Research On Biological Activity Of Platelet-rich Plasma On Fat And Its Derivatives

Part1:An experimental study on the different effects of platelet-rich plasma on fat and nanofat graft survival.ObjectiveTo investigate the effects of platelet-rich plasma on fat and nanofat graft survival and vascularization.MethodsFat was harvested from a 50-year-old healthy woman by vacuum suction,and nanofat was obtained by emulsification and centrifugation procedures.PRP was collected after two rounds of centrifugation from an autologous blood sample.206-8-week-old male nude mice were prepared,and weighed 22 to 28 g.Mice were divided into four groups.The four groups included:PRP mixed with nanofat(0.5ml),PRP mixed with fat(0.5ml),saline mixed with nanofat(0.5m1),and saline mixed with fat(0.5ml),all at a ratio of 1:4.The mixtures were administered subcutaneously into the back of the mice for self-control study.After 1 month and 3 months,the grafts were extracted and weighed.The microstructure of the fat and nanofat was examined with a scanning electron microscope.HE and immunohistochemical staining was applied to observe neovascularization.Western blot analysis was applied to analyse the expression of CD31 and VEGF.Results(1)Scanning Electron Microscope Assay:In fat tissue,fat cells had normal connections;the fat structure was complete and fibre networks were visible.In nanofat,the extracellular matrix components of were visible and their structures were intact.(2)Macroscopic observations:After 1 month,liquefaction and absorption were observed in one mouse each in the saline/fat group,and after 3 months,absorption was observed in one mouse each in the PRP/fat group and PRP/nanofat group,and in two mice in the saline/fat group.At 1 month and 3 month,the weight of the grafts in the PRP/fat group and PRP/nanofat group was significant higher than that in the saline/fat group and the saline/nanofat group.There was a significant difference.There was no significant difference between PRP/nanofat group and PRP/fat group.(3)HE staining:At 1 month,the fat cells were surrounded by numerous vacuoles and exhibited fibrosis in all groups.In the PRP/nanofat group and the PRP/fat group,the number of blood vessels was greater and the number of fibrotic and inflammatory cells was lower.At 3 month,a complete vascular lumen was observed in the periphery of the adipocytes,along with a greater degree of fibrosis in all group.A higher number of blood vessels were observed in the PRP/nanofat group and in the PRP/fat group than in the other two groups.(4)Immunohistochemical Assay:At 3 months,CD31 staining indicated that the number of new blood vessels in the PRP/nanofat group and the PRP/fat group was significantly higher than that in the other two groups.VEGF staining showed that the expression of VEGF was also significantly higher in the PRP/nanofat group than in the other three groups.(5)Western blot Assay:At 1 month and 3 month,CD31 and VEGF protein expression in the PRP/nanofat group was higher than that in the other three groups.Conclusion:PRP has no significant effect on promoting the grafts survival of nanofat and adipose tissue,however,it has significant effect on promoting vascularization.The angiogenic potential was significantly higher in the PRP/nanofat group.Part 2:Effects of adipose-derived mesenchymal stem cell and platelet-rich plasma on healing of wounds with full-thickness skin defects in mice.Objective:To investigate the effects of human adipose-derived mesenchymal stem cell(ADSC)and platelet-rich plasma(PRP)on healing of wounds with full-thickness skin defects in mice.Methods:ADSC were isolated from the lumbar and abdominal fat donated voluntarily by a healthy woman undergoing liposuction in the Department of Plastic Surgery of Guangzhou General Hospital of Guangzhou Military Area Command,and the cells were cultured and identified.ADSC of the second passage were used in the following experiments.The venous blood of the volunteer was taken,and PRP was obtained by secondary centrifugation.36 C57 BL/6 mice were divided into simple injury group(n=12),simple ADSC treatment group(n=12),and