Vol 14, No 1, 2012 11

Collagen Cross Linking Increases Its Biodegradation

Resistance in Wet Dentin Bonding

Changqi Xu
a
/Yong Wang
b
Purpose: The biodegradation of exposed dentin collagen within the adhesive/dentin (a/d) interface is one of
the main reasons for composite restoration failures and seriously affects the durability of dental restorations. In
the present study, the objective was to investigate whether the inclusion of the cross-linking reagent (glutaralde-
hyde, GA) in the adhesive would increase collagen biodegradation resistance within the a/d interface.
Materials and Methods: The model adhesive consisted of ~60 % monomers (HEMA/bis-GMA, 45/55 wt/wt) and
~ 40 % ethanol as a solvent. 5% GA was added to the above formulation. After the dentin surfaces were etched
for 15 s with 35% phosphoric acid, rinsed with water and blotted dry, adhesives both with and without GA were
applied and polymerized by visible light for 20 s. These a/d specimens were immersed in the biodegradation
solution (prepared by adding 160 mg collagenase in 1 liter of TESCA buffer solution) for up to 30 days after pro-
ceeding with the sectioning/fracture to expose the a/d interfaces. The specimens were analyzed using SEM and
micro-Raman spectroscopy.
Results: SEM results indicated that for the adhesive without GA, there were many voids and a loss of collagen
fibrils in the a/d interface after being challenged by the biodegradation solution. The Raman spectra collected
from the interface showed that the amide I of collagen at 1667 cm
-1
obviously decreased, indicating a removal
of collagen fibrils during the degradation process. For the adhesive containing GA, the collagen fibrils within the
interface did not degrade at all, which was also confirmed by the Raman results.
Conclusion: The results corroborate the previous findings that by using the current adhesive system and wet
bonding, the collagen fibrils in the a/d interface are largely unprotected and easily undergo biodegradation. Di-
rectly including cross-linking agents in the adhesive could protect collagen fibrils from degradation in situ within
the a/d interface.
Keywords: dentin, bonding, collagen cross linking, Raman.
J Adhes Dent 2012; 14: 1118. Submitted for publication: 22.06.10; accepted for publication: 01.09.10.
doi: 10.3290/j.jad.a21494
G
enerally, the longevity of ceramic or resin-composite
dental restorations is reported to be 6 to 10 years.
8
Although many factors are responsible for shortening
the longevity, recurrent or secondary caries
7,13,17
is
thought to be one of main reasons leading to the failure
of dental restorations. Caries is defined as the decom-
position process of hydroxylapatite and dentin collagen
due to the influence of organic acids generated by oral
bacteria, such as Streptococcus mutans.
4,14
Recently,
collagenase (matrix metalloproteinases, MMPs)
7,10,21
activated by acids has been proven to be involved in the
degradation process of dentin collagen, whereas oral
bacteria do not directly destroy dentin collagen.
7
Pash-
ley et al
10
have reported that the degradation of colla-
gen in the demineralized dentin is due to host-derived
MMPs that are induced by acid etching and released
slowly over time.
Several methods have been developed to control sec-
ondary caries and improve the durability of dental restora-
tions, for instance, utilizing the fluoride ion release
26
from
dental composites to prevent caries, since F
-
can improve
apatites ability to resist acid attacks. Another method is
to use chelating agents such as MMP inhibitors to stop
the activation of collagenase surrounding carious lesions.
For example, it was reported that chlorhexidine could be
used as an antimicrobial agent for disinfection and pres-
a
Research Fellow, University of Missouri-Kansas City, School of Dentistry,
Kansas City, MO, USA. Performed experiments, contributed to data analy-
sis, co-wrote mansucript.
b
Associate Professor, University of Missouri-Kansas City, School of Dentistry,
Kansas City, MO, USA. Experimental design, wrote manuscript.
Correspondence: Dr. Yong Wang, University of Missouri-Kansas City, School
of Dentistry, 650 E. 25th St., Kansas City, MO 64108, USA. Tel: +1-816-235-
2043, Fax: +1-816-235-5524. e-mail: wangyo@umkc.edu
Xu et al
12 The Journal of Adhesive Dentistry
ervation of the dentin bond over time both in vitro and in
vivo.
6
The third alternative method is cross linking dentin
collagen. It has been reported that the application of
cross-linking agents to dentin collagen could enhance
ultimate tensile strength and stiffness.
