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Effects of aging on dentin bonding and mechanical properties of restorative glass ionomer cements. Chander, Kunal 2016

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EFFECTS OF AGING ON DENTIN BONDING AND MECHANICAL PROPERTIES OF RESTORATIVE GLASS IONOMER CEMENTS by  Kunal Chander  DDS, The University of Western Ontario, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2016 © Kunal Chander, 2016   ii Abstract Objectives: To examine changes in shear bond strength to dentin (SBS), flexural strength (FS) and diametral tensile strength (DTS) of four restorative glass ionomer cements: Fuji II LC (GC/America), Equia (GC/America), Ketac Nano (3M/ESPE), and Ketac Molar (3M/ESPE) after aging in artificial saliva. Materials and Methods: For SBS testing, sound extracted human permanent molars were ground to flat occlusal dentin surfaces and fixed in circular molds with auto-cured acrylic resin..  Teeth were randomly divided into four groups: Fuji II LC, Equia, Ketac Nano, Ketac Molar. For each dentin surface, two glass ionomer cylinders were bonded. Specimens were stored in artificial saliva (37°C) and tested at 24-hour and 6-month time points (Shear Testing Machine, Bisco).  For each material, FS bars (25mm x 2mm x 2mm) and DS discs (4mm x 2mm) were fabricated, stored in artificial saliva (37°C), and tested at 24-hour and 6-month time points. (Shimadzu).  An additional FS study was conducted with glass ionomer specimens, stored either in distilled water or artificial saliva (37°C), and tested at 24-hour and 2-month time points. (Shimadzu). Data analysis included two-way ANOVA (p<0.05) with post-hoc Tukey’s tests to compare interactions. Results: There were no significant differences in SBS after 6 months storage, except for Ketac Nano, which showed a significant decrease in bond strength after aging. There were no differences in SBS among the four glass ionomers, at 24 hours or 6 months saliva storage. The diametral tensile strength values did not change significantly after aging except for Fuji II LC. All materials had a significant increase in flexural strength after aging regardless of the storage media (water or saliva). Fuji II LC had significantly higher DS and FS compared to other materials, at both 24 hours and 6 months storage.    iii Conclusions:  Aging did not affect SBS of materials except Ketac Nano. Flexural strength of all glass ionomer cements increased over time. Storage media did not affect flexural strength properties. Diametral tensile strength remained unchanged over time for all materials. Overall, Fuji II LC had superior mechanical properties compared to other materials.    iv Preface The research project was developed by Dr. Kunal Chander and Dr. Adriana Manso with guidance from the committee members, Dr. Ricardo Carvalho and Dr. Karen Campbell. The human ethics approval was obtained from the Research Ethics Board at The University of British Columbia, under ID number H14-02189.  The glass ionomer cements tested in this project were generously donated by the material’s manufacturers, 3M ESPE and GC America. The experiments were carried out by Dr. Kunal Chander.  The data collection and part of the statistical analysis was done by Dr. Kunal Chander under the supervision of Dr. Adriana Manso.  Statistical support and part of the statistical analysis was provided by Mr. Rick White (Statistician, Department of Statistics, UBC).  The thesis was prepared with guidance from Dr. Adriana Manso, Dr. Ricardo Carvalho and Dr. Karen Campbell.   v Table of Contents  Abstract .......................................................................................................................................... ii	Preface ........................................................................................................................................... iv	Table of Contents ........................................................................................................................... v	List of Tables ............................................................................................................................... viii	List of Figures ............................................................................................................................... ix List of Images ................................................................................................................................. x	List of Symbols .............................................................................................................................. xi	List of Abbreviations ................................................................................................................... xii	Acknowledgements ..................................................................................................................... xiii	Chapter 1: Introduction ................................................................................................................ 1	1.1	 Glass Ionomer Cements: Basic composition, chemical reaction and classification .......1	1.2	 Glass Ionomer Cements:  Advantages and disadvantages .............................................. 5 1.3								Glass Ionomer Cements: Restorative technique .............................................................. 8 1.4									Glass Ionomer Cements: Clinical studies ....................................................................... 8 1.5	 Glass Ionomer Cements: In vitro studies ....................................................................... 10 1.6       Glass Ionomer Cements: Methodologies for in vitro studies ........................................ 12 1.7       Glass Ionomer Cements:  In vitro artificial aging ......................................................... 14 1.8       Study rationale and hypotheses ..................................................................................... 15 Chapter 2: Matrials and Methods .............................................................................................. 17	2.1	 Tooth collection ............................................................................................................. 17	2.2	 Materials  ....................................................................................................................... 17   vi 2.3	 Artificial saliva formulation .......................................................................................... 19 2.4	 Experimental design overview ...................................................................................... 20 2.4.1 Shear bond strength to dentin .................................................................................... 20 2.4.2 Flexural strength study #1 and diametral tensile strength  ........................................ 20 2.4.3 Flexural strength study #2: Artificial saliva vs. water storage .................................. 20 2.5	 Shear bond strength to dentin ........................................................................................ 24 2.5.1 Tooth preparation ...................................................................................................... 24 2.5.2 Dentin conditioning ................................................................................................... 24 2.5.3 Bonding procedures ................................................................................................... 26 2.5.4 Specimen storage ....................................................................................................... 28 2.5.5 Shear bond strength tests ........................................................................................... 28 2.6	 Flexural strength study # 1 - Artificial saliva storage ................................................... 29 2.6.1 Flexural strength specimen fabrication ..................................................................... 29 2.6.2 Flexural strength testing ............................................................................................ 31 2.7	 Flexural strength study #2 - Artificial saliva and water storage .................................... 32 2.8	 Diametral tensile strength .............................................................................................. 32 2.8.1 Diametral tensile strength specimen fabrication ....................................................... 33 2.8.2 Diametral tensile strength testing .............................................................................. 34 2.9	 Statistical analysis ......................................................................................................... 35 Chapter 3: Results ....................................................................................................................... 36	3.1	 Shear bond strength to dentin ........................................................................................ 36	3.2	 Flexural strength study #1 ............................................................................................. 38	3.3	 Flexural strength study # 2  ........................................................................................... 40   vii 3.4	 Diametral tensile strength  ............................................................................................. 46 Chapter 4: Discussions ................................................................................................................ 49	4.1	 Shear bond strength to dentin ........................................................................................ 49	4.2	 Flexural strength ............................................................................................................ 55 4.3	 Diametral tensile strength .............................................................................................. 60 Chapter 5: Conclusion ................................................................................................................ 64	Bibliography ................................................................................................................................. 65 Appendix A:  Sample size calculations ...................................................................................... 70 Appendix B:  Shear bond strength of glass ionomers published in literature ....................... 71 Appendix C:  Flexural strength of glass ionomers published in literature ............................ 72 Appendix D:  Diametral tensile strength of glass ionomers published in literature ............. 73     viii List of Tables Table 1.1 Glass ionomer cements classification based on material composition …….………….4 Table 2.1 Restorative glass ionomer cements used in this study ..…………...………….………18 Table 2.2 Dentin conditioners and primers used in this study ..………...…………………….…19 Table 2.3 Artificial saliva composition .…………….……...…………...………………….……19 Table 2.4 Dentin surface conditioning protocols applied as per manufacturer’s recommendation…………………………………………………………………………………25 Table 3.1 Shear bond strength failure modes .….……..………………………………..…….…36 Table 3.2 Shear bond strength to dentin ……....….…………………………………….…….…37 Table 3.3 Flexural strength in artificial saliva .….…………………………….....…......…….…39 Table 3.4 Flexural strength in artificial saliva vs. water ……….……………...….….........….…41 Table 3.5 Flexural strength in artificial saliva: 24 hours vs. 2 months …………..….....…….…44 Table 3.6 Flexural strength in water: 24 hours vs. 2 months …..………….…...……....…….…44 Table 3.7 Flexural strength of Ketac Nano: Artificial saliva vs. water …..…...……..…...…..…45 Table 3.8 Flexural strength of Ketac Molar: Artificial saliva vs. water ……...…….........…...…45 Table 3.9 Flexural strength of Fuji II LC: Artificial saliva vs. water …..…….......…....….….…46 Table 3.10 Flexural strength of Equia: Artificial saliva vs. water ...…..……..........…....…..…...46 Table 3.11 Diametral tensile strength in artificial saliva ………..…..……....…..........…..…..…47   ix List of Figures Figure 1.1 Structure of glass ionomer cements …………………………………………………...3 Figure 2.1 Shear bond strength to dentin experiment overview ……………………..………….21 Figure 2.2 Flexural strength in artificial saliva experiment overview ……...………..………….22 Figure 2.3 Diametral tensile strength in artificial saliva experiment overview ……...………….22 Figure 2.4 Flexural strength in artificial saliva vs. water study experiment overview ………….23 Figure 2.5 Schematic of a 3 point bending test ……………...………………………………….32 Figure 2.6 Schematic of a diametral compression test ………………………………………….35 Figure 3.1 Box and whisker plot of shear bond strength …………………………………..…....38 Figure 3.2 Box and whisker plot of flexural strength in artificial saliva ……………..………....40 Figure 3.3 Box and whisker plot of flexural strength at 24 hours (saliva vs. water)……. ...…....42 Figure 3.4 Box and whisker plot of flexural strength at 2 months (saliva vs. water) ……...…....43 Figure 3.5 Box and whisker plot of diametral tensile strength ………………….……..……......48    x List of Images Image 2.1 Tooth embedded in PVC pipe ………………………………………………………25 Image 2.2 Ultradent bonding clamp ……………………………………………………………26 Image 2.3 Ultradent Teflon mold ………………………………………………………………26 Image 2.4 Tooth with Ultradent bonding clamp and Teflon mold ……………..………………27 Image 2.5 Shear bond tester crosshead …………………………..……………..………………29 Image 2.6 PVS flexural strength stencil mold …….……………..……………..………………30 Image 2.7 PVS diametral tensile strength stencil mold …….……………..………...….………33           xi List of Symbols ± = plus minus ° C = degree Celsius α (Alpha) = coefficient of thermal expansion      xii List of Abbreviations GIC =Glass Ionomer Cement RMGIC = Resin Modified Glass Ionomer ART = Atraumatic Restorative Treatment SBS = Shear Bond Strength FS = Flexural Strength DTS = Diametral Tensile Strength PVC = Polyvinylchloride PVS = Polyvinylsiloxane MPa = Mega Pascal SD = Standard Deviation       xiii Acknowledgements I would like to thank my supervisor, Dr. Adriana Manso, for providing continued guidance and dedication to my Master’s project.  I would like to thank Dr. Manso for being very helpful and understanding in designing the project around my busy pediatric dentistry specialty program.    I would like to thank Drs. Karen Campbell and Ricardo Carvalho for being excellent committee members in providing helpful suggestions and feedback to make this a great research project.  