dental
materials
Dental Materials 16 (2000) 311–323
www.elsevier.com/locate/dental
Effect of a sodium hypochlorite gel on dentin bonding
J. Perdigão a,*, M. Lopes b, S. Geraldeli c, G.C. Lopes d, F. Garcı́a-Godoy e
a
Division of Operative Dentistry, University of Minnesota, Minneapolis, MN, USA
Minnesota Dental Research Center for Biomaterials and Biomechanics, University of Minnesota, Minneapolis, MN, USA
c
Department of Dental Materials, University of Santo Amaro and University of Mogi da Cruzes, Sao Paulo, Brazil
d
Department of Operative Dentistry, Federal University of Santa Catarina, Florianopolis, Brazil
e
Department of Restorative Dentistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
b
Received 15 September 1999; accepted 15 November 1999
Abstract
It has been suggested that the hybrid layer (HL) does not play any important role in the mechanism of adhesion to dentin. To substantiate
this hypothetical insignificance of the HL, sodium hypochlorite (NaOCl) has been used to remove collagen from etched dentin prior to
bonding.
Objectives: The present study was conducted to determine the effect of a commercial 10% NaOCl gel on the dentin shear bond strengths
and HL ultra-morphology of two simplified dentin adhesives. The null hypothesis tested was that treatment of etched dentin collagen with
NaOCl would not compromise dentin bonding.
Methods: The labial surface of eighty bovine incisors was polished to expose middle dentin. The specimens were randomly assigned to two
total-etch adhesive systems N 40 : Prime&Bond NT (Dentsply Caulk); and Single Bond (3M Dental Products Division). After rinsing off
the etchant, one drop of 10% NaOCl (AD Gel, Kuraray Ltd.) was applied to the etched dentin surface and left for 0 (control), 15, 30, or 60 s.
The gel was rinsed off with water and the dentin surface kept visibly moist prior to the application of the adhesive as per manufacturer’s
instructions. The respective composite resin was subsequently applied and light-cured. After 24 h in water at 378C, the specimens were
thermocycled for 500 cycles in baths kept at 5 and 558C and the shear bond strengths measured. The data were analyzed with two-way
ANOVA. For TEM, sixteen dentin disks were taken from middle dentin of extracted human third molars, assigned to the eight treatment
sequences, and observed.
Results: The increase in the NaOCl application time resulted in a progressive decrease in shear bond strengths for both dentin adhesives.
For Single Bond, the application of AD Gel for 60 s resulted in a reduction of bond strengths to 38% of that obtained for the control. For
Prime&Bond NT, the mean bond strength obtained when AD Gel was applied for 60 s was 31% of that obtained for the control. The
application of AD Gel resulted in distinct morphology for each one of the two adhesives tested. For Single Bond, the general morphology of
the collagen network was maintained, regardless of the deproteinization time. The interfibrillar space within the collagen network increased
with increasing deproteinization times. For Prime&Bond NT, the general appearance of the HL was maintained for deproteinization times of
15 and 30 s. When the NaOCl gel was applied for 60 s, the morphological appearance of the HL lost its fibrillar arrangement. While remnants
of the collagen fibers were observed in one of the dentin disks, the other specimen showed an amorphous structure without any discernible
HL morphological features.
Significance: The integrity of the collagen fibrils left exposed upon acid-etching plays a major role in the mechanism of adhesion of the
specific adhesive systems tested in this study. The intermingling of the adhesive monomers with the filigree of collagen fibers or HL should
still be considered the paramount dentin bonding mechanism. q 2000 Academy of Dental Materials. Published by Elsevier Science Ltd. All
rights reserved.
Keywords: Deproteinization; Sodium hypochlorite gel; Dentin bonding
1. Introduction
Enamel is a highly mineralized tissue. The use of acidic
* Corresponding author. Division of Operative Dentistry, Department of
Restorative Sciences, University of Minnesota School of Dentistry, 8-450
Moos Tower, 515 Delaware St. SE, Minnneapolis, MN 55455, USA. Tel.:
1 1-612-625-8486; fax: 1 1-612-625-7440.
E-mail address: perdi001@tc.umn.edu (J. Perdigão).
conditioners renders enamel more receptive to fluid
adhesive resins due to an increase in surface free energy
and formation of enamel resin tags inside the microscopic
porosities created by the dissolution of hydroxyapatite
crystals [1–5]. The use of phosphoric acid has, therefore,
been widely accepted as a conditioning procedure to
increase the adhesion of resins to enamel [6]. Dentin, on
the other hand, is a dynamic substrate with a complex
0109-5641/00/$ – see front matter q 2000 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved.
PII: S0109-564 1(00)00021-X
312
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
Table 1
Adhesive System (Manufacturer)
Composition of adhesive
Prime&Bond NT Lot.9708000320 Etching gel: silica-thickened 35%
(Dentsply Caulk, Milford, DE,
H3PO4 Adhesive: PENTA,
USA)
UDMA 1 T-resin (cross-linking
agent) 1 D-resin (small hydrophilic
molecule), butylated hydroxitoluene,
4-ethyl dimethyl aminobenzoate,
cetilamine hydrofluoride, acetone,
silica nanofiller
Single Bond Lot 19980826 (3M
Etching gel: silica-thickened 35%
Dental Products Division, St.
H3PO4 Adhesive: BisGMA, HEMA,
Paul, MN, USA)
dimethacrylates, polyalkenoic acid
copolymer, initiator, water, ethanol
organic structure and biological activity that preclude the
establishment of a reliable and durable bonding [5].
