Transient acid-impairment of growth ability of oral Streptococcus, Actinomyces, and Lactobacillus: a possible ecological determinant in dental plaque.

INTRODUCTION: Dental plaque pH decreases to about 4 through bacterial fermentation of carbohydrates and this low pH is maintained for from several minutes to about an hour. Repeated acidification causes demineralization of the tooth surface, resulting in caries formation. The acidification also influences plaque bacteria. Severe acidification kills bacteria efficiently, while physiological acidification, the condition occurring in plaque, kills bacteria partially and may impair growth ability. We, therefore, investigated the effects of physiological acidification on representative caries-related bacteria. METHODS: Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, Streptococcus oralis, Lactobacillus paracasei, and Actinomyces naeslundii were used. Effects of physiological acidification at pH 4.0 on cell viability and growth ability, as well as the growth rate of these bacteria at pH 4.0-7.0, were investigated. RESULTS: Mutans streptococci and Lactobacillus grew at pH 4.0 but the growth of S. sanguinis and S. oralis ceased below pH 4.2 and pH 4.2-4.4, respectively. Acidification at pH 4.0 for 1 h killed 43-89%, 45% and 35-76% of S. sanguinis, S. oralis, and Actinomyces, respectively. Furthermore, assessment of bacterial growth curves revealed that the growth ability of the surviving cells of S. sanguinis, S. oralis and Actinomyces was impaired, but it was recovered within 2-5 h after the environmental pH had returned to 7.0. The acidification neither killed nor impaired the growth of mutans streptococci and Lactobacillus. CONCLUSIONS: These results indicate that physiological and transient acidification is not sufficient to kill bacteria, but it causes a temporary acid-impairment of their growth ability, which may function as an ecological determinant for microbial composition in dental plaque.

Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity.

Development of interglobular dentine in rat molars and its relation to maturation of enamel.

The development of interglobular dentine in the first upper and lower molars of Wistar rats aged 3, 7, 14, 21, 42 days was examined histochemically using a lectin, succinyl wheat germ agglutinin (sWGA), which is specific for N-acetyl-D-glucosamine. sWGA stained the interglobular dentine, predentin and Golgi area of odontoblasts. Interglobular dentine was not formed in the first molars of 3-day rats, but appeared in those of 7-day rats near the enamel-free area. In 14-day rats, interglobular dentine was present in most areas of the coronal dentine except the cervical area. At the interface between dentine and predentin, numerous sWGA-negative calcospherites were seen, suggesting that the interglobular dentine is formed actively there. In 21-day rats, the interlobular dentine was more numerous than in 14-day rats. Interglobular dentine was present in the cervical root dentine as well as in the coronal dentine, including the cervical area. The distribution of interglobular dentine in 42-day rats was similar to that in 21-day rats, but fluorescence of sWGA binding was less intense in the former. Because the development of interglobular dentine appeared to be time and position specific its relation to the stages of ameloblasts was analysed. Thin enamel matrix was formed at cusps in molars of 3-day rats and thickness of enamel matrix increased in 7-day rats. In these teeth, the ameloblasts were at the differentiating or secretory stage. The Golgi area and Tomes' processes of the secretory ameloblasts, the cells of intermediate layer and the enamel matrix were weakly positive with sWGA. The epithelial cells at the enamel-free area were also stained with sWGA. In 14-day rats, most of the ameloblasts in the first maxillary molars were at the maturative stage except in the cervical area, where the ameloblasts were at the transitional stage. sWGA stained the distal border and the Golgi area of the maturative ameloblasts as well as the cells of the papillary layer. The distal border of the maturative ameloblasts appeared either thick or thin, suggesting a ruffle-end and smooth-end of the cells. Ameloblasts were absent in the first molars of 21-day rats and the cervical part of the enamel was covered with the stratified epithelium like that of 42-day rats. The present study has demonstrated that interglobular dentine contains sWGA-binding glycoconjugates and the formation of the interglobular dentine is largely associated with the enamel maturation. These results suggest that matrix-to-cell interaction is important for the development of interglobular dentine.

