University of Iowa Iowa Research Online Theses and Dissertations Spring 2010 The efficacy of Novamin powered technology Oravive and TopexRenew, Crest and Prevident 5000 Plus in preventing enamel demineralization and white spot lesion formation Andre Correia Jham University of Iowa Copyright 2010 Andre Correia Jham This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/522 Recommended Citation Jham, Andre Correia. "The efficacy of Novamin powered technology Oravive and TopexRenew, Crest and Prevident 5000 Plus in preventing enamel demineralization and white spot lesion formation." MS (Master of Science) thesis, University of Iowa, 2010. http://ir.uiowa.edu/etd/522. Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Orthodontics and Orthodontology Commons
THE EFFICACY OF NOVAMIN POWERED TECHNOLOGY ORAVIVE AND TOPEXRENEW, CREST AND PREVIDENT 5000 PLUS IN PREVENTING ENAMEL DEMINERALIZATION AND WHITE SPOT LESION FORMATION by Andre Correia Jham 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
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 Andre Correia Jham 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
To my dear wife Raquel, my loving parents Alice and Gulab, and my brother and best friend Bruno for their unconditional love and support. To everyone one else who has, in one way or the other, been a part of this great journey. You are way too many to mention. ii
. Never look down on anybody unless you re helping him up. Jesse Jackson. iii
ACKNOWLEDGMENTS I would like to thank Drs. Robert Staley, James Wefel, Fang Qian, and Lina Moreno for all their valuable advice and insight. In addition, I am very thankful to Jeff Harless and Maggie Hogan for their willingness to help at all times during this project. I would also like to express my deepest gratitude to Jared Bolding and Molly Bremen for all their help and dedication since the start of this project, and to Abby Kershner and Treagan White for being available to help when needed. I would also like to thank the staff and faculty of the Department of Orthodontics, University of Iowa for providing me with a high quality education, in special to Dr. Tom Southard for believing in me. And last but not least, a big thank you for my co-residents for being this great group of people and putting up with me for the last two years. I will always consider you amongst my closest friends. iv
TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vii INTRODUCTION...1 Purpose of this Study...2 LITERATURE REVIEW......4 Demineralization Process/White Spot Lesion Formation...4 White Spot Lesions in Orthodontic Patients..........6 Methods to Decrease Demineralization during Orthodontic Treatment...7 Mechanism of Action of Fluoride....9 Fluoride Dentifrice......10 Mechanism of action of Novamin...11 Polarized Light Microscopy....13 MATERIALS AND METHODS....17 Sample Preparation...17 Experimental Procedures...19 Statistical Analysis......22 RESULTS.......23 DISCUSSION....35 SUMMARY AND CONCLUSIONS.....40 REFERENCES......42 v
LIST OF TABLES Table 1. Descriptive statistics of mean lesion depth by treatment group...31 2. Mean lesion depth by treatment group...32 vi
LIST OF FIGURES Figure 1. Clinical example of before and after treatment in a patient exhibiting severe demineralization...3 2. Diagram representing the events of demineralization and remineralization......5 3. Histologic zones of a carious lesion...16 4. Tooth painted with acid-resistant varnish to produce a 1mm x 6 mm window of exposed enamel.....17 5. Cusp tips were ground flat to facilitate the sectioning procedure......18 6. Tooth following acidic challenge..........18 7. Prevident 5000 Plus, Crest, Renew and Oravive....19 8. Polarized light microscopy image of representative lesion from the Control group.........24 9. Polarized light microscopy image of representative lesion from the Oravive group.......25 10. Polarized light microscopy image of representative lesion from the Renew group......26 11. Polarized light microscopy image of representative lesion from the Crest group......27 12. Polarized light microscopy image of representative lesion from the Prevident 5000 Plus group....28 13. Measuring technique used by Image Pro Plus computer software (Media Cybernetics, Silver Spring, Maryland) to record mean lesion depth of lesion.....29 14. Measuring technique used for eroded lesions...29 15. Composite of polarized light microscopy images of representative lesions...30 vii
16. Mean lesion depth comparison between groups..33 17. Percent (%) reduction of mean lesion depth compared to the Control group.34 viii
1 INTRODUCTION There are several benefits to orthodontic treatment, such as significant improvements in a patient s esthetics, function and overall self-esteem. However, the esthetic result of orthodontic treatment can be severely compromised by demineralization of the tooth structure around the orthodontic appliances. This could result in white, opaque areas of demineralization, also known as white spot lesions (Figure 1). An opaque white spot lesion looks chalky and could result in cavitation of tooth structure if mineral loss continues (Mitchell et al., 1992). Enamel demineralization associated with fixed orthodontics has also been shown to be a very rapid process caused by a high and continuous cariogenic challenge in the plaque developed around the brackets (Ogaard et al., 1988). Enamel demineralization associated with fixed orthodontic appliances has been observed for years and continues to be a problem even with advances in materials and techniques (Farhadian et al., 2008). The most common method of preventing demineralization around orthodontic appliances is application of topical fluoride. Topical fluoride application methods include fluoride gels, rinses and dentifrice, which are all patient compliant - dependant treatment. Strateman and Shannon (1974) found that when patients used stannous fluoride gel, only 2% developed white spot lesions, compared with 58% of the patients who did not use the gel. Geiger et al (1992) found that the use of a fluoride rinse caused a 25% reduction in the number of patients exhibiting white spot lesions. However, most topical fluoride regimens rely on patient compliance. Patients who would benefit most from supplemental fluoride due to their poor hygiene are also the least likely to comply. A viable alternative in this situation would be the use of non-compliant treatment, such as fluoride varnishes, fluoride containing bracket/band cements and light-cured sealants. In an in vitro study by Frazier et al (1996), 80% of sealed teeth exhibited no
