Associate Professor Wayne State University, United States
Introduction:: Peripheral nerve injury (PNI) due to traumatic injury resulting in a reduction or loss of neurological and motor function can have a significant negative impact on an individual’s quality of life. Damaged peripheral nerves have the capacity to regenerate and re-innervate, however, depending on the severity of the PNI, this process can take weeks to years. Schwann cells (SC) are vital to peripheral nerve regeneration, however, the influence of biomaterials on their behavior is not well characterized. This study will examine SC migration and proliferation resulting from changes in substrate stiffness.
Materials and Methods:: Altering the mechanical properties of hyaluronic acid (HA) was achieved by the degree of methacrylate substitution as previously described 1 with low modified (LMOD) indicating 30% substitution and high modified (HMOD) indicating 60% substitution. A 2% w/v MeHA, 2% w/v polyethylene oxide (PEO), and 0.05% w/v I29259 (Irgacure) solution was made as previously described2. Tensile testing was conducted on the nanofibers in a dry as spun and hydrated/submerged state (Instron 5940 w/Bio Bath) with a strain rate of 1mm/min. Samples were prepared by cutting 45mm x 10mm x 0.06mm pieces the electrospun nanofiber mat. Timelapse microscopy was used to observe the hSCs (ipn 02.8) for 8 hours using a Nikon Ti inverted microscope w/ stagetop incubation chamber. The incubation chamber was maintained as a humid environment with 5% CO2 at 37°C. NIS Elements-AR software captured images of the cells every 10 minutes. Cell pathway data was obtained using ImageJ software while MATLAB was used to quantify the cell motility coefficient and persistence time through the random walk model. To track cell proliferation over a 48-hour period, alamarBlue assay was used, with time points taken at 24 and 48 hours. For both migration and proliferation studies, cells were fixed after 48 hours.
Results, Conclusions, and Discussions:: The Young’s modulus and ultimate tensile strength values (± SD) can be found in Table 1. When comparing LMOD to HMOD, the Young’s Modulus for both states was found to be statistically significant (p< 0.05). Cell motility coefficient and cell persistence time values (± SD) can also be found in Table 1. Cells exhibited a greater tendency for movement on the stiffer substrate, but changed direction less frequently over the observation period compared to cells on a softer substrate. Between both substrates, the cell motility coefficient and cell persistence time were found to be statistically significant (p< 0.05). There appears to be a smaller difference in the effect of LMOD and HMOD MeHA fibers on hSC proliferation at 24 hours, however the effect of substrate stiffness becomes more evident at 48 hours with the hSCs proliferating more on HMOD MeHA fibers (± SD). Cell numbers can be found in Table 1.
In the early stages of regeneration, SCs migrate along axons in the proximal nerve stump and facilitate axon reconnection via the Bands of Büngner3. Considering that SC-mediated axon guidance is directional in nature, this could suggest lower cell persistence times, while increased migration in PNI would suggest a higher cell motility coefficient, both which have been observed in this study occurring on stiffer substrates. Stiffer substrates have also been linked to increased SC elongation, causing upregulation in pro-regenerative markers such as EGR14 and the release of growth factors such as TGFβ15 which regulate SC migration and growth3,5. Additional cell proliferation tests will be done to confirm behavior observed in this study, and evaluation of growth factor secretion and gene expression will be done in the future to further confirm the presence of a pro-regenerative SC phenotype.
Acknowledgements (Optional): :
References (Optional): :
Burdick, Jason A., et al. “Controlled Degradation and Mechanical Behavior of Photopolymerized Hyaluronic Acid Networks.” Biomacromolecules. 2005; 6(1): 386–391. doi:10.1021/bm049508a.
Whitehead, T.J., Sundararaghavan, H.G. Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds. J. Vis. Exp. (90), e51517, doi:10.3791/51517 (2014).
Min Q, Parkinson DB, Dun X-P. Migrating Schwann cells direct axon regeneration within the peripheral nerve bridge. Glia. 2021;69:235–254. https://doi. org/10.1002/glia.23892
Xu,Z.;Orkwis,J.A.;Harris, G.M. Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases. Int. J. Mol.Sci.2021,22,4821. https:// doi.org/10.3390/ijms22094821
R. G. Wells, D. E. Discher, Matrix elasticity, cytoskeletal tension, and TGF-β: The insoluble and soluble meet. Sci. Signal. 1, pe13 (2008).
