[Properties of gelatin-polyethylene glycol hydrogel loaded with silver nanoparticle Chlorella and its effects on healing of infected full-thickness skin defect wounds in mice].

Abstract

Objective: To explore the properties of gelatin-polyethylene glycol hydrogel loaded with silver nanoparticle (AgNP) Chlorella (hereinafter referred to as the composite hydrogel) and its effects on healing of infected full-thickness skin defect wounds in mice. Methods: The research was an experimental research. The simple gelatin-polyethylene glycol hydrogel (hereinafter referred to as the simple hydrogel) and the composite hydrogel were prepared, and the appearance and injectability of the two hydrogels were observed at 55 and 37 ℃, and under the irradiation of 808 nm near-infrared light, respectively. An electronic universal testing machine was employed to assess the tensile and compressive stress-strain properties of both types of hydrogels at room temperature. Additionally, the cyclic compressive stress-strain properties of the composite hydrogel were examined at 80% of the maximum compressive stress. Staphylococcus aureus or Escherichia coli solution was added to phosphate buffer solution (PBS), simple hydrogel, and composite hydrogel, respectively. The part of composite hydrogel containing Staphylococcus aureus or Escherichia coli solution was irradiated with near-infrared light for 5 minutes. After each sample was incubated for 6 h, the dilution plating method was used to detect and calculate the mortality rates of the two bacteria at 24 h of culture (n=5). The discarded foreskin tissue was taken from a 6-year-old healthy boy admitted to the Department of Urology of the First Affiliated Hospital of Naval Medical University for circumcision. Primary human fibroblasts (HFbs) were isolated using the enzyme extraction method, routinely cultured to the 3^rd to 6^th passages for subsequent cellular experiments. Composite hydrogel extracts with final mass concentrations of 100.0, 50.0, 25.0, 12.5, and 0 mg/mL were respectively prepared and used to culture HFbs, and the cell proliferation after 24 h of culture was detected using a cell counting kit 8 (n=3). A total of twenty 6-8 weeks old C57BL/6J female mice were utilized, and a full-thickness skin defect was surgically created on the back of each mouse. The wounds were infected with Staphylococcus aureus solution. The infected mice were divided into blank control group, simple hydrogel group, composite hydrogel group, and combined treatment group according to the random number table, and the wounds were treated with PBS, simple hydrogel, composite hydrogel, and composite hydrogel+light irradiation (under the irradiation of 808 nm near-infrared light for 5 min), respectively, with 5 mice in each group. On post injury day (PID) 0 (immediately after the first wound treatment), 3, 7, and 14, an overall assessment of wound exudation and healing were conducted, and the wound healing rates on PID 7 and 14 were calculated (n=5). On PID 14, hematoxylin-eosin staining was performed to observe histopathological changes in the mouse wound. Results: Both simple hydrogel and composite hydrogel were in a solution state at 55 ℃ and transition to a gel state when cooling to 37 ℃. After the two hydrogels were irradiated by near-infrared light, only the composite hydrogel reheated up and returned to the solution state again with injectability. The maximum tensile stress of the composite hydrogel was up to 301.42 kPa, with a corresponding strain of 87.19%; the maximum compressive stress was up to 413.79 kPa, with a corresponding strain of 91.67%, which was similar to the tensile and compressive properties of the simple hydrogel. After 10 compression cycles, the maximum compressive stress of the composite hydrogel still reached 84.1% of the first compressive stress. After 24 h of culture, the mortality rate of Staphylococcus aureus treated with simple hydrogel was significantly higher than that treated with PBS (P<0.05); the mortality rates of Escherichia coli and Staphylococcus aureus treated with composite hydrogel alone were significantly higher than those treated with simple hydrogel (P<0.05); the mortality rates of Escherichia coli and Staphylococcus aureus treated with composite hydrogel+light irradiation were significantly higher than those treated with composite hydrogel alone (P<0.05). After 24 h of culture, compared with that cultured in composite hydrogel immersion solution with final mass concentration of 0 mg/mL, the proliferation activity of HFbs cultured in composite hydrogel immersion solution with final mass concentrations of 25.0 and 50.0 mg/mL was significantly enhanced (P<0.05), while the proliferation activity of HFbs cultured in composite hydrogel immersion solution with final mass concentration of 100 mg/mL was significantly decreased (P<0.05). On PID 0 and 3, more purulent secretions were seen in the wounds of mice in blank control group and simple hydrogel group, while only a small amount of exudate was observed in the wounds of mice in composite hydrogel group, and no obvious infection was observed in the wounds of mice in combined treatment group. On PID 7 and 14, the wound healing rates of mice in simple hydrogel group were significantly higher than those in blank control group (P<0.05); the wound healing rates of mice in composite hydrogel group were significantly higher than those in simple hydrogel group (P<0.05); the wound healing rates in combined treatment group were significantly higher than those in composite hydrogel group (P<0.05). On PID 14, the wounds of mice in blank control group exhibited a high infiltration of inflammatory cells with no new epithelial layer observed; the wounds of mice in simple hydrogel group displayed a short length of newly formed epithelium with a small amount of inflammatory cells; the wounds of mice in composite hydrogel group exhibited continuous formation of new epithelium and a large amount of immature granulation tissue; the wounds of mice in combined treatment group showed continuous epithelialization with less immature granulation tissue. Conclusions: The prepared composite hydrogel exhibits excellent thermosensitivity, photothermal properties, and injectability, as well as excellent mechanical properties, antibacterial properties, and biocompatibility, and can promote the healing of infected full-thickness skin defect wounds in mice.