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Introduction Chapter 1: Dental Adhesion

1.1 Introduction 1.2 The concept of adhesion 1.3 Advantages of adhesive dentistry

1.4 Enamel and dentin-chemistry and histology 1.5 Enamel adhesion 1.6 Dentin adhesion 1.7 Classification of Dental Adhesive Systems 1.8 Chitosan

Chapter 2: Degradation of the bonded interface

2.1 Stability of the bonded interface 2.2 Degradation of collagen fibrils 2.3 Degradation of the resin

Chapter 3: Formulation of an adhesive system using Chitosan

3.1 Introduction 3.2 Materials and Methods 3.3 Results 3.4 Discussion

Chapter 4: Use of Methacrylate-Modified Chitosan in an adhesive system to

increase the durability of dentin bonding systems

4.1 Introduction 4.2 Materials and Methods 4.3 Results and Discussion

Chapter 5: Cytotoxicity tests on Chit-MA70

5.1 Introduction 5.2 Materials and Methods 5.3 Results and Discussion

Chapter 6: Summary, conclusions and future perspectives

6.1 Summary and conclusions 6.2 Future perspectives 6.3 Curriculum vitae

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Introduction The purpose of this thesis was to investigate the effect of a methacrylate-modified chitosan on

the durability of adhesive interfaces to improve the clinical performance of dental

restorations. Current research in this field aims at increasing the resin-dentin bond durability.

The aim of this project was the development of an innovative nano-engineered biocompatible

dental adhesive system capable to form covalent chemical bonds with both the dentin

collagen and the resin-based restorative materials. This research proposes a new concept of

bi-functional reactive adhesive system based on a natural biopolymer capable to create double

covalent reticulation. Chitosan is a biocompatible, biodegradable and non-toxic biopolymer1.

These features make this polysaccharide very appealing for tissue engineering and biomedical

applications.2,3 Indeed, chitosan has been proposed for different medical applications such as

topical ocular applications,4 implantation,

5,6 injection,7 and drug delivery.

8 Chitosan is also

bio-adhesive, a property that determines an increased retention at the site of application.9 In

the field of dental applications, chitosan has already been introduced in a commercial dental

adhesive to exploit its antibacterial properties. In the present work, chitosan was modified

with methacrylate moieties and a physical-chemical characterization was carried out. This

research speculated that the modified chitosan was able to covalently bind to the restorative

resin and, due to the presence of residual positive charges on the polysaccharide, to

electrostatically interact with the demineralized dentine. Chitosan bearing methacrylate

groups was blended within the primer of a three- step etch&rinse experimental dentine

bonding system to assay its bonding ability to dentine. Additionally, bonded interfaces were

challenged with a chewing simulation associated with thermo cycling to assay their stability

over time. The introduction of chitosan-modified monomers within the primer of an

experimental three-step etch-and-rinse adhesive, improved (1) immediate bond strength (T0

µTBS); (2) stability of the hybrid layer after simulated chewing and thermo cycling (TCS

µTBS). In summary, the first part of this PhD thesis aimed to review the state of the art of

dental materials currently used in dentistry and the most important problem in restorative

dentistry: the degradation of bonded interface focusing on the fundamental processes that are

responsible for the degradation of the adhesive interface (Chapter 1 and 2). The second part of

this doctoral thesis was dedicated to find the optimal formulation of a chitosan–containing

dental adhesive system.

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Several formulations (Chapter 3) were tested and the one identified as A3* was considered

the best formulation to be used as an adhesive system. This formulation was then named Chit-

MA70. Several tests were carried out, including mechanical tests such as the microtensile

bond strength test. The nanoleakage expression was evaluated using optical microscopy and

scanning electron microscopy. The evaluation of the substantivity of chitosan on the adhesive

layer was evaluated by labeling the methacrylate-modified chitosan, contained into the

primer, with fluorescein and then observing the samples by confocal laser scanning

microscopy.

In the end, cytotoxicity tests were performed to evaluate the effect on cell viability of the

adhesive system Chit-MA70. Tests carried out on gingival fibroblasts revealed a reduced if

not practically absent toxicity of the adhesive system containing methacrylate-modified

chitosan.

Considering the good results of this study, the methacrylate-modified chitosan has been

proposed as a component of a novel adhesive system.

The findings on this new dental adhesive with methacrylate-modified chitosan were claimed

into a patent in February 2014 and published in Biomacromolecules in October 2014.

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Chapter 1

Dental Adhesion

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1.1 Introduction

The advent and development of resin-based adhesive materials have allowed numerous

changes in dental clinical practice. The new concept of adhesive dentistry, based on the

proprieties of polymeric resin materials, permits “minimal invasive fillings”, restorations that

replace only the decayed tissue: the old Black’s concept ”extension for prevention” is

definitely over. These adhesive materials are: resin composites, compomers and vetroionomer

cements. The concept of adhesion to dental tissues was introduced in the field of dentistry in

1950, when the Swiss chemist Oskar Hagger developed the archetype for adhesive

monomers10. This material was first used for a dental filling in the fifties by McLean and

Kramer, who published the first paper on dentin-bonding agents (DBA) 11. The “adhesive

revolution” of the 1950s was led by Buonocore, 12 who etched human enamel with phosphoric

acid, founding that acid etching could significantly enhance the bond of restorations to enamel

because it superficially demineralized the tooth.

1.2 The concept of adhesion

The etymology of “adhesion” comes from the latin word adhaerere, which is a compound

verb of ad (to) and haerere (to stick, to attach). Adhesion is defined by Specification D907 of

the American Society for Testing and Materials as “the state in which two surfaces are held

together by interfacial forces, which may consist of covalence forces or interlocking forces or

both” 13. The adhesive, frequently fluid, joins two substrates together and solidifies. The

adherend is the material or initial substrate to which the adhesive is applied. The adhesive in

adhesive terminology is the adherent 14. The sealing properties of dental adhesives are based

on the ability to adhere to dental hard tissues, enamel and dentin, and simultaneously to bind

the lining composite with a co-polymerization of residual double bonds C=C in the oxygen

inhibition layer. The adhesion process consists of two different moments: the first phase is the

conditioning of hard dental tissues; after the removal of calcium and phosphate superficial

micro-porosities are obtained on both dentin and enamel. The second phase consists in the

resin infiltration and polymerization inside micro-porosities 15. Basic mechanism of bonding

to enamel and dentin is essentially an exchange process involving replacement of minerals

removed from the hard dental tissue by resin monomers, which, upon setting, become micro-

mechanically interlocked in the created porosities 16,17,18. Diffusion and capillarity are primary

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involved to obtain micro-mechanical retention. Microscopically, this process is called

‘hybridization’ 19. The infiltration process culminates with the formation of the hybrid layer,

the zone of interdiffusion between resin and tooth, which guarantees the seal of the

restoration. Over the hybrid layer, the adhesive layer preserves the integrity of the hybridized

dentin, protecting it from the shrinking stress of composite resin, and also acts as a stress-

absorbing layer. So the adhesive layer helps to maintain the strength of adhesion, the seal and

the bond over time 20.

1.3 Advantages of Adhesive Dentistry

The use of adhesive techniques changed operative dentistry because they restricted the

operative procedures to removal of diseased tooth tissue, thus preserving sound tissue. The

improvement of marginal sealing around bonded restoration has reduced the frequency of

unfavorable post-operative responses such as sensitivity, pulp damage, recurrent caries,

marginal discoloration, and ultimately, loss of restoration 14. The development of resin

materials makes it possible not only to fill the teeth without sacrificing sound tissue for the

mechanical retention to the tooth, but also to correct aesthetic flaws (of color, shape,

dimension) without a prosthetic approach, preserving sound tissue. The introduction of

endodontic posts, which are capable to reach a good bond with the dentin in the canal, permits

to create the core build-up for crowns. Adhesives are used to cement crowns in prosthetics. In

orthodontics adhesives are used for fast placement of brackets. Adhesive techniques are used

for periodontal or orthodontic splints. The use of adhesive materials in pediatric dentistry

permits to seal teeth fissures (dental sealants), a popular method to prevent tooth caries.

Modern adhesive techniques are used to correct unaesthetic shapes, position, dimension or

shades of the tooth. Resin composites are used to close diastema, to add length or to mask

discoloration 21,22.

1.4 Enamel and dentin-chemistry and histology

Enamel is a hard tissue that wears the dental crown, it is translucent and is the hardest and the

most mineralized tissue of the human body.

Enamel is composed of 96% of hydroxyapatite crystals; the remaining tissue is formed by a

proteic organic matrix (proteins as amelogenins and enamelins) and trace amounts of water.

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Proteins are organized in branched fibrils, the complex structures of proteoglycans. The

hydroxyapatite crystals are long up to 1 mm, 50 nm wide by 25 nm thick, extending from the

dentin toward the enamel surface 23. The enamel crystals are organized in bundles of about

1000 crystals, called enamel prisms. The cross-sectional profile of the prisms varies from

circular to keyhole-shaped. The hydroxyapatite crystals are primarily arranged with their long

axes parallel to the long axes of the prisms. At the periphery of each prism, however, the

crystals deviate somewhat from this orientation, producing an interface between prisms,

forming the interrod enamel. In this areas the crystallites run parallel to each other and

perpendicular to the surface.

Dentin results from the interaction between the ectodermal and ectomesenchymal components

of the tooth germ that induce odontoblast differentiation and dentinogenesis 14. Producing the

dentin, the odontoblasts form dentinal tubules with different densities and orientations in

distinct tooth locations. The tubular structure of dentin is responsible of its hydration due to

the communication with the pulp tissue. Dentin is composed by 76% from inorganic materials

(hydroxyapatite), 18% from organic materials (collagen) and 12% from water 20. The tubules

form about 10% of the dentin volume; they are approximately 0.63 µm in diameter near the

dentin-enamel junction and 2.5 µm in diameter near the pulp chamber 24. Dentinal tubules

contain the odontoblastic processes and form a direct connection to the vital pulp. Thus, there

are two different type of dentin: the intertubular dentin (composed from water and less

collagen fibrils) and the peritubular dentin (formed from very high concentration of collagen

fibrils). In the deepest part of the tubule length, the tubules are filled by tissue fluid (the so-

called dentinal fluid), by an organic membrane structure called lamina limitans and by

intertubular collagen fibrils of yet unknown origin and function. Dentinal fluid in the tubules

is under a slight, but constant, outward pressure from the pulp. The intrapulpal fluid pressure

is estimated to be 25 to 30 mmHg 25. Dentin is a hydrophilic and a dynamic tissue that

changes in time: there could be tertiary dentin, sclerotic dentin, sub-carious or iper-

mineralized dentin. These morphologic and structural transformation of dentin, induced by

physiologic and pathologic processes, result in a dentinal substrate that is less receptive to

adhesive treatments than normal dentin 26,27,28,29,30,31.

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A B

C

Figure 1. (A) Transverse section of dentinal tubules (SEM image, 2500×). (B) Dentinal tubules (SEM image, 5000×). (C) Tubular and peritubular dentin (SEM image, 8000×). Modified from Perdigão and Breschi (2006).

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1.5 Enamel adhesion

Adhesion to enamel is obtained by etching for 15-30 second the enamel tissue with

phosphoric acid at 30-46%. Acid etching transforms smooth enamel into an irregular surface.

A resin monomer or adhesive mixture is then applied to the enamel surface and it drawn into

surface microporosities by capillary action. The monomers in the fluid resin polymerize and

become mechanically interlocked with the enamel structure, forming microtags. A microtag

consist of the polymerized resin into enamel prisms nucleus 32. The formation of resin

microtags within the enamel structure has been considered the essential mechanism of resin-

enamel adhesion 33.

1.6 Dentin adhesion

When tooth structure is prepared with bur or other instruments, residual organic and inorganic

components form a “smear-layer” of debris on the surface 34. This iatrogenically produced

layer of debris has a great influence on any adhesive bond formed between the cut tooth and

restorative materials 35,36,37. The smear-layer obstruct the entrance of dentin tubules forming

the “smear-plugs” decreasing dentin permeability by up to 86% 38. The smear plugs are

debris tags, which may extend into the tubule to a depth of 1 to 10 µm 39,40 and are contiguous

with smear layer. The smear layer is believed to consist primarily of shattered hydroxyapatite

and fragmented and denatured collagen. Smear-layer composition changes with depth

reflecting the different composition of dentin in different areas of the tooth. The thickness and

morphology of the smear layer depend on the instrumentation used and is related to depth.

Porosity of the smear-layer still allows the flow of dentinal fluid 41. In dentin this situation

involves the exposure of collagen fibers with the creation of chemical interaction between

dentin and adhesive. This condition is possible only after etching. The smear-layer acts as a

physical obstacle that decreases the permeability of dentin. Smear-layer can be considered an

obstacle that must be removed or made permeable to permit resin impregnation. Non-acidic

adhesives, applied without prior etching, do not penetrate to smear-layer deeply enough to

establish a bond with dentin. Such bonds are prone to cohesive failure of the smear-layer 40.

