article WS

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 1 0 1 7 – 1 0 2 2

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

a r t i c l e i n f o

Article history:

Accepted 10 June 2008

Keywords:

Strontium

Fluoride

Remineralisation

Human enamel

a b s t r a c t

Previous studies showed that strontium (Sr) as well as fluoride (F) can enhance enamel

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

human premolars were demineralised to produce caries-like lesions. Half of each lesion was

covered with nail varnish as an untreated control. The specimens were then randomly

divided into F and Sr + F treatment groups. The F group was exposed to remineralising

solutions (1.5 mM CaCl2, 0.9 mM KH2PO4) containing 1 ppm, 0.1 ppm or 0.05 ppm F. The

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.

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j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / a r o b

1. Introduction

Modern glass-ionomers often contain strontium in the powder

component instead of calcium (strontium fluoroaluminosili-

cate glass) or as an additional ion to enhance radiopacity.1,2

Other studies have shown that strontium was released from

glass-ionomer cements (GICs) and dental composites.3–5 It is

interesting that strontium has, like fluoride, been considered as

a trace ion in water which is likely responsible for lower caries

* Corresponding author at: Department of Preventive Dentistry and Den 100 Kusumoto-cho, Chikusa, Nagoya 464-8650, Japan. Tel.: +81 52 751

E-mail address: [email protected] (T.T. Thuy).

0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserve doi:10.1016/j.archoralbio.2008.06.005

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

and physical properties close to calcium, so theoretically it is

able to replace Ca in hydroxyapatite.10 Analytical studies

showed higher Sr concentrations in enamel from low caries

areas.11–13 This suggests that the remineralising progress may

be facilitated by the present of strontium. Previous experiments

have given promising results although some of the data is

controversial. It is, therefore, essential to have more informa-

tion on the real efficacy of strontium on remineralisation. In a

tal Public Health, School of Dentistry, Aichi-Gakuin University, 1- 2561x1352; fax: +81 52 752 5988.

d.

Table 1 – Definition of lesion parameters

Parameter Definition

Lesion depth Ld (mm) Measure from the surface which

mineral content is 5% to where

it returned to 95% of the sound level

Mineral loss DZ (vol.%�mm) DZ = (the area under the sound enamel profile � the area under mineral profile of lesion)

Percentage enamel

remineralisation—%R

%R ¼ 1 � DZ Re DZ De

� � � 100

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 1 0 1 7 – 1 0 2 21018

previous study we evaluated the effect of strontium on enamel

remineralisation in vitro.14 Different strontium concentrations

including levels in the water associated with low caries

prevalence were tested. The results indicated that strontium

has the capacity to enhance enamel remineralisation.

Fluoride and strontium both have a positive effect on

remineralisation progress per se although the effect of fluoride is

not dependent on the presence of strontium, and neither does

the effect of strontium appear to depend on the present of

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

improved apatite crystallinity and were associated with marked

reductions in acid reactivity of synthetic carbonated apatites.15

When fluoride is available simultaneously with calcium and

phosphate, it has been suggested to accelerate the remineralis-

ing process by adsorbing to the enamel surface and attracting

calcium ions.16 When strontium is used in conjunction with

fluoride whether such phenomena occur and result in the

enhancement of remineralisation. The aim of this study was to

clarify the interaction between strontium and fluoride on

enamel remineralisation in vitro.

2. Materials and methods

2.1. Sample preparation and formation of caries lesions

Sixty enamel specimens were obtained from 1/3 mid buccal

surfaces of human premolars. The teeth were extracted for

orthodontic reasons, free from caries, any enamel malforma-

tion or hypoplasia. To avoid high variable surface fluoride,

approximately 150 mm of the outermost surface of the enamel

was removed by waterproof abrasive paper which was then

polished to produce a lustre.

To produce caries-like lesions, specimens were exposed to

demineralising gel at a pH of 5.0, 37 8C for a period of 14 days.

The gel contained 0.1 M lactic acid, 6 wt% carboxymethylcel-

lulose. At the end of demineralised period, samples were

washed carefully under running tap water for 30 min and

rinsed again with distilled water for 30 s.

2.2. Remineralising solution

The concentration of calcium was 1.5 mM (as CaCl2) and

phosphate was 0.9 mM (as KH2PO4) with a Ca:P ratio

representing hydroxyapatite stoichiometry.17 Three fluoride

concentrations (1 ppm, 0.1 ppm or 0.05 ppm) were incorpo-

rated in the remineralising solutions. Literature suggested that

strontium concentrations in water ranging from 5 ppm to

15 ppm were associated with lower caries prevalence.10,18 Our

previous study showed that strontium at 10 ppm resulted in a

higher remineralisation outcome among the tested concen-

trations.14 Hence, strontium was incorporated in remineralis-

ing solutions at a concentration of 10 ppm (as SrCl2�6H2O).

