SUMMARY Over half the children who underwent in-utero spina bifida repair are unable to walk, and over one-fourth of them require surgery for tethered spinal cord by school age. Methods to improve long-term spinal cord function and its co-morbidities for these patients, remains elusive. The long-term goal is to find in-utero spina bifida treatment approaches that address refinement of methodologies that will promote neurological and neurodevelopmental gains later in life, thereby functionally adjusting patients’ quality of life for the better. The objective of this proposal is to use a new approach to repair spina bifida in-utero in a sheep model system, which may render more beneficial outcomes for lambs over time than current methods of repair. A cryopreserved human umbilical cord (HUC), an allograft from a healthy term pregnancy following delivery, exerts anti- inflammatory and anti-scarring properties, synonymously crucial processes that directly impact whether spinal cord tethering will occur, and hence long-term outcomes. The HUC is currently FDA approved for ophthalmological diseases. In our preclinical studies, we have shown that use of the cryopreserved human umbilical cord as a meningeal patch helps regenerate the arachnoid layer, a key barrier to spinal cord infiltrating scar forming cells, ultimately reducing histological tethering and improving upon spinal cord function. Our central hypothesis is that the HUC, when used as a meningeal patch with two-layer closure during in-utero spina bifida repair, will improve long-term clinical outcomes compared to conventional repair or predicated biomaterials. This was formulated based on our preliminary data, representing a significant advancement in spina bifida treatment given clinical sequelae improvements, with the potential to vertically advance in-utero treatment. The rationale for this study is that there is an urgent critical need for an anti-scarring and regenerative matrix that can be placed as an intermediary layer between the spinal cord and skin layer to reduce tethering. The central hypothesis will be tested by pursuing three specific aims in a surgical spina bifida sheep model system with a myelotomy: 1) To compare long-term functional outcome differences between in-utero SB repair methodologies using a HUC vs. predicate (DurepairTM) biomaterial as a meningeal patch. 2) To quantify and compare long-term spatial and temporal changes of the spinal cord at defect repair sites for tethering, syringomyelia and inflammation using 3T MRI and diffusion tensor imaging. 3) To quantitatively compare the underlying mechanisms for long-term therapeutic efficacies of SB defect repair methodologies. We will pursue these aims using an innovative combination of surgical and biological manipulative techniques, since use of the cryopreserved human umbilical cord represents a new and substantive departure from the status quo by shifting focus to the anti-inflammatory, anti-scarring and regenerative...