Effect of rmEGF combined with ELF-EMF on promoting wound healing in rats

2.1ELF-EMF equipment

The ELF-EMF equipment used in the study is shown in Fig. 1. The frequency of the magnetic field was 10 Hz, and a Helmholtz coil was used to ensure the uniformity of the magnetic field strength in the coil.

2.2Animal model

Anesthesia was induced by injecting 10 ml/kg of avertin (1.25%) intraperitoneally. After successful anesthesia, the rats were shaved, disinfected, and placed with bedding. A dorsal wound 20 mm × 20 mm was made on the back of the rats (Fig. 2-A), and then grouped: The SD rats in the normal control group were not treated with rmEGF and had no ELF-EMF intervention. SD rats in EGF alone group were treated with rmEGF with no ELF-EMF intervention. SD rats in the EGF+ELF-EMF group were treated with rmEGF and exposed to 10 Hz/1.5 mT ELF-EMF (Fig. 2-B).The rats received the 10 Hz/1.5 mT magnetic field intervention for 2 hours per day. The wound healing rate of rats was calculated and the samples were stained at different time intervals (3 d, 7 d, 11 d, and 15 d) (Fig. 2-C).

Figure 2.

Establishment of the animal model and the magnetic field intervention after grouping. A is wound establishment; B is SD rats exposed to 10 Hz/1.5 mT stimulation; C is measurement of wound healing.

2.3HE staining of wound tissue

The original wound on the back of the rats was harvested with a size of about 20 mm × 20 mm. Paraffin sections were fixed with 4% paraformaldehyde solution for 12 hours, rinsed with distilled water, dehydrated by alcohol using stepwise gradient and immersed in absolute ethanol xylene solution. The paraffin was removed overnight, the slices were subjected in warm water, the slices were dewaxed, stained, and finally sealed with neutral tree glue. They were observed and photographed under a microscope.

2.4Statistical analysis

All experiments were repeated three or more times and analyzed by GraphPad Prism9.4.1(681) software. The data between multiple groups were tested for homogeneity of variance, and the data between groups were analyzed by one-way analysis of variance. If the homogeneity of variance test was not obtained, the rank-sum test was used to analyze the data between groups. ^*P< 0.05, ^** P< 0.01 indicates that the difference was statistically significant.

3.1Calculation of wound healing rate

On the third day of wound healing, the average wound healing rate was 20% in normal control group, 35% in EGF alone group, and 50% in EGF combined with ELF-EMF group. On the 7th day of wound healing, the average wound healing rate of normal control group was 60%, the average wound healing rate of EGF alone group was 73%, and the average wound healing rate of EGF+ELF-EMF group was 93%. On the 11th day of wound healing, the average wound healing rate of normal control group was 90%, the average wound healing rate of EGF alone group was 97%, and the average wound healing rate of EGF+ELF-EMF group was 99%. On the 15th day of wound healing, the wounds of all the groups were completely healed, and some wounds had scar formation. The results showed that on the 7th day of wound healing, the wound healing rate of the EGF combined with ELF-EMF group increased rapidly, compared with the control group, and the difference was statistically significant (^**P< 0.01). On the 11th day of wound healing, the wound healing rate of each group was higher, and the EGF combined with ELF-EMF group exhibited the best wound healing rate, compared with the control group, and the difference was statistically significant (^**P< 0.01; Fig. 3).

Figure 3.

Wound healing rate of different experimental groups.

3.2HE staining of wound tissue

On the third day of wound healing in the normal control group, an obvious inflammatory cell infiltration was noted with no evident endothelial cell proliferation. Further, the number of fibroblasts was less than other groups, and the arrangement was disordered (Fig. 4-3d-A). On the 3rd day after injury, there were proliferation of vascular endothelial cells, less capillary formation, more inflammatory cells, larger cell bodies of fibroblasts, and round and oval nuclei in granulation tissue in the EGF alone group, which were less than those in EGF combined with ELF-EMF group (Fig. 4-3d-B). At the same time, in the EGF combined with ELF-EMF group, the new granulation tissue showed proliferation of vascular endothelial cells, neovascularization with inflammatory cell infiltration, obvious proliferation of fibroblasts, and round or oval nuclei with deep staining (Fig. 4-3d-C).

Figure 4.

Wound healing at different time intervals in different groups. Group A is the normal control group, Group B is the EGF group, and Group C is ELF-EMF+EGF group.

On the 7th day of wound healing, in the normal control group, there were a few capillaries formed in the granulation tissue, the cells were mainly inflammatory cells, the fibroblasts were more than before, but less than other groups, and the capillaries were rare (Fig. 4-7d-A). In EGF alone group, there were more fibroblasts with darker color, active angiogenesis, and fewer inflammatory cells on the 7th day after injury (Fig. 4-7d-B). In the EGF combined with ELF-EMF group, a reduced inflammatory cell infiltration, a large number of fibroblasts, dark staining, and more capillary components were noted (Fig. 4-7d-C).

On the 11th day of wound healing, in the normal control group, the inflammatory cells were gradually reduced, the fibroblasts were significantly increased and arranged in a vortex disorderly shape (Fig. 4-11d-A). In the EGF alone group, the number of capillaries was reduced, the number of mature fibroblasts was increased, cells were arranged in bundles, and the arrangement between bundles was disordered (Fig. 4-11d-B). In the EGF combined with ELF-EMF group, the number of new capillaries and inflammatory cells was reduced, the number of mature fibroblasts was increased, the cells were arranged in bundles, and the red matrix components were uniform (Fig. 4-11d-C).

3.3Observation of wound healing in each group

After all the wounds healed (15th day), a large number of fibroblasts were observed in the wound specimens of EGF combined with ELF-EMF group. The cell body of fibroblasts was large, the nuclei were regular, the fibers were arranged in an orderly manner, and the skin epidermis was thickened. The skin appendages were significantly better than the other groups (Fig. 5-A/B/C).

Figure 5.

Differences in HE staining of fibrous tissue within the healed wound on the 15th day.Group A is the normal control group, Group B is the EGF group, and Group C is ELF-EMF+EGF group.