Substance P (SP) enhances TNF-α mRNA expression in lipopolysaccharide (LPS)-stimulated dental pulp-derived cells. We investigated the influence of SP on proinflammatory cytokine mRNA expression in TNF-α-stimulated dental pulp-derived cells. We used human dental pulp-derived cells (HDPC) collected from a first premolar extracted for orthodontic treatment. SP enhanced mRNA expression of only TNF-α among the proinflammatory cytokines expressed in response to LPS stimulation, whereas it enhanced IL-1β, TNF-α, IL-8 and IL-6 mRNA expressions (all measured cytokine mRNAs) among the proinflammatory cytokines expressed in response to TNF-α stimulation. This tendency was strong when cells were stimulated with TNF-α at a low level. The results suggest that SP contributes to the expansion of inflammation by enhancing the cytokine network.

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It has been suggested that 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a glucocorticoid-activating enzyme, plays an important role in metabolic syndrome. Although the relationship between periodontal disease and metabolic syndrome has been established, the involvement of 11β-HSD1 in periodontal disease has not been reported. This study was performed to elucidate the role of 11β-HSD1 as a new therapeutic target in periodontal disease. Periodontal tissue was obtained from patients diagnosed as having periodontal disease for the experimental group and from patients whose teeth were extracted for orthodontic treatment as the control group. The expression levels of RANKL, RANK, osteoprotegerin and 11β-HSD1 mRNA were examined by reverse transcription-PCR. There was no apparent difference in RANKL, RANK or osteoprotegerin mRNA expression between the control group and the periodontal disease group, whereas a statistically significant increase of 11β-HSD1 mRNA expression was detected in the periodontal disease group compared with the controls. Furthermore, the depth of periodontal pockets significantly correlated with the increased expression of 11β-HSD1 mRNA. These results suggest that the increased expression of 11β-HSD1 may play a role in the progression of periodontal disease.

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Using a stereotactic technique, we created a novel, acute model of Parkinson's disease (PD) in 45 female rats by high-frequency electric ablation of bilateral substantia nigra pars compacta with a high-frequency electric coagulator. Targeting was aided by anatomical coordinates from a rat brain atlas. The rats developed persistent symptoms of PD, especially akinesia and postural instability. We tried various surgical protocols. The size and shape of the lesions that were ablated in a typical setting were evaluated. We determined that the optimal surgical parameters for creating a lesion were 1 W of power for 15 sec. The long-term viability of transplanted neural stem/progenitor cells was also examined. We confirmed they were viable for more than 60 days following surgery. As the animal model was designed to suit the study of cell transplantation therapy, it did not utilize neurotoxic chemicals to make lesions, thus avoiding their harmful effects. The technique described here may provide a good acute model of PD for the research of intracerebral cell transplantation. The chronic symptoms of this model and the differentiation control of transplanted cells will be the subject of future study.

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The use of autologous bone as the graft material is the gold standard for bone tissue replacement. However, only a limited amount of bone tissue can be harvested without inducing considerable donor site morbidity. Recently, multipotent stem cells have been studied as a material for regeneration of bone defects. Embryonic stem (ES) cells are capable of differentiating into cells of all the three germ layers and are attractive for the treatment of bone defects that require numerous cells as a source of progenitors. During development, most of the craniofacial skeleton is derived from cranial neural crest (NC) cells. We attempted to establish an efficient method for induction of craniofacial bone from mouse ES(mES) cells. We differentiated ES cells into NC cells by neurosphere culture with BMP4 treatment and cultured them in osteogenic medium. Most of the clusters were positive with Alizarin Red S staining and expression of osteoblast markers including Runx2, collagen type I, osteocalcin and osteopontin were detected by RT-PCR. In addition, induced NC cells had the potential to differentiate into peripheral neurons, adipocytes, chondrocytes and myocytes. These results suggest that osteoblasts are frequently generated from mES cells through induction of NC cells, and that they are a potential source of therapeutic tissue for craniofacial repair.

