# Antimicrobial Effect of Titanium Hydroxapatite in Denture Base Resin
## 3 Results
### TiHA Resistance to Long-Time Irradiation and Soaking  
Knowing the durability of TiHA is important from a clinical application standpoint. Hence, we evaluated the impact of long-term irradiation on the antimicrobial effect of TiHA (Figure 5a). Specimens containing 9% TiHA were prepared by pre-irradiation for 0 (control), 14, 28, 42, or 56 h, which corresponded to 0, 7, 14, 21, or 28 d use, respectively, assuming that TiHA-containing dentures were UVA-irradiated 2 h a day. The CFU values for _S. sanguinis_ and _A. naslundii_ biofilms that formed on specimens that underwent 14 and 28 h pre-irradiation were not significantly different from those without pre-irradiation (Figure 5a). Unexpectedly, the CFU values for _A. naslundii_ biofilms from specimens pre-irradiated for 42 h, and for _S. sanguinis_ and _A. naslundii_ biofilms from specimens  
Figure 4: The antimicrobial effect of TiHA against biofilms formed by bacteria from the human saliva. The specimens containing 0% (control) and 9% TiHA were soaked in BHI containing 25% saliva (**a**) or 100% saliva (**b**) for 12 h to allow the biofilms to form on the specimens. After 2 h of irradiation, the bacteria were plated to determine the CFU values, which were divided by the specimen surface area. The values from specimens containing 9% TiHA were compared with those from the control for each volunteer. Data are presented as the mean \(\pm\) standard deviation for three specimens.  
pre-irradiated for 56 h, were significantly lower than those of the control (\(p<0.05\)). These observations suggested that the antimicrobial effect of TiHA was not readily reduced by UVA irradiation.  
Next, we examined the effect of soaking of the TiHA-containing specimens in ultrapure water on the antimicrobial effect of TiHA (Figure 5b). After soaking for 0 (control), 7, 14, 21, or 28 d, specimens that contained 9% TiHA were used in biofilm experiments. The CFU values for \(S\). _sanguinis_ and _A.__naeslundii_ biofilms formed on the pre-soaked specimens were not significantly different from those formed on specimens without pre-soaking (Figure 5b). Further, ultrapure water used for soaking was analyzed using ICPA (Figure 6). The concentrations of Ca\({}^{2+}\) released from the specimens containing 9% TiHA increased significantly in a time-dependent manner until 21 d after soaking (\(p<0.05\)). However, no significant differences in the Ca\({}^{2+}\) concentrations were observed between samples soaked for 21 d and 28 d. The amount of Ca\({}^{2+}\) released from specimens without TiHA was below the detection limit. The amount of Ti\({}^{4+}\) released from all the tested specimens was undetectable (data not shown). Preliminary experiments revealed that the Ca\({}^{2+}\) and Ti\({}^{4+}\) detection limits of the apparatus used in the current study were 0.087 ug/cm\({}^{3}\) and 0.013 ug/cm\({}^{3}\), respectively.  
Figure 5: The resistance of TiHA to long-time irradiation and soaking. Before the experiments, specimens containing 9% TiHA were pre-irradiated for 0 (control), 14, 28, 42, or 56 h (**a**), or soaked in sterile distilled water for 0 (control), 7, 14, 21, or 28 d (**b**) and then examined. The specimens on which _S.__sanguinis_ or _A.__naeslundii_ formed biofilms were irradiated for 2 h. The bacteria were plated to determine the CFU values, which were divided by the specimen surface area. Data are presented as the mean \(\pm\) standard deviation for three specimens. The asterisk indicates a significant difference in the CFU values of the control and the TiHA-containing specimens (\(p<0.05\)).