Using scanning electron microscopy, I investigated the morphology and development of subneural apparatuses at the neuromuscular junctions of the masticatory muscles in male Wistar rats fed a solid diet and in those fed a powdered diet. Muscle samples were taken at 2, 3, 5, 7, 12, and 20 weeks of age. The muscles were fixed for 2 hours with 3% glutaraldehyde in 0.1M phosphate buffer at pH7.3, postfixed with 2% buffered OSO_4 for 1 hour, and then treated with 8N HCI for 30 min at 60℃.The specimens were examined with a Hitachi S-4000 scanning electron microscope.The ratio of the length of the junctional folds to their maximum width(L/W)was measured as a shape factor.The number of junctional folds increased with age in both groups.The average ratio of L/W increased with age in both groups.There was a significant difference in the L/W ratio of the two groups at 5 and 7 weeks of age.These results suggest that the morphology and development of the subneural apparatuses at the neuromuscular junctions might be influenced by dietary consistency.

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I investigated radiation damage, including osteoradionecrosis, arising from tooth extraction in fields that had received radiation therapy for head and neck cancer, and evaluated the effectiveness of pilocarpine for xerostomia.Between January 1990 and April 1996, I examined 30 patients for bone changes after tooth extraction in fields irradiated at the Department of Oral Radiology, Osaka Dental University Hospital.Nineteen of the patients had been treated for nasopharyngeal cancer and 11 for oropharyngeal cancer.Between January and April 1996, 4 additional patients were given pilocarpine hydrochloride(3-mg, 6-mg and 9-mg of KSS-694 orally three times a day)for 12 weeks and evaluated every 4 weeks as a base line.One had been treated for nasopharyngeal carcinoma, two for cancer of the cheek and one for an unknown carcinoma.Eighteen of the patients(11 with nasopharyngeal carcinoma and 7 with oropharyngeal carcinoma)had extractions.Use of preoperative and postoperative radiographs indicated that damage to the bone following tooth extraction after radiation exposure was related to whether antibiotics were administered the day before the extraction, whether forceps or elevators were used, and whether the tooth was in the field of radiation.Xerostomia improved in all 4 of the patients who received 6-mg or 9-mg of pilocarpine.It improved saliva production and relieved the symptoms of xerostomia after radiation therapy for head and neck cancer, although there were minor side effects such as fever.This information can be used to improve the oral environment of patients who have received radiation therapy for head and neck cancer, and to better understand their oral environment.

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To establish an effective method for evaluating muscle fatigue after mastication, we measured the stiffness and elasticity in the masseter muscle after experimental induction of fatigue by mastication using a newly developed tactile sensor.Among the masticatory muscles, the masseter is thought to create the greatest forces of mastication and occlusion.We compared the results of the tactile sensor and the frequency analysis of the electromyogram with conventional methods of evaluating muscle fatigue.The tactile sensor indicated that increases in stiffness(decreases in the slope of the tangent of the hysteresis curve)and decreases inelasticity(increases in residual deformation)of the masseter muscle on the mastication side were proportional to the number of masticatory cycles, which were measured at 0, 50 and 100 cycles.Increases in muscle stiffness and decreases in muscle elasticity associated with an increase in the number of masticatory cycles were also observed on the non-mastication side, although they were not as pronounced as on the mastication side.A frequency analysis of simultaneously recorded electromyograms of the masseter muscle showed a decrease in the higher frequency components(300-500Hz).However, this parameter was proportional to the number of mastication cycles in only a small number of the subjects on either the mastication or non-mastication side(2 / 6 and 1 / 6, respectively).It was unclear in the other subjects.These results suggest that muscle stiffness and elasticity measured with the tactile sensor more accurately reflect muscle fatigue than conventional parameters.

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We compared the perceived quality of panoramic dental images obtained using both digital charge-coupled devices(CCD)and analog radiographic film to determine the ideal clinical exposure conditions.Images were exposed on an averaged man phantom using same machine at 60, 70 and 80kV and 2, 4, 6, 8 and 10mA.Many clinicians have noted that the visual characteristics of images made using digital radiography are different from those of film based images.We selected 15 digital radiographs and made a computer program that allowed the observers to manipulate the contrast and brightness of the image.The diagnostic image quality was evaluated by six observers, five of whom were oral radiologists, and the other a postgraduate student.The observers were asked to evaluate 20 points on both the digital and analog images.We found that the best digital radiographic images were exposed at 70kV / 8mA, and the best analog images were at 70kV / 6mA.However, the digital images deteriorated at tube currents above 8mA for all tube voltages.The observers found no difference between the two methods for the 20 anatomical points evaluated.These results indicate that the digital system is a useful diagnostic tool that also reduces radiation exposure.We concluded that digital images are clinically equivalent to conventional film images for panoramic dental radiography.

