Effect of periodontitis induced by Fusobacterium nucleatum on the microbiota of the gut and surrounding organs.

Detection of the oral bacterium Fusobacterium nucleatum in colorectal cancer tissues suggests that periodontitis may alter gut microbiota. The purpose of this study was to analyze the influence and infection route of periodontal inflammation caused by F. nucleatum, and microbiota of the gut and surrounding organs (heart, liver, kidney). Wistar female rats were orally inoculated with F. nucleatum to establish an experimental periodontitis model that was confirmed by X-ray imaging and histopathological analysis. The mandibles, gut, liver, heart, and kidneys were collected from the experimental group at 2, 4, and 8 weeks, and from the uninfected control group at 0 weeks, for DNA extraction for PCR amplification and comprehensive microbiota analysis using the Illumina MiSeq platform. Imaging confirmed the onset of periodontitis at 2 weeks post-inoculation, and histopathology showed inflammatory cell infiltration from 2 to 8 weeks. PCR and comprehensive microbiota analysis showed the presence of F. nucleatum in the heart and liver at 2 weeks, and in the liver at 4 and 8 weeks. There were changes of microbiota of the gut, heart, liver, and kidneys at 4 weeks: namely, decreased Verrucomicrobia and Bacteroidetes, and increased Firmicutes. F. nucleatum induced the onset of periodontitis and infected the heart and liver in rats. As the periodontic lesion progressed, the microbiota of the gut, liver, heart, and kidneys were altered.

Influence of IgA nephropathy on the progression of pulpitis and apical periodontitis in HIGA mice.

OBJECTIVES: Immunoglobulin (Ig)A nephropathy has been associated with oral infections such as periodontitis, but its pathogenesis is not fully understood; no treatments exist. This study analyzes the influence of IgA nephropathy, an autoimmune disease, on the pathogenesis of pulpitis and apical periodontitis. METHODS: Two groups of mice were used in pulp infection experiments: high serum IgA nephropathy model mice (HIGA) and control mice (BALB/c). Histologic analyses of the pulp and apical periodontal tissues were performed on days 3, 5, 7, 14, and 28 following oral bacterial infection. The dynamics of odontoblasts, apoptotic cells, and IgA expression were analyzed using anti-Nestin, TUNEL, and anti-IgA staining, respectively. RESULTS: Inflammatory cells infiltrated the exposed pulp at day three in both groups and by 14 days, these cells had infiltrated from the pulp to the apical periodontal tissue. The area of necrotic pulp tissue increased significantly in the control group at seven days. Odontoblasts decreased from day three onwards and disappeared by 28 days in both groups. The number of apoptotic cells in the pulp and apical periodontal tissues was significantly higher in the experimental group at day 28. The experimental group exhibited a significant increase in IgA production in the pulp after 14 days. Bone resorption in the apical periodontal tissue was significantly decreased in the experimental group at day 28. CONCLUSIONS: The results of this study suggest that IgA nephropathy may modulate the inflammatory response and sustain long-term biological defense responses in pulpitis and apical periodontitis in HIGA mice.

Effects of Rheumatoid Arthritis on the Progression of Pulpitis and Apical Periodontitis in SKG Mice.

INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease that involves joint inflammation. Although periodontal disease reportedly contributes to RA onset, the associations of RA with pulpitis and apical periodontitis have not been described. The purpose of this study was to examine the effects of immune response disruption of RA for pulpitis and apical periodontitis with SKG mice. METHODS: SKG and BALB/c (control) mice were used to establish models of pulp infection. Histologic studies of pulp and apical periodontal tissue were performed at 3, 5, 7, 14, and 28 days; odontoblast dynamics were analyzed by antinestin staining, and apoptotic cells were examined by TdT-mediated digoxygenin (biotin)-dUTP nick end labeling staining. RESULTS: Inflammatory cell infiltration into the exposed pulp was observed at 3 days in the SKG and control group groups; the infiltration extended to the apical pulp area at 14 days after surgery. Inflammatory cell infiltration and bone resorption in the apical pulp area were observed from 14-28 days in the SKG and control groups; there were significant increases in inflammatory cell infiltration and bone resorption in the control group at 28 days. The numbers of apoptotic cells in pulp and apical periodontal tissue were higher in the SKG group than in the control group at 14 and 28 days. The number of odontoblasts decreased in the SKG and control groups until 14 days and then disappeared in the SKG and control groups at 28 days. CONCLUSIONS: This study suggested that immune response disruption in RA is involved in prolonging the inflammatory state of pulpitis and apical periodontitis.

Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla.

Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.

Wnt5a plays a crucial role in determining tooth size during murine tooth development.

We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.

The effect of mineral trioxide aggregate on dental pulp healing in the infected pulp by direct pulp capping.

