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研究生:王上達
研究生(外文):Shang-Ta Wang
論文名稱:幾丁聚醣與甘油磷酸鹽混合之溫敏型水膠做為傷口敷料之特性探討
論文名稱(外文):Study of Properties and Applications of Thermosensitive Hydrogel Based on Chitosan and ��-glycerophosphate for Wound Dressing
指導教授:陳榮輝陳榮輝引用關係蔡敏郎蔡敏郎引用關係
指導教授(外文):Rong-Huei ChenMin-Lang Tsai
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:139
中文關鍵詞:幾丁聚醣溫度敏感型水膠燒燙傷口敷料活體試驗
外文關鍵詞:chitosanthermosensitive hydrogelwound dressingin vivo study
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本論文之研究主題為探討不同去乙醯程度(DD)之幾丁聚醣與甘油磷酸鹽(GP)混合形成之水膠,其做為傷口敷料之不同特性。以魷魚軟骨經由酸鹼處理而得到之��-幾丁質,利用不同條件之熱鹼處理得到四種不同DD (73%、81%、89%、95%)及分子量(453、426、361、331 kDa)之幾丁聚醣。將製備完成之幾丁聚醣溶於醋酸溶液,混合��-GP形成溫度敏感型之水膠溶液。本研究以此一水膠系統進行物性量測,並先以體外評估其做為傷口敷料之可行性後,選出較為適當之組別進行燒燙傷敷料之活體動物實驗,觀察其應用於燒燙傷傷口敷料上之特性。
將水膠溶液進行成膠溫度測定,結果顯示,其成膠溫度隨��-GP於溶液中濃度之上升,而有下降之趨勢。DD以及分子量皆會對成膠溫度造成影響,但無一定之規律可循,推測此兩種因子間存在交互作用,決定此一膠體系統之物理性質。另外,DD 81%、95%兩組別分別搭配��-GP 6%、8%時,其成膠溫度為36 oC,接近人體體溫。以流變儀測量水膠溶液隨溫度改變之黏度變化情形,可發現此一水膠於相轉換時,先有一黏度緩升之情形,至一定溫度點後則急遽上升,可得知此一水膠系統之成膠為短時間且急速之反應。
以掃瞄式電子顯微鏡觀察水膠之切面結構,可得知隨幾丁聚醣DD之改變,水膠會產生不同型態之立體孔洞結構。細胞存活率之結果顯示各組成之水膠皆無明顯之細胞毒性。水膠系統之水分穿透率介於6272.5-11160.3 g m-2 d-1之間,顯示其適宜做為燒燙傷之傷口敷料。
將DD 81%幾丁聚醣搭配6% ��-GP濃度(H81-6)與DD 95%幾丁聚醣搭配8% ��-GP濃度(H95-8)做為實驗組別,以活體動物模式進行燒燙傷敷料之評估。結果顯示,敷以幾丁聚醣/��-GP水膠之實驗組別其傷口回復有良好情形。H95-8實驗組別其傷口於第6天時面積恢復至起始值的52%,相較其他組別有顯著之差異性,顯示此一水膠做為傷口敷料可加速傷口初期之癒合。復原後28日各實驗組別傷疤面積相對於起始值分別為control:20.5%、H81-6:18%、H95-8:10.7%、正控制組(silver nitrite)組別:14.5%。H95-8之水膠組別實驗結果較其他各組為低,有顯著差異,顯示此一水膠做為傷口敷料有降低疤痕面積之功效。
組織切片以HE染色觀察可發現此一水膠系統可促進傷口組織之再上皮化,其角質層再生於第16天後可明顯觀察出。再生後之組織也較控制組為緻密且完整。以免疫組織化學染色法將傷口組織染色則可觀察出此一敷料可降低傷口過度之發炎反應,減少伴隨燒燙傷而生之疼痛感,並可縮短組織癒合時間,於癒合後抑制疤痕組織的生成。
The purpose of this thesis is to precede a feasibility study of application of chitosan/��-glycerophosphate (C/��-GP) thermosensitive hydrogel in burn wound dressing. ��-chitin had been purified by demineralization, deproteinization and decoloration from squid pen. For preparing four different degrees of deacetylation (73%, 81%, 89%, 95%; DD73, DD81, DD89, DD95) of chitosan, ��-chitin was treated by sodium hydroxide solution at 100oC in various times. Chitosan samples were mixed with different concentration of ��-glycerophosphate solution in order to prepare the hydrogel solution.
