Nanostructured hydroxyapatite, chitosan and carbon nanotube based composite biomaterials for bone tissue engineering
- Alternative Title
- 수산화인회석, 키토산, 탄소나노튜브의 나노구조 복합 생체물질 제조 및 이들 물질의 골조직 공학적 연구
- Abstract
- Over the last four decades, there is a growing interest in the field of artificial organ material isolation, preparation, transplantation, surgical reconstruction and the use of artificial prostheses to treat the loss or failure of an organ or tissue. In this regard, Hydroxyapatite (HAp) was isolated from Thunnus obesus bone with different methodologies such as thermal calcination, alkaline hydrolysis and polymer assisted thermal calcination. The obtained ceramic has been characterized by TGA, FT-IR, XRD, FE-SEM, EDX, TEM with SAED, cytotoxicity and cell proliferation analysis. The FT-IR and TGA results revealed the presence of inorganic and organic matrices in raw bone and the preserved carbonated group in derived HAp. FE-SEM results revealed the formation of nanostructured HAp (80-300 nm) at 600 ？C and crystal agglomeration was observed with increase in temperature. TEM and SAED images have signified that the thermal calcination method produces good crystallinity with dimension 0.3-1.0 ？m, whereas alkaline hydrolysis method produces nanostructured HAp crystals with length (17-71 nm) and width (5-10 nm). XRD results of derived HAps were in coherence with JCPDS data. The crystallinity of HAp isolated in the presence of polymer was lower than that obtained in the absence of polymers. Biocompatibility of HAp crystals was evaluated by cytotoxic analysis and cell proliferation of human osteoblast cell (MG-63 and MC3T3-E1).
Phosphorylated chitooligosaccharides (P-COS) were prepared using a H3PO4, P2O5, Et3PO4 and hexanol solvent system and characterized. No cytotoxicity and increased ALP activity was observed. Moreover, certain complexes of different molecular weight polysaccharide (glucosamine) with SWCNT were prepared named as SWCNT-Glucosamine, SWCNT-COS (below 1KDa), SWCNT-COS (1-3 KDa), SWCNT-Chitosan (310 KDa), SWCNT-Chitosan (510 KDa) and subjected to bone tissue engineering application in vitro. No cytotoxic effect, higher alkaline phosphatase activity and enhanced mineralization have been observed in SWCNT-glucosamine complex compared to glucosamine alone.
Further, CNT and chitosan hold great interest in recent days with respect to biomaterials, particularly those to be positioned in contact with bone such as prostheses for arthroplasty, plates or screws for fracture fixation, drug delivery systems, and scaffolding for bone regeneration. For this, low and high molecular weight chitosan with 0.25%, 0.5% and 1.0% weight of f-multiwalled carbon nanotubes (f-MWCNT) were prepared as a scaffold by freeze drying and lyophilization method and physiochemically characterized as bone graft substitutes. As compared to chitosan scaffold, greater cell proliferation, high protein content, increased alkaline phosphatase activity and mineralization were observed in the composite scaffolds due to the addition of f-MWCNT. After exploring the properties of chitosan/f-MWCNT composite scaffolds we are curious to investigate the chemical and biological properties of tri-component systems for bone tissue engineering applications. In the later chapters of the dissertation, artificial bone materials has been developed with chitosan, natural HAp and CNT. Moreover, other natural raw materials such as marine sponge collagen, gelatin, chondroitin sulfate and amylopectin have also been explored for the preparation of tricomponent scaffolds. Uniform dispersion of HAp in chitosan matrix with interconnected porosity of 70-200 ？m was observed. Cell proliferation in composite scaffolds was twice than in pure chitosan when checked in vitro using MG-63 cell line. These observations suggest that the novel composite scaffolds are promising biomaterials for matrix-based bone repair and bone augmentation. We conclude that he proper implementation of present work at clinical level would provide pave way for enhancement of bone tissue engineering applications.
