| Background and objectiveThe reduction or defect of soft tissues,such as congenital malformations,trauma and disease et al has always been a problem to be solved in plastic surgery.Therefore,the methods of soft tissue filling like Collagen injections,artificial synthetic materials implanted are widely used in clinic practice.In 1889,Vander Meulen reported of transplanted with autologous adipose tissue and has been used until now.The major advantage of the grafts is easily to obtain,simple construction and low immunoregenicity reduces the rejection of transplantation.However,despite efforts to standardize fat grafting procedures,fat graft survival rates are reported to range from 30%to 90%,and fat grafting may be accompanied by complications,such as oil cysts and calcification,which lead to unpredictable outcomes.In recent years,clinical research has made women’s age(more than or less than 45 years old)is the difference point of fat graft survival,they suggest that the ages and fluctuation of hormones could affects the activity of donor site adipose tissue.This study gives us great inspiration.Is there any difference in the structure and vascularization of the adipose tissue in different estrogen levels?And does the retention rate of fat grafts be affected by estrogen?Thus,we establish premenopausal(CON group),menopause(OVX group)and high estrogen during pregnancy(HE group)to observe morphological differences between the models before and after transplantation.We used flow cytometry,immunofluorescence,RT-PCR and gene chip technology to detect the immune response in grafts to discuss the survival mechanism of estrogen on fat graft and the immune-regulation.The results of this study provide an experimental basis for revealing the mechanisms of different physiological estrogens micro-environmental effect on the retention rate and clinical application.Methods1.The establishment and evaluation of different estrogen concentrations and effect on the retention of fat grafts.The mice were excised by sham operation,ovariectomy and placed high estrogen pills in subcutaneous.The success of surgery and estrogen treatment was confirmed by measuring serum estrogen concentrations in ELISA;Unilateral inguinal fat pad and the grafts retention were measured the weight.Hematoxylin eosin staining(H&E)and immunofluorescence were used to observe the morphological and histological changes of adipose tissue at different time points after transplantation.The expression of estrogen receptor a(ER a)in grafts was also detected by RT-PCR.2.The effect of estrogen on the regulation of inflammatory cells,vascularization and fat regeneration in fat transplantationThe SVF cells were received from the grafts were digested by collagenase.The expression of inflammatory cells,macrophage subtype(Mland M2)and local stem cells by flow cytometry.RT-PCR was used to evaluate the expression of inflammatory cytokines were secreted by macrophages.Gene chip was used to detect the trend of gene expression of fibrosis in one week after transplantation.Immunofluorescence and RT-PCR to evaluate the ability of vascularized,adipocyte proliferation and differentiation.Results1.The establishment and evaluation of different estrogen concentrations and effect on the retention of fat grafts.(1)Estrogen level negatively correlates with graft massCompared with the ERa level in the CON group,ERa levels were a mean 0.45±0.2 in the OVX group and 3.07±1.1 in the HE group(p<0.05).Before fat grafting(45 days after OVX or pellet implantation),inguinal fat pads in the OVX group were larger and more elastic than pads in the HE and CON groups.The average weight of grafts was twice as high in the OVX as in the other groups.(2)Low estrogen levels triggered early adipogenesis and adipocyte hypertrophy and estrogen receptor a.After fat grafting,the three groups differed in the rate of retention of the same mass of fat.At week 12,grafts in the OVX group looked white and felt firm and resilient,with a weight retention rate of about 52%(0.0030±0.0015%of body weight),significantly greater than the weight retention rates in the HE(22%;0.00095±0.0005%of body weight,p<0.05)and CON(28%;0.0016±0.0005%of body weight)groups.the difference was not statistically significant(p=0.175),These differences were not due to differences in body weight,suggesting that estrogen plays a role in this autologous fat grafting model.ERa expression decreased in the OVX group 1—2 weeks after fat grafting(0.15±0.03-fold)and was lower than in the other two groups at these times(p<0.05).However,ERa expression in the OVX group gradually increased,peaking 12 weeks