A calcium prerinse required to form calcium fluoride in plaque from a sodium fluoride rinse.

The purpose of this study is to determine whether a calcium (Ca) prerinse used before a 228 µg/g (ppm) fluoride (F) rinse would induce the formation of 'calcium fluoride-like' (CaF2-like) deposits in human dental plaque. Sixty minutes after the use of the Ca prerinse/F rinse, plaque samples were collected from 10 volunteers, homogenized, and split into 2 aliquots. The plaque mass from one aliquot was then extracted with a 'plaque-like' solution that extracted all the CaF2-like deposits. The total F in both aliquots was then determined and compared. The results demonstrated that, as in previous studies, the Ca prerinse induced large increases in plaque fluid and total plaque F. However, unlike previous results without the Ca prerinse, 30% of the plaque F deposits were CaF2 or CaF2-like. Given that maintaining an elevated F concentration in the vicinity of a developing lesion may play an important role in the cariostatic effect of this ion, and the potential advantages of CaF2-like deposits as an F source, these results suggest that a Ca prerinse may increase the cariostatic effect of topical agents.

No calcium-fluoride-like deposits detected in plaque shortly after a sodium fluoride mouthrinse.

Plaque 'calcium-fluoride-like' (CaF(2)-like) and fluoride deposits held by biological/bacterial calcium fluoride (Ca-F) bonds appear to be the source of cariostatic concentrations of fluoride in plaque fluid. The aim of this study was to quantify the amounts of plaque fluoride held in these reservoirs after a sodium fluoride rinse. 30 and 60 min after a 228 microg/g fluoride rinse, plaque samples were collected from 11 volunteers. Each sample was homogenized, split into 2 aliquots (aliquots 1 and 2), centrifuged, and the recovered plaque fluid combined and analyzed using microelectrodes. The plaque mass from aliquot 1 was retained. The plaque mass from aliquot 2 was extracted several times with a solution having the same fluoride, calcium and pH as the plaque fluid in order to extract the plaque CaF(2)-like deposits. The total fluoride in both aliquots was then determined. In a second experiment, the extraction completeness was examined by applying the above procedure to in vitro precipitates containing known amounts of CaF(2)-like deposits. Nearly identical fluoride concentrations were found in both plaque aliquots. The extraction of the CaF(2)-like precipitates formed in vitro removed more than 80% of these deposits. The results suggest that either CaF(2)-like deposits were not formed in plaque or, if these deposits had been formed, they were rapidly lost. The inability to form persistent amounts of CaF(2)-like deposits in plaque may account for the relatively rapid loss of plaque fluid fluoride after the use of conventional fluoride dentifrices or rinses.

Ca pre-rinse greatly increases plaque and plaque fluid F.

Previous studies demonstrated that a Ca pre-treatment greatly increases salivary F from a subsequent NaF rinse. This study examines if these increases are found in plaque and plaque fluid F. Thirteen individuals accumulated plaque before rinsing with: (1) 12 mmol/L NaF (228 microg/g F), (2) 150 mmol/L Ca rinse, or (3) the Ca rinse followed by the F rinse. One hr later, plaque samples were collected, the plaque fluid was recovered, and the plaque residues were extracted 5 times with pH 6.8 or pH 4.8 buffers, and then by acid. The F in each extract after the Ca rinse/F rinse greatly exceeded the corresponding F from the NaF rinse. Consequently, the Ca rinse/F rinse increased the total plaque F and the plaque fluid F by 12x and 5x, compared with the NaF rinse alone. These and the previous salivary results suggest that a Ca pre-treatment may increase the cariostatic effects of topical F agents.

Calcium pre-rinse greatly increases overnight salivary fluoride after a 228 ppm fluoride rinse.

