3.1. DTAanalysis
The thermal behavior of the studied glasses was explored by the DTA technique to identify the glass transition (Tg) and glass crystallization temperatures (Tc) which are useful to determine the most appropriate crystallization temperatures for each composition. Figure 1 and Table 2 represent the characteristic transition temperatures (Tg) and crystallization temperatures (Tc), for the obtained glasses as detected from DTA traces. The DTA analysis of the base composition (sample WD1) showed that an endothermic peak effect appeared at 747 oC. This effect indicative for the Tg, at which the atoms begin to arrange themselves in preliminary structure elements formerly the crystallization step. On the other hand, an exothermic peak was detected at 1038 oC represented the Tc, at which the growth of crystals has occurred completely. The recorded data revealed that additions of 10 wt.% of sodium calcium phosphate-Na4Ca(PO3)6 (sample WD2) or flourapatite-Ca5(PO4)3F (sample WD3) at the expense of diopsideCaMgSi2O6 phase in WD1 composition led to a decrease in the Tg to lower temperature from 747 oC to 733 oC (Fig. 1). Introducing of both Na4Ca(PO3)6 and Ca5(PO4)3F phases instead of 20 wt.% CaMgSi2O6 phase (sample WD4) in the base glass composition led to a reduction of both Tg and Tc to lower temperatures (721°C and 1027°C, respectively). This can be attributed to the effect of Na2O to decline the glass viscosity and accelerating the nucleation stage. Denry and Holloway [32] reported that the sodium acts as a glass network modifier capable of lowering the viscosity of silicate glasses by increasing the number of non-bridging oxygen in the glass composition. As a result of the decrease in the viscosity, the mobility and diffusion rates of the different ions and ionic complexes forming glasses during the crystallization process will be markedly increased, leading to higher crystallization tendency. Introducing of Na2O in the glass composition enhanced its crystallization tendency, leading to displacing of Tg and Tc towards lower temperatures. Similarly, P2O5 additions to the silicate glass composition led to accelerating volume nucleation and support glass-ceramic formation [33].
3.2. XRD analysis
The existence of various phases formed in the studied glasses was a function of the synthesized glass compositions and the crystallization parameters used. The XRD patterns of the investigated glass-ceramics (WD1, WD2, WD3, and WD4) are displayed in Fig. 2 and the crystalline phases developed by the reheating glasses are given in Table 2. It can be noticed from the figure that the diffraction pattern of the base glass sample (Fig. 2, pattern I), WD1 heat-treated at 745 oC/3h-1040 oC/6h, was crystallized into pyroxene members of diopsidic-type (CaMgSi2O6) as a major phase (PDF # 17–0318) beside wollastonite-CaSiO3 phase (PDF # 27-1064). Diopside and wollastonite phases are proper varieties of pyroxene minerals in which the developed glass-ceramics have high mechanical strength and great chemical durability [34]. Glass-ceramics with high Ca contents possess a high ability of good apatite mineralization with excellent bioactive properties [35].
The addition of 10 wt.% sodium calcium phosphate at the expense of 10 wt.% diopside phase in the base composition, i.e. WD2, heat-treated at 730 oC/3h-1065 oC/6h, led to the crystallization of sodium calcium phosphate phase with a new formula -Na4Ca(PO3)3 (PDF # 23–0669) in addition to diopside (as a major phase) and wollastonite phases as proved by the X-ray diffraction analysis (Fig. 2, Pattern II). Whereas, introducing of 10 wt.% fluoroapatite phase at the expense of the diopside phase (glass sample WD3 heated at 735 oC/3h-1075 oC/6h) resulted in the development of fluoroapatite (PDF # 74-4390) as a new phase together with the major diopside phase. The existence of the wollastonite phase as a secondary phase was also detected as indicated from XRD patterns (Fig. 2, pattern III). More Ca2+ ions are included in the media with P5+ ions which have a higher ionic field strength, and hence a greater tendency to shielding. It was also reported that P2O5 had a high tendency to react with CaO [36]. Thus, fluoroapatite-Ca5(PO4)3F crystallized from the droplet phase with highly concentrated Ca and P in the presence of CaF2 in the media [37]. On further decreasing the diopside components by replacing 20 wt.% of CaMgSi2O6 phase with 10 wt.% of Na4Ca(PO3)6 and 10 wt.% of Ca5(PO4)3F in the base composition (glass sample WD4 heat-treated at 720 oC/3h-1025 oC/6h) resulted in a formation of various crystalline phases, which were diopside, wollastonite, fluoroapatite and sodium calcium phosphate phases as detected by the X-ray diffraction analysis (Fig. 2, pattern IV). The high content of CaO and P2O5, by introducing both Na4Ca(PO3)6, Ca5(PO4)3F phases, leads to intense phase separation in the glass. Therefore, phase separation is likely to occur, and the phosphate-rich and silicate-rich phases are separated from the parent glass [37].
