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UNIVERSITÀ DEGLI STUDI DI TRIESTE
XXVII CICLO DEL DOTTORATO DI RICERCA IN NANOTECNOLOGIE
ROLE OF COLLAGEN CROSS-LINKERS ON THE STABILITY OF BONDED INTERFACE
Med/28 – Malattie Odontostomatologiche
DOTTORANDO ANGELONI VALERIA
COORDINATORE PROF. LUCIA PASQUATO SUPERVISORE DI TESI PROF. LORENZO BRESCHI TUTOR PROF. MILENA CADENARO
ANNO ACCADEMICO 2013 / 2014
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Summary Chapter 1: Adhesion to enamel and dentin
Introduction 6
1 Adhesion to dentin 7
1.1 Dentin 9
1.1.1 Dentinal extracellular matrix 11
1.1.2 Endogenous Enzymes 14
1.1.3 Intrinsic collagenolytic activity of mineralized dentin 20
1.2 Dentin bonding Adhesive systems 22
1.2.1 Materials 22
1.2.2 Adhesive system classification 24
1.3 Durability of the hybrid layer 29
1.3.1 Resin degradation 30
1.3.2 Collagen fibril degradation 32
References 34
2 Chapter: Collagen Cross-linkers .
Introduction 43
2.1 Cross-linking of type I collagen 44
2.2 Cross-linking agents 46
2.2.1 Synthetic Resources 46
2.2.2 Chemical agents 49
2.2.3 Natural Renewable resources 57
2.3 Use of collagen cross-linkers in clinical applications 58
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References 60
3 Chapter: Experimental Study
Introduction 65
3.1 Microtensile Bond Strength 67
3.1.1 Overview of the Technique 67
3.1.2 Storage in Artificial Aaliva 68
3.1.3 Microtensile Bond Strength test performed 69
3.2 Nanoleakage Analysis 72
3.2.1 Overview of the Technique 72
3.2.2 Nanoleakage analysis performed 73
3.3 Zymographic analysis 74
3.3.1 Overview of the Technique 74
3.3.2 Zymographic analysis performed 75
3.4 In situ Zymography analysis 78
3.4.1 Overview of the Technique 78
3.4.2 In situ Zymography performed 79
References 82
4 Chapter: Results and Discussion
4.1 Results 85
4.1.1 Microtensile Bond Strength Test 85
4.1.2 Nanoleakage analysis 88
4.1.3 Zymographic analysis 90
4.1.4 In situ Zymography analysis 95
4.2 Discussion 100
References 111
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1 Chapter
Adhesion to enamel and dentin
Introduction
There is consensus that the bonded interface remains the
weakest area of tooth-colored restorations. Contemporary
restorative dentistry is based on the adhesion between the resin
based materials and the tooth substrate. The bonded interface
between tooth substrate and adhesive materials is called hybrid
layer. Despite significant improvements in adhesive systems,
hybrid layer remains the weakest area of adhesive restorations.
Resin-dentin bonding can be consider as unique form of
tissue engineering in which a the collagen matrix of the
demineralized dentin is used as the scaffold for resin infiltration,
to produce a hybrid layer that couples adhesives/resin
composites to the underlying mineralized dentin. Thus, altered
forms of dentin in general have a negative impact on the
bonding ability of current adhesive systems. Caries-affected
dentin, a common substrate in dental preparations requiring
restorative therapies, is well-known to impair the bonding
performance of contemporary systems. The pathological and
physiological variations of dentine substrate did not received the
needed consideration during the development of new therapies.
Strengthening of type I collagen and reduced biodegradation are
likely to reinforce the dentin structure and in combination are
promising strategies to develop a strong and last longing tooth-
biomaterial interface.
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1. Adhesion to dentin
The adhesion is defined as strong molecular interactions
between two surfaces in intimate contact. In terms of influence
on achieving adhesion, the important aspects of teeth substrates
include their abundance of mineral as a potential site for
chemical interactions in both and how the adhesion is affected
by the high concentration of organic material in dentin,
specifically collagen, as well as the significant water content of
the latter. (Breschi et al, 2013)
The fundamental principle of adhesion to tooth
substrates, enamel and dentin, is based upon an exchange
process by which inorganic tooth material is replaced by
synthetic resin. This process involves three phases. Preliminary
phase, conditioning or etching, consists of removing calcium
phosphates by which microporosities are exposed at dentin tooth
surface, preparing it for the bonding procedures. The etchant is
composed of acidic molecules that, demineralizing dentin, alter
or remove the smear layer that is 1- to 5-µm–thick amorphous
layer comprised of residual organic and inorganic debris due to
mechanical caries removal.
The hybridization phase involves infiltration, primarily
based on mechanisms of diffusion, and subsequent in situ
polymerization of resin within the created surface
microporosities (Van meerbeek et al, 2003). The hybridization
phase is composed by two additional steps: priming and
bonding. The primer is a type of molecule that helps making the
dentin surface, which is very hydrophilic, more hydrophobic in
order to accept the very hydrophobic bonding resin. Thus, the
primer typically contains a molecule or molecules with both
hydrophilic and hydrophobic characteristics, i.e., amphiphilic.
After that, the bonding resin can infiltrate and, once cured,
becomes incorporated into the primed dentin (Nakabayashi et al,
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1982). Since dentin tissue is highly hydrophilic, the
development of adhesive primers with enhanced hydrophilicity
as agents for preparing the surfaces for bonding to the more
hydrophobic resin adhesive has largely solved this concern, after
many years of study and development.
The entire process lead to the formation of a zone that
consists of components of both the dentin and the resin, linked
to each other by micromechanical interlocking; this zone is
defined as hybrid layer. The hybrid layer is the structural
support of the bonded interface between the tooth and the
subsequently placed restorative material (Breschi et al, 2013)
Since bonding is created by impregnation of the dentin substrate
by blends of resin monomers, the stability of the bonded
interface, and consequently of the tooth restoration, relies on the
creation of a compact and homogenous hybrid layer. A
hydrophobic polymeric layer is more insoluble and resistant to
erosion and degradation by acids and other components of oral
fluids than a more hydrophilic one (Malacarne et al, 2006; Liu
et al, 2011) A combination of hydrophilic and hydrophobic
molecules are used in modern adhesives to effect durable
bonding. The major difference between hydrophilic and
hydrophobic adhesives is the chemistry of their monomers and
solvents. Monomers with alcohol, acid, hydroxyl, and amino
groups are more capable of enhancing chemical interactions
with the collagen and hydroxyapatite of tooth structure than are
molecules consisting predominantly of hydrocarbons. As
hydroxyl, carboxylic, phosphate, and amide functional groups
are substituted for groups containing hydrogen, methyl, ethyl,
and so forth, on the polymer chain, and as ether and ester
linkages are incorporated, the polymer becomes slightly more
hydrophilic. The monomers present in dental adhesives are
similar to those used in dental composite restoratives, thus
ensuring that there will be strong interaction between the
adhesive and the overlying composite. But critical to achieving
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bonding to the tooth is the conversion of the monomers that
penetrated the tooth structure into a rigid, strong polymer. This
process, called polymerization, involves the chemical splitting
of carbon-carbon double bonds of the monomers, followed by
the formation of carbon-carbon single bonds linking one
monomer to another, thus building a long chain of molecules
much like adding links to a chain (Stansbury JW, 2000).
1.1 Dentin
In order to understand the formation of the hybrid layer
and the aging processes in which is involved is essentially to
describe the dentin tissue.
Dentin is the tissue underlying the dental enamel that
constitutes the bulk of the tooth; dentin is composed of about 50
vol% by mineral in the form of a carbonate rich, calcium
deficient apatite; 30 vol% organic matter which is largely type I
collagen; and about 20 vol% liquid, which is similar to plasma
but is poorly characterized (Marshall et al, 1997).
Dentin it is a complex mineralized tissue (Fig.1)
arranged in an elaborate 3-dimensional framework composed of
tubules extending from the pulp to the dentin–enamel junction,
intra-tubular, and peritubular dentin. Dentin presents a
specifically oriented micro-morphology composed of tubules (≈
1–2µm diameter) surrounded by a hypermineralized layer (≈
1µm), called peritubular dentin (95 vol% of mineral), and a
softer intertubular matrix (30vol% of mineral), where the
organic material is concentrated. The intertubular matrix is
mainly composed of type I collagen fibrils (90% of the organic
matrix) with associated non-collagenous proteins and
proteoglycans (10% of the organic matrix), forming a three-
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dimensional organic network reinforced by carbonate apatite
mineral crystallites (Marshall, 1993; Bertassoni et al, 2012).
Fig 1. Distribution and hierarchical structure of dentin extracellular
matrix components relevant to dentin biomodification. (A) Tooth structure.
EN, enamel; D, dentin; PD, predentin; P, pulp. (B) Proteoglycans and
endogenous proteases distribution in coronal dentin. Endogenous proteases
include MMPs and cysteine-cathepsins with greater presence of both
enzymes in deep dentin areas and predentin and the dentin enamel junction.
Proteoglycans are also identified in deep dentin, especially predentin region.
(C) Dentinal tubule cross-section view at higher magnification representing
collagen fibrils, proteoglycans and endogenous proteases. ITD, intertubular
dentin; T, dentin tubule. Proteoglycans are bound to collagen,
immunolocalized dentin throughout and highly concentrated at the
peritubular dentin. Cathepsin B was localized in the odontoblasts and
dentinal tubules, especially in peritubular dentin. MMP-2, -9 and -3 are
localized along collagen fibrils, mainly at intertubular dentin, but no MMPs
were identified inside the tubules. (D) Collagen fibril interactions with non-
collagenous proteins in dentin organic matrix. The protein core present in
proteoglycans binds to collagen and forms aggregates of microfibrils by
holding the collagen network together. (E) Enzymatic collagen cross-links
provide tensile properties and stability to the collagen fibrils. Adapted from
Bedran-Russo et al, 2014.
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1.1.1 Dentinal Extracellular Matrix
Type I collagen
Type I collagen fibrils represents the backbone of the
dentin organic fibrillar network (Breschi et al, 2008). Type I
collagen is the most abundant of all collagen types and is a
strong and elastic biomaterial arranged into highly organized
hierarchical structures; due to its structure type I collagen is
defined as a coiled-coil trimer molecule (Bertassoni et al, 2012).
Type I collagen molecules are biosynthesized from a larger
precursor, procollagen, by cleavage at both its C- and N-
terminal ends (Bedran-Russo et al, 2014). A single collagen
molecule consists of three polypeptide chains composed of two
α1 and one α2 sequences. The resulting molecular unit has a
mass of about 285 kDa and is approximately 1.4 nm wide and
300 nm long. The basic triple-helical conformation consists of
three closepacked supercoiled helices, which requires a glycine
residue at every third position in the polypeptide chain. This
results in a (X–Y–Gly)n repeating pattern in which the X and Y
positions are frequently occupied by proline and 4-
hydroxyproline residues, respectively (Orgel et al, 2011). The
resulting polypeptide chain, therefore, assumes a left-handed
helical conformation with about three residues per turn (Ottani
et al, 2001).
Collagen fibrils are formed by spontaneous self-
assembly of the molecules into a periodic structure with 67–69
nm repeat period overlap between neighboring molecules, which
is crucial for the development of covalent inter-molecular cross-
linking. The inter-molecular cross-linking, is the basis for the
stability, tensile strength and viscoelasticity of the collagen
fibrils (Bedran-Russo et al, 2014).
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Fig 2: Representation of the progressive hydration of the collagen
Gly-Ala peptide. Top row presents the perpendicular and the bottom row
parallel view to the molecular axis at the same hydration level. (A) A view of
the non-hydrated collagen, with the three peptide chains shown in different
colors. (B) The first shell of water molecules (blue spheres), directly
hydrogen-bonded to carbonyl, hydroxyl or amide groups on the peptide
surface. (C) The second shell of water molecules, hydrogen bond to the water
in the first shell, demonstrating the filling of the super-helical groove. (D)
The third shell of water molecules. Adapted from Tjäderhane et al, 2013a
However water also plays an important role in
maintaining the conformation of native collagen molecules
indeed, as demonstrated by an important study of Bella and coll,
the lateral spacing between molecules is too long for direct
hydrogen bonds to occur between specific residues (Fig.2). It
was confirmed thereafter that these inter-chain and inter-
molecular bonds are formed by inherent water molecules which
form multispan hydrogen bonded bridges connecting
neighboring collagen molecules (Bella et al, 1994). Based on
these investigations, there is general agreement that triple-
helices are surrounded by a highly structured ‘‘cylinder of
hydration’’, and the effective diameter of these ‘‘cylinders’’
dictates the lateral separation in the macromolecular assemblies
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that form the resulting fibrillar units in collagen type I
(Bertassoni et al, 2012).
The slope of elastic stress-strain curve for collagen fibers
increases with increased degree of cross-linking, and subtle
perturbations to the cross-linking profile have been correlated
with the strength of hard tissue (Knott and Bailey, 1998). In
addition, the biodegradability and thermal stability of the tissue
is also controlled by the amount and type of collagen cross-
linking (Bedran-Russo et al, 2014). Endogenous collagen cross-
linkings are mediated by enzymatic and non-enzymatic
reactions. Enzymatic intra- and inter-molecular cross-links,
formed between telopeptides and adjacent triple helical chains
through lysine–lysine covalent bonding (Reiser et al 1992), are
controlled by a number of factors, such as lysine hydroxylation,
glycosylation, turnover rate, molecular packing, and external
forces (Yamauchi M, 2000). On the other hand non-enzymatic
collagen cross-linkings are mediated by oxidation and glycation
processes.
Proteoglycans
Proteoglycans (PGs) are a major group of non-
collagenous proteins identified in both pre-dentin and dentin.
PGs play a crucial role in dentin mineralization (Ruggeri A, et al
2009) and the structural integrity of collagen fibrils (Panwar P et
al 2013). Proteoglycans are carbohydrate-rich polyanions with a
high molecular weight (from 11,000 up to 220,000) constituted
by a polypeptide core to which is attached one or more
glycosaminoglycans, i.e. repeating disaccharide units with
sulphate ester groups linked at position 4 or 6 (Goldberg and
Takagi, l993)
They are classified into two distinct categories: the large
aggregating chondroitin/keratan sulfate family, composed of
molecules such as versican and aggregan, and the family of
small leucine-rich proteoglycans (SLRPs) (Goldberg et al, 2011;
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Nikdin et al, 2102). The presence of chondroitin-sulfate it is
claimed to regulate the biophysical properties of dentin
proteoglycans, which in turn may regulate the final collagen
fibrils three-dimensional arrangement. In other words
proteoglycans may be responsible of the three-dimensional
appearance of the dentin organic matrix due to their ability to
fill space, bind and organize water molecules, and repel
negatively charged molecules (Scott and Thomlinson, 1988;
Oyarzun et al 2000). PGs found in dentin are mainly small
leucine-rich collagen-binding carrying chondroitin-sulfate (CS,
e.g. biglycan or decorin) with a limited distribution of keratan-
sulfate (KS, e.g. fibromodulin, lumican) glycosaminoglycans
chains (GAGs). Although there are similarities in the structure
of decorin and biglycan, they differ in the pre-dentin/dentin
distribution (Orsini et al 2007) and in related gene expression
during tooth mineralization. In addition to their roles in
mineralization, PGs control the tissue hydration and molecule
diffusivity. Hence, modified forms of the tissue, such as
sclerotic dentin, can affect the distribution of PGs (Suppa et al
2006).
1.1.2 Endogenous Enzymes
Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are a family of Zn2+-
and Ca2+-dependent enzymes, which are able to degrade
practically all ECM components, making them important player
in many biological and pathological processes. Esterases and
proteases are also classified as hydrolases, because they
enzymatically add water across ester or peptide bonds. In
humans, the MMP family contains 23 members that are
frequently divided into six groups: collagenases, gelatinases,
stromelysins, matrilysins, membrane-type MMPs (MT-MMPs),
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and other MMPs– based on substrate specificity and homology
(Mazzoni et al, 2012a). Even though this classification is
commonly used both for historical and for practical reasons, it
does not fully reflect the complexity of MMP functions and
biological activities, as most MMPs can degrade several
substrates with variable specificity (Mazzoni et al, 2012a;
Hannas et al 2007). For example, collagenases-1 and -3 (MMP-
1 and -13) can also degrade gelatin at a slow rate, and gelatinase
A and B (respectively MMP-2 and -9) can degrade several types
of collagen, especially type IV collagen (Visse and Nagase
2003). Beyond their ECM degradation, MMPs also play
important roles in the conversion of non-collagenous matrix
proteins to signaling molecules that affect cell survival,
proliferation, and differentiation (Page-McCaw et al, 2007).
