Autologous fat grafting was first described in 1893, and after more than a century, autologous fat grafting has become a well-established method of soft-tissue augmentation for both reconstructive and cosmetic indications.1 Although it is considered the “ideal filler,” as it is both autologous and abundant, the efficacy of fat grafting remains under scrutiny, with unpredictable and varying survival rates reported by different surgeons.
The wide variation in fat grafting outcomes reported in the literature is accompanied by numerous descriptions of differing processing methods, including centrifugation2 and Telfa processing, each of which has been advocated without direct comparison of efficacy. According to a national consensus survey of plastic surgeons who perform fat grafting, 45 percent use gravity separation, 34 percent use centrifugation, 11 percent use gauze rolling, and 3 percent do not process.3
Fat graft survival research has focused on the importance of the heterogeneous nature of fat and on the concentration of adipocyte-derived stem cells.3–5 Recent studies show it to be an active participant in regulating both physiologic and pathologic processes, including angiogenesis, inflammation, and apoptosis. Over 100 proteins, or “adipokines,” have been identified as secretory components of the adipocyte and classified according to function.6
Adipocytes constitute approximately 35 to 70 percent of adipose tissue mass in adults and account for approximately 25 percent of the total cell population of the human body. Nonadipocyte cell types in adipose tissue include preadipocytes, fibroblasts, endothelial cells, immune cells, and, more importantly, adipocyte-derived stem cells.7 This multicellularity results in a wide array of autocrine and paracrine functions. For example, adipokines may stimulate endothelium from existing endothelial cells or through the recruitment of circulating endothelial progenitor cells.8 Many factors influence the ability of tissues to revascularize, as witnessed in pathologic conditions such as diabetes, where decreased numbers of circulating progenitor cells and decreased concentrations of angiogenic growth factors in ischemic tissues have been shown to negatively influence neovascularization and wound healing.8 These same factors likely influence the rate of fat graft revascularization and, ultimately, survival. For example, inhibiting angiogenesis in healthy adipose tissue results in a reduction in fat mass.9,10 Recent studies have found that several adipokines modulate the production of new blood vessels [e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor, insulin growth factor, adiponectin, stromal-derived factor 1, tumor necrosis factor-alpha, and leptin].11 VEGF, basic fibroblast growth factor, and platelet-derived growth factor (PDGF) are tyrosine kinase receptor–mediated growth factors that, in animal studies, have been shown to improve transplantation results.12 VEGF is also a potent angiogenic factor that influences endothelial progenitor cell proliferation, migration, and viability.13 In addition, it is up-regulated during differentiation and transplantation.14 PDGF is induced by inflammation or transplantation, is antiapoptotic, and stimulates the proliferation of preadipocytes.15
It is also widely known that the stromal fraction of adipose tissue contains a variety of progenitor cells with the ability to differentiate into both angiogenic and adipogenic pathways. For these reasons, we investigate not only the chemical composition of fat grafts but also the biological composition of fat grafts to determine how these factors influence fat graft revascularization and survival.
As hypoxia up-regulates the secretion of key angiogenic adipokines, the amount and duration of hypoxia experienced by processed lipoaspirate likely play a significant role in the production of adipokines.16 It has been proposed that hypoxia may underlie the inflammatory response in adipose tissue, and evidence that the tissue is hypoxic in obesity has been obtained in animal models.16 Cell culture studies have also demonstrated the expression and secretion of key adipokines, including leptin, interleukin-6, and VEGF.17 When fat is harvested for autologous transfer, the adipose tissue undergoes a period of relative hypoxia until the host site is revascularized. Thus, the ischemic time before reinjection may directly influence the rate of fat graft survival through differential expression of both angiogenic and apoptotic adipokines.
It is currently unclear what the relative importance of functional adipocytes is compared with the progenitor cells within lipoaspirate. In addition, no comparison exists determining the relative importance of each compositional element within differently processed lipoaspirates. We hypothesize that various processing techniques yield differently processed lipoaspirates, and that the lipoaspirate with the best survival is that which has a mixture of both functional adipocytes and progenitor cells.
