Fluoresence changes in remineralized and nonremineralized enamel adjacent to glass ionomer art restorations after ph cycling: an in-vitro study

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1 University of Iowa Iowa Research Online Theses and Dissertations 2005 Fluoresence changes in remineralized and nonremineralized enamel adjacent to glass ionomer art restorations after ph cycling: an in-vitro study Elizabeth Bowles Gaskin University of Iowa Copyright 2005 Elizabeth Bowles Gaskin This thesis is available at Iowa Research Online: Recommended Citation Gaskin, Elizabeth Bowles. "Fluoresence changes in remineralized and non-remineralized enamel adjacent to glass ionomer art restorations after ph cycling: an in-vitro study." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Other Dentistry Commons

2 FLUORESCENCE CHANGES IN REMINERALIZED AND NON-REMINERALIZED ENAMEL ADJACENT TO GLASS IONOMER ART RESTORATIONS AFTER PH CYCLING: AN IN-VITRO STUDY by Elizabeth Bowles Gaskin A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Operative Dentistry in the Graduate College of The University of Iowa May 2005 Thesis Supervisor: Professor James S. Wefel

3 Copyright by ELIZABETH BOWLES GASKIN 2005 All Rights Reserved

4 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL MASTER'S THESIS This is to certify that the Master's thesis of Elizabeth Bowles Gaskin has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Operative Dentistry at the May 2005 graduation. Thesis Committee: James S. Wefel, Thesis Supervisor Sandra Guzmán-Armstrong Steven R. Armstrong Marcos A. Vargas Marcela M. Hernández Fang Qian

5 To my Mother and Father ii

6 ACKNOWLEDGMENTS I am indebted to many people for completing this research project. I extend my sincere thanks to the United States Navy as it provided me the opportunity to pursue studies and advanced training in Operative and Preventive Dentistry. I wish to thank the entire staff of the Operative Dentistry Department for accepting me into their training program and providing me with new, invaluable skills. A special thank you to Dr. Marcos Vargas and Dr. Steven Levy, Graduate Directors of Operative Dentistry and Dental Public Health, respectively, for providing me with a three-year combined timetable. Because of their forethought, I was able to concentrate on my coursework, and this thesis project. I am well aware of the fact that it took a lot of time to formulate and was in constant revision, much to my heartfelt appreciation. This thesis project would not have been possible without my mentors, Dr. James Wefel, and Dr. Sandra Guzman-Armstrong. Their suggestions motivated me and made my project interesting, exciting, and enjoyable. I wish to thank the members of my research committee: Dr. Marcos Vargas, Dr. Steven Armstrong, Dr. Marcela Hernandez, and Dr. Fang Qian for their help in targeting a focus area, interpreting results, and providing beneficial feedback. Thanks also go to Mr. John Laffoon, Ms. Judy Heilman, and members of the Cariology Department, Ms. Maggie Hogan, Mr. Jeffrey Harless, and Ms. Pat Hancock, for training me to use the equipment required for this project. The friendship and moral support provided by fellow classmates, Ghada Maghaireh and Hidehiko Watanabe, facilitated, and enhanced the daily work of the project. Indeed, the University of Iowa was a great place to study. The climate, the atmosphere and the environment constitute one of the best. I will cherish the memories. iii

7 TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vii CHAPTER I INTRODUCTION...1 CHAPTER II REVIEW OF LITERATURE...5 Development, Prevention and Control of Dental Caries...5 Structural Elements of Enamel and their Role in Caries Formation...5 Dentin and Cementum...6 The Role of Plaque in the Formation of Caries...7 Classification Systems for Carious Lesions...8 Prevention of the Carious Lesion...8 Early Caries Detection Methods...9 Non-invasive Caries Diagnostic Methods...11 Fluorescence...12 DIAGNOdent...12 Quantitative Light Induced Fluorescence...13 Glass Ionomer Restorative Material Physical and Chemical Properties...17 Advantages and Disadvantages...19 Hybrid Forms of Glass Ionomer Cements...19 Anticariogenic Potential of GICs...20 Comparison of In-Vitro and In-Vivo Findings...22 Atraumatic Restorative Treatment (ART)...22 Restorative Treatment Approaches...22 Rationale...23 Survival and Longevity of Restorations...26 Summary...28 CHAPTER III MATERIALS AND METHODS...29 Introduction...29 Research Design...29 Study Sample...30 Inclusion and Exclusion Criteria...31 Study Group Distribution...31 Sample Preparation...32 ph Cycling...36 Quantitative Light Fluorescence...36 Calibration Area...38 Microscopy...40 Stereomicroscope...40 Polarized Light Microscope (PLM)...40 Scanning Electron Microscope...41 Statistical Analysis...41 Sample Size Calculation...41 Data Analysis...41 iv

8 CHAPTER IV RESULTS...43 Descriptive...43 Quantitative...47 Microscopy...54 CHAPTER V DISCUSSION...64 Fluorescence Changes...64 Morphological Changes...68 QLF Measurement Sessions...70 ART and Glass Ionomer Restorative Material...73 Scanning Electron Microscopy...74 Limitations...76 Clinical Implications...77 Future Directions...78 CHAPTER VI CONCLUSIONS...80 APPENDIX A QLF RAW DATA...81 APPENDIX B PROCESSING FOR SEM...84 REFERENCES...86 v

9 LIST OF TABLES Table 1. Differences between traditional and ART restorations...2 Table 2. Materials used in this study...32 Table 3. QLF measurement sessions...37 Table 4. Presentation of facial enamel surfaces before treatment (%)...43 Table 5. No treatment and precycling demineralization fluorescence...47 Table 6. Comparison of fluorescence at the start of ph cycling...49 Table 7. Treatment group mean fluorescence ( F*) values during ph cycling...50 Table 8. Tukey grouping pairwise comparisons within treatment groups*...51 Table B1. Processing for SEM...85 vi

10 LIST OF FIGURES Figure 1. Research design schematic...30 Figure 2. Distribution of teeth...31 Figure 3. Preparation and dental floss...33 Figure 4. Outline of specimen window and varnish...33 Figure 5. Presentation before (a.) and after (b.) a precycling demin solution...34 Figure 6. Completed restoration...35 Figure 7. Diagram of ph cycling...35 Figure 8. Calibration area (indicated by arrow)...38 Figure 9. An area of interest- Patch...40 Figure 10. Fluorescence differences between unrestored (a.) and restored (b.) specimens...44 Figure 11. Cervical cavitation...45 Figure 12. Glass Ionomer exhibiting pitting and porosity...46 Figure 13. Loss of restorative material...46 Figure 14. Mean fluorescence ( F) differences between no treatment and precycling demin...48 Figure 15. Mean fluorescence values by treatment group...52 Figure 16. Remineralization trend among groups...52 Figure 17. Restoration and erosion of the enamel...54 Figure 18. Outer Band of demineralization in (a) remin without fluoride and (b)...55 Figure 19. Erosion of enamel and restoration in the non-remin with fluoride group...56 Figure 20. Erosion of enamel, cracking and surface wear in the non-remin without fluoride group...56 Figure 21. Interface between restorative material and enamel for all (a) remin without fluoride, (b) remin with fluoride, (c) non-remin without fluoride, and (d) non-remin with fluoride group. The enamel is at the top and the glass ionomer restorative material at the bottom of the photograph. In the fluoride groups, there is a bond between the enamel and restorative material...57 Figure 22. Enamel margin of remin groups, (a) remin without fluoride and (b) remin with fluoride groups...58 vii

11 Figure 23. Enamel margin of non-remin groups, (a) non-remin with fluoride and (b) non-remin without fluoride. Both are irregular in appearance but the nonremin without fluoride displays more pitting and porosity...59 Figure 24. An SEM of erosion along the enamel margin...59 Figure 25. Sections from the non-remin with fluoride group (a) unetched and (b) etched...60 Figure 26. Sections from the non-remin without fluoride (a) unetched and (b) etched...61 Figure 27. Sections from the remin without fluoride group (a) unetched, and (b) etched...61 Figure 28. Sections from the remin with fluoride group (a) unetched, and (b) etched...62 Figure 29. Glass ionomer restorative material after ph cycling...62 Figure 30. Etched remin groups (a) remin with fluoride (b) remin without fluoride...63 Figure 31. Etched non-remin groups (a) non-remin with fluoride, (b) non-remin without fluoride...63 viii

12 1 CHAPTER I INTRODUCTION The Atraumatic Restorative Technique (ART) was developed by field clinicians to address dental access problems in remote regions of the world without sophisticated dental equipment or electricity. The distinguishing features of the ART approach are the removal of soft carious lesions with hand instruments followed by placement of a glass ionomer restorative material. There is no standard cavity preparation with the ART approach. Carious tissue might remain in the cavity preparation because of inadequate access with hand instruments, or a desire to conserve or possibly remineralize tooth structure. Sharp and irregular margins of the preparation are removed with hand instruments. Cotton rolls and gauze provide isolation and moisture control. The approach requires only hand instruments so electricity is not necessary. The distinctly common possibility of leaving carious tissue in a preparation is a feature found in the ART approach. In the traditional restorative approach, the entire carious lesion is removed by expansion of the cavity margins. Both restorative approaches are summarized in Table 1. Tanzania was the location for the first ART field trials during the 1980s. The use of the ART approach quickly spread to other countries due to the encouraging results from the initial trials. By the later part of the 1990s, over twenty countries were using the ART approach with many more planning field trials. Dental treatment providers included dentists, dental nurses, dental therapists, and health care workers. There were favorable survival results in clinical field trials of one, two, and three-year duration when restorations prepared using the ART approach were compared to amalgam restorations prepared using the traditional restorative approach.

