Templated agarose scaffolds for the support of motor axon regeneration into sites of complete spinal cord transection
Introduction
Axons of the central nervous system fail to spontaneously regenerate after spinal cord injury, frequently resulting in permanent deficits of motor, sensory or autonomic function. Several experimental strategies can enhance the growth of central axons, including growth factors [1], [2], stimulation of the intrinsic neuronal growth state [3], [4], neutralization of inhibitors to axon growth in the injured central nervous system environment [5], [6], and placement of a matrix to support axon growth in the lesion site [7]. Most matrices placed into spinal cord lesion sites to support axonal growth have consisted of cells, including bone marrow stromal cells, Schwann cells, fibroblasts, astrocytes or olfactory ensheathing cells [8], [9], [10], [11], [12], [13]. While these cell types support axonal attachment and extension, axons fail to retain their native organization into fascicles of related function, resulting in axonal growth into the lesion that is disorganized and circuitous in pattern [14].
Bioengineered scaffolds offer the potential to organize axonal growth to mimic the natural organization and orientation of axons as they extend through a lesion site. To accomplish this goal, scaffolds should hypothetically contain several individual compartments that bundle axons of related function, and should maintain a strictly linear configuration over distances of several millimeters in rodent models of spinal cord injury (and several centimeters for potential human translation; Fig. 1) to prevent mistargeting from one fascicle of specific function to another fascicle of different function. In addition, the scaffold should consist of a material that elicits a minimal host inflammatory response, and should be capable of releasing growth-promoting substances such as growth factors [15], [16]. Finally, the scaffold should exhibit elastic moduli resembling the normal spinal cord to minimize mechanical parenchymal damage at points of contact between the scaffold and host.
For the past several years we have been exploring the properties of bioengineered agarose scaffolds in models of spinal cord injury. Through an extrusion/fusion process, this material can be fabricated into strictly linear channels over lengths of several centimeters. When seeded with autologous bone marrow stromal cells secreting growth factors, scaffolds support host axonal growth in strictly linear arrays over the full length of a lesion site and in relatively high density [14], [17]. Growth of axons arising from intrinsic neuronal pools of the spinal cord, and of sensory axons projecting from the periphery, has been observed in partial spinal cord lesion models [14], [17]. These studies have provided important proof-of-concept indicating that bioengineered scaffolds, under ideal conditions, can support, organize and linearize axon growth through a lesion site.
The present study assessed whether bioengineered scaffolds have the potential to support and guide axonal growth in a more clinically relevant model of severe spinal cord injury. Adult rats underwent a severe lesion consisting of complete spinal cord transection, with placement of templated agarose scaffolds into the lesion site. Some implants were seeded with syngenic bone marrow stromal cells that secrete the growth factor Brain-Derived Neurotrophic Factor (BDNF). Moreover, we studied responses of axons originating in the brain that control specific components of motor function. We now report in a severe, complete transection model of spinal cord injury that templated agarose scaffolds support and guide motor axonal regeneration over the full length of the lesion.
Section snippets
Fabrication of templated agarose scaffolds
To generate agarose scaffolds, multi-component fiber bundle (MCFB) templates were fabricated from 166 μm diameter polystyrene fibers (Paradigm Optics, Vancouver, WA) arranged in a hexagonal close-packed array separated by a continuous matrix of poly (methyl methacrylate) (PMMA), as previously described [17]. Polystyrene fibers of 166 μm diameter were arranged with 66 μm interval spacing in a honeycomb array to generate final scaffolds with wall sizes of 66 μm and channel diameters of 166 μm (
Scaffold integration into lesion site
Scaffolds exhibited excellent integration into the complete transection site when examined four weeks after implantation (Fig. 1). Previously we reported the formation of reactive cell layers at host/scaffold interfaces placed in smaller, partial spinal cord lesion sites [14], [17]. In this severe, complete transection model, formation of thin reactive cell layers were detected on Nissl stains at host/scaffold interfaces, but were not qualitatively thicker or more severe in extent than previous
Discussion
To advance in clinical relevance, it is important to examine the ability of bioengineered scaffolds to support, orient and guide axonal growth in clinically relevant models of spinal cord injury. Advancing this objective, the present results indicate that in the most severe model of spinal cord injury, complete transection, bioengineered scaffolds are retained in sites of injury over long time periods and are capable of guiding axons. Consistent with previous reports in partial lesion models
Conclusion
We report that bioengineered scaffolds can support, orient and guide the regeneration of motor axons in a severe model of spinal cord injury, complete transection. Future work will focus on promoting axonal regeneration beyond the lesion, and reduction to yet more clinically relevant models, including severe contusion lesions.
Acknowledgments
This work was supported by the NIH/NIBIB (R01EB014986), the Veterans Administration, the Craig H. Neilsen Foundation, and the Bernard and Anne Spitzer Charitable Trust.
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Current address: Department of Spine Surgery, Renmin Hospital of Wuhan University, Wuhan, Hubei, China.