The crystallite dimensions (D), unit-cell parameters, crystallinity percentage (X
C
%), crystal orientations and phase compositions of the samples were evaluated by XRD. The XRD patterns of the CA-free and CA-containing HAp samples before and after soaking in SBF for 2 and 4 weeks are shown in Fig. 1. For each sample, HAp (JCPDS PDF No. 09–0432) was found to be the major crystal phase. As shown in Fig. 1, CA addition resulted in partial amorphization of HAp, which is seen by intensity decreasing of the major diffraction peaks. Taking into the consideration the well-known chelating properties of CA (Soccol et al. 2006), the presence of CA promoted HAp amorphization by partial consumption of Ca2+ ions and, thus, reducing the available amount of Ca below the Ca/P ratio 1.67, which was necessary for the stoichiometric HAp formation. Therefore, in the presence of CA, a non-stoichiometric and poorly crystalline Ca-deficient HAp (Ca10-x
(HPO4)
x
(PO4)6-x
(OH)2-x
) was precipitated. Besides, formation of the secondary phase of β-tricalcium phosphate (β-TCP, JCPDS PDF No. 09–0169) with lower intensities at around 2θ = 31.06° was detected for CA10-HAp samples with the highest content of CA, due to a thermal decomposition of the Ca-deficient HAp according to the following equation:
$$ {\text{Ca}}_{10 - x} ({\text{HPO}}_{4} )_{x} ({\text{PO}}_{4} )_{6 - x} ({\text{OH}})_{2 - x} \to {\text{Ca}}_{10} ({\text{PO}}_{4} )_{6} ({\text{OH}})_{2} + \beta-{\text{Ca}}_{3} ({\text{PO}}_{4} )_{2} $$
(1)
In addition, a presence of β-TCP was also detected in CA5-HAp sample after its soaking in SBF for 4 weeks. Therefore, one can conclude that the presence of CA influenced the formation of the secondary β-TCP phase. In the present study, the formation of the secondary β-TCP phase was observed for CA-containing HAp prepared at basic conditions.
The crystallite dimensions (D
002) of the samples along to c-axis were evaluated according to the following Scherrer equation using the line broadening of the (002) reflection (Cullity 1978):
$$ D_{002} = \frac{0.9\lambda }{{B_{1/2} \cos \theta }} $$
(2)
where λ is the wavelength of the incident X-rays, B
1/2 is the full width at half maximum (FWHM) and θ is the diffraction angle. Additionally, in order to estimate the crystallinity percentage (X
C
%) of the samples, the following relation proposed by Landi et al. (2000) was used:
$$ X_{C} \% = \left( {1 - \frac{{V_{112/300} }}{{I_{300} }}} \right)\, {\times} \,100$$
(3)
where \( V_{112/300} \) is the intensity of the hollow between (112) and (300) reflections, and I
300 is the intensity of the (300) diffraction peak. The lattice parameters (a = b and c) and unit cell volume (V) of HAp with the hexagonal structure were estimated according to the equations given in the previous study (Kokubo 1991), and these values are mentioned in Table 1. Additionally, the graphs of the lattice parameters of a and c, unit cell volume (V), crystallite dimensions (D
002) and crystallinity percentage (X
C
%) as a function of the amount of CA are shown in Fig. 2. By the analyzing the aforementioned table and figure, it can be said that all the calculated parameters related to the crystal structure of HAp were dramatically affected by both CA content and immersion period. All the parameters were found to be changed with increasing of both the CA content and soaking time, but these changes were not gradual. The crystallite dimensions were decreased by stages with increasing soaking time for the samples of CA5-HAp and CA10-HAp. With increasing soaking time in SBF, the values of the crystallinity percentage, the lattice parameter of a and the unit cell volume (V) were decreased gradually for CA0-HAp and CA10-HAp, while the X
C
% increased by stages for CA1-HAp and CA5-HAp, and there was a gradual decrease in the lattice parameter of c for CA10-HAp.
Table 1 The estimated values of the crystallite dimensions (D
002), crystallinity percentage (X
C
%) and unit cell parameters (a, c and V) for all samples before and after soaking in SBF
In the previous studies, it was reported that the immersion period in SBF caused the changes in both the lattice parameters (a and c) and the unit cell volume (V) (Hu et al. 2010; Bayraktar and Tas 1999; Kaygili et al. 2014). Therefore, the variations in these parameters are a very good agreement with the reported results in the literature.
The FTIR spectra of all the samples before and after soaking in SBF for 14 and 28 days are shown in Fig. 3. The bands observed at these spectra verify that each sample contains the functional groups of the phosphate (\( {\text{PO}}_{4}^{3 - } \)) and hydroxyl (\( {\text{OH}}^{ - } \)) belonging to HAp. The bands detected at around 566, 601, 962, 1039 and 1089 cm−1 were associated to the phosphate group (Bueno et al. 2014; Okulus et al. 2014; Fahami et al. 2011). The bands corresponding to hydroxyl groups were observed around 631 and 3571 cm−1 (Kaygili et al. 2014). The wide absorption bands about 1638 and 3450 cm−1 were related to the adsorbed water in the samples and/or in the KBr pellet (Wang et al. 2011; Torabinejad et al. 2014).
The morphologies of the samples were investigated using SEM, while the elemental analyses of them were performed by EDX. The SEM micrographs and the results of the EDX analysis for both the CA-free and CA-containing samples, performed both before and after soaking in SBF for 14 and 28 days are presented in Figs. 4 and 5, respectively. It is clearly seen that all the samples were composed of tiny crystals, which were generally smaller than 100 nm (Fig. 4). There was almost no change in their morphology with increasing amount of CA and immersion time. The microporosity was observed for all the samples, and this is one of the most desired properties for a clinical reconstructive material (Sopyan et al. 2007). Moreover, it was reported that the microporous surfaces may modulate the adsorption of proteins from serum, as well as the adhesion and proliferation of human bone cells (Rouahi et al. 2006). On account of this, it can be said that CA-assisted HAp samples synthesized by sol–gel route can be used as an implant material for biomedical applications. For each sample, the elements of Ca, P and O were detected from EDX, while no impurities were detected (Fig. 5). The presence of these elements and their Ca/P molar ratios confirmed that HAp was always formed. Both the immersion time and the amount of CA affected the elemental composition, as well as these factors influenced the Ca/P ratio. Furthermore, with increasing soaking time the Ca/P ratio was gradually decreased for each sample. This decrease was due to precipitation of a non-stoichiometric Ca-deficient HAp from SBF, which is in a good agreement with the earlier reports on the subject (Kaygili et al. 2014, 2015; Hu et al. 2010; Wan et al. 2006). The soaking results in SBF revealed that the maximum changes in the Ca/P ratio were found for the CA10-HAp samples (Fig. 5), which pointed out to the maximum amount of non-stoichiometric Ca-deficient HAp precipitation and, therefore, the highest bioactivity of these samples.