학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2015. 2. 홍병희.With the recent advances in stem cell engineering and tissue regeneration, stem cell-based regenerative medicine provides a promising strategy for the treatment of heart failure, neuronal disorders or neurodegenerative diseases, which are still one of the leading causes of human death and disability in the world. Nevertheless, the current clinical treatments for heart failure, neuronal disorders or neurodegenerative disease are quite limited, and the number of people affected by these diseases consistently increases every year. As a direct result, there is a great need to discover alternative therapies for these disorders or diseases. Recently, graphene has been recognized as a biomimetic nanomaterial and has been proposed for a number of biomedical applications because of their fascinating properties different from those of the carbon-based graphitic materials. This dissertation is the result of an effort to develop 2D and 3D platforms for controlling cell microenvironment for various cell and tissue engineering applications. The specific objectives of my thesis are as follow: (1) the development of an efficient 2D platform for the growth and differentiation of stem cells, which is crucial for autologous cell therapy and tissue engineering to treat various disorders and diseases, and the investigation of the effects of graphene on the enhanced differentiation process through analyzing the expressions of extracellular matrix (ECM) proteins and cell signaling molecules, (2) the improvement of nanoengineering approaches for controlling 3D cell microenvironment and the use of these facile techniques to regulate cell fate decisions. The main results of my dissertation research can be summarized as follows. First, we found that the 3D spheroid structure could be formed from aggregated hMSCs grown on monolayer of graphene without the use of any external factors. Second, we have provided the first demonstration that graphene can be used as a stem cell culture substrate to promote the cardiomyogenic differentiation process of MSCs without the use of any exogenous chemical inducers. Finally, the culture of hESCs on graphene promotes the stepwise differentiation of these cells into mesodermal cells and endodermal cells and their subsequent cardiomyogenic differentiation compared with their culture on glass. However, for the success of clinical regenerative application, a three-dimensional (3D) scaffold is a demanding field in terms of development of microenvironments and appropriate synergistic cell guidance cues. Thus, the following part II of the thesis presents the development and applications of carbon nanomaterial-based 3D scaffolds. Recently, graphene has been proposed as a tool for pioneering approach in the progress of designing nano-engineered cell culture platforms or scaffolds. Development of transplantable 3D hybrid graphene scaffold in vivo that can be applied in practical use for regenerative therapy is urgently needed. Especially, it is important to verify the superiority of graphenes as regenerative nanocomposites by applying to real disease animal model. Here, we developed a novel method for fabricating a hybrid bioscaffold composed of CNTs and BC for bone regeneration. Lastly, we developed a strategy to hybridize graphene with the 3D layer by layer scaffold and investigate its impact on the fundamental neuron study. This dissertation provides the details of my work on all projects related to designing scaffolds for tissue engineering and regenerativeAbstract 1
Contents 4
List of Figures 8
List of Tables and Schemes 21
Scope and Format of Dissertation 22
1. General introduction
1.1 Summary 25
1.2 Histological background. 26
1.3 Plausible mechanism of carbon nanomateiralss positive effects on the stem cell and tissue regeneration 29
1.4 2D graphene based cell engineering 37
1.5 3D graphene based cell engineering 43
1.6 References 46
Part I 2D Graphene based stem cell engineering
2. Graphene-directed spheroid formation of mesenchymal stem cells for enhanced neuronal differentiation
2.1 Introduction 50
2.2 Experimental 52
2.3 Results and Discussion 58
2.4 Conclusions 74
2.5 References 80
3. Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells
3.1 Introduction 86
3.2 Experimental 88
3.3 Results and Discussion 93
3.4 Conclusions 106
3.5 References 108
4. Graphene-regulated cardiomyogenic differentiation process of mesenchymal stem cells by enhancing the expression of extracellular matrix proteins and cell signaling molecules
4.1 Introduction 114
4.2 Experimental 116
4.3 Results and Discussion 122
4.4 Conclusions 135
4.5 References 136
Part II Engineering nature-driven three-dimensional bioscaffolds with carbon nanomaterials
5. Binding behavior of hybrid system for the APCLP coated-carbon nanotube and graphene with bacterial cellulose
5.1 Introduction 143
5.2 Experimental 145
5.3 Results and Discussion 147
5.4 Conclusions 163
5.5 References 164
6. In Situ hybridization of carbon nanotubes with bacterial cellulose for three-dimensional bioscaffolds
6.1 Introduction 166
6.2 Experimental 168
6.3 Results and Discussion 173
6.4 Conclusions 186
6.5 References 187
7. In vivo-like three-dimensional neuronal networks engineered by Graphene bioscaffolds
7.1 Introduction 192
7.2 Experimental 195
7.3 Results and Discussion 199
7.4 Conclusions 208
7.5 References 209
Abstract (Korean)
AcknowledgementDocto