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Extracellular Vesicles from Human Adipose-Derived Stem Cells for the Improvement of Angiogenesis and Fat-Grafting Application

Mou, Shan M.D.; Zhou, Muran M.D.; Li, Yuan M.D.; Wang, Jiecong M.D., Ph.D.; Yuan, Quan M.D., Ph.D.; Xiao, Peng M.D., Ph.D.; Sun, Jiaming M.D., Ph.D.; Wang, Zhenxing M.D., Ph.D.

Author Information
Plastic and Reconstructive Surgery: October 2019 - Volume 144 - Issue 4 - p 869-880
doi: 10.1097/PRS.0000000000006046

Abstract

Fat grafting has many applications, including the treatment of depressed scars, hemifacial atrophy, breast reconstruction, and face rejuvenation.1,2 In addition, it has attracted a group of scientists attempting to improve the retention rate. A generally accepted theory is that insufficient blood supply after transplantation contributes to decreasing fat volume.3 Previous studies have demonstrated that the adequacy of the blood supply early after grafting is critical for the fate of transplanted fat.4

Many strategies have been proposed to enhance vascularization, including fat grafting combined with adipose-derived mesenchymal stem cells.5,6 Adipose-derived mesenchymal stem cells have been shown to be effective for improving neovascularization and the retention rate of fat volume.7 Although the regenerative activities of mesenchymal stem cells are potentially attributed to cell-to-cell contact, they are also partly ascribed to paracrine actions.8 Recently, it has been reported that the paracrine actions of mesenchymal stem cells, at least in part, are mediated by extracellular vesicles.9

Extracellular vesicles are nanosize vesicles with lipid bilayers, which are released by many, if not all, cell types.10 Over the past decade, many studies have indicated that extracellular vesicles are key players in intercellular communication.11,12 They are carriers with numerous cargoes, including coding and noncoding RNAs, proteins, and lipids.13,14 These cargoes can be delivered to specific cell types or act in a combinatorial manner to communicate directly with other cells, which alter the recipient cell fate.15,16 Recently, an increasing number of studies have revealed that mesenchymal stem cell–derived extracellular vesicles could promote proliferation, tube formation, and migration of human vascular endothelial cells.17,18 Furthermore, the therapeutic application potential of mesenchymal stem cell–derived extracellular vesicles in bone defects and hindlimb ischemia has been demonstrated in different studies.18,19 In addition, extracellular vesicles may have a relatively superior safety profile and can be stored without loss of function compared with their parent cells, suggesting the potential of extracellular vesicles as an innovative alternative to whole mesenchymal stem cell therapies.20

Based on the proangiogenic potential of mesenchymal stem cell–derived extracellular vesicles, we hypothesized that adipose-derived mesenchymal stem cell–derived extracellular vesicles might improve the retention rate of grafted fat by improving angiogenesis. To test this hypothesis, adipose-derived mesenchymal stem cell–derived extracellular vesicles were isolated from the supernatant of cultured human adipose-derived mesenchymal stem cells by ultracentrifugation techniques. Then, the proangiogenic abilities of adipose-derived mesenchymal stem cell–derived extracellular vesicles were investigated in vitro. Subsequently, we subcutaneously co-injected human fat tissue combined with adipose-derived mesenchymal stem cell–derived extracellular vesicles nude mice and evaluated the effects at 3 months after transplantation. (See Figure, Supplemental Digital Content 1, which shows an experimental design in which human adipose-derived mesenchymal stem cells (ADSC) were cultured and passaged in culture dishes. The supernatant was collected into microfuge tubes and isolated by ultracentrifugation. Then, the isolated adipose-derived mesenchymal stem cell–derived extracellular vesicles (ADSC-EV) were added into human fat tissue at the density of 20 μg/ml. Subsequently, the fat mixture was subcutaneously implanted into a nude mouse model, http://links.lww.com/PRS/D676.)

MATERIALS AND METHODS

Acquisition of Human Fat Tissue

We obtained lipoaspirates from female patients who underwent abdominal liposuction at the Department of Plastic Surgery, Wuhan Union Hospital. The donors ranged in age between 20 and 40 years. Informed consent was provided by each participant, and the protocol was approved by the Ethics Committee of Huazhong University of Science and Technology.

