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

这项研究的总体目标是加速面神经的再生和改善功能结果。 使用新型无支架牙髓细胞结构的损伤。面神经损伤严重影响患者 通过损害运动功能和导致面瘫来提高生活质量。面神经损伤的严重程度 规定了用端到端缝合治疗轻微损伤的治疗方式，以及对更多 严重损伤包括用自体神经移植物替换受损组织；然而，这些治疗方法 需要较长的修复时间，无法实现功能完全恢复。这在一定程度上是由于 支持雪旺细胞(SC)。SCS是神经沟来源的神经胶质细胞，提供神经营养因子(NTF)， 已知可促进神经元存活和轴突延长。牙髓，在牙髓中心发现的软组织 包含一组成体干细胞/祖细胞，这些细胞也内源性地表达高水平的 NTFS，这一特征归因于它们的神经脊起源。此外，牙髓细胞(DPC)可以 诱导分化为SC样表型，进一步增强NTF的表达。我们有 开发了无支架的DPC片材，可用于促进轻度或 伤势严重。对于轻微的损伤，DPC片可以包裹在受伤的神经周围，用标准的 方法作为本地化NTF给药系统，提高疗效。在更严重的伤害中，DPC床单 可以诱骗成固体的圆柱形组织，其功能是提供生物活性的替代品 自体移植。在后者中，DPC被诱导生成对准的ECM；因此，这些管道既提供 促进轴突再生的营养线索(NTF)和定向轴突的引导线索(对齐的ECM) 成长。我们的初步数据表明，DPC膜表达高水平的NTF，足以诱导 轴突在体外培养的神经细胞中生长，并在体内促进大鼠面神经挤压伤后的再生。 此外，当在微槽基板上培养时，DPC片材会产生一种排列整齐的ECM，有效地 体外定向轴突延伸。在具体目标1中，我们将评估诱导SC分化是否会进一步 增强DPC片材的体外再生效果。我们假设SC-Differential DPC单 将在原代背根神经节细胞中有更多的NTF表达和诱导更多的轴突生长 体外培养比未诱导的DPC片。在特定的目标2中，我们将测试是否包裹了一个端到端缝合的横断 与未经治疗相比，应用DPC膜的神经损伤将促进轴突再生和功能恢复 控制。在具体目标3中，包括对准的ECM的DPC单元片将以实心、圆柱体的形式轧制 并用于桥接大鼠面神经节段性损伤。这些管道支持的能力 将对轴突再生和改善神经功能恢复进行评估。这些研究的成功将 导致开发一种新的可行的方法来加速愈合和改善功能恢复 面部神经受损，解决了与当前护理标准相关的主要临床挑战。

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.