3.3. SEM analysis
The SEM images for the WD1, WD2, WD3, and WD4 glass-ceramic samples are displayed in Fig. 3 which presents different morphological shapes as a function of oxide content and crystalline phases formed. It was clear from the figure that the volume crystallization of tiny aggregates–like growths was the distinctive microstructure formed in the WD1 sample (Fig. 3a). The introducing of 10 wt.% Na4Ca(PO3)6 was caused a change in the crystals microstructure to become dendritic like-growths with some glassy matrix in-between as seen in the micrograph of the WD2 sample (Fig. 3b). Whereas, the volume crystallization of globular grains were the distinctive shape in the WD3 sample with 10 wt.% of Ca5(PO4)3F (Fig. 3c). The replacement of diopside by both Na4Ca(PO3)6 (10 wt.%) and Ca5(PO4)3F phases (10 wt.%) in the base composition (sample WD4) led to developing bundles of oriented long rods with aggregates of spherical crystals (Fig. 3d).
3.4. Density
The obtained average values of the measuring density in the laboratory by Archimedes method for the bulk glass-ceramic species heat-treated at different temperatures are presented in Table 2 and Fig. 4a. The general trend of increasing the density for bulk glass-ceramic than that in the corresponding glass was caused by an increase in the temperature during the heat treatment process, which led to the elimination of the remaining pores or due to the growth of more amounts of denser crystalline phases [11]. The density of WD1 glass-ceramic was 2.76 g/cm3, whereas, it was 2.50 g/cm3, 2.92 g/cm3, and 2.90 g/cm3 for WD2, WD3, and WD4 glass-ceramic samples, respectively. The obtained density data revealed that the WD2 glass-ceramic was had the lowest value of the density due to less densification with increasing the free volume of the material and decreased its density [38] as a result of the presence of some glassy matrix in-between crystals of the formed microstructure as seen in Fig. 3(b). Generally, the degree of crystallinity of the glass-ceramic is more effective in their corresponding densities [39]. So, the density values of the WD3, and WD4 glass-ceramic samples were more than that of WD1, P2O5- free sample, which improved the crystallization process and increase the crystallinity of the investigated glass-ceramics and led to form denser microstructure, as shown in SEM micrographs (Fig. 3c and d).
3.5. The mechanical properties (Microhardness)
Bones in the body are constantly subject to changing mechanical stresses due to the variety of body movements. So the mechanical feature is very important and must be taken into account when designing glass-ceramic materials for use in bone tissue in bioengineering applications [11]. The mechanical properties of glass-ceramics mainly depend on the mechanical characteristics of the crystalline phases formed, including the internal microstructure of their crystals, the interfacial bonding among the crystals, and vitreous phases formed in-between, as well as, the ability of the crystals to condense. The microhardness property for the resulting glass-ceramics was determined by Vicker’s microhardness to reach the suitable data which are comparable with those of human bone (680–8000 MPa) [40]. The average hardness data measured for the prepared glass–ceramic species are displayed in Table 2 and Fig. 4b. The results showed that the synthesized glass-ceramics were presented good mechanical properties with high microhardness values of around 4560–5015 MPa. This may be attributed to the presence of high mechanical properties of pyroxene members [34], diopside, and wollastonite, as the major crystalline phases. The replacement of sodium calcium phosphate at the expense of diopside components explaining the decrease in the microhardness value observed for the resultant WD2 sample (4560 MPa) compared to the WD1 sample (4850 MPa) due to the formation of considerable volume fractions of a mechanically weaker NaCa(PO3)3 phase instead of a mechanically stronger diopside phase [12]. While the hardness values were increased significantly for WD3 and WD4 samples, reaching 5045 MPa and 5015 MPa, respectively. Kansal, et al. [13] reported that the combination of high mechanically bioactive material of pyroxene members as diopside CaMgSi2O6 and wollastonite-CaSiO3 phases with resorbable composite such as fluorapatite-Ca5(PO4)3F led to the development of glass-ceramic materials with good mechanical strength and distinct biologically active properties.
