(M-486) Developing a Biomimetic Decidua-Like Composite Hydrogel to Study Pre-Eclampsia

Introduction:: Pre-eclampsia is a life-threatening disease that is diagnosed with the rapid onset of hypertension following the 20th week of pregnancy.¹ According to the World Health Organization, between 2% and 10% of pregnancies develop pre-eclampsia worldwide.² While it is known that phenomena such as improper placentation and abnormal spiral artery remodeling play a role in the development of pre-eclampsia, the exact mechanism remains unknown.^1,3 Due to the physiological distinctiveness of placental dynamics in humans, common animal models are used sparingly in placental-related research.⁴ This presents a need for an in vitro platform to study pregnancy. Previously, homogenous gelatin methacryloyl (GelMA) hydrogels have been utilized to study the fetal and maternal mechanisms of pregnancy through 3D tissue culture and have been shown to replicate the stiffness of the endometrium and decidua.⁵ To add complexity to this model system, electrospun fibers were incorporated into the hydrogels to form fiber-reinforced hydrogel composites to augment stiffness, strength, and time-dependent properties such as hydraulic permeability. Based on the composition of the decidua, which is made up of collagen, hyaluronan, and some elastin, gelatin (denatured collagen) was used to better emulate the extracellular matrix within the uterus during pregnancy. The purpose of this project was to develop and characterize a biomimetic fiber-reinforced hydrogel composite to mimic the decidua. This material will be used as a 3D platform to investigate the role of vascularization during pregnancy in relation to preeclampsia.

Materials and Methods:: Crosslinked 12% w/v gelatin nanofibers were fabricated via electrospinning under the following conditions: 10 kV of applied voltage differential, a spinneret width of 50 mm, and a speed of 12 mm/s4 . Citric acid was used as a non-toxic method to synthesize water-stable gelatin-based nanofibers by reacting the citric acid chains with the peptide chains of gelatin through condensation. Electrospun fibers were characterized with ESEM imaging (Thermofisher Quattro ESEM) and the diameters of fibers were quantified using ImageJ. For visualization in the hydrogels, the nanofibers were fluorescently marked with 4 kDa dextran fluorescein at a concentration of 1 mg/mL and visualized with fluorescence microscopy (Keyence BZ-X810) and single photon microscopy (Leica Sp8 Lightning Single Photon). Approximately 50% degree of functionalization GelMA was synthesized utilizing the one-pot method. Nanofibers were incorporated into a 10 wt% GelMA hydrogel and the UV crosslinking time was set to between 1 – 5 minutes. Swan71 (Applied Biological Materials Inc. T0532) trophoblast cells were cultured according to company instructions and were embedded within the material. Their response was observed over a period of 3 days and live/dead staining was conducted.

Results, Conclusions, and Discussions:: Gelatin-based composite hydrogels with nanofibers were successfully fabricated and characterized to mimic the decidua. The diameter of the nanofibers was successfully quantified to be around 100 – 200 nm with ESEM. This is within the desired range of collagen fibers typically found in the extracellular matrix which is roughly 30 – 100 nm in diameter. Within the composites, dextran fluorescein fibers were visualized via confocal imaging which demonstrated the hydrogel fully permeated through the fibers. Next, the cellular viability of dissociated Swan71 trophoblast cells and Swan71 trophoblast spheroids in hydrogel-fiber composites was assessed and their viability was confirmed via live/dead staining.

Integration of gelatin nanofibers into a GelMA hydrogel shows potential to better enhance the mechanical properties of the material and to better mimic the extracellular matrix of the decidua. Viable methods were determined to examine the nanofiber network within the hydrogel matrix and mechanically characterize them. Fluorescence recovery after photobleaching (FRAP) experiments, to determine the permeability of the material, are ongoing. Other methods to mechanically test the composite material are currently ongoing, including nanoindentation, tear testing, and mass-swelling studies to evaluate the local stiffness, permeability, time-dependent properties, tensile strength, and crosslinking density of the composite material. Future directions would involve incorporating additional components found in the ECM of the decidua such as elastin or hyaluronic acid to further enhance the composite material. After further characterization, the material will be used as a 3D cell culture model to investigate the cellular mechanisms behind pre-eclampsia.

Acknowledgements (Optional): :

This material is based upon work supported by the American Heart Association under Grant No. GR0023814. The Washington University in St. Louis Biology Imaging Facility for their assistance and use of their Leica Sp8 Lightning Single Photon microscope.

References (Optional): :

 1           Phipps, E. A., Thadhani, R., Benzing, T. & Karumanchi, S. A. Pre-eclampsia: pathogenesis, novel diagnostics and therapies. Nature Reviews Nephrology 15, 275-289, doi:10.1038/s41581-019-0119-6 (2019).

2          Khan, B., Allah Yar, R., Khakwani, A. K., Karim, S. & Arslan Ali, H. Preeclampsia Incidence and Its Maternal and Neonatal Outcomes With Associated Risk Factors.

3          Brosens, I., Pijnenborg R Fau - Vercruysse, L., Vercruysse L Fau - Romero, R. & Romero, R. The "Great Obstetrical Syndromes" are associated with disorders of deep placentation.

4          Fogg, K. et al. Roadmap on biomaterials for women’s health. Journal of Physics: Materials 6, 012501, doi:10.1088/2515-7639/ac90ee (2023).

5          Zambuto, S. G., Clancy, K. B. H. & Harley, B. A. C. A gelatin hydrogel to study endometrial angiogenesis and trophoblast invasion. Interface Focus 9, 20190016, doi:10.1098/rsfs.2019.0016 (2019).