The effect of ph of etchant on the bond strength of a two-step total-etching adhesive

Transcription

1 The effect of ph of etchant on the bond strength of a two-step total-etching adhesive by Mythili Rajesh Dr John O Burgess, CHAIR Dr Firoz Rahemtulla Dr Lillian Mitchell Dr Mark Stephen Litaker Dr Mona Anabtawi A THESIS Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Master of Science BIRMINGHAM, ALABAMA 2008

2 The effect of ph of etchant on the bond strength of a two-step total-etching adhesive Mythili Rajesh MS IN CLINICAL DENTISTRY ABSTRACT Acid etchants demineralize hydroxyapatite containing substrates by creating microporosities in enamel and dentin into which a resin can penetrate retaining composite resin by micromechanical retention. The efficacy of acid etching depends on the type of acid used, its concentration, PKa, etching time and its ph. The purpose of this study was to evaluate the effect of etchant ph on the shear bond strengths and etch pattern of a twostep total-etch adhesive, Adper Single Bond Plus, on unground enamel, ground enamel and dentin. 120 extracted human molars were divided into two groups and ground to obtain flat enamel and dentin surfaces and were subdivided into five groups. The buccal surfaces of another 60 molars were lightly polished with pumice and soft bristle brush and divided into five groups. Scotchbond etching gel was diluted with NaOH solution was added to alter the ph of the etchant. Group I was left unaltered as a control (ph=0.3). In the other four groups, each substrate were etched with etchants of ph =0.8, 1.5, 2.0, 3.0, followed by rinsing with water for 5 seconds. Adper Single Bond Plus adhesive was applied to etched surfaces with agitation and light cured (Fusion, Dentlight) for 10 ii

3 seconds. Three coats of adhesive were applied. After curing the adhesives, a plastic tube (diameter=1.5mm) filled with composite-resin (Z100, 3M ESPE, St.Paul, USA) was placed over the adhesive and cured for 40 seconds. All specimens (n=10) were stored in water for 24 hours at 37 C before testing. Specimens were then placed in a special fixture, mounted on a Universal testing machine (INSTRON, model number 5562, MA, USA) and were loaded to failure at a crosshead speed of 1mm/min.Mean bond strengths were determined and intergroup differences were analyzed using one-way and two-way ANOVA. SEM analysis determined the etch pattern for all three substrates. The etchant of ph=0.8 produced higher bond strength to unground enamel and dentin whereas ph=1.5 gave higher bond strength to ground enamel. SEM analysis showed that the etching efficacy on all the three substrates correlated strongly with the ph of the etchant used. iii

4 TABLE OF CONTENTS Page ABSTRACT... ii ACKNOWLEDGMENTS... vi LIST OF TABLES... vii LIST OF FIGURES... viii INTRODUCTION...1 Fundamental principle of adhesion...1 Bonding to Enamel...2 Bonding to Dentin...3 Classification of Dental Adhesives...6 Etch-and-Rinse adhesives...6 Self-etch adhesives...8 Acid Etchants...11 Etching of Enamel...11 Etching of Dentin...11 Factors affecting the efficacy of acid etching...12 RATIONALE AND OBJECTIVE...15 SPECIFIC AIMS...17 MATERIALS AND METHODS...18 Materials...18 Teeth Preparation...19 Preparation of etchant with different ph...19 Restorative Procedures...20 Shear Bond Strength Measurement...20 Statistical Analysis...20 Scanning Electron Microscopy Evaluation...21 EXPERIMENTAL DESIGN...22 iv

5 TABLE OF CONTENTS (contd.) RESULTS Shear bond strength...26 Scanning electron microscopy evaluation...29 DISCUSSION 33 CONCLUSION...38 LIST OF REFERENCES...39 APPENDICES IRB Approval form 45 v

6 ACKNOWLEDGEMENTS I would like to express my sincere appreciation and thanks to all the persons involved in this research project. I am most thankful to my mentor, Dr. John Burgess for his guidance and dedication throughout the course of the study. His constructive critiques and his attention to detail ensured I stayed on the right path to fruitful research. I would like to express my sincere gratitude to Dr.Firoz Rahemtulla, Dr. Lillian Mitchell, Dr. Mark Litaker, Dr. Mona Anabtawi for giving useful advice and comments to me as my committee and for their full support on this project. Their trust and encouragement are sincerely appreciated from my heart. Also, I would like to thank Mr. Preston beck for his technical support and Dr.Deniz Cakir for all the help and support she gave me these two years. Also, I would like to thank 3M ESPE (USA) for their generous donation of materials used in this study. Above all, I would like to thank my husband, Rajesh Renugopal for his continuous support and encouragement, my thanks to my children, Shreeya and Nandika for giving me happiness and joy. Also, I would like to thank our parents for their love and prayers. vi

7 LIST OF TABLES Tables Page 1 Etch-and-rinse and Self-etch adhesives Materials Amounts of NaOH and water added for different ph Shear bond strengths (MPa)...26 vii

8 LIST OF FIGURES Figures Page 1 Scotchbond etchant and Adper Single Bond Plus Adhesive ph meter used to measure the etchant ph Light curing of the composite resin tube using Fusion light cure unit Specimen placed on a special fixture, to be mounted on universal testing machine for shear bond testing Universal testing machine for testing the shear bond strength Scanning Electron Microscope for the evaluation of the etch pattern Effect of etchant ph on the shear bond strength to unground enamel, ground enamel and dentin SEM evaluation of the unground enamel specimens SEM evaluation of the ground enamel specimens SEM evaluation of the ground enamel specimens...32 viii

