BMSCs-EVs Alleviate Pelvic Floor Dysfunction in Mice by Reducing Inflammation and Promoting Tissue Regeneration

Abstract

Background/Aim

Pelvic floor dysfunctions (PFDs), which encompass pelvic organ prolapse (POP), stress urinary incontinence (SUI), and anal incontinence (AI), are common degenerative diseases in women. Bone marrow mesenchymal stem cells (BMSCs) hold promise for the treatment of PFDs. Extracellular vesicles (EVs) derived from BMSCs, have displayed an extensive role in intercellular communication and tissue repair. However, efficacy of the treatment using EVs originated from BMSCs on mouse models of PFD remains unknown. This study investigated the therapeutic potential of BMSC-derived EVs in a female PFD mouse model induced by vaginal distension (VD).

Materials and Methods/Results

Flow cytometry analysis confirmed the positive expression of BMSC-related markers, and successful induction of multilineage differentiation further validated their characteristics. As expected, the EVs extracted from BMSCs exhibited typical cup-shaped and circular-shaped structures. In the PFD model, BMSC-derived EVs significantly reduced the levels of inflammatory cytokines (p<0.05), improved tissue repair, and mitigated neutrophil infiltration. Furthermore, EVs promoted cell proliferation, decreased expression of relaxin receptors, increased expression of elastin, and elevated collagen content in the anterior vaginal wall tissue (p<0.05), suggesting beneficial effects on tissue regeneration and connective tissue restoration in PFD.

Conclusion

BMSC-derived EVs effectively reduce tissue inflammation, promote tissue regeneration and connective tissue reconstruction, and improve pelvic support deficiency, thereby alleviating PFD induced by vaginal distension (VD) in vivo.

Urinary incontinence, voiding dysfunction, pelvic organ prolapse, and anal incontinence are just a few of the complicated diseases under the umbrella term of pelvic floor dysfunction (PFD) (1). These symptoms significantly impact personal health, with up to 75% of women being particularly affected (1). Pregnancy and childbirth are the most established risk factors for PFD; second prolonged labor often associated with urinary incontinence (2). The pelvic floor muscle (PFM) is essential for the maintenance of pelvic organs, and disruption of this muscle can result in altered clinicopathological features of PFD disorders, such as pelvic organ prolapse and urinary incontinence (3). Thus, therapies targeted at restoring the function of pelvic floor muscles have the potential to treat PFD.

Stem cells, particularly bone marrow mesenchymal stem cells (BMSCs), hold significant prospect in the treatment of PFD due to their regenerative capability and ability to differentiate into a range of connective tissue cell types (4). Studies in animal models of PFD or urinary incontinence have shown that BMSCs could improve the damaged external urethral sphincter, leading to symptom alleviation (5-7). Extracellular vesicles (EVs) are small extracellular vesicles containing small RNAs and proteins, which play vital roles in intercellular communication (8,9). BMSC-derived EVs have been found to regulate inflammation, reduce cell apoptosis, and promote tissue regeneration during the process of tissue repair (10), suggesting a potential role in mitigating inflammation and fibrosis in the PFM for PFD treatment.

In this study, EVs isolated from mouse BMSCs were administered to the female PFD mouse model to assess the therapeutic effects on PFD. We hypothesized that BMSC-derived EVs may play a role in improving pelvic floor dysfunction by reducing inflammatory response and fibrosis in the pelvic floor, and increasing cell proliferation and connective tissue restoration. The results of this study are expected to offer novel evidence that support the protective and regenerative functions of BMSC-derived EVs in enhancing pelvic floor support.

Materials and Methods

This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Fujian Medical University [Approval number: No.331(2021)].

BMSCs isolation and culture. Female C57BL/6J mice aged 6 to 8 weeks were euthanized by cervical dislocation, and the bone marrow was extracted from the tibia and femur. After being extracted, the BMSCs were cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin (PS, Gibco) and 10% fetal bovine serum (FBS, Gibco), and kept at 37˚C in a humidified incubator with 5% CO₂. The medium was replaced and nonadherent cells were eliminated during the three-day incubation period. The adherent cells were passaged at 1:3 ratios after reaching 80% confluence. BMSCs from the third to fifth generation were used in the subsequent experiments.

Characterization of BMSCs by Flow cytometry. The expression of BMSC surface markers was evaluated using FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). In brief, the third generation BMSCs were trypsinized, washed, and resuspended in phosphate buffer saline (PBS). After that, cells were treated for 30 min at 4˚C with isotype control and fluorescein isothiocyanate (FITC) antibodies against CD14, CD19, CD29, CD44, CD34, CD45, CD73, and CD90 (Biolegend, San Diego, CA, USA) and detected using NovoCyte™ Flow cytometry (ACEA, Cat#1300, Santa Clara, CA, USA). Flowjo (BD Biosciences) was used for data processing.