2 signs of demineralization whereas all untreated controls exhibited demineralization. Resin modified glass ionomer cements are able to continuously release fluoride over time after the initial topical fluoride application (Vorhies et al., 1998). Novamin falls into a newer category of bioactive glass-ceramic material that has been available since the 1960 s as materials to help in bone repair. The active ingredient is a calcium sodium phosphosilicate that reacts when exposed to aqueous media, thus providing calcium and phosphate ions to the applied surface. Novamin has received FDA clearance for products aiming to reduce sensitivity by blockage of dentinal tubules, and some of the products containing Novamin have also been used for reducing bleeding and gingivitis, as well as preventing demineralization and enhancing remineralization. Examples of Novamin powered technology include Oravive, a product from Natural Health Organics, which is a non-fluoridated, non-prescription dentifrice containing 5% Novamin. Another example is TopexRenew, a product of Sultan Health Care, a prescription strength 5000 ppm fluoridated dentifrice, also containing 5% Novamin. Purpose of this Study The purpose of this study was to compare in vitro the effectiveness of Novamin powered technology Oravive and TopexRenew, with Crest and Colgate s PreviDent 5000 Plus in decreasing demineralization and preventing white spot lesion formation on extracted human teeth. Teeth were cycled between demineralization and remineralization solutions and polarized light microscopy was used to analyze and compare lesion formation. Under our hypothesis, Oravive and TopexRenew would, respectively, be more effective than Crest and Prevident 5000 Plus in the prevention of enamel demineralization.
Figure 1. Clinical example of before and after treatment in a patient exhibiting severe enamel demineralization. 3
4 LITERATURE REVIEW Demineralization Process/White Spot Lesion Formation Dental caries is a multi-factorial disease that involves the interaction between 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. The organic acids are produced by plaque bacteria following exposure to fermentable carbohydrates. When a critical ph is reached, the organic acids are able to diffuse into the enamel surface through the acquired pellicle, commencing demineralization. During demineralization, less soluble phases of dicalcium phosphate dihydrate (CaHPO. 4 2H 2 O) and fluoridated hydroxyapatite (Ca 5 (PO 4 ) 3 (OH) x F (1-x) ) precipitate out of the enamel. This process continues until equilibrium is achieved between the enamel and the oral environment. Demineralization can continue as long as the oral ph remains acidic (Margolis and Moreno, 1990). When the oral ph rises above the acidic level, the remineralization process of the tooth surface can begin. Calcium and phosphate, which are present in the saliva, diffuse into the enamel with the help of fluoride to remineralize crystalline structures in demineralized areas. The rebuilt structures consist of fluoridated hydroxyapatite and fluorapatite, which are much more resistant to acid attack than the original structure (Selwitz et al., 2007). The processes of demineralization and remineralization occur several times throughout the day and if balanced, will not result in carious lesions. However, if the balance is tipped more towards demineralization rather than remineralization, the lesion will progress and eventually become a frank cavitation (Featherstone, 2000). A diagram of the demineralization and remineralization cycle is shown in Figure 2.
5 A white spot lesion is the first clinical presentation of dental caries prior to cavitation. The white opaque appearance is a result of the loss of subsurface enamel, resulting in the loss of enamel translucency (Zero, 1999). At this stage, the lesion may either progress to frank cavitation or may be arrested or reversed by modifying any of the causative factors of increasing preventive measures (Margolis and Moreno, 1990). Figure 1. Diagram representing the events of demineralization and remineralization.
6 White spot lesions in Orthodontic Patients Patients in fixed orthodontic appliances are quite susceptible to plaque accumulation and, consequently, white spot lesion formation. In vivo experiments by Ogaard et al. (1988) have shown that visible white spot lesions can develop in orthodontic patients within four weeks in the absence of any fluoride supplementation. Zachrisson and Zachrisson (1971) have shown almost a linear correlation between plaque accumulation and development of carious lesions in orthodontic patients. Visible plaque and the presence of mutans streptococci have been indicated to be good predictors of white spot lesions (Øgaard, 1989). Only at the time of removal of the appliances is the full extent of the damage realized, with the clinical appearance of white rings on a tooth around the area where the bracket once was. Once the condition is present, the patient is left with straight but esthetically unappealing teeth, counteracting the beneficial effect of the treatment. Although decalcification during orthodontic treatment is a widespread problem, published reports of prevalence vary widely. Zachrisson et al. (1971) reported that 89% of patients developed white spot lesions, while Strateman et al. (1974) observed 58% prevalence. Boersma et al. (2005), observed an even higher prevalence of decalcification, with 97% of their subjects displaying lesions after treatment. The differences in reported prevalence may be attributed to geographical location, as well as variation in the definition of a white spot lesion or the criteria applied for each individual study. However prevalent they may be, it is established that white spot lesions may persist for years, resulting in a permanent, unaesthetic result with the potential of worsening to the point of requiring permanent restoration (Øgaard, 1989; Sudjalim et al., 2006).
