Endothelial Differentiated Adipose-Derived Stem Cells Improvement of Survival and Neovascularization in Fat Transplantation

Endothelial Differentiated Adipose-Derived Stem Cells Improvement of Survival and... Abstract Background Adipose-derived stem cells (ASCs) assisted lipotransfer have been considered to facilitate the survival of fat grafts. However, emerging evidence of insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer. Objectives This study evaluated if endothelial phenotype ASCs with fat lipoaspirate improves survival and neovascularization in fat transplantation Methods ASCs were isolated from human periumbilical fat tissue and cultured in endothelial growth medium for 2 weeks. Fat lipoaspirate was mixed with fresh adipose stroma vascular fraction (SVF), endothelial differentiated ASCs (EC/ASCs), and fat lipoaspirate alone. Three fat mixtures were subcutaneously injected into the adult male Sprague–Dawley rat’s dorsum at 3 locations. At 8 weeks after transplantation, the grafted fat lipoaspirates were harvested, and the extracted fat was evaluated using photographic, survival weights measurements and histological examination. Neo-vascularization was quantified by immunofluorescence and real-time RT-PCR. Results Grafts from the EC/ASC assisted group had a higher survival rate, morphologic integrity, and most uniform lipid droplets. They also revealed less inflammation and fibrosis with increased number of vessels by histological and immunofluorescence analysis. Quantitative RT-PCR analysis indicated that the expression levels of EC-specific markers of CD31 and vWF were higher in the EC/ASC group compared with in the control and fat with SVF transplants. Conclusions These results indicated that co-implantation of fat lipoaspirate with ASCs differentiated toward an endothelial phenotype improves both survival and neovascularization of the transplanted fat lipoaspirate, which might provide benefits and represents a promising strategy for clinical application in autologous fat transplantation. Autologous fat transplantation is a useful technique during plastic and reconstructive surgery.1,2 However, the low survival rate and high reabsorption rate of the transplanted fat reduces the efficacy of this technique.3,4 To address these issues, many clinical methods have been utilized in attempts to enhance the viability and survival rate of transplanted fat tissues.5,6 A recent meta-analysis of clinical cell-assisted lipotransfer studies concluded that it was more efficacious than autologous fat grafting alone,7 and grafted fat has an increased survival rate when transplanted with the stromal vascular fraction (SVF) obtained from alternative liposuction aspirates enriched in adipose-derived stem cells (ASCs).8,9 ASCs, as an abundant source of adult mesenchymal stem cells, have the capacity to differentiate into a wide range of cell types, including chondroblasts, osteocytes, adipocytes, and endothelial cells, among others.10,11 Additionally, ASCs can secrete angiogenic factors such as VEGF, HGF, and IGF, which have proangiogenic and pro-adipogenic effects to promote the graft retention.12–14 Among these, including co-transplanting fat with SVF has been considered to facilitate the survival of fresh fat grafts.15,16 However, the ASCs present in SVF vary between both individual patients and the body sites from which lipoaspirate is obtained.17,18 Furthermore, emerging evidence has found that insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer.19 Thus, an alternative or adjunct strategy is to increase the number of endothelial cells in the transplant.20 Therefore, it remains a challenge for researchers to seek an effective solution to improve angiogenesis that would result in boosting the efficiency of survival in fat transplantation.21 New vascular network formation is critical for fat graft survival and tissue regeneration; however, the viability of fat grafting is dependent on nutrient diffusion and neovascularization, especially in the early phase after transplantation. Thus, measures that enhance vascularization of the transplanted fat tissue early after transplantation are likely to increase the viability of the transplant. Many previous studies have shown that the fate of transplanted fat tissue depends on the adequacy of blood perfusion during the early stages after transplantation.22,23 In addition, autologous fat graft survival has histologically been shown to correlate with greater blood vessel density, and expression of vascular endothelial growth factor in fat grafts has been shown to increase graft volume retention.24,25 In contrast, Philips et al have shown a strong correlation existed between stromal vascular fraction percentage of CD34+ endothelia progenitor cells and high graft retention.26 Although this technique is clinically impractical, it does highlight the importance of vascularization of the engrafted fat. Importantly, there was evidence to support that angiogenic potential of ASCs reveals differentiation toward both endothelial cell (EC) and smooth muscle cell lineages and can be used as EC substitutes in vascular tissue engineering.27–29 In addition, in animal studies, the beneficial effect of ASCs on graft survival is thought to be because of improved early vascularization, as the result of ASCs differentiation into endothelial cells.12,25 Starting from this background, we sought to address a new strategy in which more functional ASCs, namely, endothelial-like ASCs (EC/ASC) could show efficacy and possible advantages in fat grafting. We hypothesize that ASCs differentiated to an endothelial phenotype may improve the survival of fat grafting because it has the capacity to boost angiogenesis. To test this hypothesis, ASCs were cultured in a specific differentiation medium to differentiate to an endothelial phenotype. Subsequently, human fat lipoaspirate, combined with EC-differentiated ASCs or freshly isolated SVF, was subcutaneously injected into rat, and the graft volume, histologic architecture, and vascularity were evaluated 8 weeks post transplantation. METHODS Isolation and Culture of ASCs Adipose tissue was obtained by liposuction of peri-umbilical subcutaneous fat from female patients undergoing cosmetic liposuction and breast reduction (n = 7; age 28-59, 49.6 ± 10.7 years old) after informed consent was obtained in accordance with our IRB-approved protocol (Protocol # 11–015, name: Potential for Adipose-derived stem cells for Regenerative Medicine) between July 2014 and March 2015. No exclusion criteria were used. Isolation was performed as previously described.28 Briefly, adipose tissue was incubated in collagenase I (Worthington Biochemical, Lakewood, NJ) solution for 1 h at 370C. After centrifugation (1500 rpm/10 min), the stromal vascular cells were cultured in medium containing M199 (Mediatech; Manassas, VA) supplemented with 10% FBS (Gemini Bio Products; West Sacramento, CA). Non-adherent cells were removed after 24 h, and the culture medium was subsequently replaced twice weekly. Flow Cytometry Analysis The specific cell surface antigens of ACSs were characterized by flow cytometry. Approximately 5 × 105 cells were incubated with fluorescence-conjugated antibodies for 30 min. All the antibodies were obtained from BD Biosciences. Isotype identical antibodies served as controls to exclude nonspecific binding. Quantitative analysis was performed using C6 flow cytometry (Becton Dickinson). Data analysis was performed with the use of FlowJo software (FlowJo; Ashland, OR). Multipotency Differentiation of ASCs To evaluate ASC multipotent differentiation, ASCs were cultured in AdipoDiff Medium (Gbico Life Technologies) for 3 weeks to induce adipocyte differentiation. The differentiated cells were incubated with cooled methanol for 5 min and stained with fresh Oil Red-O (Sigma-Aldrich) solution for 20 min to identify lipid droplets. For osteoblast differentiation, ASC cells were cultured in OsteoDiff Medium (Gbico Life Technologies) for 3 weeks; the cells were then stained with Alizarin Red S (Sigma-Aldrich) solution for 20 min to identify calcium deposits. Endothelial Differentiation of ASCs For endothelial differentiation, ASCs were cultured in EGM-2 medium (Lonza; Walkersville, MD) with SingleQuots containing VEGF, hFGF-b, epidermal growth factor, insulin-like growth factor-1, heparin, and ascorbic acid supplemented with 2% FBS for 2 weeks. Endothelial differentiation of ASCs was confirmed by expression of EC specific markers using quantitative real-time PCR, immunofluorescence, and cord formation after plating on Matrigel. Matrigel-Based Capillary-Like Tube Formation Assays After 14 days differentiated in endothelial differentiation media (EGM-2; Lonza), ASCs were plated on Matrigel substrate (BD Biosciences) at a density of 2x105cells/100 ul Matrigel and incubated at 370C in a 5% CO2 for up to 12 h. Formation of cord-like structures was visualized by phase contrast microscopy. ASCs cultured in a non-differentiation M199 medium were used as the undifferentiated control. For immunofluorescence, cells were fixed for 15 min in 4% formaldehyde at room temperature and permeabilized with 0.3% Triton X-100 for 5 min. The cells were blocked with 10% goat serum in PBS for 1 h and incubated with a primary antibody eNOS (BD Transduction, San Jose, Calif) overnight at 40C. The secondary fluorescent antibody was used for the detection of the positive cells. Nucleus was stained with a 4,6-diamidino-2-phenylindole containing Vectashield mounting medium (Vector Laboratories Inc, Burlingame, Calif). Images were acquired using a FluoView 1000 confocal immunofluorescent microscope (Olympus, Center Valley, Pa). Quantitative Reverse Transcription Polymerase Chain Reaction Total cells and tissue RNA was extracted using TRIzol reagent. To detect the specific genes expressed, RT-PCR was performed using the following primers: Willebrand