Aesthetic Surgery Journal Advance Access published April 28, 2015
Research
Influence of Repeated Aspiration on Viability of Fat Grafts: A Comparative Study
Aesthetic Surgery Journal 2015, 1–13 © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: [email protected] DOI: 10.1093/asj/sjv047 www.aestheticsurgeryjournal.com
Rongrong Wang, MD; Jiaming Sun, MD; Lingyun Xiong, MD; and Jie Yang, MD
Accepted for publication March 4, 2015.
Autologous fat grafting has been used as a routine procedure for volume augmentation and defect restoration in cosmetic and reconstructive surgery for decades.1-4 Autologous fat tissue is considered as the ideal soft-tissue filler for its abundance, convenience, and host compatibility. In spite these benefits, the unpredictability of outcomes due to the absorption of fat grafts is still a huge limiting factor for fat grafting.5-7 Fat grafting involves harvesting, processing, and reinjection of the lipoaspirate. Each step of the process is responsible for the viability of adipocyte and long-term fat graft survival. Cheriyan et al.8 pointed out that lipoaspiration performed at a lower pressure of −250 mm Hg presented higher adipocyte viability than at a higher pressure of −760 mm Hg. Lee et al showed that fat grafts injected slowly with low shear stress significantly outperformed fat injected with high shear stress.9 A shear force of 25 atmosphere (atm) has a tremendous impact on fat graft survival, whereas a positive
pressure of 25 atm makes little influence. Additionally, in a controlled model of fat grafting with either a 5- or 3-mm aspiration cannula, the use of a larger aspiration cannula led to improved graft retention and quality.10 Appropriate suction pressure, injection rate, and cannula size could confine further mechanical damage to the fat grafts.8-10 We hypothesize that the above variables may function through mechanical forces, such that any From the Department of Plastic Surgery, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei Province, People’s Republic of China. Corresponding Author: Dr Jie Yang, Department of Plastic Surgery, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, People’s Republic of China. E-mail: [email protected]
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Abstract Background: Fat grafting has been increasingly widely used in cosmetic and reconstructive surgery. However, the long-term retention of fat grafts is still unpredictable. Many critical variables have been found to significantly affect the viability of fat grafts; still, some of the ordinary impact factors are overlooked. Objectives: We performed this study to find out whether repeated aspiration had an impact on fat grafts through an in vitro analysis and a nude mouse model. Methods: A 15 cm by 10 cm rectangle was marked at the lower abdomen. The cannula was gently advanced and retracted through the same incision in a fan fashion within the superficial layer to collect fat samples. Based on the sequence of harvesting, the collected adipose tissue was divided into five groups and labeled as syringes 1, 2, 3, 4, and 5. Part of the sample was dissociated and analyzed using cell staining, Cell Counting Kit-8 assay, and flow cytometry. The other part was injected in vivo and analyzed for weight and histology at varying time intervals. Results: Fat grafts from the former syringes were presented with a greater number of viable adipocytes and a higher level of cellular function compared to the latter syringes. Additionally, fat grafts from former syringes had higher graft retention, better vascularity, and less cystic necrosis. Neither the viability of stromal vascular fractions (SVFs) nor the ratio of CD34 + CD45− cells within the SVFs were different among the five groups. Conclusions: Repeated aspiration had a negative impact on the adipocytes, but not on the SVFs. With an increasing time of aspiration, the viability of the adipocytes and long-term retention of fat grafts decreased gradually. Harvested fat grafts from the first few syringes may be more suitable for fat grafting.
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variable that generates a certain amount of mechanical damage may negatively affect the fat grafts. Likewise, the back and forth movements of repeated suction against the adipose tissue might have a negative influence on the fat grafts. To our knowledge, there is no data available with regard to the impact of repeated aspiration on fat grafts, either with a handheld syringe or vacuum machine. The purpose of this study was designed to investigate the mechanical forces generated by repeated aspiration on the viability and long-term survival of fat grafts.
METHODS Patients
Fat Harvest Preoperatively, a rectangle with a base of 15 cm and a height of 10 cm was marked at the lower abdomen of each patient. All the adipose tissue was harvested from the marked area, into which an appropriate amount of tumescent solution (0.1% lidocaine with 1:200 000 epinephrine in normal saline) was infiltrated. A 3 mm, two-sidehole (2.5 × 5 mm), blunt-tipped cannula was attached to a 20 mL syringe for fat harvesting. A light negative pressure was provided by slowly withdrawing the plunger, not exceeding 2 cc of intra-luminal volume of the syringe. The cannula was gently advanced and retracted through the same incision in a fan fashion within the superficial layer to collect all the samples. The collected adipose tissue was divided into five groups, which were marked as syringes 1, 2, 3, 4, and 5 based on the sequence of harvesting. The volume of fat tissue from each group was about 15 mL. Lipoaspirate from each group was filtered through a 30-mesh stainless steel strainer and then washed three times with sterilized phosphate-buffered saline (PBS) to remove local anesthetics, epinephrine, oil, blood cells, and debris. Purified fat was collected for the study.
Adipocytes Staining Analysis Purified adipose tissue from each group was mixed with isometric 0.1% type I collagenase (Sigma Aldrich, St. Louis,
MO) in phosphate-buffered saline, and the mixture was incubated at 37°C in a 5% carbon dioxide incubator for 45 minutes (Thermo Fisher Scientific Inc., Waltham, MA). The digestion was terminated with complete medium [Dulbecco’s Modified Eagle Medium (DMEM)/F12 containing 10% fetal bovine serum; Sigma Aldrich, St. Louis, MO] when the particle morphology of the fat tissue disappeared. After straining through a 200-µm nylon sieve, the digested samples were centrifuged at 1000 revolutions per minute (RPM) for 10 minutes and stratified into four layers: fatty acid, mature adipocytes, culture medium, and stromal pellet. Mature adipocytes were washed four times with DMEM and centrifuged at 1000 RPM for 5 minutes. Last, mature adipocytes were stained with Calcein-AM (2 µmol/L) and Hoechst 33342 (10 µg/L; Dojindo, Kumanoto, Japan). The proportion of viable adipocytes was counted under a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Metabolic Activity of Adipocytes by Cell Counting Kit-8 Assay Cell Counting Kit-8 (CCK-8; Donjido, Kumanoto, Japan) provides sensitive colormetric assays to measure cell viability in cell proliferation and cytotoxicity assays. Compared to 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (XTT) assays, CCK-8 assay provides higher sensitivity and stability, shorter handling times, and less cytotoxicity. 105 of adipocytes from each group were prepared, which were determined by cell staining with Hoechst 33342 (flattened nucleus located on the periphery). From each group, 90 µL adipocytes were inoculated into a pre-incubated 96-well plate. CCK-8 solution (10 µL) was added to each well of the plate, and the mixture was incubated at 37°C for 3 hours. Measurements of the absorbance were made three times at 450 nm using a microplate reader (Thermo, Multiskan Spectrum).
Stromal Vascular Fractions Viability Analysis, Culture, and Multi-Differentiation Stromal pellets at the bottom of each group were collected and processed twice with red blood cell lysis buffer (Fushenbio, Shanghai, China). Then the remnant stromal vascular fractions (SVFs) were centrifuged, re-suspended by 300 µL PBS, and divided into three equal parts. A 10 µL sample of the first part was stained with Trypan Blue and counted on the hemocytometer to evaluate the viability of the SVFs. The procedure was repeated at least three times. The second part was cultured using DMEM/F12 medium supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) in a 25 cm2 culture flask (Corning, New York, NY). Cells were passaged when reaching 70-80%
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Six consecutive female patients undergoing abdominal liposuction from May 2014 to December 2014 were enrolled in this study. Informed consent was obtained from each patient, and the study protocol was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IRB no. FWA 00007304). Patients who had systemic metabolic diseases or a history of prior abdominal surgeries were not included in the study. All the procedures were performed by the same surgeon (J.Y.).
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confluence. And cells at subculture 3 were cultured in an adipogenic and osteogenic differentiation medium for 21 days, then stained with Oil Red-O and Alizarin Red S solution (Cyagen Biosciences, Santa Clara, CA).
