archives of oral biology 53 (2008) 1017–1022

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Effect of strontium in combination with fluoride on enamel

remineralisation in vitro

Tran Thu Thuy a,b,*, Haruo Nakagaki a, Kazuo Kato a, Phan Ai Hung a,b, Junko Inukai a,
Shinji Tsuboi a, Hidetaka Nakagaki a,c, Mina N. Hirose c, Seiji Igarashi c, Colin Robinson d
a
Department of Preventive Dentistry and Dental Public Health, School of Dentistry, Aichi-Gakuin University, Japan
b
Faculty of Odonto-Stomatology, HoChiMinh University of Health Sciences, Viet Nam
c
Department of Pediatric Dentistry, Health Sciences University of Hokkaido, Japan
d
Division of Oral Biology, Leeds Dental Institute, University of Leeds, UK

article info abstract

Article history: Previous studies showed that strontium (Sr) as well as fluoride (F) can enhance enamel
Accepted 10 June 2008 remineralisation. The aim of this study was to evaluate the effects of Sr in combination with
F on enamel remineralisation in vitro. Sixty enamel specimens obtained from caries free
Keywords: human premolars were demineralised to produce caries-like lesions. Half of each lesion was
Strontium covered with nail varnish as an untreated control. The specimens were then randomly
Fluoride divided into F and Sr + F treatment groups. The F group was exposed to remineralising
Remineralisation solutions (1.5 mM CaCl2, 0.9 mM KH2PO4) containing 1 ppm, 0.1 ppm or 0.05 ppm F. The
Human enamel Sr + F treatment group was exposed to the same solutions including 10 ppm Sr. After 2
weeks, lesion depth, mineral loss and percentage enamel remineralisation were determined
using transversal microradiography. There was a significant decrease in mineral loss in all
groups ( p < 0.001). Lesion depth was significantly reduced for all groups ( p < 0.05) with the
exception of group F. Remineralisation was significantly affected by F concentration
( p = 0.000). The participation of Sr resulted in a significant enhancement of remineralisation
( p < 0.001) with a synergistic effect of the Sr + F combination ( p < 0.01). It was concluded
that while the remineralising process was affected by the concentration of F, there was also
an interaction between F and Sr when they were used in conjunction.
# 2008 Elsevier Ltd. All rights reserved.

1. Introduction prevalence in several areas.6–9 This element has both chemical

and physical properties close to calcium, so theoretically it is
Modern glass-ionomers often contain strontium in the powder able to replace Ca in hydroxyapatite.10 Analytical studies
component instead of calcium (strontium fluoroaluminosili- showed higher Sr concentrations in enamel from low caries
cate glass) or as an additional ion to enhance radiopacity.1,2 areas.11–13 This suggests that the remineralising progress may
Other studies have shown that strontium was released from be facilitated by the present of strontium. Previous experiments
glass-ionomer cements (GICs) and dental composites.3–5 It is have given promising results although some of the data is
interesting that strontium has, like fluoride, been considered as controversial. It is, therefore, essential to have more informa-
a trace ion in water which is likely responsible for lower caries tion on the real efficacy of strontium on remineralisation. In a

* Corresponding author at: Department of Preventive Dentistry and Dental Public Health, School of Dentistry, Aichi-Gakuin University, 1-
100 Kusumoto-cho, Chikusa, Nagoya 464-8650, Japan. Tel.: +81 52 751 2561x1352; fax: +81 52 752 5988.
E-mail address: tranthuthuyrhm@yahoo.com (T.T. Thuy).
0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2008.06.005
1018 archives of oral biology 53 (2008) 1017–1022

previous study we evaluated the effect of strontium on enamel Table 1 – Definition of lesion parameters
remineralisation in vitro.14 Different strontium concentrations Parameter Definition
including levels in the water associated with low caries
Lesion depth Ld (mm) Measure from the surface which
prevalence were tested. The results indicated that strontium
mineral content is 5% to where
has the capacity to enhance enamel remineralisation. it returned to 95% of the sound level
Fluoride and strontium both have a positive effect on Mineral loss DZ (vol.%mm) DZ = (the area under the sound
remineralisation progress per se although the effect of fluoride is enamel profile  the area under
not dependent on the presence of strontium, and neither does mineral profile of lesion)


