Autologous fat transplantation is plagued by an unpredictable and often significant degree of graft loss. AdE4+ endothelial cells (ECs) are human endothelial cells that have been transduced with the E4ORF1 region of human adenovirus type 5, resulting in long-term preservation of EC proliferation and angiogenic capability without immortalization. We hypothesized that AdE4+ EC-enriched fat grafts would demonstrate improved volume retention secondary to enhanced angiogenesis. Three experimental groups were prepared by admixing 400 µL of patient lipoaspirate with 100 µL of AdE4+ EC suspensions (high AdE4+ EC concentration-enriched [5 × 106/mL], low AdE4+ EC concentration-enriched [1.25 × 106/mL], or PBS) and injected subcutaneously into the bilateral dorsa of nude mice. Fat transplants were explanted at 90 and 180 days for volumetric and histologic analyses. After both 90 and 180 days, AdE4+ EC-enriched fat grafts showed greater mean volume preservation compared to control grafts (p < 0.05). Regions of focal necrosis were only noticed in low AdE4+ EC concentration-enriched and control groups after 180 days. Histologic analysis demonstrated the presence of healthy adipocytes in all AdE4+ EC-enriched fat grafts in which both human and host ECs were evident after 90 and 180 days. AdE4+ EC enrichment improved fat graft volume preservation and vascularization in this murine xenograft model. Though further study is warranted, AdE4+ ECs demonstrated to be promising as a potential off-the-shelf adjunct for improving the volume, quality, and consistency of fat engraftment.

Autologous fat transplantation has become an increasingly popular procedure among plastic surgeons for both aesthetic and reconstructive applications [Bellini et al., 2017]. Despite the growing ubiquity with which the procedure is performed, many surgeons remain frustrated by the unpredictable take of grafted fat, with recent studies reporting graft survival rates varying anywhere from 15 to 80% [Parrish and Metzinger, 2010; Yu et al., 2015; Toyserkani et al., 2016; Bellini et al., 2017]. Although there are many variables that ultimately affect the survival of the grafted tissue, including patient-dependent variables (e.g., body mass index [BMI], co-morbidities), techniques used for harvest (manual vs. traditional suction-assisted lipectomy [SAL]), lipoaspirate concentration (decanting, centrifugation, Telfa rolling, etc.), perhaps the most significant factor is how quickly the transplanted tissue becomes vascularized [Parrish and Metzinger, 2010; Tremolada et al., 2010; Chang et al., 2013; Yu et al., 2015; Toyserkani et al., 2016]. The rate and degree of vascularization, in turn, is highly dependent upon the vasculogenic capacity of the recipient bed as well as the size of the individual grafted aliquots, crucial factors that determine how rapidly the cells furthest away from a blood vessel will become vascularized [Khouri et al., 2014a; Bellini et al., 2017].

Thus, there is significant interest in enhancing the survival of transplanted fat by improving the rate and degree of vascularization within fat grafts. To date, researchers have studied various means of enriching these transplants, including the addition of exogenous growth factors such as insulin and insulin-like growth factor-1 [Yuksel et al., 2000], vascular endothelial growth factor (VEGF) [Topcu et al., 2012], and the co-transplantation of adipose tissue and stromal vascular fraction (SVF) or adipose-derived stem cells (ADSCs) [Matsumoto et al., 2006; Kanthilal and Darling, 2014]. The term cell-assisted lipotransfer (CAL) was first introduced by Matsumoto et al. [2006] who first invested the role of SVF or ADSCs for enrichment of fat grafts in vivo [Matsumoto et al., 2006; Moseley et al., 2006]. While several investigators have demonstrated the benefit of CAL using SVF, platelet-rich plasma, or ADSCs to improve fat graft take, other studies have been equivocal [Yu et al., 2015; Toyserkani et al., 2016].