Ratio of CD34 + CD45- Cells Within SVFs A 100 µL sample of the third part was incubated with 5 µL CD34-PE and CD45-PerCP/Cy5.5 (PE: Phycoerythrin, PerCP/Cy5.5: Peridinin chlorophyll/Cyanin 5.5) (eBioscience, San Diego, CA) at 4°C for 40 minutes. The mixture were centrifuged, re-suspended with 500 µL PBS, and analyzed using a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data acquisition and analyses were performed by FACSDiva software (BD Biosciences, Franklin Lakes, NJ).
According to the random number table (The RAND Corporation, Santa Monica, CA),11 40 six-week-old BALB/c-nu/nu female mice (Vital River, Beijing, China) were divided into five groups and their adipose tissue was divided into syringes 1, 2, 3, 4, and 5. All the mice were housed in an specific pathogen free level laboratory and cared for in accordance with institutional guidelines. Following previously published animal models, 0.3 mL aliquots of purified lipoaspirate from each group were injected subcutaneously at 4 para-vertebral points. An 18-gauge Coleman cannula attached to a 1-cc syringe was used (Figure 1A).
Fat Graft Persistence and Histology Fat grafts were harvested at 2 and 10 weeks to determine the short- and long-term fat graft persistence (Figure 1B). Harvested fat grafts were fixed in 4% paraformaldehyde, dehydrated in graded alcohols, cleared in xylene, embedded in paraffin, sectioned at 4-μm in thickness, stained with hematoxylin and eosin, and sealed with neutral balsam. The slides were analyzed according to the following histologic parameters: (1) intact and nucleated fat cells; (2) cysts and vacuoles; (3) inflammation, the infiltration of lymphocytes, and macrophages; and (4) fibrosis and other components of connective tissue. Each of these parameters was graded on a semi-quantitative scale of 0 to 5 where 0 indicated absence; 1, minimal presence; 3, moderate presence; 4, moderate to extensive presence; and 5, extensive presence. All the slides were examined by two independent pathologists in a blind manner.12
Statistical Analysis All data were presented as mean ± standard deviation (mean ± SD) and analyzed using SPSS 17.0 (SPSS, Chicago, IL). A one-way analysis of variance test was used
to compare the proportions of viable adipocytes, absorbance, and fat graft persistence between each group. The Mann-Whitney non-parametric test was used to compare the histological parameter scaling results between the groups. Confidence level was set at 95% and a p-value < .05 was considered statistically significant.
RESULTS The patients’ average age was 32.5 years (range, 24-47 years). The mean body mass index was 23.4 (range, 21.3-26.9).
Adipocytes Staining Analysis The proportion of viable adipocytes was 94.1% in syringe 1, 93.7% in syringe 2, 86.8% in syringe 3, 81.5% in syringe 4, and 80.4% in syringe 5, respectively. In general, the proportion of viable adipocytes decreases with an increasing sequence of harvesting. There were no significant differences between groups 1 and 2 ( p = 0.72) or groups 4 and 5 ( p = 0.06). A statistically significant difference could be detected between groups 2 and 3 ( p < 0.001 and groups 3 and 4 ( p < 0.05; Figures 2A-D and 3A).
Metabolic Activity of Adipocytes The absorbance of fat cells plus CCK-8 solution is 1.989 ± 0.261 in group 1, 1.997 ± 0.215 in group 2, 1.915 ± 0.152 in group 3, 1.858 ± 0.172 in group 4, and 1.815 ± 0.240 in group 5. With an increasing sequence of harvest, the absorbance of each group decreases gradually. The variation trend is in accordance with the results of a cell staining analysis. Statistically significant differences were found among all the groups except for groups 1 and 2 ( p = 0.93), and groups 4 and 5 ( p = 0.32; Figure 3B).
SVFs Viability, Multi-Differentiation, and Surface Marker Analysis Viability of SVFs from each group were: 92.07 ± 2.66% in group 1, 91.09 ± 3.03% in group 2, 91.52 ± 1.59% in group 3, 92.40 ± 2.84% in group 4, and 92.58 ± 1.90% in group 5 (Figure 4). There is no statistically significant difference among the five groups. Additionally, SVFs isolated from any of the five groups possessed a high capability of multidirectional differentiation (Figure 5A-C). Further, the ratio of CD34 + CD45− cells in SVFs were 49.72 ± 4.45% in group 1, 50.58 ± 4.45% in group 2, 49.25 ± 5.89% in group 3, 50.32 ± 3.45% in group 4, and 50.73 ± 3.49% in group 5. No statistically significant difference could be found among these five groups (Figure 6A and B).
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Animal Model
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Figure 1. Animal model. (A) 0.3 mL aliquots of purified lipoaspirate from each group were injected subcutaneously at 4 paravertebral points. (B) Fat grafts were harvested at 2 and 10 weeks to determine the short- and long-term fat graft persistence.
Graft Persistence The average graft weight at 2 weeks of syringes 1, 2, 3, 4, and 5 were 194.75 ± 8.18 mg, 190.58 ± 7.60 mg, 179.67 ± 10.86 mg, 173.25 ± 15.04 mg, and 171.92 ± 12.20 mg, respectively. The average graft weight at 10 weeks of each group was 150.17 ± 9.45 mg, 148.58 ± 10.23 mg, 139.42 ± 8.89 mg, 130.75 ± 10.40 mg, and 128.08 ± 11.32 mg, respectively. At both 2 and 10 weeks, a greater percentage of fat grafts survived for syringes 1, 2, and 3 compared to
syringes 4 and 5 ( p < 0.001). A statistically significant difference could be found between syringes 3 and 4 and between 3 and 5 ( p < 0.05; Figure 7).
Histological Analysis Generally, the histological appearance of fat samples from syringes 1, 2, and 3 present healthier adipocytes with less vacuoles, inflammation, and fibrosis, and better vascularity
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compared to syringes 4 and 5 at 10 weeks. No significant difference could be found between syringes 4 and 5, regardless of cyst formation, inflammation scale, the level of vascularity, or development of fibrosis. There is a statistically significant difference between syringes 1 and 3 for the level of inflammation ( p < 0.05); other than that, no statistically significant difference could be found among syringes 1, 2, or 3 in terms of the established evaluation criteria (Figures 8A-T and 9A-E).
DISCUSSION Autologous fat grafting has been widely used for soft-tissue defect reconstruction for increasing indications in both cosmetic and reconstructive breast surgery; lipoatrophy; lip, nose, or tempora augmentation; correction of unsightly scars; face or hand rejuvenation; and more. However, the
survival rate and long-term retention of the fat grafts varies greatly. To date, there is still no consensus on the optimal technique for fat grafting.13-21 The approaches by which the fat tissue is harvested, processed, and re-injected all have an influence on the fat graft viability and long-term survival. According to an evidencebased review on fat grafting by Gir et al,6 techniques that use low-pressure suction by means of larger bore cannulas appear to increase adipocyte viability. Lee et al9 pointed out that adipocytes are highly resistant to forces of positive and negative pressure alone, whereas they are remarkably susceptible to the effects of shear stress. Thus, different mechanical forces may generate varying degrees of cell injuries. Likewise, might the repetitive motions of fat harvesting also have a negative influence on the fat grafts? Various studies had been conducted to evaluate the impact of different fat harvest or preparation techniques on
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Figure 2. Calcein-AM/Hoechst 33342 staining to assess the viability of the isolated adipocytes. (A) Bright field. (B) Viable adipocytes were stained green by Calcein-AM. Lipid droplets were unstained. (C) Both viable and dead adipocytes were dyed blue by Hoechst 33342. (D) Merged figure to calculate the proportion of viable adipocytes.
