Three aligned, electrospun fiber scaffolds with unique surface features were created from poly-L-lactic acid (PLLA). Fibers without surface nanotopography (smooth fibers), fibers with surface divots (shallow pits), and fibers with surface pits (deeper pits) were fabricated, and fiber alignment, diameter, and density were characterized using scanning electron microscopy (SEM). Whole dorsal root ganglia (DRG) were isolated from rats and placed onto uncoated fibers or fibers coated with laminin. On uncoated fibers, neurite outgrowth was restricted by fibers displaying divoted or pitted nanotopography when compared to neurite outgrowth on smooth fibers. However, neurites extending from whole DRG cultured on laminin-coated fibers were not restricted by divoted or pitted surface nanotopography. Thus, neurites extending on laminin-coated fibers were able to extend long neurites even in the presence of surface divots or pits. To further explore this result, individual neurons isolated from dissociated DRG were seeded onto laminin-coated smooth, pitted, or divoted fibers. Interestingly, neurons on pitted or divoted fibers exhibited a 1.5-fold increase in total neurite length, and a 2.3 or 2.7-fold increase in neurite branching compared to neurons on smooth fibers, respectively. Based on these findings, we conclude that fiber roughness in the form of pits or divots can promote extension and branching of long neurites along aligned electrospun fibers in the presence of an extracellular matrix protein coating. Thus, aligned, electrospun fibers can be crafted to not only direct the extension of axons but to induce unique branching morphologies.
There is currently no cure for individuals with spinal cord injury (SCI). While many promising approaches are being tested in clinical trials, the complexity of SCI limits several of these approaches from aiding complete functional recovery. Several different categories of biomaterials are investigated for their ability to guide axonal regeneration, to deliver proteins or small molecules locally, or to improve the viability of transplanted stem cells. The purpose of this study is to provide a brief overview of SCI, present the different categories of biomaterial scaffolds that direct and guide axonal regeneration, and then focus specifically on electrospun fiber guidance scaffolds. Much like other polymer guidance approaches, electrospun fibers can retain and deliver therapeutic drugs. The experimental section presents new data showing the incorporation of two therapeutic drugs into electrospun poly-
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.
Central nervous system (CNS) injuries persist for years, and currently there are no therapeutics that can address the complex injury cascade that develops over this time-scale. 17β-estradiol (E2) has broad tropism within the CNS, targeting and inducing beneficial phenotypic changes in myriad cells following injury. To address the unmet need for vastly prolonged E2 release, we report first-generation poly(pro-E2) biomaterial scaffolds that release E2 at nanomolar concentrations over the course of 1–10 years via slow hydrolysis in vitro. As a result of their finely tuned properties, these scaffolds demonstrate the ability to promote and guide neurite extension ex vivo and protect neurons from oxidative stress in vitro. The design and testing of these materials reported herein demonstrate the first step towards next-generation implantable biomaterials with prolonged release and excellent regenerative potential.
Digital light processing (DLP)-based three-dimensional (3D) printing offers large improvements in fabrication throughput and spatial resolution when compared to various other additive manufacturing techniques. Both properties are highly desirable when fabricating biomaterial scaffolds that require design precision. Poly(glycerol sebacate) acrylate (PGSA) is a degradable, biocompatible, and photocurable elastomer. In this work, PGSA ink was developed for DLP 3D printing of porous tubular structures. Ink formulations with varying prepolymer concentrations (10–60 wt %), diluent (dimethyl sulfoxide (DMSO), 2-butoxyethyl acetate (EGBEA), and 1:1 DMSO/EGBEA), and degree of PGSA acrylation (17–75%) were studied to optimize printing efficiency and bulk properties of the printed scaffolds. Prepolymer inks with viscosity (<5 Pa·s) and photopolymerization kinetics (exposure time <10 s) appropriate for DLP were developed. Photocrosslinked PGSA scaffolds were further exposed to postfabrication treatments including additional UV exposure or thermal curing (150 °C) to demonstrate tunability in scaffold degradation kinetics and mechanical properties. Complementary to this effort, a 3D model-generation tool was developed to enable user-friendly customization of tubular scaffold design by controlling the pore and strut size of the volumetric mesh. The resulting DLP-printed PGSA scaffolds present high mimicry to complex 3D models with a minimum feature thickness of 80 μm. The tunable properties of PGSA coupled with enhanced precision in microstructure geometry provide a fabrication platform for a variety of tissue regeneration applications.
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