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Ca, P_i, and F in the Fluid of Biofilm Formed under Sucrose

¹ Faculty of Dentistry of Piracicaba, UNICAMP, Av. Limeira 901, 13414-903, Piracicaba, SP, Brazil; and
² Paffenbarger Research Center, ADA Foundation, NIST, Gaithersburg, MD, USA

^* corresponding author, jcury{at}fop.unicamp.br

	ABSTRACT

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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.

KEY WORDS: Fluid • biofilm • sucrose • calcium • phosphorus • fluoride • demineralization • remineralization

	INTRODUCTION

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Previous studies have shown that biofilms formed under frequent exposure to sucrose have decreased concentrations of whole plaque calcium (Ca), inorganic phosphate (P_i), and fluoride (F) (Cury et al., 1997; Pearce et al., 2002; Paes Leme et al., 2004; Pecharki et al., 2005), which were not directly related to an increased concentration of insoluble extracellular polysaccharides (EPS), produced from sucrose, in the biofilm matrix (Cury et al., 2000; Ribeiro et al. , 2005). However, the pH, Ca, P_i, and F concentrations in the plaque biofilm fluid are the levels that determine the saturation of tooth mineral in the oral environment (Carey et al., 1986; Vogel et al., 1990; Margolis and Moreno, 1992), and hence govern whether tooth demineralization or remineralization is occurring. Thus, the aim of this study was to examine the relationship of the sucrose-induced decrease in whole biofilm Ca, P_i, and F concentrations to their concentrations in the biofilm fluid.

	MATERIALS & METHODS

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Experimental Design
This in situ, double-blind, crossover study was approved by the Research and Ethics Committee of FOP/UNICAMP (Protocol 015/2002). The oral health of the volunteers was assessed, and all participants signed written informed consent before being accepted into the study. During 3 phases of 15 days each, 16 adult volunteers wore acrylic palatal appliances containing 8 human enamel blocks (4 on each side), placed 1 mm below the acrylic level and covered by a plastic mesh to allow for dental biofilm accumulation (Fig.). In each phase, 8 times/day, volunteers removed the appliance from the oral cavity and dripped one of the following treatments (Cury et al., 2000) onto the dental blocks: distilled water (negative control), 10% glucose + 10% fructose solution, or 20% sucrose solution. While both glucose+fructose and sucrose test solutions will produce a pH drop in the biofilm, only sucrose will induce the formation of EPS (Cury et al., 2000). Five min after the application, the appliance was replaced in the mouth. On day 13 of each treatment phase, about 10 hrs after each experimental treatment, and after the volunteers fasted overnight, the pH of the biofilm on 2 blocks was determined in situ by the use of a metal pH micro-electrode (WPI, MEPH-3L, Sarasota, FL, USA) and a reference electrode (Orion, 9002, Boston, MA, USA), connected to a pH meter (Orion, 720-A). The acidogenicity analysis was then conducted as follows: (1) A single drop of a 20% glucose solution was applied to the blocks extra-orally; (2) after 1 min, the appliance was replaced in the mouth; and (3) the pH was measured after an additional 4 min (i.e., 5 min after the sugar challenge) (Ribeiro et al., 2005). After the measurement, these 2 blocks were removed, and the volunteers continued carrying out the treatment regimens on the remaining 6 blocks. On the following day (day 14), the dental biofilm formed was collected from 4 blocks, either after volunteers fasted (2 blocks) or 5 min after they dripped a 20% glucose solution (2 blocks), using the same procedure as above (Fig.). The volunteers then reversed the treatments: Samples previously exposed to distilled water started receiving sucrose, and those using glucose+fructose and sucrose started receiving distilled water. Twenty-four hrs later, the biofilm from the last 2 remaining blocks was collected after volunteers fasted overnight.

View larger version (40K): [in this window] [in a new window]   Figure. Illustration of the experimental design.

Analysis of the Biofilm and Fluid
Each biofilm was collected with the use of a plastic spatula and immediately placed inside an oil-filled centrifuge tube (Vogel et al., 1997). After determination of the sample weight (± 10 µg), the tube was centrifuged for 10 min (21,000 g) at 4°C to separate the fluid from the biofilm solids. The fluid was recovered with oil-filled capillary micropipettes and deposited under mineral oil on the bottom of a plastic Petri dish until the analyses. The tip of the centrifuge tube was then cut (Vogel et al., 1997), and the remaining biofilm was centrifuged into a 1.5-mL microcentrifuge tube containing 0.5 M HCl (0.5 mL/10 mg of biofilm wet weight) for extraction of acid-soluble whole biofilm Ca, P_i, and F (Cury et al., 1997, 2000). The samples were agitated by being rotated in a blood agitator at 30 rpm for 3 hrs at room temperature and centrifuged, and the supernatant was collected. The supernatant was then neutralized with NaOH and kept frozen until analyses. TISAB III (Thermo Electron, Waltham, MA, USA) was then added (1:10) just before analysis.