Isolation and Characterization of Human Adipose-Derived Mesenchymal Stem Cells

Isolation

Human adipose-derived mesenchymal stem cell isolation has been described in detail by Zuk et al.21 To isolate adipose-derived mesenchymal stem cells, lipoaspirates from five women, aged 20 to 40 years, were digested at 37°C for 1 hour with 0.1% (weight/volume) collagenase. Enzyme activity was neutralized with an equal volume of low Dulbecco’s Modified Eagle’s Medium (Hyclone, Logan, Utah), containing 10% fetal bovine serum (Gibco, Carlsbad, Calif.), after which the suspension was centrifuged at 280 g for 10 minutes. The pellet was resuspended in culture medium (Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotic) and filtered through a 70-µm nylon cell strainer. Then, the collected cells were seeded and incubated in culture medium overnight at 37°C in humidified atmosphere with 5% carbon dioxide. When the monolayer of adherent cells reached 70 to 80% confluence, cell passaging was performed.

Multilineage Differentiation of Adipose-Derived Mesenchymal Stem Cells

The trilineage differentiation potential of adipose-derived mesenchymal stem cells was assessed as described previously.17,22 All chemicals were purchased from Sigma (St. Louis, Mo.) unless stated otherwise. Osteogenic differentiation was performed by incubating adipose-derived mesenchymal stem cells in culture medium supplemented with 10 mM β-glycerophosphate, 50 μM ascorbic acid, and 0.1 μM dexamethasone. The medium was replaced every 3 days for 2 weeks and then alizarin red staining was conducted. For adipogenic differentiation, adipose-derived mesenchymal stem cells were induced in culture medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 10 μM insulin, 200 μM indomethacin, and 1 μM dexamethasone. Three weeks after induction, cells were incubated for 30 minutes in 0.5% (weight/volume) oil red O. For chondrogenic differentiation, the cells were incubated in culture medium supplemented with 1 mM sodium pyruvate, 1% insulin-transferrin sodium-selenite, 0.17 mM ascorbic acid, 0.35 mM l-proline, 1.25 mg/ml bovine serum albumin, 5.33 μg/ml linoleic acid, 0.1 μM dexamethasone, and 0.01 μg/ml transforming growth factor-β (Cell Sciences, Canton, Mass.). The medium was replaced every 3 days for 4 weeks, and then cells were assessed by toluidine blue staining.

Cell Viability Assay

A total of 1 × 103 adipose-derived mesenchymal stem cells per well were seeded onto a 96-well plate (three wells per group). At 1, 3, 5, 7, 9, and 11 days, cell viability was detected using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan), according to the manufacturer’s protocol.

Immunophenotyping

The expression of CD45 and CD29 was detected using rabbit anti-human CD45 and CD29 monoclonal antibodies (1:100; Abcam, Cambridge, United Kingdom), respectively, followed by goat anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate (Abcam). CD44, CD90, and CD105 expression was detected using mouse anti-human CD44, CD90, and CD105 monoclonal antibodies (1:100; Abcam), respectively, followed by rabbit anti-mouse immunoglobulin G conjugated with fluorescein isothiocyanate (Abcam). 4′,6-Diamidino-2-phenylindole was used for nuclear staining.

Isolation and Characterization of Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles

Isolation

Adipose-derived mesenchymal stem cell–derived extracellular vesicles were isolated by ultracentrifugation techniques as described previously by Xie et al.18 Briefly, adipose-derived mesenchymal stem cells from passages 4 to 6 were used. Once cells reached 70 to 80% confluence, they were washed three times with phosphate-buffered saline, and then serum-free Dulbecco’s Modified Eagle Medium was added. After 24 hours of incubation, the culture supernatants were collected and centrifuged at 700 g for 15 minutes to remove cells and then at 2000 g for 20 minutes to remove cell debris, followed by further centrifugation at 16,000 g at 4°C for 1 hour. The protein content of extracellular vesicles was measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Mass.), according to the manufacturer’s instructions. Then, adipose-derived mesenchymal stem cell–derived extracellular vesicles from different patients were pooled and stored for the following experiments.

