Effects of using the liquid phase method on the physicochemical, mechanical, and bioactivity properties of hydroxyapatite/calcium aluminate bioceramic nanocomposites

2.1 Materials

All chemicals used, including calcium nitrate tetrahydrate, aluminum nitrate nonahydrate, phosphoric acid (85%), ammonium hydroxide (25%), citric acid monohydrate, and ethylene glycol GR, were purchased from Merck (Stuttgart, Germany). The HA:CA weight ratio was 4:1 in all synthesized nanocomposites.

2.2 Synthesis of hydroxyapatite and calcium aluminate nanoparticles

2.3 Synthesis of nanocomposites

2.4 Characterization

Phase analysis of samples was performed by X-ray diffraction (XRD) technique on an EQUINOX Model 3000 (Inel Company, France) with Kα. λ=1.541874 Ǻ. The morphologies of the samples were investigated using a field emission scanning electron microscope (FE-SEM, Mira Model, Tskan, Czech Republic). For cooking the samples under 40 MPa and for heating SPS, an SPS 20T-10 Model was used (Serial No. EF120311, Easy Fashion Co.). SPS temperature was measured by using an optical pyrometer, RAYR312MSCL2G. Heat treatment was carried out in a vacuum condition at a heating rate of 50°C/min and at standing time of 5 min at a temperature of 1050°C. The strength of sintered samples was calculated using 1161°C ASTM standard. a SANTAM device was used in measuring the flexural strength of the sintered samples, and the Micro Vickers method was used to measure the hardness of the samples. Density, porosity, and water absorption was calculated by using Archimedes method.

2.5 Bioactivity

To verify the bioactivity properties of the samples, the SBF simulated body test was used. In this study, as suggested by Kokubo et al. [18], 1 g of each sample was immersed in the solution of a simulated body for 10 days and then rinsed with distilled water several times before drying at 70°C for 12 h. The samples were analyzed under FE-SEM.

3.1 Sample characterization

Figure 1 illustrates the XRD patterns for the as-synthesized HA and CA nanoparticles as well as the nanocomposites synthesized by three different methods. The XRD patterns of as-synthesized HA nanoparticles are shown in Figure 1(1). According to (JCPDS pdf No. 0740-086-00), the diffraction peaks at 2θ of 25.80°, 28.85°, 32.13°, 33.83°, 39.93°, 46.60°, 49.66°, 53.28°, and 63.91° prove the HA nanoparticle formation and demonstrate that other calcium salts have not been formed. After using the XRD data and Scherer equation, the average crystallite size is obtained as 9 nm. The sizes of the as-synthesized HA lattice parameters are obtained as a=9.424 Ǻ and C=6.879 Ǻ, indicating that the crystal structure of HA is hexagonal.

Figure 1:

XRD patterns for HA (1), CA, (2) and nanocomposites synthesized by method 1 (3), method 2 (4), and method 3 (5).

Figure 1(2) displays the XRD pattern of the CA nanoparticles. Diffraction peaks at 2θ positions of 21.98°, 30.07°, 35.53°, 37.29°, 47.20°, 60.49°, and 63.72° are assigned to the CA phase in accordance with (JCPDS PDF No. 0134-070-00). Average crystallite size for CA is calculated to be 15 nm. Meanwhile, based on the XRD pattern of the nanocomposite synthesized by method 1 [Figure 1(3)], the diffraction peaks of the HA phase in the 2θ positions of 25.90°, 28.16°, 29.07°, 32.02°, 34.17°, 39.83°, 46.97°, 49.58°, 53.43°, and 64.08° are clearly defined according to (JCPDS PDF No. 0740-086-00). In the XRD pattern of the nanocomposite, reflections related to CA are not recognizable due to the overlapping that occurs with the HA diffraction peaks. The remarkable thing about CA nanoparticles is that they are treated with hydration when they mix with water depending on such conditions as time and temperature durability in water. One of the CA hydration products with a metastable structure is C₂AH₈. This product is usually created at 20°C to 30°C [19]. This phase and the AH₃ phase are observed in the XRD patterns in accordance with (JCPDS PDF No. 0008-012-00) and (JCPDS PDF No. 0146-003-00), respectively. Using Scherer equation, the average crystallite size calculated by the most intense peak is 7 nm.

Figure 2:

FE-SEM images of samples before immersion in SBF.

(1) HA, (2) CA, (3) nanocomposite obtained by method 1, (4) nanocomposite obtained by method 2, and (5) nanocomposite obtained by method 3.

Figure 3:

EDX spectra for samples before immersion in SBF.

(A) HA, (B) CA, (C) nanocomposite synthesized by method 1, (D) nanocomposite synthesized by method 2, and (E) nanocomposite synthesized by method 3.

The XRD pattern of the nanocomposite obtained through method 2 [Figure 1(4)] exhibits similar phases with the samples prepared by method 1. The remarkable thing about this method is that many peaks overlap with one another. Using Scherer equation, the average crystallite size is calculated to be 6 nm. Meanwhile, in the XRD patterns of the synthesized nanocomposites by using method 3 [Figure 1(5)], new diffraction peaks at 2θ of 13.40°, 16.84°, 21.70°, 25.69°, 27.67°, 30.89°, 32.29°, 34.22°, 35.43°, 37. 23°, 39.65°, and 40.85° are observed, thus confirming the presence of tricalcium phosphate as the main phase matched with (JCPDS PDF No. 0941-001-00). This finding may be due to the calcination of the mixture at a high temperature (900°C for 3 h), which causes the conversion of a huge amount of HA to the tricalcium phosphate phase. In the XRD patterns, reflections related to CA were covered by tricalcium phosphate and HA peaks; although it is difficult to detect this phase, EDX data can confirm its presence in the nanocomposite. The average crystallite size determined from the most intense diffraction peak is 32 nm.

