Example 1
Methods
Synthesis of TiO2 Nanoparticles (TiO2 NPs) and doped TiO2 NPs
TiO2 NPs for use in the present disclosure can be synthesized using any appropriate method and are not limited to the methods disclosed herein. The NPs can be doped during production of the TiO2 NPs (Process 1), or can be doped after production of the TiO2 NPs (Process 2). For example, the doped TiO2 NPs can be produced using a two-step method wherein TiO2 NPs are produced in the first step (Process 1) and are then processed in a second step (Process 2) to produce the doped TiO2 NPs. In non-limiting embodiments, Process 1 utilizes a solvothermal method, and Process 2 utilizes the nitrogen-doping of the TiO2.
In a non-limiting version of Process 1, titanium (IV) butoxide (TB, 97%) (5-10 mmol) was added to a mixture of X mmol oleic acid (OA, 90%), Y mmol oleylamine (OM, 70%), and 100 mmol absolute ethanol (X+Y=50). X and Y can be varied while leaving the molar ratio of titanium n-butoxide (TB) and surfactants unchanged (i.e., TB/(OA+OM)=1:10) to gain different OA/OM ratios, which lead to the formation of different shapes of NPs. For example, to synthesize TiO2 with truncated rhombic shape, 5 mmol of TB was added to a mixture of 25 mmol OA, 25 mmol OM, and 100 mmol absolute ethanol. The obtained mixture in a 40 mL Teflon cup was stirred for 10 min before being transferred into a 100 mL Teflon-lined stainless steel autoclave containing 20 mL of a mixture of ethanol and water (96% ethanol, v/v). The concentration of ethanol was used at the azeotropic point so that the amount of water vapor did not change much during the crystallization process. The system was then heated at 180° C. for 18 h. The obtained white precipitates were washed several times with ethanol and then dried at room temperature. The as-synthesized TiO2 NP products were dispersed in nonpolar solvent, such as toluene. TiO2 NPs synthesized by varying the OA/OM molar ratio, When the OA/OM mole ratio is 4:6, rhombic-shaped TiO2 NPs with uniform size are obtained. By increasing the OA/OM molar ratio to 5:5, smaller TiO2 NPs with truncated rhombic shape can be produced. A further increase of this ratio up to 6:4 leads to the formation of spherical particles with an average size of 13 nm.
In a particular non-limiting example of Process 1, a solution comprised of 1.7 g of Ti(IV)-butoxide (Aldrich, 97%), 4.6 g ethanol (Decon Labs, 100%), 6.8 g oleylamine (Aldrich, 70%), and 7.1 g oleic acid (Aldrich, 90%) was prepared, then mixed with 20 mL of 4% H2O in ethanol (18-MΩ Milli-Q; Decon Labs). Each solution was clear before mixing, but the final mixture immediately clouded due to formation of micelles and likely some hydrolysis. This solution was then split into two portions (around 20 mL/portion), and each portion was placed into a high-pressure reaction vessel (Paar Series 5000 Multiple Reactor System) and reacted at 180° C. for 24-hours. The vessels were stirred via external magnetic field and Teflon-coated stir bars. The reaction vessels were Teflon-lined. Upon cooling, the solutions were decanted and rinsed 3 times with anhydrous ethanol to remove extraneous surfactants resulting in pure TiO2 NPs which were readily dispersible into 20-30 mL ethanol, but did not form clear solutions. The TiO2 NPs formed were stored in ethanol.
In a particular non-limiting embodiment of Process 2 for making doped TiO2 NPs, a portion of the TiO2 NPs in ethanol (manufactured in Process 1) are then reacted with an equal volume of triethylamine (Sigma-Aldrich Co., LLC.), also using the high-pressure reaction vessel, at 140° C. for 12 hours. Upon cooling the now-nitrogen-doped TiO2 NPs (N—TiO2 NPs) are rinsed 3 times with anhydrous ethanol. The exact concentration of TiO2 in ethanol/triethylamine varies, but in every instance there is an excess of triethylamine. The final N—TiO2 NP ethanol solution yields a gravimetrically-determined concentration of particles typically in the range of 35 mg/mL. In at least certain embodiments, the N/Ti molar ratio of the N—TiO2 NPs was in a range of 0.1% to 3.4%.
Synthesis of Co-Doped-TiO2 NPs
Nitrogen and Silver co-doping. N—Ag—TiO2 NPs can be formed following the reaction steps of Process 1 using Ti(IV)-butoxide. Ag is provided by adding silver acetlyacetonate and N is provided using tetramethyl ammonium hydroxide as the dopant sources in a wt:wt:wt N:Ag:Ti ratio of, for example, 1:1:18, which provides a 5%/5%/90% N/Ag/Ti composition. For example in one embodiment, components sufficient to provide 0.085 g N and 0.085 g Ag can be combined with a component comprising Ti (e.g., Ti(IV)-butoxide) are used. The TiO2 NPs form as in Process 1 but with the N and Ag dopants in place.
