Dental follicle is the mesenchymal tissue which surrounds developing tooth germ. During tooth root development, periodontal components such as cementum, periodontal ligament and alveolar bone are created by progenitors present in the dental follicle. Previously, we have reported the presence of periodontal ligament progenitor cells in immortalized mice dental follicle cells (MDF^E6-EGFP). Here we report the establishment of a periodontal ligament progenitor-like cell line obtained from MDF^E6-EGFP. Single cell clones were established from MDF^E6-EGFP and each of the clones was screened by the expression of tendon/ligament markers. As a result, we established a clonal cell line designated MDF-G5 which highly expressed sderaxis, growth and differentiation factor (gdf) 5, ephA4 and six-1 or type I collagen. Detectable expression of osteoblast phenotype related genes, such as bone sialoprotein, osteocalcin and osterix, was observed. To elucidate the differentiation capacity of MDF-G5 in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted MDF-G5 formed periodontal ligament-like tissue and oxytalan fiber-like fibrils were found within it. Our findings suggested that MDF-G5 possessed periodontal ligament progenitor properties, and may provide useful information for the development of not only periodontal ligament but also a regeneration therapy for patients with periodontal disease.

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With the advances in adhesive technology in recent years, the application of resin to crown restorations has increased in dental practice. Previous studies reported that the bond strength is reduced when the tooth surface is contaminated by saliva and/or blood probably because the contaminants per se may inhibit adhesion. In clinical practice, a hemostat is applied to the site of bleeding and then rinsed, dried and finally removed. However, the hemostat applied may remain on the adhered surface and affect the bond strength of bonding material to dentin. In order to confirm the tooth surface treatment effect of Bosmin® (Daiichi Sankyo Company), possible structural changes in the dentin surface after Bosmin® treatment were examined by atomic force microscopy (AFM) and compared with those after treatment with phosphoric acid and diluted hydrochloric acid used as a control in this study. We also examined whether or not the adhesive strength of Bosmin® is altered by different times of application. When the results of AFM observation were compared between Bosmin®-, phosphoric acid- and diluted hydrochloric acid-treated groups with reference to the image of the untreated group as a baseline, a concave surface in the superficial layer of dentin was encountered loss frequently in the phosphoric acid- and diluted hydrochloric acid-treated groups with more flattened AFM images. In the Bosmin®-treated group as compared with the untreated group, on the other hand, a concave surface was observed throughout the assessed superficial layer of dentin in an almost uniform manner after 15-second treatment. After 1-minute treatment with Bosmin®, a fine concave surface was observed less frequently as compared with the image after 15-second treatment but more frequently as compared with that in the untreated group. In the test on bond strength, the strength was significantly lower in the control phosphoric acid- and diluted hydrochloric acid-treated groups as compared with the untreated group, while both the Bosmin® 15-second treated group and 1-minute treated group showed a significantly higher strength as compared with the untreated group. From the evidences mentioned above, it was confirmed that Bosmin® has an excellent tooth surface treatment effect.

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Human β-defensin-2 (hBD-2) is one of the antimicrobial peptides produced by epithelial cells after stimulation by microorganisms and inflammatory mediators. Compared to Gram-positive bacteria, Gram-negative bacteria typically detected in the periodontal pockets in periodontitis elicit a stronger antibacterial peptide response of hBD-2. Differences between healthy subjects and those with periodontal disease in hBD-2 gene expression were recently reported, and hBD-2 gene expression in primary human gingival epithelial cells (HGEC) stimulated with Actinobacillus actinomycetemcomitans was described. We previously reported that expression of hBD-2 mRNA in HGEC was P. gingivalis-dependently induced according to stimulation time and was related to the initial stage of inflammatory responses after infection by pathogenic periodontal bacteria, ultimately with migration of neutrophils to infection sites. However, the role of hBD-2 is not at all clear because periodontal disease is not a chronic disease that repeatedly cycles between an advanced stage and suspended stage. We investigated the difference between the in vitro expression of hBD-2 and the production of IL-8 in HGEC in response to challenges with various concentrations of IL-1β, which is an inflammatory cytokine. mRNA expression of hBD-2 in HGEC stimulated under various conditions was assessed by the semi-quantitative reverse transcription-polymerase chain reaction, and culture supernatants from HGEC after stimulation under various conditions were analyzed quantitatively by enzyme-linked immunosorbant assay. Although the levels of hBD-2 mRNA and IL-8 production in HGEC stimulated with IL-1β tended to increase with exposure time, the levels were not significantly changed by variations in concentration. These findings suggest that the expression of hBD-2 and IL-8 was observed in IL-1β-stimulated HGEC regardless of the severity of the inflammation.

