Delivering umbilical cord mesenchymal stem cell exosomes through hydrogel ameliorates vaginal atrophy in ovariectomized rats

Characterization of ucMSC-ex

UcMSC-ex were meticulously characterized using transmission electron microscopy, which revealed a distinct cup-shaped morphology and a relatively uniform diameter (Figure 1A, 1B). Through Nanoparticle Tracking Analysis (NTA), the size distribution of exosomes was demonstrated, showcasing a predominant peak at 126.7 nm that accounted for 96.6% of the main peaks (Figure 1C). Further validation with Western blotting not only confirmed the expression of hallmark exosomal markers such as CD9, HSP70, and TSG101 but also noted the absence of Calnexin (Figure 1D). These combined results underscored the successful isolation of exosomes.

Figure 1. Characterization of umbilical cord mesenchymal stem cell-derived exosomes. (A, B) Representative transmission electron microscopy images of exosomes. (C) Nanoparticle tracking analysis showing the size distribution of exosomes. (D) Western blot analysis for the detection of specific proteins in ucMSC-ex.

Successful uptake of exosomes by vk2 cells

VK2 cells, which were stained green for the cytoskeleton and blue for the nucleus, efficiently ingested exosomes that were labeled red by PKH26, following a 12-hour co-culture. Notably, the exosomes aggregated around the nucleus, situated within the cytoskeleton (Figure 2), further confirming their successful uptake by VK2 cells.

Figure 2. Shows the fluorescent images of VK2 cells taking up ucMSC-ex.

Exosomes promote vk2 cell migration

Epithelial cell migration plays a pivotal role in the healing and recovery of injured tissues. As depicted in Figure 3F, when compared to the PBS control, our data reveal that exosome treatment markedly boosts the migration rate of VK2 cells. Flg and CK10 are established as essential markers for keratinocyte differentiation [18]. After normalization and subsequent qPCR analysis, a modest upregulation of these two markers was detected following exosome treatment. Nevertheless, it's essential to underscore that these observed changes did not achieve statistical significance (Figure 3H).

Figure 3. The effects of ucMSC-Ex on VK2 cell proliferation, migration, and differentiation. (A) CCK-8 assay measuring VK2 cell viability at different concentrations; (B) Flow cytometry analysis of cell cycle after co-culture for 18 hours; (C) Quantification of S-phase cell proportion; (D) EdU assay detecting VK2 cell proliferation after 24 hours of co-culture; (E) Quantitative analysis of EdU-positive cells; (F) Scratch assay showing ucMSC-Ex promotion of VK2 cell migration; (G) Quantification of cell migration; (H) Normalized expression levels of differentiation-related genes in ucMSC-ex treated group compared to PBS group.

Characterization of hyaluronic acid hydrogels

Post-photocrosslinking, the hyaluronic acid hydrogel took a cylindrical shape, measuring a diameter of 5 mm and a height of 1.0 cm (Figure 4A). SEM images revealed that the gel's interior hosted a loosely interconnected pore structure, affording ample space for accommodating exosomes (Figure 4B). As illustrated in the figure, the natural degradation of the hydrogel facilitated a slow, continuous release of the loaded exosomes (Figure 4C). To explore the biocompatibility of hyaluronic acid hydrogel, we conducted live/dead staining experiments. In these experiments, green fluorescence represented live cells stained with calcein AM, and red fluorescence indicated dead cells stained with PI (Figure 4D). The results indicated that the majority of VK2 cells remained viable after exposure to the hydrogel. Furthermore, additional CCK-8 analysis (Figure 4E) revealed no significant difference in cell viability between the two groups, demonstrating that the hyaluronic acid hydrogel exhibits excellent biocompatibility and is devoid of any cytotoxicity.

Figure 4. Characterization of exosome hydrogel. (A) Morphology of hyaluronic acid hydrogel post photopolymerization; (B) Cross-sectional view of hyaluronic acid hydrogel presenting a loose, porous structure under scanning electron microscopy; (C) Release curve of exosomes from the exosome hydrogel over 14 days; (D) Live/dead cell staining of VK2 cells cultured on the hyaluronic acid hydrogel. (E) Determination of cell viability by co-incubation of vk2 cells and hydrogels.

