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Title Page
ABSTRACT
국문 초록
Contents
Chapter 1. Introduction 26
1.1. Biomaterials in hard tissue engineering 26
1.2. Bioink 27
1.2.1. Hydrogel based bioink 32
1.3. Research objective 35
1.4. Scope of study 38
Chapter 2. Evaluation of enhancement of biofunctionality by multi-component bioink 40
2.1. Introduction 40
2.2. Material and Method 45
2.2.1. Materials 45
2.2.2. Synthesis of alginate-2-aminoehtyl methacrylate and fluoresce dye to gelatin 45
2.2.3. Characterization of modified alginate 46
2.2.4. Cell culture 46
2.2.5. MA-alginate/gelatin blending and fabrication 47
2.2.6. Characterization of hydrogel 48
2.2.7. Gelatin releasing and remaining test 49
2.2.8. Cell viability 50
2.2.9. Cellular activity 50
2.2.10. Osteogenic marker gene expression 51
2.2.11. Fabrication of scaffold by extrusion-based 3D printer 52
2.2.12. Alizarin Red S staining 52
2.2.13. Statistical analysis 53
2.3. Results 55
2.3.1. MA-alginate synthesized 55
2.3.2. Gelatin releasing profile 57
2.3.3. Characterization of fabricated MA-alginate/gelatin hydrogel after gelatin releasing 59
2.3.4. Effect of release gelatin to external cells 64
2.3.5. Evaluation of remaining gelatin from the hydrogel during releasing 70
2.3.6. Effect of remained gelatin to encapsulated cells 70
2.3.7. Printability test for usage as bioink 80
2.3.8. Evaluation of cell activities of printed scaffold 84
2.4. Discussion 88
2.5. Conclusion 93
Chapter 3. Effects of calcium phosphate phase transition in hydrogel-based bioink 94
3.1. Introduction 94
3.2. Materials and methods 99
3.2.1. Materials 99
3.2.2. Cell culture 100
3.2.3. α-TCP/GelMA preparation and fabrication 100
3.2.4. Characterization of the α-TCP/GelMA hydrogel 101
3.2.5. Evaluation of the effects of loaded α-TCP on cellular activity of encapsulated cells 103
3.2.6. Evaluation of osteogenic marker gene expression 103
3.2.7. Fabrication of scaffolds 104
3.2.8. Cell viability 105
3.2.9. Alizarin Red S staining 105
3.2.10. Statistical analysis 105
3.3. Results 108
3.3.1. Evaluation of possibility of α-TCP calcium phosphate phase transition in hydrogel 108
3.3.2. Characterization of hydrogel after phase transition 112
3.3.3. Evaluation of cell activities of encapsulated cells effected by loaded α-TCP and the phase transition 115
3.3.4. Optimization of printing conditions and fabrication of α-TCP/GelMA scaffolds 123
3.3.5. Evaluation of fabricated cell-laden scaffolds after 3 weeks cultivation 129
3.4. Discussion 135
3.5. Conclusion 144
Chapter 4. Synergistic effects of introduced bioactive components in hydrogel 146
4.1. Introduction 146
4.2. Materials and methods 151
4.2.1. Materials 151
4.2.2. Cell culture 151
4.2.3. Modification of sodium alginate with 2-aminoethyl methacrylate and peptides 151
4.2.4. Characterization of synthesized MA-alginate-RGD 152
4.2.5. Preparation of mixture solution and fabrication of a-TCP/MAR 153
4.2.6. Characterization of fabricated hydrogel disc 154
4.2.7. Cell adhesion test 155
4.2.8. Evaluation of cell activities including encapsulated and attached on the hydrogel 156
4.2.9. Quantitative real-time polymerase chain reaction 157
4.2.10. Alizarin Red S staining 157
4.2.11. Statistical analysis 158
4.3. Results 160
4.3.1. Characterization of hydrogels incorporating peptide and a-TCP 160
4.3.2. Evaluation of cell interaction with modified alginate with a-TCP applying in 2D cultures 167
4.3.3. Effect of incorporated peptide and CaP in 2D cultures in osteogenesis 175
