2.1. Preparation of β-tri-calcium phosphate (β-TCP)
The solid state reaction was used to synthesize β-TCP powder. Calcium carbonate (CaCO3, 99.00%) and calcium phosphate (CaHPO4, 99.00%) were combined for1/2 h in a 2:1 M ratio. At 1050°C, this combination was calcined with a heating rate of 5°C/min and kept for 6 hours in an electric oven [24].
2.2. Preparation of bioactive glass (BG)
The chemical composition of BG that has been melt quenched is given in Table 1. The starting materials of BG were silicon oxide (SiO2), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), orthoboric acid (H3BO3), ammonium dihydrogen phosphate (NH4H2PO4) and aluminum oxide (Al2O3) powders. From Table 1, the chemical composition of the prepared glass is emphasized. An appropriate amount of the starting materials, which were mechanically homogenized were melted in an electric oven at 1200–1500°C for 2 h in air using a platinum crucible. To achieve better and continuous homogeneity, every 15 min, the melted substance was turned into the crucible. At room temperature, the melted substance was cooled by putting it into a stainless-steel mold and then moved to another muffle oven and hardened there for 1 h at a temperature of roughly 360oC. At a rate of 25°C/h, the muffle was adjusted to cool to room temperature.
Table 1
Chemical composition of the prepared BG (wt%).
Glass composition | SiO2 | CaO | Na2O | B2O3 | P2O5 | Al2O3 |
BG | 50 | 20 | 15 | 7 | 4 | 4 |
2.3. Characterization of the produced samples
The prepared β-TCP and BG samples were ground on an electric milling device made by the German company Retsch GmbH PM100, to get the required particle sizes. After that, X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) were used to confirm the configurations of β-TCP and BG. The X-ray diffractometer PW 1730 from Philips was utilized for the XRD investigation. Its Ni-filtered Cu-K X-ray radiation (= 1.5406) was set at 40 kV and 30 mA. HRTEM images were captured using the JEOL JEM-2100 (Japan) microscope, which had a resolution of 1.402 Å and an accelerating voltage of 200 kV.
2.4. Preparation and characterization of the BG/ β-TCP composites
The manufactured composite samples contain 100, 95, 90, 85, or 80 weight percent of β-TCP powder and 0, 5, 10, 15, or 20 weight percent of BG powder to achieve a weight percent of 100. Their respective symbols are β-TCP, 5BG/ β-TCP, 10BG/ β-TCP, 15BG/ β-TCP, and 20BG/β-TCP. To create pastes with an appropriate consistency for working with, composite samples were mixed with distilled water at a ratio of 0.24 ml/g water to powder. Using a mold with a 10mm diameter and 2mm height, the produced pastes were cast and compressed. After one d of curing in a humidity room with a comparative humidity of around 100% and a temperature of 37 ± 0.5°C, the samples were demolded.
In order to estimate the setting time, 0.2 grams of the freshly manufactured powder were thoroughly blended with one drop of distilled water for 1/2 min before being cast into a mold with dimensions of 10 mm in height and 2 mm in thickness. After leaving the sample for 2 min in the air, the sample was gently lowered to the flat surface of the tested sample using a Gilmre needle (with a 2 mm flat end and a burden of approximately 100 g), it was gently dropped onto the flat surface of the sample being examined. The test was performed for 1/2 min until the sample under test could not be completely circled by the indenter [25].
2.5. In vitro bioactivity and mechanical tests
By soaking the samples in stimulated body fluid (SBF) solution at around 37 ± 0.5°C (the average body temperature).), a biological investigation (In-vitro) was carried out. The SBF solution's chemical makeup and manufacturing process, which is similar to how human blood plasma is generated, elsewhere [26]. To continue the sequence of HA layer production, immersion times in the biological fluid were utilized for 1, 3, 7, 14, and 28 d. Samples were described using the following tools after soaking for a variable amount of time.
A LLOYD device, Model LR 10K, was utilized to study the compressive strength of β-TCP cement and composite sample using the specific method in [27]. At room temperature, the bulk density of composites was calculated using Archimedes' method. Water was employed as the liquid [28].
In addition to the amounts of released P- and Ca- ions in the biological solution (SBF), changes in pH values were estimated. The pH value had been determined using an electrolyte-type pH meter. ICP-AES, which stands for inductively coupled plasma-atomic emission spectroscopy, was utilized to measure the amounts of Ca- and P-ions.
