Impact of Different Acid Etching Time on Microtensile Bond Streng

7/22/2019 Impact of Different Acid Etching Time on Microtensile Bond Streng

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University of Iowa

Iowa Research Online

Teses and Dissertations

2009

Impact of dierent acid etching time onmicrotensile bond strength to vital dentin

Aadarsh GopalakrishnaUniversity of Iowa

Copyright 2009 Aadarsh Gopalakrishna

Tis dissertation is available at Iowa Research Online: hp://ir.uiowa.edu/etd/291

Follow this and additional works at: hp://ir.uiowa.edu/etd

Part of the Other Dentistry Commons

Recommended CitationGopalakrishna, Aadarsh. "Impact of dierent acid etching time on microtensile bond strength to vital dentin." Master's thesis,University of Iowa, 2009.

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IMPACT OF DIFFERENT ACID ETCHING TIMES ON

MICROTENSILE BOND STRENGTH TO VITAL DENTIN

by

Aadarsh Gopalakrishna

A thesis submitted in partial fulfillmentof the requirements for the

Master of Science degree in Operative Dentistry

in the Graduate College ofThe University of Iowa

July 2009

Thesis Supervisor: Assistant Professor Saulo Geraldeli


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Graduate College

The University of IowaIowa City, Iowa

CERTIFICATE OF APPROVAL

________________________

MASTERS THESIS

_______________

This is to certify that the Masters thesis of

Aadarsh Gopalakrishna

has been approved by the Examining Committee

for the thesis requirement for the Master of Science

degree in Operative Dentistry at the July 2009 graduation.

Thesis Committee: ____________________________________Saulo Geraldeli, Thesis Supervisor

____________________________________Steve Armstrong

____________________________________Deborah Cobb

____________________________________Fang Qian


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To my parents, for their motivation, constant support and love.To my wife, who is always there for me.

To all my family and friends, who are my well wishers.

To my mentors, for their willingness to teach.


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ACKNOWLEDGMENTS

I would like to express my gratitude to Dr. Saulo Geraldeli, my thesis advisor,

who has constantly supported me throughout this thesis project. I greatly appreciate his

passion in teaching and encouragement. His continued support helped me finish this

project on time.

I would like to acknowledge Dr. Steve Armstrong for being a part of my research

committee member and sharing his research experience in my thesis project. I would like

to thank him for letting me use his biomaterial lab for this thesis. I would also like to

thank him for all the extended support in literature development.

It is my great honor to have Dr. Deborah Cobb as my graduate program director

and my committee member. I thank her for her encouragement during my program and

my thesis project. She made me feel home away from home.

I would like to acknowledge Dr. Fang Qian for her support for analyzing the

statistics of this thesis and her valuable suggestions.

I would like to thank Dr. Ricardo Atui from Guarulhos University, Brazil, for all

his clinical support in this project with patient selection and placement of the restorations

for the in vivoaspect of this research.

I would like to thank John Laffon for his help with the Scanning Electron

Microscopy procedures.

I would like to acknowledge and thank Dr. Gerald Denehy for all his support and

help to make me a better clinician and for his motivation to teach.

I would also like to thank all the friends and faculty members of the Operative

Dentistry department.


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TABLE OF CONTENTS

LIST OF TABLES -------------------------------------------------------------------------------- vi

LIST OF FIGURE -------------------------------------------------------------------------------- vii

CHAPTER

1. INTRODUCTION----------------------------------------------------------------------- 1

2. LITERATURE REVIEW-------------------------------------------------------------- 4

2.1 Statement of problem----------------------------------------------------------4

2.2 Background of tooth structures and adhesion------------------------------42.2.1 Enamel--------------------------------------------------------------------5

2.2.2 Dentin- pulp complex-------------------------------------------------- 5

2.2.3 Resin based composite (RBC)-----------------------------------------6

2.2.4 Adhesion/Bonding------------------------------------------------------72.3 Definitions and characteristics----------------------------------------------10

2.3.1 Acid etch and acids used in dentistry-------------------------------10

2.3.2 Acid etching and its effects on enamel-----------------------------102.3.3 Acid etching and its effects on dentin-pulp complex------------ 11

2.3.4 Smear layer-------------------------------------------------------------13

2.3.5 Effect of acid on dentin-----------------------------------------------132.3.7 Effect of etching time on enamel------------------------------------14

2.3.6 Effect of etching time on dentin-------------------------------------14

2.3.7 Microtensile bond strength as a method for evaluation

of resin- dentin interface----------------------------------------------152.3.8 Scanning electron microscopy---------------------------------------15

2.4 Studies supporting extended etching times on dentin-------------------15

3. MATERIALS AND METHODS-----------------------------------------------------22

3.1 Overview----------------------------------------------------------------------22

3.2 Research question------------------------------------------------------------22

3.3 Hypotheses------------------------------------------------------------------- 22

3.4 Outcome of interest--------------------------------------------------------- 233.5 Operational definitions------------------------------------------------------ 23

3.6 Variables---------------------------------------------------------------------- 233.7 IRB approval----------------------------------------------------------------- 23

3.8 Teeth samples---------------------------------------------------------------- 243.9 Scanning electron microscopy--------------------------------------------- 33

3.10 Statistical methods--------------------------------------------------------- 33

4 RESULTS------------------------------------------------------------------------------- 34

4.1 Microtenslie bond strength evaluation------------------------------------ 34


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4.2 Statistical analysis ---------------------------------------------------------- 43

4.2.1 Statistical results withoutthe pretest failure data----------------- 434.2.2 Statistical results withthe pretest failure data--------------------- 43

4.3 Results from Scanning electron microscopy---------------------------- 44

5 DISCUSSION---------------------------------------------------------------------- 56

6 CONCULSIONS------------------------------------------------------------------ 62

APPENDIX-------------------------------------------------------------------------------------- 63

BIBILOGRAPHY--------------------------------------------------------------------------------- 66


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LIST OF TABLES

Table 1: Materials, compositions and manufacturer------------------------------------------ 27

Table 2: Data of microtensile bond strength in MPa from for each group------------------35

Table 3: Mean microtensile bond strength by surfaces and etching times without

including the failure mode-------------------------------------------------------------- 36

Table 4: Mean microtensile bond strength by surfaces and etching times with

the pretest failure as 1 MPa-------------------------------------------------------------37

Table 5: Comparison of mean tensile bond strength withand withoutpre test failure--- 38

Table 6: Failure mode results--------------------------------------------------------------------- 46


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LIST OF FIGURES

Figure 1: Procedure of tooth preparation------------------------------------------------------- 28

Figure 2: Procedure of restoration--------------------------------------------------------------- 29

Figure 3: Tooth sectioning------------------------------------------------------------------------ 31

Figure 4: Dumbell sample on Dirks device----------------------------------------------------- 32

Figure 5: Graph showing data comparison of mean microtensile bond strength

withand withoutincluding pretest failures------------------------------------------ 41

Figure 6: Graph showing data comparison of mean microtensile bond strength---------- 42

Figure 7: Graph showing the fracture modes in 5 seconds group--------------------------- 47

Figure 8: Graph showing the fracture modes in 20 seconds group-------------------------- 48

Figure 9: Graph showing the failure modes in 80 seconds group--------------------------- 49

Figure 10: Graph showing a comparison of the fracture modes from each group-------- 50

Figure11: Cohesive failure in dentin------------------------------------------------------------ 51

Figure 12: Cohesive failure in composite resin------------------------------------------------ 52

Figure 13: Joint failures--------------------------------------------------------------------------- 51

Figure 14: Mixed failure-------------------------------------------------------------------------- 54

Figure 15: Adhesive failure in the hybrid layer------------------------------------------------ 55


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CHAPTER 1: INTRODUCTION

Currently there is an increase in demand for tooth colored restorations in

dentistry. With this growing demand for esthetic dentistry and decreasing demand for

amalgam restorations, these restorations are very popular as they resemble the natural

tooth in terms of color and translucency. Resins based composites (RBC) are the most

widely used tooth colored restorative materials. This material not only mimics tooth

structure in color, translucency and texture, but also exhibits adequate strength,

durability, good marginal adaptation and sealing and excellent biocompatibility compared

to other tooth colored materials. The evolution in both the physical and esthetic properties

of resin restorative materials has led to greater longevity and more esthetic restorations.

