3.4 Characterization of RHC/EGF freeze-dried dressing
Scanning electron microscopy results of RHC and RHC/EGF (1: 1) freeze-dried dressing are shown in Fig. 3. RHC and RHC/EGF freeze-dried dressing had an obvious but irregular pore structure, which facilitated substance exchange between cells and freeze-dried dressing. However, compared with RHC freeze-dried dressing, RHC/EGF freeze-dried dressing self-assemble by lyophilization to form a distinct fiber structure. This is very similar to the natural ECM structure. Conducive to the rapid integration of wound skin during wound repair, and accelerate the speed and effect of skin wound repair. In addition, solubility experiments show that RHC and RHC/EGF freeze-dried dressing have good solubility. Both can be completely dissolved in physiological saline in a short time (3~5 s), forming a transparent and uniform aqueous solution and easy-to-use.
3.5. Wound-Healing effect of RHC/EGF forfull-thickness skin defects in Vivo
We separately lyophilized EGF, RHC and RHC/EGF to obtain freeze-dried dressing. After establishing the full-thickness skin defects model, the wounds were treated with RHC and RHC/EGF freeze-dried dressing respectively. Wounds treated with EGF served as a positive control group, while physiological saline was administered to the control group. Wound closure results at each time point in each experimental group are shown in Fig. 4A. Healing time of the RHC/EGF group was obviously reduced compared with that of the other groups, and the wound closure rate of the RHC/EGF group was much higher than that of the other two groups. These results indicated that RHC/EGF increased the healing capability of wounds compared with RHC or EGF. At 10 days post-wounding, a significantly higher wound closure percentage was monitored in EGF (88.39 ± 4.13%) and RHC/EGF (90.33 ± 7.47%) versus closure in RHC (69.32 ± 6.18%) and control (65.79 ± 9.48%), showing a significant difference (P <0.01) (Fig. 4B). The appearances of the wounds indicated that RHC/EGF freeze-dried dressing was an optimal wound dressing for accelerating early healing of skin wounds. Importantly, RHC/EGF freeze-dried dressing improved wound appearance, whereas non-treatment developed noticeable large and elongate scars.
Then, the histological analysis was further investigated via H&E staining to assess the quality of the newly formed skin tissue (Fig. 4C). Early in the process of wound healing, the reduction of inflammatory cells and the presence of more capillaries in the damaged skin are beneficial to speeding up wound repair. At 3d, H&E staining results indicated relief of wound inflammation and significant capillary growth in the RHC/EGF group, while there were large amounts of inflammatory cells infiltrating into the upper layer of dermis in the control and there were almost no capillaries. Re-epithelialization is an important stage of wound repair. Through re-epithelialization, the wound area is continuously reduced until the wound is completely closed and a new dermal layer is completely formed.(Xiao, Reis, Feric, Knee, & Radisic, 2016) Evaluation of the skin-injury model after treatment for 14 and 21 d (Fig. 4D) indicated significantly increased epithelial thickness in the RHC/EGF group (41.66 ± 4.62μM,P <0.01) compared with control (26.83 ± 3.58μM) and RHC groups(32.33 ± 3.18μM).