Toshizo Toyama

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 (K1 and K2) of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy in the oral and maxillofacial region of the animal models. The decay rate constant (K1) 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.

Peptidoglycan of Actinomyces naeslundii induces inflammatory cytokine production and stimulates osteoclastogenesis in alveolar bone resorption

OBJECTIVE Actinomyces naeslundii, plays an important role in forming dental biofilms and causes gingival inflammation. Although peptidoglycan, the major cell wall component of Gram-positive bacteria, has been demonstrated to induce inflammatory cytokines, little is known about the association of peptidoglycan with alveolar bone resorption. This study investigated the involvement of peptidoglycan from A. naeslundii in osteoclast formation and bone resorption. DESIGN Osteoclast formation and function induced by peptidoglycan of A. naeslundii T14V were examined using the co-culture system of MCTC3/PA6 cells and BALB/c mouse bone marrow cells. Osteoclast formation was evaluated to count TRAP-positive multi-nuclei cells as osteoclasts. The function of osteoclasts was assessed by measuring the areas of pits absorbed. Inflammatory cytokine genes expressions, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, were examined by RT-PCR analysis using murine peritoneal macrophages. Experimental periodontitis was performed in Sprague-Dawley rats orally infected with A. naeslundii. RESULTS TRAP-positive multi-nuclei cells and the areas of pits induced by peptidoglycan were significantly greater than controls (p<0.01). Gene expression levels of IL-1β, IL-6, and TNF-α induced by A. naeslundii PGN were stronger than controls. In experimental periodontitis, bone loss of A. naeslundii-infected rats was comparable to that of rats induced by Porphyromonas gingivalis, which has been reported to be a periodontal pathogenic agent, being significantly greater than that of the sham group (p<0.01). CONCLUSIONS These results suggest that peptidoglycan of A. naeslundii is an important virulence factor in the development of periodontitis.

Involvement of HMGB1 and RAGE in IL-1β-induced gingival inflammation.

OBJECTIVE Extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promotes inflammation and participates in the pathogenesis of several disorders in peripheral organs. The role of HMGB1 and receptor for advanced glycation end products (RAGE) expressed in gingival inflammatory tissues was explored. METHODS Real time PCR was applied to assay HMGB1 and RAGE mRNA expression in gingival epithelial and fibroblast cells induced by interleukin-1β (IL-1β). A highly selective inhibitor of inducible nitric oxide (iNOS) was employed. ELISA was done for measurement of HMGB1 concentrations in cell culture media of gingival epithelial and fibroblast cells. Immunohistochemistry was performed to analyse the expression and sub-cellular localization of HMGB1, together with RAGE, in specimens obtained from patients with chronic inflammation. RESULTS A time-dependent response of HMGB1 and RAGE expression in gingival cells to IL-1β induction was observed. IL-1β promotes HMGB1 production in human gingival epithelial cells in a nitric oxide-dependent manner. HMGB1 and RAGE appeared highly expressed in gingival inflammatory tissues. CONCLUSION These results demonstrate that HMGB1 and RAGE are abundantly expressed in gingiva and promptly released during gingival inflammation. We suggest a role for HMGB1/RAGE/iNOS signalling on inflamed gingival epithelial cells.

Micromolar sodium fluoride mediates anti-osteoclastogenesis in Porphyromonas gingivalis-induced alveolar bone loss.

Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1β, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases.

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.

Inhibitory effects of Jixueteng on P. gingivalis-induced bone loss and osteoclast differentiation

