Carbon Nanotubes Induce Mineralization of Human Cementoblasts.

INTRODUCTION: Carbon nanotubes (CNT) are 1 of the allotropes of carbon with unique properties. CNT shows good bone-tissue compatibility and has been reported to induce osteogenesis; therefore, it is regarded as an ideal material in a wide range of applications. However, the therapeutic effect of CNT-containing materials in the healing of apical periodontal tissue is unknown. The purpose of this study was to clarify the effect of CNT on the proliferation and mineralization of the human cementoblast cell line (HCEM). METHODS: The proliferation of HCEM cells with CNT stimulation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed from 24-72 hours. Calcium deposition levels were evaluated by alizarin red S staining on days 7 and 10, and mineralization-related gene expression was examined by quantitative real-time polymerase chain reaction on days 3, 7, and 10. Scanning electron microscopy was used to observe the culture with CNT on day 14. RESULTS: CNT showed no cytotoxicity to HCEM cell proliferation. Treatment was performed with mineralization medium, CNT-induced HCEM mineralization on day 7, and increased calcium deposition on days 7 and 14. Messenger RNA expression of alkaline phosphatase was significantly increased throughout the experimental period, and bone sialoprotein was significantly increased on day 3 by CNT, whereas no effect was found on mRNA expression of type I collagen. CNT was observed in attachment to the cell surface on day 14. CONCLUSIONS: CNT promotes the mineralization of HCEM cells, indicating the potential as a new bioactive component for apical periodontal tissue regeneration materials through the regulation of cementoblast mineralization.

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.

Role of MAPKs in TGF-ß1-induced maturation and mineralization in human osteoblast-like cells.

OBJECTIVES: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-ß1-stimulated mineralization in the human osteoblast-like MG63 cells. METHODS: The viability of MG63 cells under TGF-ß1 stimulation was assessed by MTS assay. Western blotting determined TGF-ß1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining. RESULTS: TGF-ß1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-ß1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-ß1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-ß1-mediated upregulation of ALP and BSP. Although all three inhibitors suppressed expression of COL I, none were found to stimulate expression of OCN. CONCLUSIONS: Human osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-ß1.

Identification of Candidate Genes of Familial Multiple Idiopathic Cervical Root Resorption.

INTRODUCTION: Multiple idiopathic cervical root resorption (MICRR) is a disease with an unknown etiology that causes invasive cervical root resorption in multiple teeth. Although previous MICRR genomic studies have identified candidate gene variants, the etiology of the condition remains poorly understood. In the present study, we investigated the genetic causality of MICRR to explore candidate variants. METHODS: Saliva samples from a family containing 2 affected and two unaffected subjects with the dominant transmission of MICRR were subjected to whole-exome sequencing. RESULTS: As a result, we identified novel candidate variants of 10 genes. Each variant was confirmed by Sanger sequencing. Among them, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines classified doublecortin domain containing 1 (c.1099 C > T) and ß-defensin 114 (c.189 T > G) as "pathogenic," and solute carrier family 45 member 2 (c.152_153del) as "likely pathogenic." CONCLUSIONS: These results provide new insight to help clarify the pathogenesis of MICRR, and the variants could be applied for further investigation to understand invasive cervical root resorption.

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.

BMP-1-induced GBA1 nuclear accumulation provokes CCN2 mRNA expression via importin-ß-mediated nucleocytoplasmic pathway.

Bone morphogenetic protein (BMP)-1 is expressed by odontoblasts in the dentin-pulp complex. Although the functional effects of BMP-1 on the maturation of various preforms of proteins and enzymes involved in initiating mineralization have been widely observed, how BMP-1 affects cellular molecules remains unknown. We performed a comprehensive analysis of BMP-1-altered glycome profiles and subsequent assays to identify the target glycoproteins in human dental pulp cells (hDPCs) by a glycomic approach. In the presence of BMP-1, a lectin microarray analysis and lectin-probed blotting showed that α2,6-sialylation was significantly attenuated in insoluble fractions from hDPCs. Six proteins were identified by a mass spectrometry analysis of α2,6-sialylated glycoproteins purified using a lectin column. Among them, glucosylceramidase (GBA1) was found to accumulate in the nuclei of hDPCs in the presence of BMP-1. Moreover, BMP-1-induced cellular communication network factor (CCN) 2 expression, which is well known as the osteogenesis/chondrogenesis marker, was significantly suppressed in the cells transfected with GBA1 siRNA. Furthermore, importazole, a potent inhibitor of importin-ß-mediated nuclear import significantly suppressed BMP-1-induced GBA1 nuclear accumulation and BMP-1-induced CCN2 mRNA expression, respectively. Thus, BMP-1 facilitates the accumulation of GBA1 in the nucleus through the reduction of α2,6-sialic acid, which potentially contributes to the transcriptional regulation of the CCN2 gene via importin-ß-mediated nuclear import pathway in hDPCs. Our results offer new insights into the role of the BMP-1-GBA1-CCN2 axis in the development, tissue remodeling, and pathology of dental/craniofacial diseases.

