Efficacy and safety of a therapeutic apparatus using hydrogen peroxide photolysis to treat dental and periodontal infectious diseases.

The present study aimed to evaluate the acute locally injurious property of our most current hydroxyl radical generation system by hydrogen peroxide (H2O2) photolysis. This system, which releases 3% H2O2 with a 405-nm laser, was developed in our laboratory for the treatment of dental and periodontal infectious diseases. First, the hydroxyl radical yield generated by H2O2 photolysis was examined by applying an electron spin resonance-spin trapping technique. Second, the bactericidal effect of the device was examined under a simulant condition in which Streptococcus mutans, a pathogenic bacterial species that causes caries, was irrigated with running 3% H2O2 concomitantly with laser irradiation. Finally, the acute topical effect of the model apparatus on rat palatal mucosa was evaluated by histological examination. We found that the hydroxyl radical yield was dependent upon laser output power. The bacterial count was substantially reduced within as little as 3 min. No abnormal findings were observed in the palatal mucosa, even when rats received three treatments of 3% H2O2 with laser irradiation at an output power of 40 mW. These results suggest that our apparatus has the ability to kill bacteria via hydroxyl radical generation and is safe to use at the lesion site of dental and periodontal infectious diseases.

Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria.

The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm(2)) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment.

Synergistic interaction between wavelength of light and concentration of H2O2 in bactericidal activity of photolysis of H2O2.

The present study aimed to evaluate the interaction between wavelength of light in the range of ultra violet A-visible and concentration of H2O2 in the reaction of photolysis of H2O2 from the point of view of hydroxyl radical (·OH) generation and the bactericidal activity. Light emitting diodes (LEDs) emitting the light at wavelengths of 365, 385, 400 and 465 nm were used at an irradiance of 1000 mW/cm(2). H2O2 was used at the final concentrations of 0, 250, 500, and 1000 mM. Quantitative analysis of ·OH generated by the LED irradiation of H2O2 were performed using an electron spin resonance-spin trapping technique. In a bactericidal assay, a bacterial suspension of Staphylococcus aureus prepared in sterile physiological saline was irradiated with the LEDs. The bactericidal activity of each test condition was evaluated by viable counts. When H2O2 was irradiated with the LEDs, ·OH was generated and bacteria were killed dependently on the concentration of H2O2 and the wavelength of LED. The two-way analysis of variance revealed that the wavelength, the H2O2 concentration and their interaction significantly affected the yield of ·OH and the bactericidal activity of the photolysis of H2O2. Therefore, it is suggested that bactericidal activity of photolysis of H2O2 could be enhanced by controlling the wavelength and the concentration of H2O2, which may contributes to shortening the treatment time and/or to reducing the concentration of H2O2.

Postantibiotic effect of disinfection treatment by photolysis of hydrogen peroxide.

The purpose of the present study was to evaluate the postantibiotic effect (PAE) of the disinfection treatment by photolysis of H2O2. Postantibiotic effect was induced in Staphylococcus aureus and Streptococcus salivarius by exposing the bacteria to H2O2 at concentrations of 250-1000 mmol/l, laser irradiation at a wavelength of 405 nm, and the combination of both (photolysis of H2O2) for 10-30 seconds. The photolysis of H2O2 induced significantly longer PAE than other treatments. The PAE was augmented dependently on not only the concentration of H2O2 but the laser irradiation time. Electron spin resonance analysis showed that the hydroxyl radical was also generated dependently on both the concentration of H2O2 and the laser irradiation time, suggesting that the hydroxyl radicals contribute to the PAE. These results suggest that the disinfection treatment by photolysis of H2O2 induces PAE in S. aureus and S. salivarius even though they were treated for only 10-30 seconds.

In vitro evaluation of the risk of inducing bacterial resistance to disinfection treatment with photolysis of hydrogen peroxide.

The purpose of the present study was to evaluate the risk of inducing bacterial resistance to disinfection treatment with photolysis of H2O2 and comparing this with existing antibacterial agents. We tested seven antibacterial agents, including amoxicillin, cefepime hydrochloride, erythromycin, ofloxacin, clindamycin hydrochloride, ciprofloxacin hydrochloride, and minocycline hydrochloride, as positive controls for validation of the assay protocol. For all of the agents tested, at least one of the four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Streptococcus salivarius) was resistant to these agents by repeated exposure to subinhibitory concentrations of the agents up to 10 times. In contrast, antibacterial activity against any of the bacterial species tested (S. aureus, E. faecalis, E. coli, S. salivarius, Pseudomonas aeruginosa, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans) was not affected by repeated exposure to the disinfection treatment up to 40 times. This finding suggested that the risk of inducing bacterial resistance by disinfection treatment was low. The active ingredient of this disinfection treatment is hydroxyl radicals generated by photolysis of H2O2. Therefore, hydroxyl radicals interact with several cell structures and different metabolic pathways in microbial cells, probably resulting in a lack of development of bacterial resistance. In conclusion, disinfection treatment with photolysis of H2O2 appears to be a potential alternative for existing antimicrobial agents in terms of a low risk of inducing bacterial resistance.

