Effects of fluoridated milk on artificial enamel carious lesions: A pH cycling study

Effects of fluoridated milk on artificial enamel carious lesions: A pH cycling study

Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 817-824, Copyright © 2011 Elsevier Ltd

Abstract

Objective

To investigate the relationship between concentration of fluoride in milk and its re-mineralizing efficacy, and to test whether increasing the volume of fluoridated milk has any effect on its re-mineralizing efficacy.

Methods

Third molars were painted leaving a 1 mm window on the buccal and/or lingual surfaces. Artificial carious lesions were induced using a de-mineralizing solution. The teeth were then sectioned longitudinally. Specimens were randomly assigned to 15 groups according to different concentrations and volume; i.e. 2.5 ppm, 5 ppm, 7.5 ppm, 10 ppm, 15 ppm, 20 ppm, and 250 ppm of fluoride with 25 ml/section and 50 ml/section. De-ionized water and plain milk were used as controls. Sections were subjected to pH cycling for 20 days. Polarized light microscopy and micro-radiography were utilized to record the lesion characteristics before and after pH cycling.

Results

Mean lesion depths decreased significantly in all the fluoride groups, and increased significantly in the control groups. There were no differences in percentage of lesion depth decrease when the volume was doubled for the same fluoride concentration. There were also no statistically significant differences between percentages of decrease in lesion depth when the concentration of fluoride in milk was increased.

Conclusion

Fluoridated milk demonstrated a remineralization efficacy on early enamel carious lesions. A 2.5 ppm fluoride milk concentration provided similar remineralization potential to that of higher fluoride concentration in milk.

Introduction

Dental caries is a multi-factorial, highly transmissible disease of dental tissues. It is prevalent worldwide with individuals being susceptible to it throughout their life. Early stage of dental caries can be effectively detected by various means.

Dental caries prevalence was very high in the middle of the 20th century, with declines evident since the 1960s. However, there are suggestions that in some communities dental caries is on the increase, and this may be in part due to increasing use of refined sugars and inadequate exposure to fluoride. The progression or reversal of the caries process depends on the balance between pathological and protective factors. Fluoride has been identified as one of the protective factors, which tilts the caries balance towards the positive side. Earlier it was assumed that the beneficial effect of fluoride was mainly pre-eruptive, strengthening the tooth enamel during development, but it has now become clear that the constant presence of low levels of fluoride in the mouth inhibits de-mineralization and enhances re-mineralization.

Although water and toothpastes have been excellent vehicles for fluoride, there are parts of the world where drinking water cannot be fluoridated due to reasons such as the lack of resources, socio-economic and technical constraints, absence of political will and national policy on oral health. Fluoridated toothpastes is often unaffordable to the majority of the people in developing countries. Fluoridation of milk is recommended where fluoride concentration in the drinking water is suboptimal, caries experience is significant and programmes to provide milk to children are in place.

Whilst a recent systematic review suggested better quality research is needed regarding the effectiveness of milk fluoridation, a number of trials across the world have shown it to be effective in reducing the prevalence of dental caries in children. For example trials in Chile, Bulgaria, China, Sweden and England have shown that children who participated in the fluoridated milk programme experienced significantly less dental caries compared to children not on the programme. Adding fluoride to the milk-products as part of a National Food Supplement Programme has been found to be cost-effective in rural areas of Chile.

Though a number of in vitro studies have demonstrated the re-mineralizing efficacy of different concentrations of fluoridated milk, the re-mineralization effect of increasing the volume of fluoridated milk within the same concentration is still unclear. Thus the objectives of this study were to investigate whether increasing (1) the concentration of fluoride in milk and (2) the volume of fluoridated milk alters its re-mineralizing efficacy. The null hypothesis tested was that increasing the fluoride concentration and volume of fluoridated milk would increase its remineralization efficacy on initial enamel carious lesions.

Materials and methods

Preparation of de-mineralizing and re-mineralizing solutions

The de/re-mineralizing buffered solutions were prepared from certified chemicals and de-ionized water. The de-mineralizing solution contained 2.2 mM CaCl 2 , 2.2 mM NaH 2 PO 4 , and 0.05 M acetic acid; the pH was adjusted with 1 M KOH to 4.4. The re-mineralizing solution contained 1.5 mM CaCl 2 , 0.9 mM NaH 2 PO 4 , 0.15 M KCl at pH 7.0. These solutions were similar to those utilized by ten Cate and Duijsters. The treatment solutions were prepared from pasteurized and homogenized fresh cow’s milk, which contained calcium 110 mg/100 ml; protein 3.1 g/100 ml; carbohydrate 4.6 g/100 ml; fat 3.5 g/100 ml and was available in a tetrapack manufactured by a local Nestle plant in Hong Kong. The milk was mixed with 100 ppm and 500 ppm fluoride solutions prepared in the laboratory to make 2.5 ppm, 5 ppm, 7.5 ppm, 10 ppm, 15 ppm, 20 ppm and 250 ppm fluoridated milk solutions. The treatment groups are listed in Table 1 .

