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

Autografts are the current gold standard for large peripheral nerve defects in clinics despite the frequentlyoccurring side effects like donor site morbidity. Hollow nerve guidance conduits (NGC) are proposed alternatives toautografts, but failed to bridge gaps exceeding 3 cm in humans. Internal NGC guidance cues like microfibresare believed to enhance hollow NGCs by giving additional physical support for directed regeneration of Schwann cellsand axons. In this study, we report a new 3D in vitro model that allows the evaluation of different intraluminal fibrescaffolds inside a complete NGC. The performance of electrospun polycaprolactone (PCL) microfibres inside 5 mmlong polyethylene glycol (PEG) conduits were investigated in neuronal cell and dorsal root ganglion (DRG) cultures invitro. Z-stack confocal microscopy revealed the aligned orientation of neuronal cells along the fibres throughout thewhole NGC length and depth. The number of living cells in the centre of the scaffold was not significantly different tothe tissue culture plastic (TCP) control. For ex vivo analysis, DRGs were placed on top of fibre-filled NGCs to simulatethe proximal nerve stump. In 21 days of culture, Schwann cells and axons infiltrated the conduits along the microfibreswith 2.2 ± 0.37 mm and 2.1 ± 0.33 mm, respectively. We conclude that this in vitro model can help define internal NGCscaffolds in the future by comparing different fibre materials, composites and dimensions in one setup prior to animaltesting.