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Journal of Dental Research
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DOI: 10.1177/154405910708600715
 2007 86: 662J DENT RES
S. Paris, H. Meyer-Lueckel and A.M. Kielbassa
Resin Infiltration of Natural Caries Lesions
 
 
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INTRODUCTION
During the development of subsurface caries lesions, mineral is dissolved
out of the enamel, resulting in increased porosities that appear clinically
as the so-called 'white-spot' lesions (Ten Cate et al., 2003). Today, these
lesions are commonly treated by enhancing remineralization, e.g., by
improving the individual's oral hygiene or fluoridation. However, in non-
compliant individuals with cavitated proximal lesions and greater lesion
extension, this strategy has considerable limitations.
A promising alternative therapy for the arrest of caries lesions might be
the infiltration of subsurface lesions with low-viscous light-curing resins.
Since porosities of enamel caries act as diffusion pathways for acids and
dissolved minerals, infiltration of these lesions with resins might occlude the
pathways, thus leading to an arrest of caries progression.
Several studies have demonstrated that artificial caries lesions can be
infiltrated by commercially available adhesives and fissure sealants (Davila
et al., 1975; Robinson et al., 2001; Schmidlin et al., 2004; Meyer-Lueckel et
al., 2006). Moreover, it has been shown that infiltrated artificial lesions do
not progress in a cariogenic environment (Mueller et al., 2006; Paris et al.,
2006). Thus far, only two in vitro studies have addressed the infiltration of
natural lesions. However, these early reports were mainly descriptive
(Davila et al., 1975), or used materials which were not clinically applicable
due to their unsanitary nature (Robinson et al., 1976). Since there are
substantial structural differences between both lesion types, it is not
applicable to transfer findings from artificial to natural lesions.
The surface layer of enamel caries lesions has a lower pore volume
compared with that of the lesion body underneath (Bergman and Lind, 1966;
Silverstone, 1973). Since the infiltration of enamel caries with light-curing
resins is mainly driven by capillary forces, the pore diameter and volume
influence the penetration speed (Paris et al., 2007). Therefore, the surface
layer forms a barrier, which might hamper the infiltration of the lesion body.
From this follows that removing or perforating the surface layer might be
essential for a successful infiltration of the lesion body. In artificial lesions,
brief etching with 37% phosphoric acid enhanced resin penetration (Gray
and Shellis, 2002). With thicker and more mineralized surface layers in
natural lesions (Bergman and Lind, 1966), it was assumed that this etching
procedure would not be effective in eroding the surface layer (Meyer-
Lueckel et al., 2007). The latter study confirmed that etching with 15%
hydrochloric acid gel leads to a more effective erosion of the surface layer
compared with 37% phosphoric acid gel, but did not focus on the
subsequent infiltration of resins into the lesions.
Therefore, the aim of the present study was to evaluate the penetration
of a commercial adhesive into natural proximal caries lesions, without pre-
treatment and with prior conditioning by two different etching gels in vitro.
The working hypotheses were:
(Hypothesis 1) The surface layer of natural un-cavitated caries is a
diffusion barrier, which hampers the penetration of resin. Therefore, no
ABSTRACT
Infiltration of non-cavitated caries lesions with
light-curing resins could lead to an arrest of lesion
progression. The aim of this study was to evaluate
the penetration of a conventional adhesive into
natural enamel caries after pre-treatment with two
different etching gels in vitro. Extracted human
molars and premolars showing proximal white-
spot lesions were cut across the lesions
perpendicular to the surface. Corresponding lesion
halves were etched for 120 sec with either 37%
phosphoric acid gel (H
3
PO
4
) or 15% hydrochloric
acid gel (HCl), and subsequently infiltrated with
an adhesive. Specimens were observed by
confocal micro scopy. Mean penetration depths
(SD) in the HCl group [58 (37) �m] were
significantly increased compared with those of the
H
3
PO
4
group [18 (11) �m] (psolution until
used. Teeth were examined by 20
stereo microscopy (Stemi SV 11; Carl
Zeiss, Oberkochen, Germany), and
cavitated as well as damaged lesions
were excluded.