Osteogenic, adipogenic, and chondrogenic differentiation of BMSCs. For osteogenic differentiation, BMSCs (2×10⁴ cells/cm²) were seeded into 6-well plates. When the cell density reached 80%, the cells were transferred to the osteogenic differentiation DMEM medium containing 10% FBS, 1% PS, 10 mM β-glycerophosphate, 200 μM Glutamine, 100 nM dexamethasone, 10 mmol/l β-glycerophosphate and 50 μg/ml L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA). The medium was replaced every three days. After 21 days (d), the cells were fixed and treated with 60% isopropanol for 1 min at room temperature (RT), washed with PBS, and incubated with 10% alizarin red staining solution (Sangon Biotech, Shanghai, PR China) for 15 min at RT. Finally, the stained cells were washed with PBS and observed.

For adipogenic differentiation, BMSCs (2×10⁴ cells/cm²) were seeded into 6-well plates. When the cell density reached 80%, the cells were transferred to the adipogenic differentiation DMEM medium containing 10% FBS, 1% PS, 0.1 μM dexamethasone (Sigma-Aldrich), 5 mg/ml insulin, 50 μM rosiglitazone and 0.5 mM IBMX. The medium was replaced every three days. The cells were fixed with formalin and stained with 0.5% oil red/isopropanol staining solution (Solarbio, G1262, Beijing, PR China) for 5 min at 37˚C in the dark after 21 d. Subsequently, the staining solution was removed, and the cells were washed with ddH₂O.

For chondrogenic differentiation, BMSCs were seeded at a density of 2×10⁴ cells/cm² in 6-well plates. When the cell density reached 80%, the cells were transferred to the chondrogenic differentiation DMEM medium containing 10% FBS, 1% PS, 0.1 μM dexamethasone (Sigma-Aldrich), 50 μg/ml ascorbic acid, 2% insulin-transferrin-selenium (ITS) solution (containing 0.625 mg/ml insulin, 0.625 mg/ml transferrin, and 0.625 μg/ml selenite), 100 μg/ml sodium pyruvate, 40 μg/ml L-proline, and 10 ng/ml transforming growth factor-β3 (TGF-β3). The medium was replaced every three days. After 21 d, the cells were fixed with formalin and stained with 1% alcian blue staining solution (Sangon Biotech) and 0.1 N HCl for 30 min at RT. The cells were then washed with 0.1 N HCl and immersed in ddH₂O.

BMSCs-EVs extraction and identification. First, the culture supernatant of BMSCs was collected and subjected to 2,000 × g centrifugation at 4˚C for 30 min, and then centrifuged for 45 min at 10,000 × g (4˚C). Next, the filtrate was passed through a 0.45 μm filter membrane and centrifuged for 70 min at 100,000 × g (4˚C). The precipitate was resuspended in 10 ml of pre-cold PBS and centrifuged for 70 min at 100,000 × g (4˚C). Then, the precipitate was diluted with 100 μl of pre-cold PBS. Transmission electron microscopy and nanoparticle tracking analysis were used to identify the size distribution and shape of the isolated EVs, respectively. The protein concentration of isolated EVs was measured using bicinchoninic acid (BCA) kit (Solarbio).

Western blotting. The cells were harvested and incubated with lysis buffer containing protease inhibitors on ice for 20 min. The protein concentration was measured using BCA kit (Solarbio). Protein samples were then boiled at 98˚C for 5 min, and electrophoresis was performed at 80 V for 2 h. The isolated protein was transferred to polyvinylidene fluoride (PVDF) membranes at 350 mA for 2 h. Then, membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4˚C with primary antibodies against CD9 (ab236630, 1:1,000, Abcam, Cambridge, UK), CD81 (ab79559, Abcam, 1:1,000), CD63 (ab315108, Abcam, 1:1,000), Actin (ab8227, Abcam, 1:3,000), Elastin (15257-1-AP, Proteintech, 1:1,000, Wuhan, Hubei, PR China), and FLBN5 (ab202977, Abcam, 1:500). The membranes were washed with PBST and incubated with horseradish peroxidase conjugated goat anti-rabbit/mouse antibody (SA00001-2/SA00001-1, Proteintech, 1:5,000) for 1 h at 25˚C. After washing three times (8 min each) with PBST, the bands were detected using an enhanced chemiluminescence (ECL) kit (Solarbio) and imaged using Bio-Rad ChemiDoc machine.

