Iowa Research Online. University of Iowa. Tanner Jay Clark University of Iowa. Theses and Dissertations. Spring Copyright 2010 Tanner Jay Clark

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1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2010 The efficacy of ProSeal, SeLECT Defense, OrthoCoat, and Biscover LV resin sealants on the prevention of enamel demineralization and white spot lesion formation Tanner Jay Clark University of Iowa Copyright 2010 Tanner Jay Clark This thesis is available at Iowa Research Online: Recommended Citation Clark, Tanner Jay. "The efficacy of ProSeal, SeLECT Defense, OrthoCoat, and Biscover LV resin sealants on the prevention of enamel demineralization and white spot lesion formation." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Orthodontics and Orthodontology Commons

2 THE EFFICACY OF ProSeal, SeLECT Defense, OrthoCoat, AND Biscover LV RESIN SEALANTS ON THE PREVENTION OF ENAMEL DEMINERALIZATION AND WHITE SPOT LESION FORMATION by Tanner Jay Clark A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Orthodontics in the Graduate College of The University of Iowa May 2010 Thesis Supervisor: Professor Robert. N. Staley

3 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 Tanner Jay Clark has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Orthodontics at the May 2010 graduation. Thesis Committee: Robert. N. Staley, Thesis Supervisor James Wefel Lina Moreno Fang Qian

4 To my wife, Sarah To my family, Jeff, Barb, Frazier and MacKenzie ii

5 ACKNOWLEDGMENTS I would like to thank Drs. Robert Staley, James Wefel, Lina Moreno and Fang Qian for serving on my thesis committee and for all the help and support during this project. I would also like to thank Maggie Hogan and Jeff Harless for their patience, generous support, and insight in the laboratory procedures. I would also like to thank Abbie Kershner and Andrea Schmidt for helping me prepare and section teeth during the project. I would also like to thank Dr. Tom Southard and the rest of faculty at the University of Iowa Department of Orthodontics for giving me the opportunity to further my education and pursue a career in orthodontics. Lastly, I would like to thank my wife, Sarah, for being a great wife and friend you make me a better person. iii

6 TABLE OF CONTENTS LIST OF TABLES...v LIST OF FIGURES... vi LIST OF GRAPHS....vii INTRODUCTION...1 Purpose of this Study...4 LITERATURE REVIEW Demineralization Process...6 Polarized Light Microscopy Prevalence of Decalcification in Orthodontic Patients Decreasing Decalcification During Orthodontic Treatment The Mechanism of Action of Fluoride Fluoride Rinses...14 Fluoride Gel and Dentifrice Fluoride Varnishes Fluoride-Releasing Elastomerics Fluoride Containing Bonding Systems Enamel Coatings and Sealants...18 ProSeal...19 New Materials MATERIALS AND METHODS Tooth Preparation...24 Abrasion...25 Demineralization Tooth Sectioning and Demineralization Evaluation...25 Statistical Analysis RESULTS DISCUSSION Limitations of the Study...47 Future Directions SUMMARY AND CONCLUSIONS REFERENCES iv

7 LIST OF TABLES Table 1. Descriptive statistics for mean lesion depth by treatment group Mean lesion depths by the type of sealant materials...41 v

8 LIST OF FIGURES Figure 1. Clinical example of before (top) and after (bottom) treatment in a patient exhibiting severe demineralization Diagram representing the processes of demineralization and remineralization Histologic zones of a carious lesion Tooth mounted in Exaflex Putty Prototech Toothbrush Wear Simulator Toothbrush centered over specimen Force load application Slurry application Slurry recirculating apparatus Tooth prepared for acidic challenge Cross-sectional view of demineralized enamel window Measuring technique used by Image Pro Plus computer software (Media Cybernetics, Silver Spring, Maryland) to record mean depth of the lesion Composite of polarized light microscopy images of representative lesions from all five treatment groups vi

9 LIST OF GRAPHS Graph 1. Descriptive statistics for mean lesion depth by treatment group...39 vii

10 1 INTRODUCTION Orthodontic patients often seek comprehensive orthodontic treatment for a variety of reasons. The most common reasons for orthodontic therapy are to improve a patient s dental function and esthetics. Also, there are several psychological benefits derived from orthodontic treatment. These include an improvement in a patient s overall self-esteem and attitude. Oftentimes, the orthodontic success of an ideal Angle Class I canine occlusion is tarnished by the appearance of white spot lesions on the facial surface of teeth after removing the fixed appliances. (Figure 1). Prevention of these white, opaque areas throughout orthodontic treatment is essential to providing the patient with the most esthetic outcome. Demineralization is a complex process. It involves the loss of calcified tooth structure resulting in an altered surface appearance, often white spot lesions. An opaque white spot appears chalky, and if mineral loss continues, may result in frank cavitation of the tooth surface (Mitchell et al., 1992). A dental restoration is needed if a lesion progresses to cavitation. This is an undesirable outcome for either orthodontic or nonorthodontic patients. However, patients undergoing orthodontic treatment with fixed orthodontic appliances are often at a higher caries risk than normal because of the increased difficulty in maintaining adequate oral hygiene. Oftentimes, the patient is unable to remove all of the dental plaque and there is a continuous cariogenic challenge adjacent to the orthodontic brackets, eventually leading to demineralization (Ogaard et al., 1988). A variety of preventive treatment products and regimens have been developed in an attempt to limit the formation of these white spot lesions. Some of these methods require the cooperation of the patient while others do not. Because of the higher caries risk associated with patients undergoing orthodontic treatment, dentists and orthodontists often use adjunctive fluoride therapy to help prevent demineralization. The most