Table 1
Changes in lesion depth, by fluoride concentration and milk volume.
Treatment groups Lesion depth (Mean ± SD)
Pre t x (μm) Post t x (μm) % change
2.5 ppm F milk; 25 ml/section 103.87 ± 17.33 94.75 ± 14.81 * −7.85 ± 11.86 a
2.5 ppm F milk; 50 ml/section 104.16 ± 18.38 95.99 ± 18.30 * −7.31 ± 12.01 a
5 ppm F milk; 25 ml/section 99.05 ± 14.46 90.97 ± 16.18 * −7.78 ± 11.54 a
5 ppm F milk; 50 ml/section 98.77 ± 17.43 89.11 ± 16.35 * −8.67 ± 14.98 a
10 ppm F milk; 25 ml/section 98.37 ± 18.99 88.36 ± 16.91 * −9.55 ± 9.13 a
10 ppm F milk; 50 ml/section 99.84 ± 18.93 86.11 ± 15.97 * −12.93 ± 10.82 a
7.5 ppm F milk; 25 ml/section 98.72 ± 21.93 84.68 ± 19.66 * −13.66 ± 10.57 a
7.5 ppm F milk; 50 ml/section 97.17 ± 22.28 81.64 ± 21.15 * −15.53 ± 11.47 a
15 ppm F milk; 25 ml/section 96.80 ± 15.05 84.28 ± 19.58 * −13.14 ± 12.40 a
20 ppm F milk; 25 ml/section 95.02 ± 16.92 81.33 ± 15.13 * −14.09 ± 9.88 a
20 ppm F milk; 50 ml/section 94.41 ± 17.05 87.12 ± 16.71 * −7.56 ± 7.76 a
250 ppm F milk; 25 ml/section 95.40 ± 19.24 88.64 ± 18.04 * −6.92 ± 6.55 a
Plain milk; 25 ml/section 87.32 ± 18.78 120.47 ± 26.16 * +39.42 ± 20.58 b
Plain milk; 50 ml/section 98.95 ± 17.69 129.20 ± 24.24 * +31.70 ± 19.77 b
Water; 25 ml/section 99.89 ± 18.65 141.44 ± 21.54 * +43.71 ± 19.05 b
a,b Statistically significant differences between groups ( p < 0.05, Tukey).

* Significant changes ( p < 0.01, paired t -test) in lesion depth post pH cycling.

Lesion formation

Extracted third molars were cleaned of soft tissue debris and inspected for any cracks, caries and hypoplasia. The teeth were painted with acid resistant nail varnish (Revlon ® , New York City, NY, USA) leaving a 1 mm window on the buccal and/or lingual surfaces. They were then immersed in the de-mineralizing solution (10 ml/tooth) for 96 h to produce artificial lesions, approximately 100–150 μm deep. Subsequently, the teeth were sectioned longitudinally through the lesions to produce 100–150 μm thick sections. Three hundred and seventy five sections were selected for the study based on the uniformity of the lesion depth and mineral content. Polarized light microscopy (PLM) and micro-radiography (MRG) were used to analyse the lesion depth and mineral content of the lesions before and after the pH cycling. Prior to the pH cycling model, each section was painted, under a stereomicroscope, with the acid resistant nail varnish (Revlon ® , New York City, NY, USA), leaving only the lesion surface exposed to solutions. The painted sections were then randomly divided into 15 groups, with 25 sections per group.

The pH cycling model

The pH cycling system used in this study was modified from that of Ivancakova et al. The sections were placed in the pH cycling system on an orbital shaker for 20 days at room temperature. Each cycle involved 4 h of demineralization, 6 h of fluoride milk treatment followed by 14 h of remineralization ( Fig. 1 ). All solutions were freshly prepared each day for each cycle and the pH of demineralizing and remineralizing solutions was checked before each cycle.

Schematic illustration of the pH cycling model.
Fig. 1
Schematic illustration of the pH cycling model.

Evaluation techniques

Polarizing light microscopy

Polarized light microscopy (PLM) was employed to qualitatively evaluate the body of the lesion in the enamel samples. This was accomplished by imbibing the section in water, which normally shows a clear demarcation between sound enamel and the lesion.

Microradiography

Each section was mounted and exposed to Cu (Kα) X-rays (Softex IRS-20, JIRA, Japan) at 10 kV and 3 mA for 60 s. The films were developed using standard Kodak chemicals by developing for 60 s, rinsing in water for 60 s, fixing for 60 s and then again rinsing for 60 s.

The microradiographs of the specimens were mounted and the images acquired by Leica Qwin System (Leica Qwin Image Processing and Analysis Software, Version V2.6) and analysed for changes in mineral content and lesion depth by using computer software (Image J, USA). The results of mineral content changes were reported as the differences between the mineral content before and after the treatment compared with sound enamel ( ΔZ ) with unit of vol% μm.

Statistical analysis

Paired t -test was used to compare differences in lesion depths pre and post pH cycling in each treatment group. Differences in lesion depth and mineral content change (%) between the groups were tested for significance at the p < 0.05 level by a one-way analysis of variance (ANOVA). To assess the interaction effect between the concentration and volume of fluoridated milk tested, a two-way ANOVA was applied. The Tukey’s test was applied to make pairwise comparisons between groups. For descriptive statistics the mean and standard deviation were used.