Synergistic effect of proanthocyanidin on the bactericidal action of the photolysis of H2O2.

The in vitro antibacterial activity of the hydroxyl radical generation system by the photolysis of H2O2 in combination with proanthocyanidin, which refers to a group of polyphenolic compounds, was examined. Bactericidal activity of photo-irradiated H2O2 at 405 nm against Streptococcus mutans, a major pathogen of dental caries, was augmented in the presence of proanthocyanidin, whose bactericidal effect by itself was very poor, in a concentration-dependent manner. This combination was also proven effective against Porphyromonas gingivalis, a major pathogen of periodontitis. It is speculated that H2O2, generated from photo-irradiated proanthocyanidin around the bacterial cells, is photolyzed to the hydroxyl radical, which would in turn affect the membrane structure and function of the bacterial cells, resulting in augmented sensitivity of bacterial cells to the disinfection system utilizing the photolysis of H2O2. The present study suggests that the combination of H2O2 and proanthocyanidin works synergistically to kill bacteria when photo-irradiated.

In vitro bactericidal activity of photo-irradiated oxydol products via hydroxyl radical generation.

The oxidative power of hydroxyl radicals has been applied to disinfection systems for the purpose of oral hygiene by utilizing blue light-induced photolysis of hydrogen peroxide (H2O2) in our laboratory. In the present study, the bactericidal potential of blue light-irradiated oxydol products via hydroxyl radical generation was compared with that of 3% (w/v) H2O2. Eleven commercially available oxydol products were used in the present study. Even though a few of the products that contained ethanol, a hydroxyl radical scavenger, as an additive showed slightly lower hydroxyl radical yield as compared with 3% (w/v) H2O2, the blue-light irradiation of each oxydol product for 3 min showed similar or superior bactericidal activity against Staphylococcus aureus to that of 3% (w/v) H2O2. The results strongly suggest that any of the oxydol products tested in the present study can be used as a source of hydroxyl radicals for the disinfection technique developed in our laboratory.

Photo-irradiation of proanthocyanidin as a new disinfection technique via reactive oxygen species formation.

In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.

Corrosive effect of disinfection solution containing hydroxyl radicals generated by photolysis of H(2)O(2) on dental metals.

The purpose of the present study was to evaluate the corrosive effect of disinfection solution containing hydroxyl radicals generated by photolysis of H(2)O(2)on dental metals. Static immersion test was performed on four different dental metals: Ti, Type 316L stainless steel, Ag-Pd-Cu-Au alloy, and Co-Cr alloy. Metal specimens were immersed in 1 M H(2)O(2)(=3.4%) with or without light-emitting diode (LED) light irradiation (wavelength: 400 nm) for 1 week, and then the amounts of released ions were analyzed. Corrosive effect of the disinfection solution containing hydroxyl radicals on any dental metals tested in the present study never exceeded that of H(2)O(2) alone. Therefore, disinfection systems based on the photolysis of H(2)O(2) for the cleaning of dentures and treatment of oral infectious diseases would not cause problematic metal corrosion whenever the concentration of H(2)O(2) does not exceed 3%, which is a concentration used as an oral disinfectant.

Bactericidal action of photoirradiated gallic acid via reactive oxygen species formation.

It is known that gallic acid shows antimicrobial activity. In the present study, photoirradiation induced reactive oxygen species formation was investigated for augmentation of the antimicrobial activity of gallic acid. Staphylococcus aureus suspended in 4 mmol/L gallic acid was exposed to blue light of a LED at 400 nm. This treatment killed the bacteria, and a >5-log reduction of the viable counts was observed within 15 min. By contrast, neither the LED treatment alone nor the treatment with gallic acid alone showed substantial bactericidal effect. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of photoirradiated gallic acid was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photoirradiation of gallic acid. Thus, the present study suggests that the photo-oxidation can enhance the antimicrobial activity of gallic acid via hydroxyl radical formation.

Literature review of the role of hydroxyl radicals in chemically-induced mutagenicity and carcinogenicity for the risk assessment of a disinfection system utilizing photolysis of hydrogen peroxide.

