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研究生:陳永琇
研究生(外文):Yung-Hsiu Chen
論文名稱:幾丁聚醣及其奈米複合材料於皮膚修復之應用
論文名稱(外文):The evaluation of chitosan and its nanocomposites on wound healing applications
指導教授:徐善慧徐善慧引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:108
中文關鍵詞:幾丁聚醣酸性纖維母細胞生長因子創傷敷料幾丁聚醣-奈米銀複合材料
外文關鍵詞:chitosanaFGFwound dressingchitosan-silver nanocomposite
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中文摘要
本研究首先將幾丁聚醣溶液利用冷凍乾燥方法製得具孔洞之塊材,再以戊二醛(glutaradehyde, GA)進行交聯反應製備出具有多孔性海綿狀結構之幾丁聚醣創傷敷料,且含入可持續釋放之酸性纖維母細胞生長因子(acidic fibroblast growth factor, aFGF),測試敷料之吸水率、機械強度、表面型態,隨後進行天竺鼠傷口癒合之動物實驗。並以台鹽膠原蛋白敷料Skin Temp®做為對照組。實驗結果顯示幾丁聚醣創傷敷料之吸水率較對照組為佳,且吸水率及機械強度隨著幾丁聚醣濃度增加而增加,1 wt%幾丁聚醣創傷敷料孔洞大小會由交聯前之150 µm於交聯後變為大約245 µm,而1.5 wt%則變為355 µm。於體內動物實驗結果發現添加aFGF具有顯著刺激傷口修復,同時於動物實驗結果發現,含aFGF之幾丁聚創傷敷料之組織再生速度優於對照組,經組織學也證實在傷口上使用幾丁聚醣創傷敷料含入aFGF明顯加速天竺鼠傷口之上皮化。本研究使用此1 wt%及1.5 wt%幾丁聚醣支架作為皮膚組織工程用途,結果中顯示兩種濃度之支架皆適用於組織工程皮膚之應用開發。接著進一步探討幾丁聚醣奈米複合材料應用的可行性,本研究製備出幾丁聚醣混摻不同尺寸之奈米銀粒子(Ag # 1, 45 nm; Ag # 2, 25 nm)及濃度(Ag # 1, 30-240 ppm; Ag # 2, 10-120 ppm),並於體外實驗測試其複合材料之尺寸、分散性、機械性質、熱性質、表面型態、細胞相容性、自由基清除效率、抗菌性和type I collagen基因表現,經TEM結果得知奈米銀粒子(Ag # 1)粒徑約為45 nm,而(Ag # 2)粒徑約為25 nm,並且當奈米銀粒子(Ag # 1)與(Ag # 2)添加濃度低於120 ppm時,奈米銀粒子皆可均勻分散於幾丁聚醣基材中,但當奈米銀粒子(Ag # 1)濃度增加至240 ppm時,奈米銀粒子出現些微聚集現象,而幾丁聚醣-奈米銀(Ag # 1)與(Ag # 2)複合材料之熱性質與機械性質會隨奈米銀濃度增加而上升,但當奈米銀粒子(Ag # 1)濃度增加至240 ppm時,熱性質與機械性質則會下降,因適量奈米銀粒子(Ag # 1, 60 ppm; Ag # 2 30 ppm)添加於幾丁聚醣基材內部扮演成核劑之角色,導致平滑且均勻分布結晶之形成,進而有較好的自由基清除效率、細胞增生與type I collagen之產生,此外於抗菌測試結果中,抗菌性會隨奈米銀濃度上升而上升,且奈米銀粒子(Ag # 2)之抗菌性會優於奈米銀粒子(Ag # 1)。





關鍵字:幾丁聚醣、酸性纖維母細胞生長因子、創傷敷料、幾丁聚醣-奈米銀複合材料
Abstract
In this study, the porous chitosan sponges scaffold designed for the use of a wound dressing were successfully prepared by employing a combination of freeze drying and GA cross-linking. Furthermore, sustained release of acidic fibroblast growth factor(aFGF)was achieved by incorporated chitosan sponges scaffold. The swelling capacity, mechanical strength, morphology and animal tests of the chitosan wound dressing were investigated. Beside the control used the collagen wound dressing, Skin Temp®, by Taiwan salt biotechnology company. The water absorption and mechanical strength increased with the increased concentration of chitosan. The mean pore size increased from 150µm, the uncress-linked, to about 250-350µm, the cross-linked scaffold. The chitosan sponges containing aFGF showed a significant