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A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn−/- mice. The results showed that osteoblasts from Optn−/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1. • OPTN was identified as an important regulator of bone metabolism. • Impaired dynamic bone formation indices in Optn-/- mice indicated the role of OPTN in osteogenesis. • OPTN regulated the nuclear localization of RUNX2 by modulating the STAT1 level in osteoblasts differentiation. [ABSTRACT FROM AUTHOR]

Aim: Some patients suffering from aggressive periodontitis (Ag-P) also display neutrophil chemotaxis dysfunction. In this study, we attempted to identify the proteins involved in Ag-P associated with neutrophil chemotaxis dysfunction using proteome analysis. Material and Methods: A two-dimensional fluorescence difference gel electrophoresis system was used to detect differences in protein expression between neutrophils from four patients suffering from Ag-P combined with neutrophil chemotaxis dysfunction and those from four controls. Moreover, the mRNA levels of the proteins identified by the above method were examined in neutrophils from four types of subjects using the real-time polymerase chain reaction: twenty patients suffering from Ag-P with or without the dysfunction, 15 patients with chronic periodontitis, and 15 controls. Results: Four proteins, lactoferrin, caldesmon, heat shock protein 70, and stac, displayed a higher protein expression level in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction than in those from the control group. The caldesmon mRNA levels in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction were high compared with those in the neutrophils from the patients suffering from the other two types of periodontitis and those from the control group. Conclusion: Caldesmon may be a marker of Ag-P combined with neutrophil chemotaxis dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation. [Copyright &y& Elsevier]

Abstract: The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC. [Copyright &y& Elsevier]

Abstract: The cells of epithelial rests of Malassez (ERM) are thought to play a number of roles, such as protection against root resorption and cementoblast differentiation. However, little is known about characteristics of these cells. In the present study, we compared the expression patterns of the bone-related proteins osteopontin (OPN), osteonectin/secreted protein, acidic and rich in cysteine (SPARC), osteoprotegerin (OPG), bone morphogenetic proteins (BMP-2 and BMP-4) and the enamel matrix proteins amelogenin and tuftelin in epithelial cells derived from human periodontal ligament (ECHPL) with those of human gingival epithelial cells (HGEC) and oral mesenchymal cells (human gingival fibroblasts, human periodontal ligament fibroblasts and human pulp cells). The mRNA expression patterns were determined by the reverse-transcription polymerase chain reaction (RT-PCR). Cytokeratin 8 mRNA was expressed by oral epithelial cells but not oral mesenchymal cells. The OPN mRNA levels in ECHPL were by far the highest in the five cell types investigated. ECHPL and oral mesenchymal cells expressed OPG mRNA, whereas HGEC did not. BMP-2, SPARC and tuftelin mRNAs were detected in ECHPL and the other cells examined. The oral mesenchymal cells expressed BMP-4 mRNA much more strongly than did the oral epithelial cells. Amelogenin mRNA expression could not be detected in any of the cells. These findings suggest that cultured ERM cells are characterized by expression of the cytokeratin 8, OPG and OPN genes. [Copyright &y& Elsevier]

Objective: Both periodontal disease and obesity are risk factors for dementia, but their links to 1brain function remain unclear. In this study, we examined the effects of oral infection with a periodontal pathogen on cognitive function in a mouse model of obesity, focusing on the roles of microglia. Methods: To create a mouse model of diet-induced obesity and periodontitis, male C57BL/6 J mice were first fed a high-fat diet containing 60% lipid calories for 18 weeks, beginning at 12 weeks of age, to achieve diet-induced obesity. Then, Porphyromonas gingivalis administration in the oral cavity twice weekly for 6 weeks was performed to induce periodontitis in obese mice. Results: Obese mice orally exposed to P. gingivalis showed cognitive impairment in the novel object recognition test. Increased expression levels of inflammatory cytokines (e.g. interleukin-1β and tumor necrosis factor-α) were observed in the hippocampus of P. gingivalis-treated obese mice. Immunohistochemical analysis revealed that microglia cell body size was increased in the hippocampus and prefrontal cortex of P. gingivalis-treated obese mice, indicating microglial activation. Furthermore, depletion of microglia by PLX3397, a colony-stimulating factor 1 receptor inhibitor, ameliorated cognitive dysfunction. Conclusion: These results suggest that microglia mediate periodontal infection-induced cognitive dysfunction in obesity. [ABSTRACT FROM AUTHOR]