Twenty years ago Fusayama suggested the removal of the
smear layer from dentin with phosphoric acid to improve
adhesion [7]. In spite of relying on different agents to
dissolve the smear layer, most of the current adhesive
systems were designed to provide dentin adhesion through
the interaction of a hydrophilic monomer dissolved in an
organic solvent with a collagen-rich humid tissue [8]. It is
believed that the efficacy of the current dentin adhesives
depends upon the infiltration of those high-affinity hydrophilic monomers into the filigree of collagen fibers that
make up the structure of acid-etched dentin. This entanglement of monomers with collagen fibers and a few residual
hydroxyapatite crystals forms a hybrid tissue [9] also known
as resin–dentin interdiffusion zone [10].
The depth of dentin demineralization has become an
important issue in dentin bonding. Several authors have
expressed some concerns about deficient or incomplete
penetration of the demineralized microporous collagen
network [10,11]. The deficit of resin in the area of the
collagen network close to the unaffected dentin or around
individual collagen fibers could result in a delicate zone
inside the hybrid layer (HL) susceptible to hydrolytic degradation [11–13].
Sodium hypochlorite (NaOCl) has been used on dentin as
a deproteinizing agent [14]. NaOCl is a well-known nonspecific proteolytic agent capable of removing organic material
[15], as well as magnesium and carbonate ions [16]. Several
researchers [17–19] have studied the role of NaOCl in
dentin permeability and dentin adhesion. Depending on
each testing methodology and/or specific composition of
each dentin adhesive, the application of NaOCl upon
etching may increase or decrease bond strengths [19–21].
It has been reported that the higher the concentration of
NaOCl, the greater the dentin bond strengths until a plateau
is reached at a concentration of 10%, for an application time
of 60 s [22].
This in vitro study was conducted to determine the effects
of a commercial 10% NaOCl gel on the dentin shear bond
strengths and resin–dentin ultramorphology of two one-
bottle dentin adhesives containing different solvents. The
null hypothesis to be tested was that treatment of dentin
collagen with NaOCl upon etching would not compromise
dentin bonding.
2. Materials and methods
2.1. Bond strengths
Eighty bovine teeth were obtained at a local abattoir and
were refrigerated in a solution of 0.5% chloramine for up to
one week until use. After removing the roots, the crowns
were cleaned of debris and were mounted in phenolic rings
(Buehler Ltd., Lake Bluff, IL) with Trayresin (Dentsply
Trubyte, York, PA) cold-cure acrylic resin. The buccal
surface of each tooth was ground with a mechanical grinder
to expose middle dentin and was subsequently polished for
30 s with wet 240-, 400-, and 600-grit silicon carbide
abrasive paper. Specimens were randomly assigned to two
adhesive systems and four treatment sequences 2 × 4
design, Table 1), as follows:
Group 1 (control, NT-0)—Dentin was etched with 34%
phosphoric acid (Tooth Conditioner Gel, Dentsply Caulk,
Milford, DE) for 15 s and then rinsed with water for 10 s.
Excess water was removed by blotting with a tissue paper,
leaving dentin visibly moist. Prime&Bond NT (Dentsply
Caulk) was applied to the dentin surface in copious
amounts, leaving the surface wet and undisturbed for 20 s.
The adhesive was gently air-dried with oil-free compressed
air from an air syringe for 5 s, keeping the air syringe 2 cm
from the surface, and light-cured for 20 s.
Group 2 (NT-15)—Dentin was etched with 34% phosphoric acid (Tooth Conditioner Gel) for 15 s and then rinsed
with water for 10 s. Excess water was removed by gently
air-drying for 2–3 s. AD Gel (Lot 00176A, Kuraray Lda,
Osaka, Japan) was applied with a disposable brush to cover
the dentin surface and left undisturbed for 15 s. The gel was
rinsed for 60 s with water and the excess water removed by
blotting with a tissue paper, leaving dentin visibly moist.
Prime&Bond NT (Dentsply Caulk) was applied to the
dentin surface as in Group 1.
Group 3 (NT-30)—As in Group 2, but the AD Gel was
left on dentin for 30 s.
Group 4 (NT-60)—As in Group 2, but the AD gel was left
on dentin for 60 s.
Group 5 (control, SB-0)—Dentin was etched with 35%
phosphoric acid (Scotchbond Etching Gel, 3M Dental
Products Division, St. Paul, MN) for 15 s and rinsed for
5 s. Excess water was removed by blotting, so that the
tooth surface looked visibly moist. Single Bond (3M Dental
Products Division) was applied to the etched dentin surface
with a disposable brush. After replenishing the brush with
fresh adhesive, a second coat was applied and dried for 2 s.
The adhesive was then light-cured for 10 s.
Group 6 (SB-15)—Dentin was etched with 35% phosphoric
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
313
Table 2
Results
Prime&Bond NT
Single Bond
a
b
AD GEL
(s)
SBS (S.D.) a
(MPa)
% SBS
(0 s 100%)
Ultramorphological characteristics of the HL
HL thickness
(mm)
0 (NT-0) b
15.1 (3.3) CD
100
2.5–4.0
15 (NT-15)
10.9 (4.1) DE
72
30 (NT-30)
8.0 (4.5) EF
53
60 (NT-60)
4.8 (2.3) F
31
0 (SB-0) b
25.0 (4.1) A
100
15 (SB-15)
21.0 (5.7) AB
84
30 (SB-30)
18.5 (6.8) BC
74
60 (SB-60)
9.7 (2.4) E
38
Sprinkled layer of nanoparticles above the HL. Nano-filler
penetrated the HL in areas were the tubule entrances were
widely exposed to form triangular tag necks. Uniform
dispersion of nanofiller through the adhesive layer.