ADSC+PRP treatment group(n=12)according to the random number table.Each mouse was inflicted with a 1cm×1 cm wound with full-thickness skin defect on the back.Immediately after injury,the wounds of mice in simple injury group were subcutaneously injected with 1ml normal saline,the wounds of mice in simple ADSC treatment group were subcutaneously injected with lml phosphate buffer solution-blended ADSC suspension(with concentration of 5 ×105/ml,the same below),and the wounds of mice in ADSC+PRP treatment group were subcutaneously injected with 1ml mixture of PRP and ADSC(1:2 volume ratio).Three mice in each group were taken on post injury day(PID)3,5,7 and 14 to observe the gross condition of wound,and the wound healing rate was calculated.On PTD 3,5,and 7,the non-healing wound tissue and 0.5 cm normal skin tissue around the wound margin were taken after gross observation.The Inflammation,re-epithelialization,and angiogenesis of tissue were observed by he-matoxylin and eosin staining.Immunohistochemistry was used to observe the expression of macrophages of tissue samples collected on PID 3 and 5.Data were processed with analysis of variance of factorial design and Least-Significant Difference test.Results:(1)On PID 3,the wounds of mice in ADSC+PRP treatment group were with granulation tissue regeneration,redness,and swelling,and the wounds of mice in the other two groups were ruddy and with effusion.On PID 5,the wounds of mice in ADSC+PRP treatment group were obviously red and swollen.On PID 7,scab formed basically on wounds of mice in the three groups.On PID 14,the wounds of mice in the three groups basically healed,and their crusts were off.On PID 3,5,7 and 14,the wound healing rates of mice in ADSC+PRP treatment group were obviously higher than those of the other two groups(P<0.05 or P<0.01).On PID 5 and 7,the wound healing rates of mice in simple ADSC treatment group were obviously higher than those of simple injury group(P<0.01).(2)On PID 3,granulation tissue regeneration of wounds in ADSC+PRP treatment group was more than that in the other two groups.On PID 5,inflammatory reaction of wounds of mice was mild in the ADSC+PRP treatment group,which was severe in the other two groups.On PID 7,the re-epithelialization process of wounds of mice was almost completed in ADSC+PRP treatment group,and the number of new vessels was more in ADSC+PRP treatment group than in the other two groups.The migration diatance of regenerated epithelia around the wound edge in simple injury group and simple ADSC treatment group was short.On PID 3,5,and 7,the re-epithelialization rates of wounds of mice in ADSCs+PRP treatment group were(37.6±4.5)%,(59.1±1.3)%,and(89.2±4.3)%,respectively,significantly higher than(25.7± 1.5)%,(34.5±4.4)%,and(50.8±2.7)%in simple injury group and(29.1 ± 0.8)%,(42.6 ± 2.9)%,and(72.9 ± 3.0)%in simple ADSC treatment group(P<0.01).On PID 5 and 7,the re-epithelialization rates of wounds of mice in simple ADSC treatment group were significantly higher than those in simple injury group(P<0.05 or P<0.01).(3)On PID 3 and 5,a quite large number of new collagen fibers appeared in granulation tissue of wounds of ADSC+PRP treatment group,while the collagen fibers in the other two groups were less.On PID 7,a large number of new collagen fibers appeared in ADSC+PRP treatment group.The collagen fivers in wounds tissue of mice in simple injury group was still less.(4)On PID 3 and 5,the numbers of macrophages in wounds tissue of mice in simple ADSC treatment group were 4.7±0.6 and 5.3±0.6 respectively,obviously lower than 6.3±0.6 and 7.7±0.6 in jnjury group(P<0.05 or P<0.01);the numbers of macrophages in wounds tissue of mice in ADSC+PRP treatment group were 3.0 ± 1.1 and 2.7±0.5,significantly lower than those in the other two groups(P<0.05 or P<0.01).Conclusion:The healing speed and quality of PRP/ADSC group were better than those of ADSC group in promoting acute wound model.Human PRP and ADSC are involved in the early inflammation,metaphase of tissue proliferation,and re-epithelialization and shaping process of late stage of wounds with full-thickness skin defects in mice.