pediatric/craniofacial - projeto...

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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 sedimenta-tion1,2; external mechanical force has been used to expand the recipient tissue and the overlying

Disclosure: The authors have no financial conflict of interest.

Copyright © 2013 by the American Society of Plastic Surgeons

DOI: 10.1097/PRS.0b013e3182910a82

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 stro-mal 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 craniofa-cial 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 sup-plementation of the grafts was 5.6 × 105 and 9.9 × 105 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 supple-mentation 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.

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 Janu-ary 9, 2013.This trial is registered under the name “Clinical Trial of Fat Grafts Supplemented with Adipose-Derived Regenerative Cells,” ClinicalTrials.gov identification number NCT01674439 (http://clinicaltrials.gov/ct2/show/NCT01674439).Presented at IFATS Miami 2011: The 9th Annual Sympo-sium on Adipose Stem Cells and Clinical Applications of Adi-pose Tissue, in Miami, Florida, November 4 through 6, 2011.

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

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PEDIATRIC/CRANIOFACIAL

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Plastic and Reconstructive Surgery • July 2013

skin envelope3,4; and a recent experimental study suggested that repeated local injections of eryth-ropoietin may enhance retention of grafted fat.5

Based on the finding that aspirated fat tis-sue contains a much smaller number of adipose-derived stromal cells compared with intact tissue6–9 and that these cells play pivotal roles in the adi-pose tissue remodeling after lipoinjection,10–12 the supplementation of fat grafts with stromal vascular fraction isolated from the adipose portion of lipo-suction aspirates has been proposed as a method of compensating its relative deficiency of adipose-derived stromal cells.13

In the literature there are at least three experi-mental studies demonstrating that supplementa-tion 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 soft-tissue augmentation on the breast, face, hip, and hand.12,17–22

However, considering that these studies repre-sent level IV evidence (which corresponds to the publication of case series),23 there is a lack of ran-domized, 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 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.

PATIENTS AND METHODS

Study DesignA prospective, randomized, controlled, observer-

and 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 CriteriaFrom January of 2010 to June of 2011, every

patient with craniofacial microsomia who pre-sented at our department was assessed for enroll-ment 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 clas-sification25,26; and age between 10 and 35 years. Excluded were patients with previous facial soft-tissue 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 MaskingPatients were randomized by a computer-gen-

erated number table (http://www.randomization.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.

InterventionsPatients underwent fat grafting with either

supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group).

Surgical TechniqueFor both groups, fat harvest, preparation, and

application were performed in a standardized manner in accordance with Coleman’s recommen-dations1 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 can-nula. Syringe-assisted lipoaspirate was centrifuged at 700 g for 3 minutes (Technofat, São Paulo, Bra-zil). Following centrifugation, the blood/tumes-cent fraction was drained and the oil was removed.

For the control group, the resulting fat layer was used for grafting (Fig. 1). For the experimen-tal 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 connec-tive 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 mix-ing 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 ali-quots of fat into various depths of the soft tissue. Quantity of transplanted adipose tissue was deter-mined by attempting to obtain symmetry with the unaffected side without any overcorrection.

Clinical AssessmentPreoperative variables included age, sex, and

body mass index; and intraoperative variables were surgical time, volume of fat harvested, and

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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, surgi-cal complications were documented and patients were photographed.

Cellular AssessmentImmediately 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 Neu-bauer 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, Frank-lin 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 AssessmentComputed tomographic scans were obtained

preoperatively and 6 months postoperatively for volumetric and soft-tissue thickness measure-ments of both hemifaces using Osirix MD soft-ware (Pixmeo, Bernex, Switzerland). Volumetric augmentation was noted for each patient by com-paring 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 preopera-tive 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, http://links.lww.com/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 refer-ence points (i.e., lateral canthus, zygomatic pro-cess, angle of mandible, and mental tubercle), and symmetry scores were determined both pre-operatively 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, http://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 preopera-tively and at follow-up.)

Results ClassificationBased 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.

Fig. 1. Fat graft preparation for the control group. After centrifugation, the syringe-assisted lipoaspi-rate 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.

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Power Analysis and StatisticsStatistical and inferential analyses were

performed by means of SPSS statistics soft-ware 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 likeli-hood ratio test were used for categorical data. Relative risk and its variables were calculated

as an instrument of association between inde-pendent variables (type of intervention) and the dependent one (type of result). To identify differences between the two parallel compari-son groups and based on a previous pilot study (80 percent power, α = 0.05), it was calculated through Minitab release 14.12.0 statistical soft-ware (Minitab, Inc., State College, Pa.) that 18 patients needed to be recruited.

