Fat Grafts Supplemented with Adipose-Derived Stromal Cells in the Rehabilitation of Patients with Craniofacial Microsomia

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1 PEDIATRIC/CRANIOFACIAL Fat Grafts Supplemented with Adipose-Derived Stromal Cells in the Rehabilitation of Patients with Craniofacial Microsomia Daniela Y. S. Tanikawa, M.D. Meire Aguena, B.Sc., Ph.D. Daniela F. Bueno, D.D.S., Ph.D. Maria Rita Passos-Bueno, B.Sc., Ph.D. Nivaldo Alonso, M.D., Ph.D. São Paulo, Brazil Background: Although first reports of the clinical use of adipose-derived stromal cells suggest that this approach may be feasible and effective for soft-tissue augmentation, there is a lack of randomized, controlled clinical trials in the literature. Thus, this study aimed to investigate whether a faster protocol for isolation of adipose-derived stromal cells and their use in combination with fat tissue improve the long-term retention of the grafts in patients with craniofacial microsomia. Methods: Patients with craniofacial microsomia (n = 14) were grafted either with supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group). The number of viable cells isolated before and after the supplementation of the grafts was calculated, and these cells were examined for mesenchymal cell surface markers using flow cytometry. Computed tomography was performed to assess both hemifaces preoperatively and at 6 months postoperatively. Results: The average number of viable cells isolated before and after the supplementation of the grafts was and cells/ml of fat tissue (p = 0.015). Flow cytometric analysis revealed that the adipose-derived stromal cells were positive for mesenchymal cell markers (>95 percent for CD73 and CD105). Surviving fat volume at 6 months was 88 percent for the experimental group and 54 percent for the control group (p = 0.003). Conclusion: These results suggest that this strategy for isolation and supplementation of adipose-derived stromal cells is effective, safe, and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia. (Plast. Reconstr. Surg. 132: 141, 2013.) CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II. To overcome problems associated with fat grafting, such as unpredictable clinical results and a low rate of graft survival, many innovative efforts and refinements of surgical techniques have been reported. For example, condensation of living tissue and removal of unnecessary components have been performed by centrifugation, filtration, or gravity sedimentation 1,2 ; external mechanical force has been used to expand the recipient tissue and the overlying From the Division of Plastic and Reconstructive Surgery, University of São Paulo School of Medicine; and the Human Genome Research Center, Institute of Biosciences, University of São Paulo. Received for publication October 31, 2012; accepted January 9, This trial is registered under the name Clinical Trial of Fat Grafts Supplemented with Adipose-Derived Regenerative Cells, ClinicalTrials.gov identification number NCT ( Presented at IFATS Miami 2011: The 9th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue, in Miami, Florida, November 4 through 6, Copyright 2013 by the American Society of Plastic Surgeons DOI: /PRS.0b013e a82 Disclosure: The authors have no financial conflict of interest. Supplemental digital content is available for this article. Direct URL citations appear in the text; simply type the URL address into any Web browser to access this content. Clickable links to the material are provided in the HTML text of this article on the Journal s Web site (

2 Plastic and Reconstructive Surgery July 2013 skin envelope 3,4 ; and a recent experimental study suggested that repeated local injections of erythropoietin may enhance retention of grafted fat. 5 Based on the finding that aspirated fat tissue contains a much smaller number of adiposederived stromal cells compared with intact tissue 6 9 and that these cells play pivotal roles in the adipose tissue remodeling after lipoinjection, the supplementation of fat grafts with stromal vascular fraction isolated from the adipose portion of liposuction aspirates has been proposed as a method of compensating its relative deficiency of adiposederived stromal cells. 