Received: 9 August 2019 Revised: 21 October 2019 Accepted: 5 November 2019

DOI: 10.1111/jerd.12550

RESEARCH ARTICLE

Resin infiltration of enamel white spot lesions: An

ultramorphological analysis

Jorge Perdig~ao DMD, MS, PhD

Department of Restorative Sciences,
University of Minnesota, Minneapolis,
Minnesota
Correspondence
Jorge Perdig~ao, Department of Restorative
Sciences, University of Minnesota, 515 SE
Delaware St, 8-450 Moos Tower, Minneapolis,
MN 55455.
Email: perdi001@umn.edu
Abstract
Objective: There is not a clear understanding of the ultramorphology of enamel white
spot lesions (WSLs). The purpose of this study is to characterize resin infiltration of
enamel WSLs using electron microscopy.
Materials and methods: Enamel sections with sound enamel and WSLs were sec-
tioned from extracted teeth and assigned to three groups: (a) left untreated;
(b) etched with 15% hydrochloric acid (Icon-Etch); (c) restored with the resin infiltra-
tion sequence (Icon-Etch, Icon-Dry, and Icon-Infiltrant). Restored specimens were
demineralized to obtain replicas. Observations were carried out under a field-
emission scanning electron microscope.
Results: Icon-Etch resulted in an array of pits and funneled holes on the WSL. Rep-
licas of WSLs depicted 0.5-6.0-μm-thick shaggy resin tags up to a depth of 465 μm.
Enamel crystallites were enveloped with resin at the bottom of the WSL forming a
hybrid layer.
Conclusions: The resin infiltrant filled the spaces between the crystallites and
resulted in an enamel hybrid layer.
Clinical significance: In addition to masking enamel WSLs, resin infiltration is able
envelop residual enamel crystallites forming an enamel hybrid layer. This hybridiza-
tion makes resin-embedded enamel more resistant to acid attack than sound enamel.

KEYWORDS
acid-etching, dental adhesion, electron microscopy, resin infiltration, white spot lesion

1 | I N T RO D UC T I O N In 1908, G.V. Black described enamel WSLs as “occasional white

Current minimally invasive concepts in Operative Dentistry are
focused on the control of the etiological factors using noninvasive and
microinvasive strategies. While noninvasive strategies aim at arresting
or reverting noncavitated enamel caries lesions, microinvasive strate-
gies include barriers that prevent further dissolution of enamel by the
acidic challenge from cariogenic bacteria. Among the microinvasive
strategies, two procedures are currently used: (a) pit-and-fissure seal-
ants applied onto phosphoric acid-etched enamel and (b) low-viscosity
resins that permeate or infiltrate into hydrochloric acid (HCl)-etched
noncavitated enamel lesions (or white spot lesions [WSLs]) by capil-
1,2
lary action.
or ashy gray spots that were small and covered with the ordinary
glazed surface of the enamel, so that an exploring tine will glide over
them the same as over the perfect enamel.”3 The pioneer work of
Applebaum in 1932 with polarized microscopy, later confirmed by
Silverstone with polarized microscopy and microradiography, reported
that the surface of these early enamel lesions remained relatively
unaltered, displaying an apparently intact outer layer.4-7 Silverstone
also described a porous demineralized area underneath the external
(pseudo) intact layer known as body of the lesion.1,6,7 Further work by
Robinson and coworkers reported that the body of the lesion con-
tained 25%-50% porosity.8

J Esthet Restor Dent. 2019;1–8. wileyonlinelibrary.com/journal/jerd © 2019 Wiley Periodicals, Inc. 1