Nitrogen and Fluorine co-doping. N—F—TiO2 NPs can be formed following the reaction steps of Process 1 using Ti(IV)-butoxide. F and N are provided by adding ammonium fluoride as the dopant sources in a wt:wt:wt N:Ag:Ti ratio of, for example, 1:1:18, which provides a 5%/5%/90% N/F/Ti composition. For example in one embodiment, components sufficient to provide 0.085 g N and 0.085 g F can be combined with a component comprising Ti (e.g., Ti(IV)-butoxide) are used. The TiO2 NPs form as in Process 1 but with the N and F dopants in place.
Phosphate coating of TiO2 NPs. In this variation, undoped TiO2 NPs, N—TiO2 NPs, or co-doped TiO2 NPs are treated to provide a coating of phosphate on the outer surface of the particles. The nanoparticles are dispersed into commercial phosphate buffered saline (PBS), which is a neutral pH solution of sodium phosphate. Upon reaction as usual, the particles become phosphate-derivatized and are amenable to further mineralization. The phosphate coated TiO2 NPs are designated herein as P—TiO2 NPs. The phosphate coated N—TiO2 NPs are designated herein as N—P—TiO2 NPs. The phosphate coated N—Ag—TiO2 NPs are designated herein as N—Ag—P—TiO2 NPs. The phosphate coated N—F—TiO2 NPs are designated herein as N—F—P—TiO2 NPs.
Characterization of N—TiO2 NPs
UV-VIS Spectroscopy
TiO2 (P25, Evonik Degussa GmbH, Germany) and N—TiO2 (Oak Ridge National Laboratory, TN) nanoparticles in ethanol suspension were individually characterized regarding its optical absorbance with a Cary®50 (Agilent Technologies, Santa Clara, CA) spectrophotometer using the transmittance method. Aliquots (20 μL) of each material (either P25 or N—TiO2, 40 mg/mL in ethanol) were individually placed in a quartz microcell. Each sample was then placed inside of the spectrophotometer's chamber between the light source and the photodetector and the intensity of light that reached the photodetector was measured from 190 nm-900 nm in 2 nm increments (FIG. 1).
Scanning Electron Microscopy (SEM)
Aliquots (5 μL) of N—TiO2 NPs suspended in ethanol in the as-synthesized concentration (200 proof, 40 mg/mL, Oak Ridge National Laboratory, USA) were placed onto a polished silicon wafer. N—TiO2 NP samples were air-dried in room temperature until all the solvent had been evaporated. Individual specimens were then mounted onto standard aluminum SEM pin stubs (diameter ⅛″) and were sputter coated with a thin-layer (˜4 nm) of iridium using a sputter coater (K575D, Emitech Sample Preparation, UK) prior to the imaging process. Adhesive samples containing 50, 67 and 80% (v/v) were mounted directly onto standard aluminum SEM pin stubs using double-sided carbon tape and silver paste for electrical grounding to the stub. The adhesive samples were sputter coated using the same procedures described for the N—TiO2 in suspension. Both, N—TiO2 NPs in suspension and immobilized in dental adhesive resins were imaged using a Zeiss Neon 40 EsB SEM at 5 kV (FIG. 2). Energy dispersive X-ray spectroscopy (EDS) and EDS compositional mapping was performed using an Oxford INCA 250 microanalysis system with an analytical drift detector at 15 kV (FIG. 3).
Transmission Electron Microscopy (TEM)
N—TiO2 NPs suspended in ethanol (100%, 0.032 mg/mL, Oak Ridge National Laboratory, USA) were dispersed by brief sonication in an ultrasound bath (Bransonic 220, Branson Ultrasonics, USA). A drop of suspended TiO2 NPs was placed on holey carbon coated copper grids. The drop was allowed to adsorb for 1-2 min, then wicked with filter paper to remove excess fluid, and dried before viewing in a JEOL 2000FX transmission electron microscope. Images were made on Carestream® Kodak® electron image film SO-163 (Eastman Kodak Company, USA) and digitized with an Epson Perfection V750-M Pro scanner (Epson America, Inc. USA). X-ray spectra were collected using a Kevex thin window detector and EDS software (IXRF Systems Inc., USA) (FIG. 4).
Specimen Fabrication
Disk shaped specimens (diameter=12.00 mm, thickness ≅15 μm) of OptiBond Solo Plus (OBSP) adhesive resin (Kerr Corp., USA) and experimental adhesive resins containing 50, 67 and 80% (v/v) of N—TiO2 NPs, were manually fabricated by individually dispensing 10 μL of each material onto the surfaces of separated glass coverslips (No. 2, VWR International, LLC). Then both the unaltered and experimental adhesive resins were uniformly spread over glass coverslips using disposable flexible applicators (Kerr Corp., USA) and were polymerized using blue light irradiation (1000 mW/cm2, 1 min) emitted from a broadband LED light-curing unit (VALO, Ultradent Products, Inc., USA). Specimens of both unaltered and experimental adhesive resins were then UV-sterilized (254 nm, 800,000 μJ/cm2, UVP Crosslinker, model CL-1000, UVP, USA). Similarly, P—TiO2 NPs, N—P—TiO2 NPs, N—Ag—TiO2 NPs, N—Ag—P—TiO2 NPs, N—F—TiO2 NPs, and N—F—P—TiO2 NPs can be mixed with adhesive resins including but not limited to OBSP to form doped-TiO2 dental resins.
