journal of dentistry 39 (2011) 465–469

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journal homepage: www.intl.elsevierhealth.com/journals/jden

Influence of application time on penetration of an infiltrant

into natural enamel caries

Hendrik Meyer-Lueckel a, Andreas Chatzidakis b, Michael Naumann c,

Christof E. Dörfer a, Sebastian Paris a,*
a
Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Christian-Albrechts-Universität zu Kiel, Germany
b
Dept. of Prosthetic Dentistry, School of Dental Medicine, CharitéCentrum 3, Charité - Universitätsmedizin Berlin, Germany
c
Dept. of Prosthetic Dentistry, Center of Dentistry, University of Ulm, Germany

article info abstract

Article history: Objective: Caries infiltration is an innovative approach to treat medium stages of caries that
Received 19 January 2011 bridges the gap between preventive and invasive measures, whereby hard tissues are
Received in revised form preserved. Special low viscosity resins (infiltrants) showed almost complete penetration
8 April 2011 into natural lesions when applied for 5 min. Since shorter application times seem to be
Accepted 12 April 2011 clinically more feasible, the aim of this in vitro study was to compare the penetration of an
infiltrant (Icon pre-product; DMG, Hamburg, Germany) into natural caries lesions after
various application times.
Keywords: Methods: Extracted permanent human posterior teeth showing non-cavitated proximal
Caries caries lesions were infiltrated for either 0.5, 1, 3, or 5 min (n = 20) and light-cured. Specimens
Infiltration were prepared and lesion (LD) as well as penetration depths (PD) were analysed using dual
Infiltrant fluorescence confocal microscopy.
Hydrochloric acid Results: PD [median (Q25;Q75)] at maximum LD after 0.5 min [159 (27;340) mm] and 1 min
CLSM [152 (69;375) mm] were significantly lower compared to those after 3 min [414 (338, 518) mm]
and 5 min [407 (332;616) mm] ( p < 0.05). Deep lesion parts (PD > 500 mm) could be penetrated
almost completely after 3 min [98 (88;100)%] and 5 min [100 (81;100)%] application.
Conclusions: Thus, 3 min application of an infiltrant seems to be sufficient to achieve an
almost complete penetration of enamel caries.
# 2011 Elsevier Ltd. All rights reserved.

1. Introduction
For non-cavitated, carious pits and fissures, sealing with light
curing resins is an effective method.1–3 Progression of non-
cavitated proximal lesions extending up to the outer third of
dentine has been shown to be reduced by the application of a
commercially available adhesive.4 However, only superficial
penetration of an adhesive can be expected after etching with
phosphoric acid.5 The low porous surface layer of enamel caries
lesions acts as a diffusion barrier.6,7 Removing or perforating the
surface layer by etching with 15% hydrochloric acid gel for 2 min
resulted in a deeper infiltration of adhesives into the surface
carious enamel and of low-viscosity light curing resins into the
lesion body.5,9 Since the porosities of enamel caries lesions act
as diffusion pathways for acids and dissolved minerals,
infiltration of these pores with resins occludes the pathways
and thus lesion progression is hampered or even arrested.10 As a
positive side effect the whitish appearance is at least partially
masked as observed clinically.11 This effect seems to be stable
even after further demineralisation in vitro.12

