We elucidated the relationship among the thickness of the mandibular alveolar cortical bone, the thickness of the mandibular alveolar cancellous bone, maxillofacial morphology, occlusal force, and the masseter muscle. The subjects were 18 adult male volunteers with a mean age of 29 years who had normal occlusion. The thickness of the cortical and cancellous bone were measured using a cone beam computed tomography scan. The maxillofacial morphology was observed with lateral cephalograms and the occlusal force was measured with pressure-sensitive film. The volume of the masseter muscle was determined from MRI images and correlations were determined with Student's t-test. We found that subjects with highly developed cortical and cancellous bone have a relatively dolicofacial morphology, while those with poorly developed cortical and cancellous bone have a relatively brachiofacial morphology. The results suggest that masticatory function is related to thickness of the cancellous bone.

View full abstract

Most bacteria in natural environments form communities, which are commonly referred to as biofilms. Previously, we reported that biofilm-forming oral bacteria have an impact on pathogenesis and play a key role in development of persistent infections. We investigated how genes relate to biofilm-formation. We determined the capacity of biofilm-formation of Streptococcus intermedius (S. intermedius) clinical isolate (strain H39) by a crystal violet (CV) assay using a 96-well microtiter plate. Because CV analysis showed that the addition of glucose to the culture medium increased the biofilm-forming capacity of strain H39, we attempted to determine the gene involved in glucose metabolism and biofilm-formation in this strain. We isolated the homolog to the pgcA gene, which codes α-phosphoglucomutase in Bacillus subtilis from strain H39. Moreover, we isolated a mutant strain, which is a recombinant of this gene. Scanning electron microscopy indicated that this strain has a mesh-like structure around its cells, which is typical of biofilm-forming bacteria. Inactivation of the pgcA gene was not enough to stop biofilm-formation of strain H39. These results suggest that the genetic basis of biofilm-formation in S. intermedius is multifaceted and that the composition of the polymeric biofilm matrix is complex. Further studies are needed to elucidate the genetic basis for biofilm-formation.

View full abstract

Natural antimicrobial substances are good candidates for replacing antibiotics to which bacteria have become resistant. We evaluated the antimicrobial effect against oral microorganisms of an ethanol extract from leaves and twigs of Sciadopitys vertillata. The extract was active not only against the periodontopathic bacteria Porphyromonas gingivalis and Prevotella intermedia/nigrescens, but also against facultative cocci and rods, including biofilm-forming bacteria that possess meshwork-like structures around cells. To elucidate the effect of this activity, we examined the viability of an indicator strain after exposure to the extract. We found that the viable count was significantly reduced over time, suggesting a bactericidal effect. These results indicate that this extract may be useful as a preventive and therapeutic substance for periodontal disease.

View full abstract

Previously we used microarray analysis to demonstrate that expression of several heat shock protein genes were up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. We employed a real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy to confirm the up-regulation of these genes in strain 17 recorded by a microarray. Total RNA was isolated from 6-, 12-, 18-, 24- and 30-hour cultures of strains 17, 17-2, and ATCC 25611 (a reference strain for P. intermedia). Real-time RT-PCR was performed according to the one-step RT-PCR protocol of iScript^<TM> One-Step RT-PCR Kit with SYBR^[○!R] Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-PCR for 16 S rRNA was performed as an internal control. The target mRNA levels in strains 17, 17-2 and ATCC 25611 were defined and compared using Gene Expression Macro (Bio-Rad). The increased expression levels of heat shock protein genes, such as dnaK, grpE, dnaJ, L, groES, and clpB, were validated by real-time RT-PCR. Four out of six of the tested genes showed at least a 10-fold increase in average expression level in strain 17 as compared to that of strain 17-2 after a 12-hour culture period. Considering the up-regulation of the stress response genes in biofilm-forming strain 17, the biofilm formation in this organism might be associated with its stress response.

View full abstract

Previously we used microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to demonstrate that expression of groEL, which is a highly-conserved heat shock protein gene, was up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. The entire encoding sequence for the P. intermedia GroEL protein was cloned into pET and expressed in Escherichia coli (E. coli) BL 21 (DE 3) pLysS or BL 21 (DE 3) pLysE. GroEL was expressed in a soluble form at a high level in the E. coli cytoplasm. Recombinant GroEL was transferred from the SDS electrophoresis gel to PVDF membrane, and applied to N-terminus amino-acid sequencing. N-terminus amino-acid sequencing revealed that the alignment of 10 amino-acid residues from an N-terminus of this recombinant protein was identical to that of wild-type GroEL protein. These results show a way to generate recombinant P. intermedia GroEL that can be used for further investigation of the function of this heat shock protein in biofilm formation and for study of the development of inflammatory diseases caused by this organism.