Construction of PFD animal model. 18 C57BL/6J female mice, aged 6 to 8 weeks, were maintained in a pathogen-free environment (12 h of light and dark, 22-24˚C), with unrestricted access to food and water in the animal laboratory of the hospital. The mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, PR China) and randomly divided into three groups: Sham, Model, and Model/EVs group, with six mice in each group. The animal model of PFD was constructed in the Model and Model/EVs groups by vaginal dilatation. Briefly, the mice were anesthetized by inhalation of isoflurane. After emptying the bladder with catheterization, the catheter was slowly inserted into the vagina of mice, and a stitch was made on the anterior and posterior wall of the vaginal opening to prevent the catheter from falling off. Then, 0.3 ml sterile saline was injected into the balloon and the diameter of the balloon was adjusted to about 8 mm. After 2 h, the saline in the balloon was drained and the catheter was removed. Mice in the Model/EVs group were injected with 200 μg of EVs through the tail vein once every three days for two weeks. Mice in the Sham and Model group were injected with equal volumes of PBS.

Histopathological and immunohistochemical analysis. The tissue samples from the anterior vaginal wall of each group underwent fixation in 10% nerve buffer formaldehyde solution for 24 h. Subsequently, tissue samples were embedded in paraffin. After deparaffinization and rehydration with graded alcohol, sections with a thickness of 5 micrometers were obtained. Histopathological staining was performed using hematoxylin-eosin (HE), Masson trichrome or Sirius red staining in strict accordance with the staining protocol provided by Beyotime (Shanghai, PR China). Images of the Masson trichrome sections were captured using Nikon Eclipse Ci-L microscopes (Melville, NY, USA) and then analyzed using Image-Pro Plus 6.0 software (Media Cybemetics, Rockville, MD, USA). In the case of Sirius red staining, images of the lamina propria region were captured using a Nikon Eclipse Ci-L microscope equipped with a polarizing filter, allowing for the identification of birefringent Sirius Red stained collagen fibers. These images were then analyzed using Image Pro Plus Version 6.0 software to assess the collagen fiber composition within the vaginal muscle layer of the Sirius red stained sections. Immunohistochemical staining was performed using Ki-67 (1:200, ab15580, Abcam) or LGR7 (1:100, 18419-1-AP, Proteintech). The staining procedure was carried out overnight at 4˚C, followed by treatment with horseradish peroxidase conjugated goat anti-rabbit antibody (ab205718, 1:5,000, Abcam). The avidin-biotin-immunoperoxidase complex and 3,3-diaminobenzidine tetrahydrosalt (DAB, Beyotime) were then used. The appearance of dark brown staining suggested the presence of Ki-67 or LCR7-expressing cells. The number of positive staining cells was divided by the total number of cells in each microscopic view to calculate the percentage of positive cells. The expression intensity of Ki-67 or LGR7 was quantitatively assessed using Image Pro plus v6.0 software.

Statistical analysis. The statistical analyses were carried out using GraphPad Prism software v7.0 (GraphPad, San Diego, CA, USA). The data are presented as mean±standard deviation. The differences between groups were analyzed using either unpaired two-tailed Student’s t-test or one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple groups. p<0.05 was considered statistically significant.

Results

Successful extraction of BMSCs and BMSCs-EVs. Flow cytometry results showed that most of the isolated BMSCs did not express differentiated cell markers (CD14, CD19 and CD45) but expressed BMSC-related markers (CD29, CD34, CD44, CD73, and CD90) (11) (Figure 1A). Furthermore, the alizarin red staining, alcian blue staining, and oil red staining results showed that the isolated BMSCs could differentiate into adipogenic, osteogenic, and chondrogenic lineages, confirming their ability to differentiate into these three lineages (Figure 1B). These findings confirmed the successful extraction of BMSCs. Next, EVs derived from BMSCs displayed the typical cup-shaped and circular-shaped structures with a particle diameter ranging from 20 to 100 nm (Figure 1C and D). Western blot results showed that EV markers, such as CD9, CD63, and CD81 were more abundantly expressed in BMSCs-EVs compared to BMSC (Figure 1E), which confirmed the successful extraction of BMSCs-EVs.

Figure 1.

