臺灣博碩士論文加值系統

關節炎（OA）是一種常見的退化性關節疾病，主要由長時間坐姿不正或年齡老化等原因造成關節軟骨變薄退化而產生。由於關節軟骨缺乏血管，自我修復能力極差，目前的治療方式無法有效治癒，修復的軟骨易纖維化，造成軟骨機械性質改變。組織工程被認為是新一代可有效治療軟骨的方式，而注射型水膠可作為組織工程細胞用之支架，其具有許多的優點，包括了高含水性及具有相似於軟骨的機械性質。光交聯水膠為一種注射型水膠，其中以Poly(ethylene) glycol (PEG)組成的高分子已經被廣泛的研究及使用於組織工程。在本研究中我們藉由光交聯方式製備出PCL-PEG-PCL水膠，應用於軟骨組織工程支架，軟骨細胞(chondrocytes)及骨髓間葉幹細胞(BMSCs)被共培養於此水膠內，藉由生化分析技術、基因表現、切片組織學及動物修復實驗來觀察驗證軟骨的生長修復。研究分為兩個章節：第一章節將探討不同親疏水組成之光交聯水膠對於軟骨新生之影響；第二章節則評估軟骨及骨髓間葉幹細胞包埋於水膠內之最佳化共培養比例。
水膠支架的性質，像是生物相容性、澎潤率、機械性質及降解行為等，已被證實對於軟骨修復有極大的影響。因此第一章節著重在談討PCL-PEG-PCL水膠之性質對於包埋的軟骨生長情形之影響。首先PEG與ε-caprolactone進行共聚化及丙烯化反應，合成出可進行光交聯的PCL-PEG-PCL共軛高分子，藉由調整不同PEG及PCL的分子量，可探討不同親疏水比例對於水膠性質之影響。當PEG分子量為10,000 Da時，水膠有最高的澎潤率(9.60.2)及較低的彈性模數(0.2230.007 N/mm2)。生化分析的結果發現水膠的澎潤性與細胞分泌之多醣類(glycosaminoglycans)及膠原蛋白表現呈正相關。經過四週體外培養後之PEG分子量為10,000 Da的水膠，相較於PEG分子量為2,000 Da之水膠，細胞多糖類分泌量增加1.8倍，膠原蛋白分泌量增加2.4倍。有趣的是切片結果顯示，當疏水性PCL分子量增加時，二型膠原蛋白分泌累積量增加。研究結果證實親水性片段PEG及疏水性片段PCL的長度，對於水膠性質有極大的影響，藉由高分子親疏水鏈段調整，可增加細胞增殖能力及有效的分泌軟骨的細胞外間質。
軟骨細胞及幹細胞都可用於軟骨組織工程，共培養此兩種細胞可有效誘導細胞軟骨化，因此第二章節中，此兩種細胞以不同比例被包埋培養於PCL-PEG-PCL水膠內，觀察四週內軟骨新生情形，藉此界定出最佳化的共培養比例。結果顯示共培養系統於體外環境可有效形成軟骨化細胞並加速軟骨組織形成。1:4 CH-to-BMSCs 比例的共培養系統顯示多糖類及膠原蛋白分泌相較於單一培養一種細胞有明顯的增加。體內實驗以兔子為實驗動物，驗證共培養系統是否可用於體內環境。結果顯示共培養系統經八週後可產生透明軟骨新生，修復效果極為顯著。此研究成果證實共培養系統可於水膠內有效修復再生軟骨，其於軟骨組織工程有極大的發展潛力。

