All animals were obtained from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China) and were housed under ordinary conditions. The animals were maintained under a 12-h light/12-h dark cycle at stable temperature (22-26 °C) and humidity (50%-70%). They were given free access to food and water throughout the study period.

hUC-MSCs were purchased from Guangxi TaiMeiRenSheng Biotechnology Co. Ltd. Fourth-passage cells at logarithmic phase were inoculated in T175 air-permeable cell culture flasks with a dedicated medium (HUXUC-90011, Cyagen). The cells were cultured at 37 °C in an incubator containing 5% CO₂. When the adherent cells reached a confluence of 80%-90%, the used cell medium was discarded and replaced with a serum-free medium. After 48 h, cells were centrifuged and cell supernatants were collected. Exosomes in the cell supernatants were extracted by ultracentrifugation. Impurities in the cell supernatants were removed by centrifugation at 300 × g for 10 min, followed by the removal of dead cells by centrifugation at 2000 × g for 20 min; then, cell debris was removed by centrifugation at 10000 × g for 30 min. Subsequently, cell supernatants were filtered using a 0.22 μM membrane filter and centrifuged in an ultracentrifuge (Beckman, America) for 120 min at 120000 × g, and the supernatants were discarded. The precipitated exosomes were re-suspended in phosphate buffered saline (PBS) and centrifuged again for 120 min at 120000 × g, and the exosomes precipitate were finally re-suspended in filtered PBS. All centrifugations were performed at 4 °C.

Exosomes were identified using a nanoparticle tracking analyzer (ZetaView, Particle Metrix, Germany), which was used to measure their concentration and particle size. A transmission electron microscope (HT-7700, Hitachi High-tech Company) was used to observe the shape and size of exosomes. Western blot was used to detect the surface marker proteins, CD9, Tsg101, and Calnexin, in exosomes. The protein concentration of hUC-MSCs-derived exosomes was determined using a BCA protein quantitation kit (Zoman, Biotechnology, ZD301). After adding the required proportion of loading buffer, the sample was heated at 95 °C for protein denaturation. Protein from each sample was separated on a sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk for 2 h, the membranes were incubated overnight with anti-CD9 (20597-1-AP, Proteintech), anti-Tsg101 (28283-1-AP, Proteintech), and anti-Calnexin (10427-2-AP, Proteintech) antibodies. Subsequently, the secondary antibody (SA00001-2, Proteintech) was added, followed by incubation for 1.5 h. Band visualization was achieved using a chemiluminescence kit (P0018FM, Beyotime, China) and observed on a chemiluminescent imaging system (MiniChemi 610, Sagecreation, China).

An absorbable gelatin sponge was purchased from Jiangxi Xiangen Medical Technology Development Co., Ltd. It was sliced into appropriate sizes, and immersed in the exosome solution mixed with trehalose, to ensure that the gelatin sponge fully absorbed the exosomes. Subsequently, the loaded gelatin sponge was freeze-dried and characterized via scanning electron microscopy.

The effect of the hUC-MSCs-derived exosomes on mouse epithelioid fibroblasts (L929) was evaluated using the CCK-8 assay. Briefly, L929 cells were inoculated into 96-well plates and divided into control, sponge extract, and exosome groups. The cell density was adjusted to 10000 cells/well and then incubated at 37 °C in a 5% CO₂ incubator for 12 h. Subsequently, the medium in the exosome group was adjusted to ensure that each well contained 6 × 10⁶ particles of exosomes and then incubated at 37 °C under 5% CO₂ for a further 6 h. After 6 h, 10 μL of CCK-8 reagent was added to all groups, followed by incubation in the dark for 1 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader.

