# Antimicrobial Effect of Titanium Hydroxapatite in Denture Base Resin
## 4 Discussion  
Bacterial biofilm formation in the oral cavity starts after the adherence of early colonizers, including _S. sanguinis_ and _A. nueslundii_, to a tooth surface covered with acquired pellicles [32]. _S. aureus_ and _E. coli_ are generally used in investigations of the antimicrobial properties of photocatalytic materials [33; 34]. Therefore, _S. sanguinis_, _A. nueslundii_, _S. aureus_, and _E. coli_ were selected for the evaluation of the antimicrobial effects of TiHA-containing denture base resin in the current study. The addition of other compounds to the PMMA polymer inhibits polymerization of denture base resin, leading to a low flexural strength of the denture base resin [1; 35; 36]. The interstitial areas between PMMA particles in the TiHA-containing specimens increased with an increasing TiHA concentration (Figure 1a), suggesting an inhibition of denture base resin polymerization by TiHA. [PERSON] et al. [27] reported that the flexural strength of TiHA-containing specimens is lower than that of specimens lacking TiHA. It is hence necessary to monitor the reduction of strength of denture base resin associated with the addition of TiHA. By contrast, the concentration of the photocatalyst had a significant effect on surface roughness of the specimens [27]. The TiHA sensitivity of the planktonic bacteria tested was ranked as follows: _S. aureus_\(>\)_S. sanguinis_\(\geq\)_A. nueslundii_\(>\)_E. coli_ (Figure 2). This mostly agreed with a bacterial ranking in a previous study: _S. aureus_\(>\)_S. mutans_\(\geq\)_E. coli_[28]. These data, together with ones presented in the current study, indicate that the sensitivity of planktonic bacteria to TiHA depends on the bacterial species. Interestingly, [PERSON] et al. [28] reported that the antimicrobial effect of TiHA in tissue-conditioning materials against planktonic _S. aureus_ was ca. 1000-fold greater than that against _E. coli_. Such a high discrepancy in the TiHA sensitivity of _S. aureus_ and _E. coli_ was not apparent in the current study (Figure 2). This might be because of the different experimental base materials containing TiHA used (tissue-conditioning material and denture base resin). From a clinical viewpoint, the antimicrobial effect of TiHA against biofilm-forming bacteria is more important than that against planktonic bacteria, since the denture plaque is a bacterial biofilm. In the current study, we demonstrated that TiHA incorporated in the tested specimens killed _S. sanguinis_, _A. nueslundii_, and _S. aureus_ biofilms in a concentration-dependent manner (Figure 3). By contrast, TiHA was ineffective against planktonic _E. coli_ (Figure 2), as well as _E. coli_ biofilms (Figure 3). The photokilling mechanism of Gram-positive bacteria is different from that of Gram-negative bacteria [37; 38]. In Gram-positive bacteria, hydroxyl radicals generated by photocatalytic materials upon irradiation directly destroy the cytoplasmic membrane and abolish cellular activity. On the  
Figure 6: Ca2+ released from the TiHA-containing specimens. The concentration of Ca2+ in the ultrapure water, in which the specimens were soaked for 0, 7, 14, 21, or 28 d, was determined using ICPA. The concentration value (\(\upmu\)g/cm2) was obtained by dividing the total amount of Ca2+ in the ultrapure water by the specimen surface area. Data are presented as the mean \(\pm\) standard deviation for three specimens. The asterisk indicates a significant difference between the compared groups (\(p<0.05\)). other hand, in Gram-negative cells, the outer membrane prevents the hydroxyl radicals from severely damaging the cytoplasmic membrane. Hence, in the Gram-negative cells, the hydroxyl radicals penetrate the cytoplasmic membrane only after partially destroying the outer membrane [37; 38]. Consequently, Gram-negative cells, including _E. coli_, are relatively resistant to the photokilling.