Bacterial Strain
Streptococcus mutans strain UA159 (JM10::pJM1-ldh, luc+, Spr, luc under the control of the ldh promoter) was utilized for this project. The selection of antibiotic-resistant colonies was performed on TH plates (Todd-Hewitt, BD Difco, USA) supplemented with 0.3% yeast extract (EMB, Germany) and 800 μg/mL of spectinomycin (MP Biomedicals, USA). The plates were incubated under anaerobic conditions at 37° C. for 48 h.
Antibacterial Behavior of N—TiO2 NPs in Suspension
In order to assess the antibacterial efficacy of the N—TiO2 in ethanol suspension (200 proof, 40 mg/mL, Oak Ridge National Laboratory, USA), S. mutans biofilms were grown in sterile microcentrifuge tubes (Safe-Lock Tubes, Eppendorf North America, USA). Planktonic cultures of S. mutans (UA159-ldh, JM10) were grown in THY culture medium at 37° C. for 16 hours. Planktonic cultures having optical density (OD600) levels≥0.900 were used as inoculum to grow the biofilms. A 1:500 dilution of the inoculum was added to 0.65×THY+0.1% (w/v) sucrose biofilm growth medium.
Inoculum aliquots (1.00 mL) were added to separate sterile microcentrifuge tubes and biofilms were grown for 24-hours (static cultures, anaerobic conditions, 37° C.). After the growth period, biofilms were replenished with 1.00 mL of fresh 1×THY+1% (w/v) glucose culture medium and were incubated at 37° C. for 1 hour. Replenished biofilms (n=15/group/irradiation condition) were then exposed to the nanoparticles diluted in growth media [1×THY+1% (v/v)] in the concentrations of 19%, 25% and 30% (v/v) with or without blue light irradiation (1000 mW/cm2, 1 min) provided by a commercially available broadband LED light-curing unit (VALO, Ultradent Products, Inc., USA). Biofilms that were not exposed to the nanoparticles comprised our negative control (n=45). Positive control groups (n=15/concentration) were comprised by biofilms that were exposed to ethanol aqueous solutions (200 proof, AAPER Alcohol and Chemical Co., Shelbyville, KY) in concentrations of 19%, 25% and 30% (v/v) with or without light irradiation (1000 mW/cm2, 1 min). Following the treatment, the suspension containing either N—TiO2 NPs in suspension or ethanol aqueous solution was carefully aspirated. Immediately after, biofilms were replenished with 1.00 mL of fresh 1×THY+1% (w/v) glucose sterile culture medium. The microcentrifuge tubes containing the replenished biofilms were then sonicated to facilitate the removal of the adherent biomass using a sonicator (Q700 sonicator, QSonica, USA) connected to a water bath (4° C.; 4 cycles of 1 minute, 15 seconds interval between cycles; power 230±10 W, total energy ≈ 78 kJ).
Antibacterial behavior of N—TiO2 NPs immobilized in dental adhesive resins
In order to assess the antibacterial efficacy of experimental adhesive resins containing 50%, 67% and 80% (v/v) of N—TiO2 NPs (Oak Ridge National Laboratory, USA), S. mutans biofilms were grown against the surfaces of sterile specimens of both unaltered and experimental adhesive resins. Planktonic cultures of S. mutans (UA159-ldh, JM10) were grown in THY culture medium at 37° C. for 16 hours. Planktonic cultures having optical density (OD600) levels≥0.900 were used as inoculum to grow the biofilms. A 1:500 dilution of the inoculum was added to 0.65×THY+0.1% (w/v) sucrose biofilm growth medium. Inoculum aliquots (2.5 mL) were dispensed into the wells of sterile 24-well microtiter plates (Falcon, Corning, USA) containing sterile specimens. Biofilms were grown for either 3- or 24-hours (static cultures, anaerobic conditions, 37° C.) with or without continuous light irradiation provided by a prototype LED device (410±10 nm, 3 h irradiation=38.75 J/cm2, 24 h irradiation=310.07 J/cm2).
