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系統識別號 U0026-0108201123232500
論文名稱(中文) 探討物理性刺激對於骨膜軟骨生成的影響
論文名稱(英文) To investigate the effect of mechanical forces on periosteal chondrogenesis
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 唐逸文
研究生(英文) Yih-Wen Tarng
學號 p8891111
學位類別 博士
語文別 英文
論文頁數 78頁
口試委員 指導教授-蘇芳慶
口試委員-葉明龍
口試委員-呂佩融
召集委員-楊瑞珍
口試委員-陳理維
口試委員-曾清俊
中文關鍵字 功能性組織工程  骨膜  生物反應爐  剪應力  動態靜水壓力  生物力學測試 
英文關鍵字 Functional tissue engineering  Periosteum  Bioreactor  Shear stress  Hydrodynamic pressure  Biomechanical test 
學科別分類
中文摘要 關節軟骨一旦受損是很難自我修復的,因為關節軟骨是一個很特別的組織,它沒有血管供應,沒有淋巴系統,也沒有神經支配,因此即使是發生在年輕的病人,且只是一個局部的、範圍不大的軟骨受損,都有可能產生不可回復的退化過程;即使只是一個小小的軟骨缺損,但最後仍可能會演變成整個關節的退化性骨關節炎。組織工程是一門新的科學,它可以創造出活的組織來修補、取代受損的組織。但大多數的學者,都把重心放在細胞與組織的培養,以及強調組織學及生化學上的結果,往往忽略了軟骨組織的功能性。因此在2000年有學者提出”功能性組織工程(Functional tissue engineering)“,尤其在骨科領域裡的組織中,不論是硬骨, 軟骨或韌帶,若無法承受外在的應力,這可能是一個沒有用的組織;所以,我們深信在細胞的培養過程中,機械性的力學刺激扮演一個重要的角色,可增強生成組織的機械性質。骨膜可以說是一個天然的組織工程材料,它不但可以提供未分化的間質幹細胞來源,它還包含了自己的纖維組織鷹架(Scaffold),以及生長因子。在我們的實驗中,將骨膜的Cambium layer朝上後,四個角縫在PCL scaffold上,然後將這個組合懸吊在spinner flask生物反應爐(Bioreactor)內或放進我們自行研發的生物反應爐中,去接受水流的剪應力及動態靜水壓力的刺激。最後的結果顯示,剪應力可以刺激間質幹細胞的增生及分化成軟骨細胞,甚至可以刺激軟骨細胞分泌更多的基質,而讓新生成的軟骨擁有更好的機械性質,但完全不需要TGF-β的刺激。不幸的是,這個spinner flask bioreactor太簡單,以至於無法提供一個穩定的水流,而造成培養出來的軟骨,表面都非常不規則。因此我們必須自行研發出一套可同時提供穩定水流及可產生出動態靜水壓力的生物反應爐,意即完全模擬一般關節的情況。但我們發現動態靜水壓力,不但能夠刺激軟骨細胞分泌更多的基質,以及在組織切片染色後,更可以發現細胞的排列與分層更接近正常軟骨。總之,在我們自行研發的生物反應爐中,可以培養出表面更光滑,機械性質更好的軟骨。以上的結果也證實了,在體外培養軟骨,一定要在有物理性刺激的環境中,才能讓軟骨的機械性質更好。
英文摘要 Damaged articular cartilage has a limited ability to heal because mature cartilage is devoid of blood vessels, lymphatic channels, or neurological innervation. Even though focal and small size lesions presented in young patients, an irreversible degenerative process still occurred. Tissue engineering is a new science of creating living tissue to replace, repair, or augment disease tissue, but most researchers had focused on biochemical and biomedical application of tissues in vitro, and their overall function has been largely ignored. However, the concept of “functional tissue engineering” has become a critical issue and involves the application of physical loading in order to promote the development of tissue constructs that can adapt to the functional/mechanical demands encountered in vivo, especially bone, cartilage, and tendon tissues required in orthopedics field. These engineered tissues must resist a variety of mechanical forces, such as compression, bending or tensional force, and shear stress. Hence, the mechanical force stimulation plays an important role to increase the mechanical properties in vitro culture.
