臺灣博碩士論文加值系統

關節軟骨是一為透明且具彈性的結締組織，其覆蓋於骨頭末端以提供運動時骨頭之間潤滑及吸收壓力的保護作用，但是其一旦受到損傷，並無法自體修復。因此本研究將以組織工程方法利用明膠、透明質酸、軟骨素與纖維蛋白膠於體外培養出軟骨組織，以達到修復受損軟骨的目的。
在支架材料的製備上，選取由豬皮煉製的明膠（Gelatin），並加入透明質酸(HA)和軟骨素(C6S)，透過1-ethyl-3-(3-dimethylaminopropyl) carbodiimide（EDC）的雙式交聯反應形成共聚物（GHC6S），並藉由冷凍乾燥法移除水分，在液態氮中研磨成微顆粒；纖維蛋白原由豬血漿經冷凍沈澱後取得，另外豬血漿中的第二凝血因子加入10% CaCl2活化後與纖維蛋白原反應聚合成纖維蛋白膠。經過一週及二週的軟骨細胞培養測試，進行Hematoxylin & Eosin、Alcian blue之組織化學染色、S-100蛋白和第二型膠原蛋白免疫組織化學染色，藉以分析細胞生長分佈與基因表現的情形；配合real time-PCR分析TIMP-1, MMP-2, MT1-MMP, aggrecan, decorin, IL-b1, TGF-b1, and FADD以及一、二、十型膠原蛋白的表現與否，檢測軟骨細胞基因表現的能力及狀態。並且進行總GAGs和硫化GAGs的定量分析。
實驗結果顯示GHC6S顆粒的大小約在50-300μm左右。GHC6S顆粒所製備而成的纖維蛋白膠支架能夠有效促進細胞外基質分泌，並抑制細胞外基質的降解，適合於軟骨組織工程。

Articular cartilage provides functions of lubrication to shear stress and protection from compressive force, but it has poor ability to repair itself after suffering damage. The advanced method of tissue engineering is developed and used to maintain cell functions for tissue regeneration. Autologous fibrin glue has been demonstrated as a potential scaffold with very good biocompatibility for neocartilage formation. However, fibrin glue has been reported not to provide enough mechanical strength, but with many growth factors to interfere the tissue growth. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) tricopolymer sponge has been prepared as scaffold for cartilage tissue engineering and showed very good results, but problems of cell seeding and cell distribution troubled the researchers. In this study, GHC6S particles would be added into the fibrin glue to provide better mechanical strength, better cell distribution, and easier cell seeding, which would be expected to improve cartilage regeneration in vitro. Porcine cryo-precipitated fibrinogen and thrombin prepared from prothrombin activated by 10% CaCl2 solution were used in two groups. One is the fibrin glue group in which porcine chondrocytes were mixed with thrombin–fibrinogen solution, which was then converted into fibrin glue. The other is GHC6S-fibrin glue in which GHC6S particles were added into the thrombin-fibrinogen solution with porcine chondrocytes. After culturing for 1-2 weeks, the chondrocytes cultured in GHC6S-fibrin glue showed a round shape with distinct lacuna structure and showed positive in S-100 protein immunohistochemical stain. The related gene expressions of tissue inhibitor of metalloproteinases-1, matrix metalloproteinase- 2, MT1-MMP, aggrecan, decorin, type I, II, X collagen, interleukin-1 b, transforming growth factor-b 1 (TGF-b1), and Fas-associating death domain were checked by real-time PCR. Total GAGs and sulfated GAGs were also quantified by p-dimethylaminobenzaldehyde reaction and 1,9- dimethymethylene blue (DMMB) assay, respectively. The results indicated that the chondrocytes cultured in GHC6S-fibrin glue would effectively promote extracellular matrix (ECM) secretion and inhibit ECM degradation. The evidence could support that GHC6S-fibrin glue would be a promising scaffold for articular cartilage tissue engineering.

Contents Page
Abstract i
中文摘要 iii
Contents iv
Abbreviation list x

Chapter 1 Introduction 1
1.1 Overview 1
1.2 Osteoarthritis 1
1-2-1 Osteoarthritis prevalence 2
1-2-2 The examination of articular cartilage and its surface 3
1-3 The treatments to osteoarthritis 4
1-3-1 Drugs used for osteoarthritis 4
1-3-2 Hyaluronic acid injection 5
1-3-3 Drilling, Microfracture and Abrasion 6
1-3-4 Autologous chondrocytes transplantation 8
1-3-5 Osteochondral transplantation 9
1-4 Tissue engineering method for articular cartilage 11
1-4-1 Scaffold 11
1-4-2 Cells 13
1-4-3 Signals 14
1-5 The purpose of study 15

Chapter 2 Basic science and theory 16
2-1 The compositions of different cartilage
2-2 The structure of articular cartilage 19
2-3 metabolism of articular cartilage 24
2-4 Scaffold design by materials derived from the articular cartilage ECM 26
2-4-1 GHC6S materials 26
2-4-2 Fibrinogen polymerization 28