effect on improving wound healing and tissue repair than control. Histological examination also has demonstrated an advanced epithelialization in wound treated with aFGF incorporated chitosan sponge scaffold in guinea pigs.We also have prepared the nanocomposites of chitosan incorporated with different size (Ag # 1, 45 nm and Ag # 2, 25 nm) and amounts (Ag # 1, 30-240 ppm and Ag # 2, 10-120 ppm) of Ag nanoparticles. The nanoparticle sizes, dispersion, thermal stability, mechanical properties, surface morphology, biocompatibility, free radical scavenging ability, antibacterial properties and type I collagen gene expression of nanocomposites were characterized in vitro. Ag of contents 30-120 ppm (Ag # 1) and 10-120 ppm (Ag # 2) exhibited a homogeneous dispersion in chitosan matrix. However, when the content of Ag increased to 240 ppm, the particles tended to form aggregates. The thermostability and mechanical properties of the nanocomposites increased with Ag contents up to 120 ppm. The small amounts of Ag in the chitosan matrix could act as nucleation site for enhancing the uniform crystallization of the nanocomposites. The better biocompatibility, free radical scavenging effect and type I collagen were associated with the surface morphological change in the presence of Ag.


Key words:chitosan, aFGF, wound dressing, chitosan-silver nanocomposite
目錄……………………………………………………………..…............I
表目錄 .VI
圖目錄……………………………………………………………….......VII
中文摘要………………………………………………………………….IX
英文摘要……………………………………………………………….…XI
第一章 前言……………………………………………..1
1.1 皮膚之結構與功能…………………………………….……………...1
1.2 傷口癒合之機制…………………………………….………….……..4
1.3 傷口敷料之歷史……………………………………….………….…..5
1.4 敷料之種類…………………………………………….……….……..8
1.4.1 薄膜(Films)…………………………….………………….…...8
1.4.2 水凝膠(Hydrocolloids)…………………….………….……….8
1.4.3 水膠(Hydrogels)………………………….…………….…...…9
1.4.4 泡沫材料(Foams)………………………….…………….…….9
1.5 幾丁聚醣之來源與特色……………………….…………………….11
1.6 幾丁聚醣敷料回顧 …………………………….…………………...14
1.6.1 薄膜敷料………………………………….…………………..14
1.6.2 水膠敷料………………………………………………….…..15
1.7 纖維母細胞生長因子………………………………………....….….16
1.8 奈米技術……………………………………………………...….…..19
1.8.1 奈米…………………………………………………...……....20
1.8.2奈米材料………………………………………………...….…23
1.8.3 奈米複合材料………………………………………....……...24
1.9 奈米複合材料於生醫材料之回顧……………………………….….25
1.10 研究目的…………………………………………………….……...28
第二章 實驗藥品、儀器與方法....................................30
2.1幾丁聚醣敷料…………………………………………………....…...30