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We investigated the relationship among the expression of single-stranded DNA (SSD) and double-stranded DNA (DSD) and the p53 tumor suppressor gene in delayed neuronal death (DND) of CA1 in the gerbil hippocampus after transient brain ischemia. Although SSD proteins appeared in CA1 pyramidal cells 1 day after ischemia, p53 immunopositive cells and TUNEL positive cells were not detected, and there was no statistical difference in the number of pyramidal cells compared with the control animals. A significant reduction (29%) in the number of surviving pyramidal cells was observed compared with the control animals at day 3 after ischemia. Also, expression of SSD, p53 immunopositive cells and TUNEL positive cells were detected. At day 7 after ischemia, the number of p53 immunopositive pyramidal cells decreased along with a decrease in the number of SSD- and TUNEL-positive cells. The percentage of surviving pyramidal neurons was reduced almost 83% compared with the control animals. It was considered that this early expression of SSD with transient ischemia was a character of DND. Expression of SSD may be a trigger for progression of the death cascade.

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Osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites of the alveolar bone. RANKL, a member of the TNF family, expressed on osteoblasts stimulates RANK expressed on preosteoclasts, resulting in differentiation of preosteoclasts into mature osteoclasts. Several members of the TNF family including TNF and Fas ligand can induce apoptosis via activation of caspases (especially caspase-3) in various cells. A recent report showed that caspase-3 was activated at the early stage (within 15 min) of RANKL-induced osteoclast differentiation and that RANKL-induced osteoclast differentiation was suppressed by caspase inhibitors. Here we investigated whether caspase-3 might be activated and play a role at the late stage of RANKL-induced osteoclast differentiation. A slight but significant increase of caspase-3 activity was detected in mouse monocytic RAW264 cells differentiated into mature osteoclasts by treatment with RANKL for 3 days. In contrast with the previous report, co-treatment with Z-VAD-fmk, a pan-caspase inhibitor, augmented RANKL-induced osteoclast differentiation in RAW264 cells. Augmentation of RANKL-induced osteoclast differentiation by Z-VAD-fmk was also detected in mouse bone marrow macrophages. These results suggest that activation of caspases (presumably caspase-3) may play an inhibitory role at the late stage of RANKL-induced osteoclast differentiation.

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Trismus is a symptom of muscle malfunction and temporomandibular disorders. All cases of muscle malfunction are accompanied with hyperplasia of the tendon and aponeurosis of the masticatory muscles (HyTAM). We compared images by computed tomography (CT) and magnetic resonance imaging (MRI) to investigate the most suitable image analysis for diagnosis of malfunction of the masseter muscle. Using the best images, we analyzed the distribution of tendon and aponeurosis in the human masseter muscles. Bone, standard and soft kernel CT images were taken with 16 Row multi-detector CT and MR images of various sequences with a 1.5 Tesla MR Scanner (General Electric, Milwaukee, USA) of cow thigh muscles. Based on a previous study, we classified states of the tendon and aponeurosis of human masseter muscles. CT images of HyTAM patients and non-trismus subjects used in this study were compared with each other. The same as with the preceding experimental study, the tendons in 3D MR images were detected at low signal intensity, the same as with the 2D images. Although the 3D structures of the tendons were detected only with the multiplanar reconstruction method, they could not be observed in 3D reconstruction models because there was no signal intensity. In the CT images of the standard and soft kernel, the tendons and aponeurosis were confirmed for CT numbers between 70 and 100. Only in the standard images, could we make out the 3D reconstruction images and observe three-dimensional structures of the tendons. Based on the results of the preceding experimental study, it was determined that in the HyTAM patients, multiple tendons were connected to the mandibular angle from the zygomatic arch. There were few tendons connected to the mandibular angle from the zygomatic arch in the non-trismus subjects. MRI could detect the tendon and aponeurosis, but were not very suitable for observing three-dimensional structures, because the tendon and aponeurosis had no signal intensity. We found that it is possible to observe the conditions of tendon and aponeurosis by utilizing CT images. If hyper-tendon-aponeurosis of the masticatory muscles is the cause of trismus, it will be possible to detect this condition in the tendons of masseter and temporal muscles. By taking advantage of this diagnostic method, we can reduce the number of examinations that have up to now been necessary to diagnose HyTAM.