2,3
In these stud-
ies, demineralized dentin slabs were immersed in dif-
ferent concentrations of cross-linking agents for vari-
ous lengths of time (from 10 min to 4 or 40 h). It was
found that the mechanical properties were increased
as a function of time and concentration. It is expected
that cross linking collagen with chemical agents such
as glutaraldehyde (GA) could potentially increase stabil-
ity and biodegradation resistance. However, there have
been few studies that actually evaluated the effect of
cross-linked collagen on its biodegradation resistance.
In addition, immersing demineralized dentin into the
solution containing cross-linking agents for hours is not
feasible for clinical use.
In this study, the cross-linking agent glutaraldehyde
(GA) was added in a model adhesive for delivery to the
dentin surface during bonding. It was hoped that GA would
penetrate with adhesive monomers into the demineral-
ized dentin layer after acid etching and cross link in situ
with dentin collagen. The dentin interfaces formed with
adhesives with and without GA were challenged by a bio-
degradation solution containing collagenase. The null hy-
pothesis tested was that the cross-linking agent included
in the adhesive would not cross link with demineralized
dentin collagen or increase collagen biodegradation re-
sistance within the adhesive/dentin interface.
MATERIALS AND METHODS
Model Adhesives
The monomer mixtures used in this investigation con-
sisted of 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)
phenyl]-propane (bis-GMA, Polysciences; Warrington, PA,
USA) and hydroxyethylmethacrylate (HEMA, Acros Organ-
ics; Morris Plains, NJ, USA) with a mass ratio of 55/45.
This composition is similar to that used in commercial
dentin adhesive formulations such as Single Bond Plus
(3M ESPE; St Paul, MN, USA). The solvent used with
the model resin composition was ethanol (Fisher; Fair
Lawn, NJ, USA). The following three-component visible
light photoinitiators (all from Aldrich; Milwaukee,WI,
USA) were used in this study: camphorquinone (CQ, 0.5
wt%), 2-(dimethylamino) ethyl methacrylate (DMA-EMA,
0.5 wt%) and diphenyliodonium hexafluorophosphate
(DPI-HP 1.0 wt%); the concentration of the photoinitiator
is calculated with respect to the total amount of mono-
mer. Ethanol at a concentration of 40 wt% was added
to the above mixture to make a model adhesive with
glutaraldehyde (GA) (40 wt% is the approximate con-
centration of ethanol in the commercial bis-GMA-based
adhesives). GA of 5 wt% (Polysciences) was added to
the above formulation to make a model adhesive with
GA. Shaking and sonication were required to yield well-
mixed monomer/ethanol solutions. All the chemicals in
this study were used as received.
Enzymatic Biodegradation Solution
First of all, a TESCA (Tris, EDTA, NaCl, CaCl) buffer so-
lution was prepared by adding 11.5 g TES (Tris, EDTA,
NaCl) to 900 ml distilled water; 50 mg sodium azide
and 53 mg CaCl
2
were also added, and the pH value of
the solution was adjusted with a NaOH aqueous solu-
tion to ca 7.4 at room temperature. The final volume of
the solution was adjusted to 1 liter with distilled water.
Then, 160 mg collagenase from Clostridium histolyti-
cum (EC 3.4.24.3, P/N C-0130; Sigma Chemical; St
Louis, MO, USA) with an activity of 253 CDU/mg solid
was dissolved in 1 liter of a TESCA buffer solution.
Finally, this biodegradation solution with a 40 CDU/ml
was aliquoted and stored at -20C.
Model Adhesive/Dentin Specimen Preparation
Extracted, noncarious, unerupted human third molars,
stored at 4$C in phosphate buffered saline (PBS) con-
taining 0.002% sodium azide, were used in this study.
Teeth were collected after patients informed consent
was obtained under a protocol approved by the University
of Missouri Kansas City (UMKC) adult health sciences
institutional review board. Dentin disks were prepared by
first cutting the roots at the cementoenamel junction with
a water-cooled low-speed diamond saw (Buehler; Lake
Bluff, IL, USA), then the occlusal third of the crowns was
removed by means of a second, parallel section. Dentin
disks without any enamel remnants or exposure of the
pulp chamber were prepared. Uniform standardized smear
layers were created by wet-sanding the exposed dentin
surfaces for 30 s with 600-grit silicon carbide sandpa-
per. The prepared dentin surfaces were conditioned with
35% phosphoric acid gel (3M ESPE) for 15 s, and rinsed
with water. The etched dentin surfaces were randomly
selected for treatment with model adhesives with/without
