RESEARCH REPORTS

Biomaterials & Bioengineering

J. Kim1*, L. Gu2*, L. Breschi3,

L. Tjäderhane4, K.K. Choi1,
Implication of Ethanol Wet-
D.H. Pashley5, and F.R. Tay5,6† bonding in Hybrid Layer
1
Department of Conservative Dentistry, School of Dentistry,
Kyung Hee University, Seoul, Korea; 2Department of
Remineralization
Operative Dentistry and Endodontics, Guanghua School
of Stomatology, Institute of Stomatological Research, Sun
Yat-sen University, Guangzhou, China; 3Department of
Biomedicine, Unit of Dental Sciences and Biomaterials,
University of Trieste, Italy, and IGM-CNR, Unit of Bologna
c/o IOR, Bologna, Italy; 4Institute of Dentistry, University of
Oulu and Oulu University Hospital (OUH), Oulu, Finland;
5
Department of Oral Biology, School of Dentistry, Medical
College of Georgia, Augusta, GA 30912-1129, USA; and
6
Department of Endodontics, School of Dentistry, Medical
College of Georgia, Augusta, GA 30912-1129, USA; *authors
contributing equally to this work; †corresponding author,
ftay@mcg.edu

J Dent Res 89(6):575-580, 2010

ABSTRACT INTRODUCTION
During mineralization, unbound water within the
collagen matrix is replaced by apatite. This study
tested the null hypothesis that there is no difference
D uring hard-tissue mineralization, bulk and weakly bound water molecules
in the collagen matrix are gradually replaced by apatite crystallites (Magne
et al., 2001; Wehrli and Fernández-Seara, 2005). Likewise, during intentional
in the status of in vitro biomimetic remineralization
or pathological demineralization, dissolved intrafibrillar and interfibrillar apa-
of hybrid layers, regardless of their moisture con-
tites are replaced with a comparable volume of water (Ito et al., 2005; Toroian
tents. Acid-etched dentin was bonded with One-
et al., 2007). In the context of dentin bonding, water that fills the intrafibrillar
Step with ethanol-wet-bonding, water-wet-bonding,
and interfibrillar spaces after apatite dissolution by acids should be totally dis-
and water-overwet-bonding protocols. Composite-
placed by resin monomers during bonding (Vaidyanathan and Vaidyanathan,
dentin slabs were subjected to remineralization for
2009). Demineralized collagen is responsible for the creep behavior of hard
1-4 months in a medium containing dual bio-
tissues (Bowman et al., 1999; Pashley et al., 2003). Thus, incomplete replace-
mimetic analogs, with set Portland cement as the
ment of water introduced during adhesive application and by perfusion
calcium source and characterized by transmission
from vital dental pulps leaves behind a hydrated collagen matrix that could
electron microscopy. Remineralization was either
adversely affect the resistance of resin-dentin bonds to cyclic fatigue. To date,
non-existent or restricted to the intrafibrillar mode
complete displacement of water from the interfibrillar compartment by con-
in ethanol-wet-bonded specimens. Extensive intra-
temporary dentin adhesives has never been reported. Moreover, the ability of
fibrillar and interfibrillar remineralization was
these adhesives to replace depleted minerals from the intrafibrillar compart-
observed in water-wet-bonded specimens. Water-
ment of the collagen matrix is unknown (Pashley and Nakabayashi, 1998).
overwet specimens demonstrated partial remineral-
Ethanol-wet-bonding (Pashley et al., 2007) is an in vitro technique devel-
ization of hybrid layers and precipitation of
oped for the application of etch-and-rinse adhesives. In this technique, etha-
mineralized plates within water channels. The use
nol, a polar solvent with less hydrogen bonding capacity than water (Becker
of ethanol-wet-bonding substantiates that bio-
et al., 2007), is used to chemically dehydrate the demineralized collagen
mimetic remineralization is a progressive dehydra-
matrix (Nishitani et al., 2006). The resultant shrinkage of the collagen fibrils
tion process that replaces residual water in hybrid
in the lateral dimension and reduction in hydrophilicity of the collagen matrix
layers with apatite crystallites.
create wider interfibrillar spaces for hydrophobic resins to infiltrate the matrix
more completely (Tay et al., 2007) as a potential mechanism for extending the
KEY WORDS: biomimetics, dentin bonding, longevity of resin-dentin bonds (Hosaka et al., 2009).
ethanol, hybrid layer, intrafibrillar, interfibrillar, Biomimetic remineralization of resin-dentin bonds (Tay and Pashley,
remineralization, water. 2009) is an in vitro technique that incorporates biomimetic analogs for
sequestration of amorphous calcium phosphate nanoprecursors from a remin-
DOI: 10.1177/0022034510363380
eralization medium. The analogs also function as templates for guiding
Received September 15, 2009; Last revision January 11, the deposition of apatite crystallites within the intrafibrillar and interfibrillar
2010; Accepted January 13, 2010 compartments of incompletely resin-infiltrated hybrid layers created by