MMPs are synthesized and mostly secreted as inactive
proenzymes (zymogens), in which the so-called prodomain is
present, inhibiting the functional activity of catalytic domain. In
latent (non-activated) MMPs, the unpaired cysteine in the
prodomain forms a bridge with the catalytic zinc (referred to as
the “cysteine switch” mechanism), preventing enzymatic
activity. This conserved cysteine acts as a ligand for the
catalytic zinc atom in the active site, excluding water molecules
and rendering the enzyme inactive (Tjäderhane et al, 2013).
Activation occurs when this linkage is disrupted by proteolytic
cleavage of the propeptide by other MMPs, cysteine cathepsins
or other proteinases, or chemically, e.g. by amino phenyl
mercuric acid (APMA), replacing the thiol group with water
(Mazzoni et al 2012a). In addition to the pro- and catalytic
domains, MMPs contain other domains responsible, for
instance, for substrate specificity, recognition, and interaction
(Nagase and Fushimi 2008). In vertebrate MMP family,
collagenases (MMP-1, -8, -13), MMP-2 (gelatinase A), and
membrane-type 1-MMP (MT1-MMP, MMP- 14) can cleave
native triple-helical type I, II, and III collagens. They all cleave
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collagens into 1⁄4- and 3⁄4-fragments at the Gly-Leu/Isoleu
peptide bond where collagen peptide structure determines both
specific cleavage and binding sites for MMPs. The fragments
denature at body temperature, and are further degraded by
gelatinases and other non-specific tissue proteinases. The triple-
helical structure of the collagen makes interstitial collagens
fibrils in their native configuration resistant to most vertebrate
proteolytic enzymes (Tjäderhane et al, 2013). The substrate-
binding site of collagenases is located in a deep cleft with the
entrance being only approximately 0.5 nm wide. This is not
enough space to accommodate triple helical collagen with a
diameter that is three times larger than the active site of the
enzyme (Nagase and Fushimi 2008). Currently, two
mechanisms have been proposed to allow the degradation of
collagen fibril by MMPs. First mechanism is supported by
recent studies that demonstrated the role of MMPs in unwinding
collagen peptide helices was further supported by the cleavage
of unwound fibril by MMP-3 -1 and neutrophil elastase, which
normally do not degrade fibrillar collagen (Nagase and Fushimi
2008). The similar binding-unwinding mechanisms have later
been suggested for MMP-8, -2 and -14 (Gioia M et al, 2007).
On the other hand Perumal and coll (2008) verified that the
collagen region where cleavage begins, is fully protected by the
collagen C-telopeptide restricting the access of MMPs to the
cleavage site of the native collagen structure. They suggest that
the site must be exposed by proteolytical removal of C-terminal
telopetide by telopeptidase or, e.g. mechanical damage, before
MMP can bind to the substrate’s “interaction domain”
facilitating the triple-helix unwinding/dissociation function of
the enzyme before collagenolysis. Alternatively, damage
caused, e.g. by physical loading may induce changes in fibril
structure, such as breakage of cross-linkages at the C-terminus.
This damage may expose the first collagen peptide chain to be
cleaved by MMP, leading to a greater freedom of movement for
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other chains and their subsequent cleavage. Collagen molecules
distal to the original cleavage site would also become accessible
to MMP cleavage, leading to further degradation (Perumal,
2008). This is an inviting hypothesis, as the damages caused to
dentin collagenous matrix by demineralization in caries lesions,
phosphoric acid or acidic monomers, have been suggested to
cause changes in the collagen molecular arrangement (e.g.
breakage of cross-linkages) which may expose the catalytic
binding site (Bertassoni et al, 2012). Interestingly, telopeptidase
enzyme activity of MMP-9 has long been suggested to be
important in bone matrix degradation. MMP-9 is present in
dentin (Mazzoni et al, 2007), it is readily activated by acid pH
changes (Davis GE 1991; Tjäderhane et al 1998a), and
telopeptidase activity of MMP-9 has been suggested to have a
role in dentin matrix degradation in caries. Alternatively,
cysteine cathepsins may be involved in activation of MMPs,
also in human dentinal caries lesion processes (Nascimento et
al, 2011). Furthermore cysteine and cathepsins are able to
cleave C-terminal telopeptide of type I collagen (Garnero et al,
2003), possibly exposing the collagenase cleavage site after
being activated by acids (Fig.3). Although intermolecular cross-
links can occur along both helical and telopeptide regions, they
are especially common in the telopeptide domains. A study
conducted by Osorio and coll (Osorio et al, 2011) found that
MMP-2 may function as a “telopeptidase”. If gelatinases MMP-
2 and -9 can hydrolyze the bulky telopeptides off surface
collagen fibrils, they may create enough space for a collagenase
to bind to the proper binding site to create unwinding of the
collagen molecule that promote collagen hydrolysis. It appears
that MMPs may work in concert. Gelatinases may indeed
hydrolyze gelatin when it forms, but they may also help initiate
collagenase activity by being a “telopeptidase” (Tjäderhane, et
al, 2013).
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MMP’s activity can be regulated at multiple levels, e.g.
transcription, secretion, degranulation of enzymes from
intracellular granules, and by specific and non-specific
inhibitors (Page-McCaw et al, 2007).
Tissue inhibitors of MMPs (TIMP-1 to TIMP-4) are
specific inhibitors that participate in controlling the local
activities of MMPs in tissues. Most TIMPs inhibit active MMPs
and some TIMPs can prevent pro-MMP activation. TIMPs are
also involved in various cellular and tissue regulatory processes
(Brew K et al, 2000). There are also an ever-increasing number
of synthetic MMP inhibitors. Most of them prevent MMP
activity by chelating or replacing the active site Zn2+, or by
“coating” the substrate (Tjäderhane, et al, 2013).
Several MMPs have been identified in the dentin–pulp
complex. During tooth development, MMPs participate in the
organization of the ECM components and compartments
(Mazzoni et al 2012a). Mature human odontoblasts synthesize at
least gelatinases MMP-2 and -9, collagenases MMP-8 and -13,
and enamelysin MMP-20 (Tjäderhane et al 1998b; Sulkala et al
2002; Sulkala et al 2004; Niu et al 2011). However, the gene
expression profile is much larger, covering most of the MMP
family members. Recent studies have demonstrated the presence
of at least gelatinases MMP-2 and -9, collagenase MMP-8 and
stromelysin MMP-3 in human dentin (Mazzoni A et al, 2007,
Mazzoni et al 2011 Sulkala et al 2007; Mazzoni et al 2009);
while MMP-20 was detected in dentinal fluid. Indeed intense
gelatinolytic activity has been observed in dentinal tubules with
laser confocal microscopy (Mazzoni et al 2012b), and MMP-20
(Sulkala M et al 2002), -2 (Boushell et al 2011) and -9 (Zehnder
et al 2011) have been shown to increase in dentinal tubules of
carious teeth. In mineralized dentin, MMP-2 is probably the
most abundant MMP. TIMP-1 and -2 are also found in human
dentin (Ishiguro et al 1994; Leonardi et al 2010).
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Fig 3. A conserved cysteine residue (C) in the pro-domain
coordinates with the Zn2+ ion at the functional site of the catalytic domain.
The pro-domain is removed by cleavage in the pro-domain and between the
pro-domain and the catalytic domain. The “propeller” is a hemopexin domain
contained by most MMPs, and is attached to the catalytic domain by flexible
hinge domain. The hemopexin domain, e.g. mediates protein–protein
interactions, contributes to substrate recognition, enzyme activation, and
protease localization. Adapted from Tjäderhane et al, 2013a
Cystein Cathepsin
The lysosomal cysteine proteases belong to the clan CA
of cysteine proteases were initially considered as lysosomal
proteases, although they can also act extracellularly. Cathepsins
B, H, L, C, X, F, O and V are ubiquitously expressed in human
tissues, while cathepsins K, W and S are tissue-specific (Turk et
al 2012).
Cathepsins become active at acidic pH and most are
endopeptidases, with some exceptions like cathepsin B that can
also act as a carboxypeptidase (Turk et al 2000). Cathepsin B
endopeptidase activity has a pH optimum around 7.4, which is
expectable for an enzyme that is involved in many physiological
processes related with extracellular matrix degradation. Cysteine
cathepsin activity has been demonstrated both in intact and
carious dentin (Tersariol et al 2010; Nascimento et al 2011), and
cathepsin B has been localized in dentinal tubules (Tersariol et
al 2010). Once synthesized by odontoblast and/or pulp tissue,
secreted cathepsins can easily reach the dentinal tubules and
enter deep into dentin (Tjäderhane et a,l 2013). Moreover,
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changes in expression levels, localization and activity have been
described in carious dentin (Tersariol et al, 2010). The
significant increase of cysteine cathepsin activity in carious
dentin with increasing depth (approaching the pulp) indicates
that odontoblast or pulp-derived cysteine cathepsins may be
important in active caries lesions, especially with young patients
(Nascimento et al, 2011). The increased expression and/or
activity of cysteine cathepsins often coincide with their presence
in the extracellular environment, confirming the hypothesis that
pH is not sole factor responsible for their activity. Cathepsins
with high collagenolytic activities are known to be mainly
responsible for remodeling the extracellular matrix (Turk et al,
2012; Tjäderhane et a, 2013). The extracellular matrix consists
mostly of collagen and proteoglycans, which have all been
identified as the cathepsin substrates (Lutgens SP et al 2007).
Cathepsin K is the only cysteine cathepsin capable of cleaving
the collagen at the triple helical region (Hou et al 2002).
1.1.3 Intrinsic Collagenolytic Activity of Mineralized Dentin
Despite the adhesive approach itself, the result of resin–
dentin is often incomplete hybridization of the dentin surface,
leaving collagen fibrils unprotected and vulnerable to hydrolytic
degradation that also are susceptible to other degradation-
promoting factors such as residual solvent of the adhesive (Yiu
et al 2005) or insufficiently removed surface water. In the last
decade several studies revealed the contribution of host-derived
proteinases to the breakdown of the collagen matrices in the
pathogenesis of dentin caries (Tjäderhane et al, 1998) and
periodontal disease (Lee et al 1995), with potential and relevant
implications in dentin bonding (Pashley et al 2004). Since
Ferrari and Tay (2003) demonstrated that nanoleakage can occur
in the absence of gaps along in vivo resin–dentin interfaces, this
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suggests that the degradation of incompletely infiltrated zones
by host-derived proteinases within the dentin matrix may
proceed in the absence of bacterial enzymes (Nishitani et al,
2006). Pashley et al (2004) reported that if acid-etched dentin
matrices can be slowly degraded over time by dentin-derived
proteolytic enzymes, in the absence of bacteria. The results of
the study revealed, as bacterial collagenolytic activity was not
responsible for the dentin collagen degradation, as is frequently
advocated under in vivo conditions. This pioneer study (Pashley
et al, 2004) on the role of host-derived enzymes for the first
time supported the hypothesis that collagen degradation of
human dentin occurs over time, not only due to the activity of
bacteria-produced collagenases, but via host-derived enzymes
that are released and activated over time. The evidence of
collagenolytic/gelatinolytic activities in partially demineralized
dentin collagen matrices are indirect proofs of the existence of
matrix metalloproteinases (MMPs) in human dentin (Tjäderhane
et al, 1998a), especially both MMP-2 and MMP-9 in
demineralized mature dentin as shown by gelatin zymography
and Western blotting (Mazzoni et al 2007). Moreover, the use of
chlorhexidine, a well-know antibacterial agent with MMP
inhibiting properties (Gendron et al, 1999) when applied to acid-
etched human primary dentin resulted in the preservation of
collagen integrity within the hybrid layers in vivo after the
application of the etch-and-rinse bonding procedure (Carrilho et
al 2007), confirming the indirect involvement of MMPs in the
collagen breakdown process.
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1.2 Dentin Bonding Adhesive Systems
1.2.1 Materials
Dental adhesives consist of three main components: (1)
etchant, (2) primer, and (3) bonding resin. The latter is often
referred to as the adhesive resin, but the entire system also is
typically called an adhesive.
Etchant is composed of acidic molecules that alter or
remove the smear layer and demineralize the enamel and dentin
preparing them for bonding. The primer serves as a type of
molecule that helps make the hydrophilic dentin surface become
more hydrophobic in order to accept the bonding resin which is,
generally, very hydrophobic. Thus, the primer typically contains
a molecule or molecules with both hydrophilic and hydrophobic
components, ie, amphiphilic (Breschi et al, 2013). It functions
by penetrating the demineralized dentin and preparing it for the
bonding resin. The bonding resin then becomes incorporated
into the primed dentin and, once cured, forms the structural
support of the bonded interface between the tooth and the
subsequently placed restorative material. The entire process
forms a zone of material that consists of components of both the
dentin and the resin and, as previously mentioned, is called the
hybrid layer.
All dental adhesive systems are based on polymeric
materials which have a variety of chemical characteristics,
ranging from very hydrophilic to very hydrophobic (the
adhesive resin component of many adhesive systems).
Considering the hydrophilic nature of the tooth, especially
dentin, an adhesive also should have a hydrophilic nature when
placed in order to wet the surface and penetrate the available
microstructure (Breschi et al, 2013). On the other hand this
23
hydrophobic nature is considered to be beneficial once the
material has established a bond with the tooth substrate. A
hydrophobic polymeric layer is more insoluble and resistant to
erosion and degradation by acids and other components of oral
fluids than a more hydrophilic one (Malacarne et al 2006). The
materials available to dentistry are a combination of hydrophilic
and hydrophobic molecules which are used in modern adhesives
to effect durable bonding.
The major difference between hydrophilic and
hydrophobic adhesives is the chemistry of their monomers and
solvents. Monomers with alcohol, acid, hydroxyl, and amino
groups are more capable of enhancing chemical interactions
with the collagen and hydroxyapatite of tooth structure than are
molecules consisting predominantly of hydrocarbons. As
hydroxyl, carboxylic, phosphate, and amide functional groups
are substituted for groups containing hydrogen, methyl, ethyl,
and so forth, on the polymer chain, and as ether and ester
linkages are incorporated, the polymer becomes slightly more
hydrophilic (Van Meerbeek et al 2003). The monomers most
used in adhesive system are the hydroxylethyl methacrylate
(HEMA) and the Bisphenol glycidyl methacrylate (bis-GMA).
The first one, HEMA, is totally miscible in water and serves as
an excellent polymerizable wetting agent for dental adhesives.
Bis-GMA, instead, is the main monomer used in most dental
composites and many adhesives, is much more hydrophobic and
will only absorb about 3% water by weight into its structure
when polymerized (Sideridou et al, 2003). A mixture of the two
has intermediate characteristics and serves as a useful adhesive
for the tooth. In order to enhance the wetting, spreading, and
penetration of the polymerizable monomers into the dentin
substrate, solvents are always added to the mixture as “thinning”
agents. These solvents are typically water, ethyl alcohol, butyl
alcohol, or acetone. The first three are very hydrophilic and thus
enhance the interaction of the monomers with surface water,
24
while acetone is good at displacing water from within the
dentin. However, any solvent not displaced during the
placement procedure, such as by drying appropriately (Jacobsen
et al, 2006), will be incorporated into the bonding layer and may
serve as a weakening contaminant. The monomers present in
dental adhesives are similar to those used in dental composite
restoratives, thus ensuring that there will be strong interaction
between the adhesive and the overlying composite. Critical to
achieving bonding to the tooth is the conversion of the
monomers that penetrated the tooth structure into a rigid, strong
polymer. This process, called polymerization, involves the
chemical splitting of carbon-carbon double bonds of the
monomers, followed by the formation of carbon-carbon single
bonds linking one monomer to another, thus building a long
chain of molecules much like adding links to a chain. The
covalent bonds linking molecules together are strong and
produce a relatively stiff bonding layer that is capable of
resisting many types of mechanical and chemical forces. The
polymerization process typically is begun by exposing the
adhesive to radiation from a high-intensity light source tuned to
the same wavelength range (400 to 500 nm, blue light) that is
absorbed by the purposely added photoinitiator (Stansbuty
2000)
1.2.2 Adhesive System Classification
Current adhesive systems interact with the enamel/dentin
substrate using two different strategies, i.e. either removing the
smear layer (etch-and-rinse technique) or maintaining it as the
substrate for the bonding (self-etch technique) (Tay and Pashley
2002). The difference between the two approaches is
25
represented by the use of a preliminary and separate etching step
for etch- and-rinse systems (usually characterized by a gel of
35–37% phosphoric acid) that is later rinsed away, conversely
the self-etch/primer agent is only air-dried, thus remaining
within the modified smear layer, i.e. the self-etch approach
could be called an “etch-and-dry” approach (Breschi et al, 2008;
Van Meerbek et al, 2003). Despite differences in etching, the
other fundamental steps for adhesion are priming and bonding
that can be either separate or combined, depending on the
adhesive system. The current classification of adhesives relies
on the number of the steps constituting the system (Van
Meerbek et al, 2003). Etch-and-rinse adhesive systems can be
either three- or two-step depending on whether primer and
bonding are separated or combined in a single bottle. Similarly
self-etch adhesives can be either two- or one-step systems
depending on whether the etching/primer agent is separated
from the adhesive or combined with it to allow a single
application procedure (Van Meerbek et al, 2003).