the GA cross-linking agent. The wet bonding technique
(moisture should be visible but not pooled on the surface)
was used throughout the bonding procedures. The den-
tin adhesives were photocured for 20 s by exposure to
a visible light source (Spectrum light, Dentsply; Milford,
DE, USA). The prepared specimens were stored for 24 h
in water before being sectioned and/or fractured to ex-
pose the adhesive/dentin interfaces. The prepared dentin
disks were then sectioned perpendicularly to the adhesive
surfaces. The separated halves were either sectioned
into adhesive/dentin rectangular slabs (4 mm x 2 mm x
1.5 mm) or fractured in liquid nitrogen to expose the adhe-
sive/dentin (a/d) interfaces. The a/d interfaces from the
sectioned slabs were slightly polished for 10 s with wet
600-grit SiC sandpaper. All the specimens/sections were
stored in the buffer solution for the biodegradation study.
Enzymatic Biodegradation Process of Adhesive/Dentin
Specimens
After proceeding with the sectioning/fracturing to ex-
pose the a/d interfaces, the above a/d interface speci-
mens/sections were randomly selected for the biodeg-
radation study and immersed in the biodegradation
solution described above for 30 days in an incubator.
After degradation, the specimens were rinsed three
Vol 14, No 1, 2012 13
Xu et al
times with distilled water, with each rinse lasting one
minute. The rest of the specimens in the buffer solution
were used as controls. The specimens were classified
into four groups: A), adhesive (without GA)/dentin in-
terface specimens (without biodegradation challenge);
B), adhesive (without GA)/dentin interface specimens
(with biodegradation challenge); C), adhesive (with
GA)/dentin interface specimens (without biodegrada-
tion challenge); D), adhesive (with GA)/dentin interface
specimens (biodegradation challenge).
Micro-Raman Spectroscopy
As described above, separate adhesive/dentin slabs
(including those that were stored in biodegradation solu-
tion and those that were not) were prepared for inves-
tigation using micro-Raman spectroscopy. Since the
micro-Raman spectroscopic technique is nondestructive,
these same specimens were available for analysis using
SEM. A Lab Ram HR 800 Raman spectrometer (Horiba
Jobin Yvon; Paris, France) using monochromatic radia-
tion emitted by a He-Ne laser (wavelength 632.8 nm)
was used to collect Raman spectra. It was equipped with
a confocal microscope (Olympus BX41), a piezoelectric
XYZ stage with a minimum step width of 50 nm, and an
air-cooled CCD detector of 1024 x 256 pixels. The follow-
ing parameters were used: 600 grating, 140-m confocal
hole, and 100-m slit width. Spectra were Raman-shift
frequency calibrated using known lines of silicon.
Micro-Raman spectra were acquired by focusing the
laser beam through a 100X Olympus objective to an ap-
proximately 1 m beam diameter onto the a/d speci-
mens. Spectral maping was performed at positions cor-
responding to 1-m intervals across the a/d interfaces
using the computer-controlled stage. Two consecutive
scans of spectra (with 60 s accumulation time each)
were obtained from each site. An imaging system and
high-resolution monitor enabled visual identification of
the position at which the Raman spectra were obtained.
No post processing of the data was performed.
Scanning Electron Microscopy
Following micro-Raman analysis, the specimens de-
scribed above were prepared for SEM examination.
After dehydration in different ethanol solutions (33%,
67%, 85%, 95%, 100%), the prepared specimens were
mounted on aluminum stubs and sputter coated with
ca 20 nm of gold-palladium. The specimens were then
examined at a variety of magnifications and tilt angles
in a Philips XL30 ESEM-FEG (Philips Electron Optics;
Hillsboro, OR, USA) at 10 or 15 kV.
RESULTS
Figure 1 depicts representative SEM micrographs of
the dentin interfaces with model adhesives with and
without GA. The specimens were cross sectioned and
slightly polished to create flat surfaces. In Figs 1A and
1C, the micrographs were taken from sectioned speci-
mens not exposed to the biodegradation solution; the
Fig 1 Representative
SEM micrographs of
the dentin interfaces
with model adhesives
with and without glu-
taral dehyde ( GA) .
The specimens were
cross sectioned and
slightly polished to
create flat surfaces
before immersion in
the biodegradation
solution. A) adhesive
without GA, before
degradation; B) ad-
hesive without GA,
after degradati on;
C) adhesive with GA,
before degradation;
D) adhesive with GA,
after degradation; Ad:
adhesive; D: dentin.
Before Degradation
W
i
t
h
o
u
t