This study aimed to clarify the effect of mineral trioxide aggregate (MTA) on pulp healing in the infected pulp by direct pulp capping (DPC). Thirty-six male ICR mice were divided into infected and uninfected groups. The pulp tissue was exposed to the oral flora for 24 h after pulp exposure in the infected group, or not exposed in the uninfected group, followed by sealing with MTA, calcium hydroxide cement (CH), or no DPC. Pulpal healing process was analyzed by hematoxylin-eosin staining and immunohistochemistry for nestin and Ki67. The active cell proliferation occurred on 1 week in the both MTA and CH groups, followed by the differentiation of odontoblast-like cells on 2 weeks in the MTA group, whereas their differentiation were not facilitated in the CH group. MTA is suggested to be a useful material for DPC with the infected and uninfected pulp tissue.

The Effect of Transforming Growth Factor Beta 1 on the Mineralization of Human Cementoblasts.

INTRODUCTION: Transforming growth factor beta 1 (TGF-ß1) plays an important role in bone mineralization and has been reported to promote osteoblast proliferation and differentiation. However, there is no report about the effects of TGF-ß1 on human cementoblasts. The purpose of this study was to clarify the effect of TGF-ß1 on the proliferation and differentiation of the human cementoblast cell line (HCEM) in vitro. METHODS: HCEM cells were stimulated with TGF-ß1 at concentrations of 0.05, 0.5, 5, and 10 ng/mL. A proliferation assay was performed from 24-72 hours. The effect of TGF-ß1 on mineralization was analyzed by quantifying the area stained with alizarin red on days 7 and 14. Real-time polymerase chain reaction was used to assess the effect of TGF-ß1 on the mineralization-related genes alkaline phosphatase, bone sialoprotein, and type I collagen on days 3, 7, and 14. RESULTS: TGF-ß1 did not affect cell proliferation. TGF-ß1 together with the mineralization medium (consisting of ascorbic acid, dexamethasone, and ß-glycerophosphate) increased the alizarin red-stained area on days 7 and 14. Real-time polymerase chain reaction revealed that alkaline phosphatase messenger RNA expression was increased in TGF-ß1-stimulated HCEM cells in mineralization medium on days 3 and 7, whereas bone sialoprotein and type I collagen messenger RNA expression was increased on day 7. CONCLUSIONS: Although TGF-ß1 does not affect cell proliferation, it does promote cell differentiation and mineralization of HCEM cells.

Effects of different light sources used for dental operating microscope illumination on the visual function of operators.

OBJECTIVES: Advances in dental operative microscopes (DOMs) enable examination of root canal morphology or detection of root fractures otherwise not visible to the naked eye. However, dental therapy involving prolonged use of DOMs requires precision within a limited visual field, resulting in eye strain among users. This study examined the effects of halogen and light-emitting diode (LED) light sources on asthenopia and visual function following use of DOMs. METHODS: The study used halogen and LED light sources in DOMs. The first experiment was conducted on 6 participants with corrected visual acuity without any organic eye disease. General visual function test (calculation ability test, hand grip strength test, and ophthalmic examination) and subjective symptom questionnaire were used to evaluate the degree of fatigue before and after DOM use. The second experiment was conducted on 9 participants with spherical equivalents within ±4 diopters (D) and astigmatism of 1 D or less. Accommodative function tests (precise test for asthenopia) and a subjective symptom questionnaire (asthenopia) were used before and after use of DOM. RESULTS: No significant changes were noted in the degree of fatigue and ophthalmological parameters before and after the procedure with either light source or in between light sources. The tear firm breakup time was shortened after therapy, and a tendency toward dry eyes was observed while using the LED light source. CONCLUSIONS: The halogen and LED light sources used for DOM therapy had similar effects on asthenopia of the operators, with no significant changes in visual function.

Effect of salivary secretion with mouthguard use on seasonal allergic rhinitis symptom improvement.

OBJECTIVES: It was shown that mucosal immunity via salivary IgA may be related to the improvement of seasonal allergic rhinitis (SAR) symptoms, and improvement of SAR symptoms through saliva flow increase has been reported in patients using mouthguard (MG) in dental treatment. The purpose of this study was to analyze the effect of MG use on SAR symptom improvement and to clarify the role of saliva on SAR symptom development. METHODS: We recruited patients from the Kanagawa Dental University Hospital including 38 and 8 patients with SAR and non-SAR symptoms during two seasons from March 2017 to April 2018. We analyzed the saliva flow rate pre- and post-MG use and measured the amount of IgA and IgG4 in the saliva. We assessed the correlation between SAR symptoms and MG use. SAR symptoms were examined according to a specific clinical score. RESULTS: It was revealed that salivary IgA concentration was significantly lower in SAR patients than in controls. SAR symptoms significantly improved with MG use. The saliva flow rate and IgA levels significantly increased with MG use, although the IgG4 levels did not change. CONCLUSIONS: MG use may be beneficial for improving the symptoms of SAR patients by increasing the IgA levels. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR: UMIN000026428) on 6thMarch 2017.