4.3.4. Effect of incorporated peptide and CaP in 3D cultures 179
4.3.5. Osteogenic differentiation behavior of encapsulated cells 183
4.4. Discussion 191
4.5. Conclusion 195
Chapter 5. Effects of multi-functional hybrid bioink for osteochondral tissue regeneration 197
5.1. Introduction 197
5.2. Materials and methods 203
5.2.1. Materials 203
5.2.2. Modification of methacrylated alginate with peptides 203
5.2.3. Characterization of peptide modified methacrylated alginate 204
5.2.4. Cell culture 204
5.2.5. Fabrication of MANC hydrogel disc for pre-evaluation 205
5.2.6. Preparation of mixture solution 206
5.2.7. Fabrication of integrated structure and culturing circumstance 206
5.2.8. Characterization of fabricated integrate structures 207
5.2.9. Evaluation of chondrogenic differentiation behavior 208
5.2.10. Evaluation of osteogenic differentiation behavior 208
5.2.11. Evaluation of marker gene expression of each part of integrated structure 209
5.2.12. Histological analysis 210
5.2.13. Statistical analysis 210
5.3. Results 212
5.3.1. Characterization of peptides introduced methacrylate alginate 212
5.3.2. Evaluation of introduced N-Cadherin peptide to encapsulated cells 214
5.3.3. Fabrication of integrated structure 220
5.3.4. Evaluation of cell activities in integrated structures 222
5.3.5. Evaluation of cell differentiation om integrated structures 228
5.4. Discussion 234
5.5. Conclusion 244
Chapter 6. Conclusion 245
References 248
Figure 1.1. Schematic image of distinguished bioink by presence of cells. 30
Figure 1.2. Schematic image of research overview flow of the thesis. 37
Figure 2.1. Schematic image of multi-component bioink under blending uncrosslinked gelatin. 44
Figure 2.2. ¹H-NMR spectra of alginate and methacrylate modified alginate. 56
Figure 2.3. Releasing profile of blended MA-alginate/gelatin hydrogel. 58
Figure 2.4. Water contents of hydrogels according to different volume ratio of MA-alginate at (a) 0 h and (b) 3 weeks. * p 〈 0.05, n.s. ＝ not significant. 62
Figure 2.5. Compressive moduli during gelatin releasing of each hydrogel for 3 weeks of releasing, (a) 10:0 MA-alginate/gelatin, (b) 9:1 MA-alginate/gelatin, (c) 7:3... 63
Figure 2.6. Cell activity of external cell affected by gelatin released from hydrogel; (a) DNA quantification (b) ALP quantification. 66
Figure 2.7. qRT-PCR results for genes monitored in each group (n＝3), *p 〈0.05 n.s.＝not significant; (a) RUNX2 (b) COL-1 (c) ALP (d) OCN (e) OPN. 69
Figure 2.8. Remained gelatin in MA-alginate hydrogel while releasing. 72
Figure 2.9. Cell viability of encapsulated cells in hydrogels by live/dead image of different volume ratio of MA-alginate/gelatin hydrogels; (a) 10:0 (b) 9:1 (c) 7:3 (d) 5:5. 75
Figure 2.10. Cell activity of encapsulated cell affected by remained gelatin in hydrogel; (a) DNA quantification (b) ALP quantification. 78
Figure 2.11. In vitro osteogenic differentiation analysis by (a-e) qRT-PCR results for genes monitored in each group(n＝3), *p 〈0.05 n.s.＝not significant; (a) RUNX2 (b) COL-1 (c) ALP (d) OPN (e) OCN. 79
Figure 2.12. Printing test of alginate dominant hydrogels (a) 10:0 MA-alginate/gelatin (b) 9:1 MA-alginate/gelatin. 81
Figure 2.13. Printability test by (a) pressure/nozzle and (b) strut width. 83
Figure 2.14. Live/dead assay of fabricated scaffold of (a) 7:3 MA-alginate/gelatin and (b) 5:5 MA-alginate/gelatin. 86
Figure 2.15. Cellular activities through live/dead assay of each scaffold which cultured for 3 weeks, (a) 7:3 MA-alginate/gelatin (b) 5:5 MA-alginate/gelatin. 87