Acid diffusion through extracellular polysaccharides produced by various mutants of Streptococcus mutans.

Mutants of Streptococcus mutans V403, constructed by allelic exchange and altered in their capacity to produce enzymes involved in the production of extracellular polysaccharides from sucrose, were used to study the role of glucans and fructans in the diffusion of ions through cell concentrates. A 4.0mm diameter, 0.75 mm deep diffusion chamber with an ion-sensitive field-effect transistor electrode positioned at the base was used to monitor the diffusion of hydronium ions from a sodium lactate buffer using cell concentrates prepared from bacteria grown in various concentrations of sucrose and glucose. The wild-type strain V403 produced at least seven times as much water-insoluble glucan (ISG) from sucrose as mutants deficient in various combinations of glucosyltransferase B (GTF B), GTF C, GTF D and fructosyltransferase. The fastest diffusion of hydronium ions occurred with sucrose-grown cell concentrates of strain V403, and the time of diffusion to the bottom of the chamber was approximately 2.3 times longer when this strain was grown in glucose. The speed of diffusion with glucose-grown V403 was similar to that obtained with each of the mutants. When cells of strain V403 grown in sucrose and glucose were mixed, increases in diffusion speed were found to be directly related to the proportion of sucrose-grown cells. The mixing of ISG with several strains of S. mutans revealed that increases in diffusion speed were directly related to the quantity of ISG added.

Elevation of histidine decarboxylase activity in the mandible of mice by Prevotella intermedia lipopolysaccharide and its augmentation by an aminobisphosphonate.

Lipopolysaccharide (LPS) produced by Gram-negative bacteria is an important cause of inflammation. Aminobisphosphonates are potent inhibitors of bone resorption but have inflammatory side-effects. Here, the effects of LPS from Prevotella intermedia (a prevalent Gram-negative bacterium both in periodontitis and endodontal infections) and alendronate (an aminobisphosphonate) on the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), were examined in mouse mandible. Intravenous injection of P. intermedia LPS increased HDC activity in the mandible, maximal activity being induced within 3-6 h of the injection. The elevation of HDC activity was dependent on the dose of LPS, 10 microg/kg (0.25 microg/mouse) producing a significant elevation in enzyme activity. Intraperitoneal injection of alendronate (40 micromol/kg) also produced an increase in HDC activity. Moreover, the elevation of HDC activity induced by P. intermedia LPS was markedly augmented in mice given alendronate 3 days before the LPS injection. These results (i) suggest that P. intermedia LPS may stimulate the synthesis of histamine in the mandible and that the newly formed histamine may make at least some contribution to the development of inflammation (apical periodontitis and/or osteomyelitis); (ii) should encourage the clinical testing of antihistaminergic agents against inflammation; and (iii) confirm that care needs to be taken when administering aminobisphosphonates to patients.

Cross-sectional comparisons of caries time trends in nursery school children in Sendai, Japan.

The aim of this study was to clarify the trends in caries experience in the primary dentition of nursery school children in Sendai, Japan, over a period of 20 yr. An annual clinical dental examination of children aged 2-5 yr at 13 nursery schools in Sendai City was carried out in 1972, 1977, 1982, 1987 and 1992. Data obtained from all five surveys were compared in each age group respectively. The percentages of 2-, 3- and 4-yr-olds free from caries increased remarkably until 1977, while that in 5-yr-olds started increasing several years later. After such increments, percentages in all age groups except 3-yr-olds remained almost at the same level with slight fluctuations. The mean deft and defs values per child in 2-, 3- and 4-yr-olds decreased remarkably until 1977, but then remained almost at the same level thereafter. That in 5-yr-olds continued decreasing slightly during the 20-yr period. The marked reduction of caries experience in nursery school children shown during the 1970's did not continue through the 1980's or the beginning of the 1990's.