AdE4+ endothelial cells (ECs) are human endothelial cells that have been transduced with the E4ORF1 region of human adenovirus type 5, which are currently under investigation in FDA-approved trials for use as an off-the-shelf cell therapy, such as tendon and peri-anal fistula repair (E-CEL UVEC as an Adjunct Cell Therapy for Treatment of Anal Fistulas. ClinicalTrials.gov identifier: NCT04190862; E-CEL UVEC Cells as an Adjunct Cell Therapy for the Arthroscopic Rotator Cuff Repair in Adults. ClinicalTrials.gov identifier: NCT04057833) [Zhang et al., 2004]. Zhang et al. [2004] and Seandel et al. [2008] observed that, while native human umbilical vein endothelial cells (HUVECs) are highly sensitive to serum-deprivation, E4ORF1-transduced ECs can be maintained in the absence of serum for several days. E4ORF1-transduced ECs also exhibit improved angiogenic potential through alteration of apoptotic and inflammatory signaling, mediated via the VE-cadherin/Akt pathway [Zuk et al., 2002; Zhang et al., 2004; Seandel et al., 2008; Ucuzian et al., 2010]. Given their demonstrated angiogenic capacity and their likely regulatory approval for use in humans, in this study we investigated whether AdE4+ EC-assisted fat transfer would improve the degree of engraftment of human lipoaspirate in an immunosuppressed animal model.

Processing of Lipoaspirate

Human adipose tissue was obtained under an approved Weill Cornell Medicine protocol (IRB#1510016712) from the abdomen of healthy female patients undergoing liposuction (Table 1). Lipoaspirates from different female patients were collected and used in separate rounds of experiments. The lipoaspirate was decanted for approximately 20 min and tumescent fluid was removed. The lipoaspirate was then further processed by rolling on Telfa gauze to remove excess fluid and free lipids. Aliquots of 400 μL fat were then placed in individual Eppendorf tubes for preparation of fat grafts (Fig. 1a, b).

Table 1.

Summary of donor patient information for lipoaspirate collection

 Summary of donor patient information for lipoaspirate collection
 Summary of donor patient information for lipoaspirate collection
Fig. 1.

Schematic of AdE4+ EC-enriched lipotransfer. a A volume of 100 μL AdE4+ endothelial cells was resuspended with 400 μL processed lipoaspirate at high and low concentrations (5 × 106/mL and 1.25 × 106/mL, respectively) for fat grafting. As control, 100 μL PBS was mixed with fat grafts. b Processing and concentration of lipoaspirate with Telfa gauze. c Representative images of subcutaneous implantation of fat grafts in nude mice.

Fig. 1.

Schematic of AdE4+ EC-enriched lipotransfer. a A volume of 100 μL AdE4+ endothelial cells was resuspended with 400 μL processed lipoaspirate at high and low concentrations (5 × 106/mL and 1.25 × 106/mL, respectively) for fat grafting. As control, 100 μL PBS was mixed with fat grafts. b Processing and concentration of lipoaspirate with Telfa gauze. c Representative images of subcutaneous implantation of fat grafts in nude mice.

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Preparation of AdE4+ Endothelial Cells

AdE4+ ECs provided by Angiocrine Bioscience, Inc. (San Diego, CA, USA) were stored in liquid nitrogen and thawed at time of use. Upon thawing and centrifugation (400 g for 10 min), stored cell aliquots in cryopreservation media were resuspended in Medium 199 (Thermo Fisher Scientific, Waltham, MA, USA) to give the final concentration for the 2 experimental groups (high AdE4+ EC-enriched grafts at 5 × 106/mL and low AdE4+ EC-enriched grafts at 1.25 × 106/mL) to achieve a cell density ratio of 1:1 and 1:4 adipocytes with AdE4+ ECs that has been shown to generate extensive vascular networks in co-culture systems [Aoki et al., 2003; Hammel and Bellas, 2020; Yang et al., 2020]. A volume of 100 µL of each respective AdE4+ EC cell suspension was then admixed slowly and homogenously using a sterile spatula into individual Eppendorf tubes containing 400 μL of lipoaspirate, to achieve a final volume of 500 μL per fat graft; 400 μL fat grafts admixed with 100 μL PBS alone served as control. Each prepared fat graft was then transferred to individual 1cc syringes in preparation for fat grafting.

Animal Model

A total of 28 6-week-old NU/J nude mice weighing 20–25 g were utilized for fat graft implantation. Mice were randomly assigned to 1 of 3 experimental groups: high AdE4+ EC-enriched fat grafts, low AdE4+ EC-enriched fat grafts, and PBS control grafts. Under anesthesia with continuous inhalational Isoflurane (1–2%), each mouse received identical bilateral 500 μL subcutaneous fat grafts in accordance with its chosen experimental group. After the surgical site was prepared in sterile fashion, 1.5 mm stab incisions were made bilaterally and subcutaneous pockets (approximately 1 × 1 cm in size) off the midline were created. Following injection of the pre-loaded fat graft from the 1cc syringe, the incision was closed with 6-0 nylon suture (Fig. 1c). Each incision site was then post-operatively dressed with a Steri-StripTM for up to 14 days. The animals were euthanized 90 and 180 days after implantation, and the bilateral fat grafts were explanted for volumetric measurements as well as histologic analyses.