2 PERDIGÃO

The prevalence of enamel WSLs has increased over the last few application of a low-viscosity light-cured resin (tetraethylene glycol
decades as a result of increased susceptibility to enamel demineraliza- dimethacrylate [TEGDMA]).17,18 Studies have confirmed the effec-
tion during orthodontic treatment, as appliances make oral hygiene tiveness of the technique clinically and in vitro, not only as an esthetic
9
more challenging. In addition, excessive surplus etching of the labial treatment but also as microinvasive cariostatic procedure.16-22 This
enamel surface when placing orthodontic-fixed appliances causes iat- resin infiltration technique has also been shown to be more effective
rogenic WSLs.10 The incidence of postorthodontic WSLs has been than fluoride or amorphous calcium phosphate in improving the
11
reported to be as high as 96%. esthetic appearance of WSLs.23
Clinically, WSLs form as early as 2 weeks after plaque accumula- Resin infiltration into the porosities of WSLs has been character-
tion.12 Remineralization of WSLs may occur predominantly in the first ized using microscopy methods, especially confocal microscopy.19,24
6 months after removal of orthodontic appliances, with reduction of Nevertheless, there is not a clear understanding of the ultra-
the original WSL size to up to 50%.13 Lesions that are still present morphology inside WSLs after HCL etching, as well as after resin infil-
after 6 months are likely to remain without any further size reduction, tration. Therefore, the objective of this study is to characterize resin
becoming an esthetic concern for young patients. Compared to sound infiltration of sound enamel (control) and WSLs using field-emission
enamel, WSLs are clinically white and opaque, often observed on the scanning electron microscopy (FESEM).
labial surface of anterior teeth. In posterior teeth WSLs develop in the
interproximal contact area as incipient class II lesions.
Hydroxyapatite in sound enamel has a refractive index (RI) of
1.62.14,15 When a WSL is hydrated with saliva, the RI of saliva within
the enamel porosities is 1.33. This discrepancy in RI between saliva
and hydroxyapatite affects light scattering and makes the WSL look
slightly opaque (Figure 1). When teeth are dried, saliva is replaced
with air (RI = 1.0) within the WSL porosities. The difference in RI
between air and hydroxyapatite is wider than that between saliva and
hydroxyapatite, making the WSL more evident in dehydrated teeth
(Figure 2).14,15
The ability of replacing air in the demineralized enamel of WSLs
with a material with an RI similar to that of hydroxyapatite, such as a
methacrylate resin, has been shown to mask the WSL by preventing
light from scattering inside the WSL.14 (Figure 3). In addition, filling
the porosities with an adhesive resin reinforces the unsupported
enamel crystallites in the body of the WSL.1 This reinforced enamel
F I G U R E 2 WSLs became more evident after tooth dehydration.
becomes mechanically stronger and more resistant to acid dissolu-
The image of dehydrated WSLs is very helpful to determine the
tion.1,16 Both enamel structural reinforcement and cariostatic proper- extension of the enamel to be infiltrated with resin. WSLs, white spot
ties have been accomplished by infiltrating enamel WSLs using 15% lesions
HCl etching to make the mineralized surface layer more porous,
followed by a drying step with ethanol to remove excess water, and

F I G U R E 3 The WSLs were effectively masked after treatment

F I G U R E 1 Clinical view of a 22-year-old patient with WSLs in the with resin infiltration (Icon, Hamburg, Germany) in one session. WSLs,
maxillary anterior teeth. WSLs, white spot lesions white spot lesions
PERDIGÃO 3

2 | MATERIALS AND METHODS • Group 1—untreated:

Nine unidentified extracted intact teeth (seven maxillary premolars and
two mandibular molars) with proximal (premolars) and buccal (molars)
WSLs, as well as sound enamel, were selected for this study after
refrigeration in 0.5% chloramine T solution for up to 6 months. Teeth
had been extracted for orthodontic or surgical reasons and were exam-
ined under a stereo microscope to exclude cavitated lesions and areas
with damaged enamel. Crowns were sectioned parallel to the occlusal
surface 1 mm below the cementoenamel junction (CEJ) using a water-
cooled slow-speed diamond saw (IsoMet 5000 Precision Saw; Buehler,
Lake Bluff, Illinois). The teeth were cleaned with pumice-water suspen-
sion using a cotton pellet to avoid damaging the WSL, and then left in
distilled water for 24 hours at room temperature prior to use.
Enamel blocks of the area of interest with dimensions of
6 mm × 3 mm × 2 mm, which included sound enamel or enamel with
WSL, were sectioned from the teeth using the same water-cooled
slow-speed diamond saw.
The specimens were distributed into three groups of six enamel
sections. Compositions and detailed instructions for use are described
in Table 1:
Enamel specimens with sound enamel and with WSLs were left
untreated.
• Group 2—etched with 15% HCl (Icon-Etch; DMG, Hamburg, Ger-
many):
Enamel specimens with sound enamel and with WSLs were etched
with Icon-Etch (15% HCl; DMG), rinsed, and air-dried (Table 1). All
specimens were etched for 2 minutes without extra etching steps.
• Group 3—restored with the resin infiltration technique:
Enamel specimens with sound enamel and with WSLs were etched
with Icon-Etch (DMG), rinsed, and air-dried, followed by the appli-
cation of Icon-Dry (DMG) (Table 1). All specimens were etched for
2 minutes without the need for further etching steps. Icon-
Infiltrant (DMG) was applied (Table 1) and polymerized with an
Elipar S10 (3M Oral Care, St. Paul, Minnesota) light-curing unit.
Filtek Supreme Ultra Flowable Restorative shade A2 (3M Oral
Care) was then added to the surface of the cured Icon-Infiltrant to
facilitate handling and specimen preparation for FESEM, followed
by light-curing. These restored specimens in group 3 were further
decalcified in 6 N HCl for 24 hours25 to dissolve enamel and obtain

TABLE 1 Composition of materials used in this study and instructions for use

Commercial name (manufacturer) Composition Instructions for use

Icon-Etch (DMG) 15% hydrochloric acid, water, pyrogenic
silica, surfactant, pigments
Icon-Dry (DMG) Ethanol
Icon-Infiltrant (DMG) TEGDMA-based resin, initiators and
stabilizers
Filtek Supreme Ultra flowable Restorative TEGDMA, BisGMA, silane-treated ceramic,
(3M Oral Care) silane-treated silica, ytterbium fluoride,
diphenyliodonium hexafluorophosphate,
reacted polycaprolactone polymer,
substituted dimethacrylate
• Apply the gel and leave it for 2 min
• Remove excess material with a
cotton roll
• Rinse with water for 30 s
• Dry with oil-free and water-free air
• Apply an ample amount of material and
let it set for 30 s
• Dry with oil-free and water-free air
• When wetted with Icon-Dry, the
whitish-opaque coloration on the etched
enamel should diminish. If this is not the
case repeat the etching step once or
twice for 2 min each, and rinse and dry
the teeth again as above
• Apply an ample amount of Icon-Infiltrant
onto the etched surface
• Let Icon-Infiltrant set for 3 min
• Remove excess material with a cotton
roll and dental floss
• Light-cure Icon-Infiltrant for 40 s
• Screw a new Smooth Surface-Tip onto
the Icon-Infiltrant syringe, repeat the
application and let set for 1 min
• Remove excess material with a cotton
roll and dental floss, and light-cure for a
minimum of 40 s
• An increment of 2 mm was applied and
light-cured for 40 s

Abbreviations: BisGMA, bisphenol A diglycidyl methacrylate; TEGDMA, tetraethylene glycol dimethacrylate.

