Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth

The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell-matrix and Schwann cell-neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell-matrix and cell-cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell-matrix interactions by presenting integrin-binding RGD (Arg-Gly-Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell-neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell-neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell-matrix and cell-cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.