, compared to samples sintered at 900
C, giving an evidence for the occurrence of partial CHA decomposition and the formation of b-TCP [42]. c. Two bands appear at 495 and 425 cm1, as obviously seen from the figure, can be attributed to the bending vibration of Ca-TiO and confirming the formation of CaTiO3 due to the CHA degeneration. These results closely concord with those reported earlier [42,43]. It is interesting to observe that increasing of CHA contents, in nanocomposite samples, leads to considerable increase of the characteristic bands intensities of b-TCP. Elevation of sintering temperature up to 1300
C is responsible for disappearance of bands located at 1126 and 470 cm1 and the occurrence of positions shift for the following bands at 1040, 605 and 560 cm1 to 1035, 597 and 585 cm1 indicating the conversion of b-TCP to a-TCP. Interestingly, band located at 1420 cm1 become less intense while, the characteristic bands of CaTiO3 exhibit slight increase in their intensities referring to that possibility of reaction between TiO2 and CHA, as revealed by eqn. (1), is significantly reduced. These results strongly coincide with those obtained from XRD and agree with those reported earlier [44].

3.3. Density and porosity measurements Generally, in order to prepare dense materials, powders should be compacted under one or more factors such as pressure, temperature and dwell/holding time at the sintering temperature. Based upon those mentioned parameters, dense materials can be obtained [37]. The measured density values and porosity of all investigated samples are listed in Tables 3 and 4, respectively. As noticed from these tables, successive increase of CHA contents is responsible for remarkable decrease in density values of the sintered nanocomposites. It is worth to mention that the measured relative density values nearly reached to 93% after sintering at 900
C indicating the sintered nanocomposite samples become full dense materials. On the other hand, sintering at 1100
C for examined specimens causes an observed decrease in density values until they record 91.91% due to a partial CHA decomposition. Interestingly, further sintering of these samples to 1300
C leads to

192

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

melting point, “necks” starts to form between the particles and therefore, they can be strongly bonded with each other due to the expanding of small contact area between particles. Consequently; the density of compacts significantly increased while, the total void volume decreased. Particles, with small diameters, have high surface area as well as high surface free energy those are considered as the driving force of the sintering process. Therefore, there is a strong thermodynamic drive to decrease the surface area by bonding particles together [37]. The results point out that porosity of all examined nanocomposite samples exhibit an opposite trend for density where they record minimum value at 900
C. This result can be attributed to the growing of bonds number contributes to marked reduction of surface area and energy. Subsequently; particles cannot be seen clearly as a result of the formation of tight bonds between particles and therefore, porosity is reduced [37]. However, successive increase of sintering temperatures leads to a considerable increase of its values. Bearing in mind that porosity is desirable for implant ossesointegeration, on condition that they have appropriate mechanical properties [45]. These results support FTIR ones, those mentioned in the former section, which reveal that the characteristic band of OH associated with the HA molecule does not change for all specimens sintered at 900
C. However, it nearly disappears after sintering at 1100
C and 1300
C referring to CHA decomposition. Accordingly, density is negatively affected. These results concord with those obtained from XRD and FTIR. 3.4. Microstructure investigation by SEM Microstructure of Ti100 and Ti80 samples at 900, 1100 and 1300
C is investigated and represented in Figs. 4 and 5, respectively. The Fig. 4(aec) clarifies the formation of dense structure of Ti along with the existence of bright regions indicating the oxidation of Ti to TiO2 and confirming XRD results. It is worth to mention that the densification as well as the presence of bright regions increased significantly with increase of sintering temperature. However, number of pores is greatly decreased with increasing of sintering temperatures and reaches the minimum value after the exposure of Ti to 1300
C. As seen from Fig. 5a, the addition of CHA to Ti, at 900
C, is responsible for observed increase of porosity and detected decrease of density values. Meanwhile, microstructure of

Fig. 3. FTIR spectra of Ti-CHA sintered nanocomposites at a) 900
C, b) 1100
C and c) 1300
C for 2 h.

marked decrease in density values as a result of drastic CHA decomposition [9]. The effect of sintering temperatures on densification of the obtained nanocomposites is interpreted as the following: Sintering process can be occurred in three stages. Firstly, powders are compacted and contact with each other but they are not firmly bonded. Secondly, after sintering temperature reached 2/3 of

Fig. 4. SEM photomicrographs of Ti100 sintered nanocomposite samples at a) 900
C, b) 1100
C and c) 1300
C.

