- 學位論文
角蛋白為基底之生醫材料於組織工程及細胞工程之應用
Keratin‐based biomaterials for tissue engineering and cell engineering application
摘要
隨著新興國家醫療的普及化，使得醫療器材需求大幅增加，進而促使後端生醫材料之需求與技術要求日漸提高。然而針對生物組織材料中之天然生醫材料發展更是各國投入此領域進行創新與研發。目前常用的天然生醫材料來源有來自動物的膠原蛋白(collagen)、明膠 (gelatin)、角蛋白（keratin）、幾丁質(chitin)與幾丁聚醣(chitosan)及 其衍生物等，與來自植物的褐藻酸(alginate)與纖維素(cellulose)及其衍生物等。然而在天然生醫材料中，角蛋白之應用卻較為少人討論，故也激發起研究者之興趣。雖然角蛋白之研究與應用較鮮少人去鑽研探討。角蛋白是一具備良好細胞相容、生物降解特性之材料，由於取得來源豐富、價錢便宜，因此也是個實用的生醫材料之一。有介於此，本研究欲透過實驗探討目前角蛋白萃取技術開發進行研究與製備相關角蛋白為基底之生物醫材，能夠針對角蛋白之生醫材料發展有更多面向的瞭解。本研究分為兩個研究階段，第一部分為優化角蛋白萃取流程與探討人類脂肪幹細胞與角蛋白之生物相容性;第二以角蛋白作基底，藉由光交聯之方式與甲殼素做交聯並製備成角蛋白-甲殼素複合型薄膜生醫材料。在角蛋白萃取實驗中,我們優化過往研究者所使用之萃取過程且縮短萃取角蛋白所需時間。同一時間進行細胞相容性試驗，本研究使用人類脂肪幹細胞（hASC）探討幹細胞對於角蛋白之相容性。經由一系列之生物相相容性測試如MTT,螢光染色…等證實人類脂肪幹細胞確實在角蛋白塗層盤之中有良好的形態生長。在細胞誘導分化試驗中亦證實人類脂肪幹細胞皆可在角蛋白塗層上分化成為脂肪細胞，硬骨細胞與軟骨細胞。整體研究可直接證明角蛋白確實具備優良之細胞相容性。第二部分為製備角蛋白/甲殼素複合型薄膜，本研究已成功開發藉由光交聯之方式製備出角蛋白/甲殼素複合型薄膜，並探討該薄膜之孔隙度，吸水性與機械強度等物理性測試。在細胞相容性測試中，本實驗室發現該薄膜內所包含的角蛋白可以提供細胞貼附之功能，相較於一般之生物醫材中所不常見之功能，其優勢在於可更進一步裝載幹細胞或者常用之生長因子於該薄膜上，提供更好的細胞生長環境。最後，本研究使用ICR mice 進行皮下移植，評估角蛋白/甲殼素複合型薄膜在小鼠體內降解情況。整體來說，角蛋白不但具備良好生物相容之特性，且製備成複合型薄膜中亦可作為優良之細胞相容與生物相容之生醫材料。期許在未來角蛋白能在扮演組織工程與生醫材料開發上作為重要之角色，再朝再生醫學之臨床應用努力。
並列摘要
Clinically used biomaterials can be classified into four categories: metals & alloys, ceramics,polymers and biological materials. The first two are frequently employed as replacement of human’s hard tissue, and the last two are mostly applied to mimic human’s soft tissue. However,among all of these materials, the natural biomaterial-a branch of biological materials, became the most popular topic that attracted countries’ investment for research and innovation. The natural biomaterial such as animal resourced collagen, gelatin, keratin, chitin, chitosan and its derivatives, alginate, cellulose and its derivatives had been discovered. Most of them have been well investigated for application except for keratin. Despite of the lack of research, keratin’s great cell compatibility and biodegradability has already been proved. High accessibility and low cost also make it an ideal biomaterial. In view of the above, this project focused on extraction method of keratin and manufacturing keratin-based biomaterial for medical purpose. We expect our results can contribute to multi-faceted development of keratin-based biomaterial.The study is divided into two phases- The phase I aimed to improve extraction procedure of keratin, and investigate the biocompatibility of keratin seeded with human derived adipose stem cell (hASC). In phase II, keratin was photo-crosslinked with chitosan to produce membrane-shape keratin-chitosan complex biomaterial.In the extraction experiments, the procedure from previous researchers has been ameliorated and the required time has been shortened. Human derived adipose stem cell was seeded on keratin to access cell compatibility. After evaluated by cell morphology and series of tests like MTT, Immunocytochemistry staining and so on. It has been proved that hASC seeded on keratin coated dish showed great growth performance, and could be well differentiated into adipocyte, osteocyte and chondrocyte. These results directly proved the excellent cell compatibility of keratin.The phase II aimed to create keratin-chitosan complex membrane and explore its application in tissue engineering. The keratin-chitosan complex membrane has been successfully produced in our laboratory. And regular physical tests for mechanical strength, swelling and porosity had been performed. In cell compatibility test, we discovered a feature which is rare in general biomaterial. The content of keratin gave the membrane an advantage to be loaded with cells or regular growth factors, which may provide cells with a better niche. Finally, in vivo compatibility analyzing has been performed with ICR mice, keratin-chitosan complex membrane was subcutaneously transplanted to further evaluate its degradability. Overall, keratin can provide a perfect environment for cell attachment. In addition, with excellent biocompatibility, its composite membrane is also an idea biomaterial. Keratin is expected to play an important role in tissue engineering and biomaterial development. But it need further research to prove its potential in clinical application.
參考文獻
延伸閱讀
- Huang, T. T. (2014). 運用基因工程嵌入伴護蛋白功能區塊以建構新型的角蛋白酶 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2014.01573
- Wang, T. W., Huang, Y. C., Sun, J. S., & Lin, F. H. (2003). 以角質細胞及真皮纖維母細胞共養於雙層明膠薄膜作為類皮膚組織工程之研究. Journal of Medical and Biological Engineering, 23(4), 213-219. https://www.airitilibrary.com/Article/Detail?DocID=16090985-200312-23-4-213-219-a
- Wang, T. W. (2006). 以角質細胞及真皮纖維母細胞共養於雙層孔洞結構之明膠/葡萄胺聚醣基材作為皮膚組織工程之研究 [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2006.00978
- Lawyer, T., McIntosh, K., Clavijo, C., Potekhina, L., & Mann, B. K. (2012). Formulation Changes Affect Material Properties and Cell Behavior in HA-Based Hydrogels. International Journal of Cell Biology, 2012(), 957-965. https://doi.org/10.1155/2012/737421
- 張栩榮（2014）。Electrostatic Force-derived Biopolymeric Hydrogel applied in Tissue Engineering and Drug Delivery System〔博士論文，義守大學〕。華藝線上圖書館。https://doi.org/10.6343/ISU.2014.00007