Transmission Electron Microscopy

The isolated extracellular vesicles were applied to a formvar/carbon-coated grid and allowed to absorb onto the grid for 30 minutes. After rinsing with droplets of deionized water, the grids were fixed with 1% glutaraldehyde for 5 minutes and then stained with 2% uranyl acetate. The redundant liquid was removed, and the grid was dried at room temperature. Electron micrographs were obtained with an HT7700 microscope (Hitachi, Tokyo, Japan).

Confocal Microscopic Analysis

Isolated extracellular vesicles were labeled by PKH26 red fluorescent cell linker (Sigma) following the manufacturer’s instructions. The labeled extracellular vesicles were then observed under an A1Si confocal microscope (Nikon, Tokyo, Japan).

Nanoparticle Tracking Analysis

The size distribution and concentration of adipose-derived mesenchymal stem cell–derived extracellular vesicles were assessed by nanoparticle tracking analysis using a Nanosight NS300 equipped with a 405-nm laser (Malvern, Malvern, United Kingdom) at room temperature.

Western Blotting

Extracellular vesicles were suspended in ice-cold radioimmunoprecipitation assay buffer for 30 minutes at 4°C. The total protein of extracellular vesicles was assessed by the BCA protein assay kit, according to the manufacturer’s recommendations. Lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. Each blot was blocked and then incubated with the following primary antibodies: mouse anti–β-actin antibody (1:10,000; Sigma-Aldrich), rabbit anti-CD9 antibody (1:500; Abcam), rabbit anti-CD81 antibody (1:500; Abcam), or rabbit anti-vascular endothelial growth factor (VEGF) antibody (1:1000; Abcam). The membrane was then incubated with goat anti-mouse immunoglobulin G/horseradish peroxidase or goat anti-rabbit immunoglobulin G/horseradish peroxidase (1:2000; Abcam) and visualized with Amersham Hyperfilm ECL (GE Healthcare, Chicago, Ill.).

Cellular Uptake of Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles

Human umbilical vein endothelial cells were acquired from the American Type Culture Collection (Rockville, Md.) and incubated in culture medium at 37°C in a humidified atmosphere with 5% carbon dioxide. Human umbilical vein endothelial cells were incubated with PKH26-labeled adipose-derived mesenchymal stem cell–derived extracellular vesicles (20 µg/ml) for 4 hours and then washed three times with phosphate-buffered saline, fixed in 4% paraformaldehyde, stained with 4′,6-diamidino-2-phenylindole, and observed by structured illumination microscopy (Nikon).

Scratch Wound Healing Assay

Human umbilical vein endothelial cells were seeded at 1 × 104 cells/well in a 48-well plate (three wells per group) and cultured in culture medium. Once the monolayer of adherent cells reached 100% confluence, a wound was made in the cell field using a 200-µl pipette tip. Then, the human umbilical vein endothelial cells were cultured in Dulbecco’s Modified Eagle Medium containing extracellular vesicles (10, 20, or 40 μg/ml) or phosphate-buffered saline (control) for another 24 hours. Images were obtained under a phase-contrast microscope at 0, 6, 12, and 18 hours after the scratch wound was made. Cell migration was assessed using Image-Pro Plus 6 (Media Cybernetics, Inc., Bethesda, Md.). All experiments were repeated three or more times.

Tube Formation Assay

Matrigel (BD Biosciences, San Jose, Calif.) was added to a 96-well culture plate. Human umbilical vein endothelial cells were treated with extracellular vesicles (10, 20, or 40 μg/ml) or phosphate-buffered saline (control) for 24 hours and then seeded onto a 96-well plate at 1 × 104 per well (three wells per group). The plate was incubated at 37°C in 5% carbon dioxide for 6 hours. Tube formation was assessed under a phase-contrast microscope, and the total tube length was measured using Image-Pro Plus 6. All experiments were repeated three or more times.