Another advantage of RBC is that these restorations can be bonded to the tooth

structure instead of the traditional mechanical way of obtaining retention at the cost of

removing healthy tooth structure. This bonding to tooth structure can be obtained by

dental adhesives. These adhesives, decrease the microleakage in the tooth-restoration

interface, decrease sensitivity and improve marginal sealing. Adhesives have allowed for

esthetically restoring the teeth with minimal or no tooth preparation.

RBC adheres to enamel and dentin by adhesives. However a major shortcoming

of todays adhesive restoration is their limited durability in the mouth (Van Meerbeek et

al. 1998, 1-20) as bonding to dentin has been shown to be less reliable than bonding to

enamel and is considered to be a major cause for adhesive failure. Bonding to dentin is a

less reliable technique due to the intrinsic characteristics and composition of this

substrate, especially when compared to enamel bonding.

Whenever dentin is cut for placement of restorations, considerable quantities of

debris cover the surface of the dentin forming a smear layer. This layer is advantageous

to protect the pulp-dentin complex when non adhesive restorative materials are indicated.

However, adhesive materials such as resins require a more porous enamel and dentin

before its application in order to increase material retention.


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Resin adhesives classified as etch-and-rinse adhesives require the removal of the

smear layer. To achieve that, the bur prepared dentin surface should be treated with an

inorganic acid such as ortho phosphoric acid. When dentin is etched, the acid removes the

smear layer and modifies the outer most surface of the dentin morphologically by

dissolution of mineral content, which are predominately hydroxyapatite crystals. This

leads to opening of the dentinal tubules of dentin and the exposure of non mineral layer

of dentin made of collagen fibrils and a depleted mineral content. This is desirable with

the use of etch-and-rinse adhesives as they bridge the restoration to the tooth by forming

resin tags in the micropores created by etching and they micromechanically interlock

with the exposed collagen of the dentin to form the hybrid layer, thereby providing

retention. Most of the adhesive systems presently on the market use an acidic conditioner,

generally 30 to 40% phosphoric acid, to prepare the dentin surface to receive the bonding

components. Although the interaction of the etching agents with dentin is limited by the

mineral and non-mineral phases, there is often a discrepancy between the depths of dentin

demineralization versus monomer penetration of the adhesive, which is greatly

influenced by the etching time. This remaining unprotected mineral depleted layer at the

interface permits leakage, degradation and alters the integrity of the bond that may lead to

bonding failure. It has been suggested that the degree of resin infiltration of the exposed

collagen fibrils within the demineralized dentin has a profound influence on bond

integrity. Many in vitro studies (Abu-Hanna and Gordan 2004, 105-110; Abu-Hanna,

Gordan, and Mjor 2004, 28-33; Bolanos-Carmona et al. 2006, 1121-1129;Hashimoto et

al. 2002, 99-105; Jacques and Hebling 2005, 103-109; Sardella et al. 2005, 355-362) have

been carried out to evaluate the effect of different etching times on the dentin prior to

bonding and placing restorations, the relationship between tensile bond strength in these

restorations and the effect of etching times influence on the demineralized dentin.

Based on previous studies that excessive etching creates a deep demineralized

zone, this research will evaluate the effect of different etch times on microtensile bond


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strength of teeth which were treated in vivo. All teeth used for this research were on

patients who were scheduled for orthodontic treatment and who needed those teeth to be

extracted solely for orthodontic treatment. These teeth were treated with three different

etch times, bonded and restored with RBC. After extraction, these teeth were evaluated

for their microtensile bond strength. The bond strengths were compared between groups.

Therefore, the current study evaluates the effect of etch times on tensile bond strength in

a clinical situation versus evaluating in vitro where an ideal clinical environment is hard

to mimic. In addition to comparing bond strengths, the predominant failure mode was

determined for the different etching times. The results of this study may provide

information as to the clinical importance of avoiding extended phosphoric acid

application. On the other hand, 5 seconds application might result in equal values for

bond strength which will point out that shorter times could be used. This study could be a

valuable source of information in terms of improving the clinical longevity of the

restoration and better success of treatment with tooth colored restorations.


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CHAPTER 2: LITERATURE REVIEW

2.1 Statement of problem

The durability of the bond between adhesive materials and tooth structure is most

desired in restorative dentistry. The integrity of this interface between tooth structure and

adhesive dental materials is considered as the key to the longevity of the restoration.

However there is a broad spectrum of factors affecting the adhesion of the materials to

the tooth structure. This includes; the tooth as a substrate and its intrinsic qualities, the

type of adhesive systems, the properties of the adhesive systems, types of application

procedures, pretreatment of tooth structures and much more. The focus of this research is

to understand and evaluate the effect of acid conditioning time on dentin bond strength

using microtensile bond strength as a measure and to analyze the fracture modes with

scanning electron microscopy. Many in vitro studies (Hashimoto et al. 2002, 99-105)

(Abu-Hanna, Gordan, and Mjor 2004, 28-33) have demonstrated that increase in etching

times can reduce the bond strength; however this research project focuses on comparing

the microtensile bond strength on the vital dentin when treated with different etching

times.

2.2 Background of tooth structures and adhesion

Understanding different tooth structures is important for a successful adhesive

restorative dentistry as different tooth structures as substrate behave differently to dental

adhesive bonding systems. It would be important to understand the adhesion principal as

well.


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2.2.1 Enamel

Tooth enamel is the hardest substance in the human body. It is of ectodermal

origin and chemically composed of a highly mineralized crystalline structure with 95% to

98% inorganic matter by weight and 86% inorganic matter by volume, 2% organic matter

and 12% water by volume. Enamel is formed by ameloblasts. There is formation of

organic matrix and then calcium and phosphate in the form of hydroxyapitite are

crystallized and these crystals enlarge. The majority of enamel is in the form of

Ca10(Po4)6(OH)2, although there are other minerals present as trace elements.

Structurally, enamel is composed of millions of enamel rods or prisms. They are closely

packed crystal forms of enamel made from small elongated apatite crystals arranged in a

distinctive pattern which gives strength and structural identity to enamel rods. These

enamel rods can be described as a keyhole with a circular core and are about 5m in

diameter. Enamel is homogenous in structure except for the outer surface where the

crystals are prismless and run parallel to each other and perpendicular to the surface. The

hardness and density of enamel vary on different locations of the tooth. Enamel is very

brittle structure with low tensile strength and high modulus of elasticity making it a rigid

structure but dentin below the enamel acts as a cushion and withstands the masticatory

forces (Sturdevant 1995, 18-24)

2.2.2 Dentin-pulp complex:

Dentin and pulp tissues, in spite of the differences in structure and composition,

they are related in many physiologic and pathologic reactions. They have the same

embryonic origin and are formed from the dental papilla and maintain this relationship

throughout the life of a vital tooth. The cells of the dentin-pulp complex are the


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odontoblasts. These cells are considered as part of dentin and pulp tissues as their cell

bodies are in the peripheral part of the pulp but the cytoplasmic process called the tomes

process are in the dentinal tubules of dentin. Dentin is considered as a living tissue as it

lodges the odontoblastic cell process and can react to physiologic and pathologic stimuli.

The odontoblastic process plays a primary role in the formation of dentin and continues

to slowly form dentin even after the tooth has erupted in what is termed secondary dentin

(Sturdevant 1995, 24). After eruption localized stimuli such as caries, wear process or

restorative procedures affect the formation of dentin and this type of dentin is called the

tertiary dentin. Tertiary dentin varies in structure and components and represents the

defense mechanism of the dentin-pulp complex (Mjor, Sveen, and Heyeraas 2001, 427-

446).

The composition of human dentin is 70% inorganic, 18% organic material and

12% water by weight. Dentin is less mineralized than enamel but more than cementum or

bone. The mineral content is hydroxyapatite arranged in a less systemic manner than

enamel. The hardness of dentin is less compared to enamel and even within the dentin the

hardness decreases from superficial dentin to circumpulpal dentin. The morphologic

characteristic of dentin is the dentinal tubules which extend from pulp to dentin enamel

junction (DEJ). The dentinal tubules are filled with odontoblastic process and dentinal

fluid which is a transudate of plasma (Sturdevant 1995, 28-29). The odontoblastic

processes are extensions of odontoblasts which are present the peripheral layer of the

pulp which is responsible of dentin formation. The tubules have a highly mineralized

lining along the tubular wall termed as peritubular dentin. Dentinal tubules are separated

by hydroxyapatite embedded collagen matrix called intertubular dentin. However,


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anastamosis between tubules have been described (Mjor and Nordahl 1996, 401-412).

