Mechanism Of Platelet Rich Fibrin Promoting Fat Graft Survival And Their Optimal Mixing Ratio

ObjectivesAutologous fat graft is commonly applied to repair soft tissue defects.Various techniques have been used to increase its retention rate,but with limited effects.As the second generation of platelet concentrates,platelet rich fibrin(PRF)is easily prepared and has a long-lasting effect by releasing considerable amounts of growth factors.It has been reported than PRF can effectively promote fat graft survival,but the mechanism is unclear.The optimal mixing ratio of PRF and fat has neither been reported.Therefore,this study aims to observe the degradation of PRF,evaluate its release of growth factors,and explore the mechanism of its effect on fat graft survival as well as the optimal mixing ratio of PRF and fat.MethodsPart ?:PRF was prepared and cultured in vitro.The media were collected on days 1,2,3,4,8,12,16,20,24,and 28 to evaluate the concentration of targeted growth factors released from PRF.At the same time,0.2mL of PRF was implanted into the subcutaneous layer of BALB/c nude mice.The degradation of PRF in vivo was compared to that in vitro.Part ?:The fat graft model of BALB/c nude mice was established.Nude mice were randomly divided into PRF group and the control group.PRF granules(or normal saline in the control group)and fat granules were mixed at the volume ratio of 1:5.A total of 0.2mL mixture was injected into the subcutaneous layer of the nude mice.On days 0,3,7,14,21,and 28 after transplantation,the graft samples(n=12)were obtained to detect the concentration of target growth factors.In weeks 1,2,3,and 4 after transplantation,the graft samples(n=12)were obtained to calculate the volume and weight retention rates and evaluate the gene and protein expression of VEGF-A,PPAR-?,COL1-A1 and BAX.H&E staining,Masson's trichrome staining as well as ?-SMA and Periplipin-1 immunohistochemical staining were performed to assess the survival quality of the grafts.Part ?:The fat graft model was established.Nude mice were randomly divided into 5 groups.PRF granules and fat granules were mixed at the volume ratio of 1:5,1:10,1:15 and 1:20 respectively in PRF 1:5,1:10,1:15 and 1:20 groups.A total of 0.2mL mixture was injected into the corresponding nude mice.While 0.2mL of fat granules was injected into the control mice.In weeks 4,8,and 12 after transplantation,the samples were obtained(n=12)to calculate the volume and weight retention rates and evaluate the gene and protein expression of VEGF-A,PPAR-y,COL1-A1 and BAX.H&E staining,Masson's trichrome staining as well as ?-SMA and Periplipin-1 immunohistochemical staining were performed to assess the survival quality of fat graft.ResultsPart ?:PRF cultured in vitro showed no obvious signs of degradation and continuously released growth factors for up to 28 days.After being implanted into nude mice,it degraded rapidly.The average degradation time was 12.8± 0.97 days in vivo.Part ?:The production of growth factors by adipose tissue increased significantly after grafting.The increment of growth factors was more obvious in PRF group.In weeks 1 to 4,both the volume and weight retention rates of PRF group were significantly higher than those of the control group.Compared with the control group,PRF group had higher expression of VEGF-A and PPAR-y,and lower expression of COL1-A1 and BAX.The numbers of microvessels and viable adipocytes in PRF group were higher than those in the control group.Part ?:In week 12,both the volume and weight retention rates of groups 1:5,1:10,and 1:15 were significantly higher than those of group 1:20 and the control group.The weight retention rates of groups 1:5 and 1:10 were significantly higher than that of group 1:15.Groups 1:5 and 1:10 showed better results than other groups in terms of retention rates,related gene and protein expression,staining assessment,as well as the counts of microvessels and viable adipocytes.ConclusionsPRF can release growth factors for up to 28 days in vitro.Although it degrades within 2 weeks in vivo,PRF can still significantly improve fat graft survival through promoting autocrine of growth factors by the fat,promoting angiogenesis,enhancing adipogenic differentiation,regulating collagen deposition,as well as inhibiting apoptosis at early stage,and triggering a cascade reaction to amplify and prolong these effects.PRF can achieve a best effect when the mixing ratio of PRF and fat granules is 1:10,which is recommended here for the clinical application.