Drug‐induced gingival enlargement (DIGE) is a side effect of ciclosporin, calcium channel blockers, and phenytoin. DIGE is a serious disease that leads to masticatory and esthetic disorders, severe caries, and periodontitis but currently has no standard treatment. We recently reported that nuclear receptor 4A1 (NR4A1) is a potential therapeutic target for DIGE. This study aimed to evaluate the therapeutic effects of n‐butylidenephthalide (BP), which increases the expression of NR4A1, on DIGE. In this study, NR4A1 mRNA expression was analyzed in the patients with periodontal disease (PD) and DIGE. We evaluated the effect of BP on NR4A1 expression in gingival fibroblasts and in a DIGE mouse model. RNA sequencing (RNA‐seq) was conducted to identify the mechanisms by which BP increases NR4A1 expression. The results showed that NR4A1 mRNA expression in the patients with DIGE was significantly lower than the patients with PD. BP suppressed the upregulation of COL1A1 expression, which was upregulated by TGF‐β. BP also ameliorated gingival overgrowth in DIGE mice and reduced Col1a1 and Pai1 expression. BP also decreased Il1β mRNA expression in gingival tissue in DIGE. RNA‐seq results showed an increase in the expression of several genes related to mitogen‐activated protein kinase including DUSP genes in gingival fibroblasts stimulated by BP. Treatment with ERK and JNK inhibitors suppressed the BP‐induced increase in NR4A1 expression. In addition, BP promoted the phosphorylation of ERK in gingival fibroblasts. In conclusion, BP increases NR4A1 expression in gingival fibroblasts through ERK and JNK signaling, demonstrating its potential as a preventive and therapeutic agent against DIGE. [ABSTRACT FROM AUTHOR]

Objective: To investigate the production of leucine‐rich α‐2‐glycoprotein‐1 (LRG1) in periodontitis patients and its effectiveness as a new diagnostic marker for periodontitis. Subjects and Methods: In vitro experiments were conducted to analyze LRG1 mRNA expression in human gingival epithelial cells and fibroblasts via quantitative real‐time PCR. In vivo experiments were conducted to analyze LRG1 localization in periodontitis patients. The correlation between the serum LRG1 levels and alveolar bone resorption in the mouse periodontitis model was also investigated. Results: A positive correlation existed between the periodontal inflamed surface area and serum LRG1 levels (Spearman's rank correlation coefficient: 0.60). LRG1 mRNA expression in human gingival epithelial cells and fibroblasts was upregulated by Porphyromonas gingivalis stimulation or tumor necrosis factor‐α stimulation. Interleukin‐6 in human gingival epithelial cells and fibroblasts induced the production of LRG1 and transforming growth factor‐β. LRG1 levels in the periodontal tissue and serum in the periodontitis model were higher than those in control mice. LRG1 local administration resulted in alveolar bone resorption, whereas the administration of interleukin‐6R antibody inhibited bone resorption. Conclusions: LRG1 levels in serum and periodontal tissue are upregulated in periodontitis and are implicated in periodontal tissue destruction through interleukin‐6 production. [ABSTRACT FROM AUTHOR]