OBJECTIVE The aim of this study was to investigate the possibility of Jixueteng as a preventive and therapeutic drug for the periodontitis. We investigated the inhibitory effects of Jixueteng on Porphyromonas gingivalis-induced bone loss in mice, antibacterial activity against P. gingivalis and differentiation of osteoclast and viability of cells. MATERIALS AND METHODS Fifty-four male, 4-week-old C57BL/6N mice, were randomly divided into the following three groups of 18 mice each; group A served as the P. gingivalis non-infected control (sham group), group B was infected orally with P. gingivalis and group C was administered Jixueteng extract in drinking water and was then infected with P. gingivalis. In order to evaluate the effect of Jixueteng, the distance from the alveolar bone crest to the cemento-enamel junction was determined. P. gingivalis suspension was exposed for 1, 15 and 60 min to 5 ml of the Jixueteng extract. Furthermore, to clarify the mechanism of the inhibitory effects of Jixueteng on osteoclast formation, Jixueteng extract was added to the culture of mouse bone marrow cells, osteoclast precursor. RESULTS Administration of Jixueteng along with P. gingivalis infection significantly reduced alveolar bone loss compared to P. gingivalis infection. Jixueteng treatment at the concentration of 0.01% significantly inhibited osteoclast formation. The addition of Jixueteng extract (0.1%, 0.01%, and 0.001%) to the culture showed a significant inhibition of the number of surviving osteoclasts in a dose-dependent manner. CONCLUSION Jixueteng has an antibacterial activity against P. gingivalis and inhibitory effects on osteoclastogenesis, it may be useful as a therapeutic drug in the treatment of P. gingivalis-induced periodontitis.

Ameliorating effects of Juzentaihoto on restraint stress and P. gingivalis-induced alveolar bone loss.

OBJECTIVE Juzentaihoto (JTX) is a traditional Japanese medicine that consists of 10 herbs. The purpose of this study was to evaluate the efficacy of multi-herbal medicine JTX as a preventive and therapeutic drug for periodontal bone resorption and for reducing restraint stress. MATERIALS AND METHODS Porphyromonas gingivalis ATCC 33277 was used for testing the antibacterial activity of JTX and a rat experimental periodontitis model. To evaluate the effect of JTX against P. gingivalis infection, we determined the differences in alveolar bone loss among experimental groups. The concentrations of adrenocorticotropic hormones were measured as stress markers, and atrophy of the thymus and spleen was assessed. RESULTS JTX had antibacterial activity against P. gingivalis ATCC 33277. JTX treatment of mouse bone marrow cells at a concentration of 0.1 μg/ml significantly inhibited osteoclast formation. Administration of JTX to rats with P. gingivalis infection and restraint stress significantly reduced alveolar bone loss compared with the case with just the combination of P. gingivalis infection and restraint stress. In the restrained groups, stress markers were elevated, and the thymus and spleen were atrophied. The groups with administration of JTX showed not only inhibition of the decrease of weight but also normalization of corticosterone and cortisol values. CONCLUSION JTX effectively inhibited restraint stress and osteoclastogenesis. It appears that the effects of JTX inhibit the destruction of periodontal tissue by suppressing stress. Our study demonstrated that JTX affects the correlation between restraint stress and periodontitis.

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.

Porphyromonas gingivalis-induced alveolar bone loss is accelerated in the stroke-prone spontaneously hypertensive rat.

Porphyromonas gingivalis (P. gingivalis) is one of the prominent periodontal pathogens and is the most important bacteria involved in the onset and exacerbation of periodontitis. P. gingivalis is an anaerobic, Gram-negative coccobacillus that plays a role in the progression of periodontal disease by promoting alveolar bone resorption. The aim of the present study was to examine P. gingivalis-induced osteoclastic bone resorption in the stroke-prone spontaneously hypertensive rat (SHRSP), in which oxidative stress induced by reactive oxygen species (ROS) is increased. In the present study, we used animals orally challenged with P. gingivalis as a chronic inflammation model. Horizontal bone loss around the maxillary molars was assessed morphometrically. Animals were divided into four groups: (1) P. gingivalis-non-infected Wister Kyoto Rat (WKY), (2) orally challenged with P. gingivalis WKY (WKY + Pg), (3) P. gingivalis-non-infected SHRSP, and (4) orally challenged with P. gingivalis SHRSP (SHRSP + Pg). Alveolar bone resorption was significantly increased in the orally challenged with P. gingivalis groups, and was accelerated in the SHRSP group. Histological analysis revealed that the infiltration of inflammatory cells was absent in all groups. However, the infiltration of osteoclasts was observed in the SHRSP + Pg and SHRSP groups. We examined P. gingivalis-induced alveolar bone loss in both the SHRSP and WKY. The results obtained demonstrated that P. gingivalis-induced alveolar bone loss would be involved in hypertension and stroke animal model, such as SHRSP and/or periodontal disease.

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.