factor (vWF): 5-primers (5’-TCTTCCAGGACTGCAACAAG-3’) and 3-primers (5’-TCCGAGATGTCCTCCACATA-3’) and platelet endothelial cell adhesion molecule (CD31): 5-primers (5’-CACAGCAATTCCTCAGGCTA-3’) and 3-primers (5’- TTCAGCCTTCAGCATGGTAG-3’). mRNA levels were quantified using SYBR Green Real-Time PCR with the 7500 Fast Real-Time PCR system (Applied Biosystems). PCR reaction conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The housekeeping gene GAPDH (5-primers: 5’-GGATCTCGCTCCTGGAAGATG-3’ and 3-primers: 5’-GCACCGTCAAGGCTGAGAAC-3’) was amplified in separate tubes to normalize for variance in input RNA. For relative quantification, the efficiency of amplification for each individual primer pair was determined using cDNA target and the 2-∆∆ct method. Animal Model and Fat Transplantation All animal study protocols and procedures were approved by the Animal Care Ethics Committee of the University of the Sciences. The animals used in this study included 8 adult male Sprague–Dawley rats weighing 250-350 g. Fresh fat lipoaspirate and adipose stroma vascular fraction (SVF) were prepared onsite for same day transplant. For endothelial differentiation, ASCs were cultured in EGM2 medium for 14 days (EC/ASCs). The fat lipoaspirate (1.2g, 0.8 mL/spot) were mixed with 0.2 mL of PBS as a control, 0.2 mL of 1.5 × 105 fresh SVF, and 0.2 mL of 1.5x105 EC/ASC, and 16-gauge needles were used to inject each rat subcutaneously at 3 spots. Each rat dorsum was injected with all 3 fat mixtures, and each of the fat mixtures contained a volume of 1 mL. Assessment of the Fat Graft Survival Rates After 8 weeks, all rats were euthanized with approved IACUC protocol, and the fat grafts were carefully dissected from their back. Each fat graft was weighed and measured, and the survival ratio for transplanted fat was calculated by using the formula: survival weight/previous weight. Subsequently, each fat graft was divided into 2 equal portions. One portion was used to determine the mRNA expression of EC markers. The other portion was placed in paraformaldehyde and used for histological and immunohistochemistry examination. Histological and Immunofluorescence Analysis Formalin-fixed transplants from the 3 groups were embedded in paraffin and cryopreserved in optimal cutting temperature (OCT) media compound. Paraffin-embedded fat graft histological sections (5 μm) were prepared and then stained with hematoxylin and eosin (H&E) using standard procedures. Neovascularization was assessed by counting the capillaries in 10 fields of each H&E-stained slide and then calculating the capillary density. For immunofluorescence, the paraffin and cryostat section was stained with Cy3 conjugated anti-human nuclei antibody (Millipore Sigma), in order to detect if the human ASC proliferated in transplanted tissues. The CD31 (Santa Cruz) staining was used to detect capillary endothelial cells. After washing with PBS twice, the slides were placed under a green fluorescent beam to detect the CD31 and red fluorescence of Cy3 for human nuclear presence. Overlapping red and green fluorescence produced a yellow color, which indicated that the endothelial cells in the neovascular capillaries were derived from the transplanted human ASCs. Statistical Analysis The data are presented as mean ± standard error (SE). The Student’s t test was used to evaluate the significant differences defined as P < 0.05 between 2 groups of data in all experiments. Multigroup comparisons were determined by one-way ANOVA with Bonferroni correction. A value of P < 0.05 was considered significant. RESULTS Characterization of Human ASCs Stromal vascular fraction was isolated from human liposuction of peri-umbilical subcutaneous fat and non-adherent cells were removed after 24 h. Following culture for 5 days, ASCs showed a fibroblast-like shape on microscopy (Figure 1A). Differentiation analysis confirmed adipogenic and osteogenic differentiation potential of ASCs. Adipogenic and osteogenic differentiation levels were measured by Oil-Red-O (Figure 1B) and Alizarin Red (Figure 1C) staining, respectively. The culture-expanded ASCs were strongly positive for the mesenchymal stem cell markers of CD29, CD44, CD90, and CD105 by flow cytometry analysis (Figure 1D). Figure 1. View largeDownload slide Characterization of adipose-derived stem cells. (A) Phase contrast micrographs of ASCs cultured for 5 days. (B) ASCs stained with Oil Red O, and (C) Alizarin Red after 3 weeks cultured in adipogenic and osteogenic differentiation medium. (D) ASCs (Passage 3) were strongly positive for CD29, CD90, CD105, and CD44 consistent with mesenchymal stem cell markers by using flow cytometry analysis (with isotype control). Figure 1. View largeDownload slide Characterization of adipose-derived stem cells. (A) Phase contrast micrographs of ASCs cultured for 5 days. (B) ASCs stained with Oil Red O, and (C) Alizarin Red after 3 weeks cultured in adipogenic and osteogenic differentiation medium. (D) ASCs (Passage 3) were strongly positive for CD29, CD90, CD105, and CD44 consistent with mesenchymal stem cell markers by using flow cytometry analysis (with isotype control). ASCs Differentiation Towards an Endothelial Phenotype ASCs displayed a change in morphology after being cultured in endothelial-induced medium (EGM2) in response to differentiation (Figure 2A,B). After 14 days of being cultured in specific endothelial differentiation media, ASCs expressed EC-specific markers of CD31 and von Willebrand factor (vWF) compared with the control group by using quantitative RT-PCR analysis (Figure 2C). Immunofluorescence staining assays confirmed protein expression of eNOS in ASCs by induction (Figure 2D). Furthermore, functional assessment of the differentiated ASCs was shown by the formation of cord-like structures after plating on Matrigel (Figure 2E). Figure 2. View largeDownload slide Characteristics of ASCs differentiated into endothelial-like cells. (A, B) Phase contrast micrographs of ASCs grown in M199 medium (un-differentiation, left), and EGM2 (differentiation, right) medium for 2 weeks. (C) After 2 weeks induced in endothelia differentiation media (EGM2), ASCs expressed endothelial markers of CD31 and vWF compared with the control group by using real-time RT-PCR analysis (bars indicate ±SE, *: P < 0.05). (D) Immunofluorescence staining assays for eNOS (green) expression of ASCs (DAPI; blue). (E) Phase contrast photomicrograph of the differentiated ASCs subsequently plated onto Matrigel for 12 h demonstrating the formation of cord-like structures. Figure 2. View largeDownload slide Characteristics of ASCs differentiated into endothelial-like cells. (A, B) Phase contrast micrographs of ASCs grown in M199 medium (un-differentiation, left), and EGM2 (differentiation, right) medium for 2 weeks. (C) After 2 weeks induced in endothelia differentiation media (EGM2), ASCs expressed endothelial markers of CD31 and vWF compared with the control group by using real-time RT-PCR analysis (bars indicate ±SE, *: P < 0.05). (D) Immunofluorescence staining assays for eNOS (green) expression of ASCs (DAPI; blue). (E) Phase contrast photomicrograph of the differentiated ASCs subsequently plated onto Matrigel for 12 h demonstrating the formation of cord-like structures. Endothelial-Like ASCs Improve Fat Graft Retention Human fat lipoaspirate was co-injected with fresh isolated SVF, EC/ASCs, and the fat alone without cells as a control. One of these 3 fat mixtures was injected into each rat dorsum subcutaneously at 3 locations (Figure 3A). At the conclusion of 8 weeks post transplantation, all of the animals had survived the experiments and none of the animals died following transplantation. No inflammation or abscesses were observed in the surgical areas. The fat grafts in the EC/ASC-assisted groups appeared larger than those in the non-cell assisted and SVF-assisted group, and the dissected fat grafts in the control and SVF groups were weaker and more fragile than those in the EC/ASC group (Figure 3B). The survival ratios for the control group, SVF group, and EC/ASC group were 41.0 ± 10.1%, 41.2 ± 11%, and 56.8 ± 10.6%, respectively (n = 8). A significantly higher survival of fat grafts was observed in the EC/ASCs group than in the control or SVF groups (Figure 3C, *: P < 0.05). There was no difference between the SVF and control groups. Figure 3. View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft retention. (A) Fat lipoaspirate was mixed with PBS (control), fresh SVF, and endothelial-differentiated ASCs (EC/ASC), and each rat was injected subcutaneously in 3 spots. (B) Macroscopic images of the harvested fat grafts 8 weeks post transplantation. (C) Assisted-EC/ASCs significantly increased transplant survival. The survival ratio of transplanted fat lipoaspirate was calculated using the survival volume/previous volume (n = 8/group). Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to one-way ANOVA; *: P = 0.05. Figure 3. View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft retention. (A) Fat lipoaspirate was mixed with PBS (control), fresh SVF, and endothelial-differentiated ASCs (EC/ASC), and each rat was injected subcutaneously in 3 spots. (B) Macroscopic images of the harvested fat grafts 8 weeks post transplantation. (C) Assisted-EC/ASCs significantly increased transplant survival. The survival ratio of transplanted fat lipoaspirate was calculated using the survival volume/previous volume (n = 8/group). Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to one-way ANOVA; *: P = 0.05. Histological Evaluation of EC-Like ASC-Assisted Fat Graft The graft structure was further analyzed by masson trichrome and H&E staining after sectioning. As presented in Figure 4, the fat grafts in the EC/ASC group exhibited the best survival, most morphologic integrity, most uniform lipid droplet, least inflammation, least fibrosis, and richest blood vessels. The fat grafts in the SVF group exhibited modest survival, less integrity, less uniform lipid droplets, and connective tissue septa; and the fat grafts without cells, serving as a control group, exhibited varied sizes of lipid droplets, excessive fibrosis, and fat necrosis. Focusing on both SVF and ASC/EC transplanted specimens, we identified an additional large area composed by distinct inner and outer regions (Figure 4B). The grafts from the EC/ASC-assisted groups had more intact and nucleated adipocytes with relatively fewer fibers and vacuoles, whereas more vacuoles were observed in the SVF-assisted and control groups. In addition, newly generated blood vessels were observed in the fat grafts in the EC/ASC groups than the SVF group (Figure 4B) indicating that fat graft with EC/ASCs may promote neovascularization. Figure 4. View largeDownload slide Histological analysis of fat transplants. (A) Masson trichrome staining shows that the transplants from the EC/ASC group (right) consisted predominantly of mature adipose tissue and had lower levels of fat necrosis and fibrosis compared to those from SVF (middle) and control groups (left). Scale bar, 2000μm. (B) Hematoxylin and eosin (H&E) staining showed that the newly generated blood vessels were observed higher in EC/ASC-assisted grafts (right) compared to grafts with SVF-assisted (middle) and control groups (left). The arrows indicate neovascular capillaries. Scale bar, 400μm. Figure 4. View largeDownload slide Histological analysis of fat transplants. (A) Masson trichrome staining shows that the transplants from the EC/ASC group (right) consisted predominantly of mature adipose tissue and had lower levels of fat necrosis and fibrosis compared to those from SVF (middle) and control groups (left). Scale bar, 2000μm. (B) Hematoxylin and eosin (H&E) staining showed that the newly generated blood vessels were observed higher in EC/ASC-assisted grafts (right) compared to grafts with SVF-assisted (middle) and control groups (left). The arrows indicate neovascular capillaries. Scale bar, 400μm. EC-Like ASC Promotes Fat Graft Retention Through Neovascularization To clarify the histological reported findings, we then focused on the main parameter: neovascularization, which is an indispensable process for tissue regeneration. To determine the delivery of human ASCs promoting neovascularization, we double stained the transplant sections with anti-human nuclear (Figure 5A) and CD31 antibodies (Figure 5B), which is an endothelial cell marker. Merging the red fluorescence of anti-human nuclear with the green fluorescence of CD31 revealed 3 yellow endothelial cells, indicating that the delivery of human ASCs promoted neovascularization (Figure 5C). The CD31 and human nuclear double positive cells quantified from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC groups (right) revealed that there were more cells with positive expression of CD31 distributed to surrounding fat cells in the transplants of EC/ASC groups than that in SVF groups and fat alone groups. (9 ± 1.83, 20.75 ± 2.5 and 33.5 ± 5.57, n = 8, *: P < 0.05; Figure 5D). Figure 5. View largeDownload slide View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft angiogenesis. (A) Immunofluorescence staining analysis of the transplants with human nuclear antibody, (B) immunofluorescence for endothelial cell marker CD31, and (C) A merge of the red fluorescence of anti-human nuclear with the green fluorescence of CD31 was indicating that the delivered of human ASCs promoted neovascularization in surviving transplanted fat lipospirate. (D) The CD31 and human nuclear double positive cells were calculated from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC group (right). The positive expression of CD31 cell was higher in the transplants of EC/ASC groups when compared with SVF and control groups. These differences were significant according to a paired t test. *P < 0.05, **P < 0.01. Figure 5. View largeDownload slide View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft angiogenesis. (A) Immunofluorescence staining analysis of the transplants with human nuclear antibody, (B) immunofluorescence for endothelial cell marker CD31, and (C) A merge of the red fluorescence of anti-human nuclear with the green fluorescence of CD31 was indicating that the delivered of human ASCs promoted neovascularization in surviving transplanted fat lipospirate. (D) The CD31 and human nuclear double positive cells were calculated from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC group (right). The positive expression of CD31 cell was higher in the transplants of EC/ASC groups when compared with SVF and control groups. These differences were significant according to a paired t test. *P < 0.05, **P < 0.01. We use quantitative RT-PCR to evaluate the expression levels of angiogenic genes in transplanted fat grafts to confirm the grafts’ neovascularization. We observed increases in the expression of endothelia specific markers of CD31 (approximately 2.7-fold; *: P < 0.05) and vWF (approximately 3-fold; *: P < 0.05) mRNA in EC/ASC groups when compared with the SVF group (Figure 6). These results further indicated that ASC differentiation to endothelial phenotype may promote the neovascularization in fat grafting. The SVF group was also observed to be higher than the control group, although not significantly (1.8-fold; P = 0.36; 1.3-fold; P = 0.55). Collectively, these results proved the capacity of endothelial-induced ASC phenotypes loaded into fat tissue to be associated with new high vascularization inside the graft core early after transplantation. Figure 6. View largeDownload slide Expression of angiogenesis-related genes in transplanted fat lipoaspirate. (A, B) After 8 weeks in each group, the messenger RNA expression levels of endothelia specific markers CD31 and vWF were much higher in the EC-ASC-assisted transplants than in the other 2 transplants by quantified using real-time RT-PCR analysis. The SVF group was also observed to have increases when compared with the control group, although not significantly. Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to a paired t test. *P < 0.05, n = 8. Figure 6. View largeDownload slide Expression of angiogenesis-related genes in transplanted fat lipoaspirate. (A, B) After 8 weeks in each group, the messenger RNA expression levels of endothelia specific markers CD31 and vWF were much higher in the EC-ASC-assisted transplants than in the other 2 transplants by quantified using real-time RT-PCR analysis. The SVF group was also observed to have increases when compared with the control group, although not significantly. Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to a paired t test. *P < 0.05, n = 8. DISCUSSION In the current report, we demonstrate for the first time that supplementation of fat transplantation with endothelial cell-like ASCs can improve graft survival in a rat model. The novel findings of our study include that, first, fat grafts with endothelial differentiated ASCs produce comparable results to the SVF-assisted lipotransfer in terms of both volume of the grafts and improved angiogenesis of grafted fat tissues. Second, both messenger RNA level and immunohistochemical analyses support the notion that the observed improvement in fat graft volume retention is attributable to the induction of angiogenesis. Taken together, these data imply that ASCs induced to an EC-like phenotype with lipotransfer increased graft volume retention and revascularization, which represents potential mechanisms for adipose transplantation. Many investigators found beneficial effects of adipose-derived stroma progenitor cells on lipotransfer and have suggested that increases in the ratio of SVF/ASCs within the graft will lead to a higher retention rate in adipose transplantation.30,31 The current body of literature consists of a mixture of 2 different cell-assisted lipotransfer techniques: supplementation of fat with autologous stromal vascular fraction, and supplementation with in vitro-expanded adipose-derived stromal cells. Use of a fresh stromal vascular fraction is most easily acquired in the clinical setting; however, stromal vascular fraction consists of fibroblasts, endothelial cells, pericytes, preadipocytes, and various immune cells.26 In addition, large numbers of supplemental cells may not be needed for enhancement of fat graft retention, as Philips et al have shown a correlation between graft retention and the number of CD34+ progenitor cells present within a patient’s adipose tissue.5,26 Furthermore, Wan’s group has reported that use of enrichment for CD34+ progenitor cells of adipose-derived stromal cells showed more efficacy in volume retention than non-enrichment for CD34+ cells of SVF.32 To our knowledge, this is the first report evaluating the effect of an endothelial-like phenotype of ASCs on the retention and revascularization of adipose transplantation. Fat graft survival is mainly dependent on successful vascularization so that adipogenesis is associated with capillary angiogenesis allowing adipocyte differentiation within clusters of endothelial and stromal cells.5,26 Various approaches for accelerating angiogenesis have been successfully undertaken to enhance fat survival post transplantation, including the administration of basic fibroblast growth factors, interleukin 8 and erythropoietin, VEGF-based gene therapy, and endothelial cell and MSC therapies.21,25,33,34 In this report, we demonstrated that the ASCs cultured in differentiation medium by 2 weeks have functional capability as an endothelial phenotype and significantly reduce early necrosis, and this is associated with a better preservation of histological features and stimulates neovascularization with improved retention of transplanted fat grafts in the rat model. The immunohistochemical and qPCR analyses both supported the hypothesis that the observed