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

193

compensated by more oxidation of Ti and consequently; mechanical properties don't be greatly affected. However, when the composites sintered at 1300
C, mechanical properties are reduced remarkably as a result of CHA decomposition and consequently; mechanical properties are much affected. This result is closely concords with density results those discussed before in Section 3.3. This finding can be explained with the aid of some reports which state that mechanical properties are highly affected by CHA decomposition. Mahmoodi et al. [7] reported that the strength of HA is negatively affected by its degeneration to b-TCP. Hannora and Ataya [12] reported that the degeneration of CHA suppresses densification and accordingly; mechanical properties are greatly influenced. Que et al. [9] found that the existence of TiO2 with HA is responsible for significant improvement of mechanical properties of nanocomposites.

3.6. In vitro bioactivity assessment of sintered nanocomposites by FTIR spectroscopy

Fig. 5. SEM photomicrographs of Ti80 sintered nanocomposite samples at a) 900
C, b) 1100
C and c) 1300
C.

Table 4 The apparent porosity of sintered samples at 900, 1100 and 1300
C. Sample code

Ti100 Ti90 Ti80

Apparent porosity, % 900
C

1100
C

1300
C

6.26 7.44 7.99

4.28 8.29 7.76

3.65 10.55 13.21

nanocomposite sintered at 1100 and 1300
C exhibits less densification as a result of marked increase of pores numbers. These results agree, to good extent, with density and porosity ones those discussed former.

3.5. Mechanical properties of sintered nanocomposites Generally, sintering process positively affects the mechanical properties of the studied nanocomposites as a result of increasing of densification and reduction of porosity [37]. Therefore, hardness (Hv), longitudinal modulus (L), Young's modulus (E), shear modulus(G), bulk modulus (K) and Poisson's ratio (ʋ) are measured and listed in Table 5. Sintering of these samples at 900
C causes appropriate mechanical properties due to better densification, as discussed former, and oxidation of Ti to TiO2. Further sintering up to 1100
C is responsible for slight decrease in mechanical properties due to the effect of partial CHA decomposition which is

Generally, sintering properties of composite are highly based upon many factors such as the surface area of the powder, heating rate, Ca/P ratio and heating regime. Considering the existence of OH group, sintering of CHA is difficult due to its decomposition into tricalcium phosphate. The new formed phases exhibit different dissolution response in physiological fluid [37]. Therefore, studying the effect of sintering at different temperatures on the in vitro bioactivity of the prepared Ti-CHA nanocomposites is necessary. Fig. 6a-c illustrates the recorded FTIR absorption spectra of all sintered samples, after immersion in SBF solution for 7 days. Careful examination of these spectra reveals that the bands at 560 and 605 93% after clearly observed in sintered nanocomposite samples at 900
C with pronounced increase in their intensities referring to the development of HA-like layer on the surface of investigated samples. As expected, Ti80 and Ti100 specimens had the maximum and minimum abilities to form CHA layer, respectively. It has been reported that increasing of CHA contents, in the prepared nanocomposites, favors the apatite formation on their surfaces when soaked in SBF solution [46]. This appropriate in vitro bioactivity is considerably reduced after sintering of these nanocomposite specimens at 1100
C, Fig. 6b, as indicated by the presence of less intense bands at 1040, 605 and 560 cm1 along with the disappearance of two ill-defined bands at 1120 and 945 cm1. The most likely explanation of this result is that the investigated samples contain b-TCP phase, which is considered as high soluble one, beside the remainder HA contents. Therefore, soaking of these samples in SBF solution leads to rapid solubility for this phase. This result is in-line with those discussed before in Sections 3.3 and 3.4, respectively. It is further noted from Fig. 6c that sintered specimens at 1300
C exhibit dramatic decrease in the intensities of bands located at 1030, 975, 605 and 560 cm1 indicating the occurrence of complete CHA degeneration. Instead, they exhibit fair bioactivity