Volume Measurement

Volumetric analysis of all fat grafts after 90 and 180 days was performed immediately upon explantation using Inveon Pre-clinical MicroPET/CT/SPECT (CTI/Siemens, Knoxville, TN, USA). Regions of Interest (ROIs) were then created in digitally reconstructed images by selecting voxels using thresholds for pixel intensity (Fig. 2). A combined 3D ROI created from the coronal, axial, and sagittal views was generated in order to acquire a precise volume measurement of explanted fat grafts. At each time point, this allowed for quantitative analysis of the volume changes of fat grafts over time.

Fig. 2.

Gross and micro-CT scanning representative images of explanted fat grafts at 90 (a) and 180 days (b). Scale bars, 10 mm.

Fig. 2.

Gross and micro-CT scanning representative images of explanted fat grafts at 90 (a) and 180 days (b). Scale bars, 10 mm.

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Histologic and Immunofluorescent Analysis

After explantation, each fat graft was fixed in 10% formalin and paraffin embedded. Samples were sectioned at 8–10 μm thickness. Hematoxylin & Eosin (H&E) staining of all the explanted fat grafts was performed and compared to normal human abdominal adipose tissue in order to assess fat graft necrosis, structure, and cell infiltration. Immunofluorescent staining was conducted to assess expression of CD31 (vessels) (BioLegend, San Diego, CA, USA), perilipin (G-2) (viable adipocytes) (Santa Cruz Biotechnology, Dallas, TX, USA) and STEM121, a marker of human cytoplasm (Takara Bio Inc, Japan) to differentiate the presence and contribution of the human-derived ECs within the explanted grafts. Sections were imaged with using an Olympus 1X71 microscope (Olympus, Tokyo, Japan) with the ×4, ×10, or ×20 objective, and quantification of vessel and adipocyte parameters was analyzed using ImageJ. Serial sections of explanted fat grafts were conducted, and levels of expression were quantified for percentage area of positive staining from 3 distant sections of each sample in 5 randomly chosen high-powered (100× ) fields.

Quantitative PCR

DNA was isolated by homogenizing the cells or tissues using a Maxwell 16 Tissue DNA Purification Kit (Promega, Madison, WI, USA) and Promega Maxwell 16 Instrument. Quantitative PCR was conducted using an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). After normalization with the housekeeping gene human Apolipoprotein B (APOB), the relative quantification of the target gene E4ORF1 was performed using the 2−ΔΔCt method. The following primers and probes (ThermoFisher Scientific, Waltham, MA, USA) were used for measurement of E4ORF1 DNA levels in the explanted fat grafts and control samples: forward and reverse primers of human E4ORF1, 5′-CTGTTGTAAGACAGGCTTCTAATG-3′ and 5′-TTTGTAATCCAGAGGTTGATTATC-3′, respectively, with probe 5′-AGAATTCGGCCATTAAGGCCTCGC-3′; the human APOB primers and probe are proprietary sequences provided by ThermoFisher. The samples used in PCR test as controls were (1) human lipoaspirate sample, (2) diluted AdE4+ EC DNA (∼0.8 ng), and (3) diluted AdE4+ EC DNA (∼0.4 ng) mixed in human lipoaspirate sample.

Statistical Analysis

A Mann-Whitney U test and multiple comparison tests using a one-way ANOVA were used to compare the volume measurements of all explanted grafts between groups at 90 and 180 days using Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). Nine to 10 fat grafts were studied for each group per time point. Two fat grafts from low EC-enriched group and other grafts from the PBS-only group were excluded in volumetric quantification due to the macroscopic fat necrosis noted. The data were summarized in the form of absolute values or percentages as means ± standard deviation. p < 0.05 was considered statistically significant.

