miR-126-3p-loaded small extracellular vesicles secreted by urine-derived stem cells released from a phototriggered imine crosslink hydrogel could enhance vaginal epithelization after vaginoplasty

Abstract

Background: Due to the large area and deep width of the artificial neovagina after vaginoplasty, it takes a considerable amount of time to achieve complete epithelization of the neovagina. Currently, the clinical therapies for vaginal epithelization after vaginoplasty are still dissatisfactory. Recent studies showed that small extracellular vesicles (sEVs) derived from stem cells could accelerate wound epithelization. The sustained release of sEVs from optimized hydrogels may be a promising strategy to accelerate vaginal epithelization after vaginoplasty.

Methods: The efficacy of phototriggered imine crosslink hydrogels (piGEL) containing sEVs derived from human urine-derived stem cells (hUSC-sEVs, piGEL-sEVs) on vaginal mucosa defects in rabbits was assessed by wound closure rates, histological analysis and immunofluorescence staining analysis. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine and scratch wound assays were performed to assess the effects of hUSC-sEVs on the proliferation and migration ability of vaginal epithelial cells (VK2/E6E7). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to test the expression of epithelial differentiation markers in VK2 cells. Moreover, a microRNA (miRNA) microarray was used to find hUSC-sEVs-specific miRNAs that potentially affected the proliferation, migration and differentiation ability of VK2 cells.

Results: The in vitro release profile revealed that the piGEL could ensure sustained release of hUSC-sEVs. The in vivo results showed that piGEL-sEVs effectively promoted epithelization and angiogenesis of vaginal mucosa defects in rabbits. According to miRNA microarray and qRT-PCR results, miR-126-3p might be the crucial molecule among the various miRNAs contained in hUSC-sEVs. The data showed that hUSC-sEVs promoted the migration and differentiation of VK2 cells by delivering miR-126-3p to suppress the expression of Spred1 and PIK3R2, thereby activating the ERK1/2 and ATK signaling pathways.

Conclusion: The results indicated that piGEL-sEVs could be a novel promising approach for enhancing the epithelization of the neovagina after vaginoplasty and provided useful data for understanding the underlying mechanism of the effect of hUSC-sEVs on epithelization.

Keywords: Extracellular vesicles; Hydrogels; Mayer–Rokitansky–Küster–Hauser syndrome; MicroRNA; Stem cells.

Fig. 1

Identification of hUSCs and hUSC-sEVs. A Representative images of hUSCs observed by light microscopy. Scale bar: 100 µm. B Characteristic surface markers of hUSCs evaluated by flow cytometry. C Representative images of hUSC-sEVs under TEM. Scale bar: 50 nm. D Particle size distribution of hUSC-sEVs measured by a nanoflow cytometer. E Marker proteins of hUSC-sEVs were identified by western blot

Fig. 2

Characterization of piGEL and piGEL-sEVs. A Schematic diagram of the phototriggered imine crosslinking (PIC) mechanism for the integration of hydrogels and tissue. B Curve of release nanoparticles of piGEL-sEVs over 8 days. C Live/dead staining of VK2 cells cultured on piGEL. Scale bar: 100 µm

Fig. 3

Gross and histological evaluation of vaginal mucosa defects in each group. A Macroscopic observation of vaginal mucosa defects at days 7 and 14 after the operation. B Quantitative analysis of the wound closure ratio in each group. n = 5 per group. C H&E staining of repaired vaginal wall tissue in each group. Scale bar: 500 µm. D Masson staining of repaired vaginal wall tissue in each group. Scale bar: 100 µm. E Quantitative analysis of collagen area normalized to the connective area. *, P < 0.05, compared with the PBS group; #, P < 0.05, compared with the piGEL group; %, P < 0.05, compared with the sEVs group

Fig. 4

piGEL-sEVs promoted vaginal epithelium regeneration and angiogenesis. A IF staining for AE1/AE3 at days 7 and 14 after the operation. Scale bar: 50 μm. B Quantitative analysis of IF staining of AE1/AE3. n = 3 per group. C IF staining for CD31 at day 7 after the operation. Scale bar: 50 μm. D Quantitative analysis of new vessel number. n = 3 per group. E IF staining for α-SMA at day 14 after the operation. Scale bar: 50 μm. F Quantitative analysis of IF staining of α-SMA. n = 3 per group. *, P < 0.05, compared with the PBS group; #, P < 0.05, compared with the piGEL group; %, P < 0.05, compared with the sEVs group

Fig. 5

hUSC-sEVs promoted the migration and differentiation of VK2 cells. A Representative fluorescence micrograph of Dil (red)-labeled sEVs internalized by VK2 cells. Scale bar: 15 µm. B hUSC-sEVs did not increase the viability of VK2 cells at 24 h after coincubation, as detected by CCK-8 tests. C hUSC-sEVs did not promote the proliferation of VK2 cells at 24 h after coincubation, as detected by EdU tests. Scale bar: 50 µm. D Quantitative analysis of the proliferation rate of VK2 cells after treatment with hUSC-sEVs. n = 3 per group. E hUSC-sEVs promoted the migration of VK2 cells, as detected by scratch assays. Scale bar: 100 µm. F Quantitative analysis of the migration ability of VK2 cells after treatment with hUSC-sEVs. n = 3 per group. G mRNA expression levels of filaggrin and CK10 in VK2 cells were measured by qPCR. n = 3 per group. The PBS, sEVs1 and sEVs2 groups indicated 0, 5 and 10 × 10⁸ hUSC-sEVs particles/mL, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. indicated nonsignificant difference

Fig. 6

hUSC-sEVs activated the ERK1/2 and PI3K/AKT signaling pathways by delivering miR-126-3p. A Normalized miRNA expression levels of hUSC-sEVs measured by miRNA microarray. B The predicted miRNA expression levels of hUSC-sEVs measured by qPCR. C VK2 cells were treated with hUSC-sEVs for 6 h, and the expression of the predicted miRNAs was measured by qPCR. D The potential target genes of miR-126-3p were predicted by bioinformatics analysis. E VK2 cells were transfected with miR-126-3p for 36 h, and then the mRNA expression of Spred1 and PIK3R2 in VK2 cells was measured by qPCR. F VK2 cells were treated with hUSC-sEVs for 36 h, and the protein levels of Spred1, ERK1/2, p-ERK1/2, PIK3R2, AKT, and p-AKT in VK2 cells were analyzed by western blotting. The PBS and sEVs groups indicated 0 and 10 × 10⁸ hUSC-sEVs particles/mL. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

miR-126-3p silencing attenuated the effect of hUSC-sEVs on VK2 cells. A Representative images of the scratch migration assay of VK2 cells. Scale bar: 100 µm. B Quantitative analysis of the migration behavior of VK2 cells. n = 3 per group. C VK2 cells were treated with miR-126-3p inhibitor or control inhibitor for 36 h, and the expression of filaggrin and CK10 was measured by qPCR. n = 3 per group. D The protein levels of Spred1, ERK1/2, p-ERK1/2, PIK3R2, AKT and p-AKT were analyzed by western blotting. E Densitometric quantification of the relative band intensity in D. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Schematic diagram of therapeutic sEVs released from piGEL for epithelization of the vagina. A Fabrication and application of piGEL-sEVs. B The underlying mechanisms of hUSC-sEVs on VK2 cells