13 In the literature there are at least three experimental studies demonstrating that supplementation of progenitor cells enhances the volume or weight of surviving adipose tissue, 6,13 16 and first reports of its clinical use suggest that this approach may be feasible and effective for softtissue augmentation on the breast, face, hip, and hand. 12,17 22 However, considering that these studies represent level IV evidence (which corresponds to the publication of case series), 23 there is a lack of randomized, controlled clinical trials comparing this method to current standard techniques. 24 Thus, this study aimed to fill this gap by investigating whether a faster protocol for isolation of adiposederived stromal cells and their use in combination with fat tissue improve the long-term retention of the grafts in patients with craniofacial microsomia. PATIENTS AND METHODS Study Design A prospective, randomized, controlled, observerand patient blinded, single-center surgical trial with two parallel comparison groups was carried out. The study was approved by the institutional ethical committee (CAPPesq 1069/08). Trial Population and Eligibility Criteria From January of 2010 to June of 2011, every patient with craniofacial microsomia who presented at our department was assessed for enrollment in the study. Inclusion criteria were the presence of unilateral deformity; phenotype M0, M1 or M2, and S1 or S2 according to the Orbit, Mandible, Ear, Nerve, and Soft Tissue Plus classification 25,26 ; and age between 10 and 35 years. Excluded were patients with previous facial softtissue surgery, patients with absence of lower abdomen fat donor site, and those who refused to participate in the study. Informed consent was obtained from each subject at study entry. Randomization and Masking Patients were randomized by a computer-generated number table ( com), which was used to assign patients to one of two groups. Patients, data analysts, and the trial statistician were blinded for the trial intervention. Interventions Patients underwent fat grafting with either supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group). Surgical Technique For both groups, fat harvest, preparation, and application were performed in a standardized manner in accordance with Coleman s recommendations 1 by the same plastic surgeon. Briefly, fat was harvested from the lower abdomen using manual suction with a 10-cc syringe and a 2.5-mm blunt cannula. Syringe-assisted lipoaspirate was centrifuged at 700 g for 3 minutes (Technofat, São Paulo, Brazil). Following centrifugation, the blood/tumescent fraction was drained and the oil was removed. For the control group, the resulting fat layer was used for grafting (Fig. 1). For the experimental group, half of this volume was used for isolation of adipose-derived stromal cells, which allowed supplementation of the other half with adipose progenitor cells at a 1:1 ratio (Fig. 2). The adipose portion of liposuction aspirates was digested with 0.15% collagenase type IA (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes on a shaker at 37 C. Mature adipocytes and connective tissue were separated from the stromal vascular fraction by centrifugation at 176 g for 5 minutes, and pellets were resuspended in 1 cc of hypotonic water. Freshly isolated adipose-derived stromal cells were added to the graft material. After gentle mixing and waiting for 10 minutes for cell adherence to the aspirated fat, the cell-supplemented fat was then transferred into 1-cc syringes for injection. For both groups, multiple access sites and a fan-like pattern technique using 1.4-mm and 1.8- mm blunt cannulas were used to transfer small aliquots of fat into various depths of the soft tissue. Quantity of transplanted adipose tissue was determined by attempting to obtain symmetry with the unaffected side without any overcorrection. Clinical Assessment Preoperative variables included age, sex, and body mass index; and intraoperative variables were surgical time, volume of fat harvested, and 142