After the growth period, biofilms were replenished with 2.5 mL of fresh 1×THY+1% (w/v) glucose culture medium and were incubated at 37° C. for 1 hour. Replenished biofilms were transferred into individual sterile polypropylene tubes (3 mL, ConSert Vials, Thermo Fisher Scientific, USA) containing 1.0 mL of fresh 0.65×THY+0.1% (w/v) sucrose medium. Vials containing the specimens were sonicated to facilitate the removal of the adherent biomass using a sonicator (Q700 sonicator, QSonica, USA) connected to a water bath (4° C.; 4 cycles of 1 minute, 15-second interval between cycles; power 230±10 W, total energy ≈ 78 kJ).
Colony-Forming Units (CFU/mL)
Biofilms grown either in the microcentrifuge tubes or on the surfaces of both unaltered and experimental dental adhesive resins were sonicated to allow the antibacterial efficacy assessment using the colony-forming units method. Immediately after sonication procedures, inoculum aliquots (10 μL) from each specimen were diluted in 90 μL of 0.65× THY+0.1% (w/v) sucrose sterile culture medium. Serial dilutions were then carried out in 0.65×THY+0.1% (w/v) sucrose sterile culture medium for all samples using a multi-channel pipette (5-50 μL, VWR, USA). Aliquots (10 μL) of each dilution were then plated in triplicate (total: 30 μL/sample/dilution) using THY plates supplemented with 800 μg of spectinomycin.
Staining and Confocal Laser Scanning Microscopy
A separate set of specimens was fabricated as described above. Biofilms were then grown on the surfaces of unaltered and experimental adhesive resins, using the conditions above in preparation for staining and confocal microscopy. The biofilms on all specimens were stained using BacLight™ LIVE/DEAD fluorescent stains (1.67 μM each of Syto 9 to stain live cells and propidium iodide to stain dead/damaged cells, Molecular Probes, USA) and kept hydrated prior to confocal microscopy. The full thickness of biofilms on all specimens was imaged by confocal microscopy at three randomly selected locations per specimen, in order to gain a representative sample for each specimen, using a Leica TCS SP2 MP confocal laser scanning microscope (CLSM) with Ar (488 nm) and He/Ne (543 nm) lasers for excitation of the fluorescent stains. A 63× water immersion microscope objective lens was used and serial optical sections were recorded from the bottom of the specimen to the top of the biofilm at 0.6 μM intervals in the z-direction. Representative 3-D reconstruction images of live and dead/damaged cells in the 24-hour biofilms grown on adhesive resins were generated using Velocity software (Version 4.4.0, Velocity Software solutions Pvt. Ltd., India) to facilitate visualization of biofilm distribution in all groups investigated.
Contact Angle Goniometry
A separate set of specimens (n=4/group/concentration) was fabricated as described above in preparation for the contact angle goniometry at oral temperature (37° C.). Immediately after fabrication, specimens of each group were left undisturbed (10 min) inside of the environmental chamber of a contact angle goniometer (OCA15-Plus, Dataphysics Instruments, Germany) for thermal equilibration prior to testing. The wettability of water was tested at oral temperature by displacing a 2 μL drop of ultrapure pure water onto four random locations of each specimen (16 drops/group). The profiles of the axisymmetric drops were recorded using a high-speed and high-definition CCD camera (1 min, 25 frames/sec). The evolution of drop profiles over time was analyzed using the SCA20 software (Dataphysics Instruments, Germany) and the Laplace-Young equation was used to calculate the contact angles at time=0 s (θINITIAL) and time=59 s (θFINAL).
Results
UV-Vis Spectroscopy
FIG. 1 represents the UV-vis spectroscopy results for both the undoped and nitrogen-doped titanium dioxide nanoparticles. It is possible to observe that doped samples displayed higher absorption levels throughout the range of wavelengths considered, which confirms that nitrogen was successfully incorporated into the crystal lattice of titanic.
SEM, EDS and TEM Characterization of N—TiO2 NPs Suspended in Ethanol
FIG. 2 represents the SEM pictures of the N—TiO2 NPs at different magnifications (500× to 50,000×). Even though it is possible to observe a strong agglomeration behavior of the nanoparticles in the as-synthesized concentration (40 mg/mL in 100% ethanol), these pictures indicate that nanoparticles fabricated by the solvothermal method described above have an approximately spherical shape, smooth surfaces and most of the nanoparticles exhibit some faceting.
FIG. 3 represents the EDS pictures of the compositional analysis of the N—TiO2 NPs in the as-synthesized concentration (40 mg/mL). The mapping of elements indicates large quantities of titanium (Ti), oxygen (O), carbon (C) and silicon (Si). The visible peaks present in the EDS compositional spectrum confirm the presence, and the relative amounts (in wt %) of the elements in the samples investigated. The results of the analysis of compositional characterization of the N—TiO2 NPs by EDS revealed that Ti (40.9%), O (39.3%), C (13.3%) and Si (6.5%) were the major components found in N—TiO2 NP samples. The presence of silicon is related with the wafer substrate in which the samples were imaged. However, under the conditions used herein the doping element (nitrogen) could not be mapped. It is believed that the combination of factors like the low atomic number of nitrogen (Z=7), and the complete overlap between the Ti Lλ (0.395 keV) and the N Kα (0.392 KeV) peaks made the mapping of nitrogen in the N—TiO2 NP samples impossible. The characterization of light elements such as Be, B, N and F is difficult due to their low photon energies, low yield of x-rays and low energy to noise ratio. FIG. 4 represents the TEM pictures (500,000× magnification) and compositional analysis of N—TiO2 NPs. The TEM images presented confirm the SEM findings regarding the morphologies of the N—TiO2 NPs and demonstrated that synthesized nanoparticles had sizes varying around 10 nm. In addition, it is also possible to observe that even for a very diluted sample (1:1250 in 100% ethanol) the nanoparticles still display a strong agglomeration behavior.