Hematoxylin-Eosin (HE) analysis

Figure 5A shows that the vaginal epithelium is the thickest in the sham operation group. The nuclei of the basal layer cells are large and deeply stained, aligned neatly in a palisade arrangement. The stratum corneum is complete, with spindle-shaped granular cells and non-nucleated squamous cells observable between the basal layer and the stratum corneum. In the ovariectomy group and hydrogel group, the epithelium is thin, the arrangement of basal cells is disordered, the number of cell layers is small, and the stratum corneum disappears. Papillary-like proliferation is observed at the vaginal base in the exosome hydrogel group, the epithelial cell layers increase in number, the thickness is considerably greater than the ovariectomy group, albeit slightly less than the sham operation group. The thickness of the vaginal mucosal cells in the estrogen treatment group and the sham operation group is similar, which is consistent with previous reports: estrogen can completely reverse the atrophy of the vaginal epithelium [19]. Moreover, when analyzing uterine weight across groups, there was no statistically significant difference observed between the exosome and hydrogel groups compared to the de-ovulated group (Figure 5D). This implies that the application of exosome hydrogel does not influence uterine weight.

Figure 5. Vaginal sections and HE staining. (A) Vaginal epithelial morphology among different groups after HE staining; (B, C) Distribution of DIR-labeled exosomes in vaginal tissue; (D) Uterine weights in each group.

Immunohistochemistry

In an effort to ascertain the distribution of exosomes within the rat vagina, we examined vaginal slices marked with fluorescent DIR under a microscope. As can be discerned from the figure, green fluorescent-tagged exosomes were seen in the vaginal basal layer, sub-basal layer, and surrounding blood vessels, implying that exosome particles released from the hydrogel can be absorbed by tissue cells (Figure 5B, 5C). Ki67 is a classic marker of cell proliferation. Figure 6 reveals that the expression of epithelial cells in the vaginal mucosa is at its nadir in the ovariectomy group and the hydrogel group, yet, the exosome hydrogel treatment group exhibits the highest expression among all groups, with the sham operation group and the estrogen group showcasing similar expression levels. The expression level in the hydrogel treatment group mirrors that in the ovariectomy group, while it experiences a significant surge in the exosome hydrogel group, which underscores that the active proliferation of vaginal basal cells is primarily attributable to the effect of exosomes. Furthermore, we meticulously quantified the CD31-positive capillaries in the inherent layer, and as was expected, the count was the highest in the sham surgery group, trailed by the estrogen group. The quantity of CD31-positive microvessels diminished post-ovariectomy, but saw a revival following treatment with exosome hydrogel.

Figure 6. IHC analysis. (A) Average optical density analysis of ki67 and CD31 expression in the vaginal epithelium and lamina propria; (B, C) Quantification of ki67 and CD31 staining.

Cell source and culture

Umbilical cord mesenchymal stem cells (ucMSCs) and vaginal epithelial cells (VK2) were obtained from the Shanghai Cell Bank (ATCC, China). The ucMSCs were cultured in a medium containing 10% fetal bovine serum (Gibco, USA), 1% dual antibiotic (streptomycin + penicillin), and low glucose DMEM. UcMSCs from 2-6 generations were evenly seeded in 15 cm dishes using the complete culture medium and incubated at 37° C with 5% CO2. The culture medium was refreshed every 2-3 days. When the cells achieved 80% confluency, the culture medium was removed, washed three times with PBS, and then replaced with a 1:1 volume ratio of low glucose DMEM and exosome-specific serum-free medium (Umbibio, China). The cells were then further incubated for 48 hours and the cell supernatant was collected.

According to the cell manufacturer's instructions, VK2 cells were cultured with 10% fetal bovine serum (Absin, China), 1% double antibiotic (streptomycin + penicillin), high glucose DMEM, under similar culture conditions as the ucMSCs.

