PhD Thesis

1. King’s College London!
Thesis submitted for the degree of
PhD
Andrea Corrado Profeta
BDS Hons
Department of Restorative Dentistry
Biomaterials Science, Biomimetics and Biophotonics (B3) Research Group
King’s College London Dental Institute
at Guy’s, King’s College and St Thomas’ Hospitals
MMXIII

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Title:Hybridisation of dental hard tissues with modified adhesive systems: therapeutic
impact of bioactive silicate compounds on bonding to dentine
Author:Andrea Corrado Profeta

3. Copyright
Copyright © 2013 by Profeta, Andrea Corrado
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Recommended Citation:
Hybridisation of dental hard tissues with modified adhesive systems:
therapeutic impact of bioactive silicate compounds on bonding to dentine.
Profeta AC.
PhD Thesis 2013. King’s College London, Strand, London WC2R 2LS,
England, United Kingdom.

4. Hybridisation of dental hard tissues
with modified adhesive systems:
therapeutic impact of bioactive silicate
compounds on bonding to dentine
Andrea Corrado Profeta
Bachelor of Dental Surgery BDS Hons
Università Cattolica del Sacro Cuore (UCSC)
Class of 2006
Thesis submitted for the degree of
Doctor of Philosophy PhD in Clinical Dentistry
King’s College London (KCL)
Department of Restorative Dentistry
Biomaterials Science, Biomimetics and Biophotonics (B3) Research Group
KCL Dental Institute
at Guy’s, King’s College and St Thomas’ Hospitals
2013

5. 2
I dedicate this work to the adversities that made it so worthwhile

6. 3
Structure of the thesis, objectives and working plan
The first section of this work is a review of the literature necessary to
understand the objectives of the project; it includes general information about
dental adhesive technology as well as adhesion testing, about dentine
hybridisation and about the drawbacks of contemporary bonding systems.
Several studies revealed excellent immediate and short-term bonding
effectiveness of etch-and-rinse adhesives, yet substantial reductions in resin-
dentine bond strength occur after ageing. Degenerative phenomena involve
hydrolysis of suboptimally polymerised hydrophilic resin components and
degradation of mineral-deprived water-rich resin-sparse collagen matrices by
matrix metalloproteinases and cysteine cathepsins.
Silicate compounds, including calcium/sodium phosphosilicates, such as
commercially available bioactive glass, and calcium-silicate Portland-derived
cements are known to promote the formation of apatite in aqueous
environments that contain calcium and phosphate (e.g. saliva); thus, we have
raised questions about whether their presence at the bonded interface could
increase the in vitro durability of resin-dentine bonds through crystal formation
and self-sealing, in the presence of phosphate buffered saline or simulated
body fluid solutions.
In answering these questions, the objectives were accomplished by employing
Bioglass® 45S5 in etch-and-rinse bonding procedures either (i) included within
the composition of a resin adhesive as a tailored micro-filler, or (ii) applied
directly onto acid-etched wetted dentine. Alternative light-curable methacrylate-

7. 4
based agents containing (iii) three modified calcium-silicates derived from
ordinary Portland cement were also tested.
Confirming the relative success of bioactive materials incorporated in the
dentine bonding procedures required assessment of the potential to reduce
nano-leakage, as well as their effect upon the strength of the bond over time.
In order to explore these possibilities, which have not been previously
investigated, a combination of methods were applied in the second
experimental section. Bond strength variations were quantified using the
microtensile test while scanning electron microscopy, confocal laser scanning
microscopy and Knoop micro-indentation analysis were used to evaluate
optically and mechanically adjustments to mineral and water content within the
resin bonded-dentine interface. Initially, high microtensile values were achieved
in each tested group. All the resin-dentine interfaces created with bonding
agents containing micro-fillers showed an evident reduction of nano-leakage
and mineral deposition after the ageing period. However, only adhesive
systems containing Bioglass and two modified Portland cement-based micro-
fillers were found to reduce nano-leakage with no negative effects on bond
strength. Furthermore, specimens created with the same experimental
adhesives did not restore micro-hardness to the level of sound dentine but were
able to maintain statistically unaltered Knoop values.
The second section is also composed of a set of preliminary studies that
involved the use of up-to-date spectroscopic (attenuated total reflection Fourier
transform infrared spectroscopy) and thermoanalytical (differential scanning
calorimetry) techniques to predict the chemical-physical properties and apatite-
forming ability of the novel ion-leachable hybrid materials. Lastly, the overall

8. 5
conclusions of the present work and directions for future research are
discussed.

9. 6
Acknowledgements
According to Merriam-Webster's dictionary, adversity means “a state, condition, or
instance of serious or continued difficulty or adverse fortune” while triumph denotes “a
great victory or success.”
In any case, it is impossible to experience a sense of triumph over adversity unless you
have first stared the possibility of disaster in the face. The taste of success means little
unless you have a hint of the flavour of failure to compare it to.
Acts of great courage are only taken after terrifying fears have been acknowledged and
understood.
Against almost everyone’s predictions, this thesis is respectfully submitted to Professor
Dianne Rekow, Dean of the Dental Institute at King’s College London (KCL), and to
Professor Tim Watson, Head of the Institute’s Biomaterials, Biomimetics and
Biophotonics (B3) Research Group.
The Dental Institute at KCL is full of talented, masterful and honourable people. I am
proud to have been part of the B3 team and lucky to know so many brilliant clinicians
and scientists.
I wish past and present staff members who interacted with me throughout this project
all the best; most especially, I would like to place on record my thanks to Professors
Alistair Lax and Gordon Proctor for their direct involvement in bringing it to a successful
conclusion. Also, I would like to extend my appreciation to Dr. Richard Foxton for his
assistance in the academic and administrative requirements involved in my candidacy.
Of course I am grateful to my family for their unconditional support in everything I
choose to do and obsess over. Special mention to Agnė for helping me going through
all those years, and for so much more...
She knows the kind of pandemonium I endured in my life and that completing this work
was a pretty big deal for me. Something I am glad I experienced, but would never
welcome back again. Should somebody else ask me now, ‘Did you enjoy your PhD?’
‘Did you use your time wisely?’ I will not hand over a piece of paper with the CV and
other achievements on it to use up most of the alphabet after my name, or give an
explanation of why I might be better than others. It is not, at least for me, about looking
back or looking down, about titles, honorifics and status. I am simply going to stand up
and smile a smile which lets people know I have no regrets at all.
I was eager to be faced with all this experience had to offer, the intensity and unique
opportunity to do things at the highest level, and discover what it might show me about
myself. Unexpectedly my world was turned upside-down, my trust tested and my ego
crushed. I had to be twice as good, three times as sharp, four times as focused than all
the other PhD candidates. I had to prove myself ten times over but I never gave up and
I succeeded where others failed.
I can look at this record now and think how far I have come, and how far I have grown
and also how grateful I am for all those experiences, regardless of how difficult they
were at the time. Things I can take with me wherever I go, essential ingredients in a
better me which can never be taken away, not just material goods I own briefly. The
latin saying NIL DIFFICILE VOLENTI has certainly proved true for me and I am sure it
will hold true for anyone who believes it.

10. 7
List of contents
Structure of the thesis, objectives and working plan.............................
Acknowledgements...................................................................................
List of Figures............................................................................................
List.of Tables..............................................................................................
Section I - A review of the literature.........................................................
Chapter 1: Adhesive technology and dentine bonding
limitations................................................................................
1.1 Introduction............................................................................................
1.1.1 Coupling resin monomers to enamel...........................................
1.1.2 Adhesion to dentinal substrates...................................................
1.2 Development of dentine-resin bonding technology................................
1.2.1 Early dentine bonding agents.......................................................
1.2.2 Smear-layer removal and acid conditioning……………………….
1.2.3 Dentine hybridisation and resin-infiltrated smear-layer................
1.3 Physico-mechanical considerations of resin-bonded dentine................
1.3.1 Wettability of dentinal surfaces and contact angle.......................
1.3.2 Solubility of adhesive monomers.................................................
1.3.3 Permeability of the collagen network and
monomers diffusivity....................................................................
1.3.4 Permeability of adhesive resins and water sorption.....................
1.4 Mechanisms responsible for loss of mechanical stability.......................
3
6
14
17
19
20
21
22
23
28
29
31
32
35
36
39
41
44
47

11. 8
1.4.1 Hydrolytic degradation of dental adhesive resins.........................
1.4.2 Endogenous collagenolytic activity..............................................
1.5 Adhesion testing.....................................................................................
1.5.1 Assessment of sealing ability.......................................................
1.5.1.1 Micro-leakage and micro-permeability............................
1.5.1.2 Nano-leakage..................................................................
1.5.2 Bond strength measurement........................................................
1.5.2.1 Macro-bond strength test................................................
1.5.2.2 Micro-bond strength test.................................................
1.6 Classification of contemporary bonding systems...................................
1.6.1 Etch-and-rinse..............................................................................
1.6.2 Self-etch.......................................................................................
1.6.3 Self-adhesive...............................................................................
Chapter 2: Strategies for preventing resin-dentine bond
degradation..............................................................................
2.1 Introduction............................................................................................
2.1.1 Improvement of degree of conversion and esterase
resistance......................................................................................
2.1.2 Inhibition of enzyme-catalysed hydrolytic cleavage
of collagen.....................................................................................
2.1.3 Use of collagen cross-linking agents.............................................
2.1.4 Ethanol-wet bonding technique.....................................................
48
50
56
60
61
62
65
66
68
71
72
75
82
87
88
89
90
96
102