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I examined the effect of vitamin C deficiency and ovariectomy on the mandibular condyles.Eight-week-old female Wistar rats and non-ascorbate-synthesizing ODS rats were used in the experiment.The Wistar rats that received no operation were Group 1, and those that were ovariectomized were Group 2.The ODS rats with no operation were Group 3, and those that were ovariectomized were Group 4.All animals were sacrificed at 2, 4 and 6 weeks after operation.Group 1 is the control group.The mandibular condyles were investigated histologically and immunohistochemically using polyclonal antibodies for types I, II and X collagen.The bone and cartilage became fragile and endochondral ossification in the condylar cartilage was disrupted with vitamin C deficiency, and these effects were accelerated by the ovariectomy.

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Several biological peptides have been tested to develop new and powerful sialogogues for people with xerostomia and hyposalivation.Tachykinins have been found to elicit secretions from the salivary glands.Of these, physalaemin was determined to be the most potent.I hoped to determine if there are any new, powerful, sialogogic peptides or traditional Chinese medicines for the promotion of salivation.Several new peptides have been synthesized by the multipin method to elucidate the relation of structure and sialogogic activity of these tachykinins.I found that in physalaemin, the optimal amino acid positions were 3, 4, 7, 8, 9, 10 and 11, and that an amide group in the C-terminal end was essential to promote salivation.In contrast, sialogogic activity has characteristically been inhibited by amino acids in positions 1, 2, 5 and 6.Therefore, more powerful sialogogues can be developed if amino acids in positions 1, 2, 5 and 6 are replaced by amino acids in tachykinins, as replacement of the amino acid in position 6 clearly indicated.I also found that traditional Chinese medicines may aid the new peptides.Further study is needed before these new peptides can be used to correct hyposalivation in humans.

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There have been no examples of the use of oral fluids for screening human systemic diseases in the dental field.Since 1994 we have evaluated the possibility of using oral fluids to screen for tumor markers, Alzheimer's disease associated gene products, gene products accompanying breast cancer, and D-glucose(diabetes mellitus).We did various experiments to see if oral fluids could be used for this purpose, and concluded that it may be possible in the future to do this type of screening.

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I evaluated the effect of vertical and rotational loads on cell growth activity, intracelluar lactate dehydrogenase (LDH)activity, morphological changes, and the expression of five cytokines on human periodontal ligament cells in vitro.It was found that cell growth and intracelluar LDH activity were reduced as the load increased.Although IL-6, IL-8 and TNF-α were expressed in all samples tested, IL-1β and GM-CSF were not.Morphological changes were induced with vertical loads greater than 300g and with rotational loads of 45°.

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Calcium phosphate crystalline ceramic is a castable ceramic with good translucency and superior mechanical properties.I measured the amount of wear and loss of gloss on the surface of calcium phosphate crystalline ceramics after 40, 000 cycles of tooth brush abrasion, and determined wear resistance of test specimens using the two-body sliding wear test.Mechanically fine polished calcium phosphate crystalline ceramic showed excellent abrasion resistance and maintained its original gloss.The wear resistance of glazed calcium phosphate crystalline ceramic was best against natural tooth enamel, and was poorer against Au-Ag-Pd alloy.

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I investigated the effect of a vitamin-D deficient diet on the condylar cartilage of rats aged 3, 9, 13 and 21 weeks, using antibodies for types II, IX and X collagen, respectively.Western blot analysis indicated the existence of the three collagen types in the condylar cartilage of animals fed the vitamin-D deficient diet and in controls that were fed a normal diet.Immunohistochemical observation indicated that both types II and IX collagen were localized mainly in the maturative and hypertrophic cell layers, while type X collagen was localized in the hypertrophic cell layer.Differences in localization between the two groups were observed at 13 and 21 weeks of age.

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Since infiltration of T cells and natural killer (NK) cells is observed in inflammatory gingival tissues, local immunoresponse is believed to be involved in periodontal diseases.I investigated costimulatory effects of CD2 stimulation on IL-2-mediated NK cell proliferation and signal pathways through CD2 and IL-2 using the NK cell line, NK3.3. Costimulation of CD2 crosslinking significantly enhanced IL-2-mediated NK3.3 proliferation.Crosslinking of CD2 induced tyrosine phosphorylation of protein tyrosine kinase, Syk and the adapter proteins, Shc and Cbl. However, IL-2 stimulation enhanced tyrosine phosphorylation of Shc, but not Syk nor Cbl. Cbl constitutively associates with Grb 2 through the N-SH3 domain, whereas Shc associates with Grb2 through the SH2 domain in an activation-dependent manner.