9 1 Introduction The growing demand for tooth-colored, conservative cosmetic restorations has encouraged the widespread placement of direct composite restorations. The success of these restorations depends, in large part, on the ability of the adhesive to bond and seal the composite resin to the tooth structure. Fundamental Principle of Adhesion Adhesion or bonding is the process of forming an adhesive joint. The substrate is the adherend and the applied material, which joins the substances, prevents separation and transmits stresses across the bond, is the bonding agent or the adhesive system 1. The adhesive strength or bond strength is the measure of the load-bearing capability of the adhesive. Adhesion refers to the forces or energies between atoms or molecules at an interface that hold two phases together. 2 Adhesive restorations better transmit and distribute functional stresses across the bonding interface to the tooth and have the potential to reinforce weakened tooth structure. 1 Bonding is an exchange process, which replaces minerals removed from the tooth, by acidic resin monomers producing a micromechanical lock to tooth structure 3, 4. The process occurs in two phases: the first phase involves the removes calcium phosphate to create micro-porosities in the tooth followed by the second phase called the hybridization phase that involves infiltration and subsequent in situ

10 2 polymerization of resin within the microporosities producing a micromechanical interlocking 3,4 of the resin to the tooth. Removing the smear layer while demineralizing enamel and dentin in a predictable manner, effective wetting, diffusion, penetration and good polymerization are critical in establishing a good bond. Intimate molecular contact between the adhesive and the tooth substrate is important for the development of strong adhesive joint and wetting indicates the ability of the liquid adhesive to come in intimate contact with the solid substrate 5. Sufficient wetting by the adhesive will occur only if its surface tension is less than that of the surface energy of the adherend 1. Adhesives with higher bond strength can resist the contraction stress on the bonded interface due to the polymerization shrinkage of the composite resin 6. Adhesive bond strength depends on the physicochemical properties of the adhesive and the structural properties of the adherend 1 Bonding to Enamel Enamel is almost homogenous in structure and composition. Mature enamel is 95% by weight inorganic material, which is primarily hydroxyapatite that exists in the form of submicron crystallites. They are oriented in three dimensions in which the contiguous relationship of crystallites forms enamel rods or prisms 7. Bonding to enamel was first established by Buonocore in 1955 using acid etching technique 8. Adhesion to enamel is primarily due to micro-mechanical retention achieved through acid etching, which transforms the smooth enamel surface into an irregular surface with high surface-free energy, more than twice that of unetched enamel 1 by dissolving surface enamel prisms. Etched enamel facilitates

11 3 effective wetting of the adhesive which envelopes the individually exposed hydroxyapatite crystals followed by in situ resin polymerization. Applied adhesive penetrating into the microporosities within the etched enamel surface forms resin tags 9 that provide resin adhesion to enamel. Two types of resin tags are formed. Macrotags are formed circularly between enamel prism peripheries and microtags form at the cores of the enamel prisms, where the monomer is polymerized within the individual crypts formed where the hydroxyapatite crystals have been dissolved 10. The bond of resin to enamel is generated primarily by the resin microtags because of their greater quantity and large surface area 1. Bonding to unground enamel is different from that of ground enamel due to the presence of aprismatic layer or prismless enamel appearing in unground enamel, resulting in lower bond strength 11. This prismless enamel is hypermineralized and is approximately 30µm thick with the apatite crystals running parallel to each other and perpendicular to the surface 7.Studies showed that enamel crystallites dissolve more quickly along their c-axes, which is the principal axis of the enamel rods than perpendicular to this axes 7. Thus, prismless enamel is difficult to demineralize 12 thereby affecting the bonding mechanism. Thus, the effect of etching on enamel 13, 14 depends on whether the enamel has been instrumented before etching. Bonding to Dentin Dentin is a heterogeneous tissue consisting of 70 wt% hydroxyapatite, 18 wt% organic material, mainly type I collagen and 12 wt% water 1. Dentin contains tubules that run from the dentino-enamel junction to the dentin-pulp border. Each tubule is

12 4 surrounded by a collar of hypermineralized peritubular dentin. Intertubular dentin between the tubules is less mineralized and contains more organic collagen fibrils than the tubular dentin 1. Water content and permeability of dentin is not identical for all regions because of variations in the number of tubules in a given area of the tooth 15. Near the pulp, the tubule density is around 45,000 tubules per mm 2, the tubules are close together and the water content of deep dentin is high. Near the enamel, the tubule density is 20,000 tubules per mm 2 and the tubules are far apart, occupying less than 1% of the surface area. 16 Bonding to dentin is more difficult and less predictable due to its higher protein and water content making it a more polar substrate and much less receptive to the adhesive system 17. Unlike enamel, dentin etching lowers the surface energy of dentin due its high concentration of collagen. Thus effective wetting by the adhesive does not occur. Wetting can be improved by the use of primers, which contain bifunctional molecules. When dentin is etched, the apatite crystals are solubilized and lost from dentin during rinsing. Water occupies the space around the collagen fibers previously occupied by the apatite crystals. These exposed collagen fibers collapse during drying, decreasing the ability of the adhesive primers to infiltrate the collagen scaffold 18. This can be prevented by the moist bonding technique in which the dentin substrate is left moist after rinsing off the etchant, producing higher bond strength 19. Dentin bonding is made more complex by the presence of a smear layer. The smear layer is composed of organic debris that remains on the dentin surface after the