The number and diameter of the dentinal tubules decreases towards the dentinoenamel

junction. Superficial dentin contains about 20,000/mm2 of dentinal tubules, which are

each about 0.8m in diameter and deep dentin contains about 76,000/mm2 of dentinal

tubules which are about 2.5-3m in diameter (Fosse, Saele, and Eide 1992, 201-210);

(Pashley 1996, 104-133). This translates to more dentinal tubules close to the pulp where

are greater in diameter than the superficial dentin close to the DEJ.

2.2.3 Resin based composite (RBC)

A major problem in restorative dentistry is that dental materials do not adhere

efficiently to natural tooth structure. Classic restorative materials such as amalgam do not

bond to the tooth structure and provide little or no reinforcement of the weakened tooth

structure (Swift, Perdigao, and Heymann 1995a, 95-110). Conversely, resin bonded

composites can be adhered to the tooth surface. The adhesion of RBC to tooth structure

also has been shown to increase resistance to caries (Grogono and Mayo 1994, 89-90).

Adhesion can also reduce marginal leakage of bacterial and salivary components

at the tooth/restoration interface (Asmussen 1985, 61-73). Research suggests that bonded

resin composite restorations provide substantial reinforcement (McCullock and Smith

1986, 405-409). RBC has a wide variety of use such as direct anterior and posterior

restorations, composite veneers, and pit and fissure sealants.

2.2.4 Adhesion/Bonding

Adhesion or bonding can be described as attachment or intimate contact of two

materials. RBC can be bonded to the tooth structure by dental adhesives. Adhesion of

restorative materials to the hard components of the tooth structure has been a goal


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pursued by many researchers ever since Buonocore pioneered adhesive dentistry in 1955

(Buonocore 1955, 849-853). The 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 porosities created. This was first described by Nakabayashi, in 1992

(Nakabayashi and Takarada 1992, 125-130) and commonly referred to as hybridization

or the formation of hybrid layer.

Based upon the adhesion strategy, three mechanisms of adhesion are currently in

use. (Van Meerbeek et al. 2003a, 215-235). A review by De Munck described the

different types of adhesives (De Munck et al. 2005, 118-132). First type is the etch-and-

rinse adhesives which involve separate etch and rinse phases where acid is applied and

rinsed off followed by a application of primer and application of adhesive step or a

simplified procedure where in prime and adhesive are combined in one application

preceded by etch and rinse.

Second type is the self etch adhesives which are based on the use of non-rinse

acidic monomers that simultaneously condition and prime dentin. Regarding user-

friendliness and technique-sensitivity, this approach seems clinically most promising.

This approach eliminates the rinsing phase, which not only lessens the clinical

application time, but also significantly reduces the technique-sensitivity or the risk of

making errors during application. There are two types of self-etch adhesives: mild and

strong (Van Meerbeek et al. 2003b, 215-235; Van Meerbeek et al. 2003b, 215-235).

Strong self-etch adhesives have a very low pH of 1 and exhibit a bonding mechanism

and interfacial ultra-morphology in dentin resembling that produced by etch-and-rinse


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adhesives. Mild self-etch adhesives have a pH of around 2 and dissolve the dentin

surface only partially, so that a substantial number of hydroxyapatite crystals remain

within the hybrid layer. Specific carboxyl or phosphate groups of functional monomers

can then chemically interact with this residual hydroxyapatite (Yoshida et al. 2004, 454-

458). This two-fold bonding mechanism of mild self etch i.e., micro-mechanical and

chemical bonding is believed to be advantageous in terms of restoration durability. It has

a micro-mechanical bonding component that may provide particular resistance to

debonding stress. The chemical interaction may result in bonds that better resist

hydrolytic break-down and thus keep the restoration margins sealed for a longer period.

The third type is the Glass ionomers and glass ionomer adhesives which are

considered to self-adhere to tooth tissue (Yoshida et al. 2000, 709-714). A short

polyalkenoic acid pre-treatment cleans the tooth surface; it removes the smear layer and

exposes collagen fibrils up to about 0.5-1 m deep (Inoue et al. 2001, 237-245); therein,

glass-ionomer components inter-diffuse and establish a micro-mechanical bond following

the principle of hybridization (Lin, McIntyre, and Davidson 1992, 1836-1841); Van

Meerbeek et al., 2001). In addition to this, chemical bonding is obtained by ionic

interaction of the carboxyl groups of the polyalkenoic acid with calcium ions of

hydroxyapatite that remained attached to the collagen fibrils (Yoshida et al. 2000, 709-

714). This additional chemical adhesion may be beneficial in terms of resistance to

hydrolytic degradation. Consequently, a two-fold bonding mechanism is established,

similar to that mentioned above for mild self-etch adhesives. The basic difference with

the resin based self-etch approach is that glass ionomers are self-etching through the use

of a relatively high-molecular-weight polycarboxyl-base polymer. This limits their


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infiltration capacity, so that only shallow hybrid layers are formed. In addition, because

of this high molecular weight, they cannot infiltrate phosphoric acid decalcified dentin.

Consequently, such aggressive conditioners should not be used with glass ionomers (De

Munck et al. 2004, 73-83).

2.3 Definitions/Characteristics

2.3.1 Acid etch and acids used in dentistry

Buonocore in 1955 proposed the use of acids in dentistry. He stated industries

used acids like phosphoric acids to obtain better adhesion of paints and resin coating to

metal surface, so he proposed that acids could be used to change the surface of the

enamel and render it more receptive to adhesion. (Buonocore 1955, 849-853). He started

the use of acids on enamel. Further work suggested that a tag like extension of resins into

enamel after the acid use (Gwinnett and Matsui 1967, 1615-1620). Later it was actually

known as acid etch technique (Swift, Perdigao, and Heymann 1995b, 95-110). Ever since

then different acids have been tried, they include polyacrylic acid, citric acid, nitric acid,

with phosphoric acid most commonly used.

2.3.2 Acid etching and its effects on enamel

Acidic solutions are normally used to etch enamel and dentin in commercial

dentin adhesive systems. Bonding to enamel is a reliable technique due to the

composition of enamel. The goal of enamel etching is to increase the surface free energy

for better monomer infiltration (Nakabayashi et al., 1998) and form resin tags. Peumans

described two types of resin tags. Macro tags are formed between prism periphery in a

circular manner and micro tags which is much finer network at the core of the prisms

where hydroxyapitite crystals are been removed by the effect of the acid. Micro tags are


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responsible for most of the bond strength because of their large surface area. Generally

acid etchants remove about 10m of the enamel surface and dissolve the rod core and

periphery to form microporisities from 5 to 50 m in depth. The effect of acid etching on

enamel depends on many factors like type of acid, acid concentration, the time of etching,

rinsing time etc. There are three enamel etching patterns described. Type 1 is

predominantly dissolution of prism cores, type 2 is predominantly dissolution of prism

periphery and Type 3 in which no prism structure remain evident. Enamel surface treated

with acid has a high surface energy that allows resin monomer flow by capillary

attraction before polymerization to form resin tags. Enamel consists of mainly inorganic

hydroxyapatite which has high surface energy. Thus, bonding to enamel is easier than to

other tooth tissues and has been proven to be successful and predictable (De Munck et al.

2003, 136-140). However the solubility of enamel when exposed to acid many vary from

enamel surface to DEJ or the presence of fluoride (Sturdevant 1995, 23).

2.3.3 Acid etching and its effects on dentin-pulp complex

The composition of dentin differs markedly from that of enamel. Dentin is a

dynamic and a heterogeneous substrate which makes it more difficult to bond . Most of

the commercially available dentin bonding system use acid conditioners which remove

the smear layer and partially demineralize the intertubular and peritubular dentin. Dentin

has a higher amount of organic content than enamel and when acid demineralizes dentin,

protein rich collagen is exposed. This process changes the surface free energy of dentin.

The amount of demineralization depends on many factors like application time,

concentration and ph of acid, modifiers, surfactants and thickeners in acid.