View full abstract

We examined how periodontal disease relates to general health status. The subjects were 965 people with a mean age of 38 years who received both dental and medical examinations in 2004. The subjects were categorized by age. Their periodontal health was evaluated by the community periodontal index (CPI) and the number of missing teeth. The periodontal health of the examinees was categorized as good or poor using five different cut-off points. For case 1 a CPI=0 was considered good and a CPI &ge; 1 was poor. For case 2 a CPI &le; 1 was good and a CPI &ge; 2 was poor. For case 3 a CPI &le; 2 was good and a CPI &ge; 3 was poor. For case 4 a CPI &le; 3 was good and a CPI &ge; 4 was poor. For case 5 no missing teeth was considered good, while one or more missing teeth was poor. Medical status was evaluated by blood pressure, body mass index, total cholesterol, HDL cholesterol, triglycerides, arteriosclerosis index, blood glucose level and HbA1c level, which is a surrogate for diabetes. Based on these criteria we attempted to correlate general health with the periodontal health of the subjects. There was a significant correlation between periodontal health and general health when the triglycerides, diastolic blood pressure, and arteriosclerosis were used as the indicators of general health and CPI 0 was used as the indicator of periodontal health (case 1) for the 40-49 year age group. We concluded that it is important to consider health rather than disease, when examining the relationship between periodontal health and general health.

View full abstract

We investigated the effect of propofol on the systemic vascular absorption of lidocaine after local infiltration anesthesia in rabbits. Propofol was administered intravenously to the animals which were divided into three groups: Group L, which received 2% lidocaine solution, Group LA-L, which received 2% lidocaine solution containing 1 : 160,000 adrenalin, and Group LA-H, which received 2% lidocaine solution containing 1 : 80,000 adrenalin. The plasma lidocaine concentration was determined after submucosal infiltration of the agent. The plasma lidocaine concentration in Group L was significantly greater than that in either the LA-L or LA-H group. The results suggest that intravenous propofol administration affects the absorption of lidocaine into the vascular system because the vasodilation induced by propofol decreases the vasoconstrictor effect of adrenalin.

View full abstract

We attempted to determine the protective effect of salivary pellicle formed on enamel using the quantitative light induced fluorescence method. Ten polished bovine enamel specimens were pretreated with stimulated human saliva for 1 minute and 10 others for 60 minutes. All the specimens were then exposed to a demineralizing solution. Ten teeth that had not been exposed to the saliva were exposed to the demineralizing solution as controls. The results indicated that demineralization was decreased in the enamel of the specimens exposed to saliva for 1 minute and for 60 minutes compared with the controls. However, there was no statistically significant difference between the 1 minute group and the 60 minute group. This data suggests that the salivary protein acquired on the enamel surface affects demineralization.

View full abstract

We examined how the status of periodontal tissue affected general health. The subjects were 965 people with a mean age of 38 years who received both dental and medical examinations in 2004. The Periodontal status was evaluated using the community periodontal index (CPI), and their medical condition was determined by blood pressure, body mass index, total cholesterol, HDL cholesterol, triglycerides, arteriosclerosis index and blood glucose level. The results suggested that the periodontal health status of CPI code 0 indicates that periodontal health has not affected general health.

View full abstract

We examined the effect of fluoride dentifrice on remineralization of white spot lesions by using quantitative light-induced fluorescence (QLF). The study was done on 129 subjects who had given their informed consent. Sixty-five of the subjects were randomly allocated to a group that brushed with 950 ppm fluoride dentifrice, while 64 others used a dentifrice without fluoride. The subjects brushed their teeth twice a day with their assigned dentifrice for one year. The remineralization effect of the two dentifrices was evaluated by three QLF parameters : mineral loss measured by ΔQ (% mm^2), lesion area measured by S(mm^2), and maximum lesion depth measured by ΔMax (%). These three parameters were recorded at three, six, and twelve month recalls after the baseline examination. We concluded that the fluoride dentifrice improved remineralization of the clinical white spot lesions.

View full abstract

We attempted to clarify how age-related changes in nitric oxide (NO) in the rat supraoptic nucleus (SON) influenced salivary secretion. Although the number of the Nissl-stained neurons did not change with age, age-related significant differences were detected in the cell number, cell size and reactive density of the NADPH-d-positive neurons, which is a marker for nitric oxide synthase. We concluded that the age-related increased production in NO might be a factor that contributes to age-related inhibition of salivary secretion.