G
A
W
i
t
h

G
A
After Degradation
Xu et al
14 The Journal of Adhesive Dentistry
micrographs from the specimens exposed to the biodeg-
radation solution are shown in Figs 1B and 1D. Before
biodegradation, the appearance of the interface with
adhesive containing GA appears similar to the interface
in the adhesive without GA. The margin lines for the
interfaces can be recognized in both specimens. After
biodegradation, there is almost no change in appear-
ance of the interface with the adhesive containing GA,
but the interfacial margin line disappeared. In addition,
there are gaps and/or cracks at the a/d interface when
GA was not included in the formulation.
To minimize the effect that cutting/polishing may have
on the interfacial morphology, the specimens were frozen-
fractured to expose the interfaces before immersion in
the biodegradation solution. Figure 2 depicts representa-
tive SEM micrographs of the adhesive/dentin fractured
interface specimens from four groups. The interfaces are
not readily recognizable as compared to those in Fig 1.
However, without the effect from smear layers caused
by polishing, the collagen fibrils are visible in the dentin
interfaces with both model adhesives with and without
GA before biodegradation (Figs 2A, and 2C). After biodeg-
radation, for adhesive without GA, there are many voids
in the a/d interface (marked by circles), and the collagen
fibrils were lost (Fig 2B). Nevertheless, for the adhesive
containing GA, there was almost no change in morphol-
ogy, and the collagen fibrils did not degrade at all after
immersion in the biodegradation solution (Fig 2D). By
comparing the micrographs from Figs 1 and 2, it is evident
that fracturing provides more detailed structural informa-
tion, although the surfaces are not flat. However, cutting
and polishing diminishes the morphological differences
between specimens.
To study the chemical compositional changes, the cor-
responding Raman mapping spectra of the above a/d
interfaces with and without degradation were obtained.
Representative Raman spectra from pure dentin, inter-
face, and pure adhesive are shown in Fig 3. The intense
peaks related to the adhesive occur at 1720 cm
-1
(car-
bonyl), 1609 cm
-1
(phenyl C-C), 1453 cm
-1
(CH
2
def), and
1113 cm
-1
(C-O-C). These peaks are related to methacry-
late monomers in the model adhesives.
23
In the dentin
spectrum, the peaks related to collagen occur at 1667
cm
-1
(amide I), and 1245 cm
-1
(amide III); the peak related
to minerals occurs at 961 cm
-1
(i1 PO
4
3-
).
25
The spectrum
of the interface shows the contribution from the adhesive
and dentin. Some peaks of dentin (especially collagen)
are overlapped with the peaks of model adhesive; how-
ever, the amide I peak at 1667 cm
-1
for collagen is not
covered and is still distinguishable from the interface
spectrum.
Representative micro-Raman mapping spectra of the
dentin interfaces with model adhesives without and with
GA are shown in Fig 4, which were collected before and
after biodegradation. Figure 4A represents a series of
mapping spectra acquired at 1-m intervals across the
dentin interface with an adhesive (without GA) before
biodegradation. In the first two spectra, the relative in-
tensity of the 961 cm
-1
(PO
4
3-
) peak associated with the
mineral component suggested that the second spectrum
Fig 2 Representative
SEM micrographs of
the dentin interfaces
wi th model adhe-
sives with and with-
out glutaraldehyde
(GA). The specimens
were frozen fractured
to expose the inter-
faces before putting
in the biodegradation
solution. A) adhesive
without GA, before
degradation; B) ad-
hesive without GA,
after degradati on;
C) adhesive with GA,
before degradation;
D) adhesive with GA,
after degradation; Ad:
adhesive; HL: hybrid
layer; T: resin tag.
Before Degradation
W
i
t
h
o
u
t