In Vivo Fat Grafting Experiments

All experiments involving animals were performed in accordance with the guidelines of the Ethics Committee of Huazhong University of Science and Technology. Male BALB/c-nu nude mice (6 weeks old, weighing 16 to 18 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, People’s Republic of China). To investigate the effects of adipose-derived mesenchymal stem cell–derived extracellular vesicles on the volume retention of fat grafts in vivo, 16 mice were divided randomly into two groups (n = 8 mice per group). Recipient mice were anesthetized by means of inhaled 3% isoflurane and injected subcutaneously with 0.35 ml of human fat in the blank group. In the extracellular vesicle group, mice were injected subcutaneously with 0.35 ml of human fat and 20 µl of sterile phosphate-buffered saline containing 7 µg of adipose-derived mesenchymal stem cell–derived extracellular vesicles (pooled from five patients). Briefly, fat was injected using an 18-gauge needle, and the mini-incisions were closed with 6-0 nylon sutures. At scheduled times (1 and 3 months), four randomly selected animals in each treatment group were euthanized, and the grafts were harvested for the following analyses.

Graft Weight and Volume Measurements

We obtained a photograph of each graft and then removed residue and necrotic tissue with scissors. The weights of fat grafts were determined by a balance and their volumes were measured by the liquid overflow method.

Hematoxylin and Eosin Analyses

Paraffin-embedded tissue sections were stained with hematoxylin and eosin. Histologic parameters were examined under a light microscope.

Immunohistologic Analyses

For immunofluorescent staining, fat tissue sections were incubated with rabbit anti-human perilipin (Cell Signaling Technology, Danvers, Mass.). Nuclei were stained with 4′,6-diamidino-2-phenylindole.

For immunohistochemical staining, the primary antibodies used were rabbit anti-human CD31 (Abcam) and Ki67 (Abcam), and a rabbit monoclonal anti-CD206 antibody (Abcam). Each explant was subjected to at least five tissue slices, and we randomly selected three sections from these slices. Then, we randomly selected five fields from each section. The numbers of CD31+ vessels, Ki67+ cells, and CD206+ cells were calculated independently under the light microscope at 20× magnification by a single blinded observer. Positively stained vessels were counted by a second blinded observer.

Statistical Analysis

All values collected were presented as means ± SD. Data distributed normally were analyzed using the t test or one-way analysis of variance. If data were not distributed normally, the Mann-Whitney U test was applied. In this study, a value of p < 0.05 was considered statistically significant. The software used for statistical analysis was GraphPad Prism (GraphPad Software, Inc., San Diego, Calif.).

RESULTS

Characterization of Adipose-Derived Mesenchymal Stem Cells

Culture-expanded human adipose-derived mesenchymal stem cells presented long spindle-like adherent growth under an inverted phase-contrast microscope. As confirmed by oil red O staining, lipid vacuoles had accumulated in the cytoplasm of adipose-derived mesenchymal stem cells. Extracellular calcium deposition of the cells was assessed by alizarin red staining. After 4 weeks of culture in chondrogenic induction medium, the cells underwent chondrogenic differentiation, as revealed by toluidine blue staining. The cell proliferation assay indicated an enormous proliferative capacity of adipose-derived mesenchymal stem cells. Immunofluorescence staining demonstrated the immunophenotype of the cells that were positive for cell adhesion molecules (CD29 and CD44) and mesenchymal markers (CD90 and CD105), and negative for a hematopoietic marker (CD45). [See Figure, Supplemental Digital Content 2, which shows the characterization of human adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cell–derived extracellular vesicles, and uptake of adipose-derived mesenchymal stem cell–derived extracellular vesicles by human vascular endothelial cells. (Above, left) Representative morphology of human adipose-derived mesenchymal stem cells. Scale bar = 250 µm. (Above, second from left) Adipose-derived mesenchymal stem cells underwent adipogenic differentiation as demonstrated by oil red O staining. Scale bar = 50 µm. (Above, center) Adipose-derived mesenchymal stem cells underwent osteogenic differentiation as demonstrated by alizarin red staining. Scale bar = 100 µm. (Above, second from right) Adipose-derived mesenchymal stem cells underwent chondrogenic differentiation as demonstrated by toluidine blue staining. Scale bar = 250 µm. (Above, right) Proliferation of adipose-derived mesenchymal stem cells. Immunofluorescence staining of adipose-derived mesenchymal stem cells showed that they were positive for CD29, CD44, CD90, and CD105, and negative for CD45. Scale bars = 100 µm. (Second row) Representative transmission electron microscopic images of extracellular vesicles (arrows) at low magnification. Scale bars = 2 µm. (Third row, left) Representative transmission electron microscopic image of extracellular vesicles (arrows) at high magnification. Scale bar = 200 nm. (Third row, second from left) A spherical vesicle with a lipid layer was observed. (Third row, center) Confocal microscopic image of PKH26-stained adipose-derived mesenchymal stem cell–derived extracellular vesicles with red fluorescence. Scale bar = 50 µm. (Third row, second from right) Size distribution of adipose-derived mesenchymal stem cell–derived extracellular vesicles measured by dynamic light-scattering analysis. (Third row, right) Immunoblotting for extracellular vesicle surface markers (CD81 and CD9), a carried protein (VEGF), and actin. Human vascular endothelial cells were incubated with PKH-26 (red)–labeled adipose-derived mesenchymal stem cell–derived extracellular vesicles for 4 hours. The nuclei of human vascular endothelial cells were stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). (Below) Separate and merged channels of structured illumination microscopy are shown. Scale bars = 2 μm, http://links.lww.com/PRS/D677.]

