introduction · 1.2 the concept of adhesion 1.3 advantages of adhesive dentistry 1.4 enamel and...
TRANSCRIPT
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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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43. Perdigão J. Dent Mat 26 (2010) e24–e37
44. Pashley DH. Dentin: Scanning Microsc 1989;3:161-174
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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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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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27
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
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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
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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
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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
! ! !
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
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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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61
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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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.