- 學位論文
以微影技術製備膠原蛋白及玻尿酸複合支架之研究
Micro-Lithographic Fabrication of Collagen and Hyaluronic Acid Composite Scaffolds
摘要
組織工程中支架使用之材料多為合成高分子，因此開發以細胞外間質組成成分之天然高分子的支架極具研究潛力。本研究結合組織工程的概念以及微影成像的技術，製備有特殊圖案的天然高分子支架。從豬皮萃取膠原蛋白，藉由聚丙烯醯胺膠體電泳鑑定得到分子量分別為120、130及250 kDa的α2、α1與β 鏈，與文獻比對確認為第一型膠原蛋白。以超透析純化玻尿酸並以硫酸咔唑法確認玻尿酸含量為98 mg/g。甲基丙烯酸環氧丙酯藉由三乙基胺進行鹼催化得到環氧基(Epoxide)開環作用與玻尿酸反應鍵結後以1H NMR鑑定化學結構，可在化學位移為5.7、6.1 ppm訊號值處得到甲基丙烯酸環氧丙酯的氫質子訊號，比對文獻中玻尿酸的1H NMR圖譜，確認玻尿酸中乙醯葡萄糖胺上之甲基氫質子訊號為化學位移在2.0 ppm，以兩者為基礎計算線下面積，得接枝率為12 %。在製備甲基丙烯酸環氧丙酯接枝玻尿酸/膠原蛋白(玻尿酸：膠原蛋白 = 10 : 3 (重量比))支架中，分別使用面積為39731656及157860344 μm2的光罩圖案一及圖案二。前者為三種不同尺寸的長方形圖案組成，尺寸範圍從0.1到10 mm，其最細微處需UV照射9分鐘方能完整清晰呈現，最適當使用體積為100 μL、最佳照射焦距為40 cm；後者為長方形與弧形圖案組成，因尺寸範圍從2到5 mm，故UV照射僅需2分鐘即可製備完畢，最適當使用體積為250 μL、最佳照射焦距為 45 cm。以掃描式電子顯微鏡觀察支架表面，孔洞範圍在10-100 μm，顯示支架有較細小且多的孔洞。本研究未來可進行與細胞共培養並觀察支架圖案對細胞移動及貼附的影響。
並列摘要
In tissue engineering, most of the materials used in scaffolds were synthesized polymers, and this lead the research of applying native polymers, which were also extracellular matrix components, to be a high potential subject. In this study, utilizing lithography technology and the concept of tissue engineering to prepare the photopattern scaffolds which’s made by natural high molecular polymers. Collagen was extracted from pig skin, identified molecular weight via SDS-PAGE as 120、130、250 kDa as α2、α1 and β bands, then compared with the literature to recognize it as collagen type I. Using ultra-dialysis to purify hyaluronic acid and sulfuric acid carbazole to identify the content of hyaluronic acid which is 98 mg/g. By using 1H NMR to identify the Glycidyl Methacrylate-Hyaluronic Acid chemical structure and was informed that the grafted percentage was 12 %. Through optimizing the parameters of producing scaffolds, two photopatterns were using as photopattern 1 and 2. The former’s area was 39.73 mm2 and made by three different rectangles which dimensions were from 0.1 to 100 mm and need 9 minutes of UV irradiation, 100 μL solutions, focus 40 cm to prepare scaffold. The latter’s area was 157.86×108 mm2 ¬and made by rectangular and arc patterns which dimensions were from 2 to 5 mm and need 2 minutes of UV irradiation, 250 μL solutions, focus 45 cm to prepare scaffold. By utilizing scanning electron microscope to observe scaffold surface, the range is around 10-100 μm. In the future, this study can look for culturing scaffolds with cells and observing how pattern can affect cell migration and adhesion.