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Aggressive periodontitis (AgP), classified as Stages III or IV and grade C periodontitis, is characterized by the rapid destruction of periodontal tissue. Genetic factors contribute to the pathogenesis of this disease, and familial aggregation of periodontitis is often observed. However, the mechanisms underlying the onset or progression of AgP have not been elucidated. Previously, we performed exome sequencing and identified AgP risk factors in Japanese AgP-patients. However, the small sample size limited our scope for detecting some of the true AgP genetic risk factors. To overcome this limitation, we searched for AgP-related genes more comprehensively from the whole exome sequencing data of the Japanese AgP-patients by extending the filtering criteria range. We identified seven AgP-associated suggestive genes, including the single nucleotide polymorphism (SNP) rs854560 in paraoxonase-1 (PON-1), which is correlated with AgP. However, the mechanism(s) underlying the induction of AgP pathogenesis by the SNP rs854560 PON-1 has not been elucidated. Thus, we further analyzed the functions of the SNP rs854560 PON-1 in human periodontal ligament (HPDL) cells through transfection of the wild-type PON-1 (WT) or SNP rs854560 PON-1 (mut) into HPDL cells. Real-time PCR indicated that mut had higher mRNA expression of osteogenic related-genes and showed a higher tendency of ALP activity and proliferation. The result suggested that WT PON-1 contributes to periodontal tissue homeostasis through appropriate proliferation and cytodifferentiation of HPDL cells, while SNP rs854560 PON-1 may mediate excessive calcification of periodontal tissue due to hyper proliferation of HPDL cells, thereby increasing the risk of AgP.

Abstract Calcitonin gene-related peptide (CGRP), a neuropeptide composed of 37 amino acids secreted from the sensory nerve endings, reportedly possesses various physiological effects, such as vasodilation and neurotransmission. Recently, there have been increasing reports of the involvement of CGRP in bone metabolism; however, its specific role in the pathogenesis of periodontitis, particularly in the repair and healing processes, remains to be elucidated. Therefore, this study aimed to investigate dynamic expression patterns of CGRP during the destruction and regeneration processes of periodontal tissues in a mouse model of experimental periodontitis. We also explored the effects of CGRP on periodontal ligament cells, which can differentiate to hard tissue-forming cells (cementoblasts or osteoblasts). Our findings demonstrated that CGRP stimulation promotes the differentiation of periodontal ligament cells into hard tissue-forming cells. Experimental results using a ligature-induced periodontitis mouse model also suggested fluctuations in CGRP expression during periodontal tissue healing, underscoring the vital role of CGRP signaling in alveolar bone recovery. The study results highlight the important role of nerves in the periodontal ligament not only in sensory reception in the periphery, as previously known, but also in periodontal tissue homeostasis and tissue repair processes.

Aggressive periodontitis (AgP), Stage III or IV and Grade C according to the new periodontitis classification, is characterized by the rapid destruction of periodontal tissues in the systemically healthy population and often causes premature tooth loss. The presence of familial aggregation suggests the involvement of genetic factors in the pathogenesis. However, the genes associated with the onset and progression of the disease and details of its pathogenesis have not yet been fully identified. In recent years, the genome-wide approach (GWAS), a comprehensive genome analysis method using bioinformatics, has been used to search for disease-related genes, and the results have been applied in genomic medicine for various diseases, such as cancer. In this review, we discuss GWAS in the context of AgP. First, we introduce the relationship between single-nucleotide polymorphisms (SNPs) and susceptibility to diseases and how GWAS is useful for searching disease-related SNPs. Furthermore, we summarize the recent findings of disease-related genes using GWAS on AgP inside and outside Japan and a possible mechanism of the pathogenesis of AgP based on available literature and our research findings. These findings will lead to advancements in the prevention, prognosis, and treatment of AgP.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis. Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome

