Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

Factors associated with Japanese dentists encouraging patients to use dental floss.

OBJECTIVE: The objective of this study was to clarify to what extent Japanese dentists recommend dental floss and what factors influence dentists in encouraging their patients to use dental floss. PARTICIPANTS: The subjects in this study were 291 dentists who were directors of dental clinics, selected by stratified sampling by age. RESULTS: Dentists whose teachers at dental school had demonstrated dental flossing tended to recommend patients to use dental floss 2.2 (1.0-4.6: 95% CI) times more frequently compared with those who did not see demonstrations of flossing at dental school. Respondents who considered that using dental floss was very easy and easy, moderate, and difficult recommended patients to use dental floss 45.4 (11.2-183.9), 17.4 (6.6-45.8) and 5.9 (2.5-14.1) times more frequently, respectively, compared with those who considered it very difficult. Respondents who considered that using dental floss was effective, fairly effective or very effective in preventing dental caries recommended patients to use dental floss 3.8 (1.7-8.6), 3.8 (1.7-8.8) and 9.1 (3.6-23.0) times more frequently respectively, compared with those who considered it ineffective or only slightly effective. CONCLUSIONS: The demonstration of the use of dental floss by teachers at their dental schools gave dentists a good impression and a positive opinion of dental flossing. This was closely associated with recommendations by dentists to their patients to use dental floss.

Sonic hedgehog signaling is important in tooth root development.

Hertwig's epithelial root sheath (HERS) is important for tooth root formation, but the molecular basis for the signaling of root development remains uncertain. We hypothesized that Sonic hedgehog (Shh) signaling is involved in the HERS function, because it mediates epithelial-mesenchymal interactions during embryonic odontogenesis. We examined the gene expression patterns of Shh signaling in murine developing molar roots. Shh and Patched2 transcripts were identified in the HERS, whereas Patched1, Smoothened, and Gli1 were expressed in the proliferative dental mesenchyme in addition to the HERS. To confirm whether Shh signaling physiologically functions in vivo, we analyzed mesenchymal dysplasia (mes) mice carrying an abnormal C-terminus of the PATCHED1 protein. In the mutant, cell proliferation was repressed around the HERS at 1 wk. Moreover, the molar eruption was disturbed, and all roots were shorter than those in control littermates at 4 wks. These results indicate that Shh signaling is important in tooth root development. Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; HERS, Hertwig's epithelial root sheath; NFI-C/CTF, nuclear factor Ic/CAAT box transcription factor; PCNA, proliferating cell nuclear antigen; Ptc, patched; Shh, sonic hedgehog; Smo, smoothened.

Effects of surface modification of materials on human neutrophil activation.

The properties of a polymer surface affect the cellular functions and morphology of cells in contact with the polymer. In this paper, we will demonstrate the effects of surface modification of materials on various neutrophil markers of activation. The sulfonation of a polystyrene surface caused increases in its negative charge and hydrophilicity. The sulfonation did not affect the number of adhered neutrophils, but the shape of the neutrophils adhered on the material was different; a round shape on highly sulfonated polystyrene and a spread shape on weakly sulfonated or non-sulfonated polystyrene. Expression of the adhesion molecule, CD11b, on neutrophils was also affected by the properties of the polymer surface. CD11b was expressed in neutrophils adhered on polystyrene and the expression decreased with increasing sulfonation of the surface. The expression of CD11b on the neutrophils on highly sulfonated polystyrene was the same as that on non-adhered neutrophils. In contrast, the expression of CD11a was not affected by the properties of the material surface. The F-actin content of activated neutrophils and the production of active oxygen groups detected by means of luminol-dependent chemiluminescence were also dependent on the sulfo-group content of the material surface. Finally, the translocation of protein kinase C (PKC) was determined in neutrophils adhered to these materials. Compared to non-adhered cells, the ratio of membrane bound to cytosolic PKC increased in adhered cells, but the increase was suppressed by sulfonation of the material surface. These data suggest that activation of neutrophils on polystyrene is suppressed by surface modification with increasing negative charge and/or hydrophilicity.

Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures.

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.

Impaired polymorphonuclear leukocyte 15-lipoxygenase activity in juvenile and rapidly progressive periodontitis.