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We investigated the influence of prosthodontic treatment during the convalescent phase for care prevention of elderly patients with bone fractures resulting from falls. Eighteen non-dementia elderly patients (mean age 78 years) with bone fractures resulting from falls who were hospitalized at a convalescent rehabilitation hospital were selected as the subjects. Based on an intraoral examination upon admission, we categorized the subjects by assigning nine to a group with need of prosthodontic treatment (Prosthodontic Treated group; PT) who received the treatment, and nine to a group who needed prosthodontic treatment but had refused it (non Prosthodontic Treated group ; non-PT). Regarding the effect of care prevention, we selected four categories. Motor function, nutritional status, oral function and mental function were compared for the two groups on admission and at discharge. In motor function, oral function and mental function, significant changes were observed only in the PT group. Nutritional status was not significantly different for the PT group. The results showed that for the elderly patients with bone fractures resulting from falls, prosthodontic treatment during the convalescent phase, resulted in improvements in oral function as well as in care prevention.

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Pleomorphic adenoma (PA) is the most common salivary gland tumor and presents histological features indicative of ductal structures intermingled with mucoid, myxoid or chondroid tissues. The chondroid structure consists of chondrocyte-like cells that are associated with the cartilage lacunae of the basophilic matrix. We attempted to characterize the cartilage-like components using immunohistochemistry and gene expression analysis. We demonstrated that cartilage-like components were similar to normal cartilage in histochemistry and immnohistochemistry. Among the eleven genes with gene expression profiles that were increased by more than 8-fold in patients with PA compared with those with adenoid cystic carcinoma (AdCC), the chondrogenesis- associated YKL-39 and YKL-40 genes were selected for the present study. Quantitative analysis was performed using real-time RT-PCR for those two genes. The expression levels of the two genes were higher than those of other genes with elevated expression levels in the PA patients. Our results indicated that the structure of chondroid tissue may be similar to that of true cartilage and that cartilage formation tends to be observed in tissue types with less mesenchymal structure.

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Bacteria can accumulate on the surfaces of fixed orthodontic appliances during treatment. Bacteria on orthodontic appliances constitute a risk of white spot lesion development and lead to gingivitis. We attempted to clarify the mechanism of bacterial adhesion to orthodontic wires and understand the genetic background relating to this adhesion. The addition of 21 mM sucrose or glucose to a culture of Actinomyces oris strain MG-1 (MG-1) promoted adhesion to the orthodontic wire. The adherent strength of MG-1 was compared between media containing glucose or sucrose. In gluTSB, the MG-1 persisted even after ultrasonic treatment for 30 min, while it was easily removed when treated in sucTSB. Scanning electron micrographs revealed that MG-1 on the orthodontic wire formed meshwork-like structures around the cells in the presence of glucose. This is typically observed in a number of biofilm-forming bacterial species. However, no such unique phenotype appeared with MG-1 in sucTSB. Comparative microarray analyses indicated that expression of 18 genes was differentially altered with addition of glucose in the culture medium. They included the genes associated with regulation of biofilm formation and exopolysaccharide production, and those associated with the stress response system. These results suggest that addition of sucrose or glucose to the culture medium promotes the adhesion of MG-1 to orthodontic appliances, and that the adherence of MG-1 is particularly strong when mediated by biofilm formation enhanced by the presence of glucose.

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We developed a new approach for examining inflammation in the periodontal tissues and oral mucous membranes of patients resulting from the use of fixed orthodontic appliances. We examined the morphological changes in salivary cells. The epithelial cells of orthodontic patients exhibited changes in the cytoplasm-to-nucleus ratio. The epithelial cells were mainly neutrophils. The percentage of dead neutrophils increased in the orthodontic patients over time. These results indicated that orthodontic patients develop changes in the composition and morphology of salivary cells, the intensity of which depends on the treatment time. Salivary cells are biomarkers reflecting changes in the intraoral environment resulting from the use of the appliances.

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In the present study, crowns fabricated using the bite impression technique and using conventional methods were clinically compared to assess the efficacy of the bite impression technique. The subjects were ten patients seen at Osaka Dental University Hospital. Impression was taken using the bite impression technique and conventional methods. Evaluation criteria included the proximal contact, marginal integrity, excess crown height, occlusal adjustment time and amount of occlusal adjustment. The Wilcoxon signed-rank test was used for statistical analysis to examine the proximal contact and marginal integrity. The paired t-test was used to examine the excess crown height, occlusal adjustment time and amount of occlusal adjustment (p<0.05). The bite impression technique was superior to the conventional method in proximal contact (p<0.05). There was no significant difference between the two groups in marginal integrity. The excess crown height was significantly greater with the conventional method (p<0.05). The occlusal adjustment time was significantly shorter with the bite impression technique (p<0.05). The amount of occlusal adjustment was greater with the conventional method than with the bite impression technique (p<0.05). The results revealed the efficacy of the bite impression technique.