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Figure 4. Viability of stromal vascular fractions (SVFs) were 92.07 ± 2.66% in group 1, 91.09 ± 3.03% in group 2, 91.52 ± 1.59% in group 3, 92.40 ± 2.84% in group 4, and 92.58 ± 1.90% in group 5, respectively. No significant differences could be found among the five groups.
fat grafts. Pu et al22 conducted a comparative study between the Coleman technique and the conventional machine liposuction. It turned out that the former technique was superior to the latter one, presented with more viable adipocytes and optimal cellular function. Ferguson et al23 also demonstrated that the LipiVage System (syringe aspiration at low pressure; Lewisville, TX) was better than the conventional liposuction technique, with higher viable adipocytes count. Agostini et al24 compared the histomorphology and adipocyte viability of lipoaspirated samples harvested using wet
and dry techniques. Though the fat collected with the wet technique was more preserved than that harvested with the dry technique, no substantial differences could be found. Besides, Erdim et al25 and Ozsoy et al26 both pointed out that larger cannulas tend to increase fat cell viability compared to smaller ones. In a controlled animal model of fat grafting, Kirkham et al10 found that the use of a 5-mm aspiration cannula led to improved fat graft retention and quality compared to a 3-mm cannula. The 5-mm group presented both less infiltrate and fibrosis. Further, several published research studies all pointed out that centrifugation forces greater than 3 000 RPM caused more cellular damage.12,27-29 All the above-mentioned approaches, different as they seemed, had one thing in common: they brought about more aggressive mechanical injuries against the fat grafts, which was consistent with our initial hypothesis. In the present study, fat grafts were collected at the same frequency, under the same pressure, and from the same site. The only variable is the sequence of harvesting. We found that fat grafts from the former 1, 2, and 3 syringes were presented with a greater number of viable adipocytes, higher level of cellular function, better graft retention, and more integral architecture compared to syringes 4 and 5. With continuous aspiration in the same limited area, the viability and metabolic function of the adipocytes worsened gradually. However, neither the viability of stromal vascular fractions or the ratio of CD34 + CD45- cells within the SVFs were different among the five groups. In addition, two types of fluorescent dyes (Calcein-AM and Hoechst 33342) were applied to stain and calculate the proportion of viable adipocytes. Calcein-AM, a cell-
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Figure 3. (A) Proportion of viable adipocytes from each group. No significant differences were found between groups 1 and 2 (p = 0.72) or groups 4 and 5 (p = 0.06). *A statistically significant difference could be detected between groups 2 and 3 (p < 0.001) and groups 3 and 4 ( p < 0.05). (B) Absorbance of the mixture containing adipocytes and CCK-8 solution after 3 hours’ incubation. The variation tendency is consistent with the results of cell staining analysis.
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permanent dye, can be used to determine cell viability in most eukaryotic cells. In live cells, the non-fluorescent Calcein-AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases.30 Hoechst 33342, a nucleic acid stain, is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to double-stranded DNA. Unlike trypan blue, which is a vital stain used to selectively color dead tissues or cells blue, fluorescent dyes can easily distinguish viable cells from cell debris and oil drops.31 Further, it is unnecessary to determine the absolute number of viable adipocytes through direct cell counting. Actually, it is slightly inaccurate to compare group difference through direct adipocytes counting. First, the original weight of fat tissues to be examined cannot be guaranteed to be exactly the same, and second, 1 gram of fat tissue can generally yield a half million adipocytes, so even the very little changed weight
of fat tissue may greatly influence the ultimate absolute number of adipocytes.25,32,33 Thus, in the present study, the proportion of viable adipocytes was calculated to reflect the whole viability of fat graft. CCK-8 assay was used to determine the metabolic activity of adipocytes. CCK-8 assay, like the MTT and XTT assays, utilized a highly-soluble tetrazolium salt (WST-9) to produce a water-soluble formazan dye (generated by dehydrogenases in viable cells), which was proportional to the number of living cells. Apart from being nonradioactive and more convenient, CCK-8 assay was more sensitive than assays using other tetrazolium salts, such as MTT, XTT, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, or water soluble tetrazolium salt.34 Xiong et al35 compared CCK-8 with MTT in measuring the viability of human promyelocytic leukemia cells. He pointed out the linear correlation of optical density (OD)
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Figure 5. Stromal vascular fractions (SVFs) isolated from any group are all highly viable and possess great viability of multidirectional differentiation. Typical figures are presented. (A) Adipose-derived stem cells from passage three (100×). (B) Oil Red-O staining of adipose-derived stem cells (ASCs) under adipogenic differentiation (100×). (C) Alizarin Red S staining of ASCs under osteogenic differentiation (100×).
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Figure 7. Average weight of fat grafts harvested at 2 and 10 weeks. At both 2 and 10 weeks, a greater percentage of fat grafts survived for syringes 1, 2, and 3 compared to syringes 4 and 5 (p < 0.001). A statistically significant difference could be found between syringes 3 and 4 and syringes 3 and 5 (*p < 0.05).
value and cell viability detected by CCK-8 was better than that of MTT. In our study, OD value measured by CCK-8 correlated well with the proportion of viable adipocytes from each group (data not shown). Besides the adipocytes, the other regenerative cells also play an important role in determining the long-term maintenance of fat grafts. A large number of studies have stated that fat grafts enriched with SVFs present better grafting results than mere fat grafts.36-39 SVFs consist of at least four subgroups: blood-derived cells (CD45+), adipose-derived stem cells (ASCs) (CD31-CD34 + CD45−), endothelial (progenitor) cells (CD31 + CD34 + CD45−), and pericytes (CD31-CD34-CD45−). In particular, the concentration of
CD34 + progenitor cells within the SVFs may predict retention of the fat grafts.38 A study conducted by Yin et al39 investigated the effect of water-jet force on SVFs compared to the classic Coleman technique, and found a significantly higher ratio of CD34 + CD45− cells within the water-jet group than the Coleman technique group; meanwhile, the water-jet group presented better fat graft survival. In our study, neither the viability of SVFs nor the ratio of CD34 + CD45− cells within the SVFs were significant different among the five groups. In this sense, SVFs may be more resistant to mechanical forces than the adipocytes, just like as they are more resistant to ischemia and hypoxia.40 Further, the classic nude mouse model was used to compare the long-term retention of fat grafts of different syringes, and the outcomes were also consistent with the in vitro experiments. The first few syringes presented with better architecture, increased vascularity, decreased fibrosis, and higher long-term survival. Therefore, fat tissue from the first few syringes may be more suitable for fat grafting; in the clinical practice, harvesting a certain amount of adipose tissue from multiple areas may be preferred to obtain a higher survival of grafted fat. In the next step, we are preparing to conduct a randomized controlled clinical trial to further confirm the efficacy of our investigation. If proven true, we are more than thrilled to have it applied immediately in the clinical practice, since the approach is easily accessible to both new and skilled practitioners. Actually, whether the approach is absolutely applicable depends on the gap between fat grafts supplies and demands. Theoretically, for those large-volume fat transplantation cases, like breast and buttock augmentation, it is rather difficult to harvest a sufficient volume of fat grafts while completely avoiding repetitive aspiration. But for those small-
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Figure 6. Ratio of CD34 + CD45- cells within stromal vascular fractions (SVFs). (A) Typical flow cytometry scatter plot. (B) Ratio of CD34 + CD45− cells in SVFs were 49.72 ± 4.45% in group 1, 50.58 ± 4.45% in group 2, 49.25 ± 5.89% in group 3, 50.32 ± 3.45% in group 4, and 50.73 ± 3.49% in group 5. No statistically significant difference could be found among these five groups.
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Figure 8. Typical example photographs of Hematoxylin and eosin-stained adipose tissue harvested at 2 and 10 weeks from each group. Samples from syringe 1 at (A) 2 weeks (40×); (B) 2 weeks (100×); (C) 10 weeks (40×); and (D) 10 weeks (100×). Samples from syringe 2 at (E) 2 weeks (40×); (F) 2 weeks (100×); (G) 10 weeks (40×); and (H) 10 weeks (100×). Samples from syringe 3 at (I) 2 weeks (40×); ( J) 2 weeks (100×); (K) 10 weeks (40×); and (L) 10 weeks (100×). Samples from syringe 4 at (M) 2 weeks (40×); (N) 2 weeks (100×); (O) 10 weeks (40×); and (P) 10 weeks (100×). Samples from syringe 5 at (Q) 2 weeks (40×); (R) 2 weeks (100×); (S) 10 weeks (40×); and (T) 10 weeks (100×). Low-magnification images were further magnified in the right column. There was better integral architecture, less collagen bands, decreased areas of fibrosis, and less infiltration of macrophages in syringes 1 and 2 compared to syringes 4 and 5. There is a statistically significant difference between syringes 1 and 3 in the level of inflammation at 2 weeks; other than that, no statistically significant difference could be found among syringes 1, 2, or 3 in terms of the established evaluation criteria at either time. No significant difference could be found between syringes 4 and 5 regardless of cyst formation, inflammation scale, the level of vascularity, or development of fibrosis at either 2 or10 weeks.