11 2 common method in orthodontics is with topical fluoride. Geiger et al. (1992) found a dose response relationship between frequency of rinsing with 10ml of neutral 0.05% sodium fluoride solution and degree of enamel protection. Those who rinsed at least once every other day had 49% fewer lesions than those rinsing less frequently, where 21% exhibited white spot lesions. Alexander and Ripa (2000) reported even greater results with regular use of a high potency sodium fluoride (1.1%) dentifrice or gel. Even though these findings strongly support the use of fluoride in the orthodontic patient, each requires appropriate patient compliance for effectiveness. Poor patient compliance has always been one of the largest obstacles in orthodontic care. Geiger et al. (1988, 1992) illustrated this problem in two separate studies where they found that only 12-13% of patients reported excellent compliance with a home fluoride rinse program. This protocol also included added reinforcement with oral hygiene instructions and education to patients and parents throughout treatment. Due to the large percentage of uncooperative patients, clinicians developed alternative methods of decreasing demineralization that do not require patient compliance. One approach that allows the orthodontist to control the frequency and amount of product application are fluoride varnishes. Duraphat (Colgate-Palmolive Company, New York, New York), a viscous resinous lacquer consisting of 5% sodium fluoride that hardens into a yellowish-brown coating, is one of the most frequently used varnishes. Although varnishes provide a significant benefit to high caries risk patients, one drawback is that they often require multiple in-office applications. In an in vitro study, Frazier (1993) found that a one-time application of an unfilled resin sealant (a conventional pit and fissure sealant, Delton, Dentsply) was 80 percent effective in preventing demineralization. These results are promising, especially if the sealant could be maintained throughout treatment without the need for reapplication. However, in this study the mechanical wear of the sealant through toothbrushing was not addressed.

12 3 Sealants, especially unfilled resins, are susceptible to wear and may develop cracks in the surface or become entirely worn through (Gwinnett and Ceen, 1979). In 2004, Reliance Orthodontic Products, Inc. (Itasca, Illinois) released an enamel sealant specifically for orthodontic use. Pro Seal, a light-cured fluoride sealant, is highly filled and advertised as resisting toothbrush abrasion and normal wear for over two years. Hu and Featherstone (2005) have shown a significant decrease in enamel demineralization on teeth treated with Pro Seal and subjected to simulated mechanical abrasion. This study used enamel microhardness profiles to evaluate product performance, with teeth treated with Pro Seal demonstrating better profiles than those treated with a fluoride varnish, etchant only, or an unfilled resin. Loucks et al. (2006) found that Pro Seal provided a 92 % reduction in enamel demineralization and withstood simulated toothbrush abrasion for 24 months. In addition, Pro Seal provided significantly more protection than the unfilled sealant Delton and the topical fluoride varnish Fluor Protector. Since the release and clinical success of Pro Seal, other dental manufacturers have formulated similar sealant materials that can provide the same level of prevention against enamel demineralization. Many of these products have just been released and have not been tested clinically. Ortho-Coat is a similar product recently developed by Pulpdent that is also a light-cured fluoride releasing resin made specifically to place adjacent to and on orthodontic brackets to decrease demineralization. A new unfilled resin, called SeLECT Defense, has recently been developed by ClassOne Orthodontics. It is also used for the prevention of whitespot formation during orthodontic treatment. SeLECT antimicrobial Technology has been incorporated into brackets, archwires, molar bands, adhesive materials, and other devices, to prevent the formation of plaque on or around the bracket area. SeLECT Technology, by its antimicrobial action, will inhibit the growth of bacterial plaque around orthodontic brackets, thereby preventing demineralization (whitespot formation) around the brackets,

13 4 and can withstand the abrasion from daily toothbrushing. Specifically, SeLECT Technology utilizes the properties of selenium for its antimicrobial effect. In small doses, the selenium compound specifically kills the Streptococcus mutans and Lactobacilli bacteria that come in contact with the treated surface. The Biscover LV (BISCO, Inc.) is a low-viscosity, light-cured unfilled resin formulation mainly used to polish and seal composite resin restorations and provisional restorations. It can also be used to seal enamel prior to orthodontic bracket placement. The resin cures without an oxygen-inhibited layer and is used also to prevent enamel demineralization during orthodontic treatment. These three resins show promise in their ability to prevent enamel demineralization during orthodontic treatment. However, there long-term ability to withstand toothbrush abrasion and intra-oral wear and demineralization has yet to be studied. Purpose of this Study The purpose of this study is to compare, in vitro, the effectiveness of the resin sealants ProSeal, OrthoCoat, SeLECT Defense, and Biscover LV on the inhibition of enamel demineralization after being subjected to extensive mechanical toothbrush abrasion.

14 Figure 1. Clinical example of before (top) and after (bottom) treatment in a patient exhibiting severe enamel demineralization. 5

15 6 LITERATURE REVIEW Demineralization Process Dental caries is a multifactorial disease. It involves the interaction of diet, dental plaque containing bacteria, and host factors, such as tooth surface, saliva, and the acquired pellicle (Zero, 1999). Dental caries is initiated via demineralization of tooth mineral by organic acids. One model proposed by Harris and Garcia-Godoy (1999) described a process in which plaque microorganisms on the tooth surface produce organic acids in the presence of fermentable carbohydrates. As the plaque ph drops in response to the production of these acids, a series of complex chemical and physical events are initiated. At resting ph levels, plaque fluid is supersaturated with respect to calcium phosphate. However, as the ph falls, this level of saturation is not maintainable. When the critical ph (approximately 5.5) is reached, demineralization begins and the organic acids are able to diffuse to the enamel surface through the acquired pellicle. After the initial dissolution has occurred, less soluble solid phases of dicalcium phosphate dihydrate and fluoridated hydroxyapatite precipitate out of the enamel. This sequence occurs until an equilibrium is reached between the surrounding oral environment and the enamel. Demineralization continues as the acid penetrates deeper through the enamel rods until equilibrium is again reached. The physical degradation observed on the enamel surface is a direct result of this demineralization process. However, there is not a logical explanation why an intact surface layer is often observed with the greater structural damage deeper into the enamel. Featherstone and colleagues (2000) proposed that as the ph of the plaque falls, the proportion of undissociated acids in the plaque fluid increases. These acids can diffuse through the porous enamel matrix down a concentration gradient because they are uncharged. The acids can dissociate once they penetrate to a certain depth where the ph is higher. Here, the acids release protons, which attack the apatite lattice. The calcium

