Cells elongate on a surface with nanogrooved (NG) patterns and align along that pattern. Although various models have been proposed for how this occurs, much remains to be clarified. Studies with fixed cells do not lend themselves to answering some of these open questions. In this study, the dynamic behaviours of living mesenchymal stem cells on an NG substrate with a 200 nm groove depth, an 870 nm ridge width and a 670 nm groove width were observed using time-lapse microscopes. We found that filopodia moved as if they were probing the surroundings of the cell protrusion, and then some cell protrusions invaded the probed areas. Cell protrusions that extended perpendicular to the NG direction tended to retract more rapidly than those parallel to the grooves. From these facts, we think that the retracting phase of cell protrusions play a rule in cell alignment along the NG patterns.

Collagen hydrogel is a popular extracellular matrix (ECM) material in regenerative medicine and has an isotropic structure. In contrast, native ECM has an anisotropic structure. Electrospinning of collagen dissolved in organic solvents is widely used for fabricating anisotropic collagen nanofibres; however, such fibres are water-soluble and require cross-linking before use as scaffolds for cell culture. Herein, electrospinning using a core-shell nozzle was employed to spin an aqueous acidic solution of collagen and encapsulate it within a shell of polyvinylpyrrolidone (PVP). Subsequently, the core collagen was gelled, and the shell PVP was washed away using a basic ethanol solution to yield anisotropic collagen hydrogel nanofibres. Immunostaining and Fourier transform infrared spectroscopy revealed that the obtained fibres were composed of collagen, and surface PVP was removed completely. Circular dichroism measurements confirmed that the fibres exhibited the triple helical structure characteristic of collagen. Human umbilical vein endothelial cells cultured on the collagen hydrogel fibres were oriented along the fibre direction. Hence, this method is suitable for fabricating fibrous anisotropic collagen hydrogels without chemical and thermal cross-linking, and can facilitate the development of safe medical materials with anisotropy similar to that of native ECM.

Cell behavior can be manipulated by the topography of the culture surface. In this study, we examined the intercellular communication and osteogenic differentiation of mesenchymal stem cells (MSCs) grown on electrospun fibers with different orientations and densities. Human bone marrow-derived MSCs (hMSCs) were seeded on poly(ε-caprolactone) (PCL) electrospun scaffolds composed of aligned (1D) or cross-aligned (2D) fibers (1.0-1.2 µm diameter) with high, medium, or low fiber densities. It was found that cells preferred to adhere onto electrospun PCL fibers rather than on the flat substrate. The immunofluorescence staining showed that the expression of vinculin, a focal adhesion protein, was limited to the periphery and the two extremities of aligned cells on the edge of the fibers. Electron microscopy showed that cells extended their lamellipodia across the adjacent fibers and proliferated along the direction of fibers. Cells grown on 1D fibrous scaffolds at all fiber densities had an obvious alignment. On 2D fibers, a higher degree of cell alignment was observed at the higher fiber density. On 1D scaffolds, the gap junction intercellular communication (GJIC) quantified by the lucifer yellow dye transfer assay was significantly promoted in the aligned cells in the direction parallel to the fibers but was abolished in the direction perpendicular to the fibers. The expression of osteogenic marker genes (RUNX2, ALP, and OCN) was significantly enhanced in seven days by culture on 1D but not 2D fibers. It was thus proposed that the promoted osteogenic differentiation of hMSCs may be associated with the fiber-guided and directional induction of GJIC.

Hydrogel nanofibers derived from alginate with an anisotropic structure fabricated by using core–shell electrospinning play an important role in cell adhesion and proliferation as the extracellular matrix (ECM).

Here we show a new strategy for efficient freeze concentration-mediated cytoplasmic delivery of proteins, obtained via the endosomal escape property of polyampholyte-modified liposomes. The freeze concentration method successfully induces the efficient internalization of proteins simply by freezing cells with protein and nanocarrier complexes. However, the mechanism of protein internalization remains unclear. Here, we designed a novel protein delivery carrier by modifying liposomes through incorporating hydrophobic polyampholytes therein. These complexes were characterized for particle size, encapsulation efficiency, and cytotoxicity. Flow cytometry and microscopic analysis showed that the adsorption and internalization of protein-loaded polyampholyte-modified liposomes after freezing were enhanced compared with that observed in unfrozen complexes. Inhibition studies demonstrated that the internalization mechanism differs between unmodified and polyampholyte-modified liposomes. Furthermore, polyampholyte-modified liposomes exhibited high efficacy in facilitating endosomal escape to enhance protein delivery to the cytoplasm with low toxicity. These results strongly suggest that the freeze concentration-based strategy could be widely utilised for efficient cargo delivery into the cytoplasm in vitro not only in cancer treatment but also for gene therapy as well.