The depth of dentin also plays a role in acid demineralization. The deeper dentin

where the dentinal tubules are closer show more demineralization than the superficial

dentin as the distance between the tubules are less with less intertubular dentin. The

deeper dentin show more dentinal tubules and the diameter of the dentinal tubules

increase with lesser intertubular dentin (Sturdenant 1995, 24). The degree of


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mineralization also decreases with deeper dentin thus resulting in a more

demineralization when compared to superficial dentin when acid is applied.

Dentin is a dynamic structure which undergoes continuous physiologic and

pathologic changes. With age, dentin undergoes physiologic dentin sclerosis or reactive

sclerosis in response to mild irritation like abrasion and erosion. The result of dental

sclerosis is the formation of a precipitation of mineral crystals into the peritubular dentin

which leads to obstruction of the dentinal tubules. This process leads to less receptiveness

to adhesive treatments. With response to caries or attrition, there is formation of

hyperminerization leading to obstruction of dentinal tubules by crystalline deposits and

these dentin would respond to longer etching time for adhesive treatments and have

shown better bond strengths with extended duration of acid exposure in caries affected

dentin (Arrais et al. 2004, 458-464).

Numerous dentinal tubules are the present in the dentin which are filled with

dentinal fluid. The fuild in the dentinal tubules are under positive pressure. (Ciucchi et al.

1995, 191-194). There is no outward fluid movement from these tubules when they are

sealed with enamel and cementum but the fluid can show an outward movement when

this external seal is lost due to dental caries, tooth preparation, or tooth wear which could

interfere with adhesive procedures. This fluid due to dentin permeability caused by tooth

preparation makes dentin more challenging for bonding compared to enamel since the

fluid in these tubules can interfere with monomer infiltration of the adhesive system

(Pashley 1991, 777-781).

2.3.4 Smear Layer

The smear layer has been described as any surface debris, produced by grinding

or instrumentation of enamel, dentin, cementum or as contaminant that precludes

interaction with the underlying substrate (Ishioka and Caputo 1989, 180-185). The

thickness of the smear layer varies from 0.5- 5 m (Pashley 1992, 215-224). A smear


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plug is debris that occludes dentinal tubules. The surface smear layer and smear plugs are

porous with submicron channels but are reported to reduce dentin permeability by 86%

(Pashley 1992, 215-224). Removal of the smear layer or plug, as well as the mineral

content of intertubular dentin will increase dentin permeability.

An in vitrostudy by Spencer closely examined the smear layer, created by carbide

and diamond burs and the effect of acid etch on this smear layer (Spencer et al. 2001,

1802-1807). In this study, use of acid showed incomplete removal of the smear layer.

2.3.5 Effect of acid on dentin

Smear layer is not a stable layer and during its formation, the smear layer gets

incorporated into the underlying tissue which makes it impossible to remove by

scrubbing. On the contrary these particles are small giving them a high surface area to

mass ratio facilitating its rapid demineralization in acids (Pashley et al. 1988, 265-270).

So application of acids such as phosphoric, maleic, nitric or citric acid to dentin surface

results in removal of smear layer and demineralizing the underlying dentin (Eliades 1994,

73-81;Eliades 1994, 73-81). This acid demineralizes intertubular and peritubular dentin

and exposes the collagen along with increasing the microporosity of intertubular dentin

(Pashley 1992, 215-224; Perdigao et al. 1995, 1111-1120). Dentin is demineralized up to

7.5m depending on the type of acid used and its concentration (Chiba, Itoh, and

Wakumoto 1989, 76-85; Van Meerbeek et al. 1992, 1530-1540). The changes produced

in the mineral content of the substrate also change the surface free energy of dentin

making it more receptive to adhesive (Erickson 1992, 81-94).

2.3.6 Effect of etching time on Enamel

With the use of acids, there is microporosities created within the enamel which

promote micromechanical retention of adhesive resin material into this porosity,

however, this depends on several parameters of which etching time is one. Traditionally


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enamel was etched up to 60 seconds to produce an optimum surface for bonding but

etching times with lower etching times of 30 seconds is considered as an ideal time for

the peak quality of the etched enamel (Gardner and Hobson 2001, 64-67).

2.3.7 Effect of etching time on dentin

Acids, such as phosphoric acid, demineralize dentin, but the depth of

demineralization depends on the concentration of the acid and the duration (Chiba, Itoh,

and Wakumoto 1989, 76-85). Excessive acid conditioning for dentin pretreatment would

form a deep demineralized dentin zone within the bonded structure (Hashimoto et al.

2002, 99-105;Hashimoto et al. 2000, 406-411); (Nakabayashi, Watanabe, and Arao 1998,

379-385). There is deeper demineralization of both intertubular and peritubular dentin,

which would lead to incomplete infiltration by resin monomers.(Hashimoto et al. 2002,

99-105; Hashimoto et al. 2000, 406-411) These studies suggested that when failure was

initiated, this weaker zone created decreased bond strength. There was a direct correlation

between etching time and depth of demineralized zone. The hybrid layer thickness is

correlated directly to the etching time. Increased etching time demineralizes the dentin

surface to a depth greater than resin monomers could penetrate, producing a thick, poorly

infiltrated hybrid layer. Reducing etching time reduces the depth of the demineralized

zone and may be effective for achieving complete penetration and for sealing the dentin

surface (Abu-Hanna, Gordan, and Mjor 2004, 28-33);(Abu-Hanna and Gordan 2004,

105-110). On the contrary, the shorter etching times less than 15 seconds did not affect

the shear bond strengths of dentin as much as the longer etching times (Abu-Hanna and

Gordan 2004, 105-110).

2.3.8 Microtensile bond strength as a method for evaluation

of resin-dentin interface

Microtensile bond strength is considered as a suitable measure for the dentin

adhesive restoration joint evaluation (Pashley and Carvalho 1997, 355-372; Strang et al.

1998, 191-207; Tam and Pilliar 1993, 953-959; Eick et al. 1997, 306-335). More over


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many investigators have suggested that shear bond strength is less used as they do not

measure the adhesive properties of dentin-resin interface (Nakabayashi, Watanabe, and

Arao 1998, 379-385; Pashley and Carvalho 1997a, 355-372; Strang et al. 1998, 191-

207;Eick et al. 1997, 306-335). The dentin adhesive restoration joint is made of dissimilar

materials and when force is applied there is non-uniform distribution of stress with

adhesive showing higher strain than the adherends (Armstrong, Keller, and Boyer 2001,

201-210). This would lead to joint failure that can be measured. Microtensile bond testing

is being known to produce distinctive debonding in the joint but if the samples are tested

after a very short storage it may not produce joint failures consistently.

2.3.9 Scanning electron microscopy

Scanning electron microscopy (SEM) can demonstrate the different layers of the

dentin-adhesive-resin joint and the dentinal tubules showing resin tags formed by the

bonding systems after demineralization. SEM could evaluate the depth of dentin

demineralization and adhesive penetration into them.(Van Meerbeek et al. 1993, 1423-

1428) The specimens for SEM should be air-dried or vacuum dried prior to fixation and

examination with the SEM. Sometimes artifacts can develop generally during this process

which may provide distortion in the images (Perdigao et al. 1995, 1111-1120; Perdigao et

al. 1995, 1111-1120).

2.4 Studies supporting extended etching times on dentin

There are many reported studies comparing the effects and alterations in etch

times to microtensile bond strengths of dentin-adhesive-resin interface. Many parameters

are looked into using scanning electron microscopy, transmission electron microscopy,

optical microscopy and dye staining to better understand the interface itself.