13 Table 1. Differences between traditional and ART restorations 2 Traditional Restorative Treatment 1. Local anesthesia administered 2. Cavity preparation completed with handpiece Atraumatic Restorative Treatment 1. No local anesthesia necessary 2. Cavity preparation completed with hand instruments 3. Complete caries removal 3. May leave residual caries 4. Extension for prevention cavity preparation 4. Minimal cavity preparation 5. Isolation with rubber dam 5. Isolation with cotton rolls and gauze Glass ionomer materials incorporate the bonding mechanism and fluoride exchange of silicate cements with improved radiopacity and strength. They are the recommended restorative material for the ART approach. The development of viscous glass ionomer materials was a collaborative effort between materials manufacturers and clinical researchers. The ability to resist a caries challenge is a highly desirable quality of a restorative material. Glass ionomer restorative materials may have anti-cariogenic potential in-vitro. There are several studies demonstrating this phenomenon in-vivo. Glass ionomer materials have the capacity to transfer fluoride from restoration to tooth structure, leading to greater resistance to demineralization around the margins of the restoration. There are only a few studies on the microscopy of the excavated cavity preparation after using the ART approach. Studies of glass ionomer restorative material s effect on enamel are more numerous but they focus on traditional preparation methods. There are few published studies of its effect on demineralized enamel. Although initially created for use in remote locations, the ART approach has applicability in regular dental practice. Dental schools are incorporating the principles of ART into their curricula, primarily for rampant caries cases. Some advocate use of the

14 3 ART approach in industrialized regions for high caries populations. To include the ART approach as a permanent part of evidence-based dental practice, studies should address the applicability of ART restorations in high caries populations. The most common aids for caries diagnosis have been visual identification, tactile sensation, radiography and a thorough patient history. These are qualitative methods. The methods are ineffective in quantifying caries, particularly for very small lesions. Quantitative Light Fluorescence (QLF) is a diagnostic system for detection of small carious lesions. It calculates the size of the lesion, based on the change in fluorescence emitted from the tooth. QLF is non-destructive with uses in-vitro and in-vivo. Carious surfaces emit less fluorescence than sound tooth surfaces. Enamel comprises the outer layer of the tooth and is the hardest substance in the human body. It has a protective role, providing insulation, resistance and strength to the underlying tooth structure. A sound enamel surface occurs when there is a balance to the demineralization/remineralization cycle through a healthy diet, adequate salivary flow, good oral hygiene, and use of topical fluoride agents. A carious lesion is a disruption to the integrity of enamel. Dental caries is the primary reason for the placement of most restorations. The lesion begins as an area of demineralization on the enamel surface. The demineralization/remineralization equilibrium determines the course of the lesion. Application of topical fluoride, found in toothpastes, varnishes, gels and rinses, shifts the equilibrium towards remineralization. The additional fluoride release and recharge from glass ionomer restorative materials may benefit the surrounding enamel margins. If enamel is remineralized, it may be more resistant to demineralization. Frequent use of sugary foods and drinks, a practice of many rampant caries cases, shifts the equilibrium towards demineralization. The purpose of this in-vitro study was to compare the mean change in fluorescence of remineralized and non-remineralized enamel surrounding glass ionomer

15 4 restorations during ph cycling. This was accomplished using a non-destructive measurement system, Quantitative Light Fluorescence. There were two research questions: 1. Would specimens exposed to a remineralization period before starting ph cycling be more resistance to demineralization than specimens that were not exposed? 2. Does fluoride have any effect on remineralized and non- remineralized enamel during ph cycling?

16 5 CHAPTER II REVIEW OF LITERATURE Development, Prevention and Control of Dental Caries Structural Elements of Enamel and their Role in Caries Formation Enamel, the outer surface of the coronal portion of teeth, is the hardest substance produced by the body. It develops from the formative cells called ameloblasts and functions as a protective coating to the dentin and pulp, the internal portions of the tooth. Enamel rods are the predominant structural component of enamel. They have a major role in bonding restorative materials to tooth structure. Posterior teeth have more enamel rods than anterior teeth, twelve million to five million, respectively (Roberson & others 2002). Enamel rods, which vary in diameter from 8 µm near the external surface and 4 µm near the dentin border, are oriented from the DEJ towards the external surface of the tooth in a perpendicular direction. There are more enamel rods towards the DEJ and less near the external surface of the tooth. At the cervical margin, enamel is thinner, and the enamel rods are oriented apically. Cervical areas are more susceptible to caries because of the thin protective layer of enamel (Roberson & others 2002) or caries may reach the dentin faster. To provide sufficient strength, an underlying layer of dentin must support the enamel. Enamel is 95-98% inorganic material, predominantly calcium phosphate crystals, by weight. The calcium phosphate crystals are an impure form of hydroxyapatite because it contains carbonate, sodium, fluoride and other ions (Fejerstov & Kidd 2003). Of all the structural components of the tooth, enamel is the most resistant to acid dissolution. The application of fluoride makes enamel more resistant, presumably by lowering the rate of demineralization (Roberson & others 2002).

17 6 In the oral environment when the ph is below 5.5, calcium and phosphate ions are released from the enamel in a process known as demineralization. If these ions are not replaced and demineralization continues, a cavitation forms (Beltran-Aguilar & Beltran- Neira 2004). Healthy enamel is resistant to acid dissolution but with continued exposure to acid, it weakens and demineralization progresses through the enamel surface in a linear fashion, following the direction of the enamel rods at a constant rate (Anderson & Elliott 2000). An area of demineralization is the first sign of a carious lesion. A carious lesion that is limited to the enamel does not require restoration unless there are frank cavitations. Enamel demineralization can be improved by plaque control, dietary change and the use of fluoride (Frencken & Holmgren 1999). Dentin and Cementum The dentinoenamel junction, a hypermineralized zone, is approximately 30 µm thick and links enamel to dentin (Roberson & others 2002). Dentin, from mesoderm, is the major portion of the tooth crown and root. Its main cellular component is the odontoblast. The odontoblast is both dentin and pulp tissue as its cytoplasmic cell processes extend into the tubules in mineralized dentin and the cell bodies are in the pulp cavity (Roberson & others 2002). Dentin is more flexible than enamel and provides a cushioning effect. Dentin is, by weight, some 75% inorganic materials, predominately hydroxyapatite, and 25% organic material, water, and other materials (Roberson & others 2002). A cavitated lesion within dentin is an indication for a restoration (Frencken & Holmgren 1999). Cementum, also derived from mesoderm, covers the outer root surface and by weight is about 45% inorganic and 55% organic material and water (Roberson & others 2002). It is softer than dentin. The cementum and enamel do not meet to form the

18 7 cemental-enamel junction (CEJ) in about 10% of teeth, which can result in a sensitive area (Roberson & others 2002). The Role of Plaque in the Formation of Caries As a tooth erupts into the oral cavity, it becomes surrounded by a pellicle, a film in which bacteria attach, causing a build-up of a mass called plaque (Winston & Bhaskar 1998). Plaque bacteria hydrolyze ingested sugars producing weak acids, which travel from the plaque into surrounding tooth structure. A sucrose rich environment allows certain organisms to produce extracellular polysaccharides, which form a gelatinous material causing a diffusion-limiting barrier in the plaque. The local environment then becomes anaerobic and acidic, conditions that favor tooth dissolution (Roberson & others 2002). The acids demineralize the enamel leaching calcium and phosphate ions with eventual collapse of tooth structure and cavitations (Winston & Bhaskar 1998). Dental erosion may also result in tooth demineralization. Tooth demineralization occurs below a ph of 5.5. The phenomenon of enamel degradation is complex, however, at low ph levels; erosion is most likely the main factor (vonfraunhofer 2004). Carbonated soft drinks may operate from both processes of demineralization, the transformation of bacterial by-products and the acid in the soft drink. Grippo & others (2004) suggested the corrosive potential of an acidic drink combines several influences, ph, buffering capacity, acid chelating properties, frequency, and duration of use. Demineralization is not a continuous process as lost ions are replaced through remineralization. Remineralization occurs from plaque s ionic exchange with saliva, the buffering capacity of saliva, and the removal of the acids through diet or chemotherapeutic agents. In-vitro studies aim to simulate the intraoral environment through ph cycling systems using chemical and microbial models. Chemical models consist of acidic liquids or gels to demineralize tooth structure. Microbial models use acid producing bacteria to demineralize tooth structure.

19 8 Classification Systems for Carious Lesions There are several ways of classifying carious lesions. They may be classified by age, development, tooth structure, anatomic location, or severity. Early childhood caries and senile caries are lesions classified by age, youth, and old age, respectively. Carious lesions classified by tooth structure are enamel caries, dentinal caries, or cemental caries. Developmental caries classification includes primary and secondary caries. Primary carious lesions develop on a tooth initially free of a restoration. A secondary lesion develops around an existing restoration. Pit and fissure caries, smooth surface caries, and root surface caries classify by anatomic location (Roberson & others 2002). A classification system based on severity of the lesion is incipient, gross, and rampant caries. An incipient lesion is the smallest detectable defect and gross denotes a large cavitated defect involving most of the coronal structure of the tooth. An incipient lesion may appear as a chalky white spot on the enamel surface of the tooth, commonly called a white spot lesion. Rampant caries is a clinical situation of many gross carious lesions throughout the oral cavity (Roberson & others 2002). Prevention of the Carious Lesion The majority of adults and over sixty percent of schoolchildren in the world have some dental caries experience. Post-eruptive sugar consumption and inadequate fluoride exposure contribute to the formation of dental caries. Dental caries is a major public health problem and is expected to escalate in the future (Petersen & Lennon 2004). Dental caries has alternating remineralization and demineralization episodic phases (Roberson & others 2002). This pattern is influenced by the carcinogenicity and frequency of ingestion of foodstuffs, levels of cariogenic bacteria, exposure to fluoridation, toothbrushing frequency and duration, and oral physiology (von Fraunhofer 2004).

20 9 Preventive measures include water fluoridation, education including diet modification, toothbrushing, and topical fluorides, sealants, xylitol gum, chemotherapeutic agents, and saliva and bacterial testing. The selection of preventive measures depends upon an individualized caries risk assessment. Winston & Bhaskar (1998) stated that fluoride added to municipal drinking water supplies and toothpastes was almost certainly the most effective caries-preventive treatment available at this time. Water fluoridation, one of the greatest public health achievements, is a safe and inexpensive method of fluoride delivery for children and adults (CDC MMWR 1999). Education provides information about caries development, the role of diet and frequency of cariogenic foods, and methods that can be used to avoid caries development. The greatest benefit of toothbrushing is to introduce fluoride onto tooth structure by use of a fluoridated dentifrice. Recent scientific discussions suggested adding fluoride to milk and salt (Peterson & Lennon 2004). Topical fluorides in the form of varnishes, supplements, and mouthrinses are recommended for those at high risk for caries, regardless of age (Beltran-Aquilar & Beltran-Neira 2004). Petersen & Lennon (2004) agreed, with recommendations to enhance the preventive fluoride effect if there was high sugar consumption. The oral environment has a constant cycle of remineralization and demineralization. Strengthening the enamel surface of the tooth to attack from bacterial by-products and other acids by use of preventive methods provides the best way for tooth surfaces to remain healthy and caries free. Early Caries Detection Methods Incipient carious lesions may not be detectable clinically or radiographically. To make maximum use of remineralization therapies, caries detection techniques are needed to identify lesions earlier (Winston & Bhaskar 1998). White spot lesions are not

21 10 detectable visually until they have progressed 200 to 300 micrometers into the enamel (Winston & Bhaskar 1998). Traditional diagnostic methods, visual examination, tactile sensation, and film radiography, are more specific than sensitive and result in many false negative findings (Bader & Shugars 2004). Visual inspection, bitewing radiography and fiber optic transillumination allow qualitative, subjective interpretations of visual information (Angmar-Mansson & ten Bosch 2001). In addition to exposure to ionizing radiation, radiography lacks the ability to detect early carious lesions (Amaechi & others 2003). Sensitivity is the proportion of correctly identified cases with disease, specificity the proportion of correctly identified cases without disease (Ashley & others 1998). Identifying disease at an early stage and applying preventive therapies decreases the likelihood of needing a restoration in the future. Anusavice (2001) reviewed the sensitivity and specificity of caries detection methods and found that of all reviewed detection methods, visual inspection had the lowest sensitivity for non-cavitated occlusal lesions. Anusavice (2001) pointed out that a lack of sensitivity of diagnostic aids underestimates the true caries risk. Existing literature on caries assessment is flawed because of false negative diagnoses. Caries risk is defined as the probability that a specific number of new lesions will develop and/or a specific number of existing lesions will progress over a specified period of time (Anusavice 2001). Articles cited in this literature review reported that of all other predictors of caries, salivary levels, bacterial assays, fluoride levels, Decayed, Missing and Filled Surfaces (DMFS), and dietary controls, DMFS had statistically significant differences as the most reliable predictor for future caries. Anusavice (2001) emphasized the need for accuracy in detecting carious lesions since previous caries history is one of the most powerful predictors of future caries.