Collagen banding in the HL very evident.
Fibrillar aspect maintained, but spaces between individual
fibers inside the NaOCl-modified HL are wider than for
group NT-0. Collagen banding absent.
Fibrillar aspect maintained, but collagen fibers are
separated by electron lucent spaces indicating some
degree of dissolution. The residual collagen structures
displayed a shaggy appearance on the top the NaOClmodified HL. For EDTA-decalcified specimens, electrondense formations are identified at the transition between
the modified HL and the unaffected dentin. Collagen
banding absent.
Remains of the collagen network still observed in the
lower half of the “modified” HL. Amorphous structure
replacing the HL, without the fibrillar aspect observed in
the other groups. The upper half, close to the adhesive
layer, displayed a more digested appearance as compared
with Group NT-30. It also resulted in a wrinkled
appearance, with scattered collagen residues, and an
electron dense membrane separating this area from the
nanofiller in the adhesive. Collagen banding absent.
For EDTA-decalcified specimens, the HL is electrondense at the top and less electron-dense at the transition
with unaffected dentin. An electron-dense polyalkenoic
salt deposition like “floating bubbles” is evident in the
adhesive layer. The polyalkenoic salt forms a continuum
with the top of the HL. The morphology of the collagen
fibers within the HL is different from other adhesives—
fibers appear inflated regardless of their cross-section axis.
Fibrillar aspect maintained, but spaces between individual
fibers inside the HL are wider than for group SB-0.
Collagen banding not observed.
Interfibrillar spaces are wider than in group SB-15,
ranging from 10-20 nm. Collagen banding absent.
Fibrillar arrangement lost in most areas close to the
unaffected dentin as opposed to the corresponding area in
group SB-30. Fibrillar organization maintained in the
remaining NaOCl-modified HL but spaces between
individual fibers are now in the range of 25 to 60 nm.
2.6–4.3
2.6–4.4
2.4–5.3
2.6–3.7
2.8–3.8
2.9–4.2
4.1–5.5
Means with the same superscript letter are not significantly different at p # 0:05:
Adhesive applied as per manufacturer’s instructions.
acid (Scotchbond Etching Gel) for 15 s and rinsed for 5 s.
Excess water was removed by gently air-drying for 2–3 s.
One drop of AD Gel (Kuraray Co.) was applied and left
undisturbed on dentin for 15 s. The gel was rinsed off
with water for 60 s and the excess water removed by
blotting with a tissue paper, leaving dentin visibly moist.
Single Bond (3M Dental Products Division) was applied to
the dentin surface as in Group 5.
Group 7 (SB-30)—As in Group 6, but the AD Gel was
left on dentin for 30 s.
Group 8 (SB-60)—As in Group 6, but the AD gel was left
on dentin for 60 s.
A proprietary composite resin (Surefil for Prime&Bond
NT; Z100 for Single Bond) was condensed into a #5 gelatin
capsule (Torpac Inc., Fairfield, NJ) to fill two-thirds of the
capsule and light-cured in a Triad oven (Dentsply Trubyte)
for 2 min. A final increment of composite resin was inserted
into the gelatin capsule just before its application on the
treated surface, and the capsule was seated securely against
the flattened tooth surface. Excess material was removed
with a sharp explorer under × 2.5 magnifying loupes, and
the composite light-cured for a total of 80 s (40 s from each
opposite direction) using a Demetron 401 curing light
(Demetron/Kerr, Danbury, CT). The intensity of the light
314
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
Fig. 1. TEM resin–dentin interfaces formed by Prime&Bond NT (Group NT-0). (a) Decalcified/stained interface showing the accumulation of nano-filler
within the tags. Note a small-hybridized lateral canal (L) and the direct contact between the adhesive filler and the superficial fibers of the HL. Bar 2 mm. (b)
Non-decalcified/non-stained interface showing some diffusion of filler inside the HL (circles). Note the accumulation of a dense layer of particles on the top of
the HL, which may correspond to residual silica particles left by the etchant. Bar 1 mm. (c) Decalcified/stained interface showing a detailed view of the HL,
with a shag-carpet appearance on the top of the hybrid. Nano-filler is in intimate contact with these shagged collagen fibers. Bar 1 mm. (d) Another detailed
view of a decalcified/stained interface showing a HL with banded collagen fibers running parallel to the interface (compare to (c) where the fibers run
perpendicular to the interface). The transition between the HL and the unaffected dentin shows some electron-dense formations (ovals) which may correspond
to hydroxyapatite crystals not completely dissolved by the etchant and later enveloped by the adhesive. Bar 0.5 mm. (A Adhesive; C Composite resin;
H Hybrid layer; T Resin tag; D Unaffected dentin).
was monitored with a Curing Radiometer (Demetron/Kerr)
to be in excess of 500 mW/cm 2. After 24 h in distilled water
at 378C, the specimens were thermocycled for 500 cycles
between water baths held at 5 and 558C, with a dwell time in
each bath of 30 s, and a transfer time of 10 s. After thermocycling, the shear bond strengths were measured with an
Instron Universal Testing Machine, model 4411 (Instron
Co., Canton, MA), using the MTS Testworks software
(MTS Systems Co, Eden Prairie, MN) to record the data.