An in vitro study done by Hashimoto et al (Hashimoto et al. 2002, 99-105);

compared the over etching effects on tensile bond strength in two dentin bonding

systems. Their aim was to determine the weakest zone of resin-dentin bonds and the

relation between bond strength and failure mode to clarify the effect of demineralized


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dentin. They used human premolars which were sectioned to expose the dentin surfaces,

and the dentin surfaces were conditioned with phosphoric acid for 15, 60, 120 or 180

seconds. Resin-dentin bonded specimens were produced using two adhesives, One-Step

and OptiBond Solo and tested for microtensile bond tests. Mean bond strengths were

statistically compared using two-way ANOVA and Duncan's multiple-range test and p

value was set to 0.05. The fractured surfaces of all specimens were examined using SEM,

and the areas of failure were measured using an image analyzer. They found that for One-

Step, the bond strength decreased with increase in acid conditioning time; 15 seconds

showed bond strengths measuring 50.7+/-9.7 MPa, 60 seconds = 40.8+/-11.0 MPa, 120s

= 23.6+/-4.9 MPa and 180 seconds = 12.1+/-4.6MPa. For OptiBond Solo, the bond

strength in the case of 15 seconds acid conditioning time = 42.6+/-7.9MPa which was

significantly greater than that for the other times of 60 seconds = 31.9+/-10.3 MPa, 120

seconds = 31.8+/-14.4 MPa and 180 seconds had 31.8+/-7.4MPa. The rationale for their

study was excess etching of dentin has been shown to reduce in vitro bond strength as

adhesive may fail to penetrate the over etched demineralized collagen network. The

purpose of the present research is similar, but evaluates over etching with vital dentin

using the micro tensile bond strength as a measure. The hypothesis for their study was

that increasing acid etching time in dentin will reduce the microtensile bond strength and

a reduction in etching time may produce more functional hybrid layers. The hypothesis in

the above study was tested in vitrowhereas the current research may provide evidence of

clinical relevance regarding the relationship of bond strength to vital dentin.

Extending etching times has shown similar results on primary dentin as well. In

contrast to the permanent teeth, primary teeth have less mineral content (Sanchez-

Quevedo et al. 2001, 827-832; Sanchez-Quevedo et al. 2001, 827-832). An in vitrostudy

(Bolanos-Carmona et al. 2006b, 1121-1129) compared the tensile bond strength of

primary teeth etched at 3 different intervals and compared with a case control study. The

results revealed that 5 seconds etching time of primary dentin with 37% orthophosphoric


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acid produced a visibly demineralized layer when specimens were stained with Massons

technique using optical microscopy. Tensile bond strength was significantly lower in

primary dentin etched for 5 s compared to 15 or 30 seconds of etching. Theyconcludedthat 15 seconds and 30 seconds etch times produced better tensile bond strength

nevertheless 5 seconds still produced some demineralization. Although their primary aim

was to evaluate etch times and bond strength, they also evaluated the demineralized zone

thickness in the dentin after etching by using a dye which could stain the demineralized

dentin and compare their thickness to etch times. They used interface morphology along

with tensile bond strength determination. The interface morphology was used to

determine the associated interdiffusion zones by scanning electron microscopy and

optical microscopy. They treated the fractured /failure areas after testing for tensile bond

strength with Massons trichromic acid staining technique which stains the mineralized

type I collagen, resulting in staining collagen green. Etching of dentin with

orthophosphoric acid removes collagen, resulting in generally red stain. Specimens were

then examined in an optical microscope for presence or absence of a red band. They used

image analysis software. In each slide, three measurements of the depth of the

demineralized dentin layer were taken and5 seconds etching time of primary dentin with

37% orthophosphoric acid produced a visibly demineralized layer when specimens were

stained but the thickness of demineralization increased with increase in etching time.

Most of the studies carried out are based on the in vitrohypothesis testing which

is specific to sound dentin. A few studies comparing etch time with caries affected dentin

were done. A study (Arrais et al. 2004, 458-464) concluded that extended etching can

improve bonding to caries affected dentin; however the adhesives applied on sclerotic

showed the best results for bonding. The authors evaluated the effects of additional and

extended acid etching on microtensile bond strength (TBS) of two adhesive systems to

sound and caries-affected dentin. Additional samples were prepared for scanning electron

microscopy observations. They found that extended etching significantly increased


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microtensile bond strength in caries affected dentin but they concluded that longevity

with these results were questionable and recommended further studies.

In relation to microtensile bond strength, (Paul et al. 1999, 181-188) tested the

microtensile bond strength on extracted teeth which was stored in dry storage, wet

storage and in a dye and the results showed no significance statistically in terms of

storage but showed a significant difference with etch time. With the increase in etch

time, the bond strength decreased. The etch times used were 15s, 30s and 60s. The aim

with the study was to evaluate the influence of etch times with microtensile bond

strength of Single Bond and to verify the leakage of silver ions within the hybrid layer.

After etch, bond and resin application, the teeth were sectioned and alternate slices were

either dried for 30 minutes in air, kept wet, or they were coated with fingernail varnish

except for 0.5 mm around the bonded area. Only the varnished samples were then

stained with 50% AgNO3. Microtensile bond strength was tested using a Vitrodyne V-

1000 universal tester. The samples of the stained group were embedded in self-curing

PMMA and polished. All samples were observed with an SEM. Nanoleakage of silver

ions was measured by exposure to laser ablation with an inductively connected plasma

mass spectrometer and by electron dispersive elemental analysis. Increasing etching

times seemed to have a negligible effect on bond strength of Single Bond, producing an

average value of ca 38 MPa. However, the silver uptake increased upon prolonged

etching times.Short-term results suggested that overetching has no detrimental effect on

bond strength values of Single Bond. Increased silver uptake, depending on the etching

time, raises concern about the long-term stability of the bond.

In another study (Spencer and Swafford 1999, 501-507), they stained the

exposed collagen in various adhesive group interfaces, found that exposed protein

stained red/orange in color using a light microscope which were indentified with all the

adhesives and were obscure with transmission electron microscopcy. Simple techniques

to evaluate the hybrid layers would lead to their improvements. Here the author


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concluded that microtensile bond strength studies with One-Step as the adhesive

invariably showed most failures in the hybrid layer with the increase in etching time of

15 to 180 seconds. Their aim was to determine acid conditioning times associated with

various dentin bonding agents, that resulted in incomplete penetration of bonding areas

leaving a weak interface and to demonstrate this interface by a staining technique. Their

rationale was that other studies done to demonstrate the weak interface in bonding

created by the acid conditioning were not being effective, so developing a non

destructive staining technique to expose the collagen at this interface. Incomplete resin

infiltration of demineralized dentin can leave exposed collagen which may be penetrated

and degraded by some exogenous substances leading to weak interface. They used third

molars with various adhesive systems and the interface junction was cut for microscopic

sectioning and stained with Goldners trichrome. The exposed protein stained red/orange

in color using a light microscope was indentified with all the adhesives and were

obscure with transmission electron microscopy. They concluded that evaluation of

adhesive penetration in the decalcified dentin should be the first step in determining the

tooth resin-composite interface and simple techniques to evaluate the hybrid layers

would lead to their improvements.

On the other hand, a in vitro study (Abu-Hanna and Gordan 2004, 105-110)

compared etch times with lower and higher etching times than the recommended 15

seconds with 3 different 2 step etch and rinse adhesive system with shear bond strength

as a measure. The main aim of their study was to evaluate the effect of etching time by

lowering it to 5 seconds and increasing it to 30 seconds from the recommended etching

time of 15 seconds and evaluate their dentin bond strengths. 108 molars where distributed

equally among the 3 bonding agent groups which used a 2 step total etch system and each

group was further divided into 3 groups based on the etching times. Acetone based One-

Step (OS), ethonal based Single Bond (SB) and water based Syntac Single Component

(SSC) were the three different bonding agents. After etching the exposed flattened dentin


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based on the etching times of the groups, restoration was done with resin based

composite (Z100) using one of the three adhesive systems based on the bonding agent

groups following the manufacturers instructions. After 300 cycles of thermocycling

between 5oC and 55oC, the teeth were tested for the shear bond strength and the fracture

mode was analyzed using SEM. The results analyzed using two way ANOVA showed no

statistical differences between the etching groups in OS and SB but the SSC groups

showed higher bond strengths with 5 seconds when compared to 15 and 30 seconds. This

results may indicate that lower etch times would create an area of demineralization

enough to facilitate complete infiltration of the adhesive monomer, which produces better

bond strengths.

