2.1 Preparation of bioactive glass (BG) samples
Glass system has been prepared by melting the starting materials (SiO2, H2BO3, Na2CO3, CaCO3 and AgNO3) in a platinum crucible at 1100 °C for 2 h in air. In order to ensure a homogenous mixing of all reactants and get a bubble-free sample, the molten liquid was stirred to ensure homogenous mixing of all constituents. Then, the batches were poured in a mold with the desired shape and instantly put in another muffle to be annealed at about 380–400 °C for 1 h. Subsequently, the muffle was switched off and the temperature decreased to room temperature with a rate of 25 °C/h. The nominal composition of the prepared glass system is tabulated in Table 1.
2.2. Preparation of CHA nanopowders
CHA nanopowders have been successfully prepared and well-characterized according to the described methods in our recent published articles [18-20]. Briefly, high-energy ball mill has been utilized using calcium carbonate (CaCO3) and calcium hydrogen phosphate dihydrate (CaHPO4·2H2O) as raw materials for the preparation of HA. It is worth to note that the success of mechanochemical synthesis method to produce HA is highly dependent upon ball-to-powder ratio (BPR) and milling time as they promote the occurrence of reaction.
2.3.Preparation of CHA/BG nanocomposites powders
The CHA and BG, based on their respective wt%, as represented in Table 2, were mechanically blended for 10 h using BPR equals to 1:2 and the balls diameters were 10 mm. Then, these mixtures were milled for 5 h in a planetary ball mill (type MTI SFM (QM-3SP2)) operating at 400 rpm as a rotation speed and BPR = 20:1. It is worth to mention that the milling has been performed under dry condition.
2.4. Measurement of the physical and mechanical properties of CHA/BG nanocomposites
According to the recent articles of Taha et al. [21,22], the milled powders were pressed 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 the sintered samples at 700 °C.
According to Refs. [23-25], the microhardness of the investigated specimens was measured using Vickers indentor and calculated according to ASTM: B933-09 while, compressive strength was measured according to ASTM E9. In addition, the values of elastic moduli; Young’s modulus (L), shear modulus (G), elastic modulus (E), bulk modulus (B) and Poisson's ratio (ʋ) have been calculated according to Refs. [26,27].
2.5. Characterization of the sintered samples
The formed phases in nanocomposite samples result from sintering process, were examined using X-ray diffraction (XRD) technique, "Philips PW 1373" X-ray powder diffractometer with CuK-Ni filtered radiation. In order to confirm the molecular structure of the prepared nanocomposites, Fourier transform infrared (Vertex 80, Bruker, Germany) spectroscopy was employed. FTIR absorption spectra of the tested specimens were immediately collected by attenuated total reflection (ATR) unit, at room temperature, in the wavenumber range 4000–400 cm−1, 60 scans at resolution 4 cm−1. Scanning electron microscopy (SEM, type Quanta FEG250) with accelerating voltage of 30 kV and magnification 10× up to 300,000× was employed to examine the microstructure of these samples.
2.6. Biomedical characterization
2.6.1. In vitro bioactivity assessment of sintered nanocomposites
In vitro bioactivity of the resultant nanocomposites was evaluated by soaking samples in the simulated body fluid (SBF) prepared according to the recipe described by Kokubo et al. [28,29] while maintaining the ratio of glass grains to solution volume = 0.01 g/ml [30]. Subsequently, the soaked samples were subjected to XRD and SEM to examine the HA layer formed on their surfaces. The ionic concentrations of the prepared SBF compared to the human blood plasma concentrations are listed in Table 3.
2.6.2. Evaluation of the antibacterial effect of prepared samples
The antibacterial behavior of the prepared nanocomposites was tested against S. aureus (ATCC6538) and E. coli (ATCC25922) bacterial strains using disc-diffusion method at 37 °C for 24 h.