Briefly, 5 mL of fresh blood from New Zealand rabbits was stirred in one direction with a glass rod, followed by fibrin removal; then, 20 mL of normal saline was added. The mixture was mixed well and centrifuged at 2000 rpm for 10 min. The supernatant was discarded, normal saline was added, and the process was repeated 3-4 times until the supernatant was clear. Finally, the supernatant was discarded, and 1 mL of fresh blood was added to 49 mL of normal saline to obtain a 2% rabbit red blood cell suspension for subsequent use. The assay was divided into negative control, positive control, sponge, and exosome-loaded sponge groups. Subsequently, 500 μL of the 2% rabbit red blood cell suspension was added to each group, and an equal volume of liquid was added (normal saline was added to the negative control group, and Triton X-100 with a mass fraction of 0.5% was added to the positive control group. Gelatin sponge extract at a concentration of 10 mg/mL was added to the sponge group, and gelatin sponge extract with 1 × 10⁹ exosomes was added to the exosome-loaded sponge group). Then, the solutions were mixed evenly and incubated at 37 °C for 3 h. The samples were then centrifuged at 3000 rpm for 3 min. The optical density (OD) of the supernatant was measured at a wavelength of 545 nm, and the hemolysis rate was calculated by photography. The hemolysis rate was calculated as (OD_sam - OD_neg)/(OD_pos - OD_neg) × 100% (1)[13].

The skin irritation test was performed using three New Zealand rabbits. The hair on their backs was carefully removed. Four specific areas on their skin were identified and treated with different fluids. They were blank group without any articles, NaCl group with 0.9%NaCl solution, sponge group with 10 mg/mL sponge extract, and exosome group with 5 × 10⁸ exosomes solution. The application was repeated every 24 h until absorption was complete. The rabbits were closely monitored to assess any changes in their skin condition, such as redness, swelling, inflammation, or other related symptoms. After 72 h, skin samples were harvested from treated sites for histopathological examination to observe any signs of inflammation in the skin tissue.

Eighteen male Sprague-Dawley (SD) rats were divided into blank control, sponge, and exosome-loaded sponge groups. After anesthesia with 10% chloral hydrate, the hair on their back was removed, the subcutaneous bags were excised, and 1 cm × 1 cm × 0.5 cm of either gelatin sponges or gelatin sponges loaded with exosomes were implanted into them, while the blank control group was not implanted with any gelatin sponge. The adjacent skin was collected for pathological analysis on day 7 and 14, respectively, to evaluate the in vivo biocompatibility of the gelatin sponges.

The gelatin sponges were cut into 1 cm × 1 cm × 0.5 cm sections, and preheated in an oven at 37 °C for 5 min. In the sponge and exosome groups, the gelatin sponges were infiltrated with 100 μL of normal saline and 100 μL of exosome solution containing 5 × 10⁸ exosomes, respectively. Next, 100 μL of rat whole blood was added to each gelatin sponge in both groups for adequate absorption, followed by 10 μL of 0.2 M calcium chloride solution, and incubated at 37 °C for 15 min. Subsequently, 25 mL of deionized water was used to rinse blood from the gelatin sponge surface without coagulation. Finally, the absorbance (Abs1) of the washed liquid was measured at 540 nm by using a microplate reader. For the blank control (Abs0), 100 μL of whole blood was added to 25 mL of deionized water, and the blood coagulation index (BCI) was calculated using the formula: BCI (%) = Abs1/Abs0 × 100% (2).

The hemostatic capacity of the exosome-loaded gelatin sponge was evaluated using a rat liver defect hemostasis model[14]. Twelve SD rats were divided into sponge and exosome-loaded sponge groups (n = 6 rats/group), with an equal distribution of male and female rats. After anesthesia induction with 10% chloral hydrate, an abdominal incision was made to expose the liver, and the tissue fluid around the liver was carefully removed using a dry cotton ball to prevent inaccurate blood weight measurements. The liver was placed on a pre-weighed filter paper, and a 0.5 cm long and 0.2 cm deep wound was created on the liver using a scalpel. The gelatin sponge loaded with exosomes was immediately placed at the bleeding site and bleeding was observed. After the bleeding stopped completely, the bleeding time was recorded, and the blood-absorbed filter paper was weighed. The weight difference between the front and rear of the filter paper was considered as the weight of blood loss from the rat liver.