A study evaluated the effect of acid concentration on dentin (Perdigao et al. 1996,

262-271). Six types of phosphoric acid etching agents were evaluated and the

independent variables were two acid concentrations of 10% and 32%-37% and three

thickener conditions. The hypothesis was that the use of different etchants with similar

concentrations of phosphoric acid would result in similar depths of dentin

demineralization. They obtained thirty dentin disks from extracted human teeth by

sectioning. The dentin surfaces were etched with one of the etching agents, fixed,

dehydrated and dried. The specimens were observed with SEM. The mean deepest

demineralization of intertubular dentin was measured from the fracture surfaces of the

disks. These values were analyzed by ANOVA and Duncans test. The morphological

appearance of the dentin surfaces was compared using the presence of a cuff of

peritubular dentin; relative thickness of the layer containing residual collagen or smear

layer particles and formation of a submicron hiatus at the bottom of the exposed collagen

network. The pH of each of the etching agents was measured. A correlation analysis was

made of the pH vs. the depth of dentin demineralization. The results indicated that silica-

thickened etchants did not demineralize dentin as deeply as the polymer-thickened

etchants and unthickened etchants. High magnifications revealed three distinct zones


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within the demineralized dentin layer: An upper porous zone of residual smear layer or

denatured collagen and residual silica particles in silica thickened etchants, an

intermediate area with randomly oriented collagen fibers, and a lower zone with a

submicron hiatus, few collagen fibers, and scattered hydroxyapatite inclusions. The

results obtained suggested that similar concentrations of phosphoric acid etchants

containing distinct thickeners result in different demineralization depths as well as

different morphology of etched dentin.


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CHAPTER 3: MATERIALS AND METHODS

3.1 Overview

The aim of this study was to evaluate the influence of the different etching times

on microtensile bond strength in vital dentin associated to a simplified two-step etch-and-

rinse adhesive. Twenty-six adults in the age range of 18-25 years and who needed

extractions of premolars for orthodontic reasons were selected in this study. The subjects

were randomly divided into three groups of 5, 20 and 80 seconds. Class I cavity

preparation were made in these teeth and restored with resin-based composite. After

extraction, the teeth were sectioned to obtain two beams from each tooth and they were

subjected to microtensile bond testing and the data was recorded. Statistical analysis was

made to evaluate any difference between the three etching groups. Once the microtensile

bond strength test was complete, the type of failure mode in each sample was analyzed

with the help of scanning electron microscope (SEM).

3.2 Research Question

Based on the previous in vitro studies (Hashimoto et al. 2002, 99-105) which

found that excessive pretreatment of dentin with phosphoric acid would create a deep

demineralized poor resin infiltrated zone decreasing dentin bond strength, and

considering the lack of in vivo systematic observations on bond strength of over-etched

dentin, the following question is posed: Does increase of acid etching on vital dentin has

an impact on microtensile bond strength?

3.3 Hypotheses

It is hypothesized that due to the vital and fresh condition of dentin in vivoafter

cavity preparation, the variation on phosphoric acid time application will translate into

similar microtensile bond strength.

The specific aims of this research are:

A. Investigate the influence of different phosphoric acid application on vital dentinmicrotensile bond strength.


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The in vivo phase was conducted at Guarulhos University (Brazil) where teeth

were restored and extracted. The human subjects office at Guarulhos University (UNG

CEPPE) reviewed and approved the project.

The second and third phases, namely mechanical testing and fracture mode

evaluations, were conducted in the University of Iowa, College of Dentistry after the

approval by Iowa Human Subjects Office. The IRB approval number for this project is

200901773.

3.8 Teeth Sample

The aim of the research was to evaluate the influence of different acid etching

times on microtensile bond strength of a simplified two-step etch-and-rinse adhesive

applied on vital dentin. All in vivoprocedures were conducted at Guarulhos University,

Sao Paul, Brazil. A total of 26 subjects in the age group of 18-25 years were selected to

participate in this study. The inclusion criteria used for subject selection were:

a. Having premolars scheduled to be extracted for orthodontic reasons.

b. No caries or hypoplasia in these premolars selected.

c. Absence of history of spontaneous pain.

d. Absence of apical pathology.

e. Willingness to participate in the study.

All premolars (at least one tooth per subject) selected in this study were diagnosed

for extraction as part of the orthodontic treatment plan proposed by the department of

Orthodontics in the same university.

After receiving the informed consent signed from the patient or parent, a standard

class I occlusal cavity preparation was performed on each tooth preceded by local

anesthesia and rubber dam placement for complete isolation. Class I cavities were

designed with an occlusal depth of 1mm below the dentin-enamel junction (DEJ). The

occlusal preparation was centered at the middle of the occlusal surface with a smooth


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even pulpal floor in the dentin using a pear shaped Brassler diamond bur. The

buccolingual dimension was between 2.5-3mm and the total depth of about 2.5-3 mm.

The premolars were randomly divided based on the acid etching times of 5 (n=8),

20 (n=9) and 80 seconds (n=9).

A 34% Ortho Phosphoric acid (CAULK 34% Tooth conditioner gel,

Dentsply/Caulk, Table 1) was used to etch the enamel and dentin. The gel was applied

first on the enamel cavosurface and then extended to the dentin pulpal floor. Once in

dentin, a dental assistant with a digital watch precisely checked the time.

The acid was water rinsed for 15 seconds and the excess of water removed with

high suction from the surrounding areas of the tooth followed by blot dry of the cavity.

In the end, dentin was considered ready to receive the adhesive if superficial moisture

was present.

A generous amount of simplified two-step etch-and-rinse adhesive (Prime Bond

NT, Dentsply, Caulk, Table 1) was applied to the cavity preparation with a disposable

microbrush. The acid etched dentin was kept fully wet by the adhesive for 20 seconds.

Excess of solvent could be removed by applying a gently dry, clean air form the syringe

for at least 5 seconds. A uniform glossy appearance indicated the removal of the solvent.

The adhesive was light cured for20 seconds.

A thin layer of microhybrid resin-based composite (Esthet-X, Dentsply, Caulk,

Table 1) shade A1 was first placed in the pulpal floor of the cavity. This increment was

then light cured for 40 seconds as per manufacturers recommendations using a light-

curing unit.

To obtain consistency and to minimize inter operator discrepancies in the

procedure; a single well-trained clinician performed all restorative procedures. Shortly

after the completion of the restoration, the tooth was extracted following conventional

techniques. Patient and/or parents were given post-operatory recommendations as per

Oral Surgery Department of the same university. Existing soft tissues were removed and


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the teeth were stored in 100% humidity wrapped in water moist gauze and stored in

refrigerator at 32 F until shipping to the Unviversity of Iowa, College of Dentistry.


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Table 1 - Materials, composition and manufacturer

MATERIAL COMPOSITION MANUFACTURER

Acid etchant

Phosphoric acid 34%

Phosphoric Acid

Highly dispensed silicon dioxide

Colorant

Water

DENTSPLY Caulksimplified two-

step etch-and-

rinse adhesive

Prime & Bond NT

Di- and Trimethacrylate resins

PENTA (dipentaerythritol penta

acrylate monophosphate)Nanofillers-Amorphous Silicon

Dioxide

Photoinitiators

Stabilizers

Cetylamine hydrofluoride

Acetone

Resin-Based

Composite

Esthet- XT M

Microhybrid resin based composite


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Sequence of images d

preparation and measure

probe.

Figure 1 - Procedure of tooth preparation

epicting initial prophylaxis, rubber dam p

ent of pulpal depth of the cavity preparation

28

acement, cavity

ith a periodontal


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Sequence of images s

preparation, application

of the resin-based compo

Figure 2 - Procedure of restoration

owing application of the phosphoric acid

f the simplified two-step etch-and-rinse adhes

site layers

29

into the cavity

ive and insertion


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Upon receiving the teeth, a well-trained operator (AG) proceeded with the

microtensile sample preparation and mechanical testing.

Specimen sample preparation for microtensile bond strength analysis

Teeth were flattened horizontally at the occlusal surface by slightly grinding the tip

of the cusps. The roots were cut flat to about 2 mm below the CEJ using a saw machine

and a diamond disc (Isomet 1000, Buehler). This was done to facilitate mounting of the

teeth on 22 inch Plexy glass support (Figure 3). The Isomet trimmer uses rotary

diamond blades at varying speed to allow sectioning of the tooth to any desired thickness

by varying the distance between the cutting discs.

Using a sticky wax, each tooth was attached to the center of the Plexy-glass square

support followed by sectioning in a saw machine. The goal of the sectioning was to

obtain two beams out of each tooth, one from the mesial and one from the distal surface.

In order to achieve this, the mesial beam was distinguished from the distal by marking it

with a blue highlight marker. Each beam was further trimmed to an dumbbell shape at the

interface of resin and dentin to about 0.8 mm diameter using the CNC specimen former

(University of Iowa, IA, USA), which is a computerized system used to obtain specimens

for microtensile bond strength (Figure 3).


