Following severe neural damage, axons may fail to fully regenerate without external intervention. Although various printing and microfluidic technologies have been explored, effective nerve guidance in critical distances (> 10 mm) has remained a long-lasting challenge. Among numerous biomaterials, organic semiconductors such as poly(3,4-ethylenedioxythiophene) (PEDOT) have demonstrated promising results in nerve guidance scaffolds due to their soft mechanical properties, mixed electronic-ionic conductivity, and superb chemical stability in physiological environment, and good biocompatibility. Laminin is a major surface-bound protein in the extracellular matrix, also a well-known chemoattractant for neurite outgrowth. In this work, we introduce a novel micro-printing technique to fabricate long gradients of laminin on thin PEDOT films with various gradient profiles to potentially direct axonal regeneration.

Laminin gradients were first printed on the surface of an agarose hydrogel slab (thickness: 10 mm), using micro-scale motorized X-Y-Z stages and a nano-syringe pump. The droplets of laminin solutions with different laminin concentrations were deposited next to each other (vertical and horizontal distances of 400 µm and 800 µm, respectively). Using molecular stamping, the printed laminin pattern was then transferred onto the surface of a PEDOT film which had been previously electropolymerized on the surface of gold-coated silicon substrates in galvanostatic mode). Immunohistochemistry and fluorescent imaging were utilized to visualize and quantify various immobilized laminin gradients on PEDOT surface. 

Linear and hill gradients were created and quantified. The linear gradient consisted of 10 lines (8 mm-long) with a solution concentration range of 10-55 µg ml^-1. In the hill gradient (7.2 mm-long, 9 deposited lines), first applied solution concentration was 10 µg ml^-1 which increased linearly until it reached a maximum value of 90 µg ml^-1, then symmetrically decreased. Measured fluorescent intensity follows a linear trend (R²= 0.99) with respect to applied laminin solution concentrations in the linear gradient. Similarly, both symmetrical sides of the hill gradient show a good linear fit with almost equal absolute slope values (R²= 0.98 and R²= 0.99, respectively). These results demonstrate that a wide range of long and high-resolution gradient profiles can be generated using this technique, and neurite outgrowth to each gradient can be potentially assessed systematically to find the optimum gradient shape which results in superior axonal regeneration compared to others.