Engineered Matrices with Electrical and Chemical Stimulation for Peripheral Nerve Repair

项目概要/摘要 周围神经损伤（PNI）影响着美国数百万人，并且具有大间隙的PNI需要手术修复。 尽管生物和合成移植物被广泛用于修复具有大间隙的PNI，但它们都可能遭受 次优临床结局。自体移植是金标准治疗，但受到可用性和缺陷的限制 修复尺寸，而合成移植物具有差的生物降解性、强度、生物活性和功能性。因此 该建议的长期目标是设计具有增强的大间隙神经再生能力的移植物。 物理和化学刺激可以增强神经再生反应，因此，将这些 工程化移植物的治疗模式可以解决目前的一些治疗局限性。电刺激（ES）可以 增强神经传导、神经营养素释放和神经挤压损伤的功能恢复，但这些 尚未确定大缺口PNI的益处。使用4-氨基吡啶（4-AP; a 钾通道阻滞剂）在其对神经元的作用方面似乎与ES相似并且可以增强挤压PNI修复， 还可以与ES协同作用。实施有效的大间隙PNI的这些物理和化学线索 修复将需要外科手术插入具有适当机械强度的导电支架， 降解、导电性和孔隙性质。该提案旨在通过新颖的， 生物可降解的离子传导（IC）壳聚糖支架和混合工程神经同种异体移植物，以修复大的， 间隙神经缺损含有4-AP的生物工程IC支架可以增加体外神经营养素的释放， 大间隙PNI在体内早期修复中的髓鞘形成。初步研究表明， 4-AP和ES降低皮下植入支架周围的纤维囊厚度， 与单独的4-AP或ES相比的体外神经营养因子表达。这表明4-AP和ES的组合改善了 功能性、生物相容性和积极的免疫反应。因此，假设IC支架 结合化学和电刺激将调节细胞-材料相互作用，以增强轴突再生 与自体移植物相当的移植率和功能恢复。这将在三个具体目标中得到检验：1）发展和 表征IC支架在4-AP释放速率、电导率和生物降解方面的变化; 2）评估人类 和大鼠雪旺细胞对体外具有4-AP和/或ES的IC支架的反应以模拟体内反应， 未来的干预措施;和3）测试工程支架和同种异体移植物与4-AP +/- ES的安全性和有效性， 严重的坐骨神经缺损生物活性电化学工程修复大间隙PNI 线索将广泛影响该领域。这些研究将弥合复杂的专家系统之间的知识差距， 介导的细胞-材料相互作用微环境和研究不足的潜在再生途径。 这些发现可能会改善神经缺损的治疗，并为再生的探索性工作提供信息。 其他肌肉骨骼组织的神经支配策略。

Project Summary/Abstract Peripheral nerve injuries (PNI) affect millions of people in the US, and PNI with large gaps require surgical repair. Although biological and synthetic grafts are widely used to repair PNI with large gaps, they both can suffer from suboptimal clinical outcomes. Autografts are the gold standard treatment but are limited by availability and defect repair size, while synthetic grafts have poor biodegradability, strength, bioactivity, and functionality. Thus, the long-term objective of this proposal is to engineer grafts with enhanced large-gap nerve regeneration capabilities. Physical and chemical stimulation can enhance nerve regeneration responses, thus, incorporating these modalities into engineered grafts may address some current treatment limitations. Electrical stimulation (ES) can enhance nerve conduction, neurotrophin release, and functional recovery of nerve crush injuries, but these benefits have not been established for large-gap PNI. Chemical stimulation using 4-aminopyridine (4-AP; a potassium channel blocker) appears similar to ES in its effects on neurons and can enhance crush PNI repair, yet may act synergistically with ES. Implementing these physical and chemical cues for effective large-gap PNI repair will require surgical insertion of an electrically conductive scaffold with appropriate mechanical strength, degradation, conductivity, and pore properties. This proposal aims to deliver 4-AP and ES via novel, biodegradable, ionically conducting (IC) chitosan scaffolds and hybrid engineered nerve allografts to repair large- gap nerve defects. Bioengineered IC scaffolds with 4-AP can increase neurotrophin release in vitro and enhance myelination of large-gap PNI in vivo in early-stage repair. Preliminary studies revealed that combined application of 4-AP and ES reduced fiber capsule thickness around subcutaneously implanted scaffolds and increased in vitro neurotrophin expression compared to 4-AP or ES alone. This suggests combining 4-AP and ES improves functionality, biocompatibility, and positive immune responses. Therefore, it was hypothesized that IC scaffolds combined with chemical and electrical cues will modulate cell-material interactions to enhance axon regeneration rate and functional recovery comparable to autografts. This will be tested in three Specific Aims: 1) Develop and characterize IC scaffolds with variations in 4-AP release rate, conductivity, and biodegradation; 2) Assess human and rat Schwann cell responses to IC scaffolds with 4-AP and/or ES in vitro to model in vivo responses and future interventions; and 3) Test safety and efficacy of engineered scaffolds and allografts with 4-AP +/- ES in a critical-sized sciatic nerve defect. Engineered repair of large-gap PNI using bioactive electrical and chemical cues will broadly impact the field. These studies will bridge the knowledge gap between the complex ES- mediated cell-material interaction microenvironment and poorly studied underlying regeneration pathways. These findings may improve the treatment of nerve defects, and inform exploratory work on regenerative strategies for innervation in other musculoskeletal tissues.