Bactericidal effect of hydroxyl radicals generated by the sonolysis and photolysis of hydrogen peroxide for endodontic applications.

The aim of endodontic root canal treatment is the elimination of bacteria and their products from an infected tooth root canal. To effectively disinfect a root canal, an ultrasonic irrigation system, in which hydroxyl radicals (HO·) generated artificially by sonolysis of H2O2, was developed previously for endodontic applications and was demonstrated to have bactericidal efficacy against Enterococcus faecalis. To improve this system, we examined the in vitro bactericidal effects of HO· generated from H2O2, activated by simultaneous irradiation with ultrasound for sonolysis and dental LED light for photolysis with a peak wavelength of 405 nm. Regarding the LED irradiation, two methods were used: (i) 'ideal' experimental conditions (irradiation close to the glass tube), and (ii) simulated endodontic conditions (more distant irradiation of a masked glass tube). In these conditions, HO· generation from H2O2 was detected by electron spin resonance (ESR) spectroscopy, and bactericidal efficacy against E. faecalis was assessed by measuring the colony forming units (CFU)/mL. The results indicated that HO· generation by ESR measurements and the bactericidal effect on E. faecalis by viable count using CFU/mL were enhanced significantly in a time-dependent manner in both conditions. In a comparison of these conditions, bactericidal activity under 'ideal' experimental conditions was similar to that under simulated endodontic conditions. Moreover, the irradiation time for effective killing of E. faecalis through the sonolysis and photolysis of H2O2 under simulated endodontic conditions was shorter than that with sonolysis alone. These results demonstrate that H2O2 activated by ultrasound and LED light may be a safe and effective disinfection technique for endodontic root canal treatment.

Porphyromonas gingivalis infection modifies oral microcirculation and aortic vascular function in the stroke-prone spontaneously hypertensive rat (SHRSP).

The functional modulation of vascular endothelial cells associated with stroke and periodontal disease has not yet been clarified. The objective of this study is to analyze the vascular endothelial function of periodontitis and stroke animal models. We examined endothelial function and gingival blood flow in oral microcirculation in vivo and measured the isometric tension in vitro of the aorta in animal models for lifestyle-related diseases, such as periodontitis and stroke. Gingival reactive hyperemia (GRH) was measured using laser Doppler flowmetry. Wistar Kyoto rats (WKY) were used as control animals; Porphyromonas gingivalis (P. gingivalis) infected WKY (WKY + Pg) as the periodontitis model; stroke-prone spontaneously hypertensive rat (SHRSP) as the stroke model; and a final group consisting of P. gingivalis infected SHRSP (SHRSP + Pg). Furthermore, for each group, the relaxation of descending aortic ring preparations was measured using a force transducer. The GRH was estimated by maximum response (peak), time taken for the maximum response to fall to one half (T1/2), and increased total amount of blood flow (mass). The relative change in T1/2 and mass increased in SHRSP + Pg compared to WKY. However, mass significantly increased in WKY (758.59 ± 88.21 ml/min/100 g s to 1755.55 ± 226.10 ml/min/100 g s) and SHRSP (1214.87 ± 141.61 ml/min/100 g s to 2674.32 ± 675.48 ml/min/100 g s) after treatment with acetylcholine. In addition, T1/2 and mass significantly increased in WKY + Pg (624.18 ± 96.36 ml/min/100 g s to 2629.90 ± 612.01 ml/min/100 g s) and SHRSP + Pg (1116.36 ± 206.24 ml/min/100 g s to 1952.76 ± 217.39 ml/min/100 g s) after treatment with nitroglycerin. Furthermore, the endothelium-dependent relaxation of ring preparations, evoked by acetylcholine, was attenuated in SHRSP compared with WKY, but not in SHRSP + Pg. This attenuation effect in SHRSP could be prevented by superoxide dismutase pretreatment. Our results suggest altered endothelial function may occur in gingival tissue in animal models experiencing both periodontitis and stroke. Therefore, these results indicate the disruption of vascular function in oral microcirculation may be caused by the interaction between the oxidative stress induced by periodontitis and nitric oxide in periodontitis, similar to the interactions present in stroke cases.

Protective effects of (6R)-5,6,7,8-tetrahydro-l-biopterin on local ischemia/reperfusion-induced suppression of reactive hyperemia in rat gingiva.