Volume Preservation of Fat Grafts

After 90 days in vivo, high AdE4+ EC-enriched grafts demonstrated significantly greater volume preservation than low AdE4+ EC-enriched grafts and PBS-only fat grafts. However, there was no significant difference between low AdE4+ EC-enriched grafts and PBS-only grafts (high AdE4+ EC-enriched group: 55.5 ± 32.9%, low AdE4+ EC-enriched group: 30.3 ± 14.3%, PBS-only group: 26.6 ± 11.9%; p < 0.05) (Fig. 3a). After 180 days in vivo, low AdE4+ EC-enriched grafts showed statistically significant greater volume preservation compared to high AdE4+ E-enriched grafts and PBS-only fat grafts (high AdE4+ EC-enriched group: 36.6 ± 10.8%, low AdE4+ EC-enriched group: 53.1 ± 17.6%, PBS-only group: 26.8 ± 18.7%; p < 0.05) (Fig. 3b). Although there were no significant differences in volume preservation for either high EC or low EC enriched groups when comparing within groups between 90 and 180 days, both groups demonstrated greater volume preservation than PBS alone.

Fig. 3.

Volume preservation of explanted fat grafts after 90 and 180 days. a After 90 days, the remaining volume of grafted fat was significantly higher in high AdE4+ EC-enriched group when compared to the low AdE4+ EC-enriched group and control group (p< 0.05). b After 180 days, the remaining volume of grafted fat was significantly higher in the low AdE4+ EC-enriched group when compared to the high AdE4+ EC-enriched group and control group (p< 0.05).

Fig. 3.

Volume preservation of explanted fat grafts after 90 and 180 days. a After 90 days, the remaining volume of grafted fat was significantly higher in high AdE4+ EC-enriched group when compared to the low AdE4+ EC-enriched group and control group (p< 0.05). b After 180 days, the remaining volume of grafted fat was significantly higher in the low AdE4+ EC-enriched group when compared to the high AdE4+ EC-enriched group and control group (p< 0.05).

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Assessment of Fat Viability and Vascularization

Upon gross examination at the time of explantation of all grafts (at both the 90- and 180-day time points), excluding those found to have contained a macroscopic abscess at 180 days, healthy fat tissue was observed by both hue and consistency (Fig. 2). No obvious necrosis or inflammation was noted microscopically in serial sections of samples, but a higher amount of connective tissue composed of fibroblasts and collagen matrix between adipose cells was observed in both AdE4+ EC-enriched grafts at 90 and 180 days when compared to normal human fat tissue and PBS-only fat grafts (Fig. 4a–f, m). Immunofluorescent staining was conducted to assess adipocyte viability and the presence of ECs in explanted fat grafts. Perilipin expression demonstrated the viability of the majority of adipocytes in both AdE4+ EC-enriched grafts at 90 and 180 days, while fewer viable adipocytes were noted in control fat grafts (PBS) at both time points (p < 0.05) (90 days: high AdE4+ EC-enriched group: 23.6 ± 5.0%, low AdE4+ EC-enriched group: 19.9 ± 5.6%, PBS-only group: 15.3 ± 4.0%; 180 days: high AdE4+ EC-enriched group: 24.8 ± 4.4%, low AdE4+ EC-enriched group: 24.1 ± 6.8%, PBS-only group: 14.1 ± 5.0%) (Fig. 4g–l). CD31 expression was seen throughout AdE4+ EC-enriched grafts after 90 days with an increased density of vascular structures after 180 days. In contrast, CD31 expression was less robust in control fat grafts (PBS) after 90 days and fewer vessels were noted after 180 days (p < 0.05) (90 days: high AdE4+ EC-enriched group: 13.9 ± 4.5%, low AdE4+ EC-enriched group: 13.5 ± 2.9%, PBS-only group: 1.1 ± 1.0%; 180 days: high AdE4+ EC-enriched group: 15.6 ± 3.3%, low AdE4+ EC-enriched group: 16.0 ± 4.1%, PBS-only group: 9.1 ± 2.7%).

Fig. 4.

Adipocyte viability and vascularization assessment of explanted fat grafts after 90 and 180 days. H&E staining images show the cellular structure of fat grafts after 90 (a–c) and 180 days (d–f). There was more connective tissue formed in between adipocytes in both AdE4+ EC-enriched grafts after 90 and 180 days (red arrows) when compared to normal human fat tissue (m) and PBS-only fat grafts (c, f). The viability of adipocytes in explanted fat grafts were confirmed by expression of perilipin (green), and vascularization was demonstrated by expression of CD31 (red) after 90 (g–i) and 180 days (j–l). Putative vessels (CD31 expressing tubular structures) in fat grafts were noted in AdE4+ EC-enriched groups after 180 days (yellow arrows). Human breast fat tissue was stained as control (n). Quantification of CD31 (o) and perilipin (p) expression in explanted fat grafts is shown as percent area. n= 5, means ± SD are given. *p< 0.05.

