| Background and Objection:Soft tissue defect caused by trauma,disorder, aging and congenital malformations has been the common problems of plastic surgery.Collagen injections, dermal grafts and synthetic materials implanted in soft tissue augmentation are widely used in clinic. In1889, Meulen reported of autologous transplantation of adipose tissue. It has been over a hundred years of history. The use of body fat transplantation has major advantages:(1) low immunorejection;(2) low trauma;(3) easy to operate. Since the1930s, this technology has been widely used in plastic surgery.Although the significantly results of fat transplantation, but the long-term outcome is difficult to obtain. High absorption and low survival rate, many complications, limited its clinical application. Free fat graft has the absorption of fat liquefaction, graft, fibrous tissue replacement. It makes the graft ultimately save over time is only40-60%of the initial volume. The study found that fat graft was lack of effective blood supply due to acute ischemia and hypoxia in early transplantation. Before the graft with host to establish an adequate blood supply, adipose tissue can only rely on infiltration and infiltration of surrounding tissue fluid to maintain the nutrient supply. At that time, the central part of the adipose tissue has been ischemia and hypoxia, the occurrence of necrosis, liquefaction. Thus, early adequate blood supply and revascularization is the key to their survival after autologous granular fat transplantation. Therefore, how to enhance graft revascularization in a short time in order to improve the survival rate of transplanted fat is the focus field of present research.Recently, stem cell-based transplantation therapy has the potential to revolutionize the ability to regenerate damaged fat tissues. A promising stem cell-based transfer appears to bear to effectively promote the revascularization of the transplanted tissue.The previous studies have shown that adipose tissue-derived stem cells (adipose derived stem cells, ASCs) can promote the revascularization of the rat hind limb ischemia model, ischemic muscle tissue containing the vascular endothelial cells transformed from ASCs. The preliminary study found that human ASCs co-culture with fat particle transplanted into nude mice, can promote angiogenesis. It can significantly improve the survival of fat grafts. However, clinical application of autologous ASCs has its limitations. In vitro proliferation to obtain autologous ASCs apparently is unable to support the clinical applications. In common, it takes at least two to three weeks. It means that patients need to receive two surgeries. Moreover, cultured ASCs are complex, easily contaminated, low-security facilities and equipment demanding, higher cost.Since ASCs derived from fat in the vascular stroma cells (stromal vascular fraction is cells, SVFs), and SVFs can be isolated. Whether SVF can replace of ASCs as seed cells for the promotion early vascular of free transplanted fat? In2008, Kotaro Yoshimura reported the clinical application of SVFs with free fat transplantation. The study indicated that SVFs with fat transplantation can significantly reduce the fibrosis of fat, improve fat survival of the transplanted cells compared with the pure fat transplantation. Di jk found that SVFs intravenous injection into myocardial infarction mice can significantly promote revascularization of ischemic myocardial tissue. Another study showed that SVFs can improve the flaps survived. SVFs in human adipose tissue reserves are huge, easy isolate to the application of autologous immune rejection characteristics, ie, containing a sufficient number of adipose tissue freshly isolated SVFs. SVFs auxiliary of autologous fat transplantation, surgery can also be a complete, and without in vitro culture, safe, easier to clinic which makes SVFs is the promising cells to promote free transplanted fat revascularization seed cells.Although SVFs with fat grafts can effectively improve graft survival, it is still lack of studies on the potential mechanisms of survival after transplantation of adipose tissue. There are several questions to be future investigation:(a) morphology of SVFs with fat grafts during transfer (b) regeneration mechanism of SVF with fat grafts (c) SVFs with fat grafts what the best cell concentration can be more to promote the survival of the transplanted fat?At present study, we observe that histology of pure fat transplant. At each time point, transplantation things wet weight measurement, and analysis of HE staining, immunohistochemistry, Western blot and ELISA tests to investigate the fat cells of fat graft survival of autologous SVFs promote regeneration and revascularization mechanism, which would provide data for clinical application.Methods and materials1、Models for simply isolated fat grafts and evaluation of fat grafts survival.Each of nude mice were injected subcutaneously into the two random points on the back with0.2ml of fat particles using16gauge needle. Four indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68)2、Models for isolated fat grafts through SVFs and evaluation of fat grafts survival.Fat grafts of two groups are randomly injected subcutaneously into the back of each nude mice:suspension liquid of SVFs(1×106/ml) mixed with0.3ml of fat particles (Group S);0.3ml of DMEM mixed with0.3ml of fat particles(Group F, as a control). Five indexes are respectively observed on the lth day,4th day,7th day,14th day,30th day,60th day and90th day post transplantation:(1) General views of fat grafts;(2) Wet weight of fat grafts and percentage of wet weight;(3) Haematoxylin staining.