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I investigated radiation damage including osteoradionecrosis arising from tooth extraction in irradiated fields after radiation therapy for head and neck cancer and evaluated the effectiveness of pilocarpine for xerostomia.Using preoperative radiographs, it was found that prevention of damage to the bone following tooth extraction after radiation exposure was related to administration of antibiotics the day before the extraction, and depended on whether forceps were used and whether the tooth was in the field of radiation.Pilocarpine improved saliva production and relieved symptoms of xerostomia after radiation therapy for head and neck cancer.This information can be used to improve the oral environment of patients who have received radiation therapy for head and neck cancer, and to elevate understanding of their oral environment.

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I studied the effect of fluoride on the surface layer of dentin using an atomic force microscope(AFM)and X-ray photoelectron spectroscopy(XPS).Sound dentin and artificial white spot lesions were treated with a NaF solution (0.2, 1.0, and 2.0% NaF at pH 6.8)for 4 min at room temperature.XPS analyses indicated FAp or FHAp were produced on both dentin surfaces with increasing F concentration.Futhermore, CaF_2 was detected on both dentin surfaces that had been exposed to a high F concentration (2.0%).The AFM detected changes in the surface morphology and alterations in the dentin crystal shape on the surface of sound dentin and on artificial white spot lesions after treatment with the NaF solution.It was suggested that NaF treatment changed the surface structure of sound dentin and artificial white spot lesions by producing reaction products to the F.

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Using normalized electromyogram(EMG)linear envelopes, I investigated the efffect of aging on the activity patterns of masticatory muscles and studied the factors causing these effects.EMG signals were recorded from the bilateral masseter and anterior temporal muscles during mastication of nine different foods.Although the activity patterns changed in the dentate subjects with the different foods, there was little change in those of the complete denture wearers during mastication of various foods.Factor analysis indicated that the teeth were the most important factor in muscle activity patterns for all most all of the foods.These results suggest that although aging has little effect on the activity patterns of the masticatory muscles, the effect of tooth loss is considerable.

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I attempted to quantify occlusal relation during maximum intercuspation.Two silicone occlusal registrations were taken for each of six normal subjects and six patients with temporomandibular disorders.The silicone occlusal registration was digitized and mandibular displacement during intercuspation was estimated by the relationship of the maxillary and mandibular occlusal surfaces.The occlusal surface contact area was calculated for an intraocclusal distance of loss than 60μm.It was found that mandibular displacement during maximum intercuspation could be quantified.Also, it was possible to find the region of most frequent occlusal contact and the orientation of this contact.

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I studied of masticatory electromyographs during tapping movements, and analyzed the distribution of parameters in young adults of 22 to 23 years of age.The parameters of duration, cycle time, coefficient various and integration value showed a wide distribution, and within limits, the patterns often showed a high degree of similarity.This suggests that an individual's tapping movements reflect occlusal home and a special pattern acquired through that particular person's behavior.

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In order to determine if disturbances in mastication resulting from occlusal dysfunction affect mental stress, I created experimental occlusal interferences in rats and monitored dopamine release in their prefrontal cortex.Extracellular fluid dopamine volume in the prefrontal cortex was measured by the brain microdialysis method.When the animals with occlusal interferences were fed cubed food, there was an increase in dopamine volume in the prefrontal cortex.Experimental occlusal interference affected dopamine metabolism in the rat prefrontal cortex when the animals were fed, clearly inducing a strong stress reaction.

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I used histological and immunohistochemical methods, and enzymography to investigate the expression and localization of MMP-3 and TIMP-2 in the temporomandibular joint (TMJ) of male ICR mice, which are typical osteoarthritis models.Toluidineblue stains showed glycosaminoglycans forming proteoglycans were decreased in the surface cartilage toward the end of the experiment.Immunohistochemistry revealed MMP-3 was positive in the surface cartilage of the TMJ.The distribution of TIMP-2 was similar to that of MMP-3.ProMMP-3, the active form of MMP-3, and TIMP-2-MMP-3 complex were detected in the cartilage and / or the mixture of the disc and synovium with immunoblotting and enzymography.These results suggest that the balance between MMP-3 and TIMP-2 are disrupted in the surface of the osteoarthritic cartilage and the imbalance triggers progressive osteoarthritis.

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I examined the effect of vitamin C deficiency and ovariectomy on the mandibular condyles.Eight-week-old female Wistar rats and non-ascorbate-synthesizing Osteogenic Disorder Shionodi(ODS)rats were divided into an ovariectomized group and a control group and sacrificed at 2, 4, and 6 weeks after operation.The mandibular condyles were investigated histologically and immunohistochemically using polyclonal antibodies for types I, II and X collagen.We found that vitamin C deficiency caused weakening of the bone and cartilage and disruption of endochondral ossification in the condylar cartilage.Ovariectomy accelerated these effects when done in combination with vitamin C deficiency.

