RESUMEN
Objective. Engineered nerve conduits must simultaneously enhance axon regeneration and orient axon extension to effectively restore function of severely injured peripheral nerves. The dental pulp contains a population of stem/progenitor cells that endogenously express neurotrophic factors (NTFs), growth factors known to induce axon repair. We have previously generated scaffold-free dental pulp stem/progenitor cell (DPSC) sheets comprising an aligned extracellular matrix (ECM). Through the intrinsic NTF expression of DPSCs and the topography of the aligned ECM, these sheets both induce and guide axon regeneration. Here, the capacity of bioactive conduits generated using these aligned DPSC sheets to restore function in critical-sized nerve injuries in rodents was evaluated.Approach. Scaffold-free nerve conduits were formed by culturing DPSCs on a substrate with aligned microgrooves, inducing the cells to align and deposit an aligned ECM. The sheets were then detached from the substrate and assembled into scaffold-free cylindrical tissues.Main results. In vitroanalyses confirmed that scaffold-free DPSC conduits maintained an aligned ECM and had uniformly distributed NTF expression. Implanting the aligned DPSC conduits across critical-sized defects in the buccal branch of rat facial nerves resulted in the regeneration of a fascicular nerve-like structure and myelinated axon extension across the injury site. Furthermore, compound muscle action potential and stimulated whisker movement measurements revealed that the DPSC conduit treatment promoted similar functional recovery compared to the clinical standard of care, autografts. Significance. This study demonstrates that scaffold-free aligned DPSC conduits supply trophic and guidance cues, key design elements needed to successfully promote and orient axon regeneration. Consequently, these conduits restore function in nerve injuries to similar levels as autograft treatments. These conduits offer a novel bioactive approach to nerve repair capable of improving clinical outcomes and patient quality of life.
Asunto(s)
Pulpa Dental , Matriz Extracelular , Regeneración Nerviosa , Células Madre , Ingeniería de Tejidos , Andamios del Tejido , Pulpa Dental/citología , Pulpa Dental/fisiología , Animales , Matriz Extracelular/fisiología , Regeneración Nerviosa/fisiología , Ratas , Andamios del Tejido/química , Células Madre/fisiología , Células Madre/citología , Ingeniería de Tejidos/métodos , Células Cultivadas , Ratas Sprague-Dawley , Nervio Facial/fisiología , Traumatismos del Nervio Facial/terapia , Masculino , HumanosRESUMEN
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.
Asunto(s)
Axones , Regeneración Nerviosa , Axones/fisiología , Pulpa Dental , Matriz Extracelular , Humanos , Células MadreRESUMEN
An effective strategy for sustained neurotrophic factor (NTF) delivery to sites of peripheral nerve injury (PNI) would accelerate healing and enhance functional recovery, addressing the major clinical challenges associated with the current standard of care. In this study, scaffold-free cell sheets were generated using human dental pulp stem/progenitor cells, that endogenously express high levels of NTFs, for use as bioactive NTF delivery systems. Additionally, the effect of fibroblast growth factor 2 (FGF2) on NTF expression by dental pulp cell (DPC) sheets was evaluated. In vitro analysis confirmed that DPC sheets express high levels of NTF messenger RNA (mRNA) and proteins, and the addition of FGF2 to DPC sheet culture increased total NTF production by significantly increasing the cellularity of sheets. Furthermore, the DPC sheet secretome stimulated neurite formation and extension in cultured neuronal cells, and these functional effects were further enhanced when DPC sheets were cultured with FGF2. These neuritogenic results were reversed by NTF inhibition substantiating that DPC sheets have a positive effect on neuronal cell activity through the production of NTFs. Further evaluation of DPC sheets in a rat facial nerve crush injury model in vivo established that in comparison with untreated controls, nerves treated with DPC sheets had greater axon regeneration through the injury site and superior functional recovery as quantitatively assessed by compound muscle action potential measurements. This study demonstrates the use of DPC sheets as vehicles for NTF delivery that could augment the current methods for treating PNIs to accelerate regeneration and enhance the functional outcome. Impact statement The major challenges associated with current treatments of peripheral nerve injuries (PNIs) are prolonged repair times and insufficient functional recovery. Dental pulp stem/progenitor cells (DPCs) are known to endogenously express high levels of neurotrophic factors (NTFs), growth factors that enhance axon regeneration. In this study, we demonstrate that scaffold-free DPC sheets can act as effective carrier systems to facilitate the delivery and retention of NTF-producing DPCs to sites of PNIs and improve functional nerve regeneration. DPC sheets have high translational feasibility and could augment the current standard of care to enhance the quality of life for patients dealing with PNIs.