4 PERDIGÃO
a replica of the restored enamel surfaces, followed by rinsing with
distilled water for 5 minutes.
After drying in a vacuum desiccator for 24 hours, all specimens
were mounted on aluminum stubs with carbon adhesive tape and
F I G U R E 5 A, Micrograph of a cross-section of sound enamel etched with Icon-Etch. The depth of demineralization (arrows) varies between
0.9 and 2.0 μm. Original magnification: ×10 000. B, Micrograph of a cross section of a WSL etched with Icon-etch. The depth of demineralization
(arrows) varies between 2.1 and 92.1 μm (deepest area not shown in the micrograph). The dark arrows point to the boundary of the etched area.
Original magnification: ×10 000. WSL, white spot lesion
F I G U R E 4 A, Micrograph of unetched
sound enamel showing its aprismatic
structure and perikymata (asterisk).
Original magnification: ×5000. B,
Micrograph of unetched WSL with an
area coated with a particulate deposit
(P) and an area with exposed enamel
crystallites (E). Original magnification:
×5000X. C, Higher magnification of the
unetched WSL displayed in
Figure 4B. The enamel (E) shows
enlargement of the intercrystallite spaces
(arrows). The residual particulate deposit
(P) is also observed. Original
magnification: ×20 000. D. Micrograph of
sound enamel etched with Icon-Etch
(15% HCl) for 2 minutes. A typical enamel
etched pattern is observed with
dissolution of the interprismatic
substance (asterisk). Individualized enamel
crystallites are also observed (circle).
Original magnification: ×5000. E,
Micrograph of WSL after etching with
Icon-Etch for 2 minutes displaying deep
dissolution of enamel with an array of pits
and holes in the core of the prisms (ovals)
(compare with etched sound enamel in
Figure 4D). Original magnification:
×5000. F, Higher magnification of the
WSL etched with Icon-Etch displayed in
Figure 4E, which shows one of the holes
with a funneled aspect (wider area on the
top) as a result of 15% HCl etching.
Original magnification: ×20 000. WSL,
white spot lesion
colloidal quick-drying silver paint (Ted Pella, Inc., Redding, California).
Specimens were sputter-coated with iridium, by means of an ACE600
(Leica Microsystems, Wetzlar, Germany) sputter-coater, to reach a
thickness of 4 nm. Observations were carried out under a S4700
Hitachi FESEM (Hitachi America Ltd., Tarrytown, New York) in
PERDIGÃO 5

F I G U R E 6 A, Micrograph of a low-magnification view of a replica of proximal sound enamel etched with Icon-Etch, followed by Icon-dry and
Icon-Infiltrant, and restored with Filtek Supreme Plus Flowable Restorative. Original magnification: ×30. B, Micrograph of a higher magnification
of the same replica in Figure 6B displays the characteristic enamel etching pattern with penetration of resin tags deep into the interprismatic
areas (asterisks). Original magnification: ×5000

F I G U R E 7 A, Micrograph of a low-magnification view of a replica of a proximal WSL with an area with shaggy resin extensions resembling
resin tags (asterisks). Original magnification: ×50. B, Micrograph of a higher magnification of the area inside the rectangle of Figure 7A shows a
complex network of dense enamel resin tags (asterisks) that infiltrated the WSL to a depth between 20 and 465 μm. Original magnification:
×150. C, Micrograph of a more detailed field of the enamel resin tags, with a thickness of 0.5-6 μm. Residual enamel crystallites that were not
dissolved by 6 N HCl during the replica preparation for FESEM are observed surrounded by polymerized resin, resembling an enamel hybrid layer
(circles and ovals). Original magnification: ×2500. D, Micrograph of the resin tags displaying enamel crystallites surrounded by polymerized resin
to form an enamel hybrid layer (circles and ovals). Original magnification: ×5000. FESEM, field-emission scanning electron microscopy; WSL,
white spot lesion

secondary mode at an accelerating voltage of 2.0-5.0 kV. Micrographs 3 | RESULTS

were recorded digitally at standard magnifications. Measurements
were taken directly from the micrographs using the Quartz PCI The FESEM results are displayed in Figures 4–8. Unetched sound
(Quartz Imaging Co., Vancouver, British Columbia, Canada) FESEM- enamel (group 1) displayed the typical aprismatic feature with peri-
embedded image software. kymata (Figure 4A). The unetched WSL (group 1) in Figure 4B
6 PERDIGÃO

F I G U R E 8 A, Photograph of the enamel specimen of the buccal surface of a molar used in this study, which had a small WSL (circle) within a
large area of sound enamel (SE). A groove (Gr) was placed to separate and facilitate the visualization of different areas of enamel under the
FESEM. A dimple (asterisk) was marked with a round bur to help with the orientation of the replica. B, Micrograph of the respective replica of
enamel specimen in Figure 8A restored with the Icon resin infiltration system. The same WSL replica (circle) is surrounded by the replica of
SE. Original magnification: ×30. C, Higher magnification showing a very dense agglomerate of enamel resin tags with the same shaggy appearance
infiltrated enamel to a depth up to 370 μm, surrounded by the replica of SE. Original magnification: ×150. D, Micrograph showing a detailed view
of the enamel resin tags observed in Figure 8C. Hydroxyapatite crystallites are surrounded by polymerized resin, resembling an enamel hybrid
layer (ovals). Original magnification: ×5000. FESEM, field-emission scanning electron microscopy; WSL, white spot lesion