Figure 2.16. In vitro osteogenic cellular activities through Alizarin Res S staining of (a) 7:3 MA-alginate/gelatin (b) 5:5 MA-alginate/gelatin. 90
Figure 3.1. Schematic image of the applied α-TCP phase transition GelMA based bioink. 98
Figure 3.2. Process of calcium phosphate phase transition of α-TCP to CDHA, according to time points (a) 0 h (b) 12 h (c) 24 h and (d) 36 h. 110
Figure 3.3. The formation of CDHA during the calcium phosphate phase transition process from α-TCP, according to time points (a) 0 h (b) 12 h (c) 24 h and (d) 36 h 111
Figure 3.4. The compressive modulus of each group of α-TCP/GelMA hydrogel containing 0, 0.125, 0.25, 0.5 wt% α-TCP (a) entire process of calcium phosphate phase transition (0-36 h), (b) before the calcium phosphate phase transition (0 h)... 113
Figure 3.5. Water content of each hydrogel samples according to the amount of α-TCP at (a) 0 h and (b) 36 h (* p 〈0.05; n.s.＝not significant) 114
Figure 3.6. Cell viability of encapsulated cells in each hydrogel by (a) CCK-8 analysis during calcium phosphate phase transition and (b) DNA quantification for... 118
Figure 3.7. Cell activities of the encapsulated cells in each hydrogel groups (a) ALP analysis and (b) calcium quantification for entire cultivation 119
Figure 3.8. In vitro osteogenic differentiation of the encapsulated cells by qPCR results for genes monitored after 3 weeks of culture (n＝3). * p 〈0.05; n.s.＝not... 122
Figure 3.9. Printability test. The following results are shown: (a) pressure/nozzle, (b) strut width per pressure, (c) feed rate/nozzle, (d) strut width per feed rate. 125
Figure 3.10. Live/dead cell assay of cells in the fabricated scaffold at 0 h for (a) 0 wt% α-TCP/GelMA and (b) 0.25 wt% α-TCP/GelMA. 127
Figure 3.11. Live/dead cell assay results for the fabricated scaffolds after 3 weeks of culturing; (a) 0 wt% α-TCP/GelMA and (b) 0.25 wt% α-TCP/GelMA. 128
Figure 3.12. In vitro osteogenic cellular activities were assessed using Alizarin Red S staining; without cells (a) 0 wt% α-TCP/GelMA and (b) 0.25 wt% α-TCP/GelMA... 131
Figure 3.13. A comparison of the compressive modulus values of the fabricated scaffold after the 3 weeks of culture period. (n.s.＝not significant). 134
Figure 3.14. Optical image of 3 weeks of cultured scaffolds; (a) 0 wt% α-TCP (b) 0.25 wt% α-TCP loaded. 136
Figure 4.1. Schematic image of the hydrogel applied RGD peptide and calcium phosphate phase transition in both 2D and 3D culture. 150
Figure 4.2. ¹H-NMR spectra of alginate, methacrylate modified alginate (MA-alginate) and RGD peptide introduced MA-alginate. 162
Figure 4.3. Characterization ofRGD peptide introduced MA-alginate-by Amino acid analysis. 163
Figure 4.4. Process of calcium phosphate phase transition of α-TCP to CDHA, according to time points (a) 0 h (b) 36 h. 165
Figure 4.5. The compressive modulus of each hydrogels containing RGD peptide and 0.25 wt% α-TCP for entire process of calcium phosphate phase transition (0-36 h) 166
Figure 4.6. Presence of α-TCP on the surface of hydrogel observed in (a) FE-SEM and (b) EDS mapping. The yellow arrows indicated the α-TCP particle which appeared on the surface of hydrogel. 169
Figure 4.7. Cell adhesion ability of each composition of hydrogel on the surface of hydrogel by FE-SEM image; (a) MA-alginate (b) MA-alginate-RGD (c) α-TCP/MA-... 170
Figure 4.8. Cell adhesion ability of each composition of hydrogel on the surface of hydrogel by F-actin staining; (a) MA-alginate (b) MA-alginate-RGD (c) α-TCP/MA-... 173
