hUCMSCs were obtained and cultured according to previously described methods (Liao et al., 2023). Briefly, the umbilical cord was obtained from a healthy pregnant woman after informed consent was obtained. After the umbilical artery and umbilical vein were removed, the umbilical cord was rinsed three times with Dulbecco’s phosphate-buffered saline (D-PBS, Invitrogen), after which the soft gel tissues were cut into 0.5 cm × 0.5 cm pieces and added to gelatine-coated 10-cm tissue culture dishes. MSC NutriStem^® XF Basal Medium with MSC NutriStem^® XF Supplement Mix (Biological Industries) supplemented with 1% human platelet lysate (Sexton) was added to the culture, which was incubated at 37 °C until the hUCMSCs reached 80% confluency. The tissues were subsequently removed from the dishes, and the hUCMSCs were collected. hUCMSCs were derived from single donors. Passage 4 (P4) hUC-MSCs were used in all the experiments, and all the cells were cultured in complete medium (Biological Industries) supplemented with 1% human platelet lysate (Sexton).

Human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China), and human skin fibroblasts (HSFs) were purchased from the American Type Culture Collection (ATCC, United States). HUVECs were cultured in endothelial cell medium (ScienCell, United States) supplemented with 5% fetal bovine serum (FBS) (HyClone), 1% endothelial cell growth factor (ECGS), and 1% penicillin‒streptomycin (Gibco). HSFs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS and 0.5% penicillin‒streptomycin. All the cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂.

The phenotypes of the hUCMSCs were analysed via flow cytometry after incubation with CD90-FITC, CD29-PE, CD105-APC, CD73-PE, CD45-FITC, CD34-PE, CD105-APC, CD14-FITC, HLA-DR-PerCP, and the corresponding isotype control antibodies. All the antibodies were purchased from BD Pharmingen (San Diego, CA, United States). hUCMSCs were analysed via a flow cytometer (BD FACS Calibur™, United States). To analyse the adipogenic, osteogenic, and chondrogenic differentiation of hUCMSCs, the cells were cultured in different appropriate differentiation media (Cyagen Biosciences Inc., Guangzhou, China) for 2–3 weeks, after which they were stained with Alizarin Red, Oil Red O, and toluidine blue, respectively.

hUCMSCs were expanded from P3 to P4, and the P4 subculture supernatant was collected. Serum-free medium was used for culture during the experiment. Briefly, the culture media of the hUCMSCs were collected and centrifuged at 300 g for 10 min to remove the cells. The supernatant was transferred to a new centrifuge tube and centrifuged at 2,000 × g for 10 min. To remove dead cells and cell debris, the supernatant was transferred to another new centrifuge tube and centrifuged at 10,000 × g for 30 min. Next, the cell supernatant was ultracentrifuged at 100,000 × g for 90 min via a Beckman ultracentrifuge, and the supernatant was discarded and resuspended in precooled PBS. The exosomes were isolated by ultracentrifugation at 100,000 × g for 90 min, resuspended in the appropriate amount of PBS, and finally filtered into a new centrifuge tube with a 0.22 μm filter.

The protein content of the exosomes was quantified with a Pierce BCA protein assay kit (Thermo Scientific, United States). The surface characteristic protein markers of the exosomes, including CD9, CD81, CD63, TSG101, HSP70, Alix and Calnexin, were detected via Western blotting. All primary antibodies were purchased from Abcam and were detected by Western blotting. The morphology of the exosomes was detected with a Hitachi H-7650 transmission electron microscope (Tokyo, Japan), and the size distribution of the exosomes was measured via an NS300 instrument (Malvern Instruments Ltd., Malvern, United Kingdom) at 25 °C.

Exosomes were labelled with a PKH26 kit according to the manufacturer’s instructions (Sigma, United States). For the internalization assay, the cells were seeded in 12-well plates at the proper density and treated with 10 μg of PKH26-labelled hUCMSC-Exos. The cells were incubated in an incubator with 5% CO₂ for 24 h, washed twice with PBS and fixed in 4% paraformaldehyde for 10 min. Afterwards, the cell nucleus was stained with Hoechst (Solarbio, China), and the cytoskeleton was stained with FITC Phalloidin (Solarbio, China) according to the manufacturer’s instructions. The different fields of view were randomly imaged with laser confocal microscope (Zeiss, Germany), and statistical analysis was conducted.