Results

No statistically significant difference was noted amongst pre-treatment lesion depths between the groups ( p = 0.27, ANOVA). The lesion depth in the pre-treatment groups ranged from 87.32 μm to 104.16 μm. The post-treatment lesion depths differed statistically significantly between groups ( p < 0.001, ANOVA), ranging from 81.33 μm to 141.44 μm. The change in lesion depth varied from a reduction of 15.53% (7.5 ppm in 50 ml) to an increase of 43.71% (de-ionized water). All the test groups (groups 1–12) showed a statistically significant decrease in the lesion depth after treatment with different concentrations and volumes of fluoridated milk ( Table 1 ). Significant increases in the lesion depths were noted when plain milk and de-ionized water were used. Within the same fluoride concentration, no significant difference in percentage of lesion depth reduction was evident when the volume of fluoridated milk was doubled, except in the 20 ppm group. Even though 7.5 ppm showed a higher reduction in lesion depth than any other group, over all, there were no statistically significant differences between percentages of decrease in lesion depth when the concentration of fluoride in milk is increased from 2.5 ppm to 250 ppm (Tukey’s test).

In the two-way ANOVA, when including all groups, there was a significant relationship between fluoride concentration and lesion depth change ( p < 0.0001), no significant relationship with volume ( p = 0.320) but a significant interaction effect ( p = 0.040) was found. However when the control groups were excluded, the interaction effect was no longer significant ( p = 0.195).

Similar to the results obtained from lesion depth, ΔZ values for all the test groups (groups 1–12) were statistically lower after treatment with different concentrations and volumes of fluoridated milk ( p ≤ 0.001, ANOVA) whilst significant increases in ΔZ were noted when plain milk and de-ionized water were used ( Table 2 ). Fig. 2 graphically shows the percentage change of lesion depth and ΔZ values by groups, and comparing different concentrations of fluoride and volumes of milk and water.

Table 2
Percentage changes in ΔZ values after pH cycling.
Treatment groups ΔZ (Mean ± SD)
Pre t x Post t x % change
2.5 ppm F milk; 25 ml/section 5602.0 ± 782.84 5180.0 ± 674.94 * −6.90 ± 9.68 a
2.5 ppm F milk; 50 ml/section 5610.0 ± 831.79 5251.2 ± 850.26 * −5.99 ± 10.33 a
5 ppm F milk; 25 ml/section 5378.3 ± 666.80 4987.6 ± 736.17 * −7.02 ± 9.24 a
5 ppm F milk; 50 ml/section 5348.4 ± 761.64 4920.6 ± 747.95 * −7.26 ± 12.54 a
10 ppm F milk; 25 ml/section 5339.1 ± 855.29 4884.2 ± 781.85 * −8.12 ± 7.63 a
10 ppm F milk; 50 ml/section 5397.3 ± 848.52 4766.2 ± 702.35 * −10.99 ± 9.46 a
7.5 ppm F milk; 25 ml/section 5332.4 ± 945.82 4727.8 ± 893.37 * −10.97 ± 8.91 a
7.5 ppm F milk; 50 ml/section 5262.2 ± 976.94 4450.0 ± 1283.9 * −15.69 ± 19.71 a
15 ppm F milk; 25 ml/section 5269.8 ± 680.58 4701.6 ± 888.60 * −10.89 ± 10.44 a
20 ppm F milk; 25 ml/section 5175.7 ± 741.20 4554.5 ± 696.63 * −11.79 ± 8.18 a
20 ppm F milk; 50 ml/section 5151.3 ± 778.61 4818.4 ± 753.92 * −6.31 ± 6.06 a
250 ppm F milk; 25 ml/section 5196.8 ± 847.92 4900.9 ± 810.16 * −5.57 ± 5.51 a
Plain milk; 25 ml/section 4834.9 ± 851.08 6165.5 ± 915.80 * +29.02 ± 16.79 b
Plain milk; 50 ml/section 5357.8 ± 791.31 6529.5 ± 884.97 * +22.83 ± 14.56 b
Water; 25 ml/section 5411.0 ± 842.49 6908.6 ± 610.53 * +29.45 ± 13.9 b
a,b Different superscript letters indicate statistically significant differences between the groups ( p < 0.001, ANOVA, SNK).

* Significant changes ( p < 0.01, paired t-test) in ΔZ values post pH cycling.

Bar graph comparing percentage lesion depth and ΔZ changes.
Fig. 2
Bar graph comparing percentage lesion depth and ΔZ changes.

The PLM photomicrographs all treatment groups (groups 1–12) showed a decrease in lesion depth. Similarly, the mineral density profiles show that specimens in all the test groups show higher mineral content after pH cycling, except for the control groups. Examples of PLM micrographs, showing decreasing and increasing in lesion depth and the microradiographs showing the changes of mineral content after pH cycling are illustrated in Figs. 3 and 4 , respectively.