A knife-edge-shearing rod with a crosshead speed of
0.5 cm/min was used to load the specimens until fracture.
The distance from the probe to the dentin surface was
monitored using a spacer of two celluloid matrices. The
data were subjected to two-way ANOVA and Partial Correlation test, using the spss 8.0 for Windows (SPSS Inc.,
Chicago, IL) software package.
2.2. Transmission electron microscopy
Sixteen extracted unerupted third molars refrigerated in a
solution of 0.5% chloramine for up to three weeks after
extraction were used in this study. The occlusal enamel
was removed, and 16 dentin disks with a thickness of
800 100 mm were obtained from middle dentin by slowspeed sectioning with a water-cooled Isomet diamond saw
(Buehler Ltd.) parallel to the occlusal surface. A smear layer
was created on the top surface by wet sanding with 600-grit
silicon carbide abrasive paper for 60 s. The pulpal side of
each dentin disk was then isolated with Dentin/Enamel
Bonding Resin (Bisco Inc., Schaumburg, IL) to prevent
the organic solvents contained in the adhesives from escaping the dentinal tubules. The dentin disks were randomly
assigned to the treatment groups described for the bond
strength testing n 2 (Table 1).
After application and photo-polymerization of the respective adhesive, a 1-mm thick layer of a flowable composite
resin (Æliteflo, Bisco Inc.) was applied to the treated dentin
surface and light-cured for 40 s with an Optilux 401
(Demetron/Kerr) visible light-activation unit. A flowable
composite resin was used to facilitate ultra-microtomy and
to avoid damage to the diamond knife. The disks were then
cross-sectioned in two halves, using a water-cooled lowspeed Isomet diamond saw (Buehler Ltd.).
Four small sticks with a cross-section of 1:0 × 1:0 mm2
were cut from the center of the bonded half dentin disk using
a slow-speed Isomet diamond saw (Buehler Ltd.) under
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
315
Fig. 2. TEM resin–dentin interfaces formed by Prime&Bond NT with dentin treated with AD Gel for 15 s (Group NT-15). (a) Non-decalcified/non-stained
interface showing a NaOCl-modified HL (h). Note that this modified HL is less dense than the HL depicted in Fig. 1b for NT-0. The collagen fibers are
separated by electron lucent spaces indicating some degree of dissolution. Bar 2 mm. (b) Non-decalcified/non-stained interface showing another view of the
NaOCl-modified HL. Note that the nano-filler accumulates at the entrance of the tags to form plugs preventing the filler from penetrating the tubules
completely. Bar 2 mm. (c) Non-decalcified/non-stained interface showing another view of the NaOCl-modified HL. Note that no residual hydroxyapatite
crystals are observed in the HL in any of the fields shown in micrographs a, b, or c. Bar 2 mm. (d) Decalcified/stained interface showing a detailed view of the
transition between the HL and unaffected dentin. Electron-dense formations are identified at the transition line (ovals). This field was observed at the same
magnification used for Fig. 1d. Note that the interfibrillar spaces are wider for NT-15 than for NT-0. Bar 0.5 mm. (A Adhesive; C Composite resin;
h NaOCl-modified hybrid layer; T Resin tag; D Unaffected dentin).
water. Two sticks were partially decalcified in 10% buffered
EDTA for 72 h to facilitate ultra-microtomy, while the
remaining two sticks were left in distilled water at 48C for
72 h to control for possible artifacts during specimen
processing. After removing the sticks from the EDTA or
from water, they were immersed in 2.5% glutaraldehyde/
2% paraformaldehyde in 0.1 M sodium cacodylate buffer at
pH 7.4 for 12 h at 48C. After fixation, the bonded sticks were
rinsed with 10 ml of 0.1 M sodium cacodylate buffer at pH
7.4 for 2 h. The specimens were post-fixed with a solution of
2% osmium tetroxide in 0.1 M sodium cacodylate buffer for
1 h and washed in 0.1 M sodium cacodylate for 1 h. They
were then rinsed with deionized water four times, with a
dwell time of 5 min, and dehydrated in ascending grades of
ethanol (50% for 5 min, 70% for 5 min, 95% for 5 min and
2 × 100% for 10 min each). After the final ethanol step, the
specimens were immersed in propylene oxide for two
periods of 10 min each. The specimens were then embedded
in 50% propylene oxide/50% MedCast epoxy resin (Ted
Pella Inc., Redding, CA) in a Pelco Infiltron (Ted Pella
Inc.) rotator at 6 rpm. After 6 h of rotation, the specimens
were transferred to 100% epoxy resin at room temperature,
and placed under vacuum for 12 h to allow for resin infiltration into the specimens. Specimens were oriented in
rubber molds so the resin–dentin interface corresponding
to the central area of the dentin disk could be first exposed
for sectioning. The molds were filled with fresh
MedCast epoxy resin (Ted Pella Inc.) and left in an
incubator during 12 h at 658C. The resulting resinembedded specimen blocks were trimmed and sectioned
in an MT2-B ultra-microtome (Ivan Sorvall Inc.,
Norwalk, CT) equipped with a material-sciences type
III 3.5 mm diamond knife (Micro Star Technologies Inc.,
Huntsville, TX).
The EDTA-decalcified sticks were cut in semi-thin slices
and stained with toluidine blue under an optical microscope
to allow for the localization of the interface in each section.