DZ Re
the effect of strontium appear to depend on the present of Percentage enamel %R ¼ 1  DZ De  100
remineralisation—%R
fluoride. However, the combination of strontium and fluoride
may have advantages over either ion alone. It has been reported
that, when incorporated together, fluoride and strontium and Sr + F treatment groups. The F group (F, F1, F5) were
improved apatite crystallinity and were associated with marked exposed to remineralising solutions containing F with con-
reductions in acid reactivity of synthetic carbonated apatites.15 centrations of 1 ppm, 0.1 ppm or 0.05 ppm at pH 7.0. The Sr + F
When fluoride is available simultaneously with calcium and treatment group (SF, SF1, SF5) were exposed to the same
phosphate, it has been suggested to accelerate the remineralis- solutions, i.e. 1 ppm, 0.1 ppm or 0.05 ppm F together with
ing process by adsorbing to the enamel surface and attracting 10 ppm Sr at same pH 7.0. Each specimen was individually
calcium ions.16 When strontium is used in conjunction with immersed with 50 ml remineralising solution at 37 8C for 14
fluoride whether such phenomena occur and result in the days and the solution was replaced every 2 days.
enhancement of remineralisation. The aim of this study was to
clarify the interaction between strontium and fluoride on 2.4. Assessment of mineral content
enamel remineralisation in vitro.
Samples were analysed for remineralisation using a transversal
microradiographic technique. At the conclusion of the immer-
2. Materials and methods sion, samples were washed with distilled water and the nail
varnish was removed by acetone. Specimens were embedded in
2.1. Sample preparation and formation of caries lesions a mixture of 20% methylmethacrylate and 80% butylmetha-
crylate. 400-mm-thick sections were cut perpendicular to the
Sixty enamel specimens were obtained from 1/3 mid buccal enamel surface using a diamond blade (Isomet, Buehler Ltd.,
surfaces of human premolars. The teeth were extracted for Lake Bluff, IL) and then ground by hand on 1200-mesh abrasive
orthodontic reasons, free from caries, any enamel malforma- waterproof paper to a thickness of approximate 100 mm.
tion or hypoplasia. To avoid high variable surface fluoride, Transversal microradiographs were taken together with an Al
approximately 150 mm of the outermost surface of the enamel stepwedge on high precision photo plates (Konica Minolta,
was removed by waterproof abrasive paper which was then Japan) at 12 kV, 1.5 mA, 3 min by means of a soft X-ray generator
polished to produce a lustre. (Softex CMR-2, Kanagawa, Japan). Microradiographs were
To produce caries-like lesions, specimens were exposed to examined with a microscope (Olympus SZX9, Japan) and
demineralising gel at a pH of 5.0, 37 8C for a period of 14 days. TMR-images were captured via a camera (Olympus DP70,
The gel contained 0.1 M lactic acid, 6 wt% carboxymethylcel- Japan). The image-analysis software (Winroof, Japan) was used
lulose. At the end of demineralised period, samples were for microdensitometry measurements. The parameters of
washed carefully under running tap water for 30 min and lesion depth (Ld-mm), mineral loss (DZ, vol.%mm), percentage
rinsed again with distilled water for 30 s. of the enamel remineralisation (%R) were used to evaluate the
remineralisation of lesion. Parameters are defined in Table 1.
2.2. Remineralising solution
2.5. Statistical analysis
The concentration of calcium was 1.5 mM (as CaCl2) and
phosphate was 0.9 mM (as KH2PO4) with a Ca:P ratio Student’s and pair t-test, was used to compare lesion para-
representing hydroxyapatite stoichiometry.17 Three fluoride meters after remineralisation with untreated controls. Two-way
concentrations (1 ppm, 0.1 ppm or 0.05 ppm) were incorpo- analysis of variance was used to detect significant difference
rated in the remineralising solutions. Literature suggested that between groups and interaction between fluoride and stron-
strontium concentrations in water ranging from 5 ppm to tium.19 The difference between the fluoride concentrations
15 ppm were associated with lower caries prevalence.10,18 Our were determined by Scheffé test. A 0.1%, 1% and 5% level of
previous study showed that strontium at 10 ppm resulted in a statistical significance were applied for the analyses. The
higher remineralisation outcome among the tested concen- contribution rate was used to evaluate the impact of factors.20
trations.14 Hence, strontium was incorporated in remineralis-
ing solutions at a concentration of 10 ppm (as SrCl26H2O).
3. Results
2.3. Remineralisation procedure
Table 2 presents the parameters of the lesion before and after a
Half of each lesion was covered with nail varnish as an 2-week treatment. There was a significant decrease in mineral
untreated control. Samples were then randomly divided into F loss of all groups (paired t-test; p < 0.001) after exposure to the
 archives of oral biology 53 (2008) 1017–1022 1019

