Dorsal root ganglia neurite extension is inhibited by mechanical and chondroitin sulfate-rich interfaces

Glial scar formation plays a critical role in the regenerative failure in the central nervous system of adult mammals through the formation of mechanical or biochemical barriers as a result of its molecular composition. In this study, we report an in vitro model to study growth-cone behavior at controlled 3D interfaces using layered agarose hydrogels. The behavior of growth cones from embryonic day 9 (E9) chick dorsal root ganglia (DRGs) at interfaces that were mismatched in terms of their elasticity or chondroitin sulfate content was quantitatively determined. A mechanical barrier formed by the elasticity mismatch of layered agarose gels greatly influenced the ability of neurites from E9 DRGs to cross the 3D interface. To form chondroitin sulfate-rich interfaces, chondroitin sulfate B was covalently coupled to agarose hydrogel. Compared with unmodified agarose gels, the presence of CS-B-modified agarose gels at the interface significantly inhibited E9 DRGs neurites. After treatment of CS-B-modified agarose gels with chondroitinase ABC, the inhibitory effects of CS-B at the interface were significantly decreased. The effect of doping CS-B gels with laminin 1 (LN-1)-coupled agarose gels was investigated as a potential strategy to overcome inhibitory interfaces. When CS-B agarose gels were doped with LN-1-coupled agarose gels, DRG neurite's ability to cross 3D interfaces was significantly enhanced compared with that of non-LN-1-containing interfaces presenting equivalent CS-B. Our in vitro model may be used to study the influence of individual components of glial scar on inhibition as well as to design strategies to overcome this inhibition.