575
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© 2010 International & American Associations for Dental Research

576
etch-and-rinse (Mai et al., 2009) and self-etching adhesives
(Kim et al., 2009). As apatites are deposited in these compart-
ments, water within the microfibrils of collagen fibrils and the
interfibrillar spaces (Traore et al., 2000) is gradually replaced by
minerals (Chesnick et al., 2008). Compression of the collagen
fibrils (Bonar et al., 1985) caused by intrafibrillar remineraliza-
tion may additionally contribute to displacement of water from
resin-infiltrated collagen matrices.
The aforementioned in vitro techniques differ from the
in vivo technique of using matrix metalloproteinase inhibitors to
prevent collagen degradation (Pashley et al., 2004; Carrilho
et al., 2007), in that they recognize the significance of water
replacement by either resin or apatite as the means to prevent
degradation of resin-dentin bonds. Theoretically, if water within
the intrafibrillar and interfibrillar compartments of a collagen
matrix is completely replaced by resins by means of ethanol-
wet-bonding, intrafibrillar and interfibrillar remineralization of
collagen fibrils should not occur with the biomimetic remineral-
ization technique; but this hypothesis has never been substanti-
ated. This study tested the validity of this hypothesis by
examining the extent of biomimetic remineralization in hybrid
layers created by an etch-and-rinse adhesive under a spectrum of
moisture conditions. The null hypothesis tested was that there is
no difference in the status of in vitro biomimetic remineraliza-
tion in hybrid layers created by a two-step etch-and-rinse adhe-
sive regardless of the moisture condition under which they are
created.
MATERIALS & METHODS
Dentin Bonding
Fifteen recently extracted human third molars were collected after
donors' informed consents were obtained under a protocol
reviewed and approved by the Human Assurance Committee of
the Medical College of Georgia. A flat dentin surface was pre-
pared perpendicular to the longitudinal axis of each tooth by
means of an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA)
under water-cooling. We made a second parallel cut along the
cemento-enamel junction to expose and remove the contents from
the pulp chamber. The bonding surface was polished with 320-grit
silicon carbide paper, etched with 37% H3PO4 (Bisco, Schaumburg,
IL, USA) for 15 sec and rinsed with water for 10 sec.
The specimens were randomly divided into 3 groups (N = 5)
with an increasing spectrum of water content within the bonding
interface: (1) ethanol-wet-bonding [minimal water content]—
water-moist dentin surface rinsed with 2 mL of 100% ethanol
for 1 min, and the ethanol-saturated collagen matrix was not
allowed to evaporate to dryness to prevent surface tension forces
from collapsing the demineralized collagen network; (2) water-
wet-bonding [manufacturer’s recommended optimal water
content] — Kimwipes (Kimberly Clark, Roswell, GA, USA)
used to blot excess water that remained after rinsing of
the etchant; and (3) water-overwet-bonding [excessive water
content] — The tooth segment was connected to a Plexiglas
platform assembly to deliver 20 cm of water pressure during