13 5 preparation of the dentin during restoration 20. The smear layer is a 0.5 to 2 µm thick layer of debris with a granular substructure that entirely covers the dentin or any ground tooth substrate 20, 21. The surface of the smear layer generally appears very irregular. The orifices of the dentinal tubules are obstructed by debris tags, called smear plugs, that may extend into the tubule to a depth of 1 to 10 µm 22 and that are contiguous with the smear layer. Etchants applied to dentin results in the loss of smear layer and smear plugs and demineralization of the surface of the underlying dentin matrix to a depth of 5 to 10µm 1. This increases the porosity of the dentin permitting the infiltration of the resin into the tubules and also into the microporosities created on intertubular dentin, which after polymerization results in micromechanical retention 5. Thus, the resin-dentin hybrid layer is a reticular, intertwined hybrid tissue composed of collagen, residual mineral particles and resin 23, 24.Hybridization, or the formation of a hybrid layer, is the process of resin interlocking in the demineralized dentin surface, thereby providing micromechanical retention. This zone, in which resin of the adhesive system micromechanically interlocks with dentinal collagen, is termed the hybrid layer or hybrid zone. 1 As previously described, the dentin smear layer consists primarily of shattered and crushed hydroxyapatite and fragmented and denatured collagen. In clinical conditions, it may also be contaminated by bacteria and saliva 25. The smear layer can be a detriment to effective bonding. It has an inherently weak attachment to the underlying dentin 20 with strength of 5-6 MPa 26 and is brittle 27. Two strategies are used to overcome the low attachments strengths of the

14 6 smear layer: (1) removal of the smear layer prior to bonding (the so-called etch-andrinse approach), and (2) penetrating the smear layer and incorporate it into the bonding layer (the so-called self-etch approach) 28. Classification of Dental Adhesives Thus several factors such as etching of enamel, dentin etching, smear layer treatment resulted in the change of bonding systems from the Buonocore era to today 29. While early total-etch bonding systems produced high consistent bonding to enamel this system produced poor bonding to dentin, since the hydrophobic resins used did not wet the moist dentin substrate. As adhesives developed, bifunctional adhesive molecules were designed to chemically interact with dentin and co-polymerize with the composite resin, which limited the bond strength to the cohesive strength of the smear layer, which is relatively low 30. Incorporation of monomers with increased hydrophilicity resulting in better wetting of the moist substrate is responsible for the latest improvements in the adhesive technology 31. Based on the number of bottles and application steps, contemporary dental adhesives can be classified into three main groups namely etch-and-rinse adhesives, self-etch adhesives and glass-ionomer adhesives. 1 Etch-and-Rinse Adhesives The etch-and-rinse adhesives can be further classified into two categories namely, the three-step total-etch systems and two-step total-etch systems. The three-

15 7 step total-etch systems also known as fourth generation bonding systems are regarded as the gold standard by which the newer systems are judged 32. These systems have three steps: 1. Applying an acid etchant, which is usually phosphoric acid with a concentration between 30%- 40% for not less than 15 seconds, followed by rinsing with water for 5-10 seconds 33, The second step is the application of the primer or the adhesion promoting agent, containing hydrophilic monomers dissolved in solvents such as ethanol, acetone and/or water. Because of the volatile characteristics of acetone and ethanol, these solvents can displace water from the dentin surface and the moist collagen network, promoting the infiltration of monomers through the nanospaces of the exposed collagen network Effective primers contain monomers with hydrophilic properties that have an affinity for the exposed collagen fibril arrangement and hydrophobic properties for copolymerization with the adhesive resin 36. Most of the current systems contain HEMA (2-hydroxyethyl methacrylate) which act as surface-active agents to enhance the wettability of the adhesive resins. Thus, this step transforms the hydrophilic dentin surface to hydrophobic spongy state that allows the resin to wet and penetrate the exposed collagen network efficiently 35, The third step is the application of adhesive resin, consisting primarily of hydrophobic monomers like bis-gma, UDMA and TEG-DMA as a viscosity

16 8 regulator and hydrophilic monomers like HEMA as a wetting agent. Adhesive resin by infiltrating into the collagen network in the demineralized dentin surface forms resin tags resulting in the formation of a hybrid layer, providing micromechanical retention 1. Although three-step total-etch bonding systems provide predictable bonds, they are time-consuming. To simplify the bonding steps, fifth generation bonding systems were introduced. In these systems, the primer and adhesive application steps were combined 38, resulting in two-step etch-and-rinse adhesives. Self-etch adhesives Self-etch adhesives do not require a separate etch-and-rinse step. The self-etch adhesives can be further classified into two categories namely, the two-step self-etch systems and one-step self-etch systems. The two-step self-etching systems consist of an application of a self-etching primer followed by adhesive resin. The demineralization of dentin occurs concurrently with the infiltration of primer ensuring that the entire zone of demineralization is saturated with primer which can then be polymerized in situ 32. The self-etching systems incorporate the smear layer thereby providing a natural barrier to the pulp protecting it against bacterial invasion and limiting the outflow of pulpal fluid that might impair bonding efficiency. 27 The onestep self-etch systems consist of self-etching primer and the adhesive resin in a single bottle.

17 9 Based on their etching aggressiveness, self-etching adhesives can be subdivided into three groups: mild, moderate and strong 1. Strong self-etch adhesives have a ph of 1 or less, exhibit a bonding mechanism and interfacial ultra-morphology in dentin resembling that produced by etch-and-rinse adhesives. These adhesives dissolve the smear layer and smear plugs 1 and the hybrid layers reach a thickness of 3-4 µm. These adhesives have low bond strength values 39, 40. Moderate self-etch adhesives (ph between 1 and 2) produce a dentinal hybrid layer with a completely demineralized top layer and partially demineralized base. The deepest region of hybrid layer still contains hydroxyapatite and the transition of the hybrid layer to the underlying unaffected dentin is more gradual resulting in better micromechanical interlocking. 1, 4 Mild self-etch adhesives (ph of around 2) dissolve the dentin surface only partially, so that a substantial number of hydroxyapatite crystals remain within the hybrid layer 3.