22 11 Non-invasive Caries Diagnostic Methods Pine and Bosch (1996) reviewed non-invasive methods for diagnosis of carious lesions that would supplement a clinical examination. Non-invasive methods make use of the carious lesion s physical properties. Specifically, these methods compare healthy tooth structure with demineralized tissue within the lesion (Pine and Bosch 1996). Some non-invasive diagnostic methods were light scattering, electrical resistance, fiber-optic transillumination, Optical Coherence Tomography, and fluorescence. The Optical Caries Monitor is an example of a light scattering non-invasive diagnostic method. This instrument uses optical fiber technology and is most useful for smooth surface caries (Pine & Bosch 1996). Optical methods are suitable for in-vitro studies, in part, because they are non-destructive and quick (Anderson & others 1996). Ashley & others, (1998), compared in-vitro caries detection of posterior occlusal caries in enamel and dentin using the Electronic Caries Monitor, to caries detection by visual examination, fiber optic transillumination, and conventional and digital bitewing radiography. They determined that the Electronic Caries Monitor (ECM) was a more sensitive diagnostic method for occlusal caries detection than visual evaluation, fiberoptic transillumination, or film and digital radiographic methods. Visual evaluation of enamel caries was comparable to the Electrical Caries Monitor but there were statistically significant differences (p<0.05) when diagnosing dentinal caries; ECM had higher sensitivity (Ashley & others 1998). The high inorganic content of enamel impedes the conductance of electricity (Ashley & others 1998). Conductivity increases in an area of demineralization, for that reason, the Electronic Caries Monitor would detect an increase in electrical conductivity (Pine & Bosch 1996). The benefit of fiber optic transillumination as a cost-efficient adjunct to the clinical examination in detecting approximal dentinal lesions is questionable, since it clearly lacks the ability to detect the number of approximal enamel lesions seen on

23 12 radiographs (Pine & Bosch 1996). However, Ashley & others (1998) found the FOTI had higher specificity than ECM and would diagnose more sound surfaces correctly. Optical Coherence Tomography (OCT) is similar to ultrasound images except that light is used as the examining medium instead of sound waves. In an in-vitro study of artificial carious lesions in bovine incisor teeth, Amaechi & others (2003) correlated optical coherence tomography to quantitative light-induced fluorescence for detection and quantification of early dental caries. Exposed enamel windows were subjected to a demineralizing acidic buffer solution for 72 hours. Images were taken at baseline and 24, 48, and 72 hours. OCT collects three types of images, labeled A, B, and C. They used A images to calculate the degree of change in reflectivity, B images to longitudinally monitor lesions, and C images for transversal views. In their study, the reflectivity of the carious tissue decreased with increasing remineralization time. OCT discriminated sound from carious tissue by a decrease in reflectivity. OCT provided quantitative data and information on depth. The authors stated that OCT could identify an incipient lesion as early as 24 hours into its development (Amaechi & others 2003). While commonly used in-vivo in Ophthalmology, its use in dentistry has been for in-vitro studies. It is recommended for testing the efficacy of products that could inhibit demineralization and/or promote remineralization, in-vivo, in situ or in-vitro (Amaechi & others 2003). Fluorescence DIAGNOdent DIAGNOdent is a laser induced fluorescence early caries detection device that measures laser fluorescence within tooth structure (KaVo DIAGNOdent ). Organic and inorganic materials in teeth absorb the laser light and emit fluorescence in the infrared region (Shi & others 2001). Clean healthy tooth structure has little or no fluorescence. Scale readings on the instrument display are low. Carious tooth structure

24 13 exhibits higher scale readings on the instrument display in proportion to the degree of caries (KaVo DIAGNOdent ). The presence of a carious lesion increases the fluorescence (Shi & others 2000), presumably by an increase in the number of bacterial chromophores (KaVo DIAGNOdent ). A digital number and an audible sound record fluorescence increase. Higher numbers and higher pitched sounds indicate more demineralization and a carious lesion that may require a restoration. A systematic review of DIAGNOdent (DD) by Bader and Shugars (2004) divided twenty-five studies into four general categories: in-vitro detection of occlusal dentinal caries, in-vitro detection of occlusal enamel caries, in-vivo detection of occlusal dentinal caries, and in-vivo detection of smooth surface caries. Three studies evaluated DD in detecting residual caries and caries at the margins of restorations (Bader and Shugars 2004). DD had high sensitivity with in-vitro and in-vivo occlusal dentinal caries, variable sensitivity detecting occlusal enamel caries, questionable sensitivity on smooth surfaces, and good guarded performance in detecting caries along the margins of restorations (Bader and Shugars 2004). They suggested using DIAGNOdent to refine a questionable diagnosis, direct preventive treatment, and monitor lesion activity over time (Bader and Shugars 2004). Quantitative Light Induced Fluorescence Quantitative Light Induced Fluorescence (QLF) is a recent early caries detection method that can longitudinally monitor carious lesions over time. The set-up consists of a computer, clinical camera, and custom software. QLF appears to be a viable method of early caries detection in-vitro and in-vivo. QLF is effective in detecting carious lesions on tooth structure and adjacent to restorations. It has wide application in clinical dentistry including monitoring the change in shade during tooth whitening procedures (Amaechi and Higham 2002).

25 14 More recent studies (Gonzalez-Cabezas & others 2003, and Pretty & others 2003b) evaluated QLF s ability to detect carious lesions adjacent to restorations. Gonzalez-Cabezas & others (2003) conducted a three-part study of QLF to evaluate demineralization-surrounding tooth colored restorations in-vitro using chemical and microbial models. Gonzalez-Cabezas & others (2003) found statistically significant differences between 48 and 120 hours for lesion depth. There were also statistically significant differences between control and experimental specimens. Resin composite (Silux ) had the deepest lesions and glass ionomer (Photacfil ), the shallowest. QLF and Confocal Laser Scanning Microscope (CLSM) varied in detection of carious lesions. Lesions were in 8 of 10 specimens of the experimental group and 4 of 10 in the control group with QLF. There were lesions in 8 of 10 in the experimental group and no lesions found in the control group with CLSM. An in-vitro study by Pretty & others (2003b) evaluated the ability of Quantitative Light Induced Fluorescence to detect, quantify, and longitudinally monitor demineralization adjacent to similar restorative materials, evaluating the loss of fluorescence for restorative material, enamel margin around the restoration, and control areas. They discovered that all restorative materials except compomer showed statistically significant differences between baseline, 72 hours, and 144 hours after demineralization. Fluorescence reduction occurred with amalgam, temporary material, and glass ionomer restorative materials. Transverse microradiography (TMR) analysis confirmed subsurface lesions on all exposed areas for all restorative materials. Tranaeus & others (2001) used QLF in an in-vivo study to monitor changes in white spot lesions in caries active adolescents after fluoride varnish and/or professional tooth cleaning. They obtained consent for 31 participants, 13 and 15 years of age, with white spot lesions on the buccal surfaces of permanent molars and premolars. Patients were randomly assigned to a fluoride varnish group or professional tooth-cleaning group. Fluoride varnish was applied and patients were evaluated at baseline, one-week and then

26 15 four times after that at six-week intervals. QLF evaluation occurred at baseline and each six-week visit. There was a statistically significant difference (p = 0.03), for average change in lesion fluorescence between QLF readings with the fluoride varnish group and professional tooth-cleaning group. The fluoride varnish group demonstrated a statistically significant difference in lesion area and fluorescence change, p =0.001 and p=0.002, respectively. Shi & others (2001) compared readings and the performance of two devices, DIAGNOdent and Quantitative Light Induced Fluorescence (QLF). In their study, they used an argon laser device. The light that passed through the clinical camera was filtered by an optical bandpass filter with λ=370 nm. A light guide guided the spectrum of light at the tooth surface with a peak at 404 nm (Angmar-Mansson and ten Bosch 2001). They determined that DIAGNOdent readings increased more steeply as the lesion extended into dentin. QLF and DIAGNOdent had the same specificity. The QLF laser device had the highest correlation coefficient between mineral loss and laser fluorescence readings of enamel caries by transverse microradiography, but the QLF lamp had the highest sensitivity. The use of a laser fluorescence device may increase the sensitivity of detection of carious lesions although its specificity is reduced (Anusavice 2001). Similarly, Lagerweij & others (1999) compared the performance of three optical fluorescence systems, in-vitro, to detect small carious lesions. They also evaluated the repeatability of the measurements. The QLF set-ups included a water-cooled argon laser with illumination through a ring illuminator, an air-cooled argon laser with illumination through a beam splitter, and a clinical camera, illuminated with an arc lamp filtered to 370 nm. Correlation coefficients were 0.70 for the beam splitter, 0.63 for the clinical caries camera and 0.36 for the ring illuminator. Among QLF set-ups, Lagerweij & others (1999) found the beam emitter superior to the ring illuminator and clinical camera. Precision was independent of enamel lesion size. The beam splitter was the most precise followed by the clinical caries camera and finally, the ring illuminator. They noted the

27 16 standard deviation among the QLF systems was 3-4 times that of transverse microradiography. They suggested that validating every new system set-up, with an invitro study, would improve precision, accuracy, and uncover flaws. Post-microscopic validation occurred in studies by Gonzalez-Cabezas & others (2003), Pretty & others (2003b), Shi & others (2001), and Lagerweij & others (1999). Shi & others (2000) and Langerweij & others (1999) agreed, that the laser QLF device had a higher correlation with transverse microradiography. Transverse microradiography and histological sectioning were the gold standards for validation of QLF and one or both were used in several in-vitro studies (Gonzalez-Cabezas & others 2003, Pretty & others 2003b, Shi & others 2001, Lagerweij & others 1999, Ashley & others 1998). Authors (Gonzalez-Cabezas & others 2003, Pretty & others 2003a, Lagerweij & others 1999, Tranaeus & others 2001, Shi & others 2001) suggested that errors might occur with QLF measurements when drying and positioning specimens and from other emissions. Angmar-Mansson and ten Bosch (2001) found, in-vitro, the fluorescence loss was increased by a factor of 0.10 to 0.15 due to dehydration, and that white spot lesions were more distinguishable when teeth were air-dried. A standardized drying procedure minimized errors in recordings produced by excessive moisture (Shi & others 2001, Ashley & others 1998). Hydration of the tooth may affect the optical properties of enamel due to differences in the refractive indices of water and enamel (Amaechi & Higham 2002). Lagerweij & others (1999) stated that when using a standardized drying time, large lesions might not dry as quickly as smaller lesions. Mansson and ten Bosch (2001) recognized dehydration and errors in image reconstruction as confounding variables for QLF analysis whereas Lagerweij & others (1999) recognized moisture content, positioning and overlaps in excitation and emission wavelengths as variations having a major impact on lesion size. Other concerns were reflections, marginal color change, validation of set-ups, and cut-off points. The overlap in excitation and emission spectra, particularly with wide