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In cases in which tooth bleaching is difficult, esthetics can be improved by applying coating materials to the coronal area. It has been reported that the color of tetracycline teeth changes to dark brown with age, due to irradiation by ultraviolet rays. Therefore, if the irradiation of teeth by ultraviolet rays in natural light can be decreased by applying coating materials to tetracycline teeth, such color changes to dark brown with age can be suppressed. In this study, using two types of coating materials (White Coat™ shade OB0 (WOB), opaque (WOP), and Beauti Coat shade BW3 (BBW) , shade WO (BOP)), composite resin (UniFil® LoFlo Plus shade AO3 (UAO)), glassionomer cement (Fuji Ionomer® Type II shade YB (FYB)), and enamel, disk specimens of 0.1, 0.3, 0.5, and 1.0 mm in thickness were produced, and the light transmissivity of each specimen was measured. As the control group, disk specimens of human tooth enamel of 0.3, 0.5, and 1.0 mm were produced, and the light transmissivity was measured. After producing tetracycline discoloration models by mixing tetracycline solution with dental apatite cements, the models were covered with each disk specimen of 0.3 mm in thickness, and they were irradiated with artificial sunlight containing ultraviolet rays for 7 days. Color changes (ΔE*ab) after irradiation were measured, and the results were evaluated using one-way layout analysis of variance and Tukey's test (p<0.05). Furthermore, when coating materials are employed for suppressing the progression of tetracycline tooth color changes, they require long-term durability. Therefore, marginal leakage at the interface of coating materials (WOB, BBW) and enamel was observed by the dye-penetration method after 24 hours, and 500 and 1,000 repetitions of thermal-cycling (TC500 and TC1000, respectively) for 30 seconds at 5 and 55℃. The results obtained were evaluated using Mann-Whitney's U test (p<0.05). Also, to investigate the influence of the repeated application and removal of coating materials on the enamel surface, SEM observation of the specimens in which coating materials were removed 24 hours after application (24-hour removal), those in which coating materials were removed after TC500 (TC500 removal), and those in which the removal of coating materials was repeated 3 times after TC500 (TC500×3 removal), was performed. The following results were obtained: 1. Regarding the 0.3-mm-thick specimens, the light transmissivity was below 0.5% in WOB, WOP, and BOP, at wavelengths of around 400 nm and over, showing ultraviolet ray absorption effects. 2. Regarding the 1.0-mm-thick specimens, although the light transmissivity was below 0.5% in BBW, UAO, and FYB, at wavelengths of around 400 nm and over, suggesting ultraviolet ray absorption effects, this thickness is not suitable for clinical application. 3. Color changes of the discoloration models were significantly lower with WOB, WOP, and BOP, compared with those of enamel. These findings suggested that WOB, WOP, and BOP suppressed the progression of discoloration in tetracycline teeth. 4. In the dye-penetration test, marginal leakage was not present after 24 hours. However, since dye-leakage was found after TC500 and TC1000, it was suggested that marginal leakage would occur after long-term application of coating materials. 5. SEM observation of the enamel surface after removing coating materials showed that the coating materials were abraded at their interface with enamel in the cases of 24-hour removal, TC500 removal, and TC500×3 removal, suggesting that the influence of the repeated application and removal of coating materials on the enamel surface was low.

