Application of Reactive Oxygen Species in Dental Treatment.

Reactive oxygen species (ROS) and free radicals, which have been implicated in inflammation, pain, carcinogenesis, and aging, are actually used in dental treatments such as tooth bleaching and composite resin polymerization. Recently, numerous studies have investigated the application of ROS in the medical and dental fields. In previous studies, ROS were generated intentionally through pathways such as photolysis, photocatalytic methods, and photodynamic therapy, which are used in the medical field to target cancer. In the field of dentistry, generated ROS are applied mainly for periodontal treatment and sterilization of the root canal, and its effectiveness as an antibacterial photodynamic therapy has been widely reported.. Given this background, the present article aimed to review the basic effects of ROS in dental medicine, especially endodontic therapy, and to discuss future applications of ROS.

Examination of Calcium Silicate Cements with Low-Viscosity Methyl Cellulose or Hydroxypropyl Cellulose Additive.

The purpose of this study was to improve the operability of calcium silicate cements (CSCs) such as mineral trioxide aggregate (MTA) cement. The flow, working time, and setting time of CSCs with different compositions containing low-viscosity methyl cellulose (MC) or hydroxypropyl cellulose (HPC) additive were examined according to ISO 6876-2012; calcium ion release analysis was also conducted. MTA and low-heat Portland cement (LPC) including 20% fine particle zirconium oxide (ZO group), LPC including zirconium oxide and 2 wt% low-viscosity MC (MC group), and HPC (HPC group) were tested. MC and HPC groups exhibited significantly higher flow values and setting times than other groups (p < 0.05). Additionally, flow values of these groups were higher than the ISO 6876-2012 reference values; furthermore, working times were over 10 min. Calcium ion release was retarded with ZO, MC, and HPC groups compared with MTA. The concentration of calcium ions was decreased by the addition of the MC or HPC group compared with the ZO group. When low-viscosity MC or HPC was added, the composition of CSCs changed, thus fulfilling the requirements for use as root canal sealer. Calcium ion release by CSCs was affected by changing the CSC composition via the addition of MC or HPC.

Comparison of conventional and new-generation nickel-titanium files in regard to their physical properties.

INTRODUCTION: This study investigated the surface, fractured structure, and physicochemical properties related to cyclic fatigue in various nickel-titanium (NiTi) files. METHODS: Among a total of 10 groups of NiTi files, conventional NiTi files (ProFile [Dentsply Maillefer, Ballaigues, Switzerland] and K3 [SybronEndo, Orange, CA]) and new-generation NiTi files (ProFile Vortex [PV; Tulsa Dental Specialties, Tulsa, OK], Vortex Blue [VB; Tulsa Dental Specialties], and K3 XF [XF; SybronEndo, Orange, CA]) with the same tip diameter (ISO size 25) and 2 types of taper (0.04 and 0.06) were used in this study. Scanning electron microscopy of the file surface structure, differential scanning calorimetry, and cyclic fatigue resistance tests were conducted. RESULTS: Many mechanical grooves were recognized on the file surface. The surface in the ProFile group was extremely smooth compared with that observed for the other files. Many shallow hollows besides mechanical grooves were noted on the surface in the XF group. A smooth curve was observed in the ProFile, K3, and PV groups. Defined peaks in differential scanning calorimetry were observed in the VB and XF groups. The 0.04 taper files exhibited a statistically higher number of cycles to fracture than the 0.06 taper files in all groups (P < .05). Cracks along the mechanical grooves were observed in the NiTi files, with the exception of the XF group. The start of cracking was detected at U-shape sites in the ProFile group, the cutting edge in the PV and VB groups, and radial islands in the K3 and XF groups. CONCLUSIONS: The present findings suggest that new-generation NiTi files are not necessarily improved compared with conventional files.

Timing for composite resin placement on mineral trioxide aggregate.

INTRODUCTION: The aim of this study was to investigate the proper time to restore composite resin over mineral trioxide aggregate (MTA). METHODS: Thirty-five samples of MTA blocks were divided into 7 groups with 3 different times (10 minutes, 1 day, and 7 days) selected for restoring the composite resin over MTA with and without bonding resin, and a control group was included for comparison. After 21 days, the distances between MTA and the composite resin or between MTA and the bonding agent on sectioned planes along the long axis were measured using a scanning electron microscope (×2,000 magnification). The hardness of the MTA near the composite resin was presented as the Vickers microhardness. RESULTS: There were no gaps at the interface in the 10-minute groups, the 1-day group with a bonding agent, and the 7-day group with a bonding agent. The groups without a bonding agent at 1 and 7 days presented a separation or gap at the interface. The value of the Vickers microhardness in the 1-day groups was significantly decreased compared with those of the other groups regardless of the presence or absence of a bonding agent. CONCLUSIONS: These findings suggest that composite resin with a bonding agent over MTA can be restored almost immediately after MTA mixing during a single visit.

