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Tissue regeneration study using three-dimensional scaffold fabricated by 3D printing technology

Tissue regeneration study using three-dimensional scaffold fabricated by 3D printing technology

초록/요약

Tissue engineering has significant potential to restore the damaged bone. Bone tissue engineering, including the scaffold, cells, and growth factors as a component, can overcome the drawbacks of conventional treatment methods which comes from lowering of curative ability and pathogenic infections. Manufacturing of scaffolds using 3D bioprinting for bone tissue engineering could be a useful technology in the treatment of bone defects and a variety of studies have been conducted. Biomaterials used in 3D bioprinting must have a biocompatibility and suitable biodegradability to apply in bone tissue engineering. The 3D bioprinted scaffold must have a suitable in vivo degradation period as well as provide a good environment for cells attachment and growth. Additionally, it must fully degrade in human body with inducing osteogenesis. In this study, biodegradable polyesters (bio-inks) having various degradation periods were synthesized to apply in 3D bioprinting. Also, the scaffolds for bone tissue engineering were fabricated by using 3D bioprinting. In addition, the 3D bioprinted scaffolds with the cells and bone growth factors were implanted in bone defect site for evaluation of new bone formation. In chapter 1, the background about tissue engineering, 3D bioprinting technology, and the application of 3D bioprinted scaffolds has been described. Chapter 2 demonstrates the preparation and feasibility evaluation of three-dimensional scaffolds by using 3D bioprinting. To apply the material for 3D bioprinting, various bio-inks were designed and synthesized by ring-opening polymerization. The bio-inks composed of ε-caprolactone (CL), L-lactide (LA), and glycolide (GA) were prepared with a variety of molecular weight and monomer ratio. The obtained bio-inks were applied in 3D bioprinting to evaluate the printability as the materials for 3D bioprinting. It was confirmed that the 3D bioprinted scaffolds have a well-defined and porous structure. In addition, the degradation rate was controlled depending on the kind of bio-inks showing excellent biocompatibility. In Chapter 3, poly (PLLA-co-PGA-co-PCL) (PLGC) scaffold fabricated by 3D bioprinting and human dental pulp stem cells (hDPSCs) were used to generate neo-bone formation in bone defect. The hDPSCs showed high proliferation rate and osteogenic differentiation in presence of osteogenic factors with bone morphogenetic protein-2 (rhBMP-2). The PLGC scaffold was implanted into cranial bone defect with hDPSCs and rhBMP-2. Neo-bone formation was confirmed by micro-computed tomography (CT) and histology. Also, the PLGC scaffold was degraded gradually and it showed good correlation between scaffold degradation and new bone formation. In conclusion, the results of this study show an ideal platform of bone regeneration using the scaffold fabricated by 3D bioprinting for treatment of bone defects.

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목차

CHAPTER 1. General introduction 1
1.1. Tissue engineering 2
1.2. 3D bioprinting technology 5
1.3. Bone defect 7
1.4. Bone tissue engineering 8
1.5. Strategy of this works 10
1.6. References 11
CHAPTER 2. Synthesis and feasibility evaluation of bio-inks for 3D bioprinting 13
2.1. Introduction 14
2.2. Experimental section 18
2.2.1. Materials 18
2.2.2. Characterization 18
2.2.3. Synthesis of bio-inks 19
2.2.4. Fabrication of the 3D scaffold 20
2.2.5. Characterization of fabricated scaffolds 21
2.2.6. Animal implantation surgery 21
2.2.7. In vivo fluorescence imaging 22
2.2.8. In vivo scanning electron microscope (SEM) measurements 23
2.2.9. Histological analysis 23
2.3. Results 24
2.3.1. Preparation of bio-inks 24
2.3.2. Printability test of bio-inks 27
2.3.3. Fabrication of the 3D scaffold 31
2.3.4. In vivo implantation 31
2.3.5. The removed in vivo scaffolds 32
2.3.6. In vivo degradation 33
2.3.7. SEM morphology of the in vivo scaffolds 35
2.3.8. In vivo fluorescence imaging 37
2.3.9. In vivo immunogenicity 39
2.4. Discussion 42
2.5. Conclusion 46
2.6. References 47
CHAPTER 3. Bone regeneration study using 3D printed PLGC scaffolds with human dental pulp stem cells and rhBMP-2 50
3.1. Introduction 51
3.2. Experimental section 56
3.2.1. Culture and characterization of hDPSCs 56
3.2.2. In vitro osteogenic differentiation of hDPSCs 57
3.2.3. Determination of in vitro ALP contents of differentiated hDPSCs and hBMSCs 58
3.2.4. Characterization 59
3.2.5. Synthesis of PLGC copolymers 59
3.2.6. Synthesis of PLGC copolymers with fluorescein isothiocyanate (FITC) (PLGC-FITC) 60
3.2.7. Fabrication of the circular PLGC scaffold 61
3.2.8. Cell attachment and proliferation assays on fabricated PLGC scaffold 62
3.2.9. Animal implantation surgery 63
3.2.10. In vivo fluorescence imaging 65
3.2.11. Micro-CT analysis 65
3.2.12. Histological analysis 66
3.2.13. Statistical analysis 68
3.3. Results 70
3.3.1. Characterization of hDPSCs 70
3.3.2. In vitro osteogenic differentiation of hDPSCs 72
3.3.3. Fabrication of circular PLGC scaffold 74
3.3.4. In vitro cell attachment and proliferation on fabricated PLGC scaffold 76
3.3.5. In vivo implantation 78
3.3.6. In vivo degradation of PLGC scaffolds 78
3.3.7. Confirmation of bone formation via micro-CT 82
3.3.8. In vivo fluorescence imaging 83
3.3.9. Confirmation of bone formation via histology of in vivo tissue-engineered bone 84
3.4. Discussion 90
3.5. Conclusion 95
3.6. References 96
LIST OF PUBLICATIONS 101
LIST OF PRESENTATIONS 103
LIST OF PATENTS 104

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