Title page
ACKNOWLEDGEMENT
Contents
ABBREVIATION 11
Summary in Korean 12
General background 13
1. Embryonic stem cell 13
What is a stem cell? 13
Deriving Embryonic Stem cells 13
Characteristics of mESC 14
Applications of stem cell for medical treatment 15
2. Biomaterials 15
What is a biomaterials? 15
Thermo-sensitive Hydrogel 16
3. Cartilage 17
Cartilage 17
The Cartilage Matrix 18
Hyaline cartilage 19
Elastic cartilage 19
Fibrous cartilage 19
Ⅰ. Introduction 21
Ⅱ. Materials and Method 23
Ⅱ.1. Mouse embryonic stem cell culture : Cell culture 23
Ⅱ.2. Preparation of poly(N-isopropypacrylamide-coacrylic acid) [poly(NiPAAm-co AAc)] co-polymer 23
Ⅱ.3. mES cell encapsulation 24
Ⅱ.4. Biochemical assays for chondrocyte proliferation and GAG production 24
Ⅱ.5. RNA extraction and RT-PCR 25
Ⅱ.6. Immunohistochemistry of implanted constructs 26
Ⅱ.7. Statistical analysis 26
Ⅲ. Result and Discussion 27
Ⅲ.1. Measurement of mES proliferation 27
Ⅲ.2. RT-PCR results 27
Ⅲ.3. Immunohistochemistry of implanted construct 28
Ⅳ. CONCLUSION 31
Ⅴ. References 39
Summary in English 43
Table 1. Oligonucleotide primers used for reverse-transcription-polymerase chain reaction and expected product sizes. 32
Figure. 1. Scheme of experiment 33
Figure. 2. GAG/DNA contents from different constructs mES cells were cultured in 3-D hydrogels for one week or four weeks in basal medium. Error bars represent standard error of the mean (*p〈0.02). 34
Figure. 3A. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression from mES cells cultured in hydrogel. The expression of specific genes was analyzed by RT-PCR in mES cells differentiation against time. These data were used to investigate the... 35
Figure. 3B. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression from mES cells cultured in hydrogel. The expression of specific genes was analyzed by RT-PCR in mES cells differentiation against time. These data were used to investigate the... 36
Figure. 4. Histological sections of cell distribution stained by hematoxylin & eosin. (A), (B), & (C): hydrogel without TGF-β3 and (D), (E), & (F): hydrogel with TGF-β3 37
Figure. 5. Histology of mES cells grown for 21 days as hydrogel 3-D cultures in the presence or absence of TGF- 3 stained β with Safranin-O. (A), (B), & (C): hydrogel without TGF-β3 and (D), (E), & (F): hydrogel with TGF-β3 38