12. 9
2.1.5 Restoring the mineral phase of the collagen
matrix…………………...…………………………………………….
2.1.5.1 Guided tissue remineralisation.........................................
2.1.5.2 Top-down remineralisation via epitaxial growth…….……
2.1.5.3 Key objectives in the design of bioactive dentine
bonding systems..............................................................
2.2 Development of ion-releasing adhesives comprising
bioactive fillers........................................................................................
2.2.1 Calcium/sodium phosphate-phyllosilicates fillers..........................
2.2.2 Filler phase consisting of calcium silicate cements.......................
2.2.3 Dye-assisted confocal microscopy imaging of
remineralised hard tissues............................................................
2.2.4 Aims of the study...........................................................................
Section II - Experimental projects............................................................
Chapter 3: Chemical-physical properties and apatite-forming
ability of experimental dental resin cements
containing bioactive fillers.....................................................
3.1 Introduction............................................................................................
3.2 Materials and methods...........................................................................
3.2.1 Experimental micro-fillers and resin blends
formulation....................................................................................
3.2.2 Specimen preparation...................................................................
105
108
114
122
124
128
133
137
141
143
144
145
147
147
150

13. 10
3.2.3 Water sorption and solubility evaluation........................................
3.2.4 Differential scanning calorimetry (DSC)........................................
3.2.5 Statistics........................................................................................
3.2.6 ATR-FTIR spectroscopy................................................................
3.3 Results...................................................................................................
3.3.1 Water sorption and solubility evaluation.......................................
3.3.2 Differential scanning calorimetry (DSC).......................................
3.3.3 ATR-FTIR spectroscopy...............................................................
3.4 Discussion..............................................................................................
3.5 Conclusion.............................................................................................
Chapter 4: Bioactive effects of a calcium/sodium phosphosilicate
on the resin-dentine interface: a microtensile bond
strength, scanning electron microscopy, and confocal
microscopy study...................................................................
4.1 Introduction............................................................................................
4.2 Materials and methods...........................................................................
4.2.1 Specimen preparation..................................................................
4.2.2 Experimental bonding procedures and formulation
of resin adhesives.........................................................................
4.2.3 μTBS and SEM fractography and failure analysis.........................
4.2.4 Confocal microscopy ultramorphology and nano-leakage
evaluation......................................................................................
4.3 Results...................................................................................................
151
152
153
153
154
154
157
159
164
169
170
171
172
172
173
178
179
182

14. 11
4.3.1 μTBS and SEM fractography and failure analysis……..………….
4.3.2 Confocal microscopy ultramorphology and nano-leakage
evaluation.......................................................................................
4.4 Discussion..............................................................................................
4.5 Conclusion.............................................................................................
Chapter 5: Experimental etch-and-rinse adhesives doped with
calcium silicate-based micro-fillers to generate
therapeutic bioactivity within resin-dentine
interfaces.................................................................................
5.1 Introduction............................................................................................
5.2 Materials and methods...........................................................................
5.2.1 Preparation of the experimental bioactive
resin-base bonding agents............................................................
5.2.2 Specimen preparation and bonding procedures...........................
5.2.3 μTBS and SEM observations of the failed bonds..........................
5.2.4 Dye-assisted CLSM evaluation.....................................................
5.3 Results...................................................................................................
5.3.1 μTBS and SEM observations of the failed bonds..........................
5.3.2 Dye-assisted CLSM evaluation.....................................................
5.4 Discussion..............................................................................................
5.5 Conclusion.............................................................................................
182
186
189
195
196
197
199
199
203
205
206
207
207
211
216
222

15. 12
Chapter 6: In vitro micro-hardness of resin-dentine interfaces
created by etch-and-rinse adhesives comprising
bioactive fillers........................................................................
6.1 Introduction............................................................................................
6.2 Materials and methods...........................................................................
6.2.1 Teeth collection and preparation...................................................
6.2.2 Formulation of the comonomer resin
adhesive blend………………………………………………………..
6.2.3 Bioactive fillers and experimental bonding
systems.........................................................................................
6.2.4 Bonding procedures......................................................................
6.2.5 Knoop micro-hardness (KHN) analysis.........................................
6.3 Results...................................................................................................
6.3.1 Knoop micro-hardness (KHN) analysis.........................................
6.4 Discussion..............................................................................................
6.5 Conclusion.............................................................................................
Chapter 7: General discussion and conclusion......................................
7.1 Summary................................................................................................
7.2 Research contributions..........................................................................
7.3 Recommendations for future research...................................................
Bibliography...............................................................................................
223
224
226
226
226
229
230
231
234
234
237
242
243
244
249
251
254

16. 13
List of publications in international peer-reviewed journals
as a result of this work..............................................................................
List of abstracts in international conferences of dental research
from this work…............…….….….….….….…...........………....................
Appendix.....................................................................................................
325
326
327

17. 14
List of Figures
Figure 1.1 - Crystal structure of biogenic hydroxyapatite.…………................ 24
Figure 3.1 - ATR-FTIR spectra of the unmilled comonomer blend, of Bioglass®
45S5, HOPC, HPCTO and HPCMM powders and of the hybrid experimental
adhesives immediately after curing and following 60 days in DPBS……….. 162
Figure 4.1 - Schematic illustrating the experimental study design................. 176
Figure 4.2 - Schematic illustrating the composite-tooth matchsticks (1 mm)
prepared using a water-cooled diamond saw, stored in PBS for 24 h or 6
months, and then subjected to microtensile bond strength (μTBS) testing and
scanning electron microscopy failure analysis. This schematic also illustrates
how composite-tooth slabs were prepared, stored in PBS for 24 h or 6 months,
and evaluated by confocal laser scanning microscopy................................... 181
Figure 4.3 - Scanning electron microscopy images of failure modes of the resin-
bonded specimens created using the three different bonding approaches
tested.............................................................................................................. 185
Figure 4.4 - Confocal laser scanning microscopy (CLSM) images showing the
interfacial characterisation and nanoleakage, after 24 h of storage in PBS, of

18. 15
the resin-dentine interfaces created using the three different bonding
approaches tested......................................................................................... 187
Figure 4.5 - Confocal laser scanning microscopy (CLSM) images showing the
interfacial characterisation and nanoleakage, after 6 months of storage in PBS,
of the resin-dentine interfaces......................................................................... 188
Figure 5.1 - Chemical structures of the methacrylate monomers used in the
tested resin blends.......................................................................................... 201
Figure 5.2 - Schematic illustrating the resin-dentine match-sticks prepared
using a water-cooled diamond saw, stored in SBS for 24 h or 6 months, and
then subjected to microtensile bond strength (µTBS) testing and scanning
electron microscopy fractography. This schematic also illustrates how
composite-tooth slabs were prepared, stored in SBS for 24 h or 6 months,
immersed in fluorescein (nanoleakage) or Xylenol Orange (Calcium-binding
dye) and finally evaluated by confocal laser scanning microscopy
(CLSM)............................................................................................................ 204
Figure 5.3 - SEM failure analysis of debonded specimens............................ 210
Figure 5.4 - Confocal laser scanning microscopy (CLSM) single-projection
images showing the interfacial characterisation and nanoleakage, after 24 h of
storage in SBS................................................................................................ 213

19. 16
Figure 5.5 - CLSM single-projection images disclosing the fluorescent calcium-
chelators dye xylenol orange.......................................................................... 214
Figure 5.6 - Confocal laser scanning microscopy (CLSM) single-projection
images showing the interfacial characterisation and nanoleakage after 6 months
of SBS storage................................................................................................ 215
Figure 6.1 - Optical images obtained during the micro-hardness test along the
resin-dentine interface.................................................................................... 233

20. 17
List of Tables
Table 3.1 - Chemical structures of the constituent monomers and composition
(wt%) of the experimental adhesives used in this study................................ 149
Table 3.2 - Summary of maximum water uptake, solubility and net water uptake
data................................................................................................................ 156
Table 3.3 - Means and standard deviations for Tg initially, after the ageing
period and percentage change as determined by DSC
analysis.......................................................................................................... 158
Table 4.1 - Composition of the experimental bonding procedures/adhesive
systems used in this study............................................................................. 177
Table 4.2 - Means and standard deviations (SD) of the microtensile bond
strength values (MPa) obtained for the different experimental groups and
percentage distribution of failure modes after microtensile bond strength testing;
total number of beams (tested stick/pre-load failure)..................................... 184
Table 5.1 - Chemical composition (wt%) and application mode of the
experimental adhesive system used in this study.......................................... 202
Table 5.2 - Mean and standard deviation (SD) of the μTBS (MPa) to
dentine........................................................................................................... 209

21. 18
Table 6.1 - Chemical composition (wt%) of the experimental adhesive systems
used in this study........................................................................................... 228
Table 6.2 - The results of the micro-hardness measurements for each bonding
system after 24 hours and 6 months of PBS storage.................................... 236

22. 19
Section I - A review of the literature

23. 20
Chapter 1: Adhesive technology and dentine
bonding limitations

24. 21
1.1 Introduction
Adhesion or bonding is the process of forming an adhesive junction, which
consists of two materials joined together. Any event described as adhesion is
really an assembly involving a substrate (or ‘adherend’) with an applied
‘adhesive’ that creates an intervening ‘interface’. In reparative dentistry (Small,
2008), the adherends are enamel and dentine to which the adhesive is applied.
Dental adhesives are solutions of resin monomers that join a restorative
material with the tooth structure after their polymerisation is completed. While
most adhesive joints involve only two interfaces, dental adhesive joints may be
more complex such as the dentine-adhesive-composite interface of a bonded
composite direct restoration. The aim is to create a close relationship between
the dental substrate and restorative material, reproducing the natural
relationship of the dental tissues, and to protect the pulp. Biomimetics, or
imitating nature, is concerned with not only the natural appearance and
aesthetic aspects of the restorations but the way they work. To copy nature is to
understand the mechanics of the tooth, the way it looks and functions, and the
way every stress is distributed. Ideally, the interface should provide a secure
marginal seal and have the ability to withstand the stresses that have an effect
on the bonding integrity of the adhesives, in order to keep the restoration
adherent to the cavity walls. There are several sequential events that are
necessary to form an effective adhesive joint. Bonding between hard tissues of
the tooth and dental adhesive involves potential contributions from chemical
(e.g., ionic bonds), physical (e.g., van der Waals) and mechanical sources but
primarily relies on micro-mechanical interaction for success. For the
development of strong adhesion, good wetting and intimate contact between the

25. 22
adhesive and substrate, which must be clean and therefore in a high energy
state, are required. Films of water, organic debris, and/or biofilms are always
present in the clinical situation as dental surfaces that are prepared for
restorative procedures, which present contaminants that remain on them.
Consequently the steps for interface formation are the creation of a clean
surface and the generation of a rough surface for interfacial interlocking.
1.1.1 Coupling resin monomers to enamel
In 1955, Michael G. Buonocore reported the use of 85% phosphoric acid for 60
seconds in order to improve the retention of an acrylic resin on enamel
(Buonocore, 1955, Simonsen, 2002). Still today adhesion to enamel is achieved
by means of etching with 32-37% phosphoric acid, ideally in a gel form, for 15-
20 seconds. Acid etching creates microporosities, produces surface roughness,
reduces the surface tension of the prepared surface and forms facets on the
mineral crystals (Baier, 1992); this permits the hydrophilic monomers of the fluid
adhesive resin to penetrate into the micro-retentive spaces in-between or within
the enamel crystals. Accordingly, the micromechanical nature of the interaction
of dental adhesives with enamel is a result of the infiltration of resin monomers
into the microporosities left by the acid dissolution of enamel and subsequent
enveloping of the exposed hydroxyapatite (HAp) crystals with the polymerised
monomers (Swift et al., 1995). This makes it possible to obtain an adhesive-
composite-enamel bond strength able to resist a shearing force of more than 20
MPa, which is clinically remarkably effective (Swift et al., 1998).