View full abstract

We used β-tricalcium phosphate (β-TCP) granules to repair bone defects in crab-eating monkeys, and observed the results with LM, SEM and micro-computed tomography. During the early stages of the repair process, osteogenesis progressed more rapidly in the group treated with the large granule size than in the group treated with the small granule size. However, both experimental groups had nearly the same level of osteogenesis after 12 weeks. Furthermore, at 12 weeks the defects had been repaired in both groups regardless of the granule size.

View full abstract

We examined the metallic artifacts created in magnetic resonance images (MRI) when various orthodontic appliances were placed on study models. We used steel, ceramic and resin brackets, steel, NiTi, heat-activated NiTi and Beta-Ti orthodontic wires, and steel and elastic ligature wires. Several sequences of images were taken with a 1.5 tesla super-conductive MRI machine. Adapted steel brackets, steel wire and steel ligatures produced the largest metallic artifacts in the images. Steel brackets alone produced large metallic artifacts. Ceramic brackets and Ti materials did not produce large metallic artifacts. We concluded that orthodontists should not use steel orthodontic appliances for patients who expect to have MRI of the head and neck region.

View full abstract

I performed histological observations and measured the torque required to remove dental implants from rabbit femurs where Biopex-R^[○!R] had been placed in the gaps surrounding the implants. The removal torque was greater than 35 N・cm one week after the operation. Histological observation indicated there was no connective tissue interface between Biopex-R^[○!R] and the original bone. No inflammatory reactions were observed 8 weeks after the operation, and the biocompatibility of Biopex-R^[○!R] was very good. The results indicated that Biopex-R^[○!R] integrated directly to the original bone at an early stage. The strength of the removal torque should be adequated for initial fixation of the implant.

View full abstract

It is widely known that most bacteria produce exopolysaccaride and exist in the form of communities, commonly referred to as biofilm. Since the ability of bacteria to form biofilm is considered an important factor in persistent infections, it is important to isolate these bacteria from lesions and study their mechanism of biofilm formation. The isolation of biofilm-forming bacteria is difficult because it requires the measurement of viscosity of culture supernatants and scanning electron microscopic (SEM) observation of cell surface structures. We used Congo red agar plates (CRA) to isolate biofilm-forming bacteria from the oral cavity. Whole saliva from five healthy volunteers was diluted to 10^<-4> and used to inoculate CRA plates. The bacterial cultures were incubated aerobically for 24 hours at 37℃. Using a stereoscopic microscope, the colonies were divided into two groups, the smooth and rugose periphery types. The cell surface structures of the bacterial strains obtained from 30 arbitrarily selected colonies in each group were further studied by SEM and 16S rRNA sequencing. Bacterial strains from the smooth colony group showed biofilm-negative cell surfaces regardless of colony color. On the other hand, bacterial strains that formed rugose colonies on CRA under aerobic conditions expressed dense fibrillar structures, which is a typical phenotype for biofilm-forming bacteria. These results suggest that oral bacteria form rugose colonies cultured under aerobic condition on CRA have the capacity to form biofilm. We also found that CRA can be applied as a simple method for screening oral biofilm-forming bacteria.

View full abstract

We recently reported that wzt encoding an ATP binding protein in the O-antigen biosynthesis and export system was crucial for the biofilm formation of Escherichia hermannii. In this study, we attempted to establish the real time reverse transcription polymerase chain reaction (RT-PCR) conditions necessary to evaluate the transcriptional level of wzt along with growth of biofilm-positive and -negative strains of this organism. Transcriptional levels of wzt were normalized with a transcriptional level of the 16S rRNA gene. The relative expression levels of wzt were compared between the biofilm-positive wild type strain YS-11, the wzt-disrupted mutant 455 and the wzt-overexpressed recombinant strain 455-LM, We confirmed by melting curve analysis that the real time RT-PCR condition used in this study amplified the target gene. Also, we found that the relative expression level of wzt was greatest in strain 455-LM, while it was expressed to a lesser extent in YS-11, among the strains used. Our results suggest that this method can be used to evaluate relative transcriptional levels of wzt in E. hermannii.

View full abstract

It is well known that a few strains of Prevotella intermedia have three classes of native plasmids. Previously, the sequence alignment, eight putative open reading frames, and two gene functions were determined for one of the three plasmids, pYHBi 1, which has a molecular size of 5.0-kb. In this study, using electrophoresis and pYHBi 1 targeted PCR, we investigated the presence of native plasmids in 8 extracellular polysaccharide (EPS) producing Prevotella intermedia clinical strains and in the ATCC type strain. We found that our stock strains lacked any type of native plasmid. However, we identified some biofilm-forming clinical isolates that did not have native plasmid. The results suggest there was no correlation between EPS producibility and native plasmids.