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Sequence of images sho

over the Plexy-glass squ

different water resistantwax; beam attached to a

dumbbell sample obtaine

Figure 3 - Tooth sectioning

ing a grinded premolar prior to its stabilization

are support; sectioned premolar with two bea

shades; beam attached to attached to a Plexy bspecial device prior trimming of the resin-denti

d and ready to be mechanically tested

31

with a stick wax

identified with

y aid of a stickyn interface; final


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Out of the 26 tee

During mechanical testi

were considered pre-test

failures in the 5 seconds

seconds group.

Before mechani

stereomicroscope for any

caliper. Tensile testing

materials testing machin

tensile strength. Data we

Image of a dumbbell sam

microtensile bond strengt

th, 52 beams were obtained and trimmed to a

g of the samples 8 failures were observed b

failures. These failures were from the follo

roup, one failure in 20 seconds group and five

cal testing, each dumbbell was exa

preparation defects and their diameter measur

as performed at a crosshead speed of 1 mm/m

e (Zwick, GmbH & Co, Germany, Figure 4)

e collected and transferred to an appropriate sh

Figure 4 - Dumbbell sample on Dirks device

ple inserted on Dirks device and set in the testi

h testing

32

dumbbell shape.

fore testing and

ing groups: two

failures in the 80

ined under a

d using a digital

in in a calibrated

and subjected to

et.

g machine prior


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3.9 Scanning electron microscope (SEM)

After submitting the dumbbell to the testing microtensile bond strength, the

fractured specimens were mounted on round aluminum stubs using cyanoacrylate glue

(Zapit, Dental Ventures of America, CA). A gold sputter coater (SCD-040) was used to

sputter a layer of about 15m thickness on the samples. The failure mode was observed

under SEM (Amray 1820-D, Boston, MA, USA). The fracture surface of each specimen

was recorded as one of the four failure types: cohesive failure in the dentin, cohesive

failure in the resin composite, joint failure at the adhesive interface and mixed failure

involving both cohesive failure in dentin or resin composite and adhesive interface.

3.10 Statistical Methods

Descriptive statistics were computed using the tooth as a statistical unit. One-way

ANOVA with post-hoc Tukey-Kramers test was used to determine whether there was

significant difference in microtensile bond strength between the three etching times.

Possible association between failure type and group of etching times was assessed using

Fishers exact test. In addition, the Shapiro-Wilks test was conducted to test normality.

All tests employed a 0.05 level of statistical significance. SAS for Windows (v9.1, SAS

Institute Inc, Cary, NC, USA) was used for the data analysis.


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CHAPTER 4: RESULTS

A total of 52 beams were obtained from 26 teeth samples among the 3 groups.

There were 8 beams which failed before testing for the microtensile bond strength. These

beams were considered as pre test failures. There were two failures in the 5 seconds

group, one failure in 20 seconds group and five failures in the 80 seconds group. The

remaining intact 44 beams were used for testing the microtensile bond strength and

fracture mode analysis.

4.1 Microtensile bond strength evaluation

Descriptive statistics were computed from the recorded data using one-way

ANOVA with post-hoc Tukey-Kramers test to determine any significant differences in

microtensile bond strength among the three etching times.

Statistical analyses were applied to test the hypothesis in two types of data

collected. All data collected are included in Table 2. There were 8 specimen samples

which failed before the tensile loading and were treated as left-censored data and

assigned a bond strength of 1MPa based on half the preload value of 1 Newton applied in

material testing machine i.e. 0.5newton/0.5mm2= 1MPa (Vachiramon et al. 2008, 178-

185)

The pretest failure data were included the data as 1MPa and used in the statistical

analysis. The descriptive statistics are summaries in Tables 3 and 4 by the surface and the

etching time, including the mean and standard deviation values, and by the two

conditions with and without considering the failure mode as 1 MPa.

Table 5 includes mean bond strengths for all groups with and without the pretest

failure. For 5 seconds group the bond strengths were 32.49 +/- 9.27 MPa with the pretest

failure data included at 1MPa and 38.35 +/- 11.63 without including the pretest failure.

Mean bond strengths for 20 seconds groups was 36.41 +/- 11.17 MPa with the pretest

failure considered at 1MPa and 38.09 +/- 8.57 without including the pretest failure. Mean

bond strengths for 80 seconds groups was 19.08 +/- 10.33 MPa with the pretest failure


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included at 1MPa and 28.82 +/- 7.35 without including the pretest failure. Figure 5 and 6

shows the comparison in the mean tensile bond strength with and without including the

pretest failure.


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Table 2: Data for microtensile bond strength in MPa for each group

Tooth Group TBS_M TBS_D AVG_TBS

1 5 27.91 24.34 26.125

2 5 55.1 25.79 40.445

3 5 24.51 23.76 24.135

4 5 41.27 43.85 42.56

5 5 39.81 44.78 42.295

6 5 34.32 1 17.66

7 5 33.91 37.01 35.46

8 5 1 61.43 31.215

9 20 52.79 49.95 51.37

10 20 25.95 29.46 27.705

11 20 48.98 44.92 46.95

12 20 58.39 29.94 44.165

13 20 1 31.16 16.08

14 20 29.24 34.31 31.775

15 20 28.19 35.57 31.88

16 20 36.9 51.87 44.385

17 20 40.19 26.59 33.39

18 80 21.55 34.24 27.895

19 80 30.41 1 15.705

20 80 1 13.1 7.05

21 80 1 19.97 10.485

22 80 1 21.77 11.385

23 80 34.1 38.21 38.21

24 80 24.07 1 12.535

25 80 40.43 1 20.715

26 80 26.62 28.95 27.785


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Table 2 continued

TBS_M = mesial microtensile bond strength in MPa of mesial beam

TBS_D = distal microtensile bond strength in MPa of distal beam

Avg_TBS = Average tensile bond Strength of mesial and distal beams

Groups =Based on etching times of 5, 20and 80 seconds

Note: Pre test failures are recorded as 1 MPa


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Table3 - Mean microtensile bond strength by surfaces and etching times without

including the failure mode.

Group Mean_TBS Mean_TBS/M Mean_TBS/D Std_Dev Std_Dev/M Std_Dev/D

5 sec 38.35 36.69 37.28 11.63 10.07 13.93

20 sec 38.09 40.08 37.09 8.57 12.22 9.42

80 sec 28.82 29.53 28.63 7.35 6.96 7.83

Analysis variable: Microtensilebond strength

Groups = Based on etch times of 5, 20 and 80 seconds

Mean_TBS = Average mean microtensile bond strength in MPa

Mean_TBS/M = Mean microtensile bond strength in MPa of mesial beams

Mean_TBS/D = Mean microtensile bond strength in MPa of distal beams

Std_Dev = Standard deviation

Std_Dev/M= Standard deviation of mesial beams

Std_Dev/D= Standard deviation of distal beams


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Table 4 - Mean microtensile bond strength by surfaces and etching times withthe

pre test failure as 1MPa

Group Mean_TBS Mean_TBS/M Mean_TBS/D Std_Dev Std_Dev/M Std_Dev/D

5 sec 32.49 32.23 32.75 9.27 15.69 18.19

20 sec 36.41 35.74 37.09 11.17 17.33 9.42

80 sec 19.08 30.02 17.69 10.33 15.29 14.58

Analysis Variable: Microtensile bond strength


Mean_TBS = Average Mean microtensile bond strength in MPa

Mean_TBS/M = Mean microtensile bond strength in MPa of mesial beams

Mean_TBS/D = Mean microtensile bond strength in MPa of distal beams

Std_Dev = Standard deviation

Std_Dev/M Standard deviation of mesial beams

Std_Dev/D Standard deviation of distal beams


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Table5 Comparison of mean microtensile bond strength (MPa) withand without

pretest failures

Groups Mean_TBS PTF [1MPa] Mean_TBS without PTF

5 sec 32.49A 38.35

A

20 sec 36.40A 38.09

A

80 sec 19.08B

28.82A


Mean_TBS PTF[1MPa] = Mean microtensile bond strength in MPa

including pretest failure as 1 MPa in data

Mean_TBS without PTF = Mean microtensile bond strength in MPa without

including pre test failure

Letters A and B = Represents group comparison of the mean tensile

bond strength. Means with same letters represent

no significant statistical difference.