Introduction: Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation.Materials and Methods: Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers.Results: Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis.Conclusions: Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have gained significant attention in cell therapies due to their multipotency and immunomodulatory capacities. The transcriptional co-activators YAP/TAZ, central to the mechanotransduction system in MSCs, dominantly direct MSCs lineage commitment. However, their role in immunomodulation remains elusive. Accordingly, this present study aimed to investigate the role of mechanotransducer YAP/TAZ and their binding target transcriptional factor, TEAD, in the immunomodulatory capacities of human bone marrow-derived MSCs. Reducing YAP/TAZ activity by altering the matrix stiffness, disrupting the F-actin integrity with chemical inhibitors, or using siRNAs increased the expression of immunomodulatory genes, such as TSG-6 and IDO, upon TNF-α stimulation. Similarly, transfection of TEAD siRNA also increased the immunomodulatory capacities in MSCs. RNA-seq analysis and inhibition assays demonstrated that the immunomodulatory capacities caused by YAP/TAZ-TEAD axis disruption were due to the NF-κB signaling pathway activation. Then, we also evaluated the in vivo anti-inflammatory efficacy of MSCs in a dextran sulfate sodium (DSS)-induced mice colitis model. The administration of human MSCs transfected with TEAD siRNA, which exhibited enhanced immunomodulatory properties in vitro, significantly ameliorated inflammatory bowel disease symptoms, such as body weight loss and acute colon inflammation, in the DSS-induced mice colitis model. Our findings underscore the mechanosignaling YAP/TAZ-TEAD axis as a regulator of MSCs immunomodulation. Targeting these signaling pathways could herald promising MSCs-based therapies for immune disorders. [ABSTRACT FROM AUTHOR]

Objectives The variable region of most ACPA IgG molecules in the serum of RA patients carries N -glycan (N -glycanV). To analyse the pathogenicity of N -glycanV of ACPAs, we analysed the pathogenicity of a monoclonal ACPA, CCP-Ab1, with or without N -glycanV, which had been isolated from a patient with RA. Methods CCP-Ab1 with no N -glycosylation site in the variable region (CCP-Ab1 N-rev) was generated, and antigen binding, the effect on in vitro differentiation of osteoclasts from bone marrow mononuclear cells of autoimmune arthritis–prone SKG mice (the cell size of TRAP+ cells and bone resorption capacity) and the in vivo effect on the onset or exacerbation of autoimmune arthritis in SKG mice were evaluated in comparison with glycosylated CCP-Ab1. Results Amino acid residues in citrullinated peptide (cfc1), which are essential for binding to CCP-Ab1 N-rev and original CCP-Ab1, were almost identical. The size of TRAP+ cells was significantly larger and osteoclast bone resorption capacity was enhanced in the presence of CCP-Ab1, but not with CCP-Ab1 N-rev. This enhancing activity required the sialic acid of the N -glycan and Fc region of CCP-Ab1. CCP-Ab1, but not CCP-Ab1 N-rev, induced the exacerbation of experimental arthritis in the SKG mouse model. Conclusions These data showed that N -glycanV was required for promoting osteoclast differentiation and bone resorption activity in both in vitro and in vivo assays. The present study demonstrated the important role of N -glycanV in the exacerbation of experimental arthritis by ACPAs. [ABSTRACT FROM AUTHOR]

Aim: To retrospectively investigate the relationship between the CD4+ T‐cell counts at baseline and the efficacy of the initial periodontal treatment of patients undergoing treatment for human immunodeficiency virus (HIV) infection using the periodontal inflamed surface area (PISA). Materials and Methods: Thirty‐three patients with chronic periodontitis who had undergone periodontal examination at baseline and after the initial periodontal treatment were enrolled. PISA was calculated from the periodontal probing depth and bleeding on probing, and the ratio of PISA after treatment to that at baseline (PISA response ratio) was calculated. Groups with a response ratio of <1 and ≥1 were defined as the improvement and the non‐improvement groups, respectively. Results: PISA after the initial periodontal treatment significantly decreased compared with that at baseline (p <.05). A weak negative correlation was found between the PISA response ratio and CD4+ T‐cell counts at baseline (p <.05). The CD4+ T‐cell counts at baseline were significantly higher in the improvement group than in the non‐improvement group (p <.05). Multivariate analysis revealed that the CD4+ T‐cell counts at baseline was an independent factor that affects the PISA (p <.05). Conclusions: The higher the CD4+ T‐cell counts at baseline in patients undergoing treatment for HIV infection, the more effective the initial periodontal treatment. [ABSTRACT FROM AUTHOR]

