A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization

A plaque growth chamber was developed for long-term growth of five separate plaques from the same plaque or saliva sample under identical conditions of temperature and gas phase. Reagent addition and growth conditions for each plaque could be independently controlled, and each was accessible for sequential sampling and electrode insertion. Plaques were cultured for over six weeks on pellicle-coated Lux (TM) 25-mm diameter cover-slips at 35 degrees C under 5% CO2 in N2, and supplied with a medium containing 0.25% mucin (BMM) at 3.6 mL/h, and with periodic 5% sucrose. Electron microscopy and flora analysis of microcosm plaques showed that they had close similarities to reported characteristics of natural dental plaques. Diverse motile bacteria were present. Sucrose-induced Stephan pH curves and urea-induced pH rises were also similar to those reported for natural plaques. Changes in plaque urease, calcium, phosphate concentrations, and the flora were followed over five weeks in a plaque supplied with BMM containing additional 2.5 mmol/L calcium and 7.5 mmol/L phosphate. Despite this high environmental calcium phosphate concentration, there was no continuing increase in calcium levels, although plaque phosphate doubled. Urease levels fluctuated. Changes in the cultivable flora were minor. A urea-containing calcium phosphate/mono-fluorophosphate pH 5 solution, applied for six min every two h for seven days, increased plaque calcium, phosphate, and fluoride to high levels. Thus, plaques grown over several weeks in the multi-station artificial mouth exhibited metabolic and pH behavior typical of natural plaques, could be analyzed during development, and the system allowed manipulation of environmental variables important in plaque pH control and calcification.     

Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic pH-rise procedure

The objectives were to develop and characterize a procedure based on a ureolytic pH rise to deposit calcium phosphate into microcosm dental plaque biofilms and to test the importance of the plaque pH range. Plaque biofilms were cultured in a multiplaque culture system ('artificial mouth') with a continuous supply of a simulated oral fluid (basal medium mucin; BMM) with 146 mmol/l (5% w/v) sucrose periodically applied over 6 min every 8h. After initial plaque growth, the biofilms were periodically exposed for up to 16 days to 6-min applications of calcium phosphate monofluorophosphate urea (CPMU) solution containing 20 mmol/l CaCl(2), 12 mmol/l NaH(2)PO(4), 5 mmol/l monofluorophosphate and 500 mmol/l urea (pH 5.0). Three application regimes were examined, one included a sucrose-induced acidic pH fluctuation. Plaque hydrolysis of the urea in CPMU caused the pH to rise to between 8.2 and 8.8, depositing fluoridated and carbonated calcium phosphates, and possibly some calcium carbonate, into the plaque. Calcium, phosphate and fluoride deposition was rapid for about 4 days and then slowed. After 10 days' treatment under standard conditions (BMM containing 1 mmol/l urea and 1 mmol/l arginine), plaque calcium and phosphate concentrations had increased up to 50-fold and 10-fold to approximately 2-4 and 1-2 mmol/g plaque protein, respectively. The calcium, phosphate and fluoride content increased steadily. Calcium phosphate deposition was proportional to the plaque resting pH, increasing over four-fold when the BMM urea concentration was increased from 0 to 20 mmol/l, which raised the resting pH from 6.4 to 7.2 and yielded a mean plaque calcium concentration of 14.3 mmol/g protein, one subsample reaching 20.8 mmol/g protein. Supplementation of BMM with 20% human serum inhibited deposition. These results support the hypothesis that an alkaline pH in plaque is critical in promoting plaque mineralization and that mineral deposition is modulated by serum. These factors are likely to be important in regulating calculus formation.     