28 17 wavelength ranges, may disturb measurements by producing reflection. QLF analysis is very sensitive to the presence of stains. Gonzalez-Cabezas & others (2003) stated that marginal color change was often due to exogenous stain from food and medication. Cut off points were a concern in several studies (Ashley & others 1998, Shi & others 2001). Cut-off points determined the sensitivity and specificity of a method. Lack of software calibration was another reason for errors. As a caries detection device, Quantitative Light Induced Fluorescence has shown reliable results in-vitro and seems suitable in-vivo to monitor incipient lesions and evaluate preventive measures for caries susceptibility (Angmar-Mansson and ten Bosch 2001). Glass Ionomer Restorative Material Physical and Chemical Properties Glass ionomer cements (GIC), introduced into the dental community by Wilson and Kent in (1972), became available commercially in the United States in 1977, and received American Dental Association acceptance in 1979 (Klausner & others 1989). The uses of glass ionomer cements are for cementation of cast and indirect restorations, as liners or bases, and as a restorative material (Klausner & others 1989). In addition, Swift & others (1990) found that of the Iowa dentists responding to their survey, approximately thirty percent of their core build-ups were made with a glass ionomer restorative material. It is the only direct permanent restorative material classified as cement (Hewlett & Mount 2003). The reason the material was developed initially was to place anterior teeth restorations, repair erosion defects, cement restorations, and serve as cavity liners (Wilson & Kent 1972). They are available as a liquid and powder that are hand-mixed or encapsulated. The hand mixed version is used for the ART technique (Frencken 1999). The encapsulated form requires electricity to activate it.

29 18 Glass ionomer cements chemical composition is an aluminosilicate glass powder and copolymers of acrylic acid (Wilson & Kent 1972). The ion leachable glass powder is predominately silicon dioxide, aluminum oxide, and calcium fluoride. Glass particles can also contain aluminum phosphate, aluminum fluoride, and sodium fluoride. The glass particles were heated and fused with a fluoride flux. They were cooled and separated into particles between 20 and 50 microns (Charlton 2002). Adding tartaric acid to the liquid controls the setting reaction. The composition varies depending on the manufacturer. Fuji IX powder contains aluminum-fluorosilicate glass and dehydrated polyacrylic acid. Its liquid is polyacrylic acid, deionized water and tartaric acid with a powder liquid ratio of 3.6:1 (Palma-Dibb & others 2003). The polyacrylic acid reacts with the glass particles by an acid base reaction and calcium ions are released. Charlton (2002) described an overlapping three-phase reaction occurred when mixing the powder and liquid. In the first phase hydrogen ions from the polyacrylic acid attack the glass, liberating calcium, fluoride, and aluminum ions that result in a hydrogel around the glass particles. These leachable ions require water to react (Frencken & Holmgren 1999). In Phase II, calcium and aluminum precipitate out as polycarboxylates, which causes hardening of the cement through cross-linking. The calcium and aluminum react with the fluoride in the hydrogel. Calcium polycarbonates develop first because of efficient crosslinking ability but are not as stable or strong as the aluminum polycarbonates, which develop over the next twenty-four hours. The crosslinking process is continuous and over time improves GIC mechanical properties (Kleverlaan & others 2004). The final phase, which improves the cement s physical properties, is hydration of the hydrogel and calcium and aluminum polycarbonates. The initial setting reaction has a ph of 2.5 that increases over the next twenty-four hours (Wesenberg & Hals 1980). Protecting the restoration from water contamination is recommended for the first hour (Frencken & Holmgren 1999). The short-term water sensitivity that causes surface

30 softening and low wear resistance (Kleverlaan & others 2004) limit the full potential of GIC for dental applications. 19 Advantages and Disadvantages A significant benefit of glass ionomer cements is its ability to bond to tooth structure (Reinhardt & others 1993). There is higher bond strength to enamel in comparison to dentin (Frencken & Holmgren 1999). Glasspoole & others (2002) agreed that surface conditioning with a weak acid before placing glass ionomer resulted in improved bond strength. They examined the interface between enamel and restoration and found microporosities in the enamel suggesting that mechanical bonding may contribute to bond strength. It has high compressive strength, a coefficient of thermal expansion similar to dentin, and the ability to charge and recharge (Strother 1998) with fluoride. The anticariogenic effect of glass ionomer cements has been attributed to its fluoride release in several in-vitro experimental studies and has been assessed in in-vivo studies. Physiochemical bonding to tooth substrate is a beneficial property of glass ionomer cements (Irie & others 2003). Bonding is accomplished through the reaction of calcium ions in enamel and dentin to carboxyl groups in the cement (Zivkoviv & others 2001). Some believe the actual bonding mechanism is unknown (Charlton 2002). A national survey of general dentists listed disadvantages of the cement as poor esthetics due to staining and wear, delayed finishing and limited shade selection (Reinhardt & others 1993). Other disadvantages included short working time, moisture contamination susceptibility, desiccation, and brittleness (Dionysopoulos & others 2003). Autio-Gold & Barrett (2004) attributed the staining and roughness of glass ionomer cements to the porosity of its glass particles. Hybrid Forms of Glass Ionomer Cements Glass ionomers are available in several modified forms (Sidhu & Schmalz 2001). Hybrids of glass ionomer cements exist as giomers, resin modified glass ionomer and

31 20 compomers. Giomers are the most recent addition to the hybrid materials. Giomers consist of pre-reacted glass ionomer in a urethane resin. Giomers require a resin bonding system to adhere to tooth structure (Gonzalez & others 2004). Resin modified glass ionomer cements (RMGIC) contain methacrylate resin components and have a dual cure mechanism, an acid base reaction, and a photopolymerization reaction. Brackett & others (2003) in a two-year clinical performance study found that Class V resin modified glass ionomer restorations were more retentive than composite restorations. Dauvillier & others (2000) suggested that less contraction stress on the early setting stage of glass ionomer cements in comparison to resin composites strengthen the bond of the glass ionomer cement to the cavity walls. Compomers are resin cements that have glass components. Compomers do not have an initial acid-base reaction and are not true glass ionomer cements. These hybrid forms offer improved physical properties and overcome some of the limitations of traditional glass ionomer cements (Dionyspoulos & others 2003a). Anticariogenic Potential of GICs The anticariogenic behavior of glass ionomer cements has been attributed to its fluoride content. Glass ionomer cements contain 10 to 23 % fluoride (Charlton 2002). Some report fluoride levels as high as 28% (Frencken & Holmgren 1999). Smales & others (2000) examined the cervical and coronal demineralized areas of extracted premolar teeth restored with Class V glass ionomer and compomer restorations using light microscopy and found that, between compomer and conventional glass ionomer restorative materials, glass ionomers showed larger demineralization-free zones next to the margins of the restoration. The authors concluded that, fluoride release was responsible for the larger demineralization-free zone. Donly & others (1999) had similar results when examining resin modified and amalgam restorations in primary teeth. There was less demineralization next to the resin modified glass ionomer.

32 21 Gonzalez & others (2004) in-vitro study of direct tooth colored restorative materials demonstrated that giomer and glass ionomer material had significantly greater demineralization free zones at the enamel margins compared to compomer and composite resin material. Strother & others (1998) found that fluoride release from glass ionomer was in greater amounts and lasted longer than release from fluoride-releasing composites. Lower but consistent levels followed an initial burst of fluoride. Fluoride levels increased six to ten times more after treatment of glass ionomer specimens treated with fluoride gel. They recorded statistically significant differences in fluoride release, reuptake, and re-release among restorative materials (p < 0.001). Their study concluded that fluoride release and uptake and re- release had no effect on tensile strength or initial surface roughness. Marinelli & others (1997) demonstrated glass ionomer cement s remineralization effect on adjacent tooth structure in their comparison of three fluoride delivery systems: a glass ionomer restorative material, fluoride rinse, and fluoridated dentifrice. They found 0.05% fluoride rinse had the highest remineralization capability of tested fluoride delivery systems and glass ionomer restorative material had the remineralization capability to a fluoridated dentifrice. The fluoride content in the oral environment is related to the ability of the restorative material to leach and exchange fluoride with other ions not solely to the fluoride content in the material (Hicks & others 2003). An in-vivo clinical study by Yip & others (2002) investigating the effects of two types of cavity preparations on glass ionomer cement longevity concluded that glass ionomer material afforded some caries protection when placed into adjacent pits and fissures. This was demonstrated by the absence of carious lesions in previously sealed fissures. Wesenberg & Hals (1980) suggested that glass ionomer cements remineralized tooth structure by the release of fluoride.

33 22 Comparison of In-Vitro and In-Vivo Findings Conclusions from in-vitro studies may not be comparable with studies in-vivo (Gonzalez & others 2004). In-vivo results were not the same as in-vitro results in the dual in-vitro and in-vivo study by Papagiannoulis & others (2002). Their in-vitro results showed statistically significant differences in lesion length between glass ionomer restorations and composite restorations (p < 0.05). Lesion length was greater for composite restorations. In the in-vivo portion of the study, there was greater caries activity around glass ionomer restorations than composite restorations. They suggested that in-vitro studies could not serve as a guide for in-vivo applications. They expected to detect the release of fluoride across the interfacial gap. Fluorine was absent from the enamel margins of both glass ionomer and composite restorations analyzed by SEM. Sa & others (2004) also caution extrapolating in-vitro studies to real clinical situations. They studied the anti-cariogenic properties of different in-vitro caries models for three fluoride releasing restorative materials. Resin composite was the control material. Their chemical model showed preventive properties and inhibition of demineralization. Papagiannoulis & others (2002) in-vitro study used the chemical model and their in-vivo component the microbial model. Atraumatic Restorative Treatment (ART) Restorative Treatment Approaches Traditional preparations include sound and diseased tissue. Other approaches developed which were more conservative and reduced the amount of tooth structure necessary to place and replace restorations and restore carious lesions. One such approach, the minimal treatment approach, gained support by preserving tooth structure. A recent approach using hand instruments and adhesive restorative materials is Atraumatic Restorative Treatment.

