Electrospun fiber surface nanotopography influences astrocyte-mediated neurite outgrowth

Abstract

Aligned, electrospun fiber scaffolds provide topographical guidance for regenerating neurons and glia after central nervous system injury. To date, no study has explored how fiber surface nanotopography affects astrocyte response to fibrous scaffolds. Astrocytes play important roles in the glial scar, the blood brain barrier, and in maintaining homeostasis in the central nervous system. In this study, electrospun poly L-lactic acid fibers were engineered with smooth, pitted, or divoted surface nanotopography. Cortical or spinal cord primary rat astrocytes were cultured on the surfaces for either 1 or 3 d to examine the astrocyte response over time. The results showed that cortical astrocytes were significantly shorter and broader on the pitted and divoted fibers compared to those on smooth fibers. However, spinal cord astrocyte morphology was not significantly altered by the surface features. These findings indicate that astrocytes from unique anatomical locations respond differently to the presence of nanotopography. Western blot results show that the differences in morphology were not associated with significant changes in glial fibrillary acidicprotein (GFAP) or vinculin in either astrocyte population, suggesting that surface pits and divots do not induce a reactive phenotype in either cortical or spinal cord astrocytes. Finally, astrocytes were co-cultured with dorsal root ganglia to determine how the surfaces affected astrocyte-mediated neurite outgrowth. Astrocytes cultured on the fibers for shorter periods of time (1 d) generally supported longer neurite outgrowth. Pitted and divoted fibers restricted spinal cord astrocyte-mediated neurite outgrowth, while smooth fibers increased 3 d spinal cord astrocyte-mediated neurite outgrowth. In total, fiber surface nanotopography can influence astrocyte elongation and influence the capability of astrocytes to direct neurites. Therefore, fiber surface characteristics should be carefully controlled to optimize astrocyte-mediated axonal regeneration.

Exploring the effects of electrospun fiber surface nanotopography on neurite outgrowth and branching in neuron cultures

“…Whole DRG were cultured onto uncoated smooth (Fig 3A), pitted (Fig 3B), and divoted fibers (Fig 3C) for 4 days prior to fixation, imaging, and analysis. Based on previous studies in the Gilbert lab that showed macrophages and astrocytes exhibited a more elongated morphology on smooth fibers compared to fibers with surface topography [20,21], we hypothesized that DRG cultured onto pitted and divoted fibers would extend shorter neurites than DRG cultured onto smooth fibers. Fig 3D shows that neurites extended significantly farther from DRG cultured onto smooth electrospun fibers (1.77 ± 0.17 mm) compared to pitted (1.33 ± 0.1 mm, p = .009) and divoted fibers (1.43 ± 0.17 mm, p = 0.035).…”

“…In recent years, the Gilbert laboratory has developed methods to control the formation of nano-scale surface depressions on the surface of electrospun fibers [19–21]. Researchers in the Gilbert laboratory cultured primary bone marrow macrophages onto electrospun fibers with smooth surfaces or with surface depressions.…”

“…In a more recent study, researchers cultured primary astrocytes (derived from rat spinal cords or cortices), and a coculture of either astrocyte type and a DRG explant onto smooth, pitted, and divoted fibers. This study elucidated how fiber surface nanotopography affects astrocyte biology, and astrocyte mediated neurite extension from DRG [21]. Cortical astrocytes elongated significantly further along smooth fibers compared to fibers with pitted or divoted surfaces.…”

“…Immediately before electrospinning, once the relative humidity in the room had equilibrated to the desired level, the glove box was closed to maintain the relative humidity during electrospinning. Tight control over humidity was imperative to reproducibly fabricate electrospun fibers with the desired surface nanotopography [19–21].…”

“…Fiber surface nanotopography was created by changing the humidity of the electrospinning environment, and/or including a non-solvent of the electrospinning polymer (poly(L-lactic acid), PLLA) in the electrospinning solution. These methods of engineering electrospun fiber surface nanotopography are discussed in detail in previous laboratory publications where astrocyte and macrophage response to changes in fiber surface topography were examined [19–21]. Having already analyzed astrocyte and macrophage response to fibrous nanotopography, the emphasis of this study focused on understanding how the presence of nanotopography affected neurite extension.…”