15-lipoxygenase activity was investigated in sonicated polymorphonuclear leukocytes (PMNLs) from patients with juvenile and rapidly progressive periodontitis and adult periodontitis. The group with juvenile and rapidly progressive periodontitis had 17 patients (6 male, 11 female, mean age 27.4 years), and the age matched control group had 18 normal individuals (11 male, 7 female, mean age 26.3 years). The group with adult periodontitis had 14 patients with 9 male, 5 female, mean age 45.2 years and the age-matched control group had 6 normal subjects with 5 male, 1 female, mean age 43.7 years. 15-hydroxyeicosa-tetraenoic acid (15-HETE) synthesized in the group with juvenile and rapidly progressive periodontitis was 0.219 +/- 0.102 ng/mg protein (mean +/- S.D.), while it was 0.410 +/- 0.138 ng/mg protein in the age-matched control group. There was a significant difference between the two groups. The group with adult periodontitis produced 0.358 +/- 0.124 ng/mg protein and the age matched control group produced 0.448 +/- 0.176 ng/mg protein (no significant difference). These results are relevant to reports that PMNLs of patients with juvenile and rapidly progressive periodontitis have abnormal functions, while those of patients with adult periodontitis are normal.

Eicosapentaenoic acid abolishes the proatherogenic effects of cholesterol: effects on migration of bovine smooth muscle and endothelial cells in vitro.

It is well known that vascular endothelial cell (EC) migration plays a major role in regeneration of the injured endothelium and also that smooth muscle cell (SMC) migration is the important step for atheromatous plaque formation. In the present study, we investigated the effects of cholesterol and eicosapentaenoic acid (EPA) on bovine carotid artery EC and SMC migration using the modified Boyden chamber technique. The migration activity of the cholesterol-enriched ECs loaded with cholesterol-rich liposomes was significantly suppressed, whereas that of the cholesterol-enriched SMCs was enhanced. Next, we examined the effects of EPA pretreatment on the migration of both cell types. When ECs and SMCs were treated with EPA (5 micrograms/ml) for 2 days, the EPA content increased from 0.55 +/- 0.04% to 11.72 +/- 0.19% and 1.22 +/- 0.09% to 9.69 +/- 0.07% in cellular phospholipids, respectively. Although pretreatment of the ECs with EPA caused a significant increase in serum-induced cell migration, pretreatment of SMCs had no effect. If both cell types were concomitantly pretreated with EPA and cholesterol-rich liposomes, EPA abolished the effects of cholesterol on the migration of both cell types, but did not affect the content of cholesterol in both cells. These data indicate the possibility that EPA counteracts the atherogenic effect of cholesterol on EC and SMC migration.

Radicicol, a microbial cell differentiation modulator, inhibits in vivo angiogenesis.

Angiogenesis plays a significant role in various pathological states, including the progressive growth of solid tumors, rheumatoid arthritis, psoriasis, and diabetic retinopathy, in addition to its crucial role in embryonic development. Recent studies have revealed that an angiogenesis inhibitor is efficacious for these so-called angiogenic diseases. In the previous studies, we found that retinoids and vitamin D3 analogs, which are known to exhibit cell differentiation-modulating activity, effectively inhibit angiogenesis in vivo, thus forming the basis of our working hypothesis that a modulator of cell differentiation is capable of affecting angiogenesis. In this study, to verify this hypothesis further, radicicol (syn. monorden; 5-chloro-6-(7,8-epoxy-10-hydoxy-2-oxo-3,5-undecadienyl)-beta -resorcylic acid mu-lactone), a microbial cell differentiation modulator from a fungus, a strain of Neocosmospora tenuicristata, was examined for its anti-angiogenic activity in a bioassay system involving chorioallantoic membranes of growing chick embryos. The microbial cell differentiation modulator dose dependently inhibited embryonic angiogenesis, the ID50 value being 200 ng/egg. Radicicol also inhibited both the proliferation of and plasminogen activator production by vascular endothelial cells in the nM concentration range in a concentration-dependent manner, suggesting the possible involvement of these inhibitory effects in the anti-angiogenic action of the microbial product. These results indicate that radicicol might be a potential drug for treating different angiogenesis-dependent diseases, such as solid tumors, psoriasis, rheumatoid arthritis, and diabetic retinopathy.

The role of leukotriene B4 in neutrophil infiltration in experimentally-induced inflammation of rat tooth pulp.

