J. R. Bardill1,2, D. Park1, A. I. Marwan1,2 1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2University of Colorado Denver Anschutz Medical Campus,Pediatric Surgery,Aurora, CO, USA
Introduction:
Myelomeningoceles (MMC) are one of the most common congenital birth defects of the spinal cord. Prenatal repair of these defects became the standard of care, however, open approaches present significant risks to mother and fetus. Our laboratory has developed a novel Reverse Thermal Gel (RTG) to deliver a minimally invasive patch for MMC coverage. Preliminary studies demonstrated long-term in-vitro gel stability and minimally invasive application in a mouse MMC model with partial defect coverage.
In preparation to transition to the ovine model, an RTG patch with surface adhesion in the amniotic environment and cellular scaffolding properties to integrate with host will be needed. Here, we demonstrate the chemical synthesis and characterization of a bioadhesive RTG, in-vitro cellular scaffolding properties, and the biological applicability in a mouse MMC defect model.
Methods:
A. Synthesis of Bioadhesive RTG: Dopamine was conjugated to the RTG polymer backbone and verified using proton nuclear magnetic resonance (H NMR) spectroscopy and infrared (IR) spectroscopy. Mechanical properties were tested using rheology. B. In-vitro RTG cellular scaffold: Mouse skin fibroblasts and human epidermal keratinocytes (HEKA) were mixed (separately) with RTG-media solution, followed by gelling at 37°C to encapsulate the cells within RTG. Fibroblasts were stained for vimentin and alpha smooth muscle actin (α-SMA), and HEKAs were stained with keratin 14 (k14) antibodies. C. Adhesive RTG injection into mouse MMC defect embryos: In-vivo applicability was tested in Grhl3 mice to assess MMC defect coverage by the adhesive RTG. D. Inflammatory response: Defect tissue was immunostained for CD68 and F4/80 macrophages.
Results:
H NMR and IR verified aromatic and hydroxyl peaks from dopamine are present in the polymer backbone. The elastic strength of the adhesive RTG increased 500-1000 Pascal’s compared to the regular RTG. Dermal fibroblasts expressed vimentin and α-SMA markers. HEKAs in RTG culture expressed k14 markers. The adhesive RTG was applied to mouse MMC defects by needle injection and improved defect coverage, with an average of 72% defect coverage at harvest. CD68 and F4/80 macrophage positive cells were not significantly different when comparing adhesive RTG and saline injections in defect embryos.
Conclusion:
We synthesized and characterized a bioadhesive RTG with increased elastic strength. This adhesive RTG has in-vitro fibroblast and keratinocyte cellular scaffolding properties cultured in an RTG matrix. Finally, the adhesive RTG can be injected in a minimally invasive manner to improve mouse MMC defect coverage compared to the non-adhesive RTG with minimal macrophage response. Overall, this work has shown the adhesive RTG is a promising candidate for future application in the ovine model.