Eighteen male SD rats (220-280 g) were randomly divided into control, sponge, and exosome-loaded sponge groups. After anesthesia induction with 10% chloral hydrate, the hair on the back of rats was removed, and a full-thickness skin defect wound (approximately 1.5 cm × 1.5 cm) was created on their backs. Photographs were taken to record the original area, and the wound was covered with either a gelatin sponge or an exosome-loaded gelatin sponge, and wrapped with a transparent dressing; the control group received only a transparent dressing. On day 7 and 14 post the procedure, the gauze was opened to observe and record wound healing. All wounds were photographed, the area was analyzed using Image J software (National Institutes of Health, Bethesda, MD), and skin samples around the wounds were collected and fixed with 4% paraformaldehyde for pathological analysis.

Masson’s trichrome staining was used to determine collagen content and wound maturity. Image J software was used to analyze the areas of interest. The collagen content was quantified. Ki67 was used to evaluate local whole-cell proliferation and CD31 staining was used to evaluate angiogenesis. Sections were incubated with antibodies against Ki67 (1:600, #AF0198, Affinity) or CD31 (1:1000, Cat No.28083-1-AP, Proteintech). Subsequently, the secondary antibody was added (2118D1104, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). Six randomly selected areas were imaged using an inverted microscope for each specimen, and the number of Ki67- and CD31- positive cells/positive blood vessels was detected using image J software.

SPSS 26.0 (IBM Corp., Armonk, NY, United States) and GraphPad Prism 8.0 (GraphPad Software Inc.; San Diego, CA, United States) were used for statistical analyses. Paired or unpaired t-tests were used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among three or more groups. P < 0.05 was considered statistically significant.

We successfully isolated exosomes from hUC-MSCs by ultracentrifugation. After successful separation, the morphology of the exosomes was observed by using TEM, and a typical cup-shaped structure was observed (Figure 1A). The exosome concentration and particle size were measured using a nano-particle size analyzer. The average exosome particle size was 144.7 nm, consistent with the particle size of exosomes (30-150 nm) (Figure 1B). Western blot results showed that the extracted exosomes successfully expressed the surface marker proteins CD9 and Tsg101, but not Calnexin (Figure 1C).

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Figure 1 Characterization of human umbilical cord mesenchymal stem cell-derived exosomes. A: Morphology of the exosomes assessed by transmission electron microscopy. Scale bar = 200 nm; B: Grain size of the exosomes; C: Surface protein markers of exosomes. MSC: Mesenchymal stem cell; Exo: Exosome.

The exosomes on the gelatin sponge were detected via scanning electron microscopy. The gelatin sponge with exosomes was coarser than the sponge without exosomes, and exosome particles could be observed (Figures 2A and B).

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Figure 2 Characterization of gelatin sponge loaded with exosomes. A: SEM image of the gelatin sponge; B: SEM images of the gelatin sponge loaded with exosomes. The orange arrows show the exosomes.

The gelatin sponge loaded with exosomes did not induce significant toxicity toward L929 cells as the cells remained viable following exposure to them (Figure 3A). Moreover, the hemolytic assay showed that no hemolysis was observed with the naked eye using the gelatin sponge loaded with exosomes (Figure 3C). Measuring the OD value of the solution and calculating the hemolysis rate, we found that the hemolysis rates of the gelatin sponge and exosome-loaded gelatin sponge in whole blood were -0.01% and 0.18%, respectively, which were within the permissible range for biological materials (less than 5%), indicating that the exosome-loaded gelatin sponge was safe for use (Figure 3B). All experimental rabbits had an intact epidermis, with normal sebaceous glands, hair follicles, and hair structures; there were no notable pathological changes, such as inflammatory cell infiltration, tissue congestion, and edema (Figure 3D).