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Due to the greater awareness of esthetics in recent years, patients increasingly desire the color of composite resin restorations to match that of their natural teeth. However, in patients with large defects, such as Class III or IV cavities, even when selecting a composite resin matching a tooth color, background colors sometimes show through the resin and make restorations appear darker than natural teeth in the mouth. To block background colors, the layering technique is effective, but to the best of our knowledge, there has not been a basic study on composite resin restoration using teeth. We developed an experimental system using the classic shade guide of Vita for the direct application of nano filler composite resin as experimental teeth. By preparing standardized cavities in these experimental teeth, a colorimeter was used to measure CIE Lab and XYZ values. We then calculated ΔE*, contrast ratios and TP values and reached the following conclusions: 1. Vita classical shade guides are uniformly produced and are useful as experimental teeth. 2. With regard to ΔE*, no significant differences were seen in either white or black backgrounds for the A2 and the A3.5 assessment group. When compared to the A4D+A3B two-layer filling and the A3B single-layer filling, the ΔE* for the A2D+A3B two-layer filling in the A3 assessment group was significantly lower, thus clarifying the effectiveness of the layering technique. 3. Based on contrast ratios and TP values, there were significant differences with single-layer fillings for each group, thus clarifying that background colors show through if only a body shade resin is used. For the A3 and the A3.5 assessment groups, the two-layer technique effectively blocked background colors. The above findings suggest that in patients with large Class III or IV cavities, except for when value and chroma are high, using a dentin shade resin that is comparable to the tooth or slightly darker than the tooth for the inside layer can effectively block background colors. Furthermore, using a body shade resin comparable to the tooth for the outside layer can achieve esthetically favorable restorations. The layering technique is highly useful.

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The purpose of this study was to evaluate laser irradiation conditions suitable for the treatment of dentin hypersensitivity, the influence of the combined use of dentin hypersensitivity-inhibitory materials and two types of laser irradiation on the sealability of dentinal tubules using an in vitro model of hypersensitive dentin. The lasers used in this experiment were Erwin AdvErl® as an Er: YAG laser and Panalas®C05Σ as a CO₂ laser. The dentin discs were prepared from extracted bovine anterior teeth as hypersensitivity model samples. The samples were applied to a device produced by modifying that reported by Pashley, and the pulpal pressure was determined to be 25 mmHg. Each laser irradiated the sample surface from a distance of 0 or 1 mm, and dentin permeability was measured. Statistical analysis was performed by one-way analysis of variance and Scheffe's method. The dentin permeability inhibition rate was 27.7±10.0% -59.5±12.8% at an irradiation distance of 0 mm, showing the highest value with Co(1A). The rate was significantly higher with Er(C400F), Co(1A) , and Co (2A), in comparison with Er(Brush) (p<0.05). The dentin permeability inhibition rate was 28.9±7.2%-44.8±11.5% at an irradiation distance of 1 mm, showing the highest value with Co(1A). In cases with the combined use of MS and laser irradiation, the rate was 21.5±9.8%-72.5±10.1%, and the rate in the MS(after irradiation) group was significantly higher than that in the group in which only Co(1A) was irradiated, at an irradiation distance of 1 mm (p<0.05). In cases with the combined use of GL and laser irradiation, the rate was 32.5±5.7%-64.1±9.8%, and the rate in the GL(after irradiation) group was significantly higher than that in the group in which only Co(1A) was irradiated, at an irradiation distance of 1 mm (p<0.05). In this experiment, the dentin permeability inhibition rate increased when Er: YAG and CO₂ laser irradiation was performed. At irradiation distances of 0 and 1 mm, the rate using Er: YAG and CO₂ laser irradiation decreased when the irradiation distance from the subjects increased. Furthermore, in cases of the combined use of the MS Coat and GLUMA® desensitizer, the rate increased with Er(Brush) and Co(1A). These results suggest that symptoms of dentin hypersensitivity may be relieved when dentin hypersensitivity-inhibitory materials are used before or after irradiation with a low-output Er: YAG and CO₂ laser.