After
positive
identification,
ultra-thin
sections
85 10 nm-thick were obtained, mounted on 150-mesh
nickel grids (Ted Pella Inc.), and stained with 2% uranyl
acetate for 15 min and 3% lead citrate for 10 min. For the
non-decalcified sticks, they were sectioned in ultra-thin
slices and mounted on 150-mesh nickel grids without any
staining. After drying at room temperature, the sections
316
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
Fig. 3. TEM resin–dentin interfaces formed by Prime&Bond NT with dentin treated with AD Gel for 30 s (Group NT-30). (a) Non-decalcified/non-stained
interface showing a NaOCl-modified HL (h). The collagen fibers are separated by electron lucent spaces indicating some degree of dissolution. Bar 2 mm. (b)
Decalcified/stained interface showing the accumulation of nano-filler at the entrance of one tubule (arrow). Bar 2 mm. (c) Decalcified/stained interface
showing a detailed view of the transition between the NaOCl-modified HL and the adhesive. Note the accumulation of nano-filler at the top of the NaOClmodified HL. Compare to Fig. 1c to visualize the increase of the interfibrillar spaces with the application of NaOCl. The shaggy appearance of the residual
collagen structures is well defined on the top the NaOCl-modified HL (asterisks). Bar 0.5 mm. (d) Decalcified/stained interface showing a detailed view of
the transition between the NaOCl-modified HL and unaffected dentin. Electron-dense formations are identified at the transition line (ovals). This field was
observed at the same magnification as Fig. 2d, which allows for the observation of the increase in interfibrillar spaces. Bar 0.5 mm. (A Adhesive; C
Composite resin; h NaOCl-modified hybrid layer; T Resin tag; D Unaffected dentin).
(decalcified and non-decalcified) were analyzed under a
Philips CM-12 transmission electron microscope (Philips
Electronic Instruments Inc., Mahwah, NJ) at an accelerating
voltage of 100 kV.
3. Results
The interaction between independent variables
(“adhesive system” vs. “NaOCl application time”) was not
significant p . 0:254: When controlling for the independent variable adhesive system, the partial correlation
coefficient (20.6858) revealed a statistically significant
negative correlation between the application time of AD
Gel and mean bond strengths p # 0:0001:
3.1. Bond strengths
3.2. TEM
Mean shear bonds strengths and Duncan’s rankings are
shown in Table 2 and Graph. The application of the 10%
NaOCl gel resulted in a decrease in bond strengths for both
dentin adhesives. For Single Bond, mean bond strengths
varied from 25.0 MPa for SB-0, to 9.7 MPa for SB-60 (a
reduction to 38% of the SB-0 control). For Prime&Bond
NT, mean bond strengths varied from 15.1 MPa for NT-0,
to 4.8 MPa for NT-60 (a reduction to 31% of the NT-0
control). There was a statistically significant difference
between pairs of means at p # 0:0001: For each of the
four deproteinization times, Single Bond consistently
resulted in statistically higher bond strengths than Prime&
Bond NT.
Table 2 displays the principal morphological characteristics of the resin–dentin interfaces for the different
groups. The application of AD Gel resulted in distinct
morphology for each one of the dentin adhesives tested,
and resulted in an increase in the depth of the HL.
For NT-0 (Fig. 1), the resin–dentin interface showed
a very dense HL with fibers running either parallel or
perpendicular to the interface, which was probably a consequence of the sectioning plan. Collagen cross banding was
observed when fibers were aligned along their long axis
(Fig. 1d). The nano-filler in the adhesive was filtered into
the HL, but only in selected areas where the tubule openings
were widely exposed. No residual mineral crystals were
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
317
Fig. 4. TEM resin–dentin interfaces formed by Prime&Bond NT with dentin treated with AD Gel for 60 s (Group NT-60). (a) Non-decalcified/non-stained
interface showing a NaOCl-modified HL (h). Note that this modified HL is collapsed and without any morphological features. Compare with Fig. 3a to
visualize the difference in penetration depth between NT-30 and NT-60. Bar 2 mm. (b) A higher magnification of the micrograph shown in Fig. 1a. Note
some residual collagen filaments inside the NaOCl-modified HL. Bar 0.25 mm. (c) Non-decalcified/non-stained interface showing a NaOCl-modified HL (h)
in one specific area of the specimen depicted in (a) and (b) Note the wrinkled appearance, with scattered collagen residues, and an electron dense membrane
separating this area from the nano-filler in the adhesive (arrows). In this particular area of the specimen, the NaOCl gel resulted in a more fibrillar morphology
than in other areas of this specimen. The top of the NaOCl-modified HL is visibly more digested than the collagen in group NT-30 (compare with Fig. 3c and d
in group NT-30). Bar 0.5mm. (d) Decalcified/stained interface showing a characterless NaOCl-modified HL in another specimen. The role of the EDTA
decalcification during specimen preparation in the genesis of this amorphous structure can not be ruled out. Bar 1 mm. (A Adhesive; C Composite resin;
h NaOCl-modified hybrid layer; T Resin tag; D Unaffected dentin).
observed within the HL in non-decalcified specimens. The
general appearance of the HL was maintained for deproteinization times of 15 and 30 s, however with wider interfibrillar spaces (Fig. 2a and b). When the NaOCl gel was
applied for 60 s, the morphological appearance of the HL
varied drastically. While remnants of the collagen fibers
were observed in one of the dentin disks, the other specimen
showed an amorphous structure without any discernible
morphological features.