Figure 4.9. Cell adhesion ability analysis by DNA quantification after 5 h of culturing. (* p 〈0.05; n.s.＝not significant). 174
Figure 4.10. Cell proliferation on the hydrogel surface by (a) CCK-8 assay and (b) DNA quantification. 177
Figure 4.11. Cell activity on the hydrogel surface (a) ALP analysis and (b-d) qPCR results for gene monitored in each group; (b) RUNX2 (c) COL-1 (d) ALP, * p 〈0.05;... 178
Figure 4.12. Cell viability of cell encapsulated hydrogel disc Live/dead image of (a) 0 h (b) 36 h. 181
Figure 4.13. Cell activities of the encapsulated cells in each hydrogel groups by DNA quantification for entire cultivation. 182
Figure 4.14. Cell activities of the encapsulated cells in each hydrogel groups; (a) ALP analysis and (b) calcium quantification for 3 weeks of cultivation. 186
Figure 4.15. qPCR results for gene monitored in each group; (a) RUNX2 (b) COL-1 (c) ALP (d) OCN (e) OPN, * p〈0.05; n.s.＝not significant. 187
Figure 4.16. In vitro osteogenic cellular activities were assessed using Alizarin Red S staining (a) MA-alginate, (b) MA-alginate-RGD, (c) 0.25 wt% α-TCP loaded MA-... 190
Figure 5.1. Schematic image of fabrication of integrated osteochondral structure using multi-functional bioinks. 202
Figure 5.2. Characterization of RGD peptide and N-Cadherin modified MA-alginate by Amino acid analysis. 213
Figure 5.3. Cell activities of the encapsulated cells in each hydrogel groups for 3 weeks; (a) DNA quantification and (b) GAGs contents, * p 〈0.05; n.s.＝not significant. 216
Figure 5.4. In vitro chondrogenic cellular activities at 3-week, were assessed using (a) DMMB staining and (b) Safranin-O staining. 218
Figure 5.5. qPCR results for gene monitored in each group; (a) ACAN (b) SOX-9 (c) COL-2 (d) TGF-beta (e) COL-10 (f) PTHrP, * p 〈0.05; n.s.＝not significant. 219
Figure 5.6. Optical image of fabricated integrated structure; (a) right after fabrication and (b) after 6 weeks of incubation in cell culture media. 221
Figure 5.7. Compressive modulus of fabricated scaffold in process of calcium phosphate phase transition. 224
Figure 5.8. DNA quantification assay of the encapsulated cells in each hydrogel groups at 6 weeks, * p 〈0.05; n.s.＝not significant. 226
Figure 5.9. GAGs quantification assay of the encapsulated cells in each hydrogel groups at 6-week, * p 〈0.05; n.s.＝not significant. 227
Figure 5.10. Cell activities of the encapsulated cells in each hydrogel groups at 6-week; (a) ALP analysis and (b) calcium quantification 229
Figure 5.11. qPCR results of the top layers (cartilage targeted) for gene monitored in each group at 6-week; (a) COL II (b) COL X 231
Figure 5.12. qPCR results of the top layers (cartilage targeted) for gene monitored in each group at 6-week; (a) RUNX2 (b) ALP (c) COLI 235
Figure 5.13. qPCR results of the bottom layers (subchondral bone targeted) for gene monitored in each group at 6-week; (a) COL II (b) COL X 237
Figure 5.14. qPCR results of the bottom layers (subchondral bone targeted) for gene monitored in each group at 6-week; (a) RUNX2 (b) ALP (c) COL I (d) OPN (e) OCN. 239
Figure 5.15. In vitro osteogenic cellular activities through Alcian blue staining of (a) plain/plain (b) plain/MAR (C) MANC/plain (d) MANC/MAR (e)... 241
Figure 5.16. In vitro chondrogenic cellular activities through Alizarin Red S staining of (a) plain/plain (b) plain/MAR (c) MANC/plain (d) MANC/MAR (e)... 243
초록보기
본 연구는 하이드로겔 기반 바이오잉크의 기능성을 향상시켜 실제 조직과 유사한 생화학적, 생물리학적 특성과 높은 인쇄성을 가지는 이상적인 표적 조직 특이적인 바이오잉크의 개발을 목표로 한다.
