Title Page

Contents

Abstract 14

Chapter 1. INTRODUCTION 16

1.1. Bone & Collagen 16

1.1.1. Type I Collagen 18

1.1.2. Type I Collagen Fibrils Self-assembly in Vitro 21

1.2. Collagen Mineralization 23

1.2.1. Extrafibrillar Mineralization versus Intrafibrillar Mineralization 23

1.2.2. Mechanical Properties of Mineralized Collagen Fibrils 25

1.3. Nucleation, Growth, and Crystallization of Calcium Phosphate 26

1.3.1. Classical Nucleation Theory (CNT) of Hydroxylapatite 27

1.3.2. Non-classical Nucleation Theory of Hydroxyapatite 29

1.4. Additive /functionalization for Mineralization 30

1.4.1. Combination with Polydopamine 30

1.4.2. Polydopamine for Biomimetic Mineralization 32

1.5. Limitation of Current State Materials 33

1.6. Research Objective and Scope 34

Chapter 2. c-Axis-Oriented Platelets of Crystalline Hydroxyapatite in Biomimetic Intrafibrillar Mineralization of Polydopamine-Functionalized Collagen Type I 35

2.1. Overview 35

2.2. Introduction 36

2.3. Materials and Methods 40

2.3.1. Materials 40

2.3.2. Self-Assembly of Collagen Fibrils 40

2.3.3. Mineralization of Collagen with Polydopamine 41

2.3.4. Material Properties of Mineralized Collagen Fibrils 41

2.4. Results 42

2.4.1. Self-assembly of Col-I Fibrils with Dopamine Polymerization 42

2.4.2. Mineralization of Col-PDA Fibrils 49

2.4.3. Intrafibrillar Mineralization by Transmission Electron Microscope (TEM) 50

2.4.4. The Specific Binding Sites of PDA on Col-PDA Fibrils 61

2.4.5. Comparative Analysis of Mineralization Kinetics in Col-PDA Fibrils: TEM vs. XRD Studies 63

2.5. Discussion 66

2.6. Conclusion 68

Chapter 3. Interface-Controlled Biomimetic Intrafibrillar Mineralization of Collagen: Effect of Ca²⁺/[PO₄]³⁻ Concentration Ratio 69

3.1. Overview 69

3.2. Introduction 70

3.3. Materials and Methods 74

3.4. Results and Discussion 78

3.4.1. Col-PDA Intrafibrillar Mineralization 78

3.4.2. The Influent of Ca²⁺/[PO₄]³⁻ on Mineralization 84

3.4.3. Interface-Controlled Intrafibrillar Mineralization 96

3.4.4. The Biocompatibility of Col-PDA Fibrils 98

3.5. Conclusions 101

Chapter 4. Overall Summary 102

References 104

Appendices 117

〈Appendix 1〉 Atomic Layer Deposition of SnO₂ on C60 for Efficient Perovskite Solar Cells 117

〈Appendix 2〉 Copyright permission 135

국문초록 137

Table 2-1. The concentration of Col-I and self-assembly buffer was fixed at 50 μg/mL, and the concentration of DA added there to is listed in the table. The weight ratio of Col-I and DA listed in the... 42

Table 3-1. Zeta potential measurement of an pAsp complex in m-SBF solution as a function of [PO₄]³⁻ concentration with incubation at 37 ℃ 82

Table 3-2. Inductively coupled plasma optical emission spectroscopy (ICP-OES) of Col-PDA fibrils and after mineralization as a time series in m-SBF solution where the Ca²⁺/[PO₄]³⁻＝1/15. The results... 83

Figure 1-1. hierarchical structure of bone from collagen molecule to bone structure. Reproduced with permission. 8 Copyright 2015, Nature Publishing Group. 17

Figure 1-2. the hierarchical organization of collagen fibrils. The triple helix of collagen consists of three helical molecules. These molecules are arranged in a fibril structure, with a staggered arrangement... 20

Figure 1-3. hierarchical structure of type I collagen. 22

Figure 1-4. TEM Images of (a) extrafibrillar mineralization on collagen fibrils and (b) intrafibrillar mineralization. 25

