We investigated the relationship of stress with pain and discomfort from a localized pressure plate under an experimental acrylic plate covering the palatal mucosa. The fully dentate subjects were 6 males and 7 females with an average age of 26±2 years. All of them filled out the State-Trait Anxiety Inventory (STAI). The tests were performed with the subjects seated upright in a dental chair. Each subject was subjected to three experimental conditions for 20 minutes each: no experimental plate (N), wearing the experimental plate (D), and wearing the experimental plate with a localized pressure plate (DP). The subjects then recorded their experience of pain and discomfort on a visual analog scale (VAS) and by STAI. Saliva was collected 3 minutes and 20 minutes after the start of each test. Salivary samples were immediately frozen at -50℃ for storage prior to assay, and the cortisol concentration and α-amylase activity were measured using enzyme-linked immunosorbent assay and a kinetic measurement kit. The levels of each condition were performed with SPSS ver. 14.0J analytical software for repeated measures. A probability of less than 0.05 was considered statistically significant. The concentrations of free salivary cortisol and salivary α-amylase activity were significantly greater with DP than with N or D (p<0.01). The results showed that the stress of pain and discomfort from the experimental plate with the localized pressure plate increased the salivary cortisol concentration and α-amylase activity. These results suggest that the pain and discomfort from an ill-fitting denture can be the source of stress.

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Relapse is sometimes observed during the postoperative course following sagittal split ramus osteotomy which is widely used to correct jaw deformities. Relapse may be caused by biomechanical factors such as the postoperative occlusal force. We evaluated serial changes in the stress distribution associated with postoperative occlusal force and jaw-closing pressure on the mandible and osteosynthesis plate using three-dimensional finite element analysis. Based on CT data, we produced mandibular models 1, 3, 6, and 12 months after sagittal split ramus osteotomy, and subjected them to simulated occlusal force and jaw-closing pressure. Changes in equivalent stress in the proximal and distal segments, at the osteosynthesis site, and the fixation plate were evaluated by three-dimensional finite element analysis. The equivalent stresses in the proximal and distal segments slightly increased over time from 1 to 12 months after the operation. In particular, marked stress concentration was observed at the anterior border of the ramus at each measurement area. Stress at the osteosynthesis site increased from 1 to 6 months after the operation, but decreased after 12 months. As a result of postoperative occlusal forces and jaw-closing pressure, stress was concentrated at the anterior border of the ramus in the proximal segment. Between 3 and 6 months after the operation, tensile stress was concentrated at the upper and lower ends of the osteotomy line at the osteosynthesis site. These biomechanical findings indicate the application of clockwise stress on the distal segment up to 6 months after the operation. We concluded that sagittal split ramus osteotomy runs the risk of relapse between 3 and 6 months after the operation.

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The objective of this study was to clarify the effect of platelet-rich plasma (PRP) on alveolar bone around implant fixtures. Although various studies on PRP have been reported, it has not been clarified what growth factor contained in PRP promotes new bone formation around implants in the early bone formation process. Likewise, bone quality, new bone mass, and differences between the pre- and postoperative osteocalcification rates are not well understood. We investigated these issues using immunohistochemical staining and histological examination. We experimentally prepared alveolar bone defects in six adult female beagles, similar to defects created by implant placement, and placed fixtures in the defects. The implant regions were divided into those treated with blood clot, those treated with transplanted autologous bone alone, those treated with PRP alone, and those treated with PRP plus autologous bone. The concomitant application of PRP to bone-defective regions around implant fixtures generated the following results. Histologically, immature new blood vessels and new bone were formed in the PRP plus autologous bone graft group. Immunohistochemical examination of growth factors released in the early process of healing revealed that the transforming growth factor-β and basic fibroblast growth factor levels were high. Vascular endothelial growth factor -positivity was noted in the early phase in the PRP plus autologous bone graft group. The osseous tissue form measurement method clarified the bone quality, new bone mass, and pre- and postoperative osteocalcification rates. All of these were greater in the PRP plus autologous bone graft group than in the other groups. New bone forming capacity was greater in the autologous bone graft than in the PRP graft group. We also found that Villanueva bone staining may be useful for the observation of new bone. These findings suggest that the concomitant application of PRP and autologous bone in the bone-defect regions around implants promotes the growth of blood vessels necessary for wound healing of the tissue, and is effective for long-term bone regeneration.