For radiographic examination,
teeth were positioned in a silicone base
with the buccal aspects facing a
radiographic tube (Heliodent MD;
Siemens, Bensheim, Germany). To
simulate cheek scatter, we placed a 15-
mm wall of clear Perspex between the
tube and the teeth. Standardized
radiographs (0.12 sec, 60 kV, 7.5 mA)
were taken of each tooth (Ektaspeed;
Kodak, Stuttgart, Germany) and
developed in an automatic processor
(XR 24-II; Dürr Dental, Bietigheim-
Bissingen, Germany). The radiographic
lesion depths were independently
assessed by two examiners and scored
(Marthaler and Germann, 1970): no
translucency (R0), translucency
confined to the outer half on enamel
(R1), translucency confined to the inner
half of enamel (R2), translucency
confined to the outer half of dentin
(R3), or translucency confined to the
inner half of dentin (R4). In case of
disagreement in an assessment of
radiographic lesion depth, a consensus
rank was reached.
The roots of the teeth were
removed, and the crowns were cut
across the caries lesions perpendicular
to the surface (Band Saw; Exakt
Apparatebau, Norderstedt, Germany),
providing two halves of each lesion
(Figs. 1a, 1b). Subsequently, the cut surfaces were examined
(stereo microscope, 20 ; Stemi SV 11) and classified with respect
to the histological lesion extension, according to the radiological
grading (Marthaler and Germann, 1970): C1, extension into the
outer half of enamel; C2, extension into the inner half of enamel;
or C3, extension into the outer half of dentin. Lesions extending
into the inner half of dentin (C4) were excluded. Corresponding
lesion halves showing the same grading level (C1-C3) in
Figure 1. Representative images of a lesion treated with the adhesive. (A) Clinical aspect of the mesial
surface of a human molar showing a white-spot lesion (dotted line). The lesion was cut in two halves
along the dashed line. (B) Aspect of the cut surfaces of the same enamel lesion. (C-E) Confocal
microscopic images of resin-infiltrated lesions (E, sound enamel; SL, surface layer; LB, lesion body; R,
penetrated resin; S, lesion surface). (C) Deep resin penetration can be observed after etching with
HCl. (D) The surface layer of this H3PO4-etched caries lesion was not eroded completely. Therefore,
only superficial resin penetration occurred, as indicated by a fine rim of red fluorescence at the tooth
surface. (E) Magnified image of an HCl-etched lesion (40x objective). The outermost 50-100 �m of
prism cores are filled with resin. In non-infiltrated parts of the lesion body, the highly porous prism
centers show green fluorescence.
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664 Paris et al. J Dent Res 86(7) 2007
histological caries extension were assigned to the treatment (TRT)
group (n = 10 each). In case the corresponding lesion halves
differed in lesion extension, the deeper lesion was used as the
control (CTR; n = 10), and the remaining half was disposed of.
Subsequently, the cut surfaces were covered with nail varnish.
In the treatment (TRT) group, corresponding lesion halves were
etched either with 37% phosphoric acid gel (H
3
PO
4
; total etch;
IvoclarVivadent, Schaan, Liechtenstein), or with an experimental
15% hydrochloric acid gel (HCl). The HCl gel contained
hydrochloric acid 15%, glycerol 19%, highly dispersed silicon
dioxide 8%, and methylene blue 0.01% in aqueous solution. After
120 sec, the gels were rinsed thoroughly with water spray for 30
sec. In the control (CTR) group, no acid etching was performed.
Lesions were immersed in pure ethanol for 30 sec and
subsequently dried for 60 sec with oil-free compressed air.
A dental adhesive (Excite; IvoclarVivadent, Schaan,
Liechtenstein) labeled with 0.1% tetramethylrhodamine
isothiocyanate (TRITC; Sigma Aldrich, Steinheim, Germany) was
applied to the lesion surfaces. The resin was allowed to penetrate
the lesions for 5 min. Subsequently, excess material was removed
by means of cotton pellets, and the resin was light-cured for 30 sec
(Translux CL; Heraeus Kulzer, Hanau, Germany) at 400 mW/cm2.