Figure 1-5. The relationship between the size and energy of nanoscopic nuclei in classical nucleation theory. Adapted from ref. 28

Figure 1-6. The mechanism of nucleation based on classical nucleation theory (CNT, top) and the pre-nucleation cluster (PNC) pathway (bottom). Reproduced with permission. Copyright 2020, Elsevier... 29

Figure 2-1. Schematic of PDA-assisted mineralization with Col-I fibrillation. DA molecules added during the Col-I self-assembly process chemically were bound to the surface of Col-I molecules in... 39

Figure 2-2. Self-assembled Col-I and PDA-Col fibrils. (a), (c) TEM images of pristine Col-I and Col-PDA (fibrillation by mass ratio of Col-I and DA 5:16). Inset images are high magnification TEM image... 45

Figure 2-3. TEM images of self-assembled Col-I fibrils showed the evidence of highly ordered Col-I fibrils. High magnification TEM image exhibiting the dark/bright pattern of well-ordered structure. The... 46

Figure 2-4. AFM images of self-assembled Col-I fibrils in different amount of dopamine (DA). (a) 5:4, (b) 5:8, (c) 5:16, (d) 1:8 (e) 1:16, (f) 1:32 weight ratio of Col-I and DA. When the high concentrations... 47

Figure 2-5. CD results of Col-I molecules structure before and after self-assembly with polymerization of DA for 12 hrs at 37 ℃. It has been proven that the structure of the Col-I molecule constituting Col-... 48

Figure 2-6. (a) XRD patterns of the mineralized Col-PDA fibrils for 3 (red solid line) and 7 days (blue solid line). XRD pattern of the unmineralized Col-PDA fibrils is a control (black solid line). The merged... 50

Figure 2-7. Bright field TEM images and corresponding SAED patterns of the mineralization pristine in Col-I fibrils ((a), (c), (e), (g), and, (i)) and Col-PDA fibrils ((b), (d), (f), (h), and (j)) (scale bars: 200... 53

Figure 2-8. EDS results of pristine Col-I and Col-PDA fibrils mineralized for 12 hrs. The EDS mapping indicated the distribution of Ca and P elements. The detected Ca and P signal are found to be mostly... 54

Figure 2-9. High magnification TEM images of mineralization of Col-PDA. The diameter of mineralized Col-PDA fibrillar matrix increases with the mineralization process as the incubation time... 55

Figure 2-10. For a day, TEM images and SAED patterns of the mineralized pristine Col-I (a-c) and Col-PDA fibrils (d-f). (a-b) No intrafibrillar mineralization was found for the pristine Col-I fibrils while... 57

Figure 2-11. (a-e) Mineralization of the Col-PDA fibrils for a day. (a) Bright-field TEM image of Col-I fibrils was shown the partially mineralized ones. (b) Dark-field TEM image of Col-I fibrils after the... 59

Figure 2-12. EDS results according to mineralization time of Col-PDA (scale bars: 500 nm). The mineralization degree was calculated based on the partial area selected by the yellow line in the BF... 60

Figure 2-13. Fluorescence images of Cy5 NHS ester labelled Col-I and Col-PDA fibrils from different area on TEM grids. Col-I dyed fibrils (left panel) shown emitted red fluorescence while no almost no... 62

Figure 2-14. Mean fluorescence intensity for the evaluation of red emitting fluorescence analysis by using ImageJ software. 63

Figure 2-15. TEM results of mineralized Col-PDA fibrils for 2 days. (a-b) TEM images of the nearly complete mineralized Col-PDA fibrils. (c) SAED patterns of HAp crystals in the mineralized Col-PDA... 65

Figure 2-16. The gap zone and overlap zone between Col-I molecules that are regularly arranged, and the PDA bound to the surface of the Col-I molecule attract Ca²⁺ and PO₄³⁻ ions to induce ACP nucleation... 67

Figure 3-1. Schematic of intrafibrillar mineralization with precursor-pAsp assistance. (a) Precursor-pAsp consisting of Ca and P ions (Ca²⁺/[PO₄]³⁻ at a concentration ratio of 1/20) in m-SBF evolved into... 73

