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Womens Health

Undergraduate Lightning Talks

6 - Concentration Dependent Architecture and Biocompatibility of Vaginal Extracellular Matrix Hydrogels for Biomimetic Pelvic Organ Prolapse Repair

Saturday, October 26, 2024
1:30 PM - 1:45 PM EST
Location: Room 341

Introduction:  Pelvic organ prolapse (POP) occurs when pelvic organs descend into or outside the vagina due to weakened pelvic floor muscles and connective tissues, resulting in pelvic and back pain, incontinence, and psychological distress [1]. Approximately 50% of women over 50, particularly parous postmenopausal women, experience some degree of POP. Polypropylene (PP) mesh has been widely used for POP repair despite its association with complications, leading to life-long sequelae [2-5]. Complications have been linked to fibroblast activation and abnormal myofibroblast proliferation from the release of matrix proteins, which subsequently induce fibroblast contraction, in part due to the high material (polymer) stiffness of PP [6-8].
To facilitate healthy integration of mesh with host cells and tissue, we developed a tunable porcine vaginal extracellular matrix (vECM) hydrogel coating for PP mesh [9, 10]. We then performed a preliminary evaluation of hydrogel microstructural properties and examined in vitro biocompatibility of primary human vaginal fibroblasts within these hydrogels. Our objective was to characterize the concentration-dependent aspects of hydrogel fiber microstructure and further assess biocompatibility of vECM-based hydrogels for biomimetic POP repair, particularly regarding vaginal fibroblast response to gels of different vECM protein concentrations. We hypothesized that increased gel protein concentration would result in a more compact ECM microsctructure, and increased fibroblast to myofibroblast transformation. Additionally, we hypothesized that coating PP mesh with vECM hydrogel would not significantly change hydrogel fiber microstructure.

Materials and

Methods:  vECM hydrogels (N=30) were prepared from lyophilized decellularized porcine vagina via digestion in 0.01N HCl with 1000U/mL pepsin for 48h, followed by lyophilization and rehydration with water, 0.1N NaOH, 10X PBS, and media (DMEM; 10% FBS; 1.5% Amphotericin B, 1% penicillin/streptomycin) to a final protein concentration of either 5 (N=9) or 10mg/mL (N=9). Restorelle (Coloplast) polypropylene mesh was cut into 2x2 cm squares and placed in PDMS molds containing 1mL liquid 5 or 10 mg/mL acellular hydrogel which was then gelled at 37C to form gel-coated mesh constructs (5 mg/mL, N=2; 10 mg/mL, N=2).
Before scanning electron microscopy (SEM), all samples (5 mg/mL, N=15; 10 mg/mL, N=15) were fixed in 2.5% glutaraldehyde and washed with 1X PBS followed by a dehydrating graded ethanol wash. After critical point drying, samples were sputter-coated with a 5nm gold-palladium alloy. Each sample was imaged via JEOL JSM 6335F at 3.0 kV across three random regions at 1,000x, 7,500x, and 20,000x. Image analysis was conducted via custom MATLAB 2023a code to assess fiber porosity, average fiber diameter (AFD), and connectivity. Statistical analysis was performed using Prism 10 via a paired t-test for each condition.
vECM hydrogels were imaged via brightfield microscope to measure shrinkage of both cellular and acellular 5 mg/mL (N=18) and 10 mg/mL (N=18) gels. Image analysis was conducted using QuPath 0.5.1 with an ImageJ plugin, with outputs of Feret’s diameter, area, and perimeter. Statistical analysis was performed via Prism 10 via one-way repeated measures ANOVA followed by Tukey post-hoc test.

Results, Conclusions, and Discussions: We observed via SEM a significant increase in fiber connectivity and a decrease in AFD in the 10 mg/mL hydrogels (p < 0.001) versus 5 mg/mL. There was no statistically significant difference in porosity based on concentration (Figure 1). SEM imaging of the day 7 cellular gels showed AFD decreased in 5mg/mL gels when fibroblasts were introduced (p < 0.001) but increased in the 10mg/mL gels with the introduction of fibroblasts (p < 0.001). In all cases, fiber connectivity had an inverse correlation with AFD. Over 14 days, the 10mg/mL cellular gels showed no statistical difference in porosity, AFD, or fiber connectivity. There was also no significant difference in AFD based on fibroblast seeding.
Qualitatively, 3D seeding of fibroblasts resulted in an increase of connective protein fibers, which appeared as web-like structures and longitudinal fibrous formations on the SEM (Figure 2). Fibroblast contraction and myofibroblast proliferation can be linked to vECM-fibroblast interactions. These observed interactions induce fibroblast contraction, similar to bare mesh responses.
Hydrogels with embedded mesh showed little to no difference in their structural orientations. Additionally, both gel concentrations showed no statistical difference in porosity, AFD, or fiber connectivity when the mesh was embedded (Figure 3).
Macroscopically, the acellular hydrogels showed no statistically significant shrinkage during the 14-day period, as hypothesized (Figure 4). All cellular hydrogels shrunk exponentially (76.1% area loss) over the same period (average R²=0.8962).
Overall, we found that higher gel protein concentrations decreased the AFD but increased the fiber connectivity, with no effects on the porosity or shrinkage. Additionally, fibroblast seeding resulted in gel contraction, modified AFD and fiber connectivity, and additional longitudinal fibrous structures within the hydrogels. PP mesh with vECM hydrogel did not significantly alter hydrogel microstructure. Finally, there was no significant loss of structure in terms of porosity, fiber diameter, or fiber connectivity; instead, these values increased likely due to modification by cells. In the future, we aim to test the hydrogel-mesh constructs in rabbit models to assess the cell and tissue response.

Acknowledgements (Optional):  

References:
1. Srikrishna, S., et al., Experiences and expectations of women with urogenital prolapse: a quantitative and qualitative exploration. BJOG: An International Journal of Obstetrics & Gynaecology, 2008. 115(11): p. 1362-1368.
2. Wu, J.M., et al., Prevalence and Trends of Symptomatic Pelvic Floor Disorders in U.S. Women. Obstetrics & Gynecology, 2014. 123(1).
3. MacDonald, S., et al., Complications of Transvaginal Mesh for Pelvic Organ Prolapse and Stress Urinary Incontinence: Tips for Prevention, Recognition, and Management. Eur Urol Focus, 2016. 2(3): p. 260-267.
4. Nygaard, I., et al., Long-term outcomes following abdominal sacrocolpopexy for pelvic organ prolapse. Jama, 2013. 309(19): p. 2016-24.
5. Bueno Garcia Reyes, P. and H. Hashim, Mesh complications: best practice in diagnosis and treatment. Ther Adv Urol, 2020. 12: p. 1756287220942993.
6. Knight, K.M., et al., Mesh deformation: A mechanism underlying polypropylene prolapse mesh complications in vivo. Acta Biomaterialia, 2022. 148: p. 323-335.
7. Phogat, S., et al., 3D in vitro hydrogel models to study the human lung extracellular matrix and fibroblast function. Respiratory Research, 2023. 24(1): p. 242.
8. Marinković, A., et al., Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2012. 303(3): p. L169-L180.
9. Brown, B.N. and S.F. Badylak, Extracellular matrix as an inductive scaffold for functional tissue reconstruction. Translational Research, 2014. 163(4): p. 268-285.
10. Spang, M.T. and K.L. Christman, Extracellular matrix hydrogel therapies: In vivo applications and development. Acta Biomaterialia, 2018. 68: p. 1-14.