Gelling temperature (Tg) of the hydrogel was measured. The result indicated that the rise in the concentration of ��-glycerophosphate of this hydrogel system induced the reducing of Tg significantly. Degree of deacetylation (DD) and molecular weight (Mw) is also the key factors that influenced Tg, but the effect is not regular. Tg of DD81/6% ��-GP (H81-6) and DD95/8% ��-GP (H95-8) hydrogel groups is both in 36 oC that is similar to human body temperature. The viscosity of this hydrogel was measured by rheometer while using a cone/plate measuring system. The results were the demonstration that referred the sol-gel transition of this hydrogel system was a rapid reaction. Scanning electron microscope (SEM) graphs showed that the hydrogel present a three-dimensional porous structure which acts as the water vapor barrier. Diverse micro-structure of hydrogel made by different DD of chitosan has also been observed. Water vapour transmission rate (WVTR) of the hydrogel system was evaluated over 6h. WVTRs between 6272.5 to 11161.9 g m-2 d-1 that were reported suitable to apply in wound dressing in further research were shown in this experiment. On the other hand, cell cytotoxicity of this formulation in fibroblast was measured by MTT assay. C/��-GP hydrogel were non-toxic for human skin fibroblast and could enhance its proliferation.
Based on the data of futher study, two hydrogel groups that is DD81/6% ��-GP (H81-6) and DD95/8% ��-GP (H95-8) had been selected to use as burn wound dressing when proceed an in vivo study. H95-8 group provided a significant lower wound surface area then other groups in the 6th day. The scar surface area percentage compared to initial wound area data in the 28th day showed that negative control: 20.5%, H81-6 group: 18%, H95-8 group: 10.7%, positive control (silver nitrite): 14.5% respectively. These results indicated that the 95-8 hydrogel enhanced the initial healing rate of wounds and may inhibit the growth of scar tissue.
Histology study also demonstrated the inference of further research. Micrographs of wound tissue treated by HE stain were observed. A significant increase in re-epithelialization was represented in day 16 for H95-8 group versus those untreated wounds. In immunohistochemistry study, we discovered that the H95-8 hydrogel dressing regulated the inflammation reaction of wound and exhibit an reducing of activity of scar tissue cell.
摘要..............................................4
英文摘要…………………………………………………………………6
目錄 …8
1. 前言..............................................15
2. 文獻整理................................................19
2.1. 燒燙傷.........................................19
2.1.1. 燒燙傷程度之分級...............19
2.1.2. 深度燒燙傷之傷口處理.................20
2.1.2.1. 自體移植………………..………………………………20
2.1.2.2. 非自體移植…………………..…………………………20
2.1.2.3. 合成組織………………………….......21
2.1.2.4. 類細胞內基質結構材料………………………………..21
2.1.2.5. 合成傷口敷料................................21
2.2. 皮膚.........…………………………...22
2.3. 皮膚傷口癒合過程………………………………………………24
2.3.1. 止血期...................................25
2.3.2. 發炎期…………………………………………………….….25
2.3.3. 增生期........................................26
2.3.4. 重塑及結疤期....................................28
2.3.5. 影響傷口癒合之因素.......................…28
2.4. 傷口敷料………………………………………………………….30
2.4.1. 傷口敷料的定義與要求……………………………………..30
2.4.2. 傷口敷料的種類……………………………………………..31
2.4.2.1. 傳統型敷料……………………………………………...32
2.4.2.2. 聚胺酯敷料……………………………………………...32
2.4.2.3. 水凝膠敷料……………………………………………...32
2.4.2.4. 膠態敷料………………………………………………...32
2.4.2.5. 生物高分子敷料………………………………………...33
2.4.3. 傷口敷料市場現況與未來發展……………………………..34
2.5. 水膠………………………………………………..……………...35