* Corresponding author at: Clinic for Conservative Dentistry and Periodontology, School for Dental Medicine, Christian Albrechts-
Universität zu Kiel, Arnold-Heller-Str. 3, Haus 26, 24105 Kiel, Germany. Tel.: +49 431 597 2817; fax: +49 431 597 4108.
E-mail address: paris@konspar.uni-kiel.de (S. Paris).
0300-5712/$ – see front matter # 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2011.04.003
466 journal of dentistry 39 (2011) 465–469
From a practical point of view, proximal infiltration seems to
be advantageous compared with proximal sealing, since only
minimal tooth separation in a single visit is required for the
infiltration technique.13 The abrasion stability of infiltrated
caries lesions seems to be similar to those of sound enamel.14
Recently, in a split-mouth randomised clinical trial, caries
infiltration was shown to be a clinically feasible and efficacious
to treat non-cavitated proximal caries lesions extending
radiographically into inner half of enamel up to outer third of
dentine. Within the follow-up interval of 18 months, 11% and
39% of the infiltrated and control lesions, respectively, showed
caries progression radiographically. In this study an application
time of the pre-product infiltrant (DMG, Hamburg, Germany) of
5 min was chosen.13 With this application time, a similar
infiltrant as used in the clinical study showed almost complete
penetration into natural caries lesions.9
Since shorter application times seem to be clinically more
feasible (e.g., for patients’ convenience), the aim of this in vitro
study was to compare the penetration depths of a pre-product
infiltrant into natural caries lesions after various application
times. We hypothesised that an application time of 5 min
results in significantly higher penetration depths compared to
shorter times.
2. Materials and methods
2.1. Specimens
Eighty extracted human molars and premolars showing
‘active’ (dull surface, chalky opacity) non-cavitated proximal
white spot caries lesions (ICDAS code 2)15 were selected for
this study. Teeth were examined using a 20 stereo micro-
scope (Stemi SV 11; Carl Zeiss, Oberkochen, Germany) and
cavitated as well as damaged lesions were excluded. The study
protocol conformed to the principles outlined in the German
Ethics Committee’s statement for the use of human body
material in medical research.16 Roots were removed (Band
Saw 300cl; Exakt Apparatebau, Norderstedt, Germany), teeth
were carefully cleaned from soft tissues and stored in 0.1%
Thymol solution up to usage.
2.2. Treatment
Lesions were etched for 2 min using 15% hydrochloric acid gel
(Icon pre-product; DMG Hamburg). After drying, lesions were
stained with 0.1% ethanolic solution of tetramethylrhodamine
isothiocyanate (TRITC; Sigma–Aldrich, Steinheim, Germany)
for 12 h. Subsequently, specimens were dried using compressed
air for 10 s and an infiltrant (Icon pre-product; DMG), consisting
of triethylene glycol dimethacrylate (99%), initiators and
stabilisers was applied for either 0.5, 1, 3, or 5 min (n = 20) onto
the lesion surface. Excess material was wiped away using a
cotton roll and the resin was light cured for 60 s (530 mw/cm2,
Astralis 5, Ivoclar Vivadent, Schaan, Liechtenstein).
Teeth were sectioned perpendicular to the lesion surfaces
(Band Saw 300cl; Exakt Apparatebau) in order to obtain
specimens having lesion areas of 1 mm thickness. Unbound
red fluorophore dye was bleached by immersion in hydrogen
peroxide (30%) for 12 h at 37 8C. Subsequently, specimens were
washed with water for 60 s, fixed on object holders and
parallelised (Mikroschleifsystem 400 cs, Abrasive Paper 1200,
2400, 4000; Exakt Apparatebau). To label porous structures (i.e.
non-infiltrated lesion parts), specimens were immersed in a
50% ethanol solution of 100 mM sodium fluorescein (NaFl;
Sigma–Aldrich) for 3 min and subsequently washed in
deionised water for 10 s.
2.3. Confocal laser scanning microscopy (CLSM)
Specimens were observed with a confocal laser scanning
microscope (CLSM, Leica TCS NT; Leica, Heidelberg, Germany)
using a 10 objective in dual fluorescence mode as described
earlier.17 In confocal microscopic images lesion and penetration
depths of the resins were measured (ImageJ; NIH, Bethesda, MD,
USA) at up to nine defined points (depending on the lesion size;
indicated by a 100 mm grit). Penetration (PD) and lesion depths
(LD) were defined as the distance from the surface to the deepest
point of red and green fluorescence, respectively.
2.4. Statistical analyses
Statistical analysis was performed using SPSS software (SPSS
for Windows 11.5.1; SPSS Inc., Chicago, IL, USA). Penetration
depths (PD) at the deepest lesion sites (LDmax) were analysed.
To minimise the limiting influence of lesion depth on
penetration depth subgroup analyses for lesions having a
maximum lesion depth higher than 400 mm (PD400) as well as
500 mm (PD500) were performed. Percentage penetration was
calculated as PD/LDmax  100. These values were depicted and
compared for all lesion depths (PP) as well as for all those
lesion parts being deeper than 400 mm (PP400) as well as 500 mm
(PP500). We checked the assumption of normal distribution of
data (Shapiro–Wilk test). Differences in lesion and penetration
depths as well as percentage penetration were analysed using
Kruskal–Wallis and Mann–Whitney test. The level of signifi-
cance was set at 5% for all tests.
3. Results
Dual fluorescence images allowed analysis of the penetration
of the infiltrant and the remaining pore structures. Rather
superficial penetration could be observed after 0.5 min and
1 min application, whereas almost complete penetration of
the infiltrant could be observed after 3 min and 5 min
application (Fig. 1).
Median (25th percentile; 75th percentile) maximum lesion
depths (LDmax) were 479 (355; 651) mm for all lesions (n = 80)
evaluated. Values did not differ significantly between the
lesions of the four subgroups ( p = 0.907; Kruskal–Wallis test).
For lesions with LDmax > 400 mm (n = 51) and LDmax > 500 mm
(n = 38) medians of LDmax were 557 (489; 694) mm and 661 (525;
731) mm, respectively. No significant differences with respect
to LDmax between the four infiltrant groups were analysed
( p = 0.071 and p = 0.426).
Penetration depths (PD) at the deepest lesion site after
3 min and 5 min application were significantly higher com-
pared to those after 0.5 min and 1 min ( p < 0.05; Mann–
Whitney test). For PD400 and PD500 significantly higher values
 journal of dentistry 39 (2011) 465–469 467