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Figure 2. SEM micrograph (10,000×) of smear layer and smear plug (SP). Modified from Perdigão and Breschi (2006). The aim of etching is to remove smear-layer and smear-plug and to allow exposure and partial

demineralization of dentin tissue, leaving intact the collagen fibers. This process permits

excellent bonding of the adhesive system. The infiltration process culminates with the

formation of the “hybrid-layer” (dentin infiltrated by resin).

Figure 3. FEI-SEM micrographs of an etch-and-rinse (a) and a self-etch (b) adhesive system. Image from Dental Adhesion Review: aging and stability of the bonded interface. Breschi et al. 32

Two strategies have been used to overcome the low bond strength of the adhesive to the

smear layer. Etch-and-rinse adhesives include an acid gel to pre-treat dentin and enamel. The

acid dissolves the smear layer and the top 1-6 µm of hydroxyapatite 42. Self- etch adhesives

treat the dentin and enamel surface with a non-rinsing solution of acidic monomers in water.

These adhesive systems do not remove the smear layer, but they make it permeable to the

monomers subsequently applied.

The removal of the smear layer and smear plugs with acidic solutions results in an increase of

the fluid flow onto the exposed dentin surface. This fluid can interfere with adhesion because

hydrophobic resins do not adhere to hydrophilic substrates even if resin tags are formed in the

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dentin tubules. When dentin is covered with the smear-layer and the dentinal tubules are

occluded with smear plugs, fluid permeability is very reduced 40. After smear-layer removal

with an acid, dentinal permeability through the dentinal tubules increases by more than 90% 43. Because water contamination of bonding was known to decrease the bond strength, it was

feared that the removal of the smear-layer and the subsequent wetting of the dentin surface

would adversely affect the bond strength between dentin and resin composites because the

dentinal fluid would dilute bonding primers and other bonding resins. However, several

contemporary systems have demonstrated the ability to couple with the augmented fluid

permeability of dentin, and high and durable bond strength can be achieved 44. The main

mechanism of tooth sensitivity is based on hydrodynamic fluid movement. Removal of the

smear layer can induce tooth sensitivity in vivo. It is probable that adhesive techniques that

require smear-layer removal are associated with more post-operative sensitivity than systems

that leave the smear-layer in place. Open dentinal tubules may also permit passage to the pulp

of bacteria, their products, and toxic chemicals such as acids. Although it has been shown that

the bonding adhesive may cause transient pulpal inflammation, especially in deep cavities, a

continuous bacterial irritation due to the microgaps and microleakage is more likely to cause

pulp damage and post-operative pain 44. Complete or partial removal of the smear layer can be

obtained by applying acidic or chelating solutions, called “conditioners”. The more acidic

and aggressive the conditioners, the more completely the smear-layer and smear plugs are

removed45. Strong acids not only remove the smear-layer, but demineralize intact dentin,

remove smear-plugs, open the dentinal tubule orifice, and penetrate in tubules for a 1-5 µm

depth. The etch-and-rinse approach uses a 30% or 40% phosphoric acid gel as conditioner.

Other acids as maleic, citric, nitric and tannic may be used in various concentrations.

Figure 4. SEM images and diagrams of self-etching and etch-and-rinse adhesion strategies.

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Internal and external dentinal wetness Dentinal permeability (and consequently, the internal dentinal wetness) depends on several

factors, including the diameter and length of the tubules, the viscosity of dentinal fluid and the

molecular size of substances dissolved in it, the pressure gradient, the surface area available

for diffusion, the capacity of removal substances by pulpal circulation. Occlusal dentin is

more permeable over the pulp horns than at the center of the occlusal surface; proximal dentin

is more permeable than occlusal dentin; and coronal dentin is more permeable than root

dentin. High dentinal permeability allows bacteria and their toxins to penetrate the dentinal

tubules to the pulp if the tubules are not hermetically sealed. The variability in dentinal

permeability makes it a difficult substrate for adhesion. Removal of the smear-layer creates a

wet bonding surface on which dentinal fluid exudes from the dentinal tubules. This aqueous

environment affects adhesion, because water competes effectively, by hydrolysis, for

adhesion sites on the dentinal tissue 46. The water interferes with polymerization of adhesives,

resulting in suboptimal degree conversion. Early dentin bonding agents failed primarily

because their hydrophobic resins were not capable of sufficiently wetting the hydrophilic

substrate. In addition, bond strengths of several adhesive systems were shown to decrease as

the depth of the preparation increased, because dentinal wetness was greater 41. No significant

difference in bond strengths is observed between deep and superficial dentin when the smear-

layer is intact. Bond strengths of etch-and-rinse systems that remove the smear-layer appear to

be less affected by differences in dentinal depth 29, probably because their increased

hydrophilicity provides better bonding to the wet dentin surface. In the past the adhesive

systems were too hydrophobic to be successful, while now adhesives tend to be overly

hydrophilic, impairing adhesion. The one-step self-etch approach has been reported to

produce adhesive films that behave as semipermeable membranes and absorb moisture from

the oral envinronment as well from the tooth itself (dentinal tubular fluids). This phenomenon

is due to the highly hydrophilic nature of one step self-etch adhesives. Application of an

additional hydrophobic resin layer (as a part of a two step self-etch approach) has been shown

to improve the dentinal seal. The external wetness or environmental humidity as also

demonstrated to negatively affect bond strengths to dentin.

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1.7 Classification of Dentinal Adhesives

There are different ways for classification of dentinal adhesive system, including:

Generations: adhesives are categorized according to the order in which they were introduced

into the market. However this chronological classification is not logical, and lacks scientific

background.

Number of steps: they are classified on the number of steps necessary for a correct

application;

Solvent type: they are classified according to the solvent employed in their formulation. The

solvents used are: ethanol, acetone, water or these solvents in combinations.

The adhesive systems should be compatible with two substrates: dentin that is strongly

hydrophilic, and composite that is hydrophobic. This objective is obtained by means of three

agents:

The etching; that increases the free energy of the surface.

The primer; that is the actual adhesion-promoting agent.

The bonding; that is the agent capable to infiltrate the substrate and create the bond.

The current classification of adhesives relies on the number of the steps constituting the

system: etching, primer and bonding are always used even if in different combination: Three-step adhesives: etching, primer and bonding are applied one by one and in sequential

way.

Two-step adhesives: they are composed by etching, primer & bonding: after etching, primer

and bonding are applied together; or etching & primer, bonding: etching and primer are

applied together and air dried (self etching primer), subsequently bonding is applied.

One-step adhesives: etching, primer and bonding are mixed together in the same solution.

The first two classes of adhesives are called “etch-and-rinse” because the acid etchant is

removed with water after application. The last two classes of adhesives are characterized by

containing the etching combined with the primer or with primer and bonding together. The

acid is not removed but only air dried. This system is called “self-etch” or “etch-and-dry”.

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Figure 5. Classification of contemporary adhesives indicating their main components. Most of the adhesives contain methacrylate-based monomers. Two-step etch&rinse adhesives are often referred to as ‘one-bottle’systems. As such, one-step self-etch adhesives are often subdivided in one- and two-component systems. Etch & Rinse approach

This adhesion strategy in its most conventional form involves three steps: application of acid

etchant, followed by the primer and then by the bonding agent. The two-step version

combines the second and third step but still follows a separate etch and rinse phase. This

technique is an effective approach to achieve efficient and stable bonding to enamel and only

requires two steps. Selective dissolution of hydroxyapatite crystals by etching is followed by

in situ polymerization of resin that is easily absorbed by capillary action within the created

etch pits, enveloping individually the exposed hydroxyapatite crystals. Two types of resin tags

interlock within the etch pits. Macrotags fill up the space surrounding the enamel prisms,

while several microtags result from resin infiltration/polymerization within tiny etch pits at

the core of the etched enamel prisms. Microtags are thought to be the major contributors to

retention to enamel. In dentin the etching treatment exposes a microporous network of

collagen with elimination of most or all the hydroxyapatite. The bonding mechanism of etch-

and-rinse adhesives to dentin is primarily diffusion-based and depends upon hybridization or

infiltration of resin within the exposed collagen fibril scaffold. True chemical bonding is

unlikely, because the functional groups of monomers have only weak affinity to the

hydroxyapatite-depleted collagen. The limited monomer-collagen interaction might be the

principal reason for the so called nanoleakage phenomenon 47,48. The most critically phase in

the etch-and-rinse approach is the application of the primer. When an aceton-based adhesive

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is used, the highly technique-sensitive wet bonding-technique is mandatory 49. When an

ethanol-based adhesive is used, gentle post-conditioning air-drying of acid etched dentin and

enamel followed by a dry-bonding technique can be used 50,51. In two-step etch-and-rinse

adhesives the primer and adhesive resin are combined into one solution. In conventional

three-steps systems, the primer ensures efficient wetting of the exposed collagen fibrils,

displaces residual surface moisture, transforms a hydrophilic surface into a hydrophobic

surface, and carries monomers into the interfibrillar channels. The adhesive resin fills the

pores between the collagen fibrils, forms resin tags sealing the opened dentinal tubules,

stabilizes the hybrid-layer and resin tags, and provides sufficient methacrylate double bonds

for copolymerization with the restorative resin.

Acid etchants

Etch & Rinse adhesive involves the use of phosphoric acid with a concentration between 30%

and 40% and an etching time of not less than 15 seconds. Adequate rinsing is an essential

step. Washing times of 5 to 10 seconds are recommended to achieve the most receptive

enamel surface for bonding 52,53,54,55.

Primers

Primers serve as adhesion promoting agents and contain hydrophilic monomers dissolved in

solvents, such as acetone, ethanol, and/ or water. Because of the volatile characteristics of

acetone and ethanol, these solvents can displace water from the dentin surface and from the

moist collagen network, permitting the infiltration of monomers through the nanospaces of

exposed collagen network 56. Effective primers contain monomers with hydrophilic properties

that have an affinity for the fibril arrangement of exposed collagen and hydrophobic

properties for copolymerization with the adhesive resin 46. The objective of the priming step is

to transform the hydrophilic dentin surface into a hydrophobic and spongy state that allows

the adhesive resin to wet and penetrate the exposed collagen network efficiently 57,58,59. After

etching, the demineralized collagen network is susceptible to collapse when water is removed

by drying. Collapse and subsequent shrinkage of collagen can lead to suboptimal resin

infiltration.

The primers of many modern adhesive systems contains HEMA (2-hydroxyetil methacrylate),

which promotes adhesion due to its excellent wetting characteristics60. In addition to HEMA,

primers contain other monomers, such as NTG-GMA (N-tolyglycine glycidyl methacrylate),

PMDM (pyromellitic acid diethylmethacrylate, BPDM (biphenyl dimethacrylate), and

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PENTA (dipentaerythritol penta acrylate monophosphate). Some primers include a chemical

or photopolymerization initiator, so these monomers can be polymerized in situ. More viscous

primers have been developed to combine the primer and the bonding function, simplifying the

bonding technique (one bottle adhesives). Primers are used to prevent and treat dentinal

hypersensitivity, which is believed to depend from pressure gradients of the dentinal fluid

within patent tubules that communicate with the oral environment. The primer may induce

denaturation and precipitation of proteins from the dentinal fluid, decreasing dentinal

permeability and outward flow of pulpal fluid and reducing the clinical symptoms of

hypersensitivity 61.

HEMA

HEMA is a small monomer that is in widespread use, not only in dentistry. Uncured HEMA

presents as a fluid that is well solvable in water, ethanol and/or acetone. A very important

characteristic of HEMA is its hydrophilicity that makes it an excellent adhesion promoting

monomer. By enhancing wetting of dentin, HEMA significantly improves bond strengths.

Nevertheless, both in uncured and cured state HEMA will readily absorb water. Studies

hypothesized that HEMA containing adhesives are more susceptible to water contamination,

as the HEMA in the uncured adhesive may absorb water, which can lead to dilution of the

monomers to the extent that polymerization is inhibited. After polymerization HEMA exhibits

hydrophilic properties and leads to water uptake with consequent discoloration. HEMA is

vulnerable to hydrolysis, especially at basic pH 62.

Adhesive resin

The adhesive resin, also called bonding agent, consists primarily of hydrophobic monomers,

such as bisphenol A diglycidyl metacrylate (bis-GMA), urethane dimethacrylate (UDMA),

triethylen glycol dimethacrylate (TEG-DMA) as a viscosity regulator, and more hydrophilic

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monomers, such as HEMA as wetting agent. The principal role of the adhesive resin is to

stabilize the hybrid layer and to form resin tags. Adhesive resins can be light-cured and/or

self-cured. Self-curing has the advantage of initial polymerization at the interface due to the

higher temperature produced by body heat, but the disadvantage of a slow polymerization.

