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論文中文名稱:探討低頻電磁場對成骨組織和刺激發炎反應之影響 [以論文名稱查詢館藏系統]
論文英文名稱:The Effect of Low-frequency Electromagnetic Fields on Stimulating Inflammatory Reaction and Bone Tissue Engineering [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:97
出版年度:98
中文姓名:林佑任
英文姓名:Yu-Jen Lin
研究生學號:96688005
學位類別:碩士
語文別:中文
口試日期:2009-07-29
論文頁數:61
指導教授中文名:林忻怡
指導教授英文名:Hsin-Yi Lin
口試委員中文名:蔡偉博;黃聲東
口試委員英文名:Wei-Bor Tsai;Sheng-Tung Huang
中文關鍵詞:電磁場骨母細胞幾丁聚醣支架發炎反應
英文關鍵詞:Low-frequency pulsed electromagnetic fieldosteoblastchitosan scaffoldmacrophage inflammatory reaction
論文中文摘要:低頻電磁場(low-frequency electromagnetic field, EMF)常被應用在臨床上治療骨不癒合。超過1公分以上的骨缺損目前尚無法以電磁場做有效治療而傾向以組織工程的方式將具有生物相容性多孔的支架植入缺損處,誘導細胞長入缺陷,加速組織修復。植入手術後引起ㄧ連串發炎反應,導致纖維母細胞引入常使骨組織修復不完整。本研究探討電磁場對發炎反應之刺激作用及其後續對生長在細胞支架上的骨母細胞之生長及分化之影響。
將骨母細胞植入在幾丁聚醣多孔狀細胞支架上與巨噬細胞共同培養,利用脂多醣刺激巨噬細胞引起發炎反應後,照射9小時的電磁場(強度:18-30 Gauss、頻率:75 Hertz、脈衝寬:1.3 milliseconds、振幅:3.5±1 millivolt)。在照射後立即與照射後7天測量支架上骨母細胞之活性、增殖、分化、鹼性磷酸
論文英文摘要:Low-frequency Pulsed Electromagnetic Field (EMF) has been proved by the FDA to be used in clinical applications to treat non-union bone fractures and to release pain. Bone defects over the size of 1cm usually can’t be repaired by EMF alone and requires the used of tissue engineered scaffolds. Implanting osteogenic porous scaffolds can induce bone cell growth and promote tissue repair. However, surgical processes cause inflammatory reactions and macrophages, one of the non-specific inflammatory cells, are known to bring in fibroblast cells to the surgical sites. Fibrous tissue around implants can impede implant/tissue integration and compromise the function of implant later.
We propose to study the effects of EMF on the behaviours of macrophages during inflammation and how the effects influence the subsequent bone repair. Bone cells (7F2 osteoblast) were seeded on chitosan porous scaffolds and co-cultured with macrophage (raw264.7). Lipopolysaccharide was added to the culture medium to induce macrophage to release NO and inflammatory cytokines. The co-culture was exposed to electromagnetic field (18-30 Gauss, 75Hz, impulse width 1.3 ms) for 9 hours. Immediately and seven days after EMF exposure, osteoblast cell viability, proliferation, gene expressions (type I collagen, osteocalcin), ALP activity were measured. NO release from macrophage were measured as well.
Our results showed the concentrations of NO were lower in control than the EMF group at both time points. Osteoblasts in the EMF group had higher proliferation rate and cell viability but lower ALP activity compared to the control group. The results showed that thought EMF did not suppress the inflammatory reactions of macrophages in the co-culture, it did accelerate growth and maturation of osteoblasts with the presence of inflammatory factors. The in vivo effects of EMF suppressing pain and inflammation may not begin with macrophages but other physiological pathways.
論文目次:中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 文獻探討 2
1.2.1 生醫工程在成骨組織之應用 2
1.2.1.1 成骨組織之修復作用 2
1.2.1.2 幾丁聚醣在生醫工程之應用 5
1.2.2 電磁場在骨修復之應用 8
1.2.3 發炎反應 10
1.2.3.1 發炎反應與巨噬細胞 10
1.2.3.2 脂多醣體(LPS)與巨噬細胞之活化 10
1.2.3.3 一氧化氮NO 12
1.3 研究目的 13
1.4 研究架構 14
第二章 實驗材料與方法 15
2.1 實驗材料 15
2.1.1 實驗細胞株 15
2.1.2 實驗藥品 15
2.1.3 實驗儀器 20
2.2 實驗方法 21
2.2.1 幾丁聚醣支架的製備 21
2.2.2 電磁場產生器 22
2.2.3 細胞株培養 23
2.2.4 一氧化氮(NO) 24
2.2.5 DNA含量 25
2.2.6 細胞活性(MTT assay) 25
2.2.7 鹼性磷酸酶活性(ALP activity) 26
2.2.8及時定量反轉錄聚合酶連鎖反應(qPCR) 26
2.2.9電子式顯微鏡 (SEM) ....29
2.2.10統計分析 29
第三章 結果 30
3.1 幾丁聚醣多孔型支架 30
3.1.1 幾丁聚醣多孔型支架外觀 30
3.1.2 以SEM觀察幾丁聚醣多孔型支架 31
3.2 低頻電磁場對發炎反應之影響 32
3.2.1 發炎反應 32
3.2.1.1 LPS對巨噬細胞與骨母細胞之影響 33
3.2.1.2 LPS濃度對發炎反應之影響 34
3.2.1.3 LPS作用時間對發炎反應之影響 36
3.2.2 低頻電磁場對發炎反應之影響 38
3.3 發炎反應下低頻電磁波對成骨組織之影響 40
3.3.1 一氧化氮(NO assay) 40
3.3.2 DNA含量(DNA assay) 41
3.3.3 細胞活性(MTT assay) 42
3.3.4 鹼性磷酸酶活性(ALP activity) 43
3.3.5 同步定量聚合酶連鎖反應(Realtime PCR assay) ..44
第四章 討論 46
參考文獻 50
附錄
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