For the cell proliferation assay, HUVECs and HSFs were trypsinized and then seeded at a density of 2 × 10³ cells/well in 96-well culture plates with 100 μL of the abovementioned specific cell culture medium. After overnight incubation, 1 μg (0.01 μg/μL), 5 μg (0.05 μg/μL), or 10 μg (0.1 μg/μL) of hUCMSC-Exos were added to each well after the culture medium was replaced, and proliferation was analysed according to the manufacturer’s instructions, with MTT (Sigma, Germany) added on days 1, 3, and 5, respectively.

For the cell migration assay, HSFs were seeded at a density of 1 × 10⁵ cells/well in 6-well plates and cultured to 90% confluence. Three parallel scratches were then made in each well with a 200 μL pipette tip, and the samples were washed with serum-free medium to remove the detached cells. The width of each scratch was measured as the baseline value, and 1 mL of medium was added to each well. The cells were stimulated with 10 μg (0.01 μg/μL) or 20 μg (0.02 μg/μL) of hUCMSC-Exos for 24 or 36 h. The scratch width was photographed with a microscope (Olympus, Tokyo, Japan), and the migration rate was immediately measured with ImageJ software.

For the tube formation assay, HUVECs (2 × 10⁴ cells per well) were seeded with 1 μg (0.01 μg/μL), 5 μg (0.05 μg/μL), or 10 μg (0.1 μg/μL) of hUCMSC-Exos or PBS in 96-well culture plates that had been coated with 60 μL of Matrigel Basement Membrane Matrix (Corning, United States), and 100 μL of medium was added to each well. Tube formation was detected via microscopy (Olympus, Tokyo, Japan) at 6 and 24 h of incubation, and the formation of tubular structures was analysed via ImageJ software.

HUVECs and HSFs were seeded at a density of 3×10⁵ cells/well in 6-well culture plates, under normoxic (37 °C, 5% CO₂, 21% O₂) or hypoxic (37 °C, 5% CO₂, 5% O₂) conditions for 24 h. After which they were treated with 20 μg (0.02 μg/μL) of hUCMSC-Exos were cultured under normoxic conditions for 12 h, the cells were collected and stained with an Annexin V/7-AAD apoptosis kit (BD, USA), and the samples were analysed via a flow cytometer (BD FACS Calibur^TM, USA)

All animal experiments were approved by the ethics committee of the institutional review boards at Shenzhen Beike Biotechnology Co., Ltd. (No. BK-DWLL-2022-0001). This study adheres to the ARRIVE guideline 2.0 for the reporting of animal experiments.

All the mice were provided by Zhuhai BesTest Bio-Tech Co., Ltd. (Zhuhai, China). Fifty-four 8-week-old male BALB/c mice were divided into three groups: 1) the normal group (n = 18, not processed), 2) the saline group (n = 18, 200 μL of saline), and 3) the Exo-200 μg group (n = 18, 200 μg/200 μL of hUCMSC-Exos). The mice had unrestricted access to standard chow and drinking water and were were housed in a specific pathogen-free facility under a 12-h light/dark cycle. This experiment was performed at a constant temperature, and a bipolar electrocautery device with a 7-mm brass sheet was used as the tip. The temperature was set to 90 °C, and the weight of the tip (0.5 kg) was applied as the injury pressure to the mouse’s dorsal skin area for 5 s to obtain a scald wound of consistent area and severity. Within 24 h, Hematoxylin and Eosin (H&E) histopathological staining analysis was performed on the injured tissues of the mice to ensure that the back injury degree of each model mouse was consistent and that the injuries were deep second-degree burns (Supplementary Figure S6). The wounds were treated with subcutaneous injections of PBS or hUCMSC-Exos at five different locations, photographed on days 0, 4, 7, 10, 14, and 21 with cameras, and subsequently analysed with ImageJ software. Animals were euthanized via air displacement with 100% CO₂, with a flow replacement rate of 30%–40% maintained throughout the process. This procedure was conducted on study days 4, 7, and 21, with six animals per group. Following the cessation of respiration, death was confirmed by cervical dislocation, after which tissue samples were harvested and processed.

