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研究生:吳岳修
研究生(外文):Yueh-Hsiu Wu
論文名稱:肌肉來源細胞應用於骨組織工程之研究
論文名稱(外文):Muscle-Derived Cells as the Cell Sources for Bone Tissue Engineering
指導教授:林峰輝林峰輝引用關係
指導教授(外文):Feng-Huei Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:68
中文關鍵詞:骨組織工程明膠材料肌肉來源細胞骨形成蛋白質骨移植材料
外文關鍵詞:muscle-derived cellsbone graftgelatinbone morphogenetic protein 2bone tissue engineering
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目前自體移植骨來源受到限制,手術過程易造成病人傷口疼痛、化膿以及各種複雜因素,另一方面自體移植法其做法類似挖東牆補西牆,當遇到相當大的骨創傷或缺陷勢必無法治療。本研究使用之人工合成明膠/rhBMP-2複合材料,其優點為製造容易、成本低;並具有骨引導、骨誘導、以及生長因子等三大骨形成要素,足以取代自體移植骨,為相當適合之骨移植材料。

由人體取得之間質葉幹細胞 (MSCs) 或是骨母細胞,細胞量少而且生長速率相當緩慢,不容易在體外大量繁殖培養。而培養在二維平面(monolayer)的細胞容易因為非原來三維立體(3D)環境之緣故而改變其細胞型態(phenotype)。由肌肉衛星細胞(satellite cells)分離得來的肌肉來源細胞具有幹細胞自我修復新生、超強增生能力以及具備分化成不同中胚層細胞組織的條件,不論是何種年齡的個體所取出來之肌肉母細胞在體外都可以在短時間內大量培養繁殖。所需的經費以及時間都遠低於培養MSCs或是骨母細胞,充分符合組織工程及臨床手術上的需求。

本研究採用明膠和戊二醛交聯之材料作為具有骨引導作用的支架;在體外培養從大鼠(Wistar rat)舌頭取得之肌肉來源細胞取代MSCs來達到大量繁殖,將細胞以高濃度接種至明膠/rhBMP-2複合材料,誘使其分化為骨母前驅細胞,並同時具有骨誘導之能力。同時將材料培養在特定生物反應器中模擬體內的環境,並加入特定的調控因子(dexamethasone、β-glycerophosphate、L-ascorbic acid和1?25-dihydroxyVitamin D3),使骨母前驅細胞增生及分化。分別收集1、2、3、4星期培養液,測量鹼性磷酸脢 (alkaline phosphate,ALP)以及骨鈣蛋白 (osteocalcin) 兩種骨特異性蛋白質活性,並以H/E和von Kossa組織切片染色確定其增生及分化階段。最後測量osteocalcin的mRNA含量判斷礦化程度,期望能達到取代傳統骨移植手術上使用骨母細胞所衍生的問題,進而使新生骨組織成長之目的。
Autogenous bone graft is regarded as the gold standard for bone graft implantation as it provides three necessary elements for bone regeneration and maintenance:(1)scaffolding for osteoconduction,(2)inductive agents for osteoinduction, and(3)progenitor cells for osteogenesis. However, the availability of autograft is limited and the procedure to harvest the bone tissue is associated with complications, as well as possible donor site morbidity.

Geltain/rhBMP-2 synthetic graft offers an alternative which combines three major bone formation properties in a controlled and effective way that can be used in clinical applications. On the other hand, functional osteoblasts are obtained through bone or bone marrow. These cells are limited in supply, and difficult to obtain. Recently, skeletal muscle cells are used in bone tissue engineering. The method of harvesting muscle cells is relatively easier to perform and less painful to the patient. Moreover, muscle-derived cells have been shown to differentiate into mesenchymal tissues, such as bone and muscle, as well as cartilage. In addition, bone morphogenetic protein 2 has been identified as an inducting agent that can stimulate conversion to the osteogenic pathway.

In this study, porous gelatin material are selected as a biodegradable scaffold, combining rhBMP-2 on gelatin surface as an inductive agent, and seeding with muscle-derived cells from wistar rat as progenitor cells. The resulting bone grafts were cultured in spinnar flask with ?MEM and additives such as dexamethasone, beta-glycerophosphate, L-ascorbic acid and 1? 25-dihydroxyvitamin D3 (as the osteogenic supplements). The materials were harvested at 1, 2, 3, and 4 weeks and H&E stain, von Kossa stain, ALP assay, and osteocalcin assay were performed to confirm the proliferation and differentiation of muscle-derived cells toward osteoblasts.
中文摘要……………………………………………………………I
英文摘要……………………………………………………………II
致謝…………………………………………………………………III
目錄…………………………………………………………………IV
表目錄………………………………………………………………VI
圖目錄………………………………………………………………VII

第一章 緒論……………………………………………………1
1-1 前言……………………………………………………1
1-2 天然骨移植材料………………………………………2
1-3 人工合成骨移植材料…………………………………4
1-4 明膠骨移植材料………………………………………8
1-5 研究動機與目的………………………………………10

第二章 理論基礎………………………………………………11
2-1 骨生成理論……………………………………………11
2-1-1 骨細胞外基質……………………………………….……11
2-1-2 骨母細胞………………………………………….………13
2-1-3 生長因子的影響…………………………………….……15
2-1-4 骨重塑作用……………………………………….………16
2-2 肌肉來源細胞…………………………………………18
2-2-1 肌肉生成…………………………….……………………18
2-2-2 肌肉幹細胞及其潛力…………………………….………20
2-3 肌肉來源細胞之調控…………………………………23