2.1.1幾丁聚醣敷料之製備……………………………………….…..30
2.1.2幾丁聚醣敷料之膨潤測試……………………………….……..30
2.1.3幾丁聚醣敷料之表面型態分析………………………….……..31
2.1.4幾丁聚醣敷料之機械強度測試………………………….……..31
2.1.4.1壓縮強度測試……………………………….………….31
2.1.4.2拉伸強度測試……………………………….………….32
2.1.5傷口修復動物實驗……………………………………….……..32
2.1.5.1組織切片及染色……………………………….…....….33
2.1.5.2組織切片之製作…………………………………..….33
2.1.5.3組織染色…………………………………………..….33

2.2幾丁聚醣支架於皮膚組織工程………………………………….....34
2.2.1幾丁聚醣敷料之製備…………………………………….…....34
2.2.2細胞來源及培養.........................................................................34
2.2.3細胞於幾丁聚醣支架之植覆與培養……………………...…..36
2.2.4細胞數分析.................................................................................36
2.3幾丁聚醣-奈米銀複合材料…………………………………...…….37
2.3.1幾丁聚醣與幾丁聚醣-奈米銀複合材料薄膜之製備……..….37
2.3.2幾丁聚醣-奈米銀複合材料之TEM分析………………..…..38
2.3.3幾丁聚醣與幾丁聚醣-奈米銀複合材料之熱性質分析…..….38
2.3.4幾丁聚醣與幾丁聚醣-奈米銀複合材料之機械性質分析…...39
2.3.5幾丁聚醣與幾丁聚醣-奈米銀複合材料之表面微結構分析...39
2.3.5.1 場發射掃描式顯微鏡之分析......................................39
2.3.5.2 原子力顯微鏡之分析..................................................39
2.3.6幾丁聚醣與幾丁聚醣-奈米銀複合材料之細胞相容性測試...40
2.3.7 MTT 細胞分析方法……………………………………….....42
2.3.8幾丁聚醣與幾丁聚醣-奈米銀複合材料之自由基清除能力測定…………………………………………………………….…….…42
2.3.9 Type I collagen之基因表現......................................................43
2.3.10 抗菌測試……………………………………………………..46
2.3.11數據之統計分析………………………………………………46
第三章 結果…………………………………………..47
3.1幾丁聚醣敷料………………………………………………….……47
3.1.1幾丁聚醣敷料之製備…………………………………….……47
3.1.2幾丁聚醣表面型態之分析……………………………….……47
3.1.3膨潤測試……………………………………………….………47
3.1.4機械強度測試………………………………………….………48
3.1.5動物實驗………………………………………………….……49
3.2幾丁聚醣支架於皮膚組織工程……………………………….……51
3.2.1幾丁聚醣支架於細胞之植覆與培養……………………….…51
3.3幾丁聚醣-奈米銀複合材料………………………………...…….…51
3.3.1幾丁聚醣-奈米銀複合材料之TEM分析………………….…51
3.3.2幾丁聚醣與-奈米銀複合材料熱性質與機械性質分析……....52
3.3.3幾丁聚醣與幾丁聚醣-奈米銀複合材料之表面微結構分析....54
3.3.4幾丁聚醣與幾丁聚醣-奈米銀複合材料之細胞相容性測試....55
3.3.5幾丁聚醣與幾丁聚醣-奈米銀複合材料之之自由基清除能力測定………………………………………………………………56
3.3.6 Type I collagen之基因表現......................................................56
3.3.7抗菌測試.....................................................................................57
第四章 討論……………………………………...……58
4.1幾丁聚醣敷料………………………………………………….…….58
4.2幾丁聚醣支架於皮膚組織工程………………………………….….63
4.3幾丁聚醣-奈米銀複合材料………………………………………….64
4.3.1幾丁聚醣-奈米銀複合材料之TEM微結構分析……….….....64
4.3.2幾丁聚醣-奈米銀複合材料熱性質與機械性質分析……….....65
4.3.3幾丁聚醣-奈米銀複合材料之表面微結構分析…………...…..67
4.3.4幾丁聚醣-奈米銀複合材料之細胞相容性測試…………...…..69
4.3.5幾丁聚醣-奈米銀複合材料之之自由基清除能力測定…..…...70
4.3.6 Type I collagen之基因表現........................................................71