HUVECs and HSFs were seeded in 12-well culture plates, starved for 4 h, and then treated with 10 μg (0.01 μg/μL), 20 μg (0.02 μg/μL), or 30 μg (0.03 μg/μL) of hUCMSC-Exos or PBS for 24 h. Scar tissue samples were obtained from sacrificed mice at 4, 7, and 21 days after administration. Total RNA was isolated from cell lines or scar tissue samples with TRIzol reagent (Invitrogen, United States) following the manufacturer’s standard protocol. The RNA was reverse transcribed to cDNA via a reverse transcription kit (Takara, Dalian, China). Real-time PCR amplification was performed via a 7500 Real-Time PCR system (Applied Biosystems, United States). The expression of the target genes was assessed via the CT (2^−ΔΔCT) method and normalized to that of GAPDH. The sequences of primers used in this study are shown in Table 1.

Samples of mouse serum and wound tissue samples were collected 4, 7, and 21 days after the operation. The levels of IL-6, IL-17, IL-1β, TNF-α, and IFN-γ in scar tissues were measured separately via colorimetric sandwich ELISA kits (R&D Systems, United States). The absorbance at 450 nm was measured with a multimode microplate reader (Molecular Devices, United States). All the assays were performed following the manufacturer’s specific instructions.

Wound areas, including the surrounding skin, were collected at 4, 7, and 21 days after the operation. The samples were fixed with 4% paraformaldehyde (Phygene, China), dehydrated with a series of graded ethanol solutions, embedded in paraffin, and sliced into 5 μm-thick sections. Hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed according to standard procedures. H&E staining was used to assess the infiltration of inflammatory cells during wound repair, whereas Masson’s trichrome staining was carried out to evaluate the content and maturity of the collagen in the wound beds. Photographs were taken at ×100 and ×200 magnification via a microscope and digital camera (Leica, Germany). Statistical analysis via ImageJ software was performed on five high-powered fields per sample.

For immunohistochemical staining, three animals from each group were examined. The sections were incubated with anti-TGF-β1, anti-CD31, anti-VEGF, anti-collagen I, and anti-collagen III antibodies (Abcam, 1:1000) overnight at 4 °C. The sections were subsequently rinsed with PBS three times and incubated with a secondary antibody solution for 1 h at room temperature. A microscope was used to obtain images, which were then analysed via ImageJ software. At least 5 fields (×400 magnification) were randomly selected for analysis; the data are presented as the percentage of positive cells out of the total number of cells.

Total small RNA constitution analysis for known/unknown RNA, miRNA Upset analysis, CPM gene expression density distribution, and correlation analysis of samples were conducted to verify the miRNA quality and consistent between three hUCMSC-Exos samples. We are used Gene Ontology (GO) and KEGG pathway enrichment analysis and annotation were performed by the cluster Profiler R package for top 22 high expressed miRNAs. TargetScan, miRanda, and miRNB online databases were applied for miRNA target-gene prediction. Cytoscape was used to construct the miRNA-mRNA network for predicting the target genes of miRNAs with high expression.

Statistical analyses were conducted via GraphPad Prism 9.3.0 software (Version X, United States), and the results were compared via one-way analysis of variance (ANOVA). All quantitative data are representative of these experiments and are shown as the mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

hUCMSCs exhibited a typical fibroblast-like morphology when cultured in vitro (Supplementary Figure S1a). The cells also showed adipogenic, osteogenic, and chondrogenic differentiation capabilities under the appropriate conditions for 2–3 weeks after being stained with Oil Red O (for adipocytes), Alizarin Red S (for osteoblasts), and Alcian blue (for chondrocytes) (Supplementary Figures S1b–d). hUCMSCs were collected at P4 and subjected to flow cytometry for surface characterization. The results demonstrated that hUCMSCs were strongly positive for CD73, CD90, CD29, and CD105 but negative for CD79a, CD34, CD45, CD14, and HLA-DR (Supplementary Figure S1e).

