| BACKGROUNDRepair of soft tissue defects caused by the congenital deformity.trauma and tumorectomy has been regarded as one of considerable chanllages in plactic and reconstructive surgery.More than100years have passed by since Neuber was first to publish findings regarding the use of autologous fat transplantation in1893.He filled scars with autologous fat. Autologous free-fat transplantation has many potential advantages over other methods of soft-tissue augmentation for small to moderate-sized defects. This tissue is abundant, easily harvested, nonimmunogenic, and has very favourable physical characteristics.However.low survival rate of fat graft and unpredictability of retain volumn limit the clinical application of fat transplantation. Histologically, progressive loss of transplanted adipocytes is noted along with a conversion of the graft to fibrous tissue, often times with cyst formation.It is presumed that the portion of the graft adjacent to the native recipient blood supply nourishes the graft and sustains it. New growth of blood vessels from the surrounding tissue may take5days to infiltrate and provide adequate nutrition, which could result in graft central cell death and tissue necrosis. This concept has led to decreasing the size of fat grafts with the hope that a smaller graft will mean more fat is adjacent to viable recipient tissue, resulting in improved availability and diffusion of cellular nutrition until neovascularization occurs.The exact mechanisms that mediate fat graft survival and resorption remain unclear. One potential mechanism for graft loss is lack of adequate neovascularization within the transplanted fat. If timely neovascularization does not occur after transplantation, cells within the graft can die of ischemia, thus leading to tissue necrosis and graft loss. For autologous transplantation,Yoshimura et al. proposed cell-assisted lipotransfer to decrease resorption rates by adding stromal vascular fraction (SVF) to the fat graft. The SVF of the adipose tissue contains a group of heterogeneous cells consisting of adipose tissue-derived stem cells (ASCs), endothelial progenitor cells, hematopoietic progenitors, T cells and anti-inflammatory cells. The SVF would differentiate into mature adipocytes. stimulate in vivo regeneration by triggering migration of host stem cells to the recipient site, and promote vascularization, which is essential for fat graft take. We confirmed that cell-assisted lipotransfer based on SVF can significantly promote the viability of fat graft in rabbits attributed to its angiopoietic characteristics in our previous report. Due to its ease of harvest and the volume of tissue obtainable from liposuction, actually,freshly isolated stromal-vascular cell fraction(SVF) obtains adequate ASCs for clinical application whithout expansion in vitro.The uncomplicated enzyme-based isolation procedures make the cell therapy more attractive for researchers and clinicians.OBJECTIVES1. Modified the traditional experimental methods and optimized the technical parameters.Develop a method for the researchers in a relatively short period of time to get relatively large number of SVF cells. Investigate the feasibility of density gradient centrifugation method used in the purification of SVF cells.2. To determine whether SVF supplementation could improve long-term graft retention in patients who underwent autologous fat grafting for cosmetic improvement of facial contour.3. To determine whether SVF supplementation could improve long-term graft retention in patients who underwent autologous fat grafting for cosmetic improvement of breast augmentation.MATERIALS AND METHODS1. Concentration, digestion time and the centrifugal force parameters on the access to the SVF number.The lower abdomen or thigh in each patient was the donor site for harvesting fat grafts. Under the premise of fixed other factors constant, change the concentration, digestion time, the centrifugal force size at different levels to obtain the SVF. Erythrocyte lysis buffer lysis red blood cells. The cell counting board was used to count cells. Draw cells proliferation curve using the method of MTT.2. Using density gradient centrifugation to purify SVF cells.Ficoll liquid (1.083g/ml) was used as density gradient media. SVF cells were added to the centrifuge tube placed Ficoll solution. Centrifugation (1000g,20min), counted the number of SVF cells in the upper suspension and the bottom. The bottom of the cell pellet containing the SVF percentage(%)=(the SVF number at the bottom/SVF cell count)×1003. Supplementating Fat Grafts With Adipose Stromal Cells for Cosmetic Facial ContouringData were analyzed for38women who underwent transplantation with fat mixed with SVF (n=26) or fat grafting alone (n=12). The lower abdomen or thigh in each patient was the donor site for harvesting fat grafts. Approximately half of the collected liposuction aspirate was used for isolation of the SVF. During the processing period, the other half of the lipoaspirate was harvested as graft material. The harvested pellet was SVF. Each SVF sample underwent bacterial culture and susceptibility testing. All patients were seen at postoperative6-month intervals. Patients underwent CT and photos were taken preoperatively and6months postoperatively. For analysis of volume augmentation, we used the Philips Extended Brilliance Workspace,,which can provide objective data for various facial plastic and reconstructive surgeries4. Supplementating Fat Grafts With Adipose Stromal Cells for Cosmetic Breast Augmentation Data were analyzed for13women who underwent transplantation with fat mixed with SVF (n=8) or fat grafting alone (n=5). The lower abdomen or thigh in each patient