18 10 1, 41 Table 1: Etch-and-rinse and self-etch adhesives Retention rate of class V restorations Sensitivity to dentinwetness condition Dentin etching Bonding efficacy Three-step etch-and-rinse adhesives Range of 86%- 100% in studies upto 5 years Sensitive to overdry or overwet dentin surface Risk of overetching dentin resulting in incomplete resin infiltration Effective adhesion to enamel and dentin in vitro and in vivo. However, enamel bond strengths are more predictable than dentin bonds. Two-step etchand-rinse adhesives Range of 50%- 100% in studies upto 5 years Sensitive to dentin wetness Risk of overetching dentin resulting in incomplete resin infiltration Similar to three-step etchand-rinse adhesives provide more predictable enamel bond strengths than dentin bonds. Two-step selfetch adhesives Range of 75%- 100% in studies upto 3 years Less sensitive to dentin wetness Simultaneous demineralization and resin infiltration Insufficient bonding effectiveness to enamel, particularly mild self-etch adhesives. One-step selfetch adhesives Range of 69%- 95% in studies upto 3 years Less sensitive to dentin wetness Simultaneous demineralizatio n and resin infiltration Insufficient bonding effectiveness to enamel, particularly mild self-etch adhesives. Thus, the etch-and-rinse adhesives with their high acidity of their etchants provide higher enamel bond strengths but bonding to dentin is not predictable. Selfetch adhesives, even with high ph provide high bond strengths to dentin but enamel bonding is compromised. It can be suggested that etchants with higher ph can prevent over etching of dentin and also give good bond strengths to both enamel and dentin.

19 11 Acid Etchants Acids such as phosphoric or citric are used to remove the smear layer and to render enamel and dentin surfaces more receptive to bonding 1. Etching is defined as the dissolution of the substrate 42, 43. Etching of Enamel The main objective of acid conditioning of enamel is selective dissolution of hydroxyapatite crystals resulting in the formation of etch pits. These etch pits result in the formation of resin tags by absorbing the resin through capillary attraction thereby achieving a stable bonding to enamel 1. Generally etching enamel for 15 seconds with 37% phosphoric acid creates a surface morphology that is sufficient for micromechanical retention. 44 Acid etching removes about 10µm of the enamel surface and creates a microporous layer from 5 to 50 µm deep. There are three types of enamel-etching patterns 45. Type I enamel-etching pattern shows predominant dissolution of prism cores. Type II shows predominant dissolution of prism peripheries while in Type III enamel-etching pattern no prism structures are evident 45. Dentin Etching The objective of conditioning dentin is to simultaneously remove the smear layer and demineralize the dentin surface 1. The removal of the smear layer with an 16, 46, acid, greatly increases the permeability of the dentinal tubules by more than 90%.

20 The demineralization of the top 2-5µm of the intertubular matrix creates channels with diameter of 20nm, between the collagen fibrils of the demineralized dentin matrix, which permits resin infiltration, thereby resulting in increased intratubular dentin permeability 5. Factors affecting the efficacy of acid etching Thus, the acid etchants plays an important role in the adhesion by demineralizing the substrates thereby creating spaces into which the resin can penetrate resulting in micromechanical retention for the composite resin restoration. But the efficacy of acid etching depends on several factors like the type of acid used, 1, 13, 14. its concentration, etching time, ph, substrate etc; A great deal of research that has been published on the type of acids and acid concentration to be used as etchants 48. Many organic and inorganic acids such as phosphoric acid, nitric acid, citric acid, maleic acid and oxalic acid of various concentrations have been studied, in an attempt to obtain increased bond strength to the tooth structure. However etching with 30%-40% phosphoric acid provided better retention 34. Swift et al 49 showed that 35% phosphoric acid gave bond strength of 24.5 MPa, which was significantly higher than the bond strength obtained with the use of conditioners such as 10% maleic acid, 1.6% oxalic acid with 2.6% aluminum nitrate and 10% phosphoric acid. The lower bond strength can be due to the lower acidity of the etchants, thereby resulting in less etching efficacy of the enamel surface.

21 13 Traditionally, the recommended application time for 30-40% phosphoric acid etchants has been 60 seconds 50. Studies have demonstrated that the etching enamel for 15 seconds provided similar surface morphology, retentive properties, bond 51, 52, strength and microleakage resistance to the traditional etching time of 60 seconds 53. Mulholland and DeShazer 54 explored the effects of phosphoric acid ph on the bond strengths of orthodontic brackets cemented to unground enamel surfaces and reported a significant increase in the bond strength when the acid ph was lowered from 4 to 2. Retief 55 studied the difference in the bond strength of epoxy resin to the enamel surface etched with different concentrations of phosphoric acid that ranged from 10% to 85%. The ph values for 10, 20 and 30% phosphoric acid was in the range of and was less than zero for 40% - 85% concentrations of phosphoric acid. Higher bond strengths were recorded for the concentration of 30 %( ph=0.3) but no significant difference between the etchants with ph values of was reported. Perdigao et al 56 evaluated the effect of six phosphoric acid etching agents using FE-SEM and reported that there was a significant correlation between the depth of demineralization of dentin and the etchant ph, that etchants with higher ph produced shallower etching depths. Marshall et al 57 elucidated the importance of the ph on the etching rate of the etchants on dentin that, as the ph decreases, the etching rate increases. The more acidic and aggressive the etchant more completely the smear layer and smear plugs are removed 58.This complete removal of smear plugs results in