Etching
Etch-and-rinse adhesives: These adhesives use a strong
acid (usually 35% to 37% phosphoric acid at a pH of
approximately 0.9) to completely etch enamel and dentin,
followed by a water rinse to remove the acid from the tooth
surface (Pashley et al, 2011). On dentin, the acid demineralizes
the superficial hydroxyapatite, removes the smear layer and
smear plugs (debris which obstructs the entrances of dentinal
tubules) decreasing dentin permeability by up to 86%. Several
studies have investigated the effects of different etching times
on bond strength (Pashley et al, 2011). Although extended
etching times have been found to increase substrate porosity,
they do not necessarily increase bond strength. The
determination of an appropriate etching time should consider
either the ability of monomers to impregnate the substrate in
relation to dentin demineralization and the ability of collagen
26
fibrils to maintain integrity when exposed to phosphoric acid for
a given length of time. Excessive demineralization may produce
a weak zone comprised of suboptimally impregnated dentin at
the base of the hybrid layer consisting of exposed collagen
fibrils (Breschi et al, 2004). Etching should be limited to
superficial dentin because the viscosity of primers and bonding
agents allows only a few micrometers of impregnation. The
results of these studies thus indicate that sound dentin should be
etched for no longer than 15 seconds. After etching, the tooth
should be rinsed with an intense air or water spray to remove the
acid completely and stop the etching process. After the
conditioning of the dentinal matrix collagen fibrils is been
exposed and open the dentinal tubules completely opened.
Thereafter the primer and bonding are applied to dentin
separately (3-step etch-and-rinse) or in one single phase (2-step
etch-and-rinse) (Breschi et al, 2013).
Self-etching or etch-and-dry adhesives. These systems
use a non-rinsing solution of acidic monomers to dissolve the
smear layer on enamel and dentin surfaces. These adhesives
render the smear layer permeable to monomers rather than
removing it completely (Van Meerbeek et al, 2011), Some self-
etching adhesives simultaneously dissolve the smear layer and
infiltrate enamel and dentin (Carvalho et al, 2005), using the
mineral content of the substrate to buffer the acidic monomers
and inhibit their dentin-etching ability with increasing depth
(Salz et al, 2006). Monomers with one or more attached
carboxylic or phosphate-acid groups are considered to be self-
etching. These adhesives are classified by strength according to
chemical composition and acidity, which determine their
morphologic features (Van Meerbeek et al, 2011). Ultramild
self-etching adhesives (pH > 2.5) are able to demineralize a few
hundred nm; mild adhesives (pH ≈ 2) have a ~1-µm interaction
depth; intermediate-strength adhesives (pH = 1 to 2) have an
interaction depth of 1 to 2 µm, and strong self-etching adhesives
27
(pH < 1) have an interaction depth of several µm. The formation
of resin tags (adhesive resin infiltration within collagen network
and dentin tubules) can be assured only with the use of strong
self-etching adhesives; mild and ultramild adhesives may fail to
form such tags, only slightly demineralizing smear plugs and
allowing limited resin infiltration (Van Meerbeek et al, 2011).
Self-etch adhesive can be classified based on the number of
application steps on dentin: may be two- or one-step systems. In
two-step self-etching adhesive systems, a combined etching and
primer agent is applied on enamel and dentin and air dried,
followed by the application and polymerization of a bonding
resin, while one-step self-etching adhesives combine etching,
primer, and bonding resin in a single application. Many one-
step systems are not all-in-one solutions, and require mixing
materials from two or more bottles before application.
Nevertheless they are applied as one-step agents on the enamel
or dentin substrate (Breschi et al, 2013).
Primer
Primers are mixtures of monomers, such as hydroxyethyl
methacrylate (HEMA), triethyleneglycol dimethacrylate
(TEGDMA), bis-GMA, and urethane dimethacrylate (UDMA),
which have varying degrees of hydrophilic and hydrophobic
properties. Hydrophilic functionality facilitates monomer
permeation into the collagen matrix to form a hybridized
collagen-resin layer, and hydrophobic functionality facilitates
restoration bonding to the resin matrix. Solvents, that are
typically water, ethyl alcohol, butyl alcohol, or acetone, are
added to reduce the inherent viscosity of co-monomer blends,
allowing them to infiltrate wet demineralized dentinal matrices
(Van Landuyt et al, 2007). The type of solvent in primer
solutions has been found to significantly affect dentin bond
strengths by influencing the ability to reexpand previously dried
demineralized matrix (Carvalho et al, 2003). During rinsing of
the etching agents, the presence of water maintains the full
28
expansion of the demineralized dentinal matrix (Pashley et al,
2001; Carvalho et al, 1996). Subsequent air-drying of dentinal
surfaces removes most of the water from the matrix, causing it
to collapse in a manner similar to the collapse of collagen fibrils.
Collagen peptides come into contact with one another, forming
new interpeptide hydrogen bonds that stabilize and stiffen the
shrunken matrix (Maciel et al, 1996). Resin-dentin bonding is
compromised by the lack of sufficient interfibrillar spaces
available for resin penetration in such shrunken dentin (Pashley
et al, 1993; Breschi et al, 2008). To resolve these undesirable
situations, the dentin must be rewetted and/or primers must be
able to reexpand the collapsed matrix (Van Landuyt et al, 2007).
Although solvents facilitate the replacement of water with
adhesive monomers, primer monomer systems often fail to
displace all intrafibrillar water (Pashley et al, 2007). Primers
should be gently air dried after application to volatilize any
remaining solvent before the adhesive resin is applied, Failure to
adequately evaporate primer solvent will significantly adversely
affect the bond to dentin and can have a more adverse affect on
adhesion than just about any other application mistake.
Bonding
Hydrophobic solvent-free monomer blends (usually bis-
GMA and TEGDMA) as bonding agents. These agents should
be applied to the primed surface with a brush and thinned to an
optimal thickness of about 60 to 120 µm, depending on the
viscosity of the adhesive (Breschi et al, 2013). The upper layer
of the adhesive resin does not polymerize well because it is
exposed to oxygen, which inhibits its cure, and for this reason is
called the oxygen-inhibited layer. Therefore, the surface layer
contains unreacted methacrylate groups that may be involved in
copolymerization with the restorative resin (Endo et al, 2007).
Adequate light intensity is an important factor in curing
the resin layer; prolonged curing times that slightly exceed the
29
manufacturer recommendations have been shown to improve
polymerization and adhesive properties (Cadenaro et al, 2009).
Multi-mode or Universal Adhesive Systems
Dental manufacturers have recently made slight
modifications of dentin adhesive formulations to produce a new
class of universal adhesives. These materials are called multi-
mode or universal because they can be used as self-etch, etch-
and-rinse, or selective-etch systems. All are modeled after self-
etch systems and contain acidic monomers. Although these
universal adhesives are new and just now being studied, early
research appears to show that they are not adversely affected by
this etch-and-rinse step either in-vivo or in vitro (Perdigão et al,
2012; Muñoz et al, 2013).
1.3 Durability of the Hybrid Layer
As the hybrid layer is created by a mixture of dentin
organic matrix, residual hydroxyapatite crystallites, resin
monomers and solvents, aging may affect each of the individual
components or may be due to synergistic combinations of
degradation phenomena occurring within the hybrid layer.
Clinical longevity of the hybrid layer seems to involve both
physical and chemical factors. Physical factors such as the
occlusal chewing forces, and the repetitive expansion and
contraction stresses due to temperature changes within the oral
cavity (Gale and Darwell, 1999) are supposed to affect the
interface stability (De Munk et al, 2005; Tay and Pashley 2003).
Acidic chemical agents in dentinal fluid, saliva, food and
beverages and bacterial products further challenge the
tooth/biomaterials interface resulting in various patterns of
30
degradation of unprotected collagen fibrils (Hashimoto et al,
2003a,b), elution of resin monomers (probably due to
suboptimal polymerization) (Cadenaro et al 2005, 2006) and
degradation of resin components (Finer and Santerre 2004;
Jaffer et al, 2002). Since hydrolytic degradation occurs only in
presence of water, adhesive hydrophilicity, water sorption and
subsequent hydrolytic degradation are generally correlated
(Tanaka et al 1999; Tay et al 2002; Tay et al, 2003; Malacarne
et al, 2006). Hence, irrespective of the etch-and-rinse or the self-
etch strategy, by combining hydrophilic and ionic resin
monomers into the bonding such as in simplified adhesives (i.e.
two-step etch-and-rinse and one-step self-etch systems) the
bonded interface lacks a non-solvated hydrophobic resin coating
(Breschi et al, 2008).
1.3.1. Resin Degradation
The water “wet bonding” technique was introduced in
the early 1990s to prevent the problem of collagen collapse after
acid- etching and resulted in improved resin infiltration into
acid-etched dentin. In this bonding technique, acid-etched dentin
is kept fully hydrated throughout the bonding procedure. Two-
hydroxyethyl methacrylate (HEMA) was incorporated into
many dentin adhesives to serve as a solvent for non-water-
compatible resin monomers, to reduce phase separation of those
monomers after evaporation of the volatile solvents (Spencer
and Wang, 2002), to enhance the wetting properties of those
adhesives on acid-etched dentin (Marshall et al, 2010), and for
its purported affinity to demineralized collagen (Sharrock and
Grégoire, 2010). Increasing the hydrophilic nature of the
adhesive-dentin interface has several disadvantages (Tay and
Pashley, 2003a) and creates a weak link in the bonded assembly
(Spencer et al, 2010). Hydrophilic and ionic resin monomers are
vulnerable to hydrolysis, due to the presence of ester linkages
31
(Ferracane, 2006). Within the hybrid layer, two degradation
patterns can be observed: loss of resin from interfibrillar spaces
and disorganization of the collagen fibrils (Hashimoto et al,
2003a). Such degradation may result from the hydrolysis of
resin and/or collagen, thereby weakening the physical properties
of resin–dentin bond 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, 2005). 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.
Moreover, problems associated with water-based,
strongly acidic single-bottle self- etching adhesives arise from
both the hydrolytic instability of the methacrylate monomers
used in those systems and the side-reaction of the applied
initiator components (Moszner et al, 2005). The final goal of
bonding procedures is the complete infiltration, and
encapsulation of the collagen fibrils by the bonding resin is
recommended in order to protect them against degradation
(Hashimoto et al, 2003a; Vargas et al, 1997). It is well known
that the degree of envelopment of collagen fibrils is different
depending on the type of bonding agents, i.e. a etch-and-rinse or
a self-etch approach. For etch-and-rinse adhesives, a decreasing
gradient of resin monomer diffusion within the acid-etched
dentin (Wang and Spencer, 2002) results in incompletely
infiltrated zones along the bottom of the hybrid layer that
contain denuded collagen fibrils (Shono et al, 1999; Spencer et
al, 2004) in the demineralized zone of dentin created by the
discrepancy between the depth of acid etching and resin
infiltration. By substituting ethanol for water,
BisGMA/TEGDMA mixtures have been shown to infiltrate
32
dentin and produce high bond strengths (Pashley et al, 2007).
Thus, “ethanol-wet bonding” permits the use of hydrophobic
resins that absorb little water, for dentin bonding (Sadek et al,
2008). Using self- etch adhesives, the acidic monomers dissolve
the inorganic phase of dentin and simultaneously primes and
infiltrates the dentin matrix, resulting in fewer exposed collagen
fibrils (Spencer et al, 2004). Despite the adhesive approach
itself, the result of resin–dentin is often incomplete
hybridization of the dentin surface, leaving collagen fibrils
unprotected and vulnerable to hydrolytic degradation that also
are susceptible to other degradation promoting factors such as
residual solvent of the adhesive (Yiu et al, 2005) or
insufficiently removed surface water. This water passage was
revealed by studying the permeability of bonded interfaces and
by using a tracer detectable by electron microscopy such as
ammoniacal silver nitrate (Saboia et al, 2008). This tracer stains
pathways water-filled diffusion throughout the bonded interface
that are often manifested as creating the so-called “water trees”,
i.e. characteristic water channels at the surface of the hybrid
layer that extends into the adhesive layer, supporting the
hypothesis of complete permeation of simplified adhesive
bonded interfaces to water (Tay and Pashley, 2003b). When the
tracer was previously used to stain voids, porosities (especially
for etch-and-rinse systems) and water-filled regions and/or
hydrophilic polymer domains (especially for self-etch systems)
within hybrid layers, the silver uptake was named nanoleakage
(Breschi et al, 2008).
1.3.2 Collagen Fibril Degradation
Bond longevity may also be negatively impacted by
increased water content at the bonded interface resulting from
resin and collagen degradation. In addition, the degradation of
collagen is caused primarily by the presence of water. Resin or
33
collagen hydrolysis may degrade the hybrid layer, causing the
loss of resin from interfibrillar spaces and the disorganization of
collagen fibrils (Hashimoto et al, 2003) and physically
weakening the resin-dentin bond (Hashimoto et al, 2010). The
dentinal surface is often incompletely hybridized in resin-dentin
bonding, regardless of the adhesive system used. Such
incomplete hybridization may leave exposed collagen fibrils and
residual adhesive solvent and/or surface water that increases
vulnerability to hydrolytic degradation (Cadenaro et al, 2009;
Tjaderhane et al, 1998).
Dentin bonding may also be affected by the breakdown
of collagen matrices by host-derived proteinases released during
the development of dentinal caries and periodontal disease
(Tjaderhane et al, 1998). The presence of MMPs in human
dentin can be inferred based on evidence for
collagenolytic/gelatinolytic activities in partially demineralized
collagen matrices (Pashely et al, 2004; Mazzoni et al, 2007,
2009). When released and activated during dentin-bonding
procedures, these endogenous enzymes can degrade
extracellular matrix components (Visse et al, 2003)
34
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2 Chapter
Collagen Cross-linkers
Introduction
A cross-link is a bond that links one polymer chain to
another one. They can be covalent bonds or ionic bonds.
"Polymer chains" can refer to synthetic polymers or natural
polymers (such as proteins). When "cross-linkings" is used in
the biological field, it refers to the use of a probe to link proteins
together to check for protein–protein interactions, as well as
other creative cross-linking methodologies. Cross-linking is
used in both synthetic polymer chemistry and in the biological
sciences. They can be formed by chemical reactions that are
initiated by heat, pressure, change in pH, or radiation. For
example, mixing of an unpolymerized or partially polymerized
resin with specific chemicals called cross-linking reagents
results in a chemical reaction that forms cross-links. Cross-
linkings can also be induced in materials that are normally
thermoplastic through exposure to a radiation source, such as
electron beam exposure, gamma-radiation, or UV light.
Proteins in nature present crosslinks generated by
enzyme-catalyzed or spontaneous reactions. Such crosslinks are
important in generating mechanically stable structures such as
hair, skin and cartilage. Cross-linking reactions can be promoted
by the use of external cross- linking molecules; some examples
of cross-linking molecules will be described below.
