Understanding dentine demineralizaion and development of strategies for biomimetic remineralization of deminderalized dentine

UNDERSTANDING DENTINE DEMINERALIZATION AND DEVELOPMENT OF STRATEGIES FOR BIOMIMETIC REMINERALIZATION OF DEMINERALIZED DENTINE ZHANG XU (MCE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF RESTORATIVE DENTISTRY FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgments Acknowledgments The work leading to this thesis can not be achieved without the guidance, assistance and encouragement from my supervisors, colleagues and family. I would like to start my acknowledgment by thanking Dr. Kishen. As dentistry was not my research field for my master’s degree, I found the research to be challenging at the initial stage. With his encouragement and help, I found my research direction and also gained much knowledge in dentistry from him. I also would like express my sincere gratitude to my supervisor, Prof. Chew. As a senior professor, he has groomed many dentists and achieved much academic recognition in dentistry field. Therefore, I am honored to my research under him. Although he has heavy clinical and teaching duties, he still took time off to supervise my research work and review my manuscript. Specially, I am deeply thankful to Prof. Neoh. She met me in Beijing and introduced me to Dr. Kishen. Most importantly, Prof. Neoh provided experimental equipment for me and gave many valuable comments on my research and papers. I have benefited so much from her rigorous academic attitude and profound knowledge. I am also thankful to the Head of the Department, Assoc. Prof. Jennifer Neo and the ii Acknowledgments members of thesis committee, Dr. Zeng Kaiyang and Assoc. Prof. Hien-chi Ngo. I would also like to acknowledge the support and help from my group members: Dr. Saji, Dr. Sum, Shibi, Annie, Dr. Megha and Liza. I am also thankful to Mr. Chan and Miss Lina for their help. Finally, I would like to specially thank my wife and parents for their love, sacrifice and understanding that allowed me to finish this thesis. Zhang Xu National University of Singapore iii Table of contents Table of Contents Chapter 1: Introduction Chapter 2: Literature review 2.1 The composition and structure of dental hard tissues 2.1.1 Enamel 2.1.2 Dentine 2.1.3 Cementum 2.1.4 Inorganic phase (apatite) in dental hard tissue 2.1.5 Organic matrix in dentine 2.2 Demineralization of dentine 11 2.2.1 Mechanism of demineralization of inorganic phase in dentine caries 11 2.2.2 Organic matrix in demineralization of dentine 13 2.2.3 Different zones of dentine caries 15 2.2.4 Different methods for induction of artificial dentine caries 16 2.3 Remineralization of dentine 18 2.3.1 The role of inorganic matrix of dentine in remineralization 19 2.3.2 The role of organic matrix of dentine in remineralization 21 2.4 The clinical significance of remineralization of dentine and current clinical methodologies to repair carious dental hard tissues 2.5 Biomimetic strategies for dentine remineralization 26 29 2.5.1 Biomineralization of dentine 29 2.5.2 Heterogeneous nucleation in biomineralization 32 2.5.3 Interaction between inorganic phase and organic matrix in heterogeneous nucleation 34 2.5.4 Development of biomimetic strategies for dentine remineralization 37 2.5.5 Phosphorylation of collagen 40 2.5.6 Phosphorylation of chitosan 41 2.6 Characterization techniques 44 iv Table of contents 2.6.1 Electrical impedance spectroscopy (EIS) 44 2.6.2 FTIR 45 2.6.3 XRD 46 2.6.4 SEM and EDX 47 2.6.5 Zeta potential 48 Chapter 3: Hypothesis and Objectives 50 Chapter 4: Characterization of Acid-Demineralization of Human Dentine and Influence of Demineralization on Remineralization of Dentine 52 4.1 Introduction 53 4.2 Materials and methods 54 4.2.1 Preparation of the specimens and demineralizing solution 54 4.2.2 Demineralization 55 4.2.3 Remineralization 56 4.2.4 EIS system and its measurement 56 4.2.5 Characterization 59 4.2.6 Statistical analysis 61 4.3 Results of characterizing the demineralization process of dentine 62 4.3.1 EIS measurement 62 4.3.2 ATR-FTIR spectroscopic analysis 63 4.3.3 XRD 64 4.3.4 SEM and EDX analysis 65 4.4 Results of remineralization of demineralized dentine using fluoride 68 4.5 Discussion 71 4.6 Summary 73 Chapter 5: Formation of Calcium Phosphate Crystals on Phosphorylated Type I Collagen Substrate: In vitro 5.1 Introduction 75 77 v Table of contents 5.2 Materials and methods 77 5.2.1 Eggshell membrane preparation and phosphorylation treatment 77 5.2.2 Mineralization 77 5.2.3 Characterization 78 5.3 Results 79 5.3.1 FTIR spectroscopic analysis 79 5.3.2 SEM and EDX analysis 80 5.3.3 XRD 83 5.4 Discussion 84 5.5 Summary 87 Chapter 6: Biomimetic Remineralization of Partially Demineralized Dentine Substrate with Phosphorylation of Dentine Collagen 88 6.1 Introduction 89 6.2 Materials and methods 91 6.2.1 Preparation of dentine collagen and partially demineralized dentine 91 6.2.2 Phosphorylation treatment 93 6.2.3 Preparation of dentine and dentine collagen particles and phosphorylation treatment 93 6.2.4 Mineralization 94 6.2.5 Characterization 95 6.2.6 Statistical