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Cracking is more likely to occur in castings than in ingots in dental cobalt-chromium alloy. We investigated a casting method that prevents cracking of laser-welded regions. For the experimental material, we used cobalt-chromium alloys for denture bases and porcelain fused-to-metal crowns. Ingots and castings were analyzed. Castings were prepared employing two methods : the conventional method and a method using a sintered mold. Welding was performed using a laser for dental use. The occurrence and cause of cracking were investigated by metal microstructure observation under a light microscope, fracture surface observation under a scanning electron microscope, and element concentration analysis using an energy-dispersive X-ray spectrometer. The occurrence of cracking was reduced in castings prepared using a sintered mold. Solidification cracks were widely present on the fracture surface in the welded region of the castings prepared by the conventional method, whereas the dimple pattern was widely noted on the fracture surface in the welded region of the castings prepared using a sintered mold. On quantitative analysis, the phosphorus content was high in the fracture surface with solidification cracks. We concluded that cracking sensitivity can be reduced in dental cobalt-chromium alloy by using a sintered mold.

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Fracture healing is a special wound healing response. Wounded areas are not restored, but are regenerated and connected. There are four components to the injury site ; the cortex, the periosteum, the bone marrow, and the external soft tissues, all of which contribute to the healing process. The extent to which each component is involved depends on the conditions present at the injured tissue, such as the level of growth factors, hormones, nutrients, pH, oxygen tension, electrical environment, and mechanical stimuli of the fracture. Mechanical stimuli, applied to tissue, have various clinical effects on cells depending on the amount of stimulation. However, these have not been demonstrated by an in vitro experiment, and no standardized system has been established. In the present study, mouse preosteoblast-like cells (MC3T3-E1) were used to prepare 3D tissue cultures. The tissue cultures underwent repeated compressive loading using a cyclic compressive device, which can change the load, to examine bone differentiation markers and transcription factors. MC3T3-E1 cells were 3D cultured using a collagen scaffold to determine the conditions that induce osteoblast differentiation. The expressions of bone differentiation markers were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Cells to which no repeated compressive loading was applied were defined as the FS group. The FS group showed increased expressions of collagen Ia1 (Col I a1), alkaline phosphatase, osteopontin, and osteocalcin, over time. The day when the cells were 3D cultured was defined as day 0. Cells to which repeated compressive loading was applied on days 1-7 were defined as the EL group. This group showed decreased expressions of ALP, osteopontin, and osteocalcin in proportion to the load. The day when the 3D culture was started was defined as day 0. Cells to which repeated compressive loading was applied on days 8-14 were defined as the LL group. It showed significantly increased expressions of Col Ia1, ALP, RUNX2, osterix and Dlx5 at 5 kPa. The LL group also showed significantly increased expressions of Col Ia1, osterix, and Dlx5 at 20 kPa, and significantly decreased expressions of osteocalcin at 40 kPa. Early mechanical loading tended to decrease the expressions of differentiation markers to suppress osteoblast differentiation. Additional mechanical loading following 7 day static culture significantly increased the expressions of differentiation markers and transcription factors. Thus, the mechanical loading suppressed osteoblast differentiation at an early stage and promoted the differentiation of differentiated osteoblasts. Mechanical loading at 5 kPa promoted differentiation, suggesting that optimal cyclic compressive loading induced the bone differentiation of pre-osteoblasts.

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Male Goto-Kakizaki (GK) rats approximately 6 weeks of age were used in this study. The experimental group was injected a total of 4 times with zoledronate (ZOL) intravenously over one week. The maxillary left first molars were extracted from the animals one day after the last ZOL administration. The rats were euthanized 1, 2, 4 and 8 weeks after the extraction. Bone morphometric analysis using hematoxylin and eosin (HE) staining and tartrate-resistant acid phosphatase (TRAP) staining was performed. Although histopathological observation showed that the experimental group had delayed healing of the extraction wound compared with the controls at 1 and 2 weeks after the extraction, sequestration was not observed at 4 and 8 weeks. There were significantly fewer osteoclasts in the base of the extraction wound of the experimental group than in the controls during the experimental period. When ZOL was administered to the GK rats, which are a diabetes rat model, bisphos-phonate-related osteonecrosis of the jaws (BRONJ) was not observed. However, it was suggested that the healing process of the extraction wound was delayed by an osteoclast disorder.

