• Silk fibroin nanofiber diameter and alignment

Optimal fiber alignment was achieved at a rotation speed of 26.8 m/s.

• DRG sensory neuronal culture on fibroin fibers

• Rat sciatic nerve regeneration through implant

6 months post-implantation

• Functional recovery in rat hind limb

Motion capture analyses were performed pre-surgery, 3 months post-surgery, and 6 months post-surgery

*Fibroin functionalized with NGF and CNTF**Fibroin functionalized with NT-3

Functionalized silk fibroin-based nanofiber guidance conduitsfor peripheral nerve regeneration

Kayla Belanger,a Tony Dinis,a Bernard Devauchelle,b Christophe Eglesa

aUniversité de Technologie de Compiègne, Rue Personne de Roberval, Compiègne 60200, FrancebInstitut Faire Face, Avenue Rene Laennec, Salouël 80480, France

Kayla.belanger@utc.fr

A severe injury to a peripheral nerve leads to degeneration and loss of sensory and motorfunction. The autograft has long been considered the gold standard for nerve repair, but presentsnumerous drawbacks .1 Nerve guidance conduits (NGCs) made from biomaterials show increasingpromise as effective substitutes to the autograft. Silk fibroin has proven to be a favorablebiomaterial for tissue engineering applications due to its biocompatibility, its biodegradability, itsrobust mechanical properties, and its availability for chemical modification.2

In this study, NGCs composed of nanoscale aligned fibers achieved by electrospinning andfunctionalized with growth factors are tested both in vitro and in vivo for neuron regrowth andguidance efficiency in the peripheral nervous system of the rat.

Introduction1.

• Silk fibroin purification and biofunctionalization

Fibroin is extracted from Bombyx mori cocoons. Aqueous fibroin is functionalized withgrowth factors*.

• Silk fibroin nanofiber production and alignment

Aqueous fibroin (8%) solution is electrospun using a high speed rotating drum collector for unidirectional alignment of nanofibers.

A high voltage is applied to the polymer solution creating an electric field between the syringe needle and the collector. The droplet leaving the needle is stretched creating a solid fiber as the solvent evaporates.

• Dorsal root ganglion sensory neuronal cell culture

Sensory neurons are dissociated fromfetal Sprague Dawley dorsal root ganglia(DRG) and cultured on the nanofibrous silkfibroin materials functionalized with growth factors.

• Implantation of functionalized fibroin in rat sciatic nerve

Aligned fiber mats are rolled with small Teflon rods to achieve a multi-channel tube conduit that mimics the morphology of a native nerve.

The conduit is trimmed to 5mm and hydrated. It isthen sutured between two severed sections of a ratsciatic nerve.

• Analysis of functional recovery

Rats are subject to motion capture analysis in order to evaluate functional recovery. Reflectivemarkers are placed on several specific anatomical points on each side of the rat’s body. The rat ismade to run across a wooden plank while reflective cameras capture the hind limbs’ mobilitypatterns.

*NGF (nerve growth factor), CNTF (Ciliary neurotrophic factor), NT-3 (Neurotrophin 3)

Materials and Methods2.

Results3.

• DRG neuronal cultures show that fibroin is biocompatible and the aligned fibers supportneuron adhesion and uniaxial axon regeneration.

• Histological analyses of the implant after 6 months in vivo show successful neuronregeneration; cross sections of the implant reveal axon regrowth through the channels andaxon myelination by Schwann cells.

• Functional recovery was not optimal. 6 months may not be a sufficient timeframe for motorneurons to reinnervate muscle tissue, therefore we have begun a new in vivo study that willlast 8 months.

Conclusions4.

1. Belanger, K.; Dinis, T.; Taourirt, Sami.; Vidal, Guillaume.; Kaplan, D.; Egles, C. Macromolecular Bioscience 2016, DOI: 10.1002/mabi.201500367.2. Dinis, T.; Elia, R.; Vidal, G.; Dermigny, Q.; Denoeud, C.; Kaplan, D.; Egles, C.; Marin, F. Journal of the Mechanical Behavior of Biomedical Materials 2015, 41, 44.

Cited Literature5.

Figure A:Rotation speed: 1 m/sFiber diameter: 378 ± 190 nmScale bar: 5 µm

Figure B: Rotation speed: 26.8 m/sFiber diameter: 294 ± 150 nmScale bar: 5 µm

A B

Dissociated sensory neurons (5 days) stained with βIII-tubulin on aglass coverslip (Figure A, scale bar: 75 µm), random *functionalizedfibroin fibers (Figure B, scale bar: 50 µm), and aligned**functionalized fibroin fibers (Figure C, scale bar: 100 µm).

B D E

A

A hematoxylin and eosin stained cross section of a native ratsciatic nerve (Figure A, scale bar: 150 µm) and one of itsfascicles (Figure B, scale bar: 20 µm).

A blood vessel is circled in Figure B.

B C D

A hematoxylin and eosin stained cross section a the fibroinfiber implant (Figure C, scale bar: 150 µm) and one of itschannels (Figure D, scale bar: 20 µm) after 6 months in vivo.

Arrows in Figure D point to an axon, myelin, and a Schwanncell nucleus; A blood vessel is circled.

SEM images of DRG cultured on aligned fibroin fibers show thefavorable interaction and adherence (Figure D, scale bar: 10 µm;Figure E, scale bar: 2 µm) of the neuron extensions to the fibroinfibers.

A B

The conduit is trimmed to 5 mm and hydrated. It isthen sutured between two severed sections of a ratsciatic nerve.

Aligned fiber mats are rolled with smallTeflon-coated rods to achieve a multi-channel tube conduit that mimics themorphology of a native nerve.

Sensory neurons are dissociated fromfetal Sprague Dawley dorsal root ganglia(DRG) and cultured on the nanofibrous silkfibroin materials functionalized with growthfactors.

Aqueous fibroin (8%) solution is electrospun using a highspeed rotating drum collector for unidirectional alignment ofnanofibers.

A high voltage is applied to the polymer solution creatingan electric field between the syringe needle and thecollector. The droplet leaving the needle is stretched creatinga solid fiber as the solvent evaporates.

C

A