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The characterization of advancing bacterial front of carious lesions is important not only for the pathogenesis of pulpitis, but also for the treatment of pulpal pathosis. We immunohistologically examined the prevalence and localization of selected bacteria in human deep-carious teeth. Fourteen specimens were obtained from extracted carious third molars that showed bacterial invasion in reparative dentin and/or dental pulp. The distribution of bacterial penetration was detected nonspecifically by Brown-Brenn stain and specifically enzyme-labeled antibody method with specific rabbit antisera against six bacteria. All of the samples showed the invasion of Lactobacillus plantarum in the reparative dentin. About 90% of the examined teeth showed the invasion of Streptococcus mutans, Micromonas micros and Lactobacillus casei in the reparative dentin. These four bacteria were also detected in the dental pulp with carious exposure. In contrast, the detection frequencies of Actinomyces viscosus and Streptococcus sanguinis in reparative dentin were about 40% and 20%, respectively. The present study revealed the features of advancing bacterial front of deep-carious teeth with irreversible pulpitis.

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One of the responsibilities of a dental hygienist is dental assistance. We have investigated practical training programs for dental assistance in temporary sealing and root canal treatments at dental hygienist schools. We reported that the practical training at the school was very useful and meaningful. Students undergo clinical training just prior to graduation, but most dental hygienist schools do not have facilities to provide actual dental care. Therefore, clinical training is often done at clinics, and because it is difficult for all students in the same class to receive clinical training at one clinic, students are broken up into small groups and receive training at different clinics. However, clinical training can vary among clinics. In this paper, we report the results of a questionnaire survey for dental hygiene students in order to determine how they study the contents of school dental assistance programs and the contents of clinical training at clinics. The subjects were 139 second-year dental hygiene students studying at the Department of Dental Hygiene of the University of Shizuoka Junior College or Saitama Prefectural University Junior College in 2003 and 2004. The questionnaire survey was conducted after the completion of clinical training. The survey assessed the ratio of conservative dental treatments among daily dental treatments, and the details of two conservative treatments: composite resin restoration and root canal treatment. Valid responses were obtained from 137 students (recovery rate: 98.6%). The survey results clarified the following: 1) at most clinics, conservative treatments accounted for more than 40% of daily treatments; 2) while light cured resins were mostly used in composite resin restorations, different people were in charge of light irradiation among the clinics; and 3) different clinics utilized different sealing materials, sealing methods and techniques for filling root canals. From the viewpoint of pre-graduation clinical training, it is desirable to standardize the contents of clinical training as much as possible among participating clinics.

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This study was conducted to investigate the effects of a bleaching agent with titanium dioxide and low-concentration hydrogen peroxide on non-vital tooth and to assess a new walking bleach method. Pyrenees® (Mitsubishi Gas Chemical Company) was used as the bleaching agent. The tooth color was measured with an SE2000 spectral color-difference meter (Nippon Denshoku) and evaluated with a CIE L*a*b* color system. Hyperlightel (Kuraray Medical) was used as a light source. The bleaching agent was applied to the coronal pulp chamber of extracted human teeth and irradiated with light from the palate side. Color measurements were taken before the bleaching treatment (L*₀, a*₀, b*₀), and at 5 minutes (L*₅, a*₅, b*₅), and 10 minutes (L*₁₀, a*₁₀, b*₁₀) after the treatment. The L*₅ and L*₁₀ levels were significantly higher than the L*₀ level. The a* levels gradually increased, but did not rise significantly above the pre-treatment levels. The b*₅ and b*₁₀ levels were significantly lower than the b*₀ level. On average, ΔE*ab₅, and ΔE*ab₁₀ were 3.84 (SD±1.58), and 6.15 (SD±2.29), respectively. These levels confirm the very high effects of the bleaching. Based on these results, we concluded that Pyrenees® was found to have a high potential as non-vital tooth bleach and this bleaching method was safer than the conventional method.