26. 23
1.1.2 Adhesion to dentinal substrates
Whilst the adhesion to enamel, thanks to the etching technique, promptly
demonstrated its efficacy, convincing in the following years researchers and
clinicians, the same cannot be said for the adhesion to dentinal surfaces. The
quest for an adequate dentine bonding agent has been longer and even today
there is no confirmation of having attained the effectiveness which the adhesion
to enamel has demonstrated. Enamel is composed of 96% HAp (mineral) by
weight, whereas dentine contains a large percentage of organic material and
water. It has been found that its bulk chemical composition is about 50% in
volume made of mineral substance, 30% in volume formed of organic material,
and the residue 20 vol% represented by water (Marshall et al., 1997). This
tissue can be considered a biological composite that consists of a highly cross-
linked and insoluble in acids collagen matrix filled with mineral crystallites
located both within (intrafibrillar) and between (interfibrillar) collagen fibrils
(Kinney et al., 2003).
The mineral component is primarily a carbonated nanocrystalline hydroxyapatite
whose structure is far different from stoichiometric hydroxyapatite, represented
by the formula:
Me10(XO4)6Y2
Where Me is a divalent metal (Ca2+
, Sr2+
, Ba2+
, Pb2+
…), XO4 is a trivalent anion
(PO4
3-
, AsO4
3-
, VO4
3-
…), and Y is a monovalent anion (F-
, Cl-
, Br-
, I-
, OH-
…).
Given the unique mechanism involved in apatite crystal formation in biology,
biogenic apatite varies in several ways from the corresponding geologically
produced mineral.

27. 24
First, biogenic apatite has a hexagonal lattice structure, having a strong ability
to form solid solutions, and to accept numerous substitutions (Figure 1.1).
Figure 1.1 - Crystal structure of biogenic hydroxyapatite.
These substitutions affect the apatitic lattice parameters: the crystal size is
decreased, and thereby the surface area is increased compared to
stoichiometric HAp, thus permitting additional adsorption of ions and molecules
on the apatite surface (LeGeros, 1991).
Biological apatite contains in fact various trace elements from intrinsic or
extrinsic origins, namely significant carbonate substitutions, OH-
deficiencies,
and imperfections in the crystal lattice (Boskey, 2007). This phenomenon

28. 25
provides certain physico-chemical, biological, functional, and chemical features
important in the formation and dissolution of the crystals in dental tissues. For
example, F-
ions are readily incorporated into dental apatite, forming
fluoroapatite, a less soluble phase of calcium phosphate as compared to HAp,
confering to enamel its low dissolution properties to resist acidic attacks.
Likewise, trace elements present in extracellular fluids may have a specific role
on mineral quality and condition.
With respect to dentine apatite structure, this is represented by numerous
substitutions (i) by hydrogenophosphate (HPO4
2-
) of XO4 groups and (ii) by
carbonate (CO3
2-
) of Y2 and XO4 groups.
Finally, biological minerals tend to attain high crystallinity and a more organised
structure on the time scale of days or months rather than years (Verdelis et al.,
2007).
The dentine matrix is mainly composed of type-I collagen fibrils with associated
noncollagenous proteins, to form a three-dimensional matrix that is reinforced
by the apatite crystals (Marshall et al., 1997).
Collagen microfibrils are described as those strands of collagen that are 5-10
nm in diameter, collagen fibrils are bundles of microfibrils that are 50-100 nm in
diameter, and collagen fibres are bundles or networks of fibrils that are
approximately 0.5-1 μm thick (Eick et al., 1997).
This mineral-reinforced fibril composite is described by Weiner and Wagner as
containing parallel platelike HAp crystals with their c-axis aligned with the long
axis of the fibril (Weiner and Wagner, 1998). The location of these crystals in
the fibril was demonstrated in a study by Traub and co-workers that showed

29. 26
that mineralised collagen fibrils had the same banded pattern as negatively
stained collagen fibrils (Traub et al., 1989).
This indicated that mineral is concentrated in the hole zones of the fibril. It was
proposed that these mineral platelets were arranged in parallel like a stack of
cards within the interstices of the fibril (Palmer et al., 2008).
The quality of dentine is dependent upon the total sum of characteristics of the
tissue that influence its competence: microstructure, mineral density and
especially the particular location of the mineral with respect to organic
structures of the tissue. From a microstructural perspective, the collagen fibrils
in dentine serve as a scaffold for mineral crystallites that reinforce the matrix,
supporting the surrounding enamel. This microstructure suggests the necessity
of a hierarchical approach to the understanding of its mechanical properties
(Kinney et al., 2003).
The mineral component incorporates oriented tubules that run continuously
from the dentine-enamel junction (DEJ) to the pulp in coronal dentine, and from
the cementum-dentine junction (CEJ) to the pulp canal in the root. Each tubule
is encased in a collar of highly mineralised dentine, called peritubular dentine,
embedded in the intertubular matrix (Marshall, 1993).
These tubes are elongated cones with their largest diameters (ca. 3.0 µm) at
the pulp and their smallest diameters (ca. 0.8 µm) at the DEJ, and are filled with
a liquid that flows inside, with a pressure of about 20 mm of Hg (Van Hassel,
1971). The quantity of the tubules decreases from about 45,000 per mm2
in the
proximity of the pulp to about 20,000 per mm2
near the dentino-enamel junction.
As they converge on the pulp chamber, the surface area of the intertubular
dentine diminishes while the tubule density augments, from about 1.9 x 106

30. 27
tubules/cm2
at the DEJ to between 4.5 x 106 and 6.5 x 106 tubules/cm2
at the
dentine-pulp edge (Garberoglio and Brännström, 1976). This humid and organic
nature of dentine makes it very challenging to bond to and have an effect on the
integrity of the tooth-adhesive side of the interface. A peculiarity of dentine is
the presence of the dentinal fluid in the tubular constitution that couples the pulp
with the enamel-dentinal junction (EDJ). As stated by the hydrodynamic theory
(Neill, 1838, Gysi, 1900, Brannström and Aström, 1972), when the enamel is
lost and the dentine is exposed, external stimuli cause fluid shifts across the
dentine which activate pulpal nerves and cause pain. This fluid flux within
tubules, accountable for dentine sensitivity (Pashley et al., 1993), is also
responsible for the persistent wetness of exposed dentine surfaces due to the
outward fluid movement from the pulp, which may influence the quality of the
adhesive-dentine interface and may decrease the bond strength between resins
and dentine (Sauro et al., 2007). Furthermore, the increasing number of tubules
with depth and, consequently, the increment in dentine wetness, can make
bonding to deeper dentine even more difficult than to superficial dentine.
The fluid movement in the dentinal tubules under the influence of pulpal
pressure may in fact interfere with the penetration of the adhesive into the
conditioned dentine surface (Chersoni et al., 2004), as well as causing
deterioration of the adhesive interface with time. Another characteristic of
dentine is the presence of a coating of debris produced with mechanical
preparation, called smear-layer, consisting of shattered and crushed HAp, as
well as fragmented and denatured collagen that is contaminated by bacteria
and saliva (Brännström et al., 1981). It is revealed by scanning electron
microscopy (SEM) as a 1-2 μm adherent surface with a mainly granular

31. 28
substructure that varies in roughness, density and degree of attachment to the
underlying tooth structure according to the surface preparation (Pashley et al.,
1988). While cutting dentine, the heat and shear forces produced by the rotary
movement of the bur cause this debris to compact and aggregate. The orifices
of the dentinal tubules are obstructed by debris tags, called smear-plugs, that
are contiguous with the smear-layer and may extend into the tubules to a depth
of 1-10 μm (Prati et al., 1993).
The application of acidic agents opens the pathway for the diffusion of
monomers into the collagen network, but it also facilitates the outward seepage
of tubular fluid from the pulp to the dentine surface, leading to a deterioration in
the bonding effectiveness of some of the current adhesives. After the HAp
crystals have been removed, it is quite challenging to also maintain the spaces
created between collagen fibrils to allow monomers to diffuse into the substrate.
The demineralised dentinal matrix can actually easily collapse if the matrix
peptides, including collagen, are denatured during the conditioning, causing a
decrease in the interfibril spacing and a loss of permeability to resin monomers
(Nakabayashi et al., 1982).
1.2 Development of dentine-resin bonding technology
In the developments of dental adhesives several attempts have been made to
provide a stronger and more reliable bond as well as simplifying the clinical
procedures. These attempts have resulted in the introduction of different
generations of bonding systems which are different in chemistry, mechanism,
number of bottles, application techniques and clinical effectiveness.
In general, dentine bonding agents all contain similar ingredients, namely cross-