We have developed a new disinfection system for oral hygiene, proving that hydroxyl radicals generated by the photolysis of 1 M hydrogen peroxide could effectively kill oral pathogenic microorganisms. Prior to any clinical testing, the safety of the system especially in terms of the risk of carcinogenicity is examined by reviewing the literature. Previous studies have investigated indirectly the kinds of reactive oxygen species involved in some sort of chemically-induced mutagenicity in vitro by using reactive oxygen species scavengers, suggesting the possible involvement of hydroxyl radicals. Similarly, possible involvement of hydroxyl radicals in some sort of chemically-induced carcinogenicity has been proposed. Notably, it is suggested that the hydroxyl radical can play a role in heavy metal-induced carcinogenicity that requires chronic exposure to the carcinogen. In these cases, hydroxyl radicals produced by Fenton-like reactions may be involved in the carcinogenicity. Meanwhile, potential advantages have been reported on the use of the hydroxyl radical, being included in host immune defense by polymorphonuclear leukocytes, and medical applications such as for cancer treatment and antibiotics. From these, we conclude that there would seem to be little to no risk in using the hydroxyl radical as a disinfectant for short-term treatment of the oral cavity.

Novel denture-cleaning system based on hydroxyl radical disinfection.

The purpose of this study was to evaluate a new denture-cleaning device using hydroxyl radicals generated from photolysis of hydrogen peroxide (H2O2). Electron spin resonance analysis demonstrated that the yield of hydroxyl radicals increased with the concentration of H2O2 and light irradiation time. Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant S aureus were killed within 10 minutes with a > 5-log reduction when treated with photolysis of 500 mM H2O2; Candida albicans was killed within 30 minutes with a > 4-log reduction with photolysis of 1,000 mM H2O2. The clinical test demonstrated that the device could effectively reduce microorganisms in denture plaque by approximately 7-log order within 20 minutes.

Bactericidal action of photogenerated singlet oxygen from photosensitizers used in plaque disclosing agents.

BACKGROUND: Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of dental plaque in the field of dental care. In PDT, once the photosensitizer is irradiated with light of a specific wavelength, it transfers the excitation energy to molecular oxygen, which gives rise to singlet oxygen. METHODOLOGY/PRINCIPAL FINDINGS: Since plaque disclosing agents usually contain photosensitizers such as rose bengal, erythrosine, and phloxine, they could be used for PTD upon photoactivation. The aim of the present study is to compare the ability of these three photosensitizers to produce singlet oxygen in relation to their bactericidal activity. The generation rates of singlet oxygen determined by applying an electron spin resonance technique were in the order phloxine > erythrosine ≒ rose bengal. On the other hand, rose bengal showed the highest bactericidal activity against Streptococcus mutans, a major causative pathogen of caries, followed by erythrosine and phloxine, both of which showed activity similar to each other. One of the reasons for the discrepancy between the singlet oxygen generating ability and bactericidal activity was the incorporation efficiency of the photosensitizers into the bacterial cells. The incorporation rate of rose bengal was the highest among the three photosensitizers examined in the present study, likely leading to the highest bactericidal activity. Meanwhile, the addition of L-histidine, a singlet oxygen quencher, cancelled the bactericidal activity of any of the three photoactivated photosensitizers, proving that singlet oxygen was responsible for the bactericidal action. CONCLUSIONS: It is strongly suggested that rose bengal is a suitable photosensitizer for the plaque disclosing agents as compared to the other two photosensitizers, phloxine and erythrosine, when used for PDT.

Synergistic effect of thermal energy on bactericidal action of photolysis of H2O2 in relation to acceleration of hydroxyl radical generation.

The purpose of the present study is to evaluate the effect of thermal energy on the yield of and the bactericidal action of hydroxyl radical generated by photolysis of H(2)O(2). Different concentrations of H(2)O(2) (250, 500, 750, and 1,000 mM) were irradiated with light-emitting diodes (LEDs) at a wavelength of 400 ± 20 nm at 25°C to generate hydroxyl radical. The 500 mM H(2)O(2) was irradiated with the LEDs at different temperatures (25, 35, 45, and 55°C). Electron spin resonance spin trapping analysis showed that the yield of hydroxyl radicals increased with the temperature, as well as the concentration of H(2)O(2). Streptococcus mutans and Enterococcus faecalis were used in the bactericidal assay. The LED-light irradiation of the bacterial suspensions in 500 mM H(2)O(2) at 25°C could hardly kill the bacteria within 3 min, while the bactericidal effect was markedly enhanced with the temperature rise. For instance, a temperature increase to 55°C resulted in >99.999% reduction of viable counts of both bacterial species only within 1 min. The photolysis of 500 mM H(2)O(2) at 55°C could reduce the viable counts of bacteria more efficiently than did the photolysis of 1,000 mM H(2)O(2) at 25°C, although the yields of hydroxyl radical were almost the same under the both conditions. These findings suggest that the thermal energy accelerates the generation of hydroxyl radical by photolysis of H(2)O(2), which in turn results in a synergistic bactericidal effect of hydroxyl radical and thermal energy.