Effects of the antiepileptics phenytoin and zonisamide on dentin formation and bone mineral density of the mandible in growing rats.

This study was undertaken to examine the effects of the antiepileptics phenytoin and zonisamide on changes in the mineral density of the incisor and bone mineral density (BMD) of the mandibular head, and on the rate of dentin formation using histomorphometric measurements. After repeated administration of phenytoin or zonisamide to male growing rats, the mineral density of the lower incisors and mandibular head were determined by analyzing microradiographs and dentin formation rates were determined by histomorphometric measurements. Results showed a significant decrease in the mean values of BMD of the mandibular head and lower incisors in groups treated with phenytoin or zonisamide compared with the vehicle-treated group (p < 0.05). The percent rates of decrease in mineral density of the incisors for phenytoin and zonisamide were 6.8% and 4.0%, respectively. Phenytoin and zonisamide significantly reduced the dentin formation rate for the mesial and distal areas compared with the vehicle-treated group. Thus, epileptic children who are treated over a long period with antiepileptics, especially at primary school age, should ensure good oral hygiene so as not to suffer bone loss, edentulism or gingival overgrowth.

Dental caries in Swedish 4-year-old children. Changes between 1967 and 1987.

The aim of the study was to examine the prevalence of dental caries in 4-year-olds in the town of Umeå in 1987, and compare that data with data from 4-year-olds examined in 1967, 1971, 1976 and 1980/81, using the same diagnostic methods and criteria. The reduction in the mean dmfs value amounting to 75% between 1967 and 1980/81 did not continue up to 1987, but there was an increase in the number of children without caries from 50% in 1980/81 to 58% in 1987. There was also an increase in the proportion of children with 10 or more dmf-surfaces during the same period. The mean value for dmfs remained the same in these years. An analysis was made of the correlation between the frequency of tooth-brushing, use of fluoride toothpaste, snack consumption and the dmfs-value in 1987. A statistically significant difference in mean values for dmfs was found between children who had their teeth brushed greater than or equal to 2/day and less than or equal to 1/day. When fluoride toothpaste was used, the difference was less pronounced. There was no statistically significant difference in the mean intake frequency of snack products among children with and without caries, but a higher proportion of caries-free children was found among those with a low intake frequency of such products.

A case of Coffin-Lowry syndrome with premature exfoliation of primary teeth.

We present a case of a 5-year-old boy with premature exfoliation of primary teeth. All eight primary incisors had exfoliated by the age of 3 years, and three canines and one primary first molar were subsequently lost when he was 4 years old. None of the exfoliated teeth exhibited caries. The boy also showed characteristic facial changes, tapering of the fingers, and mental and motor retardation. Based on these findings, he was diagnosed as having Coffin-Lowry syndrome. Premature exfoliation of primary teeth in Coffin-Lowry syndrome has been described in a few reports. This manifestation of the disease would be helpful for diagnosis at an early stage as those previous reports suggested.

Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR.

AIMS: To detect quantitatively the total bacteria and Streptococcus mutans in dental plaque by real-time PCR with prbac, Sm and GTF-B primers, and to compare their presence with the prevalence of dental caries in Japanese preschool children. METHODS AND RESULTS: Human dental plaque samples were collected from the labial surfaces of the upper primary central incisors of 107 children. The dental status was recorded as dft by WHO caries diagnostic criteria. Positive dt and dft scores by the Sm or GTF-B primer were significantly higher than negative scores (P < 0.01). The proportions of Strep. mutans to the total bacteria from sound, and sound and/or filled upper primary incisors were significantly lower than those from decayed or filled, and decayed incisors, respectively (P < 0.01). CONCLUSIONS: The ratios of Strep. mutans to total bacteria in plaque detected by real-time PCR with Sm and GTF-B primers were closely associated with the prevalence of dental caries in Japanese preschool children. SIGNIFICANCE AND IMPACT OF THE STUDY: These assays may be useful for the assessment of an individual's risk of dental caries.