7 Methods to Decrease Demineralization during Orthodontic Treatment There are several methods to minimize or prevent demineralization and white spot lesion formation in the orthodontic patient. Patients should receive diet counseling that emphasizes a minimum intake of fermentable carbohydrates. Frequent consumption of sugary foods or drinks causes the ph in plaque to drop below the critical level of 5.5 (Mitchell, 1992). Especially in children and adolescents, proper nutrition is essential for maintaining overall systemic health and optimal oral health during and after treatment. Thorough oral hygiene has been shown to be an effective way to prevent and/or decrease demineralization in the orthodontic patient (Artun et al., 1986). Effective 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). Streptococcus Mutans is one of the main bacteria involved in the carious process (Van Houte, 1992). Chlorhexidine is an antimicrobial agent that is effective at reducing levels of streptococcus mutans (Ribeiro, 2007). Therefore, a protocol utilizing a chlorhexidine rinse, gel, or varnish may assist in preventing demineralization. Fluoride delivery in the oral cavity is also one of the most effective methods to reduce demineralization. Fluoride can be delivered directly to the tooth structure in multiple ways. Sources of fluoride include fluoridated dentifrice, rinses, foams, gels, varnishes, and fluoride-releasing bonding agents and cements. Fluoridated dentifrices, rinses, and gels are intended to be used by the patient at home and therefore rely on patient compliance. It can be difficult to have good levels of patient compliance. Geiger et al (1992) reported only 13% of compliance among patients participating in a fluoride rinse protocol study.
8 Thus, the use of fluoride products that do not rely on patient compliance has become increasingly popular. For instance, a fluoride varnish containing high amounts of fluoride can be applied at office visits, brackets and bands can be cemented with fluoridereleasing materials, and sealants can be placed to cover the facial surface of each tooth (Schmit et al., 2002; Todd et al., 1999; Ashcraft et al., 1997; Vorhies et al., 1998). Pro Seal (Reliance Orthodontic Products, Itasca, IL) is an example of an enamel sealant applied prior to bracket placement. This product has shown promise as a protective barrier against demineralization in non-compliant patients (Buren et al., 2008) MI Paste is a sugar-free, water-based cream containing Recaldent TM (GC Corporation, Tokyo, Japan) and was introduced to the American market in October 2004. The active ingredient of MI Paste is casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). Recaldent TM is the trademark applied to CPP-ACP technology. MI Paste has been approved for treating patients with dentinal hypersensitivity, yet is also marketed for helping prevent demineralization of tooth enamel and enhancing remineralization. The proposed anticariogenic mechanism of CPP-ACP is to localize amorphous calcium phosphate within dental plaque at the tooth surface, buffer the acidogenic challenge, and maintain a state of supersaturation of calcium and phosphate ions on the enamel surface. This process results in a decrease in demineralization during a cariogenic challenge and an increase in the subsequent remineralization of the enamel (Pulido et al., 2008). Eng (2009) found in an in vitro study that CPP-ACP showed a statistically significant reduction in lesion depth, however he could not conclude that this equates to a clinical reduction of visible demineralization.
9 Mechanism of Action of Fluoride Fluoride has several mechanisms of action to aid in the prevention of dental decay. It can incorporate into the enamel by combining with hydroxyapatite to form fluoroapatite, resulting in a less soluble and more resistant enamel when faced with an acidic challenge (Zipkin, 1970). It is also effective in inhibiting Enolase, an enzyme necessary for glycolysis, which is sensitive to fluoride at low levels. Subsequently, oral bacteria are unable to utilize fermentable carbohydrates (Levine, 1991). Besides, it can be effective in inhibiting bacterial colonization of the tooth surface via competitive binding. Due to its electronegative properties, fluoride competes with bacteria for these binding sites and prevents adhesion (Levine, 1991). However the main mechanism of action of fluoride is the remineralization of affected enamel. After an acid attack, fluoride ions react with calcium and phosphate of hydroxyapatite to form fluorapatite and can be incorporated into the partially damaged enamel structure, resulting in remineralization of the surface. This incorporation will slow, and may even stop, the progression of the lesion (Biestrock et al, 1998). Harris and Cristen (1995) actually concluded that the resulting enamel is more resistant to subsequent demineralization than the original one. While persistent, low levels of topical fluoride exhibit many beneficial properties, excessive amounts may demonstrate adverse effects. Research has indicated that increased levels of topical fluoride will lead to rapid mineral precipitation on the enamel surface and subsequent obliteration of surface enamel pores (García-Godoy et al., 2008). This limits the ability to remineralize the subsurface areas. In essence, a hard enamel shell is created with an area of demineralization remaining. For this reason, it is not recommended to apply high concentrations of fluoride (such as fluoride varnish) to areas of decalcification, as it will seal the outer enamel shell, and will prevent further remineralization from occurring, allowing the white spot lesion to remain indefinitely.
