Controlled Delivery of Growth Factors and Small Molecules for Peripheral Nerve Regeneration

Controlled local Delivery of growth factors and small molecules for peripheral nerve regeneration

 

Praima Labroo, Himanshu Sant, Scott Ho, Bruce Gale, Jill Shea, and Jayant Agarwal

Departments of Mechanical Engineering, Plastic Surgery, University of Utah, Salt Lake City, UT USA

 

Introduction

Autologous nerve grafts are the most commonly used graft to repair large nerve defects.  Despite being the current “gold standard” autologous nerve grafts cause morbidity at the donor nerve site.  Nerve conduits or tubes are a promising alternative to autografts. They act as guidance cues for the regenerating axons and allow for tension free bridging, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of growth factors or small molecules can enhance axon growth and peripheral nerve regeneration. In this work we present the design of a novel drug delivery apparatus integrated with a PLGA based nerve guide conduit. This integrated device is designed to simplify the design process and provide increased versatility for releasing a variety of different growth factors or small molecules.

Experimental methods

The bioresorbable guidance conduits were produced using 75/25 poly-lactic-glycolic-acid (PLGA). PLGA was dissolved in acetone and ethanol and conduits were then formed. Diffusion hole(s) were drilled into the inner conduit by pulsing a laser cutter. Final assembly of mold-formed conduits and end caps was done using a solvent bonding process. Tests have been performed for release of dextran (Fluorescein tagged dextran), nerve growth factor (NGF) and tacrolimus. These drugs were loaded into the conduit reservoir separately and then the conduit was placed into a receiver chamber containing DMEM F12 medium+10% FBS (fetal bovine serum) and 1% of Antimyotic solution at 37°C and 5% CO₂. This setup was used to replicate temperature and pH in physiological conditions. A series of sample collections were taken from the receiver chamber over specified time intervals and the chamber was flushed and filled with fresh media matrix each time. Fluorescence and ELISA readings were obtained and the data was analyzed to determine drug release kinetics. Collected samples were used to culture chick DRGs (dorsal root ganglia) for 72h to check the bioactivity of released samples. Drug delivering conduits loaded with NGF have been implanted in rats to test the growth across a transected nerve after 90 days.

Figure 1.  Photograph of the PLGA single-reservoir conduit prototype (left) (scale 5mm).  Schematic diagram showing diffusion hole placement (center left). An axial view of the nerve and drug delivery device and schematic diagram of the drug delivery device placement across regenerating nerve (center right), drug delivering conduit implanted across cut sciatic nerve (right)
Results

The diffusion tests indicate that 150 µm hole allows for a sustainable release of NGF and dextran for much longer than 30 days.  Five 50 µm holes were drilled for release of tacrolimus. Different hole dimensions were based on the dosage requirement and diffusion coefficient of each drug. The release was compared with the predicted outcome based on the simulations and collected samples showed bioactivity by enhancing growth of chick DRG axon length and density.

Histology of nerve and walking track data of rats is underway to confirm the success of drug delivering conduits as compared to autografts and no-drug conduits.

   

Figure 2.  Cumulative release of tacrolimus (left) cumulative release of NGF (center), cumulative release of fl dextran (right) (legends indicate the release target of the device)

 Figure 3.  Table of DRG axon growth in presence of eluates of NGF released from drug delivery conduit at different days.  The images showing DRG axon elongation after treatment with collected NGF sample (left) mage of DRG treated with 7-day collection of NGF releasing conduit (right) Image of DRG treated with 7-day collection of tacrolimus releasing conduit(right) (Scale 500 µm)

 

Conclusion

We have achieved controlled release of dextran, NGF and tacrolimus.  The initial results with Sprague Dawley rat sciatic nerve model have shown improved nerve regeneration.  Work is underway for a longer study using multiple growth factors and small molecules with rat and mouse models.