RESULTS

Study Population and Surgical VariablesA total of 29 patients were enrolled, of whom

18 could be included and randomized. During

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.


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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 compa-rable 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 har-vested, the experimental group presented 50 per-cent more lipoaspirate, but in terms of percentage yield of fat obtained after centrifugation there was no significant difference between the experi-mental and control groups (p = 0.11) (Table 2). In the early postoperative period, both groups presented discrete swelling that resolved unevent-fully, and thereafter there were no cases of surgi-cal complications.

Characterization of Adipose-Derived Stromal CellsA 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,

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 ParametersPreoperatively, both groups had similar soft-

tissue deficiencies (p = 0.41), but postoperatively, patients from the experimental group had a sta-tistically 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 vol-ume were 51 and 54 percent, respectively; for the experimental group, these indexes were 84 and 88 percent, respectively (p = 0.003 and p = 0.002) (Table 5). Regarding fat graft survival per facial anatomical region, the experimental group had statistically significantly better postoperative sym-metry scores for the lateral canthus, zygomatic process, and mental tubercle than did the control group (p = 0.002 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 = 0.001 versus p = 0.009) (Table 6 and Figs. 3 through 6).

Relative Risk and Its VariablesResults classification based on percentage of

fat graft survival regarding grafted volume showed unsatisfactory results for 57.1 percent of the con-trol group and a predominance of excellent results for the experimental group (experimental

Table 1. Descriptive Statistics of Demographic Variables

Control Group (%)

Experimental Group (%) p

Age, yr 18.7 ± 7.6 12.1 ± 2.2 0.06Sex 1.00 Male 3 (43) 2 (28) Female 4 (57) 5 (72)Body mass index 1.00 <20 kg/m2 3 (43) 2 (28) 20–25 kg/m2 4 (57) 5 (72) 25–30 kg/m2 — — >30 kg/m2 — —

Table 2. Descriptive Statistics of Intraoperative Variables

Control Group Experimental Group p

Surgical time, min <0.001 Mean ± SD 80 ± 9 125 ± 16 Median (IQR) 75 (75–90) 120 (110–140)Lipoaspirate volume, cc 0.54 Mean ± SD 103 ± 37 150 ± 44 Median (IQR) 88 (80–117) 160 (98–180)Yield after centrifugation, % 0.11 Mean ± SD 41.5 ± 5.5 46.9 ± 6.2 Median (IQR) 42.2 (37.1–46.2) 45.5 (43.7–47.5)Grafted volume, cc 0.43 Mean ± SD 29 ± 6 27 ± 7 Median (IQR) 28 (26–37) 28 (20–31)IQR, interquartile range.

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group: excellent results, 7; control group: excel-lent, 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 excel-lent result was attributable to the intervention of supplementation of adipose-derived stromal cells in 85 percent (p = 0.005) (Table 7).

DISCUSSIONThis study is the first prospective, randomized,

controlled, observer- and patient-blinded trial to objectively compare results of structural fat graft-ing with and without supplementation of adipose-derived 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

Table 3. Descriptive Statistics of Cellular Viability

Patient Age (yr) Sex BMI

No. of Viable Cells (×105/ml)

Before* After†

Control group 1 27 F 20–25 0.5 — 2 26 F <20 1.7 — 3 24 F 20–25 1.2 — 4 10 M 20–25 0.8 — 5 9 F <20 3 — 6 21 M 20–25 30 — 7 14 F 20–25 2 — Average 5.6 ± 10.8 —Experimental group 1 9 F <20 0.3 0.5 2 13 M <20 6.6 12 3 10 F 20–25 11 20 4 11 F 20–25 5.2 12 5 13 M <20 15 20 6 14 M 20–25 0.2 0.4 7 15 F 20–25 1.7 4.7 Average 5.7 ± 5.7 9.9 ± 8.4p 0.98‡ 0.015§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 CD45Average 98.6 ± 0.9 96.9 ± 2.8 98 ± 1.6 95.3 ± 4.7 0.4 ± 0.7 6.3 ± 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 ± 9.4 28.0 ± 8.7 0.41 Median (IQR) 30.0 (28.0–36.0) 28.0 (23.0–36.0)Postoperative difference between volumes of affected and

unaffected hemifaces, cc Mean ± SD 15.0 ± 8.0 4.9±2.9 0.008 Median (IQR) 15.0 (12.0–20.0) 6.0 (3.5–6.0)Percentage of fat graft survival regarding symmetry Mean ± SD 51.0 ± 21.0 84.0 ± 10.0 0.003 Median (IQR) 43.0 (42.0–61.0) 80.0 (78.0–84.0)Percentage of fat graft survival regarding grafted volume Mean ± SD 54.0 ± 20.0 88.0 ± 13.0 0.002 Median (IQR) 47.0 (46.0–62.0) 81.0 (78.0–106.0)IQR, interquartile range.


