摘要
近年來，細胞培養技術在生物科技領域具有極大的研究價值，細胞在生物體內無時無刻都受到物理刺激（Physical stimulation），而這些物理刺激透過細胞再轉換成化學信號（Chemical signal），這些化學信號對於細胞的生理行為扮演著重要的腳色，例如:細胞凋亡（Apoptosis）、細胞分化（Cell differentiation）、細胞增殖（Cell proliferation）、細胞遷移（Cell migration） 等。傳統細胞培養方式是使用聚苯乙烯為材料的培養皿（Dish）或者培養角瓶（Flask）進行細胞培養，這種培養方式雖然可以大量的培養細胞，但無法得到較貼近生理環境的細胞生理行為，因此，仿生動態培養對於生物科技的研究是必要的。
目前較常使用的細胞生物相容性彈性體，如：聚二甲基矽氧烷 （Polydimethylsiloxane, PDMS）及聚氨酯（Polyurethane, PU），多數文獻的作法是將基材浸泡於蛋白質（如:type I collagen）溶液使蛋白質沉積在基材表面，但此方法可能在動態培養過程造成材料表面原本沉積的蛋白質脫落，而導致細胞脫離基材表面而死亡。因此，本研究以濕式化學法對PDMS及PU兩種彈性體進行表面修飾，透過共價鍵（covalent bond）將生物相容性高分子或蛋白質接枝在彈性體的表面且不影響其原本物理性質（如：透光度及機械強度），同時在培養過程中結合週期性物理刺激來促進細胞的生長與分化。
本實驗以週期性物理拉伸/壓縮兩種模式來模擬生理微環境，在此環境下觀察二維PDMS基材（Two-Dimensional, 2D PDMS substrate）與三維PU支架（Three-Dimensional PU scaffold, 3D PU scaffold）表面修飾層對於類成骨細胞 （Osteoblast-like MG-63 cells）與大鼠脂肪幹細胞（rat Adipose-Derived Stem Cells, rADSCs）生長與分化之影響。PDMS透過常壓氧氣電漿將其表面導入氫氧官能基（Hydroxyl groups），接著利用戊二醛將表面修飾層（Alginate, type I collagen, poly-l-lysine, fibronectin and laminin）與基材進行共價鍵交聯並以MG-63進行動態培養且觀察其生長與分化。透過 ESCA 分析確認表面修飾層成功接枝於基材表面外，同時經由超音波震盪確認本研究接枝方法可穩定將表面修飾層接枝於PDMS表面以確保動態培養過程中細胞貼附的穩定性。由於MG-63為一類成骨細胞，其細胞所分泌的細胞外基質（Extracellular matrix, ECM）含有type I collagen，實驗結果發現PDMS表面修飾type I collagen相較於其他表面修飾層，除了具有良好的促進增殖效果外，骨分化指標：鹼性磷酸酶（Alkaline phosphatase, ALP）與骨鈣素（Osteocalcin, OCN）也有顯著上升。
本研究除了觀察表面修飾層與細胞分泌 ECM 之相容性外，也進一步利用表面修飾概念，將具有功能性的表面修飾層與細胞膜直接進行接觸，來促進rADSCs生長或分化。分別將PDMS表面接枝type I collagen與type I collagen/transforming growth factor beta-1 (TGF-β1)，利用PDMS表面的TGF-β1 直接與細胞膜上的受器receptor II serine/threonine接觸，直接對rADSCs進行軟骨誘導分化（Chodrogenesis differentiation）。常見的誘導方式為使用軟骨分化培養基（含有TGF-β1）進行長天數培養（大於7天），本研究發現TGF-β1修飾於PDMS表面並搭配週期性物理拉伸進行誘導，相較於傳統誘導方式，72小時即可觀察到軟骨形成分化基因：SOX-9、col2a1、aggrecan (ACN) 與proteoglycans蛋白質分泌。另一方面，本研究亦將抗氧化劑type I collagen/N-acetylcysteine (NAC) 接枝到 PU scaffold 孔洞表面，透過直接接觸細胞膜來調節extracellular signal-regulated kinases–mitogen-activated protein kinases (ERK-MAPK) 訊號路徑，希望藉此提升細胞的抗氧化能力。由實驗結果發現，經過72小時共培養，PU scaffold 孔洞表面修飾NAC並搭配週期性物理壓縮除可促進軟骨形成分化基因：SOX-9、aggrecan表現外，同時出現明顯的細胞增殖效果。

SUMMARY
A mild and stable process for PDMS surface modification and a bio-mimicking dynamic culturing platform were developed to evaluate the cellular responses of osteoblast-like MG-63 cells on PDMS substrate with different hydrophilic grafting layer. The experimental platform developed here, including the dynamic culture system and the modification scheme for forming PDMS-COLL, could be beneficial in evaluating the cellular responses in a more bio-mimicking and clinically relevant manner as well as in cultivating the cell/tissue needed for clinical applications.
The growth factor, TGF-β1, was co-grafted with the type I collagen onto the elastic PDMS substrate for examining the combinatory effect of surface chemistry and mechanical stimuli on rADSCs’ chondrogenesis differentiation. After 72hr static culture, the PDMS-COLL and PDMSCOLL/TGF-β1 were shown to be effective to induce the chondrogenic differentiation than control group. In contrast, if the dynamic stretching stimuli were utilized, the efficiency in chondrogenic differentiation is dependent upon the substrate as well as the dynamic stretching protocol utilized. The surface grafted with collagen and TGF-β1 layer induced more proteoglycan secretion than the one with collagen only.
For PU scaffold applications, the extracellular matrix-like type I collagen layer and the grafted antioxidative reagent NAC have improved the rADSCs adherence and enhanced the stem cell proliferation efficiency in vitro. Further, the use of dynamic compression stimulation has promoted rADSCs differentiation into the chondrogenic lineage within a short cell culture duration. This anti-oxidative PU scaffold in the combination of physical compression stimulation could become a novel approach for stem cell therapy in the future.

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