臺灣博碩士論文加值系統

本研究目的為建立具長效藥物釋放功能之多孔性幾丁聚醣支架，以作為促進皮膚創傷癒合之人工敷料。本敷料乃是透過冷凍乾燥法建立不同孔洞尺寸之幾丁聚醣多孔性支架，藉由不同冷凍溫度及時間探討孔洞大小，以掌控支架內包覆的成纖維細胞生長因子(Fibroblast growth factor‐Basic, bFGF)及轉化生長因子β1(Transforming Growth Factorβ1, TGFβ1)控制釋放特性，最後分別培養小鼠纖維母細胞(Mouse fibroblast cells, L929)與牛頸動脈內皮細胞(Bovine endothelial cells, BEC)探討細胞於支架增生結果，以判別本支架之可行性。研究結果顯示，以冷凍乾燥法製備多孔性支架可透過溫度與時間形成冰晶調控孔徑變化，前者為控制支架的初始孔徑，後者為決定支架最終孔徑，以此變化關係，本研究可製備出平均尺寸為153.25±31.87 um之最大孔洞(Pore I)及34.45±9.40 um之最小孔洞(Pore II)。透過細胞耐受性試驗發現，bFGF濃度0.005至0.1 ng/mL而TGFβ1之濃度為0.005至1ng/mL，有助於L929細胞增生；BEC細胞則在bFGF濃度為0.005 至0.05 ng/mL可被促進增生，在TGFβ1濃度0.01至10 ng/mL則出現抑制現象。藥物控制釋放結果顯示，支架孔隙尺寸為153.25±31.87 um含3.75 ng之成纖維細胞生長因子與尺寸為34.45±9.40 um含400 pg之轉化生長因子β1之釋放特徵可吻合細胞所需的生長濃度。細胞培養結果3.75 ng bFGF‐Pore I可有效促進L929細胞增生；400 pg TGFβ‐Pore II可促進L929增生，而抑制BEC增生。本支架形成bFGF/TGFβ1複合釋放材料時，將有機會控制傷口癒合過程不同種類細胞之增生，以避免特定細胞過度增生。

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by first, pre-freezing at different temperatures (-20 and -80°C) for different periods of time, then freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. It was found that the pre-freezing temperature controls the initial pore sizes, but the final pore sizes are determined by the period of time in pre-freezing. In this study, the scaffolds with the largest pore size was found to be 153.25 ± 31.87 µm and scaffolds with the smallest pores to be 34.45 ± 9.40 µm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL and the concentration for TGFβ1 was 0.005 to 1 ng/mL. In addition, a concentration of 0.005 to 0.05 ng/mL enhanced proliferation in BEC, however, for TGFβ1 it was observed that a concentration of 0.01 to 10 ng/mL reduced its proliferation. Controlled drug release of the growth factors showed that the chitosan scaffolds with pore sizes of 153.25 ± 31.87 µm (Pore I) containing 3.75 ng of bFGF and scaffolds with pore sizes of 34.45 ± 9.40 µm (Pore II) containing 400 pg of TGFβ1 showed results similar to the cell culture analysis. Cell culture of the chitosan scaffold and growth factors show that 3.75 ng of bFGF in Pore I scaffolds can effectively promote L929 cell proliferation; while 400 pg of TGFβ1 in Pore II scaffolds can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing, and will have the ability to control wound healing via regulating the proliferation of different cell types, in order to avoid cell hyperplasia.

摘要 i
Abstract iii
表目錄 viii
圖目錄 ix
第一章 前言 1
1.1 研究背景 1
1.2 研究動機與目的 2
第二章 文獻回顧 3
2.1 皮膚組織 3
2.2 傷口癒合過程 5
2.2.1 炎症期 6
2.2.2 增生期 6
2.2.3 重塑期 7
2.3 傷口敷料之簡介 7
2.3.1傳統敷料 8
2.3.2合成敷料 8
2.3.3生物性敷料 9
2.3.4人工皮膚 9
2.4幾丁聚醣之簡介 11
2.4.1多孔性幾丁聚醣 12
2.4.2幾丁聚醣傷口敷料應用 13
2.5 生長因子 15
2.5.1生長因子對傷口修復影響 15
2.5.2控制釋放 17
第三章 實驗方法與步驟 20
3.1 實驗流程圖 20
3.2 幾丁聚醣多孔支架製備 21
3.3 幾丁聚醣多孔支架孔洞大小量測 22
3.4幾丁聚醣多孔支架控制釋放試驗 22
3.4.1 bFGF與TGFβ1包覆於幾丁聚醣多孔支架 22
3.4.2酵素結合免疫吸附法(ELISA)測量生長因子控制釋放含量 23
3.5細胞培養 25
3.5.1 細胞對bFGF與TGFβ1之耐受劑量量測 26
3.5.2細胞植覆包覆bFGF與TGFβ1幾丁聚醣多孔支架 26
3.5.3細胞增生率 27
3.5.4統計分析 28
第四章 結果與討論 29
4.1幾丁聚醣多孔支架之孔洞探討 29
4.2纖維母細胞與內皮細胞對bFGF與TGFβ1之耐受濃度範圍分析 32
4.2.1 L929及BEC對bFGF之耐受範圍濃度分析 32
4.2.2L929及BEC對 TGFβ1之耐受範圍濃度分析 33
4.3 生長因子之控制釋放變化 34
4.3.1鹼性成纖維生長因子(Fibroblast growth factor-Basic)之控制釋放變化 34
4.3.2轉化生長因子β1 (Transforming growth factor beta)之控制釋放變化 37
4.4 L929及BEC培養於含bFGF與TGFβ1之多孔性幾丁聚醣支架增生影響 40
第五章 結論與未來展望 43
參考文獻 46

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