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Figure 3 Biosafety of gelatin sponge loaded with exosomes. A: Cell viability of gelatin sponge loaded with exosomes; B and C: Hemolysis ratio of gelatin sponge loaded with exosomes; D: Representative diagram of pathological condition of rabbit skin after stimulation with exosome and sponge extract. Scale bar = 50 μm. Exo: Exosome.

To investigate the in vivo biocompatibility of gelatin sponge loaded with exosomes, we implanted the gelatin sponge into the dorsal subcutaneous space of SD rats, and observed the condition of the implant on day 7 and day 14 (Figures 4A and B). A portion of the gelatin sponge was retained in the body on day 7 and day 14; however, the gelatin sponge on day 14 was obviously smaller than that on day 7, indicating that the gelatin sponge could be degraded in the body. Adjacent skin tissue samples were collected for pathological analysis. After 7 and 14 d of implantation, there was no obvious inflammatory response around the tissue, and no obvious tissue fibrosis or necrosis was observed (Figure 4C), indicating that the gelatin sponge loaded with exosomes showed good biocompatibility.

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Figure 4 Biocompatibility of gelatin sponge loaded with exosomes. A and B: The size of the gelatin sponge remaining on day 7 and day 14; C: Hematoxylin and eosin staining of the adjacent skin tissues in the dorsal subcutaneous space of SD rats after 7 and 14 d of implantation. Scale bar = 100 μm. Exo: Exosome.

We evaluated the clotting ability of gelatin sponge and gelatin sponge loaded with exosomes. Our findings showed that the BCI values of the gelatin sponge and exosome-loaded gelatin sponge groups were 45.63% and 31.47%, respectively, indicating that both groups had significant clotting ability, and the clotting ability of the exosome-loaded gelatin sponge was significantly higher than that of the gelatin sponge group (Figure 5A).

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Figure 5 Hemostatic effect of gelatin sponge loaded with exosomes. A: Clotting ability of exosome-loaded gelatin sponge; B: Experimental process of hemostasis in liver injury model; C: Hepatic blood loss in rats; D: Time required for hemostasis. ^aP < 0.05, ^cP < 0.001. n = 6. Exo: Exosome.

After establishing a liver defect model, we covered the wound with a gelatin sponge and recorded blood loss and hemostasis time. The blood loss and hemostasis time of the gelatin sponge group loaded with exosomes were significantly lower than those of the gelatin sponge group, indicating that the gelatin sponge loaded with exosomes had a better hemostatic effect than the conventional gelatin sponge (Figures 5B-D).

After establishing a rat full-thickness skin defect model, the wound was covered with a gelatin sponge, and the gauze was opened on day 7 and day 14 post the procedure to observe and record the wound healing status. The wound size of all groups decreased with time. Moreover, the wound size of the gelatin sponge group loaded with exosomes was smaller than that of the other two groups, indicating that the gelatin sponge loaded with exosomes promoted wound healing better than the conventional gelatin sponge (Figures 6A and B). Histopathological results showed that on day 14, the gelatin sponge loaded with exosomes group demonstrated better regeneration of hair follicles and sebaceous glands than the other two groups (Figure 6C).

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Figure 6 Wound healing effect of gelatin sponge loaded with exosomes. A: Representative images of rat wounds; B: Statistical analysis of the wound area; C: Hematoxylin and eosin-stained wound tissue sections as observed under a light microscope. The orange arrows indicate the stratum corneum, the yellow arrows indicate the granular layer, the green arrows indicate the sebaceous gland, the blue arrows indicate the hair follicle, the black arrows indicate the collagen fibers, the light green arrows indicate the blood vessels, and the purple arrows indicate the subcutaneous fat. Scale bar = 100 μm. ^aP < 0.05. n = 3. Exo: Exosome.