improvement in fat graft survival was attributable to the induction of neovascularization (Figures 5 and 6). Our observations are also in agreement with the findings of previous other studies; fat graft survival is mainly dependent on successful vascularization, so that adipogenesis is associated with capillary angiogenesis allowing adipocyte differentiation within clusters of endothelial and stromal cells.35,36 Compared with SVF, the purified adipose-derived stromal cells showed about 90% positive for the mesenchymal stem cell markers of CD44, CD73, CD90, and CD105 and exhibited a greater capacity for early osteogenic and adipogenic differentiation.37,38 Furthermore, important for the potential use of ASCs in regenerative medicine is their ability to obtain the functional markers associated with mature endothelial cells, which suggest that they could be used as EC substitutes in tissue engineering and cell-based therapies.28,39 However, the EC differentiation of cell strategies can take up to weeks, so to make the technology readily available for clinical use, the cryopreservation of the autologous re-endothelialization of cells also needs to conserve them for use at a later stage.28,39 Banking the EC-differentiated autologous cells and evaluate the effect of cryopreservation on ASCs maintain acquired EC characteristics as well as their ability to improve angiogenesis of grafted fat tissues will require in our future studies. Fat tissue is a large and diffuse tissue with high metabolic activity. Histological studies have suggested that revascularization of autologous fat transplants occurs only after 48 h.40 Thus, early and abundant neovasculization seems to be the key to the survival of free fat transplants. In the present study, we found that supplementation with SVF, which is a heterogeneous mixture of cells, did not result in differences in quality or in the viability of fat transfer compared with those for the control group. However, endothelial-differentiated ASCs showed improved quality and viability of fat grafts, and vascular augmentation after 8 weeks post transplantation. Our data support the following possibilities. First, endothelial-differentiated ASC may promote angiogenesis by a paracrine mechanism, as evidenced by the expression of proangiogenic factors, which enhance angiogenesis both from the host and within the graft to improve graft revascularization and survival. Second, the differences in the survival of the transplanted fat graft are attributable to the differences in the density of the mesenchymal stem cells present in the injected material. Because the SVF is composed of a heterogeneous cell population, the purification and subsequent endothelial differentiation of ASC from the SVF select for a relatively homogeneous cell population. The results and other published data support that sufficient density of mesenchymal stem cells and differentiation is necessary to improve the viability of the fat grafts.32,35 Limitations There are some limitations to this study, including the relatively small number of specimens and the comparison of SVF to endothelial-differentiated ASCs. Therefore, a comparison of ASCs to endothelial-differentiated ASC will be required to clarify whether the impact of transplanted cells into the fat graft compartment may be due to differentiation, release of soluble factors, or both. At this time, we originally chose xenografts utilize human tissue and to test our hypothesis in using non-immunocompromised animals since our main concern in this study was that to boosting the efficiency of survival and angiogenesis in lipotransfer. The most important property to demonstrate will be the improved neovascularization of the fat transplantation with the endothelial phenotype of ASCs using human fat graft extracted and handled in the same way as clinical practice. However, for our work at the conclusion of 8 weeks post transplantation, all of the animals had survived the experiments and none of the animals died following transplantation, and no inflammation or abscesses were observed in the surgical areas. It has been postulated that adipose tissue and ASCs have low immunogenicity. However, this may represent a limitation to the current study and an opportunity for further research. CONCLUSIONS The present study indicates that co-implantation of the ASCs differentiated toward an endothelial phenotype in fat transplantation improves both survival and neovascularization of the transplanted fat lipoaspirate. These effects are likely mediated by endothelial-differentiated ASC stimulation of angiogenesis and increased graft revascularization. However, our studies further suggested that co-implantation of endothelial-like phenotype of ASCs with adipose lipoaspirate holds great potential and represents a promising strategy for clinical application in autologous fat transplantation. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding This work was supported by the Cooper University Hospital Research Award in 2014 to Ping Zhang. The funding was used exclusively for laboratory and animal-related costs. Presented at: The 14th annual meeting of the International Federation for Adipose Therapeutics and Science (IFATS) in San Diego, CA, in November 2016. REFERENCES 1. Coleman SR . Structural fat grafting: more than a permanent filler . Plast Reconstr Surg . 2006 ; 118 ( 3 ): 108S - 120S . Google Scholar CrossRef Search ADS PubMed 2. Yoshimura K , Sato K , Aoi N , Kurita M , Hirohi T , Harii K . Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells . Aesthetic Plast Surg . 2008 ; 32 ( 1 ): 48 - 55 . Google Scholar CrossRef Search ADS PubMed 3. Agha RA , Goodacre T , Orgill DP . Use of autologous fat grafting for reconstruction postmastectomy and breast conserving surgery: a systematic review protocol . BMJ Open . 2013 ; 3 ( 10 ): e003709 . Google Scholar CrossRef Search ADS PubMed 4. Gimble JM , Bunnell BA , Chiu ES , Guilak F . 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Cell-assisted lipotransfer: a systematic review of its efficacy . Aesthetic Plast Surg . 2016 ; 40 ( 2 ): 309 - 318 . Google Scholar CrossRef Search ADS PubMed 9. Fu S , Luan J , Xin M , Wang Q , Xiao R , Gao Y . Fate of adipose-derived stromal vascular fraction cells after co-implantation with fat grafts: evidence of cell survival and differentiation in ischemic adipose tissue . Plast Reconstr Surg . 2013 ; 132 ( 2 ): 363 - 373 . Google Scholar CrossRef Search ADS PubMed 10. Zuk PA , Zhu M , Ashjian P , et al. Human adipose tissue is a source of multipotent stem cells . Mol Biol Cell . 2002 ; 13 ( 12 ): 4279 - 4295 . Google Scholar CrossRef Search ADS PubMed 11. Mizuno H , Tobita M , Uysal AC . Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine . Stem Cells . 2012 ; 30 ( 5 ): 804 - 810 . Google Scholar CrossRef Search ADS PubMed 12. 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 ( 2 ): 222 - 228 . Google Scholar CrossRef Search ADS PubMed 13. Yoshimura K , Sato K , Aoi N , Kurita M , Hirohi T , Harii K . Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells . Aesthetic Plast Surg . 2008 ; 32 ( 1 ): 48 - 55 ; discussion 56. Google Scholar CrossRef Search ADS PubMed 14. Moseley TA , Zhu M , Hedrick MH . Adipose-derived stem and progenitor cells as fillers in plastic and reconstructive surgery . Plast Reconstr Surg . 2006 ; 118 ( 3 ): 121S - 128S . Google Scholar CrossRef Search ADS PubMed 15. Gimble JM , Guilak F , Bunnell BA . Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells . Stem Cell Res Ther . 2010 ; 1 ( 2 ): 19 . Google Scholar CrossRef Search ADS PubMed 16. Matsumoto D , Sato K , Gonda K , et al. Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection . Tissue Engineering . 2006 ; 12 ( 12 ): 3375 - 3382 . Google Scholar CrossRef Search ADS PubMed 17. Beane OS , Fonseca VC , Cooper LL , Koren G , Darling EM . Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells . PLoS One . 2014 ; 9 ( 12 ): e115963 . Google Scholar CrossRef Search ADS PubMed 18. Jurgens WJ , Oedayrajsingh-Varma MJ , Helder MN , et al. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies . Cell Tissue Res . 2008 ; 332 ( 3 ): 415 - 426 . Google Scholar CrossRef Search ADS PubMed 19. Philips BJ , Marra KG , Rubin JP . Healing of grafted adipose tissue: current clinical applications of adipose-derived stem cells for breast and face reconstruction . Wound Repair Regen . 2014 ; 22 ( 1 ): 11 - 13 . Google Scholar CrossRef Search ADS PubMed 20. Zamora DO , Natesan S , Becerra S , et al. Enhanced wound vascularization using a dsASCs seeded FPEG scaffold . Angiogenesis . 2013 ; 16 ( 4 ): 745 - 757 . Google Scholar CrossRef Search ADS PubMed 21. Jiang A , Li M , Duan W , Dong Y , Wang Y . Improvement of the survival of human autologous fat transplantation by adipose-derived stem-cells-assisted lipotransfer combined with bFGF . ScientificWorldJournal . 2015 ; 2015 : 968057 . Google Scholar PubMed 22. Tepper OM , Galiano RD , Kalka C , Gurtner GC . Endothelial progenitor cells: the promise of vascular stem cells for plastic surgery . Plast Reconstr Surg . 2003 ; 111 ( 2 ): 846 - 854 . Google Scholar CrossRef Search ADS PubMed 23. Lee DW , Jeon YR , Cho EJ , Kang JH , Lew DH . Optimal administration routes for adipose-derived stem cells therapy in ischaemic flaps . J Tissue Eng Regen Med . 2014 ; 8 ( 8 ): 596 - 603 . Google Scholar CrossRef Search ADS PubMed 24. Eto H , Kato H , Suga H , et al. The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes . Plast Reconstr Surg . 2012 ; 129 ( 5 ): 1081 - 1092 . Google Scholar CrossRef Search ADS PubMed 25. Lu F , Li J , Gao J , et al. Improvement of the survival of human autologous fat transplantation by using VEGF-transfected adipose-derived stem cells . Plast Reconstr Surg . 2009 ; 124 ( 5 ): 1437 - 1446 . Google Scholar CrossRef Search ADS PubMed 26. 