Walsh University The Remineralization Potential of Nano-Hydroxyapatite in Hydrogen Peroxide Whitening Mouthwash A Thesis by Morgan McDermott Mathematics and Sciences Submitted in partial fulfillment of the requirements for a Bachelor of Science Degree with University Honors April, 2016 Accepted by the Honors Program ______________________________________________ Peter Tandler, Ph.D. Thesis Advisor Date ______________________________________________ Tom Freeland, Ph.D. Thesis Reader Date ______________________________________________ Ty Hawkins, Ph.D. Honors Director Date Abstract The purpose of this experiment was to analyze the demineralizing effects of Colgate Optic White mouthwash on hydroxyapatite tooth enamel. Nano-hydroxyapatite particles were used to make 10% solutions with RO water, artificial saliva, 2% hydrogen peroxide, and Colgate Optic White mouthwash. Small samples from these solutions were centrifuged to create an artificial tooth that was examined for demineralization upon being treated RO water, artificial saliva, 2% hydrogen peroxide, or Colgate Optic White mouthwash. Demineralization of the samples was measured by UV-Vis spectroscopy and calcium concentration was measured to compare demineralization among the four different solutions. Calcium standards and validation samples were prepared to make a calibration curve at a wavelength of 652 nm. The results of this experiment showed that calcium concentration was determined to be greatest in samples treated with Colgate Optic White mouthwash and least in samples treated with artificial saliva. RO water and 2% hydrogen peroxide both showed calcium concentrations greater than artificial saliva, but less than Colgate Optic White mouthwash. Colgate Optic White mouthwash significantly demineralizes more than the other three solutions and there was not any significant difference between RO water and 2% hydrogen peroxide. Artificial saliva demineralized the least out of the four groups, which was expected. These results lead to the conclusion that 2% hydrogen peroxide and RO water can demineralize to the same extent and Colgate Optic White mouthwash must contain an ingredient other than 2% hydrogen peroxide that promotes demineralization. This is significant because whitening mouthwash is commonly used by Americans to whiten their teeth, however, they are unaware that their enamel is being damaged. Having the knowledge of this and investigating the products one uses is the key to maintaining one’s oral health and preventing enamel erosion. Acknowledgements I would like to thank the entire Honors Committee for allowing me to write this thesis, and specifically Dr. Walro and Dr. Hawkins for supporting me throughout the entire process. Furthermore, I would like to express my sincere gratitude towards my thesis advisor, Dr. Tandler, and my thesis reader, Dr. Freeland, for guiding me through the writing and experimental process of this thesis. Their advice was invaluable to this research project and I cannot thank them enough for spending so much time with me and answering my endless list of questions throughout the duration of this thesis. Lastly, I would like to thank my family and friends, especially Christina McDermott and Brooke Chaney, for continuously supporting me and providing me with the motivation to make it through to the end. Table of Contents List of Figures……………………………………………………………………….Page 1 Introduction………………………………………………………………………….Page 2 Literature Review……………………………………………………………………Page 3 - Hydrogen Peroxide Whitening Agents………………………………………….Page 3 - Nano-Hydroxyapatite…………………………………………………………...Page 5 - Nano-Hydroxyapatite and Hydrogen Peroxide Whitening Agents……………...Page 8 Materials and Methods……………………………………………………..………Page 10 - Treatment Solution Preparation…………………………………………..…....Page 10 - Artificial Teeth…………………………………………………………..……..Page 11 - Ultraviolet-Visible Spectroscopy……………………………………..………..Page 12 - Microhardness Testing…………………………………………...…………….Page 13 Results…………………………………...…………………………………………Page 18 Discussion…………………………….......………………………………………..Page 22 Limitations………………………………......……………………………………..Page 23 Future Research…………………………………………..………………………...Page 24 Appendices……………………………………………..…………………………..Page 25 References ………………………………………………………..………………..Page 26 1 List of Figures Tables: - Table 1. Summary…………………………………………………...…………Page 20 - Table 2. Anova………………………………………………………...……….Page 20 - Table 3. T-test…………………………………………………………….……Page 20 Figures: - Figure 1. Spectra of calcium assay …………………….………………………Page 17 - Figure 2. Calibration curve at 415 nm…………………….......……………….Page 17 - Figure 3. Calibration curve at 652 nm……………………..…………………..Page 18 - Figure 4. Calibration curve at 652 nm with validation samples…...