vol. 92 • suppl no.

1 JDR Clinical Research Supplement

CLINICAL TRIALS

Fluoride Concentration from Dental

Sealants: A Randomized Clinical Trial
G. Campus1,2*, G. Carta1, M.G. Cagetti2, M. Bossù3, S. Sale1, F. Cocco4, G. Conti2, M. Nardone5,
G. Sanna4, L. Strohmenger2, P. Lingström6, and Italian Experimental Group on Oral Health†

Abstract: A randomized clinical trial other two groups. High-viscosity GIC into different generations according to
was performed in schoolchildren (6-7 sealants increased the fluoride concen- the mechanism of polymerization/
yrs) to evaluate fluoride concentra- trations in interproximal fluid more composition. Glass-ionomer cements
tion in interproximal fluid after the than did a Resin-based sealant con- (GIC) are alternative sealant materials,
placement of 3 different sealants. The taining fluoride (ClinicalTrials.gov with inadequate retention, but release
sample consisted of 2,776 children NCT01588210). fluoride, contributing to prevention of
randomly divided: 926 in the high- caries (Seppä et al., 1993). High-viscosity
viscosity Glass-ionomer Cement group Key Words: pit and fissure sealants, glass-ionomer materials have been intro-
(GIC group), 923 in the fluoride Resin- fluorides, saliva, dental care for chil- duced to combine the fluoride release of
based group (fluoride-RB group), and dren, community dentistry, clinical GIC and the prolonged retention of Bis-
927 in the no-fluoride Resin-based trial. GMA (MenonPreetha et al., 2007).
group (RB group). In total, 2,640 chil- The preventive effect of protracted ele-
dren completed the trial. Sealants vations of fluoride levels (from 0.03 to 0.1
were applied following manufactur- Introduction ppm) in the mouth has been well-docu-
er’s instructions. Interproximal fluid mented (Featherstone, 1999). Interdental
samples were collected at baseline and Pit and fissure caries remains a significant surfaces are prone to caries, due to a
2, 7, and 21 days after application of oral health burden, increasing dramatically more acidogenic environment (Cagetti et
sealants, by insertion of a standard- in frequency in permanent molars of al., 2011); moreover, they are less influ-
ized paperpoint into the interproxi- children and youth between the ages of 11 enced by regular toothbrushing, and
mal mesial space of the sealed tooth and 19 yrs, and is principally blamed on plaque is more easily formed in these
for 15 seconds. Fluoride concentra- the lack of sealants (Ignelzi, 2010). areas (Aspiras et al., 2010). Regular inter-
tion was evaluated by means of a flu- Several randomized clinical trials proximal fluoride applications inhibit fur-
oride ion-selective electrode. At 2 days assessed the effectiveness of sealants ther demineralization of initial lesions in
after sealant application, fluoride con- (Jokovic and Locker, 2001; Mejàre et al., situ (Särner et al., 2005). Fluoride con-
centration was significantly higher in 2003; Ahovuo-Saloranta et al., 2009). centration is higher in interproximal
GIC and fluoride-RB groups compared Sealants are more effective than fluoride areas adjacent to fillings with GIC, where
with that in the RB group (p < .01). in preventing caries in pits and fissures plaque can act as a reservoir, producing a
Mean fluoride concentrations after 7 because of their one-time application significant preventive effect (Lennon et al.,
days were 2.54 (SE 0.68) ppm, 0.85 (SE and long-term retention (Mejàre 2007). The fluoride refill of GIC with flu-
0.26) ppm, and 0.53 (SE 0.11) ppm for et al., 2003; Kervanto-Seppälä et al., oride agents (fluoride toothpaste, mouth-
the three groups, respectively. After 21 2008; Hiiri et al., 2009). rinses) prevents the decrease of fluoride
days, fluoride concentration in the GIC The most-used sealants are the Resin- levels in saliva with time. The capacity of
group remained higher than that in the based sealants (RB) (Bis-GMA), divided GIC to be a fluoride reservoir depends on
DOI: 10.1177/0022034513484329. 1Department of Surgery, Microsurgery and Medical Science, School of Dentistry, University of Sassari, Italy; 2WHO Collaborating
Centre of Milan for Epidemiology and Community Dentistry, University of Milan, “S. Paolo Hospital”, Milan, Italy; 3School of Dentistry, Sapienza, University of Rome,
Italy; 4Department of Chemistry, University of Sassari, Italy; 5Italian Minister of Health General Office, Rome, Italy; 6Department of Cariology, Institute of Odontology, The
Sahlgrenska Academy, University of Gothenburg, Sweden; *corresponding author, gcampus@uniss.it
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
© International & American Associations for Dental Research

