Development of a Vaginal Extracellular Matrix Hydrogel for Combating Genitourinary Syndrome of Menopause

Abstract

Genitourinary syndrome of menopause (GSM) is initiated by the hypoestrogenic state consequent to cessation of ovarian function and involves changes in all vaginal layers. These alterations include decreased epithelial proliferation and differentiation, impaired extracellular matrix (ECM) maintenance, pro-inflammatory immune milieu, and smooth muscle atrophy. Patient satisfaction with existing treatments for GSM is low, due in part to their lack of therapeutic effect throughout all vaginal layers and reliance on hormonal treatments. Decellularized ECM therapeutics are known to facilitate soft tissue repair in a variety of applications. Thus, a porcine vaginal tissue-derived decellularized ECM (vECM) hydrogel suitable for noninvasive topical intravaginal administration is developed specifically for vaginal repair. When applied in a rat model of surgical menopause, vECM significantly improves vaginal epithelial thickness and epithelial stem cell phenotype. Despite topical intravaginal application, vECM is also observed in the lamina propria and fibromuscularis, exerting a regenerative effect on the vaginal smooth muscle layer. Furthermore, vECM modulates macrophage density and promotes pro-regenerative M2-like phenotype within the vaginal tissue. Overall, the work herein demonstrates a new nonhormonal biomaterial treatment that counteracts pathological vaginal alterations - a hallmark of GSM - in an established preclinical model of menopause.

Keywords: acellular biomaterial; extracellular matrix; hydrogel; menopause; vaginal atrophy.

Figure 1

Production of a porcine vaginal tissue‐derived ECM hydrogel (vECM). A) Whole porcine vaginas were cleaned and frozen B) before being minced with a commercial meat grinder, C) then spun in SDS to decellularize the tissue. D) Decellularized tissue was lyophilized and E) milled to a fine and unform powder. F) Decellularized vECM powder was partially enzymatically digested with pepsin, then pH and salt balanced before incubating at 37 °C to form G) a stable hydrogel.

Figure 2

Characterization of vECM structure, composition, and material properties. Vaginal samples were taken from A) fresh tissue or tissue after either B) 1, C) 2, or D) 3 days of decellularization. Tissue samples were cryosectioned and stained with hematoxylin and eosin to demonstrate removal effective decellularization. E) Porcine vaginal tissue and decellularized vECM were analyzed via ECM targeted proteomics using QConCAT (n = 3/group). F) 6 and 8 mg mL⁻¹ vECM preparations (n = 3/group) underwent viscosity testing with a parallel plate rheometer, demonstrating shear thinning properties for both groups, and an increased viscosity in the 8 mg mL⁻¹ group at lower shear rates. G) vECM was prepared at 6 and 8 mg mL⁻¹ concentrations, and n = 3 wells per concentration underwent a turbidity assay, in which optical density (OD) was read at 405 nm for two hours or until equilibrium. H) The time to 50% equilibrium OD was determined for 6 and 8 mg mL⁻¹, demonstrating a significantly lower time for the 8 mg mL⁻¹ group.

Figure 3

Evaluation of vECM efficacy in restoring vaginal epithelium in menopausal rats. A) Experimental timeline to assess the therapeutic efficacy of intravaginal administration of vECM, alongside saline and collagen, in a rat model of surgical menopause via OVX. Vaginal tissue sections, isolated from healthy controls and OVX animals after 14 days of treatment, were stained with hematoxylin and eosin to assess vaginal epithelial thickness. Representative images are shown for B) healthy (unperturbed) controls and OVX rats treated with C) saline, D) collagen, E) 6 mg mL⁻¹ vECM, or F) 8 mg mL⁻¹ vECM. G) Data for epithelial thickness were averaged per animal and compared among groups with a one‐way ANOVA and Tukey's pairwise comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3–9/group.

