髓鞘作為神經系統的重要組成成分之一，包裹於神經元的軸突外，具有絕緣作用並提高神經衝動的傳導速度，還可為神經纖維提供營養和能量，髓鞘的破壞可能會導致運動和認知功能的喪失，大腦和脊髓中的幹細胞可通過髓鞘再生修復受損的髓鞘，然而內源性髓鞘再生過於緩慢，因此有必要設計合適的體外神經系統模型研究神經元與寡樹突膠細胞的交互作用。
本研究開發出具有高生物降解性和光聚合的材料聚癸二酸甘油酯(PGSA)，參雜導電性的高分子材料聚乙烯吡咯烷酮(PVP)成為高分子複合材料，配合3D列印技術建構出神經再生支架，並引入電刺激加速細胞再生。為了模擬細胞培養狀態，對複合材料的機械性質和3D列印成果在乾燥狀態和 DMEM浸泡狀態下進行比較，結果顯示在DMEM浸泡狀態下，殘留的乙醇會降低PGSA-PVP (10 wt.%)複合材料的相容性，而與PGSA支架相比，在PGSA-PVP (10 wt.%)複合材料支架發現較嚴重收縮，因為PVP 是比 PGSA 更親水的聚合物。此外，為了篩選出最佳塗層材料，在具有各種塗層材料的PGSA膜進行細胞黏附測試，發現PLO塗層可促進細胞增殖和分化。最後在PGSA-PVP (10 wt.%)複合材料支架建立共培養系統並外加電刺激，顯示在微結構與電刺激的協同作用下能產生明顯的神經突觸增生與誘導現象。這些結果都顯示3D列印建構的PGSA-PVP (10 wt.%)複合材料支架藉由微結構和電刺激作用在神經再生領域的可能性。

Damage to oligodendrocytes or the myelin sheath in injury and disease often leads to serious consequences on neural function. Therefore, remyelination plays a critical therapeutic strategy in the regenerative process of the central nervous system (CNS). Due to the lack of appropriate in vitro models for investigating neuron-oligodendrocyte interactions, it is necessary to develop an in vitro CNS regeneration model to allow detailed understanding. With advancements in the development and applications of biomaterials toward tissue engineering, there are various strategies to enhance remyelination in the CNS.
In this work, a biodegradable and conductive polymer composite scaffold was designed and 3D-printed. The scaffold incorporated polyvinylpyrrolidone (PVP) as a conductive material within the poly (glycerol sebacate) acrylate (PGSA) matrix. The co-cultured system of rat pheochromocytoma cells (PC12) and rat glioma cells (C6) sequential seeding on the microstructured scaffold was constructed to study neuron-oligodendrocyte interactions in vitro and examine neurite outgrowth.
Tensile tests of PGSA and PGSA-PVP (10 wt.%) were conducted and compared in both dried and DMEM-soaked states. The addition of PVP increased the compatibility of the composites, although the residual ethanol in the DMEM-soaked state reduced this property. Then, PC12 and C6 cell adhesion tests cultured on PGSA films with various coating materials were carried out respectively to identify the optimal coating materials. Among the coatings, the PLO-coated group demonstrated advantages for the co-culture of PC12 and C6 cells, enhancing both cell proliferation and differentiation.
To confirm the dimension accuracy as well as to simulate the cell culture state, the comparison of PGSA and PGSA-PVP (10 wt.%) scaffolds in both dried and DMEM-soaked states was investigated. The results showed that severe shrinkage of PGSA-PVP (10 wt.%) scaffolds could be observed in the DMEM-soaked state as PVP is a more hydrophilic polymer than PGSA.
Finally, PC12 and C6 cells were sequentially co-cultured on the microstructured scaffolds to assess the effectiveness of the compartmentalized device with electrical stimulation. PGSA-PVP (10 wt.%) microstructured scaffolds with electrical stimulation exhibited oriented stretching and neurite outgrowth of cells. As a result, the proposed device can be used as a promising tool to study neuron-oligodendrocyte interactions and show great potential in nerve regeneration.

摘要 i
Abstract ii
謝誌 iv
Table of Content vi
List of Figures viii
List of Tables xi
Chapter 1 Introduction 1
1.1 Central Nervous System and Disease 1
1.1.1 Oligodendrocytes 1
1.1.2 Demyelinating Disease 1
1.2 Central Neural System Regeneration 2
1.2.1 Mechanism of Remyelination 2
1.2.2 Oligodendrocytes in Developmental Myelination 4
1.2.3 Electrical Stimulation to Remyelination 6
1.3 Contact Guidance 7
1.4 CNS Tissue Engineering Scaffolds 8
1.5 Additive Manufacturing 8
1.6 Materials for Tissue Regeneration 10
1.6.1 Poly(Glycerol Sebacate) (PGS) 10
1.6.2 Poly(Glycerol Sebacate) Acrylate (PGSA) 11
1.6.3 Poly(VinylPyrrolidone) (PVP) 12
1.7 Motivation and Purpose 13
Chapter 2 Experimental Methods and Materials 15
2.1 Research Framework 15
2.2 Materials and Equipment 17
2.3 Synthesis of Materials 19
2.3.1 Synthesis of PGS 19
2.3.2 Synthesis of PGSA 19
2.4 Central Neural System Regeneration Scaffolds 20
2.4.1 DLP-AM Fabrication of Scaffolds 20
2.4.2 Observation of Scaffolds 21
2.5 Tensile Test of PGSA and PGSA-PVP (10 wt.%) Composite Films 22
2.6 Cell Culture of Neural Cells 22
2.7 Coating Effects on PGSA Films of PC12 and C6 cells 23
2.7.1 The Fabrication of PGSA Films 23
2.7.2 Cell Seeding on PGSA Films 24
2.7.3 Cell Viability and Morphology 24
2.7.4 Fluorescence Staining of the Films 25
2.8 PC12 and C6 cells Sequential Co-culture and Procedure 26
2.9 Electrical Stimulation to Cells 27
2.10 Immunofluorescence Staining of the Scaffolds 28
Chapter 3 Result and Discussion 29
3.2 Tensile Test of PGSA and PGSA-PVP (10 wt.%) 29
3.2 Coating Effect of PC12 cells on PGSA Films 31
3.3 Cell Culture of C6 cells in a Flask and Coating Effect of C6 cells on PGSA Films 34
3.4 Design of Scaffolds 38
3.5 Scaffolds Measurement 42
3.6 PC12 and C6 cells Sequential Co-culture on Microstructured Scaffolds with Electrical Stimulation 46
Chapter 4 Conclusion 57
Chapter 5 Future Work 59
5.1 The Co-Culture System of DRGs & OPCs with Electrical Stimulation for Myelination 59
5.2 Compartmentalized Microfluidic Platforms with Electrical Stimulation for Myelination Analysis 60
Chapter 6 Reference 62

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