Michiharu Daito

Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation

Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases. Here the cytotoxic effects of a purified preparation of a P. gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro. The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen. Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases.

Differentiation of dental pulp stem cells into a neural lineage

We previously investigated whether dental pulp-derived cells possess similar pluripotency to bone marrow cells, and reported their capacity to differentiate into osteoblasts, as well as the characteristics of the stem cells present in dental pulp. In the present study, we hypothesized that neural stem cells would also exist in rat dental pulp, similar to bone marrow and the brain, and attempted to induce their differentiation into a neural lineage by applying an in vitro study design previously reported to induce differentiation of human bone marrow cells. Before inducing differentiation, we detected cells expressing nestin (Nes), which is known to be a marker for neural stem cells, within primary cultures of rat dental pulp-derived cells, suggesting the existence of neural stem cells in dental pulp. Quantitative analyses of the mRNA and protein expression levels revealed downregulation of both the Nes mRNA and protein levels to about 68.1% and 12.4%, respectively, after the induction of differentiation compared to the corresponding levels before induction. Conversely, the glial fibrillary acidic protein (Gfap) mRNA level was elevated by 1.3-fold after the induction of differentiation compared with the level before induction. The reduced number of Nes-positive cells and decreased Nes mRNA and protein levels after the induction of differentiation may be attributed to differentiation of neural stem cells into a neural lineage. Moreover, the increased number of Gfap-positive cells and increased Gfap mRNA level after the induction of differentiation most likely support their progressive differentiation into a glial cell lineage, since Gfap is a marker that is upregulated in glial cells. Our present data demonstrate the existence of neural stem cells in tissues other than the central nervous system, and may represent a significant step toward providing more diverse and multiple sources of stem cells for future regenerative medicine.

Induction of Proliferation by 15-Deoxy-Δ12,14-Prostaglandin J2 and the Precursors in Monocytic Leukemia U937

Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several human tumors including gastric, lung, colon, prostate and breast. However, the role of PPARγ signals in leukemia is still unclear. The aim of this study is to evaluate the ability of 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂), that is a ligand for PPARγ, on proliferation of human leukemia cell line U937. 15dPGJ₂ at 5 µmol/l stimulated the proliferation. In contrast, 15dPGJ₂ at concentrations of >10 µmol/l inhibited the proliferation through the induction of apoptosis. PGD₂, PGJ₂ and Δ¹²-PGJ₂ (ΔPGJ₂), those are precursors of 15dPGJ₂, had similarly proliferative effects, whereas they showed antiproliferative effects at high concentrations. FACScan analysis revealed that PGD₂ at 5 µmol/l, PGJ₂ at 1 µmol/l, ΔPGJ₂ at 1 µmol/l and 15dPGJ₂ at 5 µmol/l, all accelerated cell cycle progression. Immunoblotting analysis revealed that PGD₂ at 5 µmol/l and 15dPGJ₂ at 5 µmol/l inhibited the expression of phospho-p38, phospho-MKK3/MKK6 and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p27. In contrast, PGJ₂ at 1 µmol/l and ΔPGJ₂ at 1 µmol/l did not affect the expression of them. These results suggest that 15dPGJ₂ and PGD₂ may, through inactivation of the p38 MAPK pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of proliferation.

