- 學位論文
以滑膜間質幹細胞合併去細胞軟骨基質進行軟骨組織工程
Applying Synovinal Mesenchymal Stem Cells and Development of Acellular Cartilage Matrix for Cartilage Tissue Engineering
摘要
軟骨組織工程是近年來修復關節軟骨的新趨勢，但目前遭遇到一些困難。由於自體軟骨細胞來源稀少又不易培養出適量的健康細胞，所以近年來大多改用間質幹細胞，但誘導間質幹細胞分化後容易走向肥大化，目前還沒有一個正確的誘導方式，在我們先前的研究中，利用軟骨碎塊可以誘導間質幹細胞進行軟骨分化，並能夠大量分泌醣胺多醣，生成類透明軟骨之結構。本研究希望能夠將軟骨去細胞化，並且保留天然的結構與成份，以期仿造天然軟骨環境，用於培養滑膜間質幹細胞，探討對幹細胞分化之效應。 本研究使用全關節置換手術取得之廢棄的軟骨組織，搭配物理、化學、酵素的方法將關節軟骨組織內的細胞打破、裂解並清洗出來。結果顯示大幅去除DNA的殘留並保留醣胺聚醣以及與生理上極為相似之纖維性結構。另外與滑膜間質幹細胞共同培養後可發現，去細胞軟骨基質能防止細胞散失，且第二型膠原蛋白表現量有上升的趨勢，顯示細胞有被誘導走向軟骨分化的跡象。使用高劑量的BMP-2誘導滑膜間質幹細胞軟骨分化會大量增加Type X Collagen的表現，而去細胞軟骨基質則能抑制此一現象。 可見去細胞軟骨基質能夠應用於軟骨組織工程，且具有些微誘導滑膜間質幹細胞軟骨分化之能力，如果生長因子配合去細胞軟骨基質使用則能抑制肥大化的現象。
並列摘要
Cartilage tissue engineering has been a new strategy for treatment of cartilage injury. However, there are some difficulties from the clinical perspective, such as the hypertrophic development and low proliferation of chondrocytes. Therefore, employing mesenchymal stem cells (MSCs) is an alternative solution. In addition, finding appropriate approaches to avoid hypertrophic differentiation of MSCs is important as well. The aim of this study was to explore the potential of discarded cartilage and synovium from total knee replacement (TKR) surgery as sources for cartilage tissue engineering by co-culturing decellular cartilage matrix and synovial-derived MSCs. The retaining cells were removed from discarded cartilage matrix through a series of physical, chemical, and enzymatic processes. The remaining DNA, glycosaminoglycans (GAGs), and collagen were determined. Cells derived from human synovium were characterized by flow cytometry and differentiation assays. After decellular treatment, the decellular cartilage powder were cultivated with synovial-derived MSCs. The effects of decellular cartilage powder on synovial-MSCs were analyzed by qPCR. The results showed that there were high portion of GAGs and collagen retained in the DNA removed matrix, and the secretion of type II collagen was increased when synovial-MSCs cultured with decellular matrix powder. The chondrogenetic differentiation of synovial-MSCs were slightly promoted by decellular matrix. Besides, decellular matrix had some supressive effect on the expression of type X collagen while synovial-MSCs were induced with growth factors. In conclusion, the cartilage ECM-derived decellular matrix shows potential as biomaterial for cartilage tissue engineering, and it promotes induction of synovial-MSCs as well.