Table 2 – Changes in lesion parameters after reminer-

alisation, compared to untreated control
Untreated control Remineralisation

Ld (mm) DZ (vol.%mm) Ld (mm) DZ (vol.%mm)

F group
F 116  3a 6021  258 114  3 4807  255***
F1 115  5 6078  344 110  5* 4948  380***
F5 114  4 6187  341 106  3*** 3727  214***

Sr + F group
SF 104  3 5407  196 99  3* 3803  260*** Fig. 2 – Enamel remineralisation (%R) in F and Sr + F groups.
SF1 106  4 5506  258 99  5** 3466  289***
SF5 102  3 5322  220 96  3*** 3305  167***

Paired t-test: *p < 0.05; **p < 0.01; ***p < 0.001.
a
Mean  S.E.

remineralising solution. Lesion depth was statistically

reduced in experimental teeth (paired t-test; p < 0.05) except
lesions exposed to 1 ppm F solution (group F).
Microradiographs of lesions exposed to solutions contain-
ing different F concentrations with and without Sr are shown
in Fig. 1. Fig. 2 shows the percentage of mineral gain in F group
and Sr + F treatment group. There was no difference in the
mineral gain when strontium was added in the solution
containing 0.05 ppm F. The inclusion of strontium in 1 ppm F
and 0.1 ppm F solutions caused a substantial increase in Fig. 3 – Average enamel remineralisation (%R) with
remineralisation. The addition of strontium to 0.1 ppm F different fluoride levels.
solution caused about a twofold increase in remineralisation
almost reaching the remineralisation seen with 0.05 ppm F
and Sr + 0.05 ppm F solutions. in conjunction with F ( p < 0.01). While F contributed 20.23% of
Average percentage remineralisation by different F con- enamel remineralisation (%R), Sr contributed 12.20% to the
centrations is shown in Fig. 3. Solutions containing 0.05 ppm F outcome. 9.22% remineralisation was added by the synergistic
produced the highest remineralisation (39%), clearly different interaction between the two ions (Table 3).
from 1 ppm (25%) or 0.1 ppm F solution (28%) ( p = 0.000 and
p < 0.01, respectively). There was no statistical difference in
the remineralising outcome between 1 ppm F and 0.1 ppm F 4. Discussion
solutions.
The average percentage remineralisation of solutions In this study fluoride was incorporated in the mineralising
containing F alone was 26%, less than that of F combined Sr solutions at concentrations of 1 ppm, 0.1 ppm or 0.05 ppm and
solutions (35%) (Fig. 4). Sr significantly affected the reminer- all levels of added fluoride resulted in a significant enhance of
alisation ( p < 0.001) with a synergistic effect when it was used mineral gain. An interesting feature revealed by Fig. 3 is that

Fig. 1 – Microradiographs after exposure to remineralising solution (— 200 mm).