뼈 조직, 특히 연골하골 조직 표적 바이오잉크의 개발 시 생체 기능성과 기계적 물성의 향상은 필수적이다. 이에 RGD 서열을 가진 비가교 젤라틴이 세포에 미치는 영향을 우선 평가 후, 세포 부착 및 증식을 개선 가능한 RGD 펩타이드를 하이드로겔에 도입하여 생체 기능성을 향상시키고자 하였다. 결과적으로, 도입된 RGD 펩타이드는 기 평가된 비가교 젤라틴 혼합 다성분 바이오잉크와 동일하게 세포 증식 및 골 형성 분화에 긍정적인 영향을 확인하였다. 또한, 기질 고분자에 알파-인산 칼슘 (α-tricalcium phosphate, α-TCP)을 첨가하여 인산 칼슘 상전이를 통한 기계적 물성을 향상시키고자 하였다. α-TCP의 인산 칼슘 상전이 반응의 결과로 칼슘 결핍 수산화인회석(calcium deficient hydroxyapatite, CDHA)이 형성되며, 이러한 반응 중 이온 교환과 결정 구조의 변화로 인해 기계적 강도와 골 형성 잠재력이 모두 크게 향상되는 결과를 확인하였다. 또한 단순 α-TCP의 적재를 통해서도 인쇄성, 구조적 안정성이 향상됨을 확인하였다.
연골조직 특이적 하이드로겔 기반 바이오잉크의 경우, 연골형성 표현형을 장기간 유지하기 위해 기질 고분자에 N-cadherin 펩타이드를 도입하였다. 도입된 N-cadherin 펩타이드는 세포 내에 존재하는 N-cadherin을 모방하여 세포와 세포 간의 상호작용을 증진시켜 연골 분화를 유도할 수 있다. 결과적으로, 펩타이드 도입으로 인하여 높은 연골 분화능을 확인하였다.
이어서, 다기능성 하이드로겔 기반 바이오잉크 개발의 다음 단계로 디지털 광 처리 (DLP) 시스템을 이용하여 복합 구조체 제작하고자 하였다. 개발 한 표적 조직 특이적 유도성 하이드로겔 기반 바이오잉크를 프린팅 시스템에 적용하여 일체형 구조의 골연골 구조체를 제작하였다. 제작 후 각 표적 조직 별 분화능을 평가한 결과, 바이오잉크의 각 부분이 골연골 조직에서 각 표적 조직에 적합한 기능 향상 가능성을 입증하였다.
결론적으로 본 연구를 통해 개발된 다기능성 표적 조직 유도 가능 하이드로겔 기반 바이오잉크의 잠재력을 입증하였으며, 이를 적용하여 환자 맞춤형 이식재 개발에 응용될 수 있을 것으로 예상된다.MARC 보기
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