Representative polarized light micrographs of specimens from experimental groups (top) and control groups (bottom).
Fig. 3
Representative polarized light micrographs of specimens from experimental groups (top) and control groups (bottom).
Representative graphs showing the relationship between lesion depth ( X -axis) and the relative percent mineral content ( Y -axis) pre and post treatment, from experimental groups (top) and control groups (bottom). Note the increase and decrease in mineral content after treatment of experimental groups and the control groups, respectively.
Fig. 4
Representative graphs showing the relationship between lesion depth ( X -axis) and the relative percent mineral content ( Y -axis) pre and post treatment, from experimental groups (top) and control groups (bottom). Note the increase and decrease in mineral content after treatment of experimental groups and the control groups, respectively.

Discussion

The anti-cariogenic properties of milk, in terms of its re-mineralization efficacy, inhibition of bacterial colonization and bio film inhibition, have been demonstrated. In our study, the caries protective effect of fluoride in milk on early enamel carious lesions was shown by significant decreases in the lesion depths and ΔZ values in all the treatment groups. It also showed that, in most concentrations of fluoridated milk, the lesion depth and ΔZ were reduced slightly more when the volume of fluoridated milk was doubled. This may be explained by having a larger volume of milk would provide a larger quantity of calcium, phosphate, other milk proteins and fluoride available to react with enamel. From data obtained, it also showed that the lesions that were treated with 2.5 ppm F in milk, 50 ml/section showed “similar” re-mineralizing efficacy to those treated with higher concentrations of fluoride in milk.

Milk is a calcium rich food, but most of the calcium in milk, is in the bound form, complexed within the milk matrix with citrate and casein. Only a small fraction of calcium is in the free form which can interact with high concentrations of fluoride to form calcium fluoride. Calcium present in high concentrations in milk is believed to interact with fluoride resulting in the precipitation of calcium fluoride, but this interaction does not occur when the concentration of fluoride in milk is low (2–5 ppm). This is because only a very small fraction of the calcium, about 80 mg/l, is free for interaction; the rest of the calcium in milk is already complexed within the milk matrix with citrate and casein. Therefore calcium fluoride does not precipitate out at low levels of fluoride. When the volume is increased the total amount of fluoride available for re-mineralization is increased whereas at higher concentrations of F, the calcium in milk may interact with F resulting in the precipitation of calcium fluoride.

In the past some reservations have been put forward regarding the use of milk as an alternative vehicle for fluoride, but it has been shown by several researchers that the availability of fluoride from milk is unaffected by the calcium in milk. Trautner and Einwag have shown sodium fluoride and di-sodium mono-fluoro-phosphate to be fully bio-available when consumed alone or with breakfast including milk. Recently, milk was shown to be an appropriate vehicle for fluoride supplementation when consumed simultaneously with solid food in young adults. However, caution must still be taken to avoid excess ingestion of fluoridated milk. Intake of high concentration of fluoridated milk together with other food and beverages with high fluoride content could cause dental fluorosis especially in children whose enamel is still developing. Toth et al. have suggested that under similar experimental conditions, physiological exposure and bio-availability of fluoride in fluoridated salt, milk and tablets are comparable.

Within the same fluoride concentration, even though the decrease in lesion depth and ΔZ values were slightly higher when the volume was double due to the higher amount of Ca, F and other milk proteins available, there is no significant differences on its overall remineralization efficacy. However when the volume of plain milk was doubled, the progression of lesions was significantly less by about 8%, indicating that at higher volumes of plain milk, the caries protective agents present in milk like calcium and phosphate ions, may exert their effect. Since these ions are limited by complexation, the increased volume of milk may also provide more free ions for inhibition of demineralization or enhancement of remineralization. The role of milk is very complex, on one hand it can act as a fluoride carrier thus preventing lesion progression; conversely milk itself contributes to maturation of the bio-film resulting in a higher de-mineralizing potential and greater volume of lesions.

The profiles of mineral content showed that all the test groups gained mineral as a result of pH cycling with fluoridated milk. Mineral was lost when plain milk and de-ionized water were used as the treatment medium.

Single section technique was used in this study to investigate re-mineralization in early enamel carious lesions. The advantage of this technique is that the same lesion is utilized pre and post experiment; this reduces the variability of the procedure. It has been stated that this is possibly the most accurate and sensitive evaluative technique.

The storage solution used for the sections in this study was thymol. The ability of thymol to penetrate bacterial cell membrane is believed to be the principle mode of action of this disinfectant substance. Normally pH lower than 5.5 is hazardous to tooth enamel and a pH lower than 6.2 hazardous to root tissues. It can be noted that the mean pH values of the treatment solutions were between 6.87 and 6.74 which are above those considered dangerous.

In vitro pH cycling model can generally be classified into progression (demineralization) or reversal (remineralization). The exposure time to the solutions and the length of the cycle can be modified according to the focus of study. A period of 20 days with 6 h treatment period was chosen for our pH cycling because it was thought that longer exposure time of the specimens to fluoridated milk would allow for greater amounts of remineralization to occur, and the 14 days of cycling used by earlier researchers might not have been sufficient to observe this remineralization.

Conclusions

Fluoridated milk appears to have a protective effect on early enamel carious lesions. According to the data obtained, it was suggested that 2.5 ppm fluoride milk provided similar remineralization potential to that of higher fluoride concentration in milk. This study has also showed that increasing the volume of fluoridated milk has little effect on its re-mineralizing efficacy.