Exosomes were separated from the culture supernatant of hUCMSCs via ultracentrifugation. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting were performed to identify the diameter and concentration, morphological characteristics, and specific proteins of the hUCMSC-Exos, respectively. NTA was performed with a NanoSight instrument to measure the diameter of the exosomes, which had an average diameter of 139 ± 16.48 nm (Figure 1A). TEM revealed that the hUCMSC-Exos had a typical “saucer-like” exosome structure (Figure 1B). Moreover, the exosome markers were strongly positive for CD9, CD81, CD63, TSG101, HSP70, and Alix but negative for Calnexin, as determined by Western blotting (Figure 1C). Taken together, these results demonstrated that hUCMSC-derived exosomes were successfully isolated from the culture supernatant, which is similar to many previous reports (Zhou et al., 2021; Xiao et al., 2021).

Exosomes can be internalized through several mechanisms, including fusion and endocytosis (Gonda et al., 2019; Montecalvo et al., 2012). To investigate whether the isolated exosomes could enter the cytoplasm of target cells, FITC Phalloidin-stained HUVECs and fibroblasts were incubated with PKH26-labelled Exos for 24 h. Both types of cells presented with red fluorescence in their cytoplasm, which suggested that the hUCMSC-Exos were readily internalized by HUVECs and HSFs (Figure 1D). The results showed that exosomes could be successfully delivered into target cells, which is the necessary process for performing their functions.

We conducted a series of assays to investigate whether the hUCMSC-Exos could promote cell proliferation, migration and angiogenesis. The proliferation of HUVECs and HSFs cultured with hUMSC-Exos for 1, 3, or 5 days. We found that hUCMSC-Exos significantly promoted HSF proliferation in a dose-dependent manner at days 3and 5; however, they did not have an obvious influence on HUVEC proliferation at different time points. Only the Exo-1 μg group on day 5 promoted proliferation compared with the control group (Figures 2A,B). As shown in Figures 2C,D, hUCMSC-Exos effectively enhanced cell migration in a dose-dependent manner compared with that in control cells, with migration increasing 1.5-fold and 2.3-fold in 10 μg and 20 μg hUCMSC-Exos groups, respectively. Moreover, an in vitro tube formation assay was conducted to evaluate the ability of the hUCMSC-Exos to promote the formation of tubular structures in endothelial cells, and the results revealed that the hUCMSC-Exos significantly promoted tube formation after 24 h of culture (Figures 2E–G). Together, these results indicate that hUCMSC-Exo treatment promotes the proliferation and migration of HSFs and enhances angiogenesis in HUVECs.

To determine whether hUCMSC-Exos affect the apoptosis of cells, we established apoptosis models in which HUVECs and HSFs were induced with Hypoxia and then incubated with exosomes. We conducted flow cytometry analyses via Annexin V/7-AAD double-staining. As shown in Supplementary Figures S2a,b, compared with that of the Hypoxia group, the apoptosis rate of the cells treated with the hUCMSC-Exos was significantly lower, suggesting that the hUCMSC-Exos inhibited the apoptosis of the HUVECs (Supplementary Figure S2a) and HSFs (Supplementary Figure S2b) in vitro. Given the positive results that we obtained in the apoptosis study, we further examined the effects of hUCMSC-Exos on the regenerative gene regulation of cells. Our results revealed that exosomes increased the expression of key factors, such as PCNA, MCP-1, TGF-β, PDGFα, PEGF, VEGF, EGF, and FGF2, in target cells; among these factors, exosomes regulate MCP-1 (4 fold)in endothelial cells most obviously, and MCP-1 can promote the chemotaxis of inflammatory cells and the proliferation and migration of vascular endothelial cells. However, the PDGF-α (2.8 fold) and FGF (2.2fold) genes of fibroblasts are significantly regulated and are involved mainly in cell proliferation and differentiation and the induction of vascular hyperplasia. These results suggest that exosomes play a significant role in regulating the expression of genes associated with tissue repair and regeneration (Supplementary Figures S2c,d).