Introduction: There has been an increasing desire for the development of predictive periodontal regenerative therapy for severe periodontitis. In this study, we investigated the effect of the combined use of fibroblast growth factor-2 (FGF-2), a drug for periodontal regeneration approved in Japan, and carbonated apatite (CO3Ap), bioresorbable and osteoconductive scaffold, on periodontal regeneration in beagle dog model of one-wall periodontal defect (severe intraosseous defect) for 24 weeks in comparison with CO3Ap or vehicle alone. Methods: One-wall periodontal defects were created (mesiodistal width × depth: 4 × 4 mm) on the mesial portion of the mandibular first molar (M1) of beagle dogs on both side. Mixture of FGF-2 and CO3Ap, vehicle and CO3Ap, or vehicle alone were administered to the defects and designated as groups FGF-2+CO3Ap, CO3Ap, and control, respectively. To assess the periodontal regeneration, radiographic analysis over time for 24 weeks, and micro computed tomography (μCT) and histological evaluation at 6 and 24 weeks were performed. Results: For the regenerated tissue in the defect site, the mineral content of the FGF-2+CO3Ap group was higher than that of the CO3Ap group in the radiographic analysis at 6–24 weeks. In the context of new bone formation and replacement, the FGF-2+CO3Ap group exhibited significantly greater new bone volume and smaller CO3Ap volume than the CO3Ap group in the μCT analysis at 6 and 24 weeks. Furthermore, the density of the new bone in the FGF-2+CO3Ap group at 24 weeks was similar to those in the control and CO3Ap groups. Histological evaluation revealed that the length of the new periodontal ligament and cementum in the FGF-2+CO3Ap group was greater than that in the CO3Ap group at 6 weeks. We also examined the effect of the combined use of the FGF-2 and CO3Ap on the existing bone adjacent to the defect and demonstrated that the existing bone height and volume in the FGF-2+CO3Ap group remained significantly greater than those in the CO3Ap group. Conclusion: This study demonstrated that the combination of FGF-2 and CO3Ap was effective not only in enhancing new bone formation and replacing scaffold but also in maintaining the existing bone adjacent to the defect site in a beagle dog model of one-wall periodontal defect. Additionally, new periodontal tissues induced by FGF-2 and CO3Ap may follow a maturation process similar to that formed by spontaneous healing. This suggests that the combined use of FGF-2 and CO3Ap would promote periodontal regeneration in severe bony defects of periodontitis patient.

Melanins are the main pigments found in mammals. Their synthesis and transfer to keratinocytes have been widely investigated for many years. However, analysis has been mainly carried out using fixed rather than live cells. In this study, we have analysed the melanosomes in living mammalian cells using newly developed scanning electron-assisted dielectric microscopy (SE-ADM). The melanosomes in human melanoma MNT-1 cells were observed as clear black particles in SE-ADM. The main structure of melanosomes was toroidal while that of normal melanocytes was ellipsoidal. In tyrosinase knockout MNT-1 cells, not only the black particles in the SE-ADM images but also the Raman shift of melanin peaks completely disappeared suggesting that the black particles were really melanosomes. We developed a deep neural network (DNN) system to automatically detect melanosomes in cells and analysed their diameter and roundness. In terms of melanosome morphology, the diameter of melanosomes in melanoma cells did not change while that in normal melanocytes increased during culture. The established DNN analysis system with SE-ADM can be used for other particles, e.g. exosomes, lysosomes, and other biological particles.