We herein investigated the regulatory mechanism in the circulation responsible for rat gingival reactive hyperemia (RH) associated with ischemia/reperfusion (I/R). RH was analyzed using a laser Doppler flowmeter. RH and I/R were elicited by gingival compression and release with a laser Doppler probe. RH increased in a time-dependent manner when the duration of compression was between 30 s and 20 min. This increase was significantly suppressed by N (ω)-nitro-l-arginine-methyl-ester (l-NAME), 7-nitroindazole (7-NI), and 2,4-diamino-6-hydroxypyrimidine (DAHP). However, RH was markedly inhibited following 60 min of compression. This inhibition was significantly decreased by treatments with superoxide dismutase (SOD), (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), and sepiapterin. The luminescent intensity of superoxide anion (O2 (•-))-induced 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazine-3-one (MCLA) was markedly decreased by SOD and BH4, but only slightly by sepiapterin. BH4 significantly decreased O2 (•-) scavenging activity in a time-dependent manner. These results suggested that nitric oxide (NO) secreted by the nitrergic nerve played a role in regulating local circulation in rat gingiva. This NO-related regulation of local circulation was temporarily inhibited in the gingiva by the I/R treatment. The decrease observed in the production of NO, which was caused by suppression of NO synthase (NOS) activity subsequent to depletion of the NOS co-factor BH4 by O2 (•-), played a partial role in this inhibition.

Bactericidal effect of hydroxyl radicals generated from a low concentration hydrogen peroxide with ultrasound in endodontic treatment.

One approach to enhance the disinfection of root canals in endodontic treatment is ultrasonic irrigation with sodium hypochlorite. Reactive oxygen species, such as hydroxyl radical, are generated by biological defense systems to kill invading bacteria. Ultrasonic irrigation with hydrogen peroxide may be a promising option to increase hydroxyl radical generation. We examined the bactericidal effects of hydroxyl radical generated from low concentration hydrogen peroxide with ultrasound in vitro. An ultrasonic tip was submerged in 0.5 or 1.0 M hydrogen peroxide in a microfuge tube. hydrogen peroxide was irradiated with the ultrasound, the tip of which was maintained centered in the tube to mimic ultrasonic irrigation. Hydroxyl radical generation was assessed by electron spin resonance spectroscopy. Subsequently, Enterococcus faecalis suspension in hydrogen peroxide was prepared and irradiated as described above. Bactericidal effects were assessed by viable counting. Electron spin resonance measurements showed that hydroxyl radical generation increased significantly in a time- and dose-dependent manner (two-way analysis of variance and Tukey's test, p<0.05). Moreover, the bactericidal effects of hydrogen peroxide against Enterococcus faecalis were enhanced by ultrasonic irradiation in a time- and dose-dependent manner. These results suggest that ultrasonic irrigation in the presence of low concentration hydrogen peroxide can serve as a disinfection strategy in endodontic treatment.

Effectiveness of curcumin-based antimicrobial photodynamic therapy against Staphylococcus aureus.

PURPOSE: This study investigated the effectiveness of curcumin-based antimicrobial photodynamic therapy (aPDT) against Staphylococcus aureus (S. aureus), the causative agent of ventilator-associated pneumonia. METHODS: Curcumin was added to S. aureus culture medium at concentrations of 25, 2.5, and 0.25 µM. After 60 min (20-25°C), each culture was irradiated for 1 and 3 min, and viable bacteria were counted. Curcumin (25 µM) was also added to a bacterial suspension with D-mannitol and sodium azide; microbial counts were determined after irradiation for 3 min. RESULTS: S. aureus was significantly reduced in the 1-min (P = 0.043) and 3-min (P = 0.011) irradiation groups in comparison to the 0-min irradiation group with 25 µM curcumin. No significant differences were observed between the curcumin alone group and the curcumin plus D-mannitol or sodium azide group. CONCLUSION: The findings of this study indicate that prolonged exposure (≥1 min) of S. aureus to LED in 25 µM curcumin solution induces cell wall injury. Curcumin-based aPDT as an adjunct to conventional oral care, employing existing dentistry equipment, offers a promising approach that does not rely on antimicrobial drugs or allows the emergence of resistant bacterial strains. This suggests its potential role in future strategies aimed at preventing ventilator-associated pneumonia.

Impact on Porphyromonas gingivalis of antimicrobial photodynamic therapy with blue light and Rose Bengal in plaque-disclosing solution.