44
2.1 Cross-linking of Type I Collagen
Type I collagen is the most abundant type of collagen
and is widely distributed in almost all connective tissues with
the exception of hyaline cartilage. It is the major protein in
bone, skin, tendon, ligament, sclera, cornea, and blood vessels
(Viguet-Carrin et al, 2005). Consistent evidences supported the
hypothesis that type I collagen molecules play a pivotal role in
mechanical properties of dentin, and consequently of the hybrid
layer (Pashley et al, 2004; Bertassoni et al, 2012; BedranRusso
et al, 2014).
In type I collagen the fibers are further stabilized by the
formation of inter- and intramolecular crosslinks. This process
occurs through the action of lysyl oxidase (LOX) (Smith-Mungo
and Kagan, 1996) This enzyme acts only on the extracellular
aggregated molecules and recognizes different binding sites that
are similar in the type I, II, and III collagens and are located in
the telopeptides and the triple-helix. The process of collagen
cross-linkings is initiated by the conversion of telopeptidyl
lysine and hydroxylysine residue to aldehyde through the action
of LOX (Viguet-Carrin et al, 2005). LOX catalyzes the
oxidative deamination of the ε-amino group on lysyl or
hydroxylysyl side chains of telopeptides, resulting in the
formation of two aldehydes, allysine and hydroxyallysine. LOX
realizes an oxidative deamination of hydroxyline residues in
position 9N (N-terminal telopeptide) and 16C (C-terminal
telopeptide), transforming them in hydroxyallysine. These
chemical reactions (Fig 1) result in the formation of crosslinks
by divalent crosslinks, which stabilize the immature collagen
fibers, further react with another telopeptide aldehyde group to
form trivalent pyridinium crosslinks (Knott and Bailey, 1998).
45
Fig.1 Pathways of collagen enzymatic crosslinks. Adapted from Viguet-
Carrin et al, 2005
The conversion of immature to mature crosslinks is a
continuous process independent of turnover rate.
The formation of trivalent mature crosslinks such as
pyridinoline and deoxypyridinoline from the hydroxyallysine
pathway is still poorly understood. Some authors have
hypothesized that they may result either in the spontaneous
condensation between two divalent ketoamine crosslinks (Eyre
and Oguchi, 1980) or a ketoamine crosslink reacting with a
hydroxyallysine residue (Robins and Ducan, 1983). Pyridinium
cross-links are predominant in mature connective tissues except
in normal skin. Pyridinoline and deoxypyridinoline are formed
during extracellular maturation of collagen fibrils, and they are
46
the predominant stabilizing bounds in mature tissues (Viguet-
Carrin et al, 2005).
2.2 Cross-linking Agents
Cross-linker agents can be dived in two different classes
of substances: synthetic resources and natural renewable
resources.
2.2.1 Synthetic Resources
Physical methods. Also called the photo-oxidative
method. It bases on synthetic biomodifiers that use light
exposure, especially ultraviolet radiation. In 1968 Christopher
Foote published the mechanisms by which photosensitised
oxidation happens in biological systems; furthermore Fujimori
in 1988 exposed a third mechanism of cross-linkage in type I
collagen that involved either oxidation by ozone or photo-
oxidation by UV light (Foote, 1968; Fujimori, 1988). Outside
biology, photopolymerisation is a comparable process that is
being used in industry to generate polymers using the free
radical- generating properties of radiant energy like UV light.
Photopolymerisation of multifunctional monomers resulting in
highly cross-linked materials suitable for applications such as
epoxy coatings, optical lenses, and dental materials are in
common use. The photo-oxidative method requires the presence
of singlet oxygen, and riboflavin (vitamin B2) is one of the most
potent producers of oxygen radicals. (Snibson et al, 2010).
47
Ribofalvin (vit B2)
Riboflavin (vitamin B2) is part of the vitamin B group.
Riboflavin is characterized by a yellow-orange color and it is
present in different foods i.e. milk, cheese, leaf vegetables, liver,
kidneys, legumes, yeast, mushrooms, and almonds (McCance
and Widdowson’s, 2002). The name "riboflavin" comes from
"ribose" (the sugar whose reduced form, ribitol, forms part of its
structure) and "flavin", the ring-moiety which imparts the
yellow color to the oxidized molecule (Fig.2); the reduced form,
which occurs in metabolism along with the oxidized form, is
colorless. Riboflavin is the central component of the cofactors
flavin adenine dinucleotide and flavin mononucleotide and as
such required for a variety of flavoprotein enzyme reactions
including activation of other vitamins. The active forms flavin
mononucleotide and flavin adenine dinucleotide function as
cofactors for a variety of flavoprotein enzyme reactions.
a b
Fig2 a: Riboflavin molecular structure; b: riboflavin powder
Due to it has poor solubility in water it is difficult to
incorporate riboflavin into many liquid products, hence the
requirement for riboflavin-5'-phosphate, a more soluble form of
riboflavin is usual. In medicine application riboflavin, in
combination with UV light, has been shown to be effective in
reducing the ability of harmful pathogens found in blood
products to cause disease (Goodrich et al, 2006; Kumar et al,
48
2004). Riboflavin and UV light treatment has also been shown
to be effective for inactivating pathogens in platelets and
plasma, and is under development for application to whole
blood. Recently, riboflavin has been used in a new treatment to
slow or stop the progression of the corneal disorder keratoconus
(Wollensak et al, 2003; Sorking et al, 2014). This is defined as
corneal collagen cross-linking. In corneal disease, riboflavin
drops are applied to the patient’s corneal surface. Once the
riboflavin has penetrated through the cornea, ultraviolet (UV) A
light therapy is applied. This induces collagen cross-linkings,
which increases the tensile strength of the cornea. The treatment
has been shown in several studies to stabilize keratoconus
disease (Wollensak et al, 2003; Snibson 2010).
In humans, there is no evidence for riboflavin toxicity
produced by excessive intakes, as its low solubility keeps it
from being absorbed in dangerous amounts within the digestive
tract. Even when 400 mg of riboflavin per day was given orally
to subjects in one study for three months to investigate the
efficacy of riboflavin in the prevention of migraine headache, no
short-term side effects were reported (Boehnke et al, 2004)
Although toxic doses can be administered by injection,
(Boehnke et al, 2004) any excess at nutritionally relevant doses
is excreted in the urine (Zempleni et al, 1996), imparting a
bright yellow color when in large quantities.
The cross-linking reaction induced by the activation of
riboflavin is schematized in Fig.3. Photo-activation of riboflavin
results in singlet-oxygen inducing chemical covalent bonds,
bridging amine groups (NH) of glycine of one chain with
carbonyl groups (CO) of hydroxyproline and proline in adjacent
chains (McCall et al, 2010).
49
F
Fig.3 Riboflavin cross-linking reaction: after riboflavin UV activation singlet
oxygen was produced, this starts the lysyl oxidase reactions which induces
chemical covalent bonds, bridging amine groups (NH) of glycine of one
chain with carbonyl groups (CO) of hydroxyproline and proline in adjacent
chains
2.2.2 Chemical Agents
Different families of substances belong to this class of
cross-linking agents; aldehyde and carbodiimide, with
respectively specific agents involved in this experimental study,
are analyzed below.
Aldehydes. Aldehyde is a family of organic compounds
containing a formyl group. Formyl group is a functional group,
with the structure R-CHO, consisting of a carbonyl center (a
carbon double bonded to oxygen) bonded to hydrogen and an R
group, which is any generic alkyl or side chain. The group
without R is called the aldehyde group or formyl group.
50
Aldehydes are common in organic chemistry. Traces of many
aldehydes are found in essential oils and often contribute to their
favorable odors, e.g. cinnamaldehyde, cilantro, and vanillin.
Because their high reactivity aldehyde molecules are involved in
many reactions (Smith, 2007). From the industrial perspective,
important reactions are condensations, e.g. to prepare
plasticizers and polyols, and reduction to produce alcohols,
especially "oxoalcohols." From the biological perspective, the
key reactions involve addition of nucleophiles to the formyl
carbon in the formation of imines (oxidative deamination) and
hemiacetals (structures of aldose sugars) (Smith, 2007).
Among these agents glutaraldehyde is the most widely
known agent of this class, anyway similar cross-linking ability is
found with other aldehydes such as formaldehyde and
glyceraldehyde. Glutaraldehyde is a dialdehyde with high
affinity for free primary amine groups of amino acids (Cheung
et al, 1985); it has been associated with a decrease in the rate of
collagen degradation (Cheung et al, 1990) and improved dentin
collagen properties (Bedran-Russo et al, 2008), reacting
primarily with the ε-amino groups of peptidyl lysine and
hydroxylysine residues of collagen fibrils. A disadvantage of
glutaraldehyde is its high cytotoxicity (Han et al, 2003), which
limits clinical applicability.
Acrolein
Acrolein (2-propenal or acrylic aldehyde) (Fig.4) is the
simplest unsaturated aldehyde. It is a three-carbon α-β-
unsaturated monoaldehyde that provides outstanding
stabilization of proteins, similar to glutaraldehyde, but
penetrates tissue more rapidly (Saito and Keino 1976),
providing excellent morphological preservation of fine structure
for electron microscopy (EM) studies (Sabatini et al, 1963). The
acrolein exists as a colorless liquid with a piercing, disagreeable,
acrid smell. Acrolein is ubiquitously present in foods, cooked or
51
not, and in the environment. It is formed from carbohydrates,
vegetable oils and animal fats, amino acids during heating of
foods, and by combustion of petroleum fuels and biodiesel
(Stevens and Maier, 2008). Chemical reactions responsible for
release of acrolein include heat-induced dehydration of glycerol,
retroaldol cleavage of dehydrated carbohydrates and lipid
peroxidation of polyunsaturated fatty acids. Smoking of tobacco
products equals or exceeds the total human exposure to acrolein
from all other sources (Bein and Leikauf, 2011). Acrolein is
metabolized by conjugation with glutathione and excreted in the
urine as mercapturic acid metabolites. The biological effects of
acrolein are a consequence of its reactivity towards biological
nucleophiles such as guanine in DNA and cysteine, lysine,
histidine, and arginine residues in critical regions of nuclear
factors, proteases, and other proteins (Aldini et al, 2011).
Fig.4 Acrolein molecular structure
Acrolein is commonly used as a tissue fixative although
can degrade enzymatic activity or antigenicity (Sabatini et al,
1963) but degradation is not excessive when fixation times are
short (Flitney, 1966). Furthermore, the rapid penetration and
strong cross-linking abilities of acrolein quickly stabilize
proteins, retaining even small peptides that can be successfully
immunolabeled (Pickel et al, 1986). Likewise other aldehydes
the acrolein can be classified as a cross-linker agent. Cross-
linking reaction is schematized in Fig.5. Acrolein reacts
52
preferentially with cysteine, lysine, and hystidine residues (the
lysine adducts being the more stable products) via Michael-type
addition reactions preserving the aldehyde functionality on the
modified protein. The reaction of acrolein with Lys may result
in β-substituted propanals (R-NH-CH2-CH2-CHO), but the
major adduct formed on reaction with protein is the Ne-(3-
formyl-3,4- dehydropiperidino) lysine adduct (Uchida et al,
1998). This compound is a reactive intermediate that can
covalently bind to thiols, such as glutathione (Furuhata et al,
2002). The amino groups (N-terminus [Phe1] or lysine29) and
the histidine residues (histidine 5 or histidine 10) were identified
as the main sites involved in the formation of inter and
intramolecular cross-linkings adducts. These results allowed the
proposal of a mechanism of protein cross-linkings by acrolein,
involving inter- and intra-molecular cross-linking adducts
between amino groups and the side chain of histidine through
Michael addition (Aldini et al, 2011).
Fig.5 Acrolein cross-linking reaction: 2 acrolein molecule reacts with a N
residue of lysine forming an intermediate that will bond to a thiolic residue of
a glutathione of another collagen chain.
53
Carbodiimide. A carbodiimide or a methanediimine is a
functional group consisting of the formula RN=C=NR.
Carbodiimides hydrolyze to form ureas, which makes them
uncommon in nature. Carbodiimides are formed by dehydration
of ureas or from thioureas. They are also formed by treating
organic isocyanates with suitable catalysts (generally based on
phosphine oxides); in this process, carbon dioxide evolves from
the isocyanate (Shennan et al, 1961). In synthetic organic
chemistry, compounds containing the carbodiimide functionality
are dehydration agents and are often used to activate carboxylic
acids towards amide or ester formation. Additives, such as N-
hydroxybenzotriazole or N-hydroxysuccinimide, are often added
to increase yields and decrease side reactions. While the cross-
linking potential is limited (Bedran-Russo et al, 2010),
carbodiimides are less toxic than aldehydes (Huang et al, 1990).
Carbodiimide hydrochloride (EDC)
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
(Fig.6) is a water soluble carbodiimide usually obtained as the
hydrochloride. It is typically employed in the 4.0-6.0 pH range
(Lòpez-Alonso et al, 2009). It is generally used as a carboxyl
activating agent for the coupling of primary amines to yield
amide bonds. Additionally, EDC can also be used to activate
phosphate groups in order to form phosphomonoesters and
phosphodiesters. Common uses for this carbodiimide include
peptide synthesis, protein cross-linkings to nucleic acids, but
also in the preparation of immunoconjugates (Lòpez-Alonso et
al, 2009). In medical filed EDC was recently studied, with
promising results, as reinforced for collagen scaffold in tissue
engineering (Li et al, 2013).
54
Fig.6: EDC molecular structure
EDC is often used in combination with N-
hydroxysuccinimide (NHS) for the immobilization of large
biomolecules.
EDC is known as a zero-length agent due to its ability to cross-
link peptides without introducing additional linkage groups. The
cross-linking mechanism is mediated by the activation of
carboxylic acid groups of glutamic and aspartic acids to form an
O-acylisourea intermediate. The latter reacts with the ε-amino
groups of lysine or hydroxylysine to form an amide cross-link,
leaving urea as the terminal by-product (Fig. 7). The addition of
N-hydroxysuccinimide to the EDC-containing solution is
effective in increasing the number of induced collagen cross-
linking and preventing the hydrolysis of activated carboxyl
groups (Staros et al, 1986; Olde et al, 1996). Cross-linkings
with EDC are especially appealing for biological applications as
the carbodiimide does not remain in the chemical bond but is
released as a substituted urea molecule.
55
Fig.7: EDC cross-linking reaction: EDC reacts with carboxylic
group of, after the elimination of O-acylisourea intermediate, the glutamic or
aspartic acid bond the N residue of lysine or hydroxylysine.
Dicyclohexylcarbodiimide (DCC)
N,N'-Dicyclohexylcarbodiimide (DCC) is an organic
compound with the chemical formula C13H22N2 (Fig.8) whose
primary use is to couple amino acids during artificial peptide
synthesis. Under standard conditions, it exists in the form of
white crystals with a heavy, sweet odor; DCC is normally used
as a condensation reagent in amide synthesis or esterification
reactions. (Chen et al, 2013; Yano et al, 2015) Differently to
EDC the DCC is insoluble in water but on the other hand it is
well soluble in other organic compounds, such as ethanol and
acetone.
During protein synthesis, the N-terminus is often used as
the attachment site on which the amino acid monomers are
added. To enhance the electrophilicity of carboxylate group, the
negatively charged oxygen must first be "activated" into a better
leaving group, and DCC is also used for this purpose. The
negatively charged oxygen will act as a nucleophile, attacking
the central carbon in DCC, thus it is temporarily attached to the
former carboxylate group forming a highly electrophilic
intermediate, making nucleophilic attack by the terminal amino
56
group on the growing peptide more efficient (Sebald et al,
1980).
Fig.8: DCC molecular structure
Similarly to the EDC cross-linker mechanism the DCC
first reacted with the carboxylic acid group of the amino acid to
form the O-acylisourea intermediate, and then the DCC adduct
underwent a relatively slow rearrangement process to form the
final derivative that possesses higher ionization efficiency and
higher molecular weight than the original amino acid (Sebald et
al,1980; Yano et al, 2015 ) (Fig.9).
Fig.9: DCC cross-linking reaction: likewise EDC, the DCC reacts
with carboxylic group of glutamic and aspartic acids, after the elimination of
O-acylisourea intermediate, the glutamic or aspartic acid bond the N residue
of lysine or hydroxylysine.
57
2.2.3 Natural Renewable Resources
Besides the family of chemical agents, naturally
compounds have received great attention in the past decade for
their potential use as cross-linking agents, especially in
dentistry.