Characterization of Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles

Observation of extracellular vesicles by transmission electron microscopy indicated that they were spheroidal vesicles with a lipid bilayer. PKH26-labeled adipose-derived mesenchymal stem cell–derived extracellular vesicles were observed by confocal microscopy. Nanoparticle tracking analysis confirmed the heterogeneity of adipose-derived mesenchymal stem cell–derived extracellular vesicles with diameters ranging from 100 to 1000 nm. Western blotting revealed that adipose-derived mesenchymal stem cell–derived extracellular vesicles contained general extracellular vesicle markers including tetraspanins (CD81 and CD9) and a cytoskeletal protein (β-actin). In addition, they contained VEGF, a signaling protein produced by cells, which stimulates the formation of blood vessels (see Figure, Supplemental Digital Content 2, third row, left, second from left, center, and second from right, http://links.lww.com/PRS/D677).

Cellular Uptake of Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles

After 4 hours of incubation, adipose-derived mesenchymal stem cell–derived extracellular vesicles (red fluorescent dye) had accumulated around the nucleus of human vascular endothelial cells, indicating that extracellular vesicles were successfully internalized by human vascular endothelial cells (see Figure, Supplemental Digital Content 2, below, http://links.lww.com/PRS/D677).

Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles Promote Migration and Tube Formation of Human Vascular Endothelial Cells In Vitro

To identify the proangiogenic potential of adipose-derived mesenchymal stem cell–derived extracellular vesicles, tube formation and cell migration assays were performed. Human umbilical vein endothelial cells migration (control, 217.8 ± 101.1 µm; 10 µg/ml extracellular vesicles, 943.6 ± 143.3 µm; 20 µg/ml extracellular vesicles, 906.0 ± 72.1 µm; 40 µg/ml extracellular vesicles, 187.2 ± 113.3 µm) was significantly enhanced by adipose-derived mesenchymal stem cell–derived extracellular vesicles. The lengths of tubes formed by human umbilical vein endothelial cells (control, 1.00 ± 0.06; 10 µg/ml extracellular vesicles, 1.47 ± 0.11.3; 20 µg/ml extracellular vesicles, 1.41 ± 0.10; 40 µg/ml extracellular vesicles, 1.07 ± 0.09; p < 0.05) were obviously increased by treatment with adipose-derived mesenchymal stem cell–derived extracellular vesicles at concentrations of 10 and 20 μg/ml. In addition, the number of complete vessel-like structures formed by human umbilical vein endothelial cells (control, 5.67 ± 1.70; 10 µg/ml extracellular vesicles, 23.00 ± 2.94; 20 µg/ml extracellular vesicles, 21.50 ± 6.50; 40 µg/ml extracellular vesicles, 5.00 ± 1.63; p < 0.05) was increased significantly in the presence of adipose-derived mesenchymal stem cell–derived extracellular vesicles at concentrations of 10 and 20 μg/ml. [See Figure, Supplemental Digital Content 3, which shows that adipose-derived mesenchymal stem cell–derived extracellular vesicles promote migration and tube formation of human umbilical vein endothelial cells. Scratch wound healing assay of human umbilical vein endothelial cells cultured in medium containing graded concentrations of adipose-derived mesenchymal stem cell–derived extracellular vesicles or phosphate-buffered saline (control) (n = 6) was performed. (Above, second row, and third row) Tube formation assay of human umbilical vein endothelial cells cultured in medium containing graded concentrations of adipose-derived mesenchymal stem cell–derived extracellular vesicles or phosphate-buffered saline (control) (n = 3). Scale bars = 250 µm. (Below, left) Quantitative analysis of the width change with graded concentrations of adipose-derived mesenchymal stem cell–derived extracellular vesicle stimulation (n = 6). Scale bars = 250 µm. Quantitative analysis of the tube length (below, center) and vessel-like structures with graded concentrations of adipose-derived mesenchymal stem cell–derived extracellular vesicle stimulation (n = 3) (below, right), http://links.lww.com/PRS/D678.]