1020 archives of oral biology 53 (2008) 1017–1022

Table 3 – ANOVA analyse for data of enamel remineralisation (%R)

Source Sum of squares d.f. Mean square F p value Contribution (%)

F 1832 2 916 10.19 0.000 20.23

Sr 1086 1 1086 12.08 0.001 12.20
F  Sr 933 2 467 5.19 0.009 9.22
Error 4316 48 90 58.35

Total 8166 53 100.00

2 2
R = .472 (adjusted R = .417).

the degree of remineralisation achieved depends on the level This was supported by the observation that incorporation of
of fluoride ions in the solution and significantly more mineral strontium, together with fluoride, retarded dissolution of
precipitation occurred in the presence of 0.05 ppm fluoride synthetic hydroxyapatite.15,27 Spets-Happonen et al. reported
than that of 0.1 ppm and 1 ppm. Silverstone reported that the that chlorhexidine-fluoride gels plus strontium were more
addition of 1 ppm fluoride to calcifying fluid markedly effective than chlorhexidine-fluoride gel in preventing soft-
enhances remineralisation while increasing the fluoride level ening of enamel in vitro.28 In contrast it was reported that the
to 10 ppm has no further effect on the degree of remineralisa- strontium additive did not improve the caries-preventive
tion.21 On the contrary, lesions treated with remineralising effect of chlorhexidine-fluoride solution in both clinical trials
solutions added 2 ppm fluoride showed less remineralisation and animal studies.29–31 In addition, strontium had no specific
than no fluoride added solutions.22 Dramatic enhancement of protective effect against enamel demineralisation in situ.32
remineralisation was also observed at 0.03–0.5 ppm fluoride.23 The reasons for the synergistic enhancement of remineralisa-
Gibbs et al. concluded that fluoride at levels of 0.058–0.138 ppm tion observed in the current study are not clear, however, they
promoted calcium uptake by artificial lesions during reminer- were dependent on the fluoride concentration (Fig. 2). Some of
alising conditions.24 This result may be due to the rapid the discrepancies may be in the actual fluoride and strontium
precipitation of minerals in the surface pores of the lesion by concentrations used. In a previous study the effect of
high fluoride concentrations thus blocking further access to strontium at different concentrations (0 ppm, 5 ppm,
the lesion interiors. Low concentrations on the other hand 10 ppm and 20 ppm) on enamel remineralisation with and
may allow deep penetration without blocking the surface. without fluoride combination was examined.14 While the
Highly fluoridated crystals are also likely to be more stable and combination of 1 ppm fluoride with strontium enhanced
less disposed to grow. During remineralisation minerals are remineralisation, the remineralisation was significantly
initially deposited in or near the surface layer and then are affected by strontium concentration. In the present study,
gradually transferred inward in the deeper part of the lesion strontium was added at only 10 ppm, in contrast to 250 ppm,
body.25 The fast rebuilding of the surface layer with a more 500 ppm or 1000 ppm in reports which reported no benefit of
stable material, fluorapatite, results in a region in the outer adding strontium.29–32 Spets-Happonen et al. also observed
part of the lesion which probably hinders or slows down the that the effectiveness of chlorhexidine-fluoride gel tended to
subsequent diffusion of minerals to the deeper part of the increase with 50 ppm strontium added whereas the effective-
lesion body. This may well be the reason why higher fluoride ness seemed to reduce with 250 ppm.31 Rapid partial reminer-
concentrations were less effective than lower ones. alisation of enamel lesions occur by solutions containing
Strontium has been considered as an element other than strontium at a concentration of 8.8 ppm (104 mol/l).33 The
fluoride which affects the behaviour of enamel mineral during result in the present study, together with previous data,
carious challenges. The mechanism is unclear and some of the suggests that concentration is a key factor which decides the
data is controversial. Dedhiya et al. proposed the formation of effect of strontium in remineralisation process.
a calcium–strontium apatite complex at the apatite crystal When strontium was combined with 1 ppm fluoride,
surface which retards the acid dissolution of hydroxyapatite.26 significant reduction of lesion depth was observed while it
was not seen with 1 ppm fluoride alone. It appears likely that
the surface inhibition is counteracted by the present of
strontium together with fluoride. Mellberg and Fletcher have
reported that strontium complexes decrease fluoride deposi-
tion in the outermost layer of artificial caries lesions in vitro
which might preclude occlusion of outer lesion pores.34
However, when appropriate levels of strontium and fluoride
are simultaneously present in solution the diffusion processes
of ions into the lesion front may be favoured resulting in
enhanced remineralisation. Calcium and phosphate in con-
junction with strontium also rapidly diffuses to inner
boundary of carious lesion to rebuilt depleted enamel.33 In
addition, during remineralisation fluoride and strontium are
Fig. 4 – Average enamel remineralisation (%R) with and most likely incorporated into the apatite structure replacing
without strontium. hydroxyl and calcium ions, respectively. These substitutions
 archives of oral biology 53 (2008) 1017–1022 1021