For light-cured adhesives agents, it is recommended to polymerize the resin before the

application of the composite. In this way, the adhesive resin is not displaced, and an adequate

light intensity is available to sufficiently cure and stabilize the bond to resist the stress

produced by polymerization shrinkage of the resin composite [37][1].

Di-methacrylate

Bis-GMA

TEGMA

Adhesive resins consist of mixtures of cross-linking monomers, and relative amounts of Bis-

GMA, TEGMA and UDMA used will have significant influence on the viscosity of the

uncured adhesive resin and on the mechanical properties of the cured polymer. Bis-GMA is

highly viscous in the uncured state. The resulting polymer of this monomer is characterized

by superior mechanical properties. UDMA is most commonly used in adhesives. This

monomer exhibit lower viscosity properties than Bis-GMA. TEGMA is usually used in

conjunction with Bis-GMA or UDMA. Both methacrylate and other dimethacrylate such as

UDMA, EGDMA or TEGMA are used as “diluents”.

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1.8 Chitosan

Chitosan is a linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine

and it is the only natural poly-cationic polysaccharide. It is commercially produced by

deacetylation of chitin obtained mainly from marine crustacean sources. Pharmaceutical and

biomedical applications of chitosan are vast, e.g. carrier in nanoparticles for gene- and drug

delivery, matrix for biomolecules, nanoparticles and cells, wound dressings, tissue

engineering, and antibacterial component in functional materials. Chitosan biocompatibility

allows its use in different medical areas such as topical ocular applications63, implantation64,65,

injection66 and drug delivery67. Chitosan is also bio-adhesive, a property that determines an

increased retention at the site of application68. Chitosan has been physically combined with

Poly(methyl methacrylate), calcium phosphates, alginate, hyaluronic acid, poly-L-lactic acid

and growth factors for potential application in orthopaedics and to create bioactive composites

endowed with osteoconductivity and enhanced mineralization67.

When chitosan is used in combination with protein-based materials (e.g. collagen),

reticulation of the mixed system can be obtained by means of crosslinking agents69-72: recent

studies are addressing the use of novel and non toxic crosslinking agents such as genipin73,74

and enzymes (transglutaminase, tyrosinase)75.

Chitin/chitosan are rich in free NH2 groups that are reactive groups for chemical

modifications to endow the polymer with further properties: for instance, bioactivity of this

polysaccharide is enhanced in the presence of oligosaccharides and peptide side chains76. The

introduction of flanking groups can be used to modulate the hydrophilic/hydrophobic

properties of chitosan.

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30. Sattabanasuk V, Shimada Y, Tagami JOper Dent 2004;29(3):333-341

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32. Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, De Stefano Dorigo E.

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33. Suppa P, Breschi L, Ruggeri A, Mazzotti G, Prati C, Chersoni S, Di Lenarda R,Pashley

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34. Bowen RL, Eick JD, Henderson DA, et al. Oper Dent Suppl. 1984;3:30-34

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36. Eick JD. Proc Finn Soc 1992;88(suppl 1):225-2423

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38. Pashley DH, Livingstone MJ, Greenhill JD. Arch Oral Biol. 1978;23:807-810

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Poorten, 1994:171-191

41. Pashley DH, Horner JA, Brewer PD. Oper Dent. 1992;Suppl 5:137-150

42. Erickson RL. Oper Dent 1992; suppl 5:81-94

43. Perdigão J. Dent Mat 26 (2010) e24–e37

44. Pashley DH. Dentin: Scanning Microsc 1989;3:161-174

45. Van Meerbeek B1, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, Van Landuyt K,

Lambrechts P, Vanherle G. Oper Dent. 2003 May-Jun;28(3):215-35

46. Meyron SD, Tobias RS, Jakeman KJ. J Prosthet Dent 1987;57:174-179 47. Sano H, Shono T, Takatsu T, Hosoda H. Oper Dent 1994;19:59-64

48. Sano H, Yoshiyama M, Ebisu S et al. Oper Dent 1995;20:160-167

49. Tay FR, Gwinnett AJ, Pang KM, Wei SH. J Dent Res 1996;75:1034-1044

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50. Van Meerbeek B, Conn LJ Jr, Duke ES, Eick JD, Robinson SJ, Guerrero D. J Dent Res

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51. Van Meerbeek B, Yoshida M, Lambrechts P, et al. J Dent Res 1998;77:50-59

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59. Nikaido T, Burrow MF, Tagami J, Tagatsu T. Quint Int 1995;26:221-226

60. Nakabayashi N, Takarada K. Dent Mater 1992;8:125-130

61. Swift EJ, Hammel SA, Perdigão J, Wefel JS J Am Dent Assoc 1994;125:571-576

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E, Suzuki K, Lambrechts P, Van Meerbeek B Biomat 2007;28:3757-3758

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2831-2841

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76. Travan, A.; Donati, I.; Marsich, E.; Bellomo, F.; Achanta, S.; Toppazzini, M.; Semeraro, S.; Scarpa,

T.; Spreafico, V.; Paoletti, S. Biomacromolecules 2010, 11 (3), 583-592

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Chapter 2

Degradation of the bonded interface

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24

2.1 Degradation of the bonded interface

The bonded interface is the weaker part of restoration because it is exposed to oral fluids.

Exposure of the dentin-adhesive interface to the oral cavity often results in marginal

discoloration, poor marginal adaptation, dentinal sensitivity, and loss of restoration retention.

Although contemporary adhesives can provide good initial bonding to enamel and dentin for

composite restorations, the long-term durability of the bonded interface remains unclear. The

stability of the bonded interface relies on the creation of a compact and homogenous hybrid

layer formed by resin monomers during dentin substrate impregnation. In the etch-and-rinse

approach a stable bond can be achieved only if the etched substrate is fully infiltrated by

adhesive, avoiding different degrees of incomplete impregnation1,2,3. Self-etch approaches use

acids that simultaneously demineralize and infiltrate dentin, and adhesive stability is related to

the effective coupling of co-monomers with the infiltrated substrate. Recent studies revealed

that 2-step self-etch systems with mild acidity (pH approximately 2) may establish chemical

bonds between specific carboxyl or phosphate group of the functional monomers and residual

hydroxyapatite crystals still present on dentin collagen scaffold due to the mild aggressiveness

of the acidic phase4. This additional interaction is claimed to enhance bond stability over time.

The clinical longevity of the hybrid-layer appears to be affected by physical and chemical

factors. Physical factors such as occlusal chewing forces, and the repetitive expansion-

contraction stresses due to temperature changes within the oral cavity are supposed to affect

the interface stability. Chemical factors, such as chemical acids in dentinal fluid, saliva, food,

beverage and bacterial products are involved in the degradation of unprotected collagen

fibers, elution of resin monomers and degradation of resin components.

2.2 Degradation of exposed collagen fibrils

The water content in the hybrid layer is considered one of the major causes for collagen

degradation. There are two processes to describe bonding degradation: loss of resin from the

interfibrillar spaces and disorganization of collagen fibrils5. The aim of the bonding approach

is the complete infiltration, and encapsulation of the collagen fibrils by the bonding agents is

recommended in order to protect them against degradation6. Etch-and-rinse adhesives may

produce an incompletely infiltrated zone along the bottom of the hybrid-layer: this layer may

contain collagen fibrils exposed by etching and not impregnated by the adhesive due to the

discrepancy between the depth of acid etching and resin infiltration7. When using self-etch

adhesives, the acidic monomers dissolve the inorganic phase of dentin and simultaneously

prime and infiltrate the dentin matrix, resulting in fewer exposed collagen fibrils8.

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25

2.3 Degradation of the resin

In the hybrid layer, both the resin and the collagen fibrils can be degradated9. Water causes

hydrolysis of resin. Hydrolysis is a chemical phenomenon that breaks covalent bonds between

the polymers by addiction of water ester bonds, resulting in resin mass loss: this is considered

as one of the main reasons for resin degradation within the hybrid layer10,11. The same process

of hydrolysis jeopardizes collagen polymers that are insufficiently coated with resin, and

enzymes enhance this degradation process12,13,14. As a result of hydrolysis, small breakdown

products that leach away from the resin are formed, promoting even more water penetration.

Resin degradation depends also on water sorption within the hybrid layer. The absorbed water

may deteriorate the mechanical properties of polymers, including elastic modulus of the resin,

which contribute to the reduction in bond strength, independently from resin hydrolysis8.

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26

Reference Chapter 2 1. Sano H, Shono T, Takatsu T, Hosada H. Oper Dent 1994;19:59-64

2. Eliades G, Vougioklakis G, Palaghias G. Dent Mater 2001;17:277-3.

3.

Yoshida Y, Van Meerbeek B, Snauwaert J, Hellemans L, Lambrechts P, Vanherle G, et al.

J Biomed Mater Res 1999;47:85-90

4. Yoshida Y, Nagakane K, Fukuda R et al. J Dent Res 2004;83:454-8

5. Hashimoto M, Ohno H, Sano H, et al. Biomat 2003;24:3795-803

6. Hashimoto M, Ohno H, Sano H, et al. J Biomed Mater Res 2002;63:306- 311

7. Wang Y, Spencer P. J Biomed Mater Res 2002;59:46-55

8. Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, De Stefano Dorigo E. Dent

Mater 2008,24:90-101

9. De Munck J, Van Landuyt K, Peumans M, et al. J Dent res 2005;84:118-132

10. Tay FR, Pashley DH. J Can Dent Assoc. 2003;69:726-31.

11. Tay FR, Hashimoto M, Pashley DH, et al. J Dent Res 2003;82:537-41

12. Hashimoto M, Ohno H, Kaga M, Endo K, Sano H, Oguchi H. J Dent Res 2000;79;1385-

13919

13. Hashimoto M, Tay FR, Ohno H, et al. J Biomed Mater Res 2003;66B:287- 298

14. Fukuda R, Yoshida Y, Nakayama Y, et al. Biomat 2003;24:1861-1867!!

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Chapter 3

Formulation of an adhesive system using Chitosan

methacrylate

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28

3.1 Introduction The objective of this research was to find a stable solution containing methacrylate-modified

chitosan to be used as a dental adhesive system.

In the present work, chitosan was modified with methacrylate moieties and a physical-chemical

characterization was carried out. The use of chitosan as a component of the adhesive system is not

new. Elsaka et al.1 previously evaluated the use of chitosan blended within the adhesive systems

due to its antibacterial properties. However, these authors reported a significantly decrease of

tensile bond strength at the dentine interface upon increasing the concentration of chitosan. We

speculated that the modified chitosan is able to covalently bind to the restorative resin and, due to

the presence of residual positive charges on the polysaccharide, to electrostatically interact with the

demineralized dentine. The chitosan bearing methacrylate groups was blended within the primer of

a three-step etch-and-rinse experimental dentine bonding system to assay its bonding ability to

dentine. Further mechanical tests were performed to evaluate the bond strength of different

formulations of the adhesive system and compared with a control without chitosan.

3.2 Materials and Methods

Chitosan was purchased from Sigma-Aldrich (St. Louis, MO) and purified as reported elsewhere2.

The relative molar mass (“molecular weight”, MW), as measured by intrinsic viscosity3, was found

to be approximately 540,000 and the residual degree of acetylation, as determined by 1H NMR, was

17%4. Lysozyme from chicken egg white (40000 units/mg protein), methacrylic acid, N-(3-

(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxy succinimmide

(NHS), bisphenol A glycerolate dimetha- crylate (BisGMA), triethylene glycol dimethacrylate

(TEGDMA), ethyl-4-dimethylaminobenzoate (EDMAB), diphenyl-(2,4,6-trimethyl- benzoyl)-

phosphine oxide (TPO), and camphorquinone (CQ) were purchased from Sigma-Aldrich (St. Louis,

MO).

Chitosan Depolymerization

Chitosan (1 g) was dissolved in deionized water (100 mL) and added with HCl 1 M (7.5 mL) and

NaNO2 10 mg/mL (1.5 mL). The solution was vigorously stirred for 50 min and reduced by means

of NaBH4.5,6 The pH of the solution was raised to approximately 5.5 with aqueous NaOH; the

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29

solution was dialyzed against NaCl (0.1 M, 2 shifts) and deionized water until the conductivity was

below 4 µS at 4°C, the pH was adjusted with HCl and the solution freeze-dried to obtain the

hydrochloride salt of chitosan. The MW value, as determined by intrinsic viscosity measurements,3

was 72,000.

Modification of Chitosan with Methacrylic Acid

Chitosan (660 mg) was dissolved in aqueous MES 0.05 M (250 mL, pH 5.5). Methacrylic acid was

added at different ratios [methacrylic acid]/[Chitosan]ru followed by the addition of NHS and EDC

where k′ and k′′ are the Huggins and Kraemer constants, respectively. The buffer used was aqueous

0.02 M HAc/NaAc, pH = 4.5, in the presence of NaCl 0.107 M.3 The polymer solutions and the

solvent were filtered through 0.8 µm Millipore filters prior to their use. Measurements were

performed at 20°C. Viscosity-average MW values were calculated according to the

Mark−Houwink−Sakurada equation, with parameters indicated by Anthonsen et al.3.

