Bcl-2 (B-cell lymphoma 2 protein) is known as an angiogenesis- and apoptosis-related protein, but its role in the pathogenesis of apical periodontitis is poorly understood. We analyzed temporal changes in the mRNA expression levels of Bcl-2 and Bax (Bcl-2-associated X protein) in experimental rat periapical lesions. Mandibular first molars of 5-week-old Wistar rats were pulp-exposed and left unsealed for 14 and 28 days. The periapical tissues were then immunostained for CD31, Bcl-2 and Bax. CD31-positive endothelial cells and CD31-negative perivascular tissues were retrieved by means of immune laser capture microdissection, and subjected to real-time PCR analysis for Bcl-2 and Bax mRNAs. At 14 days when the lesion was in the expanding phase, a significantly higher Bcl-2 mRNA expression was detected as compared with the normal periapical tissue and the lesion at 28 days (p<0.05, Mann-Whitney U-test). Bcl-2 mRNA expression in CD31-positive endothelial cells was also significantly higher than that in the CD31-negative perivascular tissues (p<0.05, Paired t test). On the other hand, Bax showed no significant difference in each observation period. It was concluded that upregulation of Bcl-2 in endothelial cells took place in the expanding phase of experimental rat periapical lesions.

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It is well-known that green tea catechin has many biological activities. We previously showed that green tea catechin inhibited inflammatory bone loss in mice. Green tea catechin includes various types of catechin components and the main component is epigallocatechin gallate (EGCG). It was reported that EGCG inhibited osteoclastogenesis. However, whether the inhibitory activities are stronger or not in comparison with other catechin components, and the effects of EGCG on osteoclastogenesis are not clear. The aim of the present study was to evaluate the inhibitory effect and intensity of EGCG on osteoclastogenesis using osteoclastogenesis assay by bone marrow macrophages cultured with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Bone marrow macrophages (BMMs) were cultured with various catechin components in the presence of M-CSF and RANKL for 72 h. To study the effects of EGCG on the differentiation of pre-osteoclast, EGCG was added at the same time as RANKL and tartarate resistant acid phosphatase (TRAP) positive mononucleated cells were checked at the 48 h time point. In another study, osteoclast formation at 72 h was checked when EGCG was added at time points of 0, 24, 48, or 60 h after stimulation with RANKL. Moreover, the osteoclast formation at 72 h was checked when BMMs were only pre-stimulated with EGCG for 24 h before being cultured with RANKL. TRAP positive multinucleated (more than 3 nuclei) cells were identified as osteoclasts. As a result, many osteoclasts were detected at 72 h, and many types of catechin components inhibited the osteoclastogenesis. EGCG is one of the catechin components that strongly inhibits osteoclastogenesis. EGCG inhibited the differentiation of pre-osteoclast induced by 48 h stimulation with RANKL. Moreover, EGCG inhibited osteoclastogenesis not only by EGCG addition at the same time as RANKL but also the addition at 24, 48 and 60 h after stimulation with RANKL. However, no influence was observed when BMMs were treated with EGCG only before RANKL stimulation. In conclusion, EGCG is a catechin component that strongly inhibits osteoclastogenesis. EGCG clearly inhibits the differentiation of pre-osteoclasts and osteoclasts from bone marrow macrophages in the presence of RANKL.