lead to changes of apatite behaviour which may also affect the 11. Curzon MEJ, Losee FL. Strontium content of enamel and
remineralisation process.35 dental caries. Caries Res 1977;11:321–6.
12. Curzon MEJ, Losee FL. Trace element composition of whole
This study clearly shows that the simultaneous presence
human enamel and dental caries. Part I. Eastern USA. JADA
of strontium with fluoride at specific concentrations
1977;94:1146–50.
enhances enamel remineralisation in vitro. While the 13. Curzon MEJ, Losee FL. Trace element composition of whole
remineralising process was affected by the concentration human enamel and dental caries. Part II. Western USA.
of fluoride, the interaction between strontium and fluoride JADA 1978;96:819–22.
induced a synergistic remineralising enhancement. These 14. Thu Thuy T, Nakagaki H, Inukai H, Tsuboi S, Robinson C.
basic data suggest the need for further study to elucidate the Effect of strontium on enamel remineralization in vitro.
Caries Res 2006;40:338.
synergy between fluoride and strontium as well as
15. Featherstone JDB, Shields CP, Khademazad B, Oldershaw
studies with restorative materials containing both fluoride MD. Acid reactivity of carbonated apatites with strontium
and strontium. While GICs have been reported to assist in and fluoride substitutions. J Dent Res 1983;62:1049–53.
the remineralisation of surrounding tooth tissues by 16. Featherstone JDB. Prevention and reversal of dental caries:
release of fluoride and possibly other ions,1,36 it is still role of low level fluoride. Community Dent Oral Epidemiol
not known whether the presence of strontium affects this 1999;27:31–40.
17. Exterkate RA, Damen JJ, ten Cate JM. A single-section model
process.
for enamel de- and remineralization studies. 1. The effects
of different Ca/P ratios in remineralization solutions. J Dent
Res 1993;72:1599–603.
Acknowledgments 18. Curzon MEJ. The relation between caries prevalence and
strontium concentrations in drinking water, plaque, and
This work was supported by the AGU High-Tech Research surface enamel. J Dent Res 1985;64:1386–8.
Center Projects from the Ministry of Education, Culture, Sport, 19. Sokal SF, Rohlf FJ. The principle and practice of statistics in
biology research. 2nd ed. New York: Freeman; 1981. p.
Science and Technology, Japan and the Overseas Training
179–453.
Projects from the Ministry of Education and Training,
20. Iwata C, Nakagaki H, Morita I, Sekiya T, Goshima M, Abe T,
Viet Nam. et al. Daily use of dentifrice with and without xylitol and
We thank the nice relationship between the Faculty of fluoride: effect on glucose retention in humans in vivo. Arch
Odonto-Stomatology, HoChiMinh University of Health Oral Biol 2003;48:389–95.
Sciences, Viet Nam and the School of Dentistry, Aichi-Gakuin 21. Silverstone LM. The effect of fluoride in the
University, Nagoya, Japan. remineralization of enamel caries and caries-like lesions in
vitro. J Public Health Dent 1982;42:42–53.
22. Lammers PC, Borggreven JM, Driessens FC. Influence of
references fluoride on in vitro remineralization of artificial subsurface
lesions determined with a sandwich technique. Caries Res
1990;24:81–5.
23. Featherstone JDB, O’reilly MM, Shariati M, Brugler S.
1. Mount GJ, Hume WR. Glass-ionomere material. Preservation Enhancement of remineralisation in vitro and in vivo. In:
and restoration of tooth structure. 2nd ed. Brisbane: Knowledge Leach SA, editor. Factors relating to demineralization and
Books and Software; 2005. p. 163–87. remineralisation of teeth. Oxford: IRL Press; 1986. p. 23–34.