Sources such as genipin (an aglycone of an iridoid
glycoside, one of the major components in the fruit of Gardenia
jasminoides) or proanthocyanidins (PACs) were objective of
studies even if their application is still limited due to its slow
cross-linking reaction (Bedra-Russo et al, 2007; Bedra-Russo et
al, 2014). Inter- and intra-molecular cross- linking is believed to
be mediated by reactions with free amino acid (lysine,
hydroxylysine, or arginine) to form a nitrogenous iridoid
derivative that undergoes dehydration to form an aromatic
monomer (Sung et al, 1999, 2003). A large variety of
bioactivities have been reported for polyphenols from plants.
Their biological functions are the foundation for their prominent
roles in plant-based dietary supplements and nutritionals, and
recent research continues to explore new applications.
PACs, belonging to a category known as condensed
tannins, are highly hydroxylated structures capable of forming
an insoluble complex with carbohydrates and proteins. The
interaction of PACs and collagen results in the formation of
complexes that are believed to be stabilized primarily by
hydrogen bonding between the protein amide carbonyl and the
phenolic hydroxyl in addition to covalent and hydrophobic
bonds (Bedran-Russo et al, 2014). The relatively high stability
of PACs-protein complexes suggests structure specificity
(Hagerman et al, 1981), which encourage hydrogen binding but
also creates hydrophobic pockets (Han et al,2003).
58
2.3 Use of Collagen Cross-linkers in Clinical
Applications
Recent in vitro studies demonstrated that the use of
cross-linking agents improved the short-term mechanical
properties of dentin collagen, reduced the susceptibility of
additionally cross-linked dentin collagen to enzymatic
degradation by collagenases, and increased the stability of the
resin-dentin interface (Bedran-Russo et al, 2008Al-Ammar et al,
2009; Macedo et al,2009;Castellan et al, 2010).
Resin–dentin bonded interfaces may be considered a
unique form of tissue engineering in which a collagen-based
dentin matrix scaffold is reinforced by resin to produce a hybrid
layer that couples adhesives/resin composites to the underlying
mineralized dentin (Liu et al, 2011). Denuded collagen matrices
of the hybrid layer are also filled with water, which serves as a
functional media for the degradation of resin matrices and
collagen (Pashley et al, 2004, Breschi et al, 2008). This pitfall
has been recognized as a major factor leading to short service
life of adhesive resin composite restorations. Strengthening of
type I collagen and reducing biodegradation are likely to
reinforce the dentin structure and in combination are promising
strategies to develop a strong and long lasting tooth-biomaterial
interface. Enhanced properties of dentin matrix have been
extensively demonstrated for various types of dentin
biomodification strategies (Bedran-Russo et al, 2008; Fawzy et
al, 2012). A pioneer study conducted by Bedran-Russo and coll
(2007) showed that naturally occurring cross-linkings agents
such as PAC and genepin are capable of stabilizing
demineralized dentin collagen improving its mechanical
properties. After that Macedo and coll (2009) demonstrated that
the application of glutaraldehyde or grape-seed extract as
chemical cross-linking agents within etched dentin prior to
59
bonding procedures significantly enhanced the dentin bond
strengths both caries-affected and sound dentin, increasing
dentin collagen stability. Another study conducted by Dos
Santos and coll in 2011(a) confirmed that biomodification of the
dentin–resin interface structures using glutaraldehyde and grape-
seed extract can increase the mechanical properties of the
interface over time and may contribute to the long-term quality
of adhesive restorations. A closer look at the interface
components shows that the mechanical properties of the
underlying dentin and hybrid layer are significantly improved
and largely attributed to agent-mediated non-enzymatic collagen
cross-linking (Dos Santos et al, 2011a; Dos Santos et al, 2011b).
In general, most dentin biomodification strategies impair
exogenous and endogenous proteolytic activity in a
concentration dependent manner. Besides that, additional recent
published studies evidenced the ability of collagen cross-linkers
in inhibition of MMPs enzymes (Tezvergil-Mutluay et al, 2010;
Tjäderhane et al, 2011). This double influence, strengthening
collagen and inhibition of native collagenases, by collagen
cross-linking agents is expected to provide the formation a more
durable hybrid layer.
60
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65
3 Chapter
Experimental Study
Introduction
In etch-and-rinse or self-etch adhesive bonding systems
the stability and integrity of collagen fibrils within the hybrid
layer are crucial for the maintenance of bond effectiveness over
time (Breschi et al, 2008; Liu et al, 2011). In the last years the
use of cross-linkers has been proposed such as bio-modifying
agents to improve the mechanical and biological properties of
type I collagen, with the aim to create a stable dentin matrix
network that, after resin infiltration, should provide a durable
hybrid layer.
The purpose of the present study was to investigate the
mechanical and biological effect of different class of collagen
cross-linking agents, used as conditioning primer, on the hybrid
layer created in association with two different 2-step etch-and-
rinse and one 1-step self-etch adhesives.
Cross-linkers tested
• EDC-HCl (ProteoChem Inc.)
• Riboflavin 5′-phosphate sodium salt hydrate
(Sigma-Aldrich)
• Acrolein (Sigma-Aldrich)
• DCC (Sigma-Aldrich)
Dentin bonding adhesive systems tested (Table1)
• Optibond FL (Kerr)
• Adper Scotchbond 1XT (3M ESPE)
• XP Bond (Dentsply)
• Clearfi SE Primer and Bond (Kurary)
66
Table.1: composition of dentin bonding adhesive systems tested
Adhesive Manufacturer Composition
Optibond FL
(3-step etch-and-rinse adhesive)
Kerr Etching: 37.5% H3PO4
Primer: TEGDMA, HEMA,
modified poly- acrylic acid,
maleic acid, ethanol, water,
photoinitiators, stabilizers
Bonding: bis-GMA, TEGDMA,
glass filler, SiO2,
photoinitiators, stabilizers
Adper Scotchbond 1 XT
Adhesive
(2-step etch-and-rinse adhesive)
3M ESPE Etching: 35% H3PO4
Primer: -
Bonding: dimethacrylates,
HEMA, poly- alkenoid acid
copolymer, 5 nm silane- treated
colloidal silica, ethanol, water,
photoinitiator
XP BOND
(2-step etch-and-rinse adhesive)
Dentsply/ Caulk Etching: 36% H3PO4
Primer: -
Bonding: PENTA, TCB,
HEMA, TEGDMA, UDMA,
tert-butanol, functionalized
amor- phous silica, ethyl-4-
dimethylaminobenzo- ate, CQ,
stabilizer, t-butanolate, CQ,
stabi- lizer, t-butanolate, CQ,
stabilizer, t-butanol
Clearfil SE Bond
(2-step self-etch adhesive)
Kuraray Self-etching primer: 10-MDP,
HEMA, photoinitiator, water
Bonding: 10-MDP, bis-GMA,
HEMA, hydrophilic
dimethacrylate, microfiller
67
3.1 Microtensile Bond Strength (µTBS)
3.1.1 Overview of the Technique
Bond strength tests are the most frequently used tests to
screen adhesives. A tensile bond strength test is defined
“microtensile” when bonded surface analyzed is 1 mm2 or less;
The microtensile bond strength test is calculated as the tensile
load at failure divided by the cross-sectional area of the bonded
interface (Breschi et al, 2013).
In these tests, large restorative material buildups are
placed on a flat dentin surface after the application of adhesive.
Multiple specimens may then be obtained from each tooth by
removing 1 × 1–mm or smaller sections (ie, sticks) from the
tooth-composite preparation. The specimens are then loaded to
failure using a universal testing machine. Later the failure of
each sticks will be analyzed and defined as “adhesive”, when
the failure occur in adhesive interface, “cohesive”, when the
failure occurs within the same material (dentin or composite), or
“mixed” failure when failure include the both typologies
(Armostrong et al, 2010).
Specimens may be prepared using trimming or non-
trimming procedures (Neves et al, 2009). Trimming procedures
create dumbbell- or hourglass- shaped specimens by trimming
them at the interface. Although this technique improves the
concentration of stress at the interface, specimen preparation is
complex and highly operator- dependent. The use of gripping
devices or glue with specimen- jig attachments, the speed of
loading, and the alignment of specimens may also affect the
results of microtensile tests.
Pre-test failure can occur during the preparation of
specimens for microtensile tests. Several approaches to the
consideration of such failures have been proposed, although
each is associated with an analytic shortcoming. The exclusion
68
of all pretest failures from statistical analyses may lead to the
overestimation of bond strength, whereas the designation of a 0-
MPa bond strength value to each failure may result in the
underestimation of bond strength. A predetermined bond
strength value, such as the lowest measured value within the test
group, may also be assigned to each pretest failure (Armostrong
et al, 2010).
3.1.2 Storage in Artificial Saliva
In order to reproduce the oral environment as
realistically as possible the specimens were stored in artificial
saliva (compounds in Table2) solution at 37°C. Many studies in
literature demonstrated that bond strength values decrease even
after brief period of storage (Kato and Nakabayashi, 1998;
Shono et al, 1999; Meiers and Young, 2001; De Munck et al,
2003). Furthermore Pashley et al (2004) demonstrated that the
degradation of the hybrid layer can occur also in absence of
bacteria. Several in vitro studies have provided morphological
evidence of resin elution and/or hydrolytic degradation of
collagen agents in resin-dentin bonds. When the samples are
stored in water or artificial saliva the degradation of adhesive
interface did not present significantly modifications (Kitasako et
al., 2000), anyway storage in artificial saliva is preferred. Since
many degradation processes are dependent on the proportion of
diffusion of the medium along the adhesive interface, it is
important to evaluate its extension in addition to the diffusion
time: the extent of the infiltration should be minimized in order
to avoid artifacts diffusion-dependent. For this reason, sticks
aged with this technique should not present an interface area too
small. Furthermore different studies highlighted that the
adhesion interface area is inversely proportional to the bond
strength maintenance, in the same time-line storage (Shono et
69
al, 1999; Armstrong et al, 2001b). Hence this technique of
storage has to be considered as a method of accelerating aging.
Components Mg
CaCl2 0,70
MgCl2·6H2O 0,20
KH2PO4 4,00
KCl 30,00
NaN3 0,30
HEPES (buffer) 20,00 Table.2: compounds of artificial saliva according to Pashley et al,
2004.
3.1.3. Microtensile Bond Strength Test Performed
The ability of the previously described cross-linkers in
improvement the bond strength of adhesive interface created
with different dentin bonding adhesive systems, was assess by
microtensile bond strength test. The cross-linkers application
period was established in 1 min according to the recent finding
published by Tezvergil-Mutluay and coll (2012), and with the
intention to test a contact time period feasible in clinical
practice.
For the microtensile bond strength tests n 160 (16 each
group) freshly extracted sound third molars were used. Tooth
crowns were flattened using a low-speed diamond saw
(Micromet, Remet, Bologna, Italy) under water irrigation and a
standardized smear layer was created with 600-grit silicon-
carbide (SiC) paper.
Specimens were then randomly assigned to the following
experimental (a) and control groups (b):
70
Experimental groups
Group 1a (G1a): dentin was etched for 15 s with 35%
phosphoric-acid gel (3 M ESPE, St. Paul, MN, USA) and rinsed
with water. acid-etched dentin was pretreated with 0.3 M EDC
water-solution for 1 min, air-dried, primed and bonded with
Optibond FL (OFL; Kerr, Orange, CA, USA) following the
manufacturer’s instructions.
Group 2a (G2a): dentin was etched and pretreated with
0.3 M EDC as described for G1a, then bonded with Adper
Scotchbond 1XT (SB1XT 3M ESPE).
Group 3a: dentin was etched and pretreated with 0.3 M
EDC as described for G1a then bonded with XP Bond ( XPB
Dentsly)
Group 4a: Clearfil SE primer (CSE Kuraray) was
applied on unetched dentin according to the manufacturers’
instructions. Then the dentin surface was pretreated with 0.3 M
EDC as described for G1a; finally the dentin was bonded with
Clearfil SE Bond (CSE Kuraray)
Group 5a: dentin was etched as described for G1a. Then
dentin was treated for 1 min with 0.1% riboflavin-5- phosphate
(2 mmol/L) water solution of, air-dried, and exposed to UVA
rays for 2 min under a UV lamp (Philips, Hamburg, Germany; λ
= 370 nm at 3 mW/cm2 or 5.4 joule/cm2) placed 1 cm from the
dentin surface. After that XPB was applied on dentin surface
Group 6a: dentin was etched as described for G1a, after
dentin was pretreated for 1 min with 0,01% acrolein water
solution. Then dentin was bonded with SB1XT
Control groups
Group 1b: OFL was applied on etched dentin without
pre-treatment in accordance with manufacturer’s instructions
Group 2b: SB1XT was applied on etched dentin without
pre-treatment in accordance with manufacturer’s instructions
71
Group 3b: XPB was applied on etched dentin without
pre-treatment in accordance with manufacturer’s instructions
Group 4b: CSE was applied on etched dentin without
pre-treatment in accordance with manufacturer’s instructions
Each bonded specimen was light-cured for 20s using a
led curing light (Demi Light, Kerr). Four 1-mm-thick layers of
microhybrid resin composite (Filtek Z250; 3M ESPE) were
placed and polymerized individually for 20 s. Specimens were
serially sectioned to obtain approximately 1-mm-thick beams in
accordance with the microtensile non-trimming technique. The
dimension of each stick (ca. 0.9 mm × 0.9 mm × 6 mm) was
recorded using a digital caliper (±0.01mm) and the bonded area
was calculated for subsequent conversion of microtensile
strength values into units of stress (MPa). Beams were stressed
to failure after 24h (T0) or 1 year (T12) of storage in artificial
buffer at 37◦C, using a simplified universal testing machine
(Bisco, Inc., Schaumburg, IL, USA) at a crosshead speed of 1
mm/min. The number of prematurely debonded sticks in each
test group was recorded, but these values were not included in
the statistical analysis because all premature failures occurred
during the cutting procedure, and they did not exceed 3% of the
total number of tested specimens and were similarly distributed
within the groups. A single observer evaluated the failure modes
under a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena
GmbH) at magnifications up to 50× and classified them as
adhesive, cohesive in dentin, cohesive in composite, or mixed
failures.
For EDC microtensile bond strength results statistical
differences among adhesive groups (i.e. XP Bond, Clearfil SE,
SB1 XT, OFL), the storage time (baseline vs. 12months) and
pre-treatment (NO-EDC vs. EDC) were assessed using SPSS
21.0 software for Mac (SPSS Inc., Chicago, IL, USA). Data
were analyzed to determine if their normal distribution
72
(Kolmogorov-Smirnov test) and equal variance (Levine test)
assumptions were violated. Since these assumptions were not
valid, data were compared by non-parametric tests (Kruskal-
Wallis test followed, if significant, by pair-wise comparison
using the Mann–Whitney U-test). Differences were considered
significant at p < 0.05. The level of significance was adjusted
according to the Bonferroni’s correction. Data are expressed as
mean ± SD.
The effects of ACR and storage time were assessed using
SPSS 21.0 software for Mac (SPSS Inc., Chicago, IL, USA).
Data were analyzed to determine if their normal distribution
(Kolmogorov-Smirnov test) and equal variance (Levine test)
assumptions were violated. Since these assumptions were not
valid, data were compared by non-parametric tests (Kruskal-
Wallis test followed, if significant, by pair-wise comparison
using the Mann–Whitney U-test). Differences were considered
significant at p < 0.05. The level of significance was adjusted
according to the Bonferroni’s correction. Data are expressed as
mean ± SD.
For RB microtensile bond strength results because a
Kolmogorov–Smirnov test determined that values were
normally distributed, data were analyzed by two-way (surface
treatment, storage) Analyses of variance (ANOVA) and post
hoc Tukey tests. P values of 0.05 were considered to indicate
statistical significance.
3.2 Nanoleakage Analyses
3.2.1 Overview of the Technique
Sano and coll (1995a) described the nanoleakage for the
first time as submicron spaces in the hybrid layer of the order of
20-100 nm in width. After that many studies have confirmed
73
that small ions or molecules can diffuse into the hybrid layer in
the absence of detectable interfacial gap formation (Sano et al,
1995a,b). Nanoleakage phenomenon has been defined as the
passage of a tracer such as silver nitrate through the hybrid
layer. According to its original definition, nanoleakage is
created by the discrepancy between dentin demineralization and
adhesive infiltration that occurs in total-etch adhesive systems,
in the absence of marginal gap formation along the resin– dentin
interface (Suppa et al, 2005). This phenomenon is not
necessarily caused by disparities between the depths of
demineralization and resin infiltration. It may also represent the
presence of areas in which the retention of residual water in
etched dentin and/or adhesive results in regions of incomplete
polymerization or increased permeability within the resin
matrices of the adhesives (Tay et al, 2002).