Primer Preparation

Methacrylate-modified chitosan (120 mg) was dissolved in aqueous MES buffer (50 mM, 6 mL, pH

5.5). The solution was a mixture of HEMA (3.6 mL) and ethanol (2.4 mL). A control adhesive

system was prepared through the same procedure, but omitting the presence of methacrylate-

modified chitosan.

R2 Resin Preparation

An experimental resin (R2), similar to the nonsolvated hydrophobic resin contained in different

commercial bonding agents of three-step etch-and-rinse and two-step self-adhesive systems, was

prepared in accordance with Cadenaro et al.7,8 In the present study, R2 was modified adding a

second photoinitiator, namely, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), to improve

the polymerization kinetic of the hydrophilic moieties blended within its formulation. The final

composition of R2 was BisGMA 70 wt %; TEGDMA 28.75 wt %; EDMAB 0.5 wt %; TPO 0.5 wt

%; and CQ 0.25%wt. Stock mixtures were prepared by incorporating all components in dark bottles

and by stirring them for 2 min by means of a vortex mixer (Vortex Classic, Velp Scientifica,

Milano, Italy).

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30

Experimental adhesive systems

In the preliminary phase of this work different solutions of methacrylate-modified chitosan were

tested to find the best composition for dental applications.

The first solution tested using methacrylate-modified chitosan contained: 50% ethanol, 1% chitosan

methacrylate, 0.25% TPO. This solution, named A1 was used as an etch-and-rinse two-step

adhesive system on human dentin. In the second solution (A2) 30% HEMA was added to reduce the

high viscosity of the solution A1.

In the second phase of the study the methacrylate-modified chitosan was used as a primer and the

experimental resin R28 as a bonding agent of a three-step etch-and-rinse adhesive system. This

formulation was named A3 and contained: primer: 1% chitosan methacrylate, 30% HEMA, 20%

ethanol; R2: 70% BisGMA, 28.75% TEGDMA, 1% EDMAB, 0.25%TPO.

The formulation A3 was also modified and 0.5% of CQ was added to R2 composition. This new

formulation was named A3*.

The methacrylate-modified chitosan was used at 2% in the formulation A4 to assess whether the

percentage of chitosan present into the primer led to a bond strength change. The formulation A4

contained: primer: 2% chitosan methacrylate, 30% HEMA, 20%; R2: 70% BisGMA, 28.75%

TEGDMA, 1% EDMAB, 0.25%TPO.

A primer solution without methacrylate-modified chitosan was used as a control (C) (Primer: 30%

HEMA, 20% ethanol; R2: 70% BisGMA, 28.75% TEGDMA, 1% EDMAB, 0.25% TPO, 0.25%

CQ). The formulation C was used as an etch-and-rinse three-step adhesive.

Human Teeth Specimen Preparation A total of 30 recently extracted, non-carious human third molars were selected, stored in 0.5%

chloramine-T solution at 4 °C, and used within one month of extraction.34 The occlusal third of

molar crowns and their roots were removed perpendicularly to the long axis of each tooth by means

of a water-cooled slow speed diamond blade (Isomet 5000, Buehler Ltd., Lake Bluff, IL, U.S.A.) to

obtain a 4 mm high dentinal section. The exposed dentine surfaces were polished with 180-grit wet

silicon carbide abrasive paper to create a standardized smear-layer.9 In all specimens the pulpal

camera was opened and filled with flowable composite to improve the stability of the specimen.

Dentine disks were then randomly and equally assigned to the six groups (N = 5): A1, A2, A3, A3*,

A4 (methacrylate-modified chitosan-containing adhesive systems) and the control formulation

without the methacrylate-modified chitosan. The exposed dentine surface was etched with 35%

phosphoric acid for 15 s, then rinsed with water and gently air-dried in accordance with the bonding

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31

technique. The formulations A1 and A2 were applied on tooth surface for 20 s and gently air-dried

to obtain a shiny surface and polymerized for 40 s with a LED curing unit (Valo, Ultradent Product

Inc. South Jordan, UT, U.S.A.; tip distance of 1 mm). The irradiance level of the light was

monitored periodically with a radiometer (Demetron 910726, Kerr Corporation. Danbury, CT,

U.S.A.) to ensure an output of at least ≥600 mW/cm2. The primer contained in the formulations A3,

A3*, A4 and the control C were applied for 20 s and gently air-dried to obtain a shiny surface and

then R2 resin was applied for 20 s, gently air-dried and polymerized for 40 s with the same LED

unit. A 2 mm increment of resin composite (Filtek Z250, 3 M ESPE, St Paul, MN, U.S.A.) was

incrementally layered on the adhesive surface to create a resin-dentin build-up and each layer was

light-cured for 20 s. The specimens were stored in artificial saliva at 37°C for 24 hours to simulate

physiological conditions.

MicroTensile Bond Strength Test (µTBS)

Resin-dentin build-ups were sectioned perpendicular to the bonded surface using a low-speed

diamond saw (Isomet 5000, Buehler Ltd., Lake Bluff, IL, U.S.A.) under water irrigation in

accordance with the non-trimming technique for microtensile bond strength test to obtain specimens

of 0.9 × 0.9 × 8.0 mm10. The dimensions of each specimen were individually measured with a

digital caliper (±0.01 mm) and recorded for subsequent bond strength calculation. Sectioned sticks

were stored for 24 h in artificial saliva at 37 °C11, then glued with cyanoacrylate (Zapit, Dental

Ventures of America Inc., Lewis Ct., Corona, CA) to the two free-sliding parts of a microtensile

testing machine. The specimens were stressed until failure under tension at a crosshead speed of 1

mm/min (Bisco Inc., Schaumburg, IL, U.S.A.). The microtensile bond strength values were then

converted into unit of stress (MPa).

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32

Interfacial Nanoleakage Analysis

Nanoleakage tests were used to verify the presence of open nanometer sized water-permeable

pathways within the hybrid layer using silver nitrate as a tracer. SEM and TEM were used to

observe the silver nitrate stains pathways created by water diffusion throughout the bonded

interface. Additional teeth were used to evaluate interfacial nanoleakage expression. Disks of

middle/ deep dentine were prepared, bonded, and aged similarly to the specimens submitted to

microtensile bond strength test. The resin-bonding build-ups were then cut perpendicular to the

bonded surface to obtain 1 mm thick specimens. Slabs were immersed in a 50% aqueous

ammoniacal silver nitrate (AgNO3) solution in the presence of laboratory light for 24 h. Specimens

were then photo developed for 8 h to reduce silver ions into metallic silver grains within voids

along the bonded interface and then polished using wet 600, 1200, and 2000-grit silicon carbide

papers12. Resin-dentin interfaces were observed under optical light microscope (Leica DMR; Leica,

Wetzlar, Germany). For scanning electron microscope analysis (SEM), specimens were polished

with 2400-grit SiC paper and 2–1-µm 59 diamond paste using a polishing cloth, then ultrasonically

cleaned, air dried, mounted on the SEM stubs, and carbon coated. Resin–dentin interfaces were

analyzed by SEM (Quanta 250; FEI, Hillsboro, OR, USA) operating in mixed

secondary/backscattered electron mode at 1 kV. Interfacial nanoleakage was scored based on the

percentage of adhesive surface showing AgNO3 deposition: 0, no nanoleakage; 1, <25% surface

with nanoleakage; 2, 25% to ≤50% surface with nanoleakage; 3, 50% to ≤75% surface with

nanoleakage; and 4, >75% surface with nanoleakage.

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33

Statistical analysis

Microtensile bond strength test data were evaluated using a commercial software for statistical

analysis (SPSS, version 20.0 for Windows; SPSS, Chicago, IL, U.S.A.). Values were normally

distributed and data were analyzed with one-way analysis of variance (ANOVA) and Tukey’s post-

hoc multiple comparison tests. A p-value lower than 0.05 was considered statistically significant.

Scores of the interfacial nanoleakage analysis were statistically analyzed using non-parametric

Kruskal−Wallis test for multiple comparisons and Mann−Whitney U-test (a p-value lower than 0.05

was considered statistically significant).

3.3 Results

After 24 h at 37 °C it was possible to submit to the microtensile bond strength test the formulations

A2, A3*, A4 and the control (C). Unfortunately, the remaining formulations A1 and A3 after for

24-h storage demonstrated a lack of ability to adhere to dental tissues. Samples obtained using the

experimental adhesive systems A1 and A3 showed a complete detachment of the adhesive interface

with a total separation between the composite material and dental tissue. The means values and

standard deviations of µTBS after 24-h are listed in Table below:

Adhesive system 24-h (MPa)

A2 17±8

A3* 26±9

A4 22±7

C 28±9

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34

The formulation A3* and the adhesive system without chitosan (control - C) showed good adhesion

values to dental tissue (respectively: 26±9 and 28±9). The lowest bond strengths were recorded for

the formulation A2 used as a two-step etch-and-rinse adhesive system. The adhesive systems

containing 2% of chitosan methacrylate used as three-step etch-and-rinse adhesives showed bond

strength values comparable to the formulations A3* and C, but better than the adhesive systems A2.

The nanoleakage expression analysis was performed on the formulations A3*, A4 and C. These

formulations were selected because A3* showed the higher values of bond strength when submitted

to the microtensile bond strength test compared to A1. A4 contained a 2% of chitosan methacrylate

compared to A3* (1%). Since the purpose of this initial phase of the study was to find the optimal

formulation to be used as adhesive system, the nanoleakage analysis was performed on the

formulations that showed the best adhesion values.

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35

Figure 1. Representative optical microscope images of resin– dentin interfaces bonded with the experimental adhesive systems after 24-h (T0). The images of A4 and C showed that AgNO3 was present in the hybrid layers.

! ! !

36

3.4 Discussion

In this first phase of the study various formulations that could be used as dental adhesive systems

were analyzed. The novelty of the study is the introduction of a biopolymer: chitosan into an

adhesive system. Previous studies on chitosan in dentistry have considered only the antibacterial

properties of this biopolymer (Elsaka et al.). In the present study the mechanical properties of

adhesive systems containing chitosan modified with methacrylate groups were considered. The

experimental formulations were used as etch-and-rinse adhesive systems. The adhesive systems A1

and A2 were used as two-step etch-and-rinse adhesives, while the successive formulations and the

control were used as three-step etch-and-rinse adhesives. The first evaluation of the tested

formulations was made after 24-h, when formulations A1 and A3 showed a complete separation

between the dentin surface, on which the adhesive system was applied, and the restorative material.

For these two experimental formulations was not possible to perform the mechanical tests. For

formulations A2, A3*, A4 and C, it was possible to perform the microtensile bond strength test.

Three-step etch-and-rinse adhesive systems showed good bond strength values, higher than the two-

step etch-and-rinse adhesive (A2). For this reason, the two-steps adhesive system was discarded and

tests were continued only for formulations A3*, A4 and C. Nanoleakage analysis was performed

using these formulations, allowing to evaluate if the hybrid layer created by the these novel

adhesive systems behaved as a semipermeable membrane. The results obtained after 24-h for the

formulation C clearly showed the presence of several silver grains both along the adhesive interface

and in the dentinal tubules. A similar result was found for the formulation A4 containing 2%

chitosan methacrylate. On the other hand, in the images obtained from the formulation A3* it was

almost impossible to trace the presence of silver grains. The hypothesis to explain this result is that

1% chitosan methacrylate contained in A3* allowed the formation of electrostatic interaction

between dentin and the composite and therefore the creation of a homogeneous and more resistant

hybrid layer.

