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.