SEM and EDS Characterization of N—TiO2 NPs Immobilized in Dental Adhesive Resins
FIG. 5 represents the SEM pictures (500× and 5,000× magnifications) of both unaltered and experimental dental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2. It is possible to observe (FIG. 5C-H) that adhesive resins containing higher N—TiO2 NP concentrations resulted in materials with rougher surfaces due to the higher presence of particles at the surface level. In addition, it is possible to observe that materials containing 67% and 80% (v/v) presented particles (FIG. 5E-H) that were not covered by the adhesive matrix when compared to the remaining groups. This finding can be observed by the presence of circular-shaped particulates of very intense brightness.
FIG. 6 represents the results of the compositional analysis of both unaltered and experimental dental adhesive resins. It is possible to observe on image A that the elements composing the unaltered adhesive resins were mainly barium, silicon, oxygen and carbon, which is in agreement with the composition expected for an unaltered dental adhesive resin. Images B to D demonstrate increasing amounts of titanium and oxygen, which can be observed on the images by the presence of increasing amounts of pink (Ti) and yellow dots (O).
Contact Angle Goniometry
The results obtained from the assessment of the wettability of water at times 0 s (θINITIAL) and 59 s (θFINAL) on both, unaltered and experimental dental adhesive resins, are presented in a self-explanatory graph of mean and standard deviation values (FIG. 7). The results demonstrate that, independent of the group considered, initial contact angles (t=0 s) had values that were consistently higher than the values of the final contact angles (t=59 s). The SNK post hoc test demonstrates that similar initial contact angle values were obtained in all groups tested. Although final contact angles were smaller in value than initial contact angles, a similar trend of wettability behavior could still be noticed, where no significant differences among the groups tested could be observed.
Antibacterial Behavior of N—TiO2 NPs in Suspension
The results of the antibacterial activity of N—TiO2 NPs against S. mutans biofilms grown in microcentrifuge tubes were determined by the colony-forming units (CFU/mL) method and are presented in FIG. 8 as mean and standard deviation values, and % survival vs. % treatment efficacy (Table 1). The results demonstrated that all N—TiO2 NPs and ethanol concentrations tested (19%, 25% or 30% (v/v)] significantly decreased the viability of S. mutans biofilms when compared to the control group (intact biofilms). It is also possible to observe that the combination of ethanol and visible light produced viability results that were higher when compared to both experimental groups (N—TiO2 NPs or ethanol only) and were comparable to the control group (intact biofilms), which suggest that blue light may act as a biomodulator in situations of low cytotoxic stress.
TABLE 1
Survival rate and Treatment efficacy
SurvivalTreatment
rate (%)efficacy (%)
Control Group (n = 45)100.00%0.00%
19% (v/v) N_TiO2 (n = 15)5.58%94.42%
25% (v/v) N_TiO2 (n = 15)0.55%99.45%
30% (v/v) N_TiO2 (n = 15)0.24%99.76%
19% (v/v) EtOH (n = 15)10.71%89.29%
25% (v/v) EtOH (n = 15)0.92%99.08%
30% (v/v) EtOH (n = 15)0.73%99.27%
19% (v/v) EtOH + light (n = 15)93.79%6.21%
25% (v/v) EtOH + light (n = 15)7.79%92.21%
30% (v/v) EtOH + light (n = 15)9.65%90.35%
Table 1: S. mutans survival rate and antibacterial efficacy of the groups were investigated. The survival rate (Sr) and Treatment efficacy (Te) were calculated using the following equations: Sr = (Nf/N0)100% and Ae = (N0 − Nf/N0)100%, where N0 is the initial population and Nf is the viable population after the treatments.