22 14 increase in dentin permeability, thereby permitting the infiltration of the resin into the tubules. When aggressive etchants are used on dentin, it may be demineralized to a depth that might not allow complete resin impregnation thereby producing a nonresin reinforced collagenous band at the base of the hybrid layer, which weakens the dentin bond and decreases its durability 59. This poor dentin seal coupled with increased permeability of bacterial products across etched dentin that might induce pulpal inflammation 30. Thus, the acidity of the etchants used on dentin should be optimized to reduce the complete removal of smear plugs which decreases the dentin permeability but results in higher bond strength. In this study bond strength is measured using micro-shear bond strength test. The small bonded surface area results in uniform stress distribution, decreased chances of encountering interfacial defects that approached the critical flaw size for spontaneous crack propagation and generates higher bond strength 60, 61. Also, preparations of the samples are relatively simple compared to microtensile bond strength specimens.

23 15 Rationale and Objective Commercially available total-etch systems are too aggressive on normal cut dentin resulting in deeper demineralization and may result in lower bond strength and decreased restoration retention rates if there is inadequate resin infiltration into the deeper layers 59. Gwinett et al 5 suggested that the depth of dentin etching may be reduced by decreasing the acidity of etchants, making it easier for resins to infiltrate the demineralized dentin. However, enamel bond strength is higher, when more acidic and aggressive etchants are used. In case of cavity restorations, if the bonding to enamel periphery exceeds bonding to dentin, the composite may partially debond from the pulpal floor. This results in gap formation between resin and dentin, so the hydraulic forces within the fluid filled gap and underlying tubules stimulate pulpal nerve endings that causes postoperative sensitivity 62. To prevent postoperative sensitivity, good bonding to all tooth substrates is critical. Thus, the best compromise between adequate enamel etching and etching without extensive dentin demineralization has to be determined. In addition, the optimum ph of the etchant, which gives good bond strength to dentin, has to be determined. There is inadequate evidence in the literature to show the effect of etchant ph on the bond strength to unground enamel, ground enamel and dentin of the two-step total-etch system. To determine the effect of ph, a single etchant with varying ph

24 16 values should be prepared and their effect on the bond strength to different substrates like unground enamel, ground enamel and dentin, and their etch pattern on them, and the association between them should be investigated. Thus, it is possible to control many variables like type of etchant, its concentration and its etching time. The purpose of this invitro study is to determine the effect etchant ph has on the bond strength of the two-step total-etching adhesive to unground enamel, ground enamel and dentin. The null hypothesis to be tested is: There is no difference in the bond strength and etch pattern of total-etching adhesive to unground enamel, ground enamel and dentin for 5 different ph values of the etchant (0.3, 0.8, 1.5, 2.0, and 3.0)

25 17 Specific Aims To determine the effect of etchant ph on the bond strength of the two-step total-etching adhesive to unground enamel, ground enamel and dentin for ph values of 0.3, 0.8, 1.5, 2.0, 3.0 using micro-shear bond strength test. To examine, using scanning electron microscopy (SEM), the etch pattern of the two-step total-etching adhesive for five ph levels on unground enamel, ground enamel and dentin.

26 18 Materials and Methods Materials One hundred and eighty extracted non-carious human molars, including first, second and third molars, were used. The teeth were collected by several departments of the University of Alabama at Birmingham, School of Dentistry under a protocol approved by the IRB (Appendix), and they were stored in 4% sodium azide solution at 4 C in a refrigerator until used. Teeth were randomly selected for the experimental and control groups. 150 teeth were used for the bond strength testing (10 teeth per group for 15 groups) and 30 teeth for SEM examination (2 teeth per group). Types of materials, product name, manufacturers and main compositions of materials used in this study are presented in Table 1. Scotchbond (3M ESPE, St. Paul, USA) was the etchant, while Adper Single Bond Plus (3M ESPE, St. Paul, USA) (Fig.1) was the adhesive and Z100 (3M ESPE, St. Paul, USA) was the resin composite. Table 2 Materials used Types of Product Name Manufacturer Composition Materials Acid Etchant Scotchbond 3M ESPE 35% phosphoric acid, Amorphous silica, water Two-step totaletching adhesive Adper Single Bond Plus 3M ESPE Bis-GMA, HEMA, ethyl alcohol, water, 10% colloidal silica nanofillers, Diurethane Dimethacrylate, Glycerol 1, 3 Dimethacrylate, copolymer of acrylic and Direct Resin Composite itaconic acids Z100 3M ESPE Bis-GMA, TEGDMA, Zirconium silicate

27 19 Teeth Preparation 120 Molars were divided into two groups and ground with 600 grit wet wheel (Wehmer, Model 108, IL, USA) to obtain flat enamel and dentin surfaces. They were then subdivided into five groups which were etched with one of the five etchants (Group I, II, III, IV, V for ph values 0.3, 0.8, 1.5, 2.0, 3.0 respectively) (n=10 for shear bond measurement, n=2 for etch pattern determination). The buccal surfaces of the other 60 molars used as the unground enamel substrate, were lightly polished with pumice and a soft bristle brush and were divided into five groups. Etchant Preparation To alter the etchant ph, it was buffered with 40% aqueous solution of NaOH (40g of NaOH in 100ml of water). By adding increments of a dilute solution of NaOH, the ph was adjusted. The ph values are measured using ph meter (Sension4, Hach, CO, USA) (Fig.2), calibrated with appropriate standards. The formula to be used to obtain different ph values are shown in Table 2. Table 3 Amounts of NaOH and water added for different ph ph Group Etchant 40% NaOH Water 0.3 I Control group 0.8 II 400 mg 0 mg 200 mg 1.5 III 400 mg 100 mg 100 mg 2.0 IV 400 mg 150 mg 50 mg 3.0 V 400 mg 200 mg 0