The effect of sucrose application frequency and basal nutrient conditions on the calcium and phosphate content of experimental dental plaque

A reduced pool of calcium in dental plaque would be expected to increase the ability of plaque fluid to dissolve the underlying enamel when the pH falls during sugar exposure. We have examined the relationship between frequency of sugar application and Ca and P(i) concentrations in artificial mouth plaque microcosm biofilms. Ten plaques were grown simultaneously from a human saliva inoculum using a continuous flow of simulated saliva, DMM, supplemented with either urea or glucose to modulate the resting pH. In addition the plaques received sucrose applications of varying frequency: 12-, 8-, 6-, or 4-hourly, or not at all. After 15 days the plaques were sampled by taking 4 full-thickness specimens of each, and acid-extractable Ca and P(i), and alkali-soluble protein and carbohydrate were determined. Ca and P(i) concentrations were in a range comparable with those in human plaque, except in the DMM + urea plaque receiving no sucrose, when concentrations were higher. Plaque Ca concentration decreased significantly as sucrose application frequency increased. Increasing sucrose application frequency also reduced the protein, i.e. the cell biomass, content of the plaques and, in the case of DMM + urea plaques, increased the water-insoluble hexose content, presumably extracellular polysaccharide. Reduced biomass was partly due to the bulking of plaque with extracellular polysaccharide, but the marked effect of urea on polysaccharide formation is not understood. This study shows that increasing frequency of sugar application alters dental plaque by reducing its mineral protection capacity.     

A comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva

The growth and pathogenic properties of dental plaque result from interactions between the microbiota and the oral environment and have been studied in laboratory experimental systems ranging from single or a few species (such as in chemostats) to dental plaque microcosms. Microcosm plaque is an in vitro version of natural plaque and has been explored as a microflora model because it is sited a more manipulable and controllable environment. It is obtained as microcosm biofilms in an 'artificial mouth' plaque culture system by culturing the bacteria in natural plaque-enriched saliva (i.e. salivary bacteria where a whole-saliva donor has abstained from oral hygiene for 24 h to increase the plaque bacteria in the saliva). The aim here was to examine whether a new, chemically defined analogue of saliva (defined medium mucin, DMM) could substitute for a previously used, chemically undefined medium (basal medium mucin, BMM) as an analogue of saliva for large-scale biofilm culturing. DMM contains various ions, mucin, amino acids, vitamins and growth factors at concentrations generally similar to those in saliva, whereas BMM contains yeast extract, peptones and mucin. To model the nutrient functions of salivary proteins, amino acids equivalent to 5 g/l casein were also included in DMM. In earlier studies, BMM-grown plaques were similar to natural plaques in structure, composition, growth rate and pH response to substrates. Their doubling-time patterns over a 20-day period were similar, except that the DMM-grown plaques showed biphasic growth patterns that were more pronounced than with BMM. Variation in enzyme profiles between BMM- and DMM-grown plaque, measured using the API-ZYM technique, provided evidence of nutritional effects on plaque composition. It was concluded that realistic growth rates and patterns are generated in microcosm plaque biofilms by supplying both DMM and BMM. However, the use of DMM enables specific modifications to be made to nutrient conditions during large-scale culture in our 'artificial mouth' biofilm system.     

Effects of bovine immune and non-immune whey preparations on the composition and pH response of human dental plaque

Colostral products from non-immunized cows (CP) and cows immunized with mutans streptococci (IP) were used as mouth rinses in a short-term human study. The acidogenic potential of the products was tested and found to be negligible in vivo before application to subsequent rinsing tests. At first, all the participants received a professional tooth cleaning, after which they rinsed with one of the solutions (IP; CP; water) three times per day for 3 d. After each rinsing period, the resting pH and decrease in plaque pH after sucrose challenge were determined, the amount of plaque was estimated, and all available plaque was collected. No significant differences were recorded in the composition or in the amounts of accumulated plaque. The resting pH values of plaques with low "innate" pH were increased after the IP rinsing period. Surprisingly, the lowest pH values after the sucrose challenge were recorded in IP plaques. The number of cultivable facultative flora or total streptococci were not affected by different rinsings, but the relative number of mutans streptococci significantly decreased after the IP rinsing period when compared to the CP period. Thus, the short term rinsing indicates favourable effects of bovine immune whey on human dental plaque.     

