An in situ hybridization and histochemical study of development and postnatal changes of mouse mandibular angular cartilage compared with condylar cartilage

To investigate the origin and postnatal changes of mouse mandibular angular cartilage, in situ hybridization for cartilaginous marker proteins, histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), and bromodeoxyuridine (BrDU) analyses were performed. Chondrocytes of the mandibular angular cartilage were derived from ALP-positive progenitor cells and first detected at embryonic day (E) 15.5. Newly formed chondrocytes rapidly differentiated into hypertrophic chondrocytes and hypertrophic cell zone rapidly extended in subsequent a few days. During this period, bone sialoprotein mRNA was more widely expressed than osteopontin mRNA in cartilage. Endochondral bone formation started at E 17.5 with the resorption of the bone collar by osteoclasts. These characteristics were consistent with those of the condylar cartilage, although developmental process was 0.5-1.5 day delayed relative to the condylar cartilage. During the postnatal period, contrast to the condylar cartilage, the angular cartilage constantly decreased in volume with advancing age. Reduction of proliferating activity estimated by BrDU incorporation accounts for this phenomenon. We demonstrate new structural features of the mandibular angular cartilage that may contribute to a coming research for the secondary cartilage.     

Nitric oxide synthase activity in neutrophils from patients with localized aggressive periodontitis

BACKGROUND: Localized aggressive periodontitis (LAgP) is associated with neutrophil dysfunction including defective chemotaxis and reduced calcium influx factor activity. Nitric oxide (NO) and its enzyme, nitric oxide synthase (NOS), have been suggested to be involved in chemotaxis. Some reports, however, were unable to detect either NO or NOS in human neutrophils. In this study, we focused on NOS activity in LAgP neutrophils and examined the involvement of NOS in chemotaxis of normal neutrophils and NOS activity in neutrophils from normal subjects and patients with LAgP. METHODS: Neutrophils from 10 normal subjects and 10 LAgP patients were isolated from peripheral venous blood. Membrane associated-NOS (MA-NOS) and soluble NOS (S-NOS) were extracted from cells with or without FMLP stimulation. NOS activity was measured using the radiolabeled L-arginine to L-citrulline conversion assay. RESULTS: N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly inhibited FMLP-induced chemotaxis (P<0.01) and dibutyryl cGMP, an activator of cGMP-dependent protein kinase, significantly attenuated the inhibition by L-NAME (P<0.01). Unstimulated and FMLP-stimulated MA-NOS activity in LAgP neutrophils was statistically significantly higher than that in normal neutrophils (P<0.05). S-NOS activity in LAgP neutrophils was higher than that in normal neutrophils. CONCLUSIONS: This study suggests that NOS is present in human neutrophils and may be involved in FMLP-induced chemotaxis in normal neutrophils. NOS activity is increased in LAgP and is negatively correlated to chemotaxis response.     

Type II collagen and aggrecan mRNA expressions in rabbit condyle following disc displacement

The aim of this investigation was to study the remodelling of cartilage in the mandibular condyle following disc displacement (DD) of the temporomandibular joint (TMJ). Forty adult Japanese white rabbits were used in this study. The right joints of 28 of the 40 rabbits had their discs surgically displaced. Four of the 28 were killed at 4 days or 1, 2, 4, 6, 8 and 12 weeks after surgery. The messenger RNA (mRNA) expression levels of aggrecan and type II collagen in cartilages were measured using in situ hybridization techniques. Results showed that aggrecan mRNA expression reduced in the first week after DD. The expression began to recover after 4 weeks and reached a normal level after 6 weeks. Type II collagen mRNA expression reduced from 4 weeks and the expression recovered after 8 weeks. This suggests that the chondrocyte reacting to the displacement of the TMJ disc, alters its matrix gene expression patterns and it is may be the cause of the shape changes of TMJ after DD.     

A searchable database for proteomes of oral microorganisms

An online database of proteomes for two-dimensional electrophoresis (2DE) gel data was constructed and it is now freely accessible through a web-based interface. Proteins from three oral bacteria, Streptococcus mutans UA159, Actinobacillus actinomycetemcomitans HK1651, and Porphyromonas gingivalis W83, whose genome databases are freely available, were separated by 2DE, and protein spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and identified. About 1000 spots from the gels of P. gingivalis W83 were extracted and analyzed by MALDI-TOF, and 330 proteins were identified. In addition, 160 of 240 spots of A. actinomycetemcomitans and 158 of 356 spots of S. mutans were identified. Information such as spot coordinates on the gels, protein names (predicted functions), molecular weights, isoelectroric points, and links to online databases, including Oral Pathogen Sequence Databases of the Los Alamos National Laboratory Bioscience Division (ORALGEN) and National Center for Biotechnology Information (NCBI) or The Institute Genomic Research (TIGR), were stored in tables accessible through the relational database management system MySQL on an Apache web server. To test for functionality of this database system, responses of S. mutans to environmental changes were analyzed using the database and 21 spots on the gel were identified as proteins whose expression had been increased or decreased by environmental pH change without in-gel trypsin digestion, protein extraction, or MALDI-TOF/TOF-MS (mass spectrometer) analysis. The identified proteins are agreement with those reported in previous papers on acid tolerance of S. mutans, demonstrating the usefulness of the system. This database is available at http://www.myamagu.dent.kyushu-u.ac.jp/~bioinformatics/index.html or http://www.bipos.mascat.nihon-u.ac.jp/index.html.     

