Engineering Nerve Constructs for Clinical Application

B.J. Pfister1, J. Huang1, E.L. Zager1, A. Iwata1, D.F.Meaney2, A.S. Cohen3,4, D.H.Smith1

Departments of (1)Neurosurgery, (2)Bioengineering, and (3)Pediatrics, University of Pennsylvania and Division of Neurology; (4)Children's Hospital of Philadelphia

In the United States, tens of thousands of peripheral nerve injuries occur each year, many resulting in the loss of bodily functions and even permanent disability. The gold standard of peripheral nerve repair traditionally relies on a surgical procedure that involves the removal and transplantation of an autograft, a separate, less important length of nerve from the patient.

In some patients the damaged nerve will regenerate, using the autograft as a guide, leading to restoration of lost bodily functions. However, some major peripheral nerve deficits that can typically be repaired are not due to the limited availability of nerves that the patient can spare (for example, an insufficient supply of donor nerves for the reconstruction of a major brachial plexus injury).

There are a number of disadvantages associated with this technique including the additional loss of neurological function associated with the harvesting other nerves for grafting (including scarring and painful neuroma formation), increased post-operative pain due to additional incisions, increased risk of infection, and increased operating room and anesthesia time. For this approach, the extent of functional recovery is largely dependent on the distance of regeneration required to bridge the damaged nerve. Typically only about 50 percent of all patients will recover some useful functions and full recovery is rare [1,2].

In the case of spinal cord injuries, there is presently no effective treatment that can restore lost function. In contrast to peripheral nerve injuries, spinal cord injuries occur in an environment that is non-permissive to regeneration. While active research is focusing on the obstacles to regeneration within the spinal cord, an effective repair strategy has yet to be uncovered [3,4].

Currently, repair of spinal cord and peripheral nerve injuries relies on the ability of axon fibers (a nerve cell process that conducts nervous signals) to regenerate across the damaged area to restore nervous system communication. Accordingly, primary repair strategies have aimed to enhance and guide axon outgrowth by bridging the damaged area with materials of biologic or synthetic origin. Several approaches have been vigorously studied including: biomaterials to act as physical guides, transplantation of various cell types to support axon growth, administration of drugs to counteract elements that inhibit axon growth, and agents that enhance axon growth [1,5]. For peripheral nerve damage, these approaches have only been successful for injuries spanning a short distance, much smaller than what autogenous grafting can repair 6. For spinal cord injury, some approaches have been able to enhance the outgrowth of a few axons, but fall far short of number that would be necessary to restore lost function [5].

Here, a distinct approach to engineering an effective man-made nerve construct for nerve repair is described. This construct consists of numerous bundles of axons, which are embedded in a collagen gel and packaged in a biocompatible conduit. Sized to the length of the damaged nerve, this construct can be directly transplanted to provide a living and functional connection. Researchers hypothesize that nerve constructs spanned by axons may establish or promote functional pathways necessary for nervous system repair that have not been achieved by any other approach. In order for this to be feasible, axons must be grown in a short period of time to lengths that can bridge any size lesion and consist of enough axons to adequately restore function.

Supporting a long held hypothesis [7], research has shown that tracts containing up to a million axons can be mechanically elongated in the laboratory at rates and to lengths that greatly exceeds what an axon can grow on its own. This process is similar to one of two distinct forms of axonal growth that occur in succession during development. First, axons grow out from the neural cell body and find their way to their final destination. After the axon integrates with its target, the growth of an animal induces the continued growth of axons as a result of mechanical stretching [7,8]. Anecdotal evidence of this form of growth can be found throughout nature. For example, the blue whale can grow an estimated 4cm per day and the giraffe’s neck increases by about 2cm per day at peak growth. It has also been shown that sensory axons in the deer antler are forced to grow at a rate of over 1cm per day 9. This process is referred to as axon stretch-growth that likely represents the primary mechanism that drives the formation of long nerves in animals.

Research has found that axons from embryonic dorsal root ganglion neurons (DRG, a type of neuron that resides in the peripheral nervous system) can sustain amazingly high stretch growth rates of up to 10mm/day and potentially much faster. Furthermore, axon tracts consisting of 105 to 106 axons can be stretch grown to 1cm in length within 4 days and up to 10cm in length in only 28 days while remaining healthy in culture and maintaining a normal structure 10. By exploiting this stretch growth process, ideal axonal tissue for transplant can be created in a matter of days for even extensive lesions.

Nonetheless, for clinical application, an embryonic source tissue for human transplantation is controversial and largely unattainable. A more ideal source of neurons would be from adult sources such as organ donors or from the patients themselves. The research team therefore examined the stretch-growth potential of axons from adult rat DRG neurons and subsequently from adult human donors for use in transplantable nerve constructs. Human DRG neurons were harvested from 18 patients, 14 who had undergone a pain management surgery, and 4 who were organ donors. It was found that human and rat DRG neurons survive in culture for more than three months and their axons can be successfully stretch-grown to a length of at least 1cm. The incorporation of elongated axonal tissue into a nerve construct offers an unexplored and potentially important new direction in bridging nerve lesions. Combining living axons derived from adult humans with other promising nerve construct designs may lead to a clinically applicable strategy for the repair of spinal cord and other nerve injuries.

Supported by Sharpe Trust and NIH grants AG21527, NS38104, NS45975.

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