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Mastication, deglutition, and respiratory movements were analyzed before and after application of topical anesthesia to the oral cavity to investigate the how sensation in the oral cavity affects swallowing and influences respiratory movement. Videofluorography was performed on 10 adult subjects using an X-ray TV system. Nasal respiratory dynamics were measured using a differential pressure flowmeter. The subjects were instructed to hold 10 mL of an oral topical anesthetic in their mouths for 5 minutes to paralyze oral sensation. Each subject freely chewed and swallowed 8 g of barium-containing corned beef as a test food. Videofluorographic images and respiratory flow waveforms were simultaneously recorded in a data recorder and analyzed on a computer. Because of the oral hypesthesia, the valleculae aggregation time (VAT) were significantly prolonged. Although VAT was less than one second before anesthesia, the time was prolonged after anesthesia in some subjects. The transport phase from the oral cavity to pharynx, starting from transport movement of the tongue to passage of the bolus tail through the posterior nasal spine, was significantly prolonged. The period from the start of hyoid bone elevation to breath-holding was significantly prolonged, showing that breath-holding for deglutition was delayed. These findings suggest that, in mastication-deglutition, oral sensation influences early movement including the occurrence of deglutition movement and breath-holding.

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α-TCP granules were mixed with crushed autologous bone, and used as a bone substitute in the alveolar bone defect area around implants. We evaluated how the ratio of α-TCP affects treatment. Female Beagle dogs were used as experimental animals. The bilateral mandibular premolars were extracted, and, after osseous healing, a bone defect was created and an implant was placed in the alveolar bone of the tooth extraction site using a trephine bar cooled with saline solution. Crushed autologous bone was used to fill the bone defect area in one group, a mixture of 75% crushed autologous bone and 25% α-TCP (α-tricalcium phosphate) (weight ratio) in another group, a 50% mixture of crushed autologous bone in another group, a 25% mixture in another, and 100% α-TCP in another crushed autologous bone and 25% and filling was performed in one mixture group, and no filling material was placed in the control group. The wound area was closed wound, and the experimental animals were sacrificed 2, 4, and 8 weeks after surgery, and the healing was histologically observed for each group. Undecalcified ground samples were observed with HE staining and Villanueva-Goldner staining to evaluate the amount of bone formation. In the autologous bone group : newly generated bone was confirmed from the second week. The amount of bone continued to increase over time after that. In the α-TCP group the remaining α-TCP granules was observed between 2-4 weeks after surgery, and the invasion of soft tissue was confirmed by the 8th week, almost all the α-TCP granules had been absorbed, and the formation of new bone around the absorption area was confirmed. The α- TCP group showed marked bone formation, in comparison with the control group. The autologous bone group showed clear bone formation, in comparison with the α-TCP group. Furthermore, in the groups where autologous bone and α-TCP were mixed, there was favorable bone formation. The amount of bone formation was dependent on the mixture ratio.

