FK506 Delivering Conduit For Peripheral Nerve Regeneration
Pratima Labroo, MS, Jill E. Shea, PhD, Brett Davis, BS, David Hilgart, BS, Christopher Lambert, BS, Himanshu Sant, PhD, Bruce Gale, PhD, Jayant Agarwal, MD.
University of Utah, Salt lake city, UT, USA.
Purpose: Following a peripheral nerve injury, nerve gaps require grafts or conduits to connect the two nerve ends. There is a clinical need to improve functional recovery following nerve injury and local release of neurotrophic factors is one way to improve outcomes. FK506, an FDA approved small molecule, has been shown to enhance axon growth in vitro and peripheral nerve regeneration in vivo. Systemic delivery of FK506 has numerous potentially serious side effects such as central nervous system toxicity, infection and nephrotoxicity. Local delivery of FK506 from a nerve guide could provide the neurotrophic benefits of FK506 but prevent the negative consequence of systemic delivery. Here we describe the in vitro and in vivo outcomes when using a novel drug delivery apparatus integrated with a biocompatible polytetrafluoroethylene (PTFE) based nerve conduit for controlled local delivery of FK506.
Methods: The device consists of two concentric PTFE tubes (Zeus Inc.) and a reservoir in between the tubes that stores FK506. A 120µm diffusion hole was drilled into the inner tube by pulsing a laser. In vitro release tests were performed over a 32-day period and the concentration of released FK506 was determined with an enzyme linked immunosorbent assay (ELISA, Abnova). The bioactivity of the released FK506 was evaluated using a chick dorsal root ganglion (DRG) cell based bioassay. In vivo testing of the FK506-delivering conduit was performed in a critical sized 9 mm mouse sciatic nerve gap model and compared with negative control (empty conduit). The animals were sacrificed at 18 weeks postoperatively and functional outcomes such as nerve histomorphometry, neuromuscular junction connectivity and compound muscle action potential were evaluated.
Results: FK506 was released at a consistent concentration, 5.2±3.4 ng/mL/day, over a period of 32 days. The released FK506 was found to be bioactive and enhanced neurite elongation by ~40% compared with DRGs exposed to control media (n=4) (p<0.05) but comparable to freshly prepared FK506 (p>0.05). In the in vivo study, we measured a 53% increase in neuromuscular junction connectivity and a 40% increase in the amplitude of muscle compound action potential when a 9mm sciatic nerve gap injury was repaired with our FK506 releasing device compared to the (no drug) device alone (n=3) (p<0.05).
Conclusion: We successfully manufactured PTFE nerve conduits that can deliver bioactive FK506 in a controlled manner for 32 days. The released FK506 enhanced neurite extension in vitro and nerve regeneration in vivo. We are currently examining how locally released FK506 impacted the nerve histomorphometry by evaluating nerve myelination and retrograde labelling compared to treatment with an empty conduit. Future work will evaluate the efficacy of this local delivery of FK506 in vivo using a bioresorbable biomaterial.
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