Adipose-Derived Mesenchymal Stem Cell–Derived Extracellular Vesicles Promote Fat Retention

At 3 months after transplantation, all mice had survived. There was no inflammation or abscesses in the injection area. All mice were euthanized, and their grafts were extirpated. Three representative images of fat grafts from each group are shown. (see Figure, Supplemental Digital Content 4, which shows macroscopic views of fat grafts 1 and 3 months after transplantation, http://links.lww.com/PRS/D679.) At 1 month after injection, grafts in the extracellular vesicle group had greater volumes (extracellular vesicle group, 0.29 ± 0.03 mm3; blank group, 0.21 ± 0.02 mm3; p < 0.05) and wet weights (extracellular vesicle group, 250.9 ± 27.6 mg; blank group, 194.6 ± 7.0 mg; p < 0.05) compared with the blank group. At 3 months after injection, larger differences in the volumes (extracellular vesicle group, 0.12 ± 0.03 mm3; blank group, 0.05 ± 0.01 mm3; p < 0.05) and wet weights (extracellular vesicle group, 105.9 ± 22.5 mg; blank group, 49.8 ± 11.1 mg; p < 0.05) of fat grafts were observed in the blank group (Fig. 1).

Fig. 1.
Fig. 1.:
Adipose-derived mesenchymal stem cell–derived extracellular vesicles (EV) improve fat-graft survival. Statistical analysis of the volume of fat grafts 1 and 3 months after transplantation (n = 8 per group) (above) and the wet weight of fat grafts (n = 8 per group) (below). *p < 0.05.

Histologic Analyses

At 1 and 3 months after injection, the fat grafts in the blank group showed excessive fat necrosis and fibrosis and infiltration with a significant number of cells of various types, whereas adipose-derived mesenchymal stem cell–derived extracellular vesicle–treated fat grafts exhibited lower levels of necrosis and fibrosis. In addition, transplants in the extracellular vesicle group exhibited better morphologic integrity (Fig. 2). (See Figure, Supplemental Digital Content 5, which shows the histological evaluation of fat grafts 1 month after transplantation. Black arrows show fibrosis and black pentagrams show cysts or vacuoles. Scale bars = 250 µm, http://links.lww.com/PRS/D680.)

Fig. 2.
Fig. 2.:
Histologic evaluation of fat grafts 3 months after transplantation. Arrowheads, fibrosis; stars, cysts or vacuoles. Scale bars = 250 µm. EV, extracellular vesicle.

Immunofluorescences Analysis

Immunofluorescences staining for perilipin was used to determine the integrity of adipose structure in fat grafts 3 months later (Fig. 3). Fat grafts in the extracellular vesicle group showed a higher integrity of adipose structure and fewer perilipin-negative crown-like structures compared with the blank group.

Fig. 3.
Fig. 3.:
Immunofluorescence staining of fat grafts 3 months after transplantation. Samples were stained for perilipin (green). Representative images of each group are shown. Stars, perilipin-negative crown-like structures. Scale bars = 50 µm.

