Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3475
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dc.contributor.advisor徐善慧zh_TW
dc.contributor.advisorShan hui Hsuen_US
dc.contributor.author鄭家昇zh_TW
dc.contributor.authorCheng, Jia shengen_US
dc.date2003zh_TW
dc.date.accessioned2014-06-06T05:32:00Z-
dc.date.available2014-06-06T05:32:00Z-
dc.identifier.urihttp://hdl.handle.net/11455/3475-
dc.description.abstractIn the first part of this study, the alkaline phosphatase (ALP) activity and calcification of osteoprogenitor cells after being stimulated with the differentiating reagent were measured. The ALP activity was found to increase significantly after ten days and decrease after eighteen days. The control osteoprogenitor cells without the differentiating reagent were not calcified. The cells with the differentiating reagent were calcified after ten days, and more after eighteen days, as demonstrated by the von Kossa stain. To enhance the cell seeding efficiency in the precision scaffolds, different methods have been compared. By dropping the cell suspensions on the scaffolds, we were able to increase the seeding efficiency to ∼50%. When the precision scaffolds had a concentric circle (i.e. loose interior) structure, the seeding efficiency could reach 66%. In the three-dimensional cell culture, the scaddolds seeded with osteoblasts were placed in a rotational bioreactor with a speed of 6 rpm. After four weeks, more cell proliferation was observed in PLLA freeze-dried scaffolds. Precision scaffolds, on the other hand, had more extracellular matrix. Based on these results, we have established a cell-precision scaffold-bioreactor model for more in-depth study in the future.en_US
dc.description.abstract本研究首先以骨先驅細胞作為研究的主題,評估以分化劑刺激的骨先驅細胞,並鑑定分化程度。實驗結果發現在平面培養分化10天後鹼性磷酸酶ALP活性明顯上升,在10-18天ALP活性有顯著的下降;在von Kossa染色方面,骨先驅細胞未加分化劑刺激者,在培養時並沒有明顯的鈣化情形,但刺激組在10天後即有著明顯的鈣化產生,18天後更為顯著。 在細胞植入支架研究方面,評估細胞植入精密支架方法以解決細胞植入率的問題,發現以滴入法植入細胞的精密支架在不同交錯角度下,有著50%以上的植入率,支架具有同心圓結構時會有更高的植入率,最高達66%。 在三度空間細胞培養方面,以轉速6 rpm進行骨母細胞-支架複合物動態培養4週後,發現冷凍乾燥支架PLLA有較佳的細胞增生現象,而精密支架則有較多的基質分泌。依據本研究之結果可建立細胞-精密支架-生物反應器的模式。zh_TW
dc.description.tableofcontents第一章 文獻回顧………………………………………………………1 1-1骨組織學……………………………………………………………1 1-2骨組織病理學………………………………………………………3 1-3骨重塑作用(Bone remodeling)….……………………………….…4 1-4組織工程…………………………………………………………....7 1-5細胞…………………………………………………………………8 1-6支架………………………………………………………………....9 1-7訊號……………………………..…………….……………..…….10 1-8生物反應器(Bioreactor)….…………………………………….….11 1-9細胞植入密度與植入法.………….…………………..…………..12 1-10總結………………………………………………………………13 第二章 實驗藥品、器材與儀器………………………………………15 2-1實驗藥品………………………………………………..…………15 2-2實驗器材與儀器………………………………………………......18 第三章 實驗方法…………………………………………………..…21 3-1高分子薄膜的製作.………………...…………………………..…21 3-2冷凍乾燥支架製作………………………………………………..21 3-3精密支架製作.…..…………………………………………….…..22 3-4精密支架含水率與孔隙度測定…………………………………..22 3-5膠原蛋白改質.…………………………………………………….23 3-6動物細胞的取得及培養(老鼠)..