Synergistic inhibition by combination of fluoride and xylitol on glycolysis by mutans streptococci and its biochemical mechanism.

The purpose of this study was to evaluate the combined inhibitory effect of fluoride and xylitol on acid production by mutans streptococci, Streptococcus mutans NCTC10449 and Streptococcus sobrinus 6715, from glucose under strictly anaerobic conditions at fixed pH 5.5 and 7.0. The bacteria were grown in a tryptone-yeast extract broth under strictly anaerobic conditions (N2: 80%; H2: 10%; CO2: 10%). Reaction mixtures for acid production from glucose contained bacterial cells with fluoride (0-6.4 mM) and/or xylitol (60 mM). Acidic end products of glucose fermentation and intracellular glycolytic intermediates were assayed. The combination of fluoride and xylitol inhibited acid production more effectively than fluoride or xylitol alone. In the presence of fluoride and xylitol, the proportion of lactic acid in the total amount of acidic end products decreased, while the proportion of formic and acetic acids increased. Analyses of intracellular glycolytic intermediates revealed that xylitol inhibited the upper part of the glycolytic pathway, while fluoride inhibited the lower part. This study indicates that fluoride and xylitol together have synergistic inhibitory effects on the acid production of mutans streptococci and suggests that xylitol has the potential to enhance inhibitory effects of low concentrations of fluoride.

Inhibition of inflammatory and bone-resorption-inhibitory effects of alendronate by etidronate.

Among the bisphosphonates (BPs), the aminobisphosphonates (aminoBPs) have much stronger bone-resorption-inhibitory activities (BRIAs) than nonaminobisphosphonates (nonaminoBPs). However, aminoBPs have inflammatory effects. We previously reported that in mice: (i) all aminoBPs tested (10-40 micromol/kg) induced various inflammatory reactions (including induction of histidine decarboxylase), whereas clodronate (a non-aminoBP) (10-160 micromol/kg) inhibited these reactions; and (ii) a clear sclerotic line (tentatively called the BP line) was detectable in the tibia by radiography a few weeks after a single injection of either alendronate (a typical aminoBP) (1.6 micromol/kg) or clodronate (160 micromol/kg), and this BP-line formation (a marker for the BRIAs of BPs) was not reduced in mice given both alendronate and clodronate. In this study, using this murine model, we compared clodronate, etidronate (another typical non-aminoBP), alendronate, etidronate + alendronate, and clodronate + alendronate in terms of their inflammatory effects and/or BP-line formation. For BP-line formation, 480 micromol/kg etidronate was needed (single injection). At 160 micromol/kg, etidronate inhibited the histidine decarboxylase induction, but not the other inflammatory reactions induced by alendronate. However, etidronate (unlike clodronate) also inhibited alendronate-induced BP-line formation (even at 40 micromol/kg). Etidronate (160 micromol/kg) also inhibited the physicochemical changes in the tibia induced by six, weekly injections of alendronate. Therefore, depending on the dose, etidronate can inhibit alendronate's inflammatory actions and its BRIA. These results, together with those reported previously, suggest that a strategy utilizing clodronate (but not etidronate) plus an aminoBP might prevent or reduce the inflammatory side effects induced by aminoBPs while preserving their powerful BRIAs. We discuss the mechanisms underlying the antagonism between aminoBPs and non-aminoBPs.

Cariogenic potential of lactosylfructoside as determined by acidogenicity of oral streptococci in vitro and human dental plaque in situ.

The cariogenic potential of lactosylfructoside [O-beta-D-galactopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside] was estimated by experiments on oral streptococci in vitro and human dental plaque in situ. Lactosylfructoside was unable to support growth of the strains of Streptococcus mutans and S. sobrinus used in this study. However, it was able to support growth of strains of S. sanguis, S. mitis and S. oralis. Acid was produced rapidly by cell suspensions of S. oralis ATCC 10557 incubated with lactosylfructoside. Application of 5% w/v solution decreased the pH of human dental plaque. The minimum pH value was below 5.3. The results suggest that lactosylfructoside is as acidogenic as lactose and could be cariogenic if it is consumed frequently and retained for a long period in the mouth.

