| Objectives:The purpose of this study was:(1) to evaluate the antibacterial efficacy of silver nanoparticles (AgNPs) solution as an irrigant against E. faecalis biofilms formed on root dentin surface;(2) to evaluate the antibacterial efficacy of AgNPs gel as a medicament against E. faecalis biofilms formed on root dentin surface and in dentinal tubules;(3) to evaluate the degree of bacterial adherence and biofilm formation on dentin treated with AgNPs gel;(4) to synthesize and characterize the mesoporous bioactive glasses (MBGs) and Ag-loaded mesoporous bioactive glasses (Ag-MBGs), and to preliminarily evaluate the antibacterial efficacy of Ag-MBGs against E. faecalis biofilms in root canal.Methods:Experiment1:Dentin sections were inoculated with E. faecalis for4weeks to establish a standard monospecies biofilm model. Ninety and six dentin sections were devided randomly into4groups (n=24), and the biofilms established on the dentin sections were irrigated with0.1%AgNPs solution,2%sodium hypochlorite (NaOC1) and sterile saline for2minutes, respectively. Control group received no irrigation. The biofilm structures of12specimens from each group were evaluated using scanning electron microscopy (SEM), while the structure and distribution of viable bacteria of the other12specimens were assessed with confocal laser scanning microscopy (CLSM) combined with viability staining. Experiment2:Dentin sections and root blocks were inoculated with E. faecalis for4weeks. Then80dentin sections and40root blocks were devided randomly into4groups, and the biofilms were treated with0.02%and0.01%AgNPs gel, calcium hydroxide (Ca(OH)2) and sterile saline for7days. The ultrastructure of the biofilms of one half of the dentin sections from each group were evaluated using SEM, while the structure and distribution of viable bacteria of the other half of the specimens were assessed with CLSM combined with viability staining. Dentinal samples at a depth of0to100?n and100to200?m from the root canal surface were obtained from the medicated canal lumens of root blocks, and the dentinal tubules disinfection with the medicaments was evaluated by counting the number of colony forming unite (CFUs). Experiment3:Two hundreds dentin sections were divided into5groups and were treated for7days with the following medicaments:sterile saline, calcium hydroxide,2%chlorhexidine (CHX) gel,0.01%and0.02%AgNPs gel. They were subsequently incubated anaerobically with E. faecalis for1and7days. The qualitative assessment of E. faecalis adherence and biofilm formation on dentin surfaces were carried out using SEM, while the cell viability and quantitative assessment of the total biovolume were determined with CLSM combined with viability staining. Twenty dentin sections were prepared and were treated with0.01%and0.02%AgNPs gel for7days, respectively. Then the dentin sections were placed in24-well cell culture plates containing1mL of deionized water. At the end of1day or7days, deionized water from one half of the wells of each group was tested for Ag+by inductively coupled plasma atomic emission spectrometry. Experiment4:MBGs were synthesized by combining an evaporation induced self assembly and sol-gel process, and Ag-MBGs were synthesized by ions adsorb method. The physicochemical properties of MBGs and Ag-MBGs were characterized. The ions release of them in Tris-HCl and the pH stability of them in simulated body fluids (SBF) was tested, respectively. Twelve human single-rooted mandibuar premolars after root canal preparation were inoculated with E. faecalis for4weeks to establish a standard monospecies biofilm model. Then the biofilms were treated with MBGs, Ag-MBGs and Ca(OH)2for7days respectively, and the antibacterial activity of them against E. faecalis biofilms were qualitatively evaluated by using SEM and CLSM combined with viability staining.Results:Experiment1:The E. faecalis biofilms were destroyed in2%NaOCl group, with very little residual biofilm structure left on dentin sections. Syringe irrigation with0.1%AgNPs solution did not disrupt the biofilm structure, and the proportion of viable bacteria in the biofilm structures were not different from that of the saline group (P>0.05), but was less than that of the control group (P<0.05). Experiment2: The biofilms treated with0.02%AgNPs gel as medicament significantly disrupted the structural integrity of the biofilm and resulted in the least number of post-treatment residual viable E. faecalis cells on dentin surfaces and the least number of CFUs in dentinal tubules at the two depths compared with0.01%AgNPs gel and Ca(OH)2groups (P<0.05), which were again less than these of the control group (P<0.05). Experiment3:Large numbers of E. faecalis cells aggregated on the saline-and Ca(OH)2-treated dentin surfaces and formed dense biofilms structures, while sparse biofilms were formed on the AgNPs gel-treated dentin surfaces, and fewer bacteria adhered to the2%CHX gel-treated dentin surfaces. The cell viability and biovolume of the2%CHX gel groups were significantly less than those of AgNPs gel groups (P <0.05), which were again significantly less than those of saline and Ca(OH)2groups (P<0.05). There were no differences in the cell viability and biovolume of biofilm between0.01%and0.02%AgNPs gel groups after1and7days incubation (P>0.05). The amount of released Ag+from the dentin sections treated with0.02%AgNPs gel after soaking in deionized water for1day or7days was higher than that of the dentin sections treated with0.01%AgNPs gel (P<0.05). The amount of released Ag+from the dentin sections after soaking in deionized water for7days was higher than that of the dentin sections after soaking in deionized water for1day, but was not different significantly (P>0.05). Experiment4:MBGs were successfully synthesized and showed typical characteristics of a mesoporous material. MBGs had a smooth and homogenous surface, and a well-ordered mesoporous structure with a pore size of4.7nm. The results of N2adsorption-desorption analyses of the MBGs showed a type IV isotherm pattern and HI-type hysteresis loops. The XRD pattern showed three diffraction peaks in the small-angle regime. Ag-MBGs kept the highly ordered mesopores, but the borders of the channels structures of Ag-MBGs were not as smooth as MBGs. Numberous nanoparticles set on the surfaces and embedded in the channels, which were confirmed the presence of Ag element by using EDS analysis. MBGs and Ag-MBGs were found to have a sustained release of ions over6days, and the pH values of SBF solution increased and maintained at approximately7.5. The biofilms treated with Ag-MBGs exhibited significant structural damages with few dead bacteria left on dentin surfaces, while the treatment with MBGs did not disrupt the biofilm structure and resulted in the most number of post-treatment residual viable E. faecalis cells than Ag-MBGs and Ca(OH)2. In addition, some MBGs and Ag-MBGs particles adhered to the root canal walls.Conclusions:The findings from this study suggested that:(1) the antibiofilm efficacy of AgNPs depends on the mode of application. AgNPs as a medicament and not as an irrigant showed potential to eliminate residual bacterial biofilms during root canal disinfection;(2)2%CHX gel provided significant residual antibacterial activity against E. faecalis. Ag+were released quickly from the dentin treated with AgNPs gel, which was not in a sustained manner. AgNPs gel treatment of dentin though produced significant E. faecalis cells death and prevented E. faecalis adherence in the early phase, displayed biofilms after7days incubation. Ca(OH)2treatment did not prevent bacterial adherence or biofilm formation on root dentin;(3) Ag-MBGs enabled a sustained Ag+released and possess a potent antibacterial effect against E. faecalis biofilms in root canal, and the antibacterial activity was induced by the release of Ag+from Ag-MBGs. |
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