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy. [ABSTRACT FROM AUTHOR]

Bone resorption inhibitors, such as bisphosphonates (BP) and denosumab, are frequently used for the management of osteoporosis. Although both drugs reduce the risk of osteoporotic fractures, they are associated with a serious side effect known as medication-related osteonecrosis of the jaw (MRONJ). Sclerostin antibodies (romosozumab) increase bone formation and decrease the risk of osteoporotic fractures: however, their anti-resorptive effect increases ONJ. Thus, this study aimed to elucidate the role of sclerostin deletion in the development of MRONJ. Sclerostin knockout (Sost Δ26/Δ26 ) mice were used to confirm the development of ONJ by performing tooth extractions. To confirm the role of sclerostin deficiency in a more ONJ-prone situation, we used the BP-induced ONJ model in combination with severe periodontitis to evaluate the development of ONJ and bone formation in wild-type (WT) and Sost Δ26/Δ26 mice. Wound healing assay using gingival fibroblasts with or without sclerostin stimulation and tooth extraction socket healing were evaluated in the WT and Sost Δ26/Δ26 mice. ONJ was not detected in the extraction socket of Sost Δ26/Δ26 mice. Moreover, the incidence of ONJ was significantly lower in the Sost Δ26/Δ26 mice treated with BP compared to that of the WT mice. Osteogenic proteins, osteocalcin, and runt-related transcription factor 2, were expressed in the bone surface in Sost Δ26/Δ26 mice. Recombinant sclerostin inhibited gingival fibroblast migration. The wound healing rate of the extraction socket was faster in Sost Δ26/Δ26 mice than in WT mice. Sclerostin deficiency did not cause ONJ and reduced the risk of developing BP-induced ONJ. Enhanced bone formation and wound healing were observed in the tooth extraction socket. The use of romosozumab (anti-sclerostin antibody) has proven to be safe for surgical procedures of the jaw. • Tooth extraction did not induce osteonecrosis of the jaw (ONJ) in Sost knockout mice. • Development of bisphosphonate-induced ONJ was reduced in Sost knockout mice. • Gingival wound healing and bone formation in tooth extraction sockets were enhanced in Sost knockout mice. [ABSTRACT FROM AUTHOR]

Cerebral hemorrhage severely affects the daily life of affected individuals. Streptococcus mutans and its adhesion factor Cnm increase the adverse effects of cerebral hemorrhages. However, the mechanism by which Cnm-positive bacteria migrate from apical lesions to cerebral hemorrhage sites is unclear. Therefore, we established an S. mutans-infected apical lesion in a rat model of hypertension and investigated the neurological symptoms associated with cerebral hemorrhage. Eighteen 12-week-old stroke-prone spontaneously hypertensive rats were randomly divided into three groups, i.e. the no infection (control), dental infection with S. mutans KSM153 wild type (Cnm positive), and KSM153 Δcnm groups. Immunofluorescent staining was performed to visualize S. mutans protein. Serum interleukin-1β levels were measured. The adhesion of S. mutans to the extracellular matrix and human fibroblast cells was also analyzed. Serum antibody titers against S. mutans were comparable between Cnm positive and knockout mutants. However, 3–10 days post-infection, neurological symptom scores and cerebral hemorrhage scores were higher in Cnm-positive rats than in knockout mutants. The localization of S. mutans-derived protein was observed in the vicinity of disrupted blood vessels. Serum interleukin-1β levels significantly increased post-KSM153 WT infection. Cnm-positive S. mutans clinical isolates showed increased adhesion to the extracellular matrix, human dental pulp cells, and human umbilical vein endothelial cells compared with the Cnm-negative S. mutans isolates. In conclusion, Cnm-positive bacteria colonize the apical lesion site using the extracellular matrix as a foothold and affect cerebral hemorrhage via the bloodstream. In this study, a dental pulp infection model with Streptococcus mutansin stroke-prone spontaneously hypertensive rats was established the progression of cerebral hemorrhage was observed. The localization of S. mutans-derived protein was observed in the vicinity of disrupted blood vessels. KSM153 WT (cnm-positive) and KSM153-cnm knockout induced similar antibody titers against S. mutans. However, the KSM153 WT strain caused more severe neurological symptoms and higher cerebral hemorrhage scores in the brain tissue than the KSM153 cnm mutant. Furthermore, the serum level of IL-1β increased significantly after infection with KSM153 WT. [ABSTRACT FROM AUTHOR]

