Engineered micro and nanotopography for neuron and glial guidance after spinal cord injury

Johnson, Christopher David Landis
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Gilbert, Ryan
Hurley, Jennifer M.
Thompson, Deanna M.
Hahn, Mariah
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Biomedical engineering
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Attribution-NonCommercial-NoDerivs 3.0 United States
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
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Spinal cord injury presents many barriers to regeneration that have yet to be solved. Guidance conduits are a promising approach that allow regenerating tissue to bypass or traverse the injury site. Peripheral nerve autografts into the spinal cord have guided neurons and glia around an injury site and resulted in some functional recovery [1]. Synthetic guidance conduits made from electrospun fibers have shown an ability to guide robust regeneration after spinal cord injury [2]–[4]. The surface topography of the electrospun fiber scaffolds is important for cell guidance, and can be modified by changing the fiber diameter, fiber alignment, and fiber surface nanotopography, among others. While the effects of these fiber physical characteristic on neuron guidance has been determined, it remains unknown how fiber diameter and surface nanotopography affect astrocytes - the most abundant cells in the CNS. Astrocytes maintain homeostasis in the healthy CNS and provide guidance and protection to neurons after injury. As a result, astrocytes should be considered an important therapeutic target for biomaterial approaches. The current thesis studied the effects of surface topography on astrocyte morphology and astrocyte phenotype.
Third, a novel biomaterial scaffold was prepared by incorporating superparamagnetic iron oxide nanoparticles (SPIONs) into the electrospinning solution. The purpose was to prepare an injectable electrospun fiber scaffold that could be magnetically oriented in situ. The surface topography was carefully controlled. The effect of incorporating increased amounts of SPIONs on the fiber diameter, fiber surface nanotopography, and fiber alignment was measured, and compared against the previous findings in this thesis and against a similar scaffold that has been successful in vivo [3]. The effect of the increasing SPION concentration on the fiber magnetization and on the speed of fiber alignment in viscous solutions was measured. From these data, the 6% SPION concentration exhibited the fastest fiber realignment times without significant changes to the fiber topography. As a result the 6% SPION fibers were tested in vitro by orienting the fibers to provide an aligned fiber scaffold within either a collagen/Matrigel or fibrin/Matrigel hydrogel. This fiber/hydrogel composite scaffold was tested by measuring the response of a primary rat dorsal root ganglion to the aligned fibers embedded within the hydrogel. The results showed that the magnetic electrospun fiber scaffold provided directional guidance to neurites, and increased both the length and alignment of the neurites within the three-dimensional hydrogel. This design proposes to unite the guidance benefits of guidance scaffolds and the minimally invasive benefits of injectable hydrogels.
Second, the astrocyte response to fiber diameter was tested. Astrocytes were cultured on large (800nm) diameter, small (386nm) diameter, and film substrates, and the astrocyte morphology was analyzed over the first four days in culture. Astrocytes became significantly more elongated on the large fibers as early as two hours after culture, and remained significantly more elongated after four days. The effect of fiber diameter on GLT-1, GLAST, and GFAP expression were examined by western blot. GLT-1 was significantly increased on the fiber substrates, compared to the film, but GLT-1 expression was not dependent on fiber diameter. A glutamate excitotoxicity assay was performed on an astrocyte/neuron coculture to determine if the increased astrocyte GLT-1 expression improved neuron protection. The findings suggest that fibrous surfaces improved cell survival, however, the differences were not statistically significant from the film control. Next neuron outgrowth was observed on the astrocyte surfaces prepared on each of the three topographies - small fiber, large fiber, and film. Neurons were cultured on the astrocyte/fiber surfaces and compared to an astrocyte/film control. The results suggest that after 4 days in culture, the astrocytes on the large fibers induced longer more directed growth, while the astrocytes on the small fibers induced comparatively shorter neurite growth with more neurite branching.
First, the astrocyte response to fiber surface nanotopography was tested. Astrocytes were cultured on three engineered surface topographies - smooth, pitted, or divoted fiber surfaces. Astrocytes isolated from the spinal cord were compared to astrocytes isolated from the cortex, to determine if there was a different response from astrocytes selected from different anatomical locations. The astrocyte morphological response was measured with immunocytochemistry, while the GFAP and vinculin protein expression was measured using Western blot techniques. The findings suggest that smooth fiber surfaces allowed for the longest cortical astrocyte extension after 4 days in culture, while the pits and divots reduced astrocyte elongation. The spinal cord astrocytes exhibited smaller elongation ratios over the times tested, compared to the cortical astrocytes, and did not have significantly different responses to the fiber surfaces tested. Since astrocyte elongation is correlated with neurite outgrowth, these data shows that the smooth fibers provide the greatest astrocyte elongation. The GFAP and vinculin expression were not significantly different among any of the surfaces or cell types tested.
May 2018
School of Engineering
Dept. of Biomedical Engineering
Rensselaer Polytechnic Institute, Troy, NY
Rensselaer Theses and Dissertations Online Collection
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