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Vital pulpotomy involves the application of a medicament or dental material to the amputated pulp in an attempt to preserve its vitality. Many materials and medicaments have been used as pulp-capping agents. Calcium hydroxide has been the standard and is generally accepted as a successful pulp-capping agent of choice. A major disadvantage of calcium hydroxide in amputated pulp is that the developmental structures in newly formed dentinal bridge including small holes or defects with degenerated and necrotic tissues are seen. The purpose of this study was to investigate histologically the tissue responses of amputated teeth pulps simultaneously by using dentin chips with calcium hydroxide. The pulp tissues of 42 sound permanent teeth with incompletely formed apices from crab eating (Macaca irus) young adult monkeys were capped with calcium hydroxide, dentin chips, or the former two agents and evaluated at 3, 7, 14, 28 days or 4 to 5 months after vital pulpotomy. Histological analysis was performed in decalcified serial paraffin sections of 6 to 8 μm thickness stained with hematoxylin-eosin, Bielschowsky-Perdrau's silver stain and Gram's stain. The results obtained were as follows. 1. Following calcium hydroxide dressing, an early calcific (dentin) bridge was well produced beneath the necrotic area of pulp and osteodentin with cell inclusions, vascular channels or finely spaced structures developed over 7 days. 2. When dentin chips were applied directly to the amputated pulp tissue, necrosis of adjacent pulp tissue and inflammation of the contiguous tissue seldom or never occurred. At the early stage, dentin particles appeared to be being phagocyted by macrophage-like cells. The proliferation of fibrous granulation tissues with many new blood vessels and calcification around dentin chips (fragments) were observed 14 days after pulpotomy. 3. Four to five months postoperatively, complete dentinal bridging with healthy radicular pulp was observed in the experimental group in whom dentin chips were applied directly to the amputated pulp areas and calcium hydroxide materials were overlaid. In the teeth, it was obviously effective for inducing continued coronal dentin bridge with fewer developmental holes and defects communicated with the coronal and pulpal surface of the dentinal bridge. These results suggested that the application of non-infected clear dentinal chips plug and calcium hydroxide material directly to the exposed pulp had biocompatibility and successfully caused the formation of a new dentin bridge.

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We have previously reported that densely calcified nodules formed from dental pulp cells are often observed in tissue culture, and that TNF-α receptor, Fas, and Fas-ligand in relation to apoptosis were detected in and around the nodules using an in situ apoptosis detection kit. However, the relationship between the nodule formation in the pulp cells and their occurrence of apoptosis has not yet been clarified. So, we tried to confirm this relationship in vitro. The dental pulp cells outgrown 5 to 9 times in the D-MEM (Dulbecco's modified Eagle Medium) harvested from a patient with periodontitis were cultured for 8 weeks and used in this study. The cells in the control group were cultured in the D-MEM culture alone, while those in the experimental group were cultured in another D-MEM containing the Caspase-3 inhibitor activated at the final stage of apoptosis. The process of nodule formation was examined and recorded using a phase contrast microscope from the start of the experiment. The Caspase-3 activity was labeled by FITC at 4 weeks and the cells containing the Caspase-3 were observed by a laser microscope. Statistical analysis of the change in number of nodules was done by a paired or unpaired t-test. As a result, the nodules were formed with time in the presence or absence of the Caspase-3 inhibitor, and were significantly increased in number. However, the number of nodules cultured in the presence of the Caspase-3 inhibitor was significantly less than that in the control group in and after three weeks. Caspase-3 activity was detected in both groups in the nodules of the 4-week culture. We had previously thought that apoptosis in cultured dental pulp cells occurred in the process of calcification. However, our findings suggest that apoptosis occurs during nodule formation.

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