3 Volume 132, Number 1 Fat Grafts for Craniofacial Microsomia Fig. 1. Fat graft preparation for the control group. After centrifugation, the syringe-assisted lipoaspirate is separated based on its density. The oil fraction at the top can be wicked away, and the bloody fraction at the bottom can be drained. The resulting fat layer is used for grafting. the ratio between the quantity of fat harvested and the quantity of fat obtained after centrifugation. Postoperatively, patients were asked to return for follow-up appointments at weeks 1, 2, and 4 and at months 3 and 6; at each follow-up visit, surgical complications were documented and patients were photographed. Cellular Assessment Immediately after the surgical procedure, the number of viable cells isolated before and after the supplementation of the grafts was counted using the trypan blue dye exclusion assay in a Neubauer chamber using a Nikon Eclipse TS100 light microscope (Nikon Instruments, Inc., Melville, N.Y.). Next, immunophenotype characterization of cell populations on passage 1 was performed by flow cytometric analysis with the anti-human antibodies CD29, CD31, CD45, CD90, CD73, and CD105 (Becton, Dickinson and Company, Franklin Lakes, N.J.) in a Guava EasyCyte flow cytometer running the Guava Express Plus software (Guava Technologies, Hayward, Calif.). Adipose-derived stem cell cultures were performed according to Aguena et al. 27 Radiographic Assessment Computed tomographic scans were obtained preoperatively and 6 months postoperatively for volumetric and soft-tissue thickness measurements of both hemifaces using Osirix MD software (Pixmeo, Bernex, Switzerland). Volumetric augmentation was noted for each patient by comparing the difference between volumes of affected and unaffected hemifaces in the preoperative and postoperative periods, which was considered the retention volume. The percentage of fat graft survival regarding symmetry was determined as a ratio of the retention volume to the preoperative difference between volumes of affected and unaffected hemifaces. The percentage of fat graft survival regarding grafted volume was determined as a ratio of the retention volume to the volume of fat grafted. (See Figure, Supplemental Digital Content 1, which shows computed tomographic volumetric measurement, PRS/A749. Using drawing tools of the navigation system, the volumes of affected and unaffected hemifaces are calculated in both preoperative and postoperative periods.) To assess fat graft survival per facial anatomical region, soft-tissue thicknesses were measured at four different reference points (i.e., lateral canthus, zygomatic process, angle of mandible, and mental tubercle), and symmetry scores were determined both preoperatively and at follow-up and calculated as a ratio between soft-tissue thicknesses of affected and unaffected sides. (See Figure, Supplemental Digital Content 2, which shows fat graft survival per facial anatomical region assessment, links.lww.com/prs/a750. At the lateral canthus, zygomatic process, angle of mandible, and mental tubercle, soft-tissue thicknesses are measured and symmetry scores are determined both preoperatively and at follow-up.) Results Classification Based on the percentage of fat graft survival regarding grafted volume, results were classified as follows: excellent, 75 to 100 percent; good, 50 to 75 percent; and unsatisfactory, 0 to 50 percent. 143

4 Plastic and Reconstructive Surgery July 2013 Fig. 2. Fat graft preparation for the experimental group. After centrifugation, 50 percent of structural fat is used for isolation of adipose-derived stromal cells (ADSC), which allows the supplementation of the other 50 percent with adipose progenitor cells at a 1:1 ratio. Power Analysis and Statistics Statistical and inferential analyses were performed by means of SPSS statistics software version 17 (SPSS, Inc., Chicago, Ill.). The assumptions of normal distribution in each group and homogeneity of variances between groups were evaluated, respectively, with the Shapiro-Wilk and Levene tests. In all inferential analyses, a type I (α) error probability at 0.05 was considered. Quantitative data were analyzed using a one-way analysis of variance for linear mixed-effect models (analysis of variance) or a t test. The Fisher s exact test and the likelihood ratio test were used for categorical data. Relative risk and its variables were calculated as an instrument of association between independent variables (type of intervention) and the dependent one (type of result). To identify differences between the two parallel comparison groups and based on a previous pilot study (80 percent power, α = 0.05), it was calculated through Minitab release statistical software (Minitab, Inc., State College, Pa.) that 18 patients needed to be recruited. RESULTS Study Population and Surgical Variables A total of 29 patients were enrolled, of whom 18 could be included and randomized. During 144