An in-dwelling electrode for in-vivo measurement of the pH of dental plaque in man.

A system for the measurement of plaque pH in vivo has been developed, using an in-dwelling miniature glass electrode. The reliability of the electrode and the closeness of the experimental conditions to natural ones compared favourably with previous techniques for measuring plaque pH. The effect on plaque pH of sucrose and saccharin rinses, and a chlorhexidine mouthwash were studied. Continuous small, rapid fluctuations in the pH of both stimulated and resting plaques were consistently observed and larger changes of pH were noted on opening the mouth and swallowing.     

Plaque pH measurements by different methods on the buccal and approximal surfaces of human teeth after a sucrose rinse

Changes in plaque pH after a sucrose rinse were simultaneously monitored by plaque sampling, touching of the plaque with antimony and glass electrodes, and telemetry. The minimum pH at approximal sites was approximately 0.7 pH units lower than that on buccal surfaces. The pH at the approximal site remained below resting levels after 120 min, and the area under the pH response curves from this site was five times greater than that from the buccal surfaces.     

A theoretical analysis of the effects of plaque thickness and initial salivary sucrose concentration on diffusion of sucrose into dental plaque and its conversion to acid during salivary clearance

A mathematical model, written in FORTRAN, has been developed to simulate the interrelated processes of salivary sucrose clearance from the mouth, diffusion of sucrose into dental plaque, and conversion of sucrose to acid and glucan. Reaction of acid with enamel is not included in the model. A total of 28 parameters can be varied by the user, and the relative importance of the different factors affecting acid formation can be assessed. The output of the program gives sucrose and acid concentrations and pH at different depths within the plaque. The initial variables studied were plaque thickness, the salivary sucrose concentration, and the duration of exposure of the plaque to sucrose. Stephan curves typical of those recorded in vivo were generated by the model. With any particular salivary sucrose concentration, there was an optimum plaque thickness at which a minimum pH was achieved at the enamel surface, with very thin or thick plaque samples producing a smaller pH fall. With thick plaque, the minimum pH was often not achieved at the inner surface but at some intermediate depth, which may explain the location of early caries lesions in fissures. The extent of the pH fall at the inner surface and the duration of the pH-minimum region of the Stephan curve were directly related to the initial salivary sucrose concentration and to the duration of exposure to sucrose prior to normal salivary clearance. Simulation of a water rinse at as short a time as two min after the beginning of normal salivary sugar clearance showed that this procedure had only a very small effect on the shape of the Stephan curve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of salivary film velocity on pH changes in an artificial plaque containing Streptococcus oralis, after exposure to sucrose

Results from a computer model suggest that following exposure of dental plaque to sucrose, the rate of clearance of acids from plaque into the overlying salivary film will be greatly retarded at low film velocities. This was investigated with an in vitro technique in which artificial plaque containing S. oralis cells was exposed to 10% sucrose for one min. The pH at the proximal (P) and distal (D) undersurfaces of the plaque (0.5 or 1.5 mm thick) was then monitored during the passage of a 0.1-mm-thick film of a sucrose-free solution over the surface. Over the range of salivary film velocities that have been estimated to occur in vivo (0.8-8 mm/min), lower minimum pH values and increased times for the pH to recover toward neutrality occurred at the lower salivary film velocity. Lower pH values were also reached with the 0.5- than with the 1.5-mm-thick plaque. P/D pH gradients, with a lower pH distally, developed at film velocities of 0.8 and 8 mm/min, and the gradients were much more pronounced at the lower velocity. No P/D pH gradients developed when the film velocity was 86.2 mm/min. Incorporation of dead S. oralis cells into the plaque at percentages up to 57% reduced the extent of the pH fall and prolonged the recovery of the pH toward neutrality. The results support the prediction that, other factors being equal, plaque located in regions of the mouth with low salivary film velocity will achieve pH values lower than those of plaque of identical dimensions and microbial composition located in areas where salivary film velocity is high.     

