- 學位論文
以幾丁聚醣為基質探討電磁場對成骨組織之影響
Utilized Chitosan as Matrix Discuss the Effect of Electromagnetic Fields on Bone Tissue Engineering
摘要
電磁場(electromagnetic field, EMF)用在骨頭治療等已經有很好的成果，而用於臨床治療骨質疏鬆、骨不癒合也有很好的療效。然而超過1公分以上的骨缺陷以電磁場治療無法有很好的效果。目前，許多研究以組織工程的方式，將具有生物相容性多孔的支架植入骨缺陷處，用以促進細胞生長及減少骨缺陷修復的時間。 本研究將探討骨母細胞生長在幾丁聚醣支架及幾丁聚醣薄膜上經過電磁場刺激的變化。將實驗分為控制組及實驗組，實驗組部份每天連續以2小時的電磁場(18-30 Gauss, 75Hz, impulse width 1.3 ms, induced electrical amplitude 3.5±1mv)刺激，連續21天，在第1、2、3週測定其細胞活性(MTT assay)、鹼性磷酸酶(ALP)、蛋白質(Total protein)、鈣質含量(Calcium)、DNA含量；並且以螢光染色(Fluorescent stain)、電子式顯微鏡(SEM)觀察骨母細胞生長形態，並以鈣質染色(Calcium stain)觀察鈣質的產生。而材料方面，實驗組部分同樣以電磁場每天2小時，連續21天，並對其壓縮、降解及接觸角作測試。 從實驗結果發現，經過電磁場刺激後，細胞活性、DNA、鈣質都有明顯的增加，而鹼性磷酸酶在第3週經過電磁場刺激的實驗組則是下降。從電子式顯微鏡及螢光染色可以發現細胞在幾丁聚醣支架上及薄膜上生長的情形(呈現多角形及紡錘體)。而在材料上，經過電磁場刺激後，其壓縮、降解及接觸角跟控制組比較並無太大的差別，説明電磁場並不會影響幾丁聚醣在物理上的性質。 由結果得知，電磁場可以促進骨母細胞培養在幾丁聚醣上的增殖及分化情況。為了更近一步模擬人體的骨組織的環境，因此未來仍需在不同細胞或動物實驗，探討電磁場與幾丁聚醣的影響。
並列摘要
Electromagnetic Field (EMF) is shown to be able to accelerate bone healing and improve on osteoporosis. However the healing effect of EMF on large size bone fracture(＞1cm) is not significant. Tissue engineering uses biocompatible porous scaffolds to fill up large defects, promote cell in-growth and can help reduce bone fracture healing time. In this study we will explore effects of EMF on cells growing on tissue engineering scaffolds. We cultivated osteoblast (7F2) on chitosan scaffold and chitosan film, and subjected the cells to EMF (Intensity 18-30 Gauss, 75Hz, impulse width 1.3 ms, induced electrical amplitude 3.5mv) for 2 hours each day for three weeks. On week 1、week 2 and week 3, tested cell viability(MTT assay), alkaline phosphatase activity(ALP), total protein, calcium content, DNA content, cell morphology (SEM、fluorescent stain) and calcium stain. For chitosan we subjected material to EMF for 2 hours for three weeks and examined its compression strength, degradation rate and contact angle. We observed increased cell viability, proliferation, calcium deposition and DNA content in cells exposed to EMF; ALP activity decreased by week 3 in cells exposed to EMF. From SEM and fluorescent stain pictures, we observed osteoblast typical and cell morphology on chitosan scaffold(Polygonal and Spindle-like shape) after EMF exposure. For chitosan, we found there is no difference in compression strength, degradation rate and contact angle after EMF exposure. In conclusion, our results show EMF promote osteoblast proliferation and differentiation on chitosan scaffold. For mimic the environment of human bone tissue, it is necessary to do in vivo tests in the future for investigating the effect of EMF on tissue engineering.