第三章 實驗材料與方法………………………………………26
3-1 明膠材料製備…………………………………………27
3-2 肌肉來源細胞培養……………………………………31
3-3 細胞seeding………………………………………….35
3-4 生物反應器……………………………………………36
3-5 分析方法………………………………………………37
3-5-1 ALP染色法…………………………….…………………37
3-5-2 von Kossa染色法………………………………….……38
3-5-3 組織切片H&E染色法…………………………….………40
3-5-4 組織切片von Kossa染色法…………………………….41
3-5-5 ALP活性分析…………………………………………….41
3-5-6 osteocalcin分析……………………………………….42
3-5-7 rhBMP-2表面接枝分析………………………………….43

第四章 結果與討論……………………………………………45
4-1 Petri-dish培養-成骨活性測試…………………….45
4-1-1 細胞培養觀察……………………………………………45
4-1-2 ALP活性分析....……………………………………….48
4-1-3 ALP染色….....…………………………………………49
4-1-4 von Kossa染色………………………………….………50
4-2 明膠材料分析…………………………………………….…51
4-2-1 明膠材料SEM觀察………………………………….……51
4-2-2 rhBMP-2接枝分析………………………….……………52
4-2-3 明膠基材培養肌肉來源細胞……………………………54
4-3 生物反應器培養-成骨活性測試……………………………55
4-3-1 H&E染色……………………………………….…………55
4-3-2 von Kossa染色………………………………………….58
4-3-3 ALP活性分析………….…………………………………60
4-3-4 osteocalcin活性分析……………………….…………61

第五章 結論……………………………………………………62

第六章 參考文獻………………………………………………63


表目錄

表.1-1 Properties of types of autologous bone grafts…3
表.1-2 Calcium-based bone-graft substitutes in the U.S or Europe…5
表.1-3 Summary of osteoconductive scaffolds…………….7
表.2-1 Systemic regulation of bone remodeling………….17
表.2-2 Isolation and characterization of MDSC……….…22
表.3-1 製備明膠材料使用藥品目錄表………………………….27
表.3-2 實驗分組表……………………………………………….29
表.3-3 細胞培養常用藥品表…………………………………….31
表.3-4 調控細胞添加藥物表…………………………………….32
表.3-5 osteocalcin EIA kit準備藥品表………………………43
表.4-1 rhBMP-2標準濃度吸光值…………………………………52


圖目錄

圖.1-1 戊二醛交聯明膠材料簡圖……………………………….9
圖.2-1 Bone cell development…………………………………14
圖.2-2 Protein expression involving in osteogenesis….15
圖.2-3 Bone remodeling…………………………………………16
圖.2-4 Myogenesis….……………………………………………19
圖.2-5 Relationship of myogenesis and MRFs………………20
圖.2-6 Proposed mechanism of myogenic differentiation...21
圖.2-7 Multi-lineage differentiation of MDSC……………22
圖.2-8 Isolation of muscle-derived cells…………………23
圖.2-9 BMP signaling pathway…………………………………24
圖.3-1 實驗設計流程圖……………………………………….…26
圖.3-2 明膠材料製備流程……………………………………….27
圖.3-3 明膠/rhBMP-2複合物製備流程………………………….28
圖.3-4 孔洞結構明膠材料……………………………………….29
圖.3-5 明膠/EDC/rhBMP-2交聯機制…………………………….30
圖.3-6 肌肉來源細胞取得流程……………………………….…33
圖.3-7 細胞接種….………………………………………………35
圖.3-8 生物反應器簡圖……………………………………….…36
圖.3-9 生物反應器實圖……………………………………….…36
圖.3-10 鹼性磷酸�J染色流程……………………………………37
圖.3-11 鹼性磷酸脢反應原理……………………………………37
圖.3-12 von Kossa染色流程…………………………………….38
圖.3-13 H&E 切片染色流程………………………………………40
圖.3-14 osteocalcin EIA kit流程………………………….…42
圖.3-15 BMP-2 EIA kit流程.……………………………………44
圖.4-1 不同階段肌肉細胞型態觀察…………………………….45
圖.4-2 desmin 與 myogenic關係圖…………………………….46
圖.4-3 pp6培養在不同血清濃度…………………………………47
圖.4-4 pp2~pp6 ALP活性分析……………………………………48
圖.4-5 pp6細胞ALP染色………………………………………….49
圖.4-6 pp6細胞von Kossa染色………………………………….50
圖.4-7 明膠材料SEM照片…………………………………………51
圖.4-8 人類海綿骨 SEM照片…………………………………….51
圖.4-9 BMP-2濃度-吸光值標準曲線…………………………….52
圖.4-10 rhBMP-2 釋放曲線………………………………………53
圖.4-11 SEM 觀察細胞生長情形…………………………………54
圖.4-12 控制組 H&E 切片染色………………………………….55
圖.4-13 實驗組 H&E 切片染色………………………………….57
圖.4-14 控制組 von Kossa 切片染色………………………….58
圖.4-15 實驗組 von Kossa 切片染色………………………….59
圖.4-16 ALP 吸光值………………………………………………60
圖.4-17 osteocalcin 吸光值……………………………………61
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