Suction-assisted lipoaspirate was harvested from thighs, flanks, and abdomen of three healthy human subjects aged 25 to 55 years undergoing elective liposuction (New York University Institutional Review Board no. H12756). Harvested fat from the same site and human subject were used for comparison between different processing methods in our experiments. Liposuction lipoaspirate separates into two layers, with the upper layer consisting of fatty tissue and the lower layer consisting of tumescent fluid. Only the fatty layer was used to perform all of our experiments. Each sample underwent some separation because of gravity before processing, with two processing techniques compared to unprocessed lipoaspirate.
Fresh lipoaspirate was loaded into 10-ml syringes and centrifuged at 3000 rpm for 3 minutes. From the resulting lipoaspirate, the blood and tumescent fraction were drained from the bottom layer and the oil was decanted and wicked with a cotton towel for 3 minutes from the top layer. From the processed lipoaspirate, the bottom 3 ml of the syringe was used as our centrifuged lipoaspirate.
Telfa was selected to investigate because of its growing popularity as a method of processing for fat grafts and proposed benefit of concentrating cellular components of the graft. To obtain our Telfa-processed lipoaspirate, the harvested fat was poured onto Telfa gauze and with the back of a scalpel it was rolled twice until most of the tumescent fraction and oil was absorbed. The Telfa rolling process took 2 to 4 minutes. This was found to be comparable to, if not more efficient than, centrifugation, limiting the amount of ischemia time (Fig. 1).
Fat Grafting Model
Using our established fat grafting model,18 the dorsal skin of male FVB wild-type mice (n = 45) aged 8 to 12 months (Jackson Laboratories, Bar Harbor, Me.) was shaved and depilated. A 3-mm incision was made caudally and 2 ml of centrifuged, Telfa-processed, or unprocessed lipoaspirate was injected into the dorsal subcutaneous tissue using a blunt-tip cannula, performing multiple passes with each injection. Bolus injection, although not ideal, allowed for graft volume persistence to be more accurately measured. Each cohort consisted of 15 mice, with all mice receiving adipose tissue from the same donor and harvest location. Differences between graft harvest sites was not a variable in this study. The decision to perform such large injections (2 ml) of lipoaspirate was made because smaller injections could result in significant margins of errors when graft persistence was measured by weight. All experiments were performed in accordance with the New York University Medical Center Institutional Animal Care and Use Committee (no. 070911).
Fat Graft Persistence
The animal was weighed before and after the 2-ml fat injection to obtain an accurate weight (in grams) of the grafted fat. Mice were killed by carbon dioxide narcosis at 2 and 10 weeks after grafting. Fat grafts were harvested and weighed immediately to determine the fat graft persistence.
Progenitor Cell Populations
Characterization of progenitor cell populations in processed lipoaspirate was determined by flow cytometry of lineage-negative cells. First, the stromal vascular fraction from each of the processed lipoaspirates was isolated performing a procedure described previously.19 Briefly, 20 g of each processed lipoaspirate was washed several times in buffered saline, digested with 0.2% collagenase, and centrifuged at 2200 rpm for 30 minutes. The resulting stromal vascular fraction was then filtered for removal of undigested tissue. Density gradient centrifugation using Histopaque 1077 (Sigma, St. Louis, Mo.) was used to obtain the mononuclear cell fraction. The lineage-negative cells were then isolated from the mononuclear cell fraction by using a magnetic cell separation lineage depletion kit (MACS Lineage Depletion Kit; Miltenyi Biotec, Inc., Auburn, Calif.). The kit labels lineage-positive cells with biotin-labeled rat anti-human antibodies [anti Gr-1 (Ly-6G/C), 7-4, CD45R (B220), Ter-119, CD5, and CD11b antibodies] and then labels these cells with anti-biotin magnetic microbeads. The remaining cell solution is processed through the magnetic cell separation column that removes lineage-positive cells, leaving only the lineage-negative or progenitor cell population. The number of progenitor cells from each processing technique is measured using a hemocytometer. From this population, flow cytometry was performed using anti-human PE-CD105, FITC-CD34, and APC-CD31 (eBioscience, San Diego, Calif.).