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Nanoscale techniques for silver production may assist a resurgence of the medical use of silver. Traditional methods for chemically synthesizing colloidal silver (CS) may lead to the presence of toxic chemical species or chemical residues, which may inhibit the effectiveness of CS as an antibacterial agent. The purpose of this study was to investigate the bactericidal effects of CS at different concentrations against key oral pathogens and the cytotoxicity to adult human gingival and periodontal ligament fibroblasts (HGF and HPDL). Streptococcus mutans (ATCC25175), Aggregatibacter actinomycetemcomitans (ATCC29522), Porphyromonas gingivalis (W83, ATCC33277), Prevotella intermedia (ATCC25611) and Fusobacterium nucleatum (ATCC25586) were selected for the bactericidal tests. HGF and HPDL were obtained from the gingival tissue around extracted adult human teeth. Each genus of bacteria (initial concentration of 1×10⁶ cell/ml) was incubated with CS for one minute at room temperature and their survival rates were measured by colony counting. Cytotoxicity of CS against HGF and HPDL was studied by cell proliferation assessment with Alamar Blue assay. The results demonstrated that a CS concentration of over 3 ppm had complete bactericidal effects on all tested bacteria. Compared with distilled water, CS concentration of 30 ppm showed significant inhibition of HGF and HPDL proliferation. However, such effects were not found when the concentration was lower than 20 ppm. The cytotoxicity (LD50) of CS determined by Alamar Blue assay was not found when the concentration was lower than 100 ppm. These results suggested that 20 ppm CS has ideal bactericidal effects against periodontal pathogens without significant cytotoxicity against host cells.

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We have considered that decreasing the surface free energy of the tooth surface may help prevent caries and periodontal disease. Recently, we successfully synthesized two antibacterial silane coupling agents, N-allyl-N-decyl-N-methyl-N-trimethoxysilylpropylammonium iodide (10-I) and N-allyl-N-methyl-N-trimethoxysilylpropyl-N-octadecylammonium iodide (18-I). The aim of this study was to investigate the cytotoxicities of these modifier agents. Glass plates were dipped in 20 mmol/l of 10-I and 18-I solutions for 1 hour at 20℃, and then were sterilized with ethylene oxide gas for 24 hours at 35℃. The modified glass plates were immersed in cell culture medium (MO5) for 24 hours at 37℃ to examine the toxicity of the extraction procedure. The extracted medium (100%) was diluted with the MO5 into ten different concentrations (0.5-50%). 0.5 ml of the V79 cell suspension (100 cells/ml) was seeded on a 24-well cell culture plate and pre-incubated for 6 hours at 37℃ in a 5% CO₂ atmosphere. After removal of MO5, 0.5 ml of the extracts containing MO5 was poured onto the cell culture of V79 cells. After 6-day cultivation, cells were stained with 0.1% methylene blue solution, and calculated. The cytotoxicities of various modified agents were evaluated according to the 50% inhibitory concentration (IC₅₀). The IC₅₀ value of 18-I was 18.8%, while that of 10-I was more than 100%. These results suggest that 10-I has no cytotoxicity, whereas 18-I has moderate cytotoxicity.

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The purpose of this study was to investigate whether the depth of post holes and the dual-cure selfetching resin bonding system affected root canal dentin. Extracted human central incisors were used in this study. Crowns of the teeth were sectioned at the cement-enamel junction using a low-speed diamond saw. Pulpal tissue was removed by using an endodontic file, and post spaces were prepared by using post drills in a low-speed handpiece to a depth of 5 or 10 mm and diameter of 1.5 mm. The roots were randomly divided into three groups, and their root canal dentin surfaces were treated with one of the following self-etching primers: 1) light-cure system G-bond (GB), 2) dual-cure system trial product (DC), or 3) dual-cure system Unifil Self-etching bond (SE). Post holes were filled with a dual-cure composite resin core material and the fiber posts were then placed in the root canal with finger pressure. Excess resin cement was removed, and cement in the root canal was light-cured. Each root was then cut perpendicular to the long axis, and the resin-dentin interfaces were observed with a scanning electron microscope (SEM) and surface profile measurement microscope. The results of surface analysis by SEM and surface profile measurement microscope revealed gaps in the resin-dentin interface in both specimens of GB. However, no gaps were found at the interface in both specimens of DC and SE. It is suggested that dual-cure bonding systems offer good sealability of build-up restoratives regardless of depth of post hole.