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 JDR Clinical Research Supplement July 2013

type and permeability of the filling mate- Figure.

rial, frequency of exposure to fluoride, as Flow chart of the study design.
well as the types and concentrations of
fluoride agents (Carvalho and Cury, 1999;
Han et al., 2002). Materials with a high ini-
tial capacity to release fluoride also have a
high capacity to act as fluoride reservoirs
(Gao and Smales, 2001).
The hypothesis for this study was that
the concentration of fluoride in interprox-
imal fluids would increase to a higher
degree after the use of high-viscosity GIC
as a pit and fissure sealant, compared
with a Resin-based (RB) sealant contain-
ing fluoride and a RB sealant without flu-
oride content. To validate this hypothe-
sis, we designed a randomized clinical
trial, including different sealants, and per-
formed it with schoolchildren.

Materials & Methods

Trial Design
The present study was carried out in
Sassari (Italy) under the supervision of
the Italian WHO Collaborating Centre for
Epidemiology and Community Dentistry
and lasted 30 mos, from January 2008
to June 2010. The study was part of a
preventive project (supported by the
Italian Minister of Health) aimed to assess
the prevalence of caries disease and
gingivitis in children aged 6 to 7 yrs and
to seal their first permanent molars.
The study was designed as a random-
ized clinical trial, approved by the Ethics
Committee of the University of Sassari
(n°128SS/2007), and registered at http://
www.clinicaltrial.gov.
Sample

The study sample was obtained from

children aged 6 to 7 yrs attending
primary schools in Sassari, the largest
district of northern Sardinia, with 127,893
inhabitants (http://www.istat.it). The
fluoride concentration in local tap water
is low (0.3 mg/L).
Power analysis was performed with
G*Power 3.1.3 for Apple, with analysis of
variance (ANOVA) for repeated measures,
within factors with an effect size of 0.06
and an error probability of 0.01; the num-
ber of participants for each group was
set at 792, with an actual power of 0.95.
The flow chart of the study is displayed
in the Fig.
Based on Italian National Institute for
Statistics data from 2008, the total number
of children aged 6 to 7 yrs living in the
area was 3,271. Parents/guardians were
contacted by mail to consent to their
child’s participation in the trial. All chil-
dren (3,271) were invited to participate,
and 2,975 (91.0%) agreed. Only chil-
dren with signed informed consent were
recruited and examined at school to eval-
uate the number of caries lesions accord-
ing to the DMFT(S)/dmft(s) index. Caries
was scored by two calibrated examiners
(SS, GiC). A hand-out about the child’s
oral condition and a request to include
the child in the study (to seal sound
first permanent molars) were submit-
ted to the parents/guardians. At the day

