Synthesis of the nanoparticles
MCSNs, Ag-MCSNs, Zn-MCSNs and Ag/Zn(1:1)-MCSNs were synthesized and characterized in our previous studies . Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs were synthesized by a template method according to our previous methods [5,8]. Different weight of silver nitrate and zinc nitrate (Reagent No.1 Factory of Shanghai Chemical Reagent Co., Ltd, China) were added (Table 1).
The prepared Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs were characterized by transmission electron microscopy (TEM, JEM-2100; JEOL, Tokyo, Japan), field emission-scanning electron microscopy (FE-SEM, 1530 VP; LEO, Germany) and energy dispersive spectrometry (EDS, I MCA 300; OXFORD, UK). The surface area, pore volume and pore size distribution according to N2 adsorption-desorption isotherms (ASAP 2020; Micromeritics, Norcross, GA, USA) were determined using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) analyses. The ions release and pH measurement of the prepared nanoparticles were tested according to our previous methods .
Antimicrobial effects of the nanoparticles
E. faecalis (ATCC 29212, Manassas, VA, USA) suspension was diluted into 1×104 colony forming units (CFUs)/mL. Then 1 mL suspension was mixed with 10 mg of all prepared nanoparticles and CH respectively, and incubated at 4°C for 24 h. Then 10 μL inoculums was plated on brain heart infusion (BHI, OXOID, Basingstoke, UK) agar plate and incubated at 37°C for 24 h. Finally, CFUs of E. faecalis were counted by Automatic colony counter (Scan 1200, Interscience, France). The test was repeated 6 times for each group.
The extracts were prepared by adding the nanoparticles to α-MEM medium (Gibco/ Thermo Scientific, Grand island, USA) at 10 mg/mL and incubated at 37 °C for 24 h respectively. Then the supernatant was sterilized using a 0.22 μm filter (Merck Millipore Ltd., Darmstadt, Germany) after centrifugated at 1000 rpm for 5 min, and supplemented with 10 % fetal bovine serum (FBS, ScienCell, SanDiego, USA).
Mouse pre-osteoblast cell line (MC3T3-E1, ATCC) were cultured in α-MEM medium containing 10% FBS and 100 IU/mL penicillin-streptomycin (HyClone, Utah, USA) at 37 °C in a 5% CO2 atmosphere. MC3T3-E1 cells were inoculated at density of 2×103 cells/well in 100 μL fresh medium for 48 h. Then, they were treated with different extracts respectively. After 1, 3 and 7 d, the cells were washed with fresh α-MEM. Then they were incubated with 100 μL α-MEM and 10 μL CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) in each well at 37 °C and 5% CO2 for 4h. The absorbance at 450 nm was measured by a spectrophotometer (Spectramax190, Molecular Devices, USA). The cells not treated by extracts were used as controls. The results of optical density (OD) were obtained in sextuplicate.
Antimicrobial activity of the nanoparticles against E. faecalis biofilms
Mature human mandibular premolars with single root were collected under the protocol approved by the Ethical Committee Department, the Affiliated Stomatological Hospital of Nanjing Medical University (PJ 2017-055-001). The crowns were removed and roots were standardized to 12 mm long from root apex. The root canals were prepared using ProTaper NiTi rotary instruments (Dentsply Maillefer, Tochigi, Japan) to F3 size according to standard processes. For sterilization, they were autoclaved at 121ºC for 20 min. Then they were placed in 3 mL of E. faecalis suspension (1×108 /mL) and were cultured under anaerobic conditions for 4 weeks at 37°C. The BHI broth was refreshed every second day to remove dead cells and to ensure bacterial viability. CH and MTA pastes (mixed with sterile ddH2O=1:1.5), MCSNs, Ag-MCSNs, Zn-MCSNs, Ag/Zn(1:1)-MCSNs, Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs pastes (mixed with sterile ddH2O=1:3) were prepared. The pastes were introduced into the root canals by lentulo-spirals (Mani Inc, Tochigiken, Japan). Then the roots were placed in sterile tubes. Five specimens were tested in each group. After 7 days, each canal was gently washed with 10 mL phosphate buffer saline (PBS) to clear the intracanal paste and dried with steriled paper points.
Then the specimens from each group were split into two halves. One root-half randomly selected from each root was scanned with a SEM (HITACHI SU3500, Tokyo, Japan) or FE-SEM (QUANTA 200F, Fei, USA) according to our previously described method . The other root-half was stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain (Molecular Probes, Eugene, OR, USA) according to the instructions of manufacturer. Three randomly selected canal wall areas of each root were scanned by a confocal laser scanning microscope (CLSM, LSM 710, Carl Zeiss, Germany) with a 5 μm step size at 20×lens. The excitation/emission wavelengths were 488/525 nm for SYTO® 9 and 561/642 nm for PI. Simultaneous dual-channel imaging was used to display the green fluorescence (live bacteria) and red fluorescence (dead bacteria) using the ZEN software (Carl Zeiss, Germany).
Bacterial colonization on root canal walls pretreated with the nanoparticles
Forty roots were prepared and autoclaved as described above. Each prepared canal was filled with 10 mg/mL suspension of CH, MCSNs, Ag-MCSNs, Zn-MCSNs, Ag/Zn(1:1)-MCSNs, Ag/Zn(1:9)-MCSNs and Ag/Zn(9:1)-MCSNs and activated by the ultrasonic device (P5XS, Satelec, Cedex, France). The ultrasonic device was set at scale 4, and two 30-second sessions of vibration were applied. All roots were stored in a 100% humid environment at 37°C for 7 days. Then each canal was washed with 10 mL PBS to remove the intracanal medication. All pretreated specimens were immersed in 3 mL E. faecalis suspension (1 × 108/mL) at 37 °C for 7 days. Afterwards, each tooth was split into two halves, one root-half randomly selected from each root was observed by the FE-SEM (QUANTA 200F, Fei, USA) and the other root-half was assessed using the CLSM (LSM 710, Carl Zeiss, Germany) according to the methods mentioned above.
Endocytosis of E. faecalis
One milliliter E. faecalis suspension (1×108/mL) was co-cultured with 10 mg of MCSNs, Ag-MCSNs, Zn-MCSNs and Ag/Zn(1:1)-MCSNs for 24 h, respectively. After diluting, the nanoparticle-treated culture material was gained by centrifugation at 3000 rpm for 10 min. Each sample was prepared for TEM observation using standard processes, including fixation, staining, dehydration, infiltration with polymer resin, oven curing, and slicing via ultra-microtome (LEICA EM UC7, Leica, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and were observed using the TEM (JEOL JEM-1010, JEOL, Japan) at 80 kV.
All data were showed as means ± standard deviation (SD) and were analyzed using One-Way ANOVA with a Post Hoc test by SPSS 22.0 (SPSS Inc., Chicago, IL). The level of significance was set at P < 0.05.