Figure 4

Characterization of vaginal epithelial stem cells in vECM treated animals. Vaginal tissue sections, isolated from healthy controls and OVX animals after 14 days of treatment, were stained against nuclei (DAPI, blue), an epithelial stem cell transcription factor (p63, magenta), and an epithelial membrane marker (filaggrin, green) to identify epithelial stem cells. Representative immunofluorescent images are shown for A) healthy (unperturbed) controls and OVX rats treated with B) saline, C) collagen, or D) 8 mg mL⁻¹ vECM. Vaginal epithelium was assessed for E) nuclei density, F) p63⁺ vaginal epithelial stem cell nuclei density, and G) percentage of nuclei within the epithelium that were p63⁺ vaginal epithelial stem cells. Data were compared among groups with a one‐way ANOVA and Tukey's pairwise comparisons **P < 0.1; ***P < 0.001; ****P < 0.0001, n = 3–9/group.

Figure 5

Evaluation of vECM efficacy in restoring vaginal smooth muscle in menopausal rats. Vaginal tissue sections, isolated from healthy controls and OVX animals after 14 days of treatment, were stained against nuclei (DAPI, blue) and smooth muscle (ɑSMA, yellow). Representative immunofluorescent images are shown for A) healthy (unperturbed) controls and OVX rats treated with B) saline, C) collagen, D) 6 mg mL⁻¹ vECM, or E) 8 mg mL⁻¹ vECM. F) Data for smooth muscle thickness were averaged per animal and compared among groups with a one‐way ANOVA and Tukey's pairwise comparisons. **P < 0.01, n = 3–9/group.

Figure 6

Assessment of vaginal smooth muscle cell proliferation following treatment. Vaginal tissue sections, isolated from healthy controls and OVX animals after 14 days of treatment, were stained against nuclei (DAPI, blue), smooth muscle actin (ɑSMA, green), and a nuclear marker of proliferating cells (Ki67, cyan). Representative immunofluorescent images are shown for A) healthy (unperturbed) controls and OVX rats treated with B) saline, C) collagen, or D) 8 mg mL⁻¹ vECM. Groups were assessed for E) Ki67+ nuclei density within the smooth muscle layer and F) proportion of Ki67+ nuclei over total nuclei within the smooth muscle layer. Data were averaged per animal and compared among groups with a one‐way ANOVA and Tukey's pairwise comparisons (F). *P < 0.05; ****P < 0.0001, n = 3–9/group.

Figure 7

vECM at two doses and collagen control demonstrate similar retention in the vaginal lumen and fibromuscularis. Two weeks following OVX, rats were treated with a single 500 µL intravaginal topical administration of fluorescently pre‐labeled A) collagen, B) 6 mg mL⁻¹ vECM, or C) 8 mg mL⁻¹ vECM. Tissue sections were harvested at 1, 2, or 3 days post‐administration (n = 2/group/timepoint). Representative tissue sections are shown for each group, in which fluorescently tagged biomaterial is shown in cyan, and tissues were stained against cell nuclei (DAPI, blue). D) Tissue sections were assessed for material retention by assessing the average area of material throughout the tissue. E) Additionally, material from all groups demonstrated material in the vaginal fibromuscularis; the frequency of this observation was reported as a percentage of sections. F) Sections were stained against macrophage marker (CD68, red), which was observed colocalized with fluorescently pre‐labeled materials in both the vaginal lumen and fibromuscularis in all groups.

Figure 8

Assessment of macrophage infiltration in vaginal tissues after various treatments. Vaginal tissue sections, isolated from healthy controls and OVX animals after 14 days of treatment, were stained against nuclei (DAPI, blue), a pan‐macrophage membrane marker (CD68, red), and an M2 macrophage membrane marker (CD163, green). Representative immunofluorescent images are shown for A) healthy (unperturbed) controls and OVX rats treated with B) saline, C) collagen, or D) vECM 8 mg mL⁻¹. Groups were assessed for E) nuclei density within the lamina propria and muscularis, F) proportion of CD68+ nuclei, indicating macrophages, and G) proportion of CD163+ nuclei, indicating anti‐inflammatory M2‐like macrophages. Data were averaged per animal and compared among groups with a one‐way ANOVA and Tukey's pairwise comparisons. *P < 0.05; ***P < 0.001; ****P < 0.0001, n = 3–9/group.