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Fluoride Release from CaF2 and Enamel DemineralizationPiracicaba Dental School, UNICAMP, Av. Limeira 901, 13414-903, Piracicaba, SP Piracicaba, SP, Brazil Correspondence: * corresponding author, litenuta{at}fop.unicamp.br
The anticaries effect of professional fluoride (F) application has been attributed to calcium-fluoride-like deposits (CaF2) formed on enamel, but this has not been clearly demonstrated. We hypothesized that CaF2 formed on plaque-free enamel by F application would reduce enamel demineralization due to the increase of F availability in fluid of subsequently formed plaque. We created distinct levels of CaF2 on enamel to evaluate a dose-response effect. Enamel blocks were mounted in contact with a S. mutans test plaque and used in situ by 10 volunteers. F released to the fluid phase of this substrate ("plaque fluid") was measured before a cariogenic challenge. "Plaque fluid" F concentration was highly correlated to the enamel CaF2 concentration (r = 0.96, p < 0.001) and to consequent enamel demineralization (r = –0.75, p < 0.001). The results suggest that F released to plaque fluid from CaF2 formed on enamel may play a significant role in the anticaries effect of professionally applied F agents.
Key Words: fluoride • topical application • plaque fluid • demineralization • calcium fluoride
Professional topical fluoride (F) application is considered an effective measure for caries prevention (Marinho et al., 2007). It increases the amount of tooth-bound F (fluorapatite, FAp) on enamel, which has been shown to enhance its resistance to demineralization (Takagi et al., 2000). However, an important effect of topical F application seems to rely on the concomitant formation of a phosphate-contaminated calcium-fluoride deposit (calcium-fluoride-like material, hereafter referred to as CaF2) on dental hard tissues, which would act as a F reservoir, slowly releasing F to interfere with de/remineralization events at the tooth/plaque interface (for review, see ten Cate, 1997). Therefore, if CaF2 formed on plaque-free enamel is an important factor responsible for the anticaries effect of professionally applied topical F treatments (Ögaard et al., 1990; Ögaard, 2001), F released from CaF2 should be found in the fluid of newly formed plaque, where it could significantly inhibit enamel demineralization. Indeed, an increase in F concentration was found in whole plaque formed on enamel treated by topical F application (Paes Leme et al., 2004), but this has never been studied in plaque fluid. Also, a dose-response effect of the concentration of CaF2 on enamel, its release to plaque fluid, and the subsequent inhibition of enamel demineralization has not been examined. Thus, we hypothesized that if CaF2 is an important source of F for plaque fluid, the amount of CaF2 formed on enamel should be related to F concentration in the plaque fluid and to the consequent inhibition of enamel demineralization.
Experimental Design This was a crossover, double-blind, short-term in situ study (Zero et al., 1992; Cury et al., 2003), approved by the Ethics Committee of the Piracicaba Dental School. Ten volunteers signed a written informed consent. In two experimental phases, they used a palatal appliance containing bovine enamel blocks with distinct levels of CaF2, in an intra-oral demineralization test (Fig.  ). Enamel blocks were treated with either a non-F solution, or an acidulated 0.5 M (9500 ppm F) NaF solution. Different concentrations of CaF2 were created on F-treated blocks by either not aging them, or by aging them for 6 hrs or 48 hrs in artificial saliva. A set of enamel blocks was used for determination of CaF2 and FAp formed and retained on enamel by these procedures. Eight blocks were mounted in a palatal appliance, 4 from each treatment in each side of the appliance, in contact with a test plaque prepared from S. mutans (Fig., a). After 30 min of intra-oral exposure, test plaque from 2 blocks of each treatment was collected for determination of baseline F, calcium (Ca), and inorganic phosphorus (Pi) concentrations in the fluid phase of the bacterial test plaque ("plaque fluid") (Fig., b). The appliances were re-inserted into the volunteers’ mouths, and a cariogenic challenge was conducted by gentle rinsing with a 20% sucrose solution during 1 min (Fig., c). Forty-five min after the cariogenic challenge, the test plaque and the other 2 blocks remaining from each treatment were collected (Fig., d). Inorganic ions were measured in the test "plaque fluid" as chemical indicators of enamel demineralization, and mineral loss was also evaluated by the percentage of loss in enamel surface microhardness (SMH) (Fig., e). CaF2 and FAp remaining in enamel were determined.
