Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets

[Image: see text] Current treatments of facial nerve injury result in poor functional outcomes due to slow and inefficient axon regeneration and aberrant reinnervation. To address these clinical challenges, bioactive scaffold-free cell sheets were engineered using neurotrophic dental pulp stem/progenitor cells (DPCs) and their aligned extracellular matrix (ECM). DPCs endogenously supply high levels of neurotrophic factors (NTFs), growth factors capable of stimulating axonal regeneration, and an aligned ECM provides guidance cues to direct axon extension. Human DPCs were grown on a substrate comprising parallel microgrooves, inducing the cells to align and deposit a linearly aligned, collagenous ECM. The resulting cell sheets were robust and could be easily removed from the underlying substrate. DPC sheets produced NTFs at levels previously shown capable of promoting axon regeneration, and, moreover, inducing DPC alignment increased the expression of select NTFs relative to unaligned controls. Furthermore, the aligned DPC sheets were able to stimulate functional neuritogenic effects in neuron-like cells in vitro. Neuronally differentiated neuroblastoma SH-SY5Y cells produced neurites that were significantly more oriented and less branched when cultured on aligned cell sheets relative to unaligned sheets. These data demonstrate that the linearly aligned DPC sheets can biomechanically support axon regeneration and improve axonal guidance which, when applied to a facial nerve injury, will result in more accurate reinnervation. The aligned DPC sheets generated here could be used in combination with commercially available nerve conduits to enhance their bioactivity or be formed into stand-alone scaffold-free nerve conduits capable of facilitating improved facial nerve recovery.