Periosteum, a natural tissue-engineering material, possesses three prerequisites over the cambium layer for tissue engineered cartilage repair. We designed an experimental model in which the periosteum was secured onto a poly-ε-caprolactone (PCL) scaffold with four corner sutures and the cambium layer facing upward. The periosteum/PCL composites were suspended into spinner flask bioreactor or fixed into our developed recirculating flow perfusion bioreactor, where the periosteal explants encountered oscillating fluid shear stress and hydrodynamic pressure stimuli simultaneously. The results demonstrate that fluid shear stress influences cell proliferation, subsequent differentiation, increase matrix synthesis and mechanical properties without TGF-βin the spinner flask bioreactor. The spinner flask bioreactor is too simple to offer hydrodynamic pressure and steady laminar flow and generated an irregular surface on the engineered cartilage due to turbulent flow. Therefore, we successfully developed a flow-perfusion bioreactor that simultaneously offered oscillating shear stress and hydrodynamic pressure, simulating the motion of a diarthrodial joint, for growing engineered cartilage in vitro. The hydrodynamic pressure plays an important role to stimulate increased more ECM production, cartilage yield, conspicuous heterogeneous cell morphology, and improved the arrangement and zonal organization of the engineered cartilage. Finally, the recirculating bioreactor manufactured smooth surface of engineered cartilage with good mechanical properties after biomechanical test, similar to native articular cartilage. According to these results, we propose that engineered cartilage must be cultured in a mechanically stimulated environment.
論文目次 中文摘要 ................................................ III
ABSTRACT ............................................... IV
誌謝 .................................................... VI
CONTENTS ............................................... VII
LIST OF TABLES ......................................... IX
LIST OF FIGURES ......................................... X
CHAPTER 1 INTRODUCTION .................................. 1
1.1 Cell and Extracelluar matrix (ECM)
1.1.1 Cell: Undifferentiated mesenchymal stem cells in the periosteum .............................................. 5
1.1.2 ECM: Proteoglycan, Collagen type II and Non-collagenous protein .................................... 8
1.1.3 Growth factor (TGF-β, IGF-1, …) .................. 9
1.1.4 Composition and Structure of Articular Cartilage .. 10
1.2 Scaffold Biomaterial: PGA, PLA and PCL ............ 11
1.3 Cell Mechanism
1.3.1 Mechanical force influences Cartilage metabolism .. 13
1.3.2 Fluid mechanism ................................... 14
1.3.3 Oxygen tension .................................... 15
1.4 Bioreactor
1.4.1 Hydrodynamic bioreactor – Dynamic fluid pressure (DFP) bioreactor ........................................ 17
1.4.2 Low shear stress – Rotating bioreactor ........... 18
1.4.3 High shear stress – Spinner flask bioreactor ......19
1.5 Biomechanical testing
1.5.1 Cartilage mechanical properties ................... 21
1.5.2 Confine compression test: Creep and stress-relaxation model .................................................. 23
1.5.3 Unconfine compression test: Stress-strain curve (Young’s modulus) ...................................... 23
MOTIVATION AND HYPOTHESIS .............................. 25
CHAPTER 2. MATERIALS AND METHODS
2.1 Periosteum preparation and culture medium preparation ............................................ 26
2.2 Scaffold: PCL ( Polycaprolactone ) scaffold ...... 27
2.3 Bioreactor:
2.3.1 Spinner flask bioreactor ......................... 28
2.3.2 Flow perfusion recirculating bioreactor .......... 30
2.4 Cartilage yield assay and histological score: ..... 35
2.5 Biomechanical testing: Confine compression test ... 37
CHAPTER 3. RESULTS
3.1 Periosteal culture in spinner flask bioreactor
3.1.1 In Gross view of engineered cartilage .......... 40
3.1.2 In Histological examination ................... 44
3.1.3 Immunohistochemistry stain (mAb S6.79) ......... 48
3.1.4 In Biomechanical test ........................ 49
3.2 Periosteal culture in novel recirculating flow-perfusion bioreactor
3.2.1. Our developed bioreactor ........................ 50
3.2.2 Cartilage yield within the engineered cartilage .. 55
3.2.3 Cellular zonal organization ...................... 57
CHAPTER 4. DISCUSSION ................................. 60
CHAPTER 5. CONCLUSION AND FUTURE WORK ................. 65
REFERENCES ............................................ 68
PUBLICATIONS ........................................... 75
BRIEF PERSONAL INTRODUCTION ............................ 77
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