Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-l-lactic acid fibers

Abstract

Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.

Glial responses to implanted electrodes in the brain

“…4). Nanostructured topographical features can increase astroglial ATP release 209 , downregulate GFAP expression 210 , and increase the expression of glutamate transporters and the clearance of extracellular glutamate 211 . Stiffer substrates have been associated with the activation of microglia (amoeboid morphology, upregulation of CD11b) and of astrocytes (hypertrophy, upregulation of GFAP), as well as their proliferation, migration and adhesion 158 .…”

The role of the surface on microglia function: implications for central nervous system tissue engineering

“…Moreover, although some authors did not find significant alterations in astrocyte activation when cells were seeded on a fibrous surface in comparison with cells cultured on flat solvent-cast films (assessed in terms of glial fibrillary acidic protein (GFAP) and vimentin protein expression) [14], others claimed that the contact of astrocytes with fibres is able to promote a decrease in GFAP expression [15] and an increase in glutamate uptake, contributing to neuroprotection in vivo [16]. Furthermore, by using micropatterned grooved scaffolds, mature astrocytes were found to revert into radial glia-like cells and consequently to a more pro-regenerative phenotype [17].…”

Nebulized solvent ablation of aligned PLLA fibers for the study of neurite response to anisotropic-to-isotropic fiber/film transition (AFFT) boundaries in astrocyte–neuron co-cultures

“…Initially, sterilized PLLA fiber scaffolds were coated with fibronectin (10 μg/mL), since we previously observed astrocytes strongly adhering to fibronectin coated fibers [34]. However, in experiments presented here, we observed astrocyte adhesion to be less at the AFFT interface, so a different coating procedure was devised.…”

“…Previously, our laboratory has shown that astrocytes cultured on aligned fibers have an aligned cytoskeleton, while the cytoskeletons of astrocytes cultured on PLLA films have no preferential orientation [34]. Similarly, others demonstrated aligned presentation of fibronectin produced by astrocytes cultured on adsorbed laminin patterns [39].…”

“…S5) makes this new biomaterial platform an attractive method to further study cellular transitions in vitro . While other studies have analyzed cellular responses to changes in fiber alignment [34], fiber diameter [32], and fiber density [42], this study is unique due to the ability of the scaffold to study cellular responses to topological transitions. The formation of isotropic fiber ablation regions in the PLLA fiber scaffolds had no discernable affects on nearby fiber orientation, density, or diameter (Fig.…”