Chapter 3 Materials and methods 32
3-1 Preparation of GHC6S particle 32
3-2 Fibrinogen cryopricipitation 37
3-3 Prothrombin activation 39
3-4 Chondrocytes isolation 40
3-5 Cells seeding 42
3-6 The morphology of GHC6S particles by scanning electrical microscopy (SEM) 43
3-7 Total DNA measurement for Examining Cell Proliferation 44
3-8 Total GAGs and non sulfated GAGs quantification 45
3-9 Sulfated GAGs measurement by DMMB 46
3-10 Histological evaluation 47
3-11 Immunohistochemical evaluation 48
3-12 Gene expression examination 49
3-12-1 RNA isolation 49
3-12-2 Reverse transcription polymerase chain reaction 50
3-12-3 Polymerase chain reaction for electrophoresis 51
3-12-4 Real-time PCR 52
3-13 Statistical analysis 56

Chapter 4 Results 57
4-1 Morphology of GHC6S particles
4-2 Histological evaluation 58
4-3 Gene expression of articular cartilage related collagens 59
4-4 Proteoglycans expression 60
4-5 Gene expression related with extra-cellular matrix degradation 61
4-6 Cytokines expression 62
4-7 GAGs synthesis 63

Chapter 5 Discussion 65
Chapter 6 Conclusion 71
References 84

Figures vii
Fig. 1-1 The arrow pointed the defect with bone marrow flowingout with mesenchymal stem cells 7
Fig. 1-2 Chondrocytes were expanded and implanted into the defect of cartilage 8
Fig. 1-3 Operation of the osteochondral graft transplantion 10
Fig. 2-1 Proteoglycan aggregate 18
Fig. 2-2 (a) The zones of articular cartilage of different collagen network 23
Fig. 2-2 (b) The zones of articular cartilage of different morphology of chondrocytes 23
Fig. 2-3 (a) The structure of fibrinogen 30
Fig. 2-3 (b) The domains of fibrinogen 31
Fig. 2-3 (c) The crosslinking of fibrinogen 31
Fig. 3-1 (a) Chemical structures of hyaluronic acid 34
Fig. 3-1 (b) Chemical structures of chondroitin-6-sulfate 34
Fig. 3-2 Diagram of crosslinking mechanism by EDC 36
Fig. 4-1 Gelatin/ hyaluronic acid/ chondroitin-6-sulfate (GHC6S) particles were examined by scanning electron microscopy. 73
Fig. 4-2 (a) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (H&E staining) 74
Fig. 4-2 (b) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (H&E staining) 74
Fig. 4-2 (c) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (H&E staining) 74
Fig. 4-2 (d) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (H&E staining) 74
Fig. 4-3 (a) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (Alcian blue staining) 75
Fig. 4-3 (b) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (Alcian blue staining) 75
Fig. 4-3 (c) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (Alcian blue staining) 75
Fig. 4-3 (d) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (Alcian blue staining) 75
Fig. 4-4 (a) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (S-100 IHC staining) 76
Fig. 4-4 (b) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (S-100 IHC staining) 76
Fig. 4-4 (c) After cultured for 1 week, constructs of chondrocytes cultured in fibrin glue (S-100 IHC staining) 76
Fig. 4-4 (d) After 1 week, constructs of chondrocytes cultured in GHC6S-fibrin glue (S-100 IHC staining) 76
Fig. 4-5 (a) The values of –△Ct of relative gene expression in real-time PCR by type II collagen 77
Fig. 4-5 (b) The values of –△Ct of relative gene expression in real-time PCR bytype I collagen 77
Fig. 4-5 (c) The values of –△Ct of relative gene expression in real-time PCR by type X collagen 77
Fig. 4-6 (a) The values of –△Ct of relative gene expression in real-time PCR by aggrecan 78
Fig. 4-6 (b) The values of –△Ct of relative gene expression in real-time PCR by decorin 78
Fig. 4-7 (a) The values of –△Ct of relative gene expression in real-time PCR by MMP-2 79
Fig. 4-7 (b) The values of –△Ct of relative gene expression in real-time PCR byMT1-MMP 79
Fig. 4-7 (b) The values of –△Ct of relative gene expression in real-time PCR by TIMP-1 79
Fig. 4-8 (a) The values of –△Ct of relative gene expression in real-time PCR by TGF-b1 80
Fig. 4-8 (b) The values of –△Ct of relative gene expression in real-time PCR by IL-1b 80
Fig. 4-8 (c) The values of –△Ct of relative gene expression in real-time PCR by FADD 80
Fig. 4-9 Total GAGs content 81
Fig. 4-10 Sulfated GAGs content 82
Fig. 4-11 Non-sulfated GAGs content 83

Tables ix
Table 2-1 The compositions of hyaline cartilage 18
Table 2-2 The identified four zones of articular cartilage 21
Table 2-3 Articular cartilage structure and the composition distribution 22
Table 2-4 The enzymes and their inhibitors to the related articular cartilage ECM 25
Table 2-5 Plasma clotting factors 29
Table 3-1 The composition of GHC6S sponge in weight percent 35
Table 3-2 CPDA-1 (500 mL Collection Bags ) compositions 38
Table 3-3 The preparation of culture medium 41
Table 3-4 Reverse transcription solution I 53
Table 3-5 Eeverse transcription solution II 53
Table 3-6 RT-PCR running solution 53
Table 3-7 The primers designed for the real-time PCRa 54

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