3.2.2 Nanoleakage Analyses Performed
42 teeth (6/groups) were processed for interfacial
nanoleakage evaluation. Middle/deep dentin was selected, acid-
etched and bonded for one of the adhesives with or without the
EDC-containing conditioner as previously described for. A 1-
mm-thick flowable composite (Filtek Flow; 3 M ESPE) was
applied on the bonded disks and light-cured. Composite-dentin
specimens were cut vertically into 1-mm- thick slabs to expose
the bonded surfaces and stored for 24 h (T0) or 1 year (T12 ) in
artificial buffer at 37 ◦ C. Specimens were covered with nail
varnish, leaving 1 mm exposed at the bonded interface, and
processed for interfacial nanoleakage evaluation. Bonded
interfaces were immersed in 50 wt% ammoniacal AgNO3
solution in darkness for 24 h according to the protocol described
by Tay et al (2002). After immersion in the tracer solution,
specimens were rinsed in distilled water and immersed in photo-
developing solution for 8 h under a fluorescent light to reduce
74
silver ions into metallic silver grain within voids along the
bonded interfaces. Nanoleakage analysis was per- formed under
light microscopy (LM - Nikon E 800; Tokyo, Japan) and the
degree of interfacial nanoleakage was scored on a scale of 0–4
by two observers as described by Saboia et al. [28]. Interfacial
nanoleakage was scored based on the percentage of the adhesive
surface showing silver nitrate deposition: 0, no nanoleakage; 1,
<25% nanoleakage; 2, 25 to ≤50% nanoleakage; 3, 50 to ≤75%
nanoleakage; and 4, >75% nanoleakage. Statistical differences
among nanoleakage group scores (i.e. percentage of specimens
falling within each score category) were analyzed using the χ2
test. All statistical testing was performed at a pre-set alpha of
0.05. Inter-observer agreement was measured using Cohen’s
kappa test.
3.3 Zymographic Analyses
3.3.1 Overview of the Technique
Zymography is an electrophoretic method used to
measure proteolytic activity. It is already widely used for
research on extracellular matrix degrading enzymes, in
particular for the matrix metalloproteases (MMPs). The method
is based on sodium dodecyl sulfate gel impregnated with a
protein substrate, gelatin, which is degraded by the proteases
resolved during the incubation period. Sites are revealed of
proteolysis as white bands on a dark blue background. Within a
certain range the band intensity can be related linearly to the
amount of protease loaded (Leber and Balkwill et al, 1997).
Zymography offers several features which render it
particularly useful with respect to alternative methods such as
ELISA: no expensive materials are routinely required (e.g.,
antibodies) and several proteases showing activity on the same
75
substrate can be detected and quantified on a single gel (Kleiner
and Stetler-Stevenson, 1994). Finally, zymography has been
shown to be extremely sensitive. Levels of less than 10 pg of
MMP- 2 have been detected on gelatin zymograms comparing
favorably with ELISAs (Kleiner and Stetler-Stevenson, 1994;
Leber and Balkwill et al, 1997).
3.3.2 Zymographic Analyses performed
Preparation of dentin powder
In the present study the zymography was used to
measure the presence and the form distribution of all molecular
forms of both MMP-2 and MMP-9 in dentin powder treated or
not with cross-linking agents tested. The zymography was
performed in according to the protocol of Mazzoni and coll
(2014), as described below.
16 sound extracted third molars were chosen. Teeth,
ground free of enamel, pulpal soft tissue, and cementum, were
reduced to fine powder when the dentin was frozen in liquid
nitrogen and triturated by means of a steel mortar/ pestle
(Reimiller, Reggio Emilia, Italy). The fine mineralized dentin
powder was pooled, dried, and kept frozen until use.
Treatment of dentin powder and group division
For each cross-linkers tested separated zymographic
analyses were performed. Dentin powder was divided in 100 mg
lots, each lots was treated as described below.
1. 0.3M EDC zymography
Group 1 (G1): dentin powder (DP) leave untreated as
mineralized control
G2: DP was treated with 1 mL of 10%/wt phosphoric
acid for 10 min to simulate the etching procedure as the
76
first step of the etch- and-rinse bonding technique and
use as demineralized control (DDP)
G3: DDP was treated with 1 mL 0.3M EDC solution for
30 min
G4: DDP was mixed with 1 mL OFL for 30 min
G5: DDP as for G3 and mixed with 1 mL OFL for 30
min
G6: DDP was mixed with 1 mL SB1XT for 30 min
G7: DDP as for G3 and mixed with 1 mL SB1XT for 30
min
G8: DDP was mixed with 1 mL XPB for 30 min
G9: DDP as for G3 and mixed with 1 mL XPB for 30
min
2. 0.1% Riboflavin zymography
G1: DP was treated with 1 mL of 10%/wt phosphoric
acid for 10 min to simulate the etching procedure as the
first step of the etch- and-rinse bonding technique and
use as demineralized control (DDP)
G2: DDP was treated with 1 mL 0.1% Riboflavin
solution for 30 min
G3: DDP was mixed with 1 mL XPB for 30 min
G4: DDP as for G2 and mixed with 1 mL XPB for 30
min
3. 0.01% Acrolein zymography
G1: DP was treated with 1 mL of 10%/wt phosphoric
acid for 10 min to simulate the etching procedure as the
first step of the etch- and-rinse bonding technique and
use as demineralized control (DDP)
G2: DDP was treated with 1 mL 0.01% acrolein solution
for 30 min
4. 0.5M DCC zymography
G1: DP leave untreated as mineralized control
G2: DP was treated with 1 mL of 10%/wt phosphoric
acid for 10 min to simulate the etching procedure as the
77
first step of the etch- and-rinse bonding technique and
use as demineralized control (DDP)
G3: DDP was treated with 1 mL 0.5M DCC/acetone
solution for 30 min
G4: DDP was treated with 1 mL 0.5M DCC/ethanol
solution for 30 min
G5: DDP was mixed with 1 mL SB1XT for 30 min
G6: DDP as for G3 and mixed with 1 mL SB1XT for 30
min
G7: DDP as for G4 and mixed with 1 mL SB1XT for 30
min
Enzyme Extraction of Demineralized Dentin Specimens
and Sample Conditioning
The demineralized dentin powder was suspended in
extraction buffer (50 mM Tris- HCl, pH 6, containing 5 mM
CaCl2, 100 mM NaCl, 0.1% Triton X-100, 0.1% non-ionic
detergent P-40, 0.1 mM ZnCl2, 0.02% NaN3) for 24 hrs at 4°C.
The specimens were then sonicated for 10 min (at ≈ 30 pulses)
and centrifuged for 20 min at 4°C (20,800 g); the supernatant
was removed and re-centrifuged. The protein content was
further concentrated by means of a Vivaspin centrifugal
concentrator (10,000-kDa cut-off) for 30 min at 20°C (15,000 g,
3 times). Total protein concentrations of dentin extracts were
determined by the Bradford assay.
Preparation and running of the zymogram gels
Dentin protein aliquots were diluted in Laemmli sample
buffer at a 4:1 ratio and subjected to electrophoresis under non-
reducing conditions in 10% sodium dodecyl sulfate-
polyacrylamide gel (SDS-PAGE) containing 1 mg/mL
fluorescently labeled gelatins. Pre-stained low- range molecular-
weight SDS-PAGE standards (Bio-Rad) were used as
molecular-weight markers. Samples run at constant of 120 V
78
until the dye front ran off the gel. After electrophoresis, the gels
were washed for 1 hr in 2% Triton X-100 and were then
incubated in activation solution (50 mmol/L Tris-HCl, 5
mmol/L CaCl2, pH 7.4) for 48 hrs. Proteolytic activity was
evaluated and registered under long-wave UV light scanner
(ChemiDoc Universal Hood, Bio-Rad). Gelatinases (MMP-2
and -9) in the samples were analyzed in duplicate by gelatin
zymography.
3.4 In situ Zymography Analyses
3.4.1 Overview of the Technique
During the past decade, in situ zymography has been
applied to localize gelatinolytic activity in tissue sections (Galis
et al, 1994). This method is based on the principle of gel
substrate zymography. The principle introduced by Galis and
coll (1995) has been modified in the procedure to demonstrate
the breakdown of gelatin differently. Instead of fluorescent
gelatin, some authors used pure gelatin that was stained after
incubation with Ponceau S (Loy et al, 2002), amido black (Ikeda
et al, 2000; Furuya et al, 2001), or Biebrich Scarlet (Wada et al,
2003). All the approaches have in common the fact that a
decrease in staining intensity is a reflection of gelatinolytic
activity. The approach of gelatin in situ zymography has been
applied to study involvement of gelatinases in many
patho/physiological processes and the involvement of
gelatinolytic activity in cancer progression (Faia et al, 2002;
Leco et al, 2001; Tarlton et al, 2000; Pirila et al, 2001).
Precise localization of gelatinase activity in sections and
cells became possible with the introduction of dye-quenched
79
(DQ)-gelatin, which is gelatin that is heavily labeled with FITC
molecules so that its fluorescence is quenched (Goodall et al,
2001; Lindsey et al, 2001; Teesalu et al, 2001; Wang and
Lakatta 2002; Mook et al, 2003; Platt et al, 2003; Lee et al,
2004). After cleavage of DQ-gelatin by gelatinolytic activity,
fluorescent peptides are produced that can be visualized against
a weakly fluorescent background (EnzCheck; Molecular Probes,
Eugene, OR). The use of DQ-gelatin instead of labeled or
unlabeled gelatin is superior for in situ zymography because
fluorescence is produced at sites of gelatinolytic activity instead
of decreased staining intensity at gelatinolytic areas.
3.4.2 In situ Zymography Performed
The in situ zymography were performed in according to
the protocol of Mazzoni et al (2014), as described below
20 freshly extracted non-carious human third molars
were selected for in situ zymography. Enamel and cementum
were removed, and 1-mm-thick disks of middle/deep coronal
dentin were obtained from each tooth by means of a slow-speed
saw (Micromet, Remet, Casalecchio di Reno, Italy). A
standardized smear layer was created with 600-grit wet silicon-
carbide paper; dentin was etched for 15 sec with 35%
phosphoric-acid gel (3M ESPE, St. Paul, MN, USA) and rinsed
with continuous water irrigation for 30 sec. Etched dentin
specimens were then equally divided into 4 groups and treated
as follows:
80
Group1 (G1): dentin was pre-treated with 0.3M EDC
water solution for 1 min, and the excess was gently blown off
with air, then bonded with OFL
G2: OFL was applied to untreated etched dentin as per
the manufacturer’s instructions
G3: dentin was pre-treated with 0.3M EDC water
solution for 1 min, and the excess was gently blown off with air,
then bonded with SB1XT
G4: dentin was pre-treated with 0.5M DCC/acetone
solution for 1 min, and the excess was gently blown off with air,
then bonded with SB1XT
G5: dentin was pre-treated with 0.5M DCC/ethanol
solution for 1 min, and the excess was gently blown off with air,
then bonded with SB1XT
G6: SB1XT was applied to untreated etched dentin as
per the manufacturer’s instructions
A 1-mm- thick flowable composite (Filtek Flow; 3M
ESPE) was applied to the resin-bonded disks and light-cured for
20 sec with a quartz-tungsten-halogen light-curing unit (Demi
Light, Kerr). Bonded specimens were then cut vertically into 1-
mm-thick slabs to expose the adhesive/dentin interfaces by
means of a slow-speed saw (Micromet); slabs were glued to
glass slides and ground down to obtain specimens ca. 50 µm
thick. In situ zymography was performed with self-quenched
fluorescein-conjugated gelatin as the MMP substrate (E-12055,
Molecular Probes, Eugene, OR, USA) the fluorescent gelatin
mixture was placed on top of each slab and covered with a
coverslip, and the slides were light-protected and incubated in
humidified chambers at 37°C for 24 hrs. The hydrolysis of
quenched fluorescein-conjugated gelatin substrate, indicative of
endogenous gelatinolytic enzyme activity, was assessed by
examination with a confocal laser-scanning microscope
[excitation (ex), 488 nm; and emission (em), lp530 nm; Nikon
81
A1-R, Tokyo, Japan]. Negative control sections were incubated
as described above, except that: (1) 250 mM
ethylenediaminetetraacetic acid (EDTA) was dissolved in the
mixture of quenched fluorescein-conjugated gelatin or (2) 2 mM
1,10-phenanthro- line or (3) standard non-fluorescent instead of
fluorescent- conjugated gelatin was used. EDTA and 1,10-
phenanthroline were used as negative controls because they are
well-known MMP inhibitors.
82
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85
4 Chapter
Results and Discussion
4.1 Results
4.1.1 Microtensile Bond Strength Test
EDC microtensile bond strength
Means and standard deviations of microtensile bond
strength (in MPa) at T0 and T12 are reported in Table1. The use
of 0.3 M EDC-containing conditioner before adhesives
application did not affect the immediate bond strength of OFL,
SB1XT and XPB (G1a 44.6 ±10.0 MPa, G1b 41.9 ±11.8 MPa;
G2a 38.8 ±8.3 MPa, G2b 40.1 ±8.2 MPa; G3a 36.5 ±7.2 MPa,
G3b 37.6 ±5.9 MPa). EDC affects the immediate bond strength
of CSE since when EDC is used the values resulted in
significant decreasing (G4a 30.1 ±6.3 MPa, G4b 32.8 ±4.4
MPa). After 12 months of storage the EDC cross-linked
specimens showed no significant loss of bond strength for OFL
and CSE (G1a 39.7 ±9.1 MPa; G4a 26.7 ±8.0 MPa respectively)
compared to T0 (p>0.05) while the bond strength of control
specimens was significantly reduced (G1b 31.9 ±5.7 MPa; G4b
21.4 ±5.7 MPa). For the SB1XT and XPBOND the EDC pre-
treatment resulted in a significant reduction of bond strength
after 1 year (G2a 31.1±7.7 MPa; G3a 28.6 ±6.4 MPa) compared
to T0 (p < 0.05), although EDC-treated specimens performed
significantly better when compared to the untreated control
specimens (G2b 24.6 ±6.0 MPa; G3b 18.1 ±4.9 MPa).
86
Table1: Microtensile bond strength values and standard deviation at
T0 and T12 are reported in MPa. Diff., significance of the difference between
pre-treatments, within adhesive groups per time point or between time points
per pre-treatment, for each adhesive groups. Diff. among adhesive groups,
significance of the difference among the adhesive groups within each pre-
treatment per time point. Note on the results on the pairwise comparisons
between adhesive groups clustered as a, b and c (within each pre-treatment
and per time point) are based on the Mann-Whitney U-Test (p<0.05) test. S,
difference statistically significant; NS, difference not statistically significant.
Percentages of the failure modes (reported in square rounds) after
microtensile test analyzed by stereomicroscopy were classified as: A,
adhesive; CC, cohesive in composite; CD, cohesive in dentin; and M, mixed
failure. Bond reduction after storage report the percentage of mean bond
reduction after 1 year of storage.
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Acrolein microtensile bond strength
Means and standard deviations of microtensile bond
strength (in MPa) at T0 and T12 are reported in Table2.
Immediate bond strength is not affected by ACR pre-treatment
(G5a 44.6±14.2, G2b 40.1±8.2). After 1 year storage in artificial
saliva ACR bonded specimens showed no significant reduction
of bond strength (G5a 46.4±6.1), while the control group
resulted in significant loss of bond strength values.
Table2. Data are expressed as mean ± SD. Differences were
considered significant at p < 0.05. The level of significance was adjusted
according to the Bonferroni’s correction. Groups with the same superscripts
are not statistically significant (p>0.05). Percentages of the failure modes
(reported in square rounds) after microtensile test analyzed by
stereomicroscopy were classified as: A, adhesive; CC, cohesive in composite;
CD, cohesive in dentin; and M, mixed failure. Bond reduction after storage
report the percentage of mean bond reduction after 1 year of storage.