I would like to thank GC America and 3M ESPE for providing all glass ionomer cements required for this project. I would like to thank the Faculty of Dentistry at UBC for funding this project. I would like to thank members of the biomaterials laboratory for their continued support and technical help.   Finally, I would like to thank my parents, family, friends and acquaintances for their continued moral support through this rollercoaster of a journey and helping me achieve my academic goals.    1 Chapter 1: Introduction Glass ionomer restorations are used in pediatric dentistry quite commonly to restore carious lesions.  Some of the indications suggested for glass ionomer use are: chemical bonding to tooth structure, less technique sensitivity when compared to composite resin, temporary measure for caries control, cases with difficult isolation, uncooperative child, treatment of hypomineralized teeth, and fluoride release to adjacent dental surfaces (American Academy of Pediatric Dentistry. Clinical Affairs Committee - Restorative Dentistry, 2012).  Additionally, glass ionomer cements have been used extensively in atraumatic restorative treatment (ART).  This technique is predominantly used in instances where due to lack of adequate patient cooperation and technical resources, the caries are excavated with hand instruments only and the cavity restored with glass ionomer restorative material (Berg, 1998; Randall & Wilson, 1999).    1.1 Glass Ionomer Cements:  Basic composition, chemical reaction and classification Glass ionomer cements (GICs) were introduced in the 1970s to provide better alternatives to silicate cements.  In simpler terms, GICs are formed by a reaction between organic acids and glass components (Anusavice, Shen, & Rawls, 2012). The powder component contains aluminum-fluorosilicate glass that dissolves upon interaction with polyacrylic acid in the liquid component.  The reaction releases calcium and aluminum ions that interact with the carboxylic acid groups. Calcium ions present in the hydroxyapatite of dental hard tissues, enamel and dentin, react with the carboxylic acid of glass ionomer cements, creating a chemical bond between the cement and the tooth structure (Anusavice et al., 2012).   The exact composition and powder to liquid ratio of the glass ionomer cements may vary depending on the manufacturer and intended use (Roberts & Berzins, 2015). However, certain   2 powder components such as silica, calcia, alumina and fluoride are always present.  The glass particle size may vary from 15 µm to 50 µm, and the powder can also contain additives such as barium, strontium or metal oxides to increase radiopacity of the material. The liquid component contains mainly copolymers of itaconic, maleic, or tricarboxylic acids. Addition of tartaric acid to the liquid component of glass ionomer provides several advantages to the glass ionomer setting reaction.  It allows the manufacturer to incorporate variety of glass particles in the powder component, affords better handling properties, decreases viscosity during initial setting reaction, lengthens working time and reduces the setting time (Anusavice et al., 2012). The conventional glass ionomer cements (GICs) set via an acid-base reaction between powder and liquid components.  To improve conventional GICs, resin-modified glass ionomer cements (RMGICs) were designed to have improved physical and mechanical properties.  They are also known as hybrid glass ionomer cements and they are formed from polyalkenoic acids, aluminum-fluorosilicate glasses, hydrophilic resin and a photoinitiator (Anusavice et al., 2012).  The addition of resin materials in RMGICs provides an increase in compressive and flexural strength compared to conventional GICs.  There is also an additional technical convenience in RMGICs, which can be first polymerized by light in its resin portion, thus, allowing a clinician the ability to partially control the setting reaction (Berg, 1998).  The acid-base reaction starts when the two parts (powder and liquid) are mixed together, and this chemical reaction continues even after the light curing procedure has been completed. More recently, high viscosity glass ionomer cements were developed to meet the clinical requirements for ART technique.  They have high powder to liquid ratio and smaller glass particle size resulting in a material with greater compressive strength without the need of light curing (Anusavice et al., 2012).     3 For a material to be considered a true glass ionomer, it is required to have an acid-base setting reaction between glass powders and polyacrylic acids (Anusavice et al., 2012; McLean, Nicholson, & Wilson, 1994).  Upon mixing of the two components, the acid from liquid component starts to dissolve glass particles in the powder.  This reaction occurs in a hydrophilic medium and releases calcium, aluminum, sodium and fluoride ions (Anusavice et al., 2012).  Initially, the carboxyl groups of polyacrylic acid chains are cross-linked by calcium ions. After the initial reaction, the glass ionomer matures over the period of 24 hours.  The glass ionomer maturation, more specifically, is a process where calcium ions linked to carboxyl groups of polyacrylic acid chains are replaced by aluminum ions. Although sodium and fluoride ions do not participate in the cross-linking with polyacrylic acids, sodium ions may replace hydrogen ions of carboxyl groups in the polyacrylic acid chains, while fluoride ions get dispersed in the set cement.  The reaction between powder and liquid results in formation of a silica gel that covers undissolved glass particles.  Figure 1.1 depicts structure of GICs after the initial setting reaction (Anusavice et al., 2012).   Figure 1.1: Structure of GICs.  Solid blue particle: unreacted glass particles; Light blue shaded area: gel with calcium and aluminum ions leached from glass/acid reaction. Figure from Phillips’ Science of Dental Materials (Anusavice et al., 2012).  Reprinted with permission from Elsevier Limited (Publisher).   4 Glass ionomer cements can be classified based on material composition or its intended clinical application. In general, all glass ionomer cements have similar basic components: the silicate glasses in the powder and the polyacrylic acids in the liquid. Resin-Modified Glass Ionomer Cements (RMGICs) have resin components added to the material to increase mechanical strength and enable light curing ability.  RMGICs such as Fuji II LC have additional hydrophilic monomers, hydroxyethyl methacrylated (HEMA), and photo-initiators to allow light polymerization (Khoroushi & Keshani, 2013). When metallic silver alloy particles are incorporated to the GIC powder to increase mechanical properties, they are called Metal Reinforced GICs. High viscosity GICs, a recent innovation in glass ionomer materials, have been developed by increasing the powder to liquid ratio and reducing the glass particle size to increase material’s viscosity, thus improving handling during restorative procedures and increasing mechanical properties after setting.  Table 1.1 shows the classification of glass ionomer cements based on their composition.   Table 1.1: Classification of GICs based on composition (Anusavice et al., 2012). GIC Type Composition Conventional GICs Silicate glass powder Polyacrylic acids Resin-modified Glass Ionomer Cements (RMGICs)  Silicate glass powder Polyacrylic acids  Water soluble methacrylate-based monomers Photoinitiators  Metal-modified Glass Ionomer Cements Silicate glass powder Polyacrylic acids  Metal fillers (silver alloy particles)  High Viscosity Glass Ionomer Cements Silicate glass powder Polyacrylic acids  Smaller glass particles sizes Higher P:L ratio   5  Glass ionomer cements are versatile with multiple applications in dentistry. They can be used for direct and indirect restorative procedures such as liners, bases, luting agents, core build-ups, pit and fissure sealants, temporary restorative materials, and as final restorations in some cases. Depending on the intended application, the GICs viscosity, handling, mechanical properties and composition may differ from one type to another.    Compomers are another type of restorative material that are used in pediatric dentistry (Berg, 1998). These materials are classified as polyacid modified resins.  These materials are essentially resin composites, however, after light curing, there is an acid-base glass ionomer-like reaction that occurs in presence of water.  Some of these materials are also capable of releasing fluoride like glass ionomers, however, the release is much lower than conventional GICs or RMGIs (Berg, 1998).  It should be noted that compomers are not true glass ionomers as the acid-base reaction is insufficient to allow material setting in absence of light.  Hence, compomers are essentially resin composites. 1.2 Glass Ionomer Cements:  Advantages and disadvantages  Glass ionomer cement restorations are able to chemically bond to enamel and dentin structures without the need of a bonding agent (Anusavice et al., 2012). To improve bonding, manufacturers have recommended the use of cavity conditioners prior to material placement to modify or partially remove smear layer, thus increasing the surface energy of the substrate.    Glass ionomers have been claimed as a “smart material” due to their ability to release fluoride after setting.  This has been suggested to reduce the risk of recurrent caries and aid in dentin and enamel demineralization (Anusavice et al., 2012; Berg, 1998).  Fluoride release has its peak in the first few days after restoration placement and continuously declines to 10% of original level in 3-4 weeks (Berg, 1998).  Studies have suggested that glass ionomer cements can   6 be recharged with fluoride when exposed to the oral environment (Arbabzadeh-Zavareh et al., 2012).  The fluoride ion source for recharge could come from topical sources (fluoridated toothpastes, oral rinses, varnishes) or from systemic fluoride intake, which may increase salivary and biofilm fluoride ion concentration. Current literature has also supported the use of glass ionomers as liners for indirect pulp capping procedures (Orhan, Oz, & Orhan, 2010), as it can aid in dentin remineralization and slow down or arrest the caries process.   The coefficient of thermal expansion of glass ionomer cement (α = 11 ppm K-1) is similar to that of enamel (α = 11.4 ppm K-1) and dentin (α = 8.3 ppm K-1) (Anusavice et al., 2012), this is advantageous in reducing marginal breakdown owing to thermal changes in the oral environment. Additionally, glass ionomer cements exhibit minimal volumetric changes and shrinkage or expansion during its setting reaction.  This is beneficial when compared to resin-based composites as the latter undergo polymerization shrinkage during setting, contributing to potential post-operative sensitivity and marginal breakdown over time (van Dijken, 2010).  Although GICs do not provide high bond strength when compared to dental adhesives, their characteristics provide excellent interfacial sealing, and are less affected by regional variances of the substrate (Hashimoto et al., 2000; Inoue et al., 2001).  Currently, multiple formulations and viscosities are available for different clinical applications of glass ionomer cements.  Resin modified light curable glass ionomers, for example, are less soluble in the oral environment, have higher fracture resistance and mechanical strength than conventional glass ionomers, which makes them a suitable restorative material. In general, glass ionomer cements require minimal resources for placement compared to other traditional restorative materials. Recently, high viscosity glass ionomer cements were developed to address the restorative needs in the atraumatic restorative technique (ART), where they may   7 have to serve as a long-term restorative material.  In this technique, hand instruments are used to excavate caries and high viscosity glass ionomers are placed in the cavity preparation.  It is a useful technique in areas with minimal infrastructure and resources (e.g. public health system, third world countries) (Frencken, 2010). Compared to resin based composites, glass ionomer cements (GICs) have low fracture strength, and low abrasion and wear resistance (Anusavice et al., 2012). It should be noted that some of the newer reinforced or resin modified glass ionomers have improved fracture resistance and long term clinical survival rate when compared with conventional GICs. However, the wear resistance still remains low. Even though multiple improvements have been made in the recent years, GICs are still not suitable for esthetic restorations due to inferior mechanical properties and poor aesthetics. Studies have reported mild initial pulpal reaction due to acidic component of glass ionomers, especially in mixes with low powder to liquid ratio (Anusavice et al., 2012; Nicholson & Czarnecka, 2008).  However, the effect is transient and resolves in days after.  The pulpal response from glass ionomer restorations is still lower than zinc oxide-eugenol based cements or adhesive resins.  Recently, human in vivo studies have shown that GICs are not irritating to the pulp, even when placed on very deep dentin (Souza, Aranha, Hebling, Giro, & Costa, 2006). During the setting reaction, GICs are sensitive to excess moisture and therefore some degree of isolation is required to reduce contamination.  In contrast, after setting reaction is complete, GICs are prone to desiccation and they can easily develop craze lines (Anusavice et al., 2012). Therefore, manufacturers recommend applying a protective coating to avoid surface crazing during maturation of the GIC reaction in the days that follow.    8 1.3  Glass Ionomer Cements: Restorative technique  The prepared tooth surface is treated with a cavity conditioner for 10-20 seconds and rinsed thoroughly with water. The conditioner is a weak polyacrylic acid that modifies or partially removes the smear layer and improves the bonding (Shafiei, Yousefipour, & Farhadpour, 2015). The dentin should ideally be kept slightly moist prior to glass ionomer material placement into the preparation.  During placement and setting reaction, isolation is critical as moisture contamination can compromise the setting reaction, bonding and mechanical properties. While setting, glass ionomer cements that are not light curable require protection of the exposed surfaces with a varnish or a coat to prevent desiccation and surface crazing.  