Inflammation was induced in rat dental pulp by applying bacterial lipopolysaccharide (LPS). Extirpated tissue samples from inflamed pulps were incubated in vitro in a Krebs buffer containing Ca2+ ionophore A23187, and leukotriene (LT) B4 released into the medium was determined by radio-immunoassay. Production of LTB4 could be detected three to 24 h after the application of LPS and showed a maximum at 12 h. Histologically, marked infiltration of neutrophils, but not other leukocytes, was characteristically observed in the LPS-applied pulps, and the temporal change in neutrophil infiltration was almost parallel, but somewhat more delayed than LTB4 production. When BW755C, a dual inhibitor of cyclo-oxygenase and lipoxygenase, was given to the animals before the application of LPS, both the production of LTB4 and the number of infiltrated neutrophils were significantly decreased, whereas administration of indomethacin had no effect. These results suggest that LTB4 may be involved in neutrophil infiltration in pulpal inflammation. It was also suggested that a major early source of LTB4 in experimental pulpitis was leukocytes, primary neutrophils, because the synthesis of LTB4 in the inflammed pulp was diminished by depletion of circulating leukocytes with cyclophosphamide prior to the application of LPS.

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides.

The purpose of the present study was to investigate the involvement of cyclooxygenase-1(COX-1) and cyclooxygenase-2 (COX-2) in PGE2 production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated from Porphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Eschericia coli (E coli) by the phenol-water procedure. The three LPS preparations produced PCE2 up to 48 h in a time-dependent manner in human gingival fibroblasts. P. gingivalis-LPS was the most potent stimulator of PGE2 production and, to a lesser extent, A actinomycetemcomitans- and E coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE2 production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE2 production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h after P gingivalis-LPS stimulation, while expression of COX-1 protein was not affected by P. gingivalis-LPS. In order to investigate the regulation of PGE2 production. P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE2 production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE2 not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE2 may be involved in the pathogenesis of periodontal diseases.

The detection of platelet-activating factor in inflamed human gingival tissue.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a biologically active lipid, like the prostaglandins, which mediates allergic and inflammatory reactions. Aggregation of washed rabbit platelets was induced by a lipid prepared from inflamed gingiva. The mobility of the active lipid was coincident with that of authentic PAF on thin-layer chromatography. The aggregation was dose-dependent and inhibited by pretreatment with a specific PAF antagonist, ONO 6240, but not by indomethacin or creatine phosphate/creatine phosphokinase, which inhibit the platelet aggregation due to arachidonic acid or ADP, respectively. Thus the active lipid was identified as PAF; the amount of PAF detected was 118.1 +/- 79.7 pg/50 mg tissue (n = 6, mean +/- SD), the amount in normal tissue being 13.0 +/- 11.3 pg/50 mg tissue (n = 6). There was therefore a significant difference between the tissues. Lyso PAF, the metabolite of PAF with acetylhydrolase, was not detectable in either gingival tissue. Thus PAF was produced more in inflamed gingival tissue than in normal tissue; PAF may be involved in the occurrence and maintenance of periodontal disease.

Involvement of arachidonic acid metabolites in increases in vascular permeability in experimental dental pulpal inflammation in the rat.

Pulp was experimentally inflamed by applying bacterial lipopolysaccharide (LPS). Changes in arachidonic acid (AA) metabolites were determined by measuring the conversion of exogenously added AA in pulp homogenates. The inflamed pulp produced 12-hydroxy-eicosatetraenoic acid (12-HETE), 6-keto-prostaglandin (PG) F1 alpha greater than PGE2, thromboxane B2 and 11-HETE, which was further identified with high-performance liquid chromatography. The LPS treatment caused a 2.0-fold increase in 12-HETE production at 1 h, a 3.8-fold increase in 6-keto-PGF1 alpha production at 12 h and increases in PGE2 and 11-HETE production of 8.8- and 5.5-fold, respectively, at 24 h. Vascular permeability in the inflamed pulp was measured by quantifying the amount of an extravasated dye; it increased markedly from 6 h and reached a peak at 12 h after the LPS application. When indomethacin (0.3-30 mg/kg, s.c.) was given before LPS, both the production of 6-keto-PGF1 alpha and PGE2 and the increase in vascular permeability were inhibited dose dependently. Exogenously applied PGE2 and PGI2 methyl ester reduced the inhibition of the increase in vascular permeability caused by indomethacin. Thus PGE2 and PGI2 may be involved in increases in vascular permeability in pulpal inflammation.

Fluoride profiles in different sites of approximal surfaces of second primary molars after topical application of acidulated phosphate fluoride gel in vivo.

Thirty-four (17 paired) extracted second primary molars were obtained from 17 individuals (9 boys and 8 girls) aged from 9 yr 2 months to 12 yr 7 months. A tooth on one side was extracted as a control, and an acidulated phosphate fluoride (APF) gel was then applied to the paired contralateral second primary molar. Three months later, the experimental tooth was extracted. Nine sites were assayed by a microsampling technique from small areas of the approximal enamel surface. The fluoride and phosphorus concentrations were determined by a fluoride electrode and by colorimetric procedure, respectively. Fluoride concentrations were higher in the teeth treated with the APF gel than in the control teeth. The highest fluoride uptake was observed in the central area of the approximal surfaces. Deeper areas (> 10 microns) had a marked uptake of fluoride as compared with surface areas (< 3 microns). It was concluded that the APF gel application increased the fluoride levels of approximal tooth surfaces, particularly the mid-central site, of second primary molars, even at 3 months after application.