Fig. 1 – Representative confocal laser scanning microscope images of lesions infiltrated. Porosities in enamel and dentine
are displayed green, whereas infiltrated lesion parts appear red. Solid material as sound enamel shows no fluorescence
and are displayed black. After 0.5 and 1 min application only superficial penetration could be observed. After 3 min and
5 min application the infiltrant penetrated more deeply.

Fig. 2 – Box plots of the percentage penetration (PD was measured at LDmax) after the four application times of the infiltrant
into all lesions evaluated (PP; dark) as well as into those lesion sites with LDmax > 400 mm (PP400; grey) or LDmax > 500 mm
(PP500; white). Statistically significant differences between application times are indicated with different letters ( p < 0.05;
Mann–Whitney test). N = number of teeth.

could be observed after 3 min and 5 min compared with

0.5 min and 1 min application ( p > 0.05) (Table 1). PP, PP400 and
PP500 were significantly higher after 3 min and 5 min com-
pared to 0.5 min and 1 min application ( p < 0.05; Mann–
Whitney test). No other significant differences between groups
were observed ( p > 0.05) (Fig. 2).
4. Discussion
This in vitro study shows that the tested infiltrant is capable to
penetrate several hundred micrometres into natural caries
lesions after an application time of at least 3 min, resulting in

Table 1 – Median (Q1, Q3) maximum lesion depths (LDmax) and penetration depths (PD) at maximum lesion depths after the
four different application times for various lesion extensions.
Application All lesions Lesions with LDmax > 400 mm Lesions with LDmax > 500 mm
time (min)
N LDmax PD N LDmax PD400 N LDmax PD500
A A A
0.5 20 523 (319;703) 159 (27;340) 11 694 (525;733) 66 (17;616) 11 694 (525;733) 66 (17;616)
1 20 447 (315;626) 152A (69;375) 12 579 (481;680) 135A (53;391) 9 633 (529;720) 148A (39;444)
3 20 471 (386;550) 414B (338;518) 14 503 (453;591) 460B (398;550) 8 562 (510;671) 537B (395;640)
5 20 487 (362;664) 407B (332;616) 14 574 (461;740) 477B (393;640) 10 662 (511;759) 615B (398;659)