16 7 and phosphate ions that are dissolved either diffuse outward into the plaque or reprecipitate before escaping the enamel. This process maintains an intact surface zone. After the ph has returned to normal levels, calcium, phosphate and fluoride reenter the enamel and repair the damaged crystallites and begin the remineralization process (Figure 2). The caries process is a dynamic cycle. There is a constant continuum between periods of demineralization and remineralization. If the negative mineral balance exceeds the rate of remineralization over an extended period of time, s carious lesion can form. Carious lesions occur in several distinct stages. The earliest stage is the incipient lesion and is characterized by both histologic, and eventually macroscopic, changes in enamel. This state is also termed a sub-surface or white spot lesion. A white spot lesion is the first clinical presentation of dental caries. The incipiency consists of an intact enamel surface with loss of mineralization deeper within the enamel. Therefore, they often present clinically with an opaque, white-chalky appearance that is due to the optical properties of the demineralized enamel. Studies regarding lesion depth disagree on the average depth of a white spot lesion. Ogaard et al. (1988) found average depth of a white spot lesion under a band following four weeks of orthodontic treatment was 100 µm. Glatz and Featherstone (1985) reported measurable demineralization, up to 75 µm deep and with 25% mineral loss, in 4 weeks, especially gingival to the bands or brackets. Zero (1999) noted that a white spot lesion must progress to a depth of 300 to 500 µm to be clinically detectable. If this process of demineralization continues, a white spot lesion will eventually progress further into a surface cavitation. This is often termed an overt, or frank lesion. Polarized Light Microscopy Polarized light microscopy is often used to evaluate carious lesions because it allows visualization of enamel demineralization and remineralization. There are several

17 8 other methods of studying demineralization, including: light microscopy, microradiography, surface hardness, electron microscopy, and confocal laser scanning microscopy (Ogaard, 1996). The main advantage with polarized light microscopy is that it permits both qualitative and quantitative evaluation due to its ease of visualization of the color spectrum (Hicks, 1981). A polarized light microscope includes a light microscope with the addition of a polarizer and analyzer that are set perpendicular to one another. Both the polarizer and the analyzer are made of prisms of calcite or a sheet of Polaroid, which only transmit light oscillation in one plane (Weyrich, 1994). When a sample is placed between the polarizer and the analyzer, the sample modifies the plane of light which produces a series of interference colors. When a crystal transmits light with equal velocity in all directions, the crystal is called isotropic. The crystal is termed anisotropic when it transmits light at different velocities and in different directions. Depending on the number of axes present, anisotropic crystals are further divided into uniaxial and biaxial. Hydroxyapatite is a uniaxial anisotropic crystal, because it has one optic axis that is coincident with its crystal ( c ) axis. Hydroxyapatite is also birefringent, which involves splitting a light ray into two components which travel at different velocities and are at right angles to one another. This results in different color and light intensities being released (Weyrich, 1994). A material that consists of non-cubic crystal is given a sign of birefringence determined by the velocity of its resultant light rays. Slow rays are deemed positive (+) and fast rays are deemed negative (-) (Theuns and Groenveld, 1977). The mineral, hydroxyapatite, composes most of the enamel structure but there is also a small amount of organic material (+) interspersed. Birefringence can be indirectly measured through polarized light microscopy. Enamel is a uniaxial birefringent object. Therefore, when enamel is oriented with its optic axis parallel to the direction of plane polarized light propagation, it will behave as

18 9 an isotropic crystal. If enamel is arranged with its optic axis in a plane perpendicular to the direction of propagation, the light will split into two beams. These beams are referred to as the extraordinary (ne) and ordinary (no) rays and they result in a series of interference colors (Silverstone, 1967). Birefringence is further subdivided into intrinsic birefringence and form birefringence. Intrinsic birefringence refers to the difference between the refractive indices of the extraordinary ray and the ordinary ray, or ne - no (Ogaard, 1996). There are several factors which influence intrinsic birefringence, including: volume of crystalline mineral, orientation of crystallites with respect to the light beam, and birefringence of crystallites such that (ne no) i = δ c(ne no) HAP where (ne no) i = intrinsic birefringence, δ = pore volume occupied by crystallites, c = crystallite orientation factor, and (ne no) HAP = birefringence of crystallites (Theuns and Groenveld, 1977). Throughout the demineralization process, spaces between the enamel crystals become larger. Form birefringence occurs when a medium that fills the voids in carious enamel has a different refractive index. The amount of form birefringence produced is determined by the size of the spaces and the refractive index of the medium. Form birefringence will not be created if the medium has the same refractive index as enamel. Therefore, different mediums are used when utilizing polarized light microscopy. The total birefringence of enamel is the sum of 1) the negative intrinsic birefringence of the inorganic material, 2) the positive intrinsic birefringence of the organic material, 3) the positive form birefringence of the spaces in relation to the mineral and possibly to the organic matter and 4) the positive form birefringence of the organic material in relation to the mineral. The organic mineral content is very small so it can be disregarded. Because there is a small amount of organic material in enamel, the observed total birefringence is made up almost entirely of the intrinsic birefringence of