4.3.7抗菌測試.......................................................................................73
第五章 結論…………………………………………....74
參考文獻……………………………………………..…75
表目錄
表1 為不同動物之間表皮層與真皮層厚度之差異……….………..…..3
表2 濕潤的傷口癒合優點………………………………….……….…...7
表3 閉合敷料之優點………………………………….………….….…..7
表4 各個敷料種類之優缺點……………………….…………….….…..9
表5 幾丁聚醣之特性及醫療之應用…………….……………………...13
表6 幾丁聚醣之之應用………………………………….………….…..13
表7 奈米尺寸下展現之基本物理效應……………………….…….…..21
表8 小尺寸效應下引起物質性質之改變………………………..……..22
表9 奈米材料之零維、一維及二維之差異…………………………….23
表10 奈米複合材料之基本性能……………………………….…..……25
表11 PCR primer…………………………………………………...……45
表12 為幾丁聚醣與幾丁聚醣-奈米銀(Ag # 1)複合材料之熱穩定性質與機械性質………………………………………………………..….97
表13 為幾丁聚醣與幾丁聚醣-奈米銀(Ag # 2)複合材料之熱穩定性質與機械性質…………………………………………………..……….97
表14 幾丁聚醣和幾丁聚醣-奈米銀複合材料之AFM表面析..…..…100


圖目錄
圖1 皮膚結構圖………………………………………………….……....1
圖2 幾丁質與幾丁聚醣之結構………………………………………....12
圖3 不同幾丁聚醣濃度所製備之創傷敷料外觀…………....................83
圖4 幾丁聚醣1 wt%及1.5 wt%之支架未交聯與交聯於SEM之表面型態..……………………………………………………………….....83
圖5 為0.5 wt%、1 wt%、1.5 wt%之幾丁聚醣創傷敷料與對照組之吸水率………………………………………………………………...84
圖6 敷料之壓縮模數…………………………………………………....84
圖7 敷料之儲存模數……………………………………………………85
圖8 含有不同aFGF量(0,10,50,100 μg)之對照組於動物實驗傷口修復觀察之照片……………………………………………..….86
圖9 含有不同aFGF量(0,10,50,100 μg)之對照組之傷口修復速率………………………………………………………………...…87
圖10 含有不同aFGF量(0,10,50,100 μg)之1 wt%幾丁聚醣創傷敷料於動物實驗傷口修復觀察之照片………………………...88
圖11 含有不同aFGF量(0,10,50,100 μg)之1 wt% 幾丁聚醣敷料之傷口修復速率……………………………………………...…89
圖12 未含aFGF之幾丁聚醣創傷敷料與對照組對傷口修復之影響..90
圖13 含aFGF之幾丁聚醣創傷敷料與對照組在第7天之傷口修復速率之影響……………………………………………………………...90
圖14 幾丁聚醣創傷敷料與對照組傷口修復14天後之組織切片圖…91
圖15 纖維母細胞於1 wt%及1.5 wt%幾丁聚醣支架之增生…………94
圖16 幾丁聚醣-奈米銀(Ag # 1)複合材料之TEM圖……………….…95
圖17 幾丁聚醣-奈米銀(Ag # 2)複合材料之TEM圖………………….96
圖18 幾丁聚醣與幾丁聚醣-複合材料之表面微結構分析…………….98
圖19 幾丁聚醣和幾丁聚醣-奈米銀複合材料之AFM表面微結構…..99
圖20 人類包皮纖維母細胞於幾丁聚醣和幾丁聚醣-奈米銀(Ag # 1)複合材料之吸附與增生……………………………………………….101
圖21 人類包皮纖維母細胞於幾丁聚醣和幾丁聚醣-奈米銀(Ag # 2)複合材料之吸附與增生…………………………………………...…..102
圖22 幾丁聚醣-奈米銀(Ag # 1)複合材料之自由基清除分析………..103
圖23 幾丁聚醣-奈米銀(Ag # 2)複合材料之自由基清除分析………..104
圖24 幾丁聚醣-奈米銀(Ag # 1)複合材料之Type I collagen之基因表現.....................................................................................................105
圖25 幾丁聚醣-奈米銀(Ag # 2)複合材料之Type I collagen之基因表現.....................................................................................................106
圖26. 幾丁聚醣-奈米銀(Ag # 1)複合材料之抗菌性分析.....................107
圖27. 幾丁聚醣-奈米銀(Ag # 2)複合材料之抗菌性分析.....................108
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