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 vol. 92 • suppl no. 1
of the dental examination, 54 children
were absent from school.
Inclusion and exclusion criteria were
evaluated according to an ad hoc pre-
pared questionnaire, completed by par-
ents together with the consent form.
Inclusion criteria were: year of birth 2000
to 2002, good general health, informed
consent signed by parents/guardians, and
presence of at least 2 sound permanent
first molars as soon after eruption as isola-
tion can be achieved.
Exclusion criteria were: carious and/
or demineralized lesions, hypomineral-
ized permanent molar, presence of filling
or sealant on the occlusal surface of the
first permanent molars, fixed orthodon-
tic appliances, and use of topical fluoride
agents except toothpaste. In total, 2,808
children were eligible for the study.
Of these, 2,776 children (93.3% of the
eligible sample) were enrolled in the
study. The participants, identified by
a serial number, were compiled into a
list. Randomization was performed (GC)
with Excel® 2003 (for Mac OsX), and
three groups were created: a first group
received fissure sealants with a high-
viscosity GIC (GIC, n = 926), a second
group was sealed with Resin-based seal-
ant containing fluoride (fluoride-RB, n =
923), and a third group received Resin-
based sealants without fluoride content
(RB, n = 927).
Each child received a fluoride tooth-
paste containing 1,450 ppm (Mentadent P,
Unilever Italy, Milan) to use twice a day
during the study period. Children were
instructed to brush teeth 3 times a day for
2 to 3 min after each of the 3 main meals;
flossing was not included.
Application of Sealants
Dental sealants were applied by five
dental hygienists in the Dental Clinic of
the University of Sassari. Before starting
the study, the hygienists underwent a
half-day course where instructions for
the 3 different materials were described
in detail. Sealing materials were: a
high-viscosity glass-ionomer cement
(GIC; KETAC™ MOLAR APLICAP, 3M
ESPE, Seefeld, Germany), a white
photopolymerizable Resin-based
sealant containing fluoride (fluoride-RB;
Helioseal F®, IvoclarVivadent AG,
Fürstentum, Liechtenstein), and a white
photopolymerizable Resin-based sealant
without fluoride (RB; Concise1930TM, 3M
ESPE, Seefeld, Germany).
Sealant materials were handled accord-
ing to manufacturer’s instructions.
Sealants were applied as follows: (1)
tooth isolation by means of cotton rolls
(Francis et al., 2008); (2) tooth-cleaning
with a rotary brush mounted on a low-
speed contra-angle with a prophylaxis
paste without fluoride; (3) enamel etch-
ing with 37% phosphoric acid (H3PO4)
for 30 sec, except for GIC, where the sur-
face was conditioned for 10 sec by the
application of Ketac Conditioner; (4) a
thorough rinse with water and drying;
(5) placement of a thin layer of sealant
material and curing; and (6) if necessary,
finally occlusion adjustment.
Interproximal Fluid Sampling
and Fluoride Evaluation
Samples of interproximal fluid (within
2 hrs after oral hygiene procedures) were
collected at 4 different times (Särner
et al., 2003): at baseline (T0) before
the sealant procedure and 2, 7, and 21
days after the sealant procedure (T1,
T2, and T3, respectively). Interproximal
fluid was collected by insertion of a
standardized (ISO no. 30) paperpoint
(Dentalica, Milan, Italy) into the mesial
interproximal space of the sealed tooth
for 15 sec. The paperpoint was then
placed in an Eppendorf® tube containing
5 mL of sterilized deionized water, after
which it was kept frozen at -80°C until
the analysis. Each sample was coded as
blue, red, or green, so that the person
conducting the analysis would not
know the group to which the samples
belonged.
Prior to analysis, the samples were kept
overnight at +4°C. Each sample was pre-
pared as follows: addition of 0.5 mL TISAB
III (Total Ionic Strength Adjustment Buffer)
to reach a pH 5, and shaking with a mag-
netic stirrer thermostatted at 25°C for 15
sec. A 100-mL quantity of the solution was
then used for the analysis.
Fluoride concentration was assessed
in an Orion model 96.09 fluoride ion-
selective electrode and an Orion model
JDR Clinical Research Supplement
900200 double-junction plastic body Ag/
AgCl reference electrode with an Orion
model 290 mV digital meter, with end-
to-end method (Brambilla et al., 1998).
All measurements were made in triplicate
and expressed as mean ± standard error.
Statistical Analysis
Data from the questionnaire were
entered into a database, FileMaker
Pro 5.0 Runtime, and then put into
an Excel® Microsoft spreadsheet. The
socio-economic status of the family was
categorized according to the SocFam
scale (Campus et al., 2007) as medium-
low and medium levels. Systemic fluoride
use via tablets/drops was categorized as
‘yes’ when children stated use for > 1 yr.
Differences in baseline demographics
and clinical characteristics among groups
were compared by c2 test with continuity
correction or by one-way ANOVA if nec-
essary (dmfs index).
To normalize the data, we logarithmi-
cally transformed fluoride concentra-
tions prior to analysis. Mean and standard
error were calculated for each product
and time-point. Fluoride concentrations
are expressed at participant level as the
mean of the fluoride concentrations mea-
sured in all teeth sealed in each partici-
pant. The mean numbers of teeth sealed
during the survey are shown in Table 1.
Data were analyzed for statistically signif-
icant differences in a mixed-effect model,
REML (REsidual Maximum Likelihood)
regression. P < .01 was considered sta-
tistically significant. All data were ana-
lyzed STATA® software (Stata Corporation,
College Station, TX, USA, version 10.1 for
Macintosh, http://www.stata.com).
Results
Of the total of 2,640 children, 861
belonging to the GIC group, 878 to the
fluoride-RB group, and 901 to the RB
group completed the RCT. The drop-out
rate was 4.9% (136 children): 65 in the
GIC group, 45 in the fluoride-RB group,
and 26 in the RB group.
Similar gender, socio-economic sta-
tus, and systemic fluoride assumption
were observed (Table 1) among the three
groups (p = .91, p = .98, and p = .77,
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 JDR Clinical Research Supplement July 2013