For analyses of total Ca and P_i in the biofilm fluid, quartz nanoliter volume pipettes (Vogel et al., 1990) were used to deposit standardized volumes of the samples or standards into Ca- or P_i-sensitive colorimetric reagents (Vogel et al., 1983). The absorbance of the mixtures, after being mixed, was then read with the use of a 50-µL micro-cuvette (Hellma, 105.202, Müllheim, Germany) in a Beckman DU-70 spectrophotometer. For the analyses of total Ca and P_i in the whole biofilm, the standards also contained TISAB III. The F analysis of all the samples was done on the surface of an oil-covered inverted F electrode with the use of a microscope and micro-reference electrode, held in a micromanipulator, to close the circuit (Vogel et al., 1997). Biofilm fluid samples were diluted with TISAB III (1:10) on the surface of the F electrode by means of micropipettes (Vogel et al., 1997).

Statistical Analyses
The statistical analysis was done with SAS software (SAS Institute Inc., version 8.01, Cary, NC, USA), with a significance level fixed at p < 0.05. We used an ANOVA to test the null hypothesis of no difference among the treatments, for each variable, considering the volunteers as statistical blocks. The assumptions of equality of variances and normal distribution of errors were checked for each variable, and when assumptions were violated, the data were transformed (Box et al., 1978). Tukey’s test was then used for post-ANOVA comparisons. For comparisons between the conditions of fasting vs. post-sugar challenge or fasting vs. reversal of treatments, paired tests were used with each treatment fixed. When the normality requirement for the t test was not satisfied, non-parametric signed tests (Wilcoxon signed-rank or sign test) were used.

	RESULTS

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The concentrations of Ca, P_i, and F in whole biofilms formed under glucose+fructose and sucrose exposure for 14 days were statistically lower than those in the distilled water group, but they did not differ statistically from each other (Table). After the sugar challenge, a significant decrease in almost all minerals in the whole biofilm was observed for glucose+fructose and sucrose groups, compared with the fasted samples. When the biofilm of the distilled water group was exposed to sucrose for 24 hrs, a significant decrease in whole biofilm Ca and P_i was found. Conversely, when the carbohydrate treatments were interrupted for 24 hrs, significant increases in biofilms Ca, P_i, and F were observed for the glucose+fructose group, while for the sucrose group, only the Ca significantly increased.

With respect to the biofilm fluid, Ca and F values were higher in the sucrose and glucose+fructose groups, compared with the negative control, but the difference was significant only for Ca in the sucrose group (Table). After the sugar challenge, Ca concentration increased and P_i decreased significantly in the biofilm fluid of each group, but the post-sugar values did not differ statistically. For F in the biofilm fluid, no significant post-sugar changes were observed, but the sucrose group had a higher concentration when compared with that of the negative control. After the reversal of treatments, a decrease in F values for the glucose+fructose and sucrose groups was observed, which was significant only for the glucose+fructose group.

	DISCUSSION

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Ca, P_i, and F ions can be stored in the biofilm either as mineral deposits (Kaufman and Kleinberg, 1973), or bound to the bacterial cell wall (Rose et al., 1993, 1996) or to proteins (Gao et al., 2001). Although the nature of the potential mineral deposits in the biofilm has not been fully explored, fasted plaque fluid is highly supersaturated with respect to hydroxyapatite and fluorapatite (Carey et al., 1986; Vogel et al., 1990), which would seem to suggest that only the more soluble calcium phosphates, such as dicalcium phosphate dihydrate (Sullivan et al., 1997) and amorphous calcium phosphate, could be important ion sources. When the pH declines during a sugar challenge, both non-mineral and mineral ion sources can release ions to the biofilm fluid. Although the measurement of total Ca in the current study prevents an accurate calculation of Ca binding or mineral saturation in the studies reported here, fluorapatite is unlikely to be an ion source, even after a challenge: If one calculates the equilibrium saturation of this mineral (Chemist version 1.0.1, Salt Lake City, UT, USA) from the average post-sugar values (Table), and if one assumes that about 50% of the total Ca is bound (Margolis and Moreno, 1994), fluorapatite will not dissolve until the pH is below 4.4.