……………………………..……23 3-6-1藥品配製……………………………………….……………...24 3-6-2新生鼠頭蓋骨骨母細胞萃取…………………………….…...25 3-6-3老鼠的骨髓基質細胞…………………………………………25 3-7細胞植入方法………………………………………………..……26 3-8骨母細胞與骨髓基質細胞體外平面分化………..………………27 3-9生物反應器(動態培養)…………………………………….…….28 3-10細胞數分析(DNA分析)…………………………………………28 3-11膠原蛋白分析(collagen定量)……………………………………29 3-12鹼性磷酸酶Alkaline phosphatase(ALP)之活性測定…………...31 3-13細胞觀察…………………………………………………………32 3-13-1亞甲藍染色觀察……………………………………………..32 3-13-2掃描式電子顯微鏡(SEM)觀察……………………………...33 3-13-3組織切片觀察………………………………………………..33 3-13-4 von kossa 染色……………………………………………...35 3-14機械性質分析……………………………………………………37 第四章 結果討論………………………………………………….….38 4-1材料性質分析……………………………………..….……..…….38 4-2材料薄膜生物相容試…….…………………………...…………..38 4-3精密支架構型析…………………………………...…….………39 4-4精密支架含水率及孔隙度分析…………………………….…….39 4-5動物細胞取得及培養…………………..…………………………40 4-6精密支架植入測試…………………….………………………….41 4-7亞甲藍染色及電子顯微鏡細胞觀察……………………………..43 4-8骨母細胞與骨髓基質細胞體外平面分化………..………………43 4-9 von kossa 染色……………………………………………………45 4-10生物反應器(3D動態培養)……………………………………..46 4-10-1支架外觀……………………………………………………...46 4-10-2生化分析-1:DNA……….……………………………………46 4-10-3生化分析-2:ALP…..…………………………………………47 4-10-4組織切片……………………………………………………47 4-11機械性質分析…………………………………………………..49 第五章 結論…………………………………………………....…51 第六章 參考文獻…………………………………………………53 圖目錄 圖1. 精密支架構型示意圖……………………………………….…57 圖2. 頭蓋骨骨母細胞培養貼附情形………………………………….59 圖3. 骨髓基質細胞2週培養貼附情形……………………………..60 圖4. 不同交錯角度精密支架植入細胞1天後,以亞甲藍染色觀察支架中細胞的分佈情形……………………………..……………..62 圖5. 表不同交錯角度支架植入骨母細胞培養2週後電子顯微鏡觀察…………………………………………………………………63 圖6. 骨母細胞體外平面分化,von kossa’s 染色……………………..65 圖7. 骨母細胞體外平面分化,von kossa’s 染色……………………..66 圖8. 骨髓基質細胞平面分化,von Kossa’s 染色…………………….67 圖9. 骨母細胞植入不同支架,培養4週後支架外觀圖……………....68 圖10. 三種不同支架植入骨母細胞分化靜態培養2週後,H&E染色組織切片圖………………………………………………………71 圖11. 三種不同支架植入骨母細胞分化動態培養2週後,H&E染色組織切片圖………………………………………………………72 圖12 三種不同支架植入骨母細胞分化靜態培養4週後,H&E染色組織切片圖…………………………………………………………73 圖13. 三不同支架植入骨母細胞分化動態培養4週後,H&E染色組織切片圖………………………………………………………....74 表目錄 表1. PCL材料在加工前後分子量分析………………………………56 表2. 骨母細胞測試材料薄膜之生物相容性測試(×104cells)………...56 表3. 不同交錯角度精密支架含水率測試……………………………58 表4. 不同交錯角度精密支架孔隙度測試…………………………….58 表5. 不同交錯角度PCL精密支架對骨母細胞之植入測試…………61 表6. 第一次骨母細胞分化培養測試,ALP活性表現………………64 表7. 第二次骨母細胞分化培養測試,ALP活性表現……………...…64 表8.骨髓基質細胞分化培養測試,ALP活性表現…………………….64 表9. 不同種類支架對骨母細胞分化培養4週生化分析結果……….69 表10. 不同種類支架對骨母細胞分化培養4週生化分析結果………70 表11. 不同支架在動、靜態培養4週後機械性質分析………………..75zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系zh_TW
dc.subject骨先驅細胞zh_TW
dc.subjectosteoprogenitor cellsen_US
dc.subjectALP活性zh_TW
dc.subject精密支架zh_TW
dc.subject動態培養zh_TW
dc.subject生物反應器zh_TW
dc.subjectALP activityen_US
dc.subjectdynamic cultureen_US
dc.subjectprecision scaffoldsen_US
dc.subjectbioreactoren_US
dc.titleEvaluation of cell seeding, differentiation and bioreactor culture for scaffolds manufactured by the fused deposition modelingen_US
dc.title熔融層積成型系統製成精密支架之細胞植入、分化與生物反應器培養zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
Appears in Collections:化學工程學系所
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