34 23 Rationale Several in-vitro and in-vivo studies demonstrated the placement, survival, and usefulness of ART restorations in primary and permanent teeth. Phantumvanit & others (1996) and Taifour & others (2003) compared the survival rates of ART restorations and amalgam restorations in different groups of children after three years. Massara & others (2002) described the first ultrastructural and chemical analysis of dentinal tissue before and after placement of ART restorations. Schriks & others (2003) evaluated the physiological and psychological aspects of ART and traditional treatment. Mjor and Gordan (1999) examined the scientific basis and cost-effectiveness of ART restorations. Yip & others (2002) investigated the effects of two cavity preparation methods on the longevity of glass ionomer cement restorations. Frencken & others (2004) conducted the first meta-analysis on the effectiveness of single surface ART restorations in the permanent dentition. Their review included two articles already noted (Phantumvanit & others, 1996 and Taifour & others, 2003). Three Thailand villages served as the study site for Phantumvanit & others (1996). Children and adults in the first village received no treatment, second village participants received glass ionomer restorations, and third village participants had amalgam restorations. Preliminary data was a baseline DMF. One dentist and two dental nurses placed the restorations. An adult was classified as anyone over thirteen years of age. Two examiners evaluated the restorations at one, two, and three years. The use of duplicate examinations on 14% of participants accessed intra- and inter-examiner agreement. The post-dmf scores were compared to the baseline DMF scores. Amalgam restorations were significantly better than ART restorations. The authors did not determine the definition of a meaningful difference beforehand; therefore, they believed their results were debatable (Phantumvanit & others 1996). Taifour & others (2003) study site was the World Health Organization Regional Centre in Syria. The sample consisted of 679-second grade children, randomly assigned

35 24 to two treatment groups. Three examiners recorded plaque scores and carious lesions initially. For the ART group, operators used hand instruments to excavate caries and placed Fuji IX and Ketac-Molar glass ionomer restorations. For the second group, Minimal Traditional Amalgam (MTA), caries was removed with a handpiece and amalgam restorations were placed into prepared cavity preparations. Eight dentists placed the Minimal Traditional Amalgams and ART restorations. Two dentists evaluated the restorations yearly. Evaluation aspects included an assessment of the physical condition, an intra- and inter-evaluator consistency test, and the presence of primary or secondary caries. There was a statistically significant finding (p=0.04) that favored the ART approach over the MTA approach. Their results were limited to single surface restorations. Marginal defects and missing restorations were primary reasons for failure of ART and MTA restorations. Both approaches produced statistically significant differences between operators (p=0.01). The survival rates of ART and MTA single surface restorations were 82.1% and 76.9%, respectively. Taifour & others (2003) stated that, although the retention rate in their study was lower than previous studies, use of ART was at least comparable to amalgam. They recommended a training course before practicing the ART approach. Massara & others (2001) described ART as a one-session approach, with gross caries removal without anesthesia followed by placement of a final restoration of glass ionomer cement. They took dentin samples from primary molar teeth of eight children after restoration with Fuji IX. Three months later, they took new dentin samples. Samples were evaluated with SEM and X-ray Energy Dispersion Spectrometry. They found that original samples were highly infected with bacteria, post-treatment samples were not, but this was not statistically significant. There was a significant increase in calcium concentration post-treatment. Massara & others (2002) attributed the arrest of caries progression to the glass ionomer materials fluoride release and physicochemical bond to dentin. Because they did not detect fluoride in pre-dentin or

36 25 post-dentin samples, they were cautious about attributing its inhibitory effect to fluoride release alone. Studies by Massara & others (2002) and Taifour & others (2003) both used conventional glass ionomer restorations. The study sample for Schriks & others (2003) was six-year old children in Indonesia. The children were randomly divided into two groups. Inclusion criteria included the presence of at least one multi-surface carious lesion in a primary molar. The control group received Minimal Cavity Preparation (MCP) restorations prepared with a handpiece, similar to MTA restorations in Taifour & others (2003). The experimental group received ART restorations prepared with spoon excavators. Chemfil glass ionomer cement was the restorative material. Psychological scoring and heart rate determined psychological and physiologic assessments, respectively. Children receiving restorations after caries excavation treated without burs had less anxiety. Children receiving ART restorations had significantly lower psychological scores than the MCP group at all treatment points, p<0.05. Girls had significantly higher psychological and heart rate scores than boys, p<0.05, at all points except matrix placement and restoration. Yip & others (2002) placed glass ionomer restorations in ART and conventional occlusal cavity preparations in 68 adult patients. Their cavity preparations were similar to Taifour & others (2003) and Schriks & others (2003), one with hand instruments, and the other with a handpiece. Three dentists placed all restorations. Restorations were evaluated after 12 months. They placed amalgam in conventional cavity preparations as controls. Yip & others (2002) found ART preparations took twice as long as conventional preparations (p<0.001). Frencken & others (2004) reviewed five clinical trials comparing single surface glass ionomer and amalgam restorations, separating the studies into an early and late group. Frencken & others (2004) reported in their meta-analysis that in earlier studies, amalgam restorations survived longer than ART restorations. In later studies, ART restorations survived longer than amalgam restorations after three years.

37 26 Mjor & Gordan (1993) did not find any clinical advantage of ART restorations over amalgam restorations. ART restorations required longer placement periods. They believed ART s greatest asset was its applicability to areas without electricity or dental equipment. The authors recommended ART for children, those with management problems, the mentally and physically disabled, elderly and as an additional treatment in school dental service. Honkala & Honkala (2002) evaluated ART restorations in an elderly population in Finland. The study population comprised homebound dentate elderly needing community based support services. They placed twenty-five ART restorations during dental home visits. One year after placement of the restorations, sixty-eight percent were labeled good, eleven percent marginal, and sixteen percent were unacceptable. The success rate of ART restorations was 89.6% at a two-year follow-up period in a pediatric population (Honkala & others 2003). Survival and Longevity of Restorations Burke & others (2000) surveyed thirty-two practitioners in the United Kingdom to determine the reasons for the placement and replacement of restorations. Restoration materials included amalgam, composite, glass ionomer, and compomer. Their study revealed that the primary reason for placement and replacement of a restoration was caries. The analysis considered such factors as oral hygiene, caries susceptibility, occlusal function, gender, and age. The mean replacement age of restorations in this study was 20.6 years for gold, 8.3 years for amalgam, 5.7 years for composite, 3.9 years for glass ionomer and 2.8 years for compomer (Burke & others 2000). Glass ionomers were replaced most often due to marginal fracture. McKenzie & others (2003) stated that flexural failure was typical of dental cements. Mount (2003) advised that the material should not be used on incisal corners or marginal ridges because of its lack of fracture resistance. Restoration longevity was described as satisfactory if

38 27 there is adequate tooth structure surrounding the glass ionomer restoration. Smales & Yip (2002) stated bulk fracture of multi-surface restorations was a short-term clinical problem with glass ionomer cement materials marketed by manufacturers for ART. Phantumvanit & others (1996) found that amalgam restorations survived longer than ART restorations and the result was statistically significant (p<0.001). In a later study, Taifour & others (2003) found no statistically significant differences in ART and amalgam restorations after 3 years (p>0.05). Mjor & Gordan (1993) noted that single surface survival rates of glass ionomer restorations on occlusal and smooth surfaces were higher than multi-surface survival rates involving proximal surfaces. Oral health practitioners restricted the use of glass ionomer restorative materials to high caries patients or those with poor hygiene. Burke & others (2000) believed this might be due to practitioner acceptance of literature claims of the caries inhibiting ability of glass ionomer materials. A study of 100 long-term patients from three dental practices in Adelaide, Australia, listed six factors that influence restoration survival. The factors were the dentist s practice and experience, and the patient s age, frequency of visits, initial or replacement restoration and change of dentist (Hawthorne & Smales 1997). Glass ionomer restorations had higher survival rates in the practice with the oldest patients. Factors with significant differences for glass ionomer restorations were the dental practice, patient age, frequency of attendance and initial, or replacement restoration. Those frequenting the practices less often had higher survival rates for glass ionomer restorations. This study determined median survival times for amalgam as years, and resin composites years. Crowns had the highest survival at 26 years. Seventy five percent of glass ionomer restorations remained at years.

39 28 Summary The number of studies examining the ART approach increased during the past few years. Many studies evaluated the longevity of restorations and found survival rates comparable to traditionally placed amalgam restorations. The approach recommends use of a fluoride releasing restorative material, a viscous glass ionomer restorative material. Most of ART applications in industrialized countries have been with high caries populations. Glass ionomer materials release fluoride and adhere to enamel and dentin, however, the exact mechanism is unclear at this time. Restorations containing a glass ionomer restorative material are often replaced due to marginal breakdown and loss of material rather than the presence of secondary caries. Secondary caries is a common reason for replacement of amalgam and composite restorations. Some authors noted that glass ionomer materials might be anticariogenic because of their bond to tooth structure, not their fluoride release. When compared to other restorative materials, glass ionomer restorative materials uptake and release fluoride at much higher levels. Several in-vitro studies and an increasing number of in-vivo studies used QLF to longitudinally monitor artificial and natural small carious lesions. Results have shown good correlation with microscopic evaluation. There is little information about ART in general dental practice. There is even less information about knowledge of the technique by general dentists. Little information is available about the use of ART restorations in high caries individuals, the role of fluoride release from glass ionomer material, tooth fluorescence, or the effect on the pulp and surrounding tooth structure when caries remains in the cavity preparation. Researchers debate whether the anticariogenic behavior of glass ionomer materials is due to its fluoride release or its bond to tooth structure.

40 29 CHAPTER III MATERIALS AND METHODS Introduction This study investigated changes in enamel adjacent to Class V glass ionomer restorations after ph cycling and fluoride treatment measured by Quantitative Light Induced Fluorescence. Remineralized and non-remineralized enamel surfaces were evaluated. The hypothesis was that a remineralization period would change the outcome of ART restorations exposed to ph cycling. The outcome variable was the change in fluorescence ( F). The instrument used to measure the change in fluorescence was the Quantitative Light Fluorescence (QLF) System (Inspektor Research Systems BV QLF/clin). The predictor variables in this study were the experimental group, measurement session, and fluoride exposure. Research Design This was an experimental, longitudinal, in-vitro study of extracted human adult molar and premolar teeth. The ph-cycling period was twenty days. There were eight QLF measurement sessions. Figure 1 illustrates a schematic of the research design. A stereomicroscope, polarized light microscope, and a scanning electron microscope were also used to evaluate selected specimens.