Topical Fluoride Application and Baseline Surface Microhardness Determination Enamel blocks (5 x 5 x 2 mm) were prepared from bovine incisors, and the enamel surface was polished flat (Zero et al., 1990). Those with a mean Knoop hardness of 332.2 ± 18.3 were randomly divided into 4 groups of 56 specimens each, comprising the different groups/treatments. Blocks from a negative control group were individually immersed for 4 min in 0.1 M phosphate-buffered solution, pH 3.5 (10 mL/block). The remaining blocks were treated for 4 min with acidulated phosphate F (APF) solution containing 0.5 M NaF in 0.1 M phosphate, pH 3.5. After treatments, blocks were washed for 30 sec with a stream of distilled water. F-treated blocks were either not aged (group APF) or aged for 6 hrs (group APF, 6-hrs-aged) and 48 hrs (group APF, 48-hrs-aged) in a flow (0.5 mL/min) of artificial saliva (remineralization solution of ten Cate and Duijsters, 1982, without F) at a volume of 6 mL/mm2, to simulate the dissolution of CaF2 that occurs in the mouth after F treatment (Dijkman et al., 1983). Enamel blocks were treated and aged just before being used in the next phase. Sixteen blocks from each group were used for determination of the baseline CaF2 and FAp concentrations, and remaining blocks were used in the in situ test. Before the intra-oral test, we determined baseline SMH on enamel blocks to be used in situ by making 8 indentations at distances of 50 to 1000 µm from one edge of the block, with a 50-g load, for 5 sec in a Future-Tech FM microhardness tester coupled to FM-ARS software (Future-Tech Corp., Kawasaki, Kanagawa, Japan). This edge was marked for subsequent mounting reference in the appliance (for details, see Cury et al., 2003).
Palatal Appliance Mounting
Analysis of Mineral Ions in "Plaque Fluid"
Analysis of Enamel Mineral Loss
Determination of CaF2 and FAp in Enamel After KOH extraction, we estimated FAp (here to represent a mixture of F-containing apatite minerals dissolved by acid) by removing 1 enamel layer in 0.75 mL of 0.5 M HCl for 30 sec under agitation. After neutralization with TISAB II containing 0.5 M NaOH, F was determined as described above. Pi was measured in the acid extract (Fiske and Subbarow, 1925), and the amount of enamel dissolved was calculated based on a Pi concentration in enamel of 17.4% and a density of 2.92. Thus, FAp concentration was expressed as µg F/g of enamel.
Statistical Analysis
Pre-treatment of enamel blocks with an APF solution significantly increased CaF2 and FAp concentrations in enamel (Table 1 , columns 2 and 3). The aging process decreased the CaF2 concentration, but did not affect FAp values.
After 30 min in situ, a significantly higher F concentration in "plaque fluid" was observed for the F-treated blocks, when compared with the negative control group (Table 1  , column 4). The highest (p < 0.05) F concentration was found in "plaque fluid" in contact with APF-treated, not aged, enamel blocks, and significantly lower F concentrations for groups aged for 6 and 48 hrs. Ca concentration in "plaque fluid" at this moment was similar for all groups, but Pi concentration was significantly higher for the negative control group when compared with groups APF and APF aged for 6 hrs (Table 1, columns 5 and 6). Forty-five min after the cariogenic challenge, F concentration in the "plaque fluid" was still high in all F-treated groups, and the differences among the groups were maintained. Ca and Pi concentrations increased significantly after the cariogenic challenge for all groups (columns 8 and 9), and were higher for the negative control group, followed by the groups APF aged for 48 hrs, APF aged for 6 hrs, and APF not aged; these last two groups did not differ statistically from each other.