Introduction: Currently, flap operation (FOP) using REGROTH® (0.3% basic fibroblast growth factor [FGF-2]) is the standard treatment for periodontal regenerative therapy in Japan. However, the periodontal tissue regenerative effect with REGROTH® monotherapy is inadequate for severe alveolar bone defects. Therefore, in this study, we evaluated the safety and effectiveness of periodontal regenerative therapy for patients with severe periodontitis using REGROTH® (test medicine) combined with Cytrans® Granules (test device: carbonated apatite granules), which is a new artificial bone. Methods: The study participants included 10 patients with severe periodontitis (mean age: 47.4 years). All participants provided written informed consents. In each patient, the intrabony defect site (mean bone defect depth: 5.7 mm) was defined as the test site. FOP was performed for the test site after the baseline investigation; moreover, the test medicine and test device were administered simultaneously. Furthermore, the observation of subjects’ general condition and test sites was conducted and the blood, urine, and periodontal tissue tests were performed up to 36 weeks after FOP. The rate of bone increase (%), clinical attachment level (CAL), probing pocket depth (PPD), bleeding on probing (BOP), tooth mobility (Mo), width of keratinized gingiva (KG), gingival recession (REC), gingival index (GI), and plaque index (PlI) were evaluated during the periodontal tissue investigation. Results: As the primary endpoint, no adverse events related to the test medicine and test device occurred during the entire observation period of this study. Regarding the secondary endpoints, there was a significant increase in new alveolar bone (p = 0.003) and CAL acquisition (p = 0.001) as well as decrease in PPD (p = 0.002) and BOP (p = 0.016) at 36 weeks after administration of the test medicine and test device compared with the preoperative values. Furthermore, at 36 weeks after surgery, the Mo, GI, and PlI decreased to preoperative levels at 40%, 60%, and 30% of sites, respectively. However, at 36 weeks after surgery, there was no difference in KG and REC compared with their preoperative values. Conclusions: The safety of periodontal regenerative therapy using the test medicine in combination with the abovementioned test device was confirmed. In addition, it was suggested that this periodontal regenerative therapy is effective for tissue regeneration in severe alveolar bone defects.This clinical trial was conducted after registering and publicizing as a specified clinical trial in the Japan registry of clinical trials (jRCTs051190045).

Introduction: Adipose-derived stem cells (ASCs) secrete various growth factors to promote wound healing and to regenerate various tissues, such as bone, cartilage, and fat tissue. Subcutaneous adipose tissue is a considerable cell source in clinical practice and can be collected relatively easily and safely under local anesthesia. Moreover, platelet-rich plasma (PRP), a plasma component containing many platelets purified by centrifuging the collected blood, also promotes wound healing. PRP can be easily gelled and is therefore attracting attention as a scaffolding material for transplanted cells. The usefulness of a mixture of ASCs and PRP for periodontal tissue regeneration has been in vitro demonstrated in our previous study. The aim of this study is to present the protocol of translation of tissue regeneration with ASCs and PRP into practical use, evaluating its efficacy. Methods: This study is a multicenter, randomized, open-label comparative clinical trial. Fifteen patients will be randomly assigned to the treatment with mixture of ASCs and PRP or enamel matrix derivate administration into periodontal tissue defects. Increase in height of new alveolar bone in the transplanted area will be evaluated. The evaluation will be performed using dental radiographs after 36 weeks of transplantation. Occurrence of adverse events will be evaluated as secondary outcome. Results: This clinical study was initiated after meeting the regulations to be complied with, including ethical review and regulatory notifications. Conclusions: If effective, this cell therapy using autologous mesenchymal stem cells can represent a useful medical technology for regeneration of periodontal defects.

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Periodontal tissue stem cells, which play a crucial role in maintaining the homeostasis of periodontal tissues, are found in the periodontal ligament (PDL). These cells have long been referred to as mesenchymal stem/stromal cells (MSCs), and their clinical applications have been extensively studied. However, tissue stem cells in the PDL have not been thoroughly investigated, and they may be different from MSCs. Recent advances in stem cell biology, such as genetic lineage tracing, identification of label-retaining cells, and single-cell transcriptome analysis, have made it possible to analyze tissue stem cells in the PDL in vivo. In this review, we summarize recent findings on these stem cell populations in PDL and discuss future research directions toward developing periodontal regenerative therapy.