Effect of the Progression of Fusobacterium nucleatum-induced Apical Periodontitis on the Gut Microbiota.

INTRODUCTION: Fusobacterium nucleatum, which is involved in the development of periodontal disease and apical lesions, can be transmitted to the colon and metastasize to colorectal cancer, suggesting a link between oral and systemic diseases. We analyzed the effects of F. nucleatum on bacterial flora in the gut and surrounding organs in a rat model of apical periodontitis and analyzed the infection route to the gut and distant organs. METHODS: We induced apical periodontitis in rat molars by infecting the dental pulp with F. nucleatum and then took X-ray images and performed histopathologic analyses. Next, we removed the maxilla, gut, heart, liver, and kidney from the rats at 0, 2, 4, and 8 weeks postsurgery and then extracted DNA samples and performed polymerase chain reaction and microbiome analyses using the Illumina MiSeq (Illumina Co, Tokyo, Japan). RESULTS: The presence of inflammatory cell infiltration confirmed apical periodontitis from 2-8 weeks. Polymerase chain reaction and microbiome analyses revealed F. nucleatum in the rat gut from 2 weeks and in the kidney from 8 weeks. The rat gut, heart, liver, and kidney exhibited altered bacterial flora, including a marked decrease in Verrucomicrobia and an increase in Proteobacteria after 2 weeks and increases in Bacteroidetes and Firmicutes after 4 weeks. CONCLUSIONS: The onset of F. nucleatum-induced apical periodontitis changed the bacterial flora in the rat gut, heart, liver, and kidney, with a confirmed progressing infection in the large intestines.

Biosynthesized selenium nanoparticles: characterization, antimicrobial, and antibiofilm activity against Enterococcus faecalis.

BACKGROUND: Control over microbial growth is a crucial factor in determining the success of endodontic therapy. Enterococcus faecalis is the most resistant biofilm-forming species leading to endodontic failure. Hence, the current researches are directed towards discovering materials with superior disinfection properties and lesser cytotoxicity. This study aimed to synthesize and characterize biogenically produced Selenium Nanoparticles, and to evaluate the antimicrobial and antibiofilm efficacy, against Enterococcus Faecalis, for the following test groups: Group I: Distilled water (control), Group II: SeNPs (1 mg/ml), Group III: Calcium hydroxide (1 mg/ml), Group IV: 2% Chlorhexidine gluconate (CHX), Group V: 5.25% Sodium hypochlorite (NaOCl). MATERIALS AND METHODS: Selenium nanoparticles were derived using fresh guava leaves (Psidium guajava) and were characterized. The antibacterial efficacy against E. faecalis was evaluated by agar well diffusion method. The antibiofilm efficacy of the test groups was observed by viable cell count, antibiofilm assay, and Anthrone and Bradford's tests. The morphology of the biofilms was analysed using the Scanning Electron Microscope and Fourier Transform Infrared spectroscopy. RESULTS: Antibacterial and antibiofilm efficacy of all tested solutions showed superior antibacterial and antibiofilm efficacy when compared to the control group. Overall, SeNPs (Group II) was the most effective against E. faecalis biofilm, followed by NaOCl (Group V), CHX (Group IV), and Ca(OH)2 (Group III). CONCLUSION: Biogenically produced SeNPs emerged as a novel antibacterial and antibiofilm agent against E. faecalis. This nano-formulation demonstrates the potential to be developed as a root canal disinfectant combating bacterial biofilm in endodontics after the results have been clinically extrapolated.

