以中胚層幹細胞及分化因子於第一型膠原蛋白介質中進行軟骨組織工程

Tissue Engineering of Cartilage: A Chondrogenic Differentiation of Mesenchymal Stem Cells in Type I Collagen Matrix

張家銘(Chia-Ming Chang)

指導教授 ： 蔡郁惠 ； 賴文福

臺北醫學大學/口腔學院/生物醫學材料研究所/碩士(2002年)

摘要

隨著生物醫學科技的進步，使組織工程無論在學理上或應用上皆進入嶄新的一頁。藉由細胞，間質(Matrix)，生長激素等三元素的調和，使組織工程在再生醫學之臨床應用跨進了一步。本研究以中胚層幹細胞，加上Transforming Growth Factor-β1 (TGF-β1)，以膠原蛋白為骨架來進行軟骨組織的組織工程體外培養。 中胚層幹細胞已被證實可在體外增殖並具有分化成軟骨細胞的能力，且骨髓抽取已有手術的標準程序，這使中胚層幹細胞成為上選的組織工程之細胞來源，並藉由幹細胞的分化能力將可彌補自體健康軟骨細胞取得不易的問題。膠原蛋白是結締組織中含量最豐富的蛋白質，也是細胞外間質中主要的組成份子之一。由於其特殊的三股螺旋結構，使得膠原蛋白不僅提供一個組織構成的骨架，也提供細胞一個十分良好的生長環境。且膠原蛋白的免疫反應極低，目前已有許多以膠原蛋白為基礎的生物醫學材料，廣泛地被運用在各種組織的修復治療之中，如心臟瓣膜、燙傷敷料等。能影響中胚層幹細胞及前趨細胞之增生及分化的因子已被廣泛地研究，其中TGF-β家族是被討論最廣泛的因子之一。已有一些報告證實TGF-β1可使中胚層幹細胞進一步分化成軟骨細胞，而近年來更有以TGF-β1在不同材料下進行的軟骨組織工程的報告。 以先前其他實驗室所發表的論文為依據，本論文將以改進過後的分離方式自紐西蘭大白兔骨髓中取得足夠數量的中胚層幹細胞來進行軟骨組織工程，並觀察細胞在繼代培養時的形態及測試繼代培養後細胞進行軟骨分化的能力，同時將細胞放入第一型膠原蛋白間質中進行組織培養，以測試最佳植入細胞之數量並觀察細胞於第一型膠原蛋白間質中的生長情況，最後在組織培養期間加入分化因子，以alcian blue組織染色法觀察軟骨醣蛋白的表現以評估軟骨分化的進程。 本論文致力於骨髓中胚層幹細胞的分離及其繼代培養，在實驗過程中，修改文獻中分離細胞的程序，使細胞取得不再是困難的工作。也確定繼代培養的可行性。近年來，已有大量軟骨組織工程的報告，多數以初級軟骨細胞作為細胞來源再配合不同的生醫材料來進行軟骨組織工程。但是，健康的軟骨細胞取得不易，更遑論由軟骨缺損病人進行自體移植在執行上的困難，由於中胚層幹細胞具有分化成軟骨的能力，可改善先前初級軟骨細胞取得的問題，大大提升自體移植的可能性。 本文依據賴文福教授實驗室先前以初級軟骨細胞製成軟骨組織的實驗流程，進一步使用中胚層幹細胞於第一型膠原蛋白間質中進行組織形成培養，在TGF-β1等分化因子於組織培養液裡進行細胞分化，再以組織染色法來確認軟骨醣蛋白的表現。在細胞分離方面，本實驗室已可取得足夠的數量，惟在新生組織評估時，我們僅藉由組織染色來確認軟骨醣蛋白的表現及軟骨細胞形態的衍生，必須再進一步的觀察第二型膠原蛋白之表現才能確切地釐清軟骨分化的適當條件，希冀這項成果將為日後體外軟骨組織工程及自體軟骨組織植入奠定新的里程碑。

關鍵字

中胚層幹細胞 ； 膠原蛋白 ； TGF-β1 ； 軟骨組織 ； 組織染色 ； 組織培養 ； 軟骨細胞 ； 組織工程

並列摘要

Tissue engineering is a new strategy to produce a biocompatible and bioactive tissue for surgical implantation to solve several clinical problems. Cells, matrix (scaffolds) and growth factors, three major components of tissue engineering can be manipulated delicately into a perfect concerto for engineering a neo-tissue to promote the life quality of human beings. By using bone marrow derived stromal cells (mesenchymal stem cells, MSCs) as the cell source, transforming growth factor-beta 1 (TGF-β1) as the chondrogenic inducer and rat-tail type I collagen as the scaffold, the goal of in vitro tissue engineering of artificial cartilage (neocartilage) was attempted in this study. MSCs have been isolated, fractionated and propagated in vitro and reported to maintain their potential to differentiate into chondrocytes. Bone marrow aspiration has been performed frequently by clinical standard procedures. Consequently, MSCs are good cell source in the matter of cartilage engineering to overcome the deficiency of healthy cartilage for primary chondrocytes. On the other hand, collagen is one of the most abundant proteins in connective tissue and is a major component of extracellular matrix. Because of its triple helix structure, collagen not only as a good scaffold but also provide a good living environment for cells. Owing to its relatively low immunogenesity, collagen has been wildly used as biomaterials in wound dressing, heart valve and so on. TGF-β1, one of the most well studied growth factors in the matter of cell proliferation and differentiation, has been reported to promote chondrogenic differentiation of MSCs. Besides, cartilage engineering by using TGF-β1 and different biomaterials has also been reported in the past years. In this thesis, modified isolation protocols were used to obtain sufficient MSCs from rabbit bone marrow for tissue engineering of cartilage. During subsequent passages, the morphology and behavior of MSCs were observed. Their potential for chondrogenic differentiation after subsequent passage was also tested. Optimal MSCs seeding density within three dimensional collagen matrix was determined in this study. In order to evaluate the progression of chondrogenic differentiation, alcian blue was used to confirm the expression of cartilage proteoglycan after tissue culture in the existence of differentiation factors. This study was focused on the isolation of and the differentiating potential of MSCs after consecutive passages. The protocols for manufacturing neocartilage composed of primary chondrocytes established in Dr. Lai’s laboratory was followed to engineer a new neocartilage from MSCs. In conclusion, sufficient MSCs would be obtained easily in this laboratory. The MSCs are cultivated in rat-tail type I collagen matrix under various culture conditions. The newly formed cell-matrix constructs was induced to differentiate into cartilage-like tissue by TGF-β1. Chondrocytic morphology and expression of cartilage proteoglycan were used to evaluate the progression of chondrogenesis. Further efforts are necessary to optimize the conditions for fabricating and evaluating neo-cartilage derived from MSCs. The obtained data will provide crucial information for the development of autogenic neocartilage implantation.

並列關鍵字

Mesenchymal stem cell ； Collagen ； TGF-β1 ； Cartilage ； tissue staining ； Tissue culture ； Chondrocyte ； Tissue engineering

參考文獻

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