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The Effect of Copper on Demineralization of Dental Enamel
1 Paediatric Dentistry and Correspondence: * corresponding author, den2aa{at}leeds.ac.uk
Previous studies have concluded that copper might inhibit enamel demineralization in vitro. Our aim was to assess the effect of copper (Cu2+), with and without amine fluoride, on human dental enamel under cariogenic challenge in situ. In a double-blind randomized four-leg crossover trial, 14 individuals wore a removable appliance containing 2 enamel slabs, 1 containing an artificial caries lesion. During each leg, the appliance was exposed twice daily to one of the test solutions: 1.25 mM CuSO4, amine fluoride (250 ppm F), copper and amine fluoride combined, or a placebo (water). A cariogenic challenge was provided in all cases by 5 daily exposures to 10% sucrose. Slabs were assessed before and after 21 days’ exposure by Knoop microhardness and transverse microradiography. Significantly less demineralization was observed with Cu2+ and fluoride in combination than with fluoride treatment alone (p < 0.05), whereas copper alone had no significant protective effect.
Key Words: amine fluoride • copper • demineralization • enamel
The association of copper (Cu2+) with lower caries prevalence has been reported by various authors (Derise et al., 1974; Duggal et al., 1991). Also, the ability of Cu2+ to inhibit cariogenesis in animals has been well-documented (Maltz and Emilson, 1988; Rosalen et al., 1996a,b). The mechanism involved has been attributed to the antimicrobial properties of Cu2+, which include acute loss of bacterial intracellular K+ and inhibition of H–-ATP synthase, inhibition of various bacterial metabolic enzymes through oxidation of key thiol groups (Maltz and Emilson, 1988), and formation of insoluble Cu-P salts on the tooth surface, thereby increasing its acid resistance. In addition, Brookes et al.(2003) reported that Cu2+ directly inhibited the acid dissolution of human enamel in vitro, suggesting that the anti-caries properties of Cu2+ could be due to a combination of its antimicrobial effects and its ability to inhibit demineralization directly. However, no in situ evidence has ever been presented to show this beneficial effect on dental enamel in response to a cariogenic challenge. The aim of this work was to compare anti-caries properties of Cu2+ alone and a combination of Cu2+ and amine fluoride with those of amine fluoride alone in situ, in human volunteers.
Study Design The modified Intra-oral Cariogenicity Test (ICT) design was used (Pollard, 1995; Duggal et al., 2001). The trial was carried out at the Leeds Dental Institute to conform to the criteria of Good Clinical Practice (ICH GCP) as set out in The Helsinki Declaration and its modified version (ICH Guideline for Good Clinical Practice, 1996).
Ethical Approval
Inclusion and Exclusion Criteria
Enamel Slab Preparation and Sterilization
Microhardness Testing
Preparation of Slabs for Transverse Microradiography The mineral density exhibited by control slabs was determined by microradiography (TMRW, 2000, version 20.1.5.1, Inspektor Research Systems, Amsterdam, The Netherlands) (de Josselin de Jong et al., 1987). The same procedure was followed for the experimental sections at the end of the in situ trial, since they were also ground to 80- to 100-µm thickness.
Microradiographic Testing
The microradiographs were analyzed by TMRW software (version 20.1.5.1, 2000) (Inspektor Research Systems, Amsterdam, The Netherlands) (de Josselin de Jong et al., 1987). The enhanced images of the microradiographs were analyzed under standard conditions of light intensity and magnification (150X), and were processed, along with data from measurement of the step wedge, by the TMR program. The mineral content of the specimens was expressed as mineral loss (
Sterilization of Slabs
Experimental Appliance The slabs were secured in position with sticky wax, care being taken to ensure that the wax did not cover the exposed surface of the enamel. The slabs were then covered with 0.15 mm Dacron gauze (Meadox Medicals, Oakland, NJ, USA), to promote plaque accumulation, and were secured with sticky wax (Pollard, 1995).
Study and Control Products
Reproducibility and Reliability
Reliability
Blindness and Randomization Test materials were coded, and the code was kept by the study’s sponsor (GABA International, AG, Münchenstein, Switzerland). Neither the study investigator nor the volunteers knew the code for the test materials during the study.
Randomization Enamel slabs were randomly allocated to each volunteer according to a randomized table. The side of the appliance used to fit the enamel slabs was allocated randomly.
Experimental Protocol/Regime
Data Handling/Statistics The normality of results was measured by a Shapiro-Wilk test and Boxplot graphs, and Student’s paired t test or the Wilcoxon test was used to measure the significant difference between groups, depending on the results distribution.
The outcome from the 14 participants (seven females and seven males; mean age, 28.79 ± 4.17 yrs) was included in the statistical analysis. The mean DMFT was 6.14 ± 5.72. All participants completed the study satisfactorily, with no adverse events reported.
