Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sprouting after spinal cord injury

Abstract

Neurotrophins have been shown to promote axonal growth and regeneration after spinal cord injury. The therapeutic utility of neurotrophins may be enhanced by using a controlled delivery system to increase the duration of neurotrophin availability following injury. Such a delivery system can be incorporated into a bioactive scaffold to serve as a physical bridge for regeneration. This study assessed the effect of controlled delivery of neurotrophin-3 (NT-3) from fibrin scaffolds implanted in spinal cord lesions immediately following 2-mm ablation injury in adult rats. Nine days after injury, fibrin scaffolds containing the delivery system and NT-3 (1000 ng/mL) elicited more robust neuronal fiber growth into the lesion than did control scaffolds or saline (1.5- to 3-fold increase). Implantation of fibrin scaffolds resulted in a dramatic reduction of glial scar formation at the white matter border of the lesion. Hindlimb motor function of treated animals did not improve relative to controls at 12 weeks post-injury. Thus, controlled delivery of NT-3 from fibrin scaffolds enhanced the initial regenerative response by increasing neuronal fiber sprouting and cell migration into the lesion, while functional motor recovery was not observed in this model.

Introduction

The ability of the adult spinal cord to regenerate after injury is limited; in order to restore functional connectivity, neurons must regenerate through a non-permissive environment that includes myelin-associated and glial scar-associated inhibitors and a cystic cavity. Neurotrophin-3 (NT-3) has been shown to promote neuronal regeneration when delivered to the site of spinal cord injury (SCI) by direct injection [1], osmotic mini-pumps [2], adenoviral vectors containing a neurotrophin gene [3], transgenic cells overexpressing a neurotrophin gene [4], and scaffolds soaked with neurotrophin [5], [6]. This paper focuses on a strategy for delivering NT-3 in a controlled manner from fibrin scaffolds that can also serve as a physical bridge for regeneration.

When using protein drugs, such as neurotrophins, as therapeutic agents, several factors, such as loss into the cerebrospinal fluid, proteolysis, or aggregation, may reduce the efficacy of the drug. The use of a delivery system that sequesters and protects the protein until the appropriate time of release can allow the drug to be available to regenerating cells over a longer period of time, which is particularly important in the case of nerve regeneration (which occurs over several weeks). In contrast to polymer-based delivery systems [7], [8], which use physical entrapment of the neurotrophin to provide sustained release in the central nervous system [9], [10], [11], in this work, an affinity-based delivery system is used to provide sustained release of NT-3 from fibrin scaffolds [12], [13].

In this delivery system (Fig. 1), a synthetic bi-domain peptide is covalently crosslinked to fibrin during polymerization [14] and can also bind to heparin via electrostatic interactions [15], thereby immobilizing heparin within the fibrin scaffold. Heparin can, in turn, bind to growth factors that contain heparin-binding domains or growth factors, such as nerve growth factor (NGF) or NT-3, with basic domains [12]. By this series of non-covalent interactions, growth factors can be immobilized within fibrin scaffolds, thereby limiting the release of growth factors by diffusion [12], [13], [16]. Following plasmin-mediated degradation of the scaffold by infiltrating cells, like neurons [17], [18], fibrin-bound growth factor is released and made available for action on nearby cells.

The delivery of growth factors from the HBDS has been shown to enhance neurite outgrowth in vitro [12], [13], [16] and neuronal fiber sprouting in vivo [16], [19]. A preliminary in vivo SCI study qualitatively showed increased neuronal fiber infiltration of the lesion when treated with fibrin containing the HBDS and NT-3 compared to those with unmodified fibrin at 9 days post-implantation [16]. A more rigorous study is necessary to examine the effect of dose of NT-3 on neuronal fiber sprouting in a quantitative manner, the effect of the delivery system on glial scar formation, and the potential of this treatment to promote functional motor recovery. Here, the neuroanatomical effects of fibrin scaffolds containing various doses of NT-3 with the delivery system were evaluated 9 days after SCI. Locomotor function was assessed for 12 weeks to investigate the ability of controlled delivery of NT-3 to enhance functional recovery in this model of SCI.