bonding. In addition, a 20-µL quantity of water was placed over
the surface of the acid-etched dentin. Two coats of One-Step
(Bisco) were applied and light-cured (600 mW/cm2) for 20 sec.
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© 2010 International & American Associations for Dental Research
Kim et al. J Dent Res 89(6) 2010
Two 2-mm-thick incremental layers of resin composite (EPIC-
TMPT, Parkell, Farmington, NY, USA) were then applied and
light-cured separately for 40 sec each. Each tooth was sectioned
occluso-gingivally to produce four 1-mm-thick slabs (5 × 4 = 20
slabs/group).
Remineralization Medium
White Portland cement (Lehigh Cement Company, Allentown,
PA, USA) was mixed with de-ionized water (water-to-powder
ratio 0.35:1) and allowed to set in silicone molds for 1 wk before
use. A biomimetic remineralization medium (Tay and Pashley,
2009) was prepared, consisting of a simulated body fluid (SBF)
with 100-5000 µg/mL of polyacrylic acid (Sigma-Aldrich, St.
Louis, MO, USA) and polyvinylphosphonic acid (Sigma-
Aldrich) as biomimetic analogs and buffered to pH 7.4.
Biomimetic Remineralization
Each specimen slab was placed over a set Portland cement block
(ca. 1 g) inside a glass scintillation vial. The latter was filled
with 15 mL of biomimetic remineralization medium and incu-
bated at 37°C. The medium was changed monthly, with its pH
monitored weekly so that it was above 9.25 to facilitate forma-
tion of apatite instead of octacalcium phosphate (Meyer and
Eanes, 1978).
Confocal Laser Scanning Microscopy (CLSM)
For each group, 4 slabs were immersed for 4 mos in the remin-
eralization medium without Portland cement blocks as the cal-
cium source to identify porous zones within hybrid layers that
were permeable to a water-soluble fluorescent dye (Kim et al.,
2009). Each slab was ultrasonicated for 5 min and immersed in
0.1 wt% Rhodamine B (Sigma-Aldrich) dissolved in phosphate-
buffered saline (pH = 7.4). After 24 hrs, the dye-infiltrated slabs
were rinsed with de-ionized water and examined by a CLSM
(LSM 510 META; Carl Zeiss, Thornwood, NY, USA) that was
coupled with a helium neon gas laser (80% of 543-nm excita-
tion, 1.2 mW). Images were captured at 5 µm beneath the pol-
ished surface to avoid superficial specimen preparation artifacts.
Transmission Electron Microscopy (TEM)
For each group, we retrieved specimens after 1-4 mos (4 slabs/
mo x 4 mos = 16 slabs) for transmission electron microscopy
(TEM) to examine the status of remineralization within the
resin-dentin interface. The slabs were fixed in Karnovsky’s fixa-
tive, post-fixed in 1% osmium tetroxide (1 hr), and rinsed with
sodium cacodylate buffer. The slab was dehydrated in an
ascending ethanol series (50-100%), immersed in propylene
oxide, and embedded in epoxy resin (Tay and Pashley, 2009).
We examined 90-nm-thick unstained sections using a JEM-1230
TEM (JEOL, Tokyo, Japan) at 110 kV.
RESULTS
Under CLSM examination, fluorescence was completely
quenched in hybrid layers from ethanol-wet-bonded specimens
that were aged for 4 mos without the use of Portland cement as
J Dent Res 89(6) 2010 Water Replacement & Biomimetic Remineralization 577

the calcium source (Fig. 1A).