2.3. Remineralisation procedure

Half of each lesion was covered with nail varnish as an

untreated control. Samples were then randomly divided into F

and Sr + F treatment groups. The F group (F, F1, F5) were

exposed to remineralising solutions containing F with con-

centrations of 1 ppm, 0.1 ppm or 0.05 ppm at pH 7.0. The Sr + F

treatment group (SF, SF1, SF5) were exposed to the same

solutions, i.e. 1 ppm, 0.1 ppm or 0.05 ppm F together with

10 ppm Sr at same pH 7.0. Each specimen was individually

immersed with 50 ml remineralising solution at 37 8C for 14

days and the solution was replaced every 2 days.

2.4. Assessment of mineral content

Samples were analysed for remineralisation using a transversal

microradiographic technique. At the conclusion of the immer-

sion, samples were washed with distilled water and the nail

varnish was removed by acetone. Specimens were embedded in

a mixture of 20% methylmethacrylate and 80% butylmetha-

crylate. 400-mm-thick sections were cut perpendicular to the

enamel surface using a diamond blade (Isomet, Buehler Ltd.,

Lake Bluff, IL) and then ground by hand on 1200-mesh abrasive

waterproof paper to a thickness of approximate 100 mm.

Transversal microradiographs were taken together with an Al

stepwedge on high precision photo plates (Konica Minolta,

Japan) at 12 kV, 1.5 mA, 3 min by means of a soft X-ray generator

(Softex CMR-2, Kanagawa, Japan). Microradiographs were

examined with a microscope (Olympus SZX9, Japan) and

TMR-images were captured via a camera (Olympus DP70,

Japan). The image-analysis software (Winroof, Japan) was used

for microdensitometry measurements. The parameters of

lesion depth (Ld-mm), mineral loss (DZ, vol.%�mm), percentage of the enamel remineralisation (%R) were used to evaluate the

remineralisation of lesion. Parameters are defined in Table 1.

2.5. Statistical analysis

Student’s and pair t-test, was used to compare lesion para-

meters after remineralisation with untreated controls. Two-way

analysis of variance was used to detect significant difference

between groups and interaction between fluoride and stron-

tium.19 The difference between the fluoride concentrations

were determined by Scheffé test. A 0.1%, 1% and 5% level of

statistical significance were applied for the analyses. The

contribution rate was used to evaluate the impact of factors.20

3. Results

Table 2 presents the parameters of the lesion before and after a

2-week treatment. There was a significant decrease in mineral

loss of all groups (paired t-test; p < 0.001) after exposure to the

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*** 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.

Fig. 2 – Enamel remineralisation (%R) in F and Sr + F groups.

Fig. 3 – Average enamel remineralisation (%R) with

different fluoride levels.

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 1 0 1 7 – 1 0 2 2 1019

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

remineralisation. The addition of strontium to 0.1 ppm F

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.

Average percentage remineralisation by different F con-

centrations is shown in Fig. 3. Solutions containing 0.05 ppm F

produced the highest remineralisation (39%), clearly different

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

solutions.

The average percentage remineralisation of solutions

containing F alone was 26%, less than that of F combined Sr

solutions (35%) (Fig. 4). Sr significantly affected the reminer-

alisation ( p < 0.001) with a synergistic effect when it was used

Fig. 1 – Microradiographs after exposure t

in conjunction with F ( p < 0.01). While F contributed 20.23% of

enamel remineralisation (%R), Sr contributed 12.20% to the

outcome. 9.22% remineralisation was added by the synergistic

interaction between the two ions (Table 3).

4. Discussion

In this study fluoride was incorporated in the mineralising

solutions at concentrations of 1 ppm, 0.1 ppm or 0.05 ppm and

all levels of added fluoride resulted in a significant enhance of

mineral gain. An interesting feature revealed by Fig. 3 is that

o remineralising solution (— 200 mm).

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

R2 = .472 (adjusted R2 = .417).