Introduction: Chewing well is essential for successful diet therapy and control of blood glucose level in patients with diabetes. In addition, long-term hyperglycemia is a risk factor for microvascular complications, which are the main cause of morbidity and mortality in these patients. Hence, it is plausible that masticatory disorder may be relevant to diabetic microvascular complications which is caused by long-term hyperglycemia. The aim of this study was to investigate whether masticatory disorders are relevant to diabetic microvascular complications. Methods: This cross-sectional study included 172 patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. Masticatory efficiency was determined quantitatively by using the GLUCO SENSOR GS-Ⅱ. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. Statistical significance was defined as a two-sided p value of < 0.05. Results: According to the bivariable analysis, masticatory efficiency was significantly correlated with duration of diabetes (p = 0. 049), number of remaining teeth (p < 0.0001), the number of moving teeth (p = 0.007) and condition of diabetic neuropathy (p < 0.0001). Moreover, the number of remaining teeth (p < 0.0001) and diabetic neuropathy (p = 0.007) remained significantly correlated with masticatory efficiency in the multivariable analysis. Conclusions: For the first time, we demonstrated that patients with type 2 diabetes who developed diabetic neuropathy had significantly reduced masticatory efficiency. Effective mastication is an important factor in successful diet therapy for diabetes. To prevent the progression of diabetic complications, especially in patients with diabetic neuropathy, it may be necessary to combine individualized therapies from dentists and nutritionists with consideration for the level of masticatory dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression. [Copyright &y& Elsevier]

Hertwig''s epithelial root sheath (HERS) is involved in the differentiation of cementoblasts. The cells of epithelial rests of Malassez (ERM) may contribute to that process. However, little is known about the role of these epithelial cells in cementum repair. In the present study, we investigated the expression of alkaline phosphatase (ALPase), osteopontin (OPN), bone morphogenetic protein (BMP)-2 and BMP-4 in epithelial cells (E cells) and fibroblastic cells (F cells) derived from the same human periodontal ligament. E cells were identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. Reverse transcription–polymerase chain reaction analysis showed that E cells have lower ALPase and BMP-4 mRNA levels than F cells. On the other hand, the expression of OPN mRNA in E cells was stronger than in F cells. No significant difference was observed in BMP-2 expression between E and F cells. Thus, they have different expression patterns of ALPase, BMP-4 and OPN, suggesting that ERM and mesenchymal cells in periodontal ligament may be cooperatively involved in cementum repair. Furthermore, E cell cultures will be useful in elucidating the role of ERM. [Copyright &y& Elsevier]

