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J Dent Res 85(11):1011-1015, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

The Effect of Copper on Demineralization of Dental Enamel

A.Z. Abdullah1,*, S.M. Strafford1, S.J. Brookes2, and M.S. Duggal1

1 Paediatric Dentistry and
2 Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, LS2 9LU, UK

* corresponding author, den2aa{at}leeds.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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
Approval was obtained from the Local Research Ethics Committee, Leeds General Infirmary (Leeds "West" Research Ethics Committee). Informed consent was obtained from each volunteer at the beginning of the study, prior to confirmation of their eligibility for the study. The participants had the right to withdraw from the study at any time and for any reason without prejudice.

Inclusion and Exclusion Criteria
The study participants were required to satisfy the following inclusion criteria:

  1. They were healthy adults, not taking any medication that would affect the microflora or the salivary flow rate (volunteers with contraindications such as epilepsy, risk of infective endocarditis, or hemophilia where excluded).
  2. They had a minimum unstimulated salivary flow rate of 0.25 mL/min.
  3. They had a Decayed, Missing, and Filled Teeth (DMFT) score ≥ 1.
  4. They had a minimum of 18 natural teeth, free of clinical signs of decay or of periodontal disease.
  5. They were available during the study period.

Enamel Slab Preparation and Sterilization
    Preparation of Slabs for Microhardness Testing
The buccal surface of each sound premolar was sectioned by means of a diamond wire to give 4 slabs (3 mm x 2 mm). Slabs were ground and polished with 5 µm and 1 µm alumina paste. Care was taken not to abrade the enamel fully.

    Microhardness Testing
Slab microhardness was measured at baseline with the use of a computer-aided Duramin Indenter Machine (Struers A/S, DK 26-10, Ballerup, Denmark). The indentations were made with a Knoop diamond under a 100-g load (Zero et al., 1990) for 30 sec. Five indentations, spaced at least 50 µm apart, were made for each slab, and the mean change in microhardness was determined. This procedure was repeated after treatment. The length of each indent was measured in microns by image analysis.

    Preparation of Slabs for Transverse Microradiography
Artificial white-spot lesions were prepared on the enamel surfaces of sound premolars (Edgar, 1983). Six enamel slabs were cut. Four slabs were sized 2 mm x 2 mm (experimental slabs), and the other 2 slabs were sized 1 mm x 2 mm (control slabs). The control slabs were ground to 80- to 100-µm thickness by anvils moving on a diamond disc with 15-µm diamond particles.

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 slabs were mounted onto transparency papers, the fabricated radiographic plate-holding cassette, and Kodak 1A photographic plates. Microradiographs were taken with a Cu(K{alpha}) x-ray source (Philips BV, Eindhoven, The Netherlands) and Kodak 1A photographic plates. Plates were exposed for 10 min (anode voltage, 25 kV; tube current, 10 mA) at a focal distance of 30 cm, and were then processed. The processing consisted of 5 minutes’ development in Kodak HR developer and 10 minutes’ fixation in Kodak Unifix before a final 30-minute wash period in water.

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 ({Delta}Z).

    Sterilization of Slabs
In all cases, slabs were stored in sealed microcentrifuge tubes kept moist with thymol (0.1% w/v). They were then sterilized by exposure to gamma irradiation (4080 Gy) at the Department of Immunology, University of Liverpool (Amaechi et al., 1998).

Experimental Appliance
A mandibular removable Hawley appliance, with a labial arch wire, C clasps, and acrylic flanges situated buccally to the first permanent molars, was used. Two enamel slabs, one for the assessment of microhardness and another for the TMR, were inserted into the left buccal flange of the appliance after randomization.

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
Study and control products were: copper sulfate (CuSO4.5H2O) (1.25 mmol/L; pH = 4.95) in plastic bottles containing 100 mL of copper sulfate solution (7.94 mg Cu2+/100 mL); amine fluoride (250 ppm fluoride) with added copper (1.25 mmol/L copper, pH = 4.21); amine fluoride as a positive control (250 ppm fluoride) (pH = 4.15); and de-ionized water as a negative control (pH = 5.11).

Reproducibility and Reliability
    Reproducibility
Fifteen percent of the enamel slabs’ microhardness and transverse microradiographic analyses were retested by the study investigator. Reproducibility agreement was found to be 90%.

    Reliability
Another member of the staff, familiar with the methods used, repeated the tests randomly for 10% of the enamel slabs, using microhardness and transverse microradiography. Ninety percent agreement was found. Fifteen percent of the data entries from the microhardness testing were rechecked randomly. A 100% level of agreement was found.

Blindness and Randomization
    Blindness
The de-ionized water (placebo) and the amine fluoride, with or without copper, solutions were all dyed with blue dye (Ariavit-Blue 0.4%, Williams Ltd., Hanslow, UK) similar to the color of the copper solution, so that all solutions were blue and indistinguishable from one another.

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
Test materials were coded according to a randomized table and then randomly allocated to each subject by the sponsor.

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
Volunteers were asked to refrain from using any other source of fluoride for the period of the study. The trial included 4 legs, with each lasting for 30 days. The first 7 days were used as a washing in/out period, at the end of which the appliance was fitted to allow plaque to accumulate on the slabs’ surfaces for 2 days. For the following 21 days, the appliance was dipped twice daily, once in the morning and once in the evening, into one of the Cu2+/fluoride/placebo or the combination of Cu2+ and fluoride solutions. In addition, participants also dipped the appliance 5 times daily into a 10% sucrose solution between the morning and the evening dips of the previous solutions. Dipping time for all solutions was 2 min. The participants used beakers provided to dip the enamel slabs in 10 mL of fresh solution each time. After slabs were dipped, the solution was collected in bottles provided, so that the participants’ compliance could be checked. The device was left intra-orally overnight and between cariogenic challenges.