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volume fat transfer cases, the approach is entirely feasible. So certain necessary conditions are required to apply this approach: (1) patients with relatively sufficient subcutaneous fat storage; (2) small volume fat transplantation, usually less than 100 mL, like facial fat grafting, hand/genital rejuvenation, touch-up procedures for breast augmentation or reconstruction with implants, etc.; and (3) patients’ willingness to bear the possible discomfort following an increased amount of tumescent solution injection. Besides, large-volume fat grafting cases should usually be avoided. Additionally, as for the sample size of this investigation, it was taken into account at the time of designing. In a study by Smith et al41 to explore the effect of harvesting and preparation techniques on adipocyte graft survival, lipoaspriates from three patients were analyzed in vitro, and five freshly harvested tissue specimens were injected into the severe combined immune deficiency mice to study the in vivo viability. The authors also pointed out that the minimum number of specimens analyzed in vivo should be
no less than five. Further, in an investigation by Li et al,42 six female patients were enrolled in a study to determine the best donor site for tissue harvesting and isolation of SVF cells for fat graft survival. Similarly, nine female patients were enrolled in another in vitro and in vivo study to compare different processing methods on fat grafting.43 After full consideration, we decided to set the number of enrolled patients at six. Besides, all the subjects were females, as male patients were lacking. Originally, a certain number of male patients were intended to be included in the investigation, however, the number of male patients who came for liposculpture procedures were rather limited. We were unable to get sufficient male patients to participate in the investigation. Interestingly, among all the references we looked up, the gender information of the enrolled subjects was either unmentioned or entirely female.10,24-26,39,41-43 Maybe these investigators suffered the same situation as we did. Admittedly, whether gender difference has an effect on fat grafting is worthy of further study.
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Figure 8. Continued
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Figure 9. Histopathology parameters of (A) integrity, (B) cysts or vacuoles, (C) inflammation, (D) vascularity, and (E) fibrosis (*P < 0.05, **P < 0.001).
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CONCLUSIONS In summary, the present study has provided cell viability assessment, metabolic activity analysis of the adipocytes, viability of SVFs, ratio of CD34 + CD45− cells within SVFs in vitro, and graft retention in vivo to demonstrate that there are substantial differences in adipose tissue harvested from different syringes. With continuous aspiration, the cellular function of adipocytes and the viability and longterm retention of fat grafts decreased gradually. Fat tissue from the first few syringes may be more suitable for fat grafting. Harvesting a certain amount of adipose tissue from multiple areas may be preferred to obtain higher survival of fat grafts.
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Acknowledgements The authors wish to thank Drs Ke Guo, Quan Yuan, and Peng Xiao for surgical assistance, and Dr Yijing Chu for computer software assistance.
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Disclosures
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The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of this article. 18.
Funding The authors received no financial support for the research, authorship, and publication of this article.
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Lewis CM. The current status of autologous fat grafting. Aesthet Plast Surg. 1993;17(2):109-112. Guerrerosantos J. Autologous fat grafting for body contouring. Clin Plast Surg. 1996;23(4):619-631. Hunstad JP, Shifrin DA, Kortesis BG. Successful treatment of Parry-Romberg syndrome with autologous fat grafting: 14-year follow-up and review. Ann Plast Surg. 2011;67(4):423-425. Largo RD, Tchang LA, Mele V, et al. Efficacy, safety, and complications of autologous fat grafting to healthy breast tissue: a systemic review. J Plast Reconstr Aesthet Surg. 2014;67(4):437-448. Coleman SR. Structural fat grafting: more than a permanent filler. Plast Reconstr Surg. 2006;118(Supp 3):108S-120S. Gir P, Brown SA, Oni G, Kashefi N, Mojallal A, Rohrich RJ. Fat grafting: evidence-based review on autologous fat harvesting, processing, reinjection, and storage. Plast Reconstr Surg. 2012;130(1):249-258. Tabit CJ, Slack GC, Fan K, Wan DC, Bradley JP. Fat grafting versus adipose-derived stem cell therapy: distinguishing indications, techniques, and outcomes. Aesthet Plast Surg. 2012;36(3):704-713. Cheriyan T, Kao HK, Qiao X, Guo L. Low harvest pressure enhances autologous fat graft viability. Plast Reconstr Surg. 2014;133(6):1365-1368. Lee JH, Kirkham JC, McCormack MC, Nicholls AM, Randolph MA, Austen WG Jr. The effect of pressure and shear on autologous fat grafting. Plast Reconstr Surg. 2013;131(5):1125-1136. Kirkham JC, Lee JH, Medina MA 3rd, McCormack MC, Randolph MA, Austen WG Jr. The impact of liposuction cannula size on adipocyte viability. Ann Plast Surg. 2012;69(4):479-481. The RAND Corporation. A million random digits with 100,000 normal deviates.. Glencoe, IL: The Free Press; 1955. Ramon Y, Shoshani O, Peled IJ, et al. Enhancing the take of injected adipose tissue by a simple method for concentrating fat cells. Plast Reconstr Surg. 2005;115(1):197-201; discsussion 202-203. Coleman SR. Hand rejuvenation with structural fat grafting. Plast Reconstr Surg. 2002;110(7):1731-1744. Cárdenas-Camarena L, Arenas-Quintana R, RoblesCervantes JA. Buttocks fat grafting: 14 years of evolution and experience. Plast Reconstr Surg. 2011;128(2):545-555. Collar RM, Boahene KD, Byrne PJ. Adjunctive fat grafting to the upper lid and brow. Clin Plast Surg. 2013;40(1):191-199. Guisantes E, Fontdevila J, Rodríguez G. Autologous fat grafting for correction of unaesthetic scars. Ann Plast Surg. 2012;69(5):550-554. Dollfus C, Blanche S, Trocme N, Funck-Brentano I, Bonnet F, Levan P. Correction of facial lipoatrophy using autologous fat transplants in HIV-infected adolescents. HIV Med. 2009;10(5):263-268. Gatti JE. Permanent lip augmentation with serial fat grafting. Ann Plast Surg. 1999;42(4):376-380. Monreal J. Fat grafting to the nose: personal experience with 36 patients. Aesthet Plast Surg. 2011;35(5):916-922.
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Moreover, a single sized cannula was applied to harvest the fat grafts. Though not all surgeons harvest fat grafts with the 3-mm sized cannula, as far as we know few studies, among all those investigations on the influence of various variables on fat grafts viability, have introduced a second variable-harvesting cannula size on the basis of their original research (processing, reinjection, cryopreservation, etc.), other than those in search of more advantageous harvesting cannulas.10,24-27,39,41-43 In the present study, we mainly focus on the influence of the repetitive motions of harvesting cannula on the viability of fat grafts; though the whole investigation may not be optimal, we assume it is sufficient to confirm our original hypothesis. Our study has its own limitations. First, gender composition. Patients enrolled in our study were entirely female, and male patients were lacking. Hormone status due to gender differences may also influence the viability of fat grafts. Second, only cell components were analyzed; however, the local vasculogenic and anti-inflammatory cytokines were neglected. Finally, the entire procedures were conducted using the wet technique: a control group of patients undergoing fat harvesting with the dry technique would enhance the strength of the design of the study.
