| Background and ObjectionAutologous fat transplantation has been used for soft tissue defect in plastic surgery, dating back to the late19th century. In1911, Bruning was the first to inject autologous fat into subcutaneous tissue for soft-tissue augmentation. With the advent of liposuction surgery, the harvesting process became easy, since when autologous fat transplantation has become popular. Autologous fat tissue, a potential soft-tissue filler, is easy and safe to harvest without producing scars on the donor or at the recipient sites, and induces no foreign-body reaction or cross-infection. However, the clinical outcome of this technique is limited by low graft survival. Free fat graft resorption is variable, with20-80%volume loss seen in long-term follow-up. The grafts only depend on the effusion of surrounding tissue to survive without adequate blood supply. A lot of adipocytes died in the central area of fat grafts response to the hypoxia condition, and then the dead adipocytes are replaced of fibrous tissue. Therefore, a sufficient blood supply early after transplantation is crucial to retaining transplanted fat tissue volume. Recently, cytotherapy has been used in autologous fat transplantation. Many researchers focus on ASCs as helper cells in autologous fat transplantation.Some researchers have developed a novel strategy called cell-assisted lipotransfer, in which the stromal vascular fraction containing ASCs are injected with fat tissues; results showed better graft retention than with control treatment. Mixing ASCs with fat tissues before transplantation enhanced neovascularization and increased the survival of fat grafts. ASCs co-transplanted with aspirated fat tissue can improve graft retention, which is confirmed. However, the mechanism of ASCs in the early stage after aspirated fat transplantation is not clear. There are several questions to be future investigation:(1) morphology of fat grafts in the early stage after ASCs co-transplanted with aspirated fat tissue,(2) the mechanism of ASCs improved angiogenesis.Aspirated fat with (group A) or without (group B) ASCs was injected subcutaneously into the back of nude mice, and the grafts were harvested at1,4,7,14,30and90days. Graft volume and histology were evaluated and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) was quantified by enzyme-linked immunosorbent assay.Methods and materials1. Cell isolation and cultureASCs were isolated from the fatty portion of the liposuction aspirates as described previously. Briefly, the aspirated fat was washed with phosphate-buffererd saline, excised and excised and digested with0.125%collagenase on a shaker at37℃for30min. An equal volume of Dulbecco’s modified Eagle medium with10%fotal bovine serum was added to neutralize the collagenase. The cell suspension was filtered through a200-mesh filter. Mature adipocytes and connective tissue were separated from pellets by centrifugation (1200g,5min). The cell pellets were resuspended, plated at a density of1×106cells per100-mm dish in Dulbecco’s modified Eagle medium with10%foetal bovine serum, and cultured at37℃in5%CO2. Primary cells were cultured for7days and were defined as passage0. The medium was replaced every3days. Cells were passaged at a ratio of1:3per week. Only cells that had been cultured for three passages were used in this study.2. Animal model and groups and HistologyThe Coleman technique was used for fat grafting. Thus, each mouse was injected subcutaneously at two spots with fat tissue (0.5ml/spot) that was mixed with5×105ASCs or not. At1,4,7,14,30and90days after fat transplantation, the grafts were excised and analysed as described below:(1) general views of fat grafts,(2) the volume and survival rate of fat grafts,(3) histology.3. ELISAThe secretion of VEGF and HGF was quantified by enzyme-linked immunosorbent assay.4. Statistical analysisThe data are expressed as means+/-S.D. A repeated measures analysis of variance was used to analyse the results. Furthermore, if it revealed statistically significant differences, paired Student’s t-test of two groups in one time point and one-way analysis of variance of one group in four time points were made. A value of P<.05was considered significant.Results1. The survival rate of aspirated fat with ASCs:(63.67±4.97)%on day1,(67.33±7.66)%on day4,(74.67±4.13)%on day7,(79.67±8.73)%on day14,(64.33±7.53)%on day30,(55.00±6.78)%on day90. The survival rate of aspirated fat without ASCs:(61.33±1.63)%on day1,(65.00±4.86)%on day4,(74.33±4.97)%on day7,(80.17±8.64)%on day14,(54.67±7.55)%on day30,(42.67±6.77)%on day90. The difference in graft volumes between the ASCs and the control groups at every time (1-14days) was not statistically significant, but the graft volumes in both groups were slightly increased over time. However, the graft survival rate declined dramatically during14-30days and continued decreasing gradually during30-90days. Fat+ASCs group had higher survival rate compared with fat-only group.2. Histological evaluation showed that on day14, the gap between adipocytes showed signs of broadening and infiltrated nucleated cells obviously disappeared in the interstitial space between adipocytes in both groups. On day4, blood vessels on the surface of the fat graft were observed in the fat+ASCs group. We also found that vessels had grown into the graft from the surface of the graft; in the fat-only group, however, this phenomenon appeared on day7. The number of vessels in the grafts increased on day14in both groups. However, the fat+ASCs group showed more compared with the control group. The grafts of the fat+ASCs group were divided into a kind of regular grid by fibrous tissue on day90, but the fat-only group showed irregular structure and more fibrous tissue.3. In group B, ELISA showed that the secretion of VEGF increased significantly on day7, and then decreased gradually. On the other hand, VEGF protein peaked4days after transplantation and as late as14days after transplantation in group A, which indicated that the secretion of VEGF by