Insufficient fat graft survival has been a challenge in autologous fat grafting for decades, and repeated procedures are often needed.1 Cell enrichment of the fat graft has been proposed as a strategy to improve the graft retention and thereby also the predictability of the technique.2,3 Enrichment with ex vivo–expanded adipose-derived stem/stromal cells and stromal vascular fraction has been investigated in numerous preclinical and clinical studies and has yielded encouraging results. However, the optimal concentration of adipose-derived stem/stromal cells has not yet been established.1,4,5
Only a few controlled clinical trials have been published to date, and the results from numerous preclinical studies are challenging to translate to humans, as these studies mainly examined mice grafted with human xenografts in very small volumes (<1 ml).1,4,5 In planning clinical therapy, dose-response studies dedicated to identifying the optimal concentration of enriched cells are needed. In the present study, we used a porcine model for the first time and used clinically relevant fat graft volumes to investigate various concentrations of adipose-derived stem/stromal cells and their effects on graft retention and quality. In addition, we compared ex vivo–expanded adipose-derived stem/stromal cells with stromal vascular fraction enrichment.
MATERIALS AND METHODS
Four-year-old obese female Göttingen mini pigs (n = 13) with weights of 65 to 75 kg were used as described previously6 in accordance with the regulations of the Danish Animal Experiments Inspectorate, permission number 2015-15-0201-00681. Eleven animals completed the study, as shown in Figure 1. Weight stability was ensured during the study period.
Harvesting, Isolation, and Culture of Adipose-Derived Stem/Stromal Cells
Adipose-derived stem/stromal cells were harvested, isolated, and cultured using previously described methods.6 In brief, approximately 300 ml of lipoaspirate was harvested from the dorsal area of the pigs. The lipoaspirate was collected in a GID-700 sterile canister (GID, Louisville, Colo.) according to the manufacturer’s instructions. The lipoaspirate was incubated with collagenase NB 4 and the cell count, size and viability were measured in triplicate before cells were seeded in a single 10-tray Cell Factory (Nunc, 6320 cm2; Thermo Fisher Scientific, Waltham, Mass.) at a density of 20,000 to 40,000 cells/cm2. Cells were cultured in culture medium containing 20% fetal bovine serum. On day 6, the culture medium was changed. Cells were passaged once on reaching 90 to 100% confluence and were reseeded in three 13-tray Cell Factories (Nunc, 8216 cm2) at a density of 20,000 to 30,000 cells/cm2 without changing culture medium until the final harvest. Figure 2 shows stromal vascular fraction cells and adipose-derived stem/stromal cells in culture.
Preparation of Adipose-Derived Stem/Stromal Cell–Enriched Fat Grafts
Washed lipoaspirates were transferred to 50-ml syringes and gently mixed with adipose-derived stem/stromal cells or stromal vascular fraction to produce 30-ml cell-enriched fat grafts. Adipose-derived stem/stromal cells were used at passage 1 at densities of 2.5 × 106, 5 × 106, 10 × 106, and 20 × 106 adipose-derived stem/stromal cells per milliliter of fat. The proportional amounts of adipose-derived stem/stromal cells were transferred to syringes and gently mixed with aliquots of fat.
Preparation of Stromal Vascular Fraction–Enriched Fat Grafts
Stromal vascular fraction enrichment in fat grafting is guided not by the stromal vascular fraction cell count but rather by the fat-to–stromal vascular fraction ratio as defined by the amount of fat needed for grafting and the amount of fat harvested for stromal vascular fraction isolation and enrichment. Stromal vascular fraction–enriched fat grafts with a fat-to–stromal vascular fraction ratio of 1:1 were chosen, because of the clinical relevance of this concentration,1 and prepared by harvesting 30 ml of lipoaspirate from the neck area of the minipig. A GID-SVF 2 canister was used for collecting the lipoaspirate before the addition of GIDzyme (collagenase). The suspension was incubated, neutralized, and centrifuged according to the manufacturer’s instructions. The isolated stromal vascular fraction cells were gently mixed with aliquots of fat, yielding a stromal vascular fraction–enriched fat graft of 30 ml.
Bolus fat grafts, 30 ml, were chosen to mimic a previous clinical trial, by our group, investigating ex vivo expanded adipose-derived stem/stromal cell enrichment.7 Six fat grafts were prepared for each minipig with the following concentrations of added cells:
- 2.5 × 106 adipose-derived stem/stromal cells per milliliter.
- 5 × 106 adipose-derived stem/stromal cells per milliliter.
- 10 × 106 adipose-derived stem/stromal cells per milliliter.
- 20 × 106 adipose-derived stem/stromal cells per milliliter.
- Stromal vascular fraction cells added in a ratio of 1:1.
- Control fat group with no added cells.
As shown in Figure 3, a randomization key for the graft placement was designed using www.randomizer.org and the surgeon performing the fat grafting (B.S.R.) was blinded to the key. A stab incision was made in the skin and the fat grafts were subsequently placed as a bolus in the subcutaneous plane under the dermis using a 50-ml Luer-Lok syringe (Becton, Dickinson & Co., Franklin Lakes, N.J.) with a blunt 10-gauge cannula. The areas were marked with needle tattooing for precise localization during magnetic resonance imaging and explantation on postoperative day 120.
