| 摘要: | 慢性傷口潰瘍是糖尿病所導致的主要併發症之ㄧ，其慢性發炎反應不僅會延緩肉芽組織成熟，且降低傷口的拉伸強度，嚴重者甚至會導致組織壞死而須截肢。因此開發多功能傷口敷料來全面性促進其傷口癒合成為刻不容緩的任務。在本研究中我們將利用先前計畫（MOST 103-2221-E-008 -118-）所發展的雙射流系統來開發褐藻酸鈣(calcium alginate)和聚乳酸羥基乙酸共聚物（PLGA）的複合奈米電紡絲。其中高保濕性的褐藻酸鈣可維持傷口的濕潤，而PLGA可增加纖維的機械強度和蛋白質吸附。為了抑制傷口感染，PLGA纖維將攜帶銀奈米粒子並進行長期釋放。另一方面，陰離子性的褐藻酸纖維可吸附帶正電的聚乙烯亞胺(PEI)/DNA奈米複合物，我們將藉此方式固定帶有血小板衍生生長因子（PDGF）基因的質體DNA，使其可原位轉染傷口的細胞並進行持續表達，由於PDGF可誘集免疫細胞，並促進纖維母細胞的增殖和分化，因此有利於胞外基質和血管的生成。除此之外，褐藻酸鈣可以釋放鈣離子至傷口部位以加速止血。而失去鈣離子的褐藻酸纖維會隨著時間逐漸降解，以提高細胞遷移並增強細胞貼附至PLGA纖維，進而促進組織再生。我們將應用共培養系統來研究轉染對細胞增殖和分化的影響，並以糖尿病小鼠模型探討所發展的纖維敷料對慢性傷口的癒合效果。 ;In Taiwan, the amount of medical resources required for the diabetes treatment steadily increases over recent years. Diabetic ulcer is a major complication of diabetes which leads chronic wound formation. The prolonged inflammatory phase may result in not only an immature granulation tissue but also a reduction of wound tensile strength, which always eventually cause amputations. Because diabetic wounds are deeper, more exudative, and more necrotic than the normal wounds, it is essential to develop a multifunctional wound dressing to promote tissue regeneration. Our previous project (MOST 103-2221-E-008 -118 -) demonstrated that a dual jet system can coelectrospin nanofibers in arbitrary ratios. Therefore, we will apply this method to fabricate a versatile composite nanofibrous matrix. Calcium alginate and poly(lactide-co-glycolide)(PLGA) will be coelectrospun as composite nanofibers. Calcium alginate fibers can be used as highly absorbent dressings to provide a moist environment in wound sites. On the other hand, PLGA will be applied to increase mechanical strength and protein adsorption. In addition, silver nanoparticles will be embedded in PLGA fibers for long-term release to inhibit the growth of microorganism. Plasmid DNA encoding platelet-derived growth factor (PDGF) will be delivered from composite fibers because this growth factor is a chemoattractant for neutrophils and can induce the proliferation and differentiation of fibroblasts. Furthermore, collagen deposition and angiogenesis can also be promoted. These PDGF plasmids will be complexed with polyethylenimine (PEI) to form cationic nanoparticles which will be adsorbed onto anionic alginate fibers through electrostatic interaction. As wound cells adhered to composite fibers, they can be in situ tranfected to continuously express PDGF. Moreover, calcium ions in alginate fibers can be released to wound sites through ion exchange to accelerate hemostasis. These calcium-insufficient alginate fibers should gradually degrade with time to allow cell infiltration, enhance cell adhesion onto PLGA fibers, and eventually improve tissue regeneration. A co-culture system will be applied to investigate the effect of transfection on cell proliferation and activation. Because complicated pathology of chronic wounds is difficult to simulate through in vitro experiments, the healing effects of these composite nanofibrous dressings will be evaluated using diabetic mouse models. Streptozocin (STZ) will be injected to C57/BL6 mice to damage insulin-producing beta cells, and the treated mice will become type I diabetic. On the other hand, mice with strain of diabetes spontaneous mutation (Leprdb) manifest morbid obesity and eventually become type II diabetic. Full-thickness skin wounds will be created in dorsal area of these diabetic mice by biopsy punches with diameters of 8 mm, which will be covered by the composite fibers. The wound tissues will be harvested and sectioned for histology analysis. Through hematoxylin/eosin (H/E) and immunohistochemical (IHC) staining, tissue regeneration in wound sites will be evaluated. In addition, Western blot and real-time polymerase chain reaction (RT-PCR) analysis will be performed to investigate the activity of re-epithelialization, remodeling, and angiogenesis in different stages. We expect this comprehensive study provides an ideal solution to facilitate diabetic-induced chronic wounds. |