A small amount of singlet oxygen generated via excited methylene blue by photodynamic therapy induces the sterilization of Enterococcus faecalis.

INTRODUCTION: The present study aimed to clarify the relationship between the amount of singlet oxygen ((1)O(2)) generated from excited methylene blue (MB) and the bactericidal effects on Enterococcus faecalis. METHODS: A diode laser was used as the laser irradiation source (λ = 660 nm, 200 mW). The laser irradiation time periods were 300, 600, and 900 seconds. In experiment 1, the amount of (1)O(2) generated from each concentration (0.0001%-1.0%) of excited MB was examined by using electron spin resonance to determine the optimal concentration of MB. In experiment 2, the bactericidal effects of (1)O(2) on E. faecalis were examined. Experimental groups were with laser irradiation, L(+); without laser irradiation, L(-); including MB, M(+); and not including MB, M(-). These were combined to form 4 groups: L(+)M(+), L(+)M(-), L(-)M(+), and L(-)M(-). After treatment, E. faecalis was incubated for 48 hours at 37°C, and the bactericidal effects of (1)O(2) on E. faecalis were determined on the basis of the number of colony-forming units per milliliter. RESULTS: The largest amount of (1)O(2) was generated from 0.01% excited MB. After 300, 600, and 900 seconds of irradiation, 35.2, 87.2, and 117.1 µmol/L (1)O(2) were detected, respectively. In group L(+)M(+),colony-forming units per milliliter of E. faecalis dramatically decreased depending on the amount of (1)O(2) generated. No other groups showed any bactericidal effects. CONCLUSIONS: Our findings suggest that 0.001%-0.01% of MB is the most effective range for generating (1)O(2) during the application of antimicrobial photodynamic therapy. At least 35.2 µmol/L generated (1)O(2) was necessary to achieve the sterilization of E. faecalis.

The effect of irradiation wavelengths and the crystal structures of titanium dioxide on the formation of singlet oxygen for bacterial killing.

Safe and effective methods for oral bacterial disinfection have been desired, since bacteria cause many infectious diseases such as dental caries, periodontal disease, and endodontic infections. Singlet oxygen ((1)O(2)) is attractive, because it is toxic to prokaryotic cells, but not to eukaryotic cells. We selected irradiation of titanium dioxide (TiO(2)) as a source of (1)O(2), because it has been used in sunscreens and cosmetic products without complications. In order to establish the optimal oral photodynamic therapy conditions, we measured the rate of (1)O(2) formation from the irradiated anatase or rutile forms of TiO(2) using 365 or 405 nm lamps. The rate of (1)O(2) formation decreased in the following order: anatase, 365 nm > rutile, 405 nm > rutile, 365 nm > anatase, 405 nm. Therefore, we concluded that irradiation of the rutile form of TiO(2) by a 405 nm lamp is the most favorable photodynamic therapy condition, because visible light is more desirable than UV light from the viewpoint of patient safety. We also confirmed that there was no direct HO(•) formation from the irradiated TiO(2).

Antioxidant effects of antioxidant biofactor on reactive oxygen species in human gingival fibroblasts.

The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O(2) (•-)) and hydroxyl radical ((•)OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O(2) (•-) and (•)OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H(2)O(2) (AOB + H(2)O(2) group), containing H(2)O(2) (H(2)O(2) group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H(2)O(2) group > H(2)O(2) group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H(2)O(2) group > H(2)O(2) group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS.

Surface changes of mineral trioxide aggregate after the application of bleaching agents: electron microscopy and an energy-dispersive X-ray microanalysis.

INTRODUCTION: The aim of this study was to investigate the changes in the surface structure and chemical composition after applying bleaching agents to completely hardened mineral trioxide aggregate. METHODS: A total of 12 samples of MTA blocks were divided into three groups, two different bleaching agents, and a control group. The surface structure was observed using a scanning electron microscope. The changes in elemental composition were analyzed by an energy-dispersive x-ray microanalysis (EDX) system. RESULTS: The surface of the MTA covered with each bleaching agent changed in terms of both color and structure compared with the control. EDX showed that both bleaching agents affected the elemental distribution. A decrease in Ca and an increase in Si were shown, and this tendency was especially pronounced in the higher hydrogen peroxide concentration group. CONCLUSIONS: The acidic conditions induced by bleaching agents brought about the deterioration of the MTA surface. These findings suggest that MTA is an insufficient barrier against tooth bleaching.