Differentiation of Rat Dental Pulp-derived Cells into an Osteoblastic Lineage

Abstract The in vitro differentiation potential of rat dental pulp-derived cells into an osteoblastic lineage was examined. Induction was carried out under in vitro osteogenesis-inducing conditions used for mesenchymal stem cells from human bone marrow. We previously reported that the level of mineralization was high at 6 weeks after induction, as determined by von Kossa staining. In the present study, we quantitatively measured the alkaline phosphatase (ALP) activity after induction. The ratios of ALP-positive cells to total cells were 44.4 ± 1.0, 46.9 ± 0.9, 40.5 ± 0.5 and 21.0 ± 1.8% at 1, 2, 3 and 6 weeks after induction, respectively. The level of ALP activity was significantly decreased at 6 weeks after induction (p Col1a2 (collagen type I) and Bglap (osteocalcin) were up-regulated by about 1.7-fold and 1.3-fold, respectively, at 6 weeks after induction compared with their corresponding levels at 1 week. These results indicate that the cells differentiated into an osteoblastic lineage in parallel with decreasing ALP activity during induction. Moreover, the expressions of Col1a2 and Bglap were inversely correlated with Alpl (ALP) expression after induction, which was correlated with Bmp2 (bone morphogenetic protein 2) expression. Taken together, the findings indicate that these molecules contribute to the differentiation of rat dental pulp-derived cells into an osteoblastic lineage.

Changes in amount of psychological palmar sweating in children at a dental office

Abstract Psychological palmar sweating is a phenomenon that occurs with emotional changes such as anxiety, fear, and anger. To identify the psychological anxiety and stress experienced by children visiting a dental office, we measured the amount of psychological palmar sweating using a sudorometer and obtained the following results. 1. Amount of sweating in dental office The amount of sweating was largest in both boys and girls when they were guided to the dental chair, followed by the period during tooth brushing and turbine sound. The least sweating occurred while they were in the waiting room. In boys, significant differences in sweating were seen between the times spent in the waiting room and when guided to the chair, and between the times when they were guided to the chair and taught tooth brushing. In girls, significant differences were seen between the times spent in the waiting room and when guided to the chair, between the times when guided to the chair and taught tooth brushing, and between being guided to the chair and turbine sound. 2. Amount of sweating by gender In the dental office, the amount of sweating was larger in girls than in boys during all times: while in the waiting room, when guided to the chair, during tooth brushing and turbine sound. However, no significant differences were seen between boys and girls. The method presented in our study maybe very useful to quantitatively understand the psychological state of children in dental offices who may be unable to express their own feelings well.

Three dimensional measurement of the palate using the semiconductor laser: On the influences of the palate of maxillary protrusion with finger sucking

The aim of the study was to measure three-dimensionally using the semiconductor laser to examine changes in palate section areas, palate projection areas and palate volumes for 5-year olds children with maxillary protrusion of primary dental arch induced by finger sucking. Because of pressure by fingers on the palate, the palate section areas between deciduous canines and between primary first molars in finger sucking children were greater than those in children with normal occlusion. The palate projection area in the anterior segment of the arch in finger sucking children was greater than that in normal occlusion children. The anterior palate volume in finger sucking children was greater than that in normal occlusion children. However, because of anterior traction of the palate by fingers in finger sucking, the palate section area between the posterior margin of the primary second molars in finger sucking children was smaller than that in normal occlusion children. The palate projection area between posterior segment of the arch in finger sucking children was smaller than that in normal occlusion children. The posterior palate volume in finger sucking children was smaller than that in normal occlusion children.

Three dimensional measurement of the palate using the semiconductor laser 2. Changes in the palate section areas, palate projection areas and palate volumes in the mixed dentition

Abstract The objective of the study was to examine changes in palate sectional areas, palate projection areas and palate volumes according to age. Using the maxillary dentition models, the palates of 7-, 8- and 9-year-old children, who were in the mixed dentition period, were measured threedimensionally using the semiconductor laser. The palate section areas, particularly between deciduous canines, primary first molars, primary second molars and first molars, increased with age, as the palate expanded vertically along with the growth and development of the maxilla. The palate projection areas increased with age, as the palate expanded laterally along with the growth and development of the maxilla. The palate volume increased with age, as the palate expanded laterally and vertically.

