The effect of inoculum source and fluid shear force on the development of in vitro oral multispecies biofilms.

AIMS: Saliva has been previously used as an inoculum for in vitro oral biofilm studies. However, the microbial community profile of saliva is markedly different from hard- and soft-tissue-associated oral biofilms. Here, we investigated the changes in the biofilm architecture and microbial diversity of in vitro oral biofilms developed from saliva, tongue or plaque-derived inocula under different salivary shear forces. METHODS AND RESULTS: Four inoculum types (saliva, bacteria harvested from the tongue, toothbrush and curette-harvested plaque) were collected and pooled. Biofilms (n ≥ 15) were grown for 20 h in cell-free human saliva flowing at three different shear forces. Stained biofilms were imaged using a confocal laser scanning microscope. Biomass, thickness and roughness were determined by image analysis and bacterial community composition analysed using Ion Torrent. All developed biofilms showed a significant reduction in observed diversity compared with their respective original inoculum. Shear force altered biofilm architecture of saliva and curette-collected plaque and community composition of saliva, tongue and curette-harvested plaque. CONCLUSIONS: Different intraoral inocula served as precursors of in vitro oral polymicrobial biofilms which can be influenced by shear. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculum selection and shear force are key factors to consider when developing multispecies biofilms within in vitro models.

History of frequent gum chewing is associated with higher unstimulated salivary flow rate and lower caries severity in healthy Chinese adults.

This was a single-center, prospective, cross-sectional study stratified by age and gender with the objective of determining the relationship between gum chewing history, salivary flow, and dental caries severity in adults. We enrolled 191 subjects aged 18-65 years who underwent assessments for gum chewing history, unstimulated salivary flow rate, salivary pH, and caries severity. Unstimulated salivary flow rate tended to decline with increasing age (p = 0.04), and significant differences in unstimulated salivary flow rate were also found for males (0.58 ± 0.32 ml/min) versus females (0.48 ± 0.30 ml/min) (p = 0.02). Weekly gum chewing frequency was greater in younger subjects (p = 0.001) while no age group differences were noted in pieces per day or chewing duration. Gum chewing habits were similar in males and females. A multivariate model demonstrated that only days per week chewing gum (p < 0.001) and gender (p = 0.007) were predictive of unstimulated salivary flow rate (R(2) = 0.40). Mean caries severity scores, assessed via ICDAS II and DMFT, increased with age. In multivariate analysis, age was positively associated with ICDAS (p = 0.001) and days per week chewing gum was negatively associated with ICDAS (p = 0.004), indicating that caries severity increased with age, and that days of chewing provided an inverse effect, with increased days of chewing being associated with decreased severity of caries. Overall, a history of frequent gum chewing is associated with higher unstimulated salivary flow rate and lower caries severity.

Delivery of active agents from chewing gum for improved remineralization.

Most surrogate measures of caries were developed to test products containing fluoride, typically at relatively high and closely controlled oral concentrations. However, since the primary mechanism for the remineralization of early enamel caries lesions by chewing gum is through stimulation of saliva, delivering Ca and Pi to the demineralized enamel lesion, established methods may lack the sensitivity to detect the additional benefit of an active agent without the strong remineralizing potential of fluoride. Issues related to the release of active agents from the gum matrix, dilution in the saliva, and limited oral retention time, along with taste, safety, regulatory, and cost concerns, impose further limitations. This paper reviews the efficacy of some active agents used in chewing gum for improved remineralization and includes results from in situ testing of calcium-containing gums, including calcium lactate, tetracalcium phosphate/dicalcium phosphate anhydrous, calcium citrate/encapsulated phosphate, and a calcium lactate/sodium phosphate blend. Despite promising in vitro data from these agents, they did not provide consistently superior results from in situ testing. There is a need to develop better predictive in vitro models for chewing gum, as well as improved sensitivity of in situ models to discriminate relatively small amounts of remineralization against a background of high biological variability.

The relationship between plaque pH, plaque acid anion profiles, and oral carbohydrate retention after ingestion of several 'reference foods' by human subjects.

