Novel nano-micro-macro multiple-nested hydrogel with gradient ciliary neurotrophic factor distribution induces directional axon regeneration of retinal ganglion cells

Ciliary neurotrophic factor (CNTF) shows potential for stimulating axon regeneration of retinal ganglion cells (RGCs), whereas unordered regeneration of axons does not have any biological significance. Given the chemotaxis characteristics of RGCs, materials with gradient CNTF distribution can chemically stimulate axon extension based on the CNTF concentration gradient. Therefore, a novel nano-micro-macro multiple-nested hydrogel with gradient CNTF distribution was developed in this study to induce directional axonal outgrowth of RGCs. The CNTF and magnetic nanoparticles (MNPs) were co-loaded into gelatin microspheres (CNTF&PEG-MNP@GMs), and subsequently suspended in a gelatin methacryloyl (GelMA) solution (viscous). Using a magnet, the CNTF&PEG-MNP@GMs were moved from the far-magnet side toward the near-magnet side forming gradient CNTF&PEG-MNP@GMs distribution inside the GelMA solution. The GelMA was then cross-linked via ultraviolet (365 nm) to form a hydrogel with gradient CNTF distribution. This hydrogel was further applied as the matrix for retinal explant culturing. The results showed that the number of axons in the segments of 200–300 µm and > 300 µm was 10.21 times and 15.02 times of those in the Gradient than those in the Without Gradient. The axons area distribution was 3.28 times and 13.41 times, respectively. 86.1% of the axons area in the Gradient were distributed in the segments > 200 µm, whereas only 56.27% in the Without Gradient. The results suggested that the hydrogel-matrix with gradient CNTF distribution could intensely induce directional axonal extension of RGCs in the retinal explant. The present study provides a new method for CNTF distribution, which could induce directional axonal regeneration of RGCs, and in turn have potential application in optic nerve injury repair to some extent.