OBJECTIVES: Antimicrobial photodynamic therapy (aPDT) in periodontal pockets using lasers is difficult to perform in some cases because of the high cost of irradiation equipment and the narrow irradiation field. The purpose of the present study was to examine the effects of aPDT in combination with a plaque-disclosing solution and blue light-emitting diode (LED), which are used for composite resin polymerization. METHODS: The reactive oxygen species generated by irradiating 0.001% RB or MB with blue light were analyzed using electron spin resonance spectroscopy. Blue-light exposure was performed at 6.92, 20.76 and 124.6 J. The microorganism to be sterilized was Porphyromonas gingivalis. After aPDT, colony-forming units (CFUs) were measured to estimate cell survival. Carbonylated protein (PC) levels were used to evaluate oxidative stress. All statistical analyses were performed with Tukey's multiple comparisons test or the unpaired t-test. RESULTS: Singlet oxygen (1O2) generation was confirmed by RB+blue LED. 1O2 production was significantly greater with the blue LED irradiation of RB than that of MB (p < 0.0001). CFUs were significantly lower in the blue LED-irradiated group than in the non-LED-irradiated group (p < 0.01). The bactericidal effect increased in a time-dependent manner. aPDT increased PC levels. No morphological changes were observed in P. gingivalis. CONCLUSIONS: The present results suggest that aPDT exerts bactericidal effects against P. gingivalis by increasing oxidative stress through the generation of 1O2 in cells. Periodontal disease may be treated by aPDT using the equipment available in dental offices.

Effect of physical stimulation (gingival massage) on age-related changes in gingival microcirculation.

The decline in circulatory function with aging may be alleviated by a combination of gingival massage (physical stimulation) and mechanical cleaning. Several studies have reported the systemic effect of physical stimulation on various parts of the body, including its therapeutic effect on pain in the neck and shoulders that becomes evident with age, and improvement in blood circulation. In contrast, few studies have reported on the changes in gingival microcirculation induced by gingival massage, while no previous study has evaluated the effect of gingival microcirculation on age-related changes in the hemodynamics of the oral cavity. This study aimed to investigate how gingival massage affects age-related changes in gingival microcirculation. Male Wistar rats (7-week, 6-month and 1-year old) were prepared for a gingival massage group and a control group. Mechanical stimulation was applied on the maxillary molar gingiva for 5 seconds twice a week for 4 weeks. Subsequently, gingival reactive hyperemia was measured using a laser Doppler flowmeter. In addition, morphological analyses were also performed by hematoxylin and eosin and Indian ink staining and a vascular resin cast model. Base Flow, maximum response (Peak), and time required for the maximum response to halve (T1/2) were reduced in 1-year-old rats compared with the other age groups. In the mechanical stimulated group, T1/2 was increased in 7-week, 6-month, and 1-year-old rats, and total blood flow (Mass) was increased in 6-month and 1-year-old rats. In addition, clear blood vessel networks and loop-like revascularization were only observed in the mechanical stimulated group. Changes in age-related decline in gingival microcirculatory function and vascular construction were reported in this study, and the results suggested that gingival massage activates both the functional and morphological aspects of gingival microcirculation and may be effective for maintaining oral health.

Singlet oxygen generated by a new nonthermal atmospheric pressure air plasma device exerts a bactericidal effect on oral pathogens.

Oral diseases generally have certain bacteria associated with them. Non-thermal atmospheric pressure plasma (NTAP), generated at atmospheric pressure and room temperature, incorporates several molecules, including reactive oxygen species, that can inactivate various bacteria including oral pathogens. For this reason, several NTAP devices have been developed to treat oral diseases. Use of noble gases can enhance the bactericidal efficacy of NTAP, but this requires additional gas supply equipment. Therefore, a new NTAP device that employs ambient air as the working gas was developed. The device generates non-thermal atmospheric pressure air plasma. Here, the singlet oxygen (1O2) levels generated, their bactericidal effects on oral pathogens (Streptococcus mutans, Porphyromonas gingivalis, and Enterococcus faecalis), and the bacterial oxidative stress they imposed were measured. 1O2 generation in phosphatebuffered saline was assessed qualitatively using electron spin resonance (ESR) spectroscopy, and bactericidal efficacy was evaluated by counting of colony-forming units/mL. Bacterial oxidative stress was determined by measurement of hydrogen peroxide (H2O2) and superoxide dismutase (SOD) activity. ESR indicated that the level of 1O2 increased significantly and time-dependently, and was inversely correlated with distance, but the bactericidal effects were correlated only with treatment time (not distance) as H2O2 increased and SOD levels decreased, suggesting that the new device has potential applicability for treatment of oral disease.

