| Autologus fat transplantation(AFT),also called autologus fat injection,refers to a kind of means that repairing subcutaneous soft tissue defect with autologus fat tissue as filling material.Compared with artificial material and dissimilar material,the autologus fat has convenience to be obtained,with low price and better biocompatibility.The survival of fat after transplantation is issue which clinicians are most concerned about.Purification of suctioned fat is directly associated with survival of fat tissue.The paper compares and analyzes the fat purified by three methods:Absorption with gauze(AWG),Centrifugation(CEN)and Decantation(DEC)in the respects of purification effect and transplant prognosis in basic research,and initially investigates specific mechanism of it influencing transplant retention and prognosis.ObjectivePurpose of the paper is to evaluate the effect of purifying fat with the AWG(Absorption with gauze)method,CEN(Centrifugation)method and DEC(Decantation)method,and make comparative analysis on the prognosis of the transplant in the animal experiments,so as to have a preliminary exploration towards the specific mechanism affecting the retention and prognosis of the transplant.MethodsFat,liquid ingredients were separated from grease and adipose tissue,and the respective volume was measured,so as to evaluate the purification effectiveness of different methods.The structure and functional integrity of the adipose tissue purified with different methods were compared through histological detection and metabolic function detection;stromal vascular fractions(SVFs)in the adipose tissue were obtained via collagen digestion,and the cellular constituents thereof wereanalyzedwith flow cytometer,the SVFs cells were cultured adherently to the third generation,adipose tissue-derived stromal cells(ADSCs)were obtained,a cell proliferation curve was drawn with CCK8 reagent,the cell cycle and apoptosis condition thereof were detected,and the differentiation activity thereof was compared through adipogenic,osteogenic and chondrogenesis differentiation;The animal model where granular fat was transplanted was constructed with immunodefic ient mice,and the transplanted fat was take n out 3 days,6 days,9 days,12 days,3 weeks,6 weeks,9 weeks and 12 weeks after the injection.The retention condition of the adipose tissue purified with different methods were compared by weighing the transplant and measuring the volume;histologically,the perilipins were used to stain the adipose cells with good activity to compare the in-vivo surviving condition of the fat purified with different methods.In-vivo prognosis of the fat transplant was observed from revascularization,substance expression of inflammatory,expression of matrix metalloproteinase,collagens remodeling and other aspects,by means of suspension array,Western blot and real-time fluorescent quantitative PCR;and then,the influence of different granular fat purification methods on the retention of the fat transplant was explored.ResultAfter being purified with different methods,the moisture content in the fat from the high to low order was respectively:DEC Group,CEN Group and AWG Group;on the contrary,the content of adipose tissue from the high to low order was respectively:AWG Group,DEC Group and CEN Group;while after high-speed centrifugation of the purified fat of the CEN Group,the grease centrifugated was higher than the DEC Group and AWG Group.After being purified with different methods,the adipose tissue was not significantly different at structural integrity,glucose metabolism ability and other aspects After being purified with different methods,the content of SVFs and ADSCs in the adipose tissue from the high to low order was respectively:AWG Group,CEN Group and DEC Group.While the content of red blood cells from the high to low order was respectively:DEC Group,CEN Group and AWG Group.The influence of the three purification methods on proliferative activity,cell cycle and apoptosis condition of ADSCs were basically consistent.Besides,the influence of the three methods on the differentiation activity of ADSCs were not obviously different.After being transplanted,the volume of the fat presented a downward trend with the time;nevertheless,as time prolonged,the descend range gradually decreased.Upon comparison of the volume of the transplant at each time point after the transplantation:AWG Group>CEN Group>DEC Group,the difference was statistically significant.Upon comparison of the weight of the transplant at each time point after the transplantation:AWG Group>CEN Group>DEC Group,and the descend amplitude was greater than the volume.Upon histological detection,it can be found that,the basic prognosis trend of the adipose tissue after the fat transplantation was:degeneration of adipose cells(dedifferentiation,disintegration and necrosis),along with the increase of the expression of inflammatory substances→formation of cystic cavity structure→collagen deposition→revascularization→fat regeneration.After being purified with the CEN method,adipose cells in the adipose tissue were mostly disintegrating and necrotic in the degenerating process,dedifferentiated adipose cells were rarely found;after the transplantation,the expression of the inflammatory substance IL-6,IL-1 were higher in the early stage,and the difference with the adipose tissue purified with DEC method and AWG method was statistically significant.Upon the detection of the early apoptosis condition of the cells in the early adi pose tissue a ftertransplantation with protein liquid-chip technology,and detection of various inflammatory factors in the transplant,it was found that,most inflammatory factors presented low expression;IL-6,IL-1,GM-CSF and IFN-g dropped after the significant increase in the early stage after fat transplantation;the expression quantity of IL-10,IL-12 and IL13 in the adipose tissue was extremely low,whose expression was not affected by whether transplanted in to the body and time variation.Although IL-2,IL-4,IL-5,IFN-G and TNF-a had certain expression in the tissue,they were not affected by different intervention factors and transplantation treatment.Conclusion1 The concentration and purification effect of the adipose tissue after aspiration is directly related to the remaining amount of the adipose tissue after the transplantation:the better the concentration and purification effect,the higher the retention rate of the adipose tissue after the transplantation will be.Through the comparison of the three purification methods,the AWG method has the best purification effect,the CEN method takes the second place,while the DEC method has the lowest concentration and purification efficiency.2 As to the CEN method(1200 g centrifugal force,3 min),critical mechanical damage was caused to the adipose cells during the purification,and it is hard to find such damage during the in-vitro function detection.However,in the malnourished environment after the transplantation,more adipose cells will be disintegrating and necrotic.3 In the fat transplanted model constructed with nude mice suffering immunodeficiency,in-vivo prognosis of,adipose tissue can be classified into three stages:(1)rapid depletion and inflammation start-up period(D1-D7)—liquid ingredients in the transplant were quickly absorbed,remaining amount of the transplant was decreased,and expression of inflammatory substances were gradually increased;(2)fat degeneration and reascularization period(W1-W3)—adipose cells were disintegrating and necrotic and dedifferentiated,inflammatory substances fall quickly after reaching the peak value,and new vessels form gradually;(3)matrix reconstruction and fat regeneration period(W3-)---in the earlier stage,a great amount of collagen deposits,and in the latter stage,it degrades gradually,newborn fat cells appear and increase gradually.4 With good concentration effect,ASCs in the transplant of the AWG Group and CEN Group gather;after the transplantation,expression of VEGFR in the fat is high,collagen remodeling occurs early,which is beneficial for the regeneration of adipose cells.5 Since activity of adipose cells is damaged,the expression of IL-1,IL-6 and other inflammatory substances increase after the transplantation of adipose tissue after being purified with the CEN method. |
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