The lack of neurotrophic factors (NTFs) following peripheral nerve injury leads to incomplete nerve regeneration and partial functional recovery . In this work, a new methodology developed by our group to produce silk fibroin conduits was used , allowing to incorporate and release bioactive Nerve Growth Factor (NGF) or Glial cell-derived neurotrophic factor (GDNF).
METHODS: Enzymatically-crosslinked silk fibroin (SF) conduits were produced by taking advantage of the tyrosine groups present in the structure of silk fibroin. In this system, hydrogen peroxide was used as the substrate and horseradish peroxidase as the enzyme. NTFs were incorporated in the silk fibroin conduits using two different methods: i) mixing the NTFs prior to silk crosslinking and ii) soaking the produced conduits in NTF solution. The release profiles were studied by means of performing ELISA. The bioactivity of the released NTFs was confirmed by in vitro assays, using neonatal rat pup dorsal root ganglion (DRGs) explants. The selected formulation was implanted in a 10 mm sciatic nerve defect in rats for 6 weeks and compared to autografts and plain SF conduits (5 rats per formulation).
RESULTS: The soaked formulations were found to release significant higher amounts of NTFs as compared to the crosslinked ones. Biological assays with DRGs allowed us to select soaked GDNF 4 µg/mL as the most promising formulation, with longer and denser axon sprouting. In the in vivo study, 80% of the animals transplanted with soaked GDNF 4 µg/mL responded positively to the nerve pinch test, as compared to 20% of animals receiving the plain SF conduit. The treatment with soaked GDNF 4 µg/mL significantly reduced muscle weight loss compared to the plain SF conduit. Furthermore, regeneration across the 10 mm nerve gap was detected in 100% of the autografts, 100% of the silk + GDNF and 80% in the SF conduits.
DISCUSSION & CONCLUSIONS: The previously described method for SF conduits production revealed to be very useful and versatile for NTFs incorporation. The incorporation was successful and preserved the bioactivity of the NTFs when released, as seen by the in vitro assays. Overall, the hypothesis that the addition of GDNF to the SF nerve conduit would yield positive results as compared to plain silk conduit was demonstrated and the nerve regeneration was found to be robust and comparable to autograft, when using the soaked GDNF at 4 µg/mL conduit.