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 1 0 1 7 – 1 0 2 21020

the degree of remineralisation achieved depends on the level

of fluoride ions in the solution and significantly more mineral

precipitation occurred in the presence of 0.05 ppm fluoride

than that of 0.1 ppm and 1 ppm. Silverstone reported that the

addition of 1 ppm fluoride to calcifying fluid markedly

enhances remineralisation while increasing the fluoride level

to 10 ppm has no further effect on the degree of remineralisa-

tion.21 On the contrary, lesions treated with remineralising

solutions added 2 ppm fluoride showed less remineralisation

than no fluoride added solutions.22 Dramatic enhancement of

remineralisation was also observed at 0.03–0.5 ppm fluoride.23

Gibbs et al. concluded that fluoride at levels of 0.058–0.138 ppm

promoted calcium uptake by artificial lesions during reminer-

alising conditions.24 This result may be due to the rapid

precipitation of minerals in the surface pores of the lesion by

high fluoride concentrations thus blocking further access to

the lesion interiors. Low concentrations on the other hand

may allow deep penetration without blocking the surface.

Highly fluoridated crystals are also likely to be more stable and

less disposed to grow. During remineralisation minerals are

initially deposited in or near the surface layer and then are

gradually transferred inward in the deeper part of the lesion

body.25 The fast rebuilding of the surface layer with a more

stable material, fluorapatite, results in a region in the outer

part of the lesion which probably hinders or slows down the

subsequent diffusion of minerals to the deeper part of the

lesion body. This may well be the reason why higher fluoride

concentrations were less effective than lower ones.

Strontium has been considered as an element other than

fluoride which affects the behaviour of enamel mineral during

carious challenges. The mechanism is unclear and some of the

data is controversial. Dedhiya et al. proposed the formation of

a calcium–strontium apatite complex at the apatite crystal

surface which retards the acid dissolution of hydroxyapatite.26

Fig. 4 – Average enamel remineralisation (%R) with and

without strontium.

This was supported by the observation that incorporation of

strontium, together with fluoride, retarded dissolution of

synthetic hydroxyapatite.15,27 Spets-Happonen et al. reported

that chlorhexidine-fluoride gels plus strontium were more

effective than chlorhexidine-fluoride gel in preventing soft-

ening of enamel in vitro.28 In contrast it was reported that the

strontium additive did not improve the caries-preventive

effect of chlorhexidine-fluoride solution in both clinical trials

and animal studies.29–31 In addition, strontium had no specific

protective effect against enamel demineralisation in situ.32

The reasons for the synergistic enhancement of remineralisa-

tion observed in the current study are not clear, however, they

were dependent on the fluoride concentration (Fig. 2). Some of

the discrepancies may be in the actual fluoride and strontium

concentrations used. In a previous study the effect of

strontium at different concentrations (0 ppm, 5 ppm,

10 ppm and 20 ppm) on enamel remineralisation with and

without fluoride combination was examined.14 While the

combination of 1 ppm fluoride with strontium enhanced

remineralisation, the remineralisation was significantly

affected by strontium concentration. In the present study,

strontium was added at only 10 ppm, in contrast to 250 ppm,

500 ppm or 1000 ppm in reports which reported no benefit of

adding strontium.29–32 Spets-Happonen et al. also observed

that the effectiveness of chlorhexidine-fluoride gel tended to

increase with 50 ppm strontium added whereas the effective-

ness seemed to reduce with 250 ppm.31 Rapid partial reminer-

alisation of enamel lesions occur by solutions containing

strontium at a concentration of 8.8 ppm (10�4 mol/l).33 The

result in the present study, together with previous data,

suggests that concentration is a key factor which decides the

effect of strontium in remineralisation process.

When strontium was combined with 1 ppm fluoride,

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

most likely incorporated into the apatite structure replacing

hydroxyl and calcium ions, respectively. These substitutions

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 1 0 1 7 – 1 0 2 2 1021

lead to changes of apatite behaviour which may also affect the

remineralisation process.35

This study clearly shows that the simultaneous presence

of strontium with fluoride at specific concentrations

enhances enamel remineralisation in vitro. While the

remineralising process was affected by the concentration

of fluoride, the interaction between strontium and fluoride

induced a synergistic remineralising enhancement. These

basic data suggest the need for further study to elucidate the

synergy between fluoride and strontium as well as

studies with restorative materials containing both fluoride

and strontium. While GICs have been reported to assist in

the remineralisation of surrounding tooth tissues by

release of fluoride and possibly other ions,1,36 it is still

not known whether the presence of strontium affects this

process.

Acknowledgments

This work was supported by the AGU High-Tech Research

Center Projects from the Ministry of Education, Culture, Sport,

Science and Technology, Japan and the Overseas Training

Projects from the Ministry of Education and Training,

Viet Nam.

We thank the nice relationship between the Faculty of

Odonto-Stomatology, HoChiMinh University of Health

Sciences, Viet Nam and the School of Dentistry, Aichi-Gakuin

University, Nagoya, Japan.

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