32. 29
linking agents, bifunctional monomers, organic solvents, curing initiators,
inhibitors or stabilisers, and sometimes inorganic filler particles.
Whereas cross-linkers have two polymerisable groups (vinyl-groups or -CQC-)
or more, functional monomers commonly have only one polymerisable group
and a functional group, which can serve different purposes, such as enhancing
wetting of dentine. Bifunctional monomers have in fact (meth)acrylate functions
at one end, in order to provide covalent bonds with the composite monomers,
and the so-called functional group, usually carboxyl, phosphate, or phosphonate
at the other end which will impart monomer-specific functions (Van Landuyt et
al., 2008).
1.2.1 Early dentine bonding agents
Since calcium is abundant in dentine, the earliest dentine bonding formulas
attempted to chemically bond to dentine by ionic bonds to this alkaline metal.
The first adhesive resin system was created and manufactured at the
Amalgamated Dental Company, England, UK, by a Swiss chemist called Oscar
Hagger: it was composed of glycerophosphoric acid dimethacrylate (GPDM)
and it was made available on the market as Sevriton Cavity Seal (The
Amalgamated Dental Company, Ltd, London, UK) (Haggar, 1951). Kramer and
McLean (1952) were among the first to investigate the bonding ability of this
material to dentine (Kramer and Mc Lean, 1952). The dentine bonded with this
adhesive system was observed by light microscopy: during the histologic
examination they demonstrated altered staining of the bonded subsurface which
took up haematoxylin more readily than did the control surfaces. It was
supposed that the resin-primer had altered the dentine. This study was followed

33. 30
by a work of Buonocore and co-workers (Buonocore and Quigley, 1958), who
etched dentine with 7% hydrochloric acid and then applied GPDM bonding
resin. These attempts were unsuccessful because of limitations in the adhesive
monomer formulations and a general lack of knowledge of dentine as a bonding
substrate. It was demonstrated that there was little evidence for the formation of
chemical bonds between resins and dentine. A few years later, coincident with
an expansion of knowledge in this area, considerable advances were made in
adhesive monomer formulations to improve resin penetration into the tissue
matrix. The development of a cross-linking dimethacrylate 2,2-bis[4(2-hydroxy-
3-methacryloyloxy-propyloxy)-phenyl] propane (Bis-GMA) reinvigorated the
research on adhesion to dentine (Bowen, 1963). Dentine adhesive products
such as Dentin Adhesit (Vivadent, Schaan, Liechtenstein), Scotchbond Dual-
Cure (3M Dental Products, St. Paul, MN, USA), Prisma Universal Bond
(Caulk/Dentsply, Milford, DE, USA) and Bondlite (Kerr, Danburry, CT, USA) did
not remove the smear-layer prior to resin application but were applied directly
to smear-layer covered dentine. The presence of smear-layer on ground
dentinal surfaces greatly reduced the permeability of tubular (Pashley, 1991)
and intertubular dentine (Watanabe et al., 1994); for this reason the resin was
unable to penetrate profoundly enough to establish a bond with intact dentine,
and hence gave very low bond strength values (ca. 3-7 MPa) (Eick et al.).
Examination of both parts of the failed resin-dentine bonds employing scanning
electron microscopy (SEM) revealed smear-layer that split into upper and lower
halves on each side. Thus, the “bond strength” was not really a measure of
bonding, but measured the strength of the cohesive forces holding smear-layer
particles together (Pashley, 1991). The actual interfacial bond strength between

34. 31
the resin and the uppermost part of the smear-layer was higher by an unknown
amount, because it did not fail.
1.2.2 Smear-layer removal and acid conditioning
Despite widespread scepticism among the dental academic community, T.
Fusayama proposed in 1980 to remove the smear-layer along with the
underlying smear-plugs that prevented resin tag formation (Fusayama, 1980).
Acid etching permitted demineralisation of the top 5-10 µm of the underlying
sound dentine allowing dentinal tubules to receive micro-tags of resin and
represented an important innovation which paved the way for the modern
concepts of dentine bonding. However, even smear-layer removal was
insufficient for high resin-dentine bond strength. The technique fell short of
expectations in practice because of the intratubular fluid’s pressure that makes
the dentine extremely humid, especially in deep cavities where a large number
of wider dentinal tubules are exposed. This phenomenon inhibited the
hydrophobic resin to efficiently adhere to the dentinal substrate and water was
regarded as a contaminant. As a result, these bonding agents required severe
air-drying of the dentine surface before application. The outcome of this
manoeuvre was frequently a layer so thin that atmospheric oxygen inhibited
their polymerisation (Erickson, 1989). Air-drying led to the evaporation of water
maintaining the collagen network expanded and its collapse due to surface
tension forces. The spaces between the collapsed collagen fibrils were
therefore greatly reduced and with this the permeability of intertubular dentine to
adhesive resins (Pashley et al., 1995a). What was also required was stripping
the mineral phase from the collagen fibrillar matrix of dentine and keeping it as

35. 32
expanded as possible in order to produce a large increase in surface and
subsurface porosity. If monomers could have infiltrated this mesh-work and
coated the fibrils with polymerised resin to improve micromechanical retention,
they would have produced high bond strength. For the mentioned reasons and
in view of the fact that a water-free environment is unachievable during clinical
procedures, dentine bonding agents were reformulated in a more hydrophilic
blend (Nakabayashi and Takarada, 1992). The introduction of low molecular
weight monomers called primers, such as 4-methacryloxyethyl-trimellitic
anhydride (4-META) and 2-hydroxyethylmethacrylate (HEMA), containing
bifunctional groups - a hydrophilic functional group with a high affinity for the
aqueous dentinal substrate, and a hydrophobic functional group having high
affinity for the bonding adhesive - along with the etch-and-rinse technique
enhanced the strength of the adhesion to dentine and provided reliable
resin/dentine bond strengths (Barkmeier and Cooley, 1992). Furthermore,
leaving the dentine wet made it possible to preserve porosity necessary for
primer penetration in the demineralised spaces (Tay et al., 1995) and led to the
formation of a acid resistant layer consisting of polymerisable hydrophilic
monomers and exposed collagen fibrils (Nakabayashi and Takarada, 1992).
1.2.3 Dentine hybridisation and resin-infiltrated smear-layer
Nakabayashi and his colleagues were the first to use transmission electron
microscopy (TEM) with sufficient resolution to show the penetration of resin
nano-tags into the demineralised dentine matrix to create an entirely new
biomaterial that was half collagen fibrils and half resin. It was neither resin nor
dentine but a hybrid of the two, and so was called hybrid layer (Nakabayashi

36. 33
and Pashley, 1998). Hybridisation thoroughly modified the physico-chemical
properties of tooth surfaces and subsurfaces and was considered a form of
tissue engineering.
With the introduction of the hybrid layer, many clinicians believed that the
mechanism of bonding had been solved. Instead, the complexities of bonding
became apparent when the same adhesive agents produced hybrid layers of
different thicknesses depending on dentine depth and dentine condition. Hybrid
layer formation was the major bonding mechanism in superficial dentine, which
incorporates fewer tubules than deep dentine, due to the amount of intertubular
dentine present in this area with little contribution from resin tags. Whereas, in
deep dentine, resin micro-tag formation remained accountable for the bond
strength, with a reduced contribution of the hybrid layer due to the limited
amount of intertubular dentine available, as the tubules become larger and
closer together.
Nano-tags seemed to be much more important to overall retention (Marshall et
al., 2010) increasing the bond strengths to 32 MPa, concurring for a better
marginal seal and acting as an elastic cushion that, thanks to its elasticity
(modulus of elasticity 3.4 GPa), was able to moderate the polymerisation
shrinkage stress of the restorative composite (Wang and Spencer, 2003).
Although the smear-layer is regarded a limiting factor in achieving high bond
strengths, nowadays it can also be considered as a bonding substrate thanks to
the development of smear-layer incorporating systems called self-etch. This
was realised by raising the amount of acidic monomers and adding 20-30%
acidic methacrylates (pH 1.9-2.8) to 20% water, 20% ethanol, 30% HEMA or
dimethacrylates. Self-etch adhesives contain high concentrations of water and

37. 34
acidic monomers (Watanabe et al., 1994). Water is a necessary ingredient
required to ionise the acidic monomers so that they can etch through smear-
layers into the underlying hard tissues (Tay et al., 2002b). Its presence entailed
the use of water-miscible hydrophilic comonomers (e.g. HEMA) and/or acetone
or ethanol as a solvent to prevent phase changes from occurring (Van Landuyt
et al., 2005). Smear-layer covered dentine is substantially drier than acid-etched
dentine. Smear-layer and smear-plugs being present, the transdentinal
permeability is greatly reduced and no significant wetness is present on the
dentine surface (Pashley, 1989). All the same, self-etching bonding systems are
applied to smear-layer covered dentine under dry conditions, since they contain
their own water. Combining acidic conditioners and resin primers did not require
a separate etch-and-rinse phase and made these agents able to simultaneously
condition and prime enamel and dentine (Chigira et al., 1994). Self-etching
systems interact very superficially with the smear-layer and the underlying
dentine. They can easily penetrate 1-2 μm of smear-layers but their penetration
is restricted just to 0.5 μm into the top portion of the underlying intact dentinal
matrix (Watanabe et al., 1994). This is due, in part, to the fact that the acidity is
partially buffered by the smear-layer during comonomer penetration (Reis et al.,
2004), and because the underlying mineralised dentine is less porous, and
hence less permeable, than smear-layers. Water is also useful for solubilising
the calcium and phosphate ions that are liberated by the etching. These ions,
released from apatite crystallites during self-etching, get incorporated into the
water of the adhesive blend or precipitate as calcium phosphates, which
become dispersed within the comonomers in the interfibrillar spaces. Some of
the calcium ions may also associate with the acidic monomers as calcium salts.