Isolation, identification, and labeling of exosomes

Using the method prescribed by Huilei Yu [42], the collected cell supernatants were first centrifuged at 300g for 5 minutes at 4° C to discard live cells. Subsequent centrifugation at 2000g for 30 minutes was used to remove dead cells, followed by another round at 10,000g for 30 minutes to exclude macromolecules and cell debris. The supernatant was then filtered using a 0.22 μm filter membrane and centrifuged at 100,000g for 70 minutes. The resulting precipitate was resuspended in PBS and centrifuged once more at 100,000g for 70 minutes to finally obtain a light-yellow precipitate.

A part of the exosome suspension was mixed with red PKH26 and green DIR (Umbibio, China) dyes, separately, in dark conditions. The excess dye was removed following the exosome extraction protocol, and the precipitate was resuspended in PBS to acquire the stained exosomes.

The morphology of the exosomes was studied using transmission electron microscopy, and the nanoparticle concentration was measured by counting the scattered particles using a nanoparticle tracking system.

Western blotting was carried out using exosome markers CD9 (1:1000; Absin, China), TSG101 (1:1000; Umbibio, China), HSP70 (1:1000; Affinity, China), and Calnexin (1:1000; Affinity, China). In brief, proteins were separated by gel electrophoresis (PAGE 10% Bis-Tris, Meilunbio, China) and transferred to a 0.22 μm polyvinylidene difluoride membrane (PVDF, Millipore, USA). They were then immunoblotted with a specific primary antibody, blocked with 5% skimmed milk powder, and subsequently blotted with a secondary rabbit antibody (1:3000; Affinity, China) for 1 hour at 37° C. Finally, the blots were visualized with an ultra-sensitive ECL kit (Meilunbio, China).

Uptake of exosomes

PKH26-labeled exosomes and VK2 cells were co-incubated at 37° C for 12 hours. Subsequently, the cells were fixed with 4% paraformaldehyde, nuclei stained with DAPI (Beyotime, China), and the cytoskeleton was stained with Actin-Tracker Green (Beyotime, China). After washing with 1% Triton ×100, images were captured using a Zeiss fluorescence microscope.

Cell viability, cell cycle, and EDU assay

Cells were seeded in 96-well plates at a density of 5 x10^3 cells/well. Control wells received PBS while experimental wells were treated with exosomes at concentrations ranging from 1μg to 16μg. After 24 hours incubation, 10μl of CCK-8 (Glpbio, USA) reagent was added to each well and incubated for an additional 2 hours. Absorbance was then measured at 450nm.

VK2 cells (5x10^5) were seeded in 6-well plates and incubated for 24 hours. Following incubation, wells received either PBS or exosomes (at concentrations of 4μg, 8μg, and 16μg). After 18 hours, cells were harvested, fixed with 75% ethanol, and stored at -20° C overnight. Subsequently, the Cell Cycle and Apoptosis Analysis Kit (Beyotime, China) was used for staining and flow cytometry analysis. Raw data were analyzed using FlowJo 10.6.2 software (FlowJo, USA).

VK2 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 3x10^3 cells/well. Control wells received PBS and experimental wells received 16μg of exosomes. After 24 hours incubation, cells were labeled with 10uM EdU (Beyotime, China) for 2 hours. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton × 100, and processed with a click reaction. Nuclei were stained with Hoechst 33342. Images were captured under a microscope, and the number of EdU-positive cells was determined. The EdU staining rate was calculated as follows: (number of EdU-positive cells/(EdU-positive cells + EdU-negative cells)) x 100%.

Cell migration

VK2 cells were seeded in 12-well plates and allowed to reach 100% confluency. A straight scratch was made across the cell monolayer using a 200μl pipette tip. After washing with PBS, cells were cultured in serum-free high glucose DMEM medium. Control wells received PBS and experimental wells received 16μg of exosomes. Cell migration was monitored and imaged at 0, 18, and 36 hours. The scratch wound healing was calculated as follows: (initial scratch area - remaining scratch area at the time of measurement) / initial scratch area x 100%.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis to detect cell differentiation

Cells were seeded in 6-well plates and allowed to reach 70% confluency. Control wells received PBS and experimental wells received 16μg of exosomes. After 72 hours incubation, the cells were harvested and the expression of Filaggrin and Cytokeratin 10 (Flg, CK10) was determined by qPCR. Related primer sequences are shown in Table 1.