analysis 97 6.3 Results 97 6.3.1 Mineralization of dentine collagen 97 6.3.1.1 FTIR spectroscopic analysis 97 6.3.1.2 XRD 99 6.3.1.3 SEM and EDX analysis 6.3.2 Remineralization of partially demineralized dentine 100 102 6.3.2.1 FTIR spectroscopic analysis 102 6.3.2.2 XRD 103 vi Table of contents 6.3.2.3 SEM and EDX analysis 104 6.3.2.4 Contact angles, surface free energy and interfacial free energy 107 6.3.2.5 Zeta potential 108 6.3.2.6 EIS measurement 109 6.4 Discussion 110 6.5 Summary 115 Chapter 7: Biomimetic Remineralization of Partially Demineralized Dentine Substrate using Phosphorylated Chitosan (P-chi) 116 7.1 Introduction 117 7.2 Materials and methods 119 7.2.1 Preparation of partially demineralized dentine sections 119 7.2.2 Preparation of dentine collagen particles 119 7.2.3 Synthesis of P-chi and modification of the dentine sections and dentine collagen particles with P-chi 119 7.2.4 Remineralization 120 7.2.5 Characterization 121 7.2.6 Statistical analysis 122 7.3 Results 122 7.3.1 FTIR spectroscopic analysis 122 7.3.2 XRD 125 7.3.3 SEM and EDX analysis 126 7.3.4 Contact angles, surface free energy and interfacial free energy 129 7.3.5 Zeta potential 130 7.3.6 EIS measurement 130 7.4 Discussion 131 7.5 Summary 135 Chapter 8: General discussion 8.1 Demineralization of dentine and its influence on remineralization 136 137 vii Table of contents 8.2 Introduction of phosphate groups onto Type I collagen to induce mineralization and its application to remineralization of partially demineralized dentine 139 8.3 Immobilization of P-chi on Type I collagen of partially demineralized dentine to induce remineralization 140 8.4 The factors influencing remineralization of partially demineralized dentine in vitro 142 Chapter 9: Conclusions and Future perspectives 146 Chapter 10: Bibliography 149 Appendix 165 viii Summary Summary Dentine remineralization is clinically significant for prevention and treatment of dentine caries, root caries, and dentine hypersensitivity. However, dentine remineralization is more difficult than enamel remineralization due to the abundant presence of organic matrix in dentine. This could be attributed to an accepted notion that dentine remineralization occurs neither by spontaneous precipitation nor by nucleation of mineral on the organic matrix, but by growth of residual crystals in the lesions. The general objective of this study was to develop a biomimetic method to facilitate remineralization of demineralized dentine. More specifically, this study aimed to study the process of demineralization in dentine and the nucleation role of phosphorylated noncollagenous proteins (NCPs) in the biomineralization of dentine. This study was designed to test the hypothesis that by mimicking the nucleating role of phosphorylated NCPs bound to collagen in biomineralization, TypeⅠcollagen in demineralized dentine when modified by phosphorylation or analogues of phosphorylated NCPs could induce marked mineralization. Attenuated total reflection fourier-transform infrared (ATR-FTIR), scanning electron microscopy (SEM), field emission electron microscope (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and electrical impedance ix Summary spectroscopy (EIS) were used to characterize the demineralization of dentine, the mineralization of Type I collagen and the remineralization of the surface of partially demineralized dentine. The biomimetic remineralization was carried out using two methods: (1) phosphorylation of Type I collagen in demineralized dentine using sodium trimetaphosphate (STMP) and (2) covalent immobilization of phosphorylated chitosan (P-chi) on the collagen in demineralized dentine. In this study, before and after demineralization and biomimetic modification, the zeta potential, the components of surface free energy of dentine surface and the interfacial free energy between dentine surface and aqueous medium were investigated. The ATR-FTIR, XRD, SEM and EIS results indicated that the effect of fluoride on remineralization of dentine was limited when less residual crystals were left on the surface of partially demineralized dentine undergoing 72-hour demineralization, whereas the biomimetic remineralization methods: phosphorylation of dentine collagen and immobilization of P-chi on dentine collagen, were able to result in favorable surface properties (i.e. high negative charge, high Lewis base (γ-, electron-donor) and low interfacial free energy between substrate and aqueous medium) for crystal nucleation and thus enhanced surface remineralization of partially demineralized dentine. The biomimetic remineralization for dental caries is in agreement with the concept of minimal intervention in caries prevention and management. Hence, it would find application in the minimally invasive management of dentine caries. x Bibliography The dissolution of apatite in the presence of aqueous metal cations at pH 2–7. 