ASCs had an earlier peak time in group A than in group B. The both groups showed low expression of VEGF after30days. After transplantation, the secretion of HGF arrived at the peak time on day7and decreased gradually during14-90days in both two groups. Group A showed more secretion of HGF compared with group B.DiscussionsAutologous fat transplantation has been used for soft tissue defect in plastic surgery. With the advent of liposuction surgery, the harvesting process became easy, since when autologous fat transplantation has become popular. Autologous fat tissue, a potential soft-tissue filler, is easy and safe to harvest without producing scars on the donor or at the recipient sites, and induces no foreign-body reaction or cross-infection. However, the clinical outcome of this technique is limited by low graft survival. Free fat graft resorption is variable, with20-80%volume loss seen in long-term follow-up.Therefore, a sufficient blood supply early after transplantation is crucial to retaining transplanted fat tissue volume. Recently, cytotherapy has been used in autologous fat transplantation. Many researchers focus on ASCs as helper cells in autologous fat transplantation.Histological changes such as lipid droplets (dead adipocytes), tissue edema, infiltration of nucleated cells, etc. result in increase of fat grafts volume in the early stage after aspirated fat transplantation. We suggest that the difference between these groups was in the graft tissue structure rather than in the graft survival rate.Although infiltrated histiocytes (maybe meaning macrophages) were once proposed to acquire lipid material and eventually replace all adipose tissue of the graft that had disappeared ("host replacement theory"), the "cell survival theory," which states that transplanted living cells partly survive and remain alive for a long time after grafting, has been generally accepted. The most superficial zone is "Surviving zone", which is less than300μm thick.In the surviving zone, both adipocytes and ASCs survive. The second zone is "Regenerating zone", of which thickness varies depending on the microenvironmental conditions such as vascularity of and attachment to the surrounding tissue. In this zone, adipocytes die as early as day1, but ASCs survive and provide new adipocytes to replace the dead ones. The most central zone is "Necrotic zone", where both adipocytes and ASCs die, no regeneration is expected, and the dead space will be absorbed or filled with scar formation.On the fourth day, graft angiogenesis occurs from the host bed vascular system. We obtained a similar result, and the histological evaluation of the fat graft indicated that mice that received ASCs had a more highly vascularized fat graft than the control mice at4days,7days and14days. We also found that there were more inflammatory cells in the area of high-density neovascularization. This phenomenon may be related partly to the macrophages, which promote angiogenesis. Other studies have indicated that in tumours, macrophages appear to undergo marked phenotypic changes when exposed to hypoxia and to switch on the expression of a number of mitogenic and pro-angiogenic cytokines and enzymes.The results of this study demonstrate that ASCs enhanced the density of neovascularization, although this had no effect on the survival rate of graft fat early after its transplantation. Angiogenic growth factors secreted by ASCs, such as VEGF and HGF, may be responsible for this result. The aspiration and injection force mechanically injures the fat, and devascularization causes ischemic injury. ASCs can secrete various angiogenic growth factors such as VEGF, HGF and bFGF in response to injury, hypoxia and other conditions. Lee et al. indicated that proliferation of ASCs and secretion of angiogenic growth factors such as VEGF and bFGF from ASCs were significantly increased under hypoxic conditions (2%O2). Similarly, other researchers report that the gene expression of angiogenic growth factors such as VEGF, HGF and bFGF were increased by1.7-to4.1-fold in response to hypoxia. Another previous study indicated that VEGF was expressed in the interstitial mononuclear cells, most significantly on day7, and then decreased in the Wistar rat model of free fat grafts, which our experiment confirmed. However, ASCs caused VEGF secretion to peak earlier in the fat+ASCs group, on day4. The secretion of HGF was maintained at a high level in group A after fat transplantation, but peak of HGF appeared in group B on14days. After mechanical injury to human adipose tissue, FGF-2, TGF-β,and PDGF are first secreted in the early stage of wound healing. Thereafter, as the above growth factors decline, VEGF and HGF secretion gradually increases during the1st week postinjury. This may explain this phenomenon. The exogenous ASCs released much HGF response to hypoxia condition to maintain the high level of HGF in group A. In vitro studies revealed that ASCs could remain viable for only3days under ischemic conditions, aspirated fat transplantation causes an ischemic condition, many of the transplanted ASCs died in the first week after transplantation. The angiogenic effect of ASCs occurred during1week post-transplantation.Conclusion1. The difference between the two groups was in the graft tissue structure rather than in the graft survival rate. Tissue edema and infiltration of inflammatory cells result in increase of fat grafts volume in the early stage after aspirated fat transplantation.2. The results of this study demonstrate that ASCs enhanced the density of neovascularization, although this had no effect on the survival rate of graft fat early after its transplantation.3. Compared with the fat-only group, the fat+ASCs group showed earlier angiogenesis and more new vessels and less fibrosis. ASCs promoted the angiogenesis and inhibited the fibrosis of fat grafts by the paracrine effect of ASCs.4. The angiogenic effect of ASCs occurred during2weeks post-transplantation. |
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