Spatial and temporal control of GDNF delivery from acellular nerve grafts and Schwann cells improves regeneration across a long nerve defect

摘要

Introduction: Peripheral nerve injury results in -200,000 surgical interventions performed annually, yet despite these repairs, a significant reduction in sensation and motor function remains leading to a need for regenerative therapies. Treating peripheral nerve injuries with growth factors, such as glial cell line-derived neurotrophic factor (GDNF), has proven useful in supporting axon survival and regeneration. Unfortunately, developing a method that delivers the appropriate spatial and temporal release profile to promote functional recovery has proven challenging. Many release mechanisms exhibit burst release profiles that are ineffective over long regeneration periods, yet prolonged exposure to GDNF can result in axonal entrapment at the site of release. Thus, a spatially and temporally-controlled GDNF delivery system was designed using a two-phase system comprised of an affinity-based biomaterial system injected into acellular nerve allografts (ANAs) and conditional lentiviral GDNF over-expression from transplanted SCs. Materials and Methods: In vitro analysis: Briefly, SCs were transduced with tetracycline-inducible (Tet-On) GDNF over-expressing lenti virus prior to transplantation. In vitro confirmation of GDNF release with doxycycline (Tet analog) treatment was performed using ELISA and GDNF bioactivity was tested by co-culturing dorsal root ganglia neurons with Tet-on, wild-type, and control vector SCs. In vivo studies: To study the effect of controlled temporal and spatial GDNF delivery on axon regeneration, 3 cm ANAs were modified by injection of a GDNF-releasing fibrin scaffold (2 week delivery) under the epineurium, and then used to bridge a 3 cm sciatic nerve defect in adult Sprague-Dawley Thy1.1 GFP~+ rats. GDNF-SCs were transplanted into the distal nerve and doxycycline was administered for 4,6, or 8 weeks to determine the optimal duration of GDNF expression. Positive control isografts, negative control ANAs, and the use of transplanted SCs and GDNF DS was controlled for. After 8 weeks, nerves were harvested and histomorphometry was performed. Muscle reinnervation was determined by muscle mass of the gastrocnemius and tibialis anterior muscles. Results and Discussion: For conditional long-term delivery of GDNF, SCs were transduced with a Tet-on GDNF overexpressing lentiviral vector. GDNF expression in GDNF-SCs was significantly higher than control SCs after doxycydine treatment (Figure 1). To confirm biological activity, SCs were cultured in Transwell plates with dissociated DRG and neurite extension was measured after 18 hrs. As shown in Figure 1, when neurons were cultured with GDNF-SCs, neurite extension was increased by ～32% versus control SCs. Histomorphometry determined both GDNF modified ANAs and 6 weeks of GONF overexpression resulted in increased axon regeneration (Figure 2). Conversely, the removal of GDNF early at 4 weeks, or prolonged expression for 8 weeks, supported poor regeneration. Increased muscle mass was observed only when GDNF was overexpressed for 6 weeks. Conclusions: GDNF is a potent factor in nerve regeneration, yet its controlled delivery has proven difficult. Through our modification of ANAs and finely tuned long-term expression from SCs, we were able to overcome significant limitations in long gap nerve regeneration.