Abstract The new 2018 classification of periodontal diseases is reported to be related to tooth loss due to periodontal disease (TLPD) during supportive periodontal therapy (SPT). However, few reports have evaluated this relationship for Asians or have analyzed the association of the new classification and TLPD by distinguishing between active periodontal therapy (APT) and SPT. In this study, we retrospectively applied the new classification to 607 Japanese periodontitis patients and examined the relationship between the new classification and annual TLPD rates per patient during the respective periods. TLPD rates were higher in patients in stage IV and/or grade C during both APT and SPT. TLPD during SPT was not associated with the presence or absence of TLPD during APT. Multivariate analysis revealed that stage IV and grade C as independent variables were significantly associated with the number of instances of TLPD not only during the total treatment period, but also during APT or SPT. Our results suggest that the new classification has a significantly strong association with TLPD during both APT and SPT, and that patients diagnosed with stage IV and/or grade C periodontitis had a higher risk of TLPD during both periods.

Abstract Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Abstract Background This cross-sectional study performed to clarify the relationship between periodontal disease and non-communicable diseases (NCDs), such as obesity, diabetes mellitus, impaired glucose tolerance (IGT), chronic obstructive pulmonary disease (COPD), and atherosclerotic cardiovascular disease (ASCVD) by introducing dental examinations into the annual health examinations conducted by Japanese companies, and to highlights the importance of a medical system that connects dental and medical professionals. Methods A total of 1.022 Hitachi Ltd. employees were enrolled in this cross-sectional study. We examined correlations and odds ratios (ORs) between the dental and overall health of employees using stratification and multiple logistic regression analyses based on the periodontal health indicators, general health indicators, and occlusal force. Results The adjusted OR of PPD for obesity (OR, 1.42; 95% confidence interval [CI], 1.09–1.84; p = 0.009), IGT (OR, 1.48; 95% CI, 1.00–2.20; p = 0.049), and COPD (OR, 1.38; 95% CI, 1.02–1.88; p = 0.038) significantly differed. The adjusted OR of body mass index (OR, 1.28; 95% CI 1.15–1.42; p

Abstract Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P

Abstract Periodontal disease is a chronic inflammatory condition that affects various peripheral organs. The periodontal inflamed surface area (PISA) quantifies periodontitis severity and the spread of inflammatory wounds. This study aimed to investigate the association between PISA and high-sensitivity C-reactive protein (hs-CRP), a systemic inflammation marker. This study included 250 community-dwelling septuagenarians (69–71 years). We collected information on their medical (e.g., diabetes and dyslipidemia) and dental examinations (e.g., measurement of the probing pocket depth). Generalized linear model analysis was used to explore the association between PISA and hs-CRP levels. There was a significant difference in hs-CRP levels between groups with PISA ≥ 500 and

Abstract Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Abstract Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.

Abstract PM2.5 has been correlated with risk factors for various diseases and infections. It promotes tissue injury by direct effects of particle components. However, effects of PM2.5 on cells have not been fully investigated. Recently, we developed a novel imaging technology, scanning electron-assisted dielectric-impedance microscopy (SE-ADM), which enables observation of various biological specimens in aqueous solution. In this study, we successfully observed PM2.5 incorporated into living mammalian cells in culture media. Our system directly revealed the process of PM2.5 aggregation in the cells at a nanometre resolution. Further, we found that the PM2.5 aggregates in the intact cells were surrounded by intracellular membrane-like structures of low-density in the SE-ADM images. Moreover, the PM2.5 aggregates were shown by confocal Raman microscopy to be located inside the cells rather than on the cell surface. We expect our method to be applicable to the observation of various nanoparticles inside cells in culture media.

