Method to Form a Fiber/Growth Factor Dual-Gradient along Electrospun Silk for Nerve Regeneration

Concentration gradients of guidance molecules influence cell behavior and growth in biological tissues and are therefore of interest for the design of biomedical scaffolds for regenerative medicine. We developed an electrospining method to generate a dual-gradient of bioactive molecules and fiber density along electrospun nanofibers without any post spinning treatment. Functionalization with fluorescent molecules demonstrated the efficiency of the method to generate a discontinuous concentration gradient along the aligned fibers. As a proof of concept for tissue engineering, the silk nanofibers were functionalized with increasing concentrations of nerve growth factor (NGF) and the biological activity was assessed and quantified with rat dorsal root ganglion (DRG) neurons cultures. Protein assays showed the absence of passive release of NGF from the functionalized fibers. The results demonstrated that the NGF concentration gradient led to an oriented and increased growth of DRG neurons (417.6 +/- 55.7 mu m) compared to a single uniform NGF concentration (264.5 +/- 37.6 mu m). The easy-to-use electrospinning technique combined with the multiple molecules that can be used for fiber functionalization makes this technique versatile for a broad range of applications from biosensors to regenerative medicine.