Since only the sucrose group induced EPS formation, the similar Ca, P_i, and F in the glucose+fructose and sucrose groups demonstrated that an increase in biofilm-insoluble EPS (Cury et al., 2000; Aires et al., 2006; Tenuta et al., 2006) was not responsible for the decrease in whole biofilm mineral ions. This decrease is in agreement with previous studies showing that pH cycling of the biofilms greatly reduced the concentration of these mineral ions in the whole biofilm (Cury et al., 1997, 2000, 2003; Pearce et al., 2002). In fact, biofilms formed during exposure to a substrate of lower acidogenic potential such as starch (Ribeiro et al., 2005), or to low sucrose concentrations (Aires et al., 2006), showed an increase in the fasted biofilm Ca, P_i, and F concentrations when tested against the sucrose concentration used here. The significant decrease in the concentrations of mineral ions in the whole biofilms for the glucose+fructose and sucrose treatments during the pH drop (Table), and the decrease in Ca and P_i during the 24 hrs of exposure of the negative control group to sucrose, are consistent with the low-pH-mediated increase in these ions in plaque fluid (below), which would facilitate their loss to saliva. Finally, it should also be noted that our previous study (Cury et al., 2003), unlike the data presented here, showed no significant changes in mineral ion concentrations when the distilled water and sucrose treatments were reversed (Table). We believe that these different results are due, as discussed in our earlier publication (Cury et al., 2003), to the difference in the ages of the biofilms (28 days vs. 14 days).

The lower baseline pH found in the glucose+fructose and sucrose biofilms (Table) could be related to the residual production of acids by bacteria during fasting (Marsh and Martin, 1992), due to bacterial consumption of intracellular polysaccharide (Tenuta et al., 2006). During the sugar challenge, the lower pH observed for glucose+fructose and sucrose groups, when compared with the negative control, could be due to the high percentage of lactobacilli in these biofilms (Ribeiro et al., 2005; Tenuta et al., 2006), as well as the much higher concentration of P_i, and hence phosphate buffering, in the distilled water biofilm. The higher Ca concentration in the fluid of the fasted biofilm formed under exposure to sucrose, compared with the control, is in accordance with its lower baseline pH. Similarly, the higher F concentration in the post-sugar sucrose biofilm fluid may be due to its lower pH.

The decrease in P_i concentration in the fluid after the sugar challenge has been observed in other studies (Margolis and Moreno, 1992) and may be due to the uptake of P_i by fermenting bacteria. In fact, an increase in phosphate in fluid has been observed after an in vitro acid treatment, when no utilization of phosphate by bacteria was expected (Vogel et al., 2000b). A dilution effect due to the applied sugar solution could also have played a role in the decreased concentration (Vogel et al., 2001). Similarly, and in accord with other studies done in the absence of F rinse or dentifrices, no significant increase in post-sugar biofilm F was observed (Pearce et al., 1999; Tanaka and Margolis, 1999; Vogel et al., 2002). Such a result may also be due to uptake by bacteria during the pH fall (Hamilton, 1990) or by enamel (Tanaka and Margolis, 1999; ten Cate et al., 2003).

Although the initial and post-sugar pHs of the distilled water group were higher than those of the other groups, a similar or larger increase in the biofilm fluid Ca was observed (Table). This suggests that some of the increased Ca in this biofilm reservoir is being released by a modest pH change. It should be noted in this regard that the total Ca concentration in the distilled water control biofilm greatly exceeded the Ca-binding capacity of oral bacteria (Rose et al., 1993), which suggests that some of this increase can be accounted for by Ca mineral reservoirs. Increases in whole biofilm mineral ions following the administration of Ca and P_i (Vogel et al., 2000a) or Ca, P_i, and F supplements (Pearce et al., 1999) have generally not produced increases in biofilm fluid concentrations, at least in fasted plaque, suggesting the existence of equilibrium or a homeostatic mechanism that maintains ion concentration in the fluid, regardless of its concentration in the whole biofilm. Mobilization of Ca to the fluid during an acidogenic challenge, however, has been observed in mineral-supplemented biofilms (Pearce et al., 1999; Vogel et al., 2000a). In the current study, the higher pH of the high-mineral distilled water group militates against the observation of such an effect.

In conclusion, the findings suggest that a carbohydrate-mediated reduction in the accumulation of Ca, P_i, and F in biofilms may not result in a reduction of the concentrations of these ions in the biofilm fluid, in either resting or challenged plaque. It appears that other factors, such as equilibrium processes in the biofilms, as well as differences in pH, may be more relevant.

	ACKNOWLEDGMENTS

 
The authors thank the volunteers for their valuable participation, Dr. Anderson T. Hara for the original figure design, FAPESP from whom the first author (Proc. 02/00261-4) and third author (Proc. 04/06624-7) received scholarships, and CNPq (Proc. 472392/2003-04) and FAPESP (Proc. 03/07926-4), which supported this study. This work is based on a thesis submitted by the first author to the Faculty of Dentistry of Piracicaba, University of Campinas, in partial fulfillment of the requirements of the Doctorate Program in Dentistry, concentration in Cariology. A preliminary report of this study was presented at the 52nd ORCA Congress.

DISCLAIMER
Certain commercial materials and equipment are identified in this paper to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by the National Institute of Standards and Technology or the American Dental Association Foundation, or that the material or the equipment identified is necessarily the best available for the purpose.

Received October 19, 2005; Last revision May 15, 2006; Accepted May 26, 2006

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