2. Billington RW, Williams JA, Pearson GJ. Ion processes in 24. Gibbs CD, Atherton SE, Huntington E, Lynch RJM,
glass ionomer cements. J Dent 2006;34:544–55. Duckworth RM. Effect of low levels of fluoride on calcium
3. Zhou M, Drummond J, Hanley L. Barium and strontium uptake by demineralized human enamel. Arch Oral Biol
leaching from aged glass particle/resin matrix dental 1995;40:879–81.
composites. Dent Mater 2005;21:145–55. 25. ten Cate JM, Arends J. Remineralization of artificial enamel
4. Czarnecka B, Nicholson J. Ion release by resin-modified lesions in vitro: III. A study of the deposition mechanism.
glass-ionomer cements into water and lactic acid solutions. Caries Res 1980;14:351–8.
J Dent 2006;34:539–43. 26. Dedhiya MG, Young F, Higuchi WI. Mechanism for the
5. Ngo HC, Mount G, Mc Intyre J, Tuisuva J, Von Doussa RJ. retardation of the acid dissolution rate of hydroxyapatite by
Chemical exchange between glass-ionomer restorations strontium. J Dent Res 1973;52:1097–109.
and residual carious dentine in permanent molars: an in 27. Herbison RJ, Handelman SL. Effect of trace elements on
vivo study. J Dent 2006;34:608–13. dissolution of hydroxyapatite by cariogenic streptococci.
6. Barmes DE. Caries aetiology on Sepik Villages—trace J Dent Res 1975;54:1107–11.
element, micronutrient and macronutrient content of the 28. Spets-Happonen S, Luoma H, Seppä L, Räisänen J. The
soil and food. Caries Res 1969;3:44–59. effect of different strontium concentrations on the
7. Curzon MEJ, Adkins BL, Bibby BG, Losee FL. Combined effect efficacy of chlorhexidine-fluoride-strontium gel in
of trace elements and fluorine on caries. J Dent Res preventing enamel softening in vitro. Arch Oral Biol
1970;49:526–9. 1993;38:107–12.
8. Curzon MEJ, Spector PC, Iker H. An association between 29. Luoma H, Seppä L, Koskinen M, Syrjänen S. Effect of
strontium in drinking water supplies and low caries chlorhexidine-fluoride applications without and with Sr and
prevalence. Arch Oral Biol 1978;23:317–21. Zn on caries, plaque, and gingiva in rats. J Dent Res
9. Vbric V, Stupar J. Dental caries and the concentration of 1984;63:1193–6.
aluminum and strontium in enamel. Caries Res 1980;14: 30. Spets-Happonen S, Luoma H, Forss H, Kentala J, Alaluusua
141–7. S, Luoma AR, et al. Effects of a chlorhexidine-fluoride-
10. Curzon MEJ, Cutress TW. Strontium. Trace elements and strontium rinsing program on caries, gingivitis and some
dental disease. London: John Wright, PSG Inc.; 1983. salivary bacteria among Finnish schoolchildren. Scand J Dent
p. 283–304. Res 1991;99:130–8.
1022 archives of oral biology 53 (2008) 1017–1022

31. Spets-Happonen S, Luoma H, Seppä L. High strontium 34. Mellberg JR, Fletcher R. Effect of a strontium complex on
addition to chlorhexidine-fluoride gel does not increase its fluoride uptake by artificial caries lesions and sound enamel
caries-preventive effect in rats. Acta Odontol Scand in vitro. Caries Res 1990;24:93–6.
1996;54:92–5. 35. Robinson C, Kirkham J, Brookes SJ, Shore RC. Chemistry
32. Brudevold F, Tehrani A, Attarzadeh F, Goulet D, van Houte J. of mature enamel. Dental enamel: formation to destruction.
Effect of some salts of calcium, sodium, potassium, and Boca Raton: CRC Press; 1995. p. 170–75.
strontium on intra-oral enamel demineralization. J Dent Res 36. Yamamoto K, Arai K, Fukazawa K, Fukui K, Nagamatsu K,
1985;64:24–7. Kato K, et al. Effect of plaque fluoride released from a glass-
33. Featherstone JDB, Rodgers BE, Smith MW. Physicochemical ionomer cement on enamel remineralization in situ. Caries
requirements for rapid remineralization of early carious Res 2005;39:157–60.
lesions. Caries Res 1981;15:221–35.