10 High amounts of topical fluoride also have the potential to be ingested, increasing the amount of systemic fluoride intake. In younger patients (under the age of six), this may lead to an increased incidence of dental fluorosis or potentially fluoride toxicity (Zero, 2006; Warren et al., 2003). Fluoride Dentifrice The most commonly used fluoride delivery method worldwide is fluoridated dentifrice. Burt and Eckland (2005) concluded fluoridated dentifrices have played a significant role in decreasing caries in industrialized countries. Marinho et al (2003) conducted a meta-analysis of seventy studies and came to the conclusion that fluoridated toothpaste is effective in preventing dental caries. Fluoride concentrations in OTC dentifrice range from 600 ppm to 1100 ppm.- look it up The low and high concentrations of fluoride are intended for patients under the age of six and those with high caries risk, respectively (Ammari, 2003). There are different sources of fluoride that can be used in dentifrice, such as sodium fluoride (NaF), monofluorophosphate (MFP) and stannous fluoride (SnF2)-SUBSCRIPT. In the United States, MFP or NaF are the leading types used (Zero, 2006). There are a few studies with prescription dentifrice such as Prevident 5000 (Colgate Oral Pharmaceuticals, Inc., Canton, Massachusetts). Baysan (2001) evaluated root caries treated with Prevident 5000 Plus (5000 ppm) versus Crest (1100 ppm) and found a greater reversal of primary root caries lesions among the group treated with Prevident 5000 Plus. In a systematic review, Twetman (2003) found that dentifrice containing 1500 ppm fluoride to have a superior preventive effect compared to that containing 1000 ppm fluoride.
11 Mechanism of Action of Novamin Bioactive glasses have been available since the late 1960 s. They contain oxides of calcium, sodium, phosphorus, and silicon in a proportion providing the material with surface activity and concomitantly with the property of forming a strong bond with bone. Bioactive glasses have been tested under different clinical situations, such as having an antibacterial effect (Allan 2001), assisting in the prevention of gingivitis, and having a scaffolding effect for new bone growth (Loty et al., 2001). Novamin is a calcium sodium phosphosilicate that belongs to a class of bioactive glasses that react when exposed to aqueous media, providing calcium and phosphate ions. It was originally developed as a bone regenerative material (Hench 1993) and contains calcium, phosphorus, sodium and silica molecules. In addition to the clinical uses mentioned above, Novamin has received approval from the FDA to be used in products that decrease hypersensitivity by the occlusion of exposed dentinal tubules. Marini et al conducted a pilot clinical study and concluded that Novamin-containing dentifrice was statistically more effective than a placebo dentifrice. There is a wide array of products containing Novamin. Oravive, a product from Natural Health Organics, is a type of non-fluoridated, non-prescription dentifrice containing 5% Novamin. NuPro is a type of prophy paste marketed by Dentsply containing 5000 ppm Fluoride and Novamin. TopexRenew, a product of SultanHealth Care, is also powered by 5% Novamin, and is a prescription strength 5000 ppm fluoridated dentifrice. Other products by Sultan Health Care include a root desensitizer called Vitalmin (100% Novamin and water) and a fluoride varnish (Durashield, 10% Novamin and 5% NaF). SootheRx is a prescription paste for hypersensitivity, containing 7.5% Novamin marketed by 3M/Omni in the United States. Dr. Collins Restore Remineralizing Toothpaste is a fluoride-free toothpaste also containing Novamin. All of
12 the above products have undergone clinical studies that support claims of decreased sensitivity through the occlusion of dentinal tubules. Burwell (2006) conducted an in vitro study comparing DenShield (Novamin containing dentifrice) and GC Tooth Mousse (Recaldent containing dentifrice) on bovine incisors following a 10-day demin/remin cycling protocol. At the end of the study the samples were analyzed with SEM (Scanning Electron Microscopy) that showed a greater number of occluded tubules with the Novamin-containing Denshield dentifrice, compared to GC Tooth Mousse. The key question to be answered is whether Novamin can prevent demineralization and promote remineralization of the enamel surface. Laboratory studies on Novamin have been released by the company. One of these studies was done by Alaudin and Fontana (2007) and compared a dentifrice containing 5% Novamin and Fluoride (MFP) to a commercially available dentifrice in remineralization of subsurface carious lesions in human tooth enamel. It used confocal laser scanning microscopy (CLSM), which is able to distinguish between sound enamel and demineralized enamel using a fluorescent dye. The authors came to the conclusion that Novamin-containing dentifrice was statistically more significant in decreasing the lesion area than the commercial dentifrice containing MFP fluoride alone. Although several studies have shown blockage of dentinal tubules when using Novamin, one must understand that this fact supports its effectiveness against hypersensitivity, not its capability to prevent demineralization and enhance remineralization. It must be kept in mind that only a few studies on the effect of Novamin on white spot lesions are available. Thus, further in vitro studies are needed to improve the delivery systems and protocols used (Wefel, 2009).