Xylitol inhibition of anaerobic acid production by Streptococcus mutans at various pH levels.

Xylitol inhibits the glycolysis and growth of Streptococcus mutans. We studied the inhibitory effect of xylitol on the acid production of S. mutans at several pH levels under the strictly anaerobic conditions found in the deep layer of dental plaque. Xylitol inhibited the rate of acid production from glucose and changed the profile of acidic end products to formate-acetate dominance, with a decrease in the intracellular level of fructose 1,6-bisphosphate and an intracellular accumulation of xylitol 5-phosphate (X5P). These results were notable at pH 5.5-7.0, but were not evident at pH 5.0. Since the activity of phosphoenolpyruvate phosphotransferase for xylitol was greater at higher pH, it is suggested that xylitol could be incorporated more efficiently at higher pH and that the resultant accumulation of X5P could inhibit the glycolysis of S. mutans more effectively.

Effects of weekly administrations of alendronate+clodronate on young mouse tibia: localized action at the proximal growth plate.

Alendronate (A), a typical aminobisphosphonate (aminoBP), has a strong bone-resorption-inhibitory activity (BRIA). However, like other aminoBPs it has inflammatory side effects. In contrast, the BRIA of clodronate (C), a non-aminoBP, is much weaker, and in animal experiments it suppresses aminoBP-induced inflammatory reactions. In the present study, we examined the effects of weekly administrations of A (1.6 micro mol/kg) + C (160 micro mol/kg) on the tibias in young mice and compared them to those induced by A or C alone. Radiophotography showed that A increased bone density at a selective site in the tibia. Indeed, one week after the final injection of A (given alone), clear sclerotic lines (tentatively called BP-lines) were visible at sites corresponding to the location of the growth plate at the time of the each injection. C also produced BP-lines, although they were weaker than those produced by A. Combined administration of A and C produced similar BP-lines as seen in mice given A alone. These results together with other physicochemical effects of A on the tibia suggest that (1) each injection of A and C inhibits bone resorption selectively and transiently at the tibial growth plate in young mice, although minor effects on other sites cannot be excluded, and (2) the combination of A and C keeps still a strong BRIA. Our findings may suggest a strategy for the prevention or reduction of some inflammatory side effects of A or other aminoBPs.

Phenytoin and its metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, show bone resorption in cultured neonatal mouse calvaria.

The effects of phenytoin and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), on bone resorption of neonatal mouse calvaria were examined in vitro. Both phenytoin and HPPH induced significant bone resorption as compared to the controls after 72 h in culture. This effect may be the cause of phenytoin-induced bone loss in vivo.

Inflammatory reactions in extraoral tissues in mice after intragingival injection of lipopolysaccharide.

Intragingival (ig) injection into mice of lipopolysaccharide (LPS) from Prevotella intermedia or Escherichia coli elevated the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), in the mandible, liver, lung, and spleen, with a time course similar to that seen with intravenous (iv) injection. The effect of i.g. injection was less than that of i.v. injection but similar to that of intraperitoneal (ip) injection. The i.g. injection also increased hepatic serotonin, reflecting platelet accumulation. In galactosamine-treated mice, the minimum ig dose of LPS needed to induce lethal hepatitis was very small (less than that needed by ip injection). These results support the idea that the LPS produced in oral tissues may be transported easily to extraoral tissues and, in some cases, may cause inflammatory or immune responses. It also may influence the pathogenesis of some systemic diseases.

The dentofacial manifestations of XXXXY syndrome: a case report.

