EUROPEAN  TISSUE  REPAIR  SOCIETY

ABSTRACT

Peripheral nerve injuries can result from mechanical, thermal, chemical, congenital or pathological etiologies. Failure to restore these damaged nerves can lead to long-term loss of muscle and sensory function. Current surgical strategies for repair of these injuries involves the transfer of normal donor nerve from an uninjured body location. Frequently, however, these 'gold standard' methods for tissue restoration are limited by tissue availability, risk of disease spread, secondary deformities and potential differences in tissue structure and size. One possible alternative to autogenous tissue replacement is the development of engineered constructs to replace those elements necessary for axonal proliferation. Despite advances and contributions in the field of tissue engineering, results to date with nerve conduits have failed to equal nerve regeneration realized with autogenous grafts for large distances. It is the purpose of this manuscript to review the current challenges to tissue engineered constructs.

INTRODUCTION

Our current understanding of nerve repair following injury, although improved, is still limited. Trauma, congenital and surgical resections all contribute to peripheral nerve defects. The current gold standard for nerve repair is primary neurorrhaphy or nerve grafting. These nerve grafts can be placed between individual nerve fascicles or more generally between severed nerve ends. Nerve grafts require autogenous donor nerves and these most commonly include the sural nerve in the lower leg or sensory nerves in the upper extremity. It is interesting to note that although nerve grafts are employed routinely, complete functional recovery is seldom obtained. With the inability to completely understand the molecular mechanism for nerve regeneration, tissue engineered constructs that attempt to mimic these interactions for nerve restoration become a technical challenge. It is the purpose of this manuscript to familiarize the reader with the current limitations in the development of tissue engineered constructs for nerve reconstruction.

APPROACH TO TISSUE ENGINEERED NERVE CONSTRUCTS

The history of nerve repair dates back to Galen who differentiated nerves from tendon in 300 AD.¹ The first documented repair however was by Ferrar in 1608.² Despite these initial efforts, amputation was the procedure of choice for severe injury. However, during WWII, nerve repair was promoted. Woodhall and Beebe noted nerve repair rarely restored function greater than 50% for the median nerve. Other nerves demonstrated poorer functional recovery.³ Although advocating repair, these early poor functional outcomes colored our perception and expectations. Many of these early results however did not utilize current microsurgical techniques. It was left to Millesi in the 1960s to advocate microsurgical repair, limited tension and the use of cable grafts for those defects that were too large for primary neurorrhaphy.⁴
When we think of the necessary elements for potential nerve constructs in nerve replacement, four major components can be identified. These include a scaffold for axonal migration, support cells (i.e., Schwann cells [SCs], macrophages), growth factors and extracellular matrix. The most appropriate combination and interaction of these individual components is currently unknown.

Nerve Architecture
A variety of conduits have attempted to be employed to bridge nerve gaps. Most of these conduits can be classified into two major components. This includes 1) natural and 2) synthetic materials. Natural materials appear to improve the biocompatibility, decrease the toxic effects and enhance the migration of support cells such as Schwann cells. A variety of natural materials have been used such as vein, laminin, fibronectin and collagen.⁵ Although apparently ideal for the use in the clinical environment, there are some inherent problems with the use of these natural materials. These include an undesirable immune response, batch to batch variations in large scale isolation procedures due to natural proteins and an inability to control the processing techniques.⁵ Nerve grafts stored for up to three weeks at 5°C acted as effective conduits for proximal regenerating fibers and resulted in values equivalent to fresh nerve grafts over moderate nerve gaps (30 mm).⁶ However, Gulati et al demonstrated that as the degeneration interval increased, the regenerative supporting ability of the stored nerve is compromised.⁷
Synthetic materials have also been employed. Variations in the chemical and engineering properties of synthetic material is attractive allowing alterations in the geometric configuration, biocompatibility, porosity, degradation and mechanical strength.^8-15 The variation in the above parameters can dramatically alter the ability of axons to proliferate. We have performed similar studies on synthetic polymers as a material for nerve conduits.^16-19 The use of PLLA poly (L-Lactic Acid) and PLGA poly (DL-glycoloic acid) conduits have been utilized in a rat sciatic nerve model.^16-19 Early and late degradation patterns have been identified and axonal regeneration does not appear to have been inhibited by the degradation products of these biomaterials. Again the ideal conduit has not yet been identified. Scaffolds for nerve conduits may ultimately include a combination of both synthetic and natural materials.