Table 2
Heat-treatment regime according to Tg and Tc of the DTA analysis, crystalline phases formed, density (g/cm3), Vicker`s microhardness (MPa), theoretical atomic Ca/P, and atomic Ca/P resulted from EDX analysis after immersion in SBF.
Sample ID. | DTA (°C) | Heat-treatment | Crystalline phases | Density (g/cm3) | Micro-hardness (MPa) | Starting Ca/P | Analyzed Ca/P |
Tg | Tc |
WD1 | 747 | 1038 | 745°C/3h 1040°C/6h | CaMgSi2O6 CaSiO3 | 2.76 | 4850 | N/A | 3.97 |
WD2 | 733 | 1065 | 730°C/3h 1065°C/6h | CaMgSi2O6 CaSiO3 NaCa(PO3)3 | 2.50 | 4560 | 6.70 | 1.91 |
WD3 | 733 | 1072 | 735°C/3h 1075°C/6h | CaMgSi2O6 CaSiO3 Ca5(PO4)3F | 2.92 | 5045 | 12.94 | 2.46 |
WD4 | 721 | 1027 | 720°C/3h 1025°C/6h | CaMgSi2O6 CaSiO3 NaCa(PO3)3 Ca5(PO4)3F | 2.90 | 5015 | 4.62 | 1.91 |
Tg; glass transation temperature, Tc; crystallization temperature |
3.6. In vitro bioactivity test
The degradation and in vitro bioactivity tests of the glass-ceramic samples under investigation were carried out in SBF. Accordingly, the possibility of the formation of a new layer of hydroxyapatite (HAp) was examined by using SEM coupled with EDX analysis. Furthermore, the pH, weight loss %, and ion concentrations (calcium and phosphate ions) were also monitored as a function of time. Figure 5 shows SEM/EDX analysis of WD1, WD2, WD3, and WD4 glass-ceramic samples after immersion in SBF for 22 d. It can be observed from the figure that newly very small spherical aggregates were formed on the glass-ceramic surfaces, which likely assigned to precipitation of a new layer of Ca-phosphate – almost HAp – crystals. This suggestion was confirmed by EDX analysis of the glass-ceramic surfaces, where the analysis showed that the atomic percentages of Ca and P became lower than the theoretical starting percentages of those atoms. Table 2 presents the comparison between the atomic Ca/P ratio of the original composition (theoretical ratio) and the ratio calculated from EDX analysis. Where, the starting Ca/P ratio was 6.70, 12.94, and 4.62 became 1.91, 2.46, and 1.91 for WD2, WD3, and WD4, respectively. While, there was no phosphate in the original composition of the WD1 sample, but, the measured Ca/P ratio was 3.97. These results indicated that the Ca/P ratio of the newly formed layer after immersion in SBF was close to the ratio in HAp (Ca/P = 1.67). Meanwhile, WD2 and WD4 samples were possessed the same Ca/P (1.91) and they were the closest ratios to that of HAp.
The in vitro degradation and dissolution study of bioactive glass-ceramic materials in the physiological fluids is important to give a simulated picture of the behavior of those materials inside the body. In this study, the change of pH, weight loss, and ion concentrations released in SBF was assessed. Figure 6a presents a change of pH of SBF solution as a function of time. It can be noted from the figure that the pH was abruptly increased to maximum values for all samples from 7.40 to 8.09, 7.95, 7.9, and 7.82 for WD1, WD2, WD3, and WD4, respectively, after 3 d of incubation. The pH value of the WD1 sample was significantly (p < 0.02) higher than other samples, whereas, WD4 was significant (p < 0.05) lower than the other samples at this time. After this increase was close to steady-state for WD2, WD3, and WD4 samples, while, the pH value of the solution incubated WD1 was slightly decreased to reach 7.83 at the end of incubation time, and it became significantly (p < 0.003) lower than the other glass-ceramic samples at this time. This difference in the pH values of incubated fluid can be explained by the weight loss and ions released from the glass-ceramic samples in the immersing solution. Figure 6b shows the cumulative wt. loss percent of WD1, WD2, WD3, and WD4 glass-ceramics as a function of time. As displayed in the figure, the weight loss % of all samples, mostly, were degraded linearly with time.