5 Volume 132, Number 1 Fat Grafts for Craniofacial Microsomia Table 1. Descriptive Statistics of Demographic Variables Control Group (%) Experimental Group (%) Age, yr 18.7 ± ± Sex 1.00 Male 3 (43) 2 (28) Female 4 (57) 5 (72) Body mass index 1.00 <20 kg/m 2 3 (43) 2 (28) kg/m 2 4 (57) 5 (72) kg/m 2 >30 kg/m 2 follow-up, two subjects from the control group dropped out (did not answer telephone calls or return for follow-up appointments), and two patients from the experimental group were excluded because of inadequacy of computed tomographic scans. Both groups were comparable in terms of age, sex, and body mass index (Table 1). For the control group, the mean surgical time was 80 minutes; for the experimental group, there was an average increase of 45 minutes (p < 0.001) (Table 2). With regard to the volume of fat harvested, the experimental group presented 50 percent more lipoaspirate, but in terms of percentage yield of fat obtained after centrifugation there was no significant difference between the experimental and control groups (p = 0.11) (Table 2). In the early postoperative period, both groups presented discrete swelling that resolved uneventfully, and thereafter there were no cases of surgical complications. Characterization of Adipose-Derived Stromal Cells A great variability in the absolute number of viable cells was found between individuals, but without correlation with age, sex, or body mass index (Table 3). For the experimental group, p before the supplementation of adipose-derived stromal cells, the average number counted was similar to the control group (p = 0.98), but after the supplementation process, there was an average increase of 73 percent for these values (p = 0.015) (Table 3). Based on the criteria of the International Society for Cellular Therapy, cells showed positive staining for mesenchymal (CD73, CD90, and CD105) and adhesion (CD29) markers and negative staining for hematopoietic (CD45) and endothelial (CD31) markers (Table 4). Radiographic Parameters Preoperatively, both groups had similar softtissue deficiencies (p = 0.41), but postoperatively, patients from the experimental group had a statistically significantly better retention volume than those from the control group (p = 0.008) (Table 5). For the control group, the percentages of fat graft survival regarding symmetry and grafted volume were 51 and 54 percent, respectively; for the experimental group, these indexes were 84 and 88 percent, respectively (p = and p = 0.002) (Table 5). Regarding fat graft survival per facial anatomical region, the experimental group had statistically significantly better postoperative symmetry scores for the lateral canthus, zygomatic process, and mental tubercle than did the control group (p = versus p = 0.58, p = 0.01 versus p = 0.18, and p = 0.04 versus p = 0.59, respectively); only for angle of mandible did both groups show significant improvement postoperatively (p = versus p = 0.009) (Table 6 and Figs. 3 through 6). Relative Risk and Its Variables Results classification based on percentage of fat graft survival regarding grafted volume showed unsatisfactory results for 57.1 percent of the control group and a predominance of excellent results for the experimental group (experimental Table 2. Descriptive Statistics of Intraoperative Variables Control Group Experimental Group p Surgical time, min <0.001 Mean ± SD 80 ± ± 16 Median (IQR) 75 (75 90) 120 ( ) Lipoaspirate volume, cc 0.54 Mean ± SD 103 ± ± 44 Median (IQR) 88 (80 117) 160 (98 180) Yield after centrifugation, % 0.11 Mean ± SD 41.5 ± ± 6.2 Median (IQR) 42.2 ( ) 45.5 ( ) Grafted volume, cc 0.43 Mean ± SD 29 ± 6 27 ± 7 Median (IQR) 28 (26 37) 28 (20 31) IQR, interquartile range. 145

6 Plastic and Reconstructive Surgery July 2013 Table 3. Descriptive Statistics of Cellular Viability No. of Viable Cells ( 10 5 /ml) Patient Age (yr) Sex BMI Before* After Control group 1 27 F F < F M F < M F Average 5.6 ± 10.8 Experimental group 1 9 F < M < F F M < M F Average 5.7 ± ± 8.4 p BMI, body mass index; F, female; M, male; ADSCs, adipose-derived stromal cells. *Before supplementation of adipose-derived stromal cells. After supplementation of adipose-derived stromal cells. Control group experimental group (before). Control group experimental group (after). Table 4. Flow Cytometric Analysis CD29 CD73 CD90 CD105 CD31 CD45 Average 98.6 ± ± ± ± ± ± 10.3 Table 5. Descriptive Statistics of Volumetric Variables Control Group Experimental Group p Preoperative difference between volumes of affected and unaffected hemifaces, cc Mean ± SD 32.1 ± ± Median (IQR) 30.0 ( ) 28.0 ( ) Postoperative difference between volumes of affected and unaffected