Spider silk (SPSI) has been established as one of natures most fascinating materials due to its unique properties. A remarkable application of the SPSI is its use in reconstructive medicine as nerve guidance structure/filament for nerve regeneration [1]. The Schwann cells (SCs), which are a crucial part of the nerve regeneration process adhere to SPSI and migrate along it to support axonal elongation[2]. SPSI degrades without inflammatory response or physiological pH changes. However, the interaction between the SCs and the silk and by that the SPSI properties, that promote SC adhesion are still unclear. The aim of this project is to elucidate material properties of SPSI, that are crucial for its unique performance in nerve regeneration. Not all spider silks show the same medical success, and we believe that properties such as composition, ultrastructure, and mechanical behavior have a pronounced influence on the acceptance of SPSI by SCs. Therefore, by combining experiments consisting of in vitro studies and the material characterization of various SPSIs, the properties, which are responsible for the advanced success of SPSI in nerve regeneration, will be clarified.

Ultrastructure investigations

The SPSI used in this experiment were: Nephila edulis and Avicularia avicularia MAG native and treated

with ethanol, UV light and autoclaved. For the tensile tests, one fibre of the major ampullate gland silk of the spider was extracted and mounted

with nailpolish on a 3D printed sample holder. Spiders were fixated and sedated to ensure a homogeneous

sample collection under forced spinning conditions. Prior to the tensile strength tests, the length of the

spider silk has been recorded with a light microscope (Keyence VHX 5000). For the measurement the thin side supports of the sample carrier are removed with a soldering iron, so that only the thread is under tension.

The spidersilk was pulled with a speed of 25μm/s until terminal rupture on an ASMEC Unat nano indenter in tensile mode. The collected rawdata was processed with numpy and pandas in python. The morphology and the size of SPSI were observed after the tensile test with scanning electron microscopy (FEI Quanta 250 FEG ESEM). A 10 nm thin gold layer was coated on the SPSI with an Edwards Scancoat 6 sputter coater. The micrographs were obtained by using the ETD. For determining the diameters of the mounted SPSI, five points were evaluated on two fibers from each silk type, leading to the average diameter from ten measurements.

We plan to go beyond the experiments in the SEM with additional spider silk variants and perform following experiments:

in-situ tensile tests on single fibres. For understanding of the spider silks crystal structure, SAXS and XRD[3] measurements will be conducted while straining the single fibres and fibre bundles in-situ on the adapted tensile stage.

Figure 1. Force-displacement curves and SEM micrographs of the native and autoclaved N. edulis MAG SPSI

References

[1] Radtke, C., et al., Spider Silk Constructs Enhance Axonal Regeneration and Remyelination in Long Nerve Defects in Sheep. PLOS ONE, 2011. 6(2): p. e16990.

[2] Kornfeld T, et al. Spider silk nerve graft promotes axonal regeneration on long distance nerve defect in a sheep model, Biomaterials, 2021 Feb 2;271:120692. doi: 10.1016/j.biomaterials.2021.120692.

[3] Riekel, C., et al., Nanoscale X-Ray Diffraction of Silk Fibers, Frontiers in Materials, 2019. 6(315).