Fig. 4.

Adipocyte viability and vascularization assessment of explanted fat grafts after 90 and 180 days. H&E staining images show the cellular structure of fat grafts after 90 (a–c) and 180 days (d–f). There was more connective tissue formed in between adipocytes in both AdE4+ EC-enriched grafts after 90 and 180 days (red arrows) when compared to normal human fat tissue (m) and PBS-only fat grafts (c, f). The viability of adipocytes in explanted fat grafts were confirmed by expression of perilipin (green), and vascularization was demonstrated by expression of CD31 (red) after 90 (g–i) and 180 days (j–l). Putative vessels (CD31 expressing tubular structures) in fat grafts were noted in AdE4+ EC-enriched groups after 180 days (yellow arrows). Human breast fat tissue was stained as control (n). Quantification of CD31 (o) and perilipin (p) expression in explanted fat grafts is shown as percent area. n= 5, means ± SD are given. *p< 0.05.

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Four abscesses were observed in the 28 fat grafts explanted at 180 days: 2 in low AdE4+ EC-enriched group and 2 in control (PBS) fat grafts. H&E staining of one representative abscess from the low AdE4+ EC-enriched group and one from the control group (PBS) demonstrated necrotic cellular debris with sparse adipocytes within the fibrous capsules (Fig. 5).

Fig. 5.

Graft abscess formation at 180 days. At 180 days, there were abscess noted in 2 grafts in the low AdE4+ EC-enriched group and 2 in the control group. Gross and micro-CT representative images demonstrated obvious abscesses formed subcutaneously where the fat grafts were implanted in low AdE4+ EC-enriched (a) and PBS-only group (b). H&E images show cellular debris with sparse adipocytes present in the fibrous capsule (c, d).

Fig. 5.

Graft abscess formation at 180 days. At 180 days, there were abscess noted in 2 grafts in the low AdE4+ EC-enriched group and 2 in the control group. Gross and micro-CT representative images demonstrated obvious abscesses formed subcutaneously where the fat grafts were implanted in low AdE4+ EC-enriched (a) and PBS-only group (b). H&E images show cellular debris with sparse adipocytes present in the fibrous capsule (c, d).

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Identification of Human Endothelial Cells

The presence of human ECs at the time of explantation of fat grafts was confirmed by colocalization of CD31 (red) and STEM121 (green) expression after both 90 and 180 days (Fig. 6). Human-derived ECs were noted to be distributed within the grafts and incorporated into vascular structures in AdE4+ EC-enriched grafts after both 90 and 180 days. Host ECs were also seen to have infiltrated into the grafts and also became incorporated into vascular structures. More human ECs were detected in high AdE4+ EC-enriched grafts at both 90 and 180 days when compared to low AdE4+ EC-enriched grafts. In comparison, there were few ECs (human and host) and vessels seen in control fat grafts (PBS) after 90 days, but some host ECs were noted to have infiltrated the grafts after 180 days. Similarly, viable human adipocytes were noted in both AdE4+ EC-enriched grafts with both murine (host) ECs and human (donor) ECs appearing around the adipocytes.

Fig. 6.

Identification of human endothelial cells in explanted fat grafts after 90 and 180 days. Human cell-specific marker (STEM121 in green) was stained to identify preserved human endothelial cells within the grafts (yellow, overlap of green and red, yellow arrows) versus mouse endothelial cells (red arrows) that had migrated and proliferated into fat grafts after implantation for 90 (a–c) and 180 days (d–f). Both human and mouse ECs were present in vascular structures contained within AdE4+ EC-enriched grafts. Human breast tissue (g) and mouse liver tissue (h) were stained as controls. i The relative DNA levels of AdE4+ ECs in 90-day explanted fat grafts (PBS control [S1], low AdE4+ EC-enriched [S2], and high AdE4+ EC-enriched [S3] groups) and PCR test controls (human lipoaspirate sample [S4], diluted AdE4+ EC DNA [∼0.8 ng; S5] and diluted AdE4+ EC DNA [∼0.4 ng] mixed in human lipoaspirate sample [S6]) normalized with the housekeeping gene APOBand compared against human lipoaspirate sample mixed with the diluted AdE4+ EC DNA. Note that no AdE4+ signal was detected in control groups (S1 and S4) as expected. Data from 180 days are not shown as no signal was detected in any of the experimental groups.