p 0.907 0.001 0.071 0.003 0.426 0.010

p Values for the comparison between the four application times of each of the six variables are given (Kruskal–Wallis test). Significant
differences for penetration depths of all lesions (PD) as well as lesions with maximum lesion depths >400 mm (PD400) and lesion depths
>500 mm (PD500) within each of the three columns are depicted with different superscript letters. N = sample size per group/subset.
468 journal of dentistry 39 (2011) 465–469
relatively homogenous resin layers. The study hypothesis that
an application time of 5 min results in significantly higher
penetration depths compared to shorter times could only
partially be confirmed, since no significant difference was
observed between 5 and 3 min of application.
We aimed to select teeth with rather ‘active’ caries lesions.
However, some lesions might have been ‘inactive’, since
surface layers of more than 50 mm in thickness could be
observed, whereby resin penetration might have been
hampered. Moreover, teeth supposed to have lesions extend-
ing histologically at least into inner parts of enamel (ICDAS
code 2) were chosen and these were allocated randomly to the
groups. Nonetheless, this procedure resulted in the selection
of lesions varying in lesion depths between groups (not
significant) and of some rather shallow lesions. To overcome
the problem of shallow lesion depths limiting resin penetra-
tion, subgroup analyses evaluating only caries lesions with
maximum lesion depths of >400 mm or >500 mm were
performed. It was assumed that these depths should be
reached by an infiltrant to enable a sustainable seal within the
lesions.
For clinical treatment of proximal caries lesions specially
designed applicators (Icon, DMG) are available to apply the
acid and the infiltrant with only minimal tooth separation. In
the present study the agents were applied directly onto the
lesions without mimicking a proximal contact point situation
and using the applicators. Moreover, dryness could be more
easily accomplished compared with the clinical situation
where rubber dam is recommended to retract the gingival and
allow for proper moist control. Moreover, in vivo remnants of
biofilm as well as proteins within the lesions might hamper
penetration of infiltrants, as well.18,19 These factors could have
resulted in higher penetration depths of the infiltrant
compared with those that can be expected clinically. None-
theless, clinical studies suggest that the established infiltrant
layer within the caries lesions seems to be efficient to inhibit
lesion progression by occluding the pores for acid and
fermentable carbohydrate diffusion.13,20 In addition, when
placing restorations use of an infiltrant before application of
conventional adhesives might be beneficial; bond strengths to
enamel seem not to be impaired.21
In previous studies using confocal microscopy either
resins,22 remaining porous structures23 or both5 were labelled
with fluorescent dyes. Rhodamine and fluorescein derivates
allow selective visualisation due to well separated excitation
and emission wavelengths.24 However, ‘direct’ staining
techniques showed limitations with respect to the visualisa-
tion of the infiltrant most possibly due to chromatographic
separation of the resin–dye-mixture during penetration into
the lesion body. Therefore, a validated ‘indirect staining
technique was used in the present study.17
As previously,5,9,17 natural lesions were shown to be
penetrated rather slowly (approximately 300 mm/90 s) com-
pared to artificial ones (>300 mm/10 s).23 Insufficient etching of
the sometimes rather thick surface layers as well as
‘contamination’ with proteins seem to be some of the factors
involved.18,19 Nonetheless, the general capability of infiltrants
to penetrate rather deeply into natural lesions (after 3 min
application) has been confirmed in the present study.
Moreover, penetration depths were shown to increase with
longer application times. Therefore, the ‘Washburn equation’,
describing the time-dependent influence of material proper-
ties as viscosity, surface tension and contact angle to enamel
on penetration abilities into porous solids,25,26 that has been
appropriate as a theoretical background in artificial lesions23
seems also be applicable to explain penetravity in natural
lesions.
Compared to solvent-containing (ethanol), solvent-free
infiltrants having TEGDMA as the main constituent seem to
be superior with respect to penetration abilities5,9,23 and
inhibition of lesion progression.10 This observation has been
attributed to incomplete polymerisation due to poor evapora-
tion of the ethanol from deeper lesion parts as well as
interference with the polymerisation process.9 Therefore, a
solvent-free infiltrant has been used in the present study.
Percentage penetration depths, as measured in the former
studies when the indirect staining technique had been used,
could be confirmed.9 Previously reported percentage penetra-
tion depths of an infiltrant (TEGDMA 90% and ethanol 10%)5
measured after direct staining should have been higher, if the
‘indirect’ staining technique had been used.
5. Conclusion
It can be concluded that 3 min application of an infiltrant
seems to be sufficient to achieve an almost complete
penetration of natural caries lesions in vitro. Further studies
should aim to evaluate, if the application time might be
reduced to 2 min and, if these established resin layers are
capable to inhibit lesion progression.
Disclosure
This study was supported by the Deutsche Forschungsge-
meinschaft (DFG; PA 1508/1-1). HML and SP receive a research
grant and royalties from DMG, Hamburg.
Acknowledgements
The authors are indebted Mr. Michael Stiebritz for his most
valuable contribution to this study and to Prof. Dr. H. Stein,
Institute for Pathology, Charité – Universitätsmedizin Berlin
for providing the CLSM. The study was partly conducted at the
Dental School at Charité – Universitätsmedizin Berlin, which is
hereby acknowledged.
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