用於神經組織工程的聚肽電解質

Abstract

本論文旨在設計一種以聚肽為基礎的高分子聚電解質，並經由靜電紡絲形成3D且順向排列的纖維狀支架，應用於神經組織工程。該材料可經由傳統化學方式所合成，具有導離性 (ionic conductivity)，還含有可刺激神經細胞的生物因子：谷氨酸，而順向排列的纖維可用於引導軸突沿著一定的方向生長，是一種可以幫助神經再生的生醫材料。 我們先合成聚谷氨酸苯酯 (poly(γ-benzyl-L-glutamate), PBG)，再進一步將PBG上的酯基進行部分水解，得到谷氨酸苯酯-谷氨酸無規共聚物 (poly(γ-benzyl-L-glutamate)-r-poly(α-L-glutamic acid), PBGA)。我們利用靜電紡絲將PBGA製成3D且順向排列的纖維狀支架，再將PBGA支架上的羧酸基與氫氧化鈉反應，得到聚肽電解質的支架: sodium salt of poly(γ-benzyl-L-glutamate)-r-poly(α-L-glutamic acid) (PBGA-Na)。當水解度為20莫耳百分率時，所產生的聚肽電解質 (即PBGA20-Na) 不溶於水，於細胞培養過程中可保持纖維的順向排列。因此，本研究將利用此支架「具導離性、順向排列、含生物因子」的優點培養神經細胞，並探討在有或無電刺激下，神經細胞在該材料上的生長以及分化情形。 三種以PBG為基礎的生醫材料，相較於常見的聚己內酯 (polycaprolactone, PCL)，具有更佳的生物相容性，可誘發更好的細胞貼附、生長以及分化。此外，細胞在含有谷氨酸的聚肽上（即PBGA20和PBGA20-Na），相對於未含谷氨酸的PBG，有更好的細胞貼附、生長以及分化能力。細胞於這些材料上的軸突分化能力，皆可透過電刺激而提升，軸突的生長還可受到順向排列的纖維所引導，呈現單一方向性生長。最後，具有導離性且含有谷氨酸的聚肽高分子電解質: PBGA20-Na，最能誘導細胞進行軸突的分化，非常適合應用於神經組織工程。

The goal of this research is to design a peptide-based polyelectrolyte containing neuronal stimulant (i.e., glutamic acid) for the fabrication of electroactive 3D fibrous scaffold with aligned fibers for neural tissue engineering. The polypeptides are designed based on biocompatible poly(γ-benzyl-L-glutamate) (PBG), which can be synthesized easily by conventional ring opening polymerization reaction. Partial hydrolysis of the benzyl groups in PBG yields a random copolymer of poly(γ-benzyl-L-glutamate)-r-poly(α-L-glutamic acid) (PBGA). By using electrospinning technique, we fabricate PBGA fibrous scaffold with aligned fibers. The scaffold made of peptide-based polyelectrolyte, sodium salt of poly(γ-benzyl-L-glutamate)-r-poly(α-L-glutamic acid) (PBGA-Na), is obtained by reacting the COOH groups in PBGA scaffold with NaOH aqueous solution. The PBGA-Na with 20 mol% of COO-Na+ side chains (i.e., PBGA20-Na) is water-insoluble, which can maintain the aligned structure of the fibrous scaffold and stimulate neurite outgrowth in alignment. Hence, in this thesis, we focus on culturing neurons on this aligned, electroactive and neuronal stimulant-contained fibrous scaffold. We also discuss the proliferation and differentiation of neurons on this scaffold with or without electrical stimulation. The biocompatibility of PBG, PBGA20 and PBGA20-Na are significantly better than polycaprolactone (PCL). Furthermore, the integration of neuronal stimulant into polypeptides (i.e., PBGA20 and PBGA20-Na) shows an enhancement of cell adhesion, proliferation and differentiation. Cells on all the materials tested in this thesis extend longer neurites with electrical stimulation than without stimulation. According to the results of the experiments, all the scaffolds with the aligned fibers can promote the growth of neurites along. Neurite outgrowth on the electroactive polypeptide containing glutamic acid (i.e., PBGA20-Na) is the longest among the four materials. In conclusion, PBGA20-Na is a unique and promising biomaterial for neural tissue engineering.