Mechanical elongation of astrocyte processes to create living scaffolds for nervous system regeneration

Abstract

Following brain injury or neurodegenerative disease, successful regeneration requires orchestrated migration of neurons and reformation of long-distance communication fibres, or axons. Such extensive regeneration does not occur in the mature brain; however, during embryonic development, pathways formed by glial cells extend several millimeters (mm) to create ‘living scaffolds’ for targeted neural cell migration and axonal pathfinding. Techniques to recapitulate long process outgrowth in glial cells have proven elusive, preventing the exploitation of this developmental mechanism for regeneration. In the current study, astrocytes were induced to form a network of interconnected processes that were subjected to controlled mechanical tension in vitro using custom-built mechanobioreactors. We discovered a specific micron (μm)-scale mechanical growth regime that induced elongation of the astrocytic processes to a remarkable length of 2.5 mm at an optimal rate of 12.5 μm/h. More rapid mechanical regimes (> 20 μm/h) caused greater incidence of process degeneration or outright breakage, whereas slow regimes (< 4 μm/h) led to adaptive motility, thus failing to achieve process elongation. Cellular phenotype for this astrocytic ‘stretch-growth’ was confirmed based on presentation of the intermediate filament glial fibrillary acidic protein (GFAP). Mechanical elongation resulted in the formation of dense bundles of aligned astrocytic processes. Importantly, seeded neurons readily adhered to, and extended neurites directly along, the elongated astrocytic processes, demonstrating permissiveness to support neuronal growth. This is the first demonstration of the controlled application of mechanical forces to create long astrocytic processes, which may form the backbone of tissue-engineered ‘living scaffolds’ that structurally emulate radial glia to facilitate neuroregeneration.

Astrocyte Structural and Molecular Response to Elevated Intraocular Pressure Occurs Rapidly and Precedes Axonal Tubulin Rearrangement within the Optic Nerve Head in a Rat Model

“…Indeed, cells rich in actin (such as fibroblasts) are exquisitely sensitive to tissue strain and readily change orientation to counter stretch [69]. In addition, the observed reversible increase in astrocyte actin bundle length 1 day after IOP elevation, may imply that astrocyte response to local ONH mechanical tissue stress involves lengthening of extensions, which is consistent with observed reports of cultured astrocyte processes lengthening under mechanical stress [70]. …”

Transplantable living scaffolds comprised of micro-tissue engineered aligned astrocyte networks to facilitate central nervous system regeneration

“…80-23; revised 2011). Primary cortical astrocytes were isolated from postnatal day 0–1 Sprague-Dawley rat pups (Charles River, Wilmington, MA) and dissociated using an established protocol [21,31]. Dissociated cells were seeded in flasks containing DMEM/F12 supplemented with 10% FBS.…”

“…Dissociated cells were seeded in flasks containing DMEM/F12 supplemented with 10% FBS. Over weeks in culture, a nearly pure population of astrocytes (>95%) was obtained through mechanical agitation to suspend non-astrocytic cell types followed by media change and passage, as described [21,31,32], with astrocytic phenotype verified using immunocytochemistry as described below. To seed astrocytes in the micro- columns, dissociated cell solution (2–12 × 10 5 cells/mL) was precisely microinjected into the micro-columns using a stereoscope for visual guidance.…”

“…Seeded micro-columns were placed in a humidified tissue culture incubator for 40 min to allow cells to adhere. Finally, the culture vessel was flooded with pre-warmed serum-free medium to induce a mature, process-bearing astrocyte phenotype [21]. This defined astrocyte medium consisted of Neurobasal medium supplemented with 2% B-27, 1% G-5, and 0.25% L-Glutamine.…”

“…Here, we are pursuing the creation of tissue engineered “living scaffolds” to facilitate nervous system regeneration [15–21]. Living scaffolds derive from a combination of biomaterial and cell-based techniques to create preformed constructs consisting of living cells in a defined, often anisotropic, three-dimensional (3-D) [15,16] architecture.…”