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cellular components in aspirated fat tissue. Plast Reconstr Surg. 2008;122(1):103-114. Lee JH, Kirkham JC, McCormack MC, et al. A novel approach to adipocyte analysis. Plast Reconstr Surg. 2012; 129(2):380-387. Keck M, Kober J, Riedl O, et al. Power assisted liposuction to obtain adipose-derived stem cells: impact on viability and differentiation to adipocytes in comparison to manual aspiration. J Plast Reconstr Aesthet Surg. 2014;67 (1):e1-e8. Cell Counting Kit-8. Technical manual. http://www. dojindo.com/ store/p/456-Cell-Counting-Kit-8.html. Accessed January 5, 2015. Xiong JW, Xiao H, Zhang ZX, et al. An experimental research on different detection conditions between MTT and CCK-8. Acta Laser Biol Sin. 2007;16:559-562. Dong Z, Peng Z, Chang Q, Lu F. The survival condition and immunoregulatory function of adipose stromal vascular fraction (SVF) in the early stage of nonvascularized adipose transplantation. PLOS O.NE 2013;8(11):e80364. Dong Z, Fu R, Liu L, Lu F. Stromal vascular fraction (SVF) cells enhance long-term survival of autologous fat grafting through the facilitation of M2 macrophages. Cell Biol Int. 2013;37(8):855-859. Philips BJ, Grahovac TL, Valentin JE, et al. Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model. Plast Reconstr Surg. 2013;132(4):845-858. Yin S, Luan J, Fu S, Wang Q, Zhuang Q. Does water-jet force make a difference in fat grafting? In vitro and in vivo evidence of improved lipoaspirate viability and fat graft survival. Plast Reconstr Surg. 2015;135(1):127-138. Suga H, Eto H, Aoi N, et al. Adipose tissue remodeling under ischemia: death of adipocytes and activation of stem/progenitor cells. Plast Reconstr Surg. 2010;126 (6):1911-1923. Smith P, Adams WP Jr, Lipschitz AH, et al. Autologous human fat grafting: effect of harvesting and preparation techniques on adipocyte graft survival. Plast Reconstr Surg. 2006;117(6):1836-1844. Li K, Gao J, Zhang Z, et al. Selection of donor site for fat grafting and cell isolation. Aesthet Plast Surg. 2013;37 (1):153-158. Salinas HM, Broelsch GF, Fernandes JR, et al. Comparative analysis of processing methods in fat grafting. Plast Reconstr Surg. 2014;134(4):675-683.
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Botti G, Pascali M, Botti C, Bodog F, Cervelli V. A clinical trial in facial fat grafting: filtered and washed versus centrifuged fat. Plast Reconstr Surg. 2011;127(6):2464-2473. Pinsolle V, Chichery A, Grolleau JL, Chavoin JP. Autologous fat injection in Poland’s syndrome. J Plast Reconstr Aesthet Surg. 2008;61(7):784-791. Pu LL, Coleman SR, Cui X, Ferguson RE Jr, Vasconez HC. Autologous fat grafts harvested and refined by the Coleman technique: a comparative study. Plast Reconstr Surg. 2008;122(3):932-937. Ferguson RE, Cui X, Fink BF, Vasconez HC, Pu LL. The viability of autologous fat grafts harvested with the LipiVage system: a comparative study. Ann Plast Surg. 2008;60(5):594-597. Erdim M, Tezel E, Numanoglu A, Sav A. Wet and dry techniques for structural fat graft harvesting: histomorphometric and cell viability assessments of lipoaspirated samples. Plast Reconstr Surg. 2012;130(2):331e-339e. Erdim M, Tezel E, Numanoglu A, et al. The effects of the size of liposuction cannula on adipocyte survival and the optimum temperature for fat graft storage: an experimental study. J Plast Reconstr Aesthet Surg. 2009;62(9): 1210-1214. Ozsoy Z, Kul Z, Bilir A. The role of cannula diameter in improved adipocyte viability: a quantitative analysis. Aesthet Surg J. 2006;26(3):287-289. Kim IH, Yang JD, Lee DG, Chung HY, Cho BC. Evaluation of centrifugation technique and effect of epinephrine on fat cell viability in autologous fat injection. Aesthet Surg J. 2009;29(1):35-39. Kurita M, Matsumoto D, Shigeura T, et al. Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation. Plast Reconstr Surg. 2008;121(3):1033-1041; discussion 1042-1043. 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(2):249-255. Vecchiatini R, Penolazzi L, Lambertini E. Effect of dynamic three-dimensional culture on osteogenic potential of human periodontal ligament-derived mesenchymal stem cells entrapped in alginate microbeads. J Periodontal Res. 2014; doi: 10.1111/jre.12225. Suga H, Matsumoto D, Inoue K, et al. Numerical measurement of viable and nonviable adipocytes and other
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Research
Influence of Repeated Aspiration on Viability of Fat Grafts: A Comparative Study
Aesthetic Surgery Journal 2015, 1–13 © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: [email protected] DOI: 10.1093/asj/sjv047 www.aestheticsurgeryjournal.com
Rongrong Wang, MD; Jiaming Sun, MD; Lingyun Xiong, MD; and Jie Yang, MD
Accepted for publication March 4, 2015.
Autologous fat grafting has been used as a routine procedure for volume augmentation and defect restoration in cosmetic and reconstructive surgery for decades.1-4 Autologous fat tissue is considered as the ideal soft-tissue filler for its abundance, convenience, and host compatibility. In spite these benefits, the unpredictability of outcomes due to the absorption of fat grafts is still a huge limiting factor for fat grafting.5-7 Fat grafting involves harvesting, processing, and reinjection of the lipoaspirate. Each step of the process is responsible for the viability of adipocyte and long-term fat graft survival. Cheriyan et al.8 pointed out that lipoaspiration performed at a lower pressure of −250 mm Hg presented higher adipocyte viability than at a higher pressure of −760 mm Hg. Lee et al showed that fat grafts injected slowly with low shear stress significantly outperformed fat injected with high shear stress.9 A shear force of 25 atmosphere (atm) has a tremendous impact on fat graft survival, whereas a positive
pressure of 25 atm makes little influence. Additionally, in a controlled model of fat grafting with either a 5- or 3-mm aspiration cannula, the use of a larger aspiration cannula led to improved graft retention and quality.10 Appropriate suction pressure, injection rate, and cannula size could confine further mechanical damage to the fat grafts.8-10 We hypothesize that the above variables may function through mechanical forces, such that any From the Department of Plastic Surgery, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei Province, People’s Republic of China. Corresponding Author: Dr Jie Yang, Department of Plastic Surgery, Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, People’s Republic of China. E-mail: [email protected]
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Abstract Background: Fat grafting has been increasingly widely used in cosmetic and reconstructive surgery. However, the long-term retention of fat grafts is still unpredictable. Many critical variables have been found to significantly affect the viability of fat grafts; still, some of the ordinary impact factors are overlooked. Objectives: We performed this study to find out whether repeated aspiration had an impact on fat grafts through an in vitro analysis and a nude mouse model. Methods: A 15 cm by 10 cm rectangle was marked at the lower abdomen. The cannula was gently advanced and retracted through the same incision in a fan fashion within the superficial layer to collect fat samples. Based on the sequence of harvesting, the collected adipose tissue was divided into five groups and labeled as syringes 1, 2, 3, 4, and 5. Part of the sample was dissociated and analyzed using cell staining, Cell Counting Kit-8 assay, and flow cytometry. The other part was injected in vivo and analyzed for weight and histology at varying time intervals. Results: Fat grafts from the former syringes were presented with a greater number of viable adipocytes and a higher level of cellular function compared to the latter syringes. Additionally, fat grafts from former syringes had higher graft retention, better vascularity, and less cystic necrosis. Neither the viability of stromal vascular fractions (SVFs) nor the ratio of CD34 + CD45− cells within the SVFs were different among the five groups. Conclusions: Repeated aspiration had a negative impact on the adipocytes, but not on the SVFs. With an increasing time of aspiration, the viability of the adipocytes and long-term retention of fat grafts decreased gradually. Harvested fat grafts from the first few syringes may be more suitable for fat grafting.
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variable that generates a certain amount of mechanical damage may negatively affect the fat grafts. Likewise, the back and forth movements of repeated suction against the adipose tissue might have a negative influence on the fat grafts. To our knowledge, there is no data available with regard to the impact of repeated aspiration on fat grafts, either with a handheld syringe or vacuum machine. The purpose of this study was designed to investigate the mechanical forces generated by repeated aspiration on the viability and long-term survival of fat grafts.
