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

Abstract

Developing robust in vitro models of in vivo environments has the potential to reduce costs and bring new therapies from the bench top to the clinic more efficiently. This study aimed to develop a biomaterial platform capable of modeling isotropic-to-anisotropic cellular transitions observed in vivo, specifically focusing on changes in cellular organization following spinal cord injury. In order to accomplish this goal, nebulized solvent patterning of aligned, electrospun poly-l-lactic acid (PLLA) fiber substrates was developed. This method produced a clear topographic transitional boundary between aligned PLLA fibers and an isotropic PLLA film region. Astrocytes were then seeded on these scaffolds, and a shift between oriented and non-oriented astrocytes was created at the anisotropic-to-isotropic fiber/film transition (AFFT) boundary. Orientation of chondroitin sulfate proteoglycans (CSPGs) and fibronectin produced by these astrocytes was analyzed, and it was found that astrocytes growing on the aligned fibers produced aligned arrays of CSPGs and fibronectin, while astrocytes growing on the isotropic film region produced randomly-oriented CSPG and fibronectin arrays. Neurite extension from rat dissociated dorsal root ganglia (DRG) was studied on astrocytes cultured on anisotropic, aligned fibers, isotropic films, or from fibers to films. It was found that neurite extension was oriented and longer on PLLA fibers compared to PLLA films. When dissociated DRG were cultured on the astrocytes near the AFFT boundary, neurites showed directed orientation that was lost upon growth into the isotropic film region. The AFFT boundary also restricted neurite extension, limiting the extension of neurites once they grew from the fibers and into the isotropic film region. This study reveals the importance of anisotropic-to-isotropic transitions restricting neurite outgrowth by itself. Furthermore, we present this scaffold as an alternative culture system to analyze neurite response to cellular boundaries created following spinal cord injury and suggest its usefulness to study cellular responses to any aligned-to-unorganized cellular boundaries seen in vivo.

Biomaterial-Supported Cell Transplantation Treatments for Spinal Cord Injury: Challenges and Perspectives

“…Given that previous work with astrocytic grafting shows a lack of regenerative potential, astrocytes, themselves may be better suited as living scaffolds, linearly guiding axons in and out of biomaterials. Comparison of cultured astrocytes on either anisotropic poly(L-lactic acid) (PLLA) fibers or isotropic PLLA films revealed linear orientation of astrocytes to the anisotropic substrate, which provided a guidance matrix for the cultured astrocytes and dorsal root ganglia (DRG) neurons (Zuidema et al, 2015 ). When tethered self-aligning collagen gels aligned both the biomaterial with astrocytes this lead to significant increased growth of adult rat DRG in the aligned portions compared to the unaligned portions (East et al, 2010 ).…”

Bioengineered cell culture systems of central nervous system injury and disease

“…Recently, an in vitro culture system using patterned electrospun fibers of poly-L-lactic acid and smooth films was developed to study how astrocytes respond to local changes in surface topography that might be similar to topographical changes following SCI (Fig. 1c) [60].…”

Biomaterial Approaches to Modulate Reactive Astroglial Response

“…The aligned fibers directed astrocyte growth, and neurons were found associated with the oriented astrocytes. Another in vitro model produced a transition boundary of aligned astrocytes with non-oriented astrocytes by removing aligned poly-L-lactic acid fibers using chloroform [Zuidema et al, 2015]. This created a gap composed of a polymer film bordering aligned fibers.…”