Antimicrobial photodynamic therapy using a plaque disclosing solution on Streptococcus mutans.

OBJECTIVES: Photodynamic therapy with a bactericidal action is called antimicrobial photodynamic therapy (aPDT),which is a method of staining an object with a photosensitizing dye and then sterilizing by irradiating the dye at it's excitation wavelength. In this study, we aimed to investigate a caries pathogenic bactericidal method in a site difficult to mechanically remove, by examining aPDT effect on Streptococcus mutans (S. mutans), which is a typical caries pathogenic bacteria by applying the plaque disclosing solution as photosensitizing dye. METHODS: The absorption wavelength spectrum of irradiating plaque staining agent phloxine B (PB) was analyzed using UV-vis. Reactive oxygen species (ROS) generated by photo excitation with blue LED irradiation was measured by electron spin resonance technique. S. mutans was cultured according to a conventional method and the effect of aPDT after PB staining was evaluated by a Colony Forming Unit (CFU). In addition, protein carbonyl (PC), an oxidative stress marker, was also measured by western blotting. RESULTS: Singlet oxygen was generated by PB with blue light. As a result of aPDT treatment on S. mutans under this condition, it was recognized that CFU was suppressed dependent on irradiation intensity of blue light. In addition, the expression of PC was enhanced by aPDT. CONCLUSIONS: aPDT is demonstrated by staining S. mutans with PB and irradiating blue light used for resin polymerization and tooth bleaching to generate ROS. Therefore, plaque-disclosing solution-based aPDT against S. mutans might represent a new method for cleaning pit and fissure grooves.

Effects of blue-light irradiation during dental treatment.

In dentistry, blue light is widely used for tooth bleaching and restoration procedures involving composite resin. In addition, many dentists use magnification loupes to enable them to provide more accurate dental treatment. Therefore, the use of light is indispensable in dental treatment. However, light can cause various toxicities, and thermal injuries caused by light irradiation are regarded as particularly important. In recent years, the eye damage and non-thermal injuries caused by blue light, the so-called "blue light hazard", have gained attention. Unfortunately, much of the research in this field has just begun, but our recent findings demonstrated that blue-light irradiation generates reactive oxygen species (ROS) and induces oxidative stress in oral tissue. However, they also showed that such oxidative stress is inhibited by antioxidants. There have not been any reports that suggested that the ROS-induced phototoxicity associated with blue-light irradiation causes direct clinical damage, but some disorders are caused by the accumulation of ROS. Therefore, it is presumed that it is necessary to suppress the accumulation of oxidative stressors in oral tissues during treatment. In the future, we have to promote discussion about the suppression of phototoxicity in dentistry, including concerning the use of antioxidants to protect against phototoxic damage.

Antimicrobial effect of blue light using Porphyromonas gingivalis pigment.

The development of antibiotics cannot keep up with the speed of resistance acquired by microorganisms. Recently, the development of antimicrobial photodynamic therapy (aPDT) has been a necessary antimicrobial strategy against antibiotic resistance. Among the wide variety of bacteria found in the oral flora, Porphyromonas gingivalis (P. gingivalis) is one of the etiological agents of periodontal disease. aPDT has been studied for periodontal disease, but has risks of cytotoxicity to normal stained tissue. In this study, we performed aPDT using protoporphyrin IX (PpIX), an intracellular pigment of P. gingivalis, without an external photosensitizer. We confirmed singlet oxygen generation by PpIX in a blue-light irradiation intensity-dependent manner. We discovered that blue-light irradiation on P. gingivalis is potentially bactericidal. The sterilization mechanism seems to be oxidative DNA damage in bacterial cells. Although it is said that no resistant bacteria will emerge using aPDT, the conventional method relies on an added photosensitizer dye. PpIX in P. gingivalis is used in energy production, so aPDT applied to PpIX of P. gingivalis should limit the appearance of resistant bacteria. This approach not only has potential as an effective treatment for new periodontal diseases, but also offers potential antibacterial treatment for multiple drug resistant bacteria.

Antimicrobial effect of titanium dioxide after ultraviolet irradiation against periodontal pathogen.