This paper presents a six-year-old patient with XXXXY syndrome, whose oral findings included a cleft soft palate, hyper- or meso-taurodontism in eight primary molars and in the mandibular permanent first molars, five congenitally missing premolars, and delayed development of the permanent tooth germs. The maxillary and mandibular primary central incisors were in a cross-bite relationship. Cephalometric findings showed a short ramus of the mandible and a short maxilla in the anterioposterior plane. The anteroposterior jaw relationship was in harmony. The cross-bite was considered to be due to the retroinclination of the maxillary primary incisors. This case emphasises the importance of regular dental care, and monitoring of facial growth and dental development in children with XXXXY syndrome.

Histopathological and immunohistochemical analysis of calcifying odontogenic cysts.

METHOD AND RESULTS: Calcifying odontogenic cysts (COCs) were examined histopathologically and immunohistochemically to characterize the histological and cytological properties of these lesions. Histopathologically, COCs showed thin or thick lining epithelium with ghost cells. COCs were classified according to proliferative type or nonproliferative type lining epithelium, the presence or absence of ameloblastomatous appearance, and the presence or absence of odontoma in the cyst walls. Immunohistochemically, amelogenin protein was expressed chiefly in ghost cells, whereas cytokeratin 19 (CK19) and bcl-2 proteins were expressed chiefly in lining epithelial cells. The proportion of cases positive for bcl-2 protein was slightly higher in COCs with odontoma than in those without odontoma. Lining epithelial cells sporadically showed positive reactions for Ki-67 antigen. Mean Ki-67 labeling index was slightly greater in COCs with proliferative type lining epithelium, COCs with ameloblastomatous appearance of the cyst walls, and COCs with odontoma of the cyst walls than in COCs without these histological features. Our results suggest that ghost cells or lining epithelial cells show ameloblastic cytodifferentiation or odontogenic epithelial characteristics, that bcl-2 protein is associated with survival of lining epithelial cells in COCs, and that high proliferation potential is associated with ameloblastomatous proliferation or combined odontoma. COCs exhibited various histological features with several transitional forms, and immunohistochemical examinations revealed little or no difference in cytodifferentiation and cellular activity among COCs. CONCLUSION: We conclude that COCs with various histological features have neoplastic potential and may not be separate entities within the same histological spectrum.

Distribution of interglobular dentine in human tooth roots.

The present study was designed to examine the distribution of interglobular dentine in human tooth roots. The material comprised 17 teeth, of which 3 were premolars extracted for orthodontic reasons from children 10-12 years of age and the other teeth (4 incisors, 3 canines and 7 molars) were extracted for periodontitis from individuals aged 32-63 years. All teeth were free of caries and cervical dentine defects. Ground sections of the teeth cut longitudinally were stained with basic fuchsin and observed by fluorescence and confocal microscopy as well as transmitted light microscopy. Basic fuchsin stained the dentinal tubules, interglobular dentine and the granular layer of Tomes. These structures appeared intense blue to faint violet with transmitted light microscopy, whereas their staining displayed intense fluorescence with fluorescence microscopy. Therefore, the interglobular dentine could be detected more sensitively with fluorescence and confocal microscopy than with transmitted light microscopy. Typical interglobular dentine was present in coronal dentine in most of the teeth. In the radicular dentin, position and size of the interglobular dentine was different among the teeth examined. Most of the teeth had the interglobular dentine in the cervical part of the roots (type A). Two premolars displayed the interglobular dentine in the coronal half of the root (type B). The types A and B contained large interglobular areas. A small amount of interglobular dentine was restricted to the apical half of the roots of two canines and one molar (type C). In contrast to types A and B which were seen at both labial or buccal and lingual sides of roots, the interglobular dentine of type C was seen only at one side, labial or lingual. Some of the tooth roots did not show any interglobular dentine (type D). Most of the incisors, canines and premolar were types A, B, and C, respectively, and the molars were mixed types A, C, and D. These results suggest that the factors affecting dentinogenesis during root formation are unique for each tooth.