Repairing peripheral nerve injury using tissue engineering techniques

Each year approximately 360,000 people in the United States suffer a peripheral nerve injury (PNI), which is a leading source of lifelong disability (Kelsey et al., 1997; Noble et al., 1998). The most frequent cause of PNIs is motor vehicle accidents, while gunshot wounds, stabbings, and birth trauma are also common factors. Patients suffering from disabilities as a result of their PNIs are also burdensome to the healthcare system, with average hospital stays of 28 days each year (Kelsey et al., 1997; Noble et al., 1998). The technique of autologous nerve grafting is considered the gold standard and the most reliable choice in repair of major defects in peripheral nerves (Chiu and Ishii, 1986; Huang et al., 2004; Lee and Wolfe, 2000; Lundborg, 2000). The introduction of an autologous axon segment provides a physical and biological scaffolding over which axonal outgrowth may occur. Complications arise, however, because of the limited supply of donor nerves and the risks associated with the harvesting surgery. Donor sites are vulnerable to infections, the formation of painful neuromas, and loss of function associated with the harvested nerve. In recent years there has been considerable interest in developing alternative strategies to repair damaged peripheral nerves through transplanted materials of biologic or synthetic origin if donor nerves are scarce (Midha et al., 1993; Hudson et al., 2000; Scherman et al., 2001; Cheng and Chen, 2002). Despite extensive research into nonautologous alternatives, the autograft has remained the gold standard for the repair of peripheral nerve injuries. Many studies have identified elements that encourage neural regeneration – from mechanisms of axon development, to experimental treatments – but individual breakthroughs remain as separate components, and even if paired, fail to completely address the complexities of real injuries. A recent article in Nature Medicine by Stuart Forbes and Nadia Rosenthal accurately captures this short-coming, and recommends that “seed-and-soil” concepts be applied to the development of future cell-based regenerative therapies. The seed-and-soil perspective posits that in failing to prepare the embedding environment (the ‘soil’) with the necessary cellular and signaling conditions, it is unreasonable to expect that stem cells (the ‘seeds’) will successfully engraft. In injury, where cellular and signaling responses diverge from the norm, addressing these numerous components is an obstacle to the development of effective treatments (Forbes and Rosenthal, 2014). At first, it is tempting to extend this notion of seed-and-soil beyond stem cells. In the case of peripheral nerve repair, where novel grafting techniques are regularly heralded, the same principles of preparing the embedding environment might apply. But unlike other organs, peripheral nerves have limited regenerative potential, and require solutions involving autologous donations, tissue-engineered nerve grafts, and other biomaterials. These sizable grafts do not fit the ‘seed’ depiction. 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 degenerate, resulting in the loss of the labeled pathway necessary to guide axon outgrowth. This factor 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 nerve damage. For example, as is commonly found with brachial plexus injury, while elbow flexion may ultimately be regained, hand function is not, resulting in significant impairment of the activities of daily living. While a primary strategy to repair major peripheral nerve injury (PNI) is to bridge the damage with axons, producing axons of sufficient length and number has posed a significant challenge. The gold standard in peripheral nerve repair, the autologous nerve graft, is limited by the availability of donor axons and complications arising from the harvesting surgery. In addition, most alternative bridges currently used for nerve damage (e.g., synthetic tubes) are limited in the length that they can span to promote repair and are typically used for gaps of less than 2–3 cm. During the past decade, our research teams have been able to utilize a novel tissue engineering technique to create transplantable nervous tissue constructs for major peripheral nerve repair. The key to this procedure is to use a specially designed motorized micro-stepper to produce continuous mechanical tension on axons spanning two initially apposed populations of cultured neurons. Using dorsal root ganglia (DRG) neurons, this technique has rapidly produced nerve tracts consisting of 10 axons grown at rates of 10 mm/day, reaching a remarkable 10 cm in length. To form transplantable nervous tissue constructs, the elongated cultures are embedded in collagen and placed into tubes composed of polyglycolic acid (PGA). In our preliminary studies, stretch-grown cultures used to repair a 1.3 cm rat sciatic nerve deficit were found to survive long-term (4 months), demonstrating robust incorporation of grafted axons within a regenerating network of host axons, including outgrowth of graft axons into host nerve (Huang et al., 2009), and improved restoration of hindlimb motor function. Ideally, nervous tissue engineered to recapitulate the geometry and orientation of the nervous system could act as a bridge across regions of damage and promote functional recovery. Towards this goal, we have been using axon stretch-growth to create long axon tracts spanning two populations of neurons to serve as living nervous tissue constructs for transplantation and repair of even extensive PNIs (Figure 1). Limiting factors of inducing axon growth for nerve repair include the length the axons can be grown, the rate of growth, the total number of axons in each preparation, and the viability of the axons. To