13 Polarized Light Microscopy Polarized light microscopy allows visualization of enamel demineralization and remineralization and is often used to evaluate carious lesions. Demineralization can also be studied by light microscopy, microradiography, surface hardness, electron microscopy, and confocal laser scanning microscopy (Ogaard et al., 1996). Polarized light microscopy is beneficial in that it permits qualitative and quantitative evaluation due to its ease of visualization of the color spectrum (Hicks, 1981). A polarized light microscope consists of a light microscope plus a polarizer and analyzer set perpendicular to one another. Both are made of prisms of calcite or a sheet of Polaroid, which 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 and produces a series of interference colors. A crystal is described as isotropic when it transmits light with equal velocity in all directions. It is anisotropic when the crystal transmits light at different velocities in different directions. Anisotropic crystals are subdivided into uniaxial and biaxial depending on the number of axes present. Hydroxyapatite is a uniaxial anisotropic crystal, meaning it has one optic axis that is coincident with its crystal axis. Hydroxyapatite is also birefringent, splitting a light ray into two components, which travel at different velocities and are polarized at right angle to one another, releasing different color and light intensities (Weyrich, 1994). A material that consists of a non-cubic crystal is given a sign of birefringence determined by the velocity of its resultant rays. Slow rays are designated positive (+) and fast rays are designated negative (-) (Theuns 1977). Enamel is composed mostly of inorganic hydroxyapatite (-) with a small amount of organic material (+) interspersed. Birefringence can be measured indirectly through polarized light microscopy. Enamel is a uniaxial birefringent object. When enamel is oriented with its optic axis
14 parallel to the direction of plane polarized light propagation, it will act as 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, which result in a series of interference colors (Silverstone, 1967). Birefringence is further categorized as intrinsic birefringence and form birefringence. Intrinsic birefringence is the difference between the refractive indices of the extraordinary ray (ne) and the ordinary ray (no), or (ne-no) (Hicks, 1981). Crstalline mineral volume, orientation of crystallites towards the light beam, and crystallite birefringence are all factors influencing intrinsic birefringence such that (n e n o ) i = δ c (n e n o ) HAP where (n e n o ) i = intrinsic birefringence, δ = pore volume occupied by crystallites, c = crystallite orientation factor, and (n e n o ) HAP = birefringence of crystallites (Theuns and Groenveld, 1977). During the demineralization process the space between the enamel crystals becomes 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. If the medium has the same refractive index as enamel, then no form birefringence will be created. Thus, when using polarized microscopy, different media are used. 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 material, and 4) the positive form birefringence of the organic versus the mineral material. The organic mineral content is so small that it can be disregarded. Therefore, the observed total birefringence is made up almost entirely by the intrinsic birefringence of the inorganic mineral and the form birefringence of the spaces in relation to the inorganic mineral (Silverstone, 1967).
15 Enamel is divided into longitudinal sections so that the crystallites of apatite are aligned along the length of the prism. Four zones of a carious lesion are visible with polarized light microscopy (Figure 3). The amount of space or tissue lost defines the zones, therefore different zones have different pore volumes. The surface zone is the outermost zone, which appears relatively unchanged and intact. It is a zone of remineralization and has a pore volume of 1-5 percent. 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 and is caused by dissolution of enamel cross striations. The dark zone has 2-4% pore volume. Like the surface zone, remineralization can occur in the dark zone. The dark zone also exhibits loss of enamel rod core to a greater extent than the fourth zone, the translucent zone. The translucent zone is the deepest of the four zones and exhibits a tenfold increase in the amount of space compared to the normal enamel. It is created by loss of enamel rod periphery and has a pore volume of 1 percent. (Silverstone 1967) Different imbibing media have different refractive indices that correspond to the zones of demineralization. Air, water, and potassium mercuric iodide dilutions (Thoulet s solution) are some of the media used in polarized light microscopy. Air (RI = 1.0) corresponds with 1 percent pore volume, water (RI = 1.33) with 5 percent pore volume, Thoulet s 1.41 with 10 percent pore volume, and Thoulet s 1.47 with a 25 percent pore volume. Different zones of a longitudinally sectioned lesion were mapped by imbibing with different media. The maps provided qualitative assessment of internal lesion pore volume. Quantitative information can be easily obtained by measuring the positively birefringent area of the lesion in the various imbibition media. Image Pro Plus (Media Cybernetics, Silver Spring, Maryland) software is used to measure a polarized light photomicrograph for statistical analysis. The polarized light microscope does allow for lesions to be quantified indirectly (Hicks, 1981).
Figure 3. Histologic zones of a carious lesion 16
17 MATERIALS AND METHODS Sample Preparation Eighty-two recently extracted non-carious human third molar teeth without observable white-spot lesions, decalcification, or dental fluorosis were selected for this in vitro study. The teeth were disinfected in Streck Tissue Fixative for two weeks, washed thoroughly with deionized water, and then stored in a 0.1% thymol solution. All remaining soft tissue, calculus, and bone were removed from the teeth utilizing razor blades and dental scalers. A small hole was drilled near the apex of the root of each tooth and a piece of waxed dental floss was tied to the root to facilitate suspension of the tooth in the experimental solutions. Each tooth was then coated with a thin layer of acidresistant varnish leaving a 1 mm x 6 mm window of exposed enamel. An example of the prepared tooth is demonstrated in Figure 4. Cusp tips were ground flat to facilitate sectioning as illustrated in Figure 5. Figure 4. Tooth painted with acid-resistant varnish to produce a 1 mm x 6 mm window of exposed enamel.
18 Figure 5. Cusp tips were ground flat to facilitate the sectioning procedure. Figure 6. Tooth following acidic challenge.