19 10 the inorganic mineral and the form birefringence of the spaces in relation to the inorganic mineral (Silverstone, 1967). The enamel is divided into longitudinal sections so that the crystallites of apatite are aligned along the length of the prism. When using polarized light microscopy, four zones of a carious lesion are visible (Figure 3). These zones represent different amounts of tissue loss, or open space, and are often compared by their percentage of pore volume. The outermost layer of enamel, the surface zone, remains relatively intact (1-5% pore volume) and is a zone of remineralization (Silverstone 1977; Gorelick and Geiger, 1982). The second zone is called the body of the lesion and displays more tissue destruction than any of the other zones. The pore volume in the body of the lesion is 5-25 percent. The third zone is the dark zone, which is caused by dissolution of enamel cross striations. This zone has a pore volume of 2-4 percent. Similar to the surface zone, remineralization can occur in the dark zone. The fourth zone, which is the deepest of the four zones, is termed the translucent zone and has a pore volume of 1 percent (Silverstone, 1967). The superficial, dark, and translucent zones act as sieves that selectively exclude imbibition media with molecular sizes greater than pore sizes present in these zones (Hicks, 1981). Air, water, and potassium mercuric iodide dilutions (Thoulet s solution) are some of the mediums used in polarized light microscopy. Air (refractive index = 1.0) corresponds with 1 percent pore volume, water (refractive index = 1.33) with 5 percent pore volume, Thoulet s 1.41 with 10 percent, and Thoulet s 1.47 with 25 percent pore volume. A map can be made of the different zones by imbibing a longitudinally sectioned lesion with different media,. These maps provide a qualitative assessment of internal lesion pore volume. Also, quantitative information is easily obtained by measuring the positively birefringent areas of the lesion in the various imbibition media. Image Pro Plus software (Media Cybernetics, Silver Spring, Maryland) allows measurement of a

20 11 polarized light photomicrograph for statistical analysis. This method does allow for lesions to be quantified indirectly with a polarized light microscope (Hicks, 1981). Prevalence of Decalcification in Orthodontic Patients Decalcification presents both biological and esthetic concerns for orthodontists. Fixed orthodontic appliances create significant plaque traps, leading to increased plaque retention and subsequent white spot lesion formation. Accumulation of bacterial plaque can also produce gingival inflammation and can contribute to degeneration of the gingival attachment. Ogaard et al., (1988) reported the presence of visible white spot lesions in orthodontic patients within as little as four weeks without fluoride supplementation. Zachrisson et al., (1971) demonstrated a linear, positive correlation between plaque accumulation and carious lesion development in orthodontic patients. In addition to presenting a significant threat to the patient s oral health, often of more concern to patients and parents is the negative impact that white spot lesions have on facial esthetics. Decalcification of the facial surfaces of anterior teeth is very common. When this occurs, the patient has a very unesthetic final result. Many studies have investigated the prevalence of white spot lesions in orthodontic patients and the findings are striking. Zachrisson and Zachrisson (1971) reported in a longitudinal study that orthodontic treatment resulted in 89% of patients developing white spot lesions. Boersma et al. (2005), observed that 97% of their subjects displaying lesions after treatment. However, Ogaard (1989) found that only 4% of orthodontically treated patients had no white spot lesions 5 years post treatment. Gorelick et al. (1982) found non-developmental lesions in 50% of treated patients in contrast to 25% of non-treated controls. Similarly, other studies have shown an increase in white spot lesions of 0-24% after treatment (Mizrahi, 1982; Stratemann, 1974). Although varying amounts of decalcification have been presented in the literature, each study

21 12 firmly agrees that decalcification poses a significant problem to orthodontists and the patients they treat. Alexander (2000), O Reily (1987) and Geiger (1988) all found that white spot lesions can appear in orthodontic patients as early as one month into treatment. Ogaard (1989) and Gorelick et al. (1982) observed a high prevalence of white spot lesions on the maxillary lateral incisors, mandibular first premolars, and mandibular canines. The lowest incidence of white spots was in the maxillary posterior segment (Gorelick et al. 1982). Mizrahi (1982) found that most white spot lesions occur in the cervical and middle thirds of the facial surface of teeth. Decreasing Decalcification During Orthodontic Treatment Immaculate oral hygiene is essential to protecting healthy enamel during orthodontic treatment. Thorough oral hygiene instruction and diet counseling that emphasizes reduced intake of fermentable carbohydrates are just a few methods to help prevent or decrease demineralization in orthodontic patients. Besides its role in the prevention of white spot lesions, proper nutrition is also essential for maintaining overall systemic health during and after treatment. The Mechanism of Action of Fluoride Thorough tooth brushing, flossing, and routine prophylactic cleanings will minimize the amount of dental plaque, thereby decreasing the probability of developing areas of decalcification (Øgaard, 1989). But because a majority of patients often have inadequate oral hygiene, orthodontists are forced to alternate approaches to help prevent demineralization. The supplemental use of fluoride has long been utilized in the dental field for caries prevention and also plays a critical role in orthodontics. The effectiveness of fluoride in this preventive role is attributable to three principal mechanisms of action:

22 13 1) inhibiting bacterial metabolism, 2) inhibiting demineralization, and 3) enhancing remineralization. To inhibit bacterial metabolism, fluoride inhibits the enzyme enolase, which is a bacterial enzyme necessary for the breakdown of carbohydrates into pyruvic acid. Without the activity of enolase, bacteria cannot utilize fermentable carbohydrates from the diet in acid production. Bacteria have proteins with acidic side chains that form bridges with calcium ions in the enamel. Fluoride competes with bacteria for these binding sites and prevents adhesion because of its electronegative properties. (Levine, 1991). Fluoride also has the ability to protect the enamel surface from demineralization during an acid attack. Systemic fluoride is incorporated into the enamel by combining with hydroxyapatite to form fluoroapatite. This allows the enamel to become less soluble and more resistant to acidic challenge (Zipkin, 1970). Although each of these mechanisms is beneficial, the primary method of action of fluoride is remineralization. Following an acid attack and the ph rising above 5.5, available calcium and phosphate are able to be integrated into the crystal structure to rebuild the damaged areas. Fluoride enhances the process by strongly adsorbing to the crystal surface, attracting calcium ions, which are then followed by phosphate ions. These three ions are able to create a new crystalline structure between hydroxyapatite (HAP) and fluorapatite (FAP), which is stronger and more acid-resistant than the previous structure (Featherstone, 2000; Moreno et al., 1977; ten Cate et al., 1991). Remineralization can occur in the presence of saliva alone. However, Arends and ten Cate (1977) found a twofold increase in the rate of remineralization in the presence of 1ppm fluoride ion. Patients with a high-caries risk, such as those with orthodontic appliances, would benefit from fluoride use because the presence of fluoride appears to push the equilibrium in favor of remineralization. The orthodontist can utilize a variety

23 14 of fluoride delivery systems during treatment. Fluoride can be introduced via dentifrice, rinses, foams, gels, varnishes, bonding agents, cements, and even elastomers. Fluoride Rinses The advantage of adding fluoride to a patient s oral hygiene protocol is welldocumented. Geiger et al. (1992) found a dose response relationship between frequency of rinsing with 10ml of neutral 0.05% sodium fluoride solution and degree of enamel protection. Those who rinsed at least once every other day had only a 21% occurrence of white spot lesions, compared to 49% for the control group. Another in vivo study was done by O Reilly and Featherstone (1987). They found that patients whose used a fluoride dentifrice and rinsed daily with a 0.05% sodium fluoride mouthrinse during the first month of treatment had significantly higher enamel microhardness than those using fluoride dentifrice alone. No visible demineralization was present at this time. However, the authors concluded that the progression of any enamel surface softening could be expected to result in observable white spots by the completion of treatment. Fluoride Gel and Dentifrice In addition to fluoride rinses, investigators have shown even superior protection from high-potency fluoride dentifrice (Prevident 5000 Plus) and gel (Prevident Neutral Sodium Fluoride Brush-On Gel), both containing 1.1% sodium fluoride. In 2000, Alexander and Ripa, showed that subjects who used either the gel after brushing with standard toothpaste (1000ppm) at bedtime, or the dentifrice only twice daily, showed significantly fewer areas of demineralization than those adding a low-potency fluoride rinse to standard brushing at bedtime (Phosflur, 0.05%APF). The difference in demineralization was clinically evident after one month of treatment and remained statistically significant throughout the study (one month post-treatment). Baysan et al. (2001) also compared the ability of two fluoridated dentifrices, one containing 5,000 ppm

24 15 (PreviDent 5000 Plus ) and the other 1,100 ppm (Winterfresh Gel ), to reverse primary root caries lesions. They concluded that the 5,000 ppm fluoride dentifrice was significantly better at remineralizing the lesions than the 1,100 ppm fluoride dentifrice. Eng (2009) found that a twice-daily treatment with either MI Paste, MI Paste Plus, or PreviDent 5000 Plus showed statistically significant reduction in lesion depth compared to controls. Recaldent, is a complex of casein phosphopeptides and amorphous calcium phosphate (CPP-ACP) and is the active ingredient in MI Paste and MI Paste Plus. He also found that CPP-ACP was effective in reducing lesion depth while integrated within a paste. However, he noted that even though CPP-ACP showed a statistically significant reduction in mean lesion depth, it remains uncertain whether this equates to a clinical reduction of visible demineralization. Fluoride Varnishes The effectiveness of fluoride dentifrices, gels and rinses in decreasing enamel demineralization is widely accepted. Acidulated fluoride gels and solutions have been used since their inception in However, these methods require patient cooperation to be effective. For example, Geiger et al. (1992) reported only 13% full compliance among study patients with a fluoridated rinse protocol. This level of non-compliance poses a problem for orthodontists. Many researchers have studied the effectiveness of professionally applied varnishes to protect non-compliant patients from white spot lesions. Two effective varnishes are Duraphat and Duraflor. They are both composed of 5% sodium fluoride by weight and have a yellowish, sticky rosin base that hardens when it comes into contact with saliva. This base serves as a protective coating that allows the fluoride to persist on the enamel surface. Todd et al. (1999) reported a 50% decrease in lesion depth in extracted teeth treated with a single application of varnish (Duraflor ) when compared to control teeth with no treatment. Wittenberger (2003) also found decreased lesion depths (53%) with the use of Duraphat.