The primary aim of this study was to rank several reference foods (apple drink, caramel, chocolate, cookie, skimmed milk powder, snack cracker, and wheat flake) according to their plaque pH response as monitored in a panel of 12 volunteers by the plaque-sampling method for comparison with data previously reported with other methods used to assess cariogenicity potential. Secondary experiments (using subsets of the panel of subjects) were undertaken in an attempt to elucidate some of the reasons for the observed plaque pH changes. Oral carbohydrate retention was measured at a single time period after food use as total anthrone-positive carbohydrate material, and as specific acidogenic sugars by gas-liquid chromatography after gel-exclusion chromatography. The concentrations of acid anions in the plaque fluid after food consumption were measured by isotachophoresis eight min after food use. According to the plaque pH response, apple-flavored fruit drink and chocolate were the most acidogenic foods and skimmed milk powder the least acidogenic. There were significant correlations (p less than 0.05) between the plaque pH data and lactate-plus-acetate concentrations in plaque fluid, but the correlations between the pH data and any of the carbohydrate retention parameters were not significant.

The effect of increased mastication by daily gum-chewing on salivary gland output and dental plaque acidogenicity.

The effect of increased mastication on plaque metabolism and salivary gland function was determined in 11 human subjects who chewed a sugarless gum for ten minutes of each waking hour for two weeks. Prior to and at the conclusion of the gum-chewing regimen, unstimulated whole saliva and 2% citric-acid-stimulated parotid saliva were collected. Flow rates, pH, and buffer capacity were determined on all saliva samples. In addition, parotid saliva was analyzed for protein concentration and the proteins further studied by SDS-PAGE. The plaque pH response to a 10% sucrose rinse was also measured before and after the regimen. Significant increases were observed in the pH and buffer capacity of unstimulated whole saliva as were similar increases in the flow rate, pH, and buffer capacity of stimulated parotid saliva. Protein concentrations and profiles remained unaffected. In addition, the resting plaque pH and minimum plaque pH reached after a sucrose challenge were both raised significantly, with a significant reduction in the cH area. The results of this study indicate that increased masticatory effort by frequent consumption of sugar-free chewing gum over a prolonged time period resulted in a functional increase in the output of stimulated parotid saliva, as well as in increases in pH and buffer capacity of whole and parotid saliva, which may help to reduce plaque acidogenicity.

Association of salivary flow rates with maximal bite force.

Mean salivary secretion and bite force decrease with advancing age. Previous studies have shown that salivary flow rates are influenced by mastication. In the present study, we examined the relationship between salivary flow rates and maximal bite force in a community-based sample of men and women 35 years of age or older. Salivary flow rates for unstimulated whole and unstimulated submandibular/sublingual (SMSL) saliva as well as citrate-stimulated parotid and SMSL saliva were measured in 399 subjects. Bite force was assessed with a bilateral force transducer. Pearson correlation analysis yielded significant positive correlations between bite force and flow rates for unstimulated whole saliva (r = 0.24, p < 0.0001), stimulated parotid saliva (r = 0.13, p < 0.03), unstimulated SMSL (r = 0.14, p < 0.0001), and stimulated SMSL (r = 0.16, p < 0.003). When adjusted for age and gender, the partial correlations between bite force and salivary flow rates remained significant for unstimulated whole saliva (r = 0.10, p < 0.05), stimulated parotid saliva (r = 0.13, p < 0.02), and stimulated SMSL saliva (r = 0.14, p < 0.006). Subjects were divided into four groups based on their maximal bite force score (low, medium low, medium high, and high). For each saliva type, the flow rate of the high-bite-force group was significantly greater than that of the low-bite-force group as well as that of the medium-high-bite-force group. These results confirm an age-related decrease in bite force and salivary flow rates and show that, regardless of age or gender, bite force is correlated with salivary flow.

Effects of dietary sucrose levels on pH fall and acid-anion profile in human dental plaque after a starch mouth-rinse.

