What if the key to better wound healing is not transplanting stem cells, but collecting more powerful messages from them? This study uses a flavonoid to make stem-cell vesicles more abundant and more effective. Wound healing is a dynamic process involving homeostasis, inflammation, proliferation, and remodeling.

Each phase is coordinated by immune cells, epithelial and mesenchymal cells, growth factors, cytokines, and chemokines. Several studies have revealed that mesenchymal stem cells play pivotal roles in all phases of wound healing. The paracrine mechanism of mesenchymal stem cells can exert therapeutic effects via extracellular vesicles, or EVs.

EVs are phospholipid-bilayer vesicles ranging in size from thirty to one hundred fifty nanometers and are secreted by cells. These vesicles play a critical role in intercellular communication by carrying growth factors, proteins, and genetic molecules derived from parent cells.

Their composition and characteristics depend on their parent cells. EVs derived from mesenchymal stem cells possess angiogenic, migratory, and immunomodulatory capacities and can promote tissue regeneration in various diseases. Recent studies have revealed that flavonoids stimulate the proliferation and differentiation of mesenchymal stem cells.

Flavonoids have also been widely investigated for antioxidant, anti-inflammatory, and wound-healing functions. Flavonoids can interact with cellular membranes, particularly lipid rafts, which present membrane proteins that regulate cellular signaling pathways and biological functions.

The signaling pathways regulated by flavonoids include the phosphatidylinositol-3 kinase and protein kinase B pathway, the mitogen-activated protein kinase pathway, and especially the MEK and ERK pathway. A previous study found that 3,2′-dihydroxyflavone significantly enhanced the proliferation rate and stemness of human induced pluripotent stem cells.

The hypothesis was that 3,2′-DHF could improve mesenchymal stem cell proliferation and activate cellular communication, while also promoting the production of extracellular vesicles. The study investigated the functional effects of EVs produced from 3,2′-DHF-treated mesenchymal stem cells in an excisional wound-healing model and examined signals related to that effect.

The growth rate of stem cells was examined after treatment with various flavonoids, including 3,2′-dihydroxyflavone. Among the various flavonoids, 3,2′-DHF-treated WJ-MSCs showed the highest cell growth. Several concentration ranges, from zero point two five to eight micromolar, were investigated, and mesenchymal stem cells were incubated with 3,2′-DHF for forty-eight hours.

Microscopy showed no differentiation or abnormality in treated cells. The MTT assay showed that mesenchymal stem cells treated with two micromolar 3,2′-DHF had a higher proliferation rate than the other groups. AKT and ERK signals, including phosphorylated AKT and phosphorylated ERK, were higher in 3,2′-DHF-treated mesenchymal stem cells than in the control.

The stemness markers CD73, CD90, and CD105 were highly expressed in both groups, while CD34 and CD45 were expressed in less than five percent of both cell populations. A concentration of two micromolar was therefore used in subsequent experiments.

Mesenchymal stem cells were cultured with two micromolar 3,2′-DHF, and the supernatant was harvested after forty-eight hours. Extracellular vesicles were isolated by conventional differential centrifugation and expressed CD63 and CD9, while calnexin and GM130 were hardly detected.

The vesicles produced with and without 3,2′-DHF were named Fla-EVs and Cont-EVs, respectively. Fla-EVs had twice the particle number of Cont-EVs: one point one one times ten to the third particles per cell versus five point five five times ten to the second.

CD63 and CD81 were expressed in more than ninety-nine percent of both EV populations. Cont-EVs and Fla-EVs had similar diameters, one hundred thirty-two nanometers and one hundred thirty point seven nanometers, and both had the cup shape of a vesicle.

Figure two first shows that 3,2-prime-DHF treatment is associated with altered WJ-MSC proliferation and increased AKT and ERK signaling, while flow cytometry confirms the expected MSC marker profile. The figure then characterizes vesicles from treated and untreated cells: nanoparticle tracking measures production and size, TEM shows their vesicle-like morphology, and Western blotting detects CD9 and CD63 but little calnexin or GM130.

Flow cytometry further confirms the EV-surface markers CD63 and CD81, establishing the identity of the isolated vesicles. To investigate anti-inflammatory effects, LPS-induced RAW 264.7 cells were used. LPS-treated macrophages displayed irregular shapes, while Cont-EV and Fla-EV treatment groups showed a smaller proportion of cells whose shape changed.

Cells had a rounder and smaller shape in the Fla-EV treatment groups than in the Cont-EV treatment groups. Nitric oxide was measured in the culture supernatant using a colorimetric assay, and pro-inflammatory cytokines were measured using ELISA.

Nitric oxide significantly decreased in the Cont-EV and Fla-EV treatment groups compared with the untreated EV group. The Fla-EV-treated group showed reduced expression of the pro-inflammatory cytokines interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha.

At one times ten to the ninth particles, Fla-EV-treated cells had significantly lower nitric oxide levels than the same particle number in the Cont-EV group, and the most dramatic cytokine decreases were observed in the Fla-EV group. These results demonstrate that Fla-EVs had a superior anti-inflammatory effect in a dose-dependent manner and were more effective than Cont-EVs.

Figure three tests whether Fla-EVs can counter inflammation in LPS-stimulated RAW two hundred sixty-four point seven macrophages. The images and relative-cell-size analysis show morphology across control, LPS, Cont-EV, and Fla-EV conditions, while the Griess assay and ELISA quantify nitric oxide and inflammatory cytokines, including interleukin-one beta, interleukin-six, and tumor-necrosis-factor alpha.