Analysis of arsenic in gray and white mineral trioxide aggregates by using atomic absorption spectrometry.

INTRODUCTION: The aim of the present study was to investigate whether the concentration of arsenic (As) released from gray or white mineral trioxide aggregates (MTAs) met the requirement of the International Standards Organization (ISO) for dental cements. METHODS: Sample preparations were carried out according to the ISO methods. After centrifugation of dissolved samples, As (III) concentration in the final supernatant was analyzed by a high-performance atomic absorption spectrophotometer. RESULTS: As (III) concentration from both MTAs was much less than the required value (2 ppm) for dental cements regulated by the ISO. An experiment simulating pulp capping by using MTA revealed that As concentration was also below the standard value of the ISO. The As concentration in white MTA was lower than the value (10 ppb) recommended for tap water and environmental standards. CONCLUSIONS: The present in vitro studies demonstrated that there is no threat to patient health in using commercially available brands of MTA for endodontic practices.

Stimulatory effects of low-concentration reactive oxygen species on calcification ability of human dental pulp cells.

The present study was conducted to investigate the effects of reactive oxygen species (ROS) on the calcification ability of human dental pulp (HDP) cells. HDP cells were treated with 100 mumol/L hydrogen peroxide (H(2)O(2)) for 5 or 10 minutes (5-min ROS group and 10-min ROS group) to investigate the mechanism of transmission to cells. Untreated cells were used as controls. Generation of free radicals was quantified by the electron spin resonance spin-trapping method and found to be increased by treatment with ROS. Formation of calcified nodules was also investigated by von Kossa staining and alizarin red S staining. Twenty-eight days after exposure, calcified nodules were present in cell cultures that had been treated with ROS for 5 or 10 minutes. Expression of mRNAs for osteopontin (OPN) and osteocalcin (OCN) was significantly greater in 10-min ROS group 6 and 9 days, respectively, after exposure than in controls. Production of OPN and OCN by 10-min ROS group was also greater 12 and 18 days, respectively, after exposure than in controls. These results suggested that calcification of HDP cells was stimulated by H(2)O(2) and by the ROS it generated.

Stimulatory Effects of CO(2) Laser, Er:YAG Laser and Ga-Al-As Laser on Exposed Dentinal Tubule Orifices.

We investigated the effects of lasers irradiation on the exposed dentinal tubule. Human tooth specimens with exposed dentinal tubule orifices were used. Three types of lasers (CO(2) laser, Er:YAG laser and Ga-Al-As laser) were employed. The parameters were 1.0 W in continuous-wave mode with an irradiation time of 30 s for the CO(2) laser, 30 mJ in continuous-wave mode with an irradiation time of 60 s for the Er:YAG laser, and 1.0 W in continuous-wave mode with an irradiation time of 60 s for the Ga-Al-As laser. A non-irradiated group was used as a control. After laser irradiation, the dentinal surface of each sample was observed using SEM. Afterwards, all samples were immersed in methylene blue dye solution in order to evaluate the penetration of the dye solution and observe the change in dentinal permeability after laser irradiation. SEM observation showed that the control group had numerous exposed dentinal tubule orifices, whereas these orifices were closed in the laser-irradiated groups. There was consistent dye penetration into the pulp chamber in the control group, whereas no dye penetration was evident in the laser-irradiated groups. Therefore, laser appears to be a promising treatment for reducing permeation through exposed dentinal tubules.

Effects of Smads and BMPs induced by Ga-Al-As laser irradiation on calcification ability of human dental pulp cells.

We investigated the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells and on Smads and bone morphogenetic protein (BMP) production as one mechanism for the transmission of laser photochemical energy to cells. HDP cells in vitro were irradiated once with a Ga-Al-As laser at 1.0 W for 500 s, and calcified nodule formation was assessed by Alizarin red S staining. The laser irradiation was greater in the laser-irradiated group than in the non-irradiated group. Both calcium production and alkaline phosphatase (ALP) activity were higher after laser irradiation. Expression of mRNAs for Smad1, Smad7, BMPs, ALP, and osteocalcin was greater after laser irradiation, whereas expression of Smad6 mRNA was inhibited. Production of BMP-2 and BMP-4 in conditioned medium was also higher after laser irradiation. These results suggest that Smads and BMPs play important roles in ALP activity and calcification upon laser irradiation of HDP cells.