Scanning electron microscopy (SEM) and (EDS) tests were carried out using a SEM-EDS Inspect S, T810, D8571, FEI Co., Japan) having an acceleration voltage of 30 kV and a magnification of 10 up to 300,000, in order to track the formation of hydroxyapatite layers on the surfaces of the samples prior their immersion in SBF solution.
Additionally, Fourier transform infrared spectra were examined by a Japanese Jasco-300E FTIR spectrophotometer. The synthesize samples were crushed, and the fine powders were combined in a 1:100 ratio with KBr. The mixture was then pressed with a weight of 5 tons per square centimeter to create a clear and homogenous disc. To prevent moisture assault, the FTIR studies were completed right away after the discs were prepared.
2.6. Drug delivery profile and release kinetic
A wide spectrum gentamicin was used in this study as a drug model. The drug was loaded into bioglass/β-TCP composite cements by dissolving gentamicin in the cement liquid phase, and prepared the cements following the previously mentioned method. The drug release profile was studied by soaking 200 mg of composite incorporated drug in 3.5 ml phosphate buffer saline (PBS) at pH 7.4 [29] up to 7 d. 1 ml of incubation solution was collected at each predetermined time (3, 6, 12 h, 1, 2, 3, 5, 7 d) and kept at -20 ºC up till the measurement. The same volume of fresh PBS was added instead of the taken solution. Standard curve of the drug was made by preparation of known concentrations of the drug and measured their absorbance by UV/VIS spectroscopy at wavelength 232 nm. This standard curve was used to determine unknown drug concentration in the collected release solutions.
The elution mechanism of gentamycin loaded into the ceramic cements was studied by using various kinetic models, such as first order, Higuchi, Hixson-Crowell, and Baker-Lonsdale models, to determine, approximately, the rate and way of drug released from such cements. Various kinetic models are represented by the equations below:
The relationship between the cumulative proportion of drug released and the square root of time is known as the Higuchi model [30]. The release of water-soluble and weakly soluble from a range of matrices, including solids and semi-solids, can be studied using this model.
Ct = KHt1/2
The Baker-Lonsdale model [31] was developed from the Higuchi model by Baker and Lonsdale (1974) and it characterized the drug release from spherical matrices using the following equation:
Korsmeyer-Peppas model is represented by the following equation:
Ct/C0 = KKtn
Where, the model is fitted on the first 60% of drug release data. Where Ct is the amount of drug released in time t, C0 is the initial amount of drug in the sample, Kh, Kb, and Kk are rate constants dervied from Higuchi, Baker-Lonsdale, and Korsmeyer–Peppas models, respectively, and n is the kinetic exponent. Korsmeyer-Peppas model depends on determination of “n” value to find out the mechanism of drug release [32]. Where, the release is considered a Fickian diffusion mechanism when n = 0.45, and it is non-Fickian transport in cases of 0.45 < n < 0.89, and when n = 0.89 the release is Case II (relaxational) transport, and in case of n > 0.89 it is super case II transport. The kinetic exponent, n, can be determined by plotting of log cumulative percentage of drug release versus log time. The regression coefficients, R2, were calculated to judge how well the data match the kinetic model.
2.7. Antimicrobial investigation
Antimicrobial ability of the designed materials combined with gentamicin and alone was evaluated using microbial pathogens those kindly obtained from Microbiology and Immunology Dep., Faculty of medicine (Boys), Al-Azhar University. One Gram-positive bacterial strain (Staphylococcus aureus), two Gram-negative bacterial strains (Pseudomonas aeruginosa, and Klebsiella pneumonia), one unicellular fungal strain (Candida albicans), and one multicellular fungal strain (Mucor racemosus) were pre-activated using Nutrient broth (for bacterial pathogens at 37oC for 24h.) and Potato Dextrose broth (for fungal pathogens at 28oC for 48h.). Inoculum size of each pathogen was justified to be constant at 106/mL approximately, spore suspension was then dispensed over the agar plate medium and circle pore was marked and the tested material was poured with different samples [33]. Moreover, screening of each of ceramic-loaded gentamicin and unloaded ceramic was performed at different concentration of ceramic and fixed gentamicin concentration. Incubation of each tested pathogen was carried out and the observed results were recorded in three replicates and compared to the standard antibacterial and antifungal agents. The observed differences between the Ceramic-loaded Gentamicin and unloaded Ceramic were evaluated based on the inhibition zone diameter (mm) around each pathogen [34]. Antibacterial and antifungal agents were compared to the tested materials efficiency. This suggestion is EXEMPT from the ETHICAL REVIEW with the number No.:EX0050072023.
2.8. Statistical analysis
The P values < 0.05 were established as statistically significant, and all data were presented as mean standard deviation (SD).