The authors report significant changes across these readouts, supporting Fla-EVs as a candidate anti-inflammatory treatment. A scratch assay was used to assess EV effects on migration in human dermal fibroblasts. The scratch gap was closed by cell migration, and pictures were taken every twelve hours at the same location.

The migration rate of the Fla-EV-treated group was visibly higher than that of the other groups, and both Cont-EVs and Fla-EVs increased fibroblast proliferation. Fibroblasts were pretreated with PD98059, a MEK and ERK inhibitor, to examine whether Fla-EVs could induce wound closure through MEK and ERK signaling.

With PD98059 treatment, inhibition of the phosphorylated ERK level in Fla-EV-treated fibroblasts was less than in Cont-EV-treated fibroblasts. The enhanced migration regulated by MSC-EVs may therefore be dependent on MEK and ERK signaling.

Fla-EVs could induce fibroblast migration despite MEK and ERK inhibition conditions, maintaining the phosphorylated ERK signal. HaCaT keratinocytes showed increased migration after EV treatment, and wound closure was fastest in the Fla-EV-treated group. Together, these results suggest that Fla-EVs exert a wound-healing effect by promoting dermal fibroblast migration and proliferation and keratinocyte migration.

Figure four tests how Fla-EVs affect wound closure in cultured cells. In scratch assays, Fla-EV treatment is associated with reduced relative wound area in NHDF fibroblasts and HaCaT keratinocytes, while the proliferation chart shows dose-dependent responses across one times ten to the power of seven, one times ten to the power of eight, and one times ten to the power of nine particles per milliliter.

Western blots examine ERK activation, including with the inhibitor PD98059, supporting a possible MEK–ERK contribution to the migration response. To assess in vivo wound repair, excisional wounds were generated in mice by punch biopsy.

Wound images were acquired at five time points within nine days, until the new skin completely closed the wound. Injection of EVs significantly reduced wound size after five days, with three animals in each group. The Fla-EV-treated group showed significant wound closure compared with the 3,2′-DHF or Cont-EV groups, suggesting more efficient wound-healing effects.

Masson’s trichrome staining was performed to determine the degree of collagen synthesis. The staining results showed that Fla-EV treatment significantly improved re-epithelialization in the epithelial layer compared with Cont-EV.

Figure five tracks punch-biopsy wounds in mice through day nine. The images and relative-area plot show that EV-treated wounds changed over time, with Fla-EV measurements marked as significantly different from control and Cont-EV at several time points. Histology adds tissue-level evidence: hematoxylin and eosin staining assesses re-epithelialization, while Masson’s trichrome visualizes collagen, and the authors report improved epithelial-layer restoration with Fla-EVs compared with Cont-EVs.

MSC therapeutic effects are primarily induced by paracrine factors through EVs containing proteins, lipids, and genetic materials, rather than by direct regenerative mechanisms. MSC-EV therapy is considered a cell-free alternative that can avoid safety issues such as tumorigenicity.

Clinical development is difficult because of low production, while 3,2′-DHF-treated WJ-MSCs secreted twofold more EVs than naïve WJ-MSCs. The EVs produced by 3,2′-DHF-treated MSCs could enhance wound recovery by activating the MEK and ERK pathway. Phosphorylated ERK was maintained in Fla-EV-treated cells during MEK and ERK inhibition, suggesting that Fla-EVs might induce ERK signaling.

EV cargo changes based on culture conditions, and preconditioning can affect scalable production and therapeutic potential. Here, 3,2′-DHF-treated MSCs produced twofold more EVs than MSCs under naïve culture conditions. Fla-EVs showed significant anti-inflammatory effects in vitro and higher wound-healing capacity in vivo, suggesting that 3,2′-DHF induces more therapeutically potent MSC-EVs than naïve MSC-EVs.

Further studies are required to discover the EV cargos, including microRNAs, proteins, and lipids, that play key roles in these effects, and to investigate a therapeutic homogeneous population of EVs. The study has several limitations. Further work should investigate the anti-inflammatory effects of Fla-EVs in vivo using animal models of wound healing and inflammation.

The hypothesis that Fla-EVs reduce inflammation through modulation of inflammatory cytokines and infiltration of immune cells requires further in vivo validation. Mice primarily heal wounds through contraction, although inflammation, angiogenesis, re-epithelialization, and collagen deposition also play critical roles.

Further application studies are needed across in vivo disease models. The mechanism of 3,2′-DHF-enhanced EV secretion demonstrated in this study was related only to the ERK-signaling pathway. Further experiments are needed to investigate changes related to the biogenesis of EV secretion in 3,2′-DHF-treated mesenchymal stem cells and to understand the underlying mechanisms of EV secretion.

The composition changes in EV cargo must be studied to assess the therapeutic effects of 3,2′-DHF. EV cargo plays a critical role in biological functions. Understanding EV composition changes after 3,2′-DHF treatment is essential to determine the efficacy and safety of enhanced EVs as a potential therapeutic agent.

The study provides insights into the potential of Fla-EVs as a wound-healing agent, but further studies are needed to address its limitations and fully understand the mechanisms underlying EV therapeutic effects. Those studies could lead to novel therapeutic strategies for inflammatory disorders and chronic wounds.

Treating mesenchymal stem cells with 3,2′-DHF produced twice as many extracellular vesicles, and those vesicles showed stronger anti-inflammatory and wound-healing effects than control vesicles. The strategy is promising, but the cargo and in vivo mechanisms still need investigation.