Most of the maturation of GICs will occur in the initial 24 hours, however the process continues over several days (Anusavice et al., 2012).    1.4  Glass Ionomer Cements:  Clinical studies Glass ionomer cements, including resin modified glass ionomers (RMGIs) and conventional glass ionomer cements (GICs) have been used in restorative treatment of primary molars as an alternative to resin composites and amalgams.  The one-year clinical follow up of primary molar interproximal lesions restored with either Ketac Molar (conventional GIC) or Dyract (compomer) resulted in similar performance (Marks, van Amerongen, Borgmeijer, Groen, & Martens, 2000).  A systematic review of randomized and quasi-randomized controlled trials examined longevity of GIC restorations using the atraumatic restorative treatment (ART) approach compared with that of equivalent placed amalgam restorations (Mickenautsch, Yengopal, & Banerjee, 2010). The results suggested that there was no difference in longevity for restorations in primary teeth, and that the longevity of ART restorations was equal to or greater   9 than that of amalgam restorations for up to 6.3 years (Mickenautsch et al., 2010). Although short-term clinical studies have suggested similar success rates of glass ionomer and compomers, few published long-term studies show conflicting results. According to Welbury et al. 2000, a 42 month follow up of primary molar carious lesions restored with either conventional GICs or compomers showed significantly higher success rate of compomer restorations (Welbury, Shaw, Murray, Gordon, & McCabe, 2000).  The primary reasons suggested for GIC restoration failure included recurrent caries and loss of restorative material.  In contrast, a 2004 investigation comparing primary molars restored with RMGICs and compomers showed similar survival rates at 7-year follow up (Qvist, Laurberg, Poulsen, & Teglers, 2004). Overall, the above studies highlight that resin modified glass ionomers have similar survival rates when compared to compomers.  Given the ease of placement and lower technique sensitivity compared to compomers or composite resins, glass ionomers are gaining popularity in primary tooth restorative procedures. A recently published review and meta-analysis suggested that RMGICs should be recommended to restore cervical lesions in permanent dentition (Schwendicke et al., 2016).   However, restorations in load-bearing situations, conventional composites present the highest probability of survival for both permanent and primary teeth when compared to compomers or RMGICs (Schwendicke et al., 2016).  For restorations in primary teeth, factors such as cavity preparation size, location in the arch, child’s level of cooperation and timing of tooth exfoliation are considered when selecting appropriate restorative materials. The clinical success rate of RMGIC restorations was assessed in a systematic review yet few studies met the inclusion criteria (Sidhu, 2010). The study has suggested that RMGI restorations perform well clinically, do not have major retention issues and   10 have low incidence of recurrent caries.  However, such restorations do show signs of marginal and surface breakdown along with surface discoloration over time (Sidhu, 2010).  1.5  Glass Ionomer Cements:  In vitro studies  There are several in vitro studies that have examined bonding performance of glass ionomer cements to dental structures using shear bond strength, microtensile bond strength and microleakage methods.  For mechanical properties of glass ionomer cements, published studies have examined flexural strength, diametral tensile strength, microhardness, compressive strength and many other performance measures (Anusavice et al., 2012; De Munck et al., 2005).  Majority of these studies have evaluated material’s performance at 24 hours, and only few studies have assessed mechanical properties and bond strengths after long-term storage.   An in vitro study of mechanical properties of glass ionomers reported no significant change in compressive strength, modulus of elasticity and diametral tensile strengths for several glass ionomers over a period of one year storage in water at 37°C (Mitra & Kedrowski, 1994). The authors suggested that given the fairly constant physical properties of GICs in a wet environment over time, glass ionomer restorations could be considered as a viable long term option in dentistry (Mitra & Kedrowski, 1994).  Another study reported that presence of water around glass ionomer cements can cause softening of the cement’s outer layer over 41 days when compared to a dry storage environment (Dupuis, Moya, Payan, & Bartala, 1996). The softening effect was limited to the surface (up to 0.6 mm deep) and the core microhardness remained unaffected. In contrast, another study looked at the effect of water aging on several restorative glass ionomer cements (De Moor & Verbeeck, 1998). Their data showed different behavior depending on the material. Some of the GICs   11 evaluated exhibited no changes in surface hardness over a 4-month period when subjected to a water bath; however few other GICs showed a decrease in surface hardness when subjected to long-term water storage. The authors attributed such changes in surface hardness due to inhibition of secondary reaction at the surface in presence of excess water and not due to the glass ionomer type (De Moor & Verbeeck, 1998).  An in vitro study examined the effects of 10-month water aging on shear bond strength of a resin modified glass ionomer cement, Vitrebond, to bovine dentin, with or without thermocycling (Mitra, 1991). The author found no significant changes in shear bond strengths (SBS) on aged samples, regardless of thermocycling, which is suggestive of stable bond strength.  Although the results of the study seem very promising relating to glass ionomer-dentin bond stability, it only concerns one type of glass ionomer cement, and it may not be applicable to other commercially available glass ionomer cements. The majority of in vitro studies that have been conducted are mainly short term. The effect of environment and time has not been studied extensively for glass ionomers bonded to tooth structure.  An in vitro study assessing shear bond strength of GICs also speculated that the bonding interface between GIC and dentin would not be greatly influenced by the 24 hours water storage (Carvalho, van Amerongen, de Gee, Bonecker, & Sampaio, 2011). However, it should be noted that the authors only assessed immediate SBS. Water storage and aging time greater than 24 hours was not considered in their study.  More recently, one study investigated the aging effect on SBS of RMGICs bonded to dentin and stored in water for 6 and 12 months (Dursun, Le Goff, Ruse, & Attal, 2013). The results demonstrated that SBS increased significantly for 6 and 12 month groups when compared to 24 hour storage. However, for chlorhexidine treated groups, the aging did not improve SBS   12 (Dursun et al., 2013). This important finding would need further investigation. From limited available studies, the bond durability of glass ionomer to tooth structure in a wet environment over time remains questionable.  1.6 Glass Ionomer Cements: Methodologies for in vitro studies  In vitro studies can assess the performance of dental restorative materials at multiple levels such as bond strength, mechanical properties and leakage susceptibility at the interfaces (De Munck et al., 2005).  More specifically, shear bond strength and microtensile bond strength to hard dental tissues (enamel or dentin), and microleakage studies assess bonding and sealing capability of dental material to tooth structure.  Mechanical property tests are designed to evaluate the material strength by itself and they include flexural strength, diametral tensile strength, compressive strength, micro hardness and several others (Anusavice et al., 2012).     When investigating dental materials bonding performance in vitro, there are several factors that may affect the outcome. Factors such as tooth selection, tooth preparation, tooth structure (enamel vs. dentin), bonding protocol, sample fabrication, material handling, testing parameters, specimen handling, specimen storage, and operator skill influence data and may produce variability among different investigators (Sirisha, Rambabu, Ravishankar, & Ravikumar, 2014; Sirisha, Rambabu, Shankar, & Ravikumar, 2014). Shear bond strength testing is used to assess bond strength between two substrates, more commonly a dental material and tooth structure.  During testing, load is applied at the interface until failure to determine shear bond strength.  Similar to other testing methods, the major limitation of the SBS method is that it is inaccurate at determining absolute bond strength (Van Noort, Cardew, Howard, & Noroozi, 1991; Van Noort, Noroozi, Howard, & Cardew, 1989).  Shear bond strength can however, be   13 used as a relative comparative factor when evaluating multiple materials in the same laboratory setting.  However, it may not necessarily yield similar absolute results when comparing data with other investigators (De Munck et al., 2005).  Therefore, the use of such a test requires appropriate controls and caution with data interpretation.  Flexural strength and diametral tensile strength tests are valid methods for assessing mechanical properties of materials, including glass ionomer cements (Anusavice et al., 2012; Darvell, 2009). Both mechanical tests have been suggested to be reproducible.  For a test of flexural strength, a rectangular bar of set dimension is subjected to a three point bending test. For diametral compression strength test, a cylindrical disc is subjected to load until failure (Anusavice et al., 2012).  Even though the recommended ISO testing specifications must be followed, the testing conditions and protocols vary slightly from one laboratory to another, thus making inter-laboratory data reproducibility challenging. In a laboratory setting, typical methodology for testing bonding performance of a new glass ionomer cements may involve 37°C distilled water storage for 24 hours prior to testing, which has been the most common protocol from the published literature (De Munck et al., 2005). Although in principle, these tests fast ways to directly compare multiple materials, they do not assess stability and effects of aging on the glass ionomer-tooth bonded interfaces over time.  It is well known that intraoral fluid provides a dynamic environment with multiple factors that constantly challenge the bonding interface between tooth and the restorative material (De Munck et al., 2005; Hashimoto et al., 2000). Therefore, the long-term bond durability between tooth and glass ionomer cements remains largely unexplored.  It becomes clinically relevant to conduct long-term aging studies when comparing multiple commercially available glass ionomer cements.   14 1.7  Glass Ionomer Cements: In vitro artificial aging  The intra oral environment provides constant challenges to any restorative material due to the presence of ions, enzymes, bacteria, pH, and temperature fluctuations.  This may result in material breakdown, discolouration, dissolution and bond degradation over time, thereby jeopardizing mechanical properties (Anusavice et al., 2012; De Munck et al., 2005).   To simulate a clinically relevant environment, laboratory studies use various biodegradation models for aging, which may provide thermal, chemical, mechanical, and/or biochemical challenges to the specimens (De Munck et al., 2005).  Some of the methods of artificial aging for dental material specimens include storage in media, thermal cycling procedures and/or occlusal loading. The major advantage of incorporating aging component to any dental materials study is to assess changes in their properties over time.    Due to the importance of long-term evaluations in laboratory studies, researchers have used different methods and media to artificially age specimens (De Munck et al., 2005).  Ideal storage media may have ionic and enzymatic components to mimic saliva.  The enzymatic component may include proteinases that have been speculated to cause hydrolytic degradation of collagen fibrils, thereby, compromising restorative dentin-resin bonding interfaces (Pashley et al., 2004).  However, it is difficult to standardize saliva composition as it may vary from one individual to another. Studies employing artificial saliva have mostly used an ionic composition to mimic oral environment.  These solutions do not incorporate enzymes and plaque biofilm that is present in a real life scenario (Cruz, Bonini, Lenzi, Imparato, & Raggio, 2015; Geramipanah, Majidpour, Sadighpour, & Fard, 2013; Pashley et al., 2004). Alternatively, several studies have used distilled water as storage media to evaluate changes in material properties over time (De Munck et al., 2005).     15  The majority of in vitro tests, whether assessing bond performance or mechanical properties, are not accurate at quantifying absolute material properties.  Therefore, adding an aging component can provide investigators with valuable information about the qualitative changes that a material may undergo over time.  One of the important aspects of extrapolating from in vitro studies is assessing stability of a material in a variety of simulated oral conditions.  1.8 Study rationale and hypotheses  Multiple studies published have demonstrated the degradation of resin-dentin bonds on resin composite restorations over time (Manuja, Nagpal, & Pandit, 2012; Spencer, Ye, Misra, Goncalves, & Laurence, 2014).  However, there is insufficient evidence in the literature to assess the impact of aging on bond strengths and mechanical properties of glass ionomer cements.  The lack of conclusive data propels one to investigate the influence of long-term storage on the bond strength and mechanical properties of restorative glass ionomer cements. The aim of the present study is to evaluate the effect aging on shear bond strengths to dentin, flexural strengths and diametral tensile strengths of four restorative glass ionomer cements: Fuji II LC (GC America), Equia (GC America), Ketac Nano (3M/ESPE), Ketac Molar (3M/ESPE). The effect of storage media, distilled water or artificial saliva, on flexural strength after 2-month storage will also be evaluated.   The research questions and null hypotheses proposed in this study are:  1. Does aging affect shear bond strength to dentin? Is shear bond strength of four restorative glass ionomers different? Null Hypothesis: Shear bond strength to dentin is not affected by storage time and material.    