Comparative study of plaque pH on enamel and cemental surfaces

This study was undertaken to compare the pH response of enamel and cemental plaques to a sucrose rinse challenge. The plaque pH readings on the enamel surfaces were consistently lower than the subjacent cemental plaque pH readings on the same teeth at the same time intervals following the sucrose exposure.     

咀嚼无糖口香糖对含漱蔗糖溶液后牙菌斑原位pH值的影响

目的 通过对牙菌斑原位pH值变化的动态监测，观察咀嚼无糖口香糖对牙菌斑原位pH值的影响。方法 采用受试者自身对照的临床试验方法，选择16名健康成人志愿者为受试者，年龄23～32岁，其中男性6名，女性10名。首先测定受试者48h菌斑的静止pH值，以及受试者用10％蔗糖溶液含漱1min后在5、10、20和30min时菌斑的pH值，取得受试者的Stephan曲线作为基线对照；而后观察咀嚼两种益达无糖口香糖对含漱10％蔗糖溶液后菌斑pH值变化的影响。菌斑原位pH值的测定采用pH微电极接触法在口内直接测量。结果 含漱10％蔗糖溶液后立即开始咀嚼无糖口香糖可使菌斑pH值在各检测时间点(含漱10％蔗糖溶液后5、10、20和30min)均维持在静止pH水平，无明显下降；含漱10％蔗糖溶液后在5min时开始咀嚼无糖口香糖则使菌斑pH值从含漱蔗糖溶液后5min时的5．59迅速回升至10min时的6．98。结论 受到蔗糖攻击后，咀嚼无糖口香糖可迅速缓冲菌斑的酸性产物，升高菌斑pH值。     

The Effect of Common Infant Foods on Plaque pH

Purpose: To evaluate the effect of cow's milk, milk plus sugar, milk plus honey and formula on plaque pH when compared to a 10% sucrose solution in 8- to 12-year-old children. Materials and Methods: Plaque pH was measured using a pH microelectrode at baseline to determine resting plaque pH and at intervals between 1 and 60 min after rinsing with the test liquids. Plaque pH data were analysed using repeated- measure ANOVA and paired t tests. Results: The study findings showed that 10% sucrose has the greatest cariogenic potential, followed by milk plus honey, milk plus sugar, formula and cow's milk. Except for the control group, the evaluated foods did not reduce the pH below 6. Conclusion: Although the test groups appear to be non-cariogenic as compared with sucrose, they still lead to considerable acidogenic response in the dental plaque. When bottle feeding is essential, it was confirmed that frequently use of these beverages may cause enamel demineralisation.     

Patterns and rates of growth of microcosm dental plaque biofilms

Rates of growth in wet weight and changes in them over time were established for microcosm dental plaques cultured from the mixed salivary bacteria in an artificial mouth. Standardized conditions included a continuous supply of medium containing 0.25% mucin and 1.5 ml of 5% w/v sucrose in 6 min every 8 h. Plaques were weighed daily. Plaque wet weight and total protein were highly correlated. Plaque doubling times were 3-7 h over day 1 and 9-21 h over day 2, which is similar to in vivo plaques. Subsequently, growth curves were either linear or between a linear and exponential increase. Evidence was obtained for plaque blooms. Methyl paraben (0.2%) applied for 15 min (3.75 ml) 6 times daily inhibited growth but only for 3 days, after which the rate was similar to control plaques, indicating that selection for resistance had occurred. It was concluded that the regulation of plaque growth rates is complex and does not conform to simple growth pattern models. Detailed studies of plaque growth and the effects of antiplaque agents can be carried out using this experimental system.     

Effect of sugarcane chewing on plaque pH in rural Kenyan children.

