| Background:Autologous fat tissue has been considered as ideal filler for soft tissue augmentation, and has a wide prospect of clinical application in the field of plastic and aesthetic surgery. However, because of the adipocytes have a low tolerance to ischemic stress; they are more susceptible to death before the original microvascular structure of adipose tissue could be re-established. Therefore, complications such as fat necrosis, absorption, indurations and cyst formation, can be frequently seen after fat grafting. Efforts at overcoming this have focused on promoting neovascularization of fat grafts. But there is no breakthrough exists to date on the best fat grafting technique and the longevity of results. Recently, a significant increase in interest in the regenerative medicine using adipose-derived stromal vascular fraction (SVF) cells is emerging as a novel therapeutic option for fat grafting.As the largest endocrine organ in the body, adipose appears to provide an ideal pool of multipotent, undifferentiated, and nucleated cell populations which are involved in homeostasis and regenerative efforts. During the procedure of liposuction, many ASCs remain in the donor site, only a few ASCs can be transfered with fat grafts. This has led researchers to seek techniques that involve digestion, isolation and concentration of adipose-derived SVF cells, to be added back to the lipoaspirated tissues to restore native cellular quantitative levels. This technique has been reported by Yoshimura, termed cell-assisted lipotransfer (CAL).In breast augmentation trials, Yoshimura and colleagues have combined SVF cells with lipoaspirates from equal volumes of adipose tissue. The authors note improved fat grafting in the presence of the SVF cells with retention of volume for>1year without evidence of fibrosis or adhesions. Complications such as cyst formation or microcalcifications occur in less than2to3%of patients. Some basic studies implicate that the potential benefits of SVF cells, possibly due to their ability to secrete growth factors that have pro-angiogenic, anti-apoptotic, and pro-adipogenic affects. However, the multipotent differentiation capability of SVF cells in the repair and regeneration process of the grafted adipose tissue is still poorly understood. Of equal importance is to understand the fate of the implanted SVF cells. Can these cells survive for a long time in the ischemic microenvironment of fat grafts? Can these cells develop into specific types of cells? To date, studies that have clearly demonstrated the survival and differentiation of SVF cells as being dynamic phenomena have not been widely reported.Objective:In this study, we will focus on the biologic outcomes of SVF cells after co-implantation with fat grafts. We aim to establish a CAL animal model using SVF cells isolated from green fluorescent protein (GFP) transgenic mice, to track and investigate the in vivo survival and differentiation of implanted SVF cells. This study may provide a theoretical basis for the application of SVF cells in autologous fat grafting.Methods:1. We isolated the SVF cells from the inguinal adipose tissue of GFP transgenic mice. Cell numbers of freshly isolated GFP positive SVF cells (GFP+SVF) were determined by trypan blue staining. BALB/c nude mice were used as recipients for fat grafting. The inguinal adipose tissue was obtained from C57BL/6J mice and finely minced.0.5ml (0.360±0.005g) of the minced adipose tissue was mixed with5×105GFP+SVF cells and injected subcutaneously onto the skulls of BALB/c nude mice. This CAL model was established for the in vivo tracking of SVF cells after co-implantation with fat grafts. BALB/c nude mice were divided into two groups:(1) C57BL/6J mice-derived adipose tissue mixed with GFP+SVF cells was used in the experimental group;(2) C57BL/6J mice-derived adipose tissue mixed with PBS was used in the control group. The fat grafts were harvested from each mouse at each time point (days1,7,14,28,35,42,56). The graft mass was compared between these two groups.2. Four nude mice of each group were subjected to fluorescence imaging for the dynamic tracking study. Survival of the implanted GFP+SVF cells in living animals was detected by in vivo fluorescence imaging on days1,7,14.28,35,42and56after CAL. Fluorescence intensity of GFP+SVF cells was quantified using the IndiGo software provided with the in vivo imaging system.3. To evaluate the in vivo adipogenic and angiogenic potential of the implanted GFP+SVF cells, the grafted adipose tissue containing GFP+SVF cells was dissected from mice at each time point (days7,14,28,35,42,56). The graft samples were embedded in paraffin, and sectioned at3μm. Immunofluorescence staining of sections was performed using anti-perilipin antibody (a specific marker for viable adipocytes) or anti-CD31antibody (a specific marker for endothelial cells). All the sections were imaged on fluorescence microscope. Images were acquired and analyzed by AxioVision software.Results:1. In both of the two groups, the graft mass decreased sharply on day14. There was no statistically significance in graft mass between the two groups at the early stage after fat grafting (p>0.05). The graft mass in GFP+SVF cells group was higher than that of control group at28,35,42and56days after fat grafting (p<0.05).2. The results of in vivo fluorescence imaging showed that there was statistically significance in fluorescence intensity of GFP+SVF cells between different time points after fat grafting (p<0.0001). The fluorescence intensity of GFP+SVF cells fell drastically on days14after the co-implantation, with47.2%average signal intensity relative to day1. The fluorescence intensity continued decrease thereafter, with17.3%average signal intensity (relative to day1) at56days.3. Immunofluorescence staining revealed that some GFP+SVF cells can spontaneously differentiate into adipocytes from day7. The shapes of GFP and Perilipin double positive (GFP+Perilipin+) cells changed over time, as they became lipid droplet-containing adipocytes at56days. At28days, GFP and CD31double positive (GFP+CD31+) vascular endothelial cells were detected in newly formed vessel wall; the microvascular structure was found that was composed of intermingled GFP+CD31+endothelial cells and GFP negative endothelial cells (GFP-CD31+).Conclusion:1. The GFP transgenic mice can be used as an effective tool for the in vivo tracking of SVF cells. This method allows us to carry out a real time and continuous observation on the in vivo survival and differentiation of SVF cells by using of fluorescence imaging and immunofluorescence histochemical staining techniques. This animal model was established for investigating the biologic outcomes of SVF cells after co-implantation with fat grafts. It would also provide a good basic for further investigation of the mechanisms of SVF cell in fat grafting. 2. The results of in vivo fluorescence imaging showed that although most implanted SVF cells died shortly at the early stage after fat grafting, some of the SVF cells remained alive in the fat grafts for a long time.3. The results of immunofluorescence histochemical staining showed convincing evidence of the multipotent differentiation capability of SVF cells in fat grafting. The results prove the principle that a few of the living SVF cells can spontaneously differentiate into adipocytes in fat grafts and contribute to adipose tissue regeneration. Meanwhile, some of the living SVF cells can differentiate into endothelial cells and participate in vascular regeneration of the fat grafts. These findings suggest that the potential benefits of SVF cells in fat grafting may be the results of concurrent effects of multiple mechanisms. |
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