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37

References Chapter 3 1. Elsaka, S. E. Dent. Dig. 2012, 2012/06/07, 603−613

2. Donati, I.; Stredanska, S.; Silvestrini, G.; Vetere, A.; Marcon, P.; Marsich, E.; Mozetic, P.;

Gamini, A.; Paoletti, S.; Vittur, F. Biomaterials 2005, 26, 987−998

3. Anthonsen,M.W.;Var̊um,K.M.;Smidsrød,O. Carbohydr. Polym. 1993, 22, 193−201

4. Var̊um,K.M.;Antohonsen,M.W.;Grasdalen,H.;Smidsrød,O. Carbohydr. Res. 1991, 211, 17−23

5. Malmo,J.;Var̊um,K.M.;Strand,S.P.Biomacromolecules2011, 12, 721−729

6. Tømmeraas, K.; Strand, S. P.; Christensen, B. E.; Smidsrød, O.; Var̊um,K.M. Carbohydr. Polym.

2011,83, 1558−1564

7. Cadenaro, M.; Pashley, D. H.; Marchesi, G.; Carrilho, M.; Antoniolli, F.; Mazzoni, A.; Tay, F.

R.; Di Lenarda, R.; Breschi, L. Dent. Mater. 2009, 25, 1269−1274

8. Cadenaro, M.; Breschi, L.; Rueggeberg, F. A.; Suchko, M.; Grodin, E.; Agee, K.; Di Lenarda, R.;

Tay, F. R.; Pashley, D. H. Dent. Mater. 2009, 25, 621−628

9. Breschi, L.; Cammelli, F.; Visintini, E.; Mazzoni, A.; Vita, F.; Carrilho, M.; Cadenaro, M.;

Foulger, S.; Mazzoti, G.; Tay, F. R.; Di Lenarda, R.; Pashley, D. J. Adhes. Dent. 2009, 11,

191−198

10. Armstrong, S.; Geraldeli, S.; Maia, R.; Raposo, L. H.; Soares, C. J.; Yamagawa, J. Dent. Mater.

2010, 26, e50−e62

11. Pashley, D. H.; Tay, F. R.; Yiu, C.; Hashimoto, M.; Breschi, L.; Carvalho, R. M.; Ito, S. J. Dent.

Res. 2004, 83, 216−221

12. Tay, F. R.; Hashimoto, M.; Pashley, D. H.; Peters, M. C.; Lai, S. C.; Yiu, C. K.; Cheong, C. J.

Dent. Res. 2003, 82, 537−541

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38

Chapter 4

Use of Methacrylate-Modified Chitosan in an adhesive system to increase the durability of dentin bonding

systems

! ! !

39

4.1 Introduction

In the second phase of the study, the optimal formulation of the adhesive was refined and subjected

to several tests. The experimental formulation A3* tested in chapter 3 was considered the optimal

formulation of the adhesive system containing chitosan methacrylate. The formulation A3* was

named Chit-MA70. Since the greatest problem of dental restorations is the reduced duration in

time1 ChitMA70 was added to dental adhesive system to improve the mechanical performance of

dental restorations. Before the mechanical tests, the adhesive system Chit-MA70 was subjected to

two types of aging: static and dynamic. The most important storage method is the second that

simulates the presence of dental restoration in the oral cavity for 5 years. To verify the substantivity

of methacrylate modified-chitosan on dental surface after bonding procedure Chit-MA70 was

marked with fluorescein and observed using Confocal Laser Scanning Microscopy (CLSM). The

introduction of chitosan-modified monomers within the primer of an experimental three-step etch-

and-rinse adhesive, improved (1) immediate bond strength (T0 µTBS); (2) stability of the hybrid

layer after simulated chewing and thermo-cycling (TCS µTBS).

4.2 Materials and Methods

Chitosan was purchased from Sigma-Aldrich (St. Louis, MO) and purified as reported elsewhere.2

The relative molar mass (“molecular weight”), MW, as measured by intrinsic viscosity,3 was found

to be approximately 540000 and the residual degree of acetylation, as determined by 1H NMR, was

17%.4 Lysozyme from chicken egg white (40000 units/mg protein), methacrylic acid, N-(3-

(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxy succinimmide

(NHS), bisphenol A glycerolate dimetha- crylate (BisGMA), triethylene glycol dimethacrylate

(TEGDMA), ethyl-4-dimethylaminobenzoate (EDMAB), diphenyl-(2,4,6-trimethyl- benzoyl)-

phosphine oxide (TPO), and camphorquinone (CQ) were purchased from Sigma-Aldrich (St. Louis,

MO).

Chitosan Depolymerization

Chitosan (1 g) was dissolved in deionized water (100 mL) and to which was added HCl 1 M (7.5

mL) and NaNO2 10 mg/mL (1.5 mL). The solution was vigorously stirred for 50 min and reduced

by means of NaBH4.5,6 The pH of the solution was raised to approximately 5.5 with aqueous

! ! !

40

NaOH, the solution was dialyzed against NaCl (0.1 M, 2 shifts) and deionized water until the

conductivity was below 4 µS at 4 °C, the pH was adjusted with HCl and the solution freeze-dried to

obtain the hydrochloride salt of chitosan. The MW value, as determined by intrinsic viscosity

measurements,3 was found to be 72000.

Modification of Chitosan with Methacrylic Acid (Chit-MA)

Chitosan (660 mg) was dissolved in MES 0.05 M (250 mL, pH 5.5). Methacrylic acid was added at

different ratio [Methacrylic acid]/[chitosan]ru followed by the addition of NHS and EDC

([EDC]/[methacrylic acid] =1.5 and [NHS]/[EDC]=1). [chitosan]ru indicates the concentration of

chitosan repeating units. The solution was stirred for 24 hours, dialyzed against NaHCO3 (0.05 M, 3

shifts), aqueous HCl (0.001 M, 2 shifts), aqueous NaCl (0.1 M, 4 shifts), deionized water until the

conductivity was below 4 µS at 4 °C and finally was freeze-dried. The degree of substitution with

methacrylic moieties was determined by 1H-NMR analyses.

Viscosity Measurements

Intrinsic viscosity ([η]) was deter- mined by analyzing the polymer concentration dependence of

the reduced specific viscosity (ηsp/c) and of the reduced logarithm of the relative viscosity

(ln(ηrel)/c) according to the Huggins (eq 1) and Kraemer (eq 2) equations, respectively:

[ ] [ ] ckcsp 2' ηη

η+= eq. 1

( ) [ ] [ ] ckcrel 2''ln

ηηη

−= eq. 2

where k′ and k′′ are the Huggins and Kraemer constants, respectively. The buffer used was aqueous

0.02 M HAc/NaAc, pH = 4.5, in the presence of NaCl 0.107 M.3 The polymer solutions and the

solvent were filtered through 0.8 µm Millipore filters prior to their use. Measurements were

! ! !

41

performed at 20 °C. Viscosity-average MW values were calculated according to the

Mark−Houwink−Sakurada equation, with parameters taken from ref 3.

1H NMR Spectroscopy

1H NMR spectra were recorded at 85 °C with a JEOL 270 NMR spectrometer (6.34 T) operating at

270 MHz for proton. The chemical shifts were expressed in ppm downfield from signal for 3-

(trimethylsilyl) propanesulfonate.

Scanning Electron Microscopy (SEM)

Specimens were analyzed using a Quanta 250 scanning electron microscope (FEI, Oregon, U.S.A.)

visualized in backscattered scanning detection mode at 2500× after sputter coating with an ultrathin

layer of carbon. The working distance was 10 mm and the accelerating voltage was 1 kV.

Attenuated Total Reflectance (ATR) InfraRed (IR) Spectroscopy

The FTIR-ATR spectra were measured using a Varian FT-660 spectrometer equipped with a

diamond crystal GladiATR accessory (Pike Technologies).

Degradation with Lysozyme

Degradation of native chitosan and of Chit-MA by means of lysozyme was followed by measuring

the time dependence of the reduced capillary viscosity (ηsp/c) at 20 °C by meansofaSchott-

GeraẗeAVS/Gautomaticmeasuringapparatusand an Ubbelohde-type viscometer. A total of 12 mL of

a 0.8 g/L solution of Chit-MA or chitosan in 0.02 M NaAc/HAc added of NaCl 0.2 M at pH 4.5

were added of 0.5 mL of lysozyme (0.5 mg/mL) dissolved in the same solvent. Prior to

measurements, all solutions were filtered through 0.8 µm Millipore filters.

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42

Primer Preparation

Chit-MA70 (120 mg) was dissolved in aqueous MES buffer (50 mM, 6 mL, pH 5.5). The solution

was a mixture of HEMA (3.6 mL) and ethanol (2.4 mL). A control adhesive system was

prepared through the same procedure but omitting the presence of Chit-MA70.

R2 Resin Preparation

An experimental resin (R2), similar to the non-solvated hydrophobic resin contained in different

commercial bonding agents of three-step etch-and-rinse and two-step self-adhesive systems, was

prepared in accordance with Cadenaro et al.8,9 In the present study, R2 was modified adding a

second photo-initiator, namely, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), to improve

the polymerization kinetic of the hydrophilic moieties blended within its formulation. The final

composition of R2 was BisGMA 70 wt %; TEGDMA 28.75 wt %; EDMAB 0.5 wt %; TPO 0.5 wt

%; and CQ 0.25%wt. Stock mixtures were prepared by incorporating all components in dark bottles

and by stirring them for 2 min by means of a vortex mixer (Vortex Classic, Velp Scientifica,

Milano, Italy).

Human Teeth Specimen Preparation

A total of 20 recently extracted, non-carious human third molars were selected, stored in 0.5%

chloramine-T solution at 4 °C, and used within one month of extraction.10 The occlusal third of the

molar crowns and the roots were removed perpendicular to the long axis of each tooth by means of

a water-cooled slow speed diamond blade (Isomet 5000, Buehler Ltd., Lake Bluff, IL, U.S.A.) to

obtain a 4 mm high dentinal section. The exposed dentine surfaces were polished with 180-grit wet

silicon carbide abrasive paper to create a standardized smear-layer.11

The dentine disks were then

randomly and equally assigned to the two groups (N = 10): (1) Chit-MA70-containing adhesive

system; (2) control formulation without Chit-MA70. The exposed dentine surface was etched with

35% phosphoric acid for 15 s then rinsed with water, gently air-dried in accordance with the wet-

bonding technique. The Chit-MA70-containing primer was applied for 20 s and gently air-dried to

obtain a shiny surface and then R2 resin was applied for 20 s, gently air-dried and polymerized for

40 s with a LED lamp (Valo, Ultradent Product Inc. South Jordan, UT, U.S.A.; tip distance of 1

mm). The irradiance level of the light was monitored periodically with a radiometer (Demetron

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43

910726, Kerr Corporation. Danbury, CT, U.S.A.) to ensure an output of at least ≥600 mW/cm2. A 2

mm increment of resin composite (Filtek Z250, 3 M ESPE, St Paul, MN, U.S.A.) was incrementally

layered on the adhesive surface to create a resin-dentine build-up and each layer was light-cured for

20 s. Specimens of the control group were similarly prepared using the primer without Chit-MA70.

Aging Process

Half of the bonded specimens of each group (N = 5) were randomly assigned to thermo-

mechanical loading to simulate in vitro aging of adhesive interface. To simulate the clinical

situation, the aging was performed by means of thermo-mechanical loading of the specimens with a

CS-4.4 Chewing Simulator coupled with a Thermo Cycling device (SD Mechatronik GmbH,

Germany). Specimens selected for occlusal loading were partially embedded in Protemp 4

(Temporisation Material, 3M ESPE) in a primary polytetrafluoroethylene (PTFE) mold (diameter of

15 mm). To ensure a perfect parallelism between the resin composite and the aluminum antagonist

of CS, specimens were gently pressed with the CS device before Protemp complete hardening.

Periodontal ligament simulation was obtained with a 2.5 mm thick layer of Even Express 2

(Impression Material, lot: F 546236, exp. date: 2015−06, 3 M ESPE) surrounding the standardized

roots (formed by Protemp including natural roots). To simulate five years of in vivo service in the

oral cavity the aluminum antagonists imprinted an occlusal load of 50 N with a frequency of 1 Hz

and a downward speed of 16 mm/s for 1200000 cycles. Simultaneously, specimens underwent 6000

thermal cycles, 5− 55 °C, of 1 min each temperature with artificial saliva. The test duration was

approximately two weeks (TCS).12,13

Static conditions (T0, no thermo-mechanical stresses) were

considered as baseline. T0 specimens underwent static storage in artificial saliva at 37 °C for 24 h.

MicroTensile Bond Strength Test (µTBS)

At the two time points, T0 and TCS, resin-dentine build-ups were sectioned perpendicular to the

bonded surface using a low-speed diamond saw (Isomet 5000, Buehler Ltd., Lake Bluff, IL, U.S.A.)

under water irrigation in accordance with the non-trimming technique for microtensile bond

strength testing to obtain specimens of 0.9 × 0.9 × 8.0 mm.14 The dimensions of each specimen

were individually measured with a digital calliper (±0.01 mm) and recorded for subsequent bond

! ! !

44

strength calculation. Sectioned sticks were stored for 24 h in artificial saliva at 37 °C,15 then glued

with cyanoacrylate (Zapit, Dental Ventures of America Inc., Lewis Ct., Corona, CA) to the two

free-sliding parts of a microtensile testing machine. The specimens were stressed until failure under

tension at a crosshead speed of 1 mm/min (Bisco Inc., Schaumburg, IL, U.S.A.). The microtensile

bond strength values were then converted into unit of stress (MPa).

