Axons navigate along specific pathways based on the gradients of guidance cues. Following substantial neural injury, axons may fail to fully regenerate without external intervention due to limited intrinsic axonal growth capacity and/or interference from scar tissue. Hence, development of platforms capable of providing gradients of guidance cues to facilitate axonal regeneration is crucial in the field of neural tissue engineering. Even though numerous technologies have already been established, effective axonal guidance over long distances is still challenging.
Here we report a novel method for creation of gradient of laminin on conducting polymer film. Conducting polymers (CPs) such as Poly(3,4-ethylenedioxythiophene) (PEDOT) have been widely used for neural interfaces, owing to their excellent biocompatibility, soft mechanical properties, relatively high conductivity and outstanding chemical stability. Laminin is a major substrate-bound molecule for axonal growth, known as a chemoattractant.
In an effort to mimic the extra-cellular environment, this study aims to provide different gradient shapes of laminin such as linear, hill, and exponential to modulate axonal regeneration in the nervous system. First, using micro-scale motorized X-Y-Z stages and nano-syringe pump, lines of laminin have been printed with various concentrations (based on the gradient profile) ranging from 20-100 ug/ml on the surface of a 2% agarose hydrogel slab (hydrogel thickness was 10 mm). The laminin concentrations can be accordingly adjusted to create different gradient shapes. Processing parameters including injection flow rate and stage velocity have been optimized to create minimum line widths (i.e. 200 um) which leads to generation of high-resolution 10mm-long laminin gradients on 10 mm length of agarose gel. The whole pattern is then transferred onto the surface of a Poly (L, Lysine)-treated PEDOT film. PEDOT was previously electropolymerized on the surface of gold coated silicon substrate using galvanostatic mode with charge density of 0.18 C/cm2. Immunohistochemistry was used to quantify various immobilized laminin gradients on the PEDOT surface.
We aim to culture dorsal root ganglion explants and cortical neurons on top of CP substrates printed with laminin gradient patterns. This will allow us to assess the neurite response to various gradients and compare these responses to find the optimum gradient type and concentration range for effective axonal regeneration. The outcome of this project will pave the way towards development of more effective engineered conduits for nerve regeneration and will help us unravel fundamental questions of axonal regeneration in the nervous system.