TNF-α enhances MMP-2 production in deciduous dental pulp fibroblasts

Abstract Inflammation in dental pulp tissue is associated with tissue degradation, and matrix metalloproteinases (MMPs) are believed to participate in this destruction. Elevated levels of some MMPs have been reported in inflamed pulp and periapical lesions. Moreover, in inflamed pulp, many kinds of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, are released from inflammatory cells like macrophages, lymphocytes and neutrophils. In the present study, we examined whether TNF-α affected the production of MMP-2 in deciduous dental pulp fibroblasts and its signaling pathways utilizing gelatin zymography and western blotting analysis. TNF-α increased the expression of MMP-2 in a dose-dependent manner in deciduous dental pulp fibroblasts. LY294002 and Wortmannin, which are PI3-K (phosphoinositide 3-kinase) inhibitors, inhibited the MMP-2 production induced by TNF-α in deciduous dental pulp fibroblasts. Moreover, in deciduous dental pulp fibroblasts cultured with TNF-α, AKT (protein kinase B) was phosphorylated in a time-dependent manner with the maximum phosphorylation at 30 min, and LY294002 and Wortmannin abolished this phosphorylation of AKT in TNF-α-stimulated deciduous dental pulp fibroblasts. These results suggest that TNF-α may enhance pulp tissue destruction during pulp inflammation in part by regulating MMP-2, and that the AKT pathway is involved in MMP-2 production in deciduous dental pulp fibroblasts.

DNA microarray analysis of dental pulp fibroblasts exfoliated from deciduous teeth

Abstract Dental pulp plays an important role in tooth vitality. Previous studies have indicated that stem cells can be isolated from dental pulp, and dental pulp exfoliated from deciduous teeth has become a useful alternative for dental tissue engineering because of its higher proliferation rate. In the present study, we analyzed the differences in gene expressions between human dental pulps exfoliated from deciduous and permanent teeth by DNA microarray assays. A scatter plot of mRNA levels based on fluorescent signals in human dental pulps from deciduous and permanent teeth indicated a dispersed distribution pattern. In a scatter plot of the genes, 2,573 genes were expressed at >2-fold higher levels in dental pulp from deciduous teeth, compared with permanent teeth. To confirm the microarray results, insulin-like growth factorbinding protein 5 (IGFBP5) and vascular endothelial growth factor A (VEGFA) showing higher mRNA expression levels were selected and analyzed for their mRNA levels by RT-PCR. Both the IGFBP5 and VEGFA mRNA levels were upregulated by about 3-fold in dental pulp from deciduous teeth compared with permanent teeth. Thus, the RT-PCR results for these genes were consistent with the microarray data. The dental pulps in deciduous and permanent teeth differ significantly with regard to their developmental processes, tissue structures and functions. The present findings using DNA microarray analyses to detect differences in the gene expressions of deciduous and permanent teeth may be useful for dental pulp tissue engineering.

An investigation of the special practitioner system of pediatric dentistry in Japan

Abstract We sent questionnaires to all the members (4,298 people) about the specialist pediatric dentistry system in Japan (Hereafter, termed specialists). The survey (1,129 dentists answered the questionnaire) revealed the following: 1.About 29% of the members who have qualifications for pediatric dentists, authorized by Japanese Society of Pediatric Dentistry (JSPD), hoped for a specialist qualification immediately. 2.Eighty point six percent of the members who do not have qualifications authorizing them as pediatric dentists answered that they wanted to take an examination to become a specialist as soon as they were qualified to take it. 3.Many members hoped that the examination to become a specialist would be strictly carried out. 4.Twenty-two point eight percent members thought that the qualifications for pediatric dentists authorized by Japanese Society of Pediatric Dentistry should be abolished after putting the specialist system into effect. 5.Fourteen percent of those that have the qualification of pediatric dentists, authorized by Japanese Society of Pediatric Dentistry, did not want to acquire the specialist title but they wanted to renew their current qualifications. 6.As dentists got older, the ratio of children patients (under 18 years old) tended to decrease. 7.However, about one-quarter, 112 members (about 10% of all) of dentists who are 50–69 years old (347 members) it occupied 34.7% of all treated exceeded 90% children patients.