BACKGROUND: Large increases in salivary fluoride were reported 1 h after a calcium pre-rinse/NaF rinse. AIMS: This study examined the persistence of these increases. METHODS: 12 subjects rinsed in the evening with water, with a 228 microg/g (ppm) F rinse or with 150 mmol/l calcium lactate followed by a 228 microg/g F rinse. In a second experiment these same patients rinsed with a 912 microg/g F rinse. Saliva samples were obtained the morning after rinsing, centrifuged and the supernatants analyzed. RESULTS: The Ca pre-rinse/228 microg/g F rinse induced an increase in overnight salivary F over the 912 microg/g F rinse (approximately 2.5 times) and a statistically significant increase over the 228 mug/g F rinse (approximately 5.5 times). CONCLUSIONS: The results suggest that a Ca pretreatment may increase the cariostatic effect of topical F agents.

Ca, Pi, and F in the fluid of biofilm formed under sucrose.

Calcium (Ca), inorganic phosphorus (P(i)), and fluoride (F) concentrations are low in the whole plaque biofilm formed under exposure to sucrose. It was hypothesized that this would be reflected in the biofilm fluid, where these low values should greatly influence the de/remineralization process. Dental biofilms were formed in situ over enamel blocks mounted in palatal appliances and exposed 8 times/day to distilled water, glucose+fructose, or sucrose solutions for 14 days. While Ca, P(i), and F concentrations in the whole biofilms were significantly lower in the glucose+fructose and sucrose groups, no effect on biofilm fluid was observed, even after a cariogenic challenge. An increase in whole biofilm mineral ions was observed 24 hrs after the carbohydrate treatments were suspended, but this effect was also not observed in the fluid. These results suggest that there is a homeostatic mechanism that maintains biofilm fluid mineral ion concentration, regardless of its total concentration in the whole biofilm.

Distribution of fluoride in saliva and plaque fluid after a 0.048 mol/L NaF rinse.

An ultramicro method has recently been described for measurement of plaque-fluid fluoride concentration (Vogel et al., 1990a). This method was used: (1) for exploration of the variation in fluoride concentration of plaque fluid collected from the same buccal tooth sites following a 0.048 mol/L NaF (0.2%) rinse, and (2) for examination of the distribution of fluoride in plaque fluid and saliva within one hour after this rinse. Results indicated an average coefficient of variation (CV) of 31% for plaque-fluid fluoride in triplicate samples recovered simultaneously from the buccal-proximal region of two teeth after the rinse. This was similar to the CV found for plaque-fluid fluoride from the same sites after separate administrations of the rinse. A strong linear correlation was found between salivary and plaque-fluid fluoride at 30 and 60 min after rinse administration, showing that plaque-fluid fluoride is influenced by the concentration of salivary fluoride after administration of this rinse. Plaque-fluid fluoride concentrations were higher than that in saliva at baseline, 30, and 60 min. Very large inter-site and intersubject variations in plaque-fluid distribution were observed, with the central incisors showing the slowest clearance. These variations suggest that an examination of plaque-fluid fluoride from specific tooth regions may be essential for understanding the effects of fluoride on the site-specificity of caries.

Permselectivity of sound and carious human dental enamel as measured by membrane potential.

A microwell technique was used for determination of the permselectivities of sound and carious enamel in the same slice of tooth. The permselectivity determination was accomplished by drilling microwells in the enamel and filling them with a simulated plaque fluid containing lactate, carbonate, and inorganic ions at concentrations similar to those in resting plaque fluid, but with different concentrations of KCl. The electrical potentials developed across the enamel membrane were measured with microreference electrodes placed in the wells or in the solution outside the tooth. The results showed that the membrane potential was a function of the composition of the solutions separated by the enamel membrane and was independent of the composition of the solutions in the adjacent wells. The enamel was found to be cation-permselective, and sound enamel was more permselective than carious enamel. The flux rate of K+ was estimated from the change in the K+ concentration of the well solution as a function of time. The flux rate and the membrane potential data were used in the Nernst-Planck Flux Equation for calculation of the diffusion coefficient of K+ through enamel. The results indicate that the permselectivity of enamel can greatly influence the diffusion of ions through enamel membranes.