We focused on the antimicrobial effects of titanium dioxide (TiO2) after stopping ultraviolet (UV) irradiation as an adjunctive treatment for peri-implantitis in this study. The aim was to determine the continuous photocatalytic effects of TiO2 after UV irradiation and its antimicrobial activity against periodontal pathogen. The continuous photocatalytic effects of TiO2 after UV irradiation were determined by electron spin resonance (ESR) spectroscopy using TiO2 particles of various sizes with various UV irradiation times. In addition, antimicrobial activity against Porphyromonas gingivalis was investigated by quantitation of colony-forming units (CFUs). The results showed that the ESR signal ratio for the UV-irradiated TiO2 was significantly higher than that of the non-irradiated TiO2. UV-irradiated TiO2 significantly reduced the number of P. gingivalis when compared with non-irradiated controls. These results suggest that TiO2 has a continuous photocatalytic effect even after stopping UV irradiation and that it showed antimicrobial activity against periodontal pathogen.

Blue light irradiation-induced oxidative stress in vivo via ROS generation in rat gingival tissue.

It has been reported that oxidative stress with reactive oxygen species (ROS) generation is induced by blue light irradiation to a living body. Only limited research has been reported in dental field on the dangers of blue light, mostly focusing on cytotoxicity associated with heat injury of dental pulp. We thus performed an in vivo study on oral tissue exposed to blue light. ROS generated upon blue light irradiation of flavin adenine dinucleotide were measured by electron spin resonance spectroscopy. After blue light irradiation, the palatal gingiva of Wistar rats were isolated. Collected samples were subjected to biochemical analysis of lipid peroxidation and glutathione. Singlet oxygen was generated by blue light irradiation, but was significantly quenched in an N-acetyl-L-cysteine (NAC) concentration-dependent manner. Blue light significantly accelerated oxidative stress and increased the oxidized glutathione levels in gingival tissue. These effects were also inhibited by NAC pre-administration. The results suggest that blue light irradiation at clinical levels of tooth bleaching treatment may enhance lipid peroxidation by the induction of oxidative stress and the consumption of a significant amount of intracellular glutathione. In addition, NAC might be an effective supplement for the protection of oral tissues against blue light irradiation-induced oxidative damage.

Passive ultrasonic irrigation in the presence of a low concentration of hydrogen peroxide enhances hydroxyl radical generation and bactericidal effect against Enterococcus faecalis.

Chemomechanical procedures can be used to eliminate bacteria from root canals. However, detectable bacteria sometimes remain because of the complexity of the root canal system. Endodontic passive ultrasonic irrigation (PUI) with hydrogen peroxide (H2O2) may be a promising option for increasing bactericidal hydroxyl radical (HO•) generation. In this in vitro experiment, we examined the effects of HO• generated using PUI and a low concentration of H2O2. An ultrasonic tip was submerged in 0.45 mol/L (1.5%) H2O2 in a microfuge tube. H2O2 was activated by an ultrasonic unit, the tip of which was kept centered in the tube, to mimic PUI. HO• generation was detected by electron spin resonance spectroscopy. An Enterococcus faecalis suspension in H2O2 was then preparedand activated as described above. Bactericidal effects were assessed by viable counting. Two-way analysis of variance and Tukey's test were used to assess the statistical significance of differences among groups (P < 0.05). HO• generation and bactericidal activity were significantly increased by PUI in H2O2 in a time-dependent manner and were significantly higher than with H2O2 alone or with PUI in a Tris-HCl suspension. These results suggest that PUI in the presence of a low H2O2 concentration is a promising new disinfection strategy.

Reactive oxygen species production in mitochondria of human gingival fibroblast induced by blue light irradiation.

In recent years, it has become well known that the production of reactive oxygen species (ROS) induced by blue-light irradiation causes adverse effects of photo-aging, such as age-related macular degeneration of the retina. Thus, orange-tinted glasses are used to protect the retina during dental treatment involving blue-light irradiation (e.g., dental resin restorations or tooth bleaching treatments). However, there are few studies examining the effects of blue-light irradiation on oral tissue. For the first time, we report that blue-light irradiation by quartz tungsten halogen lamp (QTH) or light-emitting diode (LED) decreased cell proliferation activity of human gingival fibroblasts (HGFs) in a time-dependent manner (<5 min). Additionally, in a morphological study, the cytotoxic effect was observed in the cell organelles, especially the mitochondria. Furthermore, ROS generation induced by the blue-light irradiation was detected in mitochondria of HGFs using fluorimetry. In all analyses, the cytotoxicity was significantly higher after LED irradiation compared with cytotoxicity after QTH irradiation. These results suggest that blue light irradiation, especially by LED light sources used in dental aesthetic treatment, might have adverse effects on human gingival tissue. Hence, this necessitates the development of new dental aesthetic treatment methods and/or techniques to protect HGFs from blue light irradiation during dental therapy.