Abstract Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

Energy metabolism is crucial in stem cells as they harbor various metabolic pathways depending on their developmental stages. Moreover, understanding the control of their self-renewal or differentiation via manipulation of their metabolic state may yield novel regenerative therapies. Periodontal ligament (PDL) cells existing between the tooth and alveolar bone are crucial for maintaining homeostasis in the periodontal tissue. In addition, they play a pivotal role in periodontal regeneration, as they possess the properties of mesenchymal stem cells and are capable of differentiating into osteogenic cells. Despite these abilities, the treatment outcome of periodontal regenerative therapy remains unpredictable because the biological aspects of PDL cells and the mechanisms of their differentiation remain unclear. Recent studies have revealed that metabolism and factors affecting metabolic pathways are involved in the differentiation of PDL cells. Furthermore, understanding the metabolic profile of PDL cells could be crucial in manipulating the differentiation of PDL cells. In this review, first we discuss the energy metabolism in osteoblasts and stem cells to understand the metabolism of PDL cells. Next, we summarize the metabolic preferences of PDL cells during their maintenance and cytodifferentiation. The perspectives discussed have potential applicability for creating a platform for reliable regenerative therapies for periodontal tissue.

Loeys–Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-β receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-β and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-β compared with WT MEFs. To clarify the influence of TGF-β signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-β-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-β signal dysfunction. This suggests that TGF-β signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

Periodontal disease, the most prevalent infectious disease in the world, is caused by biofilms formed in periodontal pockets. No specific bacterial species that can cause periodontitis alone has been found in any study to date. Several periodontopathic bacteria are associated with the progress of periodontal disease. Consequently, it is hypothesized that dysbiosis of subgingival microbiota may be a cause of periodontal disease. This study aimed to investigate the relationship between the subgingival microbiota and the clinical status of periodontal pockets in a quantitative and clinically applicable way with the newly developed Oral Care Chip. The Oral Care Chip is a DNA microarray tool with improved quantitative performance, that can be used in combination with competitive PCR to quantitatively detect 17 species of subgingival bacteria. Cluster analysis based on the similarity of each bacterial quantity was performed on 204 subgingival plaque samples collected from periodontitis patients and healthy volunteers. A significant difference in the number of total bacteria, Treponema denticola, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus intermedia bacteria in any combination of the three clusters indicated that these bacteria gradually increased in number from the stage before the pocket depth deepened. Conversely, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Streptococcus constellatus, which had significant differences only in limited clusters, were thought to increase in number as the pocket depth deepened, after periodontal pocket formation. Furthermore, in clusters where healthy or mild periodontal disease sites were classified, there was no statistically significant difference in pocket depth, but the number of bacteria gradually increased from the stage before the pocket depth increased. This means that quantitative changes in these bacteria can be a predictor of the progress of periodontal tissue destruction, and this novel microbiological test using the Oral Care Chip could be effective at detecting dysbiosis.

Growing evidence suggests that oral health may be an important factor associated with cognitive function in aged populations. However, many previous studies on this topic used insensitive oral indicators or did not include certain essential covariates. Thus, we examined the association between occlusal force and cognitive function in a large sample of older adults, controlling for dietary intake, vascular risk factors, inflammatory biomarkers, depression, and genetic factors.In this cross-sectional study of older community-dwelling Japanese adults, we examined data collected from 994 persons aged 70 years and 968 persons aged 80 years. Cognitive function was measured using the Japanese version of the Montreal Cognitive Assessment (MoCA-J). Oral status and function were evaluated according to the number of remaining teeth, periodontal pocket depth, and maximal occlusal force. Associations between MoCA-J scores and occlusal force were investigated via bivariate and multivariate analyses.Education level, financial status, depression score, and intake of green and yellow vegetables, as well as number of teeth and occlusal force, were significantly correlated with MoCA-J scores in both age groups. Among individuals aged 80 years, CRP and periodontal status were weakly but significantly associated with MoCA-J score. After controlling for all significant variables via bivariate analyses, the correlation between maximal occlusal force and cognitive function persisted. A path analysis confirmed the hypothesis that cognitive function is associated with occlusal force directly as well as indirectly via food intake.After controlling for possible factors, maximal occlusal force was positively associated with cognitive function directly as well as indirectly through dietary intake.