The nail varnish was carefully removed, and specimen halves were
fixed on object holders parallel to the cut surface and polished
(Exakt Mikroschleifsystem, Abrasive Paper 2400, 4000; Exakt
Apparatebau, Norderstedt, Germany). To stain remaining pores,
we immersed the specimens in 50% ethanol solution containing
100 �M/L sodium fluorescein (Sigma Aldrich) for 3 hrs.
Specimens were observed by confocal laser scanning
microscopy (CLSM Leica TCS NT; Leica, Heidelberg, Germany)
in dual-fluorescence mode and with a 10x objective. The excitation
light had two wavelength maxima, at 488 and 568 nm. The emitted
light was split by a 580-nm reflection short-pass filter and passed
through a 525/50-nm band-pass filter for FITC and a 590-nm long-
pass filter for RITC detection. Images with a lateral dimension of
1000 x 1000 �m2 and a resolution of 1024 x 1024 pixels were
recorded and analyzed by AxioVision LE software (Zeiss,
Oberkochen, Germany). Penetration depths and thicknesses of the
(residual) surface layer for the lesion halves were measured at up
to 10 defined points (depending on the lesion size; indicated by a
100-�m grit), and mean values were calculated. Additionally to
CLSM analysis, acid-etched as well as infiltrated lesion surfaces
were observed by scanning electron microscopy (APPENDIX).
Statistical analysis was performed with SPSS software (SPSS
for Windows 11.5.1; SPSS, Chicago, IL, USA). Data were checked
for normal distribution by the Kolmogorov-Smirnov test. To
analyze differences in penetration depth between lesion halves/acid
gels, we used the Wilcoxon test for paired samples. For
comparison between unpaired groups, we performed Mann-
Whitney and Kruskal-Wallis tests. Penetration depths were
analyzed with regard to possible differences between various
histological lesion extensions (C1-C3) and radiological grades
(R0-R3). The level of significance was set at 5%.
RESULTS
In the CLSM images, the penetrated resin showed a red
fluorescence, whereas remaining pores within the lesion, as
well as dentin, appeared green (Figs. 1c-1e). Solid material,
such as sound enamel or the surface layer, was displayed black.
Penetration depths varied considerably. For lesion halves
etched with HCl gel, the mean penetration depth (standard
deviation) [58 (37) �m] was significantly higher compared
with that of those lesions treated with H
3
PO
4
gel [18 (11) �m]
(p 0.05; Kruskal-
Wallis).
For radiological grading of lesion extensions, good inter-
observer agreement could be found (� = 0.804). Similar to
histological lesion extension (C1-C3), no significant
differences in penetration depth could be observed among
different radiological grades (R0-R3) (Table).
For those lesions where the surface layer was completely
removed (CTR, n = 0; H
3
PO
4
, n = 2; HCl, n = 8), significantly
higher (p 0.05; Mann-Whitney).
DISCUSSION
In previous studieswhere confocal microscopy was used, resin
penetration was visualized by labeling of the resin with
Figure 2. Mean penetration depths of resin for various pre-treatments
and histological lesion extensions (box and whisker plots with quartiles
and medians; n = 10 per group). Statistically significant differences
between groups are indicated with asterisks (*pto
Mrs. Anja Bartels and Mrs. Julia Heinrich (Dept. of Operative
Dentistry and Periodontology, CBF, Charité) for their excellent
contributions to the experiments, to Dr. Herbert Renz (Dept. of
Experimental Dentistry, CBF, Charité) for his assistance with
the SEM, and to Prof. Dr. Harald Stein (Institute for Pathology,
CBF, Charité) for providing the CLSM.
The Charité-Universitätsmedizin Berlin holds US
(US10/432,271) and European (EP06021966.4) patent
applications for an infiltration technique for dental caries lesions
in which the authors of this study are appointed as inventors.
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