Acknowledgements

The technical assistance of Mr. Yip and Mr. Shadow is gratefully acknowledged. This study was supported by a grant from the Borrow Foundation , UK.

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Effects of fluoridated milk on artificial enamel carious lesions: A pH cycling study A. Itthagarun , S. Verma , R. Lalloo , N.M. King , J.S. Wefel and R.G. Nair Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 817-824, Copyright © 2011 Elsevier Ltd Abstract Objective To investigate the relationship between concentration of fluoride in milk and its re-mineralizing efficacy, and to test whether increasing the volume of fluoridated milk has any effect on its re-mineralizing efficacy. Methods Third molars were painted leaving a 1 mm window on the buccal and/or lingual surfaces. Artificial carious lesions were induced using a de-mineralizing solution. The teeth were then sectioned longitudinally. Specimens were randomly assigned to 15 groups according to different concentrations and volume; i.e. 2.5 ppm, 5 ppm, 7.5 ppm, 10 ppm, 15 ppm, 20 ppm, and 250 ppm of fluoride with 25 ml/section and 50 ml/section. De-ionized water and plain milk were used as controls. Sections were subjected to pH cycling for 20 days. Polarized light microscopy and micro-radiography were utilized to record the lesion characteristics before and after pH cycling. Results Mean lesion depths decreased significantly in all the fluoride groups, and increased significantly in the control groups. There were no differences in percentage of lesion depth decrease when the volume was doubled for the same fluoride concentration. There were also no statistically significant differences between percentages of decrease in lesion depth when the concentration of fluoride in milk was increased. Conclusion Fluoridated milk demonstrated a remineralization efficacy on early enamel carious lesions. A 2.5 ppm fluoride milk concentration provided similar remineralization potential to that of higher fluoride concentration in milk. 1 Introduction Dental caries is a multi-factorial, highly transmissible disease of dental tissues. It is prevalent worldwide with individuals being susceptible to it throughout their life. Early stage of dental caries can be effectively detected by various means. Dental caries prevalence was very high in the middle of the 20th century, with declines evident since the 1960s. However, there are suggestions that in some communities dental caries is on the increase, and this may be in part due to increasing use of refined sugars and inadequate exposure to fluoride. The progression or reversal of the caries process depends on the balance between pathological and protective factors. Fluoride has been identified as one of the protective factors, which tilts the caries balance towards the positive side. Earlier it was assumed that the beneficial effect of fluoride was mainly pre-eruptive, strengthening the tooth enamel during development, but it has now become clear that the constant presence of low levels of fluoride in the mouth inhibits de-mineralization and enhances re-mineralization. Although water and toothpastes have been excellent vehicles for fluoride, there are parts of the world where drinking water cannot be fluoridated due to reasons such as the lack of resources, socio-economic and technical constraints, absence of political will and national policy on oral health. Fluoridated toothpastes is often unaffordable to the majority of the people in developing countries. Fluoridation of milk is recommended where fluoride concentration in the drinking water is suboptimal, caries experience is significant and programmes to provide milk to children are in place. Whilst a recent systematic review suggested better quality research is needed regarding the effectiveness of milk fluoridation, a number of trials across the world have shown it to be effective in reducing the prevalence of dental caries in children. For example trials in Chile, Bulgaria, China, Sweden and England have shown that children who participated in the fluoridated milk programme experienced significantly less dental caries compared to children not on the programme. Adding fluoride to the milk-products as part of a National Food Supplement Programme has been found to be cost-effective in rural areas of Chile. Though a number of in vitro studies have demonstrated the re-mineralizing efficacy of different concentrations of fluoridated milk, the re-mineralization effect of increasing the volume of fluoridated milk within the same concentration is still unclear. Thus the objectives of this study were to investigate whether increasing (1) the concentration of fluoride in milk and (2) the volume of fluoridated milk alters its re-mineralizing efficacy. The null hypothesis tested was that increasing the fluoride concentration and volume of fluoridated milk would increase its remineralization efficacy on initial enamel carious lesions. 