Stimulatory effects of hydroxyl radical generation by Ga-Al-As laser irradiation on mineralization ability of human dental pulp cells.

The present study was conducted to investigate the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells. HDP cells in vitro were irradiated once with a Ga-AL-As laser at 0.5 W for 500 s and at 1.0 W for 500 s in order to investigate free radicals as one mechanism for transmission of laser photochemical energy to cells. Production of the hydroxyl radical (*OH) was measured using the ESR spin-trapping method and was found to be increased by laser irradiation. The DMPO-OH was not detected in the presence of dimethyl sulfoxide (DMSO), a *OH scavenger. The formation of calcification nodule was also investigated by von Kossa staining. The number of calcified nodules was increased by 1.0 W-laser irradiation. Alkaline phosphatase (ALP) activity was higher in the 1.0 W-laser irradiation group. Expression of mRNAs for heat shock protein 27, bone morphogenetic proteins (BMPs) and ALP were greater in the 1.0 W-laser irradiation group. Expression of BMPs in the conditioned medium was also higher in the 1.0 W-laser irradiation group. In particular, DMSO decreased the number of calcified nodule produced by 1.0 W-laser irradiation. These results supposed that the mineralization of HDP cells is stimulated by laser irradiation, and that *OH generated by laser irradiation is a trigger for promotion of HDP cell mineralization.

Dentin is dissolved by high concentrations of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt with or without hydrogen peroxide.

L-Ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K(1)) is a conjugate of vitamin C and vitamin E that is water-soluble and stable at room temperature. EPC-K(1) has been developed as a hydroxyl radical (.OH) scavenger and antioxidant. In a previous tooth whitening experiment, it was accidentally found that tooth (dentin) blocks were dissolved by EPC-K(1) with H(2)O(2). In the current study, high concentrations of EPC-K(1) (2.5, 25 mM) with 3% H(2)O(2) dissolved and caused the collapse of dentin blocks. Similar concentrations of EPC-K(1) without 3% H(2)O(2), however, dissolved the dentin blocks without collapse over a 3-week period. In these cases, a.OH-like signal was detected using an ESR spin-trapping method. The volume of calcium in solution (including the dentin block) increased on the addition of EPC-K(1) in a concentration-dependent manner. In addition, the calcium : phosphorus ratio changed from 2 : 1 in sound dentin to 1 : 2 in the collapsed dentin block. High concentrations of EPC-K(1) are therefore considered to have calcium chelating and dentin dissolving activity. The dentin dissolving activity was enhanced when EPC-K(1) was used with H(2)O(2). EPC-K(1) had no protective effect when used in tooth whitening with H(2)O(2).

Effects of the hydroxyl radical and hydrogen peroxide on tooth bleaching.

The mechanisms of bleaching of discolored coronal teeth using hydrogen peroxide (H2O2) were investigated. In a scanning-electron-microscopy study, the intertubular dentin and peritubular dentin were dissolved by high concentrations of H2O2, which is used for bleaching. The X-ray diffraction study showed that hydroxyapatite was not influenced by H2O2. In an electron-spin-resonance study, more hydroxyl radical (* OH) was detected as the H2O2 concentration was increased. When amino acids that are core components of dentin proteins, such as proline and alanine, were added to H2O2, the generation of * OH decreased, but there was no change when glycine was added. A nuclear-magnetic-resonance study showed that proline was degraded completely by H2O2, the structure of alanine changed slightly, and glycine was not affected by H2O2. It is suggested that H2O2 and * OH do not influence the inorganic tissue of dentin but attack the organic component of dentin. These facts suggest that * OH has the main role in tooth bleaching with H2O2.

Generation of free radicals and/or active oxygen by light or laser irradiation of hydrogen peroxide or sodium hypochlorite.

Generation of free radical and/or active oxygen by light or laser irradiation of hydrogen peroxide (H2O2) or sodium hypochlorite (NaClO), which have been used for tooth whitening or root canal irrigation, was investigated using electron spin resonance spectroscopy combined with a spin-trapping technique. When H2O2 was exposed to light or laser radiation, the amount of hydroxyl radical generated changed according to the concentration of H2O2 and irradiation time. The amount of 5,5-dimethyl-1-pyrrolidone-(2)-oxyl-(1) (DMPO-X) also changed in accordance with irradiation time. The amounts of hydroxyl radical generated from H2O2 after irradiation were in the order: plasma lamp > halogen lamp > He-Ne laser > Yellow He-Ne laser. On the other hand, the amounts of DMPO-X generated from NaClO after irradiation were in the order: plasma lamp > Yellow He-Ne laser > halogen lamp > He-Ne laser.