19 Experimental Procedures This in vitro study compared the effectiveness of Novamin powered technology Oravive and TopexRenew with Crest and Colgate s PreviDent 5000 Plus in decreasing demineralization and preventing white spot lesion formation on extracted human teeth. Figure 7. Prevident 5000 Plus, Crest, Renew and Oravive.
20 Following preparation, the teeth were randomly divided into five groups as follows: Group 1 - Control (deionized water) Group 2 - Oravive Group 3 - Renew Group 4 - Crest Group 5 - Prevident5000 Treatment slurries for Groups 2-5 were created at a ratio of 1:3 (w/v) with deionized water. Oravive, Renew, Crest and Prevident 5000 Plus were measured and placed in separate beakers. Each product was incorporated into water by hand spatulation then stirred using a stir bar and stir plate (rpm = 400) to ensure even distribution of the active ingredient, until the slurry was uniform in color and consistency. Fresh treatment slurries were mixed twice daily for the total duration of the study (10 days). The study commenced with the treatment application protocol. The teeth were suspended fully submerged in their respective treatment slurry (Group 1 in deionized water, Group 2 in Oravive, Group 3 in Renew, Group 4 in Crest, and Group 5 in PreviDent 5000 Plus ) for two minutes. Following the treatment protocol, the teeth were rinsed with distilled water and blotted dry. They were then placed in Parafilm M- covered beakers with room-temperature, constantly-circulating (rpm = 150) artificial caries solution. The demineralizing solution consisted of of 2.2 mm Ca 2+, 2.2 mm PO 3-4, 0.05 M acetic acid and 0.25 ppm Fluoride at ph = 4.4 (ten Cate and Duijsters, 1982). Each group had separate beakers of demineralization solution. Following 4 hours of demineralization, the teeth were removed from the acid solution, rinsed thoroughly with distilled water, blotted dry, and placed in a constantlycirculating (rpm = 150), room temperature, artificial saliva (remineralization) solution consisting of 20 mm NaHCO 3, 3 mm NaH 2 PO 4, 1 mm CaCl 2 2H 2 0, at ph = 7
21 (Birkeland, 1973). Similarly to the demineralization solution, each group had separate beakers of remineralization solution. After 1 hour of remineralization, the teeth were removed, rinsed thoroughly with distilled water, blotted dry, and placed in the demineralization solution for another 4 hours. Following the second period of demineralization the treatment application protocol was repeated, which included a two minute exposure in the respective treatment slurry. Following the second treatment application protocol the teeth were rinsed thoroughly with distilled water, blotted dry, and placed in the remineralization solution for 14 hours (overnight). The cycling process proceeded for ten continuous days until significant white spot lesions were visible in the control teeth. Both demineralization and remineralization solutions were changed at the end of Day 5 to maximize the demineralization ability and the remineralization capacity of the respective solutions. Teeth were monitored for chipped acid-resistant varnish during the study and were repainted as necessary. After ten days of cycling, one control tooth was sectioned and examined under the polarized light microscope to evaluate lesion depth. Based on the depth of the lesions observed in the control samples it was determined that the study had progressed sufficiently and that cycling should be terminated. Subsequently, all teeth were removed from solution, rinsed thoroughly with distilled water, and blotted dry. Longitudinal sections were made using a Series 1000 Deluxe hard tissue microtome (Scientific Fabrication, Littleton, Colorado) to yield sections 100-140 µm thick. Sections were imbibed with deionized water (refractive index = 1.33) and photographed under a polarized light microscope (Olympus model BX-50, Olympus Corporation, Lake Success, New York). Three representative sections of every tooth were photographed and measured using Image Pro Plus computer software (Media Cybernetics, Silver Spring, Maryland). The software allowed for average depth (µm) to
22 be recorded across the length of the lesion. For most of the control teeth, the enamel surface had to be recreated due to erosion of the outer enamel surface. The resulting depths of each of the three sections were averaged together to produce an overall mean lesion depth (µm) for the tooth. Statistical Analysis Descriptive statistics were conducted. Because of lack of normality, the white spot lesion depths were analyzed using one-way ANOVA to the ranked data, followed by post-hoc Bonferroni multiple comparison test, an equivalent test statistics to the nonparametric Kruskal-Wallis test. An underlying assumption in order to use the ANOVA procedure is the normality of the residuals. Normality of residuals was checked with a non significant Shapiro-Wilk test and normal probability plots. 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).
23 RESULTS The teeth in the study were cycled for ten continuous days. Following the cycling protocol the teeth were sectioned using the hard tissue microtome producing three to five sections per tooth. This procedure led to the loss of six teeth (3 from the Control group, 1 from the Crest group and 2 from the Prevident group) due to damaged enamel and/or unusable sections. Of the remaining 76 teeth, three representative sections of each tooth were selected to be photographed and measured. Erosion of the outer enamel surface was observed in most of the teeth in the Control (Group 1) and Oravive (Group 2) groups. Representative sample lesions from each group were photographed and are displayed in Figures 8-12. In addition, a sample of the measuring technique using the Image Pro Plus computer software to determine lesion boundaries and average lesion depth is displayed in Figure 13. When measuring the teeth that displayed cavitation of the enamel surface, the surface was approximated by connecting intact enamel borders (Figure 14). This allowed for an estimated measurement of lesion depth for these teeth. A composite of all five treatment groups is depicted in Figure 15. In order to ensure the independence of samples for performing the appropriate statistical analysis, the average of three measurements was used for the data analysis. Descriptive statistics for the comparison of lesion depths between treatment groups are summarized in Table 1.
