The Effect Of Platelet-rich Plasma On Macrophage In Diabetic Wound Healing

BackgroundWith the increase of the incidence of diabetes, diabetic wound healing obstacle has become a hotspot and difficulty in the research field of wound healing. However, the mechanism of dysfunction of diabetic wound healing has not been completely elucidated. Researches showed that the phenotype and inflammatory response of macrophage can be affected by diabetes status. They also showed diabetic wound was often accompanied by abnormal inflammatory state. Some previous studies involved diabetic wound macrophage infiltration, however, few of them figured out the changes of macrophage infiltration and inflammatory reaction ability in each stage of the process of wound healing. The clinical studies show that the platelet rich plasma (Platelet-rich, plasma, PRP) could promote wound healing of diabetic wound, but the mechanism is still unclear. PRP was usually used as a vector for growth factor in wound healing, but as a mixture plasma and PRP is far more than a provider of growth factors. Studies showed that the PRP could control inflammation and has strong effect on arthritis and other inflammatory diseases. At the same time, rich in a lot of anti-inflammatory and pro-inflammatory substances and platelet is central pivot of inflammation and immune with a strong regulatory effect on macrophages. So we have enough reasons to suppose the effect of PRP on diabetic wound healing is not limited to provide abundance of growth factors, but also related to the regulation of diabetic wound inflammation response and macrophage function.ObjectiveTo observe the macrophage infusion in streptozotocin induced diabetic wound and evaluate the effect of PRP on macrophage infusion. Methods1. Difference of macrophage infusion between normal and diabetic wound healing Male Sprague Dawley rats were randomly divided into STZ induced diabetic group or the normal control group. Each group had15rats. A1cm2full-thickness skin defect on the rat dorsum was excised. Animals of each group were sacrificed on3,7,14days post operation. The differences of the wound closure rate, macrophage infiltration and the relative mRNA expression of the inflammatory factors from macrophages were observed.2. The effect of platelet-rich plasma on macrophage infusion in diabetic wound healingSTZ induced diabetic rats were randomly divided into treatment group or control group. Each group had15rats. A1cm2full-thickness skin defect on the rat dorsum was excised. Animals of each group were sacrificed on3,7,14days post operation. The differences of the wound closure rate, macrophage infiltration and the relative mRNA expression of the inflammatory factors and anti-inflammatory factors from macrophages were observed. The chemotaxis and phenotype of macrophage were observed in vitro.Results1. Difference of macrophage infusion between normal and diabetic wound healing The wound closure rate was lower in diabetic group on day3,7,14post operation (p<0.05). HE staining showed inflammatory cells infiltrated in diabetic wound tissue were on day3and sustained infiltrated on day14. The results of the CD68immunohistochemical staining showed a lower macrophage infiltration density in diabetic wound tissue on day3(p<0.01) and a significantly higher one on day14(p<0.01). The density of the wound macrophages was positively correlated with wound closure rate on day3(r=0.909, p<0.01) and negatively correlated on day14(r=-0.962, p<0.01).The relative mRNA expression of IL-12B, iNOS and TNF-alpha also presented a lower expression in diabetic wound tissue on day3post operation (p <0.05) and a higher expression on day14(p<0.05). The CCR7fluorescence staining showed more positive staining cells stayed in diabetic wound on day14post operation.2. The effect of platelet-rich plasma on macrophage infusion in diabetic woundhealing On3,7,14days after operation, wound healing rate of the treatment group were higher than those in control group and p<0.05. Active PRP group was significantly lower than that in the non-active group, p<0.01. However, wound healing rate of active PRP group was higher than that of non-active PRP group on7and14days after operation, but there was no significant difference among them. HE staining showed that after3,7,14day, the inflammatory cell infiltration was higher in treatment groups, and the non-active PRP was more pronounced. CD68staining showed a higher macrophage infiltration density in treatment groups on3,7,14day, p<0.01, with significant difference between active PRP group and non-active PRP, p<0.01. The relative mRNA expression of IL-12B and TNF-alpha presented a lower expression in treatment groups on day3and14day post operation and a higher expression of IL-10on day3,7,14. Increased infusion of macrophage in treatment groups were observed in vitro assay and a higher chemotaxis index in non-active PRP group, p<0.05. The fluorescence assay showed that PRP can promote the expression of CD206and active PRP group was more pronounced.ConclusionThere is a delayed infiltration of macrophage in diabetic wound in the early phase and a sustained infiltration in later with a corresponding change of inflammatory factors, which could contribute to the poor wound healing of the diabetic rats. Platelet-rich plasma can promote diabetic wound healing and increase the macrophage infusion in diabetic wound. Platelet-rich plasma could suppress the inflammatory factors expression and promote anti-inflammatory factors expression in diabetic wound. Platelet-rich plasma can promote macrophage migration and differentiation.