Fig. 6.

Identification of human endothelial cells in explanted fat grafts after 90 and 180 days. Human cell-specific marker (STEM121 in green) was stained to identify preserved human endothelial cells within the grafts (yellow, overlap of green and red, yellow arrows) versus mouse endothelial cells (red arrows) that had migrated and proliferated into fat grafts after implantation for 90 (a–c) and 180 days (d–f). Both human and mouse ECs were present in vascular structures contained within AdE4+ EC-enriched grafts. Human breast tissue (g) and mouse liver tissue (h) were stained as controls. i The relative DNA levels of AdE4+ ECs in 90-day explanted fat grafts (PBS control [S1], low AdE4+ EC-enriched [S2], and high AdE4+ EC-enriched [S3] groups) and PCR test controls (human lipoaspirate sample [S4], diluted AdE4+ EC DNA [∼0.8 ng; S5] and diluted AdE4+ EC DNA [∼0.4 ng] mixed in human lipoaspirate sample [S6]) normalized with the housekeeping gene APOBand compared against human lipoaspirate sample mixed with the diluted AdE4+ EC DNA. Note that no AdE4+ signal was detected in control groups (S1 and S4) as expected. Data from 180 days are not shown as no signal was detected in any of the experimental groups.

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To identify a sub-population of AdE4+ ECs among the human ECs detected by IHC within the explanted fat grafts, PCR was conducted using primers to identify a unique AdE4+ EC sequence, which demonstrated AdE4+ EC DNA present in 2/5 of the low AdE4+ EC-enriched fat grafts and in 4/5 of the high AdE4+ EC-enriched fat grafts explanted after 90 days. However, in grafts explanted after 180 days no AdE4+ EC DNA was detected in any of the AdE4+ EC-enriched fat grafts nor (as expected) in the PBS-only fat grafts at both 90- and 180-day time points (Fig. 6i).

Though known for over a century, fat grafting gained significant popularity over the past 2 decades in large part because of the contributions of Rigotti and Coleman for both reconstructive and cosmetic applications, respectively [Coleman, 2006; Khouri et al., 2014b, c]. However, despite the ubiquity with which autologous fat transfer is currently performed, the limitations of fat transplantation are well known, particularly the unpredictability of volume maintenance of the transplanted lipoaspirate [Toyserkani et al., 2016; Bellini et al., 2017]. When attempting to elucidate the causes that underlie this variability, it is essential to understand the multiple factors that may affect the engraftment of transplanted fat, such as patient-dependent and technique-dependent variables [Parrish and Metzinger, 2010; Toyserkani et al., 2016; Bellini et al., 2017]. The optimal methods of fat harvest and processing remain fervently debated among surgeons, and the literature contains many conflicting reports regarding which approaches provide the most reliable and consistent results. Furthermore, the significant body of literature evaluating adjuncts for fat transplantation stem predominantly from animal-based studies with varying methodologies, which makes direct comparison among these challenging [Parrish and Metzinger, 2010; Toyserkani et al., 2016]. Investigators have ventured to optimize each step in fat transplantation, from liposuction to injection. Conflicting evidence on the relevant parameters, including harvest cannula diameter, the use or avoidance of machine-powered suction devices, and the importance of tissue centrifugation speed on fat graft outcomes have all further complicated our understanding of techniques that are truly effective in improving fat graft viability [Ozsoy et al., 2006; Ferraro et al., 2011; Hoareau et al., 2013].