Mesenchymal stem cells (MSCs), a class of adult stem cells, have attracted scientific and medical attention due to their self-renewing properties, multipotency, and trophic factor production. Although MSCs were originally studied on classical two-dimensional (2D) plastic plates, extensive scientific efforts have developed three-dimensional (3D) MSC culture systems, including MSCs spheroids and organoids that can mimic physical conditions. Moreover, we have recently developed 3D culture clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs) for novel bone regenerative cell therapy. Of note, even though it is widely accepted that cell detachment from the culture plate causes cell apoptosis, so called anoikis, these 3D MSCs constructs can be maintained in floating culture conditions. Currently, it is unclear why 3D floating-cultured MSCs constructs can escape from anoikis. To answer this question, the present study explored trophic factor production in 3D floating-cultured C-MSCs that play a cytoprotective role against anoikis and clarified the underlying molecular mechanism in vitro. Compared with cells cultured on 2D plastic plates, PGE2 production mediated by COX2 was significantly increased, and its inhibition drastically induced cell apoptosis in 3D floating-cultured C-MSCs. In the process of C-MSCs preparation, detachment of the cell sheet from culture plate activated the p38/JNK-c-Fos signaling pathway. Moreover, blockage of this signaling by chemical inhibitors abrogated COX2/PGE2 expressions and induced severe apoptosis. These results demonstrated that cell detachment facilitates cytoprotective COX2-mediated PGE2 synthesis via p38/JNK-c-Fos signaling, revealing a possible mechanism that allows resistance against anoikis in floating-cultured 3D MSCs constructs. • COX2 and PGE2 expression levels were elevated in 3D floating cultures of C-MSCs. • COX2-mediated PGE2 synthesis plays a role in cytoprotection against floating culture-induced cell apoptosis. • Floating conditions activate the p38/JNK-c-Fos signaling pathway to induce the COX2-mediated PGE2 production in C-MSCs. [ABSTRACT FROM AUTHOR]

Objectives: Periodontal inflammation is regarded as a risk factor for drug-induced gingival overgrowth (DIGO). In order to elucidate the involvement of periodontal inflammation in DIGO, the periodontal status of subjects who do not develop DIGO despite receiving causative drugs (non-responders) needs to be examined. Therefore, the aim of the present study which was a pilot study was to assess periodontal inflammatory variables in responders (calcium channel blocker induced-GO patients), non-responders, and patients who did not receive causative drugs (non-consumers). Materials and methods: The following parameters were measured: (1) existence of gingival overgrowth, (2) number of teeth, (3) mean periodontal pocket depth (PPD), and (4) percentage of positive sites for bleeding on probing (BOP). The periodontal inflamed surface area (PISA) and periodontal epithelial surface area (PESA) and the PISA/PESA ratio which indicated the degree of periodontal inflammation in each patient were also used to evaluate periodontal inflammation. Results: Thirteen responders, 32 non-responders, and 83 non-consumers were included in the analyses. The mean PPD, percentage of BOP, PESA, and PISA, and the PISA/PESA ratio were significantly higher in responders than in non-responders and non-consumers (p < 0.01). The BOP, PISA, and PISA/PESA ratio were significantly lower in non-responders than in non-consumers (p < 0.05). A positive correlation was found between PPD and age in non-consumers. On the other hand, a negative correlation was noted between PPD and age in non-responders. Conclusions: Periodontal inflammation may be associated with the initiation of DIGO. Clinical relevance: It could be speculated that periodontal therapy before the administration of calcium channel blockers may prevent the development of gingival overgrowth. [ABSTRACT FROM AUTHOR]