Under TEM examination, 62.5%
of the specimens that were aged
in the Portland cement-block-
containing remineralization medium
showed no signs of hybrid layer
remineralization over the entire
four-month period (Fig. 1B). The
rest of the specimens exhibited
minimal remineralization around
dentinal tubular orifices (Fig. 1C),
with the mode of remineralization
being exclusively intrafibrillar in
nature (Fig. 1D).
Intense fluorescence through-
out the hybrid layers could be
detected from specimens prepared
with water-wet-bonding (Fig. 2A).
Remineralization of the hybrid
layers was apparent beyond
10,000X magnification at 2 mos,
but could be clearly discerned at
3000X after 3 mos. Remineraliza-
tion was not uniform, being more
extensive along the surface of the
hybrid layer and around the
periphery of the dentinal tubules
(Fig. 2B). Both intrafibrillar and
interfibrillar remineralization could
be identified (Fig. 2C), with the
mineral phase consistent with the
electron diffraction patterns of
poorly crystalline apatite (Fig. 2D). Figure 1. Specimens bonded with the ethanol-wet-bonding technique [minimal water content]. A, adhe-
Hybrid layers from the water- sive; between open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from a specimen
overwet bonding group also exhib- that had been immersed for 4 mos in the remineralization medium that was devoid of a Portland cement
ited intense fluorescence. Vertically block (i.e., no remineralization). Fluorescence was absent from the entire hybrid layer. Resin tags (arrows)
appeared to be longer than in the other groups. (B) TEM image of a specimen retrieved after 4 mos of
oriented water channels extended biomimetic remineralization in which there was no remineralization of the hybrid layer. A large electron-
from the resin-dentin interface dense crystal could be seen within a void (open arrowhead) along the surface of the hybrid layer. [Note:
into the overlying adhesive (Fig. Absence of remineralization within the hybrid layer could be seen in 10 out of 16 specimens over the
3A). Fifty percent of the specimens entire four-month period.] (C) TEM image of a specimen retrieved after 4 mos of biomimetic remineraliza-
examined at 3-4 mos after reminer- tion in which there was minimal remineralization of the hybrid layer along the dentin surface (arrows)
alization demonstrated filling of in regions that were adjacent to a dentinal tubule (T). AC, composite that was blended with the oxygen
those vertical water channels with inhibition layer of the adhesive. [Note: This feature was observed in 6 out of the 16 specimens over the
entire four-month period.] (D) High-magnification TEM image showing exclusive intrafibrillar remineral-
mineral plates (Fig. 3B). The rest of ization of the surface collagen fibrils depicted in (C). Electron-dense nanocrystallites could be observed
the specimens demonstrated remin- within the remineralized fibrils (arrow).
eralization of only the hybrid layers
(not shown). Apart from partial
remineralization of the collagen matrix (Fig. 3C), the smaller water surrounded by a highly ordered inner layer of tightly bound struc-
channels within the hybrid layer (i.e., terminal branches of dentinal tural water and water bridges created by hydrogen bonding
tubules that were continuous with the interfibrillar spaces) were (Ramachandran and Chandrasekharan, 1968; Bella et al., 1995). It
filled with larger hexagonal mineral platelets (Fig. 3C), similar to is unlikely that rinsing a demineralized collagen matrix with etha-
those apatite crystals identified from the cap regions of the min- nol for 1 min will remove this innermost layer of structural water
eral-filled water channels (Fig. 3D). from the collagen fibrils, which in turn results in irreversible disrup-
tion of the triple-helical structure. (Magne et al., 2001).
In water-wet-bonding, the collagen matrix is suspended in
DISCUSSION
water to prevent direct contact of the collagen fibrils via the
Water plays a pivotal role in maintaining the structural integrity of development of interpeptide hydrogen bonds (Pashley et al.,
collagen molecules (Cameron et al., 2007) and mechanical proper- 2007). Conversely, suspension of the collagen matrix in a liquid
ties of collagen fibrils (Zhang et al., 2007). Collagen molecules are chemical dehydrant (Nalla et al., 2006) removes bulk water from

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© 2010 International & American Associations for Dental Research

