Effect of Electrospun Fiber Mat Thickness and Support Method on Cell Morphology

Abstract

Electrospun fiber mats (EFMs) are highly versatile biomaterials used in a myriad of biomedical applications. Whereas some facets of EFMs are well studied and can be highly tuned (e.g., pore size, fiber diameter, etc.), other features are under characterized. For example, although substrate mechanics have been explored by several groups, most studies rely on Young's modulus alone as a characterization variable. The influence of fiber mat thickness and the effect of supports are variables that are often not considered when evaluating cell-mechanical response. To assay the role of these features in EFM scaffold design and to improve understanding of scaffold mechanical properties, we designed EFM scaffolds with varying thickness (50-200 µm) and supporting methodologies. EFM scaffolds were comprised of polycaprolactone and were either electrospun directly onto a support, suspended across an annulus (3 or 10 mm inner diameter), or "tension-released" and then suspended across an annulus. Then, single cell spreading (i.e., Feret diameter) was measured in the presence of these different features. Cells were sensitive to EFM thickness and suspended gap diameter. Overall, cell spreading was greatest for 50 µm thick EFMs suspended over a 3 mm gap, which was the smallest thickness and gap investigated. These results are counterintuitive to conventional understanding in mechanobiology, which suggests that stiffer materials, such as thicker, supported EFMs, should elicit greater cell polarization. Additional experiments with 50 µm thick EFMs on polystyrene and polydimethylsiloxane (PDMS) supports demonstrated that cells can "feel" the support underlying the EFM if it is rigid, similar to previous results in hydrogels. These results also suggest that EFM curvature may play a role in cell response, separate from Young's modulus, possibly because of internal tension generated. These parameters are not often considered in EFM design and could improve scaffold performance and ultimately patient outcomes.

Keywords: biomaterials; electrospun fiber mats; finite element modeling; glioblastoma; mechanobiology.

Figure 1

Schematic of Electrospun Fiber Mats. Electrospun fiber mats (EFMs) were either spun directly onto a stiff support (A), spun across a gap (B), or spun unto foil, removed, and then fixed across a gap (C).

Figure 2

Representative scanning electron microscope (SEM) images of EFMs. Aligned (A) and tension-released (B) 100 µm thick EFMs showing differences in fiber morphology following release of internal tension. Insets: Fourier transform of the figure, indicating degree of alignment. Scale bar = 50 µm.

Figure 3

Glioblastoma cell Feret diameter (A) as a function of polystyrene (PS)-supported EFM thickness (B). Feret diameter is indicated for a representative cell (red lines). A total of 979 cells were analyzed across at least two independent experiments. Levels connected by a star (*) are statistically significant from each other (p < 0.05).

Figure 4

Glioblastoma cell Feret diameter in response to culture on 50 µm EFMs supported on substrates of declining stiffness (A) and on polydimethylsiloxane (PDMS) supports of declining stiffness with no EFM present (B). A total of 1623 cells were analyzed across at least two independent experiments. Levels connected by a star (*) are statistically significant.

Figure 5

Glioblastoma cell Feret diameter on supported (PS) and suspended (10 mm, 3 mm) 50 µm EFMs. Scaffolds are arranged left to right by increasing indentation modulus. A total of 935 cells were analyzed across at least two independent experiments. Levels connected by a star (*) are statistically significant from each other (p < 0.05).

Figure 6

Glioblastoma cell Feret diameter measured on supports with 3 and 10 mm diameter annular gaps supporting EFMs of different thicknesses. A total of 1097 cells were analyzed across at least two independent experiments. Levels connected by a star (*) are statistically significant from each other (p < 0.05).

Figure 7

Average cell Feret diameter observed on tension-released and suspended EFM constructs, pooled data across all EFM thicknesses (A) and between tension-released mats at different fiber mat thicknesses (B). A total of 1651 cells were analyzed across at least two independent experiments. Levels connected by a star (*) are statistically significant from each other (p < 0.05).