具血管新生功能之複合型電紡支架於慢性傷口修復之應用

Abstract

不易痊癒之糖尿病慢性傷口常導致反覆感染發炎，嚴重者甚至有截肢、死亡的風險。現今已具多種糖尿病傷口癒合的研究及臨床治療，但有龐大開銷和需時間配合等缺點，且效果和治療方式仍有改進的空間。血管增生在急慢性傷口修復和人體生長發育中扮演重要的腳色，能透過細胞與細胞外基質之作用調控加速再生復原之速度，並協助維持生理平衡。 因此，本研究結合具機械強度和彈性的聚幾內酯 (PCL)、增加親水性之蠶絲蛋白 (Silk Fibroin，SF)、從新生鼠皮或大鼠血管經去細胞所得之細胞外基質(Extracellular matrix，ECM)，經電紡技術製成具高、低孔洞率之仿生結構複合型慢性傷口修復支架。並透過移植纖維母細胞、角質細胞、血管內皮細胞與間質幹細胞觀察材料中所具不同來源之去細胞基質於一般和糖尿病環境培養下，對細胞生長之調控。 經ATR-FTIR、SEM、接觸角、MTT等分析，可見具親水性及生物相容性的複合型支架為仿生微奈米結構可供細胞生長，且高孔洞率組別能使細胞長入形成3D架構；In vitro實驗培養人類角質細胞、血管內皮細胞、間質幹細胞於一般環境或糖尿病模型後，藉由支架內去細胞基質與細胞間作用可見細胞增生，且Confocal與螢光顯微鏡結果顯示，角質細胞和血管內皮細胞於相對應加入皮膚或血管之去細胞基質組別有較佳的細胞增生情形，證明細胞基質來源對不同種細胞增生之調控；而間質幹細胞於有加入去細胞基質的高孔洞率支架皆有良好生長情形； In vivo實驗，於大鼠於背部開創傷口貼附複合型支架，經天數觀察顯示高孔洞率PCL/SF/PEG-Vessel(PSG-V)組別有透過血管增生而達傷口修復之最佳成果，且載附幹細胞與去細胞基質之複合型支架能加速傷口癒合之成效。 本研究製備之高孔洞率複合型支架，能夠於只使用去細胞基質中微量生物活性物質之情況下調控各種細胞之增生機制，並誘導血管增生達到慢性傷口治療，且載附幹細胞之支架能加速癒合之速率；透過加入不同組織來源之去細胞基質，能對特定組織、器官的細胞做生長調控。結果顯示，此複合型電紡支架對未來於組織工程、血管增生及一般或慢性傷口修復之應用具有發展潛力。

Unrecoverable diabetes chronic wounds often cause recurrent infections, inflammations, and even a risk of amputation or death in severe cases. Nowadays, various studies and clinical treatments for diabetic wound ulcers are in development, but there are some shortcomings such as huge overhead and time coordination . It still has room for improvement in effects and treatments methods. Angiogenesis plays an important role in acute or chronic wound repair. It acts through the interaction between cells and extracellular matrix(ECM) to regulate, accelerate the speed of regeneration and help maintain physiological balance. Therefore, the biomimetic hybrid scaffolds with high or low porosity are prepared by electrospinning technology, which combines poly-caprolactone(PCL) with mechanical strength and elasticity, silk fibroin(SF) for increasing hydrophilic, and decellularized ECM from skins of newborn rats and blood vessels of rats. The material characteristic analysis show that the high porosity and biocompatible hybrid scaffold is hydrophilic and has biomimetic micro-nano structure, which provides sufficient surface area for cell to grow into 3D architecture. In vitro cell culture experiments indicate that keratinocytes and vascular endothelial cells have better cell proliferation in the ECM group corresponding to the skin or blood vessels, which proves that the source of extracellular matrix regulates the proliferation of different kinds of cells. Additionally, mesenchymal stem cells grow well in hybrid scaffolds with decellularized ECM. In vivo experiments, we attached hybrid scaffold on the wounds of rats, after seven days, high porosity PCL/SF/PSG-Vessel (PSG-V) scaffold achieves the best results of wound healing through angiogenesis .And the mesenchymal stem cells and decellularized matrix –drived hybrid scaffold can accelerate the wound healing ratio. In conclusion, the high-porosity hybrid electrospinning scaffold prepared in this study can regulate the proliferation mechanism of various cells by using only trace amounts of biologically active substances in the decellularized matrix, and induce vascular proliferation to achieve chronic wound healing. Besides, we can regulate the specific cell proliferation by adding extracellular matrix from different tissue sources. This investigation demonstrate that the hybrid electrospinning scaffolds have potential applications in tissue engineering, angiogenesis and general or chronic wound healing treatments.