Figure 8. Polarized light microscopy image of representative lesion from the Control group. 24
Figure 9. Polarized light microscopy image of representative lesion from the Oravive group. 25
Figure 10. Polarized light microscopy image of representative lesion from the Renew group. 26
Figure 11. Polarized light microscopy image of representative lesion from the Crest group. 27
Figure 12. Polarized light microscopy image of representative lesion from the Prevident 5000 Plus group. 28
29 Figure 13. Measuring technique used by Image Pro Plus computer software (Media Cybernetics, Silver Spring, Maryland) to record mean lesion depth. Figure 14. Measuring technique used for eroded lesions.
30 Control (n = 19) Average Depth = 128 µm Std Dev = 27 Crest (n = 14) Average Depth = 61.9 µm SD = 16.3 PreviDent 5000 Plus (n = 13) Average Depth = 38.9 µm SD = 10.4 Renew (n = 15) Oravive (n = 15) Average Depth = 66.1µm Average Depth = 127.1 µm SD = 13.7 SD = 32.2 Figure 15. Composite of polarized light microscopy images of representative lesions.
31 Table 1. Descriptive statistics of mean lesion depth by treatment group. Results of one-way ANOVA revealed a significant effect for the type of preventive treatments on the white spot lesion depths (p<0.0001). The post-hoc Bonferroni multiple comparison test indicated that the mean lesion depths observed in Control and Oravive groups were significantly greater than the other three treatment groups, while the mean lesion depth observed in Prevident was significantly lower than those in Renew and Crest. Moreover, no significant difference was found between Control and Oravive or between Renew and Crest. Table 2 presents the results of the post-hoc Bonferroni multiple comparison test.
32 *** means with the same letter are not significantly different using post-hoc Bonferroni multiple comparison test (P>.05) Table 2. Mean lesion depth by treatment group.
33 Figure 16. Mean lesion depth comparison between groups. The Oravive group showed only a 0.8% reduction in mean lesion depth compared with the Control group, while the Renew and Crest groups had 48.4% and 51.7% reduction respectively. The Prevident 5000 Plus group demonstrated a 69.7% reduction in mean lesion depth compared with the Control group and a 39% reduction, compared with the Renew/Crest group (Figure 17).
34 Figure 17. Percent (%) reduction of mean lesion depth compared to the Control group.
35 DISCUSSION The purpose of this study was to compare in vitro the effectiveness of Novamin powered technology Oravive and TopexRenew with Crest and Colgate s PreviDent 5000 Plus in decreasing demineralization and preventing white spot lesion formation on extracted human teeth. It was hypothesized that Oravive and TopexRenew would, respectively, be more effective than Crest and Prevident 5000 Plus in the prevention of enamel demineralization. Eighty-two extracted non-carious human third molar teeth without observable white-spot lesions, decalcification, or dental fluorosis were selected for this in vitro study. Throughout the study 6 teeth were lost during the sectioning process. The final n=76 proved to be adequate, since enough statistical power was achieved to show a statistically significant difference between the samples. It is not clear whether Novamin s mechanism of action is more effective in preventing demineralization or in enhancing remineralization. Thus, it was rationalized that by conducting the treatment applications before and after periods of demineralization, one would be allowing both scenarios to occur. The amount of time spent in both solutions throughout 10 days was 240 hours, divided as follows: 80 hours in the demineralization solution, 150 hours in the remineralization solution, and 10 hours in the treatment application protocol. After ten days the cycling was terminated, teeth were sectioned and the slices were examined under the microscope and measured. Statistical analysis indicated that the mean lesion depths observed in the Control and Oravive groups were significantly greater than in the Crest, Renew and Prevident 5000 Plus groups, while the mean lesion depth observed in Prevident 5000 Plus was significantly lower than those in Renew and Crest. In addition, no significant difference was found between Controls and Oravive or between Renew and Crest.