Microhardness Testing
The combination of amine fluoride and copper showed the least degree of softening in enamel, and was significantly better than amine fluoride alone in decreasing the enamel hardness [P < 0.05; (–2.31, –0.91) 95% confidence interval CI], and was also significantly better than placebo [P < 0.05; (–4.64, –2.54) 95% CI] and copper alone [P < 0.05; (3.49, 5.85) 95% CI] (Table 2  ).
Transverse Microradiography Mineral loss was the lowest in the AmF+Cu group (–253.2 ± 40.6 vol% x µm), followed by AmF alone (–275.1 ± 37.3), placebo (–378.9 ± 50.2), and Cu (–445.8 ± 50.8) groups (Table 3 ).
The AmF+Cu group showed the least mineral loss from the white-spot enamel lesions. This change was statistically significant in comparison with that in the placebo [P < 0.05; (–177.2, –74.0) 95% CI] or Cu2+ [P < 0.05; (142.4, 242.6) 95% CI] groups (Table 4 ).
In a previous study (Brookes et al., 2003), 1.25 mmol/L Cu2+ reduced the acid dissolution of human enamel by approximately 40% in vitro. In view of this, and considering the toxicological effects of copper, we used 1.25 mmol/L copper in this study. This ensured that each subject had less than 8 mg Cu2+/100 mL of Cu2+ solution, which has no observed adverse effect in humans (Araya et al., 2001). There was statistically significant demineralization (P < 0.05) in all test and control groups relative to baseline, as measured by the enamel microhardness test. This was presumably due to the severity of the cariogenic challenge used in the study. Our dipping regime in sucrose solution meant a total exposure to sucrose of some 210 min over 21 days. A previous study reported enamel demineralization in situ with a repeated two-minute cariogenic challenge administered 7 or 10 times daily over 5 days, even when the subjects used a fluoride toothpaste containing 1450 ppm NaF (Duggal et al., 2001). In contrast, the present study used 5 cariogenic challenges per day, but over 21 days as opposed to 5 days, and our subjects used a fluoride-free toothpaste for the duration of the study. Therefore, the regime used in the present study provided an intense cariogenic challenge for our experiment. Rosalen et al. (1996,b) found that plaque lactic acid concentrations decreased when rats were fed sucrose co-crystallized with copper, and this reduction was enhanced when copper was combined with fluoride. In our study, we observed a similar synergistic effect between amine fluoride and copper when used together. The nature of this synergistic effect is unclear. However, Brookes et al.(2003) suggested that the precipitation of a protective copper phosphate phase on the enamel mineral surface could reduce or inhibit demineralization, or that hetero-ionic substitution of Cu2+ stabilized the crystal lattice. In contrast, Koulourides et al.(1968) observed an inhibition of enamel remineralization by Cu2+ (CuCl2), and assumed that this was due to ionic interaction with the active enamel surface following demineralization. Stabilization of the crystal lattice would explain why Cu2+ inhibits both enamel dissolution and enamel remineralization. In the present in situ study, we observed no protective effects associated with the use of 1.25 mM Cu2+ alone. Presumably, copper reacts differently in the human mouth than it does in vitro (Brookes et al., 2003) or in animal studies (Rosalen et al., 1996a,b). Copper has antibacterial effects on oral microorganisms if it is present in the mouth at optimum levels, i.e., 150 ppm/2.36 mmol/L Cu or higher (Rosalen et al., 1996a,b). However, in our study, much lower levels of copper were used, since any future therapeutic or prophylactic use of Cu2+ in humans must take toxicity issues into account. It was possible that Cu2+ had some direct inhibitory effect on demineralization through stabilization of the enamel mineral (Brookes et al., 2003), but this had a detrimental effect on remineralization (Koulourides et al., 1968) such that, overall, there was net demineralization. When Cu2+ is used with fluoride, the two could work synergistically by fluoride counteracting the inhibitory effects of Cu2+ on remineralization, resulting in a lower net demineralization compared with when either ion is used alone. The present work suggests that a combination of amine fluoride and Cu2+ is able to reduce the enamel-demineralizing effects of a cariogenic challenge when compared with amine fluoride, placebo, or Cu2+ alone. Although the results were conclusive, they were based on experimental caries challenges designed to mimic severe caries challenges such as the dentition might face daily. While we acknowledge that these data cannot be directly extrapolated to naturally occurring caries, they nevertheless are firm data on which a more extensive clinical trial could be based in the future.
We convey our special thanks and appreciation to GABA International, AG, for sponsoring this project and for the assistance and support offered by many people in the Paediatric Dentistry Department, Leeds Dental Institute. This project was submitted in partial fulfillment of the requirements for the degree of Master, Dental Science in Paediatric Dentistry, Leeds Dental Institute. This work was presented at the 52nd ORCA Congress, held in Indianapolis, IN, USA, July 6–9, 2005. Received for publication November 18, 2005. Revision received May 18, 2006. Accepted for publication July 17, 2006.
Journal of Dental Research, Vol. 85, No. 11,
1011-1015 (2006)
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ABSTRACT
INTRODUCTION
1. 
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