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Figure 5 - Graph showing data comparison of mean microtensile bond strength with

and without including the pretest failure

PTF: Pre test failure

Y axis: Mean microtensile bond strength in MPa

X axis: 5, 20 and 80 seconds groups with and without pretest failure

0

5

10

15

20

25

30

35

40

45

PTF WITHOUT PTF

5 seconds

20 seconds

80 seconds


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Figure 6 - Graph showing comparison of mean microtensile bond strength

PTF: Pre test failure

Y axis: Mean microtensile bond strength in MPa

X axis: Groups based on etching time of 5, 20 and 80 seconds


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4.2 Statistical analysis

4.2.1 Statistical results withoutthe pretest failures data

Comparisons of microtensile bond strength for mesial surfaces

The results from one-way ANOVA procedure revealed that there was no

statistically significant effect for the etching time on the micro-tensile bond strength for

mesial surfaces, F (2, 18)=1.84, p=0.1875.That is, the data showed that there was no

significant difference in microtensile bond strength among three etching times.

Comparisons of microtensile bond strength for distal surfaces


statistically significant effect for the etching time on the micro-tensile bond strength for

distal surfaces, F (2, 18) =1.18, p=0.3298. That is, the data showed that there was no

significant difference in micro-tensile bond strength among three etching times.

Comparisons of microtensile bond strength at tooth level


statistically significant effect for the etching time on the micro-tensile bond strength at

tooth level, F(2, 22)=2.76, p=0.0851. That is, the data showed that there was no

significant difference in micro-tensile bond strength among three etching times (Table 5).

If there was a missing value at either distal or mesial surface, the average value was equal

to the one value left.

4.2.2 Statistical results withthe pretest failure data

Comparisons of microtensile bond strength at mesial surface



mesial surface, F (2, 23) =2.33, p=0.1195. That is, the data showed that there was no


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significant difference in micro-tensile bond strength among three etching times at mesial

surface.

Comparisons of micro-tensile bond strength at distal surface



tooth level, F(2, 22)=2.30, p=0.0972. That is, the data showed that there was no

significant difference in micro-tensile bond strength among three etching times on distal

surfaces

Comparisons of microtensile bond strength at tooth level

The results from one-way ANOVA procedure revealed that there was statistically

significant effect for the etching time on the micro-tensile bond strength at tooth level, F

(2, 23) =6.91, p=0.0045. The post-hoc Tukey-Kramers test indicated that the mean

micro-tensile bond strengths at distal surface observed in 20 seconds and 5 seconds

groups were significantly greater than that observed in 80 seconds group, while there was

no significant difference between 20 and 5 etching times. Table 5 shows the results from

Tukey-Kramers tests.

4.3 Results from scanning electronic microscopy

Once the bond strength was evaluated in each sample, the fracture mode was

observed under SEM (Amray 1820-D) to evaluate the predominant type of failure. The

list of fracture mode is represented in Table 6. All fractures are grouped in any of the 4

categories which include cohesive failure in dentin, cohesive failure in resin composite,

adhesive failure or joint failure and mixed failure if the fracture was in the adhesive layer

and dentin and/or resin composite.The failure mode was examined at 90 times magnification and analyzed using

Fishers exact test. The results revealed no association between groups of etching times

and failure association. Table 6 shows the failure mode in each group based on etching

time. 5 seconds (Figure 7) and 20 seconds group (Figure 8) showed majority of their


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failures as cohesive failures in resin composite and dentin followed by mixed failures and

a few failures as joint failure.

80 seconds group (Figure 9) showed about 40% of the failure as mixed failures

with about 40 % of joint failures with only about 20% of failures as cohesive failure in

dentin with no cohesive failures in resin composite.


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Table 6 - Failure mode results

Group N Type 1 Type 2 Type 3 Type 4

20 14 4 2 2 6

40 17 6 2 3 6

80 13 3 0 5 5

Groups = Based on etch times of 5, 20and 80 seconds

N = Sample size

Type 1 = Cohesive failure at dentin

Type 2 = Cohesive failure at resin based composite

Type 3 = Adhesive failure

Type 4 = Mixed (adhesive failure + dentin/ resin based composite or both)


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Figure 7 -

A chart representin

Graph showing the fracture modes in 5 second

a 5 seconds group and their percentage of fail

47

group

re mode


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Figure 8:

A chart representin

Graph showing the fracture modes in 20 second

a 20 seconds group and their percentage of fai

48

s group

lure mode


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Figure 9:

A chart representin

Graph showing the failure modes in 80 second

an 80 second group and their percentage of fai

49

group

lure mode


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Figure 10 - Graph sh

A graph comparing the f

owing a comparison of the fracture modes from

ilure mode among the 3 etching groups

50

each group.


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Cohesive fractures in den

Figure 11 Cohesive failure in dentin

tin in 5 seconds (picture1) and 20 seconds (pict

Picture 1

Picture 2

51

re 2) groups


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Pic

Figure 13 - Joint failures

ure 4 and 5 showing joint failure in the adhesiv

Picture 4

Picture 5

53

e


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Pic

Figure 14: Mixed failures

ture 6 and picture 7 showing mixed failure

Picture 6

Picture 7

54


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View of dentin

showing failure in the

specimen from 80 secon

Figure 15: Adhesive failure in the hybrid layer

side of specimen of 80 second group in lo

ybrid layer (Picture 8). Higher magnificatio

s groups showing failure in the bottom of the h

Picture 8

Picture 8 Higher magnification

55

w magnification

of picture 8 of

brid layer.


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CHAPTER 5: DISCUSSION

When performing an adhesive restoration, clinicians are daily challenged with

several steps involved that might be difficult to control, of which conditioning of enamel

and dentin with acid to a specific time is one of the variables among the many factors that

influence this adhesion process. For instance, there is a thought that phosphoric acid

should be on enamel and dentin within periods of 30 and 15 seconds, respectively as

recommended by various manufactures. However some cavity preparations for restoring

direct or indirect restorations may involve large areas of exposed dentin to which need to

be etched with acid uniformly. Conceivably, the dentin located on some surfaces of those

preparations may undergo uneven or longer periods of acid conditioning than anticipated

depending on the manner it is applied. So in this study the effect of extended etching time

on vital dentin was evaluated in comparison to some in vitro studies that have been

carried out to evaluate the same effect on the dentin (Abu-Hanna and Gordan 2004, 105-

110;Abu-Hanna, Gordan, and Mjor 2004, 28-33;Hashimoto et al. 2002, 99-105;Jacques

and Hebling 2005, 103-109) and primary dentin (Bolanos-Carmona et al. 2006a, 1121-

1129;Sardella et al. 2005, 355-362) by measuring bond strengths with either tensile bond

strength or shear bond strength tests.

Consistent with previous in vitro studies, the results of this study showed a

reduction in the bond strengths with increase in etching time on vital dentin and the null

hypothesis was rejected within the limits of the study and when pretest failure were

included in the data. In all these studies the results have been consistent that excessive

etching of dentin has an inverse effect on the bond strengths. Most of these tests were

microtensile bond testing which is a suitable approach in evaluating the joint interface.


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The groups with excessive etching time of dentin showed a drop in the bond strength

irrespective of some variation with the type of adhesive system, or methods and these

results were comparable to the results in this study.

Major limitation of this study was the sample size. When a sample and power

analysis was applied to the existing data, the total sample size needed to obtain a

detectable difference between three groups with different etching times including the

pretest failure were 375 premolars at 80% power , 0.252 effect size and standard

deviation as 11. The total sample size increased to 492 premolars when the power was

increased to 90% to detect any contrast with the same effect size and standard deviation.

It is not uncommon to have some failure of samples during trimming or sectioning of

beams before testing them for the microtensile bond strength testing and in these cases

the bond strength of these samples is considered as 1 MPa and included in the data as left

censored data for statistical analysis. On the contrary, when the pretest failures were

excluded from the data, the total sample size needed to obtain a detectable difference

between those groups were 54 at 80% power, 0.197 effect size and 10 as standard

deviations. The total sample size increased to 69 when the power was inc

Documents

Impact of Different Acid Etching Time on Microtensile Bond Streng