Peptide-guided Dental Tissue Regeneration for Oral Care Deniz Tanil Yucesoy A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2018 Reading Committee: Mehmet Sarikaya, Chair Sami Dogan Candan Tamerler Program Authorized to Offer Degree: Department of Materials Science and Engineering ©Copyright 2018 Deniz Tanil Yucesoy University of Washington Abstract Peptide-guided Dental Tissue Regeneration for Oral Care Deniz Tanil Yucesoy Chair of the Supervisory Committee: Prof. Mehmet Sarikaya Materials Science and Engineering Oral diseases are one of the most prevalent health problems that affect quality of life and well- being. These diseases include: (1) Demineralization-related dental conditions such as hypersensitivity, caries, white spot lesions; (2) Aesthetic problems e.g., tooth staining; (3) Soft tissue-related ailments that can lead to periodontitis and gingival recession; and (4) Infections and inflammations e.g., mucositis and peri-implantitis leading to implant failure. Current therapies mostly rely on replacement of the defective hard tissues with synthetic restorative materials. The implant treatment is also a common viable option in the contemporary dentistry when synthetic restorations fail and tooth extraction is indicated. The long-term success of these synthetic material-based restorations, however, is still low and questionable; indeed, they often cause post-therapeutic problems such as inflammation of gingiva or formation of secondary caries. Moreover, the failure of implants due to inflammation of the peri‐implant mucosa and peri‐implantitis as well as bacterial infections are major concerns. The primary reasons for the failure of current dental restoration therapies are four-fold: (i) Limited ability to reinstate the structure-function of the lost tissue, (ii) Lack of structural and functional integration (interface problem), (iii) Low thermal, mechanical, and chemical durability, and (iv) Tissue toxicity and low biocompatibility. The common denominator to overcome current limitations is to enable natural biomineralization in situ either on the defective hard tissues or at the tissue-material interface. The ideal approach would, therefore, be biomimetically developed repair and replacement materials, and regeneration therapies that follow the lessons from biology in dental tissue formation. Although numerous traditional biomimetic restoration approaches have been developed to reproduce biological minerals and tissues, these are still far from being feasible to use as a therapeutic and technological product due to the requirement of natural full-length proteins as the key enabling therapeutic agents. As an alternative route towards eliminating the inherent disadvantage of protein-based therapies, solid binding peptides have emerged as an effective molecular tool. These peptides are isolated through combinatorial mutagenesis and further engineered using bioinformatics and computational tools to epitomize the key functions of the natural proteins. Building upon molecular biomimetic principles, the hypothesis of this research is that “Using solid binding peptides, it could be possible to remineralize and repair the dental hard tissues; e.g., dentin, enamel and cementum, and restore the structure and function of the tooth with a well-integrated and durable mineral layer that could be administered via variety of formulations such as aqueous solution, gel and lozenges.” To demonstrate the validity of the hypothesis, it is planned to focus on four task areas including; Task-I: In vitro Peptide-guided Biomimetic Repair of Dental Hard Tissues (enamel and dentin); Task-II: In vivo Peptide-guided Remineralization to Restore Dental Caries (animal model); Task-III: Development of Mineralizing Peptide-based Restorative/Therapeutic Dental Care Products (dental lozenges); Task-IV: Improving the functionality (preventing risks associated with failure) of dental implants. The successful completion of these tasks will establish the basis of the peptide-guided remineralization therapy for developing a universal solution for oral care. Table of Contents List of Figures .................................................................................................................. iii List of Tables ...................................................................................................................xi Acknowledgements.......................................................................................................... xii Chapter 1: Introduction – Statement of the Problem and Thesis Approach ............................... 1 Chapter 2: Design Principles and Mechanism-of-Action of Amelogenin-Derived Peptides ...... 12 Chapter 3: In vitro Peptide-guided Biomimetic Dental Hard Tissue Remineralization ............. 19 3.1. In vitro Biomimetic Repair of Incipient Caries in Human Enamel.................................................. 20 3.1.1. Introduction ............................................................................................................................... 20 3.1.2. Materials and Methods .............................................................................................................. 22 3.1.3. Results ....................................................................................................................................... 28 3.1.4. Discussion ................................................................................................................................. 35 3.1.5. Conclusions ............................................................................................................................... 38 3.2. In vitro Biomimetic Repair of Hypersensitivity in Human Dentin .................................................. 41 3.2.1. Introduction ............................................................................................................................... 41 3.2.2. Materials and Methods .............................................................................................................. 44 3.2.3. Results ....................................................................................................................................... 47 3.2.4. Discussion ................................................................................................................................. 50 3.2.5. Conclusions ............................................................................................................................... 51 Chapter 4: In vivo Caries Formation and Remineralization-Rat Model ................................... 52 4.1. A Revisit to the Formation and Characterization of Carious Lesions in an in vivo Rat Model ....... 53 4.1.1. Introduction ............................................................................................................................... 53 4.1.2. Materials and Methods .............................................................................................................. 54 4.1.3. Results ....................................................................................................................................... 58 4.1.4. Discussion ................................................................................................................................. 66 4.1.5. Conclusions ............................................................................................................................... 68 4.2. Amelogenin Derived Peptide-guided Biomimetic Tooth Repair in vivo ......................................... 69 4.2.1. Introduction ............................................................................................................................... 69 4.2.2. Materials and Methods .............................................................................................................. 71 4.2.3. Results ....................................................................................................................................... 73 4.2.4. Discussion ................................................................................................................................. 79 4.2.5. Conclusions ............................................................................................................................... 80 i Chapter 5: Development of Mineralizing Peptide-based Restorative Dental Care Products ...... 82 5.1. Remineralizing Tooth Whitening Lozenges .................................................................................... 82 5.1.1. Introduction ............................................................................................................................... 82 5.1.2. Materials and Methods .............................................................................................................. 85 5.1.3. Results ....................................................................................................................................... 87 5.1.4. Discussion ................................................................................................................................. 93 5.1.5. Conclusions ............................................................................................................................... 95 Chapter 6: Enhancing the Functionality of Dental Implants .................................................. 96 6.1. Engineered Chimeric Peptides as Antimicrobial