METHODS Patients
Fat Harvest Preoperatively, a rectangle with a base of 15 cm and a height of 10 cm was marked at the lower abdomen of each patient. All the adipose tissue was harvested from the marked area, into which an appropriate amount of tumescent solution (0.1% lidocaine with 1:200 000 epinephrine in normal saline) was infiltrated. A 3 mm, two-sidehole (2.5 × 5 mm), blunt-tipped cannula was attached to a 20 mL syringe for fat harvesting. A light negative pressure was provided by slowly withdrawing the plunger, not exceeding 2 cc of intra-luminal volume of the syringe. The cannula was gently advanced and retracted through the same incision in a fan fashion within the superficial layer to collect all the samples. The collected adipose tissue was divided into five groups, which were marked as syringes 1, 2, 3, 4, and 5 based on the sequence of harvesting. The volume of fat tissue from each group was about 15 mL. Lipoaspirate from each group was filtered through a 30-mesh stainless steel strainer and then washed three times with sterilized phosphate-buffered saline (PBS) to remove local anesthetics, epinephrine, oil, blood cells, and debris. Purified fat was collected for the study.
Adipocytes Staining Analysis Purified adipose tissue from each group was mixed with isometric 0.1% type I collagenase (Sigma Aldrich, St. Louis,
MO) in phosphate-buffered saline, and the mixture was incubated at 37°C in a 5% carbon dioxide incubator for 45 minutes (Thermo Fisher Scientific Inc., Waltham, MA). The digestion was terminated with complete medium [Dulbecco’s Modified Eagle Medium (DMEM)/F12 containing 10% fetal bovine serum; Sigma Aldrich, St. Louis, MO] when the particle morphology of the fat tissue disappeared. After straining through a 200-µm nylon sieve, the digested samples were centrifuged at 1000 revolutions per minute (RPM) for 10 minutes and stratified into four layers: fatty acid, mature adipocytes, culture medium, and stromal pellet. Mature adipocytes were washed four times with DMEM and centrifuged at 1000 RPM for 5 minutes. Last, mature adipocytes were stained with Calcein-AM (2 µmol/L) and Hoechst 33342 (10 µg/L; Dojindo, Kumanoto, Japan). The proportion of viable adipocytes was counted under a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Metabolic Activity of Adipocytes by Cell Counting Kit-8 Assay Cell Counting Kit-8 (CCK-8; Donjido, Kumanoto, Japan) provides sensitive colormetric assays to measure cell viability in cell proliferation and cytotoxicity assays. Compared to 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (XTT) assays, CCK-8 assay provides higher sensitivity and stability, shorter handling times, and less cytotoxicity. 105 of adipocytes from each group were prepared, which were determined by cell staining with Hoechst 33342 (flattened nucleus located on the periphery). From each group, 90 µL adipocytes were inoculated into a pre-incubated 96-well plate. CCK-8 solution (10 µL) was added to each well of the plate, and the mixture was incubated at 37°C for 3 hours. Measurements of the absorbance were made three times at 450 nm using a microplate reader (Thermo, Multiskan Spectrum).
Stromal Vascular Fractions Viability Analysis, Culture, and Multi-Differentiation Stromal pellets at the bottom of each group were collected and processed twice with red blood cell lysis buffer (Fushenbio, Shanghai, China). Then the remnant stromal vascular fractions (SVFs) were centrifuged, re-suspended by 300 µL PBS, and divided into three equal parts. A 10 µL sample of the first part was stained with Trypan Blue and counted on the hemocytometer to evaluate the viability of the SVFs. The procedure was repeated at least three times. The second part was cultured using DMEM/F12 medium supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) in a 25 cm2 culture flask (Corning, New York, NY). Cells were passaged when reaching 70-80%
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Six consecutive female patients undergoing abdominal liposuction from May 2014 to December 2014 were enrolled in this study. Informed consent was obtained from each patient, and the study protocol was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IRB no. FWA 00007304). Patients who had systemic metabolic diseases or a history of prior abdominal surgeries were not included in the study. All the procedures were performed by the same surgeon (J.Y.).
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confluence. And cells at subculture 3 were cultured in an adipogenic and osteogenic differentiation medium for 21 days, then stained with Oil Red-O and Alizarin Red S solution (Cyagen Biosciences, Santa Clara, CA).
Ratio of CD34 + CD45- Cells Within SVFs A 100 µL sample of the third part was incubated with 5 µL CD34-PE and CD45-PerCP/Cy5.5 (PE: Phycoerythrin, PerCP/Cy5.5: Peridinin chlorophyll/Cyanin 5.5) (eBioscience, San Diego, CA) at 4°C for 40 minutes. The mixture were centrifuged, re-suspended with 500 µL PBS, and analyzed using a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data acquisition and analyses were performed by FACSDiva software (BD Biosciences, Franklin Lakes, NJ).
According to the random number table (The RAND Corporation, Santa Monica, CA),11 40 six-week-old BALB/c-nu/nu female mice (Vital River, Beijing, China) were divided into five groups and their adipose tissue was divided into syringes 1, 2, 3, 4, and 5. All the mice were housed in an specific pathogen free level laboratory and cared for in accordance with institutional guidelines. Following previously published animal models, 0.3 mL aliquots of purified lipoaspirate from each group were injected subcutaneously at 4 para-vertebral points. An 18-gauge Coleman cannula attached to a 1-cc syringe was used (Figure 1A).
Fat Graft Persistence and Histology Fat grafts were harvested at 2 and 10 weeks to determine the short- and long-term fat graft persistence (Figure 1B). Harvested fat grafts were fixed in 4% paraformaldehyde, dehydrated in graded alcohols, cleared in xylene, embedded in paraffin, sectioned at 4-μm in thickness, stained with hematoxylin and eosin, and sealed with neutral balsam. The slides were analyzed according to the following histologic parameters: (1) intact and nucleated fat cells; (2) cysts and vacuoles; (3) inflammation, the infiltration of lymphocytes, and macrophages; and (4) fibrosis and other components of connective tissue. Each of these parameters was graded on a semi-quantitative scale of 0 to 5 where 0 indicated absence; 1, minimal presence; 3, moderate presence; 4, moderate to extensive presence; and 5, extensive presence. All the slides were examined by two independent pathologists in a blind manner.12
Statistical Analysis All data were presented as mean ± standard deviation (mean ± SD) and analyzed using SPSS 17.0 (SPSS, Chicago, IL). A one-way analysis of variance test was used
to compare the proportions of viable adipocytes, absorbance, and fat graft persistence between each group. The Mann-Whitney non-parametric test was used to compare the histological parameter scaling results between the groups. Confidence level was set at 95% and a p-value < .05 was considered statistically significant.
RESULTS The patients’ average age was 32.5 years (range, 24-47 years). The mean body mass index was 23.4 (range, 21.3-26.9).
Adipocytes Staining Analysis The proportion of viable adipocytes was 94.1% in syringe 1, 93.7% in syringe 2, 86.8% in syringe 3, 81.5% in syringe 4, and 80.4% in syringe 5, respectively. In general, the proportion of viable adipocytes decreases with an increasing sequence of harvesting. There were no significant differences between groups 1 and 2 ( p = 0.72) or groups 4 and 5 ( p = 0.06). A statistically significant difference could be detected between groups 2 and 3 ( p < 0.001 and groups 3 and 4 ( p < 0.05; Figures 2A-D and 3A).
Metabolic Activity of Adipocytes The absorbance of fat cells plus CCK-8 solution is 1.989 ± 0.261 in group 1, 1.997 ± 0.215 in group 2, 1.915 ± 0.152 in group 3, 1.858 ± 0.172 in group 4, and 1.815 ± 0.240 in group 5. With an increasing sequence of harvest, the absorbance of each group decreases gradually. The variation trend is in accordance with the results of a cell staining analysis. Statistically significant differences were found among all the groups except for groups 1 and 2 ( p = 0.93), and groups 4 and 5 ( p = 0.32; Figure 3B).
SVFs Viability, Multi-Differentiation, and Surface Marker Analysis Viability of SVFs from each group were: 92.07 ± 2.66% in group 1, 91.09 ± 3.03% in group 2, 91.52 ± 1.59% in group 3, 92.40 ± 2.84% in group 4, and 92.58 ± 1.90% in group 5 (Figure 4). There is no statistically significant difference among the five groups. Additionally, SVFs isolated from any of the five groups possessed a high capability of multidirectional differentiation (Figure 5A-C). Further, the ratio of CD34 + CD45− cells in SVFs were 49.72 ± 4.45% in group 1, 50.58 ± 4.45% in group 2, 49.25 ± 5.89% in group 3, 50.32 ± 3.45% in group 4, and 50.73 ± 3.49% in group 5. No statistically significant difference could be found among these five groups (Figure 6A and B).
