Upon nerve injury, the body creates an environment consisting of permissive and non-permissive cues that instruct the function of cells involved in nerve repair. Among other roles, the developing extracellular matrix (ECM) acts as an underlying substrate to guide the union of neurites extending from the proximal stump for bridging the nerve gap. Chondroitin sulfate proteoglycans (CSPGs) are present in the nerve ECM and inhibit axon growth, potentially providing molecular cues to prevent aberrant growth and direct regeneration. In this study, we examined the potential of CSPGs to guide dorsal root ganglia (DRG) neurite outgrowth when freely available in the media or presented from a polymeric membrane. Soluble CSPGs added to the media of DRG explant cultures inhibited neurite outgrowth without spatial bias, caused retraction of axons, and decreased neurite extension in a dose-dependent manner. Poly-l-lactic acid membranes were chemically treated to enhance adsorption of CSPGs to the surface. CSPGs bound to 1,6-hexanediamine-treated membranes directed the orientation of neurite outgrowth, as neurites avoided bound CSPGs and a higher number and percentage grew on treated membranes lacking CSPGs. DRG explants cultured on CSPG-coated membranes without 1,6-hexanediamine-treatment had a smaller number of neurites and decreased neurite outgrowth, suggesting CSPGs were not retained on the membrane and were released into the culture medium. Taken together, these data demonstrate the potential of CSPG presentation to guide axonal growth. This approach offers a strategy to improve upon existing nerve guidance conduits by incorporating axon guidance molecules to direct nerve regeneration.
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Supplementary Fig. 1. Soluble CSPG inhibits neurite extension. Time-lapse phase contrast images of DRG explants cultured in basal media with a CSPG concentration of 25 μg/mL were taken at 1 h intervals for 48 h. DRG explants were cultured in normal growth media for 24 h prior to exchange with CSPG media. Images at 10× magnification. Supplementary material 1 (MOV 2105 kb)
Supplementary Fig. 2. DRG explants cultured in basal media exhibited normal neurite extension. Time-lapse phase contrast images of DRG explants cultured in basal media were taken at 1 h intervals for 48 h. DRG explants were cultured in normal growth media for 24 h prior to exchange with basal media. Images at 10× magnification. Supplementary material 2 (MOV 1555 kb)
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Man, A.J., Leach, J.K. & Bannerman, P. Redirection of Neurite Outgrowth by Coupling Chondroitin Sulfate Proteoglycans to Polymer Membranes. Ann Biomed Eng 42, 1271–1281 (2014). https://doi.org/10.1007/s10439-014-0991-y
- Dorsal root ganglia
- Chondroitin sulfate proteoglycans