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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 microvas-cular free tissue transfer has been used widely.28–31 However, although free tissue transfer is effec-tive, 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 alter-native for earlier soft-tissue improvement.33 In a volumetric outcome comparison of patients who underwent serial autologous fat grafting or micro-vascular 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 pro-cedure 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 simultane-ously targeting multiple mechanisms, including increasing revascularization, reducing apoptosis, and promoting preadipocyte differentiation.15 Furthermore, instead of using autologous cul-tured cells, there are practical reasons for our choice to evaluate the freshly isolated cell popula-tion. First, using fresh isolated cells is consistent with a smooth clinical workflow, where the patient

Fig. 3. Photographic and three-dimensional computed tomographic image corre-lation. Control group patient 1, a 24-year-old woman. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postop-eratively, with 45 percent retention volume.

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can undergo liposuction and receive a cell-supple-mented fat graft in the same visit. By contrast, use of autologous cultured cells would require a two-stage 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 proce-dure, in which a second liposuction procedure is performed to obtain the graft matrix and the cell-supplemented fat graft subsequently prepared and implanted. Significant safety considerations with cultured cells are also important because extensive cell culture can lead to the develop-ment of chromosomal abnormalities and transfor-mation of cells,34–36 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 supple-mentation of the grafts, an average increase of 73 percent for these values was observed, and by immunophenotype characterization of cells, posi-tive staining for mesenchymal and adhesion mark-ers 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 pro-tocol used for enzymatic digestion; however, fur-ther studies comparing stromal vascular fraction

Fig. 4. Photographic and three-dimensional computed tomographic image corre-lation. Control group patient 2, a 21-year-old woman. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postop-eratively, with 46 percent retention volume.


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generation with different collagenase concen-trations 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 colla-genase concentration of 0.2% and a digestion time of 1 hour as the best operating conditions.38 However, even for the comparative study of Faus-tini et al. that evaluated five different collage-nase 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 supple-mentation of adipose-derived stromal cells; how-ever, 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 wide-spread clinical application and has been adopted by many plastic surgeons.40 Thus, to understand how the supplementation of adipose-derived stro-mal 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

Fig. 5. Photographic and three-dimensional computed tomographic image correla-tion. Experimental group patient 1, a 10-year-old girl. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postop-eratively, with 100 percent retention volume.

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palpation, and photographs, but currently, more objective methods including laser scanners, three-dimensional photography, and computed tomo-graphic imaging studies are available for assessing outcomes.41 Because three-dimensional computed

tomography with volume rendering enables mea-surement of distance, area, and volume,42,43 and multiplanar three-dimensional reconstruction reduces the number of artifacts and superimpo-sition of structures (both of which can hinder

Fig. 6. Photographic and three-dimensional computed tomographic image correla-tion. Experimental group patient 2, a 13-year-old girl. (Above) Preoperative frontal images show facial asymmetry. (Below) Frontal images obtained 6 months postop-eratively, with 84 percent retention volume.

Table 6. Descriptive Statistics of Linear Variables

Control Group Experimental Group

Preoperatively Postoperatively Preoperatively Postoperatively

Lateral canthus Mean ± SD 0.78 ± 0.13 0.80 ± 0.14 0.72 ± 0.11 0.91 ± 0.10 p 0.58 0.002Zygomatic process Mean ± SD 0.87 ± 0.06 0.95 ± 0.11 0.86 ± 0.12 1.02 ± 0.08 p 0.18 0.01Angle of mandible Mean ± SD 0.67 ± 0.12 0.85 ± 0.09 0.78 ± 0.08 1.05 ± 0.20 p 0.009 0.001Mental tubercle Mean ± SD 0.75 ± 0.17 0.84 ± 0.11 0.79 ± 0.07 1.00 ± 0.11 p 0.59 0.04


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measurement),44 we used three-dimensional com-puted 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 supple-mentation 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 enrich-ment, 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 haz-ards 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 opera-tions 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 appli-cation, being effective, safe, and superior to con-ventional lipoinjection for facial recontouring in patients with craniofacial microsomia. Additional randomized controlled trials are necessary to sub-stantiate 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 MedicineAv. Dr. Arnaldo, 455, Room 1360

São Paulo, Brazil [email protected]

PATIENT CONSENTPatients or parents or guardians provided written

consent for use of the patients images.