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Animal Model
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Figure 1. Animal model. (A) 0.3 mL aliquots of purified lipoaspirate from each group were injected subcutaneously at 4 paravertebral points. (B) Fat grafts were harvested at 2 and 10 weeks to determine the short- and long-term fat graft persistence.
Graft Persistence The average graft weight at 2 weeks of syringes 1, 2, 3, 4, and 5 were 194.75 ± 8.18 mg, 190.58 ± 7.60 mg, 179.67 ± 10.86 mg, 173.25 ± 15.04 mg, and 171.92 ± 12.20 mg, respectively. The average graft weight at 10 weeks of each group was 150.17 ± 9.45 mg, 148.58 ± 10.23 mg, 139.42 ± 8.89 mg, 130.75 ± 10.40 mg, and 128.08 ± 11.32 mg, respectively. At both 2 and 10 weeks, a greater percentage of fat grafts survived for syringes 1, 2, and 3 compared to
syringes 4 and 5 ( p < 0.001). A statistically significant difference could be found between syringes 3 and 4 and between 3 and 5 ( p < 0.05; Figure 7).
Histological Analysis Generally, the histological appearance of fat samples from syringes 1, 2, and 3 present healthier adipocytes with less vacuoles, inflammation, and fibrosis, and better vascularity
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compared to syringes 4 and 5 at 10 weeks. No significant difference could be found between syringes 4 and 5, regardless of cyst formation, inflammation scale, the level of vascularity, or development of fibrosis. There is a statistically significant difference between syringes 1 and 3 for the level of inflammation ( p < 0.05); other than that, no statistically significant difference could be found among syringes 1, 2, or 3 in terms of the established evaluation criteria (Figures 8A-T and 9A-E).
DISCUSSION Autologous fat grafting has been widely used for soft-tissue defect reconstruction for increasing indications in both cosmetic and reconstructive breast surgery; lipoatrophy; lip, nose, or tempora augmentation; correction of unsightly scars; face or hand rejuvenation; and more. However, the
survival rate and long-term retention of the fat grafts varies greatly. To date, there is still no consensus on the optimal technique for fat grafting.13-21 The approaches by which the fat tissue is harvested, processed, and re-injected all have an influence on the fat graft viability and long-term survival. According to an evidencebased review on fat grafting by Gir et al,6 techniques that use low-pressure suction by means of larger bore cannulas appear to increase adipocyte viability. Lee et al9 pointed out that adipocytes are highly resistant to forces of positive and negative pressure alone, whereas they are remarkably susceptible to the effects of shear stress. Thus, different mechanical forces may generate varying degrees of cell injuries. Likewise, might the repetitive motions of fat harvesting also have a negative influence on the fat grafts? Various studies had been conducted to evaluate the impact of different fat harvest or preparation techniques on
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Figure 2. Calcein-AM/Hoechst 33342 staining to assess the viability of the isolated adipocytes. (A) Bright field. (B) Viable adipocytes were stained green by Calcein-AM. Lipid droplets were unstained. (C) Both viable and dead adipocytes were dyed blue by Hoechst 33342. (D) Merged figure to calculate the proportion of viable adipocytes.
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Figure 4. Viability of stromal vascular fractions (SVFs) were 92.07 ± 2.66% in group 1, 91.09 ± 3.03% in group 2, 91.52 ± 1.59% in group 3, 92.40 ± 2.84% in group 4, and 92.58 ± 1.90% in group 5, respectively. No significant differences could be found among the five groups.
fat grafts. Pu et al22 conducted a comparative study between the Coleman technique and the conventional machine liposuction. It turned out that the former technique was superior to the latter one, presented with more viable adipocytes and optimal cellular function. Ferguson et al23 also demonstrated that the LipiVage System (syringe aspiration at low pressure; Lewisville, TX) was better than the conventional liposuction technique, with higher viable adipocytes count. Agostini et al24 compared the histomorphology and adipocyte viability of lipoaspirated samples harvested using wet
and dry techniques. Though the fat collected with the wet technique was more preserved than that harvested with the dry technique, no substantial differences could be found. Besides, Erdim et al25 and Ozsoy et al26 both pointed out that larger cannulas tend to increase fat cell viability compared to smaller ones. In a controlled animal model of fat grafting, Kirkham et al10 found that the use of a 5-mm aspiration cannula led to improved fat graft retention and quality compared to a 3-mm cannula. The 5-mm group presented both less infiltrate and fibrosis. Further, several published research studies all pointed out that centrifugation forces greater than 3 000 RPM caused more cellular damage.12,27-29 All the above-mentioned approaches, different as they seemed, had one thing in common: they brought about more aggressive mechanical injuries against the fat grafts, which was consistent with our initial hypothesis. In the present study, fat grafts were collected at the same frequency, under the same pressure, and from the same site. The only variable is the sequence of harvesting. We found that fat grafts from the former 1, 2, and 3 syringes were presented with a greater number of viable adipocytes, higher level of cellular function, better graft retention, and more integral architecture compared to syringes 4 and 5. With continuous aspiration in the same limited area, the viability and metabolic function of the adipocytes worsened gradually. However, neither the viability of stromal vascular fractions or the ratio of CD34 + CD45- cells within the SVFs were different among the five groups. In addition, two types of fluorescent dyes (Calcein-AM and Hoechst 33342) were applied to stain and calculate the proportion of viable adipocytes. Calcein-AM, a cell-
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Figure 3. (A) Proportion of viable adipocytes from each group. No significant differences were found between groups 1 and 2 (p = 0.72) or groups 4 and 5 (p = 0.06). *A statistically significant difference could be detected between groups 2 and 3 (p < 0.001) and groups 3 and 4 ( p < 0.05). (B) Absorbance of the mixture containing adipocytes and CCK-8 solution after 3 hours’ incubation. The variation tendency is consistent with the results of cell staining analysis.
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permanent dye, can be used to determine cell viability in most eukaryotic cells. In live cells, the non-fluorescent Calcein-AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases.30 Hoechst 33342, a nucleic acid stain, is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to double-stranded DNA. Unlike trypan blue, which is a vital stain used to selectively color dead tissues or cells blue, fluorescent dyes can easily distinguish viable cells from cell debris and oil drops.31 Further, it is unnecessary to determine the absolute number of viable adipocytes through direct cell counting. Actually, it is slightly inaccurate to compare group difference through direct adipocytes counting. First, the original weight of fat tissues to be examined cannot be guaranteed to be exactly the same, and second, 1 gram of fat tissue can generally yield a half million adipocytes, so even the very little changed weight
of fat tissue may greatly influence the ultimate absolute number of adipocytes.25,32,33 Thus, in the present study, the proportion of viable adipocytes was calculated to reflect the whole viability of fat graft. CCK-8 assay was used to determine the metabolic activity of adipocytes. CCK-8 assay, like the MTT and XTT assays, utilized a highly-soluble tetrazolium salt (WST-9) to produce a water-soluble formazan dye (generated by dehydrogenases in viable cells), which was proportional to the number of living cells. Apart from being nonradioactive and more convenient, CCK-8 assay was more sensitive than assays using other tetrazolium salts, such as MTT, XTT, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, or water soluble tetrazolium salt.34 Xiong et al35 compared CCK-8 with MTT in measuring the viability of human promyelocytic leukemia cells. He pointed out the linear correlation of optical density (OD)
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Figure 5. Stromal vascular fractions (SVFs) isolated from any group are all highly viable and possess great viability of multidirectional differentiation. Typical figures are presented. (A) Adipose-derived stem cells from passage three (100×). (B) Oil Red-O staining of adipose-derived stem cells (ASCs) under adipogenic differentiation (100×). (C) Alizarin Red S staining of ASCs under osteogenic differentiation (100×).