Table 1.
Characteristics of the Three Groups at Baseline
Glass-ionomer Fluoride Resin- No-fluoride Resin- Statistical
Cement (GIC) based (fluoride- based (RB) Analysis
Groups n = 926 RB) n = 923 n = 927 p value*
Gender

 Male n (%) 462 (49.89) 458 (49.62) 455 (49.08)

 Female n (%) 464(50.11) 465 (50.38) 472 (50.92) .91

dmfs
  mean ± S.D. (median) 1.43 ± 3.09 (0) 1.48 ± 2.92 (0) 1.45 ± 2.97 (0) .64

ds
  mean ± S.D. (median) 1.38 ± 2.46 1.41 ± 2.42 1.38 ± 2.18 (0) .39

First molars sealed

  Lower jaw mean ± S.D. (median) 1.74 ± 0.26 (0) 1.68 ± 0.31 (0) 1.74 ± 0.26 (0) .70

  Upper jaw mean ± S.D. (median) 1.62 ± 0.21 (0) 1.60 ± 0.25 (0) 1.59 ± 0.25 (0) .71

Socio-economic Status

  Medium low n (%) 582 (62.85) 576 (62.40) 580 (62.57)

 Medium n (%) 344 (37.15) 347 (37.60) 347 (37.43) .98

Systemic fluoride use

 Yes n (%) 87 (9.40) 95 (10.29) 88 (9.49)

 No n (%) 839 (90.60) 828 (89.71) 839 (90.51) .77

* Comparisons among three groups were based on c2 test for proportions and ANOVA for means.

respectively). The majority of participants
(62.61%) belonged to the medium-low
socio-economic group, and only 9.73% of
the total sample confirmed having used
systemic fluoride. Caries experience was
low in all groups, with dmfs 1.43 ± 3.09 in
the GIC group, 1.48 ± 2.92 in the fluoride-
RB group, and 1.45 ± 2.97 in the RB group
(p = .64). The mean number of caries sur-
faces (ds) was similar in the three groups
(p = .39). The mean number of first molars
sealed during the trial was 1.74 in the
GIC group, 1.68 in the fluoride-RB group,
and 1.74 in the RB group in the mandibu-
lar jaw (p = .70), and 1.62, 1.60, and 1.59
in the maxillary jaw (p = .71) in the three
groups, respectively. Fewer sealants were
placed in the maxillary jaw compared with
the mandibular jaw.
Fluoride concentrations at T0 were sim-
ilar in the three groups (0.55 ppm in the
GIC and fluoride-RB groups, 0.53 ppm in
the RB group). At T1, statistically significant
differences (Table 2) were detected among
the three groups (p < .01); the fluoride con-
centration means were: 3.11 (SE0.33) ppm
fluoride in the GIC group, 1.23 (SE 0.49)
ppm in the fluoride-RB group, and 0.71 (SE
0.43) ppm in the RB group (GIC > fluoride
RB > no-fluoride RB). At T2, differences
(p = .03) in fluoride concentration means
were still evident: 2.54 (SE 0.68) ppm in the
GIC group, 0.85 (SE 0.26) ppm in the fluo-
ride-RB group, and 0.53 (SE 0.11) ppm in
the RB group. The fluoride released from
GIC decreased at T3 of about 50%, but still
remained higher compared with that from
the other groups.
Within the three groups, a statistically
significant difference in fluoride concen-
tration was found (p = .01).The individual
range of fluoride concentration measured
in the three groups was between 0.1 and
2.81 ppm.
Discussion
The study aimed to verify the fluoride
concentration in the interproximal
fluid after the application of 3 different
sealants: a high-viscosity GIC, a
fluoride Resin-based (RB) sealant, and a
no-fluoride RB sealant. Although sealants
were applied on the occlusal surface, we
used the interproximal area to analyze
the fluoride-releasing effect. At baseline,
the mean fluoride concentration was

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Table 2.
Concentration of Fluoride (ppm) in Interproximal Fluid over Time in the Three Groups
Groups Glass-ionomer Cement Fluoride Resin-based No-fluoride Resin-based
(GIC) mean (SE) value (fluoride-RB) (RB) mean (SE) value in
Times in ppm mean (SE) value in ppm ppm p value*

T0 0.55 (0.11) 0.55 (0.06) 0.53 (0.07) .36

T1 3.11 (0.33) 1.23 (0.49) 0.71 (0.43) < .01

T2 2.54 (0.68) 0.85 (0.26) 0.53 (0.11) .03

T3 1.21 (0.28) 0.75 (0.25) 0.55 (0.19) .39

Constant .02

*A mixed-effect model, REML (REsidual Maximum Likelihood).