included an area coated with a particulate deposit and an area with
exposed enamel crystallites without an obvious prismatic arrange-
ment. A higher magnification of this unetched WSL is shown in
Figure 4C. This micrograph emphasizes the area of demineralized
enamel with 50-200 nm wide pores between the enamel crystallites.
When sound enamel was etched with Icon-Etch (group 2), a typi-
cal enamel prismatic etching pattern was observed along with dissolu-
tion of the interprismatic substance, leading to exposure of enamel
crystallites (Figure 4D). Figure 4E shows a WSL after etching with
Icon-Etch (group 2) displaying marked dissolution of enamel with an
array of pits and holes observed in the core of the prisms (compare
with etched sound enamel in Figure 4D). Figure 4F, a higher magnifi-
cation of the WSL etched with Icon-Etch of Figure 4E, displays a
funneled hole in the core of an enamel prism.
A cross section of sound enamel etched with Icon-Etch for
2 minutes (group 2) is depicted in Figure 5A. The demineralization
depth varied between 0.9 and 2.0 μm. For the cross section of a WSL
(Figure 5B) etched with Icon-etch for 2 minutes (group 2), the demin-
eralization depth varied between 2.1 and 92.1 μm (the deepest is not
observed in the micrograph).
A low-magnification view of a replica of sound proximal enamel
etched with Icon-Etch for 2 minutes, followed by Icon-Dry and Icon-
Infiltrant (group 3), is shown in Figure 6A. A higher magnification of
the same replica (Figure 6B) displays the characteristic enamel etching
pattern with a penetration of the resin deep into the interprismatic
areas.
A general view of a replica of a proximal WSL (group 3) is dis-
played in Figure 7A, which depicts an area with shaggy resin exten-
sions resembling resin tags. A higher magnification of the same area
shows a complex network of enamel resin tags that infiltrated the
WSL to a depth between 20 and 465 μm (Figure 7B). A detailed field
of the interface between the polymerized Icon-Infiltrant and the intact
enamel below the WSL is shown in Figure 7C,D. These micrographs
depict an intricate network of resin tags, with a thickness of 0.5-6 μm.
In addition, enamel crystallites are surrounded by polymerized resin,
forming an enamel hybrid layer.
Figure 8A shows the buccal surface of a molar used in this study,
which had a small WSL (asterisk) within a large area of sound enamel
(group 3). The grooves were placed to facilitate the visualization of
different areas of enamel under the FESEM. After the resin infiltration
sequence, the respective replica in Figure 8B shows the same WSL
surrounded by sound enamel, allowing the comparison between the
replicas of sound enamel and WSL. A higher magnification of this
WSL area is displayed in Figure 8C. A very dense agglomerate of
enamel resin tags, with the same shaggy appearance infiltrated enamel
to a depth up to 370 μm. Figure 8D depicts a detailed view of these
enamel resin tags. Enamel crystallites are surrounded by polymerized
resin in some areas, forming an enamel hybrid layer.
PERDIGÃO
4 | DISCUSSION
The combination of HCl etching followed by the infiltration of a low-
viscosity resin was used in 1976 as an experimental enamel cari-
ostatic treatment.26 In 1987, Croll27 used a clear resin sealant on
phosphoric acid-etched enamel to saturate the enamel defects with
resin.
WSLs correspond to a phase of the caries process prior to cavita-
tion, in which hydroxyapatite has been lost from the enamel subsur-
face, leaving an apparently intact or pseudointact surface layer
covering the mineral-deprived area.28 In addition to a lower hardness,
the surface layer in natural WSLs has a mean thickness of 40-45 μm
28-30
and a mineral content of 82%-84%, and is covered with an
“acquired cuticle”31 that varies in ultrastructure and mineralization
from specimen to specimen, observed as a particulate deposit in
Figure 4B,C.
The WSL-mineralized surface layer hampers resin penetration
because of its scarce porosity (Figure 4C). While etching with a con-
ventional phosphoric acid gel does not result in adequate porosity for
30
resin to infiltrate the suface, 15% HCl is effective in partially remov-
30
ing the mineralized layer and opening wider surface porosities
boost resin infiltration of the more porous subsurface body of the
lesion.