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Figure 7. Average weight of fat grafts harvested at 2 and 10 weeks. At both 2 and 10 weeks, a greater percentage of fat grafts survived for syringes 1, 2, and 3 compared to syringes 4 and 5 (p < 0.001). A statistically significant difference could be found between syringes 3 and 4 and syringes 3 and 5 (*p < 0.05).
value and cell viability detected by CCK-8 was better than that of MTT. In our study, OD value measured by CCK-8 correlated well with the proportion of viable adipocytes from each group (data not shown). Besides the adipocytes, the other regenerative cells also play an important role in determining the long-term maintenance of fat grafts. A large number of studies have stated that fat grafts enriched with SVFs present better grafting results than mere fat grafts.36-39 SVFs consist of at least four subgroups: blood-derived cells (CD45+), adipose-derived stem cells (ASCs) (CD31-CD34 + CD45−), endothelial (progenitor) cells (CD31 + CD34 + CD45−), and pericytes (CD31-CD34-CD45−). In particular, the concentration of
CD34 + progenitor cells within the SVFs may predict retention of the fat grafts.38 A study conducted by Yin et al39 investigated the effect of water-jet force on SVFs compared to the classic Coleman technique, and found a significantly higher ratio of CD34 + CD45− cells within the water-jet group than the Coleman technique group; meanwhile, the water-jet group presented better fat graft survival. In our study, neither the viability of SVFs nor the ratio of CD34 + CD45− cells within the SVFs were significant different among the five groups. In this sense, SVFs may be more resistant to mechanical forces than the adipocytes, just like as they are more resistant to ischemia and hypoxia.40 Further, the classic nude mouse model was used to compare the long-term retention of fat grafts of different syringes, and the outcomes were also consistent with the in vitro experiments. The first few syringes presented with better architecture, increased vascularity, decreased fibrosis, and higher long-term survival. Therefore, fat tissue from the first few syringes may be more suitable for fat grafting; in the clinical practice, harvesting a certain amount of adipose tissue from multiple areas may be preferred to obtain a higher survival of grafted fat. In the next step, we are preparing to conduct a randomized controlled clinical trial to further confirm the efficacy of our investigation. If proven true, we are more than thrilled to have it applied immediately in the clinical practice, since the approach is easily accessible to both new and skilled practitioners. Actually, whether the approach is absolutely applicable depends on the gap between fat grafts supplies and demands. Theoretically, for those large-volume fat transplantation cases, like breast and buttock augmentation, it is rather difficult to harvest a sufficient volume of fat grafts while completely avoiding repetitive aspiration. But for those small-
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Figure 6. Ratio of CD34 + CD45- cells within stromal vascular fractions (SVFs). (A) Typical flow cytometry scatter plot. (B) Ratio of CD34 + CD45− cells in SVFs were 49.72 ± 4.45% in group 1, 50.58 ± 4.45% in group 2, 49.25 ± 5.89% in group 3, 50.32 ± 3.45% in group 4, and 50.73 ± 3.49% in group 5. No statistically significant difference could be found among these five groups.
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Figure 8. Typical example photographs of Hematoxylin and eosin-stained adipose tissue harvested at 2 and 10 weeks from each group. Samples from syringe 1 at (A) 2 weeks (40×); (B) 2 weeks (100×); (C) 10 weeks (40×); and (D) 10 weeks (100×). Samples from syringe 2 at (E) 2 weeks (40×); (F) 2 weeks (100×); (G) 10 weeks (40×); and (H) 10 weeks (100×). Samples from syringe 3 at (I) 2 weeks (40×); ( J) 2 weeks (100×); (K) 10 weeks (40×); and (L) 10 weeks (100×). Samples from syringe 4 at (M) 2 weeks (40×); (N) 2 weeks (100×); (O) 10 weeks (40×); and (P) 10 weeks (100×). Samples from syringe 5 at (Q) 2 weeks (40×); (R) 2 weeks (100×); (S) 10 weeks (40×); and (T) 10 weeks (100×). Low-magnification images were further magnified in the right column. There was better integral architecture, less collagen bands, decreased areas of fibrosis, and less infiltration of macrophages in syringes 1 and 2 compared to syringes 4 and 5. There is a statistically significant difference between syringes 1 and 3 in the level of inflammation at 2 weeks; other than that, no statistically significant difference could be found among syringes 1, 2, or 3 in terms of the established evaluation criteria at either time. No significant difference could be found between syringes 4 and 5 regardless of cyst formation, inflammation scale, the level of vascularity, or development of fibrosis at either 2 or10 weeks.
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volume fat transfer cases, the approach is entirely feasible. So certain necessary conditions are required to apply this approach: (1) patients with relatively sufficient subcutaneous fat storage; (2) small volume fat transplantation, usually less than 100 mL, like facial fat grafting, hand/genital rejuvenation, touch-up procedures for breast augmentation or reconstruction with implants, etc.; and (3) patients’ willingness to bear the possible discomfort following an increased amount of tumescent solution injection. Besides, large-volume fat grafting cases should usually be avoided. Additionally, as for the sample size of this investigation, it was taken into account at the time of designing. In a study by Smith et al41 to explore the effect of harvesting and preparation techniques on adipocyte graft survival, lipoaspriates from three patients were analyzed in vitro, and five freshly harvested tissue specimens were injected into the severe combined immune deficiency mice to study the in vivo viability. The authors also pointed out that the minimum number of specimens analyzed in vivo should be
no less than five. Further, in an investigation by Li et al,42 six female patients were enrolled in a study to determine the best donor site for tissue harvesting and isolation of SVF cells for fat graft survival. Similarly, nine female patients were enrolled in another in vitro and in vivo study to compare different processing methods on fat grafting.43 After full consideration, we decided to set the number of enrolled patients at six. Besides, all the subjects were females, as male patients were lacking. Originally, a certain number of male patients were intended to be included in the investigation, however, the number of male patients who came for liposculpture procedures were rather limited. We were unable to get sufficient male patients to participate in the investigation. Interestingly, among all the references we looked up, the gender information of the enrolled subjects was either unmentioned or entirely female.10,24-26,39,41-43 Maybe these investigators suffered the same situation as we did. Admittedly, whether gender difference has an effect on fat grafting is worthy of further study.
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Figure 8. Continued
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Figure 9. Histopathology parameters of (A) integrity, (B) cysts or vacuoles, (C) inflammation, (D) vascularity, and (E) fibrosis (*P < 0.05, **P < 0.001).
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CONCLUSIONS In summary, the present study has provided cell viability assessment, metabolic activity analysis of the adipocytes, viability of SVFs, ratio of CD34 + CD45− cells within SVFs in vitro, and graft retention in vivo to demonstrate that there are substantial differences in adipose tissue harvested from different syringes. With continuous aspiration, the cellular function of adipocytes and the viability and longterm retention of fat grafts decreased gradually. Fat tissue from the first few syringes may be more suitable for fat grafting. Harvesting a certain amount of adipose tissue from multiple areas may be preferred to obtain higher survival of fat grafts.
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Acknowledgements The authors wish to thank Drs Ke Guo, Quan Yuan, and Peng Xiao for surgical assistance, and Dr Yijing Chu for computer software assistance.
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Disclosures
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The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of this article. 18.
Funding The authors received no financial support for the research, authorship, and publication of this article.
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Moreover, a single sized cannula was applied to harvest the fat grafts. Though not all surgeons harvest fat grafts with the 3-mm sized cannula, as far as we know few studies, among all those investigations on the influence of various variables on fat grafts viability, have introduced a second variable-harvesting cannula size on the basis of their original research (processing, reinjection, cryopreservation, etc.), other than those in search of more advantageous harvesting cannulas.10,24-27,39,41-43 In the present study, we mainly focus on the influence of the repetitive motions of harvesting cannula on the viability of fat grafts; though the whole investigation may not be optimal, we assume it is sufficient to confirm our original hypothesis. Our study has its own limitations. First, gender composition. Patients enrolled in our study were entirely female, and male patients were lacking. Hormone status due to gender differences may also influence the viability of fat grafts. Second, only cell components were analyzed; however, the local vasculogenic and anti-inflammatory cytokines were neglected. Finally, the entire procedures were conducted using the wet technique: a control group of patients undergoing fat harvesting with the dry technique would enhance the strength of the design of the study.
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