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標題: 以不織布貼附膠原蛋白/玻尿酸/幾丁聚醣複合基質之雙層敷料製備及其生物相容性之研究
Evaluation of biocompatibility of bilayer wound dressing composed of collagen/hyaluronic acid/chitosan complex matrix and non-woven fabric
作者: 王莉芳
Wang, Li-Fang
關鍵字: Bilayer wound dressing
出版社: 動物科學系所
摘要: 皮膚之修復為臨床醫學治療上重要的課題之ㄧ。鑑於深層皮膚缺損於癒合過程中常伴隨疤痕形成(scar formation)及傷口收縮(wound contraction)等影響皮膚正常功能之缺陷,因此諸如自體移植、異種移植及異體移植等治療方式於過去數十年間陸續開發出,雖可顯著提升療效,但卻難以避免來源匱乏、感染及發炎等限制與風險。故現今研究趨向運用組織工程之概念,以天然生物材料為原料,模仿皮膚基質之形狀製備三度空間基質,使細胞依著模型進行修復並生成新的組織,而達到皮膚損傷之修復(repair)或再生(regeneration)。多孔性膠原蛋白基質具有高度的生物相容性及可接受的生物可降解性,因而廣泛應用於傷口敷料及真皮替代物之製備,但其效能卻受限於快速降解及低機械強度等缺陷。 本試驗目的在於應用萃取自雞腳的第一型膠原蛋白、公雞雞冠玻尿酸及商用幾丁聚醣為原料,利用其各自的特殊生理性質,配合冷凍乾燥及1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide(EDC)化學交聯製備膠原蛋白/幾丁聚醣/玻尿酸(Col-Chi-HA)複合基質,並以0.5%明膠黏合不織布於基質上層以建構成雙層敷料。進而探討不同比例的膠原蛋白與幾丁聚醣(10:0、9:1、8:2、7:3、6:4及5:5)對雙層敷料之物化特性與生物相容性之影響。 結果顯示:經由冷凍乾燥及EDC化學交聯可成功製備出多孔性複合基質。以掃描式電子顯微鏡觀察發現,各組雙層敷料之整體結構可區分為上下兩個明顯層次,且各組下層Col-Chi-HA複合基質之平均孔徑約介於138~210μm之間。於膨潤試驗方面,各組Col-Chi-HA複合基質皆可維持結構而無變形發生,且亦能吸收約30~40倍以上的生理緩衝食鹽溶液。而各組Col-Chi-HA複合基質之內部孔徑及膨潤率皆隨幾丁聚醣取代比例之增加而遞減。於抗拉強度方面,以10:0、9:1及8:2三組膠原蛋白對幾丁聚醣比例所製之複合基質有較高之抗拉強度。而Col-Chi-HA複合基質中幾丁聚醣之取代比例與複合基質之熱安定性及酵素耐受性成正相關。將各組複合基質與纖維母細胞共培養進行生物相容性試驗發現,各組複合基質皆能維持細胞良好活性、正常形態並持續增殖,其中以9:1的膠原蛋白對幾丁聚醣比例所製之複合基質(Col9Chi1HA)有最高之細胞生理活性表現。 綜觀以上結果顯示,各組複合基質皆具良好生物相容性,但若綜合減低製備成本、提供促進傷口癒合效用及改善膠原蛋白基質機械強度與降解速率為考量,以Col9Chi1HA複合基質較為理想。因而以不織布貼附Col9Chi1HA複合基質所製之雙層敷料可應用作為未來皮膚創傷敷料的進ㄧ步研究材料。
The skin trauma is one of the oldest problems in surgical field. In the past decades, many studies focused on some skin substitutes such as autografts, xenografts and allografts to treat the full-thickness skin defects. However, these substitutes would not prevent the wound healing from complication of scar formation and wound contraction. Therefore, the normal functions of the skin would be reduced prognosis. For solving the mentioned problems, the biological skin substitutes or biological dressings, which designed by the principle of tissue engineering already extensively developed. Nowadays, collagen-based matrices are considered to be the most promising substitutes. However, the utilization of untreated collagen matrices were limited due to the rapid biodegradation rate and lower mechanical strength. The purpose of this study was to manufacture a bilayer wound dressing consisting of an upper layer of non-woven fabric and sublayer of collagen /chitosan/hyaluronic acid (Col-Chi-HA) complex matrix to improve the low mechanical strength and rapid biodegrading rate of the uncross-linked collagen matrices. The Col-Chi-HA complex matrices were prepared by different ratios of collagen to chitosan (10: 0, 9: 1, 8: 2, 7: 3, 6: 4 and 5: 5) freeze-drying and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) cross-linking method in this study. The physico-chemical properties and biocompatibility of various bilayer wound dressings were characterized by microstructure observation, swelling, tensile strength, thermal stability, resistance of collagenase, MTS assay and the cell morphology observed for a period of coculture. The results showed that the morphology of the bilayer wound dressings significantly exhibited two different layers, and the sublayer had more porous structure and the average of pore diameters ranged from 138μm to 210μm. In swelling, all bilayer dressings could absorb more than 30 to 40 folds of phosphate-buffered saline buffer and maintained their form-stability. The Col-Chi-HA complex matrices mixed by the ratio of 10: 0, 9: 1 and 8: 2 (collagen:chtosan) exhibited the highest tensile strength, but not reached significant difference among them. The resistance of collagenase and thermal stability of various Col-Chi-HA complex matrices were increased by the proportion of chitosan. In biocompatibility experiment, the regular morphology, viability and proliferation of the fibroblasts can be maintained when them cultured with each Col-Chi-HA complex matrix. However, the Col-Chi-HA complex matrix prepared by the ratio of 9: 1 of collagen to chitosan (Col9Chi1HA) exhibited the highest cell viability. In conclusion of these results, we assumed that the 9: 1 mixing ratio of collagen to chitosan may used as the optimal ratio to manufacture complex matrix depending on the results of the physico-chemical properties, biocompatibility, reasonable cost and healing potential. Furthermore, the bilayer wound dresssing composed of non-woven fabric and Col9Chi1HA complex matrix could be developed as a suitable wound dresssing for wound repair in the future.
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