Composite elastin nanofiber-gelatin methacryloyl hydrogels for vaginal tissue engineering

Introduction::
Childbirth injury affects a significant portion of women. Over 85% of women who have a vaginal birth sustain vaginal tearing due to perineal trauma^1,2. Perineal and pelvic floor trauma can lead to numerous negative long term health effects, including pelvic organ prolapse, urinary incontinence, chronic pain, sexual discomfort, and chronic pelvic floor dysfunction^1,2. Despite the prevalence of these injuries, prevention methods remain limited and controversial because little is known regarding the mechanistic causes of perineal trauma.



A benefit of in vitro models is that direct mechanistic effects can be explored in highly tunable, high-throughput systems. Few models of the vagina exist, and current models cannot be used to study biomechanical properties of the tissue. Here, we sought to develop a tissue engineered vagina with benchmarks designed to recapitulate the vaginal microenvironment, including tissue architecture (tubular), matrix composition (collagen, elastin), biomechanical properties (viscoelastic, anisotropic), and cellular constituents (vaginal cells). The vagina is a fibromuscular tube-shaped organ spanning from the hymenal ring to the cervix and plays key roles in reproduction. The vagina is primarily composed of collagen and elastin fibers and smooth muscle^1,2. Collagen is the most abundant protein in the vaginal extracellular matrix (ECM) and allows for high tensile strength in the tissue. Elastin allows the vagina to retain its shape and withstand large deformations. As such, we developed composite biomaterials consisting of gelatin (denatured collagen) and elastin that recapitulate these key vaginal features.




Materials and Methods::
Gelatin methacryloyl (GelMA) of approximately 50% degree of modification was synthesized as described previously using the one pot method³. Lyophilized GelMA and polymerized GelMA hydrogel properties were quantified via ¹H-NMR (Bruker, Avance III HD 700 MHz), ATR-FTIR (Bruker, Alpha II), and nanoindentation (Optics11Life). Gelatin and elastin fibers were electrospun in acetic acid using the following parameters: 10 kV of applied voltage differential, a spinneret width of 50 mm and speed of 12 mm/s⁴. Electrospun fibers were characterized with ESEM imaging (Thermofisher Quattro S ESEM), ATR-FTIR (Bruker, Alpha II), fiber diameter analysis (ImageJ; n=10 fiber measurements), and tear testing (ElectroForce). Composite hydrogels were created by flooding electrospun fibers with GelMA (5 wt%) solution and polymerizing the composite hydrogels under UV light. Composite hydrogel mechanical properties were quantified via nanoindentation (OpticsIILife, Piuma). Vaginal epithelial cells (LifeLine Cell Technology; FC-0083) were cultured as per the manufacturer’s instructions.




Results, Conclusions, and Discussions::
Here, we developed gelatin-elastin electrospun fibers of varying ratios (Fig. 1). From ESEM imaging, we determined that the best ratio was 2:1 gelatin:elastin (Fig. 1A). Increasing the amount of gelatin compared to elastin prevented beading and formed fibers 170.1 ± 51.7 nm in diameter (n=10 fibers). ATR-FTIR (Bruker, Alpha II) spectra analysis demonstrated incorporation of elastin and gelatin into the fibers (Fig. 1B). We observed characteristic elastin peaks around 1600 cm^-1 associated with amide I C=O stretching vibrations,1500 cm^-1 associated amide II N-C=O stretching vibrations, and 1200 cm^-1 associated with amide III^5,6,7. We also observed characteristic gelatin peaks around 1600 cm^-1 associated with amide I C=O stretching, 1500 cm^-1 associated with N-H bending coupled to C-H stretching, and 1200 cm^-1 associated with amide III C-N stretching and N-H bending⁸. We observed differences in four peaks (highlighted; Fig. 1B). We then created hydrogel-fiber composites by flooding fibers with gelatin methacryloyl prepolymer solution and crosslinking composites under UV light. Finally, we cultured vaginal epithelial cells (encapsulated and on top) in our composites and assessed cell morphology and biocompatibility.



We created novel hydrogel-fiber composites containing gelatin and elastin fibers within gelatin methacryloyl hydrogels. We defined the material properties of these composites and describe their stiffness, viscoelastic properties, porosity, and biocompatibility. These composite nanofiber-hydrogel materials are conducive to vaginal tissue engineering but can also be utilized for other tissue systems, such as the cardiovascular and musculoskeletal systems, skin, and liver.




Acknowledgements (Optional): :
This research was funded by T32 Clinical Outcomes Research Training Program in Female Lower Urinary Tract Disorders NIH T32DK120497-01A1 (SGZ).




References (Optional): :
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