Micro-analysis of plaque fluid from single-site fasted plaque.

Despite the site-specific nature of caries, nearly all data on the concentration of ions relevant to the level of saturation of plaque fluid with respect to calcium phosphate minerals or enamel are from studies that used pooled samples. A procedure is described for the collection and analysis of inorganic ions relevant to these saturation levels in plaque fluid samples collected from a single surface on a single tooth. Various methods for examining data obtained by this procedure are described, and a mathematical procedure employing potential plots is recommended.

Increased overnight fluoride concentrations in saliva, plaque, and plaque fluid after a novel two-solution rinse.

Recent studies showed that salivary, plaque-fluid, and whole-plaque fluoride were significantly higher 120 min after subjects rinsed with a novel two-solution rinse than after they rinsed with a NaF rinse of the same fluoride concentration. In this study, the persistence of these increases was investigated overnight, a period of time that is more clinically relevant. Improved analytical techniques for the ultramicro determination of whole-plaque and plaque-fluid fluoride from the same sample are also described. Thirteen subjects abstained from toothbrushing for 48 hrs and rinsed for 1 min with a 12 mmol/L (228 ppm) NaF or the two-solution rinse before bedtime. Samples were then collected the following morning before breakfast: (1) Saliva samples were either clarified by centrifugation or acid-extracted with 1 mol/L HClO4; and (2) single-site molar plaque samples were centrifuged to obtain plaque fluid and/or extracted with 1 mol/L HClO4. Results showed that, compared with NaF, the two-solution rinse produced significantly higher fluoride concentrations in all samples: The concentration of fluoride in whole plaque and whole saliva following the new rinse exceeded concentrations found after the NaF rinse by factors of three and four, respectively, while in plaque fluid, the two-solution rinse produced about a two-fold increase over NaF values, which were near baseline levels. This increase, however, was only about 20% in centrifuged saliva. The increases in saliva and especially in plaque-fluid fluoride after the two-solution rinse indicate a greater remineralization potential, while the enhanced fluoride reservoirs found in plaque overnight after this rinse constitute a reserve that may release fluoride into the plaque fluid over an extended period of time.

Changes in the permselectivity of human teeth during caries attack.

Previous studies have shown that enamel permselectivity can influence fluid composition within caries lesions during de- and remineralization. The permselectivity of human enamel, cementum, and dentin sections was examined, in a microwell model, by measurement of the membrane potential developed by KCl diffusion while the sections were immersed in solutions simulating resting (pH = 5.6) and cariogenic plaque fluid (pH = 4.8). In a second experiment, the effects of charged compounds (phytate and Zonyl-FSC) on the tooth permselectivity were examined. The average membrane potentials (+/- SD) in "resting plaque" solution were: sound enamel, 18.9 +/- 3.2 mV, n = 66; dentin, 0.9 +/- 9.2 mV, n = 59; and cementum, -0.8 +/- 8.2 mV, n = 42, with a positive sing indicating cation selectivity. The average membrane potentials became more negative in "cariogenic plaque" solution for all types of sections: sound enamel, 5.2 +/- 2.1 mV, n = 46; dentin, -8.1 +/- 7.4 mV, n = 45; and cementum, -14.3 +/- 8.0 mV, n = 34. In lesion enamel sections, the membrane potential was reduced from the non-lesion wells in both types of test solutions, while phytate treatment caused an increase of approximately 10 mV in potential (increased cation selectivity) in every enamel well in either "resting" or "cariogenic" solution. Treatment of enamel sections with Zonyl-FSC caused the membrane potential to become more negative in both test solutions, with many of the wells showing anion selectivity in the cariogenic "plaque-like" solution. However, the changes in enamel membrane potentials induced by Zonyl-FSC slowly increased toward the initial values after treatment, while the effects of the phytate pre-treatment persisted. Most dentin sections treated with phytate also showed an increase in potential after phytate treatment; however, Zonyl-FSC seemed to have little effect on the membrane potential of dentin. The results of this study suggest that modification of tooth permselectivity by surface-active agents may be a viable method of decreasing the rate of caries progression.