Antibacterial Behavior of N—TiO2 NPs Immobilized in Dental Adhesive Resins
The results of the antibacterial efficacy of N—TiO2 NPs immobilized in dental adhesive resins against 3- or 24-hour S. mutans biofilms grown against the surfaces of specimens of both unaltered and experimental dental adhesive resins under dark or continuous light irradiation conditions were determined using the colony-forming units method (CFU/mL) and are presented as mean and standard deviation values (FIGS. 9 and 10). The results presented indicate, that regardless of the experimental groups tested or periods of time considered (either 3- or 24-hour), biofilms grown under continuous-light irradiation conditions (410±10 nm, 3-hour irradiation=38.75 J/cm2, 24-hour irradiation=310.07 J/cm2) displayed lower viability levels when compared to biofilms pertaining to either the control group or to experimental groups where biofilms were grown without light irradiation. It is also possible to observe that biofilms grown under continuous-light irradiation, displayed similar viability levels independent of the material investigated. In addition, the results of the viability levels of biofilms grown in dark conditions, indicate that experimental adhesive resins have antibacterial properties that are not dependent on light irradiation.
Confocal Laser Scanning Microscopy (CLSM)
The CLSM analysis of 24-hour S. mutans biofilms grown on the surfaces of both unaltered and experimental dental adhesive resins are presented in FIG. 11. The 3D rendering images revealed that the morphology, biovolume and viability of the cells within the investigated biofilms were significantly altered based on the N—TiO2 NP concentration (50%, 67% and 80% [v/v]) and light irradiation condition (with or without). The results clearly demonstrate that, independent of the experimental group considered, all biofilms grown under continuous-light irradiation conditions (FIGS. 11B, D, F, H) expressed higher instances of red fluorescence, which denotes that these biofilms had lower viability levels than the biofilms grown in dark conditions, which predominantly fluoresced green (FIGS. 11A, C, E, G).
These findings demonstrate that the wavelength and dose of energy used (410±10 nm, 3-hour irradiation=38.75 J/cm2, 24-hour irradiation=310.07 J/cm2) during the biofilms growth significantly impacted the ability of S. mutans to sustain viable biofilms. It is also noticeable on the CLSM results (FIGS. 11F and H) that the combination of continuous-light irradiation and experimental materials with higher nanoparticles concentration (67% and 80%) supported biofilms displaying the least amount of biovolume and viability, which can be noted on the images by the presence of extremely sparse micro colonies displaying intense red colors. The results obtained for biofilms pertaining to non-irradiated groups indicate that experimental materials containing 50%, 67% and 80% (v/v) of N—TiO2 NPs in dark conditions displayed antibacterial properties that were not dependent on light irradiation and further confirm the CFU/mL results. This finding can be observed specially in FIGS. 11E and G by the presence of colonies displaying colors that are a mix of red, green and yellow.
In addition, it is also possible to observe, that biofilms grown under dark conditions produced biofilms of similar biovolume and thickness, as noted by the large chained amorphous colonies (FIGS. 11A, C, E, G) regardless of group parameters. This finding indicates that the amounts of dead colonies present on the images are directly proportional to increasing amounts of the N—TiO2 NPs in the materials investigated.
Color Stability
The objective of the color analysis was to investigate the effect of the incorporation of 5%, 10%, 15% or 20% (v/v) of N—TiO2 into the dental adhesive resin OPTIBOND SOLO PLUS (OPTB). Disk shaped specimens were fabricated and were subjected to 500, 1000, 2500 and 5000 thermal cycles between two water baths (5° C. and 55° C., dwell time 15 sec.). Digital color analysis was then performed immediately after the fabrication of the specimens and at the completion of each thermal cycle proposed. The color stability of specimens was assessed in terms of total color change (ΔE) using the CIELab color space. The color analysis performed immediately after the fabrication of specimens demonstrated that experimental materials containing varying concentrations of N—TiO2 NPs displayed color changes that were comparable to the unaltered OPTB (FIG. 12). After the completion of 500 thermal cycles it is possible to observe that specimens pertaining to experimental groups containing higher N—TiO2 NPs had the least amount of total color change as compared to OPTB. After the completion of 5000 thermal cycles it became clear that experimental materials containing either 10%, 15% or 20% (v/v) N—TiO2 NPs have displayed the least amount of color variation when compared to OPTB. It is possible to observe that specimens containing 5% (v/v) of N—TiO2 have undergone to color changes that were similar in intensity to the color changes observed in specimens fabricated with the unaltered dental adhesive resin.
These findings indicate that incorporation of N—TiO2 NPs into OPTB rendered materials having improved color stability properties in comparison to the color stability properties of the unaltered and commercially available OPTB. The larger presence of metal oxide nanoparticles with cores resistant to degradation by water and temperature variation could explain the findings regarding the color stability reported in the present research.