Support cells
Although the exact underlying mechanisms regulating dynamic axon/SC apposition are unknown, experiments support the concept that SCs offer a highly preferred substrate for axon migration and release bioactive factors that further enhance nerve migration. For instance, SCs produce structural and adhesive extracellular matrix (ECM) molecules such as laminin and collagen, and express many cell adhesion molecules and receptors, including L1, N-cadherin, g1 integrins, and the neural cell adhesion molecule (N-CAM).20,21 Moreover, SCs synthesize and secrete a cocktail of neutrophic molecules such as NGF, brain derived neutrophic factor (BDNF), and ciliary neurotrophic factor (CNTF).^20,21 Investigators have attempted to take advantage of the cellular functions of SCs to promote regeneration in both the CNS and PNS.^22-24 In the normal nerve, SCs appear to be quiescent, having heterochromatin-rich nuclei and relatively electron-dense cytoplasm. However, upon nerve injury the SCs in the distal nerve undergo extensive change concomitant with axonal degeneration. Acellular grafts fail to support regeneration when SC migration from the nerve stumps is prevented by mitomycin C. The inability of axons to regenerate over acellular grafts longer than 40 mm has been attributable to the finite migratory ability of SCs.^25-27
Hence, SCs play an obligatory role in peripheral nerve regeneration by releasing bioactive molecules and providing a support on which axons migrate. We have also attempted to utilize Schwann cells within our previously outlined synthetic nerve guidance channels to enhance nerve regeneration by these interactive mechanisms.²⁸ Ultimately, an interaction of Schwann cells as well as other inflammatory and support cells such as monocytes, macro-phages, lymphocytes and cytokines will be important.

Soluble Substances
Soluble neurotropic and neurotrophic factors can be incorporated directly into nerve guidance conduits. Some of these factors include nerve growth factor (BGF), brain derived neurotrophic factor (BDNF), insulin like growth factor (IGF-1, IGF-2), platelet derived growth factors (PDGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF). There is increasing evidence that many neurotrop(h)ic molecules (NGF, BDNF, NT-3, NT?4/5) act directly to promote survival and indirectly on regenerating axons via non-neuronal cells. NGF has been demonstrated to prevent the death of axotomized sensory neurons completely following exogenous administration.^29-32
We have recently utilized dermal fibroblasts genetically modified to act like SCs to deliver required growth factors (e.g., nerve growth factor, NGF) through a regulatory system. In vitro and in vivo NGF secretion from transfected hDFBs was quantitatively determined as well as NGF bioactivity.^33,34 This system offers several advantages in that it allows regulated growth factory delivery for both dose and time. This may be an advantage to nerve constructs, allowing the delivery of a variety of factors at variable time sequences.

The Stuff Remaining
Insoluble extracellular matrix molecules including laminin, fibronectin, and some forms of collagen, promote axonal extension and therefore are excellent candidates for incorporation into the lumen of guidance channels.^5,35,36 Alternatively these molecules could be placed on natural biological or synthetic conduits through adhesion molecules or a similar process. Variable results have been demonstrated by the incorporation of these materials within conduits, however it appears that by reducing the concentration of protein gel or shorting the oligopeptide sequence, axonal proliferation can be promoted.^37,38 Components of the ECM and matrix analogues have also demonstrated promise in nerve replacement.³⁹ Further, lamination of alternating nonpermissive and permissive gel layers facilitated the creation of 3D neural tracts in vitro.⁴⁰

FUTURE DIRECTIONS

Although the research efforts in nerve regeneration have been extensive, we are still hindered by a lack of knowledge of the underlying mechanism for axonal proliferation. Further, we have been limited by the length of a nerve conduit that can support axonal proliferation (10-15 mm). Most of the supportive research has been conducted in the rat or mouse model, however, what may occur in the animal model may be completely different in humans. Despite these animal models, they offer the ability to regulate and test each of these four major components to nerve regeneration. The ideal combination of these four major components remains elusive. It is obvious that each act at certain time points with their ability to modulate the growth of axons. Gradients and variations in concentration must play a role, but currently these mechanisms are unknown. As our technology develops, the ability to culture and expand cell and growth factor technology increases. Further genetic engineering may allow us to alter nerve regeneration, returning to a pre-embyonic level being able to upregulate the regenerative process allowing for more active, directed axonal growth. These challenges are but the beginning in the establishment of tissue engineered constructs for nerve regeneration.