G
A
W
i
t
h

G
A
After Degradation
Vol 14, No 1, 2012 15
Xu et al
represented the bottom of the demineralized dentin. The
last two spectra were acquired from an adhesive. Vibra-
tional peaks associated with the adhesive and collagen
component of dentin were noted from the third to eighth
spectra, indicating that the model adhesive monomers
had penetrated into the interface and that the thickness
of the interfacial zone of adhesive/dentin was approxi-
mately 6 to 7 m. After biodegradation, the amide I peak
intensity at 1667 cm
-1
for collagen obviously decreased
across the whole interfacial zone, indicating damage/
removal of collagen fibrils during the degradation process
(Fig 4B). However, for the adhesive containing GA, there
was almost no change in the mapping spectra of the inter-
face before and after degradation (Figs 4C and 4D), which
indicated that the composition of the a/d interface did not
change after being challenged by the biodegradation solu-
tion. The collagen in the interface did not degrade, which
conformed to the SEM results.
The evidence of GA cross linking dentin collagen is
shown in Fig 5. Representative Raman spectra in the
region of 1000 to 1050 cm
-1
were selected from the in-
terfaces of the above four groups. The peak around 1001
cm
-1
is associated with phenylalanine of collagen,
22
and
the peak around 1031 cm
-1
is assigned to the pyridinium
ring vibration.
15
It has been found that a trivalent amino
acid based on a 3-hydroxypyridinium ring is a prominent
cross-linking residue in collagen.
15
The relative ratio of
1031/1001 can be used to measure the cross-linking
reaction between GA and collagen.
15
The higher the ratio
is, the greater the extent of collagen cross linking. As
compared to the spectra from specimens without GA, an
increase in peak intensity at 1031 cm
-1
was observed in
the spectra from the interfaces with adhesive contain-
ing GA (Fig 5). This peak intensity was the same before
and after biodegradation. The results indicated the GA
included in the adhesive cross linked demineralized den-
tin collagen.
Fig 3 Representative Raman spectra from the dentin, adhe-
sive and interface.
R
a
m
a
n

I
n
t
e
n
s
i
t
y
A
m
i
d
e

I
C
H
2
C
H
2
A
m
i
d
e

I
I
I
C
-
O
-
C
C
-
C

i
n

P
h
e
n
y
l
Dentin
Interface
Adhesive
1800 1600 1400 1200 1000 800
Raman Shift/cm
-1