25 16 Juhlin (2004) found similar results for Duraphat, with treated teeth showing a decrease in lesion depth of 65%. In the same study, Juhlin also found an 86% reduction in lesion depth obtained from the fluoride varnish Fluor Protector, which was significantly more effective than Duraphat. Part of Fluor Protector s efficacy may be attributable to its behavior similar to that of a sealant in that it remains adhered to the enamel surface for a period of time. In the study, complete removal of Duraphat was noted after approximately 9 days, while Fluor Protector was on the enamel surface after the 27 day study. Loucks (2006) found that Fluor Protector resulted in a 47% decrease in lesion depth when subjected to 24 months of simulated toothbrush abrasion and a 96 hour acid challenge. Vanish (3M Omni, St. Paul, MN) is a fluoride varnish that has recently been developed as an esthetic alternative to traditional fluoride varnish. Similar to Duraphat, Vanish contains 5 percent sodium fluoride (22,600 ppm fluoride) but in a more esthetic, clear or white color. Schemehorn et al. (2009) reported that Vanish also has an increase in fluoride-releasing efficiency over 24 hours compared with Duraphat and Duraflor. Fluoride-Releasing Elastomerics As an alternative to varnishes, clinicians have tried to use fluoride releasing elastomerics to reduce enamel demineralization. One study by Banks et al. (2000) found a 10% decrease in the number of patients presenting with decalcification at the end of treatment (as observed clinically) when utilizing fluoride releasing modules and chain (Fluor-I-Ties, Fluor-I-Chain, Ortho Arch Company, Inc., Illinois, USA). However, these modules do have several associated problems, including: have reduced elasticity, significant staining, and lack of color availability. Although no chains showed clinical failure in this study, Baty (1994) found that the Fluor-I-Chain retained only 14% of its initial force after 1 week in distilled water, as compared to 38% in a conventional chain.

26 17 Fluoride Containing Bonding Systems Fluoride releasing cement systems, including composite resins, glass ionomer, and resin-modified glass ionomers are also another method for non-compliant patients to decrease demineralization. One study by Ogaard et al. (1992) found a 48% reduction in lesion depths adjacent to brackets bonded with a fluoride releasing composite (Orthodontic Cement VP 862), compared to those observed in teeth bonded with a non-fluoride adhesive. Glass ionomer cements are another group of materials that decreases caries progression and remineralizes enamel (Donly, 1995). A unique property of this material is that they can absorb fluoride applied topically, and can therefore recharge and re-release the fluoride over a longer period of time (Voss, 1993). Although these fluoride releasing composites and glass ionomers are effective in inhibiting the caries process, there are drawbacks to their use clinically. For example, studies by Fox (1990) and Voss (1993) show that the bond strengths of these materials is lower than conventional composite resins and less than ideal for clinical treatment. The fluoride releasing properties of resin-modified glass ionomer cements is similar to glass ionomer cements in their ability to uptake fluoride and re-release it over a long period of time (Burgess and Chan, 1996). In 2002, Schmit et al. examined lesion depths in teeth bonded with resin-modified glass ionomer cement (Fuji Ortho LC, GC) and a non-fluoridated composite resin and found 50% smaller lesion depths in teeth bonded with the RMGI. The authors also found shallower depth measurements near the bracket. Pascotto et al. (2004), found the same RMGI material decreased enamel mineral loss by 12% and had a positive effect on enamel microhardness. Although the role of RMGI in preventing demineralization is clear, its use does have some clinical drawbacks, in particular its ease of use by clinicians. Lippetz et al. (1998) compared three different RMGIs (Advance, Fuji Duet, Fuji Ortho LC) with a clinically proven composite resin (Concise ). They found no difference in bond

27 18 strength of the materials 24 hours or 30 days following bonding. However, the clinical handling properties have proven less than ideal, making many practitioners hesitant to use it on their patients. Also, most RMGI are entirely or partially chemical cure. This decreases efficiency because it makes for a longer working time and longer chair time for the orthodontist. Enamel Coatings and Sealants Recently, enamel coatings and sealants have gained popularity for preventive treatment. Fornell et al. (2002) found that a hydrophobic enamel-coating polymer had no beneficial effects on gingival health, plaque and strep mutans levels, or enamel demineralization when applied immediately after bonding and at 3 month intervals throughout treatment. Enamel sealants have showed more promise in the prevention of enamel demineralization. Frazier et al. (1996) found that 80% of teeth treated with a light-cured unfilled resin (a conventional pit and fissure sealant, Delton, Dentsply) just after initial bond placement were completely protected from demineralization. The remaining 20% of experimental teeth each had small, isolated areas that had breaks in the sealant layer, leading to the associated demineralization. The author concluded that surface contamination during etching or sealant placement and incomplete application of etchant or sealant were the causes of these breaks in the sealant layer Oxygen inhibition, a lack of complete surface layer polymerization, is another cause for failure of enamel sealants. Composite resins undergo a reaction known as freeradical polymerization. This reaction occurs in three stages: initiation, propagation, and termination. During initiation, monomer particles are rapidly added to the free radical on the polymer. This allows electrons to shift to the end of the growing chain, enables more monomers to be incorporated into the polymer. This propagation continues until the freeradical is terminated. Materials that will react with the free radical can inhibit this reaction, thereby decreasing the rate of initiation/propagation and retarding the

28 19 polymerization reaction. These materials can also expedite the rate of termination and decreases the degree of polymerization or the molecular weight of the final polymer. Materials such as hydroquinone, eugenol, or large amounts of oxygen will retard the polymerization, causing an incomplete cure in the outer layer of composite resins. The degree of conversion is one method to quantify a material s polymerization and it describes the percentage of bonds that are reacting. Craig (1997) found that photoinitiated polymerization has a degree of conversion around 80%, whereas chemical cure systems can have as low as 35% conversion in the air inhibited layer. Joseph et al. (1992) found teeth with chemically activated resins showed almost total absence of a cured sealant layer. However, light-polymerizing resins did show a cured sealant layer. Although they can provide more enamel protection, they are still susceptible to oxygen inhibition and incomplete surface polymerization. Pro Seal A product receiving well-deserved attention for its ability to prevent demineralization is Pro Seal (Reliance Orthodontics Products, Inc). It is now considered the gold standard in orthodontics as a sealant material used to help prevent enamel demineralization during orthodontic treatment. Loucks (2006) found that Pro Seal provided a 92 % reduction in enamel demineralization and withstood simulated toothbrush abrasion over a 24 month period. In addition, Pro Seal provided significantly more protection than the unfilled sealant Delton and the topical fluoride varnish Fluor Protector. Pro Seal is a light-cured, fluoride containing sealant that is highly filled. It has been shown to resist toothbrush abrasion and normal wear for over 24 months, which is about the average time for comprehensive orthodontic treatment. It has a final sealant polymerization of 100% without an oxygen inhibition layer, which creates a smooth, even surface that helps prevent leakage, protects enamel and makes bonding paste