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 . Google Scholar CrossRef Search ADS PubMed 27. Cao Y , Sun Z , Liao L , Meng Y , Han Q , Zhao RC . Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo . Biochem Biophys Res Commun . 2005 ; 332 ( 2 ): 370 - 379 . Google Scholar CrossRef Search ADS PubMed 28. Zhang P , Moudgill N , Hager E , et al. Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease . Stem Cells Dev . 2011 ; 20 ( 6 ): 977 - 988 . Google Scholar CrossRef Search ADS PubMed 29. Harris LJ , Abdollahi H , Zhang P , McIlhenny S , Tulenko TN , DiMuzio PJ . Differentiation of adult stem cells into smooth muscle for vascular tissue engineering . J Surg Res . 2011 ; 168 ( 2 ): 306 - 314 . Google Scholar CrossRef Search ADS PubMed 30. He X , Zhong X , Ni Y , Liu M , Liu S , Lan X . Effect of ASCs on the graft survival rates of fat particles in rabbits . J Plast Surg Hand Surg . 2013 ; 47 ( 1 ): 3 - 7 . Google Scholar CrossRef Search ADS PubMed 31. Lee SK , Kim DW , Dhong ES , Park SH , Yoon ES . Facial soft tissue augmentation using autologous fat mixed with stromal vascular fraction . Arch Plast Surg . 2012 ; 39 ( 5 ): 534 - 539 . Google Scholar CrossRef Search ADS PubMed 32. Zielins ER , Brett EA , Blackshear CP , et al. Purified adipose-derived stromal cells provide superior fat graft retention compared with unenriched stromal vascular fraction . Plast Reconstr Surg . 2017 ; 139 ( 4 ): 911 - 914 . Google Scholar CrossRef Search ADS PubMed 33. Shoshani O , Livne E , Armoni M , et al. The effect of interleukin-8 on the viability of injected adipose tissue in nude mice . Plast Reconstr Surg . 2005 ; 115 ( 3 ): 853 - 859 . Google Scholar CrossRef Search ADS PubMed 34. Yi C , Pan Y , Zhen Y , et al. Enhancement of viability of fat grafts in nude mice by endothelial progenitor cells . Dermatol Surg . 2006 ; 32 ( 12 ): 1437 - 1443 . Google Scholar PubMed 35. Sezgin B , Ozmen S , Bulam H , et al. Improving fat graft survival through preconditioning of the recipient site with microneedling . J Plast Reconstr Aesthet Surg . 2014 ; 67 ( 5 ): 712 - 720 . Google Scholar CrossRef Search ADS PubMed 36. Bae YC , Song JS , Bae SH , Kim JH . Effects of human adipose-derived stem cells and stromal vascular fraction on cryopreserved fat transfer . Dermatol Surg . 2015 ; 41 ( 5 ): 605 - 614 . Google Scholar CrossRef Search ADS PubMed 37. Bauer-Kreisel P , Goepferich A , Blunk T . Cell-delivery therapeutics for adipose tissue regeneration . Adv Drug Deliv Rev . 2010 ; 62 ( 7-8 ): 798 - 813 . Google Scholar CrossRef Search ADS PubMed 38. Kim KI , Park S , Im GI . Osteogenic differentiation and angiogenesis with cocultured adipose-derived stromal cells and bone marrow stromal cells . Biomaterials . 2014 ; 35 ( 17 ): 4792 - 4804 . Google Scholar CrossRef Search ADS PubMed 39. Khan S , Villalobos MA , Choron RL , et al. Fibroblast growth factor and vascular endothelial growth factor play a critical role in endotheliogenesis from human adipose-derived stem cells . J Vasc Surg . 2017 ; 65 ( 5 ): 1483 - 1492 . Google Scholar CrossRef Search ADS PubMed 40. Fawcett DW . Histological observations on the relation of insulin to the deposition of glycogen in adipose tissue . Endocrinology . 1948 ; 42 ( 6 ): 454 - 467 . Google Scholar CrossRef Search ADS PubMed © 2018 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aesthetic Surgery Journal Oxford University Press

Endothelial Differentiated Adipose-Derived Stem Cells Improvement of Survival and Neovascularization in Fat Transplantation

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© 2018 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com
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1090-820X
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1527-330X
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10.1093/asj/sjy130
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Abstract

Abstract Background Adipose-derived stem cells (ASCs) assisted lipotransfer have been considered to facilitate the survival of fat grafts. However, emerging evidence of insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer. Objectives This study evaluated if endothelial phenotype ASCs with fat lipoaspirate improves survival and neovascularization in fat transplantation Methods ASCs were isolated from human periumbilical fat tissue and cultured in endothelial growth medium for 2 weeks. Fat lipoaspirate was mixed with fresh adipose stroma vascular fraction (SVF), endothelial differentiated ASCs (EC/ASCs), and fat lipoaspirate alone. Three fat mixtures were subcutaneously injected into the adult male Sprague–Dawley rat’s dorsum at 3 locations. At 8 weeks after transplantation, the grafted fat lipoaspirates were harvested, and the extracted fat was evaluated using photographic, survival weights measurements and histological examination. Neo-vascularization was quantified by immunofluorescence and real-time RT-PCR. Results Grafts from the EC/ASC assisted group had a higher survival rate, morphologic integrity, and most uniform lipid droplets. They also revealed less inflammation and fibrosis with increased number of vessels by histological and immunofluorescence analysis. Quantitative RT-PCR analysis indicated that the expression levels of EC-specific markers of CD31 and vWF were higher in the EC/ASC group compared with in the control and fat with SVF transplants. Conclusions These results indicated that co-implantation of fat lipoaspirate with ASCs differentiated toward an endothelial phenotype improves both survival and neovascularization of the transplanted fat lipoaspirate, which might provide benefits and represents a promising strategy for clinical application in autologous fat transplantation. Autologous fat transplantation is a useful technique during plastic and reconstructive surgery.1,2 However, the low survival rate and high reabsorption rate of the transplanted fat reduces the efficacy of this technique.3,4 To address these issues, many clinical methods have been utilized in attempts to enhance the viability and survival rate of transplanted fat tissues.5,6 A recent meta-analysis of clinical cell-assisted lipotransfer studies concluded that it was more efficacious than autologous fat grafting alone,7 and grafted fat has an increased survival rate when transplanted with the stromal vascular fraction (SVF) obtained from alternative liposuction aspirates enriched in adipose-derived stem cells (ASCs).8,9 ASCs, as an abundant source of adult mesenchymal stem cells, have the capacity to differentiate into a wide range of cell types, including chondroblasts, osteocytes, adipocytes, and endothelial cells, among others.10,11 Additionally, ASCs can secrete angiogenic factors such as VEGF, HGF, and IGF, which have proangiogenic and pro-adipogenic effects to promote the graft retention.12–14 Among these, including co-transplanting fat with SVF has been considered to facilitate the survival of fresh fat grafts.15,16 However, the ASCs present in SVF vary between both individual patients and the body sites from which lipoaspirate is obtained.17,18 Furthermore, emerging evidence has found that insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer.19 Thus, an alternative or adjunct strategy is to increase the number of endothelial cells in the transplant.20 Therefore, it remains a challenge for researchers to seek an effective solution to improve angiogenesis that would result in boosting the efficiency of survival in fat transplantation.21 New vascular network formation is critical for fat graft survival and tissue regeneration; however, the viability of fat grafting is dependent on nutrient diffusion and neovascularization, especially in the early phase after transplantation. Thus, measures that enhance vascularization of the transplanted fat tissue early after transplantation are likely to increase the viability of the transplant. Many previous studies have shown that the fate of transplanted fat tissue depends on the adequacy of blood perfusion during the early stages after transplantation.22,23 In addition, autologous fat graft survival has histologically been shown to correlate with greater blood vessel density, and expression of vascular endothelial growth factor in fat grafts has been shown to increase graft volume retention.24,25 In contrast, Philips et al have shown a strong correlation existed between stromal vascular fraction percentage of CD34+ endothelia progenitor cells and high graft retention.26 Although this technique is clinically impractical, it does highlight the importance of vascularization of the engrafted fat. Importantly, there was evidence to support that angiogenic potential of ASCs reveals differentiation toward both endothelial cell (EC) and smooth muscle cell lineages and can be used as EC substitutes in vascular tissue engineering.27–29 In addition, in animal studies, the beneficial effect of ASCs on graft survival is thought to be because of improved early vascularization, as the result of ASCs differentiation into endothelial cells.12,25 Starting from this background, we sought to address a new strategy in which more functional ASCs, namely, endothelial-like ASCs (EC/ASC) could show efficacy and possible advantages in fat grafting. We hypothesize that ASCs differentiated to an endothelial phenotype may improve the survival of fat grafting because it has the capacity to boost angiogenesis. To test this hypothesis, ASCs were cultured in a specific differentiation medium to differentiate to an endothelial phenotype. Subsequently, human fat lipoaspirate, combined with EC-differentiated ASCs or freshly isolated SVF, was subcutaneously injected into rat, and the graft volume, histologic architecture, and vascularity were evaluated 8 weeks post transplantation. METHODS Isolation and Culture of ASCs Adipose tissue was obtained by liposuction of peri-umbilical subcutaneous fat from female patients undergoing cosmetic liposuction and breast reduction (n = 