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Volatile sulfur compounds (VSCs) from oral plaque are known to be an important factor for oral malodor. There are a number of deodorizing agents for halitosis in the market, and studies have shown that polyphenols have an effect on reducing oral malodor. We previously reported on the bacteriostatic and deodorizing effects against Porphyromonas gingivalis, and deodorizing effect against mercaptoethanol in vitro by using Pancil PS-SP®, which is a compound of persimmon tannin and trehalose. In this study, we evaluated the suppression of clinical oral malodor with Pancil PS-M® (abbreviated as Pancil), which is an ultrafiltrated and purified form of Pancil PS-SP® with trehalose added. Oral malodor obtained from 100 participants was determined by an MS-Halimeter®. The subjects were evaluated at one hour after waking up (control), then after swallowing the distilled water. Pancil solution was applied in the mouth and was swallowed, then oral malodor was reevaluated two minutes and ten minutes later. As a result, the level of oral malodor was significantly reduced at both two minutes and ten minutes after swallowing Pancil solution compared with the control (p<0.05). It was clearly shown that Pancil strongly suppresses VSCs in vivo.

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The low level laser therapy (LLLT) effects of He-Ne laser irradiation on cultured bone-marrow-derived cells have not been well investigated. The objective of this study was to examine the LLLT effects of He-Ne laser irradiation on cell reaction of bone-marrow cells with a view to applying these effects in clinical settings. Femur-derived bone-marrow cells were obtained from Sprague-Dawley rats. He-Ne lased (1.20 J/cm²) cells in the second passage were incubated in plastic culture dishes, and the proliferation and differentiation were examined. The proliferation was evaluated by the optical density method, and the expression of alkaline phosphatase (ALP), osteopontin (OPN), and/or osteocalcin (OCN) were analyzed by real-time RT-PCR. ALP activity was also examined using culture area- and colorimetry-based assays. LLLT effects on He-Ne lased bone-marrow cells cultured on titanium plates were also examined in the same way. 1. He-Ne laser irradiation accelerated the strong proliferation and ALP (p<0.05) activity of plastic-cultured-bone-marrow cells compared with the non-irradiated cells. These results demonstrated LLLT effects on the proliferation and differentiation of He-Ne laser irradiated plastic-cultured-bone-marrow cells. 2. He-Ne lased bone-marrow cells cultured on titanium plates exhibited significant proliferation and differentiation activities. Real-time RT-PCR showed strong expression of ALP and OPN (p<0.05). These results also revealed LLLT effects on the proliferation and differentiation of He-Ne laser irradiated titanium-cultured-bone-marrow cells. Under the conditions of this study, He-Ne laser irradiation revealed LLLT effects on bone-marrow cells cultured even on titanium plates. Thus, He-Ne laser irradiation around the bone hole is expected to produce LLLT effects for the fixing of implants.

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In dentistry, ultrasonic waves have been used to remove calculus and plaque attached to the teeth and for root planing, and are an indispensable tool for removing calculus in periodontal tissue. It has been reported that plaque is removed by the cavitation effect caused by exposure to ultrasonic waves. However, little is known about the detailed effects of ultrasonic waves on periodontal tissue. In this study, we examined the influence of ultrasonic waves, which can remove the biofilm, on gingival epithelial cells. First, Streptococcus mutans biofilms were exposed to ultrasonic waves at the two frequencies of 1 MHz or 3 MHz, duty ratio of 0-90% and exposure time of 1 minute or 3 minutes, and the optimal conditions for biofilm removal were identified. We examined the effect of ultrasonic waves on the human gingival epithelial cell line Ca9-22 on cell proliferation, cell toxicity, production of IL-1β and number of cell deaths. Exposure to ultrasonic waves at 1 MHz and duty cycle of 60% for 3 minutes removed biofilms most efficiently. On the other hand, the biofilm removal rate using 3 MHz was highest at 40% duty cycle for 3 minutes. To explore the cytotoxicity of ultrasonic waves, we measured LDH in culture supernatant. Exposure to 3 MHz ultrasonic waves at 40-90% duty cycle increased LDH at all times. At 1 MHz frequency, LDH in gingival cells exposed to ultrasonic waves at 50-90% duty cycle for 1 minute or 3 minutes was significantly high. We performed the WST assay to clarify the effect of ultrasonic waves on cell proliferation. Exposure to 1 MHz and 3 MHz ultrasonic waves at 80 and 90% duty cycles for 3 minutes suppressed cell proliferation significantly. Furthermore, we examined the conditions of ultrasonic wave exposure at 1 MHz frequency for 1 minute (duty cycle 40-60%) and 3 minutes (duty cycle 50-90%), and the biofilm removal rate was more than 40%. We measured IL-1β production by ELISA and the number of dead cells stained by trypan blue under these conditions. Exposure to ultrasonic waves for 3 minutes increased IL-1β production and the number of dead cells. These data indicated that exposure to ultrasonic waves at 1 MHz frequency and 40-60% duty cycle for 1 minute had a beneficial effect on biofilm removal and was safer for gingival epithelial cells. In this study, we identified safer conditions for ultrasonic wave exposure yet with a high biofilm removal rate. It is necessary to examine not only cytotoxicity, but also the functions of cells and other cells.