Total RNA was extracted from cells using the RNA Isolation Kit V2 (Vazyme, China), which was then reverse transcribed to cDNA using the 4x reverse Transcription Master Mix (Vazyme, China). qPCR was conducted using the SYBR Green qPCR Master Mix (Glpbio, USA). GAPDH was used as an internal reference for mRNA, and relative expression was quantified using the 2^-ΔΔCt method.

Synthesis and characterization of hyaluronic acid hydrogels

Methacryloylated hyaluronic acid, sourced from Engineering for Life (China), was used to formulate a 5% hyaluronic acid gel. Initially, a 2.5% photofacial agent was resuspended in exosome precipitate (1.25 mg/ml concentration), and this suspension was combined with hyaluronic acid and stirred for 1 hour at room temperature. Following sterilization via a 0.22um sterile filter membrane, 200 ul of the mixture was transferred into a 1 ml syringe (tip removed). The mixture was then crosslinked with 395nm UV light for 6 seconds, yielding a cured exosome hydrogel. Finally, the gel was freeze-dried and its morphology was observed under a scanning electron microscope. To assess the biocompatibility of sodium hyaluronate hydrogel, an equal number of VK2 cells were seeded onto the hyaluronic acid hydrogel in a 96-well culture plate. After 24 hours of incubation, cell viability was measured using a live/dead staining kit (Beyotime, China) and the CCK-8 assay, following the manufacturer's instructions.

Release of exosomes

A certain amount of exosomes was integrated into the hyaluronic acid hydrogel, which was then transferred to a 24-well plate containing 200 μL of PBS. The liquid was replaced every other day, and the supernatant was collected for BCA protein concentration measurements prior to each liquid change. The protein concentration determined represented the mass of exosomes released.

Establishing a rat model of menopause

32 healthy 15-week-old female SD rats were purchased from Sipeifu Biotechnology (Beijing, China). These rats were randomly allocated into five groups (n = 6 in sham, ovariectomy, hydrogel, estrogen group; n = 8 in exosomes hydrogel group, 2 of them are marked with DIR). Oophorectomy surgery involved anesthetization using 1.25% tribromoethanol (250 mg/kg intraperitoneally), followed by an incision in the lower abdomen. In the model group, both ovaries were removed while in the sham operation group, some adipose tissues around the ovaries were excised. Antibiotics were administered post-operation to prevent infections.

Post-operation, vaginal smears were examined daily from the 10th day onwards for 5 days. The absence of a motility phase response indicated a successful menopause model. Hyaluronic Acid hydrogel loaded with exosomes was injected into the rats' vaginas, which were subsequently sutured to prevent gel loss. On the 3rd day, two rats implanted with DIR-labeled exosomes were sampled and processed for fluorescence microscopy. The remaining rats were euthanized on the 14th day, and vaginal tissues were collected for subsequent experiments.

Hematoxylin-Eosin (HE) staining

Specimens were fixed with 4% paraformaldehyde, dehydrated with an alcohol gradient, cleared, and embedded in paraffin blocks. 5 μm thick tissue sections were then stained with hematoxylin/eosin (Absin, China) for routine histology.

Immunohistochemistry (IHC)

Paraffin sections were deparaffinized and rehydrated through a series of xylene and alcohol washes. Endogenous peroxidases were removed with 3% hydrogen peroxide, and antigen retrieval was achieved with pH9.0 repair solution via autoclaving. Sections were then rinsed, dried, and incubated overnight at 4° C with primary antibodies (Ki67 and CD31, both 1:50 dilution, Absin, China). After rinsing, sections were incubated with a secondary antibody at 37° C for 20 min, and staining was developed with DAB. Quantification of staining in ten random fields per section was done with ImageJ software.

Statistical analysis

Each experiment was performed three times and results are presented as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 9. Comparisons between two groups were done with independent samples t-tests; comparisons among multiple groups were done using ANOVA. Differences were considered statistically significant at p < 0.05.