Effects Of VSD On Phenotypic Transformation Of Macrophages In Wound Healing

Objective:To observe the changes of M1 and M2 macrophages in the process of VSD treatment of skin wound in rats,and to explore the effect of VSD on the phenotype transformation of macrophages in the healing process of acute skin wound in rats,to explore the possible mechanism of VSD promoting early wound healing,and provide further experimental basis for the clinical application of VSD.Methods:Thirty six 8-week-old male Sprague Dawley rats were randomly divided into control group and VSD drainage group.The experimental group was treated with continuous negative pressure suction from the first day after operation,eight hours a day.The control group was covered with sterile gauze,bandaged and fixed,and the drug was changed every other day.On the 1st,3rd and 7th day after operation,the wounds were photographed and fresh specimens were taken,Hematoxylin eosin and Masson staining were used to stain the wound tissue and observe the pathological changes;Real time quantitative PCR was used to detect the relative expression of cytokines m RNA of M1 and M2 macrophages in wound tissue,and immunofluorescence was used to detect the surface markers of M1 and M2 macrophages in wound tissue.Results:1.Macroscopic view of wound:compared with the immediate wound after surgery,the wound area of each group decreased to different degrees;On the first day after surgery,the wounds of the VSD group were moist and the granulation tissue was fresh,while the wounds of the control group were dry and no granulation tissue was observed.On the third day after operation,the wound in the VSD group was significantly reduced,the wound was moist and bright red,and the granulation tissue was significantly hyperplasia.The wound in the control group also showed a tendency to shrink,the wound was drier,and the granulation tissue hyperplasia was not obvious.On the 7th day after surgery,the wound in the VSD group shrank faster,the wound was bright red and drier than before,while the wound area in the control group was smaller than on the3rd day,The scab was removed,and bright red granulation tissue was visible.The wound repair rate of the control group was significantly lower than that of the VSD group at all time points(P<0.001).On the 7th day after surgery,the wound repair rate of the VSD group was as high as71.47±2.87%,while that of the control group was only 61.97±2.47%.2.HE staining results showed that the granulation tissue area of VSD group on the first day after operation was(28.31±3.25 mm~2)was slightly higher than that of the control group(25.28±2.33 mm~2),but there was no statistical significance(P>0.05);On the 3rd day after operation,the area of granulation tissue in VSD group was 42.37±3.61 mm~2,significantly higher than the control group 33.05±2.59 mm2(P<0.01);On the 7th day after operation,the area of granulation tissue in VSD group decreased to15.46%±1.58 mm~2 and lower than that of the control group(23.14±2.03mm~2)(P<0.05)。3.Masson staining showed that the collagen fibers in VSD group increased significantly and became denser at different time points.The distribution of collagen fibers in the neogenesis of wound was also very extensive,and the arrangement was orderly and showed a sparse network structure.The volume ratio of collagen tissue in VSD group was higher than that of control group at all time points.The CVF of VSD group was49.95±2.81%on the 7th day after operation,significantly higher than that of the control group(45.04±3.57%)(P<0.001)。4.PCR results:The m RNA relative expression levels of IL-6 and TNF-αin VSD group were lower than those in control group at all time points,and it was statistically significant;The m RNA relative expression of TGF-βin VSD group was higher than that in control group at each time point,which was statistically significant;The relative expression of VEGF m RNA in VSD group was slightly lower than that in control group on the first day after operation(P>0.05),and higher than that in the control group on the third and seventh days after operation(P<0.001).5.Immunofluorescence test results:The number of CD86 labeled M1macrophages in VSD group was lower than that in control group on the 1st,3rd and 7th day after operation,which was statistically significant;On the contrary,the number of CD206 labeled M2 macrophages in VSD group was higher than that in control group on the 1st,3rd and 7th day after operation,which was statistically significant.Conclusion:VSD technology can affect the polarization of macrophages in the process of promoting wound healing:reduce the content of M1 type macrophages and increase the content of M2 macrophages,and make the wound transition from inflammation to granulation tissue regeneration as soon as possible,so as to promote the early healing of wound.