29 20 cleanup easier. Pro Seal contains a fluorescing agent that is visible under UV light that allows the orthodontist to monitor the coverage of the sealant at the time of placement and early in treatment. The sealant s added fluoride is in the form of a glass ionomer powder similar to that used in many orthodontic bonding systems. Thus, the sealant resembles a RMGI cement in that it can release fluoride to the enamel upon application. The application of Pro Seal is very straightforward. The orthodontist first begins with a prophylaxis and treatment of the enamel with conventional etchant or a self etching primer. A thin layer of sealant is then applied and cured at close range with a standard halogen bulb light or an LED light for 15 seconds per tooth. After curing, the fixed appliances can then be placed directly on the treated enamel surface with any chemical, light or dual cure paste with clinically acceptable bond strength anticipated. Bishara et al. (2005) found the bond strength on teeth treated with Pro Seal to be comparable to those bonded without the sealant. In addition, there was no significant difference found between bonds receiving a separate curing for the sealant (10 sec) and the cement (20 sec), as directed by the manufacturer, and those receiving a single curing interval (20 sec) for the sealant and cement simultaneously. New Sealant Materials Since the release and clinical success Pro Seal, other dental manufacturers have formulated similar sealant materials that they claim can provide the same level of prevention against enamel demineralization. Many of these products have just been released and have not been tested clinically. One product, OrthoCoat (Pulpdent, Inc.) is a similar product recently developed that, like ProSeal, is a light-cured fluoride releasing resin made specifically to place adjacent to orthodontic brackets to decrease demineralization. Another new resin, called SeLECT Defense, has recently been developed by ClassOne Orthodontics. It is also used for the prevention of whitespot formation during

30 21 orthodontic treatment. The product uses what the company calls SeLECT Technology. This mechanism, by its antimicrobial action, will inhibit the growth of bacterial plaque around orthodontic brackets, thereby preventing demineralization around the brackets, and can withstand the abrasion from daily toothbrushing. Specifically, SeLECT Technology utilizes the properties of selenium for its antimicrobial effect. In small doses, the selenium compound is bactericidal to the Streptococcus mutans and Lactobacilli bacteria that come in contact with the treated surface. The company has taken this SeLECT antimicrobial Technology one step further and has incorporated it into its brackets, archwires, molar bands, and adhesive material. Biscover LV (BISCO, Inc.) is a low-viscosity, light-cured unfilled resin formulation mainly used to polish and seal composite resin restorations and provisional restorations. It can also be used to seal enamel prior to orthodontic bracket placement. The resin cures without an oxygen-inhibited layer and is used also to prevent enamel demineralization during orthodontic treatment.

31 Figure 2. Diagram representing the processes of demineralization and remineralization (Harris and Christen, 1995). 22

32 Figure 3. Histologic zones of a carious lesion. 23

33 24 MATERIALS AND METHODS Tooth Preparation Seventy-five non-carious extracted human molars were disinfected in Streck Tissue Fixative (Streck Laboratories, La Vista, Nebraska) for one week. The remaining soft tissue, calculus and bone were removed with a scaler and razor blade. Cusps were then ground flat and the apices shortened to allow mounting in the tooth brush simulator. A small hole was then drilled near the apex of each tooth so that dental floss could be fed through to facilitate suspension of the teeth in solution. The buccal surfaces were then polished with a prophylaxis cup for 3 seconds with a mixture of non-fluoridated pumice and water and the teeth were randomly assigned to one of 5 groups: Group 1 (n=15) the control group, received no additional surface treatment after pumicing. Group 2 (n=15) was treated with 35% phosphoric acid and received a single application of the unfilled resin sealant, Biscover LV, per manufacturer s instructions. Group 3 (n=15) was etched with 35% phosphoric acid and received a single application of the filled resin sealant, ProSeal, per manufacturer s instructions. Group 4 (n=15) was etched with 35% phosphoric acid and received a single application of the filled resin sealant, OrthoCoat, per manufacturer s instructions. Group 5 (n=15) was etched with 35% phosphoric acid and received a single application of the unfilled resin sealant, SeLECT Defense, per manufacturer s instructions.

34 25 Teeth were then mounted in an acrylic ring and stabilized with a very high viscosity vinyl polysiloxane impression material (Exaflex Putty, GC America, Inc.) as shown in Figure 4. Abrasion Mounted teeth were placed in the Prototech Toothbrush Wear Simulator (Prototech, Portland, Oregon) and a soft bristled toothbrush (Oral-B Adult Soft 35, Oral-B Laboratories, Iowa City, Iowa) was centered over the buccal surface. Each tooth was subjected to 15,000 horizontal brush strokes at a rate of 120 strokes per minute. A constant force of 280g was also applied to each brush to simulate normal manual brushing force and a slurry of non-fluoridated toothpaste and water (1:3 ratio) was constantly recirculated by the machine (van der Weijden et al., 1996). Slurry solution and brush heads were changed between each treatment group. Figures 5-9 illustrate various components of the machine and the abrasion protocol. Demineralization Teeth from all groups were painted with a thin layer of acid-resistant varnish (nail polish), leaving an approximately 1mm window of exposed enamel on the buccal surface (Figures 10 and 11). They were then placed in a constantly circulating, roomtemperature, standard tencate Demineralizing Solution (ph = 4.4) consisting of: 2.20mM Ca +2, 2.20mM PO 3 4, 0.05M Acetic acid and 0.025ppm F - for 96 hours to generate demineralized lesions (Frazier, 1996). Tooth Sectioning and Demineralization Evaluation Each tooth was visually examined for evidence of demineralization. Longitudinal sections were then made buccolingually with a Series 1000 Deluxe hard tissue microtome (Scientific Fabrication, Littleton, California) to yield sections that were 100 to 140 µm