Table 5 Hardness (Hv), longitudinal modulus (L), Young's modulus (E), shear modulus (G), bulk modulus (K) and Poisson's ratio (ʋ) of all sintered nanocomposite specimens. Sintering Temp. (
C)

Sample code

L (GPa)

E (GPa)

G (GPa)

B (GPa)

ʋ

Hv (GPa)

900

Ti100 Ti90 Ti80 Ti100 Ti90 Ti80 Ti100 Ti90 Ti80

125.9 145.8 182.9 117.1 131.2 166.5 100.5 106.9 134.6

117.5 139.1 172.8 109.6 124.1 157.7 94.6 100.8 129.6

50.4 61 74.9 47.1 53.9 68.6 40.8 43.6 57.5

75.5 84.9 108 70 77.3 98 59.7 63.3 77.1

0.166 0.159 0.153 0.163 0.151 0.159 0.158 0.155 0.127

1.68 2.31 3.27 1.51 2.12 2.92 1.36 1.92 2.51

1100

1300

194

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195

surrounding tissues [47]. From FTIR spectra, we can conclude that the bioactivity of sintered nanocomposite samples, at 1300
C, can be only attributed to the presence of TiO2 where all HA contents are completely degenerated and converted to highly soluble phase, i.e. a-TCP which dissolves rapidly in SBF solution without the formation of HA-like layer on the surface of these specimens [37,48]. Notably, even HA-free nanocomposite specimen is able to form HA layer on its surface after immersion in SBF solution due to the biocompatible property of TiO2 as discussed former. Despite of the simplicity of SBF tests to examine the bioactivity of any material, the obtained results should be interpreted carefully. It is well-known that SBF solution is basically composed of inorganic ions with similar concentrations to the human plasma. Consequently; in vitro test doesn't always mimic the dynamic physiological environment. Therefore, this test may give false positive and false negative results. For example, samples contain TCP may not form HA-like layer after soaking in SBF solution despite its extensive bone-bonding ability in vivo. On the other hand, other materials can successively form HA layer in vitro but they don't bond to the bone once implanted in the body [49]. Accordingly; though sintered Ti-CHA specimens, at 1100 and 1300
C, don't display in vitro bioactivity, they may represent good in vivo bioactivity. 4. Conclusions A simple and cost effective method is dedicated for the preparation of titanium-carbonated hydroxyapatite (Ti-CHA) nanocomposites, with different CHA contents up to 20 wt.%. Different tools of characterizations indicate that the bioactivity of Ti-CHA nanocomposites sintered at 900
C exhibited better in vitro bioactivity character compared to those sintered at 1100 and 1300
C. Instead, the latter sintered samples may display fair in vivo bioactivity when they are implanted in living tissues. Conversely, density and mechanical properties of these nanocomposites had not been significantly affected by increasing of firing temperature to 1100
C. However, further increase of sintering temperature to 1300
C led to dramatic decrease in these properties. These results can be attributed to that as sintering temperature increased; CHA was partially or completely decomposed to b-TCP or a-TCP phases which greatly affected the previous properties. From these results, we can conclude that the optimum in vitro bioactivity, physical and mechanical properties were obtained after sintering of these nanocomposite specimens at 900
C only. Accordingly; these sintered nanocomposite specimens can be considered as promising biomaterials for many biomedical applications where they are characterized by high mechanical properties and excellent in vitro bioactivity. References Fig. 6. FTIR spectra of the investigated sintered specimens for 2 h at a) 900
C, b) 1100
C and c) 1300
C after soaking in SBF solution for 7 days.

due to the formation of Ti-OH groups, as indicated by the presence of the band located at 1117 cm1, which induce the formation of CHA layer on their surfaces after soaking in SBF solution [9,38]. This result is supported by the presence of absorption bands at 1620 and 1540 cm1 representing OH and CO2 3 groups, in apatite molecule, and reflecting the formation of CHA. This process is responsible for promoting biocompatibility and bony tissue growth in biological environment [7,9]. Furthermore, it aids inanchoring with bone or

