Mechanisms for Regenerative Healing in Intervertebral Discs

摘要 腰椎间盘(IVD)退行性变占背痛病例的40%。结构性IVD缺陷 辨别衰老和退化，起到疼痛和残疾的作用。有一种严重的未得到满足的需求 自腰椎间盘摘除以来改进的纤维环(AF)修复策略，这是取出的黄金标准治疗方法 房颤缺损所致髓核组织突出，房颤未修复及并发症 包括再突出和复发性退行性疼痛。而IVD细胞疗法的临床试验显示 承诺减少椎间盘源性疼痛和残疾他们不涉及优化的交付策略，也不是 由于对房颤细胞的多样性知之甚少，因此受到自然IVD修复过程的影响 人口及其在治疗中的作用。我们认为需要IVD再生愈合模型来确定 成功的房颤愈合策略和确定成功关键的细胞和微机械因素 治疗作为再生医学治疗的路线图。我们已经开发出一种成功的 小鼠再生性房颤愈合模型和新生儿静脉畸形伴严重房颤穿刺者完全愈合 成人修复纤维沉积和IVD丢失时IVD高度和生物力学特性的恢复 身高和生物力学功能。这个项目的前提是新生儿可以再生愈合，同时 骨骼成熟小鼠不是由于细胞外基质(ECM)硬度增加和配体改变所致 出现导致房颤前体细胞终末分化的现象。目标1决定了增长的影响， 成熟度和基质硬度对IVD愈合的影响，并决定再生窗何时关闭。我们 应用鼠标模型来确定房颤再生愈合窗口关闭的出生后年龄，如果 完全的房颤结构再生是可能的，如果改变ECM硬度可以延长再生 治疗窗，延长房颤细胞有丝分裂的年龄。目标2确定不同的房颤祖细胞 人口，他们成熟的损失，以及这些祖细胞在愈合中的作用。我们使用单个单元格和空间 用小鼠IVDS测序鉴定不同的房颤细胞群及其在再生小鼠中的定位 修复、再生后修复和再生修复组。AIM 3工程师设计了一种软合成 促进未成熟房颤细胞表型的底物。我们确定了小鼠和人类ECM的设计标准 和细胞，并使用官能化的聚乙二醇来控制底物硬度、配体类型和密度 底物。该项目的成果包括确定IVD再生修复窗口何时关闭和 如果完全再生是可能的；鉴定分散的房颤祖细胞群体及其在再生中的作用 治愈；以及确定促进未成熟房颤祖细胞表型和信息的关键设计因素 细胞递送策略。

Summary Intervertebral disc (IVD) degeneration contributes to ~40% of back pain cases. Structural IVD defects distinguish degeneration from aging and play a role pain and disability. There is a critical unmet need for improved annulus fibrosus (AF) repair strategies since discectomy, the gold standard treatment for removing herniated nucleus pulposus (NP) tissue from AF defects, leaves AF defects unrepaired and complications include reherniation and recurrent degeneration-related pain. While clinical trials of IVD cell therapy show promise to reduce discogenic pain and disability they do not involve optimized delivery strategies, and are not informed by natural IVD healing processes since remarkably little is known about the diversity of AF cell populations and their roles in healing. We believe an IVD regenerative healing model is required to identify successful AF healing strategies and to identify cellular and micromechanical factors critical in successful healing to serve as a roadmap for regenerative medicine treatments. We've developed a successful regenerative AF healing model in mice and show neonatal IVDs with severe AF puncture heal with complete restoration of IVD height and biomechanical properties while adults heal fibrotic deposition and loss of IVD height and biomechanical function. The premise of this project is that neonates regeneratively heal while skeletally mature mice do not due to increased extracellular matrix (ECM) stiffness and altered ligand presentation resulting in terminal differentiation of AF progenitors. Aim 1 determines effects of growth, maturation, and matrix stiffness on IVD healing and determines when the regenerative window closes. We apply mouse models to determines the postnatal age that the AF regenerative healing window closes, if complete AF structural regeneration is possible, and if altering ECM stiffness can extend the regenerative healing window and prolong the age when AF cells are in mitosis. Aim 2 identifies distinct AF progenitor populations, their loss with maturation, and roles of these progenitors in healing. We use single cell and spatial sequencing in mouse IVDs to identify distinct AF cell populations and their localization in mice of regenerative healing, post-regenerative healing, and regenerative restoration groups. Aim 3 engineers a soft-synthetic substrate that promotes immature AF cell phenotypes. We identify design criteria in mouse and human ECM and cells and control substrate stiffness, ligand type, and density using functionalized poly(ethylene glycol) substrates. Outcomes of this project include determining when the IVD regenerative repair window closes and if full regeneration is possible; identifying disperse AF progenitor populations and their roles in regenerative healing; and determining critical design factors that promote immature AF progenitor phenotypes and inform cell delivery strategies.