Mechanisms for Regenerative Healing in Intervertebral Discs

总结 椎间盘（IVD）退变导致约40%的背痛病例。结构性IVD缺陷 区分退化和老化，并发挥作用的疼痛和残疾。有一个关键的未满足的需要， 自椎间盘切除术以来，改善了纤维环（AF）修复策略， 从AF缺损中脱出的髓核（NP）组织，留下AF缺损未修复和并发症 包括再疝和复发性变性相关疼痛。虽然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，它们不涉及优化的输送策略， 由于对AF细胞的多样性知之甚少， 人口及其在治疗中的作用。我们认为需要一个IVD再生愈合模型来识别 成功的AF愈合策略，并确定成功治疗AF的关键细胞和微机械因素， 作为再生医学治疗的路线图。我们开发了一种成功的 在小鼠再生性AF愈合模型中，显示具有严重AF穿刺的新生儿IVD完全愈合 恢复IVD高度和生物力学特性，而成人愈合纤维化沉积和IVD丢失 身高和生物力学功能。该项目的前提是新生儿再生愈合， 由于细胞外基质（ECM）硬度增加和配体改变， 这导致AF祖细胞的终末分化。目标1确定增长的影响， 成熟和基质硬度对IVD愈合的影响，并决定再生窗口何时关闭。我们 应用小鼠模型确定AF再生愈合窗口关闭的出生后年龄，如果 完整的AF结构再生是可能的，如果改变ECM刚度可以延长再生时间， 延长AF细胞有丝分裂时的愈合窗口和年龄。Aim 2识别不同的AF祖细胞 人口，他们的损失与成熟，以及这些祖细胞在愈合的作用。我们使用单细胞和空间 在小鼠IVD中进行测序以鉴定不同的AF细胞群及其在再生性AF小鼠中的定位。 愈合、再生后愈合和再生恢复组。Aim 3设计了一种软合成材料 促进未成熟AF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）控制基底刚度、配体类型和密度 印刷受体.该项目的结果包括确定IVD再生修复窗口何时关闭， 如果完全再生是可能的;鉴定分散的AF祖细胞群体及其在再生中的作用 愈合;以及确定促进不成熟AF祖细胞表型并告知 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.