Improved Coverage of Mouse Myelomeningocele With a Mussel Inspired Reverse Thermal Gel

Abstract

BackgroundMyelomeningocele (MMC) is an open neural tube defect of the spinal column. Our laboratory previously introduced a reverse thermal gel (RTG) as the first in situ forming patch for in utero MMC application. To overcome the challenges of anchoring the RTG in the wet amniotic environment to improve MMC coverage, we modified the RTG to mimic the underwater adhesive properties of mussels. We have separated this study into three separate hypotheses-based components:1Based on mussel inspired chemistry, modification of the RTG with dopamine will increase the underwater adhesive properties to improve RTG anchoring ability in a wet environment. Methods: The dopamine-modified RTG (DRTG) was synthesized using carbonyldiimidazole chemistry and characterized with proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet-visible spectroscopy. Rheology and underwater adhesive tests measured mechanical properties. Results: DRTG synthesis was confirmed with proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet-visible spectroscopy. Rheology demonstrated increased elasticity. Underwater adhesion testing revealed DRTG has similar wet adhesive strength to Tisseel fibrin sealant.2The DRTG will support in vitro skin cell growth and will be safe for injection in a mouse animal model. Methods: Biocompatibility testing included 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DRTG-fibroblast and keratinocyte cultures, and subcutaneous injections to quantify macrophages stained with immunohistochemistry. Results: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and DRTG cell cultures revealed no cytotoxicity and demonstrated the growth of fibroblasts and keratinocytes. Subcutaneous injections cause a macrophage response that decreases after 4 wk.3In utero injection of novel DRTG into a mouse MMC model will be effective with improved patch coverage of the MMC defect. Methods: MMC coverage by DRTG was assessed using in utero mouse injections in the Grainy head-like 3 (Grhl3) mouse model. Results: In utero Grhl3 mouse injections demonstrate statistically significant (P = 0.012) improved MMC coverage of DRTG compared with previous RTG coverage with no significant macrophage response. ConclusionsThe DRTG demonstrates increased elasticity, cellular scaffolding properties, and improved MMC coverage in the Grhl3 mouse model. Future studies will be translated to the preclinical ovine model to evaluate this novel gel.