Verification of bone marrow stem cells (BMSCs) and BMSCs- extracellular vesicles (EVs). (A) Flow cytometry analysis of BMSC surface markers. Gray areas represent negative controls and red areas represent flow assays of antibodies. The percentage in the graph indicates the percentage of positive expression. (B) Verification of multi-differentiation ability of BMSCs. Alcian blue, oil red O, and alizarin red S staining can be used to demonstrate the adipogenic, osteogenic, and chondrogenic differentiation potential of BMSCs. (C) The transmission electron microscopy (TEM) picture of BMSC-EVs (scale bar: 200 nm). (D) The diameters of BMSC-EVs. (E) Western blot displaying CD9, CD81, and CD63 expression in BMSC and BMSC-EVs.

BMSCs-EVs reduced inflammation induced by vaginal distention in mice. BMSCs-EVs significantly reduced VD-induced up-regulation of inflammatory cytokines in PFD mice. Compared with the Sham group, the levels of IL-6, IL-1β, and TNF-α in the Model group were significantly increased (p<0.01), while compared with the Model group, the levels of IL-6, IL-1β, and TNF-α in the Model/EVs group were significantly decreased (p<0.05) (Figure 2A-C). Histological analysis revealed that the mucosal epithelium of the tissue in the visual field was stratified flat epithelium, with thick epithelium, local cuticular layer, irregular nuclear shape, loose and pale cytoplasm, and a small amount of inflammatory cell infiltration in the lamina propria in the Sham group (Figure 2D). The mucosal epithelium in the visual field was stratified and flat, with thick epithelial layers. The cuticle was thin, and the mucosal layer was covered with a large number of foamy cells. The cell nuclei were irregular in shape and the cytoplasm was loose and lightly stained. Scattered inflammatory cell infiltration was observed in the lamina propria of the Model group (Figure 2D). While a small number of epithelial cells showed locally loose cytoplasm and vacuolated cytoplasm, and no obvious foamy cells were observed, the connective tissue in the lamina propria was neatly arranged, and no obvious inflammatory cell infiltration was observed in the Model/EVs group (Figure 2D).

Figure 2.

Bone marrow stem cells (BMSCs)- extracellular vesicles (EVs) reduced inflammation induced by vaginal distention in mice. (A-C) ELISA data showing the levels of IL-6, IL-1β, and TNF-α in mice. (D) HE staining of anterior vaginal wall tissue of mice. Sham: no medical procedure or therapy program. Model: PFD mice caused by VD. Model/EVs: VD-induced PFD mice were treated with BMSCs-EVs. Red arrows in the figures refer to inflammatory cells infiltration. 100 μm is the scale bar.

BMSCs-EVs ameliorated vaginal distention and promoted cell proliferation of anterior vaginal wall in PFD mice model. EVs have been demonstrated to promote cell proliferation and adjust the activation and secretion of fibroblasts (12,13). Therefore, we further evaluated the therapeutic effects of BMSCs-EVs on cell proliferation and tissue elasticity within the anterior vaginal wall of PFD mice. Ki-67 is a nuclear protein mainly expressed in the active phases of the cell cycle. It plays an important role in the maintenance of chromosome dispersion during mitosis, and is closely associated with cell proliferation (14). The higher the Ki-67 positive expression rate, the faster the cell proliferation. Here, immunohistochemistry staining results revealed the significant up-regulation of Ki-67+ (%) in the Model/EVs group compared to the Model group (p<0.001), indicating the promotional effects of BMSCs-EVs on cell proliferation in the anterior vaginal wall (Figure 3A).

Figure 3.

Bone marrow stem cells (BMSCs)-extracellular vesicles (EVs) ameliorated vaginal distention and promoted cell proliferation of anterior vaginal wall in pelvic floor dysfunction (PFD) mice model. Ki-67⁺ (%) (A) and LGR7⁺ (%) (B) expression in the tissues of the anterior vaginal wall assessed by immunohistochemistry. Fibers were identified by (C) Masson’s trichrome and (D) Sirius red staining. Collagen fibers and muscle fibers in tissues are distinguished by Masson staining. Collagen fibers, cartilage, and mucus are stained blue, while muscle fibers and red blood cells are dyed red. Red or yellow type I collagen is seen by Sirius Red staining. 100 μm is the scale bar. On the left are pictures of typical tissue slices from each category. On the right are the corresponding positive-stained regions’ semi-quantitative evaluations. (E) Western blot analysis results display Elastin and FBLN5 expression. Sham: no medical procedure or therapy program. Model: vaginal distension (VD)-induced PFD mice. Model/EVs: EVs treated VD-induced PFD mice.

