Carbon Nanotubes Induce Mineralization of Human Cementoblasts.

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

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

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

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

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

Fit accuracy of resin crown on a dental model fabricated using fused deposition modeling 3D printing and a polylactic acid filament.

Purpose We considered the possibility of reducing industrial waste by fabricating and reusing dental models prepared using a fused deposition modeling (FDM) 3D printer and polylactic acid (PLA) filaments. The purpose of this study was to verify the accuracy of models fabricated using FDM and PLA.Methods The same provisional crown was used to check the marginal fit on PLA models prepared using an intraoral scanner (IOS) and FDM, plaster models made with silicone impression material and plaster, and resin models prepared using an IOS and stereolithography apparatus (SLA) 3D printer. The marginal fit was measured using micro-computed tomography at four points on the tooth: the buccal center (B), palatal center (P), mesial center (M), and distal center (D) points.Results At point B, the marginal gaps were 118 ± 21.7, 62 ± 16.4, and 50 ± 26.5 µm for the PLA, resin, and plaster models, respectively, with a significant difference between the PLA model and the other two. However, the marginal gap at all other measurement points was not significantly different between the models (P > 0.05).Conclusions We compared the accuracy of the models fabricated using the FDM, SLA, and conventional methods. The combination of FDM and PLA filaments showed no significant differences from the other models, except at point B, indicating its usefulness. Therefore, FDM and PLA may become necessary materials for dental treatment in the future.

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

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

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

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

Cell Biological Assays for Measuring Odontogenic Activities of CCN Proteins.

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

Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc.

Tumor necrosis factor α stimulates expression and secretion of urokinase plasminogen activator in human dental pulp cells.

Plasminogen activator (PA) is the enzyme responsible for converting plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. PA exists in two forms: urokinase-type PA (uPA) and tissue-type PA (tPA). Here we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNF-α) on PA production and secretion in human dental pulp cells. When the cells were stimulated with TNF-α (10 ng/mL), PA activity in the medium clearly increased in a time-dependent manner, and this activity was reduced after immunoprecipitation with anti-uPA antibody, but not with anti-tPA antibody. In TNF-α-stimulated cells, the expression of uPA mRNA was enhanced, but was lower than that of tPA mRNA. The expression of uPA mRNA and PA secretion stimulated by TNF-α were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFκB inhibitor pyrrolidine dithiocarbamate, but were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. In the presence of another inflammatory cytokine, interleukin 1ß (IL-1ß, 100 pg/mL), TNF-α-stimulated expression of uPA mRNA and secretion of uPA were enhanced. These observations suggest that TNF-α stimulates uPA production and secretion, and that this effect is regulated via activation of NFκB and tyrosine phosphorylation, apparently in conjunction with IL-1ß, during inflammation in human dental pulp.

Role of CTGF/CCN2 in reparative dentinogenesis in human dental pulp.

Connective tissue growth factor/CCN family 2 (CTGF/CCN2) has been considered to participate in tooth development. To date, the expression and role of CTGF/CCN2 in reparative dentinogenesis have been unclear. Our previous study revealed that matrix metalloproteinase-3 (MMP-3) stimulates cell migration via CTGF/CCN2 expression and secretion in human dental pulp cells, and that this is dependent on dynamin-related endocytosis and independent of protease activity. The objective of the present study was to determine the expression of CTGF/CCN2 in reparative dentin in human carious teeth and to examine the effect of CTGF/CCN2 on mineralization in cultured human dental pulp cells. Minimal expression of CTGF/CCN2 was evident in odontoblasts subjacent to the dentin-pulp junction in healthy teeth, whereas strong expression was detected in odontoblast-like cells lining the reparative dentin subjacent to dental caries. In human dental pulp cells, CTGF/CCN2 promoted mineralization but failed to induce proliferation, suggesting that this molecule has the ability to induce the differentiation of human dental pulp cells. Taken together, the data suggest that CTGF/CCN2 is likely involved in reparative dentinogenesis through formation of hard tissue in human carious teeth.

MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration.

Matrix metalloproteinase-3 (MMP-3) expression is promoted after pulpotomy, and application of MMP-3 to dental pulp after pulpotomy accelerates angiogenesis and hard tissue formation. However, the mechanism by which MMP-3 promotes dental pulp wound healing is still unclear. Connective tissue growth factor/CCN family 2 (CTGF/CCN2), a protein belonging to the CCN family, is considered to participate in wound healing, angiogenesis, and cell migration. In this study, we examined the involvement of CTGF/CCN2 in MMP-3-induced cell migration in human dental pulp (fibroblast-like) cells. In human dental pulp cells, MMP-3 promoted cell migration, but this effect was clearly blocked in the presence of anti-CTGF/CCN2 antibody. MMP-3 provoked mRNA and protein expression and secretion of CTGF/CCN2 in a concentration- and time-dependent manner. The MMP-3 inhibitor NNGH failed to suppress MMP-3-induced CTGF/CCN2 protein expression. The potent dynamin inhibitor dynasore clearly inhibited MMP-3-induced CTGF/CCN2 expression. These results strongly suggest that MMP-3 induces CTGF/CCN2 production independently of the protease activity of MMP-3 and dependently on dynamin-related endocytosis, which is involved in cell migration in human dental pulp cells.