hemifaces, cc Mean ± SD 15.0 ± ± Median (IQR) 15.0 ( ) 6.0 ( ) Percentage of fat graft survival regarding symmetry Mean ± SD 51.0 ± ± Median (IQR) 43.0 ( ) 80.0 ( ) Percentage of fat graft survival regarding grafted volume Mean ± SD 54.0 ± ± Median (IQR) 47.0 ( ) 81.0 ( ) IQR, interquartile range. group: excellent results, 7; control group: excellent, 1; good, 2; unsatisfactory, 4). Through the calculation of relative risk and its variables, it was observed that the experimental group would be seven times as likely as the control group to obtain an excellent result, and an excellent result was attributable to the intervention of supplementation of adipose-derived stromal cells in 85 percent (p = 0.005) (Table 7). DISCUSSION This study is the first prospective, randomized, controlled, observer- and patient-blinded trial to objectively compare results of structural fat grafting with and without supplementation of adiposederived stromal cells. Patients with craniofacial microsomia were chosen because this condition is usually unilateral. Through the use of data from the contralaterally preserved healthy hemiface, we 146

7 Volume 132, Number 1 Fat Grafts for Craniofacial Microsomia Fig. 3. Photographic and three-dimensional computed tomographic image correlation. Control group patient 1, a 24-year-old woman. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postoperatively, with 45 percent retention volume. were able to obtain control values for estimating facial volume and to evaluating volume changes on the affected side. Moreover, this study is the first to correlate the cellular yield of a protocol for isolation of adipose-derived stromal cells with clinical results and demographic variables. For correction of soft-tissue deficiency in patients with craniofacial microsomia, a microvascular free tissue transfer has been used widely However, although free tissue transfer is effective, it is not without risk and morbidity in this patient population, 32 and because of this, serial fat grafting has recently been proposed as an alternative for earlier soft-tissue improvement. 33 In a volumetric outcome comparison of patients who underwent serial autologous fat grafting or microvascular free flap surgery, it was reported that although the mean number of procedures needed to obtain symmetry was less for the microvascular free flap group compared with the fat-grafting group (2.2 versus 4.3), the combined surgical time was greater for the microvascular free flap group (490 versus 280 minutes). 33 In this study, for patients undergoing structural fat grafting with supplementation of adipose-derived stromal cells, symmetry scores were high, with only a single procedure and a mean surgical time of 125 minutes. Because adipose-derived stromal cells contain multiple types of stem and regenerative cells, a cooperative interaction among these cells and the factors that they produce may allow these cells to enhance graft survival and quality by simultaneously targeting multiple mechanisms, including increasing revascularization, reducing apoptosis, and promoting preadipocyte differentiation. 15 Furthermore, instead of using autologous cultured cells, there are practical reasons for our choice to evaluate the freshly isolated cell population. First, using fresh isolated cells is consistent with a smooth clinical workflow, where the patient 147

8 Plastic and Reconstructive Surgery July 2013 Fig. 4. Photographic and three-dimensional computed tomographic image correlation. Control group patient 2, a 21-year-old woman. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postoperatively, with 46 percent retention volume. can undergo liposuction and receive a cell-supplemented fat graft in the same visit. By contrast, use of autologous cultured cells would require a twostage procedure: the first to harvest tissue from which the cells needed to initiate the cultures are procured, and a delay of a few weeks during which these cells are cultured before the second procedure, in which a second liposuction procedure is performed to obtain the graft matrix and the cellsupplemented fat graft subsequently prepared and implanted. Significant safety considerations with cultured cells are also important because extensive cell culture can lead to the development of chromosomal abnormalities and transformation of cells, properties that are not seen in the freshly isolated population. In addition, cell culture for clinical use requires application of expensive reagents and good manufacturing practices infrastructure, and this motivation is to understandably generate an adequate cell dose. However, as shown by several authors, it does not appear to be necessary with adipose tissue, where the stem cell frequency is approximately 2500-fold higher. 