Fluoride analysis in nanoliter- and microliter-size fluid samples.

A variety of techniques is described for measuring fluoride in volumes of from 0.005 to 5 microL, including: (1) micropipette procedures for transference and dilution of samples, (2) construction of miniature and micro fluoride-selective electrodes, and (3) methods for adapting standard electrodes for micro- and semi-micro volumes. These described techniques have a number of advantages, including speed of analysis, high accuracy, and adaptability to many types of fluid samples. Recent studies involving use of these procedures include the analysis of fluoride in: (1) plaque fluid samples from single sites before and after topical fluoride administration, (2) tooth mineral samples recovered by acid-etch or microdrill biopsy of enamel, and (3) fluid recovered from the interior of the tooth during simulation of the caries process.

The determination of extracellular fluid volume in whole dental plaque using potassium- or chloride-selective micro-electrodes.

This paper describes a procedure for the determination of the volume of water available for diffusion in whole plaque samples collected from single sites. In this procedure, known micro volumes of H2O were added to single-site plaque samples with calibrated nanoliter pipettes and the potassium or chloride concentration at each dilution measured with ion-selective micro-electrodes. By plotting the inverse of the concentration versus the volume of H2O added, one can determine the original volume of plaque fluid available for diffusion. Volumes determined in simultaneous measurements performed with chloride and potassium micro-electrodes were statistically indistinguishable. From the observed constancy in the initial potassium concentration in rat and human plaque fluid, a simplified macro method is suggested which allows for estimation of the extracellular fluid volume within 20% error in plaque samples.

Composition of plaque and saliva following use of an alpha-tricalcium-phosphate-containing chewing gum and a subsequent sucrose challenge.

Previous studies demonstrated that the chewing of a 2.5% (mass fraction) alpha-tricalcium-phosphate-fortified (alpha-TCP) experimental chewing gum released sufficient calcium and phosphate to eliminate any fall in the tooth mineral saturation of plaque fluid after a sucrose rinse (Vogel et al., 1998). In contrast, the chewing of a conventional sugar-free gum did not eliminate this decrease in saturation. The purpose of this study was to examine if the release of ions from plaque calcium-phosphate pools induced by this gum could provide protection during subsequent exposure to cariogenic conditions. Fourteen subjects accumulated plaque for 48 hrs, fasted overnight, chewed a control or experimental gum for 15 min, and subsequently rinsed 1 min with a mass fraction 10% sucrose solution. Before gum chewing, and at 7 min and 15 min afterward, whole plaque, plaque fluid, and salivary samples were obtained and analyzed by micro-analytical techniques. Additional samples were collected and analyzed at 25 min (7 min after the sucrose rinse). Although the results confirmed the deposition of large amounts of calcium and phosphates in plaque seen in the previous study, only a small increase was seen in plaque-fluid-free calcium and phosphate before sucrose administration. This suggests that few of the mineral ions were mobilized under non-cariogenic conditions. However, 7 min after the sucrose rinsing, an increase in these concentrations was seen which, based on hydroxyapatite ion activity product calculations, indicated a decrease in the driving force for demineralization compared with that seen with the control gum. These results suggest that the chewing of the experimental gum deposits a labile mineral reservoir in plaque that can resist a subsequent cariogenic challenge.

Micro-analytical determination of pH, calcium, and phosphate in plaque fluid.

