Industrial Research Institute Swinburne (IRIS) and Department of Chemistry and Biotechnology, Swinburne University of Technology , Hawthorn, 3122 Victoria, Australia.
ACS Appl Mater Interfaces. 2015 Mar 4;7(8):4979-89. doi: 10.1021/acsami.5b00107. Epub 2015 Feb 20.
Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.
有序表面纳米结构因其能够以可控的方式调节细胞表面相互作用而在生物技术和生物医学工程中引起了广泛关注。然而，由于制造成本高和制造速度慢，大面积有序纳米结构的制造能力受到限制。在这里，我们使用包括胶体自组装、胶体光刻和掠入射沉积（GLAD）在内的多种简便技术组合，制造了具有大面积（1.5×1.5cm(2)）的有序纳米结构。将聚苯乙烯（722nm）胶体在水-空气界面自组装成六方密堆积（hcp）晶体阵列，转移到生物相容性钽（Ta）表面上，并用作掩模以生成有序 Ta 图案。通过通过晶体掩模溅射涂覆 Ta，形成约 60nm 高的特征尺寸。使用 GLAD 分别进一步将特征尺寸增加约 200nm 高度，从而制造了四种不同的表面（FLAT、Ta60、GLAD100 和 GLAD200）。在这些有序纳米结构上研究了原代人脂肪来源干细胞（hADSCs）的细胞黏附、增殖和成骨分化长达 2 周。我们的结果表明，hADSCs 的细胞铺展、焦点粘附形成和丝状伪足延伸在 GLAD 表面受到抑制，而每个表面的生长速度相似。I 型胶原蛋白（COL1）和骨钙素（OC）的免疫染色显示，在成骨培养 1 周后，与 Ta60 和 FLAT 表面相比，GLAD 表面沉积了更高的成骨成分。在成骨培养 2 周后，GLAD 表面的碱性磷酸酶（ALP）活性和钙含量更高。此外，成骨样细胞在 Ta60 和 FLAT 表面上融合，而 GLAD 表面未完全覆盖，这表明 GLAD 表面上细胞-细胞相互作用强于细胞-基底相互作用。在 GLAD 表面可以发现可见的富含氮的多孔表面装饰的细胞外基质沉积物。使用新型 Ar 团簇源和 X 射线光电子能谱（XPS）对表面成分进行深度剖析表明，GLAD 表面上沉积的细胞外基质富含氮。所制造的有序表面纳米形貌具有在多个领域应用的潜力，并证明人干细胞的行为可以在这些有序纳米形貌上得到指导，为生物材料和组织工程中的应用提供了新的知识。