The Icon instructions recommend etching the WSL for 2 minutes
with 15% HCl (Table 1). However, if the whitish-opaque aspect per-
sists immediately after applying Icon-Dry, the manufacturer recom-
mends one or two additional etching steps of 2 minutes each. It is
likely that the extra etching steps are recommended because it has
been reported that 29% of the WLSs have a surface layer thicker than
50 μm.30 It is difficult to create enough porosities on thick surface
layers with just one application of 15% HCl, resulting in poor capillary
infiltration of the resin into the body of the lesion. In addition, when a
WSL is still fully visible on a hydrated tooth surface (or on a tooth that
has been wet with ethanol in Icon-Dry), this lesion has likely extended
through the enamel and possibly into the dentin,15 which may also
preclude the resin from completely infiltrating the WSL.11
WSLs are highly variable, not only regarding the surface layer
thickness, but also the depth of the body of the lesion,32 as shown in
Figures 7A-D with a length of enamel resin tags ranging from
20-465 μm. The broad range of the length of resin tags in our study
may also be caused by wide variations of the mineral content in the
29
body of the lesion below the mineralized surface layer. The low-
viscosity TEGDMA-based infiltrant is more effective than dentin
adhesives in masking and infiltrating WSLs.33,34 The ability of the low-
viscosity resin to infiltrate the full depth of the WSL is also crucial for
preventing the progression of early caries lesions,17,35 as the enamel
hybrid layer forms when the adhesive resin reaches the transition
between the WSL and normal enamel. Nakabayashi et al36 elegantly
described the enamel resin tags as pure resin, but the authors also
noted a thin zone at the bottom side of the resin tags in which the
resin impregnated the interprismatic enamel and formed a resin-
reinforced tissue (hybrid layer) that was part enamel and part resin.
7
Enamel hybrid layers are distinct from dentin hybrid layers because
they lack the typical collagen fibers present in the latter.37
Our findings that enamel crystallites are surrounded by polymer-
ized resin in areas of the resin-infiltrated WSL are clinically relevant
because hybridization makes resin-embedded enamel more resistant
to acid attack than sound enamel.36-40 In our study, the enamel crys-
tallites resisted the decalcification method to obtain replicas (6 N HCl
for 24 hours) because those crystallites were enveloped by the resin
used in Icon-Infiltrant. This type of replica preparation method
removes nonimpregnated enamel and also enamel that is poorly
encapsulated by resin.40
One of the limitations of this in vitro study is that the presence of
saliva and organic material inside the WSL in clinical situations may
prevent a complete infiltration of the lesion with resin.
WSLs have become an esthetic challenge especially when they
occur in anterior teeth, as WSLs were still observed on the labial sur-
faces of anterior teeth more than 5 years after the orthodontic treat-
ment was concluded.41 The successful treatment of the resulting
esthetic imbalance depends on many factors, including the intrinsic
characteristics of the WSL and the complete infiltration of the low-
viscosity resin (Icon-infiltrant) to the bottom of the WSL to form an
to
enamel hybrid layer. Further studies should evaluate the long-term
clinical behavior of resin-infiltrated enamel WSLs and the risks of
treating deep WSL with resin infiltration without reaching the full
depth of the lesion.
5 | C O N CL U S I O N S
Within the limitations of any in vitro study, the infiltration of
demineralized enamel in WSLs with a low-viscosity light-cured resin
resulted in an enamel hybridization process similar to the one that
occurs in etched dentin, along with the formation of resin extensions
into the hollow spaces inside the demineralized enamel.
DISCLOSURE OF INTERESTS
The author does not have any financial interest in the companies
whose materials are included in this article.
OR CID
~
Jorge Perdigao https://orcid.org/0000-0003-1841-6365
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How to cite this article: Perdig~ao J. Resin infiltration of
enamel white spot lesions: An ultramorphological analysis.
J Esthet Restor Dent. 2019;1–8. https://doi.org/10.1111/jerd.
12550