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Mehri Behbehani, et al.

motor nerve lesions with commonly used sensory microfibres) can be directly conducted in whole nerve
sural nerve grafts [3-5] . conduits in order to provide a prospective platform for
In recent years, bioengineers have focused on the comparison of different internal NGC scaffolds. The
development of NGCs to provide an alternative effectiveness of the model was determined by
treatment to autografts, where different materials and neuronal cell culture and primary rat dorsal root
designs were explored resulting in a number of ganglion explants in conjunction with cellular responses
devices that have seen FDA approval (Neuroflex/ investigated using three-dimensional z-stack confocal
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Neuromatrix , NeuraGen , AxoGuard , Avance , and two-photon imaging.
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Neu-rolac ® or SaluTunnel ). However, these 2. Methods
commercial NGCs lack internal guidance structures
and cannot exceed injury gaps greater than 3 cm, 2.1 Conduit Fabrication
which is shorter than the defined critical nerve gap of Three-dimensional conduits were fabricated by
4 cm in humans [6,7] . To this date, no other commercial microstereolithography. Polyethylene glycoldiacrylate
solution has been presented. Therefore, key challenges (Mn = 575 g/mol, Sigma, Poole, U.K.) was mixed with
now focus on enhancement of NGCs by including 4% diphenyl-(2,4,6-trimethylbenzoyl)-Phosphineoxide/2
internal conduit guidance structures for targeted axon -hydroxy-2-methylpropiophenone 50/50 photoinitiator
regeneration through the nerve gap to maximise the (wt/wt, Sigma) to obtain a photocurable version of the
number of regenerating axons and therefore the target polymer. A cross sectional image of the required
regeneration outcome. Aligned fibres of a natural or conduit size (1.2 mm internal diameter, 250 µm wall
synthetic material can be one approach to improve thickness) was uploaded to a digital micromirror
hollow nerve guides [8-11] by mimicking the natural device (Texas Instruments Incorporated, USA, associated
regeneration guidance cues of bands of Büngner, where software: ALP-3 Basic, ViALUX GmbH), which
comprised of a range of mirrors, where each 20 µm
Schwann cells and fibroblasts form aligned bands between size mirror represented a 1:1 object-to-image size ratio.
the proximal and distal nerve end to guide axons to their The beam of a 10 mW 405 nm laser (Vortran Laser
target effectors [12] . With adjustment of the fibre Technology Inc, USA) was expanded and aligned
density in the NGC lumen, specific regeneration needs while running through a spatial filter and a mirror set.
can be addressed like varying axon numbers in nerves. The beam then reached the digital micromirror device
In comparison, hollow nerve guides only provide and was reflected in the shape of the uploaded cross
outer guidance for the injured nerve to keep both section image. The liquid prepolymer was placed
nerve stumps connected but cannot provide relevant under a motorized z-axis translation stage (Thorlabs
internal guidance support for regenerating cells and Ltd, UK, associated software: APT software), which
tissue. Furthermore, fibre containing conduits would was mounted at the liquid-air interface. Controlled by
not require an adaption of nerve fascicles and blood velocity and acceleration, the z-stage moved down
vessels between the proximal stump and the conduit into the bulk material where the irradiated regions
which is necessary when using autografts [13] . were polymerized. Once the final length of the
Newly developed NGC scaffold designs, which structure was reached, the laser power was turned off
have seen in vivo implantation, have compared and the stage returned to its origin, carrying the
favourably to hollow conduits and autografts [8,14-16] . complete 3D conduit structure. Unreacted prepolymer
However, new designs have, for the most part, was washed off with isopropyl alcohol (IPA, Fisher
not been directly compared to previous generation Scientific UK). All fabricated structures were washed
NGCs, or to currently used nerve conduits. Furthermore, in IPA for 7 days to ensure full removal of unreacted
with advanced NGC improvements more adequate prepolymer and added photoinitiator.
evaluation techniques are required. More complex 2.2 Microfibre Fabrication
NGC structures, such as those that comprise of two
components, like an additional internal scaffold, would The fabrication of 5 µm PCL fibres (referred to as
greatly benefit from novel assessment techniques like in microfibres) was conducted using electrospinning. A
vitro evaluations in three dimensions, instead of plastic syringe (Becton Dickinson) was filled with
relying on data retrieved from in vitro analysis on flat 10% PCL solution (wt/wt, Sigma Aldrich Co, U.K.) in
material films. We therefore hypothesized that the dichloromethane (Fisher Scientific U.K.) and topped
evaluation of intraluminal nerve guide scaffolds (e.g. with a 20G blunt stainless-steel needle. The needle

2 International Journal of Bioprinting (2018)–Volume 4, Issue 1