16 2. Does flexural strength and diametral tensile strength of materials change after aging? Is flexural strength and diametral tensile strength of materials different? Null Hypothesis: Flexural strength and diametral tensile strength are not affected by storage time and material.  3. Does type of storage (water vs. saliva) affect flexural strength of materials?  Null Hypothesis: Flexural strength is not affected by storage time, storage media and material.      17 Chapter 2:  Materials and Methods 2.1 Tooth collection This study was approved by the Clinical Research Ethics Board of the University of British Columbia, ID H14-02189.  Non-carious human permanent molars with intact crowns were collected from two Oral and Maxillofacial Surgery clinics in Vancouver, British Columbia.  The teeth were extracted only for potential pathological reasons and not for the purposes of this study.  The teeth were stored in a jar containing 0.1% thymol solution immediately after extraction.  Once collected from the clinics, the teeth were stored at 4°C in the refrigerator.  Prior to use, the teeth were cleaned with hand instruments to remove any tissue debris and calculus.     2.2 Materials  Four restorative glass ionomer cements were used in this study: Fuji II LC (GC America Corp.), Equia (GC America Corp), Ketac Nano (3M/ESPE), and Ketac Molar (3M/ESPE). The materials classification and setting reaction, composition, and manufacturer’s lot numbers are listed in Table 2.1.  The corresponding dentin conditioners or primer required for each material are listed in Table 2.2 with their purposes, composition and manufacturer’s lot numbers. .           18 Table 2.1:  Restorative Glass Ionomer Cements used in this study. Material Classification Composition Manufacturer Fuji II LC (Capsule) Resin Modified Glass Ionomer  Light curable and auto curable. Liquid:  Distilled Water, Polyacrylic acid, 2-Hydroxyethylmethacrylate, Urethanedimethacrylate, Camphorquinone (<1%).  Powder: Fluoro Aluminosilicate glass GC Corp. Lot #s: 1410221, 1409031, 1408018 Equia (Capsule) Reinforced Glass Ionomer  Auto curable only Liquid:  Water, Polyacrylic Acid, polybasic carboxylic Acid  Powder:  Fluoro Aluminosilicate Glass GC Corp. Lot #s: 1408082, 1312121, 1404111, 1405871 Ketac Nano (Quick Mix Capsule) Resin Modified Glass Ionomer  Light Curable only Aqueous paste: Silane-treated ceramic, Silane-treated silica, water, HEMA, acrylic/itaconic acid copolymer.   Non aqueous paste: silane-treated glass, silane-treated ZrO2 silica, silane treated silica, PEGDMA, HEMA, Bis-GMA, TEGDMA.  3M ESPE Lot #s: N637872, N511985 Ketac Molar (Quick Aplicap) Reinforced Glass Ionomer  Auto curable only Liquid:  Polycarboxylic acid, tartaric acid, water.  Powder:  Alumino-fluoro-silicate glass, spray-dried polycarbonate acid. 3M ESPE Lot #s: 518442, 520397, 568914         19 Table 2.2:  Dentin conditioners and primers used in this study. Material Purpose Composition Manufacturer GC Cavity Conditioner Dentin surface conditioner prior to Fuji II LC and Equia placement 20% polyacrylic acid, 3% AlCl3   GC Corp. Lot #: 1312091, 1303121 Ketac Conditioner Dentin surface conditioner prior to Ketac Molar placement 25% Polyacrylic acid 3M ESPE Lot #: 477305 Ketac Primer Light curable primer prior to Ketac Nano placement Water, HEMA, acrylic-itaconic acid copolymer, photo initiators.   3M ESPE Lot #: N573827  2.3  Artificial Saliva formulation  The formulation for artificial saliva was based on previous long-term aging studies from Dr. David Pashley research group (Pashley et al., 2004). The artificial saliva was prepared fresh for the experiment and the final pH was adjusted to 7.0 with 1 N NaOH solution. It was kept in the refrigerator until used at 4ºC.  The composition of artificial saliva used in this study is shown in Table 2.3. Table 2.3  Artificial Saliva composition (Pashley et al. 2004). Artificial Saliva Composition  CaCl2                      (0.7 mmoles/L) MgCl2·6H2O           (0.2 mmoles/L) KH2PO4                  (4.0 mmoles/L) KCl                         (30 mmoles/L) HEPES buffer         (20 mmoles/L) pH 7.0   20 2.4 Experimental design overview  2.4.1 Shear bond strength   Sample size calculations (Appendix A) using mean shear bond strengths and standard deviations from a pilot study resulted in a minimum sample size of 12 specimens per group in order to detect a statistically significant difference in shear bond strength to dentin, with 80 % power and 95 % confidence (SigmaPlot 11, Systat Software Inc.).  An overview of the experimental design for the shear bond strength experiment employed in this study is outlined in Figure 2.1.  2.4.2 Flexural strength study #1 and diametral tensile strength   Sample size calculations (Appendix A) using a pilot study resulted in a minimum sample size of 12 (FS) and 3 (DTS) per group in order to detect a statistically significant difference between groups with 80 % power and 95 % confidence (SigmaPlot 11, Systat Software Inc.). Figures 2.2 and 2.3 provide an overview of flexural strength study #1 and diametral tensile strength studies respectively.    2.4.3 Flexural strength study #2: Artificial saliva vs. water storage  Sample size calculations (Appendix A) using mean flexural strength and standard deviations from a pilot study resulted in a minimum sample size of 10 per group in order to detect a statistically significant difference between groups (saliva vs. water) with 80 % power and 95 % confidence (SigmaPlot 11, Systat Software Inc.). Figure 2.4 provides an overview of the flexural strength study #2 comparing storage media.     21                       80 Teeth Fuji II LC 20 Teeth 2 specimens bonded per tooth Test 1st specimen at 24 hours per tooth Test 2nd specimen at 6 months per tooth Ketac Nano 20 Teeth 2 specimens bonded per tooth Test 1st specimen at 24 hours per tooth Test 2nd specimen at 6 months per tooth Ketac Molar 20 Teeth 2 specimens bonded per tooth Test 1st specimen at 24 hours per tooth Test 2nd specimen at 6 months per tooth Equia 20 Teeth 2 specimens bonded per tooth Test 1st specimen at 24 hours per tooth Test 2nd specimen at 6 months per tooth Figure 2.1:  Shear Bond Strength to Dentin experiment overview.   22    Fuji II LC 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Ketac Nano 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Ketac Molar 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Equia 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Fuji II LC 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Ketac Nano 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Ketac Molar 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Equia 30 specimens Test 15 specimens at 24 hr. Test 15 specimens at 6 months Figure 2.2:  Flexural Strength Study #1 overview.  Note that specimens were aged in Artificial Saliva only. Figure 2.3:  Diametral Tensile Strength experiment overview.   Note that specimens were aged in Artificial Saliva only.   23 	  Fuji II LC 40 specimens AT 24 hours: Test 10 water aged specimens Test 10 saliva aged specimens  AT 6 months: Test 10 water aged specimens Test 10 saliva aged specimens 20 specimens stored in WATER 20 specimens stored in SALIVA Ketac Nano 40 specimens AT 24 hours: Test 10 water aged specimens Test 10 saliva aged specimens  AT 6 months: Test 10 water aged specimens Test 10 saliva aged specimens 20 specimens stored in WATER 20 specimens stored in SALIVA Ketac Molar 40 specimens AT 24 hours: Test 10 water aged specimens Test 10 saliva aged specimens  AT 6 months: Test 10 water aged specimens Test 10 saliva aged specimens 20 specimens stored in WATER 20 specimens stored in SALIVA Equia 40 specimens AT 24 hours: Test 10 water aged specimens Test 10 saliva aged specimens  AT 6 months: Test 10 water aged specimens Test 10 saliva aged specimens 20 specimens stored in WATER 20 specimens stored in SALIVA Figure 2.4:  Flexural Strength Study #2 in Artificial Saliva vs. Water Study overview   24 2.5 Shear bond strength to dentin  2.5.1 Tooth preparation  The tooth specimens were prepared on the day of use for bonding.  The occlusal surface of each molar was ground flat to mid-coronal dentin using a 180-grit silicon carbide (SiC) paper under copious water-cooling, in a wheel spinning at 350 rpm (Unipol-1210 Precision Lapping/Polishing machine).  Teeth were inspected under a light microscope for remaining enamel on the surface and for pulp exposure. In case of pulp exposure after grinding, teeth were discarded.  In case of remaining enamel, tooth surface was additionally grounded. Each tooth was fixed in a round polyvinylchloride (PVC) mold with orthodontic acrylic resin (Caulk Orthodontic Acrylic Resin, Dentsply) (Image 2.1).  The tooth specimen embedded in PVC mold was then re-polished with a 180-grit SiC paper under water cooling to remove any acrylic resin debris.  The tooth specimen was re-inspected for pulp exposures and acrylic resin debris and subsequently stored in distilled water at room temperature until ready for use. Immediately before bonding, the flat dentin surface was manually polished with low pressure using a 320-grit sandpaper in clockwise motion for 30 seconds and then counter clockwise motion for additional 30 seconds to create a standardized smear layer.  Subsequently, the specimens were subjected to an ultrasonic bath in distilled water for 2 minutes to remove gross particulate matter.   The dentin surface was rinsed with copious amounts of water and gently air-dried.   2.5.2 Dentin conditioning Manufacturer’s recommended protocols for dentin conditioning were followed (Table 2.4).  For a prepared tooth specimen, the dentin surface was treated with the recommended dentin conditioner following the recommended time, rinsed with copious amounts of water for 20 seconds, and blotted dry as per manufacturer’s instructions for Fuji II LC, Equia and Ketac   25 Molar glass ionomer cements. For Ketac Nano, Ketac primer was applied to the dentin surface for 15 seconds, air thinned until a shiny dentin surface was obtained and light cured for 10 seconds on high setting (900 to 1000 mW/cm2). Table 2.4: Dentin surface conditioning protocols applied as per manufacturer’s recommendation. Material Protocol Fuji II LC • GC Cavity Conditioner applied for 10 seconds, rinsed thoroughly for 20 seconds and blot dried. Equia • GC Cavity Conditioner applied for 10 seconds, rinsed thoroughly for 20 seconds and blot dried. Ketac Nano • Ketac Primer applied, left on surface for 15 seconds, air dried for 10 seconds and then light cured for 10 seconds. Ketac Molar • Ketac Conditioner applied for 10 seconds, rinsed thoroughly for 20 seconds and blot dried.   Image 2.1:  Tooth with flat occlusal dentin surface embedded in PVC pipe with orthodontic acrylic resin.           PVC pipe  Flat Dentin Surface   Acrylic Resin    26  2.5.3 Bonding procedures  Each pre conditioned dentin surface was secured in the Ultradent bonding clamp (Image 2.2) with the Ultradent bonding Teflon mold insert with 2.38 mm diameter (Image 2.3) (Ultradent Products, Inc.). The internal surface of the mold was lightly coated with petroleum jelly to avoid bonding of the glass ionomer cement to the mold. The Teflon mold was cleaned between each bonding procedure, and the same mold was not used for different materials. Subsequently, the mold was placed in tight contact with the dentin surface to provide a scaffold for glass ionomer material placement (Image 2.4).    Image 2.2:  Ultradent Bonding Clamp (Ultradent Products Inc.)  Image 2.3: Ultradent Teflon Mold for bonding cylinders to tooth structure, with inner diameter of 2.38 mm.   2.38 mm diameter   27  A.    B.  C.  Image 2.4:  Tooth assembled with Ultradent Jig apparatus (A), Teflon mold and bonding clamp views (B and C).   Each pre-treated dentin specimen secured in the Ultradent Jig was individually bonded with one of the glass ionomer cements.  For Fuji II LC and Equia, each GIC capsule was activated and triturated for 10 seconds in an amalgamator at 4400 rpms (Henry Schein Touch Pad Amalgamator TP-103).  For Ketac Molar, each aplicap was activated for 4 seconds and then triturated for 10 seconds in an amalgamator at 4400 rpms (Henry Schein Touch Pad Amalgamator TP-103).  Ketac Nano capsule did not require trituration and was dispensed as per manufacturer’s instructions.  Each material was carefully syringed into the Teflon mold with the   28 manufacturer supplied capsule applicator and gently packed.  For Ketac Nano and Fuji II LC specimens, the material was immediately light activated for 60 seconds on high setting (900 to 1000 mW/cm2) using a LED light-curing unit (Bluephase 20i, Ivoclar Vivadent).  For the self-cured GICs (Equia and Ketac Molar), the recommended self-curing time of 10 minutes was allowed. The specimen was then carefully removed from the Teflon mold. If any excess or flash was present around the cylinder on the dentin surface, it was gently removed with a sharp #15 scalpel blade.  The remaining dentin surface was rewetted with a moist tissue paper (except for Ketac Nano) and the dentin specimen was returned to the Ultradent Jig bonding clamp for the bonding procedure of the second cylinder on the same dentin surface, performed in a similar fashion as previously described.    2.5.4  Specimen storage Immediately after bonding two cylinders to the dentin surface, the specimens were stored in artificial saliva (Table 2.3) at 37°C (Precision Scientific Co. Model 17 incubator) until testing.  Any additional protective coating recommended by some of the manufacturers was not applied. The artificial saliva was systematically replaced with fresh solution every 60 days. 2.5.5  Shear bond strength test For each dentin surface, one of the glass ionomer cylinders was randomly selected and tested at 24 hours, and the remaining cylinder was tested after 6 months of storage.  The shear bond strength tests were performed using the Bisco Shear Bond tester (Bisco Inc., IL, USA) with a crosshead speed of 1 mm/minute (Image 2.5).  The shear bond strength values were calculated by dividing the load at failure (Newton) by the area of the cylindrical cross-section (4.45 mm2), and expressed in Mega Pascal (MPa). After testing, each glass ionomer cylinder was examined   29 under light microscope (10x magnification, Carl Zeiss Jenna) to inspect for voids at the bonding surface. Specimens with voids or defects at glass ionomer bonded surface were eliminated. Each corresponding dentin surface was examined under 10x magnification in a powered light microscope (Carl Zeiss Jena) to determine mode of failure.  