In 5 rural Kenyan children, the effect of sugarcane chewing on plaque pH was compared with the effect of a mouthrinse with 10% sucrose at various intraoral sites. They all had poor oral hygiene and at least two carious cavities in occlusal surfaces of molars. pH measurements were conducted under field conditions using paladium touch microelectrodes connected to a battery-operated pH meter. There was a marked difference in pH response of non-carious approximal sites between maxilla and mandible, with the lowest values in the maxilla. However, the pH recovery following the instantaneous drop occurred in parallel even if most pH values had not returned to baseline values 30 min after the sucrose rinse. Following the sugarcane chewing, the pH fall was less pronounced on all sites, and within 5-10 min the values had returned to resting pH and even exceeded this. In carious cavities, a similar pattern was observed, although the acidity in these sites was more pronounced, also reflected in a lower resting mean pH. The main conclusion from this study is that sugarcane chewing yields a less pronounced pH drop and a quicker pH recovery in dental plaque than is seen following a mouthrinse with 10% sucrose. This difference probably results from stimulation of salivary flow associated with the chewing.     

Assessment of denture plaque pH in subjects with and without denture stomatitis

abstract — To evaluate the "resting" pH and induced pH changes in denture plaque, soft deposits were collected from the fitting surface of the denture, pooled and suspended in water. Plaque pH was determined with microelectrode equipment before and after mouth rinsing with a sucrose solution. A characteristic level in the "resting" pH of denture plaque was found in most of 12 subjects tested. pH values below the baseline level were recorded for more than 2 h after a rinse. The pH depressions were more pronounced in maxillary than in mandibular plaque. Further, the pH minima tended to be lower in subjects with denture stomatitis than in. controls. No clear relationship could be established between the "resting" pH and the concentration of Candida hyphae in denture smears or palatal inflammation.     

Effects of salivary bicarbonate content and film velocity on pH changes in an artificial plaque containing Streptococcus oralis, after exposure to sucrose

Chewing-gum stimulation of salivary flow (at the time of the pH minimum following exposure of plaque to carbohydrate) has been shown to cause a rapid increase in plaque pH. The objective of this study was to determine whether the rise in plaque pH is primarily due to the increased buffering capacity of stimulated saliva, or to the fact that an increased flow rate increases the concentration gradient for acid to diffuse from the plaque into the overlying salivary film, which will be moving at a higher velocity. This was investigated with an in vitro technique in which artificial plaque (0.5 or 1.5 mm deep) containing S. oralis cells was exposed to 10% sucrose for one min. The pH values at the proximal and distal undersurfaces of the plaque were then monitored during the passage of a 0.1-mm-thick film of a sucrose-free artificial saliva over the surface, at a range of film velocities (0.8-8 mm/min) that have been estimated to occur in vivo. When a minimum plaque pH had been achieved, the salivary film velocity was either (a) kept the same, with or without 15 mmol/L HCO3 (the concentration measured in chewing-gum-stimulated saliva), (b) increased to 86.2 mm/min, or (c) increased to 86.2 mm/min with 15 mmol/L HCO3 added to the artificial saliva. The findings suggest that after sucrose ingestion, the rapid rise from minimum plaque pH values, which can occur with gum-chewing stimulation of salivary flow, is due to the combined effects of the increase in salivary film velocity, and of a greater availability of bicarbonate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of denture plaque pH in subjects with and without denture stomatitis.

To evaluate the "resting" pH and induced pH changes in denture plaque, soft deposits were collected from the fitting surface of the denture, pooled and suspended in water. Plaque pH was determined with microelectrode equipment before and after mouth rinsing with a sucrose solution. A characteristic level in the "resting" pH of denture plaque was found in most of 12 subjects tested. pH values below the baseline level were recorded for more than 2 h after a rinse. The pH depressions were more pronounced in maxillary than in mandibular plaque. Further, the pH minima tended to be lower in subjects with denture stomatitis than in controls. No clear relationship could be established between the "resting" pH and the concentration of Candida hyphae in denture smears or palatal inflammation.     

