Page 103 - IJB-4-1

Pre-clinical evaluation of advanced nerve guide conduits using a novel 3D in vitro testing model

multi-cell type source and their anatomical morphology critical gap injury in a mouse model [17] and provide
is suitable to simulate the proximal nerve stump after a basis for longer injury gaps of rat sciatic (>10mm),
nerve injury when placed on top of an NGC. and human trials thereafter. The microfibres used in
Additionally, DRGs contain three main cell types: this study were fabricated by electrospinning.
sensory neurons, which are surrounded by satellite Electrospinning is a controlled and fast fibre fabrication
glial cells, Schwann cells and fibroblasts, which are method, which allows the spinning of highly aligned
arranged in a collagen-dominant extracellular matrix [30] . or random oriented nano and microfibres, which have
Evaluating NGC scaffolds using DRGs therefore [37–39]
allows axon-glia communication, approximates the seen different applications in tissue engineering .
native peripheral nerve environment and contains the A range of fibre diameters (1, 5 and 8 µm) were
major cell components, which take part in peripheral extensively studied by Daud et al., where NG108–15
nerve regeneration. During Wallerian degeneration, neuronal cells formed the longest neurites in
Schwann cells and fibroblasts proliferate and migrate co-culture experiments together with primary Schwann
from both the proximal and the distal nerve stump into cells when grown on 5 µm fibres . Additionally, PCL
[18]
the nerve lesion and provide a chemical and physical was chosen in respect to its bioresorbable properties
guide for regenerating axons [1,31] . The close association of and FDA approval [40] as well as its great suitability for
Schwann cells and axons was also observed in this peripheral nerve repair [18,41–43] . Besides PCL microfibres,
study (yellow fluorescent signal in Figure 6B). In a range of other internal NGC scaffolds have been
addition, Schwann cells proliferated further than the suggested as being suitable candidates to enhance hollow
axons regenerated (Figure 6D), suggesting Schwann nerve conduits in nerve repair (summarised by [44,45] ).
cells direct extending axons. The use of DRGs as an
evaluation tool for peripheral nerve repair studies is The comparison of different NGC scaffold candidates
not a new idea and was presented by several research would be beneficial for the broad research community
groups in the past [18,32–34] . However, the described in to identify an optimal internal guide for hollow NGCs.
vitro assessment methods are mainly confined to the In this context, the developed 3D DRG model is of
evaluation of a single internal NGC scaffold. The value in the process of identifying internal NGC
current study suggests using DRGs to evaluate scaffolds. It is of note that more than 90% of all DRG
multiple scaffold structures inside the NGC, where the explants attached to NGCs in the developed model. In
model is not restricted to the evaluation of a single our experience, the attachment of DRGs to scaffolds is
conduit design. Additionally, this study showed the normally poor, typically around 20%. Furthermore,
use of DRGs to simulate the proximal nerve stump the proposed removal step of the microfibres from the
when placed on top of the NGC device. It should conduit is an easy and fast method for sample imaging
also be mentioned that several NGCs can be tested at and is to our knowledge a new technique to image
once by only sacrificing one animal compared to the
direct in vivo implantation of a single NGC in a single fibre scaffolds in NGCs. With this technique common
animal. The presented model may therefore act in confocal microscopy is still feasible without the need
terms of the refinement and the reduction of the 3Rs in of time consuming sectioning processes like
animal testing [35] , showing an advantage from an cryosectioning or paraffin embedding. The advantage
ethical point of view, but is also advantageous in terms of this technique is the maintenance of the intraluminal
of time and resources. guide complexity where z-stack microscopy can
The hollow nerve conduits in this study were reveal Schwann cell migration and axon sprouting
fabricated by microstereolithography from PEG. through the whole depth of the internal scaffold,
Microstereolithography is a micro-structuring technique together with simple but accurate assessment of cell
for complex 3D structuring with advantages of high health using live/dead analysis along the full length of
resolution and fast manufacturing times for nerve a scaffold to be investigated. Therefore, not only can
regeneration [17,36] . Herein, PEG conduits were used as internal fibre scaffolds of different materials and
a non-degradable test-conduit device for the analysis diameter be investigated but also different fibre
of the intraluminal microfibre scaffold. This model densities inside NGCs. Scaffolds, which are too highly
can be used with any other kind of hollow nerve guide packed can result in cell necrosis in the scaffold core
and is not restricted to the use of PEG NGCs. which can lead to direct failure of cell ingrowth [26–29] .
Combinations of different hollow NGCs and internal In this model, such packing densities could easily be
scaffolds can also be studied. The size of the conduits identified prior to in vivo implantation and might
studied were directly relevant to a common fibular therefore also reduce the number of animals.

International Journal of Bioprinting (2018)–Volume 4, Issue 1 9