For SB-0, the HL was electron-dense at the top, and less
electron-dense at the transition to the unaffected dentin for
EDTA-decalcified specimens (Fig. 5). An electron-dense
polyalkenoic salt deposition like “floating bubbles” was
observed in the adhesive layer. The polyalkenoic salt
formed a continuum with the top of the HL. The fibers
within the HL appear swollen regardless of their crosssection axis (Fig. 5d). No residual mineral crystals were
observed within the HL in non-decalcified specimens (Fig.
5e). For groups SB-15, SB-30, and SB-60 (Figs. 6–8) the
general morphology of the collagen network was maintained, regardless of the AD Gel application time. The interfibrillar spaces increased with the application of NaOCl, to
reach a maximum range of 25–60 nm for a deproteinization
time of 60 s. Also, the accumulation of the polyalkenoate
salt was constant for all the specimens, but more apparent
for specimens subjected to EDTA-decalcification that had
been stained with heavy metals (uranyl acetate and lead
citrate). This polyalkenoate salt deposition was more
evident with increased NaOCl application time, and lined
the wall of the tubules for groups SB-30 and SB-60 (Figs.7b,
c, and 8a, b), which was not frequently observed for the
control group. The effect of NaOCl on collagen fibers was
readily observed in the area of the transition between the HL
and the unaffected dentin. When comparing Fig. 7d with
Fig. 8c, shown at similar magnifications, it is observed that
the lower area of the NaOCl-modified HL in group SB-60 (Fig.
8c) lost its fibrillar arrangement as opposed to the corresponding area in group SB-30 (Fig. 7d), which still shows a welldefined fibrillar structure. For group SB-60, the lower third of
the dentin tubules showed an accumulation of electron dense
deposits (Fig. 8a and b). This electron-density was very similar
to that of unaffected dentin, but heavier than that of the polyalkenoate salt (Fig. 8a and b).
4. Discussion
The interfacial integrity of the resin–dentin bond may
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J. Perdigão et al. / Dental Materials 16 (2000) 311–323
Fig. 5. TEM resin–dentin interfaces formed by Single Bond (Group SB-0). (a) Decalcified/stained interface showing a HL with a variable electron density
gradually fading from the top to the transition to unaffected dentin. Electron-dense polyalkenoic salt deposition like “floating bubbles” (arrows) in the adhesive
layer. Bar 3 mm. (b) Higher magnification of the HL of a decalcified/stained specimen. Bar 2 mm. (c) Non-decalcified/non-stained interface of the same
specimen shown in (b) Note that the electron-density of the HL is uniform, with well-defined collagen fibers. Also, the “floating bubbles” from the polyalkenoic
salt are not electron-dense as in (a) and (b), because they were not exposed to heavy metal staining. Bar 2 mm. (d) Close-up view of the HL in a decalcified/
stained specimen. The polyalkenoic salt intermingles with the top of the HL like a continuous structure. For Single Bond, the morphology of the collagen fibers
within the HL is different from other adhesives—fibers appear inflated regardless of their cross-section axis (compare with Fig. 1c and d). Interfibrillar spaces
are virtually absent. Bar 0.5 mm. (e) Close-up view of the HL in a non-decalcified/non-stained specimen showing the transition between the Hl and
unaffected dentin. Note that no hydroxyapatite crystals are observed within the HL. The collagen fibers within the HL continue with the fibers in the
unaffected dentin (arrows). Bar 0.25 mm. (A Adhesive; C Composite resin; H Hybrid layer; T Resin tag; D Unaffected dentin; Pk
Polyalkenoate salt).
depend upon the contribution of both inter- and peritubular
dentin [23,24]. The contribution of the peritubular part has
been more controversial. The triangular-shaped hybridization at the transition between peri- and intertubular
dentin has been described as resin-tag hybridization and
described as fundamental for reliable bonding [25,26].
Additional retention and hermetic sealing may also be
provided by the formation of microscopic resin tags in
the lateral canals that radiate from the main tubules.
Such nanometer-sized tags in lateral tubule branches are
hybridized, a phenomenon described as lateral tubule
hybridization [27].
The contribution of the HL to bond strength was reported
by Gwinnett in 1994 [28]. He hypothesized that the collagen
layer offers no quantitative contribution to the interfacial
bond strength. For some adhesive systems, such as
Prime&Bond 2.1, the removal of collagen fibers with a
standard laboratory NaOCl solution has been reported to
actually increase bond strengths [21]. This increase in
bond strengths may be the result of a more severe interaction of this laboratory-prepared NaOCl solution with
dentin, even though a recent morphological study using
FE-SEM and AFM [29] did not show any residual collagen
after the application of a laboratory solution of 5% NaOCl.
For another acetone-based adhesive, One-Step (Bisco Inc.),
the HL may not play any important role in the effectiveness
of the bonding [21,30]. For a multi-bottle adhesive system,
All-Bond 2 (Bisco Inc.), one study reported that the
presence or absence of the HL does not affect fracture
toughness of resin–dentin interfaces [20]. In the case of
All-Bond 2, a different mechanical behavior of the adhesive
interface would be expected. The Young Modulus of the
adhesive resin is 1.8 GPa, while the Young modulus of
the All-Bond 2-infiltrated HL was estimated to be 3.6 GPa
[31]. Dentin has a Young modulus in the range of 11–18 GPa
[18]. The presence of the collagen layer would presumably
allow for the establishment of an elastic gradient at the
interface.