Immunohistochemical Analyses

To investigate the vascularization, cell proliferation, and extent of inflammatory reaction in fat tissue, tissue slices were stained with CD31, Ki67, and CD206, respectively. At 1 month, an observably increased number of CD31+ vessels (16.4 ± 4.0/field versus 5.0 ± 2.0/field; p < 0.05), Ki67+ cells (15.2 ± 4.3/field versus 5.8 ± 1.5/field; p < 0.05), and CD206+ M2 macrophages (14.4 ± 4.9/field versus 5.0 ± 1.7/field; p < 0.05) was evident in the extracellular vesicle group. (See Figure, Supplemental Digital Content 6, which shows immunohistochemical staining for CD31, Ki67, and CD206 1 month after transplantation. Representative images of CD31+ vessels (red arrows), Ki67+ cells (green arrows), and CD206+ cells (black arrows) in fat grafts from each group 1 month after transplantation are shown. Scale bars = 50 µm, http://links.lww.com/PRS/D681.) At 3 months, the number of CD31+ vessels (20.0 ± 4.9/field versus 9.8 ± 1.7/field; p < 0.05), Ki67+ cells (19.4 ± 9.9/field versus 8.2 ± 3.1/field; p < 0.05), and CD206+ M2 macrophages (26.2 ± 5.5/field versus 16.8 ± 5.2/field; p < 0.05) was increased in the extracellular vesicle group (Fig. 4).

Fig. 4.
Fig. 4.:
Immunohistochemical staining for CD31, Ki67, and CD206. Representative images of CD31+ vessels (red arrows), Ki67+ cells (green arrows), and CD206+ cells (black arrows) in fat grafts from each group 3 months after transplantation are shown. Quantitative analysis showed an obvious increase of CD31+ vessels, Ki67+ cells, and CD206+ cells in the extracellular vesicle (EV) group 1 and 3 months after transplantation. Three samples from two groups and five fields of each sample were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars = 50 µm.

DISCUSSION

Autologous fat grafting is widely used in plastic and reconstructive surgery. Many theories have been proposed to improve graft retention rate and overall outcome. Using techniques that adhere to the principles of fat grafting, large-volume grafting can be consistently performed with high retention rates,23 and fat grafting supplemented with angiogenic products to promoting vascularization also attained good results.24,25 To the best of our knowledge, this is the first study to assess the effect of adipose-derived mesenchymal stem cell–derived extracellular vesicles on fat-graft retention, angiogenesis, and inflammation.

We investigated the proangiogenic activities of adipose-derived mesenchymal stem cell–derived extracellular vesicles both in vitro and in vivo. We discovered that they were internalized by human umbilical vein endothelial cells in vitro and promoted their migration and tube formation. In this study, we used phosphate-buffered saline as a control, because ultracentrifugation-based techniques can provide a relatively high purity of extracellular vesicles. These findings revealed the proangiogenic effects of adipose-derived mesenchymal stem cell–derived extracellular vesicles. We also found that human umbilical vein endothelial cells added a higher concentration of extracellular vesicles had no significant differences in cell migration compared with the control group. This might be explained by the different extracellular vesicle contents, which could either positively or negatively regulate cell migration.26 To further investigate whether adipose-derived mesenchymal stem cell–derived extracellular vesicles improve vascularization in fat grafts, in vivo experiments were performed. Consistently, fat grafts co-transplanted with the vesicles had a significantly increased density of CD31+ vessels (Fig. 4). Histologic evaluation (Fig. 2) and serial volumetric analyses (Fig. 1, above) suggested that adipose-derived mesenchymal stem cell–derived extracellular vesicles improved the volume retention of transplanted fat. These observations confirmed that the vesicles promoted the volume retention of grafts by enhancing neovascularization (Fig. 5), which is in accordance with many previous studies addressing the close relationship between neovascularization and fat transplantation retention rate.27

Fig. 5.
Fig. 5.:
A model in which adipose-derived mesenchymal stem cell–derived extracellular vesicles (ADSC-EVs) make a prominent contribution to angiogenesis and inflammation regulation in the application of fat transplantation.

In addition, we found that extracellular vesicles may have the potential to regulate the inflammatory response. In this study, fat grafts in the extracellular vesicle group showed a higher density of CD206+ M2 macrophages (Fig. 4). It has been revealed that adipose-derived mesenchymal stem cell–derived extracellular vesicles transfer into macrophages and induce the antiinflammatory M2 phenotype by transactivating arginase-1 with extracellular vesicle–carried active STAT3.28 Numerous studies have demonstrated that M2 macrophages secrete various proangiogenic factors (e.g., VEGF) and express high levels of interleukin-12, indicating that M2 macrophages play an important role in inflammation suppression and wound healing.29–31 M2 macrophages have also been shown to improve fat-graft retention rate because of their reparative abilities.24 We hypothesized that adipose-derived mesenchymal stem cell–derived extracellular vesicles could enhance M2-like polarized activation, which might play an important role in the increased retention rate of fat grafts (Fig. 5).