1

P
O
4
3
-
Fig 4 Representa-
t i ve mi cr o- Raman
mappi ng spect r a
of the dentin inter-
faces wi th model
adhesives without (A
and B) and with GA
(C and D), collected
before and after bio-
degradation.
After degradation Before degradation
PO4
3-
PO4
3-
1600 1400 1200 1000 800 1600 1400 1200 1000 800
1600 1400 1200 1000 800 1600 1400 1200 1000 800
A
C
B
D
4
4
2
2
-4
-4
-2
-2
0 0
X
(

m
)
X
(

m
)
Amide I
Amide I
Dentin
Dentin
Interface
Interface
Adhesive wo GA
Adhesive wo GA
Raman Shift/cm
-1
Raman Shift/cm
-1
Raman Shift/cm
-1
Raman Shift/cm
-1
PO4
3-
4
2
-4
-2
0
X
(

m
)
Amide I
Dentin
Interface
Adhesive w GA
PO4
3-
4
2
-4
-2
0
X
(

m
)
Amide I
Dentin
Interface
Adhesive w GA
Xu et al
16 The Journal of Adhesive Dentistry
The effects of adding GA to the adhesive on the degree
of conversion and penetration of adhesive in the interface
were evaluated. Representative spectra of polymerized
adhesives with and without GA are shown in Fig 6. The
peak appearing at 1637 cm
-1
is associated with the C=C
of methylacrylate, and the peak at 1608 cm
-1
is related
to C-C in phenyl of the adhesive monomer.
9,27
The adhe-
sive degree of conversion can be calculated based on
the intensity ratio of 1637/1608,
9,27
which did not show
any difference between the adhesives with and without
GA. This indicates that adding GA to an adhesive does
not induce a negative effect on the degree of conver-
sion. However, adding GA may slightly affect the adhesive
penetration into the a/d interface. To determine the dif-
ferences in adhesive penetration, the relative intensity
ratios of 1113 cm
-1
(C-O-C, adhesive)/1667 cm
-1
(amide I,
collagen) were calculated using the spectral subtraction
technique.
23
The ratio of 1113/1667 shows a gradual de-
cline for both adhesives with and without GA as a function
of position (Fig 7). The ratio for the adhesive with GA is
slightly lower than that for the adhesive without GA, while
the difference is not significant (p > 0.05).
DISCUSSION
Current dentin bonding strategies rely on micromechani-
cal retention between collagen and infiltrated resin in the
demineralized dentin layer. The strength of interlocking
via monomers/resin penetration and entanglement of
exposed collagen fibrils depend on the quality and lon-
gevity of both resin and collagen phases. Since it has to
be formed in the presence of water (wet bonding), there
is substantial evidence to suggest that the quality of this
layer is very poor.
5,11,12,18-20,23-25
Instead of serving as a
stable connection between the bulk adhesive and subja-
cent intact dentin, the layer has been called the weakest
link in the a/d bond.
18
Results from both in vitro and in
vivo studies have indicated that the poor quality of infil-
trated resin (due to inadequate monomer/polymer con-
version, phase separation, hydrolysis) and unprotected/
exposed collagen fibrils (inducing degradation) are two
major factors inhibiting the formation of a durable a/d
bond when using current adhesive systems.
Numerous efforts have been made to improve the qual-
ity of infiltrated resin by introducing new materials or tech-
niques. Only recently, improving the stability of collagen
fibrils by cross linking has been attempted.
1,2
In these
Fig 5 Representa-
ti ve Raman spec-
tra in the region of
1000 to 1050 cm
-1
,
selected from the in-
terfaces of the above
four groups.
Fig 6 Representative spectra of polymerized adhesives with
and without GA, showing the information on the degree of con-
version.
C-C in Phenyl
C = C
Adhesive without GA
Adhesive with GA
1
6
3
7
c
m
-
1
1
6
0
8
c
m
-
1
Without GA With GA
0.932 0.009 0.936 0.008
1680 1660 1640 1620 1600 1580 1560 1540
R
a
m
a
n