Characterization of excess hard tissue occurring in the mesio-buccal surface of the mandibular first molar in microphthalmic mouse

OBJECTIVE: The aim of the present study was to characterize the excess hard tissue on the mandible of the microphthalmic mouse having a mutation at the mitf locus. DESIGN: Homozygous mutant (mi/mi) and wild-type (+/+) mice were obtained by mating a breeding pair (strain name, B6C3Fe a/a-Mitf(mi)/J). We used mi/mi and +/+ mice at ages 6, 7, 8, 9, 28, and 49 days for micro-computed tomographic and histologic analyses. RESULTS: Excess hard tissue was found on the mesio-buccal surface of the mandibular first molar in all 11mi/mi mice, but none was found in the 8mi/+ or 14 +/+ mice. The excess hard tissue was located in the mental foramen connected to the mandibular canal. The mandibular canal passed near the basal part of the incisor and the root of the mandibular first molar due to aberrant development of the teeth and mandible. The excess hard tissue contained predentine immunostained for dentine sialoprotein, a marker for early stages of dentinogenesis, which was first observed at about 7 days of age. Dentine, predentine, pulp, and root-like structures were observed in the excess hard tissue, but neither enamel nor enamel organ was observed. CONCLUSION: Odontogenic cells in the basal part of the incisor and/or the mandibular first molar with the ability to develop into odontoblasts and pulp cells appeared to migrate through the mandibular canal to the mental foramen, where they developed into odontoblasts and pulp-like cells, and then formed dentine and predentine-like structures.     

Immobilization of simulated reducing agent at the surface of SiO2 fillers in dental composite resins

To reduce the leachability of reducing agents from composite resins, immobilization of a simulated reducing agent at the surface of SiO2 fillers was examined. SiO2 plates were immersed in 2% 3-aminopropyltriethoxy silane/ethanol solution, and then immersed in dimethyl sulfoxide with 0.25 wt% 4-dimethyl amino benzoic acid (DMABA), 2.0 wt% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 0.5 wt% N-hydroxysuccinimide. Wide-scan spectrum of X-ray photoelectron spectroscopy did not detect carbon contamination. However, narrow scan detected an O=C-N peak at 399.8 eV, suggesting that DMABA could be immobilized on silane-coupled SiO2 plates. Further, surface plasmon resonance analysis indicated the adsorption of MMA at the surface of reducing agent-immobilized plate.     

Age-related differences in expression of vascular endothelial growth factor by periodontal ligament cells in vitro

The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150 g each in the young group and 350 g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan. The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.     

An ultrastructural analysis of the prototype single-step adhesive applied on enamel and dentin surfaces

A new type of self-etching agent, the single-step adhesive, was developed. The purpose of this study was to investigate the difference in the ultrastructural features of enamel and dentin surfaces following application of the prototype single-step adhesive (EXM618, 3M, MN, USA). Extracted caries-free human premolars were used in this study. Occlusal enamels of teeth--to be used as cut surface specimens--were removed perpendicular to the long axis of the specimens. The mesial and distal surfaces of these teeth, on the other hand, were used as uncut surface specimens in the test. In addition, Mega Bond (Kuraray Medical Inc., Tokyo, Japan) and Xeno CFII Bond (Dentsply Sankin, Tokyo, Japan) were used as controls. After covering half of the tooth surfaces with nail varnish (for control), the other half of each surface was treated with one of the three test adhesives--EXM618, Mega Bond, or Xeno CFII Bond--according to the manufacturer's recommendations. Conditioned enamel and dentin surfaces (i.e., decalcified depth and rugged surface) were observed with a scanning confocal laser microscope (SCLM 1100, Olympus, Tokyo, Japan; henceforth abbreviated as SCLM). Based on the findings of this study, the prototype single-step adhesive EXM618 appears to be suitable for use in dental clinics.     

High prevalence of Helicobacter pylori detected by PCR in the oral cavities of periodontitis patients

BACKGROUND: Helicobacter pylori has been associated with the development of peptic ulcers and gastric cancer. Although it may be transmitted through the oral cavity, it is unknown whether the oral cavity acts as a permanent reservoir for this bacterium. The purpose of this study was to use nested polymerase chain reaction (PCR) to clarify whether the oral cavity acts as a reservoir for H. pylori. METHODS: The existence of H. pylori in the oral cavity was determined by nested PCR in 57 subjects and by culture method in 18 subjects. The presence of periodontopathic bacteria was also determined by 16S rRNA-based PCR method. RESULTS: Although H. pylori was rarely detected in the oral cavity by culture technique, it was frequently detected (35.1%) by nested PCR in the oral cavity, especially among periodontitis patients who had the bacterium in the gastrointestinal tract (46.4%). Among the subjects who harbored H. pylori in the stomach or duodenum, 41.2% of patients with periodontal pockets > or = 4 mm and 9.1% of subjects without pockets showed H. pylori in dental plaque, although a statistically significant difference was not observed. One patient who had periodontal pockets retained H. pylori in the oral cavity even after eradication of the bacterium from the stomach and duodenum. Most (8/10) of the patients who had H. pylori in dental plaque harbored Bacteroides forsythus in their oral cavities. CONCLUSION: Close attention should be given to periodontitis patients who harbor H. pylori in the oral cavity.