- Issued Date
- 2011
- Awarded Date
- 2011. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Department
- 대학원 화학과
- Advisor
- Se-Kwon Kim
- Table Of Contents
- Table of Contents
Abstract i
Table of Contents iii
List of Figures xi
List of Tables xvii
List of Abbreviations xviii
Chapter 1. An ode to tissue engineering 1
1.1. Introduction 1
1.2. Stem cell therapy 1
1.3. Scaffolds in tissue engineering 3
1.4. Bone tissue engineering 4
1.5. Biopolymers and bioceramics in bone tissue engineering 5
1.6. Scope of the thesis 8
1.7. Plan of the proposed work 9
Chapter 2. Effect of temperature on isolation and characterization of hydroxyapatite from Tuna (Thunnus obesus) bone 10
2.1. Introduction 11
2.2. Materials and methods 12
2.2.1. Sample preparation 12
2.2.2. General characterization 13
2.2.2.1. Thermogravimetric analysis 13
2.2.2.2. Fourier transform infrared spectroscopy 13
2.2.2.3. X-Ray diffraction analysis 13
2.2.2.4. Field emission - scanning electron microscopy 14
2.2.2.5. Optical microscopy analysis 14
2.2.2.6. Cell viability assay 14
2.3. Results and discussion 14
2.3.1. Thermal analysis of Thunnus obesus bone 15
2.3.2. Fourier transform infrared spectroscopic analysis 16
2.3.3. X-Ray diffraction analysis of Thunnus obesus bone 18
2.3.4. Field emission- scanning electron microscopic analysis 21
2.3.5. Electron dispersive X-ray analysis 22
2.3.6. Cytotoxicity assay 23
Conclusion 24
Chapter 3. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone 26
3.1. Introduction 27
3.2. Materials and methods 30
3.2.1. Fish bone preparation 30
3.2.2. Alkaline hydrolysis method 30
3.2.3. Thermal calcination method 30
3.2.4. Characterization techniques 30
3.2.5. In vitro cell proliferation assay 31
3.2.6. Morphological studies with optical microscopy and Hoechst stain 33342 31
3.3. Results and discussion 32
3.3.1. General examination of raw bone and derived HAp 33
3.3.2. Thermal stability of isolated HAp 34
3.3.3. Stretching frequency of isolated HAp 35
3.3.4. Phase and crystallinity of the isolated HAp 38
3.3.5. Morphological study of derived HAp by SEM 41
3.3.6. Transmission electron microscopy and selected area diffraction analysis 43
3.3.7. Energy dispersive X-ray analysis 44
3.3.8. Cytotoxicity and cell proliferation of macro and nanocrystals of HAp 45
3.3.9. Morphological studies with optical microscopy and Hoechst stain 33342 46
Conclusion 48
Chapter 4. Polymer assisted isolation of hydroxyapatite from Thunnus obesus bone 49
4.1. Introduction 50
4.2. Materials and methods 51
4.2.1. Materials 51
4.2.2. Thermal calcination method 51
4.2.3. Polymer assisted isolation of HAp 52
4.2.4. Characterization techniques 52
4.2.5. In vitro cytotoxicity and cell proliferation assay 52
4.2.6. Morphological studies with optical microscopy and Hoechst staining 33342 53
4.3. Results and discussion 53
4.3.1. Thermo gravimetric analysis of derived HAp 53
4.3.2. Stretching frequency of derived HAp 54
4.3.3. X-ray diffraction analysis 56
4.3.4. Field emission Scanning electron microscopic analysis 57
4.3.5. Cytotoxicity of derived HAp 58
4.3.6. Morphological studies with optical microscopy and Hoechst 33342staining 59
Conclusion 61
Chapter 5. Biocompatibility and alkaline phosphatase activity of phosphorylated chitooligosaccharides on osteosarcoma MG-63 cell line 62
5.1. Introduction 63
5.1.1. Preparation of chitosan by chemical methods 63
5.1.2. Enzymatic hydrolysis method 64
5.1.3. Preparation of chitooligosaccharide by UF membrane system 65
5.1.4. The manufacture of chitosan and its oligomers 67
5.1.5. Properties and application of chitosan 68
5.2. Experimental Section 71
5.2.1. Materials 71
5.2.2. Synthesis of P-COS 71
5.2.3. Characterization 72
5.3. Results and Discussion 73
5.3.1. General description 73
5.3.2. Solubility of P-COS 73
5.3.3. Stretching frequency P-COS 74
5.3.4. Thermal stability of P-COS 75
5.3.5. 13C NMR spectra of P-COS 76
5.3.6. 31P NMR spectra of P-chitooligosaccharide 77
5.3.7. Biocompatibility study of P-COS on osteoblast like MG-63 cell line 78
5.3.8. ALP activity of P-COS 78
Conclusion 79
Chapter 6. Stimulation of minerals by carbon nanotube grafted glucosamine in mouse mesenchymal stem cells for bone tissue engineering 80
6.1. Introduction 81
6.2. Materials and method 84
6.2.1. Functionalisation of SWCNT 84
6.2.2. Acylation of CNT with thionyl chloride (SOCl2) 85
6.2.3. Amidation with glucosamine, COS and chitosan 85
