奈米碳管/幾丁聚醣之導電複合材料結合電刺激於PC12細胞生長與分化之影響

Abstract

神經導管是一種很有潛力能夠替代自體神經移植修復的方法，可當作斷裂神經兩端接口處的橋樑，引導斷裂神經的修復過程。而藉由低頻電流刺激能夠使神經細胞的膜電位與離子通透性改變並增加神經衝動(nerve impulse)的發生機會，進而促進神經細胞再生的說法亦在許多研究中被證實，若結合上述兩種假設，我們預期能夠得到更好的神經修復效果。 本研究中利用共凝集法(coagulation)製備直徑約為200μm的多壁奈米碳管/幾丁聚醣纖維，此複合材料纖維具有良好的導電性、生物相容性、生物分解性等優點，適合應用於電刺激促進神經再生之研究。經過各種材料性質之評估後，選用0.5wt% 多壁奈米碳管/2wt% 幾丁聚醣製備之纖維作為體外實驗之材料，於體外實驗中將材料與PC12類神經細胞共同培養藉以評斷神經再生之效果，實驗中控制參數為頻率20Hz、duty time為50%、刺激時間為每天一小時，分別施加直流與交流電流等刺激方式，並觀察電刺激對於細胞生長與分化之影響。 在體外生物相容性評估的結果顯示，多壁奈米碳管/幾丁聚醣纖維能夠提供細胞良好的生長環境，並於電刺激實驗中量化的數據證實，電刺激治療後神經細胞突觸之平均長度與生長方向都有顯著的差異，而使用正弦交流電對細胞於體外施加電刺激治療時，其促進細胞生長與分化的效果都明顯優於直流電流刺激的治療，因此評估使用正弦交流電刺激於神經導管的應用中能夠達到良好的促進神經再生效果。

Nerve conduit is a method with potential to replace autologous nerve grafts, serving as the bridge to connect interface of two fracture nerves, and conducting the repairing procedure of the fracture nerves. It is also verified by many researches that by the low-frequency electrical stimulation, the cell membrane potential and permeability will change and possibility of nerve impulse will increase and further facilitate the nerve cell regenerating. If combining the assumptions stated above, it is expected to get better result of the nerve repairing. In this research, coagulation method is used to make the multi-walled carbon nanotube(MWCNT)/ chitosan compound fiber with the diameter in around 200μm and because the chitosan fiber equips with several advantages including conductivity, biocompatibility, Biodegradable and so on, it is suitable to be applied in the research of how the electricity stimulation facilitate the nerve regeneration.Through the evaluation of different kinds of materials, 0.5wt% MWCNT/ 2wt% chitosan compound is selected as the materials for the in vitro experiment, and the materials and the PC12 cell are co-cultured to evaluate the result of the nerve regeneration. In the experiment, the control parameters are as the following: 20Hz frequency, 50% duty time, one hour stimulation time per day by implementing of the direct current(DC) / alternating current(AC) electricity separately to observe the effect of electricity stimulation to the cell generation. The result of evaluating the biocompatibility in vitro manifests that multi-walled carbon nanotube/chitosan can provide the cells a great environment to grow up and it is verified that the median neurite length and the direction of growing up have obvious difference after the electricity stimulation experiment, and achieve the great result of the nerve regeneration.