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

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The “ATCC” cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while “UKN” cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2326-5) contains supplementary material, which is available to authorized users.

The use of nerve guidance conduits (NGCs) to treat peripheral nerve injuries is a favorable approach to the current "gold standard" of autografting. However, as simple hollow tubes, they lack specific topographical and mechanical guidance cues present in nerve grafts and therefore are not suitable for treating large gap injuries (30− 50 mm). The incorporation of intraluminal guidance scaffolds, such as aligned fibers, has been shown to increase neuronal cell neurite outgrowth and Schwann cell migration distances. A novel blend of PHAs, P(3HO)/P(3HB) (50:50), was investigated for its potential as an intraluminal aligned fiber guidance scaffold. Aligned fibers of 5 and 8 μm diameter were manufactured by electrospinning and characterized using SEM. Fibers were investigated for their effect on neuronal cell differentiation, Schwann cell phenotype, and cell viability in vitro. Overall, P(3HO)/P(3HB) (50:50) fibers supported higher neuronal and Schwann cell adhesion compared to PCL fibers. The 5 μm PHA blend fibers also supported significantly higher DRG neurite outgrowth and Schwann cell migration distance using a 3D ex vivo nerve injury model.