578 Kim et al. J Dent Res 89(6) 2010

from those water-contaminated

ethanol-wet-bonded specimens
indirectly showed that low-
molecular-weight ethanol-solvated
resin monomers could infiltrate the
intrafibrillar compartment of a
dehydrated collagen network (Tay
et al., 2007), despite the presence of
a size exclusion effect (Toroian et al.,
2007). By replacing most of the
water from the collagen matrix, the
ethanol-wet-bonding technique has
the potential to create durable resin-
dentin bonds (Hosaka et al., 2009).
The concept that biomimetic
remineralization of hybrid layers
can occur only in the presence of
incomplete water replacement
by adhesive resins was further for-
tified by the fairly extensive
intrafibrillar and interfibrillar rem-
ineralization in the water-wet-
bonding group. In our previous
studies, we were surprised by the
extent of intrafibrillar remineral-
ization observed in hybrid layers
created by both etch-and-rinse and
self-etching adhesives (Mai et al.,
2009; Tay and Pashley, 2009).
Results from the ethanol-wet-
bonding group in the present study
helped to clarify our concern and
Figure 2. Specimens bonded by the water-wet-bonding technique [manufacturer’s recommended optimal water implied that contemporary adhe-
content]. A, adhesive; between open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from sives, when used according to the
a specimen that had been immersed for 4 mos in the remineralization medium that was devoid of a Portland manufacturers’ instructions, are
cement block. Intense fluorescence present within the hybrid layer and dentinal tubules indicates that the polym- incapable of displacing water from
erized adhesive was permeable to the water-soluble fluorescent dye after water sorption. (B) TEM image of the intrafibrillar compartment of
a specimen retrieved after 4 mos of biomimetic remineralization, showing non-uniform remineralization of
the hybrid layer. Regions that were more heavily remineralized are indicated by asterisks corresponding to
the collagen matrix. Incomplete
the dark regions depicted in the CLSM image in (A). T, dentinal tubule. (C) High-magnification TEM image of water replacement from the interfi-
(B) showing both intrafibrillar remineralization (arrow) and extrafibrillar remineralization (pointers) within the brillar compartment, dilution of
hybrid layer. Mineral nanocrystals were arranged in an ordered manner within the collagen fibril, giving a water-soluble monomers with
vague, banded appearance (open arrowheads). (D) Selected-area electron diffraction of those nanocrystals water, or subsequent water sorp-
depicted in (C) yielded ring patterns that were consistent with the characteristics of poorly crystalline apatite. tion during aging could have addi-
tionally accounted for the
the dentinal tubule anastomosis and the loosely bound water lay- susceptibility of hybrid layers to interfibrillar remineralization.
ers from the intrafibrillar compartments and results in increased The inclusion of a water-overwet-bonding group, although
intramolecular hydrogen bonding among the collagen molecules far removed from clinical practice, broadens our understanding
(Mogilner et al., 2002). Intramolecular hydrogen bonds cause of the biomimetic remineralization scheme. Identification of
shrinkage of the fibrils by reduction in the lateral spacing of the large mineral plates in the basal region of the filled water chan-
collagen molecules (Miles and Ghelashvili, 1999). There was no nels signifies the absence of an appropriate scaffold for the
biomimetic remineralization of the hybrid layers when ethanol deposition of nanocrystals. Conversely, the conglomerates of
wet-bonding was meticulously performed. However, in the apatite nanocrystals within the cap region of the water channel
absence of tubular occlusion (Sadek et al., 2007), ethanol-wet- probably represent hierarchical three-dimensional precipitation
bonding replaced water from the interfibrillar compartment effec- of the mineral phase within a water-entrapped polymer hydrogel
tively, but incompletely removed the loosely bound water from matrix (Liu et al., 2009). The close adaptation of the large
the intrafibrillar compartment of collagen fibrils around the tubular mineral plates to the conformation of the basal region of the
orifices. The exclusive intrafibrillar remineralization identified water channel provides support for the involvement of a liquid