Effect of dental plaque age and bacterial composition on the pH of artificial fissures in human volunteers

Changes in sucrose-induced plaque pH profiles and the microbial composition of occlusal tooth surface fissures were analyzed using wire telemetry and bacterial culturing techniques. Four human volunteers wore appliances containing artificial fissures constructed with ion-sensitive field-effect transistor (ISFET) electrodes for 1, 2 and 4 days; 1 subject kept the electrode for 3 weeks. After monitoring the plaque pH response at the base of the fissure to a 10% (w/v) sucrose rinse the plaque was removed and analyzed for total viable bacteria, total and specific streptococci, lactobacilli and Actinomyces spp. One-day-old plaque showed a rapid drop in plaque pH to a minimum of 4.8 +/- 0.2, with 2-day-old plaque showing the most acidogenic pH profile (minimum pH 4.6 +/- 0.2). The 4-day-old plaque response was less acidogenic (minimum pH 5.0 +/- 0.3) than the results from days 1 and 2. Responses from 13- and 21-day-old fissure plaques showed greatly decreased acidogenic responses (day 21 minimum pH 5.7). Viable bacteria recovered from the fissure increased from approximately 4 x 10(6) colony-forming units on day 1 to 1.2 x 10(7) on days 2 and 4 and 1.7 x 10(7) on day 21. Streptococci (greater than 50%) and Actinomyces (greater than 10%) dominated in the fissure plaques and their levels were related to minimum pH. Since fissure plaque of all ages tested contained high concentrations of acidogenic bacteria, the decreased acidogenic response at the base of fissures with increasing plaque age suggests that maturing fissure plaques provide an increasingly greater diffusion barrier to fermentable carbohydrates.     

Effect of urea concentration on human plaque pH levels in situ

Urea solutions, varying in concentration between 0 and 16.7% (w/v), were applied to dental plaques in situ for different lengths of time, and the resulting changes in plaque pH were measured for periods up to two hours. The pH was measured with antimony micro-electrodes in subjects who had not brushed their teeth for 3 days and had not eaten for at least 12 hr. Application of urea solutions to the plaques for short periods of time resulted in an initial rapid rise in pH followed by a slow pH fall. When the urea solutions were applied for longer time periods, the pH rose higher than previously and, if added for a sufficiently long time, the pH reached an asymptote, the level of which was a function of the urea concentration. Once an asymptote was reached, failure to apply further urea solution to the plaque resulted in an immediate fall in the pH towards the base-line. Applications of solutions of high urea concentration for short periods of time resulted in pH curves similar to those observed when solutions of low urea concentration were applied for longer time periods. The data showed that urea levels equivalent to those in saliva could produce a rise in plaque pH equivalent to the difference in pH between plaque and saliva which has been reported previously in fasting subjects.     

蔗糖浓度对牙菌斑生物膜细菌组成及pH值影响的体外实验

目的 研究不同蔗糖质量浓度对体外牙菌斑生物膜细菌组成和pH值的影响,探讨口腔微生物在蔗糖存在下的致龋能力.方法 采集有龋者(龋失补牙面数＞3)集合牙菌斑样本5例,混合后在不同质量浓度(0、1.5、3.0、5.0及20.0g/L)蔗糖中体外培养生物膜;对生物膜进行聚合酶链反应-变性梯度凝胶电泳分析和细菌量计数,并行pH值监测.结果 随着培养基中蔗糖质量浓度增加,生物膜细菌种类减少;随着蔗糖质量浓度的升高(0、1.5、3.0、5.0、20.0g/L),生物膜培养36 h后所能达到的最低pH值呈降低趋势,分别为5.4、4.7、4.5、4.2及4.2;在有蔗糖时,最终12 h生物膜培养达到pH最小值约需6h,相比最初12h(约需10 h)明显缩短;对生物膜细菌量进行单因素方差分析结果显示,在蔗糖质量浓度为5.0g/L时细菌量最大,向下蔗糖质量浓度依次为3.0、20.0、1.5及0g/L,各组间相比差异有统计学意义(P＜0.05).结论 蔗糖浓度可影响体外牙菌斑生物膜的细菌组成和pH值,含蔗糖的培养基可筛选出具有潜在强产酸和耐酸能力的菌群.