View full abstract

Recent studies have indicated that bacteria producing a large amount of extracellular polysaccharides cause persistent infections. However, the exact distribution of the biofilm-forming bacteria in acute periodontal lesions remains poorly understood. To estimate the number of biofilm-forming bacteria in periodontal pockets, we isolated and identified the bacteial strains of plaque samples collected from four patients with acute periodontitis. Forty bacterial strains that were isolated produced viscous materials in their spent culture media and possessed meshwork-like structures on their cell surfaces. These included three anaerobic rods, one anaerobic coccus, three facultative anaerobic rods, one facultative anaerobic coccus, and one microaerophilic rod. These results suggest that many oral bacteria found in subgingival plaque have the ability to produce viscous materials resulting in the formation meshwork structures.

View full abstract

[in Japanese]

View full abstract

We evaluated the relationship between the stability of intercuspation and cranio-mandibular disorders. Occlusal contact area, the number of occluding teeth and asymmetry of the occlusal contact area were measured on video images of the occlusal contacts during strong and weak occlusal forces. A premature contact index (PCI) was constructed based on changes in the contact area between the weak and strong forces. Occlusal contact area, the number of occluding teeth, and asymmetry were significantly different among the subjects. These differences were greater with weak rather than strong forces. There were also significant differences in PCI.

View full abstract

Previously we used microarray analysis to demonstrate that expression of several heat shock protein genes were up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. We employed a real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy to confirm the up-regulation of these genes in strain 17 recorded by a microarray. Total RNA was isolated from 6-, 12-, 18-, 24- and 30-hour cultures of strains 17, 17-2, and ATCC 25611 (a reference strain for P. intermedia). Real-time RT-PCR was performed according to the one-step RT-PCR protocol of iScript^<TM> One-Step RT-PCR Kit with SYBR^[○!R] Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-PCR for 16 S rRNA was performed as an internal control. tThe target mRNA levels in strains 17, 17-2 and ATCC 25611 were defined and compared using Gene Expression Macro (Bio-Rad). The (Increased expression levels of heat shock protein genes, such as dnaK, grpE, dnaJ, groEL, groES, and clpB, were validated by real-time RT-PCR. Four out of six of the tested genes showed at least a 10-fold increase in average expression level in strain 17 as compared to that of strain 17-2 after a 12-hour culture period. Considering the up-regulation of the stress response genes in biofilm-forming strain 17, the biofilm formation in this organism might be associated with its stress response.

View full abstract

Previously we used microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to demonstrate that expression of groEL, which is a highly-conserved heat shock protein gene, was up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. The entire encoding sequence for the Prevotella intermedia GroEL protein was cloned into pET and expressed in Escherichia coli (E coli) BL 21 (DE 3) pLysS or BL 21 (DE 3) pLysE. GroEL was expressed in a soluble form at a high level in the E. coli cytoplasm. Recombinant GroEL was transferred from the SDS electrophoresis gel to PVDF membrane, and applied to N-terminus amino-acid sequencing. N-terminus amino-acid sequencing revealed that the alignment of 10 amino-acid residues from an N-terminus of this recombinant protein was identical to that of wild-type GroEL protein. These results show a way to generate recombinant P. intermedia GroEL that can be used for further investigation of the function of this heat shock protein in biofilm formation and for study of the development of inflammatory diseases caused by this organism.

View full abstract

Most bacteria in natural environments form communities, which are commonly referred to as biofilms. Previously, we reported that biofilm formation by oral bacteria have an impact on pathogenesis and plays a key role in development of persistent infections. Using a 96-well microtiter crystal violet (CV) plate assay, we determined the capacity of biofilm-formation of Streptococcus intermedius clinical isolates. The CV analysis showed that glucose increased biofilm-formation of the organism. Furthermore, we isolated the homolog to the pgcA gene, which codes α-phosphoglucomutase in Bacillus subtilis. However, we found that pgcA gene inactivation did not stop biofilm-formation of Streptococcus intermedius.

View full abstract

Natural antimicrobial substances are good candidates for replacing antibiotics to which bacteria have become resistant. We evaluated the antimicrobial effect against oral microorganisms of an ethanol extract from leaves and twigs of Sciadopitys vertillata. The extract was active not only against the periodontopathic bacteria Porphyromonas gingivalis and Prevotella intermedia/nigrescens, but also against facultative cocci and rods, including biofilm-forming bacteria that possess meshwork-like structures around cells. To elucidate the effect of this activity, we examined the viability of an indicator strain after exposure to the extract. We found that the viable count was significantly reduced over time, suggesting a bactericidal effect. These results indicate that this extract may be useful as a preventive and therapeutic substance for periodontal disease.

View full abstract