Masson’s trichrome staining was performed on the skin adjacent to the wound to evaluate the formation and distribution of collagen, which is important for promoting wound healing. Microscopic observations showed that collagen fibers in the skin of all three groups were relatively few on day 7. However, on day 14, the collagen fibers in the skin of the three groups gradually increased along with gradual healing of the wound (Figure 7A). Statistical analysis showed that the collagen fibers in the exosome-loaded gelatin sponge group were significantly higher than those in the other two groups on day 14; on day 7, the collagen fibers in the gelatin sponge and exosome-loaded gelatin sponge groups were significantly higher than those in the control group (Figure 7B). These findings suggested that the exosome-loaded gelatin sponge promoted wound healing by promoting collagen fiber synthesis.

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Figure 7 Gelatin sponge loaded with exosomes promotes collagen formation, cell proliferation, and angiogenesis during the wound healing process. A: Masson-stained wound tissue sections as observed. Scale bar = 100 μm; B: Statistical analysis of the percentage of collagen volume with respect to tissue volume; C: CD31-positive vessels in wound tissue sections. Scale bar = 100 μm; D: Statistical analysis of CD31-positive vessels; E: Ki67-positive cells in wound tissue sections. Scale bar = 50 μm; F: Statistical analysis of Ki67 positive cell area percentage. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Exo: Exosome.

To explore the possible mechanism of action of gelatin sponges loaded with exosomes, we employed immunohistochemical techniques to evaluate angiogenesis and cell proliferation in the granulation tissue. To evaluate angiogenesis, we stained the skin near the wound for CD31. On day 7 and day 14, the number of CD31-positive blood vessels in the exosome-loaded gelatin sponge group was significantly higher than that in the control group; on day 14, this number was significantly higher than that in the gelatin sponge group (Figures 7C and D). Subsequently, we detected the proliferation of whole cells of granulation tissue in the skin near the wound and found that the proportion of Ki67-positive cells in the exosome-loaded gelatin sponge group was significantly higher than that in the control and gelatin sponge groups on day 7 and day 14. However, there was no statistically significant difference in the proportion of Ki67-positive cells between the gelatin sponge and control groups (Figures 7E and F). These results indicated that the gelatin sponge loaded with exosomes could promote cell proliferation and angiogenesis, thereby accelerating wound healing.

Hemostasis and wound healing are one of the common problems in clinics which need to be paid attention to. Gelatin sponge is often used as a hemostatic material in clinics. Exosomes have been proved to play an important role in wound repair. Therefore, it is worth studying whether the combination of exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) and a gelatin sponge could promote hemostasis and wound healing more efficiently.

Poor wound healing would contribute a negative impact on patients’ quality of life and aggravate pain, stress and depression, so it is of great significance to find ways to effectively promote wound healing.

The present study aimed to investigate the hemostatic and wound healing efficacy of a gelatin sponge loaded with hUC-MSCs-derived exosomes.

After the exosomes were extracted and characterized by ultracentrifugation, we loaded the exosomes on a gelatin sponge. Then, in vitro and in vivo experiments, including cell viability assay, hemolysis assay, skin irritation test, and histocompatibility assay were performed to verify the safety of the exosome-loaded gelatin sponge. Subsequently, whole blood coagulation index test, hemostatic assay using a rat liver defect hemostasis model, and full-thickness skin defect healing promoting test were performed to verify the effects of the exosome-loaded gelatin sponge in hemostasis and wound healing.

We successfully extracted exosomes from hUC-MSCs. The safety experiments showed that the gelatin sponge loaded with exosomes would not cause abnormal proliferation of L929 cells, hemolysis, or irritation to skin and tissues. In addition, the exosome-loaded gelatin sponge had a better hemostatic effect than the traditional gelatin sponge, which can promote the formation of collagen fibers and blood vessels around the wound and increase the proportion of Ki67-positive cells, thus promoting the wound healing.

In a word, gelatin sponge loaded with hUC-MSCs-derived exosomes is safe. It is better than traditional gelatin sponge in stopping bleeding and promoting wound healing.

The gelatin sponge loaded with exosomes derived from hUC-MSCs may be a potential material to stop bleeding and promote wound healing.