Human body fat transplant to nude mice

Original Contribution Kitasato Med J 2011;41:57-62 Human body fat transplant to nude mice Akio Nanjo, 1 Akira Takeda, 2 Eiju Uchinuma 2 1 Department of Plastic Surgery, Kitasato University Kitasato Institute Medical Center Hospital 2 Department of Plastic and Aesthetic Surgery, Kitasato University School of Medicine Objective: We transplanted two forms of human fat, en bloc and minced fat, onto nude mice, in accordance with fat transplants clinically performed, then evaluated the results as to whether or not any differences emerged in the fat engraftment. Materials and Methods: Fats derived from the human body were processed into two forms, en bloc and minced fat and transplanted to nude mice to make study models. The models were analyzed over an 8-week period, based on the findings from the following evaluations: 1. macroscopic, 2. quantitative, 3. histologic, and 4. 3-dimensional computer graphics (3DCG). Results: Macroscopically, the pieces of fat in the en bloc model were significantly engrafted. Quantitative changes were 82% or lower in the en bloc model and were 32% or lower in the minced model in the 8th week. There were histologic differences in collagenous fiber overgrowth and neoangiogenesis. The engraftment ratio in the volume evaluation using 3DCG was similar to that in the quantitative evaluation. Conclusions: Differences were observed depending on the forms of the fat to be transplanted, and the en bloc model was clearly significant. Key words: fat engraftment, human body fat, nude mouse, quantitative evaluation, 3DCG (3- dimensional computer graphics) Introduction A ugmentation effects due to fat injection are clinically used to improve tissue defects and facial morphology and for anti-aging procedures in plastic and cosmetic surgery. 1-8 In 1981, a fat suction method was established and the removed fats are reused for cosmetic surgery. 1,8 Recently, given that the amount of transplanted fat that will be absorbed is unpredictable, this method has been discontinued as a procedure. Research on engraftment of injected fat has been conducted on rats, rabbits, nude mice, and humans to analyze quantitative changes and histological evaluations. 2-5,8-29 To date, there have been few studies where the status of engraftment of human body fat is clearly evaluated. We evaluated the findings from the following evaluations on the engraftment status of the transplanted en bloc and minced fat that were processed in accordance with the clinical procedures over a specific period: 1. macroscopic evaluation, 2. quantitative evaluation, 3. histologic evaluation, and 4. 3-dimensional computer graphic (3DCG) evaluation. We also proposed applications of fat transplantation for clinical use. Materials and Methods Animals: Four-week-old BalbC nude mice (Charles River Laboratories Japan Inc., Yokohama), weighing around 20 g each, were used in this experiment. Management of the mice followed the policy of the experimental animal facility at Kitasato University School of Medicine. Fat: Excess human body fat removed during normal surgery was used. The study was approved by the Ethics Committee of Kitasato University Hospital. Fat Graft: The collected fat was processed into en bloc fat and minced fat by using a feather knife and the processed fat was stored in saline while being cooled in ice at 4. The total amount of fat to be transplanted was measured to 0.5 g by using a microelectronic scale balance (Shimadzu Corporation, Kyoto). The fat was then transplanted to the nude mice while the mice were Received 12 October 2010, accepted 14 October 2010 Correspondence to: Akio Nanjo, Department of Plastic Surgery, Kitasato University Kitasato Institute Medical Center Hospital 6-100 Arai, Kitamoto, Saitama 364-8501, Japan E-mail: nanjo-a@insti.kitasato-u.ac.jp 57

Nanjo, et al. under ether inhalation anesthesia (Figure 1). Analysis of grafted fat Samples were taken, 2 to 8 weeks after the transplant. Quantitative evaluation: A microelectronic scale balance (Shimadzu Corporation, Kyoto) was used. It evaluated statistics based on the result. Histological analysis After the samples were fixed by paraformaldehyde, they were embedded in paraffin. Then, the samples were cut into sections (4μmm), and H&E staining was done. Image analysis 3DCG software (Ratoc System Engineering Co., LTD., Tokyo): Tissue sections were traced and their images were created using the 3DCG software. 28-29 Statistical analysis Paired t-test and Wilcoxon t-test were used for fat reduction of each en bloc model and minced model and statistically compared at weeks 4 and 8. A P value of <0.01 was considered to indicate statistical significance. Results Macroscopic evaluation In both the en bloc and minced models, inflow blood vessels beneath the skin and from dartos were observed. In the minced model, significant contraction of the fat tissues were observed at week 8, and necrotic or degenerated tissues were observed during the observation period. 2,8 Quantitative evaluation In the en bloc model, the fat reduced to 82% or smaller at week 8 compared with immediately after the transplant. In the minced model, the fat rapidly reduced to 32% or smaller at week 8 compared with what it was at the time of the transplant. When fat reduction of each en bloc model and minced model was statistically compared at weeks 4 and 8, statistically significant differences were observed between the en bloc model and the minced En bloc fat was transplanted at 3 areas under the skin on the left side of the back of each nude mouse. Minced fat was also transplanted at 3 areas under the skin on the right side of the back of each of the same mice. Figure 1. Method of fat graft fat transplant Fat reduction of each en bloc model (A) and minced model (B) was statistically compared from weeks 2 to 8. 58 Figure 2. The temporal change of the transplant fat.

