5.1. Surface morphology
Broad face SEM micrographs of the cements after soaking in a solution with different pH values for 1 and 30 days are shown in Fig. 1, in addition to Ca/P ratio.The Ca/P ratios of the cement specimen after 1 day of soaking in pH 7.4 and 4.0 solutions were 1.44 and 2.56 (Fig.
1a), respectively; on day 7, the Ca/P ratios became 1.68 and 1.84. Before soaking, the cement specimen essentially appeared rather smooth looking with particle entanglement and several micropores (Fig. 1b). After soaking in a physiological solution, i
t was
clear that precipitation took place on the cement surfaces, which were covered with clusters of precipitated apatite spherulites (Fig. 1cf). However, it is worth noting that after soaking in a pH 7.4 solution for 1 day (Fig. 1c), the size of apatite spherulites was greater than that formed in the pH 4.0 solution(Fig. 1d). With increasing soaking time, spherulites coalesced to form a surface apatite layer.
Greater spherule aggregates appeared on the cement surface under a pH 7.4 condition (Fig. 1e) compared to that under a pH 4.0 condition (Fig. 1f).
5.2. Diametral tensile strength
The changes in diametral tensile strength value of the cements before and after soaking in a pH 7.4 or 4.0 solution as a function of time are shown in Fig. 2. The cement specimen gradually increased in strength with an increase in soaking time, achieving a maximum on day 7, and thereafter decreased. The values at 0- and 30-day soaking in a pH 7.4 solution were 4.7
± 0.4 and 4.8 ± 0.8 MPa, respectively, indicating no significant difference (P > 0.05). In the case of pH 4.0, day 30 samples could achieve a value of 3.8 ± 0.6 MPa, which was comparable to that obtained in the environment of pH 7.4. Scheffé post hoc tests revealed that the difference between the strength values of cement specimens exposed to pH 7.4 and pH 4.0 was not statistically significant (P > 0.05) at the same soaking time point, although the cement soaked in a pH 7.4 solution had a higher strength than the pH 4.0 solution. Soaking time significantly (P < 0.05) affected the strength of the cement soaked in either pH 7.4 or 4.0 solution.
5.3. Weight change
There were statistically significant differences (P < 0.05) in the weight change among the groups because of solution pH and soaking time. Figure 3 shows the weight changes for the cements after exposure to the physiological solution. In terms of solution pH, the cement gained weight of 2.0% and 1.2% after 3 days of soaking in a pH 7.4 and 4.0 solution, respectively; afterwards, the sample weight reduced to -0.2% and 0.8% on day 30. Not only the initial solution pH values significantly (P < 0.05) affected the weight change of the cement to some extent, but soaking time did also.
5.4. Porosity
Results shown in Fig. 4 indicate that the porosity increased from the initial 23% to approximately 32% after 30-day soaking; these values were significantly different (P < 0.05).
Concerning solution pH, the cement had a lower porosity in a solution with pH 7.4 at the same soaking time point.
5.5. pH variation of the solution
Figure 5 presents the time-dependent pH changes of the cement-immersed solutions. While soaked in a pH 7.4 solution, the cements caused the pH of the solution to increase at a steady state and attained alkaline values of 8.5 on day 30. In contrast, after soaking in a pH 4.0 solution the cement produced a lower pH value at the same time point. On day 1, the pH (7.3) of the solution originating from the pH 4.0 condition was significantly (P < 0.05) lower than the pH (7.8) of the solution starting from the pH 7.4 condition. The initial solution pH and soaking time significantly (P < 0.05) affected the sequent pH variation.
5.6. Setting time
The setting times (15-24 min) increased significantly (P < 0.05) with increasing Bi2O3
content (Table 1). These values were significantly (P < 0.05) lower than that of WMTA (168 min).
5.7. pH variation in the cement
The pH value of all cements during setting is presented in Fig. 6. After 20 min, all of the
cements reached a pH value of about 12.0. By 1 h, they approached a steady-state pH value.
The variations in pH value slightly decreased with increasing Bi2O3 content.
5.8. Phase composition
Figure 7 shows the XRD patterns of β-Ca2SiO4 cements with and without Bi2O3. The strongest peak at 2θ = 27.4º was ascribed to Bi2O3. It indicates that the products of the hydration process were calcium silicate hydrate (C–S–H) at 2θ = 29.4º overlapping with calcite. In addition, an incompletely reacted inorganic component phase of β-Ca2SiO4 at 2θ between 32−34º was found. The higher the Bi2O3 content, the lower the C–S–H content was in the cement.
5.9. Effect of Bi2O3 on DTS
The relationship between the Bi2O3 content and DTS of the cements is shown in Table 1.
The strength of the cements did not change significantly, although a decreasing trend with increasing Bi2O3 content was found. WMTA had a strength value of 4.2 MPa, which was significantly (P < 0.05) higher than those of all dicalcium silicate cements.
5.10. Radiopacity
The radiopacity (as an equivalent thickness of Al) of the CS control was recorded as 1.7 mm of Al (Table 1). After the addition of 5, 10, and 20 wt% Bi2O3, the radiopacity of the cement became significantly (P < 0.05) higher, with values of approximately 3.3, 5.8, and 8.4 mm of Al, respectively.
5.11. Solubility
The solubility of the four dicalcium silicate cements ranged from 0.8% to 1.1%. The incorporation of Bi2O3 did not significantly (P < 0.05) enhance the solubility of dicalcium silicate cements. WMTA had a solubility of 1.4% and was significantly different with P < 0.05 for all comparisons with dicalcium silicate cements with and without Bi2O3.
5.12. Cell proliferation
Figure 8 shows that the proliferation of MG63 cells cultured on Bi2O3-containing cement surfaces was lower than that on the surface of the cement control at all culture time points. For example, on day 7, the OD value for CSB20 cement was approximately 46% lower than that of the control.
5.13. Cell differentiation
The intracellular ALP level was measured to observe the functional activity of cells. The results are shown in Fig. 9. The ALP level decreased with increasing Bi2O3 content of the cements at all incubation times. On day 7, a significant 21% reduction (P < 0.05) in the ALP level was measured for CSB20 compared to the CS control. The reduction became 24% after 14 days of culture.
5.14. Mineralization
Quantification of calcium mineral deposits by the Alizarin Red S assay showed that the calcium content in the blank groups (day 0) was much lower than that obtained for the cement seeded with cells due to the lack of positive red staining (Fig. 10). This explains why calcium contents in the material composition did not appreciably affect the quantification of calcium mineral deposits. On day 7, more mineral deposition was found in MG63 cells cultured on the cement specimens than those obtained on day 0. With increasing culture time, mineral
deposition increased for the cells cultured on all cements, but less mineral deposition was found for cements with higher Bi2O3 content. By day 14, a significant 28% reduction (P < 0.05) of calcium content was observed for the 20 wt% Bi2O3-containing cement (CSB20) compared to the CS control.