Interfacial Nanoleakage Analysis

Eight additional teeth were used to evaluate interfacial nanoleakage expression. Disks of middle/

deep dentine were prepared, bonded, and aged similarly to the specimens submitted microtensile

bond strength test. The resin-bonding build-ups were then cut perpendicular to the bonded surface

to obtain 1 mm thick specimens. Slabs were immersed in a 50% aqueous ammoniacal silver nitrate

(AgNO3) solution in the presence of laboratory light for 24 h. Specimens were then photodeveloped

for 8 h to reduce silver ions into metallic silver grains within voids along the bonded interface and

then polished using wet 600, 1200, and 2000-grit silicon carbide papers.16 Resin-dentine interfaces

were observed under optical light microscope (Leica DMR; Leica, Wetzlar, Germany) and scanning

electron microscopy operating in backscattered scanning detection mode at 1 kV. The amount of

silver nitrate deposit was evaluated and scored by two investigators using the method of Saboia et

al.17 Interfacial nanoleakage was scored based on the percentage of the adhesive surface showing

AgNO3 deposition: 0, no nanoleakage; 1, nanoleakage on <25% of adhesive surface; 2,

nanoleakage on 25−50% of the adhesive surface; 3, nanoleakage on 50−75% of the adhesive

surface; and 4, nanoleakage on >75% of the adhesive surface. Intraexaminer reliability was assessed

using the kappa (κ) test.

Statistical Analysis

Microtensile bond strength test data were evaluated using commercial software (SPSS, version 20.0

for Windows; SPSS, Chicago, IL, U.S.A.). Values were normally distributed and data were

analyzed with one-way analysis of variance (ANOVA) and Tukey’s post hoc multiple comparison

tests. A p-value <0.05 was considered statistically significant. Scores of the interfacial nanoleakage

! ! !

45

analysis were statistically analyzed using nonparametric Kruskal−Wallis test for multiple

comparisons and Mann−Whitney U-test (p-value <0.05 was considered statistically significant).

Confocal Laser Scanning Microscopy (CLSM)

Fluorescein- labeled Chit-MA18

was used for visualization of the polysaccharide on the resin and in

the restored tooth by means of CLSM, respectively. In the second case, human molars were

prepared as in the interfacial nanoleakage analysis, avoiding immersion in ammonia silver nitrate

solution. The specimens were placed over a coverslip and mounted on the stage of an inverted microscope LEICA TCS SP2 associated with a confocal argon-ion laser-scanning microscope.

Confocal data have been processed by means of Image Pro Plus 6.2 software extended with the 3D

Constructor package. Laser excitation light was provided at 488 nm and fluorescent emissions were

collected in the wavelength range between 510 and 580 nm. For image acquisition, an exposure

time of 0.8 s was adopted with a binning of 2 × 2 on the charge- coupled device camera, yielding a

pixel size of 1.46 µm. The lens magnification was 60×.

4.3 Results and Discussion

Derivatisation of Chitosan with Methacrylic Acid and Characterization

Methacrylate moieties have been anchored onto the chitosan chain by exploiting the carbodiimide

chemistry. The presence of side-chain moieties was verified and quantified by means of ATR-IR

and 1H NMR. Initially, the derivatization was carried out on the sample with MW = 540000. The

degree of modification of the polymer chain was measured by means of 1H NMR (Figure 1a)

comparing the areas of the signals arising from the vinyl protons of the inserted moieties (at around

5.7 and 5.5 ppm, respectively) with the signal arising from the anomeric protons of the

polysaccharide chain (from 4.5 to 5.1 ppm).

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46

Figure 1. (a) 1H NMR of Chit-MA. (b) Dependence of the degree of substitution of the chitosan with

methacrylate units from the molar ratio between methacrylic acid and chitosan repeating units. Lines are

drawn to guide the eye. In both cases, chitosan with MW ∼ 540000 was used.

The degree of substitution thus obtained is in good agreement with the one calculated comparing

the signal of the methyl groups of the residual acetyl moieties on chitosan (at approximately 2.1

ppm) to the signal of the methyl group of the methacrylate moiety on the polymer chain (at

approximately 1.9 ppm), which are partially superimposed (Figure 1a). The degree of substitution

(ds), that is, the amount of methacrylate moieties, was found to depend on the [MA]/[Chitosan]ru

ratio (Figure 1b). In particular, with a [MA]/[Chitosan]ru ratio up to 2, the percentage of bonded

methacrylate groups increases and reaches the highest value of 23%. Further increase of the amount

of methacrylic acid used for the modification produces a decrease of the degree of substitution,

together with visual inhomogeneity and partial precipitation of the chitosan sample excessively

modified with hydrophobic moieties on amino groups.19

The intrinsic viscosity of the methacrylate-

modified chitosan was measured and compared to the one of the unmodified sample (Figure 2a).

The insertion of the methacrylate groups increases the rigidity of the polysaccharide chain, as

revealed by the dependence of the intrinsic viscosity of the modified polymer from the ratio

[MA]/[Chitosan]ru (Figure 2a). The increase in [η] upon increasing the degree of substitution up to

! ! !

47

19% indicates stiffening of the polymer chain, in line with the results reported for the rigidity of the

chitosan chain at different values of the residual degree of acetylation.3 It is interesting to note that,

upon exceeding the ratio [MA]/[Chitosan] of 1, a decrease in the intrinsic viscosity was detected.

This is basically due to the limited solubility of the sample, which was noted during the filtration

procedure prior to analysis. One key requirement for a primer to be used in dental restoration is its

stability to withstand the environment of the mouth with limited degradation. It is important to

underline that biological fluids contain notable amounts of lysozyme. This enzyme has been

reported to be around 1.53 µg/mL for the stimulated parotid saliva and around 6 µg/mL for the

stimulated submandibular/sublingual saliva.20

The methacrylate-modified chitosan was treated with

lysozyme and the rate on enzymatic cleavage of the polymer chain was compared to the one

recorded for the unmodified sample (Figure 2b). Although hydrolysis proceeds slowly for both

samples tested, it an be seen that the presence of the methacrylate units reduces the rate of

polysaccharide chain cleavage21,22

from 6.7 × 10−6 g

2 dL−2 min

−1 to 3.1 × 10−6

g2

dL−2

min−1 for

unmodified chitosan and Chit-MA, respectively. Hence, although the methacrylate moiety is linked

to the chitosan backbone through an amide bond, much similar to the acetyl groups, it impairs its

recognition in the enzyme cleft. It can be concluded that there is little impact of the lysozyme on the

degradation of the methacrylate-modified chitosan that can then be safely proposed as a component

of the primer for dental restoration. It should also be noted that the cleavage of chitosan by

lysozyme can be further reduced by using polysaccharide samples with lower degree of residual

acetylation.21

Figure 2. (a) Dependence of the intrinsic viscosity ([η]) of methacylate-modified chitosan samples from the

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48

molar ratio between methacrylic acid and chitosan repeating units ([MA]/[Chitosan]ru). Lines are drawn to

guide the eye. (b) Variation of the reduced specific viscosity of chitosan (■) and Chit-MA (□) with time upon

treatment with lysozyme. Lines represent the linear best-fit of the initial experimental data (R2 > 0.99 for

both cases).

Chitosan Depolymerization

The very high viscosity of chitosan with a relative molar mass, MW, of approximately 540000

might hamper its potential use in adhesive systems. One of the features required for a primer is the

possibility to flow and, once applied to the demineralized tooth, to infiltrate within the dentine

tubules. This provides mechanical anchoring to the dental restoration prolonging its duration. For

this reason, methacrylate-modified chitosan was synthesized starting from a sample with a lower

molecular weight obtained through degradation by means of NaNO2 which cleaves the

polysaccharide chain by reacting with its free amino groups leaving a 2,5-anhydro-D-mannose

reducing end.23

This process allowed obtaining a chitosan sample of approximately 70000, as

determined by means of intrinsic viscosity measurements. This sample has been indicated as Chit-

MA70. The reduction of the molecular weight has a notable influence on the rheological properties

of the polysaccharide, as the zero-shear viscosity decreases from approximately 3.8 Pa· s for the

chitosan sample with MW of 540000 to 0.004 Pa·s for the sample at 70000, both measured at a total

polymer concentration of 15 g/L (data not shown). The reduction in zero-shear viscosity did not

affect the degree of substitution with methacrylic acid. Indeed upon using a ratio [MA]/[Chitosan]ru

= 1, a 16% of methacrylate flanking groups were introduced. This sample, at lower molecular

weight, was indicated as Chit-MA70 and it was used to formulate an aqueous-based primer

containing HEMA as acrylic monomer.

Formulation of the Primer Containing Chit-MA70

For the use in a primer formulation, the Chit-MA70 sample should maintain a good solubility in

mixed water/ethanol environment. A complete solubility was attained for Chit-MA70 dissolved in

acetic acid at pH 5.5 up to an ethanol concentration of 60% (V/V) with a total polysaccharide

concentration of 1% (w/V; see Supporting Information, S1). In order to highlight the reactivity of

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49

the acrylic bonds on the modified chitosan in the primer, the photoreticulating agent TPO was

added to the Chit-MA70 primer and laid over the commercial Filtek Z250 resin used for dental

restoration and exposed to photoreticulation for the polymerization. The reticulated specimens was

washed extensively with water and analyzed by means of ATR-IR to detect the presence of the

modified chitosan on the surface of the restorative material (Figure 3a). The peak of the restorative

resin at 1254.2 cm−1 is shifted to 1269.8 cm−1 as a consequence of the presence of Chit-MA70 on

the surface of the material. In addition, the peaks at 1545 and 1660 cm−1, that are visible for Chit-

MA70 alone, are also detected when the modified chitosan sample is reticulated over the surface of

the restorative material, while the same peaks are completely absent on the resin itself. The

presence of Chit-MA70 was checked also by means of CLSM using a fluorescein labeled sample in

the primer (Figure 3b). As expected, there seems not to be an extensive mixing upon the two

components, that is, restorative material and primer, and a clear interface was found where the

reticulated chitosan sample is present. The interaction between Chit-MA70 and demineralized

dentine was analyzed by means of SEM. Specifically, the Chit-MA70 adhesive system was placed

over a dentine disk for 2 h and, after extensive washing of the specimen, SEM images were taken

(Figure 3c,d). From the comparison between Figure 3c and d, it can be noted that in the latter one

the SEM images show the presence of a polysaccharide layer on the surface of the dentine. In this

case, the Chit-MA70 in the primer covers and fills also some dentine tubules. Considering that in

this case there was no reticulation of the methacrylate moieties prior to the rinse and analysis, it can

be safely concluded that electrostatic interactions took place between the layer of Chit-MA70 and

the organic component of demineralized tooth, composed basically by collagen and

glycosaminoglycans.24

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50

Figure 3. (a) ATR-IR of the composite restorative resin (light blue), Chit-MA70 as such (red), and chemically bound on the restorative material (purple). (b) CLSM of the restorative composite resin with Chit-MA70 marked with fluorescein linked on the surface. (c, d) Scanning electron microscopy pictures of the surface of the tooth upon etching prior (c) to and after (d) the deposition of Chit-MA70.

Testing of the Adhesive System on Human Molar Teeth

The primer containing Chit-MA70 was used to simulate a dental restoration and it was applied

according to the Materials and Methods. In order to verify the presence of Chit-MA70 at the

interface between the restoration and the demineralized tooth, a CLSM analysis was performed

using a fluorescein-labeled sample. Fluorescein-labeled Chit-MA70 was included in the primer and

the restoration on human molar tooth was analyzed by means of CLSM (Figure 4a,b). The modified

chitosan can be detected at the interface between the dentine and the restoration material. In

addition, Chit-MA70 intrudes within the dentine tubules up to a depth of approximately 100 µm

after bonding and storage procedures. To assess the ability of the primer containing Chit-MA70 to

! ! !

51

increase the bonding to dentine, human teeth were subjected to two different aging methods,

namely, dynamic storage with a chewing simulator coupled with a thermal treatment (CS) or a

static storage in artificial saliva for the same time.25 The specimens were analyzed performing

microtensile bond strength tests (µTBS) after static (T0) and dynamic storage (Tcs). The results of

µTBS are reported in Figure 4c. After CS, the bond strength of adhesive interfaces created with the

Chit- MA70-containing primer did not show any significant decrease in the bond strength (Chit-

MA70: T0 µTBS = 26.0 ± 8.7 MPa; Tcs µTBS = 28.4 ± 8.8 MPa), while the control specimens (i.e.,

the same formulation without the addition of Chit-MA70) showed a reduction of 30% in the bond

strength with respect to the values of the static storage (control: T0 µTBS = 25.5 ± 8.7 MPa; Tcs

µTBS = 18.0 ± 6.0 MPa). Conversely, no significant difference in bond strength was found between

the Chit-MA70 containing primer and the control after storage in static conditions (26.0 ± 8.7 and

25.5 ± 8.7 MPa, respectively). These data suggest that the presence of Chit-MA70, by chemically

binding the R2 adhesive through covalent links and by physically binding the organic part of the

dentine through electrostatic interactions, increases stability of the bond and stabilizes the hybrid

layer. Figure 5 presents the extent of AgNO3 deposition (interfacial nanoleakage expression) along

the bonded interface for the restoration in the presence of Chit-MA70 containing primer and of the

control primer. Adhesive interfaces treated with Chit-MA70 containing primer showed lower

interfacial nanoleakage expression after either chewing simulation (CS) or static storage in artificial

saliva. Conversely, adhesive interfaces created with control primer demonstrated a significant

increase in interfacial nanoleakage expression (irrespective of aging). The increased interfacial

nanoleakage in etch and rinse adhesive systems is due to the degradations of collagen fibrils and the

hydrolysis of resin from interfibrillar spaces within the hybrid layer. These processes weaken the

strength and durability of resin−dentine bond.26 The analysis of interfacial nanoleakage expression

of the bonded interfaces showed an increased stability of the hybrid layer when Chit-MA70 was

included in the primer.