Bioactivity of N—TiO2 NPs and N—P—TiO2 NPs
The in vitro testing of the bioactivity of experimental dental adhesive resins containing 20% (v/v) of either N—TiO2 NPs or N—P—TiO2 NPs was conducted to demonstrate the ability of experimental materials to spontaneously deposit a crystalline layer of amorphous calcium phosphate upon exposure to Dulbecco's phosphate buffer solution (DPBS). Specimens fabricated with OPTB-only served as the control group. SEM and EDS analyses were used to characterize the bioactivity of experimental dental adhesive resins. The SEM and EDS representative images presented in FIGS. 13-15 represent the results obtained with the in vitro bioactive testing of unaltered OPTB, as well as with N—P—TiO2 NPs and N—TiO2 NPs, respectively. It is possible to observe from the EDS compositional analysis (FIG. 13E) that specimens fabricated with unaltered OPTB were able to promote the precipitation of very small amounts of calcium (Ca, 0.4%), and phosphorous (P, 0.7%). FIG. 14E represents the EDS compositional analysis results of specimens fabricated with experimental dental adhesive resins containing 20% (v/v) of N—P—TiO2 NPs. It is possible to observe that these materials promoted the highest precipitation of Ca (6.6%) and P (5.6%). FIG. 15E shows the EDS compositional analysis results of specimens fabricated with 20% (v/v) of N—TiO2. The results have demonstrated that these materials promoted an intermediate precipitation of Ca (4.2%) and P (3.0%). In addition, the EDS mapping of individual elements further confirmed the findings of the compositional analysis performed.
Discussion
In at least certain embodiments, doped TiO2 NPs were obtained via a two-step fabrication process. In a first step, undoped TiO2 NPs were synthesized. In a second step, nitrogen-doping, or co-doping of the TiO2 NPs was carried out. After the doping process, the obtained single-doped NPs had their initial visual aspect altered from a bright white—into a yellow—pale suspension, which indicates that the doping process was carried out successfully. The results of the UV-vis spectroscopy of both undoped and N—TiO2 NPs are presented in FIG. 1, which shows that N—TiO2 NPs had higher levels of light absorption when compared to the behavior observed for undoped TiO2 NPs. The nanoparticles had significant absorption behavior in the visible region (between 400 nm and 600 nm).
The SEM analysis of nanoparticles presented in FIG. 2 revealed important aspects related to morphologies and agglomeration levels of nanoparticles. The layered and amorphous structures visible in the images suggest that N—TiO2 NPs have spherical shapes, smooth surfaces and display a strong agglomeration behavior in ethanol (40 mg/mL). The use of surfactants is one approach that could be used to improve the dispersability behavior of N—TiO2 NPs. However, the use of surfactants decreases the possibility of oxidation reactions taking place on the N—TiO2 NP surfaces due to the creation of a physical barrier, thereby diminishing their antibacterial behavior. Thus the methods of the present work were designed to maximize the photocatalytic behavior of N—TiO2 NPs. The SEM images demonstrate a physical association of nanoparticles due to the drying process that is required for SEM imaging.
The results of the compositional characterization of the nanoparticles using EDS are presented in FIG. 3. The analysis revealed that Ti (40.9%), O (39.3%), C (13.3%) and Si (6.5%) were the major components found in N—TiO2 NP samples. However, under the conditions of the present work, the doping element (nitrogen) could not be mapped. Apparently the combination of factors like the low atomic number of nitrogen (Z=7), and the complete overlap between the Ti Lλ, (0.395 keV) and the N Kα (0.392 KeV) peaks made the mapping of nitrogen in the N—TiO2 NP samples excessively difficult.
Characterization of the NPs by TEM is presented in FIG. 4. The images demonstrated that N—TiO2 NPs have mostly spherical shapes, smooth surfaces and a homogeneous distribution of sizes, with individual NP sizes ranging around 10 nm. It is also possible to observe that N—TiO2 NPs still tend to have strong agglomeration behaviors even for very diluted samples (1:1250 or 0.032 mg/mL). Control over the nanoparticle agglomeration levels is a key factor in the optimization of photocatalytic reactions, because agglomeration can decrease the nanoparticle surface to volume ratio, decrease the amount of free surface area that is actually available for oxidative reactions to take place and, increase the amount of recombination centers present in the bulk of the photocatalyst, thereby adversely impacting the overall photocatalytic behavior of any light responsive material.
SEM and EDS analyses were used to characterize the surface properties and compositions of specimens fabricated with both unaltered and experimental dental adhesive resins. The SEM results demonstrated the successful incorporation of nanoparticles into the polymer matrix, which can be observed by the presence of increasing amounts of particulates on the surfaces of specimens that were fabricated with higher nanoparticles concentrations. In addition, specimens fabricated with higher nanoparticles content displayed rougher surfaces when compared to specimens of OPTB resin due to the strong presence of exposed particulates.
The compositional analysis performed using EDS further corroborates our SEM findings regarding the successful incorporation of nanoparticles into the OPTB. It is possible to observe on the EDS images, that specimens of OPTB displayed barium (Ba), Si, O and C as its major chemical components, which is an expected composition. The compositional mapping of specimens fabricated with experimental dental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2 NPs clearly demonstrate higher concentrations of Ti and O, which can be noticed by observing increasing amounts of pink (Ti) and yellow (O) dots on the images. These results agree with the compositions expected for samples fabricated with experimental materials.
