蔡偉博臺灣大學：化學工程學研究所王鵬元Wang, Peng-YuanPeng-YuanWang2007-11-262018-06-282007-11-262018-06-282006http://ntur.lib.ntu.edu.tw//handle/246246/52210本研究的目的在探討，短期的連續機械壓縮與長期的間歇機械壓縮，對於關節軟骨組織工程的影響。我們以冷凍-凝膠法(freeze-gelation method)製作出彈性(elastic)的孔洞幾丁聚醣-明膠(chitosan-gelatin)鷹架，此鷹架的孔隙度(porosity)約為95％，孔洞直徑(pore size)約為25-50 μm。 我們將出生一週的紐西蘭大白兔關節軟骨取下，分離細胞後於體外初級培養(primary culture)至第二代之後，植布於彈性鷹架中。一天後，細胞呈現圓形的貼附，細胞數量最多可達到初始種植數量的二分之一。靜態培養兩周後，細胞形態依然呈現圓形的貼附，數量亦無顯著的增加。在壓縮實驗中，我們將細胞於彈性鷹架上培養三天後，放入自行設計之可壓縮生物反應器中，進行動態的機械壓縮刺激。壓縮的振幅設為40％，頻率設為0.1 Hz。經過短期連續的壓縮刺激後，利用反轉錄酶連鎖反應(RT-PCR)分析細胞的第一型、第二型膠原蛋白與Aggrecan的基因表現；經過長期間歇的壓縮刺激後，利用Hoechst 33258螢光法定量細胞數、二甲基亞甲基藍法(1,9-Dimethylmethylene-blue, DMMB)定量total GAGs含量、氯胺法(Chloramine T)定量膠原蛋白(total Collagen)含量。 結果顯示，短期連續的機械壓縮，能快速的影響軟骨細胞的基因表現。在壓縮三小時後，三種基因的表現量均向上增加，其中，第一型膠原蛋白與Aggrecan表現可持續增加至六小時。壓縮九小時後，三種基因均與未壓縮的複合物無明顯差異。長期間歇的機械壓縮，在壓縮刺激三週後，複合物中細胞數與GAGs的含量有明顯的增加，但是對於膠原蛋白的含量則無影響。經過本研究的評估後，物理的壓縮刺激與彈性鷹架系統，對於軟骨組織工程確實有著顯著的影響。The aim of this study is to estimate the effects of dynamic mechanical compression, inculding short-term continuous and long-term intermittent compression, on articular cartilage tissue engineering. The porous elastic chitosan-gelatin scaffold was produced by freeze-gelatin method. The porosity of scaffold is around 95% and pore size is between 25 to 50 μm. The seeded cells were isolated from New Zealand whit rabbit knee cartilage and primary cultured until passage two. After one day cultured, the cell can adhere to the scaffold but the amount of the cells only half of original seeded number. Sequentially, one week culture, the amount of the cells just increases slightly. In the compression studies, constructs were pre-cultured for three days. After pre-culture, constructs were puted into the mechanical compression bioreactor and stimulated by dynamic mechanical compression. The amplitude of compression is 40% and frequency is 0.1 Hz. After the continue-short-term stimulation, using reverse transcription polymerase chain reaction (RT-PCR) to analyze the level of type I collagen, type II collagen, and aggrecan gene expression. Furthermore, after the intermittence-long-term stimulation, using Hoechest33258 fluorescence method to quantitative the cells number, 1,9-Dimethylmethylene-blue (DMB) method to quantitative the total GAGs content and Chloramine T method to quantitative the total collagen content. The results show that chondrocytes expression is accessible by mechanical compression during shot-term stimulation. In the short-term study, three kinds gene were increased after three hours compression. The level of type I collagen and aggrecan gene expression is continue increased to sixth hour. After nine hours stimulation, the gene expression is not obvious different from the free-sweling condition. In the long-term study, cells number and total GAGs content were distinct improved by compression during three weeks, but total collagen content is not incresd by compression. In this study, we propose the physical mechanical compression has a huge potential to affect cartilage tissue engineering.誌 謝 i 摘 要 iii Abstrat v 目 錄 vii 圖 目 錄 ix 表 目 錄 xi 中英文對照表 xiii 第一章 緒 論 1 1.1 關節軟骨簡介 1 1.2 關節軟骨的受損與修復 3 1.3 組織工程三要素 5 1.4 幾丁聚醣與明膠 7 1.5 週期性壓縮在關節軟骨組織工程的效應 8 1.6 研究目的 12 1.7 研究架構 13 第二章 實驗材料、儀器與方法 27 2.1 實驗材料 27 2.2 實驗儀器 28 2.3 藥品配置 29 2.4 可壓縮生物反應器介紹 32 2.5 彈性鷹架的製作 37 2.6 細胞初級培養 39 2.7 幾丁聚醣與明膠細胞貼附測試 39 2.8 鷹架細胞植布 40 2.9 掃描式電子顯微鏡 40 2.10 反轉錄聚合酶連鎖反應 (Reverse Transcription Polymerase Chain Reaction, RT-PCR) 41 2.11 DNA定量 44 2.12 GAGs定量 44 2.13 膠原蛋白定量 45 2.14 鷹架機械性質測試 46 2.15 統計分析 46 第三章 結果與討論 47 3.1第二代可壓縮生物反應器設計與製作 47 3.2材料生物相容性測試 49 3.3 彈性鷹架(幾丁聚醣-明膠)的結構與性質 50 3.3.1 鷹架的巨觀性質 50 3.3.2 鷹架的孔洞大小 51 3.3.3 鷹架的機械性質 51 3.3.4 鷹架的孔隙度 53 3.4 鷹架細胞靜態培養增生數量與細胞形態 54 3.5 壓縮實驗 55 3.5.1 短期刺激 － 基因表現 56 3.5.2 長期刺激 － 細胞生長 57 3.5.3 長期刺激 － 胞外間質分泌量 59 第四章 結論 75 第五章 未來計畫 77 第六章 參考文獻 79 附錄 A 基因序列與編號 91 附錄 B 網路文獻 93 附錄 C 軟骨細胞與胞外間質分子示意圖 1012739264 bytesapplication/pdfen-US多孔性幾丁聚醣-明膠彈性鷹架機械壓縮式生物反應器短期與長期週期性機械壓縮porous chitosan-gelatin elastic scaffoldmechanical compression bioreactorshort-term continuous and long-term intermittent mechanical compressionthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52210/1/ntu-95-R93524030-1.pdf