38. 35
This mixture fills the interfibrillar spaces and some free ions may still diffuse up
into the overlying adhesive layer (Bayle et al., 2007). The primed surfaces are
not rinsed with water, leaving the dissolved smear-layer and demineralisation
products to reprecipitate within the diffusion channels created by the acid
primers. Compared with etch-and-rinse adhesives, many advantages have
been attributed to self-etch adhesives. It has been suggested that they improve
the efficiency of clinical procedures by omitting the obligatory rinse phase in
etch-and-rinse adhesives and thus reducing the chairside time. Conditioning,
rinsing and drying steps, which may be critical and difficult to standardise in
clinical conditions, are eliminated in self-etch adhesives. Technique sensitivity
correlated with bonding to dehydrated demineralised dentine is eliminated, as
rinsing and drying phases are no longer needed. Since monomers infiltrate
concomitantly as they demineralise, the collapse of the collagen network is
prevented (Peumans et al., 2005). For the same reason, incomplete resin
infiltration should be avoided. As the smear-layer and smear-plugs are not
removed before the actual bonding procedure, rewetting of dentine by dentinal
fluid should be disallowed too (Van Meerbeek et al., 2005). However, some
leakage observations in the hybrid layer, and especially beyond the hybrid
layer, have shed doubt on the concept that self-etch adhesives guarantee
complete resin infiltration (Carvalho et al., 2005).
1.3 Physico-mechanical considerations of resin-bonded dentine
One factor that could be easily overlooked is the requirement for the bonding
system to act as a means of transferring load from one part of a structure to
another. This generates stresses and strains within the resin-bonded dentine

39. 36
and it is important that the adhesive has the necessary physico-mechanical
properties to withstand these stresses and strains. Thus, the assessment of a
bonding system should be based on its ability to carry load and contribute to the
structural integrity of the whole unit.
The durability of the resin-dentine bond is related to the depth of
demineralisation versus the depth of monomer penetration, and the ability of the
polymer not only to envelope each fibril but also to do so without leaving any
gap or space between the resin and the fibril. That is, the resin-infiltrated layer
must be free of any porosity or defects that can act as stress raisers under
function or permit hydrolysis of collagen fibrils (Nakabayashi et al., 1982).
1.3.1 Wettability of dentinal surfaces and contact angle
Wetting is a general term used to indicate the ability of a liquid to come into
intimate contact with a solid substrate and to maintain contact with it. The
balance between adhesive and cohesive forces dictates the degree of wetting
(wettability). Adhesive forces between the liquid and the solid cause a drop to
spread across, whereas cohesive forces within the liquid cause the drop to ball
up and avoid contact with the surface. If a liquid can spread across a surface, it
is said to “wet the surface”. This wetting ability of a liquid for a surface is usually
characterised by measuring the contact angle (resultant between adhesive and
cohesive forces) of a droplet on the surface.
Resin contact angle measurement on dentine provides information on the
interaction between adhesives and dentine, and it also indicates the affinity of
dentine for the adhesive resin (Rosales-Leal et al., 2001).

40. 37
Low contact angles imply good wetting while a contact angle greater than 90°
usually means that wetting of the surface is unfavourable: the fluid does not
spread over a large area of the surface but tends to minimise contact with it
forming a compact liquid droplet. The tendency of a drop to spread out over a
flat, solid surface hence increases as the contact angle decreases. For water, a
wettable surface may also be termed hydrophilic and a non-wettable surface
hydrophobic. Superhydrophobic surfaces have contact angles greater than
150°, showing almost no contact between the liquid drop and the surface (Feng
et al., 2002).
Wettability of dentine is an important topic to take into consideration as good
spreading of monomers on this tissue is very important for successful bonding.
For a liquid to spread uniformly across a solid surface, the surface tension of
the liquid must be less than the free surface energy of the substrate. Substrates
for bonding may present low or high surface energy. HAp is a high-energy
substrate while collagen has a low-energy surface (Akinmade and Nicholson,
1993). Accordingly, acid etching increases the surface energy of enamel but
decreases that of dentine. Unlike enamel, acid-etched dentine does not
increase its surface energy to facilitate spreading of adhesive resins (Attal et al.,
1994). Thus, for hybridisation of demineralised dentine with resin to occur, it is
necessary to match the surface tension of the primer with that of the
demineralised dentinal surface, depending on whether it is wet or dry.
Commonly used bonding monomers such as HEMA have excellent spreading
properties (Bowen et al., 1996) and could be considered to be surface-active
comonomers (Rosales-Leal et al., 2001). That is, they are considered to

41. 38
improve the ability of the monomers to wet the surface of acid-etched dentinal
substrate.
Wetting of the surface of dentine by monomers is a necessary initial step in
bonding, but it alone is not sufficient to establish a successful bond, because it
does not guarantee monomer penetration into the subsurface. The permeability
of the demineralised intertubular dentinal network to monomers is a critical
variable in dentine bonding (Nakabayashi and Takarada, 1992). To attain
intimate association between resin monomers and collagen fibrils, the primers
and bonding agents must be able to “wet” the collagen fibrils. If the fibril is
enveloped by water, the monomers must be able to successfully compete with
water for the fibril surface.
Barbosa and collaborators found that dentine permeability was also intensified
by the removal of organic materials (Barbosa et al., 1994). Sodium hypochlorite
(NaOCl) is a well-known nonspecific proteolytic agent and its collagen removal
ability after acid conditioning has been evaluated (Wakabayashi et al., 1994).
After NaOCl treatment, the extent to which the primer wets the dentine surface
is increased because the interactions between the primer and the deproteinised
dentine are greater than before (Toledano et al., 2002). Deproteinisation leads
to a hydrophilic surface (Attal et al., 1994) and eliminates the exposed collagen
fibres. Besides, dentine becomes a porous structure with multiple irregularities
which allows good mechanical retention (Vargas et al., 1997). However,
complete removal of the collagen matrix with NaOCl as an adjunctive step of
restorative and adhesive dentistry is still a subject for debate. Sauro et al.
(Sauro et al., 2009a) evaluated the efficacy of a 12% w/v NaOCl solution for
complete removal of exposed collagen matrices from acid-etched dentine

42. 39
surfaces within a maximum clinically possible period of 120 seconds and a
longer period of application (10 minutes) using confocal reflection/immuno-
fluorescence microscopy and ESEM. An extended period (45 minutes) of
NaOCl application was also performed as a negative control. This study
demonstrated that complete removal of the exposed collagen matrix from the
etched dentine surface can be achieved by applying a 12% w/v NaOCl solution,
but at this concentration, it required a far longer reaction time than is clinically
acceptable.
1.3.2 Solubility of adhesive monomers
Solubility is the property of a substance called solute to dissolve in a liquid
solvent to form a homogeneous solution. It is measured as the saturation
concentration where adding more solute does not increase the concentration of
the solution.
The term “solubility parameter” was first used in dentistry by Asmussen
(Asmussen et al., 1991). They regarded demineralised collagen as a porous
solid polymer and reasoned that for primers to penetrate demineralised dentine,
the primer should have a solubility parameter that is similar to the polymeric
substrate, as is generally true in polymer chemistry.
The concept was extended to Hansen’s triple solubility parameters so as to
calculate the relative contribution of dispersive force (δd), polar force (δp),
hydrogen bonding force (δh), and the total cohesive energy density of adhesive
(δt). As Hoy’s triple solubility parameters are more widely used on dentine
bonds, chemical structures modify the calculated Hoy’s triple solubility
parameters for δd, δp, δh and δt (Mai et al., 2009).

43. 40
Solubility parameter calculations have been used to quantify the degrees of
hydrophilicity of polymers, important for the adhesive penetration into exposed
collagen fibrils, and predict dentine-adhesive bond strengths (Asmussen and
Uno, 1993).
When a primer that has a low solubility in water is applied to moist
demineralised dentine, the result is a limited distribution of the monomers into
the water-filled three-dimensional network between the collagen fibrils, with a
consequent low bond strength. Some hydrophilic monomers, such as HEMA,
are very solubile in either water or acetone. Replacing water in the spaces
around collagen fibrils, HEMA acts like a polymerisable solvent for the adhesive
monomers placed thereafter. The uptake of adhesive monomers into these
nano-spaces is contingent on their solubility in the solvent that occupies the
spaces, hence this theory is very useful in predicting how miscible monomers
should be in demineralised matrices saturated with various solvents (Sadek et
al., 2007). Furthermore, the diffusion of the monomers is also determined by the
size of the spaces between collagen fibrils and by the depth that they must
reach from the surface. Wet demineralised dentine exhibits a fully expanded
collagen network that offers maximal volumes between its fibrils. Under similar
conditions, the bonding substrate has high permeability. At the other extreme,
when there are no spaces between the collagen fibrils, as in air-dried, fully
collapsed dentine (Carvalho et al., 1996), the permeability to monomer is
extremely low. The ideal condition exists when there is both high permeability of
the substrate (dentine) and high diffusivity of the solute (resin monomer)
(Nakabayashi and Takarada, 1992).