7; age 28-59, 49.6 ± 10.7 years old) after informed consent was obtained in accordance with our IRB-approved protocol (Protocol # 11–015, name: Potential for Adipose-derived stem cells for Regenerative Medicine) between July 2014 and March 2015. No exclusion criteria were used. Isolation was performed as previously described.28 Briefly, adipose tissue was incubated in collagenase I (Worthington Biochemical, Lakewood, NJ) solution for 1 h at 370C. After centrifugation (1500 rpm/10 min), the stromal vascular cells were cultured in medium containing M199 (Mediatech; Manassas, VA) supplemented with 10% FBS (Gemini Bio Products; West Sacramento, CA). Non-adherent cells were removed after 24 h, and the culture medium was subsequently replaced twice weekly. Flow Cytometry Analysis The specific cell surface antigens of ACSs were characterized by flow cytometry. Approximately 5 × 105 cells were incubated with fluorescence-conjugated antibodies for 30 min. All the antibodies were obtained from BD Biosciences. Isotype identical antibodies served as controls to exclude nonspecific binding. Quantitative analysis was performed using C6 flow cytometry (Becton Dickinson). Data analysis was performed with the use of FlowJo software (FlowJo; Ashland, OR). Multipotency Differentiation of ASCs To evaluate ASC multipotent differentiation, ASCs were cultured in AdipoDiff Medium (Gbico Life Technologies) for 3 weeks to induce adipocyte differentiation. The differentiated cells were incubated with cooled methanol for 5 min and stained with fresh Oil Red-O (Sigma-Aldrich) solution for 20 min to identify lipid droplets. For osteoblast differentiation, ASC cells were cultured in OsteoDiff Medium (Gbico Life Technologies) for 3 weeks; the cells were then stained with Alizarin Red S (Sigma-Aldrich) solution for 20 min to identify calcium deposits. Endothelial Differentiation of ASCs For endothelial differentiation, ASCs were cultured in EGM-2 medium (Lonza; Walkersville, MD) with SingleQuots containing VEGF, hFGF-b, epidermal growth factor, insulin-like growth factor-1, heparin, and ascorbic acid supplemented with 2% FBS for 2 weeks. Endothelial differentiation of ASCs was confirmed by expression of EC specific markers using quantitative real-time PCR, immunofluorescence, and cord formation after plating on Matrigel. Matrigel-Based Capillary-Like Tube Formation Assays After 14 days differentiated in endothelial differentiation media (EGM-2; Lonza), ASCs were plated on Matrigel substrate (BD Biosciences) at a density of 2x105cells/100 ul Matrigel and incubated at 370C in a 5% CO2 for up to 12 h. Formation of cord-like structures was visualized by phase contrast microscopy. ASCs cultured in a non-differentiation M199 medium were used as the undifferentiated control. For immunofluorescence, cells were fixed for 15 min in 4% formaldehyde at room temperature and permeabilized with 0.3% Triton X-100 for 5 min. The cells were blocked with 10% goat serum in PBS for 1 h and incubated with a primary antibody eNOS (BD Transduction, San Jose, Calif) overnight at 40C. The secondary fluorescent antibody was used for the detection of the positive cells. Nucleus was stained with a 4,6-diamidino-2-phenylindole containing Vectashield mounting medium (Vector Laboratories Inc, Burlingame, Calif). Images were acquired using a FluoView 1000 confocal immunofluorescent microscope (Olympus, Center Valley, Pa). Quantitative Reverse Transcription Polymerase Chain Reaction Total cells and tissue RNA was extracted using TRIzol reagent. To detect the specific genes expressed, RT-PCR was performed using the following primers: Willebrand factor (vWF): 5-primers (5’-TCTTCCAGGACTGCAACAAG-3’) and 3-primers (5’-TCCGAGATGTCCTCCACATA-3’) and platelet endothelial cell adhesion molecule (CD31): 5-primers (5’-CACAGCAATTCCTCAGGCTA-3’) and 3-primers (5’- TTCAGCCTTCAGCATGGTAG-3’). mRNA levels were quantified using SYBR Green Real-Time PCR with the 7500 Fast Real-Time PCR system (Applied Biosystems). PCR reaction conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The housekeeping gene GAPDH (5-primers: 5’-GGATCTCGCTCCTGGAAGATG-3’ and 3-primers: 5’-GCACCGTCAAGGCTGAGAAC-3’) was amplified in separate tubes to normalize for variance in input RNA. For relative quantification, the efficiency of amplification for each individual primer pair was determined using cDNA target and the 2-∆∆ct method. Animal Model and Fat Transplantation All animal study protocols and procedures were approved by the Animal Care Ethics Committee of the University of the Sciences. The animals used in this study included 8 adult male Sprague–Dawley rats weighing 250-350 g. Fresh fat lipoaspirate and adipose stroma vascular fraction (SVF) were prepared onsite for same day transplant. For endothelial differentiation, ASCs were cultured in EGM2 medium for 14 days (EC/ASCs). The fat lipoaspirate (1.2g, 0.8 mL/spot) were mixed with 0.2 mL of PBS as a control, 0.2 mL of 1.5 × 105 fresh SVF, and 0.2 mL of 1.5x105 EC/ASC, and 16-gauge needles were used to inject each rat subcutaneously at 3 spots. Each rat dorsum was injected with all 3 fat mixtures, and each of the fat mixtures contained a volume of 1 mL. Assessment of the Fat Graft Survival Rates After 8 weeks, all rats were euthanized with approved IACUC protocol, and the fat grafts were carefully dissected from their back. Each fat graft was weighed and measured, and the survival ratio for transplanted fat was calculated by using the formula: survival weight/previous weight. Subsequently, each fat graft was divided into 2 equal portions. One portion was used to determine the mRNA expression of EC markers. The other portion was placed in paraformaldehyde and used for histological and immunohistochemistry examination. Histological and Immunofluorescence Analysis Formalin-fixed transplants from the 3 groups were embedded in paraffin and cryopreserved in optimal cutting temperature (OCT) media compound. Paraffin-embedded fat graft histological sections (5 μm) were prepared and then stained with hematoxylin and eosin (H&E) using standard procedures. Neovascularization was assessed by counting the capillaries in 10 fields of each H&E-stained slide and then calculating the capillary density. For immunofluorescence, the paraffin and cryostat section was stained with Cy3 conjugated anti-human nuclei antibody (Millipore Sigma), in order to detect if the human ASC proliferated in transplanted tissues. The CD31 (Santa Cruz) staining was used to detect capillary endothelial cells. After washing with PBS twice, the slides were placed under a green fluorescent beam to detect the CD31 and red fluorescence of Cy3 for human nuclear presence. Overlapping red and green fluorescence produced a yellow color, which indicated that the endothelial cells in the neovascular capillaries were derived from the transplanted human ASCs. Statistical Analysis The data are presented as mean ± standard error (SE). The Student’s t test was used to evaluate the significant differences defined as P < 0.05 between 2 groups of data in all experiments. Multigroup comparisons were determined by one-way ANOVA with Bonferroni correction. A value of P < 0.05 was considered significant. RESULTS Characterization of Human ASCs Stromal vascular fraction was isolated from human liposuction of peri-umbilical subcutaneous fat and non-adherent cells were removed after 24 h. Following culture for 5 days, ASCs showed a fibroblast-like shape on microscopy (Figure 1A). Differentiation analysis confirmed adipogenic and osteogenic differentiation potential of ASCs. Adipogenic and osteogenic differentiation levels were measured by Oil-Red-O (Figure 1B) and Alizarin Red (Figure 1C) staining, respectively. The culture-expanded ASCs were strongly positive for the mesenchymal stem cell markers of CD29, CD44, CD90, and CD105 by flow cytometry analysis (Figure 1D). Figure 1. View largeDownload slide Characterization of adipose-derived stem cells. (A) Phase contrast micrographs of ASCs cultured for 5 days. (B) ASCs stained with Oil Red O, and (C) Alizarin Red after 3 weeks cultured in adipogenic and osteogenic differentiation medium. (D) ASCs (Passage 3) were strongly positive for CD29, CD90, CD105, and CD44 consistent with mesenchymal stem cell markers by using flow cytometry analysis (with isotype control). Figure 1. View largeDownload slide Characterization of adipose-derived stem cells. (A) Phase contrast micrographs of ASCs cultured for 5 days. (B) ASCs stained with Oil Red O, and (C) Alizarin Red after 3 weeks cultured in adipogenic and osteogenic differentiation medium. (D) ASCs (Passage 3) were strongly positive for CD29, CD90, CD105, and CD44 consistent with mesenchymal stem cell markers by using flow cytometry analysis (with isotype control). ASCs Differentiation Towards an Endothelial Phenotype ASCs displayed a change in morphology after being cultured in endothelial-induced medium (EGM2) in response to differentiation (Figure 2A,B). After 14 days of being cultured in specific endothelial differentiation media, ASCs expressed EC-specific markers of CD31 and von Willebrand factor (vWF) compared with the control group by using quantitative RT-PCR analysis (Figure 2C). Immunofluorescence staining assays confirmed protein expression of eNOS in ASCs by induction (Figure 2D). Furthermore, functional assessment of the differentiated ASCs was shown by the formation of cord-like structures after plating on Matrigel (Figure 2E). Figure 2. View largeDownload slide Characteristics of ASCs differentiated into endothelial-like cells. (A, B) Phase contrast micrographs of ASCs grown in M199 medium (un-differentiation, left), and EGM2 (differentiation, right) medium for 2 weeks. (C) After 2 weeks induced in endothelia differentiation media (EGM2), ASCs expressed endothelial markers of CD31 and vWF compared with the control group by using real-time RT-PCR analysis (bars indicate ±SE, *: P < 0.05). (D) Immunofluorescence staining assays for eNOS (green) expression of ASCs (DAPI; blue). (E) Phase contrast photomicrograph of the differentiated ASCs subsequently plated onto Matrigel for 12 h demonstrating the formation of cord-like structures. Figure 2. View largeDownload slide Characteristics of ASCs differentiated into endothelial-like cells. (A, B) Phase contrast micrographs of ASCs grown in M199 medium (un-differentiation, left), and EGM2 (differentiation, right) medium for 2 weeks. (C) After 2 weeks induced in endothelia differentiation media (EGM2), ASCs expressed endothelial markers of CD31 and vWF compared with the control group by using real-time RT-PCR analysis (bars indicate ±SE, *: P < 0.05). (D) Immunofluorescence staining assays for eNOS (green) expression of ASCs (DAPI; blue). (E) Phase contrast photomicrograph of the differentiated ASCs subsequently plated onto Matrigel for 12 h demonstrating the formation of cord-like structures. Endothelial-Like ASCs Improve Fat Graft Retention Human fat lipoaspirate was co-injected with fresh isolated SVF, EC/ASCs, and the fat alone without cells as a control. One of these 3 fat mixtures was injected into each rat dorsum subcutaneously at 3 locations (Figure 3A). At the conclusion of 8 weeks post transplantation, all of the animals had survived the experiments and none of the animals died following transplantation. No inflammation or abscesses were observed in the surgical areas. The fat grafts in the EC/ASC-assisted groups appeared larger than those in the non-cell assisted and SVF-assisted group, and the dissected fat grafts in the control and SVF groups were weaker and more fragile than those in the EC/ASC group (Figure 3B). The survival ratios for the control group, SVF group, and EC/ASC group were 41.0 ± 10.1%, 41.2 ± 11%, and 56.8 ± 10.6%, respectively (n = 8). A significantly higher survival of fat grafts was observed in the EC/ASCs group than in the control or SVF groups (Figure 3C, *: P < 0.05). There was no difference between the SVF and control groups. Figure 3. View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft retention. (A) Fat lipoaspirate was mixed with PBS (control), fresh SVF, and endothelial-differentiated ASCs (EC/ASC), and each rat was injected subcutaneously in 3 spots. (B) Macroscopic images of the harvested fat grafts 8 weeks post transplantation. (C) Assisted-EC/ASCs significantly increased transplant survival. The survival ratio of transplanted fat lipoaspirate was calculated using the survival volume/previous volume (n = 8/group). Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to one-way ANOVA; *: P = 0.05. Figure 3. View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft retention. (A) Fat lipoaspirate was mixed with PBS (control), fresh SVF, and endothelial-differentiated ASCs (EC/ASC), and each rat was injected subcutaneously in 3 spots. (B) Macroscopic images of the harvested fat grafts 8 weeks post transplantation. (C) Assisted-EC/ASCs significantly increased transplant survival. The survival ratio of transplanted fat lipoaspirate was calculated using the survival volume/previous volume (n = 8/group). Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to one-way ANOVA; *: P = 0.05. Histological Evaluation of EC-Like ASC-Assisted Fat Graft The graft structure was further analyzed by masson trichrome and H&E staining after sectioning. As presented in Figure 4, the fat grafts in the EC/ASC group exhibited the best survival, most morphologic integrity, most uniform lipid droplet, least inflammation, least fibrosis, and richest blood vessels. The fat grafts in the SVF group exhibited modest survival, less integrity, less uniform lipid droplets, and connective tissue septa; and the fat grafts without cells, serving as a control group, exhibited varied sizes of lipid droplets, excessive fibrosis, and fat necrosis. Focusing on both SVF and ASC/EC transplanted specimens, we identified an additional large area composed by distinct inner and outer regions (Figure 4B). The grafts from the EC/ASC-assisted groups had more intact and nucleated adipocytes with relatively fewer fibers and vacuoles, whereas more vacuoles were observed in the SVF-assisted and control groups. In addition, newly generated blood vessels were observed in the fat grafts in the EC/ASC groups than the SVF group (Figure 4B) indicating that fat graft with EC/ASCs may promote neovascularization. Figure 4. View largeDownload slide Histological analysis of fat transplants. (A) Masson trichrome staining shows that the transplants from the EC/ASC group (right) consisted predominantly of mature adipose tissue and had lower levels of fat necrosis and fibrosis compared to those from SVF (middle) and control groups (left). Scale bar, 2000μm. (B) Hematoxylin and eosin (H&E) staining showed that the newly generated blood vessels were observed higher in EC/ASC-assisted grafts (right) compared to grafts with SVF-assisted (middle) and control groups (left). The arrows indicate neovascular capillaries. Scale bar, 400μm. Figure 4. View largeDownload slide Histological analysis of fat transplants. (A) Masson trichrome staining shows that the transplants from the EC/ASC group (right) consisted predominantly of mature adipose tissue and had lower levels of fat necrosis and fibrosis compared to those from SVF (middle) and control groups (left). Scale bar, 2000μm. (B) Hematoxylin and eosin (H&E) staining showed that the newly generated blood vessels were observed higher in EC/ASC-assisted grafts (right) compared to grafts with SVF-assisted (middle) and control groups (left). The arrows indicate neovascular capillaries. Scale bar, 400μm. EC-Like ASC Promotes Fat Graft Retention Through Neovascularization To clarify the histological reported findings, we then focused on the main parameter: neovascularization, which is an indispensable process for tissue regeneration. To determine the delivery of human ASCs promoting neovascularization, we double stained the transplant sections with anti-human nuclear (Figure 5A) and CD31 antibodies (Figure 5B), which is an endothelial cell marker. Merging the red fluorescence of anti-human nuclear with the green fluorescence of CD31 revealed 3 yellow endothelial cells, indicating that the delivery of human ASCs promoted neovascularization (Figure 5C). The CD31 and human nuclear double positive cells quantified from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC groups (right) revealed that there were more cells with positive expression of CD31 distributed to surrounding fat cells in the transplants of EC/ASC groups than that in SVF groups and fat alone groups. (9 ± 1.83, 20.75 ± 2.5 and 33.5 ± 5.57, n = 8, *: P < 0.05; Figure 5D). Figure 5. View largeDownload slide View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft angiogenesis. (A) Immunofluorescence staining analysis of the transplants with human nuclear antibody, (B) immunofluorescence for endothelial cell marker CD31, and (C) A merge of the red fluorescence of anti-human nuclear with the green fluorescence of CD31 was indicating that the delivered of human ASCs promoted neovascularization in surviving transplanted fat lipospirate. (D) The CD31 and human nuclear double positive cells were calculated from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC group (right). The positive expression of CD31 cell was higher in the transplants of EC/ASC groups when compared with SVF and control groups. These differences were significant according to a paired t test. *P < 0.05, **P < 0.01. Figure 5. View largeDownload slide View largeDownload slide Endothelial phenotype of ASC-assisted transplanted fat lipoaspirate improved fat graft angiogenesis. (A) Immunofluorescence staining analysis of the transplants with human nuclear antibody, (B) immunofluorescence for endothelial cell marker CD31, and (C) A merge of the red fluorescence of anti-human nuclear with the green fluorescence of CD31 was indicating that the delivered of human ASCs promoted neovascularization in surviving transplanted fat lipospirate. (D) The CD31 and human nuclear double positive cells were calculated from 5 randomly selected fields in each sample from the control group (left), SVF group (middle), and EC/ASC group (right). The positive expression of CD31 cell was higher in the transplants of EC/ASC groups when compared with SVF and control groups. These differences were significant according to a paired t test. *P < 0.05, **P < 0.01. We use quantitative RT-PCR to evaluate the expression levels of angiogenic genes in transplanted fat grafts to confirm the grafts’ neovascularization. We observed increases in the expression of endothelia specific markers of CD31 (approximately 2.7-fold; *: P < 0.05) and vWF (approximately 3-fold; *: P < 0.05) mRNA in EC/ASC groups when compared with the SVF group (Figure 6). These results further indicated that ASC differentiation to endothelial phenotype may promote the neovascularization in fat grafting. The SVF group was also observed to be higher than the control group, although not significantly (1.8-fold; P = 0.36; 1.3-fold; P = 0.55). Collectively, these results proved the capacity of endothelial-induced ASC phenotypes loaded into fat tissue to be associated with new high vascularization inside the graft core early after transplantation. Figure 6. View largeDownload slide Expression of angiogenesis-related genes in transplanted fat lipoaspirate. (A, B) After 8 weeks in each group, the messenger RNA expression levels of endothelia specific markers CD31 and vWF were much higher in the EC-ASC-assisted transplants than in the other 2 transplants by quantified using real-time RT-PCR analysis. The SVF group was also observed to have increases when compared with the control group, although not significantly. Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to a paired t test. *P < 0.05, n = 8. Figure 6. View largeDownload slide Expression of angiogenesis-related genes in transplanted fat lipoaspirate. (A, B) After 8 weeks in each group, the messenger RNA expression levels of endothelia specific markers CD31 and vWF were much higher in the EC-ASC-assisted transplants than in the other 2 transplants by quantified using real-time RT-PCR analysis. The SVF group was also observed to have increases when compared with the control group, although not significantly. Data are shown as mean ± standard error of the mean (error bars). These differences were significant according to a paired t test. *P < 0.05, n = 8. DISCUSSION In the current report, we demonstrate for the first time that supplementation of fat transplantation with endothelial cell-like ASCs can improve graft survival in a rat model. The novel findings of our study include that, first, fat grafts with endothelial differentiated ASCs produce comparable results to the SVF-assisted lipotransfer in terms of both volume of the grafts and improved angiogenesis of grafted fat tissues. Second, both messenger RNA level and immunohistochemical analyses support the notion that the observed improvement in fat graft volume retention is attributable to the induction of angiogenesis. Taken together, these data imply that ASCs induced to an EC-like phenotype with lipotransfer increased graft volume retention and revascularization, which represents potential mechanisms for adipose transplantation. Many investigators found beneficial effects of adipose-derived stroma progenitor cells on lipotransfer and have suggested that increases in the ratio of SVF/ASCs within the graft will lead to a higher retention rate in adipose transplantation.30,31 The current body of literature consists of a mixture of 2 different cell-assisted lipotransfer techniques: supplementation of fat with autologous stromal vascular fraction, and supplementation with in vitro-expanded adipose-derived stromal cells. Use of a fresh stromal vascular fraction is most easily acquired in the clinical setting; however, stromal vascular fraction consists of fibroblasts, endothelial cells, pericytes, preadipocytes, and various immune cells.26 In addition, large numbers of supplemental cells may not be needed for enhancement of fat graft retention, as Philips et al have shown a correlation between graft retention and the number of CD34+ progenitor cells present within a patient’s adipose tissue.5,26 Furthermore, Wan’s group has reported that use of enrichment for CD34+ progenitor cells of adipose-derived stromal cells showed more efficacy in volume retention than non-enrichment for CD34+ cells of SVF.32 To our knowledge, this is the first report evaluating the effect of an endothelial-like phenotype of ASCs on the retention and revascularization of adipose transplantation. Fat graft survival is mainly dependent on successful vascularization so that adipogenesis is associated with capillary angiogenesis allowing adipocyte differentiation within clusters of endothelial and stromal cells.5,26 Various approaches for accelerating angiogenesis have been successfully undertaken to enhance fat survival post transplantation, including the administration of basic fibroblast growth factors, interleukin 8 and erythropoietin, VEGF-based gene therapy, and endothelial cell and MSC therapies.21,25,33,34 In this report, we demonstrated that the ASCs cultured in differentiation medium by 2 weeks have functional capability as an endothelial phenotype and significantly reduce early necrosis, and this is associated with a better preservation of histological features and stimulates neovascularization with improved retention of transplanted fat grafts in the rat model. The immunohistochemical and qPCR analyses both supported the hypothesis that the observed improvement in fat graft survival was attributable to the induction of neovascularization (Figures 5 and 6). Our observations are also in agreement with the findings of previous other studies; fat graft survival is mainly dependent on successful vascularization, so that adipogenesis is associated with capillary angiogenesis allowing adipocyte differentiation within clusters of endothelial and stromal cells.35,36 Compared with SVF, the purified adipose-derived stromal cells showed about 90% positive for the mesenchymal stem cell markers of CD44, CD73, CD90, and CD105 and exhibited a greater capacity for early osteogenic and adipogenic differentiation.37,38 Furthermore, important for the potential use of ASCs in regenerative medicine is their ability to obtain the functional markers associated with mature endothelial cells, which suggest that they could be used as EC substitutes in tissue engineering and cell-based therapies.28,39 However, the EC differentiation of cell strategies can take up to weeks, so to make the technology readily available for clinical use, the cryopreservation of the autologous re-endothelialization of cells also needs to conserve them for use at a later stage.28,39 Banking the EC-differentiated autologous cells and evaluate the effect of cryopreservation on ASCs maintain acquired EC characteristics as well as their ability to improve angiogenesis of grafted fat tissues will require in our future studies. Fat tissue is a large and diffuse tissue with high metabolic activity. Histological studies have suggested that revascularization of autologous fat transplants occurs only after 48 h.40 Thus, early and abundant neovasculization seems to be the key to the survival of free fat transplants. In the present study, we found that supplementation with SVF, which is a heterogeneous mixture of cells, did not result in differences in quality or in the viability of fat transfer compared with those for the control group. However, endothelial-differentiated ASCs showed improved quality and viability of fat grafts, and vascular augmentation after 8 weeks post transplantation. Our data support the following possibilities. First, endothelial-differentiated ASC may promote angiogenesis by a paracrine mechanism, as evidenced by the expression of proangiogenic factors, which enhance angiogenesis both from the host and within the graft to improve graft revascularization and survival. Second, the differences in the survival of the transplanted fat graft are attributable to the differences in the density of the mesenchymal stem cells present in the injected material. Because the SVF is composed of a heterogeneous cell population, the purification and subsequent endothelial differentiation of ASC from the SVF select for a relatively homogeneous cell population. The results and other published data support that sufficient density of mesenchymal stem cells and differentiation is necessary to improve the viability of the fat grafts.32,35 Limitations There are some limitations to this study, including the relatively small number of specimens and the comparison of SVF to endothelial-differentiated ASCs. Therefore, a comparison of ASCs to endothelial-differentiated ASC will be required to clarify whether the impact of transplanted cells into the fat graft compartment may be due to differentiation, release of soluble factors, or both. At this time, we originally chose xenografts utilize human tissue and to test our hypothesis in using non-immunocompromised animals since our main concern in this study was that to boosting the efficiency of survival and angiogenesis in lipotransfer. The most important property to demonstrate will be the improved neovascularization of the fat transplantation with the endothelial phenotype of ASCs using human fat graft extracted and handled in the same way as clinical practice. However, for our work at the conclusion of 8 weeks post transplantation, all of the animals had survived the experiments and none of the animals died following transplantation, and no inflammation or abscesses were observed in the surgical areas. It has been postulated that adipose tissue and ASCs have low immunogenicity. However, this may represent a limitation to the current study and an opportunity for further research. CONCLUSIONS The present study indicates that co-implantation of the ASCs differentiated toward an endothelial phenotype in fat transplantation improves both survival and neovascularization of the transplanted fat lipoaspirate. These effects are likely mediated by endothelial-differentiated ASC stimulation of angiogenesis and increased graft revascularization. However, our studies further suggested that co-implantation of endothelial-like phenotype of ASCs with adipose lipoaspirate holds great potential and represents a promising strategy for clinical application in autologous fat transplantation. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding This work was supported by the Cooper University Hospital Research Award in 2014 to Ping Zhang. The funding was used exclusively for laboratory and animal-related costs. Presented at: The 14th annual meeting of the International Federation for Adipose Therapeutics and Science (IFATS) in San Diego, CA, in November 2016. REFERENCES 1. Coleman SR . Structural fat grafting: more than a permanent filler . Plast Reconstr Surg . 2006 ; 118 ( 3 ): 108S - 120S . Google Scholar CrossRef Search ADS PubMed 2. Yoshimura K , Sato K , Aoi N , Kurita M , Hirohi T , Harii K . Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells . Aesthetic Plast Surg . 2008 ; 32 ( 1 ): 48 - 55 . Google Scholar CrossRef Search ADS PubMed 3. Agha RA , Goodacre T , Orgill DP . Use of autologous fat grafting for reconstruction postmastectomy and breast conserving surgery: a systematic review protocol . BMJ Open . 2013 ; 3 ( 10 ): e003709 . Google Scholar CrossRef Search ADS PubMed 4. Gimble JM , Bunnell BA , Chiu ES , Guilak F . 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Aesthetic Surgery JournalOxford University Press

Published: May 28, 2018

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