Micro-analytical techniques for the determination of calcium, phosphate, and pH in a small volume (less than 0.25 microliter) of plaque fluid are described and evaluated. The accuracy and the precision of the techniques were compared with those for standard macrotechniques applied to a large pooled plaque fluid sample. The results obtained for the micro-analysis of pooled plaque fluid were in excellent agreement with those obtained by macromethods. The described techniques were also used to analyze plaque fluid obtained from single quadrants of the oral cavities of five individuals. In this fashion, it was determined that, although a significant variation in plaque fluid composition exists between the quadrants, a greater variation exists between subjects. Analyses of plaque fluid obtained from six individuals, following sucrose exposure, were also conducted. The pH value of the fluid changed with time, following a typical Stephan curve, with a minimum value occurring between 15 and 30 minutes; following this, the pH increased to a value near that for resting plaque. An inverse relationship between pH and calcium and phosphorus concentrations was observed. It is noted that the described techniques are sensitive enough to carry out the above analyses both accurately and precisely using plaque obtained from a single quadrant.

Micro-electrode techniques for the analysis of oral fluids.

This paper describes the use of micro-electrodes for the analysis of small fluid volumes recovered from the oral environment. The analysis has several advantages: (1) It directly measures the activity of ions, a quantity more relevant to mineral saturation than the conventionally measured concentration, (2) minimum fluid volume for analysis is usually less than 0.005 microL, small enough to avoid sample pooling in most analyses, (3) numerous ions can be measured simultaneously, (4) the analysis time is very short, and (5) the use of mineral oil to isolate specimens provides a simple method for controlling the CO2 tension and humidity over the specimens.

Composition of plaque and saliva following a sucrose challenge and use of an alpha-tricalcium-phosphate-containing chewing gum.

Calcium phosphate concentrations in plaque, plaque fluid, and saliva play an important role in caries prevention. In this study, we used a microanalytical technique to examine the anticaries potential of a 2.5% (mass fraction) alpha-tricalcium-phosphate-fortified experimental gum by measuring the pH, free and total calcium, and total phosphate in plaque fluid, whole plaque, and saliva, and centrifuged saliva from 14 subjects who (1) accumulated plaque for 48 hours, (2) fasted overnight, (3) rinsed for 1 min with sucrose, and (4) chewed a control or experimental gum for 15 min. From these data, the hydroxyapatite (HAp) ion activity products (IAP[HAp]) of saliva and plaque fluid were calculated as a measure of tooth mineral saturation. Results, compared with those of the control gum, show significant increases in pH and in free calcium and phosphate concentrations in plaque fluid and saliva when the experimental gum was chewed following sucrose ingestion. These increases result in a rise in fluid saturation with respect to tooth mineral that, for plaque fluid, nearly cancels the decrease seen with the control gum after the sucrose rinse. This suggests that the experimental gum may be more effective than a conventional gum in ameliorating the cariogenic effects of sucrose. Similar statistically significant increases were also seen in the total calcium content of the plaque fluid, centrifuged saliva, whole saliva, and whole plaque, and in the total phosphate of whole plaque and whole saliva. These results suggest that the deposition of a mineral reservoir in plaque and saliva by the experimental gum may help resist future cariogenic challenges.

Micro-analysis of mineral saturation within enamel during lactic acid demineralization.

In this study, the physicochemical factors responsible for caries-like lesion propagation were investigated by means of a micro-analytical system used to study the fluid within a lesion during a simulation of the decay process. Four 500-microns-thick serial sections prepared from a single human molar were mounted between glass plates with only the natural surface of the tooth exposed. Microwells were then drilled into sound and pre-existing carious regions of the section through one of the plates. These microwells were then filled with fluid under mineral oil, and after a week of equilibration, the natural surface of the section was exposed to a lesion-producing fluid. The concentrations of calcium, phosphate, and hydrogen ions of the fluid in the wells were then followed as a function of time as the lesion advanced. The results of this study, in which lactic acid was used to demineralize enamel, were consistent with those previously reported (Vogel et al., 1987a): The solution within the lesion remained saturated during the acid attack. Differences in initial mobilities of the calcium and phosphate and other ions, a result of the permselectivity of the enamel, increased the concentrations within the lesion and permanently changed the ratio of these ions in the lesion solution. Based on these results, we suggest that the ionic permselectivity of tooth enamel can have a profound effect on the transport of mineral from a caries lesion.