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The main goal of root canal treatment is to disinfect the root canal. Proper cleansing of the root canal space is considered essential for success in endodontics. The disinfection of the root canal is performed by mechanical enlargement and chemical irrigants. Then an intracanal medicament is applied in the root canal. However, it is very difficult to completely eliminate bacteria from the root canal system, including those regions where access is difficult, such as canal irregularities, bifurcation, isthmus, fin, and the like. Excess enlargement of the root canal causes root fracture and may necessitate future tooth extraction and has a great influence on a prognosis. Thus, this study was performed to develop a novel method for complete disinfection of the root canal system. Optimal conditions for ultrasound and nanobubbles were examined to deliver medicaments deeper to the dentinal tubules of root canals. Nanobubbles of 0.2-0.3 μm in diameter and with a final concentration of 5% were superior to those of 10 %, and a ultrasound voltage 30 V was superior compared with 31 V to deliver medicaments. The root canals of extracted dog's teeth were infected by GFP-labeled Enterococcus faecalis in the dentinal tubules for 72 hours, and then ultrasound treatment was performed. Disinfection was examined in sections, 150 μm in thickness, after 48 hours-cultivation by confocal laser microscopy. As a result, a significant reduction of GFP fluorescence was seen by application of medicaments into the root canal with ampicillin together with 5% nanobubbles and ultrasound at a voltage of 30 V for 120 seconds compared with medication alone or in conjunction with nanobubbles and ultrasound. These results suggested that medicaments could be delivered deeply in the dental tubules using nanobubbles together with ultrasound leading to disinfection of the intracanal space in a short time.

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The purpose of this study was to evaluate the effect of remaining dentin thickness (RDT) on resin composite bond strength to flat dentin surfaces using various adhesive systems. The occlusal enamel of human molars was ground to expose flat dentin surfaces and finished using a carbide bur. Each specimen was restored with one of three adhesives: Clearfil Mega Bond (Kuraray Medical), Single Bond (3M ESPE) or Clearfil Tri-S Bond (Kuraray Medical), followed by buildup (3×5×2 mm, C-factor=0.3) of resin composite using Z100 (3M ESPE). After light-curing at 600 mW/cm^2 for 40 s, teeth were stored in the dark for 24 hours in water. The teeth were sectioned perpendicularly to the flat dentin to obtain beams having cross-sectional areas of approximately 1 mm^2. The remaining dentin thickness (RDT) was measured for all specimens. The micro-tensile bond strength μ-TBS, MPa) was determined. Data were divided into two groups: 1 flat: RDT<2 mm, 4 flat: 4 mm. Data (n=10) were analyzed using the Bonferroni test. The results of bond strength were Clearfil Mega Bond: 1 flat: 59.1 (6.2), 4 flat: 82.9 (7.0) ; Single Bond: 1 flat: 63.6 (5.2), 4 flat: 63.8 (7.0) ; Clearfil Tri-S Bond: 1 flat: 39.0 (3.4), 4 flat: 50.0 (4.7). The RDT 4 mm group using Clearfil Mega Bond showed the highest μ-TBS in all data. Clearfil Mega Bond and Clearfil Tri-S Bond showed a correlation of μ-TBS with RDT. For Single Bond, there was no significant difference in μ-TBS for all RDT. Thus, the bond strength of resin composite to flat dentin with different RDT was affected by the adhesive system used.