To evaluate the therapeutic potential of the hUCMSC-Exos in terms of the wound healing rate and scar formation, a mouse wound model with scalded skin was used. Our model produces a deep partial-thickness burn (deep second-degree), where signs of dermal injury included vascular damage or blockage, collagen damage, and dermal appendage damage but does not involve the complete destruction of skin appendages, making it a suitable model for studying repair processes (Supplementary Figure S6). Cutaneous wounds were created on the dorsal skin of male BALB/c mice, followed by local injection of 200 μg of hUCMSC-Exos (n = 18/group) or an equal volume of diluent (saline) around the wound area once daily for three consecutive days (Figure 3A). The body weight changes of the mice during the modelling and treatment phases were documented, and the Exos group at days 4 and 7 presented greater body weights than did the saline group and significantly differed from the saline group in terms of wound healing (Figure 3B). As shown in Figures 3C,D, cutaneous wound closure was greater in the Exos group than in the saline group after the subcutaneous administration of the hUCMSC-Exos. Notably, there was a significant difference between the two groups, especially on day 4.

During the wound healing process, hUCMSC-Exos may stimulate nearby stem cells to proliferate, differentiate and repair tissue through paracrine signalling (Zhou et al., 2023). Moreover, reepithelialization and angiogenesis occur (Liu et al., 2021). As shown by the H&E staining results in Figure 3E, the histological structure of the regenerated dermis showed that the structure of the normal mouse dermis and epidermis was regular; dermal cell destruction, epidermal thickening, and hyperkeratosis were observed in the saline group at days 4 and 7; on the other hand, the dermal cell structure was noticeably more regular in the Exos group than in the saline group, and there was a significant improvement in stratum corneum hyperkeratosis. On day 21, skin injury repair was assessed in both groups of mice, and better results were observed in the Exos group (Figures 3G,H). The Masson staining results revealed skin fibrosis: the Exos group presented normal collagen deposition and more skin appendages, whereas the saline group presented excessive collagen deposition at days 7 and 14 (Figures 3F,I). Similarly, we detected high expression of the PCNA and α-SMA genes at the site of the injured skin, which are highly correlated with cell proliferation and the degree of fibroblast-to-myofibroblast transformation and wound contraction (Supplementary Figures S4c,d). Taken together, these results indicate that exosomes strongly promote wound repair and skin regeneration.

Angiogenesis is an essential physiological process for maintaining provisional granulation tissue. Our experiments revealed that hUCMSC-Exos effectively promoted angiogenesis in a wound mouse model in vivo (Figures 4A–D; Supplementary Figures S3a,b). Immunostaining for TGF-β1 (Figure 4A), CD31 (Figure 4C) and VEGF (Supplementary Figure S3a) in pathological skin tissues was performed at days 4, 7, and 21 after treatment with the hUCMSC-Exos to investigate their proangiogenic effects in vivo. Compared with the saline group, the Exos group presented notable increases in the expression of TGF-β1-, VEGF- and CD31-positive microvessels in skin tissue at days 4 and 7 (Figures 4B,D; Supplementary Figure S3b), which indicates that Exos significantly stimulate angiogenesis. However, the expression of CD31 and VEGF in the Exos group had already decreased on day 21 (Figure 4D; Supplementary Figure S3b). This finding indicated a tendency toward normalization at an earlier process stage, and the Exos group underwent skin remodelling at a more accelerated rate. These findings closely resemble the typical angiogenic patterns observed in the skin of wounded mice (Supplementary Figure S3e). In addition, we detected the expression of the VEGF and PDGF genes, which are highly correlated with angiogenesis, in injured tissues and reached similar conclusions (Supplementary Figures S4a,b). Specifically, the Exos group exhibited a more robust angiogenic response in the injured skin, characterized by earlier onset, increased vessel numbers, and higher vessel densities. In contrast, the saline group exhibited a less pronounced angiogenic response, with fewer vessels and a greater amount of inflammatory exudate. These results indicate that hUCMSC-derived exosomes have superior proangiogenic properties.