2 Materials and methods 2.1 Preparation of de-mineralizing and re-mineralizing solutions The de/re-mineralizing buffered solutions were prepared from certified chemicals and de-ionized water. The de-mineralizing solution contained 2.2 mM CaCl 2 , 2.2 mM NaH 2 PO 4 , and 0.05 M acetic acid; the pH was adjusted with 1 M KOH to 4.4. The re-mineralizing solution contained 1.5 mM CaCl 2 , 0.9 mM NaH 2 PO 4 , 0.15 M KCl at pH 7.0. These solutions were similar to those utilized by ten Cate and Duijsters. The treatment solutions were prepared from pasteurized and homogenized fresh cow's milk, which contained calcium 110 mg/100 ml; protein 3.1 g/100 ml; carbohydrate 4.6 g/100 ml; fat 3.5 g/100 ml and was available in a tetrapack manufactured by a local Nestle plant in Hong Kong. The milk was mixed with 100 ppm and 500 ppm fluoride solutions prepared in the laboratory to make 2.5 ppm, 5 ppm, 7.5 ppm, 10 ppm, 15 ppm, 20 ppm and 250 ppm fluoridated milk solutions. The treatment groups are listed in Table 1 . Table 1 Changes in lesion depth, by fluoride concentration and milk volume. Treatment groups Lesion depth (Mean ± SD) Pre t x (μm) Post t x (μm) % change 2.5 ppm F milk; 25 ml/section 103.87 ± 17.33 94.75 ± 14.81 * −7.85 ± 11.86 a 2.5 ppm F milk; 50 ml/section 104.16 ± 18.38 95.99 ± 18.30 * −7.31 ± 12.01 a 5 ppm F milk; 25 ml/section 99.05 ± 14.46 90.97 ± 16.18 * −7.78 ± 11.54 a 5 ppm F milk; 50 ml/section 98.77 ± 17.43 89.11 ± 16.35 * −8.67 ± 14.98 a 10 ppm F milk; 25 ml/section 98.37 ± 18.99 88.36 ± 16.91 * −9.55 ± 9.13 a 10 ppm F milk; 50 ml/section 99.84 ± 18.93 86.11 ± 15.97 * −12.93 ± 10.82 a 7.5 ppm F milk; 25 ml/section 98.72 ± 21.93 84.68 ± 19.66 * −13.66 ± 10.57 a 7.5 ppm F milk; 50 ml/section 97.17 ± 22.28 81.64 ± 21.15 * −15.53 ± 11.47 a 15 ppm F milk; 25 ml/section 96.80 ± 15.05 84.28 ± 19.58 * −13.14 ± 12.40 a 20 ppm F milk; 25 ml/section 95.02 ± 16.92 81.33 ± 15.13 * −14.09 ± 9.88 a 20 ppm F milk; 50 ml/section 94.41 ± 17.05 87.12 ± 16.71 * −7.56 ± 7.76 a 250 ppm F milk; 25 ml/section 95.40 ± 19.24 88.64 ± 18.04 * −6.92 ± 6.55 a Plain milk; 25 ml/section 87.32 ± 18.78 120.47 ± 26.16 * +39.42 ± 20.58 b Plain milk; 50 ml/section 98.95 ± 17.69 129.20 ± 24.24 * +31.70 ± 19.77 b Water; 25 ml/section 99.89 ± 18.65 141.44 ± 21.54 * +43.71 ± 19.05 b a,b Statistically significant differences between groups ( p < 0.05, Tukey). * Significant changes ( p < 0.01, paired t -test) in lesion depth post pH cycling. 2.2 Lesion formation Extracted third molars were cleaned of soft tissue debris and inspected for any cracks, caries and hypoplasia. The teeth were painted with acid resistant nail varnish (Revlon ® , New York City, NY, USA) leaving a 1 mm window on the buccal and/or lingual surfaces. They were then immersed in the de-mineralizing solution (10 ml/tooth) for 96 h to produce artificial lesions, approximately 100–150 μm deep. Subsequently, the teeth were sectioned longitudinally through the lesions to produce 100–150 μm thick sections. Three hundred and seventy five sections were selected for the study based on the uniformity of the lesion depth and mineral content. Polarized light microscopy (PLM) and micro-radiography (MRG) were used to analyse the lesion depth and mineral content of the lesions before and after the pH cycling. Prior to the pH cycling model, each section was painted, under a stereomicroscope, with the acid resistant nail varnish (Revlon ® , New York City, NY, USA), leaving only the lesion surface exposed to solutions. The painted sections were then randomly divided into 15 groups, with 25 sections per group. 2.3 The pH cycling model The pH cycling system used in this study was modified from that of Ivancakova et al. The sections were placed in the pH cycling system on an orbital shaker for 20 days at room temperature. Each cycle involved 4 h of demineralization, 6 h of fluoride milk treatment followed by 14 h of remineralization ( Fig. 1 ). All solutions were freshly prepared each day for each cycle and the pH of demineralizing and remineralizing solutions was checked before each cycle. Fig. 1 Schematic illustration of the pH cycling model. 2.4 Evaluation techniques 2.4.1 Polarizing light microscopy Polarized light microscopy (PLM) was employed to qualitatively evaluate the body of the lesion in the enamel samples. This was accomplished by imbibing the section in water, which normally shows a clear demarcation between sound enamel and the lesion. 2.4.2 Microradiography Each section was mounted and exposed to Cu (Kα) X-rays (Softex IRS-20, JIRA, Japan) at 10 kV and 3 mA for 60 s. The films were developed using standard Kodak chemicals by developing for 60 s, rinsing in water for 60 s, fixing for 60 s and then again rinsing for 60 s. The microradiographs of the specimens were mounted and the images acquired by Leica Qwin System (Leica Qwin Image Processing and Analysis Software, Version V2.6) and analysed for changes in mineral content and lesion depth by using computer software (Image J, USA). The results of mineral content changes were reported as the differences between the mineral content before and after the treatment compared with sound enamel ( ΔZ ) with unit of vol% μm. 2.5 Statistical analysis Paired t -test was used to compare differences in lesion depths pre and post pH cycling in each treatment group. Differences in lesion depth and mineral content change (%) between the groups were tested for significance at the p < 0.05 level by a one-way analysis of variance (ANOVA). To assess the interaction effect between the concentration and volume of fluoridated milk tested, a two-way ANOVA was applied. The Tukey's test was applied to make pairwise comparisons between groups. For descriptive statistics the mean and standard deviation were used. 3 Results No statistically significant difference was noted amongst