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.

Bactericidal Effect of a Novel Alkaline EDTA Root Canal Cleaning Solution.

OBJECTIVES: In this study, we aimed to evaluate the bactericidal effect and cytotoxicity of an ethylenediaminetetra-acetic acid (EDTA)-based root canal irrigant solution capable of efficiently removing both the organic matter and the smear layer. We prepared a strong alkaline EDTA (AE) solution with an acid buffer capacity similar to that of sodium hypochlorite. MATERIALS AND METHODS: AE was used at concentrations of 1%, 2%, and 3%. The bactericidal effect of AE on Enterococcus faecalis was evaluated by determining the colony number and biofilm removal rate. Biofilms were visualized using a Live/Dead BacLight bacterial viability kit. Viability of AE-treated cells were determined using a CCK-8 cell counting assay. STATISTICAL ANALYSIS: One-way analysis of variance followed by a Dunnett's multiple comparison test were used for comparisons among groups. RESULTS: Significant reduction in cell viability and biofilm formation were observed in case of 3% and 2% AE. AE exerted bactericidal effects in a concentration-dependent manner. Damage of normal human fibroblasts was not observed at any of the AE concentrations. CONCLUSIONS: Our results suggest that the AE solution could be used as an effective canal irrigant for the removal of bacterial biofilm.

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.

Low-Intensity Pulsed Ultrasound Prevents Development of Bisphosphonate-Related Osteonecrosis of the Jaw-Like Pathophysiology in a Rat Model.

We developed a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ) by removing a maxillary molar tooth (M1) from ovariectomized rats after treatment with alendronate. To mimic periodontitis, some of the rats were administered Porphyromonas gingivalis (p. gingivalis) at the M1 site every 2 to 3 d for 2 wk. Rats pretreated with alendronate plus p. gingivalis showed delayed healing of socket epithelia, periosteal reaction of alveolar bone formation and lower bone mineral density in the alveolus above adjacent M2 teeth. These abnormalities were prevented by tooth socket exposure to 20 min/d low-intensity pulsed ultrasound (LIPUS), which restored diminished expression of RANKL, Bcl-2, IL-6, Hsp70, NF-κB and TNF-α messenger ribonucleic acids in remote bone marrow, suggesting LIPUS prevented development of BRONJ-like pathophysiology in rat by inducing systemic responses for regeneration, in addition to accelerating local healing. Non-invasive treatment by LIPUS, as well as low-level laser therapy, may be useful for medication-related osteonecrosis of the jaw patients.

Delayed diagnosis of odontogenic infection: a lesson from altered mental status and abnormal breathing.

Altered mental status is a common, yet challenging, clinical presentation encountered by physicians. Here, we report a case of a 68-year-old Japanese female who was transferred to the emergency department due to faint. The laboratory results showed hyponatremia, ketonuria, hyperglycemia and acute kidney injury without fever or inflammatory findings. Although these abnormalities were corrected, her mental status was exacerbated, and apnea/tachypnea appeared. She was eventually diagnosed with acute apical abscess and recovered immediately after dental extractions. This case suggests that odontogenic infection should be considered in the differential diagnosis of altered mental status and that interdisciplinary dental management, including surgical treatment, should be considered for patients with predisposing factors.

Cell Biological Assays for Measuring Odontogenic Activities of CCN Proteins.

In postnatal dentin formation, odontoblast differentiation occurs in the pulp tissue regenerative process under pathological condition. Odontoblasts and newly differentiated odontoblast-like cells beneath the caries lesion form tertiary dentin and are highly odontogenic. To observe the activity of dentinogenesis occur within the hard tissue, a combination of immunohistological analysis and immunodetection of dentinogenesis specific molecules, such as dentin sialophosphoprotein (DSPP) and/or its cleaved products dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), is a reliable approach. Besides, recent studies have revealed that the expression of CCN family member 2 (CCN2), a member of the CCN family protein, is confirmed in accordance with tooth development and reparative dentin formation. Therefore, CCN2 could serve as a marker for dentinogenesis. Here, we describe a method for visualizing the CCN2 signal as an odontogenic activity in formalin-fixed paraffin-embedded (FFPE) sections of demineralized human teeth and human dental pulp cells.

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.

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)-1ß, 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-1ß, 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.

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.