Additionally, we conducted experiments to determine the potential impact of hUCMSC-Exos on collagen synthesis at the wound site. As determined by Masson’s trichrome staining, wounds treated with hUCMSC-Exos exhibited greater collagen maturity than those in the other groups did (Figures 3F,I). To examine the influence of the hUCMSC-Exos on collagen, IHC staining was performed to detect the expression of collagen I and collagen III (Figures 4E,F; Supplementary Figures S3c–e). This analysis revealed that the hUCMSC-Exos had a stimulatory effect on collagen deposition, which is consistent with previous reports (Lin et al., 2024). Notably, increased collagen maturity was observed in the wound area, accompanied by a reduced ratio of collagen III to collagen I, which became more apparent with repeated hUCMSC-Exos treatments than in the saline groups. In essence, the wound healing process involves a pivotal transition in collagen types; type III collagen is initially predominant but is subsequently gradually replaced by type I collagen (Gelse et al., 2003). Furthermore, we obtained similar results by testing the gene expression of collagen III to collagen I in the injured tissues (Supplementary Figures S4e–g). Thus, these results demonstrated that hUCMSC-Exos regulate collagen deposition and organization, promote skin repair.

Our study investigated the impact of hUCMSC-Exos on wound inflammation in mice. To evaluate the anti-inflammatory potential of these exosomes, we employed ELISA to quantify the expression levels of inflammatory markers, such as IL-1β, IL-17, IL-6, TNF-α, and IFN-γ, in tissue homogenates from mice treated with exosomes or saline. Our findings revealed that inflammation occurred during the early stages of wound healing (Figures 5A–E). Compared with that in the normal group, the expression of inflammatory factors in the saline-treated group significantly increased. Importantly, we observed a significant reduction in the expression of these inflammatory markers after hUCMSC-Exos treatment, especially at the 7-day time point. Similarly, we found that the expression of inflammatory cytokines in mouse serum samples was comparable (Supplementary Figures S5a–d). These results collectively indicate that hUCMSC-Exos possess robust anti-inflammatory properties, thus accelerating the skin repair process.

Exosomes can posttranscriptionally regulate coding genes through miRNAs, thereby affecting the biological activities of recipient cells (Li B. et al., 2021; Li C. et al., 2021). To explore the potential molecular mechanisms of the hUCMSC-Exos RNA cargo in wound healing, the small RNAs from the hUCMSC-Exos and their target genes were examined via RNA sequencing. We observed a significant correlation in the enrichment patterns of miRNA molecules across the three batches of exosomes, indicating a high degree of consistency and reproducibility in our findings (Figure 6A). Moreover, let-7a-5p, miR-26a-5p, let-7f-5p, miR-126-3p, miR-127-3p, miR-222-3p, let-7i-5p, miR-100-5p, and miR-92a-3p were highly expressed in the hUCMSC-Exos (Figure 6B). Interestingly, the high levels of several specific miRNAs, such as miR-26a-5p, miR-126-3p, miR-146a-5p, and miR-127-3p, may suggest a potential inhibitory effect of hUCMSC-Exos on wound healing.

Additionally, functional enrichment analysis revealed the dominant pathway enriched in the hUCMSC-Exos. We performed a functional enrichment analysis of 22 selected miRNAs via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. GO analysis of the selected miRNAs revealed similar affected processes, such as the cell cycle, cell motility and differentiation (Figure 6C). KEGG pathway enrichment analysis revealed that the miRNAs enriched in the PI3K‒Akt signalling pathway, MAPK signalling pathway, and Wnt signalling pathway (Figure 6D). The interactions of known miRNAs and mRNAs with respect to cell proliferation and migration, signalling molecules and interactions, signal transduction, and the immune system were observed via Cytoscape (Figure 6E). The most abundant miRNAs, such as miR-126-3P, miR-26-5P, and miR-146a-5P, were found to target genes encoding ULK2, COL19A1, IL6ST, SLC7A5, CDKN2AIP, MED1, KLF7, GABPA, and IRAK1, which are associated with skin injury repair. Notably, these results are consistent with the finding that the most common miRNAs in hUCMSC-Exos and their target genes promote cell proliferation and reduce inflammatory responses. Together, these bioinformatic results suggest that exosome-derived miRNAs can contribute to promoting skin repair and regeneration.