Human body fat transplant to nude mice We used the paired t-test: (A) the result was P < 0.01, mean ± 2SD was 0.62 ± 0.24 in the en bloc model (n = 15), P < 0.01, mean ± 2SD was 0.42 ± 0.24 in the minced model (n = 15). We used the Wilcoxson t- test: (B) the result was P < 0.01, mean ± 2SD was 0.51 ±0.34 in the en bloc model (n = 15), P < 0.01, mean ± 2SD was 0.14 ± 0.05 in the minced model (n = 15). Figure 3. Comparison of fat decrease between the en bloc model and the minced model at week 4 (A) and week 8 (B). Figure 4. Histological findings In the en bloc model, the development of new capillary vessels entering the surrounding area of the coating layers was observed. Collagenous fiber overgrowth occurred encompassing the surrounding area. In the minced model, collagenous fiber overgrowth occurred in a netted manner while wrapping fat cells and neoangiogenesis were observed in the collagenous fiber. In the en bloc model, fat cells of uniform size were observed, and the same findings were observed after several weeks. In the minced model, differences in the sizes of the cells became clearer after several weeks. The view 2 weeks after transplantation of fat (A) en bloc model, (B) minced model. The view 8 weeks after transplantation of fat (C) en bloc model, (D) minced model. Hematoxilin and eosin (H&E) staining. 59

Nanjo, et al. When pure fat and collagenous fiber overgrowth are compared using 3DCG, volume in the minced model significantly decreased. Two weeks after transplantation of fat: (A) the en bloc model and (B) the minced model. Eight weeks after transplantation of fat: (C) the en bloc model and (D) the minced model Figure 5. 3DCG findings model, P < 0.01 (Figures 2 and 3). Histologic evaluation In both the en bloc and minced models, there were coating layers composed of flat cells on H&E staining, and large amounts of fat cells were observed inside the models. The fat cells decreased over time. Atypical cells were not observed during the observation period. Clear differences were observed in newly formed capillary vessels, collagenous fiber outgrowth, and the dimensions of the cells in both models (Figure 4). 3DCG evaluation The fat in the minced model significantly decreased, even visually, over time. The whole volume corresponded to the quantitative changes. As indicated in Figure 5, decrease in fat volume had an impact on the whole volume. Discussion According to clinical procedures, 2 forms of fat tissues, en bloc and minced, were transplanted onto the backs of nude mice. The engraftment ratio of each model was quantitated and evaluated as to the volume ratio of inner engraftment fat and collagenous fiber overgrowth over time. The status of the inner fat tissues and of collagenous fiber was demonstrated using 3DCG. In addition, evaluation of the fat engraftment ratio onto the backs of 60 nude mice where there is moderate stretching movement, appeared to justify evaluating the engraftment ratio in consideration of the momentum equivalent to that of the movement of human facial expressions. Comparison with previous reports Quantitative residual ratio of minced transplanted fat after fat suction surgery in clinical experiments ranges from 10%-60%. A survey done by the American Society of Plastic Surgeons presented that the quantitative residual ratio of fat was about 30% 1 year after the transplant. A report where the external appearance volume was evaluated by using magnetic resonance images suggested that volume will effectively increase by about 50%. 1-8 From previous studies with animal experiments, the engraftment ratio was shown to be 40%-45% in an en bloc model and 10%-40% in a minced model using the suction method. In the experiment in the present study, the engraftment ratio of the minced model fell under the range of the previous reports; however, the ratio of the en bloc model exceeded that of the previous reports. 9-14,18-21 The 3DCG evaluation demonstrated that the previous reports not only evaluated the transplanted fat engraftment but also collagenous fiber overgrowth. 9-21 Various attempts for fat conservation have been suggested. Even the atraumatic fat processing method used in this experiment corresponds to the engraftment ratio in previous reports. These attempts (e.g., suction at 1 atmosphere or lower, centrifuge separation, and cleansing

