藉由範圍性奈米結構控制H9c2心肌纖維母細胞功能、增生、型態、和貼附

Abstract

奈米尺度表面形態會直接的影響細胞的行為。為了明確定義出調控細胞生長的結構 確切尺度範圍和型態，本研究將H9c2 心肌纖維母細胞養在10-nm 至200-nm 的奈米點 陣列上。奈米點陣列結構是藉由陽極氧化鋁製程(AAO)在TaN 表面的矽晶圓上製備而 成。 實驗結果發現，即使表面細胞數已經達飽和，cardiomyoblasts 在50-nm 尺度上的nanodots 仍有最佳的存活能力；100-nm 以及200-nm 表面，細胞培養三天後數量明顯減少了53.7%以及72.6%。50-nm 表面的細胞於表面有較大的貼附面積、並且較多的伸展細胞本體、細胞較快的成長速率；100-nm 和200-nm 表面細胞數目有明顯減少，並且伴隨著凋亡的產生。 根據螢光染色的vinculin 和actin filament 分布探討奈米表面對focal adhesion 的影 響，結構尺寸低於50-nm(含50-nm 尺度結構)會促進細胞的貼附、細胞骨架的完整，尤 其是在50-nm 結構上的細胞具顯著差異。Bromodeoxyuridine (BrdU) proliferation assay 也指出50-nm 結構也會促進細胞的增生。 我們也更進一步使用RT-PCR 和Western blot，針對基因還有蛋白質的分析，結果指出，在100-nm 的奈米點表面會誘發心肌細胞肥大和纖維化，我們推測可能是因為此奈米結構促成細胞的收縮功能降低，所引起的細胞肥大，纖維化的部分則是因為細胞間基質的降解所導致；高vinculin 表現量則是發生在50-nm nanodots，表示心肌纖維母細胞的貼附狀況良好。 根據實驗結果顯示，nanodots 結構調控cardiomyoblasts 生長是與奈米結構尺寸大小有關的。心肌纖維母細胞生長在50-nm 結構表面具有最佳的生存能力、型態、貼附、和增生能力；細胞生長在100-nm 以上的結構表面則會降低細胞的生長和貼附。未來，預期可以利用此研究的發現，應用到人工植體之生物醫學層面。

Surface topology at nanoscale encodes information that directs cell behavior. To identify the exact ranges in size and shape that modulates cell growth, H9c2 cells were cultured onto nanodot arrays with dot diameters ranging from 10 to 200-nm. The nanodot arrays were fabricated by anodic aluminum oxide (AAO) processing on TaN-coated wafers. Optimized growth occurred when cardiomyoblasts were cultured on nanodot arrays with dot size at 50-nm, on which maximum viability was maintained even when the cell density reached saturation. Nanodots of 100-nm and 200-nm prevented viable growth of cardiomyocytes with 53.7% and 72.6% reduction on day 3, respectively. Cells seeded on 50-nm nanodots showed flat morphology, with largest surface area, most extended lamellipodia, and fastest growth rate. Cell grown on flat surface remained stable in culture dish, while apoptosis-like growth was observed with 100-nm and 200-nm nanodots with significant reduction in the surface area. Immunostaining against vinculin and actin filament indicated that nanodots smaller than 50-nm promoted cell adhesion and cytoskeleton organization. Best adhesion occurred at 50-nm. Nanodots of 100-nm retarded the formation of focal adhesions while 200-nm inhibited the organization of cytoskeleton. Incorporation of Bromodeoxyuridine (BrdU) indicates proliferating growth of the cells. BrdU was applied to differentiate the newly proliferated cells from pre-existing culture. Maximum proliferation occurred for cells grown on 50-nm nanodots, which is approximately 2-fold compared to flat surface. We also utilize RT-PCR and Western blot to indicate fibrosis and hypertrophy were induced on 100-nm nanodots. We hypothesize the cardiomyoblasts seeded on 100-nm nanodots induced extracellular matrix-degrading metalloproteinases and promote fibrosis. High expression of vinculin was occurred in 50-nm nanodots. It means that 50-nm nanodots have good focal adhesion for cardiomyoblasts. Here we show that the ability of nanodot arrays to modulate the growth of cardiomyoblasts is size-dependent. Optimized growth with the best viability, morphology, adhesion, and proliferation occurred at size of 50-nm. Retardation of growth was observed when the dot size was larger than 100-nm. Possible application of nanostructure on the artificial implants is expected.