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J Dent Res 89(6) 2010 Water Replacement & Biomimetic Remineralization 579

amorphous precursor pathway as a

biomineralization strategy (Gower,
2008). The fragmented plates are
likely to be artifacts created dur-
ing ultramicrotomy of a single
brittle mineral sheet.
Identification of mineral plate-
lets within the horizontally ori-
ented water channels of the hybrid
layer suggests that these channels
were intricately connected to the
vertical water channels on the
hybrid layer surface, as confirmed
in previous nanoleakage studies
(Tay et al., 2005a,b). Mineral-filled
water channels had never been
identified within hybrid layers cre-
ated with etch-and-rinse adhesives
by the conventional water-wet-
bonding technique. These channels
represent the terminal branches of
the dentinal tubule anastomosis
and are continuous with the inter-
fibrillar compartments of the col-
lagen matrix. They were also
identified when a non-resin-
containing glass-ionomer cement
was placed over moist acid-etched
dentin (Kim et al., 2010). During
adhesive application without per-
fusion, and with blot-drying, these
terminal branches collapse due to
shrinkage of the collagen matrix
that is induced by the polar adhe- Figure 3. Specimens bonded with water-overwet-bonding [excessive water content]. A, adhesive; between
sive solvents with chemical dehy- open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from a specimen that had been
dration potential (Nakajima et al., immersed for 4 mos in the remineralization medium that was devoid of a Portland cement block. Intense
fluorescence could be observed within the entire hybrid layer, the dentinal tubules beneath the hybrid
2002; Nalla et al., 2006; Becker et al., layer, and the vertically oriented water channel (pointer). (B) TEM image of a specimen retrieved after
2007). During water-overwet- 4 mos of biomimetic remineralization. Numerous mineral-filled water channels could be seen on top of
bonding, continuous replenishment the partially remineralized hybrid layer. Each water channel consisted of a basal region that was filled
of water via perfusion and an with large mineral plates (open arrowhead) and a cap region (arrow) that was filled with very fine nano-
excessive amount of water enables crystals. (C) High-magnification TEM image of a similar remineralized hybrid layer in this group. Two
these terminal branches to remain modes of mineralization could be identified within the hybrid layer: (a) remineralization of the collagen
fibrils (arrows) with nanocrystals similar to those depicted in Figs. 1D and 2C [This mode of remineral-
fully extended, even in the pres-
ization occurred predominantly along the top of the hybrid layer surface, but could also be seen, albeit
ence of the dehydrating adhesive sparsely, from the base of the hybrid layer.]; and (b) the presence of electron-dense crystals in channels
solvents. On polymerization of the (open arrowheads) that were oriented roughly parallel to the mineralized dentin base. The large basal
adhesive, these fully extended regions of the vertically oriented water channels above the hybrid layer were densely filled with much
channels were stabilized in their larger mineral plates (asterisk). (D) High-magnification TEM image of the cap region of a mineral-filled
fully extended conditions. Lacking water channel revealed a dense conglomerate of 5-20 nm hexagonal nanocrystals within the cap region.
an appropriate scaffold, only larger Selected-area electron diffraction of those nanocrystals yielded spotted diffraction rings that were charac-
teristic of apatites with a higher degree of crystallinity (inset).
mineral platelets could be formed
within these water channels.
The null hypothesis—that there is no difference in the status of to less-porous hybrid layers by the elimination of denuded colla-
in vitro biomimetic remineralization in hybrid layers created by a gen matrices with progressive dehydration and water replacement
two-step etch-and-rinse adhesive, regardless of the moisture con- (Fernández-Seara et al., 2004). Collectively, they represent an
dition under which they are created—is rejected. Biomimetic alternative philosophy to the use of matrix metalloproteinase
remineralization of the hybrid layer and the ethanol wet- inhibitors to prevent collagen degradation and may be combined
bonding technique achieved similar results, in that both led with the latter to extend the longevity of resin-dentin bonds.

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© 2010 International & American Associations for Dental Research

580
ACKNOWLEDGMENTS
This study was supported by Grant R21 DE019213-01 from the
National Institute of Dental and Craniofacial Research (PI.
Franklin R. Tay). The dentin adhesive used in this study was a
generous gift from Bisco Inc. We thank Bob Smith for TEM
technical assistance, Thomas Bryan for resin embedding, and
Michelle Barnes for secretarial support.
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