Whereas most of studies investigating relationship between oral health and atherosclerosis have focused on periodontitis, very few of them were examined about occlusal status of natural teeth which possibly influence dietary habit. The primary aim of this cross-sectional study was to investigate the association between the occlusal support of posterior teeth and the prevalence of atherosclerosis in community-dwelling septuagenarians. Also, the second aim was to test the hypothesis that the intake of key nutrients for atherosclerosis prevention would have a mediating effect on the relationship between the occlusal status and atherosclerosis. The study population included 468 community-dwelling dentate persons aged 69-71 years recruited from the local residential registration in Japan. Participants were divided into three groups, according to the number of occlusal support zones (OSZ) in the posterior area: Complete (four OSZ), Moderate (three or two OSZ), and Collapsed (one or no OSZ). Dietary intakes were assessed using a brief-type self-administered diet history questionnaire. Atherosclerosis was defined as carotid intima-media thickness ≧1.10 mm by using carotid ultrasonography test. The logistic or linear regression model was used in multivariate analysis to assess relationship between occlusal status and atherosclerosis, and the mediating effect of key nutrients within the relationship. Multivariable analysis showed a significant association between occlusal status and atherosclerosis (odds ratio for Collapsed group to Complete group: 1.87; 95% CI: 1.45-2.41), independent of periodontal status (odds ratio: 2.01, 95%CI: 1.46-2.78). Fish and shellfish, vitamin B6 and n-3PUFAs were significantly related to both of occlusal status and atherosclerosis, and also was indicated a mediating effect on the association between occlusal status and atherosclerosis. This study implied that, within the limitation of the cross-sectional study design, the reduced posterior occlusion was related to the increased prevalence of atherosclerosis via the decline of key dietary intakes among Japanese community-dwelling dentate individuals.

Periodontal ligament (PDL) plays crucial roles in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In attempt to understand the molecular and genetic basis of PDL functions, we investigated the expression profile of active genes in human periodontal ligament obtained by collecting sequences with 3′-directed cDNA library, which faithfully represents composition of the mRNA population. We succeeded to obtain a total of 1752 cDNA sequences by sequencing randomly selected clones and a total of 1318 different species was identified as gene signatures (GS) by their sequence identity. The resulting expression profile showed that collagen types I and III were the most abundant genes and osteogenesis-relating genes, such as osteonectin and periostin were highly expressed. In the gene expression profile of human PDL, we found a novel gene which was highly expressed in PDL, but not in other tissue-cDNA libraries. We cloned a full-length cDNA of the gene and identified that it codes a novel protein, which is a new member of class I of small leucine-rich repeat proteoglycan (SLRP) family. We designated it periodontal ligament associated protein-1 (PLAP-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. In situ mRNA hybridization analysis using mouse periodontium revealed that PLAP-1 was expressed only in PDL tissues. Over-expression of PLAP-1 in PDL-derived clone cells interfered with both naturally and bone morphogenetic protein 2 (BMP-2)-induced mineralization of the PDL cells. On the other hand, knockdown of PLAP-1 transcript levels by RNA interference enhanced BMP-2-induced differentiation of PDL cells. Furthermore, co-immunoprecipitation assays showed a direct interaction between PLAP-1 and BMP-2 in vitro. These results suggest that PLAP-1 plays a specific role(s) in the periodontal ligament as a negative regulator of cytodifferentiation and mineralization, probably by regulating BMP-2 activity to prevent the periodontal ligament from developing non-physiological mineralization, such as ankylosis.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

Cigarette smoking is a major lifestyle-related risk factor for periodontal diseases. However, the pathophysiological role of cigarette smoking in periodontal disease has yet to be fully elucidated. Here we report that the systemic administration of cigarette smoke condensate or nicotine, which is the major ingredient of cigarette smoke, augmented alveolar bone loss. Concomitantly, the number of osteoclasts in periodontal tissues increased and the expression of receptor activator of nuclear factor κB ligand was upregulated at the ligated side in mice with periodontitis. Nicotine also attenuated alveolar bone repair after ligature removal. These observations highlight the destruction of periodontal tissue by smoking and the unfavorable clinical course of periodontal disease in patients with a cigarette smoking habit. The present study demonstrates that periodontal disease models are useful for elucidating the pathogenesis of cigarette smoking-related periodontal diseases.

