本研究目的希望能使用天然性的藻膠粉末所製作出來的alginate beads為一暫時性的細胞附著支架，把本實驗室培養的初代軟骨細胞種植進去，經過生物反應器的作用，再把暫時性的支架溶解，把細胞離心轉移到多孔性的人造過濾膜上，評估此方式對於豬膝蓋軟骨細胞的影響。本研究主要利用alginate beads為暫時性支架的目的有二: 1.支架在組織工程實驗中的角色，相當重要，可提供細胞力學上的支持，可以提供細胞攀附的著力點，更可以提供因有利學上的支持後所衍生出的細胞產物 2.我們在實驗後其要溶解alginate beads的目的，是把我們藉由經過暫時性支架的細胞的衍生物，再行離心，此為我們所預期的實驗結果。海藻酸鈉(sodium alginate)為一線性多醣類可溶性聚電解質，容易與二價陽離子Ca2+ 、Cu2+ 、Co2+等發生鍵合作用，產生分子間的架橋作用。由於凝膠中同時存在海藻酸鈉、海藻酸和海藻酸鈣三種分子結構的互相交錯連接 ;一開始的交聯作用因為結合力的不均衡，產生較大的空隙，因此可容納大量的水分。之後使用化學性的染色方式以及RT-PCR，來進行培養物的檢驗，針對β-actin、collagen type 2、aggrecan進行實驗前後的比較，實驗結果後collagen type 2 以及aggrecan在含量上為相對性的減少，本研究討論結果為生物反應器的力學環境以及暫時性的支架尚需改進，但在整體實驗流程上面已是一個穩定的組織工程結果，在追求更進一步的質量上面的研究，尚需更為長久的研究人力加入，以期能對於下一階段的醫學工程，提供一己之力。

We use a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult porcine chondrocytes without the aid of a synthetic matrix. The first step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cellassociated matrix (CM). Then we use cyclic static pressures on chondrocytes cultured in alginate beads, which is physiologically closer to the in vivo environment of cells in cartilage. Articular cartilage chondrocytes cultured in alginate beads were exposed to 1.9 MPa cyclic and static loadings via a custom-made chamber based machine. The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. These tissues, easily recovered from the insert, were then subjected to biochemical and staining analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with Safranin O,fast green and Sirius Red. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue. Compared with controls (unloaded cells), cyclic loading decreased the collagen and aggrecan content of cells at 1.9 MPa in cellular matrix and further removed matrix. In alginate matrix, chondrocytes released a metalloprotease, which required Mn2+ for activity. Cyclic load levels inhibited its specific activity in cellular matrix.

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