after fat grafting(p<0.05).Hematoxylin and eosin staining and immunofluorescence staining showed that in the OVX group had a large number of oil droplets followed by adipocytes were severe destroyed compared with the CON and the HE groups.At 2 weeks,a lot of small multilocular fat cell began to show up in the OVX group.ERa expression decreased in the OVX group 1-2 weeks after fat grafting(0.15±0.03-fold)and was lower than in the other two groups at these times(p<0.05).However,ERa expression in the OVX group gradually increased,peaking 12 weeks after fat grafting(p<0.05).2.The effect of estrogen on the regulation of inflammatory cells,vascularization and fat regeneration in fat transplantation(1)Insufficient inflammation infiltration and abnormal M1 to M2 switch.During the period of inflammation following fat transplantation(1-2 weeks),OVX graft had a more severe CD 11 b+(monocytes,granulocytes marker)leukocytes infiltration(43.7±9.17%)than HE group(25.19±1.06%)and CON group(21.77±6.38%)(p<0.05).M1 were found to account for～70%of infiltrating macrophages in the CON group(M1/M2 = 2.35± 1.20),but only～57%of that in the HE group(M1/M2 = 1.38±0.49).By contrast,83%of infiltrating macrophages in the OVX group were M1(M1/M2 = 5.18± 1.62),a finding consistent with the early presence of oil cavities in these grafts.By week 2,both OVX and CON group had a switch from M1 to M2,however the switch was the other way around in HE group(M2 to Ml).These data suggested insufficient of pro-inflammatory macrophages in the HE group,which led to impaired initial tissue destruction and inadequate later scavenging ability.Cellular quantification correlated with the level of transcription of the pro/anti-inflammatory cytokines TNF-α/IL-10,which are released mainly by M1 or M2 respectively.From the gross views,Microarray showed altered transcriptions of fibrosis-related genes during the inflammatory stage in OVX,demonstrating mild but consistent fibrosis tendency.(2)Early Angiogenesis level didn’t parallel with fat retention in estrogen manipulation model.We found only appropriate estrogen concentration could induce early angiogenesis in fat grafting model.The CD31+ area and the expression of vascular endothelial growth factor(VEGF)were significantly larger/higher in the CON group than the other two at 12 weeks(p<0.05).Even that there was rich primitive vascularization in HE group,eventually the capillary degenerated,as well as VEGF regressed simultaneously with delayed tissue necrosis.From the fat retention respect,weaker early capillarization due to estrogen-deficiency could not overcome the pro-adipogenic effect of OVX.(3)High estrogen protected local stem cells but inhibited adipogenic differentiation.Although grafts in the HE group owned more local stem cells,adipogenesis was inhibited.During thelst month after grafting,the number of Sca1+/CD45-cells(i.e.,mouse local stem cells of non-hematopoietic origin)was considerably higher in the HE(19.17±3.45,1w;21.23±2.176,4w)than in the OVX(6.972±1.395,1w;6.162±1.972,4w)group and CON group(18.23±4.78,1w;10.67±3.991,4w,p<0.05).Cell proliferation is a key element to adipose regeneration.The percentage of proliferating cells(Ki67+)and the expression of the adipogenesis transcription factor PPAR-γwere significantly higher in the OVX than in the CON and HE groups from week 4 to week 12(p<0.05).The percentage of Ki67+ cells increased markedly in the HE group from week 4 to week 12(p<0.01),whereas the expression of PPAR-γ was low at 12 weeks,suggesting that adipogenesis was poor in the HE group.Conclusions1.An animal model was successfully constructed to simulate different estrogen physiological concentrations of ovariectomy and implantation of estrogen pellets.2.Although the graft is poorly vascularized in the ovariectomy group.However,the low estrogen environment can promote fatty acid synthesis,recruit amount of granulocyte and Mlmacrophages to remove necrotic adipose tissue immediately that is helpful for fat regeneration and tissue reconstruction.3.The retention rate of the grafts is not well as we expected in the high estrogen environment,nonetheless,the stem cell content is the highest,which may be used in the application of the wound therapy.However,it is an idealized model that ignores the age,sexual,and the other factors in mice.In addition,the observation of graft is 3 months,the long-term retention rate of graft needs to be further studied.This study reveals the mechanism of graft in different estrogen environments,providing more clues and theoretical basis for clinical research. |
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