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Chem Rev 2008; 108:4628-4669 166 Appendix (3) Preparation of P-chi To the mixture of chitosan (2.0 g) in methanesulphonic acid (20 ml) was added phosphorus pentoxide and the mixture was stirred at 0-5℃ for 3h. After that, the mixture was stood overnight at -20℃, and the product then was precipitated with methanol and collected by centrifugation. Next, the product was washed with acetone and collected by centrifugation (3 circles). Finally, the product was dried in fume hood. The yield was 1.0-1.6 g. Adapted from Ref. [188] (4) FTIR spectra peak assignments of unmodified and phosphorylated chitosan Adapted from Ref. [191] 167 [...]... 82 Fig 5.3 XRD of Type I collagen membrane from ESM before and after mineralization 84 Chapter 6 Fig 6.1 Flowchart of remineralization of completely demineralized dentine 90 Fig 6.2 Flowchart of remineralization of partially demineralized dentine 91 Fig 6.3 ATR-FTIR analysis of the surface of dentine collagen before and after STMP treatment 98 Fig 6.4 ATR-FTIR analysis of the surface of dentine collagen... phosphorylationsites, and contains an RGD domain The roles of inorganic phase and organic matrix in demineralization and remineralization of dentine will be reviewed based on the available literature in the following sections 2.2 Demineralization of dentine The characterization of changes in the inorganic phase and organic matrix of dentine is important to understand the principle of demineralization of dentine and. .. accomplish remineralization of collagen Based on previous studies, two biomimetic strategies have been proposed in this study: (1) introduction of functional groups of NCPs onto dentine collagen and (2) development of analogues of NCPs, which would facilitate remineralization of dentine With these biomimetic strategies, collagen matrix can work as a scaffold to be remineralized, thereby enhancing the remineralization. .. mineralization 99 Fig 6.5 XRD of the samples before and after mineralization treatment 100 Fig 6.6 SEM and EDX results of mineralization of dentine collagen 101 Fig 6.7 ATR-FTIR analysis of the surface of partially demineralized dentine section 102 Fig 6.8 XRD of the surface of partially demineralized dentine section 104 Fig 6.9 SEM and EDX results of mineralization of dentine collagen 106 Fig 6.10... Demineralization of dentine is the process of removing mineral ions from the apatite lattice structure resulting in dissolution of the inorganic matrix, while the term remineralization of dentine refers to the process of restoring the inorganic matrix [1] Dentine remineralization is a clinically significant treatment approach for the prevention and management of dentine caries, root caries, and dentine hypersensitivity... scaffold to be remineralized, thereby enhancing the remineralization of dentine caries The detailed review of previous and on-going research on de- and remineralization of dentine, biomimetic mineralization and the techniques to characterize demineralization and remineralization of dentine will be presented in the Chapter 2 (Literature review) of this thesis 4 Literature review Chapter 2 ... Literature Review Dentine is a biocomposite of inorganic phase and organic matrix, which exhibited a complex behavior during demineralization and remineralization process In this section, the roles of inorganic phase and organic matrix in the demineralization and remineralization of dentine is reviewed Moreover, the current methodologies to treat dentine caries, biomimetic mineralization strategies and important... apparent resistance (Ra) of the dentine specimens measured by EIS system during remineralization 109 Chapter 7 Fig 7.1 Flowchart of remineralization of partially demineralized dentine 118 Fig 7.2 FTIR analysis of phosphorylation of chitosan 122 Fig 7.3 ATR-FTIR analysis of the surface of partially demineralized dentine section 124 Fig 7.4 XRD of the surface of partially demineralized dentine section 125... 7.5 SEM results of remineralization of partially demineralized dentine section 128 Fig 7.6 Change in apparent resistance of the dentine specimens measured by the EIS system in the process of remineralization 131 Chapter 8 Fig 8.1 Mechanism of HAP nucleation on residual crystal, phosphorylated dentine collagen and dentine collagen cross-linked with P-chi 145 xiii List of abbreviation List of Abbreviation... SEM, ATR-FTIR and XRD results of remineralization of demineralized Dentine 70 Fig 4.11 Change in apparent resistance of the dentine specimens measured by the EIS system in the process of remineralization 71 Chapter 5 Fig 5.1 FTIR analysis of the surface of Type I collagen membrane from ESM 80 Fig 5.2 SEM micrographs of Type I collagen membranes from ESM and mineral crystals on xii List of figures their . UNDERSTANDING DENTINE DEMINERALIZATION AND DEVELOPMENT OF STRATEGIES FOR BIOMIMETIC REMINERALIZATION OF DEMINERALIZED DENTINE ZHANG XU (MCE) A THESIS SUBMITTED FOR. artificial dentine caries 16 2.3 Remineralization of dentine 18 2.3.1 The role of inorganic matrix of dentine in remineralization 19 2.3.2 The role of organic matrix of dentine in remineralization. introduction of functional groups of NCPs onto dentine collagen and (2) development of analogues of NCPs, which would facilitate remineralization of dentine. With these biomimetic strategies,