ACKNOWLEDGMENTSThe State of São Paulo Research Foundation/Center

of Excellence for Production Innovation and Develop-ment and the National Council for Scientific and Tech-nological 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;30:249–255.

3. Khouri R, Del Vecchio D. Breast reconstruction and aug-mentation using pre-expansion and autologous fat trans-plantation. Clin Plast Surg. 2009;36:269–280, viii.

4. Kato H, Suga H, Eto H, et al. Reversible adipose tis-sue enlargement induced by external tissue suspension: Possible contribution of basic fibroblast growth factor in the preservation of enlarged tissue. Tissue Eng Part A 2010;16:2029–2040.

5. 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:e13986.

6. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipo-transfer: Supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382.

7. Suga H, Matsumoto D, Inoue K, et al. Numerical measure-ment of viable and nonviable adipocytes and other cellu-lar components in aspirated fat tissue. Plast Reconstr Surg. 2008;122:103–114.

8. Eto H, Suga H, Matsumoto D, et al. Characterization of struc-ture and cellular components of aspirated and excised adi-pose tissue. Plast Reconstr Surg. 2009;124:1087–1097.

9. Kuroda Y, Kitada M, Wakao S, et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA 2010;107:8639–8643.

10. Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover in humans. Nature 2008;453:783–787.

Table 7. Descriptive Statistics of Relative Risk and Its Variables

RR 95% CI p AR NNT

Excellent vs. (good plus unsatisfactory) 7.14 1.14–42.97 0.005 0.85 1.17(Excellent plus good) vs. unsatisfactory 2.38 0.99–5.49 0.07 0.57 1.75RR, relative risk; AR, attributable risk; NNT, number needed to treat.

mailto:[email protected]

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11. Suga H, Eto H, Shigeura T, et al. IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogen-esis through a c-Jun N-terminal kinase-dependent mecha-nism. Stem Cells 2009;27:238–249.

12. Yoshimura K, Suga H, Eto H. Adipose-derived stem/progeni-tor cells: Roles in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med. 2009;4:265–273.

13. Moseley TA, Zhu M, Hedrick MH. Adipose-derived stem and progenitor cells as fillers in plastic and reconstructive sur-gery. Plast Reconstr Surg. 2006;118(Suppl):121S–128S.

14. Masuda T, Furue M, Matsuda T. Novel strategy for soft tis-sue augmentation based on transplantation of fragmented omentum and preadipocytes. Tissue Eng. 2004;10:1672–1683.

15. Zhu M, Zhou Z, Chen Y, et al. Supplementation of fat grafts with adipose-derived regenerative cells improves long-term graft retention. Ann Plast Surg. 2010;64:222–228.

16. Li LT, Teng SC, Lin YH, Fang HW. Dose-dependent effect of adipose-derived stromal vascular fraction cells improve angiogenesis and anti-inflammation of human fat graft. Paper presented at: IFATS Miami 2011: The 9th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; November 4-6, 2011; Miami, Fla.

17. Yoshimura K, Matsumoto D, Gonda K. A clinical trial of soft tissue augmentation by lipoinjection with adipose-derived stromal cells (ASCs). Paper presented at: IFATS 2005: The 3rd Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; September 10–15, 2005; Charlottesville, Va.

18. Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K. Cell-assisted lipotransfer for cosmetic breast augmenta-tion: Supportive use of adipose-derived stem/stromal cells. Aesthetic Plast Surg. 2008;32:48–55; discussion 56.

19. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted lipotransfer for facial lipoatrophy: Efficacy of clinical use of adipose-derived stem cells. Dermatol Surg. 2008;34:1178–1185.

20. Yoshimura K, Asano Y, Aoi N, et al. Progenitor-enriched adi-pose tissue transplantation as rescue for breast implant com-plications. Breast J. 2010;16:169–175.

21. Tiryaki T, Findikli N, Tiryaki D. Staged stem cell-enriched tissue (SET) injections for soft tissue augmentation in hos-tile recipient areas: A preliminary report. Aesthetic Plast Surg. 2011;35:965–971.

22. Karaaltin MV, Akpinar AC, Baghaki S, Akpinar F. Treatment of “en coup de sabre” deformity with adipose-derived regenerative cell-enriched fat graft. J Craniofac Surg. 2012;23:e103–e105.

23. Rohrich RJ. Introduction to the facial soft-tissue fillers con-ference supplement. Plast Reconstr Surg. 2011;127:3S–5S.

24. Tabit CJ, Slack GC, Fan K, Wan DC, Bradley JP. Fat graft-ing versus adipose-derived stem cell therapy: Distinguishing indications, techniques, and outcomes. Aesthetic Plast Surg. 2012;36:704–713.