Data Handling/Statistics
The sample size for this study was estimated to be 14 volunteers. SPSS statistical software (version 11.5.0, SPSS Inc., Chicago, IL, USA) was used for data analysis. A significance level of P < 0.05 was accepted. We used Confidence Interval Analysis software (version 2.1.1, Trevor Bryant, 2000) to measure the confidence interval.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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
Microhardness measurements showed that all groups had significant enamel softening, compared with baseline, with all 4 test and control solutions (P < 0.05). The AmF+Cu group showed the least softening of enamel, with a change of 2.33 ± 1.07 µm from baseline, followed by AmF alone (3.95 ± 1.57), placebo (5.93 ± 2.48), and, finally, copper (7.00 ± 2.89) (Table 1Go).


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  		Table 1. Baselines (Mean ± SD), Test Values (Mean ± SD), Differences (Mean ± SD) (µm), and Statistical Analysis (Wilcoxon paired test) for Enamel Slabs Tested for Microhardness with the 4 Test and Control Solutions
 
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 2Go).


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  		Table 2. Results of the Statistical Analysis (Wilcoxon paired test) for the Change in Enamel Microhardness in All 4 Test and Control Groups (in pairs)
 
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 3Go).


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  		Table 3. Baselines (Mean ± SD), Test Values (Mean ± SD), Differences (Mean ± SD) (vol% x µm), and Statistical Analysis (paired Student’s t test) for Enamel Slabs Tested for Mineral Loss with Transverse Microradiography with All 4 Tests and Control Solutions
 
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 4Go).


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  		Table 4. Results of the Statistical Analyses (paired Student’s t test) for the Change in Mineral Loss Tested with Transverse Microradiography among All 4 Tests and Control Groups (in pairs)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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.


   ACKNOWLEDGMENTS
 
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 November 18, 2005; Last revision May 18, 2006; Accepted July 17, 2006


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amaechi BT, Higham SM, Edgar WM (1998). Efficacy of sterilisation methods and their effect on enamel demineralisation. Caries Res 32:441–446.[ISI][Medline]

Araya M, McGoldrick MC, Klevay L, Strain JJ, Robson P, Nielsen F, et al. (2001). Determination of an acute No-Observed-Adverse-Effect Level (NOAEL) for copper in water. Reg Tox Pharm 34:137–145.

Brookes SJ, Shore RC, Robinson C, Wood SR, Kirkham J (2003). Copper ions inhibit the demineralisation of the human enamel. Arch Oral Biol 48:25–30.[ISI][Medline]

de Josselin de Jong E, van der Linden AH, ten Bosch JJ (1987). Longitudinal microradiography: a non-destructive automated quantitative method to follow mineral changes in mineralised tissue slices. Phys Med Biol 32:1209–1220.[ISI][Medline]

Derise NL, Ritchey SJ, Furr AK (1974). Mineral composition of normal human enamel and dentin and the relation of composition of dental caries. I. Macrominerals and comparison of methods of analyses. J Dent Res 53:847–852.[Abstract/Free Full Text]

Duggal MS, Chawla HS, Curzon ME (1991). A study of the relationship between trace elements in saliva and dental caries in children. Arch Oral Biol 36:881–884.[ISI][Medline]

Duggal MS, Toumba KJ, Amaechi BT, Kowash MB, Higham SM (2001). Enamel demineralization in situ with various frequencies of carbohydrate consumption with and without fluoride toothpaste. J Dent Res 80:1721–1724.[Abstract/Free Full Text]

Edgar WM (1983). Distribution of acetic acid in enamel during demineralisation in HEC/lactic acid system. In: Demineralization and remineralization of the teeth. Leach SA, Edgar WM, editors. Oxford: IRL Press, pp. 145–152.

ICH Topic E 6 (R1) Guideline for Good Clinical Practice (1996). CPMP/ICH/135/95; 17th July 1996. http://www.emea.eu.int

Koulourides T, Feagin F, Pigman W (1968). Effect of pH, ionic strength, and cupric ions on the rehardening rate of buffer-softened human dental enamel. Arch Oral Biol 13:335–341.[ISI][Medline]

Maltz M, Emilson CG (1988). Effect of copper fluoride and copper sulfate on dental plaque, Streptococcus mutans and caries in hamsters. Scand J Dent Res 95:390–392.

Pollard MA (1995). Potential cariogenicity of starches and fruits as assessed by the plaque-sampling method and an intraoral cariogenicity test. Caries Res 29:68–74.[ISI][Medline]

Rosalen PL, Bowen WH, Pearson SK (1996a). Effect of copper co-crystallized with sugar on caries development in desalivated rats. Caries Res 30:367–372.[ISI][Medline]

Rosalen PL, Pearson SK, Bowen WH (1996b). Effects of copper, iron and fluoride co-crystallized with sugar on caries development and acid formation in desalivated rats. Arch Oral Biol 41:1003–1010.[ISI][Medline]

Zero DT, Rahbek I, Fu J, Proskin HM, Featherstone JD (1990). Comparison of the iodide permeability test, the surface microhardness test, and mineral dissolution of bovine enamel following acid challenge. Caries Res 24:181–188.[ISI][Medline]





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