Neural Tissue Engineering
Neural tissue engineering offers tremendous promise to combat the effects of disease, aging, or injury in the nervous system. The Cullen Lab is developing tissue-engineering strategies to directly replace damaged or diseased neural tissue, or to augment the capacity for nervous system regeneration and restore lost function. Our overall strategy is to engineer living three-dimensional (3-D) neural tissue that is “pre-engineered” outside the body with controlled neuroanatomical and functional characteristics, that upon delivery into the body will promote neuroregeneration or recreate lost neuroanatomy. One application being investigated is developing constructs of axonal tracts that, upon transplantation, may facilitate nervous system repair by directly restoring lost connections or by serving as a targeted scaffold to promote host regeneration by exploiting axon-mediated axonal regeneration.
Concept: Three-dimensional constructs for neuroregeneration. (a-b) Tissue engineered constructs were “pre-engineered” in vitro to contain long axonal tracts, thus recapitulating the architecture of lost neural tissue to directly replace lost axonal tracts or to serve as living targeted scaffolds axonal regeneration. Constructs may be (a) unidirectional or (b) bidirectional depending on the application specific to trauma or disease condition.
Techniques to achieve longitudinal axonal extension and/or growth. Confocal reconstructions of neuronal constructs achieved via (a) stretch-growth, (b) microconduit containment, or (c) along a microfiber. (a) Axon stretch-growth results in two neuronal populations spanned by long axonal tracts. (b) A dense cluster of neuronal somata was located at one end of the micro-conduits with axonal projections extending longitudinally projecting several millimeters in the interior (outer diameter denoted by dashed lines). (c) Preferential longitudinal growth along a conductive polymer micro-fiber.
Neuronal survival and maintenance of architecture in engineered nervous tissue constructs. Representative confocal reconstructions of transplanted GFP+ engineered nervous tissue constructs used to bridge an excised segment of sciatic nerve (6 weeks post-implantation). (A) Continuous proximal (top) and distal portions (bottom) from a GFP+ construct immunolabeled for NF-200 (red) (scale bars = 0.5 mm). Multiple transplanted ganglia were evident on the proximal and distal ends (arrow heads) with aligned axonal tracts spanning these neuronal populations. Remnants of the PGA tube were observed bordering the transplant at this time-point (note arced border material autofluorescing red). (B-C) Higher magnification regions from (A) rotated 90° (scale bars = 100 µm). (B) Major bundles of neurites projected from the proximal ganglia across the constructs as well as into host nerve towards the spinal cord (white arrow). (C) Similarly, neuritic bundles also projected from the distal ganglia across the constructs and distally into the distal nerve segment (yellow arrow). (D-F) Increased magnification from specified regions; GFP+ (left column), NF-200+ (center column), with overlay (right column); scale bars = 20 µm. (D) Central axonal tracts co-labeled for GFP and NF-200. (E) Transplanted ganglia became dense, three-dimensional clusters of neurons. (F) Neurites growing from the host into the proximal end of the constructs were observed as NF-200+ axons that were not co-localized with GFP (arrows).
Host axons growing along transplanted axons. Axons from the surviving cluster of transplanted neurons at the graft interior (blown up region from above figure). Axons from the transplanted neuronal constructs are labeled green (GFP+) and transplant and host neurofilament-positive axons are immunostained red. These axons are a mix of the transplanted axons and host axons, suggesting that host axonal growth occurs via axon-mediated axon regeneration.
In addition, advances in neural tissue engineering have resulted in the development and implementation of 3-D neural cellular constructs, which may serve as neurofidelic in vitro investigational platforms. Cells in the brain interact within a complex, multicellular environment with tightly-coupled 3-D cell-cell/cell-extracellular matrix interactions; yet most in vitro models utilize planar systems lacking in vivo-like ECM. As such, neural cultures with cells distributed throughout a thick (>500microns), bioactive extracellular matrix may provide a more physiologically-relevant setting to study neurobiological phenomena than traditional planar cultures. Dr. Cullen has pioneered the development of a range of 3-D neural cellular constructs and has applied these to increase our understanding of factors influencing neural cell survival, function, and network formation.
Tissue Engineered 3-D Neural Cell Cultures for Neurobiological Investigations In Vitro. Cellular constructs consist of (a) neurons or (b) neurons mixed with astrocytes, distributed throughout the full thickness of a 3-D matrix/scaffold (>500 µm). (c) Neurons in 3-D culture, shown to express neuronal-specific proteins MAP-2 (green) and tau-5 (red) with nuclear marker Hoechst 33258 (blue), assume complex morphologies with 3-D neurite extension. (d) These cultures are useful to study neuron-astrocyte interactions in 3-D. (e) Live neurons (green) demonstrating survival and network formation throughout the thickness of the 3-D cultures. (f) Volumetric rendering of live neurons in 3-D culture. Neurobiological studies of neural cells within 3-D matrices provide enhanced fidelity to in vivo while affording all advantages of traditional (planar) in vitro systems.