在美國估計每年已經有超過五十萬的補骨手術案例，而且呈現倍數增加的趨勢。為了改善這個現象，許多文獻已經有探討研究補骨的方法：自體骨移植、異體骨移植、異種骨移植、異質成形骨粉、骨形成蛋白，都已經被開發並應用在人體治療。到目前為止，自體骨移植一直是補骨治療裡的黃金準則，然而口腔內部能取得骨粉的位置有限，而且從口腔外部來的自體骨可能會造成供體部位受傷或是感染的後遺症。有鑑於此，異體骨移植、異種骨移植、異質成形骨粉、骨形成蛋白替代方法也慢慢被開發。但可惜的是，這些替代方法缺乏直接骨誘導作用，而且無法保證他們的效力。為了解決上述的方法的缺陷，新穎的補骨方法：組織工程一直在研究進行中。將骨母細胞播種至適當的細胞載體，配合適當生長因子使細胞在載體內增生、分化、基質成形，並隨著載體裂解達到修復骨頭缺損的能力。
本次實驗目的是運用組織工程的方法：結合大鼠骨髓間葉幹細胞和海藻凝膠的方式。透過二甲基藍來測試不同的二價離子(鈣離子和鋇離子)形成的海藻凝膠裂解性質。再透過微電腦斷層掃描和掃描式電子顯微鏡來觀察海藻凝膠內部的孔洞分布、大小以及連接性。接著，從大鼠股骨骨髓抽取間葉幹細胞，加入維生素C、甘油磷酸、地塞米松誘導其形成骨分化細胞，運用免疫細胞化學染色證實骨分化生物標記：鹼性磷酸酶、骨鈣蛋白的表現後，再將骨分化細胞播種至海藻凝膠，運用細胞存活率分析 (MTT assay)測試細胞的生長情形。此外，運用G4RGDY短肽修飾海藻酸鈉的結構，改善細胞和海藻酸鈉之間的貼附能力。最後，再透過掃描式電子顯微鏡和能量散射光譜儀，觀察細胞在載體內部的形狀以及骨基質分泌。我們的實驗結果顯示：鋇離子海藻凝膠的裂解程度顯著小於鈣離子海藻凝膠。此外，載體的孔洞大小平均是150~300μm、孔洞連接性都超過95%。接著，骨化細胞也能在鋇離子海藻凝膠內部隨著時間慢慢增生、表現些微貼附並呈現圓形且聚集成堆的景象、並能分泌鈣離子和磷離子複合物。綜合上述結果：代表鋇離子和海藻酸鈉的鍵結強度比鈣離子強，形成的海藻凝膠載體穩定性較佳。而且細胞也適合在鋇離子海藻凝膠生長，而且能獲得營養供給，還能在內部表現出骨基質所需的元素。經實驗證實，透過鋇離子和海藻酸鈉形成的海藻凝膠是能讓骨分化細胞在內部生存。希望透過這次研究能提供另一個選擇方向，未來還能運用在動物實驗中，提升骨組織工程的治療效率。

關鍵字 : 骨髓間業基質幹細胞、海藻凝膠、鋇離子

In previous studies, we can see that there have been several studies about bone tissue regeneration composed of bone marrow mesenchymal stem cell, calcium-alginate, and growth factor. In this study, we aimed to use tissue engineering made up of bone marrow mesenchymal stem cell, barium-alginate, and growth factor to compare the alginate hydrogel integrity and osteoid secretion with calcium-alginate. First, we induced mesenchymal stem cell into osteoblast by osteogenic medium. Second, we observed the alginate hydrogel degradation by DMMB assay. Third, we calculated alginate hydrogel pore size and porosity by SEM and Micro CT. Fourth, we detected the cell viability within the alginate hydrogel by MTT assay. Finally, we observed cell morphology and osteoid secretion by SEM and EDS. The results show the degradation of barium-alginate is smaller than calcium-alginate, the pore size is 150~300μm, the porosity is greater than 95%, the cell can proliferate within barium-alginate through MTT assay, and the cell morphology within the hydrogel is round and cluster, and the calcium ion and phosphorus ions is detected by EDS. In summary, the composite of bone marrow mesenchymal stem cell and barium-alginate hydrogel can be a novel tissue engineering technique for bone
regeneration.

Key words : Bone marrow mesenchymal stem cell, barium-alginate hydrogel

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