Abstract Diabetic patients are at an increased risk of developing severe and progressive periodontitis. Periodontal disease also increases the severity of diabetes by enhancing insulin resistance. Therefore, the regulation of periodontal inflammation in diabetic patients may contribute to the control of both diseases. Glycyrrhizic acid exerts anti-inflammatory effects by inhibiting high mobility group box 1 (HMGB1). HMGB1, one of the ligands of the receptor for advanced glycation end products (RAGE), is a damage-associated molecular pattern and induces inflammatory cytokine production. In the present study, we examined the effects of glycyrrhizic acid on ligature- and Porphyromonas gulae infection-induced periodontitis as well as the involvement of the HMGB1-RAGE axis in diabetic model mice. The molars of diabetic model mice, established by feeding HFD32 to KK/TaJcl mice, were subjected to silk thread ligation and P. gulae was then intraorally applied in the presence or absence of glycyrrhizic acid given topically. The topical application of glycyrrhizic acid suppressed ligature/ P. gulae -induced increases in interleukin (IL)-6 and tumor necrosis factor (TNF)-α at the mRNA level in the gingiva and at the protein level in serum. Furthermore, glycyrrhizic acid suppressed ligature/ P. gulae -induced increases in serum amyloid A (SAA) in serum and fasting blood glucose levels. It also suppressed ligature/ P. gulae -induced increases of HMGB1 and RAGE at the mRNA level in the gingiva and at the protein level in serum. A mouse anti-HMGB1-neutralizing antibody inhibited increases in serum glucose levels. In conclusion, topical treatments with glycyrrhizic acid may suppress periodontal and systemic inflammation and reduce blood glucose levels through the HMGB1-RAGE axis in diabetic mice. Highlights • The regulation of inflammation in diabetes is known to improve blood glucose level. • Murine periodontitis increased cytokine and glucose levels in serum in diabetic mice. • Topical application of glycyrrhizic acid reduced cytokine and glucose levels in the mice. • The regulation by glycyrrhizic acid may be mediated through HMGB1-RAGE axis. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the anti-inflammatory effect of brain-derived neurotrophic factor (BDNF) on human dental pulp cells (HDPCs) and identify the intracellular signaling pathway involved. We investigated the effect of BDNF (50 ng/ml) on interleukin (IL)-6 and IL-8 expression in peptidoglycan (PGN)-treated HDPCs. An inhibition assay was performed with MAPK or NF-κB inhibitors to determine the possible signaling pathway. IL-6 and IL-8 mRNA, IL-6 and IL-8 protein, and phosphorylated p38 kinase activity were determined using real-time PCR, ELISA, and Western blot analysis, respectively. BDNF significantly attenuated PGN-induced IL-6 and IL-8 mRNA and protein levels in HDPCs. A p38 inhibitor also inhibited IL-6 and IL-8 mRNA transcription. PGN stimulated phosphorylated p38 kinase activity in HDPCs, which was inhibited by BDNF. Suppression of phosphorylated p38 kinase activity by BDNF in HDPCs inhibited increased IL-6 and IL-8 expression induced by PGN. Our findings suggest that BDNF regulates intracellular signaling molecule activities to exert its anti-inflammatory effect. [ABSTRACT FROM AUTHOR]

Cherubism (OMIM#118400) is a genetic disorder in children characterized by excessive jawbone destruction with proliferation of fibro-osseous lesions containing a large number of osteoclasts. Mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for cherubism. Analysis of the knock-in (KI) mouse model of cherubism showed that homozygous cherubism mice ( Sh3bp2 KI/KI ) spontaneously develop systemic autoinflammation and inflammatory bone loss and that cherubism is a TNF-α-dependent hematopoietic disorder. In this study, we investigated whether bone marrow transplantation (BMT) is effective for the treatment of inflammation and bone loss in Sh3bp2 KI/KI mice. Bone marrow (BM) cells from wild-type ( Sh3bp2 +/+ ) mice were transplanted to 6-week-old Sh3bp2 KI/KI mice with developing inflammation and to 10-week-old Sh3bp2 KI/KI mice with established inflammation. Six-week-old Sh3bp2 KI/KI mice transplanted with Sh3bp2 +/+ BM cells exhibited improved body weight loss, facial swelling, and survival rate. Inflammatory lesions in the liver and lung as well as bone loss in calvaria and mandibula were ameliorated at 10 weeks after BMT compared to Sh3bp2 KI/KI mice transplanted with Sh3bp2 KI/KI BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20 weeks in 6-week-old Sh3bp2 KI/KI mice transplanted with Sh3bp2 +/+ BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2 KI/KI mice. Thus our study demonstrates that BMT improves the inflammation and bone loss in cherubism mice. BMT may be effective for the treatment of cherubism patients. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF), recognized as essential in the developing nervous system, is involved in differentiation and proliferation in non-neuronal cells, such as endothelial cells, osteoblasts, and periodontal ligament cells. We have focused on the application of BDNF to the regeneration of periodontal tissue and indicated that BDNF promotes the regeneration of experimentally created periodontal defects. Cementoblasts form cementum, mineralized tissue, which is key to establishing a functional periodontium. The application of BDNF to the regeneration of periodontal tissue requires elucidation of the mechanism by which BDNF regulates the functions of cementoblasts. In this study, we examined how BDNF regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase (ALP), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2)) in cultures of immortalized human cementoblast-like (HCEM) cells. BDNF elevated the mRNA levels of ALP, OPN, and BMP-2 in HCEM cells. Small interfering RNA (siRNA) for TRKB, a high affinity receptor of BDNF, siRNA for ELK-1, which is a downstream target of ERK1/2, and PD98059, an ERK inhibitor, obviated the increase in the mRNA levels. BDNF increased the levels of phosphorylated ERK1/2 and Elk-1, and the blocking of BDNF signaling by treatment with siRNA for TRKB and PD98059 suppressed the phosphorylation of ERK1/2 and Elk-1. Furthermore, BDNF increased the levels of phosphorylated c-Raf, which activates the ERK signaling pathway. These findings provide the first evidence that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the BDNF-induced mRNA expression of ALP, OPN, and BMP-2 in HCEM cells. [ABSTRACT FROM AUTHOR]

Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial–mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial–mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial–mesenchymal interaction during odontoblast differentiation. [Copyright &y& Elsevier]

Background: Periodontal disease (PD) is a risk factor for systemic diseases, including neurodegenerative diseases. The role of the local and systemic inflammation induced by PD in neuroinflammation currently remains unclear. The present study investigated the involvement of periodontal inflammation in neuroinflammation and blood–brain barrier (BBB) disruption. Methods: To induce PD in mice (c57/BL6), a ligature was placed around the second maxillary molar. Periodontal, systemic, and neuroinflammation were assessed based on the inflammatory cytokine mRNA or protein levels using qPCR and ELISA. The BBB permeability was evaluated by the mRNA levels and protein levels of tight junction-related proteins in the hippocampus using qPCR and immunofluorescence. Dextran tracing in the hippocampus was also conducted to examine the role of periodontal inflammation in BBB disruption. Results: The TNF-α, IL-1β, and IL-6 levels markedly increased in gingival tissue 1 week after ligation. The IL-6 serum levels were also increased by ligature-induced PD. In the hippocampus, the IL-1β mRNA expression levels were significantly increased by ligature-induced PD through serum IL-6. The ligature-induced PD decreased the claudin 5 expression levels in the hippocampus, and the neutralization of IL-6 restored its levels. The extravascular 3-kDa dextran levels were increased by ligature-induced PD. Conclusions: These results suggest that the periodontal inflammation-induced expression of IL-6 is related to neuroinflammation and BBB disruption in the hippocampus, ultimately leading to cognitive impairment. Periodontal therapy may protect against neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: Multiple external root resorption (MERR) has been reported in systemic sclerosis (SSc) patients in Japan and Spain. To establish whether MERR is a new manifestation, we investigated the prevalence of MERR and systemic and oral manifestations to be associated with MERR in patients with SSc. Methods: Root resorption was detected by dental X-rays, panoramagraphy or cone beam computed tomography (CBCT). The prevalence of systemic and oral manifestations was examined by rheumatologists and dentists, respectively. Autoantibodies were investigated using laboratory tests. Results: MERR was detected in four out of the 41 patients (9.8%) who participated in the present study. The prevalence of digital ulcers was significantly higher in patients with MERR (MERR vs. non-MERR, 75% vs. 16.2%, p < 0.05), whereas that of other systemic manifestations was not. The prevalence of face skin sclerosis (100% vs. 10.8%, p < 0.01), calcinosis at the facial region (75% vs. 0%, p < 0.01), limited mouth opening (75% vs. 18.9% p < 0.05), temporomandibular disorder symptoms (50% vs. 2.7%, p < 0.05), and tongue rigidity (75% vs. 2.7%, p < 0.05) was significantly higher in patients with MERR. Conclusion: SSc patients with MERR had highly homogenous maxillofacial manifestations. Further clinical and basic studies are needed to elucidate the mechanisms underlying MERR in SSc patients. [ABSTRACT FROM AUTHOR]

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer's disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD. [Copyright &y& Elsevier]