Reevaluation of analytical methods for photogenerated singlet oxygen.

The aim of the present study is to compare different analytical methods for singlet oxygen and to discuss an appropriate way to evaluate the yield of singlet oxygen photogenerated from photosensitizers. Singlet oxygen photogenerated from rose bengal was evaluated by electron spin resonance analysis using sterically hindered amines, spectrophotometric analysis of 1,3-diphenylisobenzofuran oxidation, and analysis of fluorescent probe (Singlet Oxygen Sensor Green®). All of the analytical methods could evaluate the relative yield of singlet oxygen. The sensitivity of the analytical methods was 1,3-diphenylisobenzofuran < electron spin resonance < Singlet Oxygen Sensor Green®. However, Singlet Oxygen Sensor Green® could be used only when the concentration of rose bengal was very low (<1 µM). In addition, since the absorption spectra of 1,3-diphenylisobenzofuran is considerably changed by irradiation of 405 nm laser, photosensitizers which are excited by light with a wavelength of around 400 nm such as hematoporphyrin cannot be used in the 1,3-diphenylisobenzofuran oxidation method. On the other hand, electron spin resonance analysis using a sterically hindered amine, especially 2,2,6,6-tetramethyl-4-piperidinol and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, had proper sensitivity and wide detectable range for the yield of photogenerated singlet oxygen. Therefore, in photodynamic therapy, it is suggested that the relative yield of singlet oxygen generated by various photosensitizers can be evaluated properly by electron spin resonance analysis.

Photolysis of hydrogen peroxide, an effective disinfection system via hydroxyl radical formation.

The relationship between the amount of hydroxyl radicals generated by photolysis of H(2)O(2) and bactericidal activity was examined. H(2)O(2) (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H(2)O(2) resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 µM hydroxyl radicals would result in reductions of viable counts of >99.99%.

A retrospective study of fixed dental prostheses without regular maintenance.

PURPOSE: The purpose of this study was to evaluate survival rate and the reasons of failure of fixed dental prostheses (FDPs) without having regular maintenance in a long-term after insertion. MATERIALS AND METHODS: The subjects were 55 patients (69 FDPs, 142 abutment teeth) who had received no regular maintenance after insertion of FDPs at the Prosthodontic Clinic, Tohoku University Dental Hospital in Japan from 1983 to 1989. Extraction of abutment teeth or removal of FDPs was considered as failure. Abutment teeth and FDPs requiring additional treatments were considered as the complications. The survival curves were analyzed using the Kaplan-Meier method. The reasons for failures and their frequency as well as complications were also examined. RESULTS: The average follow-up period was 16.5 years. 14 out of 142 abutment teeth were extracted, resulting in 10% failure rate. 22 out of total 66 FDPs were removed; the failure rate of FDPs was 33%. The most common reason for the failure and complication was periodontal disease. The mean value of plaque index was 43.2%. The analysis of the survival curves with the Kaplan-Meier method showed that the survival rates of FDPs at 15 years after insertion were 74%. As the status before treatment was not clearly stated, we cannot make an easy comparison; however, our data presented very similar results with reports from previous long-term clinical studies. CONCLUSION: This study suggested that the abutment teeth and FDPs in patients without regular maintenance had many periodontal problems due to dental plaque.

[A review of clinical follow-up studies focusing on pretreatment conditions of abutment and clinical examination parameters].

PURPOSE: When patients have their teeth taken care of, one of their biggest concerns is how long the solution will last. Studies on the longevity or prognosis of bridges published so far have mainly reported on the evaluation of the survival rate for each kind of bridge. We believe that abutments need to be researched more carefully and clinically in the future. STUDY SELECTION: A Medline search was conducted, as well as manual searching of bibliographies from relevant articles. Studies reporting the survival rate were selected from the reports mentioning the prognosis of bridges. We mainly reviewed 1) the conditions of abutments before treatments and 2) applicable parameters in each case. RESULTS: We found no study that mentioned the detailed conditions of abutments before treatment using systematic evaluation parameters. Most parameters concerned the prosthodontics itself and bacteria. Only 21. 2% described objective parameters. CONCLUSION: This review indicates the necessity for establishing a classification of abutments based on some evidence so that the after-treatment conditions can be predicted from the pre-treatment conditions. It also indicates that we have to consider objective parameters carefully when conducting prospective research on the prognosis of fixed partial dentures or when establishing a new clinical evaluation system.