28 20 Restorative procedures The modified etchants at varying ph were applied to the prepared tooth surfaces for 15 seconds, rinsed with water for 15 seconds and then blot dried with cotton. Immediately after blotting, 2-3 consecutive coats of adhesive was applied to etched surfaces for 15 seconds with gentle agitation using a fully saturated applicator. The applied adhesive was gently air thinned for five seconds to evaporate solvents. Fusion (Dentlight Inc. TX, USA) a LED light-curing unit (Fig.3) with a light intensity of 600 mw/cm 2 was used throughout the restorative procedure. After light irradiation of the adhesives, a plastic tube (diameter=1.5mm) filled with composite-resin (Z100, 3M ESPE, St. Paul, USA) was placed over the adhesive and cured for 40 seconds. Samples (n=10) were stored in water for 24 hours at 37 C before testing. Shear Bond Strength Measurement Specimens were then placed in a special fixture (Fig.4), mounted on a Universal testing machine (INSTRON, model number 5562, MA, USA) (Fig.5) and loaded to failure at a crosshead speed of 1mm/min. The stress at failure was converted to bond strength by dividing by the bonding area. Bond strength was calculated in megapascals. Statistical Analysis Mean bond strength was compared among ph and substrate groups using twoway analysis of variance (ANOVA). Significance interaction effects were further

29 21 evaluated using one-way ANOVA. Thus, bond strength was compared among ph groups separately for each of the three substrates, and bond strength was compared among dentin, ground enamel and unground enamel surfaces separately for each ph group using a separate one-way ANOVA. Pairwise post-hoc comparisons were conducted using Tukey s test. The correlation between ph and the bond strength for each substrate was determined using Pearson s correlation coefficient. SEM (Scanning Electron Microscopy) Evaluation To observe the effect of conditioning with etchants of different ph on unground enamel, ground enamel and dentin surfaces, the prepared tooth surfaces were etched with the prepared etchants for 15second followed by rinsing with water for 15 seconds and blot dried with cotton. Then the specimens were fixed with 10% formaldehyde for 24 hours followed by rinsing with Cacodylic acid buffer for hours. They were dehydrated by being rinsed with an ascending series of ethanol (70, 80, 95 and 100%) for 15 minutes each and then subjected to critical point drying. The specimens were mounted on aluminum stubs using colloidal silica and sputter coated with gold/palladium and examined using a scanning electron microscope (ISI, MX40A, International Scientific Instruments, Tokyo, Japan) (Fig.6)operating at 15 kilovolt.

30 22 Experimental Design 180 Human Molars Unground Enamel 60 Molars Ground Enamel 60 Molars Dentin 60 Molars Bond Strength 50 Molars (n=10) Etch Pattern 10 Molars (n=2) Bond Strength 50 Molars (n=10) Etch Pattern 10 Molars (n=2) Bond Strength 50 Molars (n=10) Etch Pattern 10 Molars (n=2)

31 23 Fig.1 Scotchbond etchant and Adper Single Bond Plus Adhesive Fig.2 ph meter (Sension 4, Hach, CO, USA) used to measure the ph of the etchant

32 24 Fig.3 Light curing of the tube filled with composite using Fusion LED light curing unit Fig.4 Specimen placed on a special fixture, to be mounted on universal testing machine for shear bond testing

33 25 Fig.5 Universal testing machine for testing the shear bond strength Fig.6 Scanning Electron Microscope for the evaluation of the etch pattern

34 26 Results Shear bond strength The means and standard deviations of the shear bond strengths obtained for the different ph groups for all substrates are given in Table 3. Table 4. Shear bond strengths given in MPa: mean±sd, n=10 Substrates ph=0.3 ph=0.8 ph=1.5 ph=2.0 ph=3.0 Unground 25.4 ± ± ± ± ± 5.3 Enamel Ground 28.6 ± ± ± ± ± 4.1 Enamel Dentin 29.2 ± ± ± ± ± 5.5 Two-way ANOVA (ph and substrates) showed that the interaction between the factors was statistically significant (P< 0.001), indicating that the effect of different etchant ph on the bond strength was dependent upon the substrate tested. One-way ANOVA showed that there are no significant differences in the bond strength to all the three substrates when the etchant ph is 0.3 (P>0.05). For all the ph groups there was significant difference in the bond strength to the three substrates. (P=0.03 for ph=1.5 and P<0.001 for ph=0.8, 2.0, 3.0). One-way ANOVA showed significant differences in bond strength among substrates for all ph values. (P<0.001) Tukey s multiple comparison tests showed the following results: For unground enamel, the bond strengths ranged from 9.8 MPa to 31.9 MPa with ph=0.8 showing statistically significant higher bond strength than ph=1.5, 2.0, 3.0. ph=3.0 showed statistically significant lower bond strength than all the etchant ph values.

