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Ca, Pi, 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 Correspondence: * corresponding author, jcury{at}fop.unicamp.br
Calcium (Ca), inorganic phosphorus (Pi), 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, Pi, 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
Previous studies have shown that biofilms formed under frequent exposure to sucrose have decreased concentrations of whole plaque calcium (Ca), inorganic phosphate (Pi), 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, Pi, 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, Pi, and F concentrations to their concentrations in the biofilm fluid.
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.
In all experimental phases, the volunteers used a silica-based dentifrice containing 1100 µg F/g (NaF) and consumed fluoridated water (0.76 ± 0.03 mg F/L). A washout interval of 7 days separated each experimental phase. No restrictions were made with regard to the volunteers’ diet, but they were instructed to remove the appliances during meals (Cury et al., 1997, 2000).
Analysis of the Biofilm and Fluid For analyses of total Ca and Pi 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 Pi-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 Pi 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 concentrations of Ca, Pi, 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 Pi was found. Conversely, when the carbohydrate treatments were interrupted for 24 hrs, significant increases in biofilms Ca, Pi, and F were observed for the glucose+fructose group, while for the sucrose group, only the Ca significantly increased.
The fasted biofilm pHs of the 3 groups differed statistically, with sucrose showing the lowest and distilled water showing the highest values (Table  ). After the cariogenic challenge, the pHs of all the groups decreased significantly, with five-minute values of the glucose+fructose and sucrose groups differing statistically from those of the distilled water, but with no difference between them.
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
Ca, Pi, 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, Pi, 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, Pi, 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
The lower baseline pH found in the glucose+fructose and sucrose biofilms (Table The decrease in Pi 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 Pi 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 In conclusion, the findings suggest that a carbohydrate-mediated reduction in the accumulation of Ca, Pi, 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.
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 Received for publication October 19, 2005. Revision received May 15, 2006. Accepted for publication May 26, 2006.
Journal of Dental Research, Vol. 85, No. 9,
834-838 (2006) This article has been cited by other articles:
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