Thus far, numerous investigations have reported that mesenchymal stem cell–assisted lipotransfer has a higher retention rate of grafts.32 Although tremendous advances have been made in these studies, there are still some limitations such as immunogenicity and tumorigenicity. Mesenchymal stem cell extracellular vesicles are prevailingly released from the endosomal compartment, which makes them azoic entities.33 In this case, they may possess advantages over mesenchymal stem cells, such as durability and lower immunogenicity.

Numerous studies are currently underway for the application of mesenchymal stem cell extracellular vesicles as therapeutic agents in multiple diseases, including kidney disease, osteoarthritis,34 cardiovascular diseases,35 cartilage defects,36 neurodegenerative diseases,37 full-thickness skin defects,38 organ transplantation, and others.39 In addition, multiple clinical trials (Table 1) relevant to extracellular vesicles have been completed, which could provide reference for future clinical trials of our therapy.40–42 Furthermore, extracellular vesicles have shown their priority regarding safety profile and storage compared with their parent cells. In addition, the protocol to generate and test clinical-grade extracellular vesicles has been proposed. Previous studies have revealed the bioreactor culture of bone marrow–derived mesenchymal stem cells was adapted to enable the generation of large amounts of exosomes from mesenchymal stem cells.43 Although this study refers to pancreatic cancer treatment, it provides the possibility to generate a large amount of clinical-grade extracellular vesicles from adipose-derived mesenchymal stem cells, which are indeed mesenchymal stem cells. Extracellular vesicle products can be mixed with fat tissues for transplantation or injected separately as regenerative medicine.44 These advantages suggest the potential of extracellular vesicles as innovative medical products.

Table 1. - Relevant Clinical Trials of Extracellular Vesicle–Based Therapies
Vesicle Type Disease EV Source Administration Side Effects Reference No.
Autologous exosomes Colorectal cancer Ascites fluid Subcutaneous No major toxicity; moderate pain, swelling, pruritus at injection site; fever, fatigue and nausea 40
Allogeneic EVs Type 1 diabetes mellitus Umbilical cord blood–derived MSCs Intravenous ND 41
Allogeneic exosomes Graft-versus-host disease MSCs Intravenous No major side effects 42
EV, extracellular vesicle; ND, not determined; MSC, mesenchymal stem cells.

However, many difficulties need to be overcome before clinical translation of extracellular vesicles. Good medical practice compliance and a developed understanding of the advantage of regulatory requirements is important to extracellular vesicle–based therapeutics development. Also, increasing attention has been paid regarding the safety of fat grafting.45,46 Although extracellular vesicles showed a superior safety profile, the various risks associated with extracellular vesicles have not been fully elucidated. Because extracellular vesicles are important factors in cellular communications,35 transplantation of extracellular vesicles may exert unintended paracrine effects on peripheral tissues.47 Further investigations are needed to clarify the safety issue.

The next frontier in fat grafting is translating the well-described regenerative potential of products from fat tissues (e.g., adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cell–derived extracellular vesicles) to the clinical arena.48 In this study, we determined the proangiogenic and antiinflammatory effects of extracellular vesicles, which would be helpful in regenerative medicine.

CONCLUSIONS

This study demonstrated that adipose-derived mesenchymal stem cell–derived extracellular vesicles possess a capacity for enhancing the volume retention of fat grafts in a long-term nude mouse fat transplantation model by means of improving angiogenesis and regulating immune responses. These findings, based on adipose-derived mesenchymal stem cell–derived extracellular vesicles, could offer a promising addition or alternative to autologous fat grafting once the clinical translation to the patient shows sufficient effectiveness, and may potentially show its regenerative potential for future clinical applications.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (no. 81501688 to Q.Y., no. 81601701 to J.W., and no.81701922 to Z.W.), the National Public Welfare Industry Research Foundation of China (201502029 to J.S.), the Natural Science Foundation of Hubei Province (2017CFB263 to Z.W.), and the Science Foundation of Wuhan Union Hospital (2016ZYCX034 to Z.W.).

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