The wettability analysis using the measurement of contact angles was made necessary in the present work to investigate the impact of the incorporation of nanoparticles on the wettability characteristics of OPTB. The measurement of contact angles at the solid-liquid-vapor interface is considered to be the most widely known technique used to investigate the wettability of solid surfaces. The hydrophobicity behavior of dental composites is an important factor in the longevity of resin-based materials because it affects the initial absorption of water, which regulates the attachment of oral bacteria. The wettability findings reported in the present work demonstrated that the incorporation of N—TiO2 NPs into OPTB promoted the attainment of experimental materials with wettability properties that were not significantly different when compared to the control group. From the clinical perspective, the fact that there were no statistical differences between the groups is important because in order to promote the establishment of an adequate adhesive layer, dental adhesive resins must compete with water from the dentine substrate to wet the collagen fibrils. Adhesive materials must come into intimate contact with the dentine substrate to allow for the proper micromechanical surface attachment.
When observing results of the assessment of the antibacterial efficacy of N—TiO2 NPs in suspension (FIG. 8 and Table 1), it is possible to see that S. mutans biofilms displayed similar CFU/mL values regardless of treatment with either nanoparticles or ethanol. This indicates that the N—TiO2 NPs suspended in 100% ethanol did not present a strong antibacterial effect against S. mutans biofilms in the conditions investigated. The analysis of the survival rates (Sr) and treatment efficacy (Te) for the same experimental groups discussed (Table 1) further corroborates this finding.
Oxidative photocatalytic reactions are inhibited in the presence of ethanol because some reactive species of oxygen, such as hydroxyl radicals, are strongly quenched. In the same direction, Hydroxyl radicals and hydrogen peroxide appeared to be the major species associated with the antibacterial effects observed against Staphylococcus epidermidis.
Results from antibacterial assays performed herein with experimental adhesive resins containing 50%, 67% or 80% (v/v) of N—TiO2 NPs against S. mutans biofilms grown for either 3-hour or 24-h, with or without continuous-light irradiation, are presented in FIGS. 9-10. These results have demonstrated that, independent of growth time (either 3-hour or 24-h), or light irradiation conditions (with or without light), experimental groups containing higher N—TiO2 NP concentrations were more antibacterial in nature when compared to the control group, which indicates the establishment of a concentration-dependent antibacterial mechanism.
The CLSM images presented in FIG. 11 illustrate and further corroborate the results of the antibacterial assays performed on dental adhesive resins. These results confirm a decrease in cells viability and biovolume when specimens were fabricated with higher concentrations of doped TiO2 NPs while also being irradiated with continuous visible light irradiation, and therefore align the expected results with the representative CSLM images. It is interesting to note, that while it was expected that the N—TiO2 NPs would affect the viability of S. mutans biofilms when exposed to visible light, it is apparent that there is a toxicity effect (“dark toxicity”) absent exposure of the adhesive to light. Although many photosensitizers are able to increase bactericidal effects, they usually require an irradiation light source in order to elicit reduced viability. However, the dark toxicity observed was statistically significant due to decrease in viability between the OPTB control and the 80% N—TiO2 NP adhesive resin in dark conditions in both the 3-hour and 24-hour biofilms. It is also supported by the 24-hour CLSM images that visually show a change in viability, but not necessarily the structure of the biofilm.
Both our CLSM images as well as our CFU/mL results show not only a decrease in biovolume, but also a highly toxic effect when biofilms are grown on an adhesive containing N—TiO2 NPs in the presence of blue light. Since it was previously established that the N—TiO2 NPs do have some degree of dark toxicity, the high degree of bactericidal effects may have been due to a two-fold mechanism; the restriction of EPS in the formation of the biofilm, as well as the toxicity of the nanoparticles themselves.
In the present work, a titanium dioxide-based photocatalyst was successfully prepared by doping TiO2 NPs with nitrogen using a simple solvothermal method. These NPs were demonstrated to have superior visible light absorption levels when compared to pure TiO2 NPs due to the contribution of substitutional nitrogen in the crystal lattice of titania. The visible light-driven antibacterial efficacy of N—TiO2 NPs was investigated for nanoparticles suspended in ethanol and incorporated in a commercially available dental adhesive resin (OPTB). It was demonstrated that nanoparticles in suspension have only a limited antibacterial behavior against S. mutans biofilms probably due to the use of ethanol as a solvent, which is a well-known potent hydroxyl scavenger. The present work has shown for the first time that specimens fabricated with experimental dental adhesive resins containing either 50%, 67% or 80% (v/v) of N—TiO2 NPs were shown to have strong antibacterial behavior in both, dark and light irradiated conditions, when compared to the antibacterial behavior of unaltered dental adhesive resins (e.g., OPTB). This indicates that N—TiO2 NPs comprise a feasible antibacterial agent against oral cariogenic biofilms. The present work has also demonstrated that experimental materials had similar wettability behaviors when compared to the unaltered adhesive resins, which is important from the clinical perspective.