44. 41
Unfortunately, many adhesive monomers are not very soluble in water. That is
why marketed adhesives are generally solvated in ethanol or acetone. When
solvated adhesives are placed on water-saturated acid-etched dentine, their
solvents attempt to penetrate into the water-filled spaces and some of the water
in these spaces diffuses into the solvent. This culminates in too little solvent
remaining in the infiltrating adhesive with the capacity to keep hydrophobic
dimethacrylates like BisGMA (2,2-bis [4-(2-hydroxy-3-methacryloyloxypropoxy)]
- phenyl propane) in solution. The net result is partial penetration of BisGMA
into water-saturated matrices (Spencer and Wang, 2002). When BisGMA-
HEMA mixtures are placed on water-saturated dentine, the applied
concentrations changes as the much more water-soluble HEMA diffuses to the
base of the demineralised zone. This can result in final molar ratios of BisGMA
and HEMA in the hybrid layer that are very different from the applied molar
ratio.
1.3.3 Permeability of the collagen network and monomers diffusivity
Permeability quantifies the effort with which a substance can penetrate a
membrane or diffusion barrier. The permeability of dentinal substrate to
monomers and their diffusivity are extremely important for the creation of the
hybrid layer.
After the dentinal surface is acid etched and subsequently rinsed, intertubular
spaces are filled with water and are presumed to be still as wide as when they
were occupied by apatite crystallites (Van Meerbeek et al., 1996).
Maintaining the permeability of the substrate as high as possible allows the
achievement of good monomer infiltration because it is through these 15 to 20

45. 42
nm wide diffusion pathways that adhesive monomer must move to fill the
demineralised dentinal matrix and envelop every fibril. As these molecules
diffuse into demineralised dentine, they may encounter some very small or
narrow constrictions within the interfibrillar spaces, especially if the permeability
of the collagen network has not been maintained. This reduces the rate of
inward diffusion of adhesive monomers. If the strength of the bond is
proportional to the sum of the cross-sectional areas of the resin-infiltrated
interfibrillar spaces, then reductions in the size of these spaces should lead to
lower bond strengths. Therefore it is essential to increase monomer
concentration in demineralised dentine and to ensure that it becomes fully
polymerised, to produce strong, durable hybrid layers.
The ability of resins to infiltrate the exposed collagen mesh of dentine and to
create a molecular-level intertwining within the fibril network depends upon their
concentration and uniformity of penetration (Eick et al., 1996), their degree of
polymerisation and cross-linking, and the amount of water that should be
replaced in the demineralised dentinal substrate (Jacobsen and Soderholm,
1995).
The mechanism available for resin infiltration involves the diffusion of the
monomer into the solvent present in the spaces of the substrate and along
collagen fibrils. That is the reason why this zone is also known as the resin
interdiffusion zone (Van Meerbeek et al., 1996). The rate of diffusion depends
on the affinity of the monomer for the substrate and is proportional to the
concentration, temperature and viscosity of the solution (Cussler, 1976). The
intrinsic diffusivity of the molecule, namely, its intrinsic free diffusion coefficient
in the solvent, which is inversely related to its molecular weight or size, is also

46. 43
an important variable. As the diffusion rate is proportional to the square root of
the molecular weight, the smaller molecules diffuse faster and deeper than the
larger ones (Nakabayashi and Pashley, 1998). On this account, whenever a
blend of monomers of widely differing molecular weights is used in a primer or
bonding agent, the rate of diffusion into the underlying substrate may vary to a
considerable extent. This can result in final molar ratios of monomers in the
hybrid layer that are very different from the initial applied concentrations (Eick et
al., 1997).
It has been mentioned how the presence of water during bonding procedures
may come from several sources (i.e. tubular fluid, relative humidity, rinsing
procedures). Post-etching rinsing thoroughly sponged out the dissolved dentine
minerals and left approximately 70% of the demineralised dentine occupied by
water (Nakabayashi et al., 2004). One of the assumptions with the 'wet-bonding'
technique is that exposed collagen is not dried out thoroughly after etching to
prevent its collapse to a thinner less permeable layer and the consequent
restriction of the spaces around fibrils through which resins had to diffuse
(Nakaoki et al., 2000). One way to avoid more than necessary and desirable air
drying of dentine is to add water-miscible solvents in the primer solutions to
chemically remove water from demineralised dentine (Suh, 1991). During the
priming phase, the solvent (which exceeds the water) diffuses through the
spaces between the collagen fibrils to reach the bottom of the demineralised
zone in conjunction with the monomers that therefore have less water to
challenge with (Eick et al., 1996). After evaporation of the solvent, the resin
infiltration is thought to take the place of all the water present between the
collagen fibrils.

47. 44
However, when it was demonstrated that acid-etching lowered the stiffness of
dentine from 18000 MPa to 1-5 MPa (Eddleston et al., 2003), also the
susceptibility of the demineralised matrix to collapse became evident. It was
discovered that even after primer infiltration (35% HEMA in 65% water) into the
matrix, this was still so compliant that evaporation of the solvent was enough to
cause it to collapse and extrude much of the monomers it had taken up
(Eddleston et al., 2003).
Solvents such as ethanol or acetone have much higher vapour pressures and
generate less surface tension forces on the collagen fibrils network compared
with aqueous primers while they evaporate (Maciel et al., 1996). Despite this,
the use of ethanol-solvated primer mixtures also seems to stiffen the matrix
enough to lower, but not to completely prevent, matrix collapse (Agee et al.,
2006).
1.3.4 Permeability of adhesive resins and water sorption
Ideally, polymer networks should be insoluble materials with relatively high
chemical and thermal stability. Unfortunately, very few polymers are absolutely
impermeable to water. Water movement in a polymer system is related to the
availability of molecular-sized pores in its structure, and the affinity of the
polymer components with water (Van Landingham et al., 1999). The availability
of nanopores depends on the polymer microstructure, morphology and cross-
link density, which are functions of degree of cure, relationship between the
relative quantities of substances forming the compound, molecular chain
stiffness and the cohesive energy density of the polymer (Soles and Yee, 2000).
The affinity of the polymer to water is related to the presence of hydrogen

48. 45
bonding sites along the polymer chains which create attractive forces between
the polymer and water molecules (Soles and Yee, 2000). Incorporation of high
concentrations of hydrophilic functional groups and methacrylate-based resin
monomers in contemporary bonding systems, to achieve immediate bond
strength to an intrinsically wet substrate such as dentine, also increased their
attraction of water (Nishitani et al., 2007). The more hydrophilic the polymer, the
greater is also the likelihood of formation of micro-cavities of different sizes in
the polymeric network (Van Landingham et al., 1999). Many in vivo and in vitro
studies have shown that resin-dentine interfaces become much weaker over
time (Hashimoto et al., 2003). Sauro and collaborators (Sauro et al., 2007)
showed that continued water flow under simulated pulpal pressure increased
convective fluid movement through polymerised resins. It was also
demonstrated that the higher is the dentine permeability, the lower is the tensile
bond strengths of simplified adhesives. The presence of hydroxyl, carboxyl and
phosphate groups in monomers and their resultant polymers make them more
hydrophilic and, as a result, more prone to water sorption. In the manners now
being exemplified, when water sorption is sufficiently high, macromolecular
polymer chains undergo a relaxation process as they swell to absorb the water.
Most of the unreacted methacrylate groups trapped in the polymer network
should not be released into aqueous environments, because they are still part
of dimethacrylate molecules that have reacted and therefore are covalently
bonded to the main polymer chain. Despite this, significant amounts of
unreacted monomer or small chain polymer are released to the surrounding
environment at a rate that is controlled by the swelling and relaxation capacities
of the polymer (Santerre et al., 2001).

49. 46
A number of studies have shown that elution for resin-based materials ranged
from 0.05% to 2.0% of the weight of the specimen into aqueous media, with
elution into alcohol and other organic solvents being higher in most cases (2-
6%) (Ferracane, 1994, Hume and Gerzina, 1996, Pelka, 1999, Munksgaard et
al., 2000, Tanaka et al., 1991). It has been demonstrated that the movement of
water from hydrated dentine may cause the formation of water filled channels
within the polymer matrices of contemporary hydrophilic dentine adhesives (Tay
et al., 2004b). More hydrophilic polymer networks permit a faster release of
unreacted monomers through nanovoids in the material (Brazel and Peppas,
1999). Accordingly, these water filled channels may accelerate elution of
unreacted monomers from polymerised resins (Ito et al., 2005), as well as
further the progress of weakening of the polymers by plasticisation (Wang and
Spencer, 2003).
This phenomenon decreases the stiffness of the polymers (Ito et al., 2005),
produces stresses on the interface with the cavity wall and reduces bond
strengths (Carrilho et al., 2005b).
Water sorption/solubility investigations of hydrophilic adhesives in common use
demonstrated that these systems have much higher water sorption than the
more hydrophobic BisGMA/TEGDMA resins employed to seal multi-step
adhesives (Ito et al., 2005). The hybrid layer created by simplified adhesives,
containing high percentages of hydrophilic monomers, resulted in the formation
of a porous interface (Wang and Spencer, 2003). This interface behaved as a
permeable membrane (Tay et al., 2002a) that allowed water sorption, polymer
swelling, resin hydrolysis and elution of unreacted monomers (Malacarne et al.,
2006). When 3-step etch-and-rinse and 2-step self-etch adhesives were

50. 47
challenged with thermomechanical loading between 5 and 55°C and up to 100
000 cycles, their microtensile bond strengths fell 25-30%. Conversely, the
microtensile bond strengths of 1-step self-etch adhesives fell 50-80% after
thermomechanical loading (Frankenberger et al., 2005). When dentine,
respectively bonded with 3-step etch-and-rinse, 2-step etch-and-rinse, 2-step
self-etch and 1-step self-etch adhesives, was directly exposed to water using
miniature specimens that accelerate water sorption, the microtensile bond
strengths of 3-step etch-and-rinse and 2-step self-etch adhesives did not lessen
remarkably after one year of direct water storage. In contrast, the bonding
effectiveness values of the 2-step etch-and-rinse and 1-step self-etch adhesives
were reduced to almost zero after the same period of direct water exposure (De
Munck et al., 2006). Clearly, the more hydrophilic the resins, the more water the
polymers absorb, the more the polymers become plasticised and the more they
lose their mechanical properties. Thus, water plasticisation of resins contributes
to a reduction in resin-dentine bond strength durability.
1.4 Mechanisms responsible for loss of mechanical stability
Despite successful immediate bonding, the longevity of resin-bonded
restorations remains questionable due to physical (occlusal forces, expansion
and contraction stresses related to temperature changes) and chemical factors
challenging the adhesive interface (Breschi et al., 2008). Today, the most
difficult task in adhesive dentistry is to make the adhesive-tooth interface more
resistant against ageing, thereby rendering the restorative treatment more
predictable in terms of clinical performance in the long term. Despite the
enormous advances made in adhesive technology during the last 50 years, the

51. 48
bonded interface itself remains the weakest area of composite restorations and
none of the current adhesives or techniques is able to produce an interface that
is absolutely resistant to degradation (Breschi et al., 2008). The degradation of
the adhesive interface, which may occur in a relatively short term, depends on
the way the adhesive has been manipulated, on the actual adhesive approach
and on the adhesive composition.
Hydrolysis of interface components, such as dentinal collagen and resin, due to
water sorption, potentially enhanced by enzymatic degradation, and subsequent
elution of the break-down products are the major factors thought to destabilise
the adhesive-dentine bond (De Munck et al., 2009).
1.4.1 Hydrolytic degradation of dental adhesive resins
Dental polymer networks have been shown to be susceptible to hygroscopic
and hydrolytic effects to varying extents dependent upon their chemistry and
structure (Ferracane, 2006).
In the evolution of dentine adhesives, manufacturers have incorporated
increasing concentrations of hydrophilic and ionic monomers to make these
adhesives more compatible for bonding to intrinsically moist, acid-etched
dentine (Van Landuyt et al., 2007).
Increasing the hydrophilic nature of the adhesive-dentine interface has several
disadvantages (Tay and Pashley, 2003a) and affects the integrity and durability
of the adhesive/dentine interfacial bond (Spencer et al., 2010).
Hydrophilic and ionic resin monomers are vulnerable to hydrolysis, due to the
presence of ester linkages, typical of all methacrylates (Ferracane, 2006).