2.5.1. 酸鹼敏感型水膠……………………………………………..36
2.5.2. 溫度敏感型水膠…………………………………………..…37
2.5.3. 其他智慧型水膠……………………………………………..38
2.5.4. 幾丁聚醣/聚醇磷酸鹽或醣磷酸鹽水膠系統…………….…39
2.6. 幾丁聚醣簡介…………………………………………………….40
2.6.1. 幾丁質與幾丁聚醣的發現……………………………...…...40
2.6.2. 幾丁質與幾丁聚醣的分佈……………………………...…...40
2.6.3. 幾丁質與幾丁聚醣的結構…………………………...……...41
2.6.4. 幾丁質與幾丁聚醣的溶解性質………………………...…...41
2.6.5. 幾丁聚醣的特性……………………………………...……...42
2.7. 幾丁聚醣作為傷口覆材…………………………………...….….43
2.7.1. 生物相容性.……………………………………………...…..43
2.7.2. 生物降解性……………………………………………….….44
2.7.3. 免疫刺激作用. ………………………………………...…….44
2.7.4. 凝血作用. ………………………………………………...….45
2.7.5. 加速傷口癒合...................................45
2.7.5.1. 幾丁質及其衍生物對炎症期之影響...........46
2.7.5.2. 幾丁質及其衍生物對增生期之影響............46
2.7.5.2.1. 角質細胞..............................46
2.7.5.2.2. 血管內皮細胞...........................47
2.7.5.2.3. 纖維母細胞..............................47
2.7.5.3. 幾丁質及其衍生物對重塑期之影響..............48
2.7.5.3.1. 傷口收縮..............................48
2.7.5.3.2. 疤痕組織................................48
2.7.6. 抑菌性.........................................48
2.8. 幾丁聚醣溫敏型水膠做為傷口覆材之評估.…............50
3. 實驗流程………………………………………………………...…….51
4. 實驗材料與方法………………………………………………...…….52
4.1. 材料………………………………………………………...……..52
4.1.1. 材料來源………………………………………………...…...52
4.1.2. 溶劑與藥品………………………………………...………...52
4.2. 實驗方法……………………………………………………...…..54
4.2.1. 幾丁聚醣之製備……………………………………...……...54
4.2.1.1. 幾丁質之製備……………………………………...……54
4.2.1.2. 幾丁聚醣之製備……………………………………...…54
4.2.2. 幾丁聚醣之物性測定………………………………...……...54
4.2.2.1. 幾丁聚醣去乙醯度之測定……………………...………55
4.2.2.2. 幾丁聚醣分子量之測定……………………………...…55
4.2.3. 幾丁聚醣溫敏型水膠之製備及特性分析.........56
4.2.3.1. 幾丁聚醣溫敏型水膠之製備…………………...………56
4.2.3.2. 幾丁聚醣溫敏型水膠成膠溫度之測定....…....57
4.2.3.3. 幾丁聚醣/�狴怐o磷酸鹽溫敏型水膠隨溫度變化之流變性質測定…………………...................…………………………...…57
4.2.3.4. 幾丁聚醣溫敏型水膠孔洞性質之分析……….…...58
4.2.4. 幾丁聚醣溫敏型水膠做為傷口敷料之體外評估……..……58
4.2.4.1. 幾丁聚醣溫敏型水膠對於人類皮膚纖維母細胞存活率之測定……………………………………............…………...…58
4.2.4.1.1. 實驗原理………………………………………...….58
4.2.4.1.2. 人類皮膚纖維母細胞(CCD-966SK cells)之培養.................................................58
4.2.4.1.3. 細胞之繼代培養………………………………...….58
4.2.4.1.4. 細胞解凍與保存………………………………..…..60
4.2.4.1.5. MTT assay……………………………..….60
4.2.4.2. 幾丁聚醣溫敏型水膠之水蒸氣穿透率測定………..….61
4.2.5. 幾丁聚醣溫敏型水膠做為燒燙傷傷口覆材之有效性及發炎反應評估……………………………………………..…..............…….62
4.2.5.1.傷口敷料之活體動物試驗…………………………….…62
4.2.5.1.1. 活體動物試驗傷口製造與處理…………….……...62
4.2.5.1.2. 傷口與疤痕面積之量測…………………….……...63
4.2.5.2.組織切片實驗…………………………………….………63
4.2.5.2.1. 灌流…………………………………………………64
4.2.5.1.2. 冷凍組織切片………………………………………64
4.2.5.1.3. HE染色………………………………………………64
4.2.5.1.4. 免疫組織化學染色………………………………....65
5. 實驗結果與討論………………………………………………………66
5.1. 幾丁聚醣之原料製備………………………………………….66
5.2. 幾丁聚醣溫度敏感型水膠之成膠溫度…………………….…67
5.2.1. ��-甘油磷酸鹽之濃度效應………...……....68
5.2.2. 幾丁聚醣去乙醯程度及分子量之效應…………………..69
5.3. 幾丁聚醣溫敏型水膠於不同溫度下之黏度變化…………….71
5.3.1. 不同去乙醯程度之組別……………………………….….71
5.3.2. 不同濃度之甘油磷酸鹽之組別…………………………..72
5.4. 幾丁聚醣溫敏型水膠之切面孔洞性質觀察……………….…74
5.4.1. 不同去乙醯度實驗組別之切面觀察……………………..74
5.4.2. 不同放大倍率之切面觀…………………………………..75
5.5. 幾丁聚醣溫敏型水膠對於人類皮膚纖維母細胞(CCD-966SK)之存活率測定…………………………………………….……76
5.5.1. 幾丁聚醣去乙醯程度之效應……………………………..76
5.5.2. ��-甘油磷酸鹽濃度之效應………………………...………78
5.6. 幾丁聚醣溫敏型水膠之水蒸氣通透率……………………….78
5.7. 幾丁聚醣溫敏型水膠於37 oC下之黏度測定……………...….79
5.8. 幾丁聚醣溫敏型水膠應用於燒燙傷敷料之活體動物實驗.....81
5.8.1. 傷口癒合巨觀之實驗結果………………………………..81
5.8.1.1. 傷口復原情形之觀察....................81
5.8.1.2. 傷口復原及疤痕面積之觀察....................84
5.8.2. 傷口癒合微觀之實驗結果.........................87
5.2.8.1. 組織切片HE染色之觀察......................87
5.2.8.2. 組織切片免疫組織化學染色之觀察...........89
5.2.8.2.1. 巨噬細胞之觀察及發炎反應之評估.........91
5.2.8.2.1. 血管內皮生長因子之觀察..............91
6. 結論……………………………………………………………………93
7.參考文獻………………………………..………………………………94
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