Although surgeons may disagree on the ideal techniques for lipoaspirate harvest and processing, it is commonly understood that a key factor contributing to high resorption rates of transplanted fat is cell death and apoptosis secondary to inflammatory conditions post transplantation, both caused and exacerbated by ischemia [Moseley et al., 2006]. While previous studies have demonstrated that adipose-derived stem cells and their associated angiogenic and antiapoptotic properties can improve survival of grafted fat in mice [Matsumoto et al., 2006; Moseley et al., 2006; Kanthilal and Darling, 2014], these results have not been widely recapitulated in humans. ADSCs secrete cytokines and growth factors that promote angiogenesis and improve the efficacy of autologous fat grafting via paracrine signaling [Moseley et al., 2006; Kanthilal and Darling, 2014; Li et al., 2019]. As such, many investigators have sought to augment the rate of revascularization of grafted fat by enriching lipoaspirate with cells that express growth factors known to promote more rapid angiogenesis from the recipient bed into the grafted tissue, or cells that promote EC formation of vascular structures to support the grafted tissue [Matsumoto et al., 2006; Tremolada et al., 2010; Toyserkani et al., 2016]. Among the most commonly studied cell populations used to enrich lipoaspirate have been SVF, ADSCs, and bone marrow-derived mesenchymal stem cells (BMSCs); however, the results have been variable [Wang et al., 2015; Toyserkani et al., 2016]. For example, Wang et al. [2015] demonstrated that fat grafts enhanced by SVF in breast augmentation showed no significant advantage over non-CAL technique at 6 months postoperatively. In another study, the benefit of the CAL technique seemed to be correlated with injected volume, and significant improvement of fat graft survival was only seen when the injection volume was less than 100 mL [Laloze et al., 2018]. Additionally, there is a theoretical risk that supplemental SVF and/or ADSCs may promote cancer development and progression in studies [Zimmerlin et al., 2011; Zhang et al., 2012; Kamat et al., 2015; Toyoda et al., 2018].

AdE4+ ECs are endothelial cells that have been transduced with the E4ORF1 region of human adenovirus type 5, which supports long-term survival of primary ECs in serum-free conditions and preserves in vivo angiogenic potential of ECs for tubulogenesis and sprouting by Akt activation [Zhang et al., 2004; Seandel et al., 2008; Chen et al., 2021]. To date, AdE4+ ECs are being investigated in ongoing clinical trials to restore damaged vascular niches (phase 1b/2) (Trial of AB-205 in Adults With Lymphoma Undergoing High-Dose Therapy and Autologous Stem Cell Transplantation. ClinicalTrials.gov identifier: NCT03925935.), non-healing peri-anal fistula (phase 1b; E-CEL UVEC as an Adjunct Cell Therapy for Treatment of Anal Fistulas. ClinicalTrials.gov identifier: NCT04190862), and tendon repair (phase 1b; E-CEL UVEC Cells as an Adjunct Cell Therapy for the Arthroscopic Rotator Cuff Repair in Adults. ClinicalTrials.gov identifier: NCT04057833). The engineered AdE4+ ECs can respond to cytokines produced by damaged cells and release angiocrine factors to support microvasculature repair in a juxtacrine and paracrine matter through interactions with intact ECs (Trial of AB-205 in Adults With Lymphoma Undergoing High-Dose Therapy and Autologous Stem Cell Transplantation. ClinicalTrials.gov identifier: NCT03925935) [Zhang et al., 2004; Seandel et al., 2008]. Unlike SVF, ADSCs, and BMSCs, which are by definition a heterogenous cell population which have multi-differentiation potentials and whose behavior is quite variable depending upon the donor patient, AdE4+ ECs are a well-characterized homogenous cell population. AdE4+ ECs are supplied by a commercial vendor and are currently being studied in human subjects; the ability to supplement fat grafts with off-the-shelf cellular adjuvants that improve the quantity and consistency of fat graft take would have great value to surgeons and their patients. Further, with increased efficiency of take, the volume of harvested lipoaspirate needed is smaller, which would benefit thin patients with less available donor tissue.

Given the significant angiogenic capabilities demonstrated by AdE4+ ECs and the fact that they have already been used safely in patients and may have a future off the shelf availability, we sought to determine if AdE4+ EC enrichment of lipoaspirate would improve the degree of fat engraftment. In the current study, we incorporated 5 × 106 cells/mL in high AdE4+ EC-enriched grafts and 1.25 × 106 cells/mL in low AdE4+ EC-enriched grafts (a calculated cell density ratio of 1:1 and 1:4 AdE4+ ECs to adipocytes in a total volume of 500 μL) based upon the applications for which ECs have been previously proven to enhance fat grafting survival [Hammel and Bellas, 2020; Yang et al., 2020]. According to the cell size range of mature adipocytes (20–240 μm) with a mean cell size of 100 μm, the approximate cell number of adipocytes included within 400 μL human lipoaspirate was calculated to be 2.5 × 106, thus the total cell number of AdE4+ ECs that need to be added was calculated accordingly to achieve the chosen ratios [McLaughlin et al., 2014]. In our study, the increased number of vessels and healthy adipocytes, as evidenced by CD31 and perilipin expression, respectively, within grafts supplemented with AdE4+ ECs validated our hypothesis that addition of these cells would produce an improved angiogenic response resulting in more robust adipocytes.