Analysis of the %SMC data (Table 1
CaF2 significantly decreased and FAp significantly increased after the in situ test for groups APF and APF aged for 6 hrs when compared with the values after treatment/aging (Table 1
F released to the "plaque fluid" before cariogenic challenge was highly correlated to the concentration of CaF2 formed and retained on enamel (r = 0.96, p < 0.001) (Table 2
Most F formed on enamel in this study was CaF2, in agreement with the expected effect of the low pH of the acidulated phosphate F solution used (Larsen and Richards, 2001). Also, the aging protocol used was effective in creating enamel with dissimilar concentrations of CaF2, due to the undersaturation of saliva with respect to CaF2 (McCann, 1968). FAp dissolution was probably suppressed by the small amount of F being released to the saliva-like solution. Thus, CaF2 concentration decreased 10 times after 48 hrs in the continuous flow of artificial saliva used, and no change in FAp levels was observed. Although the experimental model used did not rely on natural biofilm formation, which is the main limitation of the present study, it facilitated the evaluation of the dynamics among F reservoirs on enamel, their dissolution, and the inhibition of enamel demineralization. Thus, F released to the "plaque fluid" was significantly correlated to the CaF2 concentration created on enamel. Also, a dose-response effect was observed between the concentration of CaF2 reservoirs on enamel and F released to "plaque fluid" and the subsequent inhibition of enamel demineralization. In fact, the %SMC was inversely correlated to F concentration in the "plaque fluid" before the cariogenic challenge. The higher Ca and Pi concentrations in the "plaque fluid" 45 min after the challenge appear to be the result of tooth dissolution, since they were significantly correlated to the %SMC (for Ca, r = 0.71, p < 0.0001; for Pi, r = 0.65, p < 0.0001). Thus, F release to "plaque fluid" from CaF2 dissolution significantly inhibited enamel demineralization, at a greater extent for the groups with a higher CaF2 content. Enamel dissolution would have been impaired by F available in the "plaque fluid", and at the same time that hydroxyapatite from enamel dissolves, there is a concomitant gain of FAp (ten Cate, 1997). Indeed, an increase of FAp in enamel was observed after the cariogenic challenge in enamel blocks from groups not aged and aged for 6 hrs. Considering the high F concentration in "plaque fluid" at the moment of the cariogenic challenge, some inhibition of acid production by test plaque bacteria cannot be overruled as an additional effect decreasing enamel demineralization (Bradshaw et al., 2002). A continuous dissolution of CaF2 during the intra-oral test was apparent, since "plaque fluid" F values were not different before and 45 min after the cariogenic challenge. The CaF2 concentration remaining on the blocks after the in situ test was about half of the initial values. It could be observed that, after the 75-minute intra-oral test, a similar CaF2 concentration remained on enamel treated but not aged, in comparison with that exposed for 6 hrs in the artificial saliva used to age the samples, even considering that the access to the artificial saliva in vitro was greater than that to natural saliva in the mouth. This suggests that the low pH environment created by sugar exposure increased the dissolution rate (Rölla and Ögaard, 1986). Although it is not known how long CaF2 enamel reservoirs would last, a higher F concentration in whole plaque was still found 14 days after topical F application, under a high cariogenic challenge in situ (Paes Leme et al., 2004), but such evaluation of F availability is still lacking for plaque fluid. Since the concentration of FAp in enamel was not altered by the aging protocol, the correlation of FAp with F in the "plaque fluid" or mineral loss observed was weak. However, FAp in enamel increased after the cariogenic test for the groups APF and APF aged for 6 hrs, and the significance of this increase in the inhibition of mineral loss in subsequent demineralization events remains to be tested. In conclusion, the findings showed a very clear relationship among concentration of CaF2 on enamel, F release to plaque fluid, and the consequent reduction of demineralization.
We thank the volunteers for their valuable participation, and FAPESP (05/04703-0) and CNPq for the scholarships granted to the first and second authors, respectively. This paper is based on a thesis submitted by the second author to the Faculty of Dentistry of Piracicaba, UNICAMP, SP, Brazil, in partial fulfillment of the requirements for the Master’s Program in Dentistry (Pediatric Dentistry). A preliminary report was presented at the 54th Orca Congress, in Helsingör, Denmark, July, 2007. Received for publication February 1, 2008. Revision received June 17, 2008. Accepted for publication August 1, 2008.
Journal of Dental Research, Vol. 87, No. 11,
1032-1036 (2008)
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