Log-restricted likelihood = -130.26 c2 = 17.13, p < .01.

Random-effects Parameters Estimate (SE) 95% Confidence Interval

Group: Independent

SD (individual) 0.02 (0.02) 0.01 / 0.20

SD (constant) 0.24(0.25) 0.14 / 0.43
SD (residual) 0.44 (0.03) 0.40/ 0.50

similar to those reported in the literature
(Naumova et al., 2012).
Fluoride concentration from the GIC
group was significantly higher compared
with that from the other two groups. It is
well-known that fluoride promotes rem-
ineralization (ten Cate, 1999), and inhibits
bacterial acid production in vitro (Jenkins,
1999) and in vivo (Vogel et al., 2002).
The presence of fluoride in the oral fluids
plays a major role in combating enamel
dissolution. Fluoride concentration is
higher in interproximal areas adjacent to
fillings with GIC, acting as a reservoir and
producing a significant preventive effect
(Lennon et al., 2007). According to the
present study, high-viscosity GIC demon-
strated the capacity to increase and main-
tain high fluoride levels in the interproxi-
mal fluids, showing the capacity to act as
a fluoride reservoir. Fluoride release from
GICs and Resin-based materials is a com-
plex process that is not fully understood.
In GICs, there is an acid-base reaction
resulting in a F-polysalt matrix. In fluoride
Resin-based materials, the source of fluo-
ride would come from the glass filler par-
ticles, resulting in a slow diffusive release
(Chan et al., 2006). It is difficult to deter-
mine if fluoride was leaching or recharg-
ing. Only the fluoride concentration in the
interproximal fluids was recorded, influ-
enced by the fluoride release. For GIC, flu-
oride release occurs by means of 3 mech-
anisms: surface loss, diffusion through
pores and cracks, and bulk diffusion.
Components of saliva, acquired pellicle,
pH, ion concentration, and temperature
might decrease the fluoride diffusion from
the restorative materials (Markovic et al.,
2008), but these factors were not analyzed
in this study, nor was the addition of other
fluoride sources such as toothpaste or diet.
This study benefits from several
strengths, including its design (three-arm
study) and the large sample size. The sam-
ple came from an age group (6-7 yrs) dur-
ing which the highest risk of occlusal car-
ies of the first molar exists (Carvalho et
al., 1989). However, during the erup-
tion period, it is difficult to reach the ade-
quate moisture control needed during
sealant application (Baseggio et al., 2010).
High-viscosity GIC seemed an interest-
ing alternative, because they tolerate some
humidity (Baseggio et al., 2010). Since the
retention rate influences the caries rate
(Kervanto-Seppälä et al., 2008), evidence
is accumulating that high-viscosity GIC
and Resin-based sealants may be equally
effective in preventing lesion development
(Beiruti et al., 2006).
In this study, no comparison about the
retention rates of the 3 types of seal-
ants was carried out, because of the short
experimental period; nevertheless, after 3
wks, all sealants were still in place. A lon-
ger follow-up period would have been
beneficial. Many authors considered GIC an
unsuitable material for sealing pits and fis-
sures because of their low fracture strength
and high wear, but their physical charac-
teristics have clearly improved with the
introduction of the high-viscosity material
(Frencken and Wolke, 2010). Even when
it has clinically disappeared, glass-ionomer
material may still be present in the deeper
part of the pit and fissure system, continu-
ing to exercise a caries-preventive effect
(Frencken and Wolke, 2010).
Conclusion
High-viscosity glass-ionomer cements,
as pit and fissure sealants, increase the
interproximal fluoride concentration more
than do Resin-based fluoride-containing

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 JDR Clinical Research Supplement
sealants, representing an effective
intervention for increasing fluoride in the
oral environment.
Acknowledgments
†Italian Experimental Group on Oral
Health: M. Bossù, G. Campus, M.G. Cagetti,
G.P. Caria, G. Carta, F. Cocco, G. Congiu,
G. Conti, D. Corridone, P. Lingström, S.
Mastrobernardino, A. Mura, A. Polimeni,
S. Sale, G. Sanna, and L. Strohmenger. The
authors thank the manager of the Sassari
school district and all the teaching staff. The
authors received no financial support and
declare no potential conflicts of interest
with respect to the authorship and/or
publication of this article.
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