The volume preservation was 2.09-fold and 1.36-fold at 90 and 180 days, respectively, in the high AdE4+ EC-enriched group, and 1.14-fold and 1.98-fold at 90 and 180 days, respectively, in the low AdE4+ EC-enriched groups when compared to the control group, which is similar to other studies that have demonstrated the benefit of the CAL technique in fat grafting [Laloze et al., 2018]. While greater mean volume preservation was observed in the AdE4+ EC-enriched groups as compared to control grafts (PBS) in all instances, these gains were not always statistically significant. High AdE4+ EC-enriched grafts demonstrated greater volume preservation than low AdE4+ EC-enriched grafts and PBS-only fat grafts after 90 days in vivo; however, there was no significant difference between low AdE4+ EC-enriched grafts and PBS-only grafts. After 6 months, the low AdE4+ EC-enriched had the greatest volume preservation, whereas the high AdE4+ EC-enriched group demonstrated a non-significant decrease in volume in comparison to the 90-day time point; although the mean volume preservation in both AdE4+ EC-enriched groups was greater than PBS-only fat grafts, this was not statistically significant.

The lack of consistency between high and low AdE4+ EC-enriched fat grafts at 90 and 180 days may be attributed to several possible causes, all which manifest the several limitations of this study. First, the mouse model is not an ideal analogue due to the “looser and thinner” skin than human skin, even though it can still generate significant contractile forces which was confirmed with our result of volume loss in the PBS-only group that was consistent with clinical observations [Parrish and Metzinger, 2010; Toyserkani et al., 2016]. Further, subcutaneous placement into a “potential space” between the undersurface of the thin rodent dermis and the underlying fascia is not directly comparable to most clinical practice. Additionally, immunocompromised mice exhibit a decreased immune reaction to the graft, the proper amount of which is necessary to begin the angiogenic response to support fat engraftment. Although the volume of fat grafts injected in this pre-clinical study is similar to other experimental study designs, they are much greater than used in the Coleman technique where only 100-200 μL aliquots are placed that require less time for revascularization. Finally, the heterogeneity of the donor patient’s age and body mass index might have affected the viability of transplanted fat grafts and contributed to the high variability in volume retention between groups and time points. A larger sample size might reduce variation among those variables.

Interestingly, histologic analysis of explanted fat grafts demonstrated a significantly higher EC density as well as greater adipocyte viability in EC-enriched grafts when compared to the PBS-only group, which indicates a potential benefit to their use that did not consistently translate to improved graft take for the reasons listed above.

AdE4+ EC-assisted fat transfer demonstrated better fat graft volume preservation in AdE4+ EC-enriched groups compared to control groups at 90 and 180 days in our mouse model. These data suggest that AdE4+ EC-assisted fat transfer decreases resorption and volume loss of autologous fat grafts via the pro-survival influence of AdE4+ ECs. Though further investigation is warranted, AdE4+ ECs demonstrated to be promising as a potential off-the-shelf adjunct for improving the volume, quality, and consistency of fat engraftment. Future studies with a larger sample size should better control for donor patient age and BMI as well as target the optimal EC-to-adipocyte ratio to optimize rapid revascularization and engraftment in transplanted adipose tissue.

All animal care and experimental procedures were in compliance with the Guide for the Care and Use of Laboratory Animals and were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee (protocol #2019-0012). All studies involving human adipose tissues and cells were approved and obtained under an approved Weill Cornell Medicine protocol (IRB#1510016712).

The authors declare that they have no conflicts of interest to disclose.

This research was supported by Angiocrine Bioscience, Inc. (Grant # SRA200923-01).

Xue Dong and Ishani D. Premaratne carried out the experiment and analyzed the data. Mariam Gadjiko and Nabih Berri assisted with patient consenting and animal surgery. Xue Dong wrote the manuscript with input from all authors. Jason Spector supervised the project.

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Additional information

Xue Dong and Ishani Premaratne contributed equally to the work and final publication.