The long-term stability and qualitative characteristics of periodontium regenerated by FGF-2 treatment were compared with normal physiological healing tissue controls in a Beagle dog 2-wall periodontal defect model 13 months after treatment by assessing tissue histology and three-dimensional microstructure using micro-computed tomography (μCT). After FGF-2 (0.3%) or vehicle treatment at the defect sites, serial changes in the bone mineral content (BMC) were observed using periodic X-ray imaging. Tissues were harvested at 13 months, evaluated histomorphometrically, and the cortical bone volume and trabecular bone structure of the newly formed bone were analyzed using μCT. FGF-2 significantly increased the BMC of the defect area at 2 months compared with that of the control group, and this difference was unchanged through 13 months. The cortical bone volume was significantly increased by FGF-2, but there was no difference between the groups in trabecular bone structure. Bone maturation was occurring in both groups because of the lower cortical volume and denser trabecular bone than what is found in intact bone. FGF-2 also increased the area of newly formed bone as assessed histomorphometrically, but the ratios of trabecular bone in the defect area were similar between the control and FGF-2 groups. These results suggest that FGF-2 stimulates neogenesis of alveolar bone that is of similar quality to that of the control group. The lengths of the regenerated periodontal ligament and cementum, measured as the distance from the defect bottom to the apical end of the gingival epithelium, and height and area of the newly formed bone in the FGF-2 group were larger than those in the control group. The present study demonstrated that, within the limitation of artificial periodontal defect model, the periodontal tissue regenerated by FGF-2 was maintained for 13 months after treatment and was qualitatively equivalent to that generated through the physiological healing process.

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.

The periodontal ligament has a rich sensory nerve supply which originates from the trigeminal ganglion and trigeminal mesencephalic nucleus. Although various types of mechanoreceptors have been reported in the periodontal ligament, the Ruffini ending is an essential one. It is unknown whether the distribution of periodontal nerve fibers in deciduous teeth is identical to that in permanent teeth or not. Moreover, morphological changes in the distribution of periodontal nerve fibers during resorption of deciduous teeth and eruption of successional permanent teeth in diphyodont animals have not been reported in detail. Therefore, in this study, we examined changes in the distribution of periodontal nerve fibers in the cat during changes in dentition (i.e., deciduous, mixed and permanent dentition) by immunohistochemistry of protein gene product 9.5. During deciduous dentition, periodontal nerve fibers were concentrated at the apical portion, and sparsely distributed in the periodontal ligament of deciduous molars. During mixed dentition, the periodontal nerve fibers of deciduous molars showed degenerative profiles during resorption. In permanent dentition, the periodontal nerve fibers of permanent premolars, the successors of deciduous molars, increased in number. Similar to permanent premolars, the periodontal nerve fibers of permanent molars, having no predecessors, increased in number, and were densely present in the apical portion. The present results indicate that the distribution of periodontal nerve fibers in deciduous dentition is almost identical to that in permanent dentition although the number of periodontal nerve fibers in deciduous dentition was low. The sparse distribution of periodontal nerve fibers in deciduous dentition agrees with clinical evidence that children are less sensitive to tooth stimulation than adults.