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There are many methods for regeneration of periodontal ligament damaged by periodontal disease. Although enamel matrix derivative (EMD) is one of the most effective methods, its mechanism is not well understood. We explored how the cell proliferation, differentiation and gene expression of periodontal ligament fibroblasts (HPdLF) are influenced by peptides derived from EMD. In addition, we studied hybridization using microarray analysis. Microarray analysis showed that the expression of genes related to osteogenesis increased with the use of EMD. Our study showed that peptides derived from EMD are beneficial to periodontal ligament cells.

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We created periodontal defects on the palatal roots of normal and type 2 diabetes model rat maxillary first molars in order to examine the relation between advanced glycation end products (AGEs) and type III collagen. We observed the pathology and histology of the early wound healing. The experimental group showed poor formation of type III collagen and increased amorphous blood capillary localization of AGEs in vascular endothelium. It is suggested that AGEs inhibit formation collagen and interfere with the early wound healing process.

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To prevent alveolar bone resorption in periodontitis, it is important to understand the changes in expression of various molecules during differentiation of osteoclasts from their precursor cells. Since osteoclasts originate from monocytes and macrophages, we looked at changes in mRNA expression of osteoprotegerin (OPG), receptor activator of NF κ-B ligand (RANKL) and receptor activator of NF κ-B (RANK) during differentiation of the THP-1 cell, a human monocytic leukemia cell line, into macrophages. We demonstrated that OPG, a secretory protein and a competitive inhibitor of RANK binding to RANKL, was induced when THP-1 monocytes differentiated into macrophages. In contrast, expression of RANK or RANKL did not change during the differentiation. The induction of OPG mRNA in macrophages possibly influences the regulation of the differentiation of osteoclasts. This work was supported in part by Grant 07-05 from Osaka Dental University Research Funds.

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Mesenchymal stem cells (MSCs) are highly proliferative and can differentiate into multiple lineages. It has been demonstrated that S100 A4, a S100 calcium-binding protein, can act as an inhibitor of mineralization. We investigated the effect of S100 A4 inhibition by short interfering RNA (siRNA), which is a modified siRNA with improved silencing longevity. We also studied the specific effect of siRNA on the expression of many genes related to osteogenic differentiation. Inhibition of S100 A4 by siRNA resulted in increased expression of many genes related to osteogenic differentiation. This study provided a tool for better understanding of osteogenesis differentiation in MSCs.

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We investigated the relationship between discomfort and stress from an experimental acrylic plate covering the palatal mucosa (exp. plate) using localized pressure plates of varying thickness. The fully dentate subjects were examined under the following five experimental conditions: without an exp. plate (N), with an exp. plate (D), and with a combination of an exp. plate and localized pressure plates of thickness of 0.1mm, 0.2mm and 0.5mm (DP 1, DP 2, and DP 3, respectively). These apparatuses were placed in the oral cavity under pressure for 5min and then left in place at rest for 15min. Cortisol concentration and α-amylase activity were measured using Enzyme-Linked Immunosorbent Assay (ELISA) and a kinetic measurement kit. We found a significant increase in the salivary cortisol concentration and α-amylase activity caused by the increase in discomfort resulting from localized pressure.

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We measured the retentive force and negative pressure generated between the inner and outer crowns of electroformed telescope crowns having 0°, 2°, 4° and 6° taper with glycerin solutions of different viscosities. The peak retentive force was significantly greater at a glycerin concentration of 20% or more, when compared with no solution. The increase in maximum negative pressure was greater at 0° or 2° than at 4° or 6°, and greater increases were observed with glycerin concentrations of 60% and 80% compared with lower concentrations. At 2°, the retentive force did not increase significantly as the glycerin concentration increased when holes were present in the outer crowns. The above findings suggest that the viscosity of saliva and the negative pressure generated between the inner and outer crowns effectively increases the retentive force in a 2° tapered electroformed telescope crown.

