含有聚胜肽纖維與褐藻酸之新穎複合水凝膠之設計、合成、製備與性質鑑定

Abstract

位在神經系統的損傷往往是永久性且不可逆的，而傳統的異體移植治療方法目前是供不應求的。因此，為了幫助修復受損的神經系統，神經組織工程是最有前瞻性的技術之一。生物支架在神經組織工程中扮演著重要的角色，其應包含高生物相容性、高孔隙度以進行養分交換、異向性的結構以利神經細胞的生長導引、生物化學界面以增強細胞的表現、以及合適的機械性質。在我們的研究中，我們選用水凝膠作為神經組織工程中的生物支架材料。

水凝膠是能夠吸收大量水分的親水性網狀聚合物。水凝膠通常具有良好的生物相容性和高孔隙度，說明其在生物醫學應用方面具有很高的潛力。在神經組織工程的研究中，異向性纖維複合水凝膠在體外或體內實驗中顯示了良好的結果。在本研究中，我們設計了一種新穎的纖維複合水凝膠，其獨特的交聯機制是由帶正電的聚胜肽纖維水溶液和帶負電的褐藻酸水溶液，透過靜電吸引力與氫鍵之物理交聯，形成網狀結構。

通過N-carboxyanhydrides的開環聚合反應，合成了具備神經傳遞物質谷氨酸和幫助細胞貼附之胜肽L-麩氨酸的共聚物。首先將最佳化組成之共聚胜肽(6BG4bocL)通過靜電紡絲製成聚胜肽纖維(fib-6BG4bocL)，然後透過化學反應選擇性地去掉boc保護基使聚胜肽纖維形成帶有正電荷之纖維(fib-6BG4L)。部分水解的聚胜肽纖維在去離子水中溶解，通過共注射技術與褐藻酸形成異向性纖維複合水凝膠。共注射纖維複合水凝膠已經在偏光光學顯微鏡下被證實其具有異向性的結構。

我們同時也使用簡單的滴落法製備等向性水凝膠，以評估纖維複合水凝膠之機械性質和生物相容性。纖維複合水凝膠的複數模數約為180-3,008帕斯卡，其機械強度適合最為應用於神經組織工程之生物支架。除了機械性質，我們還對等向性水凝膠進行了細胞存活率和細胞毒性測試。經過6天的體外細胞培養，在Alamar Blue試驗中，聚胜肽的奈米纖維水凝膠顯著增強了PC12細胞的存活率，並且在Live/Dead試驗中也表現出低的細胞毒性。在此研究中，聚胜肽纖維複合水凝膠顯示出其作為應用於神經組織工程之生物支架的潛力。

Injuries to the nervous system are often irreversible and permanent, and there is an urgent need to develop an effective treatment for recovery. Repair the damaged nervous system, neural tissue engineering stands out as one of the most promising techniques. Scaffolds play an important role in neural tissue engineering. Ideal scaffolds of neural tissue engineering should exhibit high biocompatibility, high porosity for nutrient exchange, physical cues for orientational cell growth, chemical cues to enhance cell growth and differentiation, and adequate mechanical properties. In our study, we chose hydrogel as the biomedical scaffold for neural tissue engineering. Hydrogels have hydrophilic polymeric network structures that can absorb a large amount of water. Hydrogel usually has good biocompatibility and high porosity, indicating its potential in biomedical applications. Anisotropic fibrous composite hydrogels are proposed to enhance neurite outgrowth directly, which is necessary for neural tissue engineering. In this study, we designed and fabricated a novel polypeptide-based fibrous composite hydrogel by gelling the positively charged polypeptide fiber solution with the negatively charged alginic acid solution through electrostatic interactions. The copolypeptide of neurotransmitter glutamate and cell-adhesive L-lysine were synthesized via ring-opening polymerization of N-carboxyanhydrides. The optimal composition copolypeptide (6BG4bocL) was first fabricated into fiber (fib-6BG4bocL) by electrospinning, and then the fiber underwent selective deprotection of the Boc-protecting group to form positively charged polypeptide fiber fib-6BG4L. The partially-hydrolyzed fiber was dissolved in DI water and formed an anisotropic fibrous composite hydrogel with alginic acid by co-extrusion technique. The anisotropic structure of the co-extruded polypeptide-based fibrous composite hydrogel was confirmed under a polarized optical microscope. The isotropic hydrogels were also fabricated by a simple dropped method for the characterization of their mechanical properties and biocompatibility. The complex moduli of fibrous composite hydrogels were about 180-3008 Pa, which is adequate for application in neural tissue. Cell viability and cytotoxicity tests were also performed on isotropic hydrogels. The peptide-based fibrous composite hydrogel significantly enhanced the cell viability of PC12 cells in the Alamar Blue assay after 6 days of in vitro cell culture and also showed low cytotoxicity by Live/Dead assay. The polypeptide-based fibrous composite hydrogel should be a good candidate material for biomedical scaffolds for neural tissue engineering.