Arachidonic-acid metabolism in normal and experimentally-inflamed rat dental pulp.

Pulp homogenates were incubated with [14C]-arachidonic acid and the metabolites separated by thin-layer chromatography. The main products of normal pulp were 6-keto-prostaglandin (PG) F1 alpha and 12-hydroxy-eicosatetraenoic acid (12-HETE), further identified by high performance-liquid chromatography. Thromboxane (TX) B2, and PGD2, E2 and F2 alpha were also detected at less than 30 per cent of 6-keto-PGF1 alpha. When the pulp was inflamed by applying bacterial lipopolysaccharide, production of all these metabolites increased; in particular, PGE2 was increased 9.3-fold compared with normal, and 6-keto-PGF1 alpha and HETE 3.8- and 2.0-fold, respectively. An unidentified product, slightly more polar than 12-HETE, was also markedly produced by the inflamed pulp. Thus arachidonic-acid metabolites including lipoxygenase products may be involved in the development of pulpal inflammation.

Immunohistochemical detection of prostaglandin I2 synthase in various calcified tissue-forming cells in rat.

Localization of prostaglandin (PG) I2 synthase immunoreactivity was examined in demineralized sections of rat pulpal, periodontal and skeletal tissues using isn-1, a monoclonal antibody raised against the enzyme. Various calcified tissue-forming cells, i.e. odontoblasts, osteoblasts, osteocytes, cementoblasts, cementocytes and chondrocytes, were similarly immunoreactive for PGI2 synthase, suggesting that they are capable of producing PGI2. In odontoblasts and chondrocytes, the reactivity increased gradually with maturation. Weak immunoreactivity was also observed in endothelial cells and fibroblast-like cells in pulpal and periodontal tissues. However, no reactivity was seen in ameloblasts. These results suggest the possible involvement of PGI2 in the regulation of the metabolism of various calcified tissues. Monoclonal antibodies such as isn-1 may become useful markers of the maturation of calcified tissue-forming cells of mesenchymal origin.

Daily use of dentifrice with and without xylitol and fluoride: effect on glucose retention in humans in vivo.

The effect of daily use of three different dentifrices on glucose retention after glucose mouth rinsing was tested in this study regarding xylitol and fluoride. Six experimental groups used three different dentifrices produced by two different companies: xylitol- and fluoride-containing dentifrice (XF), non-xylitol- and fluoride-containing dentifrice (F), and non-xylitol- and non-fluoride-containing dentifrice (NonX-NonF). Subjects were divided at random and rinsed their mouths for 15s with 20ml of 0.5M glucose solution. Glucose and lactate retention were determined by collecting samples of saliva from the approximal areas of the maxillary and mandibular anterior teeth and using the enzyme membrane test. Samples were collected 0, 1 and 2 months after the start of regular dentifrice use. There were significant differences in glucose retention in relation to the dentifrice used, month of sampling, site of sampling, and time since start of rinsing. Their contribution ratios were 2.0, 4.4, 11.7 and 7.4%, respectively (P<0.01). There were significant differences observed between the XF and NonX-NonF groups, with the XF group presenting lower glucose retention than the NonX-NonF group. The XF group presented lower glucose retention than the F group. The F group showed lower glucose retention than the NonX-NonF group. There were significant differences in lactate retention in relation to the month and site of sampling, and their contribution ratios were 3.3 and 2.8%, respectively (P<0.01). There were, however, no significant differences in glucose and lactate retention in relation to the dentifrice manufacturer. It was concluded that the XF dentifrice was the most effective, and the F dentifrice was more effective in reducing glucose retention than the NonX-NonF dentifrice.

Glucose retention on the surfaces of primary teeth in 3- and 4-yr-old children.

Glucose retention was determined in 38 kindergarten children ages 3-4 yr. The children rinsed their mouths with 10 ml of a 0.5 mol/l glucose solution for 15 s and then spat out. Three minutes after they put the solution in their mouths, a small paper-point was used to collect samples of saliva from the labial and buccal surfaces of the maxillary and mandibular primary teeth. The concentration of glucose in the small amount of saliva collected was measured with an immobilized enzyme system. Glucose retention was highest on the maxillary central primary incisor, second highest on the maxillary first primary molar and third highest on the maxillary lateral primary incisor. An intermediate value was seen on the maxillary and mandibular second primary molars, the mandibular first primary molar and the maxillary primary canine. A lower value was observed on the mandibular primary canine and the lowest on the mandibular incisors. It was concluded that there were site differences in glucose retention on primary teeth of 3- and 4-yr-old children.