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We examined the detection of the human papillomaviruses (HPV) p53, mutant type p53, Rb and p16 in oral dysplasias and oral squamous cell carcinomas. We used 10 samples each of irritation fibromas (IF), low grade dysplasias (LGD), high grade dysplasias (HGD) and squamous cell carcinomas (SCC) obtained from Osaka Dental University Hospital. We detected the DNA of HPV16 and HPV18 by polymerase chain reaction (PCR) and the mRNA of HPV by in situ hybridization (ISH). We also studied the immunohistochemical expression of p53, mutant type p53, Rb and p16. HPV16 was detected in almost all cases of LGD and SCC, but in no cases of ISH. Marked expression of mutant type p53 and Rb was seen in HPV16 positive LGD and SCC. HPV infections were detected by PCR in almost all cases of HGD and SCC. Specimens that showed over expression of mutant type p53 and Rb were almost all positive for HPV16. It is suggested that HPV16 may play an important role in the transformation of p53 and Rb.

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Natural killer (NK) cells exhibit their cytotoxicity through adhesion to the target cells via adhesion molecules. We investigated the effect of the adhesion molecules on NK cell cytotoxicity. Cytotoxicity assay revealed that cells of the NK cell line NK 92 MI kill the cells of the melanoma cell line WM 35. Fluorescence-activated cell scanning (FACS) analysis confirmed the expression of CD 11 a/CD 18 (LFA-1) by NK 92 MI cells and CD 54 by WM 35 cells. Next, we investigated whether the cytotoxicity was dependent on the adhesion molecule CD 54. The anti-CD 54 neutralizing antibody significantly suppressed the killing of WM 35 cells by NK 92 MI cells. These results suggest that the killing of WM 35 cells by NK 92 MI cells might be CD 54-dependent.

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It is known that the N-methyl-D-aspartate (NMDA) receptor is important for learning/memory capability and neuropathic pain. Although it has been reported that NMDA receptor antagonists cause learning/memory disorder, they are effective for neuropathic pain. We investigated the relation between the NMDA receptor and learning/memory capability using neuropathic pain chronic constriction injury (CCI) model rats on the 8-arm radial maze and the step-through type passive avoidance apparatus. The CCI model rats performed better on the tests than either the normal animals or those that had received NMDA receptor antagonists. These findings suggest that the neuropathic pain of the CCI rats raised their learning/memory ability through the NMDA receptor.

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During anesthesia, muscarinic parasympathetic antagonists can be used as a preoperative medication on order to reduce salivary flow, tracheobronchial secretions, and pharyngeal secretions, as well as to decrease the acidity of gastric secretions. We did an in vivo study to examine whether muscarinic receptor subtypes play a role in the exocrine function of the rat submandibular gland. The effects of atropine, pirenzepine, methoctramine, 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) and glycopyrrolate were examined on secretion from the submandibular gland evoked by acetylcholine in pentobarbital-anesthetized rats. Glycopyrrolate caused less elevation in heart rate compared with other anticholinergics. Atropine, 4-DAMP and glycopyrrolate markedly inhibited acetylcholine-evoked fluid responses. Pirenzepine showed significantly lower inhibitory potency than atropine, 4-DAMP or glycopyrrolate, while methoctramine had even less of an inhibitory effect. Pirenzepine, 4-DAMP and glycopyrrolate significantly inhibited both protein concentration and amylase activity in the acetylcholine-evoked submandibular saliva, while methoctramine did not affect the responses. The reduction of the protein concentration and amylase activity in submandibular saliva caused by pirenzepine, 4-DAMP and glycopyrrolate were inhibited by N^G-Nitro-L-arginine methyl ester (L-NAME). Thus, it might be effective to administer anticholinergic drugs together with L-NAME as premedication. These results suggest that glycopyrrolate significantly decreases the salivary secretion, and has significantly fewer side effects than other muscarinic parasympathetic antagonists. Further controlled studies are required to determine the safety, efficacy and patient tolerance of glycopyrrolate.

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Brief exposure to volatile anesthetics before prolonged ischemia exerts a cardioprotective effect. We investigated whether extracellular signal-regulated kinase (ERK) is involved in sevoflurane-induced preconditioning. Isolated perfused guinea pig hearts were subjected to 30min global ischemia and 120min reperfusion (CTL). Sevofluraneinduced preconditioning was elicited by administration of sevoflurane at one minimum alveolar concentration with 10min washout before ischemia (SEVO). Infarct size was significantly reduced in SEVO compared to CTL. Phosphorylation of ERK at 10min after reperfusion was significantly increased in SEVO compared with CTL. Activation of ERK plays an important role in sevoflurane-induced preconditioning. This work was supported in part by Grant 07-06 from Osaka Dental University Research Funds.

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