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52

Figure 4. CLSM image (a) and 3-D reconstruction (b) of the human tooth restoration using fluorescein-labeled Chit-MA70 in the primer. (c) Microtensile bond strength (µTBS) of human tooth restoration using the primer as such (control) and with Chit-MA70 after static (open bars) and dynamic (filled bars) aging. Measures are reported as mean ± s.d. (n = 100); *p < 0.05. (d) Distribution of nanoleakage within the different treatment groups. Different gray scale indicate the respective degree of nanoleakage (0% to >75% of the adhesive joint) observed at 100× magnification using optical microscope.

! ! !

53

Figure 5. Representative images of nanoleakage analysis for each tested group using optical microscope at 100× magnification [(A) [TCS] and (C) [T0] for Chit-MA70 containing primer; (E) [TCS] and (G) [T0] for the control primer] and backscattered scanning electron microscopy (SEM) at 2500× [(B) [TCS] and (D) [T0] for Chit-MA70 containing primer; group 2 = (F) [TCS] and (H) [T0] for the control primer].

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54

References Chapter 4

1. Bohaty, B. S.; Ye, Q.; Misra, A.; Sene, F.; Spencer, P. Clin. Cosmet. Invest. Dent. 2013, 5, 33−42.

2. Donati, I.; Stredanska, S.; Silvestrini, G.; Vetere, A.; Marcon, P.; Marsich, E.; Mozetic, P.; Gamini, A.; Paoletti, S.; Vittur, F. Biomaterials 2005, 26, 987−998.

3. Anthonsen,M.W.;Var̊um,K.M.;Smidsrød,O. Carbohydr. Polym. 1993, 22, 193−201.

5. Var̊um,K.M.;Antohonsen,M.W.;Grasdalen,H.;Smidsrød,O. Carbohydr. Res. 1991, 211, 17−23.

6. Malmo,J.;Var̊um,K.M.;Strand,S.P.Biomacromolecules2011, 12, 721−729.

7. Tømmeraas, K.; Strand, S. P.; Christensen, B. E.; Smidsrød, O.; Var̊um,K.M. Carbohydr. Polym. 2011,83, 1558−1564.

8. Cadenaro, M.; Pashley, D. H.; Marchesi, G.; Carrilho, M.; Antoniolli, F.; Mazzoni, A.; Tay, F. R.; Di Lenarda, R.; Breschi, L. Dent. Mater. 2009, 25, 1269−1274.

9. Cadenaro, M.; Breschi, L.; Rueggeberg, F. A.; Suchko, M.; Grodin, E.; Agee, K.; Di Lenarda, R.; Tay, F. R.; Pashley, D. H. Dent. Mater. 2009, 25, 621−628.

10. Perdigaõ,J.Dent.Mater.2010,26,24−37.

11. Breschi, L.; Cammelli, F.; Visintini, E.; Mazzoni, A.; Vita, F.; Carrilho, M.; Cadenaro, M.; Foulger, S.; Mazzoti, G.; Tay, F. R.; Di Lenarda, R.; Pashley, D. J. Adhes. Dent. 2009, 11,191−198.

12. Lutz, F.; Krejci, I.; Barbakow, F. J. Dent. Res. 1992, 71, 1525− 1529.

13. Steiner, M.; Mitsias, M. E.; Ludwig, K.; Kern, M. Dent. Mater. 2009, 25, 494−499.

14. Armstrong, S.; Geraldeli, S.; Maia, R.; Raposo, L. H.; Soares, C. J.; Yamagawa, J. Dent. Mater. 2010, 26, e50−e62.

15. Pashley, D. H.; Tay, F. R.; Yiu, C.; Hashimoto, M.; Breschi, L.; Carvalho, R. M.; Ito, S. J. Dent. Res. 2004, 83, 216−221.

16. Tay, F. R.; Hashimoto, M.; Pashley, D. H.; Peters, M. C.; Lai, S. C.; Yiu, C. K.; Cheong, C. J. Dent. Res. 2003, 82, 537−541.

17. Saboia, V. P. A.; Silva, F. C. F. A.; Nato, F.; Mazzoni, A.; Cadenaro, M.; Mazzotti, G.; Giannini, M.; Breschi, L. Eur. J. Oral Sci. 2009, 117, 618−624.

18. Donati, I.; Feresini, M.; Travan, A.; Marsich, E.; Lapasin, R.; Paoletti, S. Biomacromolecules 2011, 12, 4044−4056.

19. Einbu,A.;Var̊um,K.M.Structure-PropertyRelationshipsin Chitosan. Tomasik, P., Ed.; CRC Press: New York, 2004; pp 217−229.

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20. Yeh, C. K.; Dodds, M. W. J.; Zuo, P.; Johnson, D. A. Arch. Oral Biol. 1997, 42, 25−31

21. Nordtveit,R.J.;Var̊um,K.M.;Smidsrød,O.Carbohydr.Polym. 1994, 23, 253−26

22. Var̊um,K.M.;Myhr,M.M.;Hierde,R.J.N.;Smidsrød,O. Carbohydr. Res. 1997, 299, 99−101

23. Tømmeraas,K.;Var̊um,K.M.;Christensen,B.E.;Smidsrød,O. Carbohydr. Res. 2001, 333, 137−144

24. Breschi, L.; Gobbi, P.; Lopes, M.; Prati, C.; Falconi, M.; Teti, G.; Mazzotti, G. J. Biomed. Mater. Res., Part A 2003, 67, 11−17

25. De Munck, J.; Van Landuyt, K.; Peumans, M.; Poitevin, A.; Lambrechts, P.; Braem, M.; Van Meerbeek, B. J. Dent. Res. 2005, 84, 118−132

26. Malacarne, J.; Carvalho, R. M.; de Goes, M. F.; Svizero, N.; Pashley, D. H.; Tay, F. R.; Yiu, C. K.; Carrilho, M. R. Dent. Mater. 2006, 22, 973−980

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Chapter 5

Cytotoxicity tests on Chit-MA70

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5.1 Introduction

Methacrylic monomers such as bisphenol-A-glycidyl methacrylate (BisGMA) and urethane-

dimethacrylate (UDMA) are widely used in dentistry. In fact, materials used for the restoration of

compromised teeth due to caries, secondary caries, fractures, etc., contain good percentages of

methacrylic monomers. Unfortunately, when not fully cured these monomers may result in a high

toxicity on oral tissues, especially on gingival fibroblasts and pulp cells.

Numerous studies have evaluated the toxicity of methacrylic monomers. These studies compared

the toxic effect of BisGMA, TEGMA, UDMA and HEMA1 both as pure substance and as binary

mixtures.2

In this last part of the research several cytotoxicity tests were performed in order to evaluate the

biocompatibility and toxicity of the new adhesive system Chit-MA70. Tests were performed in

order to have a complete assessment from a biological standpoint. Tests were performed using the

primer of the adhesive system Chit-MA70 and the primer of an adhesive system already present on

the market. For the cytotoxicity tests cultures of gingival fibroblasts were used.

Initially, the primers were diluted and placed in culture to assess the maximum toxicity of the

adhesive system. Subsequently, samples were prepared using a method similar to that used for the

nanoleakage analysis, and the slabs were then placed in the culture of gingival fibroblasts.

5.2 Materials and Methods

Culture of Human Gingival Fibroblasts

Human gingival fibroblasts (HGFs) were obtained from fragments of healthy marginal gingival

tissue taken from the retromolar area withdrawn during surgical extraction of impacted third molars

in adult subjects following regularization of the surgical flap before sutures. The tissue fragments

were immediately placed in Dulbecco’s modified Eagle’s medium (DMEM, EuroCloneSpA Life-

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Sciences-Division, Milano, Italy) for at least 1 h, rinsed three times in phosphate-buffered saline

solution (PBS, EuroCloneSpA), minced into small tissue pieces, and cultured in DMEM, containing

10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, and 1% fungizone (Sigma-Aldrich,

Milan, Italy)3. Cells were maintained at 37uC in a humidified atmosphere of 5% (v/v) CO2. After

one week, the fungizone was removed from the culture medium. Cells were used after 4–8

passages.

Cytotoxicity test of Chit-MA70 on gingival fibroblast culture

Gingival fibroblasts were used in DMEM/F12 soil, sown in 96-well plates (3000 cells /cockpit).

Each sample was tested in six replicates. The polymer (Chit-MA) was dissolved in water and

diluted to 2% in soil. As a reference for the cytotoxicity, a primer of a commercial adhesive (Adper

Scotchbond Multipurpose, 3M ESPE) at the same concentrations of the experimental primer was

used. The commercial primer was dissolved in water and diluted to 1% v/v in cell’s culture medium

(maximum dilution that allows buffering the medium to physiological values. At higher

concentrations, the solubility was very poor and the pH was very low). For concentrations of 1 %

and 0.66 %, the mixture + ground polymer was added to the serum, NaCl and HEPES to balance

the ionic strength and pH. As a control reference, cells in complete medium and cells in medium +

water with added salt, serum and buffer reference checks of the two higher concentrations were

used. The test was carried out after 24 and 72 hours after treatment using the kit Promega CellTiter

96 ® Aqueous One Solution Cell Proliferation Assay (MTS).

The primer of Chit-MA70 and commercial primer were tested in different concentrations: 1%,

0.7%, 0.07%, and 0.01%.

Cytotoxicity assay on Chit-MA70 simulating a dental restoration

Other cytotoxicity tests were performed simulating an adhesive dental restoration. To perform this

cytotoxicity assay the occlusal third of the molar crowns and the roots were removed by means of a

water-cooled slow speed diamond blade (Isomet 5000, Buehler Ltd., Lake Bluff, IL, U.S.A.) to

obtain a dentinal section 4 mm-high.

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The exposed dentine surfaces were polished with 180-grit wet silicon carbide abrasive paper to

create a standardized smear-layer4 and the adhesive system Chit-MA70 was applied. Bonded

specimens were cut using a low-speed diamond saw under water irrigation into 1-mm-thick slabs to

expose the bonding interface area.

The slabs obtained were placed on human gingival fibroblasts culture medium.

5.3 Results and Discussion

Tests were performed on gingival fibroblasts cultures at 24 and 72 hours and demonstrated that the

addition of methacrylate-modified chitosan into the primer did not affect cell viability.

The cytotoxicity test on Chit-MA70 demonstrated that there were no significant differences for any

concentration of the methacrylate-modified chitosan if compared to the control. On the contrary, the

cytotoxicity test performed on the commercial primer showed a reduction of viability of gingival

fibroblasts if compared both to the control and the methacrylate-modified chitosan.

The results of the cytotoxicity test of the chitosan-methacrylate polymer and the commercial primer

are shown in figure below:

Comparison of the cytotoxicity of Chit-MA and a commercial primer in gingival fibroblast culture after 24h and 72h.

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The cytotoxicity tests performed by simulating a dental restoration showed an increased mortality

of gingival fibroblasts after 24 hours. Unfortunately in this case it was not possible to assess

whether the cell viability depended on the methacrylic groups content of the experimental primer or

on the methacrylate groups present in the R2 resin and in the composite. The tests were repeated

using the control adhesive system (without methacrylate-modified chitosan) and with the

commercial adhesive system. The results obtained were the same as those observed for the adhesive

system Chit-MA70. The cytotoxic effects on human gingival fibroblasts caused by biomaterials

commonly used in restorative dentistry, have been the subject of many studies.2

The cytotoxicity effects obtained with the primer of the adhesive system Chit-MA70, when

positioned in a human fibroblasts culture, allow speculating that the presence of methacrylate-

modified chitosan determines a lower citotoxicity compared to the commercial adhesive system

tested in the study.

We can speculate that the high cell mortality observed when simulating a dental restoration may

have been determined in part by the unpolymerized monomers of the adhesive system Chit-MA70

and in part by the high percentage of uncured monomers of composite resins and the presence of

high percentage of HEMA. 5,6,7 Further studies are needed to verify the concentration of residual

uncured monomers in the experimental adhesive.

Despite further tests are needed to assess in vitro and in vivo the full effectiveness of the new

adhesive system, the results obtained in this research demonstrated that the methacrylate-modified

chitosan did not reduced the bond strength values to the dental tissue. It also has a good

biocompatibility and cytotoxicity to human cells comparable to that of commercial adhesives.

