Title: Tissue-Engineered Constructs for Peripheral Nerve Injury

Title: Tissue-Engineered Constructs for Peripheral Nerve Injury

Authors: Michael J. Brenner, MD, Susan E. Mackinnon, MD, Shelly E. Sakiyama-Elbert, PhD, Annie C. Lee, and Daniel A. Hunter, RA

Introduction: Attempts to restore function after peripheral nerve injury date back to the thirteenth century; unfortunately, full functional recovery is, even today, seldom achieved after severe injuries. Patients who suffer such injuries may be unable to work or perform basic activities of daily living, and many suffer permanent functional losses and chronic pain. Advances in nerve guidance conduits hold considerable promise for improving clinical outcomes in this population of patients.

Background: Tissue-engineered nerve conduits provide an unlimited source of nerve graft material in the form of a fabricated microenvironment. Crucial components of this microenvironment include a scaffold for axonal proliferation, support for Schwann cells, a basement-membrane matrix or matrix analogue, and a delivery system capable of providing controlled release of growth factor. Nerve conduits avoid the morbidity associated with nerve autografts, such as scars, anesthesia, or painful neuromas and circumvent the need for immunosuppression that arises with nerve allografting. The ideal conduit closely mirrors the normal, physiologic conditions under which nerve regeneration is most successful.

Methods: A novel neurotrophin delivery system was investigated in a rodent sciatic nerve injury model (Fig. 1). The key component of this delivery system is a heparin-peptide complex that mediates neurotrophin release. The peptide component of the delivery system is covalently bound to the fibrin matrix, whereas the heparin component of the delivery system binds growth factor and peptide through electrostatic interactions (Fig. 2). The delivery system was engineered to provide gradual, localized release of growth factor in a manner that is triggered by cellular activity. By controlling the ratios of peptide to heparin and heparin to growth factor, the diffusion-based release of growth factor was dramatically slowed. This allowed cell-activated matrix degradation to act as the dominant factor in controlling growth factor release. The components of this delivery system were combined in different permutations to optimize nerve regeneration at varying neurotrophin concentrations. A total of 54 rodents were enrolled in 9 groups of 6 animals each. All nerves were harvested at 6 weeks and analyzed by quantitative histomorphometry (Fig. 3).

Results: The fully equipped conduits, which contained delivery system and growth factor incorporated into a fibrin matrix, showed a dose-dependent increase in nerve regeneration with higher concentrations of growth factor (Fig 4). Only very modest nerve regeneration (mean fiber counts <1500) was observed in the partially equipped conduits and virtually no nerve regeneration was observed in empty conduits or conduits with fibrin matrix alone. Thus, all components of the delivery apparatus were shown to be essential for nerve regeneration in a rodent model. Ongoing studies investigate the effects of Schwann cells on the delivery system and assess functional recovery and nerve regeneration at 16 weeks (Fig. 3).

Summary: These results suggest that a heparin-based delivery system can provide controlled release of neurotrophins within nerve conduits. Advances in controlled release may eventually allow for more broad application of nerve conduits. A variety of injuries that severely impair patients’ quality of life could potentially be addressed with conduit-based therapies. Novel conduit constructs may eventually be applied to traumatic nerve injuries that now end the careers of many adults. Certain cranial nerve lesions resulting from morbid head and neck cancer surgery might also be more successfully managed, as well as peripheral nerve deficits following urologic or gynecologic cancer surgeries. While many challenges remain, nerve conduits that mimic physiologic mechanisms of nerve regeneration hold considerable promise for restoration of function in patients with peripheral nerve injuries.

Fig. 1 Schematic of a silicone conduit equipped with Schwann cells and a heparin-based delivery system that provides controlled release of -nerve growth factor.

Fig. 2 .Fibrin matrix containing delivery system and growth factor. Fibrin, peptide, heparin, and growth factor are bound to one another to form an interlinking structural complex:. The delivery system refers to the heparin- peptide unit () of this complex. The peptide component of the delivery system () is covalently linked to fibrin (), whereas the heparin component of the delivery system () is bound to the peptide and to growth factor (G) by non-covalent electrostatic interactions.

Fig. 3 Experimental design. Initially, rodent nerves are harvested for Schwann cells, and these Schwann cells are then cultured and expanded in vitro. Just prior to surgery, the conduit is equipped with the heparin-based delivery system elements and/or Schwann cells, depending on group. At t0, surgery is performed. Walking tracks are recorded weekly thereafter, and animals are sacrificed at 6 or 16 weeks from time of initial surgery. Nerve regeneration is evaluated by quantitative histomorphometry and functional recovery is determined based on walking track parameters.

Fig. 4 Dose-response relationship between -nerve growth factor (NGF) concentration and nerve regeneration. The number of nerve fibers regenerating across a 15mm silicone conduit is shown for sections taken from mid-conduit (left) and from the distal end of the conduit (right).