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In the mid-1970s, adhesive systems (phosphoric acid etching/unfilled resin) exhibited poor resin bonding to dentin instead of good bonding to enamel, leading to occasional pulpal irritation. To avoid such reactions, glassionomer cements (GIC) were sometimes placed on the dentinal walls. In deep cavities with a large volume, GICs were also placed as a dentin substitute to control polymerization shrinkage. This so-called "Sandwich Technique" is still adopted, even though the development of liner/base materials has progressed dramatically. However, resin bonding to these recent materials is unknown. The aim of this study was to examine resin bonding to recent liner/base materials conditioned by self-etching priming systems. A conventional high-strength GIC (FujiIX_GP, GC), two resin-modified GICs (Fuji Lining LC, GC and Vitre Bond, 3M ESPE) and a flowable composite (MI Flow, GC) were used as liner/base materials with four self-etching priming adhesives, a phosphoric acid or a silane coupling agent. Under the conditions of the present study, excellent adhesive properties were obtained between resin and self-etching primed GIC or resin-modified GICs. Phosphoric acid etching and/or silane coupling inhibited resin bonding to GICs under certain conditions, which require special attention.

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Objectives: The purpose of the present study was to evaluate the effect of sugar-free chewing gums containing Enterococcus faecium (E. faecium) on periodontal health and oral microbial flora. Material and methods: Thirty generalized chronic periodontitis patients (57.3 y ± 8.4) who signed an informed consent were recruited in this study. The subjects were grouped double-blindly into two groups: 1) fifteen subjects chewing E. faecium gum or 2) fifteen subjects chewing placebo gum. Before and after the four-week experiment period, saliva was collected after 5 minutes of stimulation and then used for biochemical measurements (lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities, free hemoglobin (F-Hb), pH) and microbiological measurements for periodontopathic bacteria: Porphyromonas gingivalis gingivalis), Treponema denticola (T denticola), Tannerella forsythia (T forsythia), Prevotella intermedia (P intermedia), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and total bacterial counts by using the real-time polymerase chain reaction (PCR) method. The microbiological quantitative measurements were also taken from the deepest periodontal pocket of each patient. To evaluate whether E. faecium was fixed in the oral cavity or not, saliva and periodontal pocket samples were also analyzed by conventional bacterial culture of E. faecium. Clinical periodontal measurements were done for Ramfjord's six representative teeth including plaque index (PlI), probing pocket depth (PPD), gingival index (GI), and bleeding on probing (BOP). Statistical analyses were performed with Student's t-test for subjects' distribution and Wilcoxon's signed-ranks test for microbial, biochemical and clinical data. A P value of below 5 % was considered significant. Results: The E. faecium gum group showed a significant decrease in red complex periodontal pathogens including 7: forsythia, 7: denticola and P.gingivalis compared to the base-line, especially in saliva samples (p<0.05). However, this tendency was not seen in subgingival plaque samples from each deepest periodontal pocket. Both groups had significantly improved clinical status in PH, GI, PPD, BOP and biochemical markers for ALP and F-Hb in saliva at four weeks after the experiments compared to the base-line. None of the patients showed E. faecium fixation or acidific change of pH. Conclusion: The present study suggests that sugar-free chewing gums containing the lactic acid-producing bacteria, E. faecium, could have beneficial effects on periodontal health by inhibiting periodontal red complex pathogens including P. gingivalis, T. denticola and T. forsythia.

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