(4) Immunohistochemical staining (S100, CD68).(5) Statistical analysis.3、Detection of revascularization in isolated fat grafts through SVFs.Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (CD31, CD34)and analysis of densities and trends of blood vessels by using system software;(3)Detect HGF vascular factor using Western Blot;(4) Detect VEGF and bFGF using ELISA;(5) Statistical analysis.4、Detection of regeneration of adipocyte in isolated fat grafts through SVFs.Examinations of both Group S and Group F are carried out in each time point mentioned above:(1) Haematoxylin staining;(2) Immunohistochemical staining (S100, CD68) and analysis of liabilities and trends of naive fat cells and mature adipose cells by using system software;(3)Detect expression and differentiation of human-derived adipokine by using PPARγ Fluorescence in situ hybridization.(4) Statistical analysis.5、Detect the optimal concentration of SVFs0.3ml of fat particles are respectively mixed with SVFs of different concentrations:5×105/ml(Group A);1×106/ml(Group B);2×106/ml(Group C),and with0.3ml of DMEM complete medium(Group D,as a control). Four indexes are observed at3months post transplantation:(1)Wet weight of fat tissue;(2) Haematoxylin staining;(3)’ Point-counting’ technique is applied to assessed fibrous necrosis of grafts and adipokine survival.(4)The numbers of capillary vessels.(n=6)6、Statistical analysis.Results were expressed as the mean standard error. SPSS13.0software was used for data analysis. The data was statistically analyzed by using a Two-way ANOVA. LSD test was used for the multiple comparisons of the mean values. Between the two groups were compared using a paired t-test; differences of the different time points using analysis of covariance. A p-level of0.05was considered to represent a statistical significance.Results: 1、Simply isolated fat grafts survivals at different time points after transplantion:(82.98±3.01) percentage on lth day;(81.91±2.931) percentage on4th day;(82.27±1.74) percentage on7th day;(82.98±1.90) percentage on14th day;(48.58±3.13) percentage on30th day;(47.16±2.49) percentage on60th day;(47.52±1.74) percentage on90th day.NO significant changes of wet weight of the fat grafts appeared within two weeks, but wet weight approximately reduced to half of the first day by14-30days,also subsequently, increasingly reduced slowly,47percentage of the tissues survived on90th day. Haematoxylin staining indicated:large numbers of necrosis vacuoles were shaped in center of early fat grafts and reached a peak by14days,wides of gap among adipocytes increased, regional fibrosis was obvious; a small number of naive fat cells were found by7days, subsequently increasingly increased and tended to mature,these naive fat cells differentiated into mature adipocytes late in transplantation and the number gradually reduced, organizational structures of fat grafts and normal tissues looked basically the same. Both positive effect of S-100protein and negative effect of CD68in naive cells demonstrated that the newborn naive cells were adipoblasts.2、Percentage of wet weight in isolated fat grafts through SVFs:(82.98±2.95) percentage on1th day;(84.04±2.93) percentage on4th day;(84.40±2.58) percentage on7th day;(87.23±3.01) percentage on14th day;(56.03±2.20) percentage on30th day;(54.61±1.74) percentage on60th day;(56.38±3.50) percentage on90th day. Compared with control group, it presents no significances on1th day,4th day and7th day using Two-sample t-test (P>0.05),but other groups are si gnificantly larger than control group, The difference of multiple comprare between ex perimental groups is significant(P<0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).Compared with control group, survival of fat grafts through SVFs raise d slightly on4th day,reduced by27percentage of the first day on30th day,56percent age of the grafts survived on90th day. Haematoxylin staining indicated that necrosis vacuoles and cyst-like changes appeared in center fat grafts through SVFs in early tim e,but less than control group,the degree of fibrosis was also milder compared with co ntrol group.On7th day,it appeared larger number of naive multilocular fat cells than control group, companied with existence of newborn fat cells of varying degrees of m aturity. It apperred more mature adipocytes in late time. Immunohistochemical stainin g indicated positive effect of S-100protein and