Glycerol-3-Phosphate Dehydrogenase Assay
A glycerol-3-phosphate dehydrogenase assay (Takara Bio, Kyoto, Japan) was used to measure intracellular enzyme level of adipocytes, reflecting the functional status of adipocytes, and allowing determination of the number of functional adipocytes.20 Briefly, a 1-g sample from unprocessed, centrifuged, or Telfa-processed lipoaspirate was mixed with 0.25-M sucrose solution to a total of 5 ml and homogenized. The mixture was then centrifuged at 1200 rpm for 5 minutes. A 1-ml aliquot of the middle aqueous layer was centrifuged again at 10,000 rpm for 10 minutes. The supernatant from the second centrifugation was diluted 10 times with enzyme extracting reagent, and the optical absorption was measured at 340 nm for 10 minutes on a 96-well UV plate after addition of twice the volume of substrate reagent. Glycerol-3-phosphate dehydrogenase activity was calculated based on the following formula: glycerol-3-phosphate dehydrogenase (U/ml) = ΔOD × 0.482 × 10, where ΔOD is change in optical density per minute.
Secretory Protein Analysis
Sample Preparation for Protein Analysis
Briefly, 0.3 g of adipose tissue sample was placed in 3 ml of tissue extraction reagent (Invitrogen, Calif.) and protease inhibitor (Roche Applied Science, Indianapolis, Ind.). The sample was then homogenized for 30 seconds on ice at 4°C using a Polytron PT-35 homogenizer (Kinematica, Inc., Bohemia, N.Y.) with a PTA 10 generator (Kinematica). The homogenized suspension was centrifuged at 1200 rpm for 5 minutes at 4°C. The supernatant was separated and again centrifuged at 10,000 rpm for a further 10 minutes at 4°C. The supernatant was then divided into aliquots and stored at −80°C until protein analysis.
Enzyme-Linked Immunosorbent Assay
The content of VEGF and PDGF-BB from each sample of processed lipoaspirate and from the harvested fat grafts was measured using Quantikine Colorimetric Sandwich enzyme-linked immunosorbent assays (R&D Systems, Inc., Minneapolis, Minn.) with standard curves generated by means of absorbance readings for known concentrations. The samples were read in a 96-well microplate reader Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.) and the data were analyzed with SoftMax Pro software (Molecular Devices).
Histology and Immunofluorescence
Mice were killed by carbon dioxide narcosis at post–fat grafting weeks 1, 2, and 10 for fat graft harvest. Fat grafts for frozen sections were placed in 10% formalin for 10 minutes, followed by 30 minutes in 15% sucrose, and then left in 30% sucrose overnight. Subsequently, using a double-bubble chamber with 2-methylbutane and liquid nitrogen, the tissue was slowly frozen on optimum cutting tissue medium (Fisher Scientific, Pittsburgh, Pa.) and stored at −80°C.
Frozen sections (10 μm) were stained with rat anti-mouse CD31 primary antibody (platelet-endothelial cell adhesion molecule; BD Biosciences, Franklin Lakes, N.J.) and goat anti-rat immunoglobulin G secondary (Alexa Fluor 594; Invitrogen, Carlsbad, Calif.). Control samples were prepared without primary antibody. Slides were mounted with 4′,6-diamidino-2-phenylindole (Sigma) and viewed on an Olympus BX51 epifluorescent microscope (Olympus, Tokyo, Japan). Immunofluorescent CD31 staining identified vascular structures (red staining) within the fat graft, whereas the 4′,6-diamidino-2-phenylindole was used to outline the architecture of the fat graft. Dual-filter images were superimposed to illustrate the vascular staining within the graft structure. Adobe Photoshop CS4 (Adobe Systems, Inc., San Jose, Calif.) was used to quantify positive CD31 staining per high-power field. The vascular density of the fat grafts was determined by quantifying the total area of CD31 staining (red) per megapixel of stained graft.
Unprocessed and processed lipoaspirate was harvested from the same human subject for fat graft persistence and histology to avoid variations in properties among patients. Protein analysis and cellular characterization were also analyzed using lipoaspirate harvested from the same human subject for comparison. The data are presented as mean ± SEM. One-way analysis of variance with the post hoc Tukey-Kramer test was used for comparison between groups, with statistical significance considered to be p < 0.05. The number of mice per processing technique was determined using G*Power (Melbourne, Australia) to provide a power greater than 0.80.
