SilkNerve: Bioactive Implant for Peripheral Nerve Regeneration
T.Dinis 1, 2,*, G. Vidal 1, F. Marin 1, D. Kaplan 2, C.Eglès 1
1 BioMécanique et BioIngénierie, UMR 7338, Université de Technologie de Compiègne
2 Department of Biomedical Engineering, Tufts University, Medford – MA (USA)
Keywords: Silk nanofibers; biofunctionalization; nerve regeneration; nerve guidance conduit.
1. Introduction
Severe peripheral nerve damage affects 400 000
people each year. There are currently no effective
biomaterials for nerve repair after injury or trauma.
Silk proteins belong to a class of unique, high
molecular weight proteins that have found
widespread use in biomaterials and regenerative
medicine. These protein characteristics have robust
mechanical properties, biocompatibility and
biodegradability which can be enhanced with a
variety of chemical modifications. These
modifications provide tools for the attachment of
growth factors, cell binding domains and other
molecules of interest to silk. To manage and
stimulate the nerve regeneration, we propose to
develop a new type of biofunctionalized material
consisting on aligned silk nanofibers, produced by
the electrospinning technique.
2. Methods
Silk Fibroin (SF) proteins are extracted from
Bombyx mori cocoons to obtain 8% SF (w/v)
solution by dialysis in salt solution, then
electrospinned at 12kV to get biofunctionalized
aligned nanofibers. Before spinning, all of the silk
fibroin aqueous solutions were blended with 5%
PEO (w/v) in the volume ratio 1:4 to improve the
spinnability of the fibers, increasing the viscosity of
the solution. Two different design collectors were
used to align silk fibers, The manufacture of the
electrospun scaffold in glass coverslip for cell
culture are performed using
an oscillating
deposition system of 2 collectors whereas the
fibers from the nerve guidance conduits are aligned
using a mandrel (rotating speed : 9,5 m/s).
Different concentrations (5, 50, 500 ng/mL) of
Nerve Growth Factor (NGF) are added to the
fibroin solution to confer a bioactivation of SF
electrospun (2D). Moreover, one part of these
samples are rolled around teflon skewers (diameter
= 0,3mm) in order to manufacture regeneration
tubes (3D) also called nerve guidance conduit.
The morphology and structure of SF electrospun
nanofibrous scaffold and SF nerve guidance
conduits were analyzed by Scanning Electron
Microscopy (SEM) and Fourier Transformed
Infrared Spectroscopy (FTIR).
Neuron cells isolated from Sprague Dawley (rat)
Dorsal Root Ganglions (DRGs) are used for cellbased assays culture on this biomaterial.
Neuron cell adhesion and outgrowth on nanofibers
as well as drug release are assessed by
immunostaining and ELISA after 3, 5 and 7 days of
incubation.
3. Results and Discussion
Using a specific design of the electrospinning’s
collectors, SF nanofibers can be oriented and
confer a regular diameter. After spinning, annealing
treatments were performed on the samples to induce
β-sheet structure on silk fibroin nanofibers. SF
electrospun on glass cover slides was composed of
nanofibers with 892±97 nm in diameter. 98% of
the fibers presented a degree of deviation less than
2°.
Nerves guides conduits were obtained and
characterized by SEM to analyse the porosity,
diameter and alignment of the nanofibers inside the
tubes. Tubes present fibers aligned and also regular
diameters outside and inside. Thanks to the
manufacture process of the tube, we are able to
realize a multi- channel conduit composed by only
silk fibroin biomaterial.
Figure 1 Characterization of the silk biomaterial by
SEM. The nanofibers from the silk electrospun
scaffold 2D on a glass coverslip are regular and
E
well aligned (A). Manufacturing the nerve guidance
conduit allows a 3D configuration with porosity
(B), where fibers from the tube are aligned around
the pores (C) and inside the tube as well (D).
DRGs are treated with enzymes to obtain individual
neuron cells. This dissociation is validated by cell
viability studies and has showed a larger number of
viable neurons to monitor the cytotoxicity and
adherence effect on silk nanofibers.
Thanks to the configuration of our nanofibrous
scaffold containing aligned fibers, we are able to
manage the orientation of the axonal outgrowth in
two directions. The morphology of the cells do not
show abnormality after 3, 5, 7 and 10 days of
incubation.
Although ELISA assay hasn’t shown a release of
the NGF on the cell medium, we proved by
cultivating primary rat neurons on SF
biofunctionalized nanofibers that NGF increase the
adherence and survival of the neurons. The axonal
outgrowth
is
also
stimulated
by
this
functionalization.
Figure 2: Neuron cells outgrowth isolated from rat
DRGs (after 5 days) on SF nanofibers, random (A),
aligned (B and C), aligned and biofunctionalized
with NGF (D). Immunostainning tubulin βIII. SF
Nanofibers are biocompatible and allow the
adherence for neuron cells (E and F).
4. Conclusions
The alignment of silk fibers allows physical
guidance for neuronal growth, stimulated by
biofunctionalization. NGF seems bioavailable for
neuron cells, despite this; we have not measured
release on the culture medium. We are currently
working with other functionalizations of nanofibers
and we are improving the conception of the
biomimetic nerve guides 3D. Neuron cell's behavior
*Corresponding author. Email: tony.dinis@utc
through the nanoscale engineering of these
materials surfaces will be studied by cell cultures
and organotypic cultures of DRGs. A previous
study has shown that NGF-biofunctionalization
promotes the axon regeneration in central nervous
system; it seems that motor neurons behave
similarly, and even better.
Soon, we will realize the first tests of functional
rehabilitation after a rat sciatic nerve injury and
grafting of nerve guide, using a motion analysis
system VICON.
Acknowledgments
The authors thank Picardie Region for its financial
support and the Tissue Engineering Resource
Center (TERC) from Tufts University.
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