This was classified as adhesive (failure occurred between dentin and glass ionomer cement), cohesive (failure occurred exclusively in the glass ionomer cement), or mixed (failure have a mixed mode, including adhesive and cohesive in the same tested surface).  Image 2.5:  Shear Bond Tester crosshead shape. 2.6 Flexural strength study # 1 – Artificial saliva storage  Flexural strength bars used for both flexural strength studies (#1 and #2) were fabricated and tested following the same protocol, according to the specification in the International Standard ISO 9917-2:2010.  2.6.1 Flexural strength specimen fabrication  Acrylic resin bars with the specimen dimensions (25 mm x 2 mm x 2 mm) were fabricated in the laboratory as templates.  The acrylic bars were then used to produce a polyvinyl siloxane (PVS) stencil mold, using light and medium body PVS combined (Image 2.6). Those stencil molds with the exact dimensions were then used to fabricate glass ionomer bars (25 mm x 2 mm x 2 mm) for the flexural strength test.     30          Image 2.6:  Polyvinyl siloxane (PVS) mold used to fabricated rectangular glass ionomer cement bars of 25 mm x 2 mm x 2 mm for flexural strength testing.    A glass slab covered with plastic film was used as a solid and flat base for holding the PVS stencil mold during the insertion of the material. Each glass ionomer cement capsule (Fuji II LC, Equia, and Ketac Molar) was activated and triturated for 10 seconds in an amalgamator at 4400 rpms (Henry Schein Touch Pad Amalgamator TP-103). Ketac Nano, however, is the only glass ionomer cement used in the study that did not require trituration prior to dispensing.  Immediately after trituration, glass ionomer cement was carefully dispensed into the mold and any excess was gently removed.  Another piece of clear plastic film was overlaid on the PVS mold already filled with the GIC material followed by a glass microscopic slide. Gentle finger pressure was applied to completely pack the material into the mold and to obtain a smooth flat surface.  When recommended by the manufacturer (Fuji II LC and Ketac Nano), the material was light cured using a LED light-curing unit (Bluephase 20i, Ivoclar Vivadent) for 120 seconds at 900 to 1000 mW/cm2 output.  Specimens of self-cured glass ionomer cement (Equia and Ketac Molar), were kept under pressure created by the glass slab weight then allowed to set for 20 minutes prior to removal from the mold. The bar was then manually polished under finger   31 pressure on wet 800 grit sandpaper, followed by wet 1500 grit sandpaper until the surface was flat, smooth and devoid of any defects.  The final dimensions of the flexural bars were measured with a Mastercraft® digital caliper to comply with ISO 9917-2:2010 specified dimensions described above.  The bars were examined under 10x magnification (Carl Zeiss Jenna) microscope to exclude any specimens with surface defects. In study #1, the specimens were immediately placed into a vial containing artificial saliva and stored at 37°C.  No additional protective coating was applied on the GIC surfaces prior to storage. In total, 30 flexural bars were fabricated.  The artificial saliva was replaced every 60 days. 2.6.2  Flexural strength testing  Flexural strength bars were subjected to a three point bending test (Figure 2.5) after 24 hours and 6 months storage in a universal testing machine (Shimadzu AutoGraph AGS-X Series, Shimadzu Corporation, Kyoto, Japan) with a cross-head speed of 1 mm/min until failure. The supports were set at 20 mm apart and load applied at midpoint of the specimen (Figure 2.5).  The flexural strength was calculated based on the formula in figure 2.5 (Anusavice et al., 2012).  The specimens were then examined under a light microscope using 10x magnification (Carl Zeiss Jenna) at the fracture interface to check for presence of any defects.  Specimens with obvious voids at the failure interface were discarded.     32  Figure 2.5:  Schematic representation of a three point bending test with equation for calculating flexural strength (Anusavice et al., 2012).   Reprinted with permission from Elsevier Limited (Publisher).  2.7 Flexural strength study #2 – Artificial saliva and water storage  An additional study was conducted to examine the effect of storage media, either artificial saliva or distilled water, on the flexural strength of the materials after aging. For each glass ionomer cement used previously, 40 additional flexural strength bars were fabricated according to the protocol described in section 2.6. For each material, the flexural strength bars were randomly divided, 20 specimens allocated for artificial saliva storage, and 20 specimens for distilled water storage, both at 37°C.  After 24 hours storage, half of the specimens in each storage media were tested following the same testing protocol outlined in section 2.6.3. The remaining samples were then tested after 2-month storage.  All specimens with obvious voids at the failure interface were discarded from the final sample.  2.8 Diametral tensile strength test  Cylindrical discs were fabricated following thickness: diameter ratio of 1:2 for diametral tensile strength tests. Discs measuring 4 mm diameter and 2 mm thick were produced based on the proposed dimensions from previous studies (Daifalla & Mobarak, 2015; Mitra & Kedrowski, 1994)    33  2.8.1 Diametral tensile strength specimen fabrication   An initial resin composite disc with the expected dimensions for the diametral tensile strength discs was fabricated in the laboratory as a template to produce the molds.  The disc was then used to produce a polyvinyl siloxane (PVS) stencil mold, using light and medium body PVS combined (Image 2.7). Those stencil molds with dimensions were then used to fabricate glass ionomer discs (4 mm diameter x 2 mm thickness) for the diametral tensile strength test.    Image 2.7:  PVS stencil mold to fabricate disc with dimension of 4 mm diameter and 2 mm height.   The PVS stencil mold was placed over glass slab and clear plastic film.  Fuji II LC capsule was activated and triturated for 10 seconds in an amalgamator at 4400 rpms (Henry Schein Touch Pad Amalgamator TP-103).  The material was placed in the PVS mold and excess material was removed from the mold.  A clear plastic film was overlaid on both sides of the PVS mold followed by a glass slide.  Gentle finger pressure was applied to completely pack the material in the mold and to obtain a smooth flat surface.  The material was light cured on high setting (900 to 1000 mW/cm2) using an Ivoclar Vivadent Bluephase 20i light curing unit for 60 seconds.  The disc was removed from the mold and immediately wet sanded with finger pressure on a wet 800 grit and 1500 grit sandpaper until the surface was smooth and devoid of any defects.  The disc dimensions were measured with a Mastercraft® digital caliper to comply with   34 the 4 mm diameter and 2 mm thick dimensions.  The disc was immediately placed in a vial containing artificial saliva and stored at 37°C.  In total, 30 discs were fabricated for each material.  The artificial saliva was replaced every 60 days.  Ketac Nano diametral tensile strength discs were fabricated and stored similarly as above, except that Ketac Nano Quick Mix capsule did not require trituration prior to dispensing.  Equia diametral tensile strength discs were fabricated and stored similarly as above except the material does not required light curing and it was allowed to self-cure.  The material was overlaid with a glass slab to provide extra weight and allowed to set for 20 minutes prior to removal from the mold. Ketac Molar discs were fabricated and stored similarly as above except: the capsule was activated for 4 seconds and triturated for 10 seconds, and material was allowed to set for 20 minutes.    2.8.2  Diametral tensile strength testing   Specimens were subjected to a diametral tensile strength test with load applied parallel to the disc diameter (Figure 2.6) after 24 hours and 6 months artificial saliva storage using a universal testing machine (Shimadzu AutoGraph AGS-X Series, Shimadzu Corporation, Kyoto, Japan) with a cross head speed of 1 mm/min until failure.  The diametral tensile strength was calculated based on the formula in figure 2.6 (Anusavice et al., 2012).  After testing, the specimens were examined under 10x magnification (Carl Zeiss Jenna) microscope at the fracture interface to check for defects.  Specimens with obvious voids at the failure interface were discarded from the final sample.   35  Figure 2.6:  Schematic representation of a diametral compression test with the equation for calculating diametral tensile strength (Anusavice et al., 2012).  Reprinted with permission from Elsevier Limited (Publisher).  2.9 Statistical analysis  For shear bond strength, the data was subjected to a mixed effect ANOVA analysis with tooth specimen as a random factor to control for repeated measurements on each tooth.  Statistical significance was examined at p<0.05.  Post-hoc Tukey test was used to evaluate interactions between materials and time.   For flexural strength study #1 and diametral tensile strength studies in artificial saliva, the data were subjected to a two-way ANOVA followed by a post-hoc Tukey test to evaluate interactions between materials and time.  Statistical significance was examined at p<0.05. For the flexural strength study #2 in water and saliva storage, the data was subjected to a three-way ANOVA with time, storage media and materials as comparative factors, followed by a post-hoc Tukey tests to evaluate interactions among them.  Statistical significance was examined at p<0.05.        36 Chapter 3: Results 3.1 Shear bond strength to dentin The mode of failure analysis (Table 3.1) demonstrated that while the majority of the failure modes for Ketac Nano were adhesive, for the remaining materials, the most frequent failure mode was the mixed failure. Conversely, cohesive failures were not found for any tested material and storage time.  The shear bond strength to dentin, in Table 3.2, shows descriptive statistics with post-hoc pairwise statistical comparison results indicated as superscript letters. It was determined that shear bond strength did not demonstrate statistically significant differences among the four materials used in this study, after 24-hour and 6-month storage.  Thus, the first null hypothesis that there is no difference among materials in each storage period is accepted. After 6 months of artificial saliva storage, Ketac Nano presented a significant decrease in shear bond strength to dentin (p = 0.0046). However, for the remaining materials tested, the storage did not show significant effect in shear bond strengths.  Thus, the null hypothesis that there is no difference in shear bond strength after 6-month storage can be partially rejected. Table 3.1: Failure mode of SBS tested samples, classified as adhesive, cohesive, or mixed. Material Failure Mode at 24hrs Failure Mode at 6 months  Adhesive Cohesive Mixed Adhesive Cohesive Mixed Ketac Nano 15 0 3 18 0 0 Ketac Molar 4 0 10 4 0 10 Fuji II LC 4 0 10 5 0 9 Equia 0 0 15 2 0 13       37 Table 3.2: Shear bond strength (SBS) to dentin at 24 hours and 6 months in saliva storage.   Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n). Material 24 hours storage 6 months storage Ketac Nano 6.8 (4.8)a,A n=18 4.4 (3.7) b,A n=18 Ketac Molar 4.0 (1.8) a,A n=14 3.6  (2.1) a,A n=14 Fuji II LC 7.6 (4.2) a,A n=14 7.0 (5.7) a,A n=14 Equia 4.8 (2.1) a,A n=15 5.7 (3.2) a,A n=15 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.  Figure 3.1 shows box and whisker plots for the shear bond strength data.  The box plot appears skewed as the medians are not near the middle of interquartile range.  Relatively larger interquartile ranges for Ketac Nano and Fuji II LC data suggest widely distributed data for shear bond strength.  The circles past the whiskers (Figure 3.1) suggest possibility of outliers in the Ketac Molar (24 hour) and Ketac Nano (6 month) data.    38  Figure 3.1:  Box and whisker plot of shear bond strength for four glass ionomers.  Note that circles on the plot represent possible outliers in the Ketac Molar (24 hr.) data and Ketac Nano (6 month) data.   3.2 Flexural strength study # 1 - Artificial saliva storage   Flexural strength results suggested increases that were statistically significant after 6 months of artificial saliva aging for all materials (p < 0.001) with an overall statistical power of 1.000.  At 24 hours and 6 months individual analysis, Fuji II LC showed significantly higher flexural strength when compared to the other three materials (p < 0.001).  Ketac Nano had significantly higher flexural strength at 24 hours and 6 months when compared to Ketac Molar (p < 0.001).  However, no statistically significant difference was found in flexural strength of Ketac   39 Molar and Equia for both storage times.  Table 3.3 shows the flexural strength results with descriptive statistics and post-hoc pairwise statistical comparison indicated as superscript letters.  Thus, the null hypothesis for no differences in materials and storage times must be rejected.   Table 3.3:  Flexural strength at 24 hours and 6 months in artificial saliva storage.   Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n). Material Storage period % Increase 24 Hours 6 Months Ketac Nano 16.0 (3.8)a,B n=15 30.2 (15.6)b,B n=13 88.8% Ketac Molar 6.2 (1.2)a,C n=14 18.3 (4.8)b,C n=14 195.2% Fuji II LC 23.2 (7.7)a,A n=15 50.4 (6.0)b,A n=15 117.2% Equia 11.5 (1.5)a,BC n=15 21.8 (4.3)b,C n=12 89.6% Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.   Figure 3.2 shows a box plot of the flexural strengths of four materials in artificial saliva at 24 hours and 6 months.  It appears that Ketac Molar and Fuji II LC flexural strength data at 24 hours may have outliers (dots beyond whiskers in figure 3.2).  Ketac Nano appears to have some skewness and large distribution of flexural strength data at 6 months.   40  Figure 3.2:  Box and whisker plot of flexural strength for four glass ionomers aged in artificial saliva.  Note that circles on the plot represent possible outliers in the Ketac Molar (24 hour) data and Fuji II LC (24 hour) data.  3.3 Flexural strength study # 2 - Artificial saliva and water storage  The statistical power at α = 0.05 was 1.000 for the overall sample.  