I
n
t
e
n
s
i
t
y
Raman Shift/cm
-1
NH
2
+ 3CHO-(CH
2
)
3
-CHO
glutaraldehyde
collagen
pyridinium ring
1050 1040 1030 1020 1010 1000
Without GA before degradation
Without GA after degradation
With GA before degradation
With GA after degradation
R
a
m
a
n

I
n
t
e
n
s
i
t
y
Raman Shift/cm
-1
1
0
3
1
.
2

c
m
-
1
1
0
2
9
.
3

c
m
-
1
1
0
0
1
.
3

c
m
-
1
(CH
2)2
CHO
(CH
2)2
CHO
(CH
2)3
CHO N +
Vol 14, No 1, 2012 17
Xu et al
studies, dentin collagen is cross linked by immersing in
the cross-linking solution for various time periods (up to
72 h).
1,2
Although the application of cross linkers did im-
prove mechanical strength and stability of dentin collagen,
to date there has been no feasible way to clinically deliver
cross-linking agents to the dentin bonding surface, since
effective cross-linking induction usually requires a long in-
cubation period in the cross-linking solution (> 1 h). In the
previous studies, after acid etcing, the demineralized den-
tin surfaces were immersed in the respective cross-linking
solutions for 1 h,
1,16
which makes this approach clinically
unfeasible. In this study, we investigated the potential of
adding a cross-linking reagent (glutaraldehyde, GA) to the
adhesive for collagen cross linking. Our results indicated
that the cross-linking agent included in the model adhesive
would not only cross link demineralized dentin collagen in
situ, but also increase collagen biodegradation resistance
within the adhesive/dentin interface formed under wet con-
ditions. The null hypothesis was thus rejected.
In the control group (adhesive without GA), collagen
fibrils in the a/d interface were obviously degraded or
removed after immersion in the biodegradation solution
(Figs 2B and 4B). Based on the Raman spectra, collagen
in mineralized dentin did not degrade after the biodeg-
radation challenge, since it was protected by minerals
(data not shown). It is very likely that collagen fibrils in
the demin eralized dentin layer are not entirely protected
or sealed by adhesive resin, which is consistent with
our previous study.
23
In that study, we used a novel mi-
croscopic staining technique to characterize the ideal or
optimum hybrid layer as compared with the a/d interface
prepared by the wet-bonding technique. The ideal resin-
collagen hybrid structure was prepared under controlled,
optimum conditions. Using a histomorphological staining
technique, any collagen that is not encased in adhesive
resin is available for reaction with the Goldners trichrome
stain.
20,23
The results indicate that the section of the
ideal resin-collagen hybrid specimen does not pick up any
stains, but that the a/d interface always picks up stains,
indicating that the adhesive does not encapsulate the
collagen fibrils throughout the width of the demineralized
dentin. Thus, it is almost impossible to form an optimum
hybrid structure under wet bonding conditions. The results
of this study further confirmed that collagen fibrils in the
a/d interface were not protected by resin. After being chal-
lenged by the biodegradation solution, the unprotected,
exposed collagen fibrils were digested by collagenase.
In the past, the difficulty in recognizing the fact that the
collagen is not protected or sealed by infiltrated resin un-
der wet bonding conditions might be partly due to the SEM
techniques used. The most popular SEM techniques for
determining the quality of the interface have relied on mor-
phological characterization of the polished a/d specimens.
Using the polishing preparation technique, the existence of
smooth, acid-resistant interfacial layers has been consist-
ently reported for most adhesive systems. Our previous
studies have indicated that polishing the a/d interface
during specimen preparation for SEM can adversely af-
fect and even obscure the morphological detail of the a/d
specimens, which actually possess a porous interfacial
structure. The quality of the a/d interface can be easily
overestimated due to polishing.
24
In the present study,
there is almost no change in appearance of the polished
interfaces before and after challenge by the biodegrada-
tion solution, except the after degradation, there are some
cracks at the a/d interface when GA is not included in the
formulation (Fig 1). It once again shows that the polishing
techqnique could mask the morphological detail and shield
the interface from attack by the collagenase.
The artifacts just described can be avoided by using
the fracture techqnique. Before the degradation chal-
lenge, dentin collagen fibrils could be clearly observed in
the fractured interfaces (Figs 2A and 2C). After degrada-
tion, the changes in interfaces were able to be monitored
easily. It showed that collagen fibrils were degraded in or
had disappeared from the interface with adhesive (with-
out GA) (Fig 2B), but were still visible in the interface
with adhesive (containing GA) after the biodegradation
challenge (Fig 2D). These results are confirmed by the
confocal Raman mapping results (Fig 4). For the adhesive
Fig 7 Raman intensity ratios of
1113/1667 as a function of spa-
tial position across the dentin inter-
faces with adhesive containing and
adhesive not containing GA.
Collagen
A
m
i
d
e