Riboflavin microtensile bond strength
Means and standard deviations of microtensile bond
strength (in MPa) at T0 and T12 are reported in Table 3. The use
of the experimental primer containing UVA-activated riboflavin
before XP Bond application (G6a 44.4±10.4 MPa,) increased
the immediate bond strength compared with control specimens
(G3b 37.6±5.9 MPa). After storage for 12 months, the RB cross-
linked specimens showed a significant loss of bond strength
(G6a 30.9±12.2 MPa) although the values of the experimental
group resulted significantly higher compared to untreated
control (G3b 18.1±4.9)
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Table3: Values of microtensile bond strength are means ± standard
deviations. Groups with the same superscripts are not statistically significant
(p>0.05). Percentages of the failure modes (reported in square rounds) after
microtensile test analyzed by stereomicroscopy were classified as: A,
adhesive; CC, cohesive in composite; CD, cohesive in dentin; and M, mixed
failure. Bond reduction after storage report the percentage of mean bond
reduction after 1 year of storage.
4.1.2 Nanoleakage Analyses
EDC nanoleakage Analysis
The extent of silver nitrate depositions along the bonded
inter- faces is shown in Fig.1.
No difference was found in the interfacial nanoleakage
expression between the EDC-treated specimens and controls or
between the adhesives. In vitro aging for 1 year in artificial
saliva caused an increase in interfacial nanoleakage expression
compared to T0 (p < 0.05) for all tested groups.
89
Fig.1 Distribution of nanoleakage within the EDC pre-treated groups
and control groups. The numbers indicate the percentage of specimens with
respective nanoleakage values observed (from 0 to >75% of the adhesive
joint). N = number of sections analyzed. Different letters over the column
indicate statistical differences between the groups (chi-square test p < 0.05).
Different capital letters indicate statistical differences in interfacial
nanoleakage expression.
Riboflavin nanoleakage analysis
The Fig.2 summarizes the extent of interfacial
nanoleakage. The quantity of interfacial silver uptake did not
differ between groups at T0 (p>0.05; Fig.2). At T12, fewer
interfacial silver deposits were found in the RB-treated
specimens (G6a) than in control specimens (G3b; p<0.05;
Fig.1).
Fig.2 Distribution of nanoleakage within the different treatment
groups. The numbers indicate the percentage of specimens with respective
nanoleakage values observed (from 0 to >75% of the adhesive joint). N =
number of sections analyzed. Different letters over the column indicate
statistical differences between the groups (p < 0.05)
90
4.1.3 Zymographic Analyses
EDC zymographic analysis
Results of EDC zymographic analysis are shown in Fig3.
Proteins extracted from mineralized and demineralized dentin
powder (Lane1; Lane2) showed the presence of MMP-2 pro-
and active-forms (72- and 66-kDa, respectively) and pro-MMP-
9 (95kDa), and an additional band of approximately 50 kDa;
after demineralization an increase in the expression of MMP-9
and of the additional band at 50kDa was observed. The
incubation of demineralized dentin with 0.3M EDC resulted in
complete inhibition of dentin gelatinases (Lane3).
Fig.3. EDC zymographic analysis. Lane 1: mineralized dentin
powder showing the presence of MMP-2 pro- and active-form (72- and 66-
kDa, respectively) and pro-MMP-9 (95 kDa) and an additional band around
50 kDa. Lane 2: proteins extracted from dentin powder demineralized with
10% phosphoric acid, showing similar presence of MMP-2 pro- and active-
form and an increase in the expression of MMP-9 and of the additional band
at 50 kDa. Lane 3: demineralize dentin powder after incubation with 0.3M
EDC showing complete inhibition of enzymatic activity.
Demineralized dentin powder treated with OFL and
SB1XT resulted in enzymatic activation (respectively Fig.4,
lane 4; Fig5 lane 6). Pre-treatment with EDC followed by the
application of OFL resulted in complete inactivation of dentinal
91
gelatinases (Fig.4, lane 5). Whereas pre-treatment with EDC
followed by the application of SB1XT resulted in almost
complete inhibition of MMPs, although the presence of a band
corresponding to the active MMP-2 (66-kDa) is still detectable
(Fig5 Lane 7).
Fig..4. EDC zymographic analysis. Lane 4: Demineralized dentin
powder treated with OFL showing enzymatic activation of both MMP-2 and -
9 and of the additional band at approx. 50 kDa. Lane 5: Proteins extracted
from demineralized dentin powder pre-treated with 0.3M EDC followed by
OFL application showing complete inactivation of dentinal gelatinases.
Fig.5. EDC zymographic analysis. Lane 6: Demineralized dentin
powder treated with SB1XT showing enzymatic activation of both MMP-2
and -9 and of the additional band at approx. 50 kDa. Lane 7: Proteins
extracted from demineralized dentin powder pre-treated with 0.3M EDC
followed by SB1XT application showing reduced activation of MMP-9,
92
MMP-2, and of the 50-kDa band, and presence of the MMP-2 intermediate
form compared with lane 6.
The application of XPB on demineralized dentin
revealed activation of both pro- and active-form of MMP-9
(respectively 95 kDa and 86 kDa) (Fig6 lane 8,); the application
of EDC before XPB resulted in complete inhibition of
gelatinolytic activity (Fig.6; lane 9).
Fig..6. EDC zymographic analysis. Lane 8: demineralized dentin
powder treated with XPB showing enzymatic activation of both pro- and
active-form of MMP-9. Lane 9: Proteins extracted from demineralized dentin
powder pre-treated with 0.3M EDC followed by XPB application showing a
complete inhibition of MMP’s activity.
Riboflavin zymographic analysis
Results of RB zymographic analysis is showed in Fig.7.
Demineralized and XPB dentin extract showed forms of
gelatinolytic enzymes, including a 72-kDa MMP-2 pro-form, a
fainter 86-kDa band corresponding to the active form of MMP-
9, and other minor gelatinolytic band around 50 kDa :lane 1 and
Lane 3 Pretreatment of demineralized dentin with 0.1% RB
resulted in the partial inhibition of the MMP-2 pro- form and
complete inhibition of the 86-kDa active MMP-9 even when
XPB was applied before the pretreatment: Lane 2 and Lane 4.
93
Fig.7. Riboflavin zymographic analysis. Lane 1: phosphoric-acid-
demineralized dentin extracts showed multiple forms of gelatinolytic
enzymes, including a 72-kDa MMP-2 proform, a fainter 86-kDa band
corresponding to the active form of MMP-9, and other minor gelatinolytic
bands with lower molecular weights. Lane 2:, incubation of phosphoric-acid-
demineralized dentin powder treated with 0.1% riboflavin solution plus
irradiation with UVA resulted in the partial inactivation of the 72-kDa MMP-
2 proform and complete inhibition of the 86-kDa MMP-9. Lane 3: dentin
treated with XPB showed intense activity related to the MMP-2 proform and
fainter activity related to MMP-9. Lane 4: incubation of demineralized dentin
powder with riboflavin followed by incubation with XPB also resulted in
partial inhibition of the 72-kDa MMP-2 proform and complete inactivation of
the 86-kDa MMP-9.
DCC Zymographic analysis
Results of DCC zymography are showed in Fig.8 Fig.9.
Mineralized and demineralized dentin extracts resulted in
enzymatic activation of pro- and active-form of MMP-2. The
application of DCC/A and DCC/E pretreatment resulted in
complete inactivation of gelatinolytic activity Lane 3, 4. Similar
results were obtained when both DCC solutions are applied in
association to SB1XT (Fig.9 Lane 6, Lane 7)
94
Fig.8: DCC zymographic analysis. Lane 1: mineralized dentin
powder showing the presence of MMP-2 pro- and active-form (72- and 66-
kDa, respectively) and pro-MMP-9 (95 kDa) and an additional band around
50 kDa. Lane 2: proteins extracted from dentin powder demineralized with
10% phosphoric acid, showing similar presence of MMP-2 pro- and active-
form and an increase in the expression of MMP-9 and of the additional band
at 50 kDa. Lane 3: demineralized dentin powder incubated with 0.5M
DCC/acetone showing complete inhibition of dentin gelatinases. Lane 4:
likewise Lane 3 demineralized dentin powder incubated with 0.5M
DCC/ethanol showing complete inhibition of dentinal MMPs.
Fig.9: DCC zymographic analysis. Lane 5: Demineralized dentin
powder treated with SB1XT showing enzymatic activation of both MMP-2
and -9 and of the additional band at approx. 50 kDa. Lane 6, 7: Proteins
extracted from demineralized dentin powder pre-treated with 0.5M
DCC/acetone and DCC/ethanol respectively followed by SB1XT application
showing complete inactivation of gelatinolytic activity.
95
Acrolein Zymographic analysis
Results of are showed in Fig.10. Pretreatment of
demineralized dentin powder with 0.01% ACR resulted in
almost complete inactivation of pro- and active-form of MMP-2
and active MMP9, although a band around 100 kDa is still
detectable, and it could be attributed to a complexed pro-form of
MMP-9.
Fig.10: Acrolein zymographic analysis. Lane 1: demineralized
dentin powder showing activity of pro-form of MMP-9 (92 kDa) and active
form of MMP-2 (66 kDa). Lane 2: demineralized dentin powder after
incubation with 0.01% ACR showing complete inactivation of MMP-2, and
reduced MMP-9 activity, although a complexed form of MMP-9 around 100
kDa is still detectable.
4.1.4 In situ Zymographic Analyses
For control specimens bonded with OFL and SB1XT, the
in situ zymography analysis revealed an intense green
fluorescence within the HL after incubation, indicating that the
fluorescein-conjugated gelatin was strongly hydrolyzed at these
sites (Figs 11d; 12d; 13 d: 14 d).
Hybrid layers created by either OFL or SB1XT, which
were pre-treated with 0.3M EDC-containing primer or with
96
DCC/A and DCC/E, exhibited almost no fluorescence signal at
the adhesive-dentin interface (Figs. 11c; 12c; 13c; 14c,
respectively).
Fig.11: In situ Zymography analysis. Resin-bonded dentin interfaces
prepared with Optibond FL (OFL) with or without EDC pre-treatment,
incubated with quenched fluorescein-labeled gelatin. D = Dentin; HL =
Hybrid Layer; R = Resin Composite; bar = 5 µm. (a) Image of OFL without
EDC pre-treatment, obtained by merging differential interference contrast
image (showing the optical density of the resin- dentin interface) and image
acquired in green channel (showing enzymatic activity). (b) Image of HL
created with OFL after EDC pre-treatment obtained by merging differential
interference contrast image and image acquired in green channel (c)
Acquired image in green channel, showing fluorescence within the HL
created with OFL. (d) Image acquired in green channel of hybrid layer
created with OFL applied to acid-etched dentin pre-treated with EDC
showing absence of fluorescence.
97
Fig12: In situ Zymography analysis. Resin-bonded dentin interfaces
prepared with SB1XT with or without EDC pre-treatment, incubated with
quenched fluorescein-labeled gelatin. D = Dentin; HL = Hybrid Layer; R =
Resin Composite; bar = 5 µm. (a) Image of SB1XT without EDC pre-
treatment, obtained by merging differential interference contrast image
(showing the optical density of the resin- dentin interface) and image
acquired in green channel (showing enzymatic activity). (b) Image of HL
created with SB1XT after EDC pre-treatment obtained by merging
differential interference contrast image and image acquired in green channel.
(c) Image acquired in green channel, showing fluorescence (identifying
intense endogenous enzymatic activity) in dentinal tubules and within the
hybrid layer (HL) created with SB1XT without EDC pre-treatment. (d)
Image acquired in green channel of hybrid layer created with SB1XT applied
to acid-etched dentin pre-treated with EDC showing absence of fluorescence.
98
Fig.13: In situ Zymography analysis. Resin-bonded dentin interfaces
prepared with SB1XT with or without EDC pre-treatment, incubated with
quenched fluorescein-labeled gelatin. D = Dentin; HL = Hybrid Layer; R =
Resin Composite; bar = 5 µm. (a) Image of SB1XT without DCC/acetone
(DCC/A) pre-treatment, as Fig.12 (a). (b) Image of HL created with SB1XT
after DCC/A pre-treatment obtained by merging differential interference
contrast image and image acquired in green channel. (c) As Fig.12(c) (HL)
created with SB1XT without DCC/A pre-treatment. (d) Image acquired in
green channel of hybrid layer created with SB1XT applied to acid-etched
dentin pre-treated with DCC/A showing absence of fluorescence.
99
Fig.14: In situ Zymography analysis. Resin-bonded dentin interfaces
prepared with SB1XT with or without EDC pre-treatment, incubated with
quenched fluorescein-labeled gelatin. D = Dentin; HL = Hybrid Layer; R =
Resin Composite; bar = 5 µm. (a) Image of SB1XT without DCC/ethanol
(DCC/E) pre-treatment, as Fig.12(a). (b) Image of HL created with SB1XT
after DCC/E pre-treatment obtained by merging differential interference
contrast image and image acquired in green channel. (c) As Fig.12(c) (HL)
created with SB1XT without DCC/E pre-treatment. (d) Image acquired in
green channel of hybrid layer created with SB1XT applied to acid-etched
dentin pre-treated with DCC/A showing absence of fluorescence.
100
4.2 Discussion
Over the last few years, the experimental use of collagen
cross-linking agents to increase the longevity of resin-dentin
bonds gained increased popularity. The use of cross-linkers can
be considered as a biological tissue engineering approach where
dentin tissue repair/regeneration is the development of a
biomimetic strategy to enhance the tissue properties by
modifying the chemistry of the tissue (Bedran-Russo et al,
2014). The biomodification of the existing tooth hard tissue
structures is a novel approach to improve the biomechanical
properties of the tissue for preventive and reparative/restorative
purposes. The approach was thought to be determined by non-
enzymatic inter- or intra-molecular collagen cross-linking
(Stenzel et al, 1974). However, the multiple interactions
between bioactive agents with various extracellular components
of the dentin matrix are likely the determinants of the tissue
enhanced biomechanics and biostability. Therefore, the term
biomodification is more appropriate to define the bioactivity of
these highly bioactive chemical mediators.
The present study demonstrated that increased bond
strength can be obtained by the additional use of different
biochemical cross-linkers in association to different dentin
bonding adhesive systems. Furthermore, the cross-linker pre-
treatment before the bonding procedure resulted in an almost
complete inactivation of dentinal gelatinases.
Previous studies investigated the use of different cross-
linkers, such as glutaraldehyde, genepin, proanthocidin and
EDC, as biomodifier agent, although the application time
required to be effective could not be considered acceptable:
from 10 min up to several hours (Bedran-Russo et al,
2007;Macedo et al; 2009; Castellan et al, 2010; Dos Santos et
al, 2011). Among these studies only one study concerning the
101
use of EDC was conducted to evaluate the capabilities to
increase the mechanical properties of the etching-dentin matrix
in 1 min application time, and the results revealed that also a
short application time was sufficient to inactivate endogenous
protease activity of dentin without significantly stiffening the
collagen matrix (Tezvergil-Mutlua et al, 2012). According to
this finding in the present study all the cross-linking agents
tested were applied for 1 min on the dentin surface.
The µTBS analyses revealed that the use of EDC,
riboflavin (RB) and acrolein (ACR) pretreatments can improve
the durability and the structural integrity of the resin/dentin
interfaces created either with etch-and-rinse or self-etch
adhesive systems.