35 26 thick Before sectioning, digital photographs (Hitachi KP-D50 Digital Camera, Tokyo, Japan) were made of one tooth from each group at 15x magnification (Nikon SMZ-10 Microscope, Tokyo, Japan) to grossly compare the exposed enamel window of each treatment. Sections were then soaked in deionized water and examined under polarized light microscopy (Olympus BX50, Melville, New York). Three sections from each tooth were photographed under maximum illumination and 4x magnification (SPOT, Diagnostic Instruments, Inc., Sterling Heights, Michigan). On teeth that displayed demineralization, Image Pro Plus 4.1 (Media Cybernetics, Silver Spring, Maryland) were utilized to obtain an average lesion depth (µm) for each section. This program registered the largest and smallest areas within the lesion and calculated an average depth for the section (Figure 13). The values for each of the three sections were then averaged to determine the lesion depth reported for each tooth. Statistical Analysis Descriptive statistics were compiled from the results of the study. The one-way analysis of variance (ANOVA) with post-hoc Ryan-Einot-Gabriel-Welsch multiple range test was used to determine whether there was a significant difference in lesion depth between sealant materials. All tests employed a 0.05 level of statistical significance. SAS for Windows (v9.1, SAS Institute Inc, Cary, NC, USA) was used for the data analysis.

36 Figure 4. Tooth mounted in Exaflex Putty. 27

37 Figure 5. Prototech Toothbrush Wear Simulator. 28

38 Figure 6. Toothbrush centered over specimen. 29

39 Figure 7. Force load application. 30

40 Figure 8. Slurry application. 31

41 Figure 9. Slurry recirculating apparatus. 32

42 Figure 10. Tooth prepared for acidic challenge. 33

43 Figure 11. Cross-sectional view of demineralized enamel window. 34

44 Figure 12. Measuring technique used by Image Pro Plus computer software (Media Cybernetics, Silver Spring, Maryland) to record mean depth of the lesion. 35

45 36 RESULTS Seventy-five extracted non-carious third molars were randomly selected and included in the study. They were divided into five experimental groups, comprising 15 teeth per group. Three sections were made from each tooth. During the sectioning process, some enamel surfaces did not remain intact. Some of this can be attributed to the sectioning process inadvertently removing the enamel surface. Graph 1 shows the average lesion depths for each of the 15 teeth in each of the five groups. The mean lesion depth (microns), standard deviation, and minimum and maximum measurements for each group are reported in Table 1. Descriptive statistics were conducted with the study data. The one-way ANOVA with post-hoc Ryan-Einot-Gabriel-Welsch multiple range test was used to determine whether there was a significant difference in lesion depth between sealant materials. An underlying assumption in order to use the ANOVA is the normality of the residuals. Normality of residuals was checked with a non-significant Shapiro-Wilk test and normal probability plots. Since the assumption of normality was valid, the one-way ANOVA was used to evaluate the performance of the sealant materials. All tests employed a 0.05 level of statistical significance. Statistical analyses were carried out with the statistical package SAS System version 9.1(SAS Institute Inc, Cary, NC, USA). Results of the one-way ANOVA revealed a significant effect for type of sealant materials on the lesion depth (p<0.0001). The post-hoc Ryan-Einot-Gabriel-Welsch multiple range test indicated that the mean lesion depth observed in ProSeal was significantly lower than in the other four groups. Moreover, no significant difference was found between SeLECT Defense, OrthoCoat, and Biscover LV. Table 2 presents the results of the post-hoc Ryan-Einot-Gabriel-Welsch multiple range test. Results from this study indicated that ProSeal reduced enamel demineralization by 82% when compared to controls. This was a significant reduction compared to the

46 37 other three sealant materials and the control. Results also indicated that Biscover LV, OrthoCoat, and SeLECT Defense all reduced enamel demineralization by 67%, 64%, and 64% compared to controls, respectively. However, there was no significant difference between these three sealant materials. Figure 13 are colored photomicrographs of representative samples from each of the five groups. As seen in the illustrations, most of the teeth, with the exception of the ProSeal group, had most of the sealant material worn away from the enamel surface.

47 38 Graph 1. Mean lesion depths and standard deviation for each of the five treatment groups.

48 39 Group N Mean Depth (µm) Std Dev Min Max Control SeLECT Defense OrthoCoat Biscover LV ProSeal Table 1. Descriptive statistics for mean lesion depth by treatment group.

49 40 Sealant Materials Mean Lesion Depth (SD) Control (14.61) SeLECT Defense (20.45) OrthoCoat (18.85) Biscover LV (13.29) A Group Comparisons*** B B B ProSeal (14.22) C ***means with the same letter are not significantly different using post-hoc Ryan-Einot-Gabriel-Welsch multiple range test (P>.05). Table 2. Mean lesion depths by the type of sealant materials.

50 41 Control (n = 15) Average Depth = µm Std Dev = Biscover LV (n = 15) Average Depth = 35.28µm Std Dev = SeLECT Defense (n = 15) Average Depth = 38.81µm Std Dev = Figure 13. Composite of polarized light microscopy images of representative lesions from all five treatment groups.

51 42 OrthoCoat (n = 15) Average Depth = 38.67µm Std Dev = 2.41 ProSeal (n =15) Average Depth = 19.55µm Std Dev = Figure 13 - continued