pre-treatment lesion depths between the groups ( p = 0.27, ANOVA). The lesion depth in the pre-treatment groups ranged from 87.32 μm to 104.16 μm. The post-treatment lesion depths differed statistically significantly between groups ( p < 0.001, ANOVA), ranging from 81.33 μm to 141.44 μm. The change in lesion depth varied from a reduction of 15.53% (7.5 ppm in 50 ml) to an increase of 43.71% (de-ionized water). All the test groups (groups 1–12) showed a statistically significant decrease in the lesion depth after treatment with different concentrations and volumes of fluoridated milk ( Table 1 ). Significant increases in the lesion depths were noted when plain milk and de-ionized water were used. Within the same fluoride concentration, no significant difference in percentage of lesion depth reduction was evident when the volume of fluoridated milk was doubled, except in the 20 ppm group. Even though 7.5 ppm showed a higher reduction in lesion depth than any other group, over all, there were no statistically significant differences between percentages of decrease in lesion depth when the concentration of fluoride in milk is increased from 2.5 ppm to 250 ppm (Tukey's test). In the two-way ANOVA, when including all groups, there was a significant relationship between fluoride concentration and lesion depth change ( p < 0.0001), no significant relationship with volume ( p = 0.320) but a significant interaction effect ( p = 0.040) was found. However when the control groups were excluded, the interaction effect was no longer significant ( p = 0.195). Similar to the results obtained from lesion depth, ΔZ values for all the test groups (groups 1–12) were statistically lower after treatment with different concentrations and volumes of fluoridated milk ( p ≤ 0.001, ANOVA) whilst significant increases in ΔZ were noted when plain milk and de-ionized water were used ( Table 2 ). Fig. 2 graphically shows the percentage change of lesion depth and ΔZ values by groups, and comparing different concentrations of fluoride and volumes of milk and water. Table 2 Percentage changes in ΔZ values after pH cycling. Treatment groups ΔZ (Mean ± SD) Pre t x Post t x % change 2.5 ppm F milk; 25 ml/section 5602.0 ± 782.84 5180.0 ± 674.94 * −6.90 ± 9.68 a 2.5 ppm F milk; 50 ml/section 5610.0 ± 831.79 5251.2 ± 850.26 * −5.99 ± 10.33 a 5 ppm F milk; 25 ml/section 5378.3 ± 666.80 4987.6 ± 736.17 * −7.02 ± 9.24 a 5 ppm F milk; 50 ml/section 5348.4 ± 761.64 4920.6 ± 747.95 * −7.26 ± 12.54 a 10 ppm F milk; 25 ml/section 5339.1 ± 855.29 4884.2 ± 781.85 * −8.12 ± 7.63 a 10 ppm F milk; 50 ml/section 5397.3 ± 848.52 4766.2 ± 702.35 * −10.99 ± 9.46 a 7.5 ppm F milk; 25 ml/section 5332.4 ± 945.82 4727.8 ± 893.37 * −10.97 ± 8.91 a 7.5 ppm F milk; 50 ml/section 5262.2 ± 976.94 4450.0 ± 1283.9 * −15.69 ± 19.71 a 15 ppm F milk; 25 ml/section 5269.8 ± 680.58 4701.6 ± 888.60 * −10.89 ± 10.44 a 20 ppm F milk; 25 ml/section 5175.7 ± 741.20 4554.5 ± 696.63 * −11.79 ± 8.18 a 20 ppm F milk; 50 ml/section 5151.3 ± 778.61 4818.4 ± 753.92 * −6.31 ± 6.06 a 250 ppm F milk; 25 ml/section 5196.8 ± 847.92 4900.9 ± 810.16 * −5.57 ± 5.51 a Plain milk; 25 ml/section 4834.9 ± 851.08 6165.5 ± 915.80 * +29.02 ± 16.79 b Plain milk; 50 ml/section 5357.8 ± 791.31 6529.5 ± 884.97 * +22.83 ± 14.56 b Water; 25 ml/section 5411.0 ± 842.49 6908.6 ± 610.53 * +29.45 ± 13.9 b a,b Different superscript letters indicate statistically significant differences between the groups ( p < 0.001, ANOVA, SNK). * Significant changes ( p < 0.01, paired t-test) in ΔZ values post pH cycling. Fig. 2 Bar graph comparing percentage lesion depth and ΔZ changes. The PLM photomicrographs all treatment groups (groups 1–12) showed a decrease in lesion depth. Similarly, the mineral density profiles show that specimens in all the test groups show higher mineral content after pH cycling, except for the control groups. Examples of PLM micrographs, showing decreasing and increasing in lesion depth and the microradiographs showing the changes of mineral content after pH cycling are illustrated in Figs. 3 and 4 , respectively. Fig. 3 Representative polarized light micrographs of specimens from experimental groups (top) and control groups (bottom). Fig. 4 Representative graphs showing the relationship between lesion depth ( X -axis) and the relative percent mineral content ( Y -axis) pre and post treatment, from experimental groups (top) and control groups (bottom). Note the increase and decrease in mineral content after treatment of experimental groups and the control groups, respectively. 4 Discussion The anti-cariogenic properties of milk, in terms of its re-mineralization efficacy, inhibition of bacterial colonization and bio film inhibition, have been demonstrated. In our study, the caries protective effect of fluoride in milk on early enamel carious lesions was shown by significant decreases in the lesion depths and ΔZ values in all the treatment groups. It also showed that, in most concentrations of fluoridated milk, the lesion depth and ΔZ were reduced slightly more when the volume of fluoridated milk was doubled. This may be explained by having a larger volume of milk would provide a larger quantity of calcium, phosphate, other milk proteins and fluoride available to react with enamel. From data obtained, it also showed that the lesions that were treated with 2.5 ppm F in milk, 50 ml/section showed “similar” re-mineralizing efficacy to those treated with higher concentrations of fluoride in milk. Milk is a calcium rich food, but most of the calcium in milk, is in the bound form, complexed within the milk matrix with citrate and casein. Only a small fraction of calcium is in the free form which can interact with high concentrations of fluoride to form calcium fluoride. Calcium present in high concentrations in milk is believed to interact with fluoride resulting in the precipitation of calcium fluoride, but this interaction does not occur when the concentration of fluoride in milk is low (2–5 ppm). This is because only a very small fraction of the calcium, about 80 mg/l, is free for interaction; the rest of the calcium in milk is already complexed within the milk matrix with citrate and casein. Therefore calcium fluoride does not precipitate out at low levels of fluoride. When the volume is increased the total amount of fluoride available for re-mineralization is increased whereas at higher concentrations of F, the calcium in milk may interact with F resulting in the precipitation of calcium fluoride. In the past some reservations have been put forward regarding the use of milk as an alternative vehicle for fluoride, but it has been shown by several researchers that the availability of fluoride from milk is unaffected by the calcium in milk. Trautner and Einwag have shown sodium fluoride and di-sodium mono-fluoro-phosphate to be fully bio-available when consumed alone or with breakfast including milk. Recently, milk was shown to be an appropriate vehicle for fluoride supplementation when consumed simultaneously with solid food in young adults. However, caution must still be taken to avoid excess ingestion of fluoridated milk. Intake of high concentration of fluoridated milk together with other food and beverages with high fluoride content could cause dental fluorosis especially in children whose enamel is still developing. Toth et al. have suggested that under similar experimental conditions, physiological exposure and bio-availability of fluoride in fluoridated salt, milk and tablets are comparable. Within the same fluoride concentration, even though the decrease in lesion depth and ΔZ values were slightly higher when the volume was double due to the higher amount of Ca, F and other milk proteins available, there is no significant differences on its overall remineralization efficacy. However when the volume of plain milk was doubled, the progression of lesions was significantly less by about 8%, indicating that at higher volumes of plain milk, the caries protective agents present in milk like calcium and phosphate ions, may exert their effect. Since these ions are limited by complexation, the increased volume of milk may also provide more free ions for inhibition of demineralization or enhancement of remineralization. The role of milk is very complex, on one hand it can act as a fluoride carrier thus preventing lesion progression; conversely milk itself contributes to maturation of the bio-film resulting in a higher de-mineralizing potential and greater volume of lesions. The profiles of mineral content showed that all the test groups gained mineral as a result of pH cycling with fluoridated milk. Mineral was lost when plain milk and de-ionized water were used as the treatment medium. Single section technique was used in this study to investigate re-mineralization in early enamel carious lesions. The advantage of this technique is that the same lesion is utilized pre and post experiment; this reduces the variability of the procedure. It has been stated that this is possibly the most accurate and sensitive evaluative technique. The storage solution used for the sections in this study was thymol. The ability of thymol to penetrate bacterial cell membrane is believed to be the principle mode of action of this disinfectant substance. Normally pH lower than 5.5 is hazardous to tooth enamel and a pH lower than 6.2 hazardous to root tissues. It can be noted that the mean pH values of the treatment solutions were between 6.87 and 6.74 which are above those considered dangerous. In vitro pH cycling model can generally be classified into progression (demineralization) or reversal (remineralization). The exposure time to the solutions and the length of the cycle can be modified according to the focus of study. A period of 20 days with 6 h treatment period was chosen for our pH cycling because it was thought that longer exposure time of the specimens to fluoridated milk would allow for greater amounts of remineralization to occur, and the 14 days of cycling used by earlier researchers might not have been sufficient to observe this remineralization. 5 Conclusions Fluoridated milk appears to have a protective effect on early enamel carious lesions. According to the data obtained, it was suggested that 2.5 ppm fluoride milk provided similar remineralization potential to that of higher fluoride concentration in milk. This study has also showed that increasing the volume of fluoridated milk has little effect on its re-mineralizing efficacy. Acknowledgements The technical assistance of Mr. Yip and Mr. Shadow is gratefully acknowledged. This study was supported by a grant from the Borrow Foundation , UK. References 1. Selwitz R.H., Ismail A.I., Pitts N.B.: Dental caries. 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