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研究生:李建德
研究生(外文):Chien-TeLee
論文名稱:發展三維之聚乙二醇水膠作為組織工程支架的應用
論文名稱(外文):Development of Three Dimensional Polyethylene Glycol Based Hydrogel for Tissue Engineering Scaffold Application
指導教授:葉明龍葉明龍引用關係
指導教授(外文):Ming-Long Yeh
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:65
中文關鍵詞:軟骨修復雙光子雷射掃描式顯微鏡聚乙二醇二丙烯酸酯微井脂肪幹細胞組織工程共培養
外文關鍵詞:Cartilage degenerationTwo-photon laser scanning microscopyPoly (ethylene glycol) diacrylateMicro-wellAdipose-derived stem cellTissue engineeringCo-culture
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關節軟骨退化已成為高齡化社會的普遍健康問題。由於缺乏神經及血管分布,導致退化性關節炎難以自我修復。許多治療方式無法重建較能承受應力的透明軟骨,反而快速生成纖維軟骨填補受損區域。回顧過往的研究,治療成果受限於質傳障礙、非正確誘導分化、無法固定細胞在受損處,或是具功能性細胞的數量不足。為了要更加瞭解軟骨的修復過程,需要建立在近似生理環境的監測系統以評估細胞間電訊號及化學訊號的溝通過程。本研究中使用聚乙二醇二丙烯酸酯做成三種工具。採集大鼠之脂肪幹細胞用來評估這三種工具的可行性。第一種是使用雙光子雷射掃描顯微鏡製做出細微圖案在薄膜上,並且成功接枝上胜肽,使細胞可以部份貼附在材料表面。第二種是微井結構,可以承載數量多且族群小的細胞。細胞在微井結構中進行完刺激後,便可以被微井包覆並脫離載台,以進行植入步驟。第三種是為了軟骨組織工程開發出的合理設計支架,該支架包覆的細胞在共培養策略下可以成功被誘導軟骨化,表現出軟骨細胞特有的蛋白質。
大致而言,由不同製程給予了各自的監測、乘載、誘導分化功能。而選用的脂肪幹細胞是理想的自體細胞來源。這三種工具所使用的材料相同,於是未來目標是先完整建立各別工具的評估結果。終極目標是將三種工具結合一起,當組織工程使用時,可以即時監測生長分化情形並給予適當誘導環境之細胞支架。

Articular cartilage degeneration which is hard to repair by itself because of no nerves and blood vessels has become a public health issue since average of lifespan is increasing. Many repairing approaches did not restore the normal hyaline cartilage but fibrocartilage instead. Previous researches were limited by barrier of mass transfer, improper induction sequence, cell anchorage, or few amount of available cells. Buildup of the monitoring systems that simulate physiological conditions enables us to inspect the events of electrical and chemical communications between cells. In this study, we fabricated poly (ethylene glycol) diacrylate (PEGDA) into three kinds of devices by customized mold. PEGDA is hydrophilic, biocompatible and photo-crosslink-able. In addition, adipose-derived stem cells (ASC) were harvested as the standard cells to evaluate whether the systems were functional.
First product was thin film of PEGDA featuring micro-pattern created by two-photon laser scanning microscopy. Surface of the film was grafted RGD peptide so that cells could partially adhere to the surface of polymer. Second product was micro-well which hosted high quantity but small population of cells. The bonding between silane and PEGDA was competed by protein; thus, we were able to control the delamination speed of micro-well from culture stage. The last product was scaffold for cartilage tissue engineering with novel design to reduce barrier of mass transfer. ASC were encapsulated in PEGDA and induced by co-culture strategy to stimulate the protein express which are the characteristic of chondrogenesis. Compared with treatment of exogenous supplement of growth factors, co-culture strategy achieved the same results.
In general, different manufacturing process granted three devices which shared the same material with the abilities to monitor, house, and induce chondrogenesis. ASC is an ideal autologous cell source because it is abundant in quantity and has outstanding differential potential. Future work is to fully utilize these devices to acquire information of regulating chondrogenesis. Ultimate goal is to integrate three devices into tissue engineering scaffold with real-time surveillance on proliferation and differentiation status.

摘要 i
Abstract ii
Acknowledgements iv
Table of contents v
List of tables viii
List of figures ix
Chapter 1 Introduction 1
1.1 Background 1
1.1.1 Compositions and properties of cartilage 1
1.1.2 Cartilage degeneration and approaches of repair 2
1.2 Scaffolds 4
1.2.1 Design of scaffold 4
1.2.2 Natural biomaterials 5
1.2.3 Artificial biomaterials 6
1.3 Two-photon laser scanning microscopy 8
1.4 Micro-well array 10
1.5 Cell Source 10
1.5.1 Differentiated cells of normal tissue 10
1.5.2 Stem cells 11
1.5.3 Bone marrow stem cells 12
1.5.4 Adipose-derived stem cells 12
1.6 Environment 14
1.6.1 Mechanical stimulation 14
1.6.2 Biochemical stimulation 14
1.6.3 Co-culture system 15
1.7 Purpose and specific aims 16
1.7.1 Purpose 16
1.7.2 Aims 17
Chapter 2 Materials and methods 21
2.1 Materials and instruments 21
2.2 Cell culture 24
2.2.1 Harvest of ASC 24
2.2.2 Harvest of chondrocytes 24
2.2.3 Cell maintenance and passage 25
2.3 Gelation of PEGDA 25
2.3.1 Customized mold to load PEGDA solution 25
2.3.1.1 PEGDA film with micro-pattern created by TPLSM 25
2.3.1.2 Micro-well array with controllable delamination hosted cells 25
2.3.1.2 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 26
2.3.2 Parameters to crosslink PEGDA 26
2.4 Mechanical property of PEGDA gel 26
2.5 Cell membrane fluorescent stain-Cell linker 27
2.6 Biochemical evaluation 27
2.6.1 Cell viability assay 27
2.6.2 Western blot 28
2.7 Histological section 29
Chapter 3 Results 30
3.1 PEGDA film with micro-pattern created by TPLSM 31
3.2 Micro-well array with controllable delamination hosted cells 33
3.3 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 35
3.3.1 Geometric structure of scaffold 35
3.3.2 Young’s modulus of PEGDA hydrogel 35
3.3.3 Cell morphology within scaffold 38
3.3.4 Cell viability in hydrogel 39
3.3.5 Subcutaneous implant of hydrogel loaded with cell 41
3.3.5.1 Observation at the surface of PEGDA after implantation 41
3.3.5.2 Fluorescence image of cell within hydrogel 42
3.3.6 Sections of PEGDA hydrogel scaffold 43
3.3.7 Change of protein secretion of ASC upon treatment 47
Chapter 4 Discussion 48
4.1 PEGDA film with micro-pattern created by TPLSM 48
4.2 Micro-well array with controllable delamination hosted cells 48
4.3 PEGDA encapsulate ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 50
4.4 Integration of three tools 57
Chapter 5 Conclusion 59
References 60
Curriculum vitae 66

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