negative effect of CD68in naive cells.3.Haematoxylin staining indicated:neovascularization of higher vascular density appeared on4th day in capsule of the grafts(SVFs Group) compared with control Group (appeared on7th day),also neovascularization gradually extended into center of the grafts; We detected the expressions of VEGF and bFGF at different time points by using ELISA. The levels of both angiogenic factors in SVFs Group were higher than control Group on4th day,7th day and14th day,also they reached a peak on7th day. Two-sample t-test represents the significant difference(P<0.05),30days after transplantation,the levels went down and there was no significant difference(P>0.05). The expressions of HGF in SVFs group were higher than those in the control group except30th day detected by using Western blot,there were significant differences(P<0.05). Immunohistochemical staining indicated:The levels of CD34expressed by hematopoietic cell lines were low,but begun to increase from7th day and reached a peak by30days,then gradually reduced. Small interstitial-like cells differentiated mature luminal-like vascular structures in morphology. The density of blood vessel with positive effect of CD31was analysed through Immunohistochemistry system software.The density gradually raised on4th day in SVFs group(on7th day in control group) and it reached a peak by14-30days,then leveled off.The vascular density in SVFs group was higher than that in control group, Two-sample t-test represents the significant differences on7th day,14th day and30th day(P<0.05) and there was no difference in other groups(P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05). 4.①HE staining after transplantation:To observe the dynamic evolution of pure fat transplantation group, the mother of neonatal fat cells to become the spindle lipoblasts is followed into preadipocytes, round lipoblasts:from primitive mesenchymal organization Yan, multivesicular lipoblasts, single bubble signet ring-like cells, and mature fat cells. The SVFs group to a variety of different differentiation status of the cells in the form, and more than the control group.②The immunohistochemical staining system software S100positive changes in the density of mature fat cells and neonatal fat cells at different time points:7days after cell transplantation of neonatal fat mother began to increase60days reached a peak, followed by a stable density. The SVFs higher than the control group, independent sample t-test respectively in14days,30days,60days difference between the two groups was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Mature fat cells in the transplant density gradually decreased by14-30days reduced to a maximum value, is relatively stable after60days, the density is slightly increasing trend, SVFs group was higher. Two groups at14days30days60days the difference was statistically significant (P<0.05), the remaining time points were no significant differences (P>0.05). Different time points the SVFs group covariance analysis that there is statistical difference between the number of show days(P<0.05).③mmunohistochemical staining expression of CD68early less concentrated in the capsule, advanced gradually increased the expression of most of the negative region of new fat cells.④The PPARy fluorescence in situ hybridization confirmed that the nucleus of newborn fat-like a little green fluorescent expression confirmed that most of the nascent fat cells derived from human to further exclude macrophages.5. Different concentrations of group:①wet weight:group A (60.0±6.325) mg, group B (81.67±7.528) mg, group C (68.33±7.528) mg, group D (48.33±7.528) mg,B group fat survival rate were higher than A, C, D group (P<0.05), A and C, between the two groups comparison was no significant difference (P>0.05).②ensity of blood vessels:A and C and group B blood vessel density higher than in group D and group B was significantly higher than the other three groups (P<0.05), A and C, between the two groups was no significant difference (P>0.05).③point count:survival of group B the fat cell counts were higher than A, C and D group (P <0.05), fibrous tissue counts were lower than group D (P<0.05), and two groups A and C, There was no difference betweenstatistically significant (P>0.05).DISCUSSIONSAt present, the absorption rate after transplantation of autologous fat particles, is from5%to100%after the fat transplantation, the absorption rate of about50%after1year. In general, the volume of fat absorbed the fastest within one month, beginning to stabilize after2to6months, almost no absorb any more after one year. In recent, the clinic results of fat grafting and revascularization were concerned. This study showed that the survival rate of transplanted fat pure fat in2weeks to1month significantly decreased and remained stable after three months. The presence of new fat cells in the transplant early, and mature fat cells increased over time, ultimately survived.A new approach was developed for the preparation of soft tissue repair material. SVFs