The effects on the metabolism of starch by plaques formed in human subjects during periods of dietary sucrose limitation or supplementation were studied. High sucrose (HS) plaques showed lower resting pH and pH minima, and higher concentrations of lactate ion after the starch mouth-rinse than low sucrose (LS) plaques. Plaque samples incubated with starch solutions in vitro showed no differences in final pH or acid-anion concentrations between HS and LS diets. Plaque-amylase activity was higher on HS than LS diets. Thus starch was more acidogenic when consumed as part of a diet already rich in sucrose, but it is unclear whether this was a specific or non-specific effect of starch on plaque metabolism.

Influence of mastication on saliva, plaque pH and masseter muscle activity in man.

An earlier study showed that frequent gum chewing may enhance parotid gland function and reduce the acidogenicity of dental plaque. The aim now was to determine whether these effects would be observed after a 2-week period of diet altered to increase masticatory effort, and secondarily to assess the effects of chewing gum on masseter muscle activity. Ten subjects took part in the first experiment. Saliva was collected before and after the diet change and the plaque pH response to a sucrose challenge was measured. Subjects completed 3-day diet histories and wore electromyographic (EMG) devices to record masseter activity. In the second experiment, 10 subjects wore EMG devices for 3 days to record masseter activity on three daily regimens: baseline (no gum chewing), hourly gum chewing (sugar-free gum chewed for 10 min every hour) and chewing five sticks of gum each for 20 min during the day. Data were analysed by paired t test or repeated-measures analysis of variance. For the first experiment, EMG data indicated significant increases in chewing activity (p < 0.05), although there were no changes in salivary flow rates or the plaque pH response to sucrose. The second experiment showed that total EMG activity increased significantly on both gum-chewing regimens (p < 0.01), the magnitude of the increase being greater for hourly chewing. Overall, masseter EMG activity was increased 41% by diet alteration, compared to increases of 96 and 152% on the five-stick and hourly gum-chewing regimens, respectively.

Effect of donor age on the concentrations of histatins in human parotid and submandibular/sublingual saliva.

Histatins are small proteins of human glandular saliva that have antifungal properties. Recent studies show that oral candidal infections increase with age, suggesting an age-associated compromise in oral host defence. Here, the effect of age and of physiological gland stimulation on the concentration and secretion of salivary histatins was investigated. Parotid and submandibular/sublingual salivas were collected from six young adults under unstimulated, mechanical (chewing) and gustatory (0.025 M and 0.1 M citric acid) stimulation, and the concentration and secretion of histatins was measured by cationic polyacrylamide gel electrophoresis with subsequent densitometric scanning of the stained gels. With gland stimulation, parotid saliva showed no significant increase in histatin concentration (microg/ml); however, histatin secretion (microg/min) increased up to 26-fold (p<0.005; ANOVA). Stimulation of submandibular/sublingual saliva resulted in significant increases in both histatin concentration (p<0.005) and secretion (p<0.0005). Ageing effects on salivary histatins were determined in citric acid (0.1 M)-stimulated parotid and submandibular/sublingual saliva samples collected from 80 individuals (divided into four age groups having approximately equal numbers of males and females: 35-44 years; 45-54 years; 55-64 years and 65-76 years). None of the patients was taking medications or wore dentures. ANOVA showed no sex differences in histatins. Regression analysis showed significant age-associated decreases for parotid saliva histatin concentration (p<0.002) and secretion (p<0. 002) as well as for submandibular/sublingual saliva histatin concentration (p<0.0001) and secretion (p<0.0001). Both saliva types showed significant (p<0.0001) decreases in the histatin concentration per mg of total protein, suggesting a preferential decrease in salivary histatins compared to total salivary protein. These results suggest that the salivary histatin component of the oral host defence system is compromised with increasing age.

A population-based study of salivary lysozyme concentrations and candidal counts.