[Work load analysis for oral care in special nursing homes].

The work load analysis for oral care in special nursing homes. Dental hygienists undertook oral care of 100 person who were confined to special nursing homes in Aichi prefecture, Japan. The time required, the physical load, the psychological burden were analysed. The results were summarized as follows; 1) Oral care by dental hygienist was incorporated as one of the 59 care tasks given in special nursing. 2) The working time for required oral care was 25.7 minutes per resident on an average. 3) In general, speaking to the elderly, recording on an assessment card, and observing conditions were undertaken most frequently. In oral care, tooth cleaning, gargling, cleaning of dentures, wiping of the mouth and advising those caring for the elderly, was performed most frequently. 4) In general, time taken for advising those caring for the elderly, report to the home dentistry, advising the elderly, recording, and evaluation was most common. In oral care by a dental hygienist, tooth cleaning, cleaning of the dentures, cleaning the mouth, gargling, prevention of dryness of the mouth were performed most frequently. 5) In physical activity, raising the person, moving him from the wheelchair to the bed and helping him to lie down were most frequent. In oral care by a dental hygienist, wiping the mouth, inserting dentures, tooth cleaning, cleaning the mouth and removing dentures were most common. 6) The psychological burden when lifting the person, moving him from the bed to the wheelchair and helping him to lie down was the greatest. In burden involving oral care by a dental hygienist, tooth cleaning, removing dentures, inserting dentures and cleaning the mouth were the greatest.

[An oral health scoring system for promoting 8020 achievement in residents].

The present study was undertaken to develop a self-scoring system which can be used by a resident to check lifestyle. The oral health scoring system which we used in Tobishima village, Aichi-ken, was named SAWAYAKA score. A total of 777 subjects were examined. The subjects responded to a questionnaire regarding their past individual lifestyles and dietary habits. Oral health conditions were also examined by dentists. The odds ratio and 95% confidence interval were calculated both from retained tooth numbers and the questionnaire. Questions with significant odds ratio were selected and the partial regression coefficients of quantification II method by Hayashi were calculated. The results are as follows; 1) Eleven questions showed a significant odds ratio between retained tooth numbers and past lifestyle and dietary habits. The questions involved the frequency of snack intake, tooth brushing frequency, having own tooth brush, smoking, drinking, having a hobby, having a family dentist, consulting a dentist before a problem got serious, gum bleeding, swollen gums and toothache caused by sensitivity to cold water. 2) The eleven items were analysed by using Hayashi's quantification II method. 3) The results showed that unswollen gums affected the retention of teeth by the range of 1.240. Toothache caused by sensitivity to cold water affected the retention of teeth by the range of 0.765. Having a hobby affected the retention of teeth by the range of 0.691. 4) The "SAWAYAKA" score was used to select important items, excluding drinking. 5) When results were analysed with the SAWAYAKA score, an average of 9.6 was obtained. It was concluded that the scoring list could be used for checking resident's lifestyles, and for promoting the preservation of more than 20 teeth at the age of 80.

[Matrix vesicles in membranous ossification].

Calcification process in membranous ossification was examined histologically by light and electron microscopies in comparison with that in enchondral ossification. The following results were obtained: 1) In the rat tibial articular cartilage, the extracellular matrix proceeding from the transitional to the hypertrophic zone consisted of the following three layers from the proximal: the first layer in which ruthenium red positive granules were densely populated and thin collagen fibrils diffusely arranged but few matrix vesicles were observed; the second one containing numerous matrix vesicles and slightly thick collagen fibrils; and the last one in which the thick collagen fibrils were observed while matrix vesicles and ruthenium red positive granules were few. 2) In the calcified regions in the extracellular matrix of MC3T3-E1 cells, the regions of alveolar new bone formation due to orthodontic tooth movement and the calcified regions in the interparietal suture expanded mechanically in vitro, the number of matrix vesicles was less than those in the regions of enchondral ossification. 3) Ruthenium red positive granules were fewer in the regions of alveolar new bone formation after the orthodontic tooth movement than in the calcified regions of cartilage. 4) The observation on MC3T3-E1 cells revealed the two different possibilities of the processes of calcification: the calcification induced by matrix vesicles as an initiator and that directly related to mineral deposit on the collagen fibrils.