Figure 4:

FE-SEM images for samples after immersion in SBF.

(1) HA, (2) CA, (3) nanocomposite obtained by method 1, (4) nanocomposite obtained by method 2, and (5) nanocomposite obtained by method 3.

Figure 5:

EDX spectra of samples after immersion in SBF.

(A) HA, (B) CA, (C) nanocomposites synthesized by method 1, (D) nanocomposites synthesized by method 2, (E) nanocomposites synthesized by method 3.

The FE-SEM images for the as-synthesized HA and CA nanoparticles and the nanocomposites synthesized by the three different liquid phase methods are presented in Figure 2. As shown in Figure 2(1), the as-synthesized HA has a narrow particle size distribution in the range of 30 nm–40 nm. In addition, porosity is low and the morphology of particles is spherical. EDX results of as-synthesized HA (Figure 3A) are very close to the calculated values, suggesting that HA is synthesized with high purity. In the HA formula, Ca₅(PO₄)₃(OH), the molar ratio of calcium to phosphate is 1.67, and the molar ratio obtained from EDX data is calculated as 1.60.

The FE-SEM image of the CA nanoparticles [Figure 2(2)] represent spherical particles with narrow size distribution between 35 nm–45 nm. The EDX spectrum of the sample is exhibited in Figure 3B. The Ca/Al molar ratio from the EDX analysis of as-synthesized CA (0.57) is in good agreement with this ratio in the CA formula, CaAl₂O₄ (0.5). Meanwhile, the FE-SEM image of the nanocomposite synthesized by method 1 (Figures 2 and 3) displays low porosity, good particle size distribution in the range of 35 nm–40 nm, and spherical morphology. The EDX analysis of the sample (Figure 3C) represents elements that form CA and HA.

According to the FE-SEM image of the nanocomposite prepared by method 2 [Figure 2(4)], particle size distribution is in the range of 45 nm–50 nm, and the morphology of the particles is spherical. In the EDX spectrum of the nanocomposites (Figure 3D), the existence of Ca, Al, and P elements are well confirmed. The FE-SEM image [Figure 2(5)] and EDX spectrum (Figure 3E) of the nanocomposite synthesized by method 3 can be described as follows. The structure and morphology of this nanocomposite is different from those of the two other nanocomposites. This difference is due to the formation of tricalcium phosphate and high-temperature calcination. The composite particles are agglomerated with size distribution of 80 nm–110 nm. They do not have a specific morphology. In the EDX analysis of the nanocomposites, components are well specified, but the calculation of the ratios is difficult due to other phases.

3.2 Physical and mechanical properties

The physical and mechanical properties of HA and other nanocomposites are listed in Table 1. According to Table 1, with the formation of the HA/CA composite, the physical and mechanical properties of HA are changed. Moreover, the nanocomposites have higher density and lower porosity, compared with HA. Reducing porosity in nanocomposites reduces the amount of water absorption in them. The mechanical properties of the nanocomposites are also higher compared with those reported for CA nanoparticles. Among nanocomposites, the nanocomposite synthesized by method 1 has better mechanical properties. Due to the presence of calcium phosphate in the structure of the nanocomposite obtained by method 3, its applications is different from the nanocomposite synthesized by method 1. For example, method 3 can be used as a drug-delivery system, whereas method 1 can be used for implant covers.

For comparative purposes, the structural and mechanical properties of the synthesized HA/CA bioceramic nancomposites and other samples reported in previous studies are summarized in Table 2. As shown in Table 2, all the HA/CA nanocomposites synthesized by liquid phase methods 1, 2, and 3 have smaller particles and better mechanical properties compared with the HA/CA micro-scale composites reported in previous studies [14], [15].

3.3 Bioactivity behavior

By comparing the FE-SEM images of HA nanoparticles before [Figure 2(1)] and after soaking in SBF solution [Figure 4(1)], we can conclude that apatite has grown on HA. The morphology of HA nanoparticles before the bioactivity test is spherical, but after soaking in the SBF solution, it appears as a sponge that represents the growth of apatite. It should be noted that determining the bioactivity behavior of samples by using FE-SEM images is qualitative, although semi-quantitative EDX analysis is commonly used to confirm such a behavior. As the base material in this sample consists of HA nanoparticles and apatite in the SBF test is created on the sample surface, we cannot use semi-quantitative EDX analysis to calculate the approximate amount of apatite growth; however, we can infer from the FE-SEM images that the apatite has grown well on HA nanoparticles and that the nanoparticles have a very good bioactivity.

Meanwhile, the morphology of CA nanoparticles before soaking in the SBF solution [Figure 2(2)] is spherical; after the bioactivity test [Figure 4(2)], it appears in sheeted form, which indicates the presence of apatite. As specified in EDX analysis (Figure 5B), in the nanoparticles of CA that are under the SBF test, some amount of phosphorus is observed due to growth of apatite on its surface. The FE-SEM images of the nanocomposites synthesized by method 1 [Figure 4(3)] show the presence of apatite with sheeted morphology. Such images indicate the good bioactivity properties of the nanocomposites. SEM images of the nanocomposite synthesized by method 2 [Figure 4(4)] also show the presence of apatite, and the morphology of the formed apatite is sheeted. Based on the images, apatite formation is sporadic, irregular, and incomplete. The FE-SEM images of the nanocomposites synthesized by method 3 before [Figure 2(5)] and after [Figure 4(5)] the bioactivity tests indicate that the cavities in the images are filled with very small spherical particles. These particles are most likely the apatite formed due to the sample immersion in the SBF solution. Figure 5C–E shows the EDX spectra for the synthesized nanocomposites after immersion in the SBF solution.