37 In this study, by counting the number of viable cells isolated before and after the supplementation of the grafts, an average increase of 73 percent for these values was observed, and by immunophenotype characterization of cells, positive staining for mesenchymal and adhesion markers was detected, which confirms the effectiveness of this strategy for isolation and supplementation of adipose-derived stromal cells. However, in spite of a statistically significant augmentation of progenitor cells in the grafts (p = 0.015), we did not find the intuitive twofold higher number of cells that would be expected with the supplementation process at the 1:1 ratio. This could be attributable to the 20-minute protocol used for enzymatic digestion; however, further studies comparing stromal vascular fraction 148

9 Volume 132, Number 1 Fat Grafts for Craniofacial Microsomia Fig. 5. Photographic and three-dimensional computed tomographic image correlation. Experimental group patient 1, a 10-year-old girl. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postoperatively, with 100 percent retention volume. generation with different collagenase concentrations and digestion times are still required to elucidate which is the optimized method. In the literature, Matsumoto et al. described a 0.075% collagenase digestion for 30 minutes, 6 Tiryaki et al. reported an incubation period of 40 to 60 minutes, 21 and Faustini et al. suggested a collagenase concentration of 0.2% and a digestion time of 1 hour as the best operating conditions. 38 However, even for the comparative study of Faustini et al. that evaluated five different collagenase concentrations ranging from 0.05 to 0.2%, 38 there are limitations with regard to different incubation times that ranged from 1 to 12 hours, but not below it. In this study, striking differences in clinical results could be appreciated because of supplementation of adipose-derived stromal cells; however, in agreement with the findings of Padoin et al., a great variability in the absolute number of these cells was detected between individuals, and age, sex, and body mass index of the donor seemed not to influence this cellular yield. 39 Because we previously demonstrated a significant higher concentration of nucleated cells and an enriched subpopulation of cells of mesenchymal origin in samples from the lower abdomen when compared with others, 27 we did not consider any other donor sites. Although no consensus exists regarding the best technique and the longevity of results, the Coleman technique has gained widespread clinical application and has been adopted by many plastic surgeons. 40 Thus, to understand how the supplementation of adipose-derived stromal cells significantly enhanced graft survival, we have focused our attention on the comparison of this technique with and without the cellular enrichment. Results of fat-grafting procedures typically are assessed by observation, examination with 149

10 Plastic and Reconstructive Surgery July 2013 Fig. 6. Photographic and three-dimensional computed tomographic image correlation. Experimental group patient 2, a 13-year-old girl. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postoperatively, with 84 percent retention volume. palpation, and photographs, but currently, more objective methods including laser scanners, threedimensional photography, and computed tomographic imaging studies are available for assessing outcomes. 41 Because three-dimensional computed tomography with volume rendering enables measurement of distance, area, and volume, 42,43 and multiplanar three-dimensional reconstruction reduces the number of artifacts and superimposition of structures (both of which can hinder Table 6. Descriptive Statistics of Linear Variables Control Group Experimental Group Preoperatively Postoperatively Preoperatively Postoperatively Lateral canthus Mean ± SD 0.78 ± ± ± ± 0.10 p Zygomatic process Mean ± SD 0.87 ± ± ± ± 0.08 p Angle of mandible Mean ± SD 0.67 ± ± ± ± 0.20 p Mental tubercle Mean ± SD 0.75 ± ± ± ± 0.11 p

