Improved Wound Healing and Skin Regeneration Ability of 3,2'-Dihydroxyflavone-Treated Mesenchymal Stem Cell-Derived Extracellular Vesicles

Abstract

Flavonoids enhance the self-renewal and differentiation potential of mesenchymal stem cells (MSCs) and have therapeutic activities, including regenerative, anti-oxidative, and anti-inflammatory effects. Recent studies have revealed that MSC-derived extracellular vesicles (MSC-EVs) have therapeutic effects on tissue regeneration and inflammation. To facilitate further research on the therapeutic potential of MSC-EVs derived from flavonoid-treated MSCs, we surveyed the production of EVs and their therapeutic applications in wound regeneration. MSCs treated with flavonoids enhanced EV production twofold compared with naïve MSCs. EVs produced by MSCs treated with flavonoids (Fla-EVs) displayed significant anti-inflammatory and wound-healing effects in vitro. The wound-healing capacity of EVs was mediated by the upregulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Interestingly, the protein level of p-ERK under inhibition of MEK signals was maintained in Fla-EV-treated fibroblasts, suggesting that Fla-EVs have a higher therapeutic potential than naïve MSC-EVs (Cont-EVs) in wound healing. Moreover, the in vivo wound closure effect of the Fla-EVs showed significant improvement compared with that of the flavonoid-only treatment group and the Cont-EVs. This study provides a strategy for the efficient production of EVs with superior therapeutic potential using flavonoids.

Keywords: EVs; MEK/ERK signal; MSCs; anti-inflammation; flavonoid; wound healing.

Figure 1

Structure of 3,2′-DHF and screening of several flavonoid treatments in WJ-MSCs. (A) Schematic illustration of therapeutic effects of Fla-EV. (B) Structure of 3,2′-dihydroxyflavone. (C) Comparison of time-dependent cell growth in several flavonoid treatment conditions derived from various sources.

Figure 2

Characterization of 3,2′-DHF-treated WJ-MSCs and EVs. (A) The proliferation rate of WJ-MSC treated with various concentrations of 3,2′-DHF. WJ-MSCs have the best proliferation rate with 1–2 μM 3,2′-DHF. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ** p < 0.05, **** p < 0.0001. (B) Protein expression of MEK/ERK signaling proteins in WJ-MSCs with and without 3,2′-DHF treatment. The bar graph shows the protein expression of AKT and MEK in 3,2′-DHF-treated and untreated WJ-MSCs. (C) Flow cytometry analysis to confirm the characteristics of WJ-MSCs with or without 3,2′-DHF. The expression of the positive markers CD73, CD90, and CD105, and the negative markers CD34 and CD45, was assessed. (D) The relative EV production amounts of Cont-EV and Fla-EV were measured using nanoparticle tracking analysis (NTA). Statistical analysis was determined using two-way ANOVA: **** p < 0.0001. (E) Transmission electron microscopy (TEM) images of Cont-EV and Fla-EV. Scale bar: 100 nm. The size of the EVs was determined with NTA. (F) Characterization of EV-positive markers CD9 and CD63, and negative markers calnexin and GM130, in Cont-EV and Fla-EV using Western blotting. (G) Representative plot of EV-surface markers CD63 and CD81 in Cont-EV and Fla-EV via flow cytometry analysis.

Figure 3

Anti-inflammatory effects of Fla-EVs in RAW 264.7 cells. (A) Schematic anti-inflammatory assay of RAW 264.7 cells with LPS and EVs. (B) Representative images of RAW 264.7 cells incubated with LPS and EVs for 24 h. Scale bar: 200 μm. (C) Relative size of each cell as analyzed with image J. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ## p < 0.01, ** p < 0.01, *** p < 0.001. (D,E) RAW 264.7 cells were co-treated with 10 ng/mL of LPS and EVs (1 × 10⁷ and 1 × 10⁹ particles, respectively). (D) Nitric oxide levels as determined with the Griess assay. (E) Expression level of inflammatory cytokines, including mIL-1β, mIL-6, and mTNF-α, as measured with ELISA. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: # p < 0.05, #### p < 0.0001, *** p < 0.001, **** p < 0.0001. # compared with the LPS group and * indicate comparisons between Cont-EV and Fla-EV.

Figure 4

In vitro wound-healing effects of Fla-EVs. (A) Representative image of NHDFs incubated with EVs after scratching. The bar graph shows the relative wound area in each group. * indicates comparison with control and # indicates comparison with Cont-EV. Statistical analysis was determined using two-way ANOVA with Tukey’s multiple comparisons test: # p < 0.05, ## p < 0.01, #### p < 0.0001, *** p < 0.001, **** p < 0.0001. * indicates comparison with control and # indicates comparison with Cont-EV. (B) Proliferation rate of NHDFs treated with 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ EV particles. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ** p < 0.01, *** p < 0.001, **** p < 0.0001. * indicates comparison with control. (C) Western blot image showing p-ERK and ERK expression levels in EV-treated NHDFs. (D) Representative Western blot image of EV-treated NHDFs with or without PD98059. (E) Representative image of HaCaTs incubated with EVs after scratching. The bar graph shows the relative wound area in each group. Statistical analysis was determined using two-way ANOVA with Tukey’s multiple comparisons test: #### p < 0.0001, **** p < 0.0001. * indicates comparison with control and # indicates comparison with Cont-EV.

Figure 5

In vivo wound-healing effects of Fla-EVs. (A) Representative images of wounds monitored every two days until day 9. (B) Graphical data describing relative wound areas calculated to the wound area on day 0 in each group. Statistical analysis was determined using two-way ANOVA with Tukey’s multiple comparisons test: #### p < 0.0001, *** p < 0.001, **** p < 0.0001. * indicates comparison with control and # indicates comparison with Cont-EV. (C) Histological analysis of the wound area via hematoxylin and eosin and Masson’s trichrome staining. * p < 0.05, *** p < 0.001, **** p < 0.0001 compared with control; #### p < 0.0001 indicates comparison between Cont-EV and Fla-EV.