Figure 3-2. Determination of the regions of mineralized fibrils Ca²⁺/[PO₄]³⁻＝1/10 10 h. 77

Figure 3-3. (a) AFM images of self-assembled Col-PDA fibrils confirming the approximate band gap characteristic fibrils. (b) FTIR shows the IR bands characteristics of peptide linkages are as follows.... 79

Figure 3-4. Bright-field TEM images with low- (left panels) and high-magnification (right panels) and corresponding SAED patterns, as insets, of the self-assembled Col-PDA fibrils in the presence of pAsp... 80

Figure 3-5. DLS of the SBF solution in the presence of pAsp. DLS shows the initial size of the primary precursor at around ~5 nm in diameter after incubation at 37 ℃ for 12 h before the... 82

Figure 3-6. DLS spectra and zeta potential analysis of precursors in the m-SBF solution. (a) DLS spectra show the increased diameters of complexes with greater [PO₄]³⁻ concentrations and incubation... 83

Figure 3-7. ICP-OES plot of Col-PDA fibrils and after mineralization as a time series in an m-SBF solution when Ca²⁺/[PO₄]³⁻＝1/15. 84

Figure 3-8. EDS mapping of Col-PDA fibrils at an early-stage of mineralization in m-SBF with Ca²⁺/[PO₄]³⁻＝1/15 for 1.5 and 3 h. The EDS mapping indicated the distribution of Ca and P elements... 86

Figure 3-9. The early mineralization stage of Col-PDA fibrils with a Ca²⁺/[PO₄]³⁻ ratio of 1/15 in an m-SBF solution for 6 h. (a) Dark-field images of TEM showed regions with heavier elements containing... 86

Figure 3-10. EDS spectrum of Ca²⁺/[PO₄]³⁻＝1/15 for 6 h. Spectra showing the presence of Ca and P elements are highlighted in red and green colors. The detected Ca signal was of relatively higher... 87

Figure 3-11. XRD patterns of Col-PDA fibrils mineralized in m-SBF solutions with Ca²⁺/[PO₄]³⁻ ratios of (a) 1/5, (b) 1/10, (c) 1/15, (d) 1/20, and (e) Col-PDA before mineralization with JCPDS of HAp as... 89

Figure 3-12. FTIR spectra of Col-PDA fibrils mineralized with m-SBF at Ca²⁺/[PO₄]³⁻ ratios of (a)1/5, (b) 1/10, (c) 1/15, (d) 1/20 as a series of different mineralization times. The spectra of a phosphate band... 91

Figure 3-13. FIIR spectra of Col-PDA fibrils mineralized at various Ca²⁺/[PO₄]³⁻ ratios, 1/5, 1/10, 1/15, and 1/20 for (a) 6, (b) 12, (c) 24, and (d) 48 h, respectively. The spectra show intensity differences... 92

Figure 3-14. Bright-field TEM images and corresponding SAED patterns of mineralized Col-PDA fibrils in different m-SBF solutions (a) Ca²⁺/[PO₄]³⁻＝1/1 incubated for 7 days, (b) Ca²⁺/[PO₄]³⁻＝1/5... 94

Figure 3-15. Bright-field TEM images and corresponding SAED patterns of mineralized Col-PDA fibrils. The self-assembled Col-PDA fibrils were incubated in m-SBF with Ca²⁺/[PO₄]³⁻＝1/10 for (a) 6... 95

Figure 3-16. Degree of mineralization of Col-PDA fibrils calculated from the mineralized regions in TEM images (right-panel) of each stage of mineralization. The upper TEM image shows full... 97

Figure 3-17. Proliferation of MC3T3-E1 cell lines cultured on different substrates. The MTT assay was used to measure mineralization after 24 and 48 h. Data are expressed as mean values ± standard... 99

Figure 3-18. Live/dead fluorescent images of MC3T3-E1 cells cultures on bare glass, TC-plated Col-PDA fibrils for 24 and 48 h. Calcein shows green fluorescence in live cells, while EthD-1 shows red... 100