The relationship between salivary lysozyme concentration and oral candida load was examined in 595 adults. Unstimulated whole saliva, and citrate-stimulated parotid and submandibular/sublingual saliva were collected from each participant. Candida colony-forming units (c.f.u.) in unstimulated whole saliva were determined. An enzyme-linked immunosorbent assay for lysozyme using commercially available antibodies was developed. This assay showed a linear relation of salivary lysozyme concentrations from 0.5 to 4.0 ng/ml. Significant negative relations were observed between lysozyme concentration and flow rate: r = -0.16 (p < 0.001) for stimulated parotid and r = -0.22 (p < 0.0001) for stimulated submandibular/sublingual saliva. The lysozyme concentration in stimulated submandibular/sublingual saliva was higher in males than in female, but no sex difference was observed for stimulated parotid saliva. The lysozyme concentration of stimulated parotid saliva was positively correlated with candida counts (r = 0.18: p < 0.005). Further study of groups according to their levels of candida in whole saliva revealed that lysozyme concentrations were higher in the high candida (> or = 1000 c.f.u./ml) group than in the zero and moderate candida categories in stimulated parotid saliva (p < 0.001): there were no concentration differences in stimulated submandibular/sublingual saliva. These results suggest that parotid lysozyme concentration increases as candida load increases.

Salivary alterations in type 2 (non-insulin-dependent) diabetes mellitus and hypertension.

OBJECTIVES: The aim of this study was to determine whether saliva output and composition are altered in type 2 diabetes mellitus by comparison with a healthy, non-medicated control group, and also a group of hypertensives. METHODS: From a community-dwelling cohort of Mexican American and European American subjects enrolled in the OH:SALSA oral aging study, we identified 233 subjects with type 2 diabetes mellitus, 227 with hypertension, and 240 healthy control subjects. We collected unstimulated whole (UW) and submandibular/ sublingual (US) saliva, as well as stimulated parotid (SP) and submandibular/ sublingual (SS) saliva. Flow rates were determined, yeast carriage was assayed in UW saliva, and SP and SS saliva samples were analyzed for protein composition. ELISA was used to determine concentrations of an array of specific protein components, with both antimicrobial and other activities. RESULTS: Both diabetic and hypertensive subjects had reduced output of both stimulated and unstimulated submandibular/sublingual saliva. 30% of the diabetic subjects had high oral yeast counts (> or =1000 cfu/mL) compared with 17% of the healthy subjects and 20% of the hypertensives. Significant increases in the concentrations of a number of the protein components were found in the diabetic subjects, specifically, SP lactoferrin, myeloperoxidase (MPO), and salivary peroxidase (SPO), as well as SS total protein, albumin, lactoferrin and secretory IgA. CONCLUSIONS: The pattern of decreased flow rates and increased protein concentrations were similar, but consistently greater in diabetics than hypertensives, suggesting that disease-specific mechanisms may be responsible. Diabetics may be more prone to oral dryness and infections than non-diabetics.

Effects of glycemic control on saliva flow rates and protein composition in non-insulin-dependent diabetes mellitus.

OBJECTIVE: The objective of this study was to determine whether improvements in the level of diabetic control in a group of subjects with poorly controlled non-insulin-dependent diabetes mellitus influence salivary output and composition. STUDY DESIGN: Repeated whole unstimulated and stimulated parotid saliva samples were collected from diabetic patients attending an outpatient diabetes education program and a matched nondiabetic control group. Saliva was analyzed for flow rates, parotid protein concentration and composition, and amylase activity. Subjective responses to questions about salivary hypofunction were tested. RESULTS: There were no significant differences in whole unstimulated and stimulated parotid flow rates or stimulated parotid protein concentration and composition between diabetics and the control group. Amylase activity was higher in diabetics and decreased with improved glycemic control. Subjects reporting taste alterations had higher mean blood glucose levels than subjects with normal taste sensation. CONCLUSIONS: Poorly controlled non-insulin-dependent diabetes mellitus has no influence on saliva output, although amylase activity may be elevated, and there may be taste alterations.

Parotid saliva protein profiles in caries-free and caries-active adults.

OBJECTIVE: The objective of this study was to determine if there were any differences in the parotid saliva output and composition related to caries activity. STUDY DESIGN: Stimulated parotid saliva samples were collected from 85 healthy young adults, caries-active or caries-free. Flow rates were determined, and samples were analyzed for pH and buffer capacity, total protein, electrolytes, proteins with a high performance liquid chromatography method, and histatins. RESULTS: There were no differences in flow rates or pH, but buffer capacity was higher in women than in men, and K+ and Cl- were both slightly higher in the caries-active group. The women had a significantly higher total protein concentration, as well as higher concentrations of each of the individual protein components assayed. There were no differences attributable to caries activity. CONCLUSIONS: Significant sex differences in salivary protein concentrations exist. Caries activity may be related to some salivary electrolyte alterations, but not to protein composition.

