| BackgroundFat grafting is widely used for soft tissue filling and regeneration.However,low survival rate of graft is a bottleneck problem which limits the wide application of fat grafting.Fat harvesting,processing and reinjection would affect fat survival,among which processing is one of the key factors.The lipoaspirates contain fat particles,tumescent fluid,broken adipocytes,oil,cytokines and inflammatory factors released during the operation and blood components released from the blood vessels after mechanical damage,etc.Previous studies have confirmed that lidocaine and adrenaline in tumescent fluid,oil and inflammatory factors do harm to graft survival.However,literature at home and abroad suggested that there is no sufficient evidence on the effect of blood on graft outcome.The blood in fat for grafting is in the form of whole blood,whose comprehensive effect on graft outcome is unclear.Thus,we designed in vivo experiments to investigate the effect of blood on graft outcome.Then,we investigated the effect of red blood cells(RBCs)on the biological behavior of ASCs,a key component in fat graft.The study provides data support for blood removal during purification of lipoaspirate,and explores possible mechanism.The study provides basis for improving graft quality and further improving surgical effects.Objective1.To confirm the effects of blood content on the volume retention and quality of fat grafts,and to provide evidence for clinical fat purification;2.To study the effect of Hb on biological viability of ASCs and possible mechanism,and discuss the mechanism of blood on fat survival.MethodsFor in vivo experiments,the lipoaspirate was washed and purified and then mixed with different proportions of whole blood from the same patient,resulting in 20%blood group(group A),10%blood group(group B),5%blood group(group C)and normal saline(NS)group(group D),respectively.Fat grafts were injected into the back of 48 nude mice,with 12 mice for each group.The grafts were extracted at 2 and 8 weeks.The gross volume retention of the samples was calculated.HE staining was performed to assess tissue structure.Then,immunohistochemical staining was performed to assess viable adipocytes and angiogenesis in the grafts.For in vitro experiments,ASCs were extracted via collagenase digestion and underwent multilineage differentiation cultures.The peripheral venous blood and fat samples were collected from the same patient.ASCs were treated with Hb.Then,the proliferation,migration,adipogenic differentiation and reactive oxygen species(ROS)levels of ASCs was detected.All data were statistically analyzed using SPSS 25.0 and ImageJ software.Results1.At 2 and 8 weeks after fat grafting,the volume retention of the 3 blood groups was statistically lower than NS group,with statistical difference(p>0.05).2.The results of score of HE staining showed that the score in the NS group was better than that in the three blood groups,with statistically significant difference at 2 and 8 weeks after fat grafting(p<0.05).Immunohistochemical staining for perilipin and CD31 showed that the number of viable adipocytes and CD31 positive blood vessels in the NS group was greater than that in the three blood groups,with statistically significant differences at 2 and 8 weeks after fat grafting(p<0.05).3.ASCs undergone decreased proliferation,migration,and adipogenic differentiation ability increased intracellular ROS generation after Hb treatment,with statistically significant differences compared with the control groups(p<0.05).Conclusions1.The blood in fat grafts leads to decreased long-term graft volume retention and quality.Blood should be removed in clinical fat processing,which may result in improvement in long-term volume retention and graft quality.2.Hb treatment results in a decrease in biological activity of ASCs(possibly by enhanced oxidative stress of ASCs leading to cell damage),which further affected ASCs role in fat survival.This pathway may be one of the mechanisms of the effect of Hb upon fat survival. |
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