Dental Stem Cells and Scaffold-free Tissue Engineering to Enhance Facial Nerve Regeneration

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The overall goal of this study is to accelerate regeneration and improve functional outcomes in facial nerve injuries using novel scaffold-free dental pulp cell constructs. Facial nerve injuries severely impact patient quality of life by impairing motor function and causing facial paralysis. The severity of facial nerve injury dictates treatment modality where mild injuries are treated with end-to-end suturing, and the treatment of more severe injuries involves replacing damaged tissue with autologous nerve grafts; however, these therapies require a prolonged repair time and full functional recovery is not achieved. This is due in part to diminished Schwann cell (SC) support. SCs, neural crest-derived glial cells, provide neurotrophic factors (NTFs), which are known to promote neuron survival and axon extension. The dental pulp, the soft tissue found at the center of the tooth, contains a population of adult stem/progenitor cells that also endogenously express high levels of NTFs, a characteristic attributed to their neural crest origins. Furthermore, dental pulp cells (DPCs) can be induced to differentiate towards a SC-like phenotype, which further enhances NTF expression. We have developed scaffold-free DPC sheets that can be applied for enhancing nerve regeneration in both mild or severe injuries. For mild injury, DPC sheets can be wrapped around injured nerves treated using standard methods to act as localized NTF delivery systems to enhance outcomes. In more severe injuries, DPC sheets can be coaxed into solid, cylindrical tissues that function as conduits that provide a bioactive alternative to autografts. In the latter, DPCs are induced to generate an aligned ECM; therefore, these conduits provide both trophic cues (NTFs) that promote axon regeneration and guidance cues (aligned ECM) that orient axonal growth. Our preliminary data establishes that DPC sheets express high levels of NTFs sufficient to induce neurite outgrowth in neuronal cells in vitro, and enhance regeneration in rat facial nerve crush injury in vivo. Moreover, when cultured on a micro-grooved substrate, DPC sheets produce an aligned ECM that effectively orients neurite extension in vitro. In Specific Aim 1, we will evaluate if inducing SC differentiation will further enhance the regenerative effects of DPC sheets in vitro. We hypothesize that SC-differentiated DPC sheets will have greater NTF expression and induce greater neurite outgrowth in primary dorsal root ganglion cells in vitro than un-induced DPC sheets. In Specific Aim 2, we will test if wrapping an end-to-end sutured transection nerve injury with a DPC sheet will enhance axon regeneration and functional recovery relative to untreated controls. In Specific Aim 3, DPC cell sheets, comprising an aligned ECM, will be rolled in solid, cylindrical conduits and used to bridge segmental injuries in the rat facial nerve. The ability of these conduits to support axonal regeneration and improve nerve functional recovery will be evaluated. The success of these studies will lead to development of a novel and feasible method to accelerate healing and improve functional recovery of damaged facial nerves addressing the major clinical challenge associated with the current standard of care.