Osteoarthritis (OA) is a common type of degenerative arthritis marked by the thinning of the articular cartilage and affects sedentary or older individuals in general. Owing to the absence of blood vessels and cells, articular cartilage has very limited self-regeneration capacity following injury. Current methods to repair articular hyaline cartilage often lead to fibrocartilage formation, which is inferior for the application intended. Therefore, tissue engineering methods have been considered for cartilage repair and restoration. Injectable hydrogels are attractive candidates as cartilage engineering tissue scaffolds due to their advantages such as high water content and mechanical similarity with the cartilage. Photocrosslinked PEG-based hydrogels are one of the most widely investigated and utilized systems in tissue engineering. In this study, we fabricated PCL-PEG-PCL hydrogels via polymerization for use as cartilage reconstruction scaffolds. Chondrocytes and mesenchymal stem cells were photo-encapsulated and co-cultured. Cartilage regeneration was evaluated by biochemical assay, gene expression, histology, and in vivo tests. The study was divided into two sections, first discussing the effect of different hydrophobic/hydrophilic ratios on neocartilage regeneration, and then evaluating the different ratios of mesenchymal stem cells to chondrocytes on cartilage repair.
Hydrogels have been investigated as scaffolds for cartilage tissue engineering with the properties of biocompatibility, swelling ratio, mechanical strength, and degradation behavior. First section was to design the most suitable PCL-PEG-PCL hydrogel to allow optimal proliferation and differentiation of encapsulated chondrocytes for cartilage regeneration. Poly(ethylene) glycol (PEG) was copolymerized with ε-caprolactone (PCL) and then acrylated to confer photocrosslinking capacity. Chondrocytes were encapsulated within photocrosslinked poly(ethylene glycol)-co-poly(caprolactone) hydrogels prepared with varying composition. The effects of the composition of the hydrogel on its properties and cell behavior were studied by varying the molecular weights of PEG and PCL. Hydrogels with higher PEG molecular weights (10,000) were associated with higher swelling ratios (9.6) and reduced elastic modulus (0.223 N/mm2). Biochemical analysis showed a positive correlation between swelling ratio and expression levels of glycosaminoglycans and total collagen. There was a 1.8-folds increase in glycosaminoglycan and 2.4-folds increase in total collagen content in hydrogels with the highest molecular weight (10,000) PEG compared to lowest molecular weight (2,000) PEG after 4 weeks of culture. Interestingly, histological examinations revealed more extensive type II collagen secretion and accumulation around chondrocytes when molecular weights of the hydrophobic PCL segment increased. The alternation of hydrophilic and hydrophobic segment lengths of PCL resulted in changes in hydrogel properties, which achieved maximum proliferation and production and distribution of extracellular matrix for cartilage regneration.
Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culturing of CH and BMSCs is a promising strategy for promoting chondrogenic differentiation. In second section, CH and BMSCs were encapsulated in PCL-PEG-PCL photo-crosslinked hydrogels for four weeks. Various ratios of CH: BMSCs co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CH and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culturing with a 1:4 CH-to-BMSCs ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with better regeneration than achieved in spontaneous repair. This finding suggests that the co-culture of these two types of cells promotes cartilage regeneration, and the system, including the hydrogel scaffold, has potential in cartilage tissue engineering.

摘要 I
Abstract III
CHAPTER ONE Introduction 1
CHAPTER TWO Literature review 4
2.1. Hydrogels in tissue engineering 5
2.2. Polymers for injectable hydrogel systems 6
2.3. Injectable hydrogel systems 7
2.4. Cells for cartilage tissue engineering 11
CHAPTER THREE Materials and Methods 15
3.1. Synthesis of amphiphilic PCL-PEG-PCL block copolymers 15
3.2. Acrylation of amphiphilic block copolymers 16
3.3. Aggregation and phase behavior measurement 16
3.4. Photo-crosslinking hydrogel preparation 16
3.5. Swelling, mechanical and degradation testing 17
3.6. Cell isolation and expansion 17
3.7. Encapsulation of cells in hydrogel 18
3.8. Biochemical analysis 19
3.9. Histological examination and immunohistochemical staining 19
3.10. qRT-PCR measurement for marker genes expression levels 20
3.11. Surgical procedure and in vivo evaluation 21
3.12. Statistical analysis 22
CHAPTER FOUR Results and Discussion Cartilage formation through alterations of amphiphilicity of poly (ethylene glycol)-poly(caprolactone) copolymer hydrogels 26
4.1. Synthesis of PEG-PCL copolymers 26
4.2. Aggregation and phase behavior 27
4.3. Characterization of photocrosslinked hydrogels 28
4.4. Formation of cartilage ECM by chondrocytes in hydrogels 29
4.5. Histological analysis 29
4.6. Cell differentiation as revealed by qRT-PCR 31
4.7. Discussion 31
CHAPTER FIVE Results and Discussion In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photo-crosslinked PCL-PEG-PCL hydrogels enhances cartilage formation 49
5.1. Biochemical and histological examination chondrogenesis of BMSCs inhydrogel: effect of PCL block length 49
5.2. Effect of ratio of chondrocytes to BMSCs on cartilaginous phenotype 51
5.3. In vivo neocartilage formation with co-culture system 53
CHAPTER SIX Conclusions 66
References 68

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