製備溫感型胺基酸水膠包覆聚乳酸甘醇酸微粒控制釋放生長因子作為關節軟骨修復之研究

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研究生：	詹芸臻 Chan, Yun-Chen
論文名稱：	製備溫感型胺基酸水膠包覆聚乳酸甘醇酸微粒控制釋放生長因子作為關節軟骨修復之研究 Study of Growth Factor-loaded Microspheres in mPEG Polypeptide Hydrogel System for Articular Cartilage Repair
指導教授：	朱一民 Chu, I-Ming
口試委員:	蔡德豪 Tsai, De-Hao 林世傑 Lin, Shih-Jie
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2020
畢業學年度：	107
語文別：	英文
論文頁數：	60
中文關鍵詞：	溫度敏感型胺基酸高分子、細胞支架、聚乳酸甘醇酸微粒、軟骨細胞、緩慢釋放、TGF-β3
外文關鍵詞：	chondrocytes, methoxy poly(ethylene glycol)-poly(alanine), PLGA microspheres, tissue engineering, thermosensitive hydrogel, TGF-β3
相關次數：	點閱：2 下載：0
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退化性膝關節炎(osteoarthritis)通常來自膝關節老化、運動損傷等因素，此種情形膝關節軟骨再生能力差，許多醫療團隊與研究者致力於膝關節修復的技術開發，目前有藥物注射、關節鏡手術、置換關節等方法，未來人們期望以侵入性最小的手術或組織工程技術達到膝關節再生的最大效益。
本研究探討可注射溫度敏感型胺基酸高分子水膠添加聚乳酸甘醇酸微粒包覆生長因子的緩釋模型，結合注射型載體與持續提供生長因子的優點，期望未來能應用至膝關節軟骨修復手術，當水膠承載生長因子載體與自體軟骨細胞(或幹細胞)時，注射至膝關節軟骨缺陷，可提升手術的便利性，同時有助於膝關節軟骨長期修復。此研究所使用溫度敏感型胺基酸高分子水膠(methoxy poly(ethylene glycol)-poly (alanine), mPA) 已有相關研究發表其對於軟骨細胞生長的助益，在體外培養實驗中，顯示軟骨細胞確實與此mPA水膠有良好的生物相容性，mPA水膠可模擬膝關節軟骨組織的生長環境，具有三維結構、高含水率、體內自發降解等特性，可作為優良的細胞載體。本研究在此mPA水膠中添加生長因子載體(TGF-β3-loaded PLGA microspheres)，進行生長因子緩慢釋放行為的討論，在此模型中生長因子的釋放曲線可維持2個月以上，改善以mPA水膠包覆蛋白質而有第一天突然釋放的缺點；在體外細胞實驗中，軟骨細胞與生長因子載體同樣被包覆於mPA水膠中進行3周的培養，以即時聚合酶連鎖反應(real-time PCR)結果發現延長釋放生長因子對比鄰的軟骨細胞生長有助益，可避免軟骨細胞在長時間培養過程中朝向肥大、細胞凋亡的方向生長。
期望有新穎的組織工程技術持續被提出，與醫學技術相輔相成，為大眾提供更多醫療的選擇。

Co-cultured scaffolds for the cartilage repair have been widely developed for decades, but the diffusion of limitation of nutrients resulted in the poor cell proliferation and the lack of ECM production. Transforming growth factor beta (TGF-β) is a potent promoter for chondrogenesis and ECM formation. In this study, the aim is to develop a type of thermosensitive hydrogel matrix to encapsulate chondrocytes together with TGF-β3-loaded PLGA microspheres for cartilage repair. Methoxy poly(ethylene glycol)-poly (alanine), mPA, which is able to gel in situ under physiological temperature, is used to deliver cells to defect area by injection with minimum invasiveness. Poly (lactic-co-glycolic acid) (PLGA) microspheres were used for sustained release of growth factor TGF-β3. It was found that the release can be extended for over 2 months. This hydrogel plus microspheres composite system can be used to deliver cells with proper growth factors for long-term repair in an easy and effective manner.

Abstract    I
Contents    III
List of Tables    VI
List of Figures    VII
Abbreviation    IX
Chapter 1. Introduction and Literature Review    1
1.    Articular cartilage damage    2
2.    Surgery    4
2.1.    Arthroscopic surgery    4
2.2.    Autologous chondrocyte implantation (ACI)    4
3.    Articular cartilage tissue engineering    6
3.1.    Hydrogels for articular cartilage repair    7
3.2.    Cell strategies    8
3.3.    Bioactive additives (biofactors)    9
4.    Injectable hydrogels    11
4.1.    Natural materials    12
4.2.    Synthetic polymer    13
5.    Thermosensitive injectable hydrogels    15
5.1.    Poly(peptide)-based hydrogels    17
5.2.    mPEG-poly(alanine) hydrogels    19
6.    PLGA microspheres    20
7.    Composite scaffolds for cartilage repair    23
8.    Specific aims    24
Chapter 2. Materials and Methods    25
1.    Flow chart of experiment    25
2.    Materials    26
3.    Equipment    28
4.    Synthesis of the diblock copolymers    29
5.    Characterization of diblock copolymers    31
6.    Preparation and sol-gel transition of hydrogels    31
7.    Degradation study of hydrogels    32
7.1 In vitro degradation study    32
7.2 In vivo degradation study    32
8.    Fabrication of protein-loaded PLGA MS    33
9.    In vitro cytotoxicity    35
10.    In vitro protein release from hydrogel composite system  36
10.1.    Release study of macromolecular proteins    36
10.2.    Release study of chondrogenic growth factors, TGF-β3  37
11.    Gene expression of encapsulated chondrocytes    38
Chapter 3. Results and Discussion    39
1.    Characterization of diblock copolymers mPA    39
2.    Sol-gel transition profile of mPA hydrogels    42
3.    Morphology of hydrogels and PLGA MS    44
4.    Degradation study of hydrogels    46
5.    In vitro cytotoxicity    48
6.    In vitro protein release from hydrogel composite system  49
7.    Gene expression of encapsulated chondrocytes    51
Chapter 4. Conclusion    53
Future Work    54
Reference    55

                                

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