MG63類成骨母細胞在聚己內酯立體支架中的生長

Abstract

摘要 細胞在三維空間(3D)中環境較在二維平面(2D)複雜，例如在細胞貼附、細胞聚集、細胞移動、細胞形狀、細胞增生分化、細胞基因表現、細胞和細胞外間質之間的影響及細胞與細胞之間的訊息傳遞等行為表現上都可能有所差異。 人體組織細胞大多數是處於由細胞外間質所建構的三維空間中，為模擬細胞在體內的情形，設計體外實驗將細胞培養在3D空間中，並與2D平面的生長情形作比較。 本實驗使用的細胞是類成骨母細胞MG63，材料選擇是聚己內酯polycaprolactone (PCL)，基材的型式為薄膜及3D立體支架。支架是採用快速原型技術中的fused deposition method(FDM)所製造，設計的支架孔洞有三種孔徑尺寸，分別為#300um、#250um、#200um，上述尺寸表示3D支架中相鄰二條PCL fiber的中心線距，而每條PCL fiber的寬徑平均為130um。 MG63種植在2D的實驗結果顯示：Tissue culture plate及PCL薄膜上的形態為紡錘狀；反觀在3D的支架上，細胞有聚集現象且被細胞外間質包覆而呈現立體架構，而後漸漸的將孔洞表面完全覆蓋填滿。三種支架孔徑尺寸的細胞活性測試結果以細胞生長在孔徑尺寸為#200um最良好。MG63培養在二層膜系統中，上下層薄膜都能檢測到細胞活性及觀察到細胞附著形態。 生長於鈣化誘導培養液中的MG63細胞，其鹼性磷酸酶(Alkaline phosphatase)，基因表現隨著時間遞增而增加，且在3D PCL支架比在2D PCL薄膜明顯。

Abstract The mechanism of the cells grow in the three dimensional environment (3D) is more complicate than that two dimensional surface (2D), The behavior of the cells is different, in terms of cell attachment, cell aggregation, cell migration, cell shape, cell proliferation and differentiation, gene expression, the interaction between cell and extracellular matrix etc. In living tissue, the cells mostly exist and connect with ECM components, which integrated into 3D framework. To simulate the growth of the cells in vivo, we cultured the MG63 osteoblastic cells in 3D scaffold, which is polycaprolactone (PCL) and fabricated via fused deposition method. The diameter of PCL fiber of 3D scaffolds is 130um. The size of interconnected pore of 3D scaffold is expressed as the distance between two adjacent PCL fibers and three sizes of #300um, #250um, and #200um were designed. The morphology of MG63 cells seeded into PCL scaffold were examined by SEM. The MG63 cells cultured on 2D surface, including tissue culture plate and PCL membrane is flat and spindle in shape. However, the cells cluster intermingled with extracellular matrix was noted in 3D scaffold. The cell proliferation and connection of the cells attached on adjacent PCL fibers increased with culture period, followed by filling-in of the superficial pores of 3D scaffolds. The cell viability was aided by MTT assay, which indicated the growth of MG63 cells in our study. The results of MTT assay demonstrated the cellular growth in #200um scaffold was the best among the three scaffolds with different pore-sizes in our study. To provide the substrate for cell adhesion in the 2-dimensional culture surface, the cells grown on the PCL membrane were covered with another PCL membrane, which made MG63 cells interposed between upper and lower membranes. The cell viability and cell attachment to the upper membrane was detected. Moreover, the total viability on upper and lower membranes was higher than that on single membrane. The gene expression of osteogenic marker, alkaline phosphatase, of MG63 cells was assayed by polymerase chain reaction (PCR) technique, after the MG63 cells were cultured in osteogenic medium for 2 weeks and 4 weeks. Our preliminary results showed that the expression of ALP increased with culture time and was significantly higher in 3D PCL scaffold than on PCL membrane.