In detail the results of the µTBS of the EDC experimental
group showed that bond strength values were significantly
higher compared to the values of the control group. Furthermore
in terms of percentage of bond strength reduction, the 2-step
self-etch adhesive (CSE) and the 3-step etch-and-rinse (OFL)
seem to be more responsive to the EDC pretreatment. These
data confirm previous in vivo and in vitro findings of etch-and-
rinse dentin bonding agents (Peumans et al, 2005; De Munck et
al, 2012) supporting the improved stability of the three-step
systems vs the two-step etch-and-rinse systems due to their
increased hydrophobicity (Malacerne et al, 2006) and curing
ability (Cadenaro et al, 2005; Breschi et al, 2007). The observed
decline in bond strength can be related to the loss of integrity of
resinous components within the hybrid layer (HL) due to
polymer swelling and resin leaching that occur after water/oral
fluid sorption, which is recognized to be more pronounced for
simplified (two-step) etch-and-rinse adhesives than unsimplified
systems (three-step) (Pashley et al, 2011; De Munck, et al,
2003; Van Meerbeek et al, 2011). The 2-step self-etch adhesive
is considered the most durable bond (Van Meerbeek et al,
2011), however doubts could be rise concerning the
102
effectiveness of the dentin substrate since unlike the etch-and-
rinse technique, the self-etch does not completely expose the
dentin collagen matrix (Van Meerbeek et al, 2011; Suppa et al,
2005). For the self-etch adhesives the maintenance of the smear
layer with hydroxyapatite residues in the HL is considered
crucial to protect collagen fibrils and generate chemical
interaction. In case of CSE chemical interaction is achieved
through specific functional monomer: the 10-MDP (10-
methacryloyloxydecyl dihydrogen phosphate) (Islam et al,
2014). The µTBS results revealed that bonded interface created
with the EDC pre-treatment and CSE is positively influenced by
EDC suggesting that the presence of smear layer and the
partially denude collagen matrix induced by acidity of the self-
etching primer, allows the infiltration of the EDC within the
dentin collagen network. This results coincide with a recent
study (Zheng et al, 2015) where 3-step etch-and-rinse (OFL)
and 2-step self-etch adhesives (CSE) were tested using as
conditioning primer different MMPs inhibitors. Results of this
recent study showed that the use of MMPs inhibitors stabilized
the bond strength values over time both for OFL and CSE.
These data confirmed that the presence of smear layer does not
affect the activity of additional primer applied during bonding
procedures.
Considering the EDC collagen cross-linking reaction
described in chapter 2, it is possible to affirm that the dentin
collagen reinforcement and strengthening obtained with the use
of EDC through the formation of inter- and intra- molecular
crosslinks (Bedran-Russo et al, 2007) might be of crucial
importance in the improvement of the bond strength of the
resin/dentin interface over time against the enzymatic and/or
hydrolytic degradation.
In relation to the results obtained with the use of RB as
cross-linker agent showed that the application of UVA-
activated RB before bonding increased the immediate bond
103
strength and preserve the mechanical properties of the HL
created by XPB. In cross-linking therapy with RB/UVA, yellow
riboflavin works as a photosensitizer that stimulates the
formation of reactive oxygen species, and simultaneously acts
also as a shield from the penetration of UVA, as previously
described in chapter 2 (Snibson, 2010). After the demonstration
of the ability of RB/UVA in increasing the stiffness of type I
collagen, RB/UVA therapy has been started to be used as a
common procedure in keratoconus diseases treatment. Our
results demonstrated that the use RB/UVA for dentin collagen
conditioning is effective in the preservation of the durability of
the HL, probably due to the formation in the dentin collagen
matric of covalent cross-links through oxidation (Sionkowska,
2006).
The use of ACR as collagen cross-linker within the
bonding procedure, represents the first study evaluating the
effectiveness of its cross-linking reaction in dentin HL creation.
Previous studies widely investigated the effect of glutaraldehyde
(GD) on dentin matrix; as well as ACR, the GD is commonly
used as tissue fixative and it is a well-known cross-linking
agent. It has been proved that GD increased biological tissue
mechanical properties and lower degradation rates (Nimni et al,
1988; Sung et al, 1999). GD increases type I collagen covalent
bonding by bridging the amino groups of lysine and
hydroxylysine residues of different collagen polypeptide chains
by monomeric or oligomeric cross-links; the presence of
exogenous cross-links induced by GD had demonstrated to be
sufficient to positively affect the mechanical properties of the
exposed dentin matrix (Bedran-Russo et al, 2007;Macedo et al;
2009; Al-Ammar et al, 2009). Similarly, ACR is included in the
present experimental project in order to investigate the influence
of a diverse aldehyde molecule on dentin tissue cross-linking.
According to the data the use of ACR as exogenous
collagen crosslinking agent, resulted in the preservation of the
104
µTBS values of the HL created by SB1XT. The results confirm
previously outcomes obtained with GD, although in these cases
the pre-treatment time was significantly longer as previously
mentioned (Castellan et al, 2010; Dos Santos et al, 2011). Our
results showed that the bond strength of the tested adhesives was
completely maintained over time when ACR was used as
additional primer. This result could be related to the ability of
ACR to fix tissues avoiding their degradation. As well as GD, a
disadvantage of ACR compared to the other crosslinking agents
used is its high cytotoxicity that may arise from residues of
unreacted or degraded crosslinking agents (Han et al, 2003).
Looking overall at µTBS results, with the limitation of
this in vitro study it possible to conclude that changes within the
dentin matrix promoted by the different cross-linkers tested are
not adhesive-system-dependent, since each cross-linker is able
to preserve the durability of the bond strength over time.
Additionally, the different extend of bond preservation of
cross-linkers treated specimens might also be related to the
interaction of tested cross-linking agents with the dentin
collagen matrix and their susceptibility to endogenous MMPs,
that could result in different mode of MMPs activation
(Mazzoni et al, 2006; Nishitani et al, 2006a; Mazzoni et al,
2012). The zymography and in situ zymography support the
hypothesis that the stabilization of HL created after treatment
with cross-linkers derived from inactivation and inhibition of
dentin gelatinolytic activity (Calero et al, 2002; Matchett et al,
2005 Tezvergil-Mutluay et al, 2012). Evidence of collagenolytic
and gelatinolytic activities in dentin treated with both etch-and-
rinse and self-etch denting bonding agents was previously
shown (Mazzoni et al, 2006; Nishitani et al, 2006a; Breschi et
al, 2008), confirming the involvement of these endoproteases in
the disruption of collagen fibrils within hybrid layers being
responsible for the poor durability of resin-dentin bond
durability over time.
105
Zymographic analyses performed in the present work
confirmed previous findings (Mazzoni et al, 2006, 2012, 2013)
that the use of adhesive systems tested results in increased
MMP-2 and -9 activity but also revealed that when cross-linkers
tested (EDC, ACR, RB, DCC) are used as conditioning primers,
the gelatinolytic activity is reduced or almost complete
inhibited. Results of EDC zymography are showed in
Fig.3,4,5,6. When demineralized dentin powder was pre-treated
with 0.3M EDC before the OFL or XPB application complete
inactivation of dentinal gelatinases was obtained. Similar
inhibitory findings were recorded for SB1XT after pre-treatment
with the EDC-containing primer, although an intermediate form
of MMP-2 activation could be seen (Fig.5 Lane 7). This
intermediate form (68-kDa) can be considered as a transient
phase (Atkinson et al, 1995) infrequently seen in zymography
(because it is rapidly changed into active form with slightly
lower molecular weight) (Mazzoni et al, 2014), and its
activation from intermediate to fully active MMP-2 may be due
to autolytic activity by other MMPs, or may be controlled by
tissue inhibitors of metalloproteinases (TIMPs) (Tezvergil-
Mutluay et al, 2012; Tjäderhane et al, 2013; Mazzoni et al,
2014).
Similarly, dentin powder pre-treatment with RB/UVA
resulted in the partial inactivation of the 72-kDa MMP-2
proform and complete inhibition of the 86-kDa MMP-9 active;
likewise when XP Bond were applied after RB/UVA treatment
gelatinolytic activity resulted in partial inhibition of the 72-kDa
MMP-2 proform and complete inactivation of the 86-kDa
MMP-9 (Fig.7).
Likewise, DCC/acetone (DCC/A) and DCC/ethanol
(DCC/E) resulted in complete inhibition of MMPs activity when
they are used as pre-treatment before SB1XT application (Fig.8)
because no activity was detectable for both solutions tested.
Same results occurred when DCC/A - /E were directly applied
106
on demineralized dentin confirming the results showed in Fig.9.
Hence, it possible to speculate that the influence of DCC on
MMPs activity is not related to the DCC solvent. At the same
time the inhibition ability could be also correlated to the
solvents used, especially for the ethanol. In 2011 Tezvergil-
Mutluay and coll showed that many alcohols can inhibit both
soluble rhMMPs and matrix-bound MMPs. The molecular
structures of the catalytic domains of MMP- 2 and 9 are similar
(Visse et al, 2003). The domain consists of a 5-stranded β-
pleated sheet, with three α-helices and connective loops. The
protease domain contains one catalytic zinc, one structural zinc
and three calcium ions. The substrate-binding cleft is formed by
strand IV, helix B, and the extended loop region after helix B.
Three histidines coordinate the active site zinc. The fourth
ligand of the catalytic zinc is a water molecule (Farkas et al,
2004) that hydrolyzes the specific peptide bond. It was also
shown that the more hydrophilic is the alcohol, lower is its
ability to inhibit rhMMP-9 (Tezvergil-Mutluay et al, 2011). This
finding validates the hypothesis that water exclusion is crucial to
reduce gelatinolytic activity. Thus we can speculate that
alcohols that can inhibit MMPs by forming a coordinate
covalence bond between the catalytic zinc and the oxygen atom
of the hydroxyl group of the alcohol. According to the study of
Tezvergil-Mutluay and coll, we can support the hypothesis that
inhibitory effect of DCC/E is due to both interactions of DCC
and the solvents.
As well as the cross-linkers described above, ACR
results in decrease endogenous enzymes’ activity in
demineralized dentin. However the results in Fig.10 showed a
presence of activity around 100 kDa that could be ascribed to a
pro-MMP-9 complex form. Different studies (Aldini et al, 2011;
Guglicci et al, 2007) hypothesized that enzyme inhibition
involves modification of Cys residues critical for the catalytic
site, but no definitive structure characterization was provided.
107
Hence, the inhibit capability of ACR is associate to the
modification of Cys residues of MMPs, preventing their
activation. Unfortunately we were not yet able to perform a
zymography of ACR pre-treatment in association of SB1XT,
due to the difficulties to extract proteins from this treatment
group. Thus, we may speculate that proteins were not
extractable due to high cross-linking created by ACR.
Because homogenization of tissues for gelatin
zymography is mandatory, in situ zymography analysis was
performed on four groups chosen as model (described for extent
in chapter 2), to localize the MMP activity previously detected
by zymography. Gelatinolytic activity was clearly detectable
within the HLs (Figs11c, 12c) and along the tubular wall dentin
extending from the dentinal tubules in the control groups. The
location of the activity in these groups well correlates with the
demineralized uninfiltrated collagen layer simplified etch-and-
rinse adhesives at the bottom of the HL, an area also known for
nanoleakage expression and the presence of naked collagen
fibrils (Suppa et al, 2005; Breschi et al, 2008). The effectiveness
of EDC and DCC/A - /E used as conditioner primers before the
bonding application was evident by the reduced protease activity
detectable within the HL (Figs.11d, 12d, 13d, 14d). These
correlative results confirmed and validated the zymography
analysis outcomes.
Based on the findings of the present project the double
effectiveness of cross-linkers tested in improve mechanical
properties and in inhibit gelatinolytic activity within the HL has
been demonstrated. Previous studies suggested that this MMP
resistance may be attributed to an alternative silencing
mechanism of MMPs and probably other exogenous collagen
degradation enzymes via conformational changes in the enzyme
3-D structure (Busenlehner and Armstrong, 2005). The use of
cross-linking agents may create multiple cross-links between
amino acids within the catalytic site that irreversibly alter the 3-
108
D conformation or flexibility of the cleft-like catalytic domain
and prevent its optimal recognition and complexing with the
type I collagen substrate (O’Farrell et al., 2006). Although there
is no evidence that the catalytic domain of collagenolytic MMPs
can be cross-linked to inactivate their functions, it has been
hypothesized that the use of cross-linking agents may also
contribute to MMP silencing via allosteric control of non-
catalytic domains (Liu et al, 2011). For example, the catalytic
domains in collagenolytic MMPs can cleave non-collagen
substrates, but the hemopexin-like domain of these enzymes is
crucial to initially unwind and subsequently cleave the three
triple-helical fibrillar elements of the collagen molecule in
succession (Lauer-Fields et al, 2009). For MMP-2, there are
three fibronectin-like repeats that form a domain for binding to
collagen or gelatin substrates. This collagen-binding domain
binds preferentially to the α1 chain and mediates local
unwinding and gross alteration of the triple helix prior to the
cleavage of the β2 chain (Gioia et al, 2007). Regardless of
which of the two collagen-binding mechanisms is involved,
cross-linking of either the hemopexin-like or fibronectin-like
domains may contribute to inactivation of the associated MMPs
and reduction in their collagenolytic efficacy.
Cross-linking may also affect MMP activities known to
be modified by non-collagenous proteins (Malla et al, 2008). In
dentin, MMP activities and resistance to degradation may be
regulated by fetuin-A (Ray et al, 2003) and the SIBLINGs Bone
Sialoprotein (BSP) and Dentin Matrix Protein-1 (DMP-1)
(Fedarko et al, 2004), all of them being present in dentin. Thus,
cross-linking of these non-collagenous proteins may indirectly
silence MMPs via inactivation of the functional domains of
these glycoproteins. Since MMPs do not turn over in dentin,
their inactivation by cross-linking agents should last for a long
time and may be even more effective than inhibitors (Cova et al,
2011; Tjäderhane et al, 203).
109
Nowadays in the clinical practice chlorhexidine is used
as therapeutic primer to prevent the degradation of HL caused
by endogenous enzymes (Carrilho et al, 2007; Breschi et al,
2009, 2010) however it is known that after 18–24 months
chlorhexidine may leach out. The use of cross-linkers could be
considered as a long-lasting alternative to preserve the integrity
of HL over time. The use of ACR should be avoided due to its
high cytotoxicity; likewise the use RB/UVA results no
convenient in term of clinical time and operator depending
procedure: in fact use of RB requires the use of an additional
UVA lamp and light activation of 2 min (to be added to 1 min
application of RB). EDC and DCC are the more favorable cross-
linkers for the use in clinical practice. DCC in particular could
be very promising since its hydrophobicity and its solubility in
acetone or ethanol. The water molecules present in the HL are
responsible of the ester-bonds in adhesive polymers and peptide
bonds in collagen causing the failure of resin-tooth interface.
For this reason, in last years many studies were conducted with
the aim to create possible “ethanol-wet bonding procedures”
(Hosaka et al, 2009; Sadek et al, 2010a,b; Sauro et al, 2010).
Wet- bonding with ethanol instead of water provides an
opportunity for coaxing hydrophobic monomers into a
demineralized collagen matrix without sacrificing any additional
matrix shrinkage. Infiltration of hydrophobic monomers into a
collagen matrix decreases water sorption/solubility, resin
plasticization, and enzyme-catalyzed hydrolytic cleavage of
collagen (Hosaka et al, 2009; Sadek et al., 2010a), thereby
creating more durable resin bonds. Sauro and coll (2009a,b)
demonstrated that ethanol wet-bonding is capable of increasing
resin uptake and producing better sealing of the collagen matrix,
even with the use of hydrophilic adhesives. The presence of
ethanol probably also increases the degree of conversion of the
hydrophilic adhesives. Sadek and coll (2010b) also
demonstrated that hybrid layer created with ethanol wet-
110
bonding showed almost complete absence of nanoleakage as
well as excellent durability of bond strength. Additionally the
presence of water is essential for the activation of MMP
enzymes, on the other hand ethanol is showed to prevent the
gelatinolytic activity (Tezvergil-Mutluay et al, 2001). Anyway
ethanol wet-bonding is considered as a bonding philosophy
rather than a bonding technique (Liu et al, 2011), due to its
clinical impracticality (Nishitani et al, 2006b; Sauro et al, 2010;
Sadek et al, 2007, 2010b): when ethanol replacement is not
meticulously performed to prevent water-saturated collagen
from exposure to air, the surface tension present along the air-
collagen interface can easily result in collapse of the collagen
matrix and prevent optimal infiltration of the adhesive
monomers (Osorio et al, 2010). In this background the use of
collagen cross-linker, such as DCC, in ethanol solution could
strengthen the collagen matrix and creating an HL in absence of
water. Nevertheless the application of ethanol on dentin matrix
does not completely avoid the presence of water caused by
outward fluid flows without the use of adjunctive tubular
occlusion agents (Sadek et al, 2007; Cadenaro et al, 2009).
In conclusion further studies are needed to well
understand the structure of the HL created using DCC/E or /A as
conditioning primer, considering the preliminary but
encouraging results obtained from this study. Additionally in
vivo studies will be essential to better understand the feasibility
of the DCC/E or /A as dentin conditioning primer during
bonding procedures.
111
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