35 27 For ground enamel, the bond strengths ranged from 12.4 MPa to 28.7 MPa with group V (ph=3.0) showing significant lower bond strength than the other groups. All the other groups showed statistically similar bond strengths. For dentin, the bond strengths ranged from 24.1 MPa to 38 MPa with group II (ph=0.8) showing significant higher bond strength than groups I&V (ph=0.3, 3.0), while group V (ph=3.0) showing significantly lower bond strength than groups II, III & IV (ph=0.8, 1.5, 2.0). Additionally, statistical analysis revealed that only Group I (ph=0.3) resulted in statistically similar bond strengths for unground enamel, ground enamel and dentin. In group II (ph=0.8), ground enamel showed significantly lower bond strength than unground enamel and dentin. For ph=1.5, unground enamel showed significantly lower bond strength than dentin. In group IV (ph=2.0), all the substrates showed significant differences with dentin showing highest value. For ph=3.0, dentin showed significantly higher bond strength than unground enamel & ground enamel. A significant correlation was found between the ph of the etchant and the bond substrates obtained for all the three substrates. For unground enamel, r = -0.85; p < , while for ground enamel, r = -0.78; p< Dentin showed r = -0.54; p<0.03.

36 28 Bond Strength (MPa) Unground Enamel Ground Enamel Dentin ph of Etchant Fig.7 Effect of Etchant ph on the shear bond strength to unground enamel, ground enamel and dentin

37 29 SEM Evaluation SEM observation of the etched specimens showed different morphological features. The lower the ph, more aggressive the demineralization. Unground enamel specimens (Fig.8A and 8B) etched with ph=0.3 etchant showed type II etch pattern where the prism peripheries were lost. Specimens (Fig.8C, D, E, F, G, H, I, J) etched with etchants of ph= 0.8, 1.5, 2.0, 3.0 did not show any etching pattern topography. SEM analysis showed that the deeper enamel etching patterns were obtained with the use of lower etchant ph. Higher ph etchants resulted in visibly shallower enamel etching patterns (Fig.9). Ground enamel specimens (Fig. 9A, B, C, D, E, F) showed the characteristic prism-end structure when etched with etchants of ph=0.3, 0.8, 1.5. The prism cores were affected to a greater extent than the peripheral regions, thus exhibiting the type I etch pattern. A higher magnification demonstrated the characteristic honeycomb appearance. Specimens (Fig.9G, H) etched with etchants of ph=2.0 showed type I etch pattern but shallower than the other groups. Specimens (Fig.9I, J) etched with ph=3.0 etchant showed a pattern that was difficult to relate surface topography to prism structure. Dentin specimens (Fig.10A, B, C, D) etched with ph=0.3, 0.8 etchants showed completely open tubules, while specimens (Fig.10E, F, G, H) etched with ph=1.5, 2.0 etchants showed partially open tubules and the smear layer was completely removed in all the specimens. Specimens (Fig.10I, J) etched with ph=3.0 etchants showed fully occluded tubules but the smear layer was removed.

38 30 A B ph= x C 3000x D ph= x E 3000x F ph= x G 3000x H ph= x I 2000x J ph= x 3000x Fig. 8 SEM Evaluation of the Unground enamel specimens. A. Specimen etched with ph=0.3 etchant showing type II etch pattern, where the prism peripheries are lost. B. same as A at higher magnification. C. Specimen etched with ph=0.8 etchant showing microporosities along the entire aprismatic surface and D shows higher magnification. E. Specimen etched with ph=1.5 etchant showing an uneven etching pattern and F shows higher magnification. G. Specimen etched with ph=2.0 etchant showing faint relief of aprismatic layer and H. shows higher magnification. I. Specimen etched with ph=3.0 etchant showing shallow etch pattern and the surface is not porous and J. shows higher magnification

39 31 A B ph= x C 3000x D ph= x E 3000x F ph= x G 3000x H ph= x I 2000x J ph= x 2000x Fig.9 SEM Evaluation of the ground enamel specimens. A. Specimen etched with ph=0.3 etchant showing type I etch pattern, where the prism cores are lost. B. same as A at higher magnification. C. Specimen etched with ph=0.8 etchant showing predominant type I etch pattern and D shows higher magnification. E. Specimen etched with ph=1.5 etchant showing type I etching pattern but shallower than the groups etched with ph=0.3 and 0.8 and F. at higher magnification shows honeycomb appearance. G. Specimen etched with ph=2.0 etchant showing type I etch pattern but the craters are shallower than obtained with lower ph groups and H. shows higher magnification. I. Specimen etched with ph=3.0 etchant showing no regular etching pattern and J. shows higher magnification

40 32 A B ph= x C 2000x D ph= x E 5000x F ph= x G 2000x H ph= x I 2000x J ph= x 2000x Fig.10 SEM Evaluation of the dentin specimens. Specimen etched with ph=0.3 etchant showing wide open dentinal tubules. B. same as A at higher magnification. C. Specimen etched with ph=0.8 etchant also shows wide open dentinal tubules and D shows higher magnification. E. Specimen etched with ph=1.5 etchant showing partially occluded tubules and F shows higher magnification. G. Specimen etched with ph=2.0 etchant also shows partially occluded tubules and H. shows higher magnification. I. Specimen etched with ph=3.0 etchant shows tubules occluded with smear plugs and J. shows higher magnification

41 33 Discussion The microshear bond strength of a two-step total-etch adhesive to unground enamel, ground enamel and dentin when etched with etchants with ph ranging from 0.3, 0.8, 1.5, 2.0 and 3.0 showed statistically significant differences. Thus, the null hypothesis is rejected in this study. Furthermore, etch patterns observed from specimens treated with etchants of different ph also showed a significant difference with lower ph etchants producing deeper and more distinct etch patterns. In the present study, to determine the effect of etchant ph on the bond strength and etch pattern, Scotchbond etchant gel was modified with varying amounts of 40% NaOH and water to obtain five etchants with different ph levels. Thus, many factors that influence the efficacy of etchants such as type of acid, concentration of the acid, etching time were controlled. By adding the NaOH solution and water a change in viscosity was produced with greater amounts, producing a solution with low viscosity compared to the original etchant. However, the influence of this change in viscosity on the bond strengths obtained in this study would be minimal as studies show that the phosphoric acid etchants of different viscosities showed similar demineralizing effects on dentin 63. Similarly, the enamel bond strengths obtained with enamel etched with different viscosities were not statistically different 64. In addition, the etchant did not have to flow into a pit or fissure since only flat surfaces were used in this study, so the change in viscosity should have minimal impact on the study. Bonding to unground enamel showed decreased bond strength when treated with etchant of higher ph values. In addition, the bond strengths were significantly