[1] A. Manikandan, M.P. Mani, S.K. Jaganathan, R. Rajasekar, Morphological, thermal, and blood-compatible properties of electrospun nanocomposites for tissue engineering application, Polym. Compos (2017) 1e8. [2] M. Aminzare, A. Eskandari, M.H. Baroonian, A. Berenov, Z. Razavi Hesabi, M. Taheri, S.K. Sadrnezhaad, Hydroxyapatite nanocomposites: synthesis, sintering and mechanical properties, Ceram. Int. 39 (2013) 2197e2206. [3] R.A. Youness, M.A. Taha, H. Elhaes, M. Ibrahim, Molecular modeling, FTIR spectral characterization and mechanical properties of carbonatedhydroxyapatite prepared by mechanochemical synthesis, Mater. Chem. Phys. 190 (2017) 209e218. [4] A. Refaat, R.A. Youness, M.A. Taha, M. Ibrahim, Effect of zinc oxide on the electronic properties of carbonated hydroxyapatite, J. Mol. Struct. 1147 (2017) 148e154. [5] B.N. Tabrizi, E. Zalnezhad, A.M.S. Hamouda, W.J. Basirun, B.P. Murphy, A. Fahami, M. Sarraf, A.R. Rafieerad, Gradual mechanochemical reaction to produce carbonate doped fluorapatite-titania composite powder, Ceram. Int. 40 (2014) 15623e15631.

R.A. Youness et al. / Journal of Molecular Structure 1150 (2017) 188e195 [6] B.N. Tabrizi, A. Fahami, R.E. Kahrizsangi, A. Khazraei, M.R. Yazdani, M.J. Kajbafzadeh, A study on mechanochemical behavior of CaO-P2O5-CaF2ZrO2 system to produce fluorapatite-zirconia composite nanopowders, Powder Technol. 243 (2013) 59e70. [7] M. Mahmoodi, P.M. Hashemi, R. Imani, Characterization of a novel nanobiomaterial fabricated from HA, TiO2 and Al2O3 powders: an in vitro study, Prog. Biomater. 3 (25) (2014) 1e10. [8] R.J. Kane, H.E. Weiss-Bilka, M.J. Meagher, Y. Liu, J.A. Gargac, G.L. Niebur, D.R. Wagner, R.K. Roeder, Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties, Acta Biomater. 17 (2015) 16e25. [9] W. Que, K.A. Khor, J.L. Xu, L.G. Yu, Hydroxyapatite/titania nanocomposites derived by combining high-energy ball milling with spark plasma sintering processes, J. Eur. Ceram. Soc. 28 (2008) 3083e3090. [10] S. Vahabzadeh, M. Roy, A. Bandyopadhyay, S. Bose, Phase stability and biological property evaluation of plasma sprayed hydroxyapatite coatings for orthopaedic and dental applications, ActaBiomater 17 (2015) 47e55. [11] H. Zhou, J. Lee, Nanoscale hydroxyapatite particles for bone tissue engineering, ActaBiomater 7 (2011) 2769e2781. [12] A.E. Hannora, S. Ataya, Structure and compression strength of hydroxyapatite/ titania nanocomposites formed by high energy ball milling, Alloys Compd. 658 (2016) 222e233. [13] S. Wu, Z. Weng, X. Liu, K.W.K. Yeung, P.K. Chu, Functionalized TiO2 based nanomaterials for biomedical applications, Adv.Funct. Mater. 