…………..Page 18 - Figure 5. Comparison of demineralization potential among test groups…...….Page 19 Images: - Image 1. Artificial tooth produced by centrifugation……..……...…………….Page 10 - Image 2. Jasco UV-Vis 650 spectrophotometer…………….………………….Page 11 - Image 3. Shimadzu type M Vickers microhardness tester……………………..Page 13 - Image 4. Microhardness tester diamond indenter on aluminum……….....……Page 14 - Image 5. Indentation diagram………………………………………………….Page 14 2 Introduction Tooth enamel is composed of hydroxyapatite (Ca5 (OH) (PO4)3), which is a matrix of calcium and phosphate [8]. When bacteria such as Streptococcus mutans stick to the enamel, they form large masses, referred to as plaque, which create acid buildup. These acid attacks lower the pH of the oral environment and cause mineral loss, or the dissolving of calcium and phosphate, and lead to porous enamel [8]. Enamel that contains large pores due to acid-induced mineral loss, called demineralization, allows bacteria to easily infect the deeper portion of the enamel. This infection creates a lesion, which is referred to as a carie, and is the primary stage of cavity development [8]. As demineralization continues, the lesion grows, reaching deeper layers such as dentin and eventually forms a cavity, which can result in total loss of the tooth if not treated properly [8]. In dentistry, remineralization is defined as the process of gaining mineral content in the enamel [9]. Saliva serves as a source of calcium and phosphate ions, so it remineralizes tooth enamel naturally when the oral environment is at a neutral pH. However, under acidic conditions, saliva cannot overcome the acidity and demineralization occurs regardless because the rate of demineralization is much faster than the rate of remineralization [9]. Several factors including microhardness, lesion depth, surface roughness, and mineral composition can be measured to determine whether or not remineralization has occurred. Microhardness increases with remineralization and decreases with demineralization because enamel weakens with increased pore size. Lesion depth increases with demineralization and decreases with remineralization because as the lesion progresses, it reaches deeper layers in the enamel. Surface roughness is decreased with demineralization and increased with remineralization because as new mineral is formed on the enamel surface, the morphology is altered. Mineral content 3 increases with remineralization and decreases with demineralization because calcium and phosphate concentration are lowered when the pH decreases [9]. This process of remineralization and demineralization is constantly occurring in the mouth, which is why everyday oral hygiene must be performed to prevent caries formation. Literature Review Hydrogen Peroxide Whitening Agents The use of whitening agents, or the “bleaching” of teeth, has increased in popularity recently due to its ability to reduce stains on the surface enamel, creating a brighter, more appealing smile. When hydrogen peroxide is applied to teeth, it diffuses into the enamel and decomposes into free radicals that react with pigment molecules. Pigment molecules are found in beverages, foods, and medications, and adhere more easily to rough surfaces, which are created on enamel after bleaching [3]. Pigment molecules appear as stains or yellowness in tooth enamel and when they react with hydrogen peroxide, they oxidize to hydrolyze and become more hydrophilic, allowing them to be easily removed with water [3]. Hydrogen peroxide is the most common whitening agent used because it is inexpensive and readily available, and its low pH enables it to easily enter the enamel [4]. However, studies have shown that bleaching via hydrogen peroxide can increase susceptibility to caries and cavities, enhance the progression of cavity formation, promote tooth sensitivity, and even cause further discoloration [4]. Hydrogen peroxide causes the enamel surface to weaken, allowing the formation of caries, which are lesions that eventually turn into cavities. Caries are caused by bacteria such as Streptococcus mutans that can easily adhere to the enamel and infect the tissue [3]. This easy adherence to enamel surface increases the rate of demineralization, or mineral loss, and allows for caries lesions to form. 4 There is often confusion regarding bleaching because dentists typically recommend hydrogen peroxide to promote gum health. However, high concentrations of hydrogen peroxide can cause negative side effects such as tooth sensitivity [4]. Healthy enamel reacts differently to hydrogen peroxide than demineralized enamel does because healthy enamel does not allow hydrogen peroxide to penetrate as deeply. Eroded or decaying enamel is very porous and weak, making it more susceptible to damage because hydrogen