41 Teeth Group 1 n=25 Class V prep Group 2 n=25 Class V prep Group 3 n=25 Class V prep Group 4 n=25 Class V prep 1* Precycling demineralization All Groups 72 hours demin solution 2* Groups 1 and 2 100% humidity 7 days ART Restoration Non-remin groups Groups 3 and 4 ART Restoration Precycling remineralization 7 days remin fluid Remin groups 3* 4* Group 1 (Fluoride) Group 2 (no fluoride) Experimental Negative control All groups proceeded to ph cycling. Group 3 (no fluoride) Experimental Group 4 (Fluoride) Positive control *Indicates a QLF measurement session Figure 1. Research design schematic Study Sample One hundred extracted, human, permanent, molar and premolar teeth were selected for the study. Teeth were obtained from extraction procedures at the University of Iowa College of Dentistry and were stored at the Dow s Institute of Dental Research Laboratory in 1% thymol. Teeth were selected and then disinfected in Streck tissue fixative (Streck Laboratories, LaVista, Nebraska) for two weeks. The coronal and root surfaces of each tooth were hand scaled to remove soft tissue and debris. A wet toothbrush (Sonicare ), removed extrinsic stains from the facial surface of specimens. Specimens were placed in deionized water after cleaning. They were visually evaluated.

42 31 Inclusion and Exclusion Criteria Inclusion criteria included unrestored permanent molar and premolar teeth with intact buccal surfaces and three millimeters or more apical root diameter. Exclusion criteria included cavitated carious lesions on any surface, intrinsic staining, and the presence of restorations, visible cracks, fractures, or depressions on the middle or cervical third of the buccal surface. Study Group Distribution Teeth were randomly divided into four groups (n=25) using random numbers (Microsoft Excel). Figure 2 represents the distribution of teeth for Groups 1 through 4. Distribution of teeth Percentage Group 1 Group 2 Group 3 Group 4 Tooth Category Max Molar Man Molar Premolar Figure 2. Distribution of teeth A 2 mm hole was placed in the apical third of each tooth with a #330 bur in a Kavo high-speed handpiece in order to insert a 15 mm length of waxed dental floss. The dental floss was used to suspend specimens in beakers. Groups were color-coded and teeth numbered 1 through 25. There were two remineralization groups (remin), and two non-remineralization groups (non-remin). Two experimental groups and two control groups comprised the

43 32 study sample. Two groups, one remin group and one non-remin group had a fluoride rinse during ph cycling. Table 2 is a list of materials used in this study. Table 2. Materials used in this study Material Ingredients Streck Tissue Fixative Precycling demineralizatio n solution Remin fluid (Synthetic saliva) GC cavity conditioner 2-bromo-2 nitropropane-1,3 diol, diazolidinyl urea, zinc sulfate, formaldehyde 2.20 mm calcium 2.20 mm phosphate, 0.05 acetic acid ph mm sodium bicarbonate, 3 mm sodium phosphate basic, 1mM calcium carbonate dihydrate 20% polyacrylic acid Mountain Dew Carbonated water, high fructose corn syrup, concentrated orange juice, citric acid, sodium benzoate, caffeine, sodium citrate, gum arabic, erythorbic acid, calcium disodium, brominated vegetable oil, yellow 5 Fluoride rinse 5,000 ppm NaF Fuji IX GP Fast restorative material Powder aluminum fluorosilicate glass, dehydrated polyacrylic acid Liquid polyacrylic acid, deionized water, tartaric acid Sample Preparation Each tooth was prepared for a Class V cavity preparation (3 mm x 2 mm x 1.6 mm). The preparation was placed in the middle third of the buccal surface of each specimen with a sharp #330-carbide bur (Brasseler, Atlanta, Georgia, USA) in a highspeed handpiece (Kavo ). A six- inch piece of dental floss was inserted through a 2 mm hole in the apical third of each specimen. A color-coded and numbered label attached to the end of the dental floss identified the specimen group (See Table 2). Specimen preparation is shown in Figure 3.

44 33 Figure 3. Preparation and dental floss Two layers of acid-resistant varnish (Nailslicks, Classic Red, Novell Corporation, Hunt Valley, Maryland) were placed on each specimen, except for the preparation and a one-millimeter perimeter of enamel around the preparation. All specimens were placed in a pre-demineralizing solution (2.20 mm calcium 2.20 mm phosphate, 0.05 acetic acid, ph 4.5) for 72 hours to demineralize the exposed enamel perimeter. A ph meter (ph Meter Orion Research, Inc., Boston, MA) verified that the ph was 4.5. The preparation and one millimeter perimeter of enamel, known as a window, is shown in Figure 4. Figure 4. Outline of specimen window and varnish

45 34 Figure 5. Presentation before (a.) and after (b.) a precycling demin solution There was daily evaluation of specimens. After three days, white spot lesions were visible around the perimeter of the preparations. These enamel lesions simulated the presentation of lesions restored using the ART approach. Figure 5 shows the difference in specimen presentation at the start and after 72 hours in a precycling demineralization solution. All restorations in remin and non-remin groups were placed following manufacturer s instructions. Preparations were conditioned with GC cavity conditioner (20% polyacrylic acid, GC Corporation, Tokyo, Japan) for 10 seconds, followed by a water rinse. Preparations were dried with a cotton pellet leaving a moist surface. A capsule of Fuji IX GP Fast (GC Corporation, Europe NV) restorative material was mixed in an amalgamator (Kerr Automix Amalgamator Class II) at 4000 rpm for 10 seconds followed by placement into the preparation in a single layer using digital pressure. One capsule supplied enough restorative material for four random specimens. A thin layer of petroleum jelly (Fougera USP) was placed on the restorations ten minutes after setting to avoid desiccation or water intake that could modify the physical properties (Palma-Dibb & others 2003). Rotary instruments were not used to finish the restoration to avoid abrading the enamel structure and to simulate the ART approach. Figure 6 illustrates a completed restoration and surrounding demineralized enamel.

46 35 Figure 6. Completed restoration Groups 3 and 4 were placed in remin fluid for one week. At the same time, unrestored groups 1 and 2 were kept at 100% humidity (beakers lined with water moistened towels covered with paraffin wax) at room temperature for one week. The restoration of the non-remin groups was delayed until the remin groups had completed 6-7 days of pre-cycling remineralization. A twenty- day cycling sequence followed. All groups started ph cycling simultaneously. Figure 7 shows a diagram of ph cycling. Rinse with Deionized water All Groups Start here Overnight in in clockwise Remin saliva fluid All All Groups Mountain Dew 4 hours All Groups Rinse with Deionized water All Groups Groups 1 and 4 Join Groups 2 and 3 In Saliva Remin fluid ph cycling 20 days Remin Saliva fluid* 4 hours All All Groups Rinse with Deionized water Groups 1 and 4 Fluoride rinse** Groups 1 and 4 Remin Saliva fluid Groups 2 and 3 Rinse with Deionized water All Groups *Remin fluid is synthetic saliva **Fluoride rinse is a 5,000 ppm sodium fluoride rinse applied for four minutes. Figure 7. Diagram of ph cycling

47 36 ph Cycling Specimens followed a routine regimen in solutions for twenty consecutive days. Each group was designated three beakers, labeled 1, 2, and 3. Each beaker was colorcoded as indicated in Figure 1, and contained 7 or 8 specimens. Specific beakers were allotted to each group, rinsing beakers after each solution change. All groups completed the same ph cycling sequence. Specimens from all groups were placed in Mountain Dew (Pepsi Bottling Company, ph 2.9) for four hours followed by four hours in a remin fluid (20 mm sodium bicarbonate, 3 mm sodium phosphate basic, 1mM calcium carbonate dihydrate). After removal from remin fluid and rinsing with water, specimens from Groups 1 and 4 were placed in a beaker with a 5,000-ppm solution (11.05 g NaF in 1L water) of NaF (Ozark- Ma, Honing Pennwalt Chemicals Lot # PR ) for four minutes. Specimens were rinsed with water and returned to beakers of fresh remin fluid. All solutions were stirred (Cole-Parmer 5 Position Stirrer Model Series) at a low setting. Each group of specimens had its own beaker of solutions (remin fluid, Mountain Dew ), 20 milliliters per tooth. Deionized water was the only water used throughout the experiment. Remin fluid was prepared daily. Mountain Dew was replaced daily. The sodium fluoride solution was replaced every five days. Solutions and specimens were kept at room temperature, in beakers, covered with paraffin wax. Quantitative Light Fluorescence A clinical camera (Inspektor Quantitative Light-Induced Fluorescence System version , Inspektor Research Systems, Netherlands) connected to a personal computer (Gateway VX920 ) was used for lesion analysis. The clinical camera captured the image that was stored on the hard drive of the computer for further analysis by the software quantitative program. A QLF measurement was obtained after a period in remin fluid (specimens in remin fluid >=4 hours). QLF measurements were recorded at eight

48 37 periods: no treatment, precycling demineralization, precycling remineralization period for Groups 3 and 4, baseline, restoration, and days 5, 10, 16, and 20 during ph cycling. The intervals of QLF measurements are listed in Table 3. Table 3. QLF measurement sessions Session # Step name Groups 1 No treatment 1,2,3,4 2 Precycling demineralization 1,2,3,4 3 Restoration (remin groups) 3,4 4 Baseline-start of cycling 1,2,3,4 Restoration (non-remin groups) Precycling remineralization (remin groups) 5 Day 5 ph cycling 1,2,3,4 6 Day 10 ph cycling 1,2,3,4 7 Day 16 ph cycling 1,2,3,4 8 Day 20 ph cycling 1,2,3,4 There were two training sessions at Dow s Institute, College of Dentistry before using the QLF system. The training sessions reviewed hardware and software components, positioning of specimens, image capture guidelines, and transfer of quantitative data. QLF software program (Inspektor Research Systems) settings were inspected and adjusted at each measurement session, following manufacturer guidelines. A validation pilot study preceded the present study, which measured repositioning accuracy and variation. A specimen was measured five times, starting a new session for each measurement. The pilot study demonstrated that QLF measurements were

49 38 repeatable with little variation when using the software positioning tools and external positioning devices while maintaining standardized drying and lighting protocols. Specimen evaluation took place in a dark room with no overhead lighting. Each specimen was held in position with red beading wax exposing the facial surface to the clinical camera. Standardized drying of each specimen consisted of placing a drop of deionized water on the buccal surface, blotting it once with tissue paper and then drying the surface for 5 seconds with compressed air at its lowest setting. The clinical camera, controlled by the principal investigator and another trained evaluator, captured the image immediately after drying. Calibration Area A standardized calibration area was established at the first measurement session. It was a one-millimeter region of sound enamel adjacent to the mesial or distal surface of the window. An example of a specimen with an exposed calibration area is represented in Figure 8. Figure 8. Calibration area (indicated by arrow) Nail varnish was removed from the area indicated by the white arrow in Figure 8 with a hand scaler to expose a sound enamel surface. The nail varnish was reapplied after each measurement session. The calibration area contained sound enamel not