Three-way ANOVA (p<0.05) with Post-hoc Tukey tests suggested significant differences in flexural strength after 2 months aging in saliva and water.  The three-way interaction was not significant (F = 0.7225), thus it was removed from the statistical model. Table 3.4 shows flexural strength values in saliva and water storage, at 24 hours and 2 months aging periods.   41 Table 3.4:  Flexural strength at 24 hours and 2 months in artificial saliva and water storage.  Data represented by means (MPa), standard deviation (SD) in parenthesis and sample size (n). Material Artificial saliva Distilled water 24 hours 2 months 24 hours 2 months Ketac Nano 15.6 (4.9)b,B n=10 * 44.1 (7.7)a,B n=10 ◊ 17.6 (2.9)b,B n=10 * 40.6 (12.7)a,B n=9 ~ Ketac Molar 16.7 (7.7)b,B n=10 * 28.2 (7.0)a,C n=10 ◊ 24.1 (7.2)b,B n=10 # 34.1 (5.1)a,B n=9 ◊ Fuji II LC 47.2 (3.8)b,A n=10 * 59.8 (5.2)a,A n=9 ◊ 43.8 (4.1)b,A n=9 * 56.9 (12.5)a,A n=9 ◊ Equia 22.4 (6.2)a,B n=9 * 21.0 (2.8)a,C n=10 ◊ 21.1 (2.8)a,B n=9 * 17.1 (3.1)a,C n=10 ◊ Note: Lower case letters compare columns per storage media independently. Upper case letters compare rows in each storage media and time independently. Symbols are used to compare storage media per material/time independently. The * & # symbols compares saliva and water in 24 hours storage, and ◊  & ~ symbols compares saliva and water in 2 months storage.  Same symbols and same superscript letters indicate no significant difference.   Figure 3.3 shows box plot of flexural strengths of materials stored at 24 hours with storage in either water or saliva.  Figure 3.4 shows box plot of flexural strengths of materials at 2 months with storage in either water or saliva.  Fuji II LC showed higher flexural strength compared to the other three materials.        42   Figure 3.3: Box and whisker plot of flexural strength of materials in at 24 hours with storage in either saliva or water.    43  Figure 3.4: Box and whisker plot of flexural strength 2 months with storage in either saliva or water.  Note that circles on the plot represent possible outliers in Equia (saliva) and Ketac Molar (saliva) data.    Considering storage in artificial saliva only, the flexural strength significantly (p<0.05) increased after 2 months of storage for all materials except Equia (Table 3.5).  Similarly, considering water storage independently, the flexural strength increased significantly after 2 months of storage for all materials except Equia (Table 3.6). Overall, the flexural strength values significantly increased for each material after 2 month storage except for Equia, thus the null hypothesis that there are no changes in flexural strength after aging can be rejected.  Fuji II LC had significantly higher flexural strength at 24 hours and 2 months (p <0.001) either for both   44 water and saliva storage when compared to the other three materials (Tables 3.5 and 3.6). Therefore, the result allows us to reject the second null hypothesis that there is no difference among the materials evaluated. Table 3.5:  Flexural strength in artificial saliva at 24 hours and 2 months.   Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Material Artificial Saliva  Storage % Change  24 hrs. 2 months  Ketac Nano 15.6 (4.9)b,B n=10 44.1 (7.7)a,B n=10 183% Ketac Molar 16.7 (7.7)b,B n=10 28.2 (7.0)a,C n=10 69% Fuji II LC 47.2 (3.8)b,A n=10 59.8 (5.2)a,A n=9 27% Equia 22.4 (6.2)a,B n=9 21.0 (2.8)a,C n=10 -6% Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.  Table 3.6:  Flexural strength in water at 24 hours and 2 months.  Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Material Water Storage % Change  24 hrs. 2 months  Ketac Nano 17.6 (2.9)b,B n=10 40.6 (12.7)a,B n=9 131% Ketac Molar 24.1 (7.2)b,B n=10 34.1 (5.1)a,B n=9 41% Fuji II LC 43.8 (4.1)b,A n=9 56.9 (12.5)a,A n=9 30% Equia 21.1 (2.8)a,B n=9 17.1 (3.1)a,C n=10 -19% Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.   45  Considering each material on its own and comparing saliva and water storage, Ketac Molar showed significantly (p = 0.021) higher flexural strength in water at 24 hours (Table 3.8) and Ketac Nano showed significantly (p = 0.045) higher flexural strength in water at 2 months (Table 3.7).  The remaining materials did not show statistically significant difference in flexural strengths when stored in either water or saliva (Tables 3.9 and 3.10).  Therefore, it seems that the effect of storage media, saliva or water, on flexural properties of GIC is material and storage time dependent.   Table 3.7: Flexural strength (FS) for Ketac Nano at 24 hours and 2 months in artificial saliva and water storage.  Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Storage Ketac Nano FS in MPa (SD)  24 hrs. 60 days Saliva 15.6 (4.9)b,A n=10 44.1 (7.7)a,A n=10 Water 17.6 (2.9)b,A n=10 40.6 (12.7)a,B n=9 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.  Table 3.8: Flexural strength (FS) for Ketac Molar at 24 hours and 2 months in artificial saliva and water storage.  Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Storage Ketac Molar FS in MPa (SD)  24 hrs. 60 days Saliva 16.7 (7.7)b,B n=10 28.2 (7.0)a,A n=10 Water 24.1 (7.2)b,A n=10 34.1 (5.1)a,A n=9 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.      46 Table 3.9: Flexural strength (FS) for Fuji II LC at 24 hours and 2 months in artificial saliva and water storage.  Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Storage Fuji II FS in MPa (SD)  24 hrs. 60 days Saliva 47.2 (3.8)b,A n=10 59.8 (5.2)a,A n=9 Water 43.8 (4.1)b,A n=9 56.9 (12.5)a,A n=9 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.   Table 3.10: Flexural strength (FS) for Equia at 24 hours and 2 months in artificial saliva and water storage.  Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Storage Equia FS in MPa (SD)  24 hrs. 60 days Saliva 22.4 (6.2)a,A n=9 21.0 (2.8)a,A n=10 Water 21.1 (2.8)a,A n=9 17.1 (3.1)b,A n=10 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.  3.4  Diametral tensile strength   Two-way ANOVA (p<0.05) with Post-hoc Tukey tests suggested significantly higher diametral tensile strength of Fuji II LC when compared to the other three materials (p <0.001) at 24 hours and 6 months storage (Table 3.11). Six-month aging demonstrated a significant increase in diametral tensile strength for Fuji II LC (p = 0.049).  However, no significant changes were observed over time for the other three materials. Thus, the null hypothesis as diametral tensile   47 strength of all materials is not different, can be rejected. However, the null hypothesis as diametral tensile strength of materials does not change over time can only be partially rejected. Table 3.11:  Diametral tensile strength at 24 hours and 6 months in artificial saliva storage.   Data represented by mean (MPa), standard deviation (SD) in parenthesis and sample size (n).  Material 24 hours 6 months Ketac Nano 10.3 (1.9)a,B n=15 12.2 (2.9)a,B n=15 Ketac Molar 8.4 (2.8)a,B n=15 9.9 (2.4)a,B n=15 Fuji II LC 19.9 (4.0)b,A n=15 22.1 (3.1)a,A n=15 Equia 9.9 (2.7)a,B n=15 11.6 (3.3)a,B n=15 Note: Lower case letters compare columns (storage time for each material) and Upper case letters compare rows of data (materials at a specific storage time).  Similar letters indicate no significant difference.   Figure 3.5 shows box plot of the diametral tensile strength data.  The medians were near the middle of interquartile limits and Fuji II LC had higher DTS compared to the other three materials.   48  Figure 3.5:  Box and whisker plot of diametral tensile strength for four glass ionomers aged in artificial saliva.       49 Chapter 4:  Discussion 4.1 Shear bond strength to dentin Shear bond strengths values observed in this study were comparable to those previously published in literature (Appendix B). It is well known that several factors such as smear layer preparation, specimen fabrication method, specimen dimension, testing machine, crosshead shape and speed, and storage vary significantly from one research laboratory to another (Sirisha, Rambabu, Ravishankar, et al., 2014; Sirisha, Rambabu, Shankar, et al., 2014).  The great variability in bonding protocols and testing conditions make it difficult to compare results from one research laboratory to another.  This has been a common issue when compared with GIC shear bond strengths to dentin. The results of the present study showed no significant change in shear bond strength (SBS) of GICs after 6 months of artificial saliva aging, except for Ketac Nano, which showed a significant decrease in SBS values after aging.  Recently, Lawson et al. examined the role of light curing on the dentin bond strength of Ketac Nano (Lawson, Cakir, Beck, Ramp, & Burgess, 2012). They found that light activation is required as uncured specimens resulted in a 0 MPa bond strength to dentin.  Additionally, Ketac Nano requires the application of a light cured primer to the dental surface prior to bonding and when omitted results in low to minimal bonding to dentin (El-Askary & Nassif, 2011; Imbery et al., 2013).  However, when the dentin surface is demineralized prior to Ketac Nano primer application, there is a significant increase in bond strengths (El-Askary & Nassif, 2011).   Scanning electron microscopy (SEM) analysis also shows the penetration of Ketac Nano primer into the demineralized dentinal tubules to form resin tags and a hybrid layer (El-Askary & Nassif, 2011).  Based on these studies, it could be speculated that Ketac Nano perhaps behaves more like a resin composite rather than a true glass   50 ionomer.  It is currently questionable whether Ketac Nano is a true glass ionomer due to its resin content, which may retard and compromise the polyalkenoate chemical reaction (Roberts & Berzins, 2015). A conventional or reinforced glass ionomer cement only set via acid-base polyalkenoate setting reaction (Anusavice et al., 2012).  In resin-modified glass ionomer cements, there is also predominance of polyalkenoate reaction in the set material, which differentiates resin-modified glass ionomers from compomers or resin composites.  Recently, two fourier transform infrared (FTIR) spectroscopy studies have confirmed presence of the acid-base polyalkenoate reaction along with the formation of calcium-polycarboxylate bond in Ketac Nano (Falsafi, Mitra, Oxman, Ton, & Bui, 2014; Roberts & Berzins, 2015).  Therefore, Ketac Nano does technically fit a description of a resin-modified glass ionomer material.  However, in comparison to conventional glass ionomers and even other resin modified glass ionomers, the extent of polyalkenoate reaction is significantly lower in Ketac Nano (Roberts & Berzins, 2015).   Furthermore, compared to the conventional GICs and other RMGIs, Ketac Nano does not self-cure and requires an additional light cured primer step.  It has been suggested that a material should only be regarded as a true glass ionomer if there is sufficient acid-base reaction to allow self curing in dark (McLean et al., 1994).  However, Ketac Nano is currently regarded as a RMGI that is not able to self-cure in absence of light.  Further, it would be reasonable to propose that Ketac Nano could be classified as a compomer rather than a RMGI (McLean et al., 1994).  As the light curable primer composition for Ketac Nano is mostly water (40-50%), HEMA (35-45%), and a small percentage of polyacrylic acids (10-15%), it is possible that the bonding interface between Ketac Nano and the dental substrate is more susceptible to degradation over time, similar to the trends seen in resin composites bonded to dentin (Pashley et al., 2011).  Additionally, the competitive nature between the polyalkenoate reaction and resin matrix   51 polymerization could have a direct impact on Ketac Nano’s chemical bond to dentin, thus contributing to the bond degradation over time (Roberts & Berzins, 2015). This may explain a decrease in Ketac Nano bond strength after 6 months in this study. As there are no other long-term studies evaluating SBS of Ketac Nano to dentin, further studies are needed to confirm this hypothesis. It was interesting to see that the failure mode for Ketac Nano was predominantly adhesive, which was not the trend among the other three materials tested. This may suggest that the interface between the light cured primer and Ketac Nano could be relatively prone to degradation, resulting in decrease in SBS over time.  Among the four materials evaluated in this study, Fuji II LC has been extensively studied in the past 20 years, and significant volume of data on shear bond strength to dentin is available. However, there has only been one study published that examined the effects of long term storage on SBS of Fuji II LC to dentin (Dursun et al., 2013).  The investigators found  significant increases in SBS after 6-month storage in distilled water.  However, this study observed stability of the bond after aging. It is possible that some differences in their protocol yielded different results when compared to the present study.  In contrast, their protocol created the smear layer with 800-grit SiC paper and the cross-sectional diameter of the Fuji II LC bonded cylinders was wider at 3 mm. In this study, different storage media provided an ionic environment, with potential for ion exchange within the bonded interface. This may have affected the long term results. In fact, the effect of storage media on SBS of GICs to dentin has never been fully investigated.   Nevertheless, since the protocol used in this study differs from most published studies, the data still provides an important contribution to our current understanding and reinforces the need to further investigate the role of saliva in the performance of GICs over time.   52 The Ketac Molar SBS results in this study were similar to previously published studies (Almuammar et al. 2001, Yesilyurt et al. 2008). One of them also used encapsulated Ketac Molar formulation (Aplicap) (Almuammar, Schulman, & Salama, 2001).  Carvalho et al. reported higher SBS when compared to the values reported in this study; however, their protocols were significantly different.  They maintained temperature at 37°C on the tooth surface during bonding and used a hand-mixed version (Ketac Molar EasyMix) as opposed to the aplicaps (Carvalho et al., 2011).  These differences in protocols could have contributed to the differences found in this study.  Even though Ketac Molar has been available in the market for a relatively long period of time, there are no published long-term studies on shear bond strengths to dentin, and our findings add this important information to the literature. Equia, a new reinforced glass ionomer cement system, has no studies published examining shear bond strength to human dentin.  