6.2.4. FTIR spectrometry 85
6.2.5. Cell culture 85
6.2.6. Alkaline phosphatase activity 86
6.2.7. Mineralization Assay 86
6.3. Results and discussions 86
6.3.1. General observation 86
6.3.2. FT-IR spectroscopy 87
6.3.3. Solubility studies 88
6.3.4. Morphology studies of SWCNTs derivatives 89
6.3.5. Effects of glucosamine and SWCNT-Glucosamine on mesenchymal stem cell 91
6.3.6. Alkaline phosphatase activity 92
6.3.7. Determination of mineralization through optical microscopy 93
Conclusion 95
Chapter 7. Biomimetic and biocompatible chitosan-carbon nanotube composite scaffolds for bone tissue engineering 96
7.1. Introduction to carbon nanotubes for bone tissue engineering 97
7.2. Materials and methods 100
7.2.1. Functionalization and purification of carbon nanotubes (CNTs) 100
7.2.2. Preparation of chitosan and chitosan ？ MWCNT scaffolds 100
7.2.3. General characterization 101
7.2.4. Porosity measurement 101
7.2.5. Water uptake and retention abilities 102
7.2.6. Cell culture 102
7.2.7. In vitro cytotoxicity and cell proliferation assay 103
7.2.8. Hoechst 33342 staining 103
7.2.9. Total protein measurement 104
7.2.10. Alkaline phosphatase activity 104
7.3. Results and discussion 104
7.3.1. Material selection and scaffold preparation 104
7.3.2. Gross Examination of the scaffolds 105
7.3.3. Porosity 106
7.3.4. Water uptake and retention abilities 107
7.3.5. Thermal stability of the scaffolds 108
7.3.6. Stretching frequency of the scaffolds 110
7.3.7. X-ray diffraction studies 112
7.3.8. Morphology studies of the scaffolds 113
7.3.9. Optical microcopy analysis 114
7.3.10. Hoechst 33342 staining of MG-63 cells 114
7.3.11. In vitro cytotoxicity and cell proliferation 116
7.3.12. Protein estimation 118
7.3.13. Alkaline phosphatase assay 119
7.3.14. Mineralization Assay 120
7.3.15. General discussion 122
Conclusion 124
Chapter 8. Preparation and characterization of carbon nanotube-grafted-chitosan - natural hydroxyapatite composite for bone tissue engineering 125
8.1. Introduction 126
8.2. Materials and methods 128
8.2.1. Isolation of HAp from Thunnus obesus 128
8.2.2. Functionalization and purification of carbon nanotubes (CNTs) 128
8.3. Preparation of scaffold materials 128
8.3.1. Chitosan scaffold preparation 128
8.3.2. Chitosan/hydroxyapatite (Chitosan/HAp) scaffold 128
8.3.3. f-MWCNT-g-chitosan/HAp scaffold 129
8.3.4. General characterization techniques 130
8.4. Results and discussion 130
8.4. 1. Material selection and scaffold preparation 130
8.4. 2. Gross Examination of the scaffolds 132
8.4. 3. Porosity measurement 133
8.4. 4. Water uptake and retention abilities 134
8.4. 5. In vitro degradation with PBS 135
8.4. 6. Thermal stability of the scaffolds 136
8.4. 7. Stretching frequency of the scaffolds 137
8.4. 8. X-ray diffraction studies 140
8.4. 9. Compressive energy 141
8.4. 10. Morphology studies of the scaffolds 142
8.4. 11. Optical microcopy analysis 143
8.4. 12. In vitro cytotoxicity and cell proliferation 144
Conclusion 146
Chapter 9. Biophysicochemical evaluation of chitosan-natural hydroxyapatite based tricomponent systems for bone tissue engineering 147
9.1. Introduction 148
9.2. Materials and methods 152
9.2.1. Isolation of HAp 152
9.2.2. Isolation of marine sponge collagens (MSCol) 152
9.2.3. Preparation of scaffolds 153
9.2.3.1. Chitosan scaffold 153
9.2.3.2. Chitosan-HAp (chitosan/HAp) scaffold 153
9.2.3.3. Chitosan-HAp-Marine Sponge Collagen (chitosan/HAp-MSCol) scaffold 153
9.2.3.4. Chitosan-HAp-Gelatin (chitosan/HAp-Gel) scaffold 154
9.2.3.5. Chitosan-HAp-Chondroitin sulfate (chitosan/HAp-Chon) scaffold 154
9.2.3.6. Chitosan-HAp-Amylopectin (chitosan/HAp-Amy) scaffold 154
9.2.4. Characterization techniques 154
9.3. Results and discussion 154
9.3.1. Gross examination of the scaffolds 156
9.3.2. Porosity measurement 156
9.3.4. FT-IR spectrometry 159
9.3.5. X-ray diffraction analysis 162
9.3.6. Thermogravimetric analysis 165
9.3.7. Scanning electron microscopy analysis 168
9.3.8. Optical microscopy evaluation 170
9.3.9. In vitro cell proliferation 172
Conclusion 173
Chapter 10. Cumulative Summary and conclusion 174
10.1. Novelty of the present scientific piece of work. 181
10.2. Publications from the present work 182
10.3. Challenges and future directions for bone biomaterials. 183
Bibliography 184
Acknowledgements 204
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- Doctor
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