42 34 lower than the bond strengths to ground enamel and dentin for all the ph values except for the ph value of 0.8. The bond strength obtained was consistent with another study reported by Perdigao et al 65, who demonstrated that the microtensile bond strength to intact enamel with Adper Single Bond was 31.5MPa. It has been suggested that shear bond strength of MPa is adequate for bonding orthodontic brackets to teeth 66, 67. In this study even a higher ph = 3.0 showed greater bond strength than this optimal range. SEM evaluation, except for the control group did not show much mineral loss. Thus, it can be suggested that for bonding orthodontic brackets to teeth, etchants of higher ph can be used to reduce mineral loss due to etching and decrease enamel damage during debonding. It has been postulated that minimum bond strength of 17 to 20 MPa to enamel and dentin is needed to resist contraction stresses of resin composite materials 68. The shear bond strength of total-etch adhesives to enamel ranged from 26.9±1.17 and ±1.73 MPa and studies confirm this bond strength is sufficient for successful retention and marginal integrity of resin restorations 53, Bond strengths to ground enamel obtained in this study for all the ph groups was well within the optimal range suggested for successful retention except for the group treated with etchant ph=3.0, which showed a significant lower bond strength of 12.4 MPa. Bond strengths to ground enamel did not show significant difference when treated with etchants of ph= 0.3, 0.8, 1.5 and 2.0. The relationship between the enamel etching pattern and the shear bond strength of resin to enamel is still unclear. It is considered that, for mechanical

43 35 retention to occur, an ideal etchant should produce a uniform pattern of mineral loss free of surface precipitates 72, 73. SEM etch patterns seen on ground enamel with different ph values showed that higher the ph, more conservative the mineral loss. The type I etch pattern was most commonly seen for enamel. Groups etched with etchant ph of 0.3 and 0.8 showed complete dissolution of the prism cores while etchant ph of 1.5 and 2.0 produced shallow craters. Higher magnification of the etch pattern for all the four groups showed the characteristic honeycomb appearance as shown by Silverstone et al 45. Evaluation of specimens etched with etchant ph=3.0 did not show any pattern which also correlates with its lower bond strength. In the present study bond strengths to dentin for all the ph levels ranged from 24 to 38 MPa. The control group (ph=0.3) gave the bond strength of 29.2±6.7 MPa, which was consistent with a study by Lee et al 74. They showed that the tensile bond strength of Adper Single Bond Plus was 24.4 MPa and its unfilled version, Adper Single Bond also gave similar bond strength. However, Nunes et al 75 using microtensile bond strength of Adper Single Bond had values ranging from 76 MPa for Adper single bond to 58 Mpa for its filled version (mentioned by authors as exp Single bond). The higher bond strength can be attributed to the testing method and preparation of specimens is very difficult. Bond strength to dentin when treated with different ph levels showed that groups etched with etchant ph of 0.3 and 3.0 produced significantly lower bond strength. The lower bond strength produced by etchant of ph=0.3 may be due to its higher acidity, which resulted in deeper demineralization and subsequent inadequate

44 36 infiltration of the resin into the deeper layers. SEM evaluation also showed complete dissolution of the smear layer and smear plugs and demineralized peritubular dentin resulting in wide open dentinal tubules. The lower bond strength of ph=3.0 may be due to its higher ph. SEM evaluation showed that only the smear layer was removed and the tubules were occluded with smear plugs, thus limiting the penetration of the bonding agent into the tubules. Bond strength to dentin when treated with etchants of different ph showed that ph=0.8, 1.5, 2.0 obtained significantly higher bond strength than ph=0.3, 3.0, suggesting that ph of the etchant which is not too low or too high is optimal for dentin bonding. ph=0.8 showed the highest bond strength of 38±5.9 MPa. At this ph, SEM analysis showed complete dissolution of smear layer and smear plugs and widened dentinal tubules, which could have resulted in larger diameter resin tags, which could have resulted in the highest bond strength to dentin. Bond strengths to different substrates within each group when analyzed revealed that dentin showed higher bond strength than unground enamel and ground enamel. The higher bond strength to dentin can be attributed to the fact that the adhesive contains HEMA/alcohol mixture that is able to better wet the etched the dentin surface and maintain the collagen fibers in an expanded condition thereby improving infiltration of the resin 76. Application of three coats of adhesive with agitation followed by air drying after every coat according to the manufacturer s instructions also could have resulted in improved infiltration of the resin. SEM analysis of the penetration of resin into the tubules, if done could have revealed this.

45 37 Thus the etchant ph of 0.8 gave higher bond strength to unground enamel and dentin. Also ph of 1.5 gave higher bond strengths to ground enamel. Thus, the ph of range of seems to be the optimum ph to obtain higher bond strengths to all the three substrates. This study investigated only the immediate bond strengths. This study investigated only 24 hours bond strengths. The effect of etchant ph on the long-term bond strengths should also be studied, as it determines the durability of the bond. In addition, etchant ph effect on microleakage should also be studied. The bonding performance to other substrates like sclerotic dentin and primary teeth are different from the substrates used in this study. Therefore, the effect of etchant ph on these substrates should also be studied.