Only one study has examined Equia’s immediate SBS to bovine dentin, which was found to be comparable to the 24 hour SBS results to the present study (Poggio, Beltrami, Scribante, Colombo, & Lombardini, 2014).  Although impossible to discuss our data with other published data, our findings establish pioneering information on this material to be analyzed against in future studies. As discussed earlier, the predominant failure mode in the Ketac Nano samples were the adhesive type. . However, the reinforced glass ionomers (Ketac Molar and Equia) and Fuji II LC had mixed failures for the majority of specimens.  In cases of mixed failure modes, the residual glass ionomer on the tooth surface was less than 5% of the bonding area.        53 The large standard deviation for Fuji II LC and Ketac Nano within SBS data found in this study is comparable to previously published studies (Khoroushi et al., 2012; Lawson et al., 2012). Glass ionomer cements are known for being highly technique sensitive, which has a direct impact on bond strengths to dentin. Beyond the material characteristics itself, other factors such as dentin moisture control, porosities incorporated during mixing and insertion, and variations in maturation of the material all play a significant role in the final SBS. The present study found large standard deviation in several groups due to multiple specimens having very low SBS (< 1PMa) despite standardization of dentin surface preparation, cylindrical specimen fabrication and testing conditions. Post-testing examination under light microscope facilitated the identification of sample specimens with large voids at the bonding interface, thereby providing the opportunity to discard those specimens from the final data analysis. Yet there were a number of specimens that had no obvious defects at the bonding interface despite exhibiting very low bond strength. Several authors have suggested similar issues when conducting shear bond strength tests to human teeth in vitro (Kelly, Benetti, Rungruanganunt, & Bona, 2012; Lawson et al., 2012; Salz & Bock, 2010; Sirisha, Rambabu, Ravishankar, et al., 2014; Sirisha, Rambabu, Shankar, et al., 2014).  It is well known that dentin permeability and depth along with the volume of intertubular dentin structure available for bonding plays a significant role in SBS to dentin (Sirisha, Rambabu, Ravishankar, et al., 2014; Sirisha, Rambabu, Shankar, et al., 2014). Depending on patient’s age at the time of the extraction, the grinding process to remove occlusal enamel could result in dentin surfaces in close proximity to the pulp, which are less favorable for bonding. Such factors, which are difficult to control, can likely result in a large variation in shear bond strength values, even with all other variables being well controlled by the operator. Nevertheless, for ethical scientific reporting, all SBS values without any detectable defects were   54 included in the statistical analysis, which resulted in large standard deviations and inability to reject null hypotheses.  Another possible explanation for the large standard deviation in SBS is the use of the Ultradent bonding jig used in this study to form and bond glass ionomer cylinders to the dentin surface.  After the material was bonded to the tooth surface, disassembling the jig was a challenging, technique sensitive step.  Even though careful removal of the specimens was performed, it is possible that this step may have introduced stresses to some of the sample’s freshly bonded interfaces.  This variability in jig disassembly could explain the large variation in the shear bond strength values.  In order to minimize discrepancies inherent from hand mixing and powder-to-liquid ratio, in the present study, encapsulated versions were favoured for all materials tested.  However, other studies used manual powder/liquid mixing methods, which may have contributed to results different than this study (Carvalho et al., 2011; Yesilyurt et al., 2008). It has been suggested that cross sectional area may also influence shear bond strength values (Phrukkanon, Burrow, & Tyas, 1998).  Larger cross-sectional areas usually correlated with low shear bond strength values. The cross-sectional diameter of specimens in this study was 2.38 mm; however, the majority of the comparative studies (Appendix B) used a 3 mm cross-sectional diameter mold. This discrepancy may account for the differences found in the present study.  Most studies assessing immediate and long-term GICs shear bond strength to dentin used distilled water as storage media, which differs from the artificial saliva used in this study. Even though it has been demonstrated that storage media does not have influence on the mechanical properties of glass ionomer cements (Nicholson & Wilson, 2000), there are no studies assessing   55 the storage media effects on SBS to dentin. For future studies, it would be a reasonable approach to evaluate the immediate and long-term effects of different storage media on SBS to dentin.   4.2 Flexural strength  The first flexural strength (FS) study examined the effects of artificial saliva aging at 24 hours and 6 months on flexural strength of glass ionomers.  The second FS study was designed to examine the potential effects of storage media (saliva vs. water) on FS of GICs after aging.   The first FS study clearly demonstrated that 6 months aging resulted in an increase of FS values for all GICs tested. In the second FS study, an increase in FS over time was also observed for all testing conditions (storage media and storage time), with Fuji II LC having statistically significant higher values.  Therefore, the corresponding null hypotheses tested can be rejected. In this study, Fuji II LC (RMGI) had significantly higher FS than the other three materials tested.  This finding is in agreement with previously published studies (Mitra & Kedrowski, 1994; Miyazaki, Moore, & Onose, 1996).  Considering FS study #1, the 24 hours FS values were significantly lower than what has been previously reported in the literature for all GICs tested (Appendix C).  These findings could be attributed to critical differences in protocols in this study compared to the published literature.  In this study, the specimens were immediately subjected to storage once the material was set.  Also, intentionally, no protective coating was applied to the specimens to evaluate the effects of media on FS values. In contrast, several studies (Appendix C) protected the GIC specimens during maturation by storing them in a high relative humid environment (Mitra & Kedrowski, 1994), by coating with paraffin wax (Molina, Cabral, Mazzola, Lascano, & Frencken, 2013), or by applying a resin coat on the specimens’ surface (Bonifacio, Werner, & Kleverlaan, 2012; Zoergiebel & Ilie, 2013).  Significantly lower   56 FS for glass ionomer cements immediately immersed in media were found when compared to the samples that were allowed to mature in a dry environment (Moreau & Xu, 2010).  Changes in flexural strength due to application of a protective resin coat on Fuji II LC specimens were examined (Miyazaki et al., 1996).  The investigators found that Fuji II LC specimens protected with a resin coat had significantly higher initial flexural strength when compared to specimens that were not coated.  However, after 6 months, both coated and uncoated specimens had similar flexural strengths.  Molina et al. reported higher FS values for Ketac Molar and Equia at 24 hours compared to this study.  However, they stored specimens in paraffin wax for 24 hours prior to testing.  Their Equia specimens were coated with a layer of G-coat to add protection during maturation (Molina et al., 2013). Other studies have also suggested that manufacturer supplied G-coat is critical in improving the flexural strength of Equia (Bonifacio et al., 2012; Molina et al., 2013; Zoergiebel & Ilie, 2013).  In another study, Ketac Molar specimens were stored in distilled water at different time points after the initial setting reaction (Piwowarczyk, Ottl, Lauer, & Buchler, 2002).  The investigators found Ketac Molar FS at 15 minutes, 1 hour, and 24 hours to be 12.1 MPa, 12.6 MPa and 24 MPa respectively.  Their results demonstrated a clear time-dependent maturation process for reinforced glass ionomer cements (Piwowarczyk et al., 2002). The protection of glass ionomers during maturation likely improved flexural strength at 24 hours in the above mentioned studies.  Thus, the absence of a protective feature to allow material maturation could potentially explain the lower initial flexural strengths for the first FS study.  When comparing 24-hour results of two FS studies in this research, the first study (artificial saliva storage only) had lower flexural strength values than those of the second flexural strength study (Appendix C). However, after 6 months of storage, the flexural strength values reached comparable levels to both previously reported values (Appendix C) and the second FS   57 study.  The protocol for both FS studies was identical although the products batch numbers were different and the specimens were fabricated at different seasons of the year.  It is possible that discrepancies between the 24 hour FS values of two FS studies may have been due to differences in batch numbers, changes in room temperature and humidity, potential exposure to outside freezing temperatures during product shipping and operator error.  However, this hypothesis is lacking support from previously published literature, making it difficult to explain the exact cause of discrepancy. Nevertheless, the results found in the second FS study also support improvements in flexural strength of glass ionomer cements over time. Future studies are required to better understand the effects of storage temperatures, material batch numbers, room temperature and humidity on the final mechanical properties of multiple GICs.   The reported flexural strength values for several GICs vary widely from one research group to another (Appendix C).  Fuji II LC, Equia and Ketac Molar immediate (24 hour) flexural strength values observed in this study were in the general range of those reported in the literature (Marovic et al., 2013; Howard et al., 2014; Miyazaki et al., 1996; Mitra et al., 1994; Molina et al., 2013; Piwowarczyk et al., 2002; Bonifacio et al., 2012).  It is important to emphasize that glass ionomer cement flexural strength studies are very technique sensitive and any irregularities (cracks, porosities) in the specimens could result in dramatically lower flexural strengths. It is apparent that for FS specimen fabrication, the following factors may be critical role: using rigid split and sealed molds, applying coating on the specimens, and polishing procedures.  In this study, polishing procedures were carried out immediately after the material was set. It is possible that delaying polishing will reduce the likelihood of adding stresses in the initial phase of glass ionomer maturation and is likely to minimize the possibility of crack formation.  As every laboratory has a different approach to specimen fabrication, this factor may be significant when   58 comparing studies.  Future studies are needed to assess the effects of resin coating and delayed polishing on flexural strength values of multiple GIC materials.  Therefore, even though the data in this study may not correspond to absolute values published in the literature, it is useful for suggesting trends in changes in flexural strengths of GICs over time.  The trends observed in both flexural strength studies were similar; however, the hypothesis that the storage media could have an effect on the flexural strength of GICs was not proven.  The results of the second FS study (saliva vs. water) demonstrated a significant increase in flexural strengths for most of the GIC materials, either stored in artificial saliva or distilled water.  The results suggested improvements in flexural strength to be independent of storage media.  These findings are consistent with previously published studies suggesting that the type of media does not have a significant effect on the mechanical properties of glass ionomer cements (Nicholson & Wilson, 2000; Zoergiebel & Ilie, 2013).  Nicholson et al. examined compressive strength of materials in several media (distilled water, saline, artificial saliva, ionic solutions, etc). These investigators did not find significant differences in compressive strengths when GIC specimens were stored in different solutions.  Similarly, Zoergieble et al. did not find significant differences in the flexural strength of glass ionomers when stored in either distilled water or saliva (Zoergiebel & Ilie, 2013). In the present study, it is clear that aging resulted in an increase in flexural strength of the restorative glass ionomers.  Earlier studies on glass ionomer chemistry argued a continued reaction after the initial acid-base reaction between polyacrylic acids and glass powder (Crisp, Pringuer, Wardleworth, & Wilson, 1974).  Crisp et al. suggested exchange of calcium ions by aluminum ions in the carboxyl group cross-links, which continues from a period of 24 hours to several days (Crisp et al., 1974; Crisp & Wilson, 1974a, 1974b).  After initial powder/liquid   59 mixing, the calcium ions are more easily available for crosslinking with the carboxyl groups compared to the aluminum ions.  The delayed exchange of aluminum ions is attributed to its lower mobility, slower release from glasses and higher cross-linking requirement (3 carboxyl groups for a favourable steric configuration) when compared to calcium ions (Crisp & Wilson, 1976).  The delayed incorporation of aluminum ions in the reaction likely improves material strength over time. The addition of tartaric acid to the liquid component was suggested to increase the rate of extraction of ions from glasses after mixing, thereby, speeding the rate of hardening of the mixed cement (Crisp & Wilson, 1976).  However, tartaric acid doesn’t seem to have a direct role in continued increase in strength of GICs over time even though it makes aluminum ions available for cross-linking after the initial reaction.  Water, either bound or unbound, is also present in the set cement (Nicholson, 1998).  With aging, the ratio of bound:unbound water increases in the set cement, which has been suggested to increase glass ionomer strength over time (Nicholson, 1998).  