title: Transplantable living scaffolds comprised of micro-tissue engineered aligned astrocyte networks to facilitate central nervous system regeneration.
abstract: 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 180μm or 300-350μm but not 1.0mm, 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 (9-12×10(5)cells/mL) but not lower (2-6×10(5)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 150μm in diameter yet extending to a remarkable length of over 2.5cm. 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.
  
  
  
  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.
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, United States; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, United States.
authors.forename: Carla C
authors.initials: CC
authors.lastname: Winter
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, United States; School of Biomedical Engineering, Drexel University, Philadelphia, PA, United States.
authors.forename: Kritika S
authors.initials: KS
authors.lastname: Katiyar
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Nicole S
authors.initials: NS
authors.lastname: Hernandez
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Yeri J
authors.initials: YJ
authors.lastname: Song
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, United States; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, United States.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Struzyna
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, United States.
authors.forename: James P
authors.initials: JP
authors.lastname: Harris
authors.affiliation: Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, United States; Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: dkacy@mail.med.upenn.edu.
authors.forename: D Kacy
authors.initials: DK
authors.lastname: Cullen
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000637:transplantation*; D002490:Central Nervous System / Q000293:injuries / Q000502:physiology; D020007:Implants, Experimental; D051381:Rats; D017207:Rats, Sprague-Dawley; D012038:Regeneration; D023822:Tissue Engineering; D054457:Tissue Scaffolds
keywords: Axon pathfinding; Biomaterials; Cell migration; Glial cell transplant; Living scaffold; Neural stem cells; Neurodegeneration; Neurotrauma; Regeneration; Tissue engineering; Traumatic brain injury