11 Volume 132, Number 1 Fat Grafts for Craniofacial Microsomia Table 7. Descriptive Statistics of Relative Risk and Its Variables RR 95% CI p AR NNT Excellent vs. (good plus unsatisfactory) (Excellent plus good) vs. unsatisfactory RR, relative risk; AR, attributable risk; NNT, number needed to treat. measurement), 44 we used three-dimensional computed tomography for blinded and comparative evaluation of facial volume during preoperative and 6-month postoperative periods. In contrast to satisfactory outcomes observed in patients who underwent structural fat grafting with supplementation of adipose-derived stromal cells, such results were not achieved in patients who received conventional microfat grafting alone, for whom resorption rates were at least threefold higher. In agreement with these results, fat graft survival per facial anatomical region were also significantly better for patients receiving the cellular enrichment, even in areas such as the temporal region, considered one of the poorest recipient sites. 45 Many investigators are concerned about the potential complications of stem cell therapy. We therefore kept monitoring clinical parameters, and thus far (for up to 1 year) no complications or hazards associated with this new therapy modality have been detected in either donor or recipient sites. At our institution, the mean additional cost for patients receiving the supplementation of adipose-derived stromal cells, including facility, equipment, and consumables, was $180, but we believe this increased cost is well offset by the cost reduction from the decreased number of operations necessary to obtain satisfactory results. Our study has the limitation of a substantial dropout rate, with fewer patients included than intended, but with the number of patients balanced between both treatment groups. Whereas we think this has not influenced the results, this is of course not certain. Another possible limitation is that the nature of treatment made it impossible to blind the surgical team. To overcome this drawback, we have blinded patients, data analysts, and the trial statistician. Although longer follow-up is still required, our results showed that this strategy for isolation and supplementation of adipose-derived stromal cells enhanced fat survival in a relevant clinical application, being effective, safe, and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia. Additional randomized controlled trials are necessary to substantiate the evidence for this strategy for increasing fat graft survival in various clinical circumstances. Nivaldo Alonso, M.D., Ph.D. Division of Plastic and Reconstructive Surgery University of São Paulo School of Medicine Av. Dr. Arnaldo, 455, Room 1360 São Paulo, Brazil nivalonso@gmail.com PATIENT CONSENT Patients or parents or guardians provided written consent for use of the patients images. ACKNOWLEDGMENTS The State of São Paulo Research Foundation/Center of Excellence for Production Innovation and Development and the National Council for Scientific and Technological Development sponsored this study. REFERENCES 1. Coleman SR. Structural fat grafting: More than a permanent filler. Plast Reconstr Surg. 2006;118(Suppl):108S 120S. 2. Condé-Green A, Baptista LS, de Amorin NF, et al. Effects of centrifugation on cell composition and viability of aspirated adipose tissue processed for transplantation. Aesthet Surg J. 2010;30: Khouri R, Del Vecchio D. Breast reconstruction and augmentation using pre-expansion and autologous fat transplantation. Clin Plast Surg. 2009;36: , viii. 4. Kato H, Suga H, Eto H, et al. Reversible adipose tissue enlargement induced by external tissue suspension: Possible contribution of basic fibroblast growth factor in the preservation of enlarged tissue. Tissue Eng Part A 2010;16: Hamed S, Egozi D, Kruchevsky D, Teot L, Gilhar A, Ullmann Y. Erythropoietin improves the survival of fat tissue after its transplantation in nude mice. PLoS One 2010;5:e Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12: Suga H, Matsumoto D, Inoue K, et al. Numerical measurement of viable and nonviable adipocytes and other cellular components in aspirated fat tissue. Plast Reconstr Surg. 2008;122: Eto H, Suga H, Matsumoto D, et al. Characterization of structure and cellular components of aspirated and excised adipose tissue. Plast Reconstr Surg. 2009;124: Kuroda Y, Kitada M, Wakao S, et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA 2010;107: Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover in humans. Nature 2008;453:

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