25. Horgan JE, Padwa BL, LaBrie RA, Mulliken JB. OMENS-Plus: Analysis of craniofacial and extracraniofacial anoma-lies in hemifacial microsomia. Cleft Palate Craniofac J. 1995;32:405–412.

26. Gougoutas AJ, Singh DJ, Low DW, Bartlett SP. Hemifacial microsomia: Clinical features and pictographic representa-tions of the OMENS classification system. Plast Reconstr Surg. 2007;120:112e–120e.

27. Aguena M, Fanganiello RD, Tissiani LA, et al. Optimization of parameters for a more efficient use of adipose-derived stem cells in regenerative medicine therapies. Stem Cells Int. 2012;2012:303610.

28. Longaker MT, Siebert JW. Microvascular free-flap cor-rection of severe hemifacial atrophy. Plast Reconstr Surg. 1995;96:800–809.

29. Longaker MT, Siebert JW. Microsurgical correction of facial contour in congenital craniofacial malformations: The marriage of hard and soft tissue. Plast Reconstr Surg. 1996;98:942–950.

30. Iñigo F, Jimenez-Murat Y, Arroyo O, Fernandez M, Ysunza A. Restoration of facial contour in Romberg’s disease and hemi-facial microsomia: Experience with 118 cases. Microsurgery 2000;20:167–172.

31. Koshima I. Short pedicle superficial inferior epigastric artery adiposal flap: New anatomical findings and the use of this flap for reconstruction of facial contour. Plast Reconstr Surg. 2005;116:1091–1097.

32. Saadeh PB, Chang CC, Warren SM, Reavey P, McCarthy JG, Siebert JW. Microsurgical correction of facial contour defor-mities in patients with craniofacial malformations: A 15-year experience. Plast Reconstr Surg. 2008;121:368e–378e.

33. Tanna N, Wan DC, Kawamoto HK, Bradley JP. Craniofacial microsomia soft-tissue reconstruction comparison: Infra-mammary extended circumflex scapular flap versus serial fat grafting. Plast Reconstr Surg. 2011;127:802–811.

34. Rubio D, Garcia-Castro J, Martín MC, et al. Spontaneous human adult stem cell transformation. Cancer Res. 2005;65:3035–3039.

35. Josse C, Schoemans R, Niessen NA, et al. Systematic chro-mosomal aberrations found in murine bone marrow-derived mesenchymal stem cells. Stem Cells Dev. 2010;19:1167–1173.

36. Zhang S, Han Z, Kong Q, et al. Malignant transforma-tion of rat bone marrow-derived mesenchymal stem cells treated with 4-nitroquinoline 1-oxide. Chem Biol Interact. 2010;188:119–126.

37. Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue: An underappreciated source of stem cells for biotechnology. Trends Biotechnol. 2006;24:150–154.

38. Faustini M, Bucco M, Chlapanidas T, et al. Nonexpanded mesenchymal stem cells for regenerative medicine: Yield in stromal vascular fraction from adipose tissues. Tissue Eng Part C Methods 2010;16:1515–1521.

39. Padoin AV, Braga-Silva J, Martins P, et al. Sources of pro-cessed lipoaspirate cells: Influence of donor site on cell con-centration. Plast Reconstr Surg. 2008;122:614–618.

40. Kaufman MR, Bradley JP, Dickinson B, et al. Autologous fat transfer national consensus survey: Trends in techniques for harvest, preparation, and application, and perception of short- and long-term results. Plast Reconstr Surg. 2007;119:323–331.

41. Kaufman MR, Miller TA, Huang C, et al. Autologous fat transfer for facial recontouring: Is there science behind the art? Plast Reconstr Surg. 2007;119:2287–2296.

42. Fontdevila J, Serra-Renom JM, Raigosa M, et al. Assessing the long-term viability of facial fat grafts: An objective measure using computed tomography. Aesthet Surg J. 2008;28:380–386.

43. Kim G, Jung HJ, Lee HJ, Lee JS, Koo S, Chang SH. Accuracy and reliability of length measurements on three-dimensional computed tomography using open-source OsiriX software. J Digit Imaging 2012;25:486–491.

44. Goldenberg DC, Alonso N, Goldenberg FC, et al. Using computed tomography to evaluate maxillary changes after surgically assisted rapid palatal expansion. J Craniofac Surg. 2007;18:302–311.

45. Mojallal A, Shipkov C, Braye F, Breton P, Foyatier JL. Influence of the recipient site on the outcomes of fat graft-ing in facial reconstructive surgery. Plast Reconstr Surg. 2009;124:471–483.

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