I

a
t

1
6
6
7
C
-
O
-
C

a
t

1
1
1
3
Raman Shift/cm
-1
R
a
m
a
n

I
n
t
e
n
s
i
t
y
Adhesive
1700 1600 1100
1,2
1
0,8
0,6
0,4
0,2
0
1 2 3 4 5 6 7
Depth/um
R
a
t
i
o
n

o
f

1
1
1
3
/
1
6
6
7
Adhesive without GA
Adhesive with GA
Penetration of BIS-GMA
Xu et al
18 The Journal of Adhesive Dentistry
containing GA, there was almost no change in the amide
I peak intensity for collagen before and after degrada-
tion, indicating the preservation of collagen fibrils dur-
ing the biodegradation process (Fig 4). However, for the
adhesive without GA, there was a dramatic decrease in
the collagen amide I peak after biodegradation (Fig 4),
which indicated that collagen fibrils were removed after
being challenged by the collagenase solution. The confo-
cal Raman technique has many advantages. It not only
provides information on chemical compositional changes,
but also can detect information from the sub-surface of
the specimen due to its confocal setup. SEM is a surficial,
morphological technique. As discussed above, its results
are very sensitive to the surface preparation techniques.
Unlike SEM, the surface influence or interference from the
preparation techniques can be eliminated in the confocal
Raman studies by adjusting the focusing positions.
CONCLUSION
In summary, the results of this study corroborate the
previous findings that when using current adhesive
systems and wet bonding, the collagen fibrils in the a/d
interface are largely unprotected and easily undergo
biodegradation. Collagen cross linking shows promise
as a way to improve and preserve the durability of bond-
ing. Based on the Raman results, GA included in the
adhesive uniformly cross linked the demineralized col-
lagen (Fig 5). Cross-linked collagen fibrils survived in a
strong biodegradation solution. Directly including cross-
linking agents in the adhesive could be a good, clinically
practicable method of protecting collagen fibrils from
degradation in situ within the a/d interface, as well as
improving the durability of adhesively luted dental res-
torations. Although GA has been used in Gluma desen-
sitizer clinically, due to the toxicity concerns about GA,
the delivery of more biocompatible cross-linking agents
should be studied in the future.
ACKNOWLEDGMENTS
This investigation was supported in part by USPHS Research
Grants DE 015281 and DE 021023 from the National Institute of
Dental and Craniofacial Research, National Institutes of Health,
Bethesda, MD 20892, USA. The authors would like to acknowl-
edge the SEM technical support of Dr. Vladimer Dusevich and the
secretarial support of John Fife from UMKC School of Dentistry.
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Clinical relevance: Directly including cross-linking
agents in the adhesive could protect collagen fibrils
from degradation in situ within the adhesive/dentin
interface.
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