Although researchers have recently demonstrated a relationship between oral health and arterial sclerosis, the genetic contribution to this relationship has been ignored even though genetic factors are expected to have some effect on various diseases. The aim of this study was to evaluate oral health as a significant risk factor related to arterial sclerosis after eliminating genetic confounding through study of older Japanese twins.Medical and dental surveys were conducted individually for 106 Japanese twin pairs over the age of 50 years. Maximal carotid intima-media thickness (IMT-Cmax) was measured as a surrogate marker of arterial sclerosis. IMT-Cmax > 1.0 mm was diagnosed as arterial sclerosis. All of the twins were examined for the number of remaining teeth, masticatory performance, and periodontal status. We evaluated each measurement related with IMT-Cmax and arterial sclerosis using generalized estimating equations analysis adjusted for potential risk factors. For non-smoking monozygotic twins, a regression analysis using a "between within" model was conducted to evaluate the relationship between IMT-Cmax and the number of teeth as the environmental factor controlling genetic and familial confounding.We examined 91 monozygotic and 15 dizygotic twin pairs (males: 42, females: 64) with a mean (± standard deviation) age of 67.4 ± 10.0 years. Out of all of the oral health-related measurements collected, only the number of teeth was significantly related to arterial sclerosis (odds ratio: 0.72, 95% confidence interval: 0.52-0.99 per five teeth). Regression analysis showed a significant association between the IMT-Cmax and the number of teeth as an environmental factor (p = 0.037).Analysis of monozygotic twins older than 50 years of age showed that having fewer teeth could be a significant environmental factor related to arterial sclerosis, even after controlling for genetic and familial confounding.

Transforming growth factor beta (TGF-β) is a multi-functional growth factor expressed in many tissues and organs. Genetic animal models have revealed the critical functions of TGF-β in craniofacial development, including the teeth and periodontal tissue. However, the physiological function of TGF-β in the periodontal ligament (PDL) has not been fully elucidated. In this study, we examined the roles of TGF-β in the cytodifferentiation of PDL cells using a TGF-β receptor kinase inhibitor, SB431542. Mouse PDL cell clones (MPDL22) were cultured in calcification-inducing medium with or without SB431542 in the presence or absence of various growth factors, such as bone morphogenetic protein (BMP)-2, TGF-β and fibroblast growth factor (FGF)-2. SB431542 dramatically enhanced the BMP-2-dependent calcification of MPDL22 cells and accelerated the expression of ossification genes alkaline phosphatase (ALPase) and Runt-related transcription factor (Runx) 2 during early osteoblastic differentiation. SB431542 did not promote MPDL22 calcification without BMP-2 stimulation. The cell growth rate and collagen synthesis during the late stage of MPDL22 culture were retarded by SB431542. Quantitative reverse transcription polymerase chain reaction analysis revealed that the expressions of Smurf1 and Smad6, which are negative feedback components in the TGF-β/BMP signaling pathway, were downregulated in MPDL22 cells with SB431542 treatment. These results suggest that an endogenous signal from TGF-β negatively regulates the early commitment and cytodifferentiation of PDL cells into hard tissue-forming cells. A synthetic drug that regulates endogenous TGF-β signals may be efficacious for developing periodontal regenerative therapies.

Suppression of antigen-induced late airway obstruction associated with neutrophilic inflammation by selective and non-selective phosphodiesterase (PDE) inhibitors was investigated in mice. Respiratory resistance (Rrs) increased in sensitized BDF1 mice 4-6 h after antigen provocation, whereas no obvious immediate reaction was observed. This reaction was associated with marked airway neutrophilia without significant infiltration of eosinophils. A selective PDE IV inhibitor, T-440 (10-30 mg/kg), and a non-selective PDE inhibitor, theophylline (10 mg/kg), significantly inhibited airway obstruction and neutrophilia when administered orally. An anti-allergic drug, ketotifen (1 mg/kg), caused slight inhibition of airway obstruction, whereas it did not affect airway neutrophilia. These results suggest that neutrophilic inflammation plays a role in the airway obstructive reaction and that PDE has a regulatory role in obstructive airway disease associated with airway inflammation.

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation.We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured > or = 3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 microL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified.Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis.ClinicalTrials.gov NCT00514657.