The presence of inorganic network with silicon and phosphorus ions has also been speculated to increase strength of GICs (Nicholson, 1998).  Previous research suggests that such inorganic networks form in between already cross-linked organic acids after the initial reaction (Wilson, 1996).  The formation of silicate/phosphate inorganic network likely happens over the period of several days, thereby, increasing material strength over time (Wasson & Nicholson, 1993).  It is evident from the above studies that the exact mechanism of time dependent improvement in glass ionomer strength is complex and its setting reaction has not been fully elucidated.  There are likely multiple mechanisms involved that contribute to an increase in mechanical strength of GICs over time.        60 4.3 Diametral tensile strength  In this study, the diametral tensile strength (DTS) of Fuji II LC was found to be significantly higher than Equia, Ketac Molar and Ketac Nano at 24 hours and after 6-month aging in artificial saliva.  Except for Fuji II LC, the diametral tensile strength did not change significantly after aging. Thus, the null hypothesis of lack of effect of material and storage time on DTS must be partially rejected. The DTS in this study did not differ significantly from those previously published in the literature (Appendix D).  However, majority of the studies published have not investigated long-term storage (greater than 3 months) and distilled water was used as storage media.   The results of this study showed that there was indeed a trend in increase for diametral tensile strength values for all materials aged in artificial saliva, and only Fuji II LC demonstrated  statistically significant differences.  The Fuji II LC diametral tensile strength values increased by 11% over time.  Similarly, other investigators have reported no significant changes in the diametral tensile strength of glass ionomers after long-term aging (Mitra & Kedrowski, 1994; Uno, Finger, & Fritz, 1996).  It seems that this particular mechanical property of glass ionomers is least affected by aging and this finding is somewhat consistent with other investigations (Appendix D).  It is possible that the DTS test itself is not very sensitive at detecting changes in material properties over time when compared to the flexural strength test.  The DTS test uses compressive forces on cylindrical specimens to determine the ultimate tensile strength of brittle materials such as GICs (Anusavice et al., 2012). The test is only valid if the specimen fractures at the center in two equal halves due to tensile stress.  It is possible that shear stresses may be introduced at the compression contact areas during testing.  This may lead to specimen failure primarily due to shear stresses as opposed to tensile stresses (Darvell, 2009).  The inherent nature   61 of the test may not be adequate to show significant improvements in material properties over time.  Nevertheless, this test is still favoured in the literature as it is easy to conduct and reproducible.    The diametral tensile strength (DTS) values of Fuji II LC reported in the literature have great variability from one laboratory to another (Appendix D). Reports for DTS of Fuji II LC at 24 hours are in the range of 16 MPa to 40 MPa.  These variations are likely due to different methods of specimen preparation and handling. Mitra et al. allowed specimens to mature at 37 °C and 100 % relative humidity for 1 hour prior to storing them in distilled water (Mitra & Kedrowski, 1994).  Similarly, Howard et al. allowed specimens to mature for 15 minutes at 37 °C and 100 % relative humidity prior to immersion in the storage media (Howard et al., 2014).  Interestingly, both studies (Mitra et al. and Howard et al.) reported Fuji II LC DTS to be around 41MPa.  In contrast, Cattani-Lorente et al. showed significant differences in DTS when specimens were aged in relative humidity of 80-90% versus specimens immediately immersed in water (Cattani-Lorente, Dupuis, Moya, Payan, & Meyer, 1999).  They found that Fuji II LC specimens stored in a wet environment had lower DTS (21.2 MPa) than specimens stored in high relative humidity (31 MPa).  It seems evident from previous research that specimen fabrication and handling protocols can significantly influence in vitro diametral tensile strength tests, as seen by the large range of reported values in the literature (Appendix D).    In this study, ranges of DTS for Ketac Molar and Equia were consistent with previously published studies (Appendix D). The literature is still lacking long-term aging studies for diametral tensile strength of these two materials.  However, it is evident that DTS of reinforced glass ionomers (as Ketac Molar and Equia) is significantly lower than DTS of resin modified glass ionomers, such as Fuji II LC (Appendix D). Others have reported similar trends when   62 comparing diametral tensile strengths of resin modified glass ionomers to conventional glass ionomers (Mitra & Kedrowski, 1994; Uno et al., 1996).  RMGIs are likely superior to conventional GICs due to incorporation of resinous materials in addition to the conventional GIC components (Anusavice et al., 2012).  Manufacturers recommend coating for the reinforced and conventional glass ionomers to allow protection of the material during the maturation phase (Anusavice et al., 2012).  GC America supplies reinforced glass ionomers (Equia) with G-Coat Plus (GC Corp.).  G-Coat is a nano-filled resin, which is to be applied to glass ionomer restorations after initial setting reaction is complete. As discussed in previous sections, a protective coat on the reinforced GIC specimens (Equia and Ketac Molar) was intentionally omitted in this protocol, prior to immersion in artificial saliva.  This may have resulted in lower diametral tensile strength than what has been suggested by the manufacturers.  As already discussed in the flexural strength section, there is a significant effect of GIC coating on mechanical properties of some glass ionomers (Bonifacio et al., 2012; Lohbauer et al., 2011; Molina et al., 2013; Zoergiebel & Ilie, 2013).   At this time, no suitable studies are available examining the DTS of Ketac Nano.  According to the product brochure (3M ESPE Ketac Nano), the diametral tensile strength of Ketac Nano is reported to be greater than 45 MPa.  However, specimen fabrication protocols and testing conditions were not disclosed.  Given the absence of adequate data in the literature, there is no suitable comparison for Ketac Nano DTS values obtained in this study.  While no claims can be made about the absolute DTS of Ketac Nano, it is evident that the 6-month aging had no significant effect on Ketac Nano DTS.  Ketac Nano is classified as a resin modified glass ionomer, however, its DTS was lower than Fuji II LC (RMGI).  This finding may be attributed to   63 different material composition of Ketac Nano (particle size, particle type, additional polymers, etc.) when compared to the other three glass ionomers.  Fuji II LC may have superior mechanical properties due to its resin content and higher filler content compared to Ketac Nano or conventional GICs (Moreau & Xu, 2010).   64 Chapter 5:  Conclusions This study aimed to answer the following research questions: 1) Does aging affect shear bond strength to dentin? 2) Does flexural strength and diametral tensile strength of restorative glass ionomers change over time? 3) Does the storage media affect flexural strength of glass ionomers? 4) Do mechanical properties and bond strength to dentin vary among different glass ionomer cements?  The following conclusions can be drawn from this research: 1. There was stability in shear bond strength to dentin over time for all materials tested, except Ketac Nano, which had a significant decrease in shear bond strength after aging. As Ketac Nano is a relative new product which requires a light curing primer, further studies are still needed to better understand its bonding mechanism and long-term bonding performance. 2.  Aging significantly increased the flexural strength values for all restorative glass ionomer cements evaluated. However, storage media did not play a significant role in flexural strengths of restorative glass ionomer cements evaluated. 3.  Diametral tensile strength of glass ionomer cements studied did not change after aging.   4.  Among the restorative glass ionomers evaluated, Fuji II LC had significantly higher flexural and diametral tensile strength. No significant differences among the materials were observed in shear bond strength to dentin.  Generally, restorative glass ionomer cements do not demonstrate bond degradation over time and, more importantly, they showed improved mechanical properties after aging. 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Clin Oral Investig, 17(2), 619-626. doi:10.1007/s00784-012-0733-1   70 APPENDIX A:  Sample size calculations For each experiment, a pilot study with tests done at 24 hours was used to calculate sample size needed to achieve a power of 80% with alpha of less than 5% to detect differences between the groups.  “Sample size” function in SigmaPlot 11 statistical software yielded the following sample sizes: Pilot	Study:	SBS	 SBS	@	24	hours	(Water	storage)	 SigmaPlot	11:	Sample	Size	Calculator		 		 N	 MPa	(St.	Dev)	 Difference	in	means:	3.49	Average	St.	Deviation:	2.47	Number	of	Groups:	4	Sample	size	needed	per	group:	12			Ketac	Molar	 5	 6.04	(1.21)	Ketac	Nano	 9	 5.36	(3.79)	Fuji	II	LC	 5	 16.43	(3.81)	Equia	 5	 2.55	(1.08)		Pilot	Study:	FS	 FS	@	24	hours	(Water	Storage)	 SigmaPlot	11:	Sample	Size	Calculator	 	 		 N	 MPa	(St.	Dev)	 Difference	in	means:	9.90	Average	St.	Deviation:	6.90	Number	of	Groups:	4	Sample	size	needed	per	group:	12			Ketac	Molar	 5	 19.78	(9.22)	Ketac	Nano	 5	 29.68	(5.83)	Fuji	II	LC	 5	 57.56	(8.82)	Equia	 5	 20.17	(3.75)		Pilot	Study:	DTS	 DTS	@	24	hours	(Water	Storage)	 SigmaPlot	11:	Sample	Size	Calculator		 		 N	 MPa	(St.	Dev)	 Difference	in	means:	14.10	Average	St.	Deviation:	3.41	Number	of	Groups:	4	Sample	size	needed	per	group:	3			Ketac	Molar	 5	 10.83	(3.77)	Ketac	Nano	 5	 12.23	(2.42)	Fuji	II	LC	 5	 22.94	(1.22)	Equia	 5	 8.84	(4.03)		Pilot	Study:	FS	in	Saliva	&	Water	 FS	@	24	hours	 SigmaPlot	11:	Sample	Size	Calculator	 	 		 N	 MPa	(St.	Dev)	 Difference	in	means:	7.24	Average	St.	Deviation:	5.20	Number	of	Groups:	2	(Saliva	vs.	Water)	Sample	size	needed	per	group:	10			Fuji	II	LC	(Saliva	storage)	 5	 25.38	(7.54)	Fuji	II	LC	(Water	Storage)	 5	 32.61	(2.81)	   71 APPENDIX B:  Shear bond strength of glass ionomers published in literature.  Material Published Literature SBS ± SD (MPa) at 24 hours unless specified Present Study SBS ± SD (MPa) Ketac Nano 7.07 ± 4.21 (Lawson et al., 2012)  3.72 ± 1.44 at 48 hours (Imbery et al., 2013)  5.5 ± 2.2 (El-Askary & Nassif, 2011)  2.04 ± 0.81 (Korkmaz, Ozel, Attar, & Ozge Bicer, 2010) 6.8 ± 4.8 (24 hours) 4.4 ± 3.7 (6 months) Ketac Molar 7.6 ± 1.5 (Carvalho et al., 2011)  3.4 ± 0.4 at 6 weeks (Yesilyurt, Bulucu, Sezen, Bulut, & Celik, 2008)  1.9 ± 1.4 (Czarnecka, Deregowska-Nosowicz, Limanowska-Shaw, & Nicholson, 2007)  3.77 ± 1.76 (Almuammar et al., 2001) 4.0 ± 1.8 (24 hours) 3.6 ± 2.1 (6 months) Fuji II LC 8.3 ± 0.6 at 24 hours, 12.7 ± 3.4 at 6 months, 12.6 ± 3.8 at 12 months (Dursun et al., 2013)  4.99 ± 2.07 (Imbery et al., 2013)  12.46 ± 5.06 (Lawson et al., 2012)  13.79 ± 5.18 (Khoroushi, Karvandi, & Sadeghi, 2012)  11.56 ± 3.15 (Hajizadeh, Ghavamnasiri, Namazikhah, Majidinia, & Bagheri, 2009)  6.38 ± 1.44 (de Souza-Gabriel, do Amaral, Pecora, Palma-Dibb, & Corona, 2006)  10.56 ± 2.67 (Wang, Sakai, Kawai, Buzalaf, & Atta, 2006)  9.55 ± 1.06 (Almuammar et al., 2001) 7.6 ± 4.2 (24 hours) 7.0 ± 5.7 (6 months) Equia 3.51 ± 1.22 (bovine dentin) (Poggio et al., 2014) 4.8 ± 2.1 (24 hours) 5.7 ± 3.2 (6 months)        72 APPENDIX C:  Flexural strength of glass ionomers published in literature.  Materials Published Literature FS ± SD (MPa) at 24 hrs. unless specified FS Study #1: Artificial Saliva FS ± SD (MPa)  FS Study #2: Artificial Saliva vs. Water FS ± SD (MPa) Ketac Nano ~ 40 MPa at 84 days in pH 7.0 (Moreau & Xu, 2010) 16.0 ± 3.8 (24 hrs.) 30.2 ± 15.6 (6 months) Saliva: 15.6 ± 4.9 (24 hrs.) 44.1 ± 7.7 (2 months) Water: 17.6 ± 2.9 (24 hrs.) 37.8 ± 15.0 (2 months) Ketac Molar 28.9 ± 5.4 (Molina et al., 2013)  44.1 ± 5.6 (Bonifacio et al., 2012)  34.5 ± 7.2 (Bonifacio et al., 2009)  51 ± 5 (Peez & Frank, 2006)  ~24 (Piwowarczyk et al., 2002)  21.2 ± 3.1 at 7 days (Xie, Brantley, Culbertson, & Wang, 2000) 6.2 ± 1.2 (24 hrs.) 18.3 ± 4.8 (6 months) Saliva: 16.7 ± 7.7 (24 hrs.) 28.2 ± 7.0 (2 months) Water: 24.1 ± 7.2 (24 hrs.) 34.1 ± 5.1 (2 months) Fuji II LC 33.1 MPa (Marovic et al., 2014)  51.9 ± 1.8 (Howard, Weng, & Xie, 2014)  71.1 ± 3.6 at 7 days (Xie et al., 2000)  54 ± 10 at 24 hours (dry storage), 10 ± 3 at 24 hours (wet storage) (Cattani-Lorente, Dupuis, Payan, Moya, & Meyer, 1999)  50.1 ± 4.4 at 24 hours, 59.3 ± 4.7 at 6 months (Miyazaki et al., 1996)  56.6 ± 3.8 (Mitra & Kedrowski, 1994) 23.2 ± 7.7 (24 hrs.) 50.4 ± 6.0 (6 months) Saliva: 47.2 ± 3.8 (24 hrs.) 59.8 ± 5.2 (2 months) Water: 43.8 ± 4.1 (24 hrs.) 56.9 ± 12.5 (2 months) Equia 12 ± 7.4 at 1 week, 9.2 ± 5.0 at 1 month in saliva (Zoergiebel & Ilie, 2013)  49.8 ± 6.4 (Molina et al., 2013)  20.2 ± 4.1 (Bonifacio et al., 2012) 11.5 ± 1.5 (24 hrs.) 21.8 ± 4.3 (6 months) Saliva: 22.4 ± 6.2 (24 hrs.) 21.0 ± 2.8 (2 months) Water: 21.1 ± 2.8 (24 hrs.) 17.1 ± 3.1 (2 months)      73  APPENDIX D:  Diametral tensile strength of glass ionomers published in literature.  Material Published Literature DTS ± SD (MPa) at 24 hrs. unless specified This Study DTS ± SD (MPa) Ketac Nano ~ 47 MPa (3M Ketac Nano Brochure)  10.3 ± 1.9 (24 hours) 12.2 ± 2.9 (6 months) Ketac Molar 11.0 ± 5.2 (Daifalla & Mobarak, 2015)  7.5 ± 0.7 (Molina et al., 2013)  12.46 ± 0.62 (Troca et al., 2011)  9.5 ± 1.3 (Piwowarczyk et al., 2002) 8.4 ± 2.8 (24 hours) 9.9 ± 2.4 (6 months) Fuji II LC 41.2 ± 2.7 (Howard et al., 2014) 31.2 ± 2.2 (Zhao & Xie, 2011) 21.2 ± 1.1 (Xie, Zhao, & Park, 2007)  21 ± 5 (24 hours), 16 ± 4 (3 months) (Cattani-Lorente, Dupuis, Moya, et al., 1999)  16.1 ± 3.1 (24 hours), 14.8 ± 2.1 (6 months) (Uno et al., 1996) 40.7 ± 0.5 (24 hours), 40 ± 2.1 (6 months) (Mitra & Kedrowski, 1994) 19.9 ± 4.0 (24 hours) 22.1 ± 3.1 (6 months) Equia 10.0 ± 0.7 (Molina et al., 2013) 9.9 ± 2.7 (24 hours) 11.6 ± 3.3 (6 months)  

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