52. 49
These ester linkages are theoretically susceptible to several esterases in body
fluids (Soderholm et al., 1984).
Adhesive hydrophilicity, water sorption, and subsequent hydrolytic degradation
have been considered as highly correlative, because hydrolytic degradation
occurs only in the presence of water (Carrilho et al., 2005a).
Several studies have established a direct relationship between the presence of
hydrophilic and acidic resin monomers in adhesive blends with decreased
longevity of resin-dentine bonds (Peumans et al., 2005), owing to the fact that
resin composition and hydrophilicity expedite water sorption in hydrophilic
resins (Malacarne et al., 2006).
Even the inclusion of small amounts of water may culminate in nano-phase
separation of the adhesive components in the form of nanoscopic worm-like
structures between the polymerised hydrophilic and hydrophobic resin phases
(Ye et al., 2009b). Nano-phase separation reduces the dynamic mechanical
properties of the polymerised adhesives (Park et al., 2010) and increases their
susceptibility to esterase-catalysed hydrolysis (Kostoryz et al., 2009).
Esterases known to activate ester hydrolysis include salivary esterase,
cholesterol esterase, pseudocholinesterase, porcine liver esterase, and
acetylcholinesterase. In contrast to HEMA, Bis-GMA has greater susceptibility
to hydrolysis by cholesterol esterase and acetylcholinesterase. Biodegradation
of HEMA/Bis-GMA adhesives in the presence of either enzyme appear to be
more clinically relevant, since they simulate salivary enzyme activity (Yourtee et
al., 2001).
Previous work has shown that human saliva contains sufficient esterase activity
to attack resin composites (Lin et al., 2005).

53. 50
Nevertheless, it is not known whether there are similar esterases in dentinal
fluid and how they could reach resin-dentine interfaces.
Hydrolysis of methacrylate ester bonds caused either by the increase in acidity
of monomer components (Aida et al., 2009) or by salivary esterases (Shokati et
al., 2010) can break covalent bonds between the polymers by the addition of
water to the ester bonds.
Apart from water, the interfibrillar spaces in acid-etched dentine also include
highly hydrated negatively charged proteoglycans that constitute a hydrogel
within that space (Scott and Thomlinson, 1998). If these hydrogels continue to
be hydrated in interfibrillar spaces, they may be responsible for “molecular
sieving” of larger dimethacrylates like BisGMA, allowing only smaller molecules
such as HEMA to infiltrate the base of the hybrid layers. Since HEMA forms a
linear polymer that does not cross-link, HEMA-rich regions of hybrid layers may
undergo large strains during function that prompt further degradation and
compromise the longevity of resin-dentine bonds (Liu et al., 2011c).
1.4.2 Endogenous collagenolytic activity
Collagen serves as a structural barrier between tissues, and thus collagen
catabolism (collagenolysis) is required to be a tightly regulated process in
normal physiology. The turnover of connective tissue and degradation of nearly
all extracellular matrix components has been ascribed to different members of
the matrix metalloproteinase (MMP) family, due to their ability to catalyse the
hydrolysis of type I collagen triple helical structure. MMPs are a group of zinc-
and calcium-dependent enzymes operating in homeostatic and reparative

54. 51
processes, but unregulated catalysis by these extracellular proteinases leads to
the pathological destruction of the tissues to which they are bound.
In soft tissues, these collagenases are either secreted in a latent form or
inhibited by tissue inhibitors or metalloproteinases (TIMPs). In mineralised
tissues, these enzymes may be active, secreted in a latent form or inhibited by
TIMPs as well as being incorporated by apatite crystallites that fossilise them
and enable their activity.
It has been mentioned that resin-dentine bonding could be considered a unique
form of tissue engineering in which dentists utilise the natural collagen fibril
matrix of demineralised dentine, which is continuous with the underlying
mineralised matrix, as a scaffold for resin infiltration. The collagen fibrils of the
hybrid layer, by being anchored into the underlying mineralised matrix, provide
micromechanical retention of adhesive resins that, in turn, retain resin
composites. The only continuity between adhesively retained restorations and
the hybrid layer are the resin tags in the tubules, along with the nanometre-wide
resin extensions that pass around and between collagen fibrils.
Nevertheless, unprotected type I collagen fibrils situated at the bottom of the
hybrid layer are subjected to deterioration over time due to the activation of
endogenous collagenolytic enzymes (Mazzoni et al., 2006).
Several studies reported that mineralised dentine contains in fact bound MMPs
such as MMP-2, -3, -8, -9 and -20 (Toledano et al., 2010). Even though the
quantitative analysis of different MMPs in dentine remains to be completed, the
currently available data indicate that MMP- 2 may be the prevalent MMPs in
human dentine matrix (Mazzoni et al., 2007). Although classified as a gelatinase

55. 52
(gelatinase A), MMP- 2 is also an effective collagenase (Aimes and Quigley,
1995).
These host-derived proteases contribute to the breakdown of collagen matrices
in the pathogenesis of dentinal caries (Chaussain-Miller et al., 2006) and
periodontal disease (Hannas et al., 2007). In addition, non-collagen-bound
MMPs are also present in saliva (Sulkala et al., 2001), in dentinal tubules, and,
presumably, in dentinal fluid (Boushell et al., 2008).
Proof of degenerative modifications in hybrid layers was offered by De Munck
and collaborators (2003) with long-term in vitro TEM studies that indicated loss
of staining and loss of cross-banded collagen after 4-5 years of water storage
(De Munck et al., 2003). The degradation was irregular and variable but also
extensive. The high resolution provided by TEM examination suggested that
collagen had been converted into gelatin. That is, the hybrid layer was not
empty but still contained organic material not pigmented with heavy metal stains
which are typically taken up by native cross-banded collagen fibrils (García-
Godoy et al., 2007).
When normal hybrid layers receive tensile stressing, the collagen fibrils share
the stress with the resin network by being loaded in parallel. Subsequent to
cleavage of collagen and its conversion to weaker gelatin (i.e. loss of cross-
banded collagen), the stresses applied to the weakened hybrid layer are carried
only by the stiffest surviving material. In this way the resin meshworks pull out of
the "gelatinised" hybrid layer, producing lower bond strengths (De Munck et al.,
2003).
To demonstrate the degradation of dentine matrices by endogenous MMPs,
Pashley and collaborators (2004) acid-etched disks of dentine with 37%

56. 53
phosphoric acid for 15 s, then placed them in buffered calcium- and phosphate-
containing media with or without four protease inhibitors, normally utilised in
biochemistry to prevent MMPs during collagen extraction and purification
(Pashley et al., 2004). Since MMPs are technically hydrolases, that is to say
they catalyse specific peptide bonds in presence of water, half of the etched
specimens were incubated in mineral oil. Specimens were removed and
processed for TEM observation of the quality of the collagen after 24h, 90 days
and 250 days. The naked collagen fibrils had degraded down to the mineralised
base after the period of incubation in the absence of protease inhibitors. By
contrast, in specimens incubated in the presence of protease inhibitors, the
collagen fibrils appeared normal. Similarly, specimens incubated in oil looked
normal over the 250 days, as in the absence of water MMPs could not cleave
collagen.
Mazzoni and collaborators (2006) reported that when etch-and-rinse systems
were applied on dentine their intrinsic acidity (i.e. pHs between 2.6 and 4.7) was
enough to demineralise dentine but not to denature the collagenases. Hence,
the pH of the adhesives was sufficient to expose and set in motion dentinal
MMPs, initiating autolytic phenomena that ultimately affected the hybrid layer
(Mazzoni et al., 2006).
Such results were consistent with a previous study showing that exposure of
MMPs to an acidic pH (c. pH 4.5) activates MMPs in carious dentine
(Tjäderhane et al., 1998).
Furthermore, when normal mineralised human dentine powder was mixed with
different self-etch adhesives with pHs between 1.5 and 2.7, the gelatinolytic and
collagenolytic activity of dentine increased more than 10-fold (Nishitani et al.,

57. 54
2006). Following application of self-etching primers, increases of collagenolytic
activity were also reported for root canal dentine shavings produced during
rotary instrumentation with Gates-Glidden burs (Tay et al., 2006).
This body of increasing evidence indicates that endogenous MMPs are
uncovered and/or activated by many, if not all, dentine bonding procedures.
It was also suggested that mildly acidic resin monomers can activate MMPs by
inhibiting TIMPs (Ishiguro et al., 1994) in TIMP-MMP complexes, thereby
producing active MMPs (Tjäderhane et al., 1998, Sulkala et al., 2001).
Alternatively, acidic resin monomers may set in motion latent forms of MMPs
(pro-MMPs) via the cysteine-switch mechanism that uncovers the catalytic
domain of these enzymes that were blocked by propeptides (Tallant et al.,
2010).
Cysteine cathepsins are papain-like endopeptidases having a vital role in
mammalian cellular turnover, e.g. bone remodelling and resorption.
Most of these peptidases become activated at the low pH found in lysosomes.
Thus, their activities occur almost entirely within those organelles, playing a part
in intracellular proteolysis within the lysosomal compartments of living cells
(Dickinson, 2002).
However, they also exist as exopeptidases and participate in extracellular
matrix degradation through the breakdown of type I collagen and proteoglycans
(Obermajer et al., 2008). For example, cathepsin K, highly expressed in type I
collagen degradation, works extracellularly after secretion by osteoclasts during
bone homeostasis.
The different members of this family of proteases are distinguished by their
structure, catalytic mechanism, and which proteins they cleave. Cathepsins B,