In vivo fluoride concentrations measured for two hours after a NaF or a novel two-solution rinse.

The concentrations of fluoride in various samples from the oral environment were measured at timed intervals after a novel rinse or a NaF rinse, both containing a total of 12 mmol/L (228 ppm) fluoride. The novel rinse consisted of two solutions mixed just before application: Part A contained calcium chloride and sodium acetate; part B contained a hydrolyzable source of fluoride (sodium hexafluorosilicate) and sodium phosphate. Samples were obtained as follows: Single-site plaque-fluid samples were obtained by centrifugation of first-molar plaque; pooled whole-plaque samples were collected from second molars; centrifuged, pooled whole-saliva was collected by vacuum. All samples were analyzed by micro-analytical methods. Results showed that, compared with NaF, the two-solution rinse produced significantly higher salivary fluoride concentrations, plaque-fluid fluoride concentrations, and acid-extractable fluoride in the whole plaque by factors of about 4, 2, and 6, respectively, at 120 min. The results of this study suggest that the new rinse may provide a greater cariostatic effect at the same fluoride dosage than does a NaF rinse.

Effect in vitro acidification on plaque fluid composition with and without a NaF or a controlled-release fluoride rinse.

Plaque fluid ion concentration changes, especially fluoride, in response to the pH decrease associated with a cariogenic episode are important components of the caries process. A "controlled-release" (CR) fluoride rinse, based on the controlled release of fluoride in the presence of calcium, has been shown to form large fluoride reservoirs in resting plaque. In this study, the in vitro acid-induced release of fluoride, and other ions, was examined in 48-hour-fasted plaque fluid from subjects (n = 11) who received no rinse, or who used a 228-ppm CR or NaF fluoride rinse 1 hr before being sampled. After collection, the plaque was centrifuged to yield plaque fluid, acidified (0.1 microL of 0.5 mol/L HCl per milligram plaque), and then re-centrifuged before a second sample was obtained. Although previous studies indicated a higher plaque fluid fluoride after the new rinse relative to NaF, no statistically significant difference was observed here. Average fluoride release after acidification (average pH, 5.2) was statistically greater following the use of the CR rinse (153 micromol/L) compared with the NaF rinse (17 micromol/L). No fluoride release was seen in the no-rinse samples. The pH, free calcium, phosphate, acetate, propionate, and buffer capacity were not affected by the different amounts of fluoride deposited in the plaque. However, following acid addition, an increase in free calcium and phosphate was observed, which was also independent of the rinse. The large release of fluoride following acidification suggests that the new rinse may provide an improved cariostatic effect.

The buffer capacity of single-site, resting, human dental-plaque fluid.

A carbonate equilibration method was used to measure the buffer capacity of resting plaque fluid collected from single buccal or interproximal sites of upper and lower first molars or anterior teeth. The maximum buffer capacity was 26 m-equiv./l at pH 7.1. The buffer contribution from the measured concentrations of phosphate and carbonate was calculated for each sample. These values were compared with the buffering actually measured and with that expected from organic acids, proteins, and amino acids at average values, as taken from reports in the literature. Relative contributions of buffer species at the average pH of the samples (6.86) were: 35 per cent phosphate, 10 per cent carbonate, 10 per cent protein, 10 per cent organic acids, 2 per cent amino acids, 30 per cent unidentified. There were no significant differences in the buffer capacities of samples originating from sites that differ in their accessibility to saliva. Buffering in resting plaque fluid is more than twice that in saliva and did not show differences correlated with the intra-oral location of the samples.