不同來源幾丁聚醣當生物支架在軟骨組織工程之應用比較

The Application of Different Chitosan Sources as Scaffolds for Cartilage Tissue Engineering

徐琴舒(Chin-Shu Hsu)

指導教授 ： 許明照

臺北醫學大學/口腔醫學院/生物醫學材料研究所/碩士(2007年)

摘要

中文摘要
本篇論文的目的是希望製作出適合軟骨細胞生長的支架，使軟骨細胞正常分泌出細胞外間質，維持細胞的生長，進而達到軟骨再生，修復受損軟骨的目的。利用電氣紡絲技術和冷凍乾燥技術製作幾丁聚醣生物支架，分析支架機械性質，並將軟骨細胞植入支架中培養，觀察軟骨細胞的生長表現。以MTT assay觀察軟骨細胞在不同時間的生長情形，利用H＆E stain觀察軟骨細胞在支架中的分布，再以RT-PCR分析軟骨細胞特有的蛋白質表現量。在電氣紡絲的技術中使用市售不同分子量的幾丁聚醣為材料；而在冷凍乾燥的部份則是使用從真菌中萃取出的RHIZOCHITOSAN及SACCHACHITOSAN和市售的幾丁聚醣做為材料一起比較。
結果顯示，在電氣紡絲的實驗中，以醋酸水溶液當溶劑來開發水系的幾丁聚醣電紡膜是較困難的，因為電紡過程中所產生的熱能，無法順利將溶劑中的水分完全蒸發，可能是由於水的沸點較高的原因，無論添加其他的溶劑或介面活性劑，像乙醇、PEG、DMF、NMP等，仍然無法順利使纖維成形。
另一部份，從真菌中萃取出的RHIZOCHITOSAN及SACCHACHITOSAN和市售的幾丁聚醣，在經冷凍乾燥技術製作出的海綿狀支架，在SEM觀察下，支架是成相互連結的孔洞，顯示是適合細胞生長；在支架孔洞大小的測量發現，純的RHIZOCHITOSAN孔徑較小，加了膠原蛋白後，孔徑變大了；當RHIZOCHITOSAN比例越多時，孔徑則又縮小。在市售不同分子量的幾丁聚醣中，隨著分子量越大，則孔隙度越大。支架的機械性質發現，加了EDC做交聯的組別拉長百分比都明顯較低；在市售的幾丁聚醣中，隨著分子量越來越高，拉長百分比也越來越低；RHIZOCHITOSAN部分拉長百分比並沒有明顯的趨勢性。在拉張力部分，加了EDC的組別顯示需要較大的力氣才有斷裂點，尤其在市售幾丁聚醣的部份；靜態應力中，RHIZOCHITOSAN是較小的，約在2.5~4.7*105 (Pa)之間；市售的幾丁聚醣則較大，約在7.7~11.6*105 (Pa)之間。目前已將軟骨細胞植入，然而有關觀察細胞生長的實驗，仍在進行中，期望細胞能有良好的生長表現。

關鍵字

幾丁聚醣；支架；軟骨組織工程

並列摘要

The purpose of the study is to make the scaffolds fitting for chondrocyte to grow and let them secrete Extracellular matrix regularly to keep the development, which could attain the goal of regenerating and repaired the damaged cartilage. We construct chitosan scaffolds and analyze their Mechanical properties by electrospinning and freeze-drying, then installing chondrocyte into scaffolds to cultivate, at last, observing their development. We observe the growth state of them in different time by MTT assay, and observe their distribution in the scaffolds with H & E stain and then we analyze their characteristic of protein expresses by RT-PCR way. We use chitosan with deferent molecular weight for the material in electrospinning; and take RHIZOCHITOSAN and SACCHACHITOSAN extracting from fungus comparing to chitosan in freeze-drying.
The result shows that it is much tougher using acetic acid solution for solvent to originate Hydrophilic chitosan membrane in the experiment of electrospinning. For the boiling point of water is higher, the thermal energy generated from electrospinning process couldn’t evaporate the moisture content in the solvent completely to bring about fiber whatever even though we add other solvent or surfactant such as alcohol、PEG、DMF and NMP.
In the other aspect, sponge made by chitosan and the RHIZOCHITOSAN and SACCHACHITOSAN extracting from fungus in freeze-drying show the structure which are composed of the connected holes under SEM observe. It means that the environment is suit for developing chondrocyte; and we also found that the pore size of pure RHIZOCHITOSAN are smaller in scaffold pore size to measure. The pore size became bigger after collagen added and they became tiny when more RHIZOCHITOSAN joined. And the size of them changed with the molecular weight of chitosan. We found that adding EDC crosslinking conditions, Strain is lower obvious; in scaffold Mechanical properties and strain moved contradictorily with the molecular weight and irrelatively to RHIZOCHITOSAN. In the Force part, especially for chitosan, groups EDC added shows the fracture point occurs with the increasing strength; and in Static Stress, the RHIZOCHITOSAN is smaller（2.5~4.7*105 (Pa) ）and the chitosan is bigger (7.7~11.6*105 (Pa). Now we’ve installed the chitosan into scaffold, and the experiment is just ongoing. We look forward to the chitosan progress be prosperous.

並列關鍵字

Chitosan；Scaffold；Cartilage Tissue Engineering

參考文獻

林士凱。利用匍枝根黴菌(Rhizopus stolonifer)細胞壁組成（RHIZOCHITOSAN）結合血小板（RegenplexTM ）作為創傷敷材之探討。台北醫學大學生物醫學材料研究所碩士論文。中華民國九十五年。

劉慧如。醣化去乙醯幾丁質與不同來源之幾丁聚醣之特性探討與基因轉染率之比較。台北醫學大學生物醫學材料研究所碩士論文。中華民國九十年。

Ghosh, P. and M. Smith (2002). "Osteoarthritis, genetic and molecular mechanisms." Biogerontology 3(1-2): 85-88.

Aigner, T., B. Kurz, et al. (2002). "Roles of chondrocytes in the pathogenesis of osteoarthritis." Current Opinion in Rheumatology 14(5): 578-584.

Bhattarai, N., D. Edmondson, et al. (2005). "Electrospun chitosan-based nanofibers and their cellular compatibility." Biomaterials 26(31): 6176-6184.