58. 55
L, and S cleave the non-helical telopeptide extensions of collagen molecules,
while cathepsin K cleaves the collagen molecules along their triple helix region
(Liu et al., 2011c).
Unlike the collagenolytic MMPs (MMP-1, -2, -8, and -13) that cleave type I
collagen into a ¾ N-terminal fragment and ¼ C-terminal fragment at a single
site within the triple helix (between amino acids 775 and 776 from the first GXY
triplet of the triple helix domain), cathepsin K cleaves collagen molecules at
multiple sites within the triple helix, thereby giving rise to fragments of various
sizes (Garnero et al., 1998).
Tersariol et al. reported for the first time the presence of cysteine cathepsins in
dentine demonstrating their expression by mature human odontoblasts
(Tersariol et al., 2010). However, these collagen-degrading enzymes are
thought to be more abundant (approximately 10-fold) in carious dentine (Liu et
al., 2011c).
Like MMPs, cysteine cathepsins may be activated in mildly acidic environments.
Acid activation of dentine-bound cathepsins may also coincide with the
conversion of matrix-bound MMPs into their reactive form. On top of that,
glycosaminoglycans (GAGs) can promote further conversion of the latent forms
of the cathepsin enzyme family into their mature forms at neutral pH (Obermajer
et al., 2008). Consequently, GAG-cathepsin activation allows active cathepsins
to be functional even in neutral pH environments.
The existence of cysteine cathepsins in dentinal tubules (Tersariol et al., 2010)
indicates that they are derived from the dental pulp via the dentinal fluid and
may be activated by mildly acidic resin monomers. They may subsequently

59. 56
interact with GAGs and assist salivary MMPs in the degradation of incompletely
infiltrated collagen fibrils within the hybrid layer.
1.5 Adhesion testing
Several aspects should be considered when testing the strength and durability
of the bond to dentine. These include the heterogeneity of its structure and
composition, the features of the dentinal surface exposed after cavity
preparation, and the characteristics of the adhesive itself, such as its strategy of
interaction and basic physicochemical properties. Laboratory experiments
conducted on dental adhesives can be classified into two types, namely
behavioural tests and structural integrity tests. In the behavioural tests the focus
is on understanding how the material behaves and how one might be able to
change the properties of the material by changing such things as its
composition. These experiments are not designed to assess the clinical
performance of the material used. Examples of the sorts of things one might
measure are tensile/shear bond-strength, thus enabling bond strength to be
measured as a material property, elastic modulus, fracture toughness,
coefficient of thermal expansion and translucency. However, all sorts of
chemical and mechanical challenges that are inherent to the oral environment
should also be taken into account, such as moisture, masticatory stresses,
changes in temperature and pH, and dietary and chewing related habits (Mjör
and Gordan, 2002). Structural integrity tests aim to provide an experimental
arrangement that mimic the performance of the material during function. In
other words, the material is being applied in a situation in an attempt to provide
some insight into how the material might respond to a clinical environment and

60. 57
to learn what makes the structure fail. This will be a complex interaction
between material, design and environment. Thus, the structural integrity test is
seeking to establish a link between the material and its performance in a clinical
situation. Typical examples of such tests are represented by fatigue tests.
Besides static bond-strength tests, theoretically clinically more relevant is in fact
to test adhesive interfaces dynamically, as in the clinical situation tooth-
composite bonds are seldom subjected to acute tensile/shear stresses. It is,
however, exposed to cyclic sub-critical loadings produced during chewing (De
Munck et al., 2005). Although fatigue tests are more labour intensive and time-
consuming than static bond-strength tests, a steadily growing, but still only low
number of fatigue tests have been tried out throughout recent years with regard
to their potential to predict clinical effectiveness. In the literature, six different
fatigue tests have been reported on, as there are, chronologically: (i) a macro-
push-out fatigue test (Frankenberger et al., 1999); (ii) a macro-shear fatigue test
(Erickson et al., 2009); (iii) a micro-rotary fatigue test (Van Meerbeek et al.,
2003); (iv) a micro-shear fatigue test (Braem, 2007); (v) a micro-4-point-bend
fatigue test (Staninec et al., 2008); and (vi) a micro-tensile fatigue test (Poitevin
et al., 2010). Despite the alleged need for more fatigue testing of adhesives and
even though several typical fatigue phenomena can be observed, little new
information on bonding effectiveness is provided than that revealed by the
easier and faster static bond-strength tests (Van Meerbeek et al., 2010). For
example, micro-rotary as well as micro-tensile fatigue testing revealed a similar
superior bonding effectiveness of the 3-step ‘gold-standard’ etch-and-rinse
adhesive OptiBond FL (Kerr, West Collins Orange, CA) over the 2-step ‘gold-
standard’ self-etch adhesive Clearfil SE Bond (Kuraray, Tokyo, Japan), that in

61. 58
turn bonds significantly better than the 1-step adhesive G-Bond (GC, Tokyo,
Japan). In addition, these fatigue tests have largely been applied to dentine with
bonding to enamel being much more difficult to assess in fatigue (Van
Meerbeek et al., 2010).
The longevity of the bond upon ageing of the specimens is another aspect of
the performance of dental adhesives that requires particular attention. Several
studies highlighted very good instantaneous and short-term bonding
effectiveness either to enamel or dentine (Inoue et al., 2001), but durability and
stability of the resin-dentine bonded interfaces created by current adhesive
systems still remain unconvincing (De Munck et al., 2005). This shifted the
focus of researchers’ investigations to the evaluation of ageing mechanisms.
Accordingly, besides determining ‘immediate’ bond strength values, measuring
the ‘aged’ bond strength was decisive in order to estimate the clinical
effectiveness of this type of material (Breschi et al., 2008).
In vivo studies are ideally suited to assess both the performance and the
longevity of restorative materials (Hebling et al., 2005, Carrilho et al., 2007b),
but their feasibility is complicated or even precluded by the associated
bureaucratic requirements, they also require much more time to collect
significant information and a higher cost is involved in the procedure (Reinke et
al., 2012). Laboratory studies, on the other hand, offer the advantages of lower
costs, shorter duration, greater standardisation due to the possibility of isolation
of variables and have been widely used to predict the performance and
longevity of adhesive materials (De Munck et al., 2005, Van Noort, 1994,
Amaral et al., 2007).

62. 59
Most of the knowledge we have about the longevity of dentine bonds are based
on in vitro studies, in which some kind of ‘ageing’ factor is added to the
investigation design (De Munck et al., 2005). This could range from examining
the effects of long-term storage in water, or some more aggressive solutions
(Lee et al., 1994, Yamauti et al., 2003, De Munck et al., 2007, Toledano et al.,
2006) along with the use of pH (Peris et al., 2007, Passalini et al., 2010),
thermal (Price et al., 2003, Nikaido et al., 2002, Bedran-de-Castro et al., 2004,
Lodovici et al., 2009), and mechanical loading cycling (Bedran-de-Castro et al.,
2004, Lodovici et al., 2009, Li et al., 2002, Osorio et al., 2005) as well as their
combinations (Grande et al., 2005, Bedran-de-Castro et al., 2004, Lodovici et
al., 2009) in order to recreate some of the challenges that these restorations are
prone to under clinical service for prolonged periods of time.
The immersion of micro-specimens in water is a well-validated method to
assess resin-dentine bond strength durability (De Munck et al., 2006). It usually
requires 6 months to detect drops on the μTBS values (De Munck et al., 2005),
but this period of time may be even shorter when daily water exchange is
performed (Skovron et al., 2010).
Doing so, it was reported that all classes of adhesives exhibited mechanical and
morphological evidence of degradation that resembled in vivo ageing (Shono et
al., 1999). Other water-storage studies confirmed that immediate resin-dentine
bond strength values do not always correlate with long term bond stability since
deterioration throughout the dentine bonded interface occurs at a fast pace
(Carrilho et al., 2005b, Garcia-Godoy et al., 2010, Hashimoto et al., 2010a).
The introduction of pH, thermal, and mechanical loading cycling are attempts to
simulate clinically relevant conditions; however, they still lack standardisation in

63. 60
the number of cycles, temperature, dwell time, immersion time, load and load
frequency and this may hinder comparison of study results and lead to
contradictory findings (Amaral et al., 2007, Reinke et al., 2012).
Recently, an in situ model has been used for the evaluation of ageing
mechanisms involved in the degradation of resin-dentine bonded interfaces
created with two simplified etch-and-rinse adhesives [Adper Single Bond 2
(3MESPE, St. Paul, MN, USA) and Optibond Solo Plus (Kerr, Danburry, CT,
USA)] under more realistic conditions (Reinke et al., 2012).
Compared to the immediate results, where no restorations were included in the
intra-oral appliances used by volunteers and no ageing method was performed,
rapid deterioration in resin-dentine bond strength were observed after the 14-
day simulated cariogenic challenge accountable for a more intense and rapid
degradation rate of the collagen.
However, the findings of the present investigation could not be compared to
other durability studies since this was the first one that employed an in situ
model to investigate the degradation of resin-dentine bonds that occurs with
etch-and-rinse adhesives.
1.5.1 Assessment of sealing ability
The seal of a restorative material against the tooth structure, and the quality and
durability of the seal, are major considerations for the longevity of adhesive
composite restorations. Since the longevity of an adhesive composite
restoration is mainly affected by the leakage of oral fluids along the interface
between the restorative material and the tooth substrate (De Almeida et al.,
2003), it is very important to evaluate the capacity of a bonding system to