Effects Of Double-network Hydrogel Biomaterials On The Process Of Burn Wound Healing By Promoting Local Infiltration And Functional Expression Of Skin-resided Regulatory T Cells

Objective:To determine the effect of skin-resided regulatory T cells（Tregs）on burn wound healing and the functional effects of double-network hydrogel biomaterials on skin-resided regulatory T cells in vivo.Method:The experiment was divided into two parts according to the purpose.To determine the effect of skin-Treg cells on burn wounds in the first part,transgenic mice(Foxp3^DTR)-diphtheria toxin（DT）injection group（F-DT group）（n=40）and wild type（WT）mice-diphtheria toxin injection group（C-DT group）（n=40）.The mice were then divided into the early group and the late group according to the random number table method.Mice in the early group were intraperitoneally injected with DT on the day before the burn(D_-1),and 1 days（D₁）and 3 days（D₃）after the burn;Mice in the late group of DT infection were injected at 5 days（D₅）,7 days（D₇）,and 9 days（D₉）after burn.For measurement of wound closure,wounds were photographed at D₀,D₃,D₅,D₇,D₉and D₁₁after burn respectively,and the area of wounds at different time was measured from scanned images using Image J software.Using flow cytometry analysis to determine the infiltration and functional expression of skin Treg cells of F-DT groups when compared with control group.Mice were sacrificed at 7th day after burn,and using microscope to watching the proliferation of granulation tissue and the process of re-epithelialization by H&E staining.To investigate the effects of ethylene glycol chitosan（GC）/sodium alginate（Alg）double-network biomaterials on the functional activities of skin Tregs in vivo in the next part,C57BL/6 wild-type mice were divided into two group according to the random number table method.Namely,control group（n=40）and double-network hydrogel biomaterial（DN）group（n=40）.Mice in DN group were fixed with GC/Alg gel on the wound surface after burn.The wounds were photographed at D₀,D₃,D₅,D₇,D₉and D₁₁after burn,respectively,and calculated the wound area and healing rate.Using flow cytometry to analyze the local infiltration and functional expression of skin Tregs at each time between control group and DN group.Results:Firstly,when compared with C-DT early group[D₃（-0.16±0.04）%],the wound healing rate of the early F-DT group was significantly lower[D₃（-0.23±0.03）%]（P<0.05）;and when compared with C-DT late group[D₃（-0.15±0.01）%],F-DT late group[D₃（-0.16±0.03）%]were observed no significant difference in wound healing rate（P>0.05）.The effect of the early F-DT group on wound healing at 7-11 days was more pronounced than that in the late F-DT group.After fixed GC/Alg double-network biomaterials to burn wounds to stimulate skin-resident Tregs in vivo,when compared with control group[D₅（-0.09±0.04）%],the wound healing rate of DN group[D₅（0.18±0.03）%]was significantly increased（P<0.05）;When using flow cytometry to analysis local infiltration of Tregs within control group,the expression of CD4⁺Foxp3⁺T cells in D₅group[（10.59±1.77）%]was significantly higher than that in D₀group[（1.5±0.65）%],and the expression of CD25⁺Tregs in D₃group[（18.77±3.49）%]was higher than in D₀[（10.04±0.39）%];After stimulation with GC/Alg in vivo,when compared with D₃[CD4⁺Foxp3⁺T cells（8.21±2.61）%,CD25⁺Tregs（18.77±3.49）%]in control group.Compared with the DN group,CD4⁺Foxp3⁺T cells showed a significant increase in local wound infiltration[（22.44±2.83）%]after 3 days burn in DN group,and the infiltration of CD25⁺Tregs show significant increase too after 3 days burn[（64.74±3.31）%]in DN group.Besides,the functional expression of CTLA-4 in CD4+Foxp3+T cells in control group was significantly higher in D₃group[（18.33±4.78）%]when compared with D₀[（11.76±0.5）%],and the functional expression of ICOS was significant higher in D₃group[（19.27±2.15）%]when compared with D₀[（5.74±0.83）%];After GC/Alg stimulation in vivo,when compared with D₃[CTLA-4（18.33±4.78）%,ICOS（19.27±2.15）]in control group,the functional expression of CTLA-4 was significantly increased(（58.09±4.74）%]in the DN group,and the expression level of ICOS[（54.08±4.33）%]increased too.Generally speaking,the difference between the results was statistically significant（P<0.05）.Conclusion:1.Skin-regulating T cells are important components in the healing process of skin wounds,and they mainly act on burn skin wounds in the early stage（3days after burn）and facilitate the healing rate in the late healing process;2.Dual-network biological material GC/Alg It can promote the healing process of burn wounds by promoting local infiltration and functional expression of skin Tregs.