Furthermore, the WD1 sample showed the highest weight loss %, while, WD4 sample presented the lowest one. However, at the initial periods of incubation, the differences in weight loss % among all samples were insignificant (p > 0.05), but, they became significant at the end of incubation time (p < 0.05). Accordingly, the introduction of Na4Ca(PO3)6 or Ca5(PO4)3F into the glass-ceramic (sample WD2 and WD3, respectively) was decreased the degradation, and consequently, the reaction with SBF of the resulted glass-ceramics, and this effect become obvious when both phases were introduced in the glass composition. It has been reported that the fluoride ions decrease the glass reactivity in the solution. Accordingly, the addition of fluoride, as a fluorapatite apatite phase, into the glass composition was acted as a corrosion inhibitor [41]. And so, the existence of fluorapatite crystals in the glass-ceramic was decreased its degradation in SBF.
Figure 6. weight loss % (.g− 1.d− 1), pH, Ca and P ions concentrations (mM), a, b, c and d, respectively, for WD1, WD2, WD3 and WD4 samples immersed in SBF up to 22 d.
Table 3
The weight loss % rate (g− 1.d− 1), release rates (mM∙d− 0.5) of Ca2+ and PO43− from the glass-ceramic samples immersed in SBF. R2 is the coefficient of determination.
| Wt. loss (%.g− 1.d− 1) | R2 | Ca2+ (mM.d− 0.5) | R2 | PO43− (mM.d− 0.5) | R2 |
WD1 | 1.00 | 0.9669 | 2.00 | 0.9648 | 1.93 | 0.9978 |
WD2 | 0.58 | 0.9907 | 2.08 | 0.9911 | 1.86 | 0.997 |
WD3 | 0.74 | 0.99 | 1.79 | 0.9921 | 1.87 | 0.9952 |
WD4 | 0.42 | 0.9926 | 2.13 | 0.994 | 1.98 | 0.9949 |
3.7. Cell viability test
The in vitro cytotoxicity of WD1,WD2,WD3 and WD4 samples was evaluated against normal fibroblast Vero cells using the MTT assay [42]. Figure 7a shows the cell viability % of WD1, WD2, WD3, and WD4 samples at different concentrations (500, 250, 125, 62.5, 31.25 and 15.63 mg.ml− 1). From the figure it was generally noticed that the cell viability was decreased at high concertations, 500 and 250 mg.ml− 1 for all samples, while, the viability of cells was increased with a decrease of concentrations (from 125 mg.ml− 1 to 15.63 mg.ml− 1). On the other hand, the glass-ceramic combined fluorapatite phase (sample WD3) was showed higher cell viability than other samples at 62.5 mg.ml− 1, whereas, the glass-ceramic contained sodium calcium phosphate phase (WD2) demonstrated higher cell viability than the other glass-ceramics at high concentrations (> 62.5 mg.ml− 1). The CC50 values are shown in Fig. 7b as common parameters for the cytotoxicity of the investigated compounds. It is the cytotoxic concentration of the material to cause death to 50% of viable cells. The calculated CC50 values were about 141, 162, 121, and 116 mg.ml− 1 for WD1, WD2, WD3, and WD4, respectively. Where, WD2 (162 mg.ml− 1) sample was demonstrated the highest CC50 value, while, WD4 was showed the lowest one (116 mg.ml− 1). And so, combining of sodium calcium phosphate (Na4Ca(PO3)6) in wollastonite-diopside glass-ceramic was presented less cytotoxic effect than that included fluorapatite (Ca5(PO4)3F), and a combining of both crystalline phases has increased the cytotoxicity to the highest value. This can be explained by the degradation behavior of the samples in the aqueous solution. According to the degradation results, WD4 was showed a lower degradation rate and higher released Ca2+ ion concentration than WD2 and WD3 samples. This released high Ca2+ ion was increased the calcium ion concentration in the cell culture medium which may result in cell death, as reported previously [43, 44].
The photos seen under an inverted microscope for the Vero cells after incubation with all samples for 24 h confirmed the results of MTT assay in which different morphological changes and loss of adhesion to a substratum were observed at a higher concentration for WD1, WD2, WD3, and WD4. Unlike, the control cells showed adherent growth and a regular polygonal shape, While at lower concentration the adherent ability increased with normal cell morphology and few round cells representing cellular outgrowth. Studies were found that suitable divalent ions such as Ca2+and Mg2+ ions can promote cell attachment, growth, and differentiation due to cell adhesion molecules on cell surfaces [45–48].