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References Chapter 5

1. Barbosa MO1, de Carvalho RV, Demarco FF, Ogliari FA, Zanchi CH, Piva E, da Silva AF. J

Mater Sci Mater Med. 2015 Jan;26(1):5370. doi: 10.1007/s10856-014-5370-6. Epub 2015 Jan 15

2. Durner J1, Wellner P, Hickel R, Reichl FX. Dent Mater. 2012 Aug; 28 (8): 818-23. Doi: 10.1016

/ j.dental. 2012.04.031. Epub 2012 May 10

3. Sancilio S1, di Giacomo V1, Di Giulio M1, Gallorini M1, Marsich E2, Travan A3, Tarusha L3,

Cellini L1, Cataldi A1. PLoS One. 2014 May 7;9(5):e96520. doi: 10.1371/journal.pone.0096520.

eCollection 2014

4. Breschi, L.; Cammelli, F.; Visintini, E.; Mazzoni, A.; Vita, F.; Carrilho, M.; Cadenaro, M.;

Foulger, S.; Mazzoti, G.; Tay, F. R.; Di Lenarda, R.; Pashley, D. J. Adhes. Dent. 2009,

11,191−198

5. Tadin A1, Marovic D, Galic N, Kovacic I, Zeljezic D.Acta Odontol Scand. 2014 May;72(4):304-

11. doi: 10.3109/00016357.2013.824607. Epub 2013 Aug 22

6. Furche S1, Hickel R, Reichl FX, van Landuyt K, Shehata M, Durner J. Dent Mater. 2013

Jun;29(6):618-25. doi: 10.1016/j.dental.2013.03.009. Epub 2013 Apr 6

7. Falconi M, Teti G, Zago M, Pelotti S, Breschi L, Mazzotti G. Cell Biol Toxicol. 2007;23:313–22

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Chapter 6

Summary, conclusions and future perspectives

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6.1 Summary and Conclusions

Dental caries is still a major public health problem in most high-income countries as the disease

affects 60-90% of school-aged children and it affects nearly 100% of the adult population in the

majority of countries1. The placement of effective long-lasting restorations is crucial to reduce the

long-term cost of dental treatments. Longevity of dental restoration is function of many different

factors, including material, patient and in particular dentist who performs the restoration2. The

materials used for direct and indirect restorations are adhesive systems and resin-based composites.

In 2010 in Italy € 18 million were spent for restorative materials.1

The stability of the bonded interface over time is strictly related to the creation of a stable and

homogeneous hybrid layer, where there is an effective coupling of the monomers within the

infiltrated substrate3. An efficient adhesive system must establish interactions with both the

hydrophobic layer (the resins of the restoration material) and the hydrophilic counterpart

(demineralized dentine). Nowadays, the durability of the adhesive system represents the major

limitation regarding resin-based dental restorations where the hybrid layer between dentin

(hydrophilic component) and the restorative resin (hydrophobic component) is the weakest point.

The average duration of composite resin restorations is limited to about 6 years.5 Hence, innovative

strategies to overcome this limitation are continuously sought. Several studies have related bond

failure over time to the degradation of resin polymers, initiated with the elution of unreacted

monomers and leading to water sorption and polymer swelling. 3

Chit-MA70 shows both hydrophilic and hydrophobic features, coupled with the possibility of

chemically or physically interacting with both the restorative material and the organic part of the

demineralized tooth, which makes it a good candidate to enhance the sealing potential and extend

dental restoration durability.4 The aim of this project was the development of an innovative nano-

engineered biocompatible dental adhesive system capable to form covalent chemical bonds with

both the dentin collagen and the resin-based restorative materials. In this research, chitosan was

included in the composition of an adhesive system. The use of chitosan as a component of the

adhesive system is not new. Elsaka et al.6 previously evaluated the use of chitosan blended within

the adhesive systems due to its antibacterial properties. However, these authors reported a

significantly decrease of tensile bonding strength at the dentine interface upon increasing the

concentration of chitosan. We speculated that the modified chitosan was able to covalently bind to

the restorative resin and, due to the presence of residual positive charges on the polysaccharide, to

electrostatically interact with the demineralized dentin. The chitosan with methacrylate groups was

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64

blended within the primer of a three-step etch-and-rinse experimental dentine bonding system to

assay its bonding ability to dentin. The modified chitosan was able to covalently bind to the

restorative resin and, given the presence of residual positive charges on the polysaccharide, to

electrostatically interact with the demineralized dentin. The three-steps etch-and-rinse experimental

dental adhesive with modified chitosan showed a good infiltration within the dentin tubules of

human molar teeth. Chitosan was introduced into the primer of the adhesive system because it has

hydrophilic properties, similar to hydrophilic monomers content of commercial primers, such as 2-

hydroxyethyl methacrylate (HEMA). Chitosan and HEMA are responsible for improving the

wettability and promoting the re-expansion of the network of collagen fibers collapsed after the

etching step. The present work demonstrated that the presence of the methacrylate-modified

chitosan in the adhesive system allowed obtaining a dental restoration that did not show a decrease

in the bond strength and reduced the interfacial nanoleakage after aging with a chewing simulator

coupled with a thermal-cycler. The effect of chewing simulation associated with thermo-cycling on

resin–dentin interfaces has not been well described yet. In this study the effect of this storage

method was investigated to determine if such stresses affect the bond strength and the nanoleakage

analysis. Indeed, the aging of the dental restoration with a chewing simulator combined with the

thermal treatment (CS group) showed no variation of the bond strength compared to the control

dental restoration when the methacrylate-modified chitosan was introduced in the adhesive system.

Although additional work on the performance of the modified chitosan is needed (currently

ongoing), it might be an interesting component to be added to water-based adhesive systems to

increase the durability of dental restoration. In addition, the use of chitosan specimens with

different degrees of substitution with methacrylate moieties and with the presence of spacers

between the polysaccharide and the reactive methacrylate group is foreseen for forthcoming work.

Furthermore, other cytotoxicity analyses will be conducted and zymography analysis will be

performed to evaluate the effects of chitosan on endogenous dentin.

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References Chapter 6

1. Petersen, P. E.; Baez, E.; Kwan, S.; Ogawa, H. Report of the meeting convened at WHO; Data,

W. L., Ed.; WHO: Geneva, Switzerland, 2009

2. Manhart, J.; Chen, H.; Hamm, G.; Hickel, R. Oper. Dent. 2004, 29 (5), 481-508

3. Breschi, L.; Mazzoni, A.; Ruggeri, A.; Cadenaro, M.; Di Lenarda, R.; De Stefano Dorigo, E.

Dent. Mater. 2008, 24, 90−101

4. Sano, H.; Yoshikawa, T.; Pereira, P. N. R.; Kanemura, N.; Morigamui, M.; Tagami, J.; Pashley, D. H. J. Dent. Res. 1999, 78, 906− 911

5. Bohaty, B. S.; Ye, Q.; Misra, A.; Sene, F.; Spencer, P. Clin. Cosmet. Invest. Dent. 2013, 5,

33−42

6. Elsaka, S. E. Dent. Dig. 2012, 2012/06/07, 603−613

7. Toledano M, Cabello I, Aguilera FS, Osorio E, Osorio R. J Biomech. 2015 Jan 2;48(1):14-21.

doi: 10.1016/j.jbiomech.2014.11.014. Epub 2014 Nov 20

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Curriculum vitae

Date of birth :June 6th, 1986 Place of birth: Catania (CT), Italy

Civil status: Unmarried

Citizenship: Italian

Address: Clinica Odontoiatrica e Stomatologica, Dipartimento di Scienze Mediche Chirurgiche e

della Salute, Università degli Studi di Trieste, Piazza Ospedale 1, I-34129 Trieste, Italia.

E-mail: marina.diolosà@phd.units.it

2011: Degree in Dentistry, University of Trieste, Trieste, Italy

Research activity

2012-to date: PhD Program in School of Nanotechnology

Membership in Dental Societies

2010-to date: member of ADM (Academy Dental Materials) 2010-to date: member of IADR

(International Association of Dental Research)

Publications

• Diolosà M, Donati I, Turco G, Cadenaro M, Di Lenarda R, Breschi L, Paoletti S. Use of

methacrylate-modified chitosan to increase the durability of dentin bonding systems.

Biomacromolecules. 2014 Oct 27. [Epub ahead of print]

• Marchesi G, Frassetto A, Mazzoni A, Apolonio F, Diolosà M, Cadenaro M, Breschi M.

Adhesive performances of a multi-mode adhesive system: 1-year in vitro study.J Dent. 2014

May;42(5):603-12. doi: 10.1016/j.jdent.2013.12.008. Epub 2013 Dec 25.PMID: 24373855

[PubMed - in process]

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67

• Navarra CO, Reda B, Diolosà M, Casula I, Di Lenarda R, Breschi L, Cadenaro M. The

effects of two 10% carbamide peroxide nightguard bleaching agents, with and without a

desensitizer, on enamel and sensitivity: an in vivo study. Int J Dent Hyg. 2013 Oct 9. doi:

10.1111/idh.12054. [Epub ahead of print]

• Marchesi M, Frassetto A, Visintini E, Diolosà M, Turco G, Salgarello S, Cadenaro M,

Breschi L. Influence of ageing on self-etch adhesives: one step vs. two-step systems. Eur J

Oral Sci. 2013 Feb;121(1):43-9. doi: 10.1111/eos.12009. Epub 2012 Dec 21.

• Navarra CO, Breschi L, Turco G, Diolosà M, Fontanive L, Manzoli L, Di Lenarda R,

Cadenaro M. Degree of conversion of two-step etch-and-rinse adhesives: In situ micro-

Raman analysis. J Dent. 2012 Sep;40(9):711-7. Epub 2012 May 11.

Publications/abstracts in conferences/congresses

• Microshear bond strength test of four commercial adhesives. Diolosà M, Marchesi G, Cadenaro M,

Di Lenarda R, Breschi L. XIX National Meeting of the College of Dentistry Teachers “L’high tech

come supporto alla ricerca, alla didattica ed alla clinica in odontostomatologia”. Turin, April 12-14,

2012

• In situ interfacial MicroRaman analysis of three commercial of two-step etch-and-rinse adhesives.

Navarra CO, Diolosà M, Di Lenarda R, Breschi L, Cadenaro M. XIX National Meeting of the

College of Dentistry Teachers “L’high tech come supporto alla ricerca, alla didattica ed alla clinica

in odontostomatologia”. Turin, April 12-14, 2012

• Microtensile bond strenght test of one-step adhesive on dentin. Frassetto A, Diolosà M, Di Lenarda R, Cadenaro M, Breschi L. Dental Materials Vol. 28 Supplement 1, Page e8. Lake Buena Vista, FL, USA, September 19-22, 2012

• Valutazione della forza di legame di un nuovo sistema adesivo sperimentale. Diolosà M, Donati I, Breschi L, Paoletti S, Di Lenarda R, Cadenaro M. VI Expo di Autunno “Le terapie mini-invasive in odontoiatria, risparmio biologico ed economico”. Milan, November 30 – December 1, 2012

• Bonding effectiveness of chitosan-containing experimental adhesive. Diolosà M, Donati I,

Turco G, Frassetto A, Breschi L, Di Lenarda R, Paoletti S, Cadenaro M. IADR Meeting. Seattle, March 19-23, 2013

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68

• Bonding effectiveness of a chitosan-containing experimental adhesive. Diolosà M, Donati I, Turco G,

Paoletti S, Breschi L, Cadenaro M. XIX National Meeting of the College of Dentistry Teachers.“Evidenza scientifica Interdisciplinarietà e Tecnologie applicate”. Rome, April 18-20, 2013

• Bonding effectiveness of a chitosan-containing experimental adhesive. Diolosà M, Donati I,

Paoletti S, Di Lenarda R, Breschi L, Cadenaro M. IADR-CED Conference. Florence, September 4-7, 2013

• Bonding effectiveness of a new universal simplified adhesive on dentin. Frassetto A,

Diolosà M, Di Lenarda R, Cadenaro M, Breschi L. IADR-CED Conference. 2013 Florence, September 4-7, 2013

• Stability of the bond created by a chitosan-containing experimental adhesive. Diolosà M,

Donati I, Paoletti S, Breschi L, Cadenaro M. ADM Conference. Vancouver, October 9-12, 2013

• Bonding effectiveness of four self-etch adhesive systems. Diolosà M Frassetto A, Turco G, Di

Lenarda R, Cadenaro M, Breschi L. . XX National Meeting of the College of Dentistry Teachers.“Odontoiatria traslazionale: dalla ricerca di base alla clinica”. Rome, April 10-12, 2014

• Evaluation of cytotoxicity of chitosan methacrylate polymer introduced in a bio-dental adhesive system. M. Diolosà, E.Marsich, I. Donati, G.Turco, S. Paoletti, R. Di Lenarda, L. Breschi, M. Cadenaro. ADM Conference. Bologna, October 8-11, 2014.