Effect Of Ngf Delivering Conduit On Peripheral Nerve Regeneration
Pratima Labroo, MS, Isak Goodwin, MD, Brett Davis, BS, Kyle Edwards, MD, Scott Ho, MS, Himanshu J. Sant, PhD, Bruce K. Gale, PhD, Jill E. Shea, PhD, Jayant Agarwal, MD.
University of Utah, Salt lake city, UT, USA.
Purpose: Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons and allow for tension free bridging, without the need to harvest donor nerve. It has also been shown that locally delivering nerve growth factor (NGF) can enhance peripheral nerve recovery. There are major benefits in providing a guidance conduit that can independently deliver localized drugs to the injury site. A bulk diffusion delivery device will provide flexibility in easily altering the drug of choice and precision in using traditional fluid mechanics to control delivery rather than complex polymer degradation. This paper describes the in vitro and in vivo outcomes of a novel drug delivery apparatus integrated with a PLGA (poly lactic-co-glycolic acid) based nerve guide conduit for controlled local delivery of NGF.
Methods: The device consists of two concentric tubes and a reservoir in between the tubes that stores NGF. All device components were manufactured using 75/25 poly-lactic-glycolic-acid (Evonik). A 120µm diffusion hole was drilled into the inner conduit by pulsing a laser cutter. In vitro release tests were performed over a 4-week period. Concentration of NGF released from conduits was determined with an ELISA (R&D systems). The released NGF was collected every 3-4 days, and the bioactivity of the released NGF was determined using a chick dorsal root ganglion (DRG) based bioassay. In vivo testing of the NGF-delivering conduit (n=8) was performed in a rat sciatic nerve gap model and compared with negative control (empty conduits, n=8). The animals were sacrificed at 6 months postoperatively and sciatic nerve, gastrocnemius muscle and soleus muscle samples were harvested for histomorphometric analysis.
Results: The release of NGF from the conduit was within the concentration range of 0.5~10 ng/ml/day (n=5) without an initial burst. The released NGF stimulated in vitro neurite growth of cultured DRGs (823.4±24.3 µm using day 28 release sample) comparable to fresh NGF (845.7±25.7 µm using 10 ng/ml fresh NGF) (p>0.05), demonstrating that the released NGF from the conduit at 37°C for 4 weeks was bioactive. In the in vivo study, reduced muscle atrophy was observed in the NGF delivery group (49.4±1.4 %) as compared to the empty conduit (65.1±5.1 %) (p<0.05). ~20% more myelination was observed in the distal sciatic nerve of the NGF nerve conduit compared to the empty conduit (p<0.05). The NGF-delivering conduit had a higher percentage of re-innervated neuromuscular junctions (28.1±2 %) compared to empty conduits (9.7±1.3 %) (p<0.05).
Conclusions: The in vitro diffusion tests demonstrated that our drug delivering nerve guide was capable of releasing bioactive concentrations of NGF over a 4-week period. Additionally, the in vivo testing of the device in a 15-mm rat sciatic nerve defect model demonstrated that locally releasing NGF from our device resulted in improved muscle weight, myelinated nerve growth, and neuromuscular junction connectivity to a greater degree than with the device alone (no NGF).
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