Mononuclear Lineage-Negative Cells
The centrifugation technique creates a processed lipoaspirate with a greater number of progenitor cells (×10,000) per gram of tissue compared with the Telfa-processed and the unprocessed lipoaspirate (19.0 versus 12.3 versus 9.35) (Fig. 2).
Adipocyte-Derived Mesenchymal Stem Cells
Within the population of lineage-negative cells isolated, there was a greater percentage of adipocyte-derived mesenchymal stem cells, which were CD105/CD34+, in the centrifuged lipoaspirate (16.55 percent) compared with the unprocessed (13.9 percent) and Telfa-processed lipoaspirate (10.8 percent) (Fig. 3).
Telfa creates a processed lipoaspirate with a greater number of functional adipocytes measured by glycerol-3-phosphate dehydrogenase assay (0.104 ± 0.01 U/ml) compared with the centrifuged (0.080 ± 0.004 U/ml; p < 0.05) and unprocessed lipoaspirate (0.083 ± 0.005 U/ml; p < 0.05) (Fig. 4). Following the same protocol described previously, Telfa-processed lipoaspirate had approximately 4.8 × 104 cells/ml of lipoaspirate compared with 3.7 × 104 cells/ml in the centrifuged lipoaspirate.20
Fat Graft Persistence
Telfa creates a processed lipoaspirate with greater fat graft persistence by weight than centrifuged and unprocessed lipoaspirate both at 2 weeks and at 10 weeks after grafting. At 2 weeks, Telfa-processed lipoaspirate achieved significantly greater fat graft persistence by weight (99.8 ± 1.36 percent) than centrifuged (83.14 ± 0.77 percent) and unprocessed lipoaspirate (55.48 ± 3.1 percent) (Fig. 5, above). Even at 10 weeks, Telfa-processed lipoaspirate had greater fat graft persistence (70.9 ± 6.2 percent) than centrifuged (56.7 ± 5.5 percent) and unprocessed lipoaspirate (42.2 ± 2.7 percent) (Fig. 5, below).
At baseline, concentrations of VEGF in lipo aspirate samples (unprocessed, centrifuged, and Telfa-processed lipoaspirate) were not statistically different. In fat harvested at 1 week after grafting, there was a significant increase in VEGF secretion among all processing techniques; however, the Telfa-processed lipoaspirate had the greatest VEGF increase (110.29 ± 4.7 mg/ml) compared with the centrifuged (82.83 ± 5.7 mg/ml; p < 0.05) and unprocessed lipoaspirate (83.18 ± 7.7 mg/ml; p < 0.05) (Fig. 6). Similarly, at week 2, the Telfa-processed lipoaspirate harvested fat had statistically greater VEGF secretion (138.69 ± 5.8 mg/ml) than the centrifuged (68.58 ± 2.4 mg/ml; p < 0.05) and the unprocessed lipoaspirate (63.96 ± 9.0 mg/ml) (Fig. 6).
Baseline analysis reveals that, following processing, both centrifuged (25.54 ± 1.7 pg/ml) and Telfa-processed lipoaspirate (28.47 ± 1.0 pg/ml) had a statistically greater concentration of PDGF-BB than the unprocessed lipoaspirate (17.56 ± 2.0 pg/ml) (Fig. 6). After grafting, at both weeks 1 and 2, Telfa-processed lipoaspirate was found to have the highest concentration of PDGF (48.41 ± 3.8 pg/ml and 31.89 ± 1.0 pg/ml, respectively) (Fig. 7).
On immunofluorescent staining of CD31, which stains endothelial cells red, Telfa-processed lipoaspirate (36.9 ± 1.6 percent per high-power field) had significantly greater vascularity compared with the centrifuged lipoaspirate (14.0 ± 4.3 percent per high-power field) (p < 0.05) (Fig. 8).