24 (35) (2014) 5464e5481. [14] J. Forsgren, F. Svahn, T. Jarmar, H. Engqvist, Formation and adhesion of biomimetic hydroxyapatite deposited on titanium substrates, ActaBiomater 3 (6) (2007) 980e984. [15] H. Li, K. Khor, P. Cheang, Impact formation and microstructure characterization of thermal sprayed hydroxyapatite/titania composite coatings, Biomaterials 24 (2003) 949e957. [16] A. Manikandan, M. Durka, K. Seevakan, S.A. Antony, A novel one-pot combustion synthesis and opto-magnetic properties of magnetically separable spinel MnxMg1-xFe2O4 (0.0 x 0.5) nano-photocatalysts, J. Supercond. Nov. Magn. 28 (2015) 1405e1416. [17] A. Manikandan, E. Hema, M. Durka, M.A. Selvi, T. Alagesan, S.A. Antony, Mn2þ doped NiS (MnxNi1-xS: x ¼ 0.0, 0.3 and 0.5) nanocrystals: structural, morphological, opto-magnetic and photocatalytic properties, J. Inorg. Organomet. Polym. Mater. 25 (2015) 804e815. [18] A. Manikandan, E. Hema, M. Durka, K. Seevakan, T. Alagesan, S.A. Antony, Room temperature ferromagnetism of magnetically recyclable photocatalyst of Cu1-xMnxFe2O4-TiO2 (0.0x0.5) nano-composites, J. Supercond. Nov. Magn. 28 (2015) 1783e1795. [19] D.K. Manimegalai, A. Manikandan, S. Moortheswaran, S.A. Antony, MagnetoOptical and photocatalytic properties of magnetically recyclable Zn1-xMnxS (x ¼ 0.0, 0.3 and 0.5) nano-catalysts, J. Supercond. Nov. Magn. 28 (2015) 2755e2766. [20] V. Umapathy, A. Manikandan, S.A. Antony, P. Ramu, P. Neeraja, Synthesis, structural, morphological and opto-magnetic properties of Bi2MoO6nanophotocatalyst by sol-gel method, T. Nonferr. Metal. Soc. 25 (2015) 3271e3278. [21] N.C.S. Selvam, A. Manikandan, L.J. Kennedy, J.J. Vijaya, Comparative investigation of ZrO2nano and microstructures for structural, optical and photocatalytic properties, J. Colloid Interface Sci. 389 (2013) 91e98. [22] K. Niespodziana, K. Jurczyk, J. Jakubowicz, M. Jurczyk, Fabrication and properties of titanium-hydroxyapatite nanocomposites, Mater. Chem. Phys. 123 (2010) 160e165. [23] K. Jurczyk, G. Adamek, M.M. Kubicka, J. Jakubowicz, M. Jurczyk, Nanostructured titanium-10 wt.% 45S5 bioglass-Ag composite foams for biomedical applications, Materials 8 (2015) 1398e1412. [24] A. Fahami, R.E. Kahrizsangi, B.N. Tabrizi, Mechanochemical synthesis of hydroxyapatite/titanium nanocomposite, Solid State Sci. 13 (2011) 135e141. [25] D. Bovand, M. Yousefpour, S. Rasouli, S. Bagherifard, N. Bovand, A. Tamayol, Characterization of Ti-HA composite fabricated by mechanical alloying, Mater. Des. 65 (2015) 447e453. [26] M.E. Jazi, M.S. Hashjin, A. Nemati, F. Bakhshi, Synthesis and characterization of hydroxyapatite/titania nanocomposites using in situ precipitation technique, Superlattice Microst 51 (2012) 877e885. [27] M.A. Taha, A.H. Nassar, M.F. Zawrah, Effects of milling parameters on sinterability, mechanical and electrical properties of Cu-4 wt.% ZrO2 nanocomposite, Mater. Chem. Phys. 181 (2016) 26e32.