Elastin plays a crucial role in tissue elasticity, enabling tissues to stretch and recoil without requiring external energy input (15). Numerous studies have highlighted the influence of elastin metabolism in PFD, characterized by enhanced degradation, aberrant synthesis, and disruptions in elastin homeostasis (16,17). LGR7, the relaxin receptor, has been identified as a marker of collagen alterations associated with diminished ligament strength (18). Notably, in the present study, LGR7+ (%) was significantly decreased in the Model/EVs group compared to the Model group (p<0.05) (Figure 3B).

Alterations in collagen isoforms and impaired collagen cross-linking have been implicated in the pathogenesis of PFD (19). Correspondingly, Masson trichromatic staining (Figure 3C) and Sirius red staining (Figure 3D) results revealed decreased tissue fibrosis in the Model/EVs group compared to the Model group (p<0.05), suggesting the protective effect on fibrosis caused by tissue injury. In addition, western blot results showed that compared with the Sham group, the Model group had significantly lower expression of Elastin and FBLN5, but compared with the Model group, the protein expression of Elastin and FBLN5 was increased in the Model/EVs group (Figure 3E).

Discussion

PFDs encompass a variety of ailments and have a major negative impact on women’s quality of life (20,21). BMSCs have become an attractive option for the treatment of PFD. We performed a systematic evaluation of the therapeutic potential of BMSC-EVs in a VD-induced female PFD mouse model. The results showed that the EVs could promote cell proliferation, regulate tissue inflammation, restore tissue elasticity, and reduce damage-induced tissue fibrosis in the vaginal walls. The extracted EVs exhibited typical exosomal characteristics, supporting their role in inter-cellular communication and tissue repair. In the mice model of PFD induced by vaginal distension, BMSC-EVs displayed significant effects, including effective reduction of inflammatory cytokine levels, improvement in tissue morphology, and inhibition of neutrophil infiltration. Additionally, the EVs promoted cell proliferation, alleviated fibrillation, decreased the expression of LGR7, and increased the content of collagen in the anterior vaginal wall tissue, indicating their effectiveness in tissue regeneration and connective tissue restoration in PFD.

PFD animal models are essential for studying the molecular mechanisms, histopathology, biomechanics, and for evaluating different treatments, including both surgical and medical methods (22). While non-human primates and large quadrupeds are suitable for model generation resembling the physiological characteristics of human patients and can be applied for surgical evaluation (23,24), rodent models are more appropriate for mechanistic analyses and medical therapy evaluation. VD based models can be easily generated on both rats and mice with handy equipment and friendly cost and are suitable for simulating the muscle wall tissue damage and treatment applications (25,26). To further assess the feasibility and therapeutic potential of BMSC-derived EVs, it is important to include animal models of other species, particularly the commonly used rat models, to test their universality (27).

The amelioration of proliferation inhibition, fibrillation, and collagen alterations in the anterior vaginal wall tissue following BMSC-EV treatment underscores the potential of these EVs in promoting tissue regeneration and restoring tissue integrity. BMSCs over-expressing elastin displayed alleviated collagen structure defects in both in vitro and in vivo rat PFD models (7). Thus, the inhibition of LGR7 signaling and the collagen alteration may originate from the elastin signals in BMSC-EVs. On the other hand, while tissue fibrosis in response to muscle injury can serve as structural compensation for damage, increased differentiation of fibroblasts into non-functional cells can impair the functionality of the wound tissue (28,29). Thus, BMSC-EVs may serve as an external signals that promote fibroblast self-renewal and ordered differentiation. Further in-depth studies should be conducted to explore the protein content and signaling molecules within the EVs to validate these hypotheses.

In conclusion, this study demonstrated the promising therapeutic potential of BMSC-EVs in alleviating pelvic floor dysfunction in a VD-induced PFD mouse model. Through inhibiting tissue inflammation, preventing tissue damage, promoting tissue repair, improving recovery from acute injury, and facilitating tissue regeneration, BMSC-EVs offer a novel approach to enhancing pelvic floor supportive function and addressing PFD-related pathological changes. Further research and clinical studies are warranted to evaluate the full therapeutic effect of BMSC-EVs in the management of PFD, in order to translate these findings into clinical applications and to ultimately improve women’s health and quality of life.

Conflicts of Interest

The Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors’ Contributions

Linlin Hu and Caihong Chen contributed to the conceptualization, formal analysis, methodology, validation, writing, reviewing and editing of the article. Linlin Hu worked in data curation, investigation, visualization and writing of the original draft. Caihong Chen was involved in project administration, resources and supervision. All Authors read and approved the final manuscript.

Funding

This research was supported by the Quanzhou Science and Technology Plan project (2021N029S).