To investigate the effects of exosomes derived from human epidermal stem cells (ESCs) on fibroblasts of hypertrophic scars (HS).

The foreskin tissues were collected from 20 healthy young children (7~12 years old) who underwent foreskin resection in the Department of Pediatric Surgery, the Hospital, from July 2021 to January 2022. FBs were isolated by the collagenase digestion method, while ESCs were extracted by the rapid attachment method from the foreskin tissues and identified by flow cytometry. Six HS specimens were collected from patients with hyperplasia of HS in the Department of Burn Surgery, the Hospital, from October 2021 to May 2022. Fibroblasts derived from hypertrophic scars (HSFs) were isolated by the collagenase digestion method. ESC exosomes (ESC-Exo) and FB exosomes (FB-Exo) were extracted by differential high-speed centrifugation. The morphology was observed by projection electron microscopy, the particle size was detected by nanoparticle tracking analyzer, and the protein expression of CD9, CD63, and α-tubulin was detected by Western blotting. PKH67-stained ESC-Exo and HSFs were co-cultured for 24 hours to observe the phagocytosis of human ESC exosomes by HSFs. All HSFs were randomly divided into ESC-Exo group (including 5 μg/ml ESC-Exo, 10 μg/ml ESC-Exo, 20 μg/ml ESC-Exo and 30 μg/ml ESC-Exo group), FB-Exo group (including 20 μg/ml FB-Exo, 30 μg/ml FB-Exo group), and control group, with 3 wells in each group. The number of HSFs was measured by CCK8 at 24, 48 and 72 h to determine the effect of exosomes on their proliferative ability, as well as to determine the minimum treatment time and minimum effective concentration. After that, HSFs were randomly divided into the control group, ESC-Exo group, and FB-Exo group, with 3 wells in each group (the same applies to the following). The number and the ratio of HSF cells in the proliferative phase were detected by Ki67 fluorescence staining. Scratch tests were performed according to the lowest effective concentration screened by the CCK8 experiment. The scratch area of cells was observed at 0, 12, 24, and 48 h after treatment, and the cell migration rate was calculated. The collagen contraction experiment wasused to observe the remaining area of collagen at 0, 24, 48, and 72 h after treatment, and the ratio of collagen contraction area was calculated. After 48 hours of treatment, RNA and total protein were collected, and the mRNA and protein expressions of collagen type Ⅰ (COLⅠ) and α-smooth muscle actin (α-SMA) were detected by real-time fluorescence quantitative (RT-PCR) and Western blotting. The relative fluorescence expression intensity of COLⅠ and α-SMA was confirmed by fluorescence staining. Data were compared by repeated measure analysis of variance, one-way analysis of variance, and independent sample t-test.

At 24 hours of culture, FBs showed a typical fusiform structure, and ESCs arranged in "paving stone" like clusters. ESCs and FBs were identified by flow cytometry. The exosomes were vesicular and positively expressed CD9 and CD63 but did not express α-tubulin. After 24 hours of co-culture, human ESC-Exo was swallowed into HSFs. After 24 to 72 hours of culture, CCK8 tests showed the proliferation of HSFs in groups with 20 μg/ml and 30 μg/ml ESC-Exo were significantly lower than in the control group. After 24 hours of culture, the absorbance of CCK8 in the 5 μg/ml ESC-Exo group, 10 μg/ml ESC-Exo group, and 30 μg/ml FB-Exo group were not different from that in the PBS group (t-values were 0.45, 2.04, 0.39, P>0.05). The absorbance of CCK8 in the 20 μg/ml FB-Exo group was slightly higher than that in the PBS group (t=4.52, P<0.05), while the absorbance of CCK8 in 20 μg/ml ESC-Exo and 30 μg/ml ESC-Exo groups was significantly lower than that in PBS group (t-values were 7.06, 15.26, P<0.001). These results indicated that the ESC-Exo at this concentration could significantly inhibit the proliferation of HSF, and the inhibitory ability became more obvious with the extension of treatment time (interaction F=5.19, P<0.001). In addition, 30 μg/ml ESC-Exo showed more significant inhibition than 20 μg/ml ESC-Exo at 24, 48, and 72 h (t-values were 5.88, 5.18, 13.64, P<0.05). The shortest effective time was 24 hours, and the lowest effective concentration was 20 μg/ml. Therefore, 20 μg/ml was selected as the following experimental concentration. After 24 hours of treatment, the ratio of Ki67-positive cells in the ESC-Exo group was obviously decreased (t=16.97, P<0.001), while there was no significant difference in the FB-Exo group (t=1.01, P>0.05). After a co-culture with ESC-Exo for 12, 24, and 48h, the migration ability of HSF decreased significantly. The migration rates of scratched cells in the ESC-Exo group were (52.86%±5.02%), (59.10%±7.45%), and (70.78%±11.33%), respectively. They were lower than the control group (60.87%±3.35%), (92.15%±3.61%), and (100.00%±0.00%). The difference was statistically significant (t-values were 2.57, 10.60, 9.37, P<0.05), and the effect increased with the extension of time (interaction F=33.26, P<0.001). The cell mobility of the FB-Exo group was (58.78%±5.46%), (89.69%±5.80%), and (98.40%±1.28%), respectively, which showed no significant difference compared with the control group (t-values were 1.08, 1.28, 0.83, P>0.05). The results of the collagen contraction experiment showed that after treatment for 24, 48, and 72 h, the ESC-Exo inhibited collagen gel shrinkage significantly, and the cell shrinkage rates were (23.07%±8.69%), (30.68%±6.18%), (45.92%±3.74%), respectively. They were lower than the control group (46.18%±2.21%), (66.80%±7.34%), and (76.65%±3.47%). The difference was statistically significant (t-values were 5.68, 8.88, 7.55, P<0.001), and the effect increased with the extension of time (interaction F=10.28, P<0.001). The cell shrinkage rates in the FB-Exo group were (41.19%±12.33%), (63.54%±4.20%), and (73.05%±3.22%), respectively, showing no significant difference compared with the control group (t-values were 1.09, 0.71, 0.79, P>0.05). The relative mRNA expressions of COLⅠ and α-SMA in ESC-Exo treated for 48 hours were significantly lower than those in the control group (t=6.25, 3.07, P<0.05). Nevertheless, the relative mRNA expression of COLⅠ in the FB-Exo group was slightly higher than that of the control group (t=3.78, P<0.05), while there was no significant difference in α-SMA mRNA between FB-Exo group and control group (t=1.44, P>0.05). Western blot and fluorescence staining results also showed that ESC-Exo inhibited the expression of COLⅠ and α-SMA in HSFs (t-values were 7.00, 9.79, P<0.001), while FB-Exo didn′t (t-values were 2.59, 3.23, P>0.05).

A stable and reliable system for human ESCs culture and exosome isolation and extraction was established to verify the ability of ESC-Exo to inhibit the proliferation, migration, and contraction of fibroblasts derived from hypertrophic scars and suppress their transformation into myofibroblasts.