The optimal technique with which to harvest and process fat for microstructural fat grafting remains a topic of great debate. Current research in fat grafting revolves around the idea of augmenting the number of stem cells of harvested lipoaspirate by centrifugation and the importance of these pluripotent cells in tissue engineering.21–25 Meanwhile, recent studies have shown adipocytes to be an active participant in regulating both physiologic and pathologic processes by secreting proteins involved in angiogenesis, inflammation, and apoptosis.6 Although in each processing technique adipose-derived stem cells are lost in the fluid layer, we believe each processing technique creates a different chemical and biological composition within the lipoaspirate, and that the interplay of these different components contributes to the revascularization and potential survival of the fat graft. Although centrifugation of the harvested lipoaspirate remains the processing technique most commonly used among plastic surgeons,26 and likely derives its clinical outcomes from the increased number of progenitor cells in centrifuged fat, many studies postulate that the process of centrifugation may negatively influence graft survival.27–29 We believe that reabsorbing the tumescent fraction of the fat on Telfa gauze creates a processed lipoaspirate with a unique set of characteristics secondary to the increased adipocyte concentration, and that the interplay of these different components contributes to the revascularization and potential survival of the fat graft.
Previous studies in our laboratory using an established murine model of fat grafting have shown revascularization of the grafted fat as early as 2 weeks after grafting.12 We used this model to investigate the contribution of each processing technique on graft survival and found that, at 2 weeks, Telfa-processed fat had the greatest survival by weight persistence, followed by centrifuged and unprocessed lipoaspirate. This trend was found to continue at 10 weeks as well, with Telfa-processed lipoaspirate demonstrating greater graft persistence over time, suggesting that the method of fat processing may play an intricate role in facilitating the revascularization of the graft.
The proposed mechanism by which centrifugation creates an optimal processed lipoaspirate is by concentrating stem cells into the most dense fraction of the lipoaspirate. To elucidate the mechanism by which each of the processing techniques contributes to fat graft survival, we performed a compositional analysis into the progenitor and adipocyte components that result after each technique. As expected, we found that centrifugation created a processed lipoaspirate with the greatest number of progenitor cells per gram of tissue compared with “Telfa-rolled” and unprocessed fat. In particular, centrifuged lipoaspirate had a greater number of CD105+/CD34+ adipocyte-derived mesenchymal stem cells compared with the other processing techniques. However, the Telfa-processed lipoaspirate had a significantly greater number of functional adipocytes than the centrifuged or the unprocessed lipoaspirate when measured by glycerol-3-phosphate dehydrogenase assay.
To determine whether this difference in composition affects adipokine secretion, we next quantified expression of several key vasculogenic adipokines. VEGF appears to be one of the most endothelial cell–specific angiogenic factors characterized to date30 and has been shown to play an important role in enhancing the survival of transplanted fat31 and stimulating adipogenesis.32 We found that, after week 1 and week 2 after fat grafting, there is a significant increase in VEGF concentration compared with baseline, with the concentration in the Telfa-processed lipoaspirate fat grafts being significantly greater compared with the centrifuged and unprocessed lipoaspirate at both time points, suggesting that the improved survival for the Telfa-processed lipoaspirate is from an enhanced ability for neovascularization.
To further support this, we found that PDGF-BB expression was significantly higher in the Telfa-processed lipoaspirate at all time points in the study. PDGF-BB is an angiogenic adipokine with chemotactic and antiapoptotic properties28 shown to stimulate the proliferation of adipocytes and improve survival.15,29 In addition, it is a potent endothelial progenitor cell chemotactic agent. Considered together, this increased concentration of VEGF and PDGF, particularly after grafting, suggests that the increased functional adipocyte concentration in Telfa-processed lipoaspirate plays an important role in enhancing the revascularization and survival of the Telfa-processed lipoaspirate fat grafts over centrifuged and unprocessed lipoaspirate. Using immunofluorescent staining of CD31 on fat grafts harvested at week 2, which stains endothelial cells red, we saw that Telfa-processed fat had significantly increased vascularity compared with the centrifuged lipoaspirate, confirming that the increased VEGF expression correlates to increased vascularity on histology.
Clear differences exist in the fat derived from different processing techniques. Although centrifugation produces processed fat with the greatest number of progenitor cells, Telfa-processed fat produces fat with greater cytokine (i.e., VEGF and PDGF) secretion and greater fat graft persistence. These differences suggest that although maximizing progenitor cells within a lipoaspirate is important, the synergistic activity between these cells plus the cytokine production from viable adipocytes may optimize fat graft survival. The significance of these findings remains to be determined in humans.
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