36 Some of the results encountered were expected, such as the Prevident 5000 Plus (69.7%) showing a higher reduction in mean lesion depth when compared to the Crest toothpaste (51.7%). Since the amount of fluoride present in the Crest toothpaste is significantly less than the amount present in the Prevident 5000 Plus, which makes F much less available to prevent demineralization and promote remineralization of white spot lesions in the Crest group. Through comparison of the mean lesion depth reduction in the Control (0 ppm F), Crest(1100 ppm F) and Prevident 5000 Plus (5000 ppm F)groups, it can be concluded that the fluoride dose response was effective under the model utilized in this study. It was also expected that Renew (48.4%) showed superior performance than Oravive (0.8%). Although both products contain 5% Novamin, Renew also contained 5000 ppm F. Some of the results, however, were quite different from those expected. It was hypothesized that Oravive (5% Novamin) would be more effective than Crest (1100 ppm F) in preventing demineralization and/or enhancing remineralization, and the results showed that the Crest group had a significantly higher reduction in mean lesion depth when compared to the Oravive group. In fact, perhaps the most unexpected finding was the fact that the Oravive group performed almost exactly the same as the Control group, with no statistically significant difference between both groups. Due to the combination of Novamin and fluoride, and according to the literature and manufacturer advertisements, it was expected that the Renew group (5% Novamin; 5000 ppm Fl) would be more effective and have stronger results than the Prevident 5000 Plus group (5000 ppm Fl), hence our hypothesis. However, based on the results when comparing the Control group against both groups containing Prevident 5000 Plus or Renew, Prevident 5000 Plus had a significantly more effective performance in reducing mean lesion depth, compared to Renew (69.7% reduction versus 48.4% for the Renew group). The Renew had also been expected to be more effective than the Crest group,
37 since it has a higher F concentration in addition to 5% Novamin, but no statistically significant difference was found between these two groups. A common pattern observed showed that the Novamin-containing products used in this study (Oravive and Renew) did not perform as expected, indicating that, under the model chosen in this study, Novamin was not an effective agent in preventing demineralization and/or enhancing remineralization. This could also be explained due to the fact that when Calcium and Phosphate ions (existing in Novamin) are applied in conjunction with fluoride, their low solubility could lead to precipitation of these ions, which in turn could make it more difficult for these ions to reach the tooth surface (Wefel, 2009). In disagreement with these findings, Alaudin and Fontana (2007) compared dentifrice containing 5% Novamin and fluoride (MFP) to a commercially available dentifrice with MFP only, and concluded that the Novamin-containing dentifrice was statistically more significant in decreasing the lesion area than the commercial dentifrice containing MFP alone. One of the differences between our study and Alaudin s is that we used NaF instead of MFP fluoride. When considering the clinical application of these particular products, some important factors must also be taken into account. While Crest, Renew, and Prevident 5000 Plus have all been shown in this study to decrease mean lesion depth, Oravive did not present any statistically significant difference, compared to the Control group; thus, under the model utilized in this study, it would be fair to say that we do not recommend its use in the prevention of demineralization and/or enhancement of remineralization. While both Crest and Renew proved to be effective agents in the prevention of demineralization and/or enhancement of remineralization, no significant difference was found in lesion depth reduction between them (51.7% versus 48.4%, respectively). Therefore, it can be concluded that it is more cost effective and convenient to use the
38 non-prescription Crest toothpaste rather than the more expensive, prescription-only Renew toothpaste. When comparing both prescription-only toothpastes Prevident 5000 Plus and Renew, our study showed a statistically significant difference in the reduction of lesion depth (69.7% versus 48.4%, respectively). As the cost of both products is comparable, one might lean towards the use of Prevident 5000 Plus. However it should be taken into consideration that despite Renew being less effective than Prevident 5000 Plus in the prevention of white spot lesions, and equally as effective as the non-prescription Crest toothpaste, some Novamin-containing products have also been shown to be effective desensitization agents, according to Burwell (2006). An orthodontic patient with sensitivity, in particular an adult, could profit from this added benefit and may be more compliant using a single preventive product rather than multiple products. There were also several inherent limitations to this study. The fact that it was conducted in vitro is one of them. It is quite difficult to recreate the oral environment in a laboratory study outside of the mouth. Saliva has several components that are not part of the remineralization solution used in this study, such as proteins and bacteria. It is also impossible to precisely account for the salivary flow that continuously remineralizes the oral environment. Naturally the cycling simulated in our study happens in a much more varied manner in the oral cavity, being affected not only by schedule but also by diet. Certain foods such as sugary snacks and drinks can potentially increase demineralization, while other calcium-rich products such as milk can inhibit demineralization. Also, the absence of plaque on the teeth used in the study makes it difficult to fully evaluate the effectiveness of Novamin. Tai BJ et al (2006) conducted a randomized, double-blinded pilot clinical trial to evaluate the anti-gingivitis and anti-plaque effects of a Novamin containing dentifrice. 100 patients had a baseline measurement of their Plaque Index(PLI) and Gingival Bleeding Index (GBI) and 6 wks later following use of the
39 Novamin containing dentifrice or a placebo control toothpaste. Patients were told not to perform any oral hygiene for 8h prior to the baseline and 6 wks examination. Following the clinical assessments, all subjects received a supragingival prophylaxis and were given either the test-dentifrice or the placebo formulation. The results showed a statistically significant difference in the reduction of gingival bleeding (58%) and reduction in plaque growth (16%) in the Novamin group. Another limitation relates to the short term period in which it was conducted (10 days). For that reason, the experimental conditions were intentionally more extreme in order to create lesions within this short period of time. Ogaard et all (1988) has shown that white spot lesions can normally develop anywhere from 4 wks to several months. Both this study and Ogaard s study are examples of harsher acidogenic challenges in significantly shorter periods of time, when compared to the naturally slower and milder demin/remin cycle over the course of 2 years in the orthodontic patient. Novamin s mechanism of action might require a longer treatment time period and/or a less acidogenic challenge to be effective. The accelerated and stronger conditions in our study might have led to a limitation of Novamin s ability to prevent demineralization and/or enhance remineralization. Certainly additional research is warranted to help understand the exact optimal conditions under which Novamin-containing products can be more effective in the prevention of demineralization and enhancement of remineralization. Ideal formulation and delivery methods need to be further developed and investigated and then tested in randomized clinical trials, in order to fully explore the potential of this promising agent.