The effect of sweeteners on acid production in plaque.

In studies of the pH response of dental plaque in situ to rinses with conventional sugars, glucose and maltose give similar falls in pH, and fructose only slightly smaller effects compared with sucrose. Lactose and galactose are less acidogenic, while the pH response to glucose syrups varies according to their composition. Of the sugar alcohols, sorbitol, mannitol, maltitol and lactitol are all slowly fermented to acid by oral bacterial, and xylitol is virtually non-fermentable. Acid formation in plaque by fermentable sugar alcohols can be too slow to overcome the buffering power of plaque and saliva and plaque pH can rise following exposure to these compounds. Lycasin, a synthetic sweetener containing sorbitol, maltitol and some higher sugar alcohols gives effects similar to its major constituents. Palatinit, L-sorbose and trichlorogalactosucrose are of low acidogenicity, but coupling sugar is more fermentable and can give rise to a substantial pH response, albeit less than sucrose. Non-nutritive intense sweeteners may affect plaque pH by their sialogogue effects.

The effect of stannous fluoride on dentinal hypersensitivity.

Many agents have shown varying degrees of effectiveness on pain resulting from exposed dentine. One which has shown some promising results is stannous fluoride (SnF2). The purpose of the following paper is twofold: to review and summarise the clinical literature pertaining to the relative effectiveness of solutions or gels containing SnF2 in controlling pain associated with dentinal hypersensitivity; and to statistically re-evaluate these studies in combination, in order to develop recommendations for the optimal use of SnF2 for hypersensitivity. Seven blinded clinical studies were identified and reviewed. Five of these compared 0.4 per cent SnF2 gel solution to an identical placebo. One compared a 0.4 per cent SnF2 gel solution and a 0.717 per cent F solution to an aqueous placebo. The final study compared a 0.717 per cent F solution to an aqueous placebo. Statistical power analysis and a combined meta-analysis were used to ensure adequate internal consistency and to contribute to an overall consensus of the efficacy across time. It was concluded that the 0.717 per cent F solution provides a virtually immediate and definable effect, which seems to continue for several months. This effect was present in all subjects used in the study. This solution was applied directly to the sensitive area for one minute and allowed to remain for 3-5 minutes. An additional one minute application was applied if needed. The effect of the 0.4 per cent SnF2 gel appears to be more gradual, perhaps involving a different mechanism of action. This solution requires approximately two to four weeks of continuous treatment to be effective. It was concluded that an effective strategy involving the use of stannous fluoride gel includes the application of the 0.717 per cent F solution in the office, effectively providing immediate relief. The patient would then use the 0.4 per cent SnF2 gel at home in order to achieve the long-term effect. In order to control episodic pain while the gel is developing its effect, a small amount of the 0.717 per cent F solution could be given to the patient for occasional symptomatic application.

Interactions between fluoride and plaque in the remineralization of enamel caries-like lesions.

The effect of fluoride and plaque, separately and together on the remineralization of early artificial caries lesions was studied in a group of volunteers in a double-blind randomized study design. Artificial caries lesions produced in slabs of human enamel were mounted buccally in silver bands cemented bilaterally to the mandibular first molar teeth. Volunteers wore the bands for two 2-week periods, rinsing daily with either a placebo or a 500 ppm fluoride mouthrinse. Plaque accumulation was encouraged by placing Dacron gauze over one of each pair of slabs. Sections were cut from the lesions and analyzed by microradiography followed by microdensitometry and image analysis. Microdensitometric data revealed that non-significant increases in mineralization had occurred in all of the test groups. However, the image analysis results indicated that significant remineralization had occurred in lesions after 2 weeks on the fluoride rinse regimen, with no significant remineralization in lesions exposed to the placebo. The presence or absence of plaque over the lesions had no effect on the amount of remineralization induced on the fluoride rinse regimen.