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I studied whether the drug-efflux mechanism is related to streptomycin (SM) and gentamicin (GM) resistance in anaerobic gram negative rod Prevotella intermedia MA1 (MA1) and MA1-V2(V2).In MA1 24hrs after inoculation, the ratio of growth with treatment by with carbonyl cyanide m-chloro phenyl(CCCP)to growth without was 0.87.The ratios of growth with 32, 64 and 128μg / mL SM combined with 5μg / mL CCCP to the growth with SM alone (growth ratios)were 0.58, 0.45 and 0.58, respectively.These ratios with 5μg / mL CCCP and 32, 64 and 128μg / mL GM were 0.47, 0.51 and 0.43.There was growth inhibition of MA1 with exposure to CCCP alone.The growth ratios for V2 with 10μg / mL CCCP-SM or CCCP-GM were the same as those for MA1.The accumulations of SM and GM in MA1 and V2 cells whose growth ratio decreased with 5 to 10μg / mL CCCP were larger than those without CCCP.The cell strains whose growth ratio decreased after exposure to CCCP were frequently collapsed in transmission electron micrographs.These results suggest that the drug-efflux mechnism is closely related to the SM and GM resistance of Prevotella intermedia MA1 and MA1-V2.

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I studied β-lactamase, outer membrane permeability (OMP), and efflux of drugs to elucidate the mechanism of β-lactam antibiotic resistance of Prevotella intermedia MA1-V2.0.25〜8μg / mL of CVA, SBT, and TAZ, and 1 / 16 MIC of PIPC, CEZ, and CFTM were added to cultures at the middle logarithmic phase and the treatment continued for 30 minutes.The β-lactamase activity in the periplasmic space of these cells was inhibited 80〜96%.The percentage varied depending on the β-lactamase inhibitor and β-lactam combination.The inhibitors inhibited β-lactamase activity in the periplasmic space, and decreased the viable count and MIC of 3 β-lactams.I studied the effect of OMP on the MIC of β-lactams, and also measured the viable count with carbonyl cyanide m-chlorophenyl-hydrazone to study efflux of drugs.No decrease was detected in either the MIC or viable count.These results suggest that β-lactamase of this strain play a direct role in the resistance of β-lactam antibiotics.

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Although the use of propofol for sedation during dental treatment is on the increase, there have been few reports on its effect on the central nervous system.I used microdialysis to examine the effect of propofol on dopamine metabolism in the rat prefrontal cortex to study its effect on the central nervous system.The dopamine level of the animals receiving propofol was significantly increased with stimulation compared with those receiving physiological saline.Administrating both propofol and bicuculine with noxious stimulation did not significantly change the dopamine level.This suggests that the mechanism of propofol might be related to the GABA_A receptor.

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The elk-1 oncogene is a member of the est family and regulates gene expression in a variety of biological processes.We used molecular biological techniques to investigate the expression pattern of the elk-1 gene in mice and in human oral precancerous lesions and squamous cell carcinomas.elk-1 mRNA was moderately expressed in erythroplakia and leukoplakia, weakly expressed in normal gingiva, and strongly expressed in squamous cell carcinomas.Furthermore, elk-1 mRNA was detected in mice From 8 to 12 days of gestation.It was weak in the ovaries, and was not detected in the intestine, stomach, uterus, kidney, spleen, liver or testes.These results suggest that elk-1 may play an important role in the development of oral precancerous lesions and squamous cell carcinomas.

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In order to evaluate deformation of the cervical vertebra during unilateral occlusion, we placed strain gauges on the right and left laminae of the first, second and third cervical vertebrae in monkeys.We electrically stimulated the bilateral masseter muscles of the anesthetized animals to produce mild, intermediate and strong occlusal forces by inserting 3mm and 7mm thick wood chips between the left maxillary and mandibular first molars.As the thickness of the chip and the occlusal force increased, the first cervical vertebra tended to lean toward the working side while the second cervical vertebra leaned in the opposite direction.The results showed that the odontoid process of the second cervical vertebra plays an extremely important role in maintaining posture of the head and neck during occlusion and mastication.

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It is difficult to evaluate muscle fatigue in masticatory muscles because they differ considerably from other skeletal muscles in structure and function.We analyzed muscle fatigue in the masseter muscle after mastication using a newly developed tactile sensor and clarified that the muscle stiffness increases and elasticity decreases as the number of masticatory cycles increases.Frequency analysis of the electromyogram to evaluate muscle fatigue showed that increasing the number of masticatory cycles decreased the higher frequency components of the electromyogram.However, changes in this parameter were not as pronounced as those in the muscle stiffness and elasticity as measured with the tactile sensor.

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