J. R. Bardill1, D. Park1, U. Shabeka3, A. I. Marwan1,2,3 3University Of Colorado Denver,Laboratory For Fetal Regenerative Biology,Aurora, CO, USA 1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2Children’s Hospital Colorado,Pediatric Surgery,Aurora, CO, USA
Introduction: Myelomeningocele (MMC) results in lifelong neurological and functional deficits. In-utero repair closes the defect, resulting in a 50% reduction in post-natal ventriculoperitoneal shunting. However, it is associated with maternal and fetal risks. Novel fetoscopic approaches using 2-3 ports, pre-fabricated patches, and skin closure demonstrate promising results, however, they are performed late and have a prolonged operative time. Our novel, Reverse Thermal Gel (RTG) has posed a unique solution for an early, minimally invasive delivery of a patch. So far, we have shown fast in-situ gelling properties and RTG stability after long term exposure in amniotic fluid. Moreover, RTG is non-toxic to cells and embryos in culture. Here, we hypothesize that RTG can serve as an in-vitro 3D cellular scaffold for mouse skin fibroblasts and will be successfully injected and localized to the MMC defect of mouse embryos with minimal inflammatory response.
Methods: 1. In-vitro 3D-RTG cellular scaffold: Mouse skin fibroblasts were mixed with RTG-media solution, followed by gelling at 37°C to encapsulate the cells within the RTG. The fibroblasts were cultured for 3 days and then viability was assessed using a live/dead stain and confocal microscopy. 2. RTG injection into mouse MMC defect embryos: Grainyhead like-3 (Grhl3) mice express MMC phenotypes. Pregnant E16.5 Grhl3 mice underwent laparotomy to expose uterine horns. A RTG solution or saline was applied onto the MMC defect by transuterine injection. On E19, the embryos were harvested to assess RTG coverage of the MMC defect. 3. Inflammatory response to RTG injection: Defect tissue was stained for CD68 and F4/80 macrophage antibodies and imaged on a fluorescent microscope.
Results: Fibroblast 3D-RTG culture live/dead staining reveals 90-95% live cells after 3 days. The RTG successfully applies to mouse MMC defects and forms a stable gel covering the defect. A total of 6 MMC defect embryos are recovered at E19 harvest: 3 defect embryos demonstrate more than 50% coverage of the defect, while the other 3 show less than 50% coverage. CD68 and F4/80 macrophage response demonstrates little to no difference in inflammation comparing RTG and saline injections in defect embryos.
Conclusion: Using a mouse model, we demonstrate that by simple needle injection, a RTG can cover a mouse MMC defect and remain at the site of the defect until harvest with minimal inflammatory response. However, RTG defect coverage decreases over time. Moreover, the RTG has in-vitro fibroblast scaffolding properties, which could allow for native cellular integration into the patch and/or allow cellular treatments to be incorporated into the patch. Based on our results, we are currently modifying the RTG to enhance the adhesive properties. Overall, our work demonstrates that a RTG is a promising candidate for a minimally invasive approach to patch MMC and will be conducive to transitioning to a fetoscopic approach in a sheep model.