Expression of toll-like receptor 2 and 4 in dental pulp.

Toll-like receptors (TLRs) are important factors in innate immune responses because they mediate signals from bacterial cell wall components during inflammatory reactions. However, the role of TLR in dental pulp, which is bounded by hard tissues, is little understood. The present study investigated the expression of TLR-2 and TLR-4 in experimentally inflamed pulp by quantitative real-time polymerase chain reaction and immunohistochemistry. Total RNA isolated from pulp tissue from 0 to 72 hours after bacterial dentinal infection. The TLR-2 messenger RNA (mRNA) level was 30-fold higher than the TLR-4 mRNA level at 9 hours. The TLR-2 mRNA level in pulp began to increase by 3 hours after bacterial infection, reaching a maximum level after 9 hours and gradually decreasing from 9 to 72 hours. Numerous TLR-2- and CD64-positive cells detected on macrophage and dendritic-like cells, TLR-4-positive cells detected a little in the pulp at 9 hours. These results suggest that TLR-2 may be mainly regulated during the early stage of pulp inflammation triggered by bacterial infection.

Evaluation of the biocompatibility of resin-based root canal sealers in rat periapical tissue.

We evaluated the biocompatibility of resin-based root canal sealers (RCSs) in the periapical tissues of rats. Wistar rats underwent tooth replantation for reproducing the response of periapical tissue with RCSs. The resin-based Epipany SE, AH Plus Jet, the eugenol-based sealer (Canals) and a control group were employed. The upper right first molar was extracted and applied with RCSs on apices, and then the tooth was repositioned. Histological evaluation demonstrated that mild inflammation occurred in the periapical tissue with Epiphany and AH Plus Jet sealers on day 7, whereas Canals induced severe-to-moderate inflammation. The statistical analyses demonstrated that the significant differences were observed between Canals and the other groups on day 7 regarding inflammatory response. On day 14, the lesions induced by all sealers were healed and replaced predominantly by fibrous connective tissue. Our results suggest that Epiphany SE and AH Plus Jet are good biocompatible materials.

A biocompatible model for evaluation of the responses of rat periapical tissue to a new zinc oxide-eugenol sealer.

We aimed to establish an experimental animal model to evaluate materials for endodontic therapy. We focused on the biocompatibility of new paste-type zinc oxide-eugenol (ZOE) sealer. The results of this sealer were compared with those of conventional powder/liquid ZOE and eugenol-free sealers. The molars of Wistar rats were extracted and repositioned in the original socket after application of the sealers on the root apices. Mild inflammation occurred in the periapical tissue of the replanted teeth with both ZOE sealers on day 7, whereas the eugenol-free sealer induced severe inflammation. On day 14, the lesions induced by all types of sealers were healed and replaced predominantly by fibrous connective tissue. Thus, all endodontic materials showed high biocompatibility, although the extent of inflammatory reactions during the early stages varied depending on the types of materials. We demonstrated that our animal model was useful for the assessment of the biocompatibility of endodontic materials.

Expression of Toll-like receptor 2 and 4 in inflamed pulp in severe combined immunodeficiency mice.

INTRODUCTION: Toll-like receptors (TLRs) are important receptors mediating innate immune responses because they detect factors released from bacterial cell wall components during inflammatory reactions. However, the role of TLRs in dental pulp, which is bounded by hard tissues, is poorly understood. The purpose of this study was to investigate the relationship between the innate immune system and the defense of pulp tissue by using immunodeficient mice that lack an adaptive immune response METHODS: Mice with severe combined immunodeficiency (SCID) were used as a model because they lack an adaptive immune response. The expression of TLR-2 and TLR-4 in experimentally inflamed pulps in SCID mice was measured by quantitative real-time polymerase chain reaction and immunohistochemistry. Total RNA was isolated from pulp tissues at 0 to 24 hours after bacterial dentinal infection. Anti-TLR-2, anti-TLR-4 cells, anti-CD64, and antinestin cells were detected with labeled streptavidin-biotin methods. RESULTS: TLR-2 messenger RNA was detected at 3 hours after bacterial infection and then gradually increased from 9 to 24 hours. Numerous TLR-2- and CD64-positive cells detected on macrophages and dendritic-like cells, and TLR-4- and CD64-positive macrophages were detected in the early stage of pulpitis. CONCLUSION: These results suggest that the expression of TLR-2 and TLR-4 may be triggered by bacterial infection in irreversible pulpitis without a need for an adaptive immune response. Those signals may relate to pulpal responses to bacterial infection.