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A strong interface between the denture-base resin and the metal frame of removable partial dentures is necessary to prevent fractures. Until now, thermoplastic acrylic resin of the denture base has only been mechanically joined. We investigated the application of a technique that directly joins the metal and resin materials called the Laser-Assisted Metal and Plastic (LAMP) joining method. This technique has been developed at the Joining and Welding Research Institute, Osaka University, for joining thermoplastic resin used in denture bases to a metal frame. We also confirmed that the LAMP joining method can be used to prepare dentures. In this study, we first investigated the strength of single-lap joint samples of thermoplastic acrylic resin for the denture base (acrylic resin) and a cobalt-chromium alloy prepared employing the LAMP joining method. A high-power laser diode was used for LAMP joining, with the irradiation condition set at an output of 160, 170, 180, 190, and 200 W. Five samples were prepared for each condition. The joined samples were subjected to a shear test, and the tensile load (N) was compared. In addition, we observed the cross-sectional and fracture surfaces of the joined region under a scanning electron microscope. To investigate application of the LAMP joining in dentistry we experimentally joined an existing palatal bar (cobalt bar wire) made of a cobalt-chromium alloy to an acrylic resin, and investigated the joint strength. The cobalt bar wire surface was joined to the acrylic resin using LAMP joining, with the laser focal position set at the bar surface. Cobalt bar wires were prepared with and without surface treatment with sandblasting. The irradiation condition was set at 150, 160, and 170 W. Five samples were prepared for each condition. Cobalt bar wires in the joint samples were subjected to a pull-out test, and the loads (N) were compared with those of non-irradiated samples. The fracture surface of the joined region of the cobalt bar wire that was pulled out was observed under a scanning electron microscope. Testing single-lap joint samples revealed that a predictable, high joint strength was obtained at 180 W. Analysis of variance with the irradiation condition as a factor clarified that the irradiation condition significantly influenced the joint strength. Observation of the cross-sectional and fracture surfaces showed that the, acrylic resin filled the uneven surface of the alloy without a break. This experiment of joining the cobalt bar wire encased in acrylic resin showed that the joint strength of samples without sandblasting was low regardless of the use of laser irradiation. Although laser irradiation increased the joint strength in sandblasted samples, variations were large. Observation of the fractured surface of the joined region showed that while only a small amount of acrylic resin was present in the non-sandblasted sample, the sample was tightly joined with the acrylic resin. This suggests that it is possible to have a strong bond of the cobalt bar wire embedded in the acrylic resin.

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We constructed a three-dimensional finite element model of the mandible, performed mechanical analysis at the time of corticotomy in the mandibular anterior segment, and evaluated tooth displacement and the stress distribution in the mandibular anterior teeth and anterior alveolar region. Based on computed tomographic (CT) data, a three-dimensional mandibular model was produced, and, using this model, we made the following three models : a model after extraction of the mandibular first premolars (control model) and models after horizontal corticotomy 2 mm below the tooth apices of the mandibular anterior teeth followed by vertical corticotomy only on the labial side (labial model) or on both the labial and lingual sides (labiolingual model). In the control model, stress in the alveolar region below the root apex was the greatest for the central incisors and decreased distally. After corticotomy, stress decreased in the body of the bone below the corticotomy line. This tendency was more marked after bilateral vertical labiolingual corticotomy. In the region above the osteotomy line, stress was greatest in the labiolingual model, followed in order by the labial and control models. Concerning stress distribution in the 6 mandibular anterior teeth, although no marked difference was observed in the coronal labial area between the labiolingual and labial models, stress at 1/3 the distance from the root apex as well as at the root apex was elevated in the labiolingual model. Displacement on the incisal margin was marked in the labiolingual model. Our results suggest that corticotomy is useful for tooth movement because the continuity of cortical bone is interrupted, resulting in the concentration of orthodontic forces. These forces were dispersed to the surrounding bone and on the teeth, while the stress was concentrated in the surrounding alveolar region. We found that In addition, corticotomy on both the labial and lingual sides, as opposed to only on the labial side, may be more effective.

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There are few reports concerning the effects of cigarette-smoke on orthodontic tooth movement. By quantitatively measuring the components present in the cigarette smoke, we hoped to be able investigate their effects on tooth movement. This study was done to evaluate a practical method of obtaining mainstream cigarette smoke. We used distilled water, physiological saline solution and ethanol as scavenging solvents. The mainstream smoke was allowed to flow into a gas-washing bottle. The solutions were isolated using a separation process and analyzed to determine the amounts of nicotine and tar present. Samples were analyzed using ^1H nuclear magnetic resonance (^1H NMR). The chemical 1,2-diphenylethane was added to the ethanol solutions for use during NMR measurements. Each of the solvents was scavenged. Ethanol, in particular, provided the greatest amount of cigarette smoke components by weight. Nicotine and tar, which have characteristic signals in the NMR spectra, were isolated for each solvent. The NMR showed the standard amount of nicotine in the ethanol solvent. From these results, we concluded that ethanol is the most suitable solvent for collecting mainstream cigarette-smoke. Based on these results, we can quantify the concentration of the solution administered to an animal as we like.

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