50 39 exposed to any type of solution. The order of groups for QLF measurement sessions were selected by random numbers (Microsoft Excel). The QLF clinical system captured the fluorescent image of each specimen on the computer by a camera mounted on a stand projected towards each specimen. A standard procedure used by Amaechi & others (2003) was used in this study. Two investigators acquired QLF specimen images. The QLF set-up consisted of a special intraoral camera device connected to a computer fitted with a frame grabber and to which the QLF software was installed. To visualize and capture the tooth image, white light from a special arc lamp based on xenon technology was filtered through a blue-transmitting bandpass filter with a peak intensity of λ=370 nm and a bandwidth of 80 nm, to illuminate the tooth with a blue-violet light with an intensity of 13 mw/cm². The fluorescence loss is obtained by reconstructing the fluorescence of sound enamel at the site of the lesion from the fluorescence of the surrounding sound enamel (assumed 100%). The decrease in fluorescence was determined by calculating the percentage difference between the actual and reconstructed fluorescence surface. Any area with a fluorescence radiance drop of more than 5% was considered to be a lesion (Amaechi & others 2003). Pretty & others (2003) used a similar system and defined the absolute decrease in fluorescence as the F, defined as the percentage loss between actual and reconstructed fluorescence. The QLF quantitative program analyzed saved computer images. An area of interest or patch was placed over a segment of enamel on the occlusal aspect of the window. The patch ended on sound enamel. It did not include the glass ionomer restoration or nail varnish (Figure 9). Lesions below 95% of the reference threshold were considered demineralization (Gonzalez-Cabezas & others 2003). The principal investigator, at the start of the study, set cut-off points for QLF threshold levels for identification of demineralization. Previous studies were used as a reference (Pretty & others 2003b, Amaechi & others 2003). Images of each specimen were retained for future analysis. The principal

51 40 investigator conducted the quantitative analysis of all images. Random numbers were used to select the order of groups for each measurement session and quantitative analysis. Figure 9. An area of interest- Patch Microscopy Three specimens per group were randomly selected at the conclusion of ph cycling for viewing with a stereomicroscope, polarized light microscope, and scanning electron microscope. Stereomicroscope At the conclusion of ph cycling, three whole specimens per group were removed from remin fluid. Specimens were rinsed with water, and viewed at 1X, 3X and 10 X magnifications (Nikon Optiphot-POL). Polarized Light Microscope (PLM) Three specimens per group were longitudinally sectioned with a water-cooled diamond saw (Scifab Series 1000 Deluxe Hard Tissue Microtome, Lafayette, CO). Sections were microns in thickness, four sections per specimen, and twelve sections per group. Sections were viewed with a Polarized Light Microscope (Olympus BH2, Japan) at 2X and 4X magnifications.

52 41 Scanning Electron Microscope Two sections per group, previously sectioned for PLM, were placed overnight in 3% glutaraldehyde, and then dehydrated with % ethanol. The sections were placed overnight in a dessicator, and sputter coated with gold palladium (Balzers Union SCD 040) for examination under a Scanning Electron Microscope (Amray 1820 Scanning Electron Microscope). One of the specimens was conditioned with GC cavity conditioner prior to dehydration with ethanol to remove surface debris (standard procedure used at Dow s Institute, University of Iowa). A third specimen was sectioned leaving the entire window intact. The window was dehydrated with ascending alcohol solutions, %. Appendix B shows the steps of processing each section. Following overnight placement in hexamethyldisilazane (HMDS), sections were sputter coated with gold palladium (Perdigao & others 1999). Statistical Analysis Sample Size Calculation A sample size calculation based on a pilot study preceded the start of the study. Using a Student s t-test, a sample size of 22 specimens per group, with a 10% difference of means between two groups, provided 80% power for comparison between visits at no treatment and precycling demineralization. A determination of a sample size of specimens per group would produce 80% power or higher with the significance level set at Data Analysis Mean fluorescence values were compared using one-way ANOVA with repeated measures and Tukey post-hoc tests to estimate group fluorescence changes over five measurement sessions during ph cycling. A two-way ANOVA with repeated measures assessed fluorescence differences between remin and non-remin groups and fluoride

53 42 exposure during ph cycling. Paired and two-sample t-tests were used to compare fluorescence differences between no treatment and precycling demineralization and precycling demineralization and precycling remineralization sessions, respectively. Fluorescence differences between groups at baseline were measured by the Wilcoxon Rank Sum tests. Fluorescence differences between groups at precycling demineralization were evaluated by the Kruskal-Wallis test. Significant effects were set at the p<0.05 level.

54 43 CHAPTER IV RESULTS Descriptive All specimens had sound or slightly demineralized facial enamel surfaces before treatment. Most specimens had sound enamel surfaces as indicated in Table 4. All specimens satisfied inclusion criteria. Table 4. Presentation of facial enamel surfaces before treatment (%) Group Sound Demineralized Non-remin with fluoride Non-remin without fluoride Remin without fluoride 92 8 Remin with fluoride The enamel margin contained white spot lesions after three days in precycling demineralization solution. The enamel was chalky white and dull in color. Some areas were soft. The dentin within the preparation was leathery in consistency and had a dull yellow color. There were no visible differences in the presentation of the remin groups and nonremin groups during QLF measurement sessions. Cavitated cervical lesions appeared on some specimens after day 10 of ph cycling. Generally, besides minor chipping of glass ionomer restorative material from specimens in the remin with fluoride group, restorations remained intact. An unrestored specimen had more fluorescence than a restored specimen did. A restored specimen effectively blocks some light reflection. Figure 10 depicts

55 44 fluorescence ( F) differences between an unrestored and a restored specimen. A restoration placed in a preparation was similar to demineralization, F decreased. a. Unrestored b. Restored Figure 10. Fluorescence differences between unrestored (a.) and restored (b.) specimens The occlusal enamel adjacent to the restoration remained intact throughout cycling. There were no cavitated lesions in this area in any group. All specimens experienced enamel surface erosion around the perimeter of the restoration and on the restorative material. There were cavitated lesions in the cervical enamel of the non-fluoride groups. The non-remin without fluoride and remin without fluoride groups had frank cavitated lesions, five in the non-remin without fluoride group, and three in the remin without fluoride group. The non-remin with fluoride group had only one cavitated lesion. There were no cavitated lesions in the remin with fluoride group. A cavitated cervical lesion in a specimen from the non-remin without fluoride group is shown in Figure 11.

56 45 Figure 11. Cervical cavitation The restorative material developed pits and porosities similar to that displayed in Figure 12. The porosities increased throughout cycling. Some restorations had a scooped out concave appearance within the preparation. The presence of pits and porosities was uniform across all groups. There was more restorative material loss in the remin groups. In all examined specimens, cracks were found within the body of the restoration, and minimal loss along the margins. The entire surface of the restoration was covered with a white surface layer that was not removed by washing in deionized water. Figure 13 is a photograph of a specimen from the remin without fluoride group.

57 46 Figure 12. Glass Ionomer exhibiting pitting and porosity The occlusal enamel margin of the restoration did not display cavitation in any specimen. There was no difference in this area of the restoration among remin, nonremin, fluoride or non-fluoride groups. Figure 13. Loss of restorative material

58 47 Quantitative The specimens in all groups completed twenty days of ph cycling, however, specimens unresponsive to precycling demineralization ( F=0), were removed from further statistical analysis. The number of specimens included in the final quantitative analysis were twenty-three for the non-remin with fluoride group (n=23), twenty-five for the non-remin without fluoride group (n=25), twenty-four specimens for both remin without fluoride group (n=24), and twenty-one for the remin with fluoride group (n=21). The significance level was set at p<0.05 for all calculations. The percentage of mean fluorescence changes ( F) was analyzed at the 5% threshold level similar to QLF evaluations in other studies (Pretty & others 2003b, Gonzalez-Cabezas & others 2003). Fluorescence change ( F) is the loss between actual and reconstructed fluorescence and is depicted as a negative value. The fluorescence of the enamel surrounding the unrestored preparations was similar among the four groups. Table 5 indicates smaller amounts of demineralization at the no treatment session, and more demineralization after the precycling demineralization session. Table 5. No treatment and precycling demineralization fluorescence Group No treatment ( F) Precycling demineralization ( F) Non-remin with fluoride -2.48± ±4.12 Non-remin without -3.13± ±2.66 fluoride Remin without fluoride -2.19± ±5.92 Remin with fluoride -3.08± ±3.55 Figure 14 is a comparison of fluorescence differences between no treatment and precycling demineralization sessions within individual groups. Table 5 and Figure 14

59 48 indicate that there was much greater demineralization after three days in a precycling demineralization solution No treatment Predemin Mean Fluorescence (-) Nonremin/Fluoride Non-Remin/No Fluoride Remin/No Fluoride Remin/Fluoride Treatment Groups Figure 14. Mean fluorescence ( F) differences between no treatment and precycling demin There were statistically significant differences in lesion detection within each group after three days in precycling demineralization solution. Paired-sample t-tests and Wilcoxon Signed-Rank tests revealed fluorescence values at no treatment were higher than values at precycling demineralization within each group (p<0.0001), which indicated that demineralization had occurred. Table 5 lists fluorescence values with standard deviations for no treatment and precycling demineralization sessions. The Kruskal- Wallis Test measured comparisons of fluorescence among groups after three days in precycling demineralization solution. The test revealed no statistically significant

60 49 differences among groups (p=0.9316). The amount of demineralization was comparable among groups. Two sample t-tests revealed no statistically significant differences in remineralization between Group 3 and Group 4 from restoration to precycling remineralization measurement session (p=0.8353). The measurement occurred after the groups had been in a remin fluid for seven days. There were no differences between the non-remin, remin, or non-fluoride groups at the start of ph cycling. The only statistically significant difference recorded was between the designated fluoride groups, remin with fluoride, and non-remin with fluoride groups (p=0.0095). The precycling remineralization period might be responsible for the difference since there was no fluoride rinse application at this point. The Wilcoxon Rank-Sum test revealed that the remin with fluoride group had significantly greater remineralization (-8.33±5.83) than the non-remin with fluoride group (-13.95±5.95). The non-remin with fluoride and non-remin without fluoride groups (p=0.3847), and remin with fluoride and remin without fluoride groups (p=0.2249) were not significantly different statistically at the start of ph cycling. This was an expected result since cycling had not yet occurred. Table 6 compares fluorescence at the start of ph cycling prior to any fluoride application. Table 6. Comparison of fluorescence at the start of ph cycling Pairs Non-remin groups Remin groups *Significance set at 0.05 p-value* Start

61 50 A one-way ANOVA with repeated measures tested the progression of ph cycling for each group. Mean fluorescence change values with standard deviations for each group are listed in Table 7. There was a relative increase in remineralization from the start of ph cycling to day 20 for all groups except for Group 4; from day 16 to day 20 there was a decrease in remineralization but this was not statistically significant. Table 7. Treatment group mean fluorescence ( F*) values during ph cycling Group ph cycling Non-remin with fluoride Non-remin without fluoride Remin without fluoride Remin with fluoride Baseline ± ± ± ±5.83 Day ± ± ± ±4.04 Day ± ± ± ±4.20 Day ± ± ± ±3.61 Day ± ± ± ±3.99 * F is defined as the percentage loss between actual and reconstructed fluorescence and is recorded as a negative number. Higher negative values indicate more demineralization. The measurement session had a significant effect for all four groups: non-remin with fluoride group (p<0.0001), non-remin without fluoride group (p=0.0004), remin without fluoride group (p=0.0026), and remin with fluoride group (p=0.0020). There were differences in remineralization depending upon the measurement session. The greatest difference in remineralization occurred at Day 20. The experimental groups (non-remin with fluoride and remin without fluoride) had significant differences throughout cycling. The changes in remineralization were more gradual. In the control groups (non-remin without fluoride and remin group with fluoride), baseline was

62 51 significantly different from Day 5, 10, 16, and 20. Tukey post-hoc test results are provided in Table 8. Table 8. Tukey grouping pairwise comparisons within treatment groups* Measurement Session Group Non-remin with fluoride Non-remin without fluoride Remin without fluoride Remin with fluoride Start A A A A Day 5 B B A B B Day 10 BC B AB B Day 16 BC B B B Day 20 C B B B *Similar letters within groups not statistically significant Figure 15 is a bar graph of mean fluorescence changes within groups. This illustrates that there were smaller lesions at the end of ph cycling than at baseline. The greatest increase in remineralization occurred between baseline and Day 5 with gradual increases through Day 20. Some groups started cycling with lower fluorescence change values, indicating more demineralization. The non-remin groups had greater demineralization than the remin groups; however, this difference was not statistically significant. The rate of remineralization appeared similar among groups. The line graph in Figure 16 demonstrates this trend. Corresponding fluorescence values are located in Table 6.

