以胺基酸修飾幾丁聚醣/膠原蛋白複合基材之製備、特性分析及其在組織工程、生物反應器之應用

本研究採用冷凍凝膠法並以胺基酸(丙胺酸、甘胺酸或麩胺酸)為交聯架橋劑，製作多孔狀之幾丁聚醣與膠原蛋白複合基材，隨後分析基材的親水性、顯微結構、吸水能力、熱性質分析、機械性質、酵素降解特性，並將細胞培養於基材上進行細胞相容性測試。研究結果發現添加胺基酸之多孔狀之幾丁聚醣與膠原蛋白複合基材可顯著提高最大拉伸應力兩倍，到達73.5 kPa，拉伸應變則維持不變。若將同樣成分的幾丁聚醣與膠原蛋白溶液使用冷凍凝膠法來製成多孔基材，其機械強度與傳統冷凍乾燥法製成之基材相近。觀察基材接觸角發現，加入膠原蛋白後可大幅增加幾丁聚醣基材的親水性，而添加胺基酸後會使得親水性下降，但是仍然比幾丁聚醣基材佳。研究中亦觀察基材的吸水能力，發現添加胺基酸的複合基材吸水率為337~473%，顯著比幾丁聚醣基材吸水率108%多出數倍，本研究亦將基材置於溶菌酶分解作用下觀察降解情況，發現沒有添加胺基酸的基材，會比較容易受到溶菌酶的分解而使其在四周內減重23%，而有添加胺基酸的複合基材僅被分解減重15%。另在平面培養狀態下觀察皮膚纖維母細胞於各基材上貼附增殖情況，發現在添加胺基酸的基材表面上細胞貼附情況較佳，其中以麩胺酸為交聯架橋劑之幾丁聚醣/膠原蛋白複合基材最適合皮膚纖維母細胞貼附增殖。本研究隨後針對添加麩胺酸的複合基材將之置入填充床式循環灌流生物反應器內進行肝細胞培養，經由電子顯微鏡觀察，得知肝細胞在該基材上可形成類似肝小球結構，另對於培養液中氨、尿素和白蛋白濃度等肝細胞的生理代謝功能指標進行分析，確認肝細胞具有將氨轉化為尿素以及合成白蛋白的能力。本研究中也嘗試利用乳化/冷凍凝膠法製備多孔幾丁聚醣/膠原蛋白複合基材微載體，並分析其粒徑分佈、zeta potential、顯微結構，探討複合基材微載體在spinner flask生物反應器之應用，觀察細胞貼附於微載體上情況，可做為後續在微載體上進行大量細胞培養的參考。綜言之，本研究將幾丁聚醣與膠原蛋白兩種天然高分子，以胺基酸做為交聯架橋劑形成共價鍵結製成新型複合基材。此複合基材具有較高機械強度、較佳親水性、較佳之細胞相容性、低生物毒性等優點，預期它在生醫材料及其相關領域頗具應用潛力。

關鍵字

幾丁聚醣；膠原蛋白；胺基酸；交聯劑；微載體；生物反應器

並列摘要

In this research, porous chitosan/collagen composite scaffolds were made by a freeze-gelation method and amino acids (alanine, glycine, and glutamic acid) were used as cross-linking bridges in the scaffolds. The properties of the composite scaffolds including hydrophilicity, microstructure, water uptake ability, thermal stability, mechanical properties, and in vitro enzymatic degradability were analyzed. Cells were cultured on the composite scaffolds to examine their cytocompatibility. The mechanical test results indicated that the maximum tensile stress of the composite scaffolds using amino acids as cross-linking bridges was enhanced by two times and reached 73.5 kPa. The maximum tensile strain remained unchanged. The chitosan/collagen composite scaffolds made by the freeze-gelation method had comparable mechanical strength to that of the scaffolds made by a freeze-drying method. The measurement of contact angle revealed that the addition of collagen into chitosan scaffold substantially reduced the contact angle, but the addition of amino acids into the composite scaffolds increased the contact angle but the value still lower than that of the chitosan scaffold. The measurement of water uptake ability indicated that the composite scaffolds with the addition of amino acids had high water uptake percentage, reaching 337~473%. In contrast, the water uptake percentage was only 108% for chitosan scaffold. The enzymatic degradability of the scaffolds was investigated in a lysozyme solution. The composite scaffolds without the addition of amino acid were degraded by 23%, significantly higher than the scaffolds with the addition of amino acid which was degraded by 15% after 4 weeks. The cytocompatibility test of the composite scaffolds indicated that it was enhanced by the addition of amino acids. The proliferation of skin fibroblast cells on the chitosan/collagen composite scaffolds with the addition of glutamic acid was the best among all types of scaffolds. Besides, we placed the composite scaffolds with the addition of glutamic acid into a packed-bed perfusion bioreactor for the cultivation of hepatocyte cells. The scanning electron microscope images revealed that the hepatocytes could form spheroid structure after 7 days. The physiological functions of hepatocytes were also examined and the hepatocytes were able to coverrt ammonia into urea and produce albumin. This research also tried to use emulsification/freeze-gelation method to produce chitosan/ collagen composite microcarriers. The size distribution, zeta potential, microstructure were also examined. Besides, the composite microcarriers were used for the cultivation of CHO cells in a spinner flask. In summary, this research used amino acids as cross-linking bridges to prepare a novel composite scaffold. This scaffold had advantages including higher mechanical strength, better hydrophilicity, better cytocompatibility, and low toxicity, and thus it has great potential in tissue engineering-related applications.

並列關鍵字

chitosan ； collagen ； amino acids ； cross-linking reagent ； microcarrier ； bioreactor

參考文獻

428. Wang YC, Kuo CH, Hsieh HJ. Enhancement of cell growth on microcarriers immobilized with insulin. Journal of the Chinese Institute of Chemical Engineers 2001 Mar;32(2):125-133.

1. Hsieh CY, Tsai SP, Ho MH, Wang DM, Liu C, Hsieh CH, Tseng HC, Hsieh HJ. Analysis of freeze-gelation and cross-linking processes for preparing porous chitosan scaffolds. Carbohydrate Polymers 2006;(in press).

2. Wang YC, Lin MC, Wang da M, Hsieh HJ. Fabrication of a novel porous pga-chitosan hybrid matrix for tissue engineering. Biomaterials 2003 Mar;24(6):1047-1057.

3. Wang YC, Kao SH, Hsieh HJ. A chemical surface modification of chitosan by glycoconjugates to enhance the cell-biomaterial interaction. Biomacromolecules 2003 Mar-Apr;4(2):224-231.

4. Hsieh CY, Tsai SP, Wang DM, Chang YN, Hsieh HJ. Preparation of gamma-pga/chitosan composite tissue engineering matrices. Biomaterials 2005 Oct;26(28):5617-5623.

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以胺基酸修飾幾丁聚醣/膠原蛋白複合基材之製備、特性分析及其在組織工程、生物反應器之應用

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