with fat particles, it has a high survival rate, low absorption, safe to use, low-cost advantages and reduce pollution. It can be directly used in autologous transplantation. SVFs have the potential of osteogenic, chondrogenic, adipogenic, vascular and nerves differentiation, and promote revascularization, improve graft survival rate. At present study, we evaluate the dynamic evolution of morphological and histological differences between SVFs with fat graft and fat graft alone. The data shows that SVFs can increase the survival rate of fat graft transplantation significantly reduced the fibrosis of the fat liquefaction, the early emergence of a more naive multilocular fat cells in the more pure fat group, and accompanied by varying degrees.As is well known clinic, any transplant must take into account the blood supply, a sufficient blood supply support the graft survive. However, the early nutrition of cells also rely on the infiltration of the interstitial fluid, but the maximum nutrition distance of this penetration is150μm. More than this distance from the cell can not get essential nutrients to support cell metabolism. Therefore, how to quickly rebuild the blood supply is the key to the success of fat grafting. SVFs have to promote vascularization would reflect a promising potential applications. The mechanism of promote revascularization after free fat transplantation is still need further study. The present study demonstrated for the first four days of revascularization of the earliest of SVFs with fat grafts after transplantation, compared with pure fat grafts to early vascular density than pure fat group. The SVFs group of early vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and basic fibroblast cell growth factor (bFGF) expression of vascular endothelial growth factor than those of pure fat group, which SVFs in the lack of early transplantation oxygen state, the secretion of angiogenic factors through paracrine mechanisms, thereby accelerating neovascularization "budding" from around the graft recipient site formation, improved graft survival.Autologous fat transplantation, the tough challenge is to maintain its long-term result. At present, there are two academic point of view:(1) replaced on the host, the host macrophage phagocytosis of necrotic fat cells release lipid droplets, into mature fat cells, to replace the transplanted cells.(2) cell survival theory, the host macrophages only clear part of the necrotic fat cells release lipid droplets and the formation of fat cells to replace the transplantation of adipose tissue. The present study demonstrated that the SVFs transplantation more than the simple steatosis group to promote the regeneration of fat cells. Neonatal fat cells in the dynamic evolution of pure fat transplantation is followed by the spindle fat cell into a round fat cell, a single bubble the sexual signet ring-like cells and mature fat cells. SVFs group is different differentiation status of the cell survival, most of new fat cells derived from human SVFs in the interstitial cell differentiation and dedifferentiation of fat cells and then differentiation, a small part is original and some fat cells into fat differentiation from, reveals a potential mechanism of fat cell regeneration.The previous studies demonstrated that SVFs with fat grafts and fat particles in the volume ratio is1:1, to obtain the ideal result of fat transplantation.1:1volume ratio for transplantation is not accurate. Collagen digestion protocol is too complicated. The assessment of the transplant the proportion of fat transplantation is crucial. The previous study showed that per gram fat can be harvested approximately0.5×106-2.0×106of SVFs. At present study, the data indicated that lml of human adipose tissue can be harvested0.9×106-3.0×106SVFs extracted. It showed that the concentration of SVFs is one important factor for transplantation. Concentration is more accurate than transplant.However, to date, the effective concentration of SVFs auxiliary fat transplantation is yet to be reported. Therefore, the objective of this study was to investigate that the role of the concentration of SVFs if enhance transplantation of adipose tissue survival and angiogenesis.Conclusion1. During the entire process of transferred fat survived, graft volume and histology were accompanied changes.2. SVF with fat grafts can improve fat graft revascularization, fat cell regeneration, increase graft survival more than only fat grafts3. SVFs auxiliary fat graft revascularization occurred more pure fat transplantation early autologous SVFs secretion of angiogenic factors through paracrine mechanisms to improve fat graft revascularization.4. The data showed that cell survival, dedifferentiation and re-differentiation maybe the mechanism of fat graft fat cell regeneration.5.1×106/ml in magnitude has the highest survival rate of SVFs transplanted fat It is more suitable for fat transplantation, has a promising clinical application. |
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