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Mehri Behbehani, et al.

Green fluorescence from axons βIII tubulin could be Additionally, there is a regulatory and ethically driven
seen simultaneously where Schwann cells were effort to reduce the number of animals used in medical
located. The co-localization could be identified by research.
an overlay of green and red fluorescent signals 3D in vitro models are of increasing interest because
appearing in a yellow-orange colour (Figure 6B). of their “closer-to-in-vivo” behaviour with higher
The distance that neuronal and Schwann cells physiological relevance. In 3D models, cells can be
infiltrated into the conduit was defined as the last cultivated in three dimensions like in their natural
detectable signal seen distant from the DRG body. environment in tissues and organs. In neurosciences,
The distance of outgrown cells was of particular 3D models are mostly devoted to the central nervous
importance as demonstrating the microfibres system to study the neuronal network and signalling
efficiency to support cell infiltration in a simulated pathways, brain barriers, disorders and the effects of
nerve lesion in vitro. Schwann cells migrated and neurotoxins (reviewed by [24,25] ). Studies on peripheral
proliferated 2.2 ± 0.37 mm along the aligned nerve injuries, and nerve guides in particular,
microfibre scaffold inside the NGCs, where would benefit from in vitro 3D models due to the
neuronal cells showed the extension of axons by discussed limitations of animal injury models, 2D in
2.1 ± 0.33 mm (Figure 6D). Together, Schwann vitro cultures and the lack of published data. Today
cells and axons infiltrated 43 % of the 5 mm long there is an increased need for 3D models on internal
conduit in vitro in 21 days. NGC scaffolds. In particular, hollow NGCs have
shown their limitations in repairing nerve gaps
4. Discussion exceeding 3 cm and improvement on this matter is
urgently needed. Different attempts have been made to
Most cell-based research is acquired via experiments study internal scaffolds for NGCs in a three-dimensional
in monolayer cultures and largely assumes that these environment but documentation on a 3D in vitro
monolayer cultures reflect the physiology of tissue. To model to test different scaffolds in one setup prior to
this day, cell cultivation in a monolayer is the animal implantation is still missing. In support of this
traditional way of culturing cells. Cultivation in notion, we developed an in vitro 3D DRG model that
monolayers has proved to be simple, convenient and allows the investigation of different internal fibre
lead to relatively fast results. However, for some time, scaffolds inside an NGC. In order to ensure that the
only in vitro 2D cultivation models and in vivo animal microfibre scaffold supported cell growth and viability
models were available and used for early stage throughout the entire conduit length and depth, cell
medical device evaluation [19–21] . Between both models culture was conducted prior to DRG cultures. The
there is a large gap in relation to the physiological developed system showed an in vitro 3D environment,
relevance. Monolayer cultures are far away from which was based on the final implantable product and
the biological behaviour in the human body, especially therefore more likely mimicked physiological relevant
when cell lines were chosen over primary cells, and conditions. The morphology and viability of neuronal
animal studies, whilst representing a whole organism cells on microfibres inside a tubular nerve guide using
and metabolism, are not human. Though animal the proposed setup was examined, where neuronal
studies can mirror human biological responses, they morphology remained unchanged throughout the entire
do not always reproduce human physiology [22] . nerve guide and where cells showed similar and not
Especially the dominant use of rat sciatic nerve injury significantly different viability rates compared to the
models to study NGCs, which have limitations when flat TCP control. This is of particular interest as it was
translating results to humans [23] . Rats are too small in previously mentioned that even thin 3D scaffolds
size to enable the study of human critical nerve gap showed a much lower cell growth in the centre of
lengths of > 4 cm and homogenous in-bred strains those structures due to the lack of nutrient and oxygen
reduce the variability between results. Another diffusion to the scaffold core [26–29] . Herein, the number
concern that may result in mistranslation is the of living cells inside the centre of the microfibre
attempt to compare experiments of healthy animals scaffold measured more than 90%.
versus sick patients, who normally suffer from DRGs are an effective tool for the evaluation of
multiple injuries besides the injury to a peripheral internal scaffolds in NGCs as described in this study.
nerve due to traffic, industrial or domestic accidents. They are easy to harvest, demonstrate a primary

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