Ideally, the intermingling of resin with acid-etched dentin
should be thoroughly accomplished from the top to the
bottom of the intertubular area [32]. That entanglement, however, may depend on several factors, such as
the aggressiveness of the acid, the post-etching moisture
conditions of the dentin surface, the type of adhesive, and its
diffusibility into the exposed micro-porous collagen network
[8,26]. It has been demonstrated that the utilization of different
phosphoric acid gels on dentin results in different thickness of
the HL. The varying thickness does not, however, affect the
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
319
Fig. 6. TEM resin–dentin interfaces formed by Single Bond with dentin treated with AD Gel for 15 s (Group SB-15). (a) Non-decalcified/non-stained interface
showing a NaOCl-modified HL (h). If compared with the specimens in Group SB-0, the morphological differences are not observed at this magnification. The
accumulation of the polyalkenoic salt is visible at the top of the NaOCl-modified HL. Bar 2 mm. (b) Non-decalcified/non-stained interface of the second
specimen. At this magnification, some differences are already discernible between the morphology of the NaOCl-modified HL (h) and that of the HL (H) in
group SB-0 (compare with Fig. 5c). In SB-15, the collagen fibers are thinner and arranged more randomly. Bar 2 mm. (c) Decalcified/stained interface
showing a NaOCl-modified HL with wider interfibrillar spaces than those observed for group SB-0. The surface of the HL is coated with the polyalkenoate salt
precipitate. Bar 1 mm. (d) Higher magnification of the NaOCl-modified HL in a decalcified/stained specimen. If compared with Fig. 5d, one can easily
discern the increase in the interfibrillar spaces. Bar 1 mm. (A Adhesive; C Composite resin; h NaOCl-modified hybrid layer; T Resin tag; D
Unaffected dentin; Pk Polyalkenoate salt).
bond strengths associated with one-bottle adhesive systems
when dentin is etched for 15 s [33]. Likewise, dentin bond
strengths associated with Single Bond do not depend upon
the etching time [34]. In the present study, treatment of the
collagen with NaOCl resulted in a decrease in bond
strengths in spite of a deeper penetration of the adhesive.
Consequently, while the depth of dentin demineralization
may not be an important factor for dentin adhesion, the
quality and/or integrity of the collagen available for resin
infiltration may be of fundamental importance. As a result of
presumed changes in the collagen fibrils upon application of
NaOCl, dentin bond strengths significantly decreased for
both adhesives.
One question brought up by the recent introduction of
nano-filled dentin adhesives on the market is the penetration
of filler into the HL [35]. Although a controversial issue, the
nano-filler has been described as being able to penetrate the
HL in some areas. This is particularly noticeable when
observing non-decalcified unstained interfaces like in Fig.
1b. The clinical significance of this finding remains to be
discovered.
Under the TEM, the application of AD Gel for 60 s did
not remove the collagen layer, but altered the morphological characteristics and spatial configuration of the
collagen fibrils in the HL. Overall, the deposition of the
polyalkenoate in the tubules increased with the NaOCl
Gel application time (Figs.7b, c, and 8a, b). This phenomenon may have been a result of the depletion of organic
components by the NaOCl gel below the area etched by
the acid, and consequent exposure of calcium on the
surface of the dentin unaffected by the acid. This calcium
may have attracted the polyalkenoic acid and formed a
precipitate. For group SB-60, the lower third of the dentin
tubules showed an accumulation of electron dense deposits (Fig.8a and b). This electron-density was very similar
to that of unaffected dentin, but heavier than that of the
polyalkenoate salt (Fig. 8a and b). It is speculated that
these deposits are remnants of hydroxyapatite that resulted
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J. Perdigão et al. / Dental Materials 16 (2000) 311–323
Fig. 7. TEM resin–dentin interfaces formed by Single Bond with dentin treated with AD Gel for 30 s (Group SB-30). (a) Non-decalcified/non-stained interface
showing a NaOCl-modified HL (h) that lost part of its reticular morphology when compared to Fig. 6a. The long strand of polyalkenoate salt on the top of the
NaOCl-modified HL forms a uniform coating. Bar 2 mm. (b) Decalcified/stained interface that corresponds to the non-decalcified specimen in micrograph
“a”. The collagen fibers lost their characteristic disposition observed in the control group (Fig. 5b). Note how the polyalkenoate salt forms a uniform layer on
the NaOCl-modified HL and within the tubules. Overall, the deposition of this polyalkenoate in the tubules increased with the NaOCl Gel application time.
Bar 3 mm. (c) Higher magnification of a decalcified/stained interface. The polyalkenoate salt lines the wall of the tubule. This inner lining was not frequently
observed for the control group (SB-0). Bar 1 mm. (d) Close-up view of the transition between the NaOCl-modified HL and unaffected dentin for a
decalcified/stained interface. Note that the fibrillar arrangement was not disrupted at the base of the NaOCl-modified HL. The transition to the unaffected
dentin displays an electron-dense formation that may be a result of denudation of hydroxyapatite crystals from collagen fibers and enveloping of these crystals
with resin (ovals). Bar 0.5 mm. (A Adhesive; C Composite resin; h NaOCl-modified hybrid layer; T Resin tag; D Unaffected dentin; Pk
Polyalkenoate salt).
from the proteolytic action of NaOCl on unaffected
dentin.
