Best Paper by a Resident or Student Award: Vaginal Extracellular Matrix-derived Hydrogel: The Future of Vaginoplasty

Objectives: Genitoplasty improves quality of life of many transgender individuals, but clinically viable strategies for generating genital tissue de novo for genitoplasty do not exist. We generated and characterized decellularized vaginal extracellular matrix (vECM) and vECM hydrogels and cultured primary neonatal human foreskin (HFF) or adult vaginal fibroblasts (HVF) on these gels in estrogenized conditions to test viability and behavior as an augment to vaginoplasty.

Methods: Porcine vECM hydrogels were created at 5 mg/mL (Figure 1A). dsDNA (PicoGreen) and sGAGs (DMMB) were quantified in tissue, vECM, and gels. Gel protein content was confirmed via BCA. Gelation at 37°C was confirmed via rheometer. HFFs or HVFs were seeded on gels in phenol red free DMEM containing 14% FBS and allowed to adhere overnight. 200 or 400pg/mL 17-B-estradiol (E2) containing media was then added in the presence (FBS) and absence (LAH) of serum for 4 days with gel diameter measured at day 4 (N=4/line/condition). t-tests and one-way ANOVA/Bonferroni post-hoc were performed. 14mm gels seeded with HFFs or HVFs were cultured in DMEM/FBS for 1 or 7 days and stained via H&E and picrosirius red. Growth of HFFs and HVFs cultured under identical media conditions without hydrogels (N=3/line) were compared. Cell viability was assessed via ethidium homodimer assay. Mann-Whitney and Kruskall-Wallis/Dunn were used.

Results: 69 hydrogels were generated from 138mg lyophilized vECM. dsDNA was reduced by 75.8% in vECM and 100% in gel. sGAGs were 1.6+/-0.4% (tissue), 1.4+/-0.4% (vECM), and 1.8+/-0.6% (gel) dry weight. Protein content was 3.4+/-0.8mg/mL. Gelation at 37°C was distinguished by sustained rise in elastic modulus followed by plateau at 86.0+/-1.6Pa. Significant differences in diameter at day 4 are displayed in Figure 2A-C. H&E and picrosirius red images of gels at day 7 showed thickened and contracted gel fibers at the site of cell seeding (Figure 1B). HFFs not cultured on gels grew slower than HVFs regardless of E2 presence and no significant differences in viability were observed.

Conclusions: vECM and hydrogels retained native sGAG content and gels retained protein content after gelation, ideal for a vaginal biomimetic material. No dsDNA was detected in gels. Our samples exhibited gelation and mechanical behavior similar to literature values for other ECM-derived hydrogels. Gel shrinkage was used as a measure of cellular activity and proliferation within the gel matrix. Serum supplementation enhanced gel shrinkage regardless of cell line. 400pg/mL E2 supplementation eliminated significant differences in gel shrinkage between lines regardless of serum supplementation. Contraction of gels seeded with HFFs was significantly higher in the presence of E2, an effect not seen with HVFs. This represents a promising material for enhancing vaginoplasty. Additional work is ongoing to verify retention of other matrix components, quantify fibroblast ingrowth into gels, and culture primary adult penile dermal fibroblasts using this model.