Human body fat transplant to nude mice with saline 2,6-8 ) do not seem to have a major impact on the increase of the engraftment ratio. A pure fat engraftment ratio seems to be determined by the fat dimensions and forms, rather than the variety of attempts. Histological evaluation According to the histological results of this report, definite differences in accordance with the transplanted fat forms are how abnormally collagenous fiber grows. Regardless of the transplanted fat forms, coating layers enclose the whole tissue of the transplanted fat; however, in the minced model, collagenous fiber overgrowth became activated, entering the transplanted fat inside of the coating layers. The fiber then attached to the surrounding transplanted fat in a netted manner. Previous studies showed that when conditions were not met and fat cells did not graft, degeneration necrosis occurred in the whole fat capsules which change into inflammatory cells, necrotic tissue formed inside the coating layers of collagenous fiber, fat volume decreased, as well as necrotic tissue being absorbed. 2,8 In the present study, reduction was observed in 2 cases of the minced transplanted fat at week 5. From this experiment, the en bloc model will increase the engraftment ratio under conditions where fat of 0.5 g (10 mm), a moderate amount for good blood circulation, is used. There are some reports that sufficient blood circulation is achieved when the transplanted fat dimensions are from 3 to 6 mm. 1,2,8 The important point is how to transplant normal fat tissues with minimal damage in moderate dimensions. This experiment demonstrated that as lobule division by the existing fascia was maintained in the en bloc model, the density of blood vessels was non-dense, and good mobility of the muscle movement was formed, as in the LAFS (lubricant adipofascial system) of deep fat structures. On the other hand, the transplanted fat grafted in the developed collagenous fiber was observed in the minced model. This finding showed that the minced model had a structure similar to a PAFS (protective adipofascial system), which is a shallow fat structure and also protects the inside against external forces. 24-29 Fat transplant and clinical application When a characteristic such as fat softness is required, as in an augmentation mammaplasty, a transplant of en bloc fat, in which areas that contact surrounding tissues increase to secure blood circulation, fibrillation is prevented, and pure fat tissue grafts appear to be efficient. In the minced model, the transplanted fats are rapidly absorbed and degeneration necrosis capsules are made at an early stage when injected with large amounts of fat. Thus, transplantation of en bloc fats is considered to be ideal, when there is good blood circulation to a small dimension of fat (volume of 0.5 g and 10 mm or smaller as used in this experiment). When augmentation effects are focused on, the minced fat transplant with an increase in volume due to collagenous fiber overgrowth and tightening effects, as well as ease of formation, is usually clinically effective in some cases. This is not engraftment of pure fat cells but involves the engraftment ratio hidden by collagenous fiber overgrowth. The conclusion is that there are differences in the engraftment ratio, as in animal experiments of previous studies, depending on where the fats are injected. The previous studies reported that the engraftment ratio of a transplanted fat of 0.5 g (10 mm or shorter) was 40%- 50%, especially, in the en bloc model, but no mention was made that collagenous fiber overgrowth was a part of the components of the engraftment ratio. When it is taken into consideration that the engraftment ratio in the en bloc model of this experiment was 82%, the amount of fat transplant in an en bloc shape would be ideal to increase the engraftment ratio. Actually, tissue findings, collagenous fiber overgrowth, and blood flow findings, in evaluation using a 3DCG, indicated that en bloc fat transplant could maintain stable and pure fat tissues. For clinical application, the en bloc model is effective when the engraftment ratio, sensitivity, and movement are important. The en bloc fat would be the most suitable for the nasolabial fold, breasts, upper lids, and cheeks. The minced model would be ideal for areas with less movement where a small amount of augmentation effects is sufficient. And the minced model appears to be effective in areas where a small amount of fat would be sufficient, such as the temple, the lower part of inferior eyelids, forehead, and mentum except for upper eyelids where formation is considered. 2-5 Fat injection is likely to be considered as a treatment with volume adjustment. When fat injection is used for the treatment of facial wrinkles when a small amount of fat is injected, the face can be tightened with internal adjustments. Fat injection is highly regarded because of its positive effects on external appearance. Acknowledgments The authors thank Dr. Shohei Yamashina for valuable suggestions in the preparation of the manuscript. 61

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