In the NT-60 group, most of the residual collagen was
dissolved leaving an amorphous layer, probably gelatinized
collagen (Fig. 4), but the morphology was not consistent
between specimens. This variance in morphological
results is in agreement with the difference in bond
strengths obtained with Prime&Bond NT in the present
project as compared to a previous report [35]. Similarly,
dentin bond strengths associated with Prime&Bond 2.1
applied on moist dentin vary in a wide range from 7 to
23 MPa [36–39]. This discrepancy can be attributed not
only to the variability in individual’s interpretation of
the concept of “wet-bonding technique”, but also to
differences in testing assemblies. In spite of being
consistently applied to moist dentin, the level of moisture may be different for each research experiment and
therefore may result in a wide variation in adhesion
strengths. In other words, this wet-bonding technique
is difficult to standardize clinically and thus is sensitive
to errors of inaccurate clinical handling [40,41]. This incon-
sistency makes acetone-based adhesives very technique
sensitive.
The decrease in bond strengths upon the application of
AD Gel on etched dentin might be a consequence of several
mechanisms acting individually or simultaneously:
1. Partial dissolution of intertubular collagen—The integrity of collagen fibers upon etching has been deemed
essential for obtaining reliable and durable dentin
bonds because collagen denaturation has been thought
to play a negative role in dentin adhesion [42]. In result,
some authors have advocated the use of 10% citric acid
with 3% ferric chloride as a dentin conditioner [9], as
ferric chloride could prevent etched collagen from
undergoing denaturation. Nevertheless, such preventive effect has never been substantiated with sound
research.
2. De-stabilization of the collagen molecule—Dentin
collagen, after demineralization, is in a “de-stabilized”, but not denatured state, susceptible to proteolytic degradation [43,44]. The durability of collagen
J. Perdigão et al. / Dental Materials 16 (2000) 311–323
321
Fig. 8. TEM resin–dentin interfaces formed by Single Bond with dentin treated with AD Gel for 60 s (Group SB-60). (a) Non-decalcified/non-stained interface
showing a NaOCl-modified HL (h). Note that the polyalkenoate salt deposition is not as apparent as in decalcified/stained specimens, but still arranged as
“floating bubbles” within the adhesive layer. Bar 3 mm. (b) Close up of the area shown in the lower left corner of Fig. 8a. Note the hydroxyapatite deposition
in the tubules (long arrows) and the polyalkenoate accumulation (star). The arrowheads point at residual peritubular dentin. Bar 1 mm. (c) Close-up view of
the transition between the NaOCl-modified HL and unaffected dentin for a decalcified/stained interface. Compare with Fig. 7d. The fibrillar arrangement in
group SB-60 is partially lost at the base of the NaOCl-modified HL. Also, the electron-dense formation at the transition to the unaffected dentin is wider and
more prominent for group SB-60 than for group SB-30 (arrows), which may be a result of a more severe effect of NaOCl on the hydroxyapatite crystals of
unaffected dentin. Bar 0.5 mm. (d) Close-up view of the transition between the NaOCl-modified HL and unaffected dentin for a non-decalcified/non-stained
interface. Besides the presence of loose hydroxyapatite crystals at that transition (circles), note that there is an electron-lucent halo between the NaOCl
modified HL and unaffected dentin (asterisks). This halo may correspond to the electron-dense formation shown in Fig. 8c. Bar 0.25 mm. (A Adhesive;
C Composite resin; h NaOCl-modified hybrid layer; T Resin tag; D Unaffected dentin; Pk Polyalkenoate salt).
fibers in proteolytic solutions is dependent on both
the degree of cross-linking and the extent of denaturation [45]. In the present study, the lack of
collagen banding in NaOCl-treated collagen may
have been a result of the deproteinization effects of
NaOCl on collagen fibrils de-stabilized by the etchant. Some peptide bonds in collagen fibrils become
very susceptible to cleavage after denaturation takes
place [45].
3. Volumetric contraction of NaOCl-treated dentin—It
has been recently reported that 10% NaOCl results
in a contraction of demineralized dentin up to 12%
and decreases the N/Ca ration of a dentin lesion by
at least 20% [15]. This change in mineral profile and
volumetric contraction of demineralized dentin may
be partially responsible for the decrease in bond
strengths observed in this project.
4. Changes in the crystallinity of dentin apatite upon
NaOCl treatment—X-ray ion diffraction studies
have suggested that recrystallization takes place
after NaOCl application. The apatite crystals undergo
substitution of certain ions in the crystal lattice [15].
This re-crystallization might be responsible for
changes in the surface tension of the substrate, therefore it may compromise the bonding ability of the
dentin surface.
The null hypothesis was rejected—the application of a
10% NaOCl gel for 30 or 60 s resulted in a significant
decrease in bond strengths for both adhesives. Further
studies should concentrate on the potential use of NaOCl
as a remineralizing agent for the areas of the dentin cushion
susceptible to microleakage [46,47]. By removing organic
components from dentin, NaOCl also removes mineralization inhibitors, presumably phosphoproteins, and increases
the porosity of the residual dentin [15,16]. NaOCl has been
shown to increase the mineral content of a carious lesion in
vitro, regardless of the presence of F 2 ion [15,47]. Additionally, it would be paramount to study the “etching”
effects of NaOCl on the unaffected dentin underneath the
exposed collagen network. It was clear from the present
project that the application of NaOCl induces morphological changes beyond the area corresponding to the deepest
penetration of the phosphoric acid gel.
At this point, it is still unclear what are the long-term
effects of the application of NaOCl on etched dentin in
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J. Perdigão et al. / Dental Materials 16 (2000) 311–323
vivo. Therefore, its use on a clinical setting is strongly
discouraged.
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