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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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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.

  87  

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)

  88  

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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