195

[28] M.A. Taha, A.H. Nassar, M.F. Zawrah, Improvement of wettability, sinterability, mechanical and electrical properties of Al2O3-Ni nanocomposites prepared by mechanical alloying, Ceram. Int. 43 (2017) 3576e3582. [29] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity, Biomater 27 (15) (2006) 2907e2915. [30] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramics A-W, J. Biomed. Mater. Res. A 24 (1990) 721e734. [31] R.L. Siqueira, E.D. Zanotto, The influence of phosphorus precursors on the synthesis and bioactivity of SiO2-CaO-P2O5 sol-gel glasses and glass-ceramics, J. Mater. Sci. Mater. Med. 24 (2013) 365e379. [32] W. Shih, J. Wang, M. Wang, M. Hon, A study on the phase transformation of the nanosized hydroxyapatite synthesized by hydrolysis using in situ high temperature X-ray diffraction, Mater. Sci. Eng. C 26 (2006) 1434e1438. [33] A.E. Hannora, Preparation and characterization of hydroxyapatite/alumina nanocomposites by high-energy vibratory ball milling, J. Ceram. Sci. Tech. 5 (4) (2014) 293e298. [34] K. Chinnaraj, A. Manikandan, P. Ramu, S.A. Antony, P. Neeraja, Comparative study of microwave and sol-gel assisted combustion methods of Fe3O4 nanostructures: structural, morphological, optical, magnetic and catalytic properties, J. Supercond. Nov. Magn. 28 (2015) 179e190. [35] A. Manikandan, L.J. Kennedy, J.A. Mary, A. Dinesh, J.J. Vijaya, Structural, optical and magnetic properties of Fe3O4 nanoparticles prepared by a facile microwave combustion method, Ind. Eng. Chem. Res. 20 (2014) 2077e2085. [36] A. Manikandan, L.J. Kennedy, J.J. Vijaya, Comparative investigation of NiO nano and microstructures for structural, optical and magnetic properties, Phys. E Low. Dimens. Syst. Nanostruct. 49 (2013) 117e123. [37] M. Prakasam, J. Locs, K. Salma-Ancane, D. Loca, A. Largeteau, L. Berzina-Cimdina, Facbrication, properties and applications of dense hydroxyapatite: a review, J. Funct. Biomater. 6 (2015) 1099e1140. [38] K. Kavitha, M. Prabhu, V. Rajendran, P. Manivasankan, P. Prabu, T. Jayakumar, Optimization of nano-titania and titania-chitosan nanocomposite to enhance biocompatibility, Curr. Nanosci. 9 (2013) 308e317. [39] W. Dong, G. Zhao, Q. Bao, X. Gu, Solvothermal preparation of CATiO3 prism and CaTi2O4(OH)2 nanosheet by a facile surfactant-free method, Mater. Sci. 21 (4) (2015) 583e585. [40] V. Vetrivel, K. Rajendran, V. Kalaiselvi, Synthesis and characterization of pure titanium dioxide nanoparticles by sol-gel method, Int. J. Chem. Tech. Res. 7 (3) (2015) 1090e1097. [41] C. Piccirillo, M.F. Silva, R.C. Pullar, I.B. da Cruz, R. Jorge, M.M.E. Pintado, P.M.L. Castro, Extraction and characterization of apatite- and tricalcium phosphate-based materials from cod fish bones, Mater Sci. Eng. C 33 (2013) 103e110. [42] N. Douard, R. Detsch, R. Chotard-Ghodsnia, C. Damia, U. Deisinger, E. Champion, Processing, physic-chemical characterization and in vitro evaluation of silicon containing b-tricalcium phosphate ceramics, Mater. Sci. Eng. C 31 (2011) 531e539. [43] I.M. Hung, W.J. Shih, M.H. Hon, M.C. Wang, The properties of sintered calcium phosphate with [Ca]/[P]¼1.50, Int. J. Mol. Sci. 13 (2012) 13569e13586. [44] J. Venkatesan, Z.J. Qian, B. Ryu, N.V. Thomas, S.K. Kim, A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus Obesus bone, Biomed. Mater. 6 (3) (2011) 035003. [45] W.N.S.W. Nawai, N.A. Kasani, R. Nordin, Z.A. Ahmad, S. Shamsuddin, Effect of sintering temperature on microstructure and mechanical properties of Ti-NbSn-HA composites produced by powder metallurgy, Appl. Mech. Mater 625 (2014) 180e183. [46] D.K. Pattanayak, V. Mathur, B.T. Rao, T.R.R. Mohan, Synthesis and characterization of titanium-calcium phosphate composites for bio applications, Trends Biomater. Artif. Organs 17 (1) (2003) 8e12. [47] L. Kong, Y. Gao, G. Lu, Y. Gong, N. Zhao, X. Zhang, A study on the bioactivity of chitosan/nano-hydroxyapatite composite scaffolds for bone tissue engineering, Eur. Polym. J. 42 (12) (2006) 3171e3179. [48] L.J. Fuh, Y.J. Huang, W.C. Chen, D.J. Lin, Preparation of micro porous bioceramic containing silicon-substituted hydroxyapatite and beta-tricalcium phosphate, Mater. Sci. Eng. C 75 (2017) 798e806. [49] D. Bellucci, A. Sola, A. Anesi, R. Salvatori, L. Chiarini, V. Cannillo, Bioactive glass/hydroxyapatite composites: mechanical properties and biological evaluation, Mater. Sci. Eng. C 51 (2015) 196e205.