63 Start Day 5 Day 10 Day 16 Day 20 Mean Fluorescence (-) Nonremin/Fluoride (1) Non-remin/No Fluoride Remin/No Fluoride (2) (3) Remin/Fluoride (4) Treatment Groups Figure 15. Mean fluorescence values by treatment group Fluorescence (-) Start Day 5 Day 10 Day 16 Day 20 Day Nonremin with Fluoride (1) Nonremin without Fluoride (2) Remin without Fluoride (3) Remin with fluoride (4) Figure 16. Remineralization trend among groups

64 53 There were no significant interactions between group and measurement session when all pairwise comparisons were considered. An interaction occurs when the effect of one independent variable fluctuates at different levels of the second independent variable. A main effect was interpreted for non-significant interactions. There must be a difference between at least two levels of the predictor variable (group, measurement session) concerning mean values on the outcome variable (fluorescence). This study s emphasis was on the remin and non-remin groups; however, fluoride and non-fluoride groups are briefly described. For the nonremin groups, results from a two-way ANOVA with repeated measures on one factor (measurement session) showed there was no significant interaction between the type of group (non-remin with fluoride and non-remin without fluoride) and the time of the measurement session (p=0.8147). This result suggested differences in fluorescence changes among groups were consistent over the measurements sessions during ph cycling. The main effect of group was not significant (p=0.1306). The measurement session showed a significant main effect (p<0.0001). Tukey post-hoc test revealed that the values of fluorescence change at Day 5 to Day 20 were significantly higher than values at baseline, and values at Day 16 and Day 20 were significantly higher than Day 5. Values were not significant between all others for the non-remin groups. For the remin groups, a two-way ANOVA determined that the interaction between group and measurement session was not significant (p=0.5695). These results suggested consistent differences in fluorescence changes over the five sessions. The main effect of group (remin with fluoride and remin without fluoride) was significant (p<0.0001). Tukey post-hoc tests indicated that values of fluorescence change were significantly higher for the remin with fluoride group. There was also a significant main effect for measurement session (p<0.0001). Tukey post-hoc tests indicated that

65 54 fluorescence change values at Day 5 to Day 20 were higher than baseline. Other differences were not significant. There were no significant interactions between group and measurement session for the fluoride groups (non-remin with fluoride and remin with fluoride) during ph cycling (p=0.3191). Group had a significant main effect (p<0.0001), as the remin with fluoride group had higher fluorescence change values than the non-remin with fluoride group. Measurement session also had a significant main effect (p<0.0001); values at Day 16 and 20 were significantly higher than those at Day 5 were. There were no significant interactions between group and measurement session for the non-fluoride groups (non-remin without fluoride and remin without fluoride) during ph cycling (p=0.8917), nor a significant main effect for group (p=0.3240). A significant main effect occurred for measurement session (p<0.0001). Tukey post-hoc tests indicated that fluorescence change values from Day 5 to Day 20 were significantly higher than values at baseline and values at Day 20 were significantly higher than Day 5. Microscopy Erosion was commonly found in specimens within all groups. Polarized light microscopy confirmed surface erosion and demineralization. Figure 17 is a polarized light microscope photograph showing the restoration and eroded enamel margin. Figure 17. Restoration and erosion of the enamel

66 55 The photograph shows evidence of demineralization on the occlusal and cervical enamel, indicated by the dark band outlining the enamel. The remin groups had less demineralization on the occlusal enamel adjacent to the restoration. The thin outer band in Figure 18(a) is from a remin group and the thicker outer band in Figure 18(b) is from a non-remin group. The occlusal enamel is to the right of the restoration. There appears to be de/remineralization occurring within dentin beneath the restoration. The loss of restorative material exhibited in Figure 18(b) might be due to specimen processing rather than the cycling process. a. Remin Group b. Non-remin Group Figure 18. Outer Band of demineralization in (a) remin without fluoride and (b) non-remin with fluoride group Full views of specimens in all groups showed surface erosion of the restoration and enamel. The erosion of the enamel surface is clearer in the scanning electron microscope photograph of the restoration and enamel margin. The restorative material is concave within the preparation in Figure 19, which is a section from the non-remin with fluoride group. This concavity might occur from erosion from the demineralizing solution, Mountain Dew. Its low ph might dissolve restorative material. Group specimens displayed a wide variety of patterns in porosity, cracking and pitting. A

67 56 possible reason for this is SEM processing, specifically dehydration in ethanol. A section from the non-remin without fluoride group, seen in Figure 20, has cracks indicative of processing stresses. The restoration surface is also eroded but less than the section in Figure 19. The surface of the restoration in Figure 20 is convex. The remin groups exhibited similar surface characteristics, some restoration surfaces were concave, others convex. Figure 19. Erosion of enamel and restoration in the non-remin with fluoride group Figure 20. Erosion of enamel, cracking and surface wear in the non-remin without fluoride group

68 57 The enamel-restorative interface revealed additional information. The enamel surface in the non-remin without fluoride group had the greatest amount of porosity. The enamel surface of the remin with fluoride group had normal structural anatomy. The other groups, non-remin with fluoride and remin without fluoride were more difficult to characterize. The interface between restorative material and enamel for all specimens are displayed in Figure 21. Figure 21. Interface between restorative material and enamel for all (a) remin without fluoride, (b) remin with fluoride, (c) non-remin without fluoride, and (d) non-remin with fluoride group. The enamel is at the top and the glass ionomer restorative material at the bottom of the photograph. In the fluoride groups, there is a bond between the enamel and restorative material.

69 58 The interface between the restoration and enamel in the remin with fluoride group might contain crystals. The arrows point to crystalline structures in the interface in Figure 21(b). Similar crystalline structures are seen in 22(b). These crystals may be an apatite. The incorporation of fluoride in synthetic and biological apatites causes an increase in crystallinity reflecting the increase in crystal size and thickness (LeGeros 1999). Fluoride incorporation causes the growth of larger and thicker crystals. No other group contained these crystal structures. The enamel margins of the remin groups in Figure 22 display normal enamel architecture. In the remin without fluoride group, the enamel was irregular in places, unlike the uniform pattern found in healthy enamel. This might be due to the presence of demineralization on the surface. The enamel margin of the non-remin groups, Figure 23, shows an irregular pattern compared to the pattern of the remin groups. This group also displays more porosity. a. Remin without fluoride b. Remin with fluoride Figure 22. Enamel margin of remin groups, (a) remin without fluoride and (b) remin with fluoride groups

70 59 a. Non-remin with fluoride b. Non-remin without fluoride Figure 23. Enamel margin of non-remin groups, (a) non-remin with fluoride and (b) non-remin without fluoride. Both are irregular in appearance but the non-remin without fluoride displays more pitting and porosity. The pattern of the non-remin groups is similar to that of an etched surface. The etched pattern is more apparent in the non-remin without fluoride group. A longitudinal section shows erosion of the enamel surface more clearly. Figure 24 is a photograph of an eroded enamel margin in the remin with fluoride group. Figure 24. An SEM of erosion along the enamel margin SEM sections were prepared by two SEM preparation methods previously described. The first method was a standardized method used in the Dows Institute of

71 60 Clinical Research. The first specimens prepared for SEM may have a smear layer over the enamel. This was assumed because a film was visible when viewed with SEM. This observation is illustrated in Figures 25 through 30. Before submitting the second sections to a drying method, sections were etched for 10 seconds with 20% polyacrylic acid. Figures 25a, 26a, 27a, and 28a were the first SEM sections. Figures 25b, 26b, 27b, and 28b were the second SEM sections. Appendix B lists processing for each SEM section. Unetched refers to the first section; etched to the second section. In Figure 25 (a) and (b), enamel is located to the right, glass ionomer to the left. Figure 25 (a) has a covering over the enamel surface not present in (b) after etching. The enamel rods were more visible in the non-fluoride groups than the fluoride groups. The fluoride groups had a surface layer over the enamel that was not removed by etching with polyacrylic acid. This observation was observed for the fluoride group in the remin and non-remin groups. a. Section unetched b. Section etched Figure 25. Sections from the non-remin with fluoride group (a) unetched and (b) etched.

72 61 a. Section unetched b. Section etched Figure 26. Sections from the non-remin without fluoride (a) unetched and (b) etched. The glass ionomer restoration had a similar appearance for both non-remin and remin groups at the magnifications used for this project. In Figure 26(a) and 27(a), enamel is located on the right and glass ionomer on the left. Figure 26 (b) and 27 (b) shows the enamel to the left and glass ionomer to the right. The enamel rods are more visible in (b) after etching. a. Section unetched b. Section etched Figure 27. Sections from the remin without fluoride group (a) unetched, and (b) etched.

73 62 Figure 28 (a) and (b) are photographs of the remin with fluoride group enamel. The surface covering seen in (a) was not removed by etching as shown in (b). a. Section unetched b. Section etched Figure 28. Sections from the remin with fluoride group (a) unetched, and (b) etched A glass ionomer SEM is displayed in Figure 29. It consists of glassy particles surrounded by a non-crystalline matrix. Figure 29. Glass ionomer restorative material after ph cycling.

74 63 A comparison of the remin groups after etching in Figure 30 demonstrated a covering over the remin with fluoride group but not over the remin without fluoride group. A similar pattern occurred for the non-remin groups in Figure 31. a. Remin with fluoride b. Remin without fluoride Figure 30. Etched remin groups (a) remin with fluoride (b) remin without fluoride a. Non-remin with fluoride b. Non-remin without fluoride Figure 31. Etched non-remin groups (a) non-remin with fluoride, (b) non-remin without fluoride