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可供細胞生長的膠原蛋白間質之特性研究 • 膠原蛋白具有生物相容性，生物可降解性，無毒性及低過敏性反應性，近十年來逐漸成為重要的生物醫材，應用的範圍很廣，包括用於燒燙傷的治療，或作為皮下植入劑，也是細胞生長的良好間質材料。本研究選擇台灣目前常被丟棄的黑豬皮當作萃取純化膠原蛋白的來源，萃取出膠原蛋白Type I 作為細胞生長之間質材料，進而對此些材料進行特性研究。豬皮經過去毛、脫脂及均質化的前處理過程後，以兩種方式進行膠原蛋白的萃取：其一是以改變酸儉鹼值，利用等電點的原理使膠原蛋白沈析出來；另一種是改變鹽份的濃度，以鹽析的方式使膠原蛋白析出。以電泳比較純化出的膠原蛋白，顯示兩者之間無明顯的差異性，而批次之間具有良好的再現性。為了提高膠原蛋白廣泛的適用於細胞培養上，利用戊二醛 (Glutaraldehyde) 當作交鏈劑，以增加膠原蛋白纖維之間的強度。當濃度 1% 的膠原蛋白溶液，加入不同濃度的戊二醛，並充分反應24小時後，以動態機械分析儀 (Dynamic mechanical analysis，DMA) 分析其黏彈性模式，進而解析其 Maxwell 和 voigt 組元的組合形式。其結果顯示，隨交鏈劑之增加，膠原蛋白液體的黏彈性模式無太大的差異，但黏彈度隨之而增強。上述交鏈的膠原蛋白溶液，經過冷凍乾燥法，形成厚度在0.2-0.3 mm的膠原蛋白海綿組織，進而解析其特質。其結果顯示戊二醛濃度越高，交鏈的程度則越高，而游離胺基含量明顯隨之下降。以DMA測膠原蛋白海綿組織的拉張力，並以 break modulus 來比較膠原蛋白海綿組織的強度，結果發現交鏈的程度越高，膠原蛋白海綿組織的剛性越強，但以酸鹼值及鹽析萃取出的膠原蛋白Type I 並沒有顯著的不同。掃描式電子顯微鏡 (SEM) 的鏡檢結果也顯示膠原蛋白纖維經過戊二醛交鏈的纖維之間結構更為緊密，且纖維會形成片狀結構。利用此些膠原蛋白海綿組織作為細胞培養之基質，將由裸鼠皮取出培養的纖維母細胞，分別注入 3105個/ml 的纖維母細胞於不同濃度的戊二醛交鏈的膠原蛋白海綿組織，結果顯示交鏈劑戊二醛的濃度高達0.2%時，膠原蛋白海綿組織仍可與纖維母細胞有很好的相容性。
The Characterization of Collagen Matrix for the Cell Culture • Collagen possesses several characteristics such as bio--compatible, biodegradable, less toxic and least immunological reactions, making it become an important biomaterial in extensive domains such as wound dressings and collagen implant, and matrix for cell culture. This study adopted discarded porcine skin as the sources to isolate type I collagen. Porcine skin was cleaned off hair, defatted and homogenized before performing the further treatment. There are two ways to isolate collagen type I. One was to change pH value by repeated precipi--tation at its PI value. Another one was to change NaCl concen--tration by salt out method. Based on the comparison of SDS electrophoresis, it was found that there was no significant difference between different batch and from different sources.In order to improve the usefulness of collagen as the matrix for cell culture, glutaraldehyde was adopted as a cross-linker to increase the strength of collagen fiber. With the addition of glutaraldehyde at different concentration into 1 % collagen solution for 24 hours, the viscoelasticity of collagen solution measured by Dynamic mechanical analysis demonstrated to follow the same model pattern for all collagen solution examined. A creep model that combined by one Maxwell and one Voiget was satisfactorily to describe the change of viscoelas--ticity expressed by these collagen solution .These crosslinked collagen solution were freeze-dried to form collagen sponge with a thickness of 0.2 to 0.3 mm for the further characterization study. Results showed that the content of free amine group decreased with increasing glutaraldehyde concentrations as a result of increasing the degree of cross--linking. The break modulus measured by Dynamic Mechanical Analyzer (DMA) demonstrated that the higher the degree of crosslinking, the higher the break modulus was. Also, there is no obvious difference between collagen that isolated by different methods. From the results of Scanning Electron Microscopy showed that collagen fibers that crosslinked by glutaraldehyde was much closer and fibers becomes a sheet-like structure.Using these collagen sponge as the matrix for cell culture, the compatibility of fibroblast isolated from nude mouse was tested at a cell density of 3 x 105 fibroblast/ml in 1x1 cm2. The result showed that fibroblast still had good compatibility with collagen sponge even when glutaraldehyde concentration was up to 0.2%.
