Development of transplantable nervous tissue constructs comprised of stretch-grown axons

Abstract

Pursuing a new approach to nervous system repair, fasciculated axon tracts grown in vitro were developed into nervous tissue constructs designed to span peripheral nerve or spinal cord lesions. We optimized the newfound process of extreme axon stretch growth to maximize the number and length of axon tracts, reach an unprecedented axon growth-rate of 1 cm/day, and create 5 cm long axon tracts in 8 days to serve as the core component of a living nervous tissue construct. Immunocytochemical analysis confirmed that elongating fibers were axons, and that all major cytoskeletal constituents were present across the stretch-growth regions. We formed a transplantable nervous tissue construct by encasing the elongated cells in an 80% collagen hydrogel, removing them from culture, and inserting them into a synthetic conduit. Alternatively, we induced axon stretch growth directly on a surgical membrane that could be removed from the elongation device, and formed into a cylindrical construct suitable for transplant. The ability to rapidly create living nervous tissue constructs that recapitulates the uniaxial orientations of the original nerve offers an unexplored and potentially complimentary direction in nerve repair. Ideally, bridging nerve damage with living axon tracts may serve to establish or promote new functional connections.

Introduction

With insufficient techniques to repair peripheral nerve damage and no clinically effective approach to bridge spinal cord lesions, millions of patients endure devastating lifelong disabilities. For peripheral nerve injury (PNI), autologous nerve grafting remains the gold standard for repair. However, this approach is limited by the availability of donor nerve and complications arising from the harvesting surgery (Evans, 2000, Lee and Wolfe, 2000). As an alternative, synthetic conduits made from collagen or polyglycolic acid (PGA) have been increasingly used to reconnect severed nerves in patients (Belkas et al., 2004, Evans, 2001, Meek and Coert, 2002, Schmidt and Leach, 2003). These conduits provide a physical guide for axons sprouting from the proximal nerve stump to reach the disconnected nerve segment. Thereafter, chemical and physical cues from the disconnected nerve form a labeled pathway, which then directs the continued growth of regenerating axons to ultimately reinnervate the tissue (Belkas et al., 2004, Hall, 2001, Lee and Wolfe, 2000). However, synthetic conduits have only been clinically successful for the repair of short nerve lesions and are typically used for gaps less than 1–2 cm (Belkas et al., 2004, Evans, 2000, Hall, 2001, Lee and Wolfe, 2000, Meek and Coert, 2002). Regardless of whether nerves are grafted with donor nerves or synthetic conduits, the axons and many supportive cells of the disconnected portion of the nerve rapidly degenerate, resulting in the loss of the labeled pathway necessary to guide axon outgrowth (Belkas et al., 2004, Evans, 2000, Hall, 2001, Lee and Wolfe, 2000, Meek and Coert, 2002). This degeneration, coupled with the relatively slow growth of sprouting axons (approximately 1 mm/day), commonly results in poor functional recovery of extremities that are far away from the nerve damage (Belkas et al., 2004, Hall, 2001, Lee and Wolfe, 2000).

Even more daunting than PNI repair is the restoration of axonal pathways transected due to spinal cord injury (SCI). To successfully bridge spinal cord lesions, new intraspinal circuits or ‘relays’ must be formed (Bareyre et al., 2004, Bunge, 2001, Fawcett, 2002). However, SCI lesions are commonly several centimeters long and therefore require extensive axon growth in an environment that is notoriously non-permissive for axon outgrowth (Bunge, 2001, Fawcett, 2002, Fry, 2001, Hall, 2001, McKerracher, 2001). While many promising preclinical studies have demonstrated techniques that facilitate axon growth in animal models of SCI, producing axons of sufficient length and number for clinical application remains an enormous challenge (Bunge, 2001, Fawcett, 2002, Fry, 2001, McKerracher, 2001).

Recently, we have identified a new mechanism of sustained axon growth that can far exceed the rate of axon growth cone extension (Pfister et al., 2004, Smith et al., 2001). Unlike axon outgrowth in response to chemical cues, established axon tracts can grow under the application of mechanical forces. Since these axons no longer have growth cones, this distinct growth process is driven by mechanical stretch applied to the central portion of the axon cylinder. We have recapitulated this natural axonal “stretch-growth” through the progressive mechanical distraction of axons bridging two populations of cultured neurons. Even at rates of stretch up to 1 cm/day, this process results in axon expansion in both length and caliber, producing uniaxially oriented tracts.

Here we exploited this axon stretch growth process to develop nervous tissue constructs comprised of living axons. Rather than enticing axons to regenerate in vivo, axons are rapidly stretch grown ex vivo to a length sufficient to span the nerve damage. These stretch-grown axon tracts serve as the core element in a three-dimensional transplantable nervous tissue construct that recapitulates the uniaxial orientations of the original nerve. This approach may establish or promote functional connections necessary for nervous system repair that have not been achieved by other methods.