| Background and ObjectiveChronic wounds are defined as wounds that have not heal completely after 30 days of standard medical treatment and are characterized by impaired remodeling of the extracellular matrix(ECM),prolonged inflammation and periods of relatively steady healing interposed with plateaus in which the wound does not improve.Chronic wounds such as diabetic foot ulcers(DFU)have been recognized as a major global public health problem associated with diabetes.Recent studies have shown that platelet-rich plasma(PRP)treatment can accelerate wound healing.PRP promotes the healing of diabetic foot ulcers mainly by various protein substances and cellular components.Activated platelets in PRP can release a variety of high concentrations of growth factors and cytokines,promote cell division,accelerate cell proliferation,promote angiogenesis and nerve repair.In addition,the white blood cells contained in PRP provide the basis for its antibacterial effect.Compared with traditional antibiotic therapy,PRP can not induce antibiotic resistance,and the antimicrobial and wound healing characteristics of PRP have a synergistic effect on the prevention of infection.However,the variability and heterogeneity of PRP separation system,PRP end-product components and related terms have brought great confusion to the application standardization of PRP products,and the related fields urgently need the progress of related research and the formulation of standards.Based on the preparation method of PRP,the preparation technique of was improved,and the therapeutic effect of modified PRP(M-PRP)on the wound of diabetic foot was detected,and its mechanism was further explored.Firstly,the differences in composition and growth factor content between M-PRP and PRP were compared,and the biological functions of M-PRP were discussed.Secondly,the physical and chemical properties and biological functions of M-PRP from different blood sources(DM-PRP:blood preparation from DiabetesMellitus patients;NM-PRP:blood preparation from normal non-diabetic patients)were analyzed.Third,through retrospective analysis of the clinical efficacy of M-PRP on wound healing in patients with diabetic foot;finally,the granulation tissue of diabetic patients was sequenced to analyze the mechanism of M-PRP and verify it.This study aims to provide a new method for the treatment of diabetic foot wounds,explore the mechanism of platelet concentrate on wound healing and standardize the current chaotic situation in the field of PRP.Methods1.The changes of platelet,red blood cell and white blood cell in different blood preparations(whole blood,Platelet-Poor Plasma(PPP),PRP,M-PRP)were detected by blood routine test.2.The changes of fibrinogen(Fib),albumin(Alb)and total protein(TP)among different platelet concentrates were detected by blood biochemistry.3.Enzyme linked immunosorbent assay(ELISA)was used to detect the cumulative release of different platelet concentrates(PRP,M-PRP)growth factors within seven days,including transforming growth factor β(TGF-β),platelet derived growth factor(PDGF),vascular endothelial growth factor(VEGF)and fibroblast growth factor(FGF)and epidermal growth factor(EGF).4.The proliferation of human umbilical vein endothelial cells(HUVEC)and human skin fibroblasts(HSF)was detected by MTS proliferation assay,the effect of M-PRP on the migration of HSF cells was detected by scratch test in vitro,the adhesion ability of HUVEC cells was detected by adhesion test,and the angiogenesis of M-PRP on HUVEC cells was detected by tubule formation test.5.Clinical retrospective study analyzed the wound healing of patients with diabetic foot treated with M-PRP in the past two years,and counted the healing rate,healing days and amputation rate.6.Transcriptome sequencing was used to study the changes of genes and functions before and after M-PRP treatment of diabetic foot wounds.Real time-qPCR experiments were used to verify.Results1.Blood routine results show that compared with whole blood(platelets:100-300 × 109/L;red blood cells:100-300 × 1012/L;white blood cells:100-300 × 109/L),the red blood cells in platelet concentrates(PPP,PRP,M-PRP)Compared with the white blood cell content far below the normal range,the platelet count in PPP is the lowest,the platelet enrichment in PRP is about 2-3 times,the platelet count in M-PRP has no significant decrease,and the platelet count in the normal range and M-PRP from different sources has no significant difference.2.Blood biochemical detection of Fib(2-4 g/L),Alb(35-50 g/L),TP(60-80 g/L)content changes,there is no significant difference in the content of each protein component of PRP and M-PRP;The changes in the contents of various protein components were more obvious between the two groups.Except for the content of Fib,the contents of Alb and TP in the non-diabetic group were higher than those in the diabetic group.3.ELISA was used to detect the cumulative release of five growth factors contained in PRP and M-PRP within seven days.The results showed that the total amount of growth factor released within seven days was higher in PRP than M-PRP,and M-PRP in non-diabetic group was higher than M-PRP in diabetic group.There was no significant difference in the total amount of EGF released.4.The basic experimental studies show that,first,M-PRP can promote cell proliferation,and the promoting effect of different sources of M-PRP on cell proliferation is different;second,MPRP affects cell migration and the source of M-PRP has no significant effect on the migration of HSF cells;third,M-PRP enhances cell adhesion,and NM-PRP adhesion is significantly better than DM-PRP.F ourth,M-PRP increased the number of neovascularization,but different sources of MPRP had no significant effect on angiogenesis of HUVEC cells.5.Clinical retrospective analysis showed that after the application of M-PRP to the wounds of diabetic foot patients,the amputation rate was significantly reduced,the wound site status of the patients stable,the number of days of healing and the healing rate increased significantly,and the plateau period of chronic wounds was greatly accelerated,proving that M-PRP is essential for wound healing.There is a clear promoting effect,but the specific link of M-PRP to wound healing is unclear.6.Transcriptome sequencing of the granulation tissue of patients before and after treatment,GSVA functional enrichment showed that M-PRP can affect cell proliferation,migration,adhesion and angiogenesis;differential gene analysis found that M-PRP mainly induces macrophage differentiation by affecting IL-4 affect wound healing.ConclusionThis thesis demonstrates the role of M-PRP in wound healing of diabetic foot ulcers and confirms that M-PRP improves wound healing mainly through improving the immune microenvironment of the wound site:M-PRP promotes the secretion of inflammatory factor IL-4 inducing the differentiation of macrophages into repairing macrophages,and recruits fibroblasts and endothelial cells to enhance cell proliferation,migration,adhesion,and angiogenesis,thereby improving wound healing.Clinical analysis validates the superior efficacy of M-PRP in the treatment of diabetic foot ulcer wounds,which makes it possible for the large-scale clinical application of M-PRP.This study is expected to provide an effective new method for the treatment of diabetic foot wounds. |
