The development of suitable scaffolds for bone tissue engineering requires an in-depth understanding of the interactions between osteoblasts and scaffolding biomaterials. Although there have been a large amount of knowledge accumulated on the cell-material interactions on two-dimensional (2D) planar substrates, our understanding of how osteoblasts respond to a biomimetic nanostructured three-dimensional (3D) scaffold is very limited. In this work, we developed an approach to use confocal microscopy as an effective tool for visualizing, analyzing, and quantifying osteoblast-matrix interactions and bone tissue formation on 3D nanofibrous gelatin scaffolds (3D-NF-GS). Integrin β1, phosphor-paxillin, and vinculin were used to detect osteoblasts responses to the nanofibrous architecture of 3D-NF-GS. Unlike osteoblasts cultured on 2D substrates, osteoblasts seeded on 3D-NF-GS showed less focal adhesions for phospho-paxillin and vinculin, and the integrin β1 was difficult to detect after the first 5 days. Bone sialoprotein (BSP) expression on the 3D-NF-GS was present mainly in the cell cytoplasm at 5 days and inside secretory vesicles at 2 weeks, whereas most of the BSP on the 2D gelatin substrates was concentrated either in cell interface toward the periphery or at focal adhesion sites. Confocal images showed that osteoblasts were able to migrate throughout the 3D matrix within 5 days. By 14 days, osteoblasts were organized as nodular aggregations inside the scaffold pores and a large amount of collagen and other cell secretions covered and remodeled the surfaces of the 3D-NF-GS. These nodules were mineralized and were uniformly distributed inside the entire 3D-NF-GS after being cultured for 2 weeks. Taken together, these results give insight into osteoblast-matrix interactions in biomimetic nanofibrous 3D scaffolds and will guide the development of optimal scaffolds for bone tissue engineering.
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