was the donor site for harvesting fat grafts. Approximately half of the collected liposuction aspirate was used for isolation of the SVF. During the processing period, the other half of the lipoaspirate was harvested as graft material. To eliminate any remaining collagenase, the pellets were washed3times in PBS by repeated suspension and centrifugation. The harvested pellet was SVF. Each SVF sample underwent bacterial culture and susceptibility testing. All patients were seen at postoperative6-month intervals. Patients underwent Chest CT. breast mammography and photos were taken preoperatively and6months postoperatively. For analysis of volume augmentation, we used the Philips Extended Brilliance Workspace,,which can provide objective data for various facial plastic and reconstructive surgeries5. STATISTICAL ANALYSISThe data obtained was represent by mean±standard deviation (x±s), using SPSS13.0software package for statistical analysis. P<0.05represent significant differences.RESULTS:1. The SVF number of0.10%concentration group were (0.72±0.12)×106,0.20%group were (0.95±0.16)×106,0.25%group were (1.91±0.56)×106,0.40%group were (1.88±0.55)×1O6,0.50%group were (1.86±0.53)×106, using SPSS13.0software for multiple comparison between means of random design samples. Three groups (0.25%、0.40%、0.50%) with the first two groups (0.10%、0.20%) mean difference was significant (P<0.05).The mean difference between the three groups was not significant (P>0.05). There are significant differences between mean of the two groups (0.10%、0.20%)(P<0.05). The collagenase digestion concentration greater than or equal to0.25%, adipose tissue has been fully digested, can obtain a higher order of magnitude of the SVF cells.2. The SVF number of lOmin digestion group were (0.10±0.06)×106,20min group were (0.20±0.08)×1O6,30min group were (1.96±0.42)×106,40min group were (1.95±0.35)×1O6,50min group were (1.90±0.29)×106, using SPSS13.0software for multiple comparison between means of random design samples. Three groups(30min、40min、50min) with the first two groups (10min、20min) mean difference was significant (P<0.05).The mean difference between the three groups was not significant (P>0.05). There are significant differences between mean of the two groups (P<0.05). Collagenase digestion time is greater than or equal to30min (30min,40min,50min), adipose tissue has been fully digested, can obtain a higher order of magnitude of the SVF cells continue to extend the digestion time can not get more SVF cells.3. The SVF number of1000g group were (1.89±0.50)×106,2000g group were (1.88±0.55)×106,3000g group were(1.89±0.55)×106, The mean difference between the three groups was not significant (P>0.05). Centrifugal force change on SVF access number has no effect.4. After gradient centrifugation most of the red blood cells in the SVF has been removed. But still visible SVF cells mixed with some red blood cells. After erythrocyte lysis RBC almost invisible in cell pellets. The ratio of the bottom sediments in the SVF accounting for the total number of SVF was (11.30±5.10)%. Detected by MTT cell proliferation and growth capacity between the two experimental methods,OD value of the difference was not significant (P>0.05),5. All patients showed cosmetic improvements, but the degree varied among patients. Bacterial culture of each SVF sample was negative. Patients showed slight bruising and swelling during the first week after surgery but no scars during follow-up and no obvious contour irregularity. No further complications were evidenced during follow-up.38cases of patients with head CT showed no abnormal. In the SVF group, the mean fat transplanted was17.54±7.30ml and in the fat alone group16.25±6.34ml. Six months later, in the SVF group, the mean final fat volume was11.50±5.29ml and in the fat alone group7.56±3.26ml. The fat survival rate in SVF group was64.82±10.23(%) and control group was46.41±9.34(%).The difference in fat take between the2groups was statistically significant(P<0.05).6. All patients showed cosmetic improvements, but the degree varied among patients. Bacterial culture of each SVF sample was negative. Increased breast tenderness, chest curve compared with the preoperative improvement. Patients showed slight bruising and swelling during the first week after surgery but no scars during follow-up and no obvious contour irregularity.13cases of patients before operation and after6months of breast mammography, chest CT showed no abnormal. In the SVF group, the mean fat transplanted was151.25±35.63(ml) and in the fat alone group124.00±18.17ml. Six months later, in the SVF group, the mean final fat volume was94.68±24.62ml and in the fat alone group59.82±12.27ml. The fat survival rate in SVF group was63.06±10.47(%) and control group was47.95±4.20(%).The difference in fat take between the2groups was statistically significant(P <0.05).CONCLUSIONS1. The use of a concentration of0.25%type I collagenase and digestion time of30minutes available to get a relatively large number of SVF cells. Density gradient centrifugation is able to remove most of the red blood cells in cell pellets.2. Supplementing fat grafts with SVF for cosmetic facial contouring can improve long-term retention of fat as compared with fat grafting alone and provides satisfactory outcomes without any major complications.3. Supplementing fat grafts with SVF for breast augmentation can improve long-term retention of fat as compared with fat grafting alone and provides satisfactory outcomes without any major complications. Large sample of long-term controlled follow-up study of the safety and effectiveness of the evaluation of this technology is very necessary. |
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