糖尿病患之傷口癒合不易，故其傷口敷料成為一個重要研究課題。海藻酸 (Alginate, AL) 作為傷口敷料支撐層、幾丁聚醣 (Chitosan, CS) 為結構穩定材料，及聚麩胺酸為高親水性吸收層 (Polyglutamic acid, PGA) ，所以本研究使用三種天然生醫材料建構多層型水膠敷材，並對不同組成之水膠AL-CaCl2、AL-PGA及AL-CS-PGA進行多項測試。為觀察多層型水膠結構分佈，在各組成高分子鏈接上螢光標記，於共軛聚焦顯微鏡下發現PGA和AL形成雙層結構，CS則分佈於PGA層及AL層界面，形成多層型結構。接著對多層型水膠進行保水率、膨潤度及水氣透過率等物理性質測試。在這些實驗結果發現，保水率、膨潤度及水氣透過率等，其由高至低之順序皆為AL-PGA > AL-CS-PGA > AL-CaCl2。AL-PGA因為結構成份含有大量高親水性的聚麩胺酸，故其具有最佳保溼能力 (以轉速為15000 rpm處理5分鐘後其保水率為84.6 ± 1.97 %)，與較高之水氣透過率。AL-CaCl2的結構是三組中最緊密的，水膠行為 (如保水率、膨潤度及水氣透過率等) 則較AL-PGA和AL-CS-PGA來的低。AL-CS-PGA在各個測試結果的表現皆位於兩者之間，除了有良好的保水率 (轉速為15000 rpm處理5分鐘後其保水率為79.69 ± 1.05 %) 與膨潤度 (1044 ± 20 %) 外，其水氣透過率也較為緩慢可以保持傷口的濕潤。
在水膠穩定度的評估中，運用奧氏黏度計評估多層型水膠在模擬正常生理環境下的穩定度，結果發現，在第五天時AL-PGA的黏度提高，為了了解是何種物質造成外在環境黏度的提升，因此，運用鈣離子釋放與螢光定量對多層型水膠做更深入的分析。將各組別放置在生理食鹽水環境下，其螢光定量試驗結果顯示，AL-PGA水膠在90分鐘後快速釋放出38 %的PGA，AL-CS-PGA水膠僅釋放出10 %的PGA。此外，將AL-CS-PGA水膠置於細胞培養液中觀察其釋放情形發現，水膠中各成份的釋放速度為PGA > AL > CS。生理食鹽水的鈣離子釋放結果顯示，鈣離子釋放的速度為AL-PGA > AL-CS-PGA > AL-CaCl2。三種水膠中，AL-PGA擁有最快的釋放速度 (3.8 ± 0.2 mg/g)，而AL-CS-PGA則受到CS與AL和PGA結構的纏繞牽制使得釋放速度減緩 (2.2 ± 0.4 mg/g)。
另外進行生物相容性的相關實驗如凝血性質測試、血液常規檢測、血小板吸附及蛋白質吸附等測試。在血液常規檢測的結果顯示，AL-CS-PGA水膠不會造成血液成份改變，同時相對於其他兩組AL-CaCl2與AL-PGA有促進凝血及血小板吸附的功能。此外，AL-CS-PGA與AL-PGA皆具有吸附蛋白質的能力。細胞相容性的實驗發現AL-CS-PGA不會產生細胞毒性，甚至會促進纖維母細胞的增生。根據細胞行為儀的測試結果也發現AL-CS-PGA可促進細胞遷移。
進一步以糖尿病大白鼠做為水膠之皮膚傷口癒合測試。結果發現傷口癒合程度為AL-CS-PGA > AL-PGA > AL-CaCl2。根據組織切片染色及膠原蛋白定量得知，AL-CS-PGA較其他兩組增加較多之膠原蛋白在新生組織中的含量。以免疫染色方式也發現AL-CS-PGA也比較其他兩組更促進表皮分化。綜合上述的實驗結果證明，此多層型水膠具有發展成先進傷口敷料的潛力。

Diabetes mellitus wound is one of the medical problems, making the development of advanced wound dressings an important issue. In this study, alginate (Alginate, AL), chitosan (chitosan, CS), and PGA (polyglutamic acid, PGA) were employed to prepare multi-layered hydrogels. For comparison, AL-CaCl2 and AL-PGA were also prepared. To observe multi-layered structure, the constituents were labeled with different fluorescent molecules. The images from confocal laser scanning microscope (CLSM) revealed that the PGA and AL formed two layers, with CS distributed in PGA layer and the interface of the AL layer. The hydrogels were subject to characterization including water retention, hydrogels stability, swelling ratio and water vapor transmission rate. The results showed that these properties of AL-CS-PGA was lower than those of AL-PGA. Biocompatibility tests were performed including blood coagulation, hematological parameters, platelet adhesion and protein adsorption. The results of hematological parameters show that AL-CS-PGA hydrogel had no adverse reaction, at the same time to promote coagulation and platelet adhesion. In addition, AL-CS-PGA and AL-PGA can adsorb serum proteins such as HSA and HPF. Furthermore, cell compatibility evaluations of these hydrogels showed no cytotoxicity response, and were able to promote proliferation of fibroblasts. Electric cell-substrate impedance sensing (ECIS) showed that AL-CS-PGA can promote cell migration.
Effect of the hydrogels on wound healing was evaluated using diabetic mellitus rat model. The results showed that AL-CS-PGA exhibited higher rate of wound healing than conventional AL hydrogels. Tissue section staining and collagen quantification indicated that AL-CS-PGA can increase collagen regeneration without causing abnormal cells in the apoptotic response, thus helping epithelialization. These experimental results demonstrated that this multilayer hydrogel would be feasible as advanced wound dressings.

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