PROJECT SUMMARY Low back pain is the second most common cause of doctor visits and intervertebral disc (IVD) herniation is a direct cause of pain. Lumbar discectomy is the standard of care for herniation, yet this very common procedure has 5-25% complication rates including re-herniation and recurrent back pain at the same level. Discectomy complications cannot be further reduced by optimizing the amount of tissue removed during procedures, but instead discectomies require a reparative component to greatly reduce complications. This project develops, optimizes and validates biomaterials that seal the annulus fibrosus and restore nucleus pulposus swelling following discectomy procedures. The first funding period of this grant resulted in 34 papers that developed human and bovine organ culture models of IVD degeneration and determined that methods to repair large IVD defects are lacking and a critical scientific barrier that must be addressed to limit degeneration following IVD herniation and injury. We also developed novel hydrogels with promise to seal the annulus fibrosus, restore nucleus pulposus swelling, and return IVD biomechanical behaviors to the healthy state. Additional acellular biomaterial optimization, modification of biomaterials for use as cell carriers, and pre-clinical evaluations are required before clinical translation. Aim 1 optimizes in situ performance of acellular biomaterials for IVD repair with biomaterial refinements and TGFβ3 dose studies using bovine organ culture discectomy models. Aim 2 optimizes in situ performance of biomaterials for mesenchymal stem cell delivery using these same models. Aim 3 validates these IVD repair strategies in pre-clinical human organ culture and sheep in vivo studies. We apply a genipin- crosslinked fibrin hydrogel capable of sealing annulus fibrosus defects without risk of herniation under rigorous biomechanical loading. We also apply a novel carboxymethylcellulose/methylcellulose hydrogel formulation capable of restoring nucleus pulposus swelling and returning IVD biomechanical behaviors to intact conditions. The investigative team closely collaborates with extensive biomaterials, biomechanics, tissue engineering, and spine surgery expertise. All methods are well-established in the labs of this team. This project is highly significant because of the tremendous health burden of IVD herniation and injury, because discectomy procedures are among the most common spine surgery procedures, and because this project has a clear translational trajectory. This project is innovative because it uses novel biomaterial formulations and approaches for discectomy repair that are capable of transforming current surgical interventions and thinking since no IVD repair strategies exist. The approach is robust because it addresses fundamental questions for IVD repair in a systematic manner that allows iterative optimization with evaluation tests that increasingly challenge the repair strategies.