Neural tissue engineers are exploiting key mechanisms responsible for neural cell migration and axonal path finding during embryonic development to create living scaffolds for neuroregeneration following injury and disease. These mechanisms involve the combined use of haptotactic, chemotactic, and mechanical cues to direct cell movement and re-growth. Living scaffolds provide these cues through the use of cells engineered in a predefined architecture, generally in combination with biomaterial strategies. Although several hurdles exist in the implementation of living regenerative scaffolds, there are considerable therapeutic advantages to using living cells in conjunction with biomaterials. The leading contemporary living scaffolds for neurorepair are utilizing aligned glial cells and neuronal/axonal tracts to direct regenerating axons across damaged tissue to appropriate targets, and in some cases to directly replace the function of lost cells. Future advances in technology, including the use of exogenous stimulation and genetically engineered stem cells, will further the potential of living scaffolds and drive a new era of personalized medicine for neuroregeneration.
Neurotrauma, stroke, and neurodegenerative disease may result in widespread loss of neural cells as well as the complex interconnectivity necessary for proper central nervous system function, generally resulting in permanent functional deficits. Potential regenerative strategies involve the recruitment of endogenous neural stem cells and/or directed axonal regeneration through the use of tissue engineered “living scaffolds” built to mimic features of three-dimensional (3-D) in vivo migratory or guidance pathways. Accordingly, we devised a novel biomaterial encasement scheme using tubular hydrogel-collagen micro-columns that facilitated the self-assembly of seeded astrocytes into 3-D living scaffolds consisting of long, cable-like aligned astrocytic networks. Here, robust astrocyte alignment was achieved within a micro-column inner diameter (ID) of 180 μm or 300–350 μm but not 1.0 mm, suggesting that radius of curvature dictated the extent of alignment. Moreover, within small ID micro-columns, >70% of the astrocytes assumed a bi-polar morphology, versus ~10% in larger micro-columns or planar surfaces. Cell–cell interactions also influenced the aligned architecture, as extensive astrocyte-collagen contraction was achieved at high (9–12 × 105 cells/mL) but not lower (2–6 × 105 cells/mL) seeding densities. This high density micro-column seeding led to the formation of ultra-dense 3-D “bundles” of aligned bi-polar astrocytes within collagen measuring up to 150 μm in diameter yet extending to a remarkable length of over 2.5 cm. Importantly, co-seeded neurons extended neurites directly along the aligned astrocytic bundles, demonstrating permissive cues for neurite extension. These transplantable cable-like astrocytic networks structurally mimic the glial tube that guides neuronal progenitor migration in vivo along the rostral migratory stream, and therefore may be useful to guide progenitor cells to repopulate sites of widespread neurodegeneration. Statement of Significance This manuscript details our development of novel micro-tissue engineering techniques to generate robust networks of longitudinally aligned astrocytes within transplantable micro-column hydrogels. We report a novel biomaterial encasement scheme that facilitated the self-assembly of seeded astrocytes into long, aligned regenerative pathways. These miniature “living scaffold” constructs physically emulate the glial tube – a pathway in the brain consisting of aligned astrocytes that guide the migration of neuronal progenitor cells – and therefore may facilitate directed neuronal migration for central nervous system repair. The small size and self-contained design of these aligned astrocyte constructs will permit minimally invasive transplantation in models of central nervous system injury in future studies.
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