Dental resin curing blue light induced oxidative stress with reactive oxygen species production.

Dental resin curing blue light has been used in the treatment of tooth bleaching and to restore teeth with resin-based composite fillings. However, there has been little consideration of its effect on oral tissues such as dental pulp and oral mucosa. The aim of this study was to investigate whether dental resin curing blue light irradiation affects the dental pulp, especially the blood vessels that are known as the first target of reactive oxygen species (ROS), which play an important role in vascular reactivity. We found that blue light irradiation increased the level of lipid peroxidation in isolated rat aorta blood vessels by measuring malondialdehyde. Furthermore, cell proliferative activity was decreased in a time-dependent manner and apoptosis of human aorta vascular smooth muscle cells (VSMCs) was induced. These results indicated that (ROS) such as hydrogen peroxide and hydroxyl radicals were generated in VSMCs by irradiation with blue light, and they induced cytotoxicity associated with oxidative stress, which increased lipid peroxidation and apoptosis. In addition, N-acetyl-l-cysteine, which is a typical intracellular antioxidant, protected VSMCs against cytotoxicity associated with oxidative stress. These findings suggested that antioxidants may be used to prevent oxidative stress in dental pulp by repeated and/or multiple treatments with blue light irradiation in future dental treatments.

Assessments of salivary antioxidant activity using electron spin resonance spectroscopy.

OBJECTIVE: In recent years, the function of saliva has been focused on evaluation of general status. The relationship between salivary antioxidant activity and periodontal disease progression is unclear. The aim of this study is to assess the relationship between periodontal disease and salivary antioxidant activity towards various reactive oxygen species (ROS) using electron spin resonance (ESR) technique. METHODS: We demonstrated that whole saliva derived rats or human subjects scavenged ROS such as superoxide (O(2)(·-)) and hydroxyl radical (HO(·)) using ESR spectroscopy with spin trapping agent. In addition, we assessed the relationship between antioxidants activity towards ROS and periodontal index with superoxide dismutase (SOD) activity in human subject saliva. RESULTS: Antioxidant activity towards O(2)(·-) was increased by Porphyromonas gingivalis (P. gingivalis) infection in rat, although antioxidant activity towards HO(·) was not changed. In human, a strong correlation (r = 0.88, p < 0.01) recognized between salivary antioxidant activity towards O(2)(·-) and probing pocket depth (PPD). In addition, the intensity of salivary antioxidant activity depended on SOD activity level. SOD activity was also correlated with PPD. CONCLUSIONS: Rat salivary antioxidant activity towards O(2)(·-) was up-regulated by the inflammatory response caused by P. gingivalis infection. Similar response was recognized in human saliva with periodontal index. Additionally, a linear correlation between antioxidant activity towards O(2)(·-) and SOD activity was verified by ESR technique. Therefore, evaluation of the salivary antioxidant activity towards O(2)(·-) might be an effective parameter for the objective assessment of periodontal disease progression.

Gingival vascular functions are altered in type 2 diabetes mellitus model and/or periodontitis model.

The association of vascular reactivity between diabetes and periodontal disease has not been clarified. Gingival blood flow was measured by laser Doppler flowmetry for 31 weeks in Wistar rats, Wistar rats orally challenged with Porphyromonas gingivalis (Wistar rats + Porphyromonas gingivalis), Goto-Kakizaki rats, and Goto-Kakizaki rats orally challenged with Porphyromonas gingivalis (Goto-Kakizaki rats + Porphyromonas gingivalis). Effects of alveolar bone resorption on periodontal tissue was enhanced in Wistar rats + Porphyromonas gingivalis, and Goto-Kakizaki rats, with this effect being significantly enhanced by Goto-Kakizaki rats + Porphyromonas gingivalis. Using the L-band electron spin resonance technique, we succeeded in measuring oxidative stress as decay rate constant (K(1) and K(2)) of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy in the oral and maxillofacial region of the animal models. The decay rate constant (K(1)) of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy was significantly greater in the oral and maxillofacial region of Goto-Kakizaki rats + Porphyromonas gingivalis compared to Wistar rats, Wistar rats + Porphyromonas gingivalis and Goto-Kakizaki rats groups. Gingival reactive hyperemia was attenuated by periodontal disease, and this effect was also remarkable in the diabetes mellitus model. Taken together, we found that vascular endothelial function was decreased in diabetes mellitus and/or periodontal disease animal models due to increasing oxidative stress in the gingival circulation.