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研究生：	陳玟伶 Chen, Wen-Ling
論文名稱：	以聚乳酸/甘醇酸支架結合內皮前驅細胞應用於兔子之骨軟骨再生 Osteochondral regeneration by combining PLGA scaffolding and endothelial progenitor cells in rabbits
指導教授：	葉明龍 Yeh, Ming-Long
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	50
中文關鍵詞：	內皮前驅細胞、聚乳酸甘醇酸、支架、骨軟骨再生
外文關鍵詞：	endothelial progenitor cells (EPCs), Poly-lactic-glycolic acid (PLGA) scaffold, osteochondral regeneration
相關次數：	點閱：201 下載：1
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許多學者與臨床研究已針對骨軟骨修復的治療做許多研究，但治療結果往往形成纖維性組織。本研究探討內皮前驅細胞是否能如實驗假設，藉由內皮前驅細胞增進血管新生並且促進骨軟骨修復。聚乳酸甘醇支架除了能提供受損組織暫時性的力學支撐，其孔洞性構造還能提供內皮前驅細胞貼附的空間。本研究在兔子股骨內上髁(medial femoral condyle)處製造直徑3mm、深3mm圓柱狀的骨軟骨缺陷，在此模型架構下分別比較下列三組實驗在4周與12周後的結果: 結合內皮前驅細胞貼附於聚乳酸甘醇支架組、僅植入聚乳酸甘醇支架組與缺陷組。
4周後病理切片與免疫組織化學染色結果中MMP-13, TGFβ2, 與 TGFβ-3的表現顯示內皮前驅細胞確實在早期骨軟骨修復期間提供了一個較佳的環境。在這樣穩定的復原環境下，結合內皮前驅細胞貼附於聚乳酸甘醇支架組的修復組織型態最終接近於正常骨軟骨，而僅植入聚乳酸甘醇支架組和缺陷組則趨向纖維化組織的結果。本研究以micro-CT觀察組織中骨質密度比例來判斷骨組織修復的結果，其結合內皮前驅細胞貼附於聚乳酸甘醇支架組在12周的結果不僅都高於僅植入聚乳酸甘醇支架組與缺陷組，而且還接近於正常骨組織的骨質密度比例。各實驗組於12周的骨小梁厚度不但明顯高於4周的結果，還高過正常骨組織的骨小量厚度。綜合各組於12周時骨質密度比例和骨小梁厚度的資料顯示骨軟骨缺陷處仍處於修復狀態，所以在後續的研究中將會進行更長時間的觀察。
總體而言，本研究成功地利用內皮細胞貼附於聚乳酸甘醇支架來修復骨軟骨缺損，為一前瞻性修復骨軟骨組織再生的方法。

Treatments for osteochondral lesions have been worldly explored but most outcomes resulted in fibrous tissue regeneration. In our study, we investigated whether endothelial progenitor cells (EPCs) not only increase angiogenesis and but also promote osteogenesis and chondrogenesis as hypothesized. Poly-lactic-glycolic acid (PLGA) scaffold was designed to provide temporary mechanical support in the early recovery and keep implanted autologous EPCs as a carrier during implantation. In this study, a defect hole of 3mm in diameter and 3mm in depth was created on the medial femoral condyle of the rabbit. EPCs seeded PLGA scaffold (EPC-PLGA group) were implanted and compared with PLGA scaffold (PLGA group) implantation and empty defect (ED group) with no treatments in this rabbit model.
Histological results at 4 weeks showed that the presence of EPCs do speed up the early recovery with a better microenvironment containing more cytokines such as MMP-13, TGFβ2, and TGFβ-3 confirmed by immunohistochemical staining. Within the stable development, the osteochondral regenerative regions in EPC-PLGA group present similar compositions and morphologies as normal articular cartilage while ED group and PLGA group present fibrous-tissue-like results. Bone volume/tissue volume (BV/TV) ratio obtained by micro-CT of EPC-PLGA group is higher than ED group and PLGA group at 12 weeks. In particular the BV/TV ratio in EPC-PLGA group is similar to the Sham group. The thickness of trabecular bone (Tb.Th) was obviously higher from 4 week time point to 12 week time point, quantified values of all groups at 12 weeks are higher than the Sham group which demonstrate the regenerative regions were still under recovery and longer time of observation are needed in the future study.
In summary, this study successfully proposes a promising solution for osteochondral regeneration by implanting EPC-PLGA scaffold.

中文摘要...................................................II
Abstract.................................................III
誌謝.......................................................V
CHAPTER ONE INTRODUCTION...................................1
1	Anatomy and Composition of Knee Joints.............1
2	Osteochondritis Dissecans(OCD) and Osteoarthritis........................................	2
3	Cartilage Repair in Tissue Engineering............4
4	Endothelial Progenitor Cells (EPCs)...............7
5	Motivation and Aim................................8
CHAPTER TWO MATERIALS AND METHODS.........................9
1	Ethics Statement..................................9
2	Research Design...................................9
3	PLGA Fabrication and Characteristics Measurement..	10
4	Scanning Electron Microscope......................11
5	Isolation of EPC..................................11
6	Seeding EPCs into PLGA scaffolds..................12
7	Rabbit Model of EPC-attached PLGA Scaffold Implantation- Surgical Procedure..........................14
8	Macroscopic and Histological Evaluations..........15
9	Micro-CT Scanning.................................18
10	Histological Techniques...........................19
10.1	Scaffold Frozen Section...........................19
10.2	Pathological Observation..........................19
10.3	Immunohistochemical Staining......................19
10.4	Statistical Analysis..............................20
CHAPTER THREE RESULTS.....................................21
1	EPC Culture Status................................21
2	The Characteristics of PLGA Scaffold..............22
3	Adherence Confirmation After Seeding EPCs into PLGA Scaffold..................................................23
4	Macroscopic Appearance............................24
4.1	Macroscopic Appearance at 4 weeks.................24
4.2	Macroscopic Appearance at 12 weeks................25
4.3	Quantitative Scores of Macroscopic Appearance.....25
5	Bone Regeneration Assessment by Micro-CT Analysis....................................	26
5.1	Micro-CT Image Analysis at 4 weeks................26
5.2	Micro-CT Image Analysis at 12 weeks...............27
5.3	Quantitative Scores of Micro-CT Images............27
6	Histological Examination..........................29
6.1	Histological Examinations of Normal Cartilage.....29
6.2	Histological Examinations of Each Group...........30
CHAPTER FOUR DISSCUSSION..................................39
1	EPC Culture Status................................39
2	The Functional Role of PLGA Scaffold in Osteochondral Regeneration................................40
3	The Combination of EPCs and PLGA Scaffold.........40
4	Cytokine Expression Triggered by EPCs.............42
5	The Successful Factors of Osteochondral Regeneration in EPC-PLGA Group.........................................43
5.1	Bone Marrow Derived Chondrocytes..................43
5.2	Excellent Microenvironment Promoted by EPCs.......43
CHAPTER FIVE CONCLUSION and FUTURE WORK...................45
CHAPTER SIX REFERENCES....................................46

                                    

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校內：2017-09-06公開
校外：2017-09-06公開

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