Magnetic Resonance Imaging/Volumetric Measurements
Magnetic resonance imaging was performed immediately after grafting (postoperative day 0) and again at postoperative day 120. A 3.0-T magnetic resonance imaging scanner with an abdominal coil (Magnetom Verio; Siemens, Erlangen, Germany) was used. Volumetric analyses of the fat grafts were performed on an axial breathhold Dixon sequence with a 1.5-mm slice thickness. All scans recorded on postoperative day 120 were also performed with intravenous contrast (gadobenate dimeglumine, 0.1 mmol/kg) to visualize any larger encapsulated fat necrosis. The volume of the retained fat grafts was calculated by drawing a region of interest around the bolus graft in images containing the fat graft at postoperative day 0 and at postoperative day 120, as described previously8 and as shown in Figure 4. The areas of the regions of interest were then multiplied by slice thickness and summed to determine the graft volume. Graft retention on postoperative day 120 was calculated as a percentage of the initial volume recorded on postoperative day 0.
Pigs were euthanized on postoperative day 120 and the fat grafts were explanted and fixed with 10% formalin. Fat grafts were measured and cut into 3-mm slices. Slices from the center and the periphery of each fat graft were stained with hematoxylin and eosin. An Olympus BX51 light microscope (Olympus Corp., Tokyo, Japan) was used for the microscopic analysis performed by an experienced consultant pathologist (M.M.T.) in a blinded fashion. Fat graft morphology was assessed using the method previously described in several rodent studies,9–14 with some modifications. The four parameters assessed were as follows: (1) fat graft integrity, as manifested by the presence of intact and nucleated adipocytes; (2) necrosis, as manifested by the presence of lipophages, denucleated lipocytes, and cystic spaces; (3) inflammation, manifested by infiltration of lymphocytes and macrophages; and (4) fibrosis, as manifested by levels of collagen and elastic fibers. The parameters were combined and graded as a total score on a semiquantitative scale of 0 to 5, with 0 representing no changes from normal fat morphology, 1 representing minimal changes, 2 representing minimal to moderate changes, 3 representing moderate changes, 4 representing moderate to extensive changes, and 5 representing extensive changes and complete deterioration of the fat morphology.
We used a linear normal mixed model with the outcome log(retention) to compare fat graft retention rates among the various groups. We used a fixed effect of the treatment group and random effects of pigs. The model fit was assessed graphically using residual plots, and the normality of standardized residuals and standardized estimated random effects were analyzed using the Kolmogorov-Smirnov test. The effects of the concentration and type of enriched cells were evaluated using likelihood ratio tests at a 5 percent significance level. The estimates and 95 percent confidence intervals presented in this study were obtained from the restricted maximum likelihood estimation. Pairwise comparisons were adjusted for multiple testing by the single-step adjustment.15
In a within-pig pairwise comparison of doses based on a 0.01 significance level, 11 pigs, a standard deviation of log(retention) on 0.50, and a within-pig correlation of 40 percent, a significant relative change of at least 45 percent in retention comparing specific doses is declared significant by a paired t test with probability more than 80 percent. The Wilcoxon signed rank test was used to compare the histology scores and overall graft morphology.
All calculations were performed in R, version 3.3.2 (www.r-project.org) using the add-on packages lme4 and multcomp. Unless indicated otherwise, the results are presented as the means ± SD.
Effect of Cell Enrichment on Fat Graft Volume Retention
The absolute volumes and retention rates of the retained fat grafts enriched with various concentrations of cells are presented in Table 1 and Figures 5 through 7. No encapsulated fat necrosis was observed on magnetic resonance imaging.
Table 1. -
Fat Graft Volumes Assessed by Magnetic Resonance Imaging (n = 11)*
||POD 0 (ml)†
||POD 120 (ml)†
||Retention Rate (%)‡
||29.7 ± 1.0
||2.7 ± 1.6
|2.5 × 106 ADCs/ml
||29.6 ± 1.0
||3.8 ± 1.5
|5 × 106 ADCs/ml
||29.5 ± 1.6
||3.7 ± 1.8
|10 × 106 ADCs/ml
||30.0 ± 0.8
||4.3 ± 1.8
|20 × 106 ADCs/ml
||29.5 ± 1.6
||3.5 ± 1.6
||29.6 ± 0.6
||3.9 ± 1.5
OD, postoperative day; ADCs, adipose-derived stem/stromal cells; ns, nonsignificant; SVF, stromal vascular fraction.
Retention rate, residual volume of the retained graft reported as a fraction of the initially injected volume on POD 0; Relative retention, improvement in the residual volume on POD 120 as cell-enriched graft retention relative to the nonenriched control.
Data are displayed as means ± SD.
Data are displayed as geometric means (geometric SD factor).
Isolation of stromal vascular fraction yielded on average 9.8 × 105 ± 3.1 × 105 cells/ml, with a viability of 72 ± 7.7 percent. The overall efficacy of cell enrichment was evaluated by comparing a pool of the five treatment groups with the control group. The cell-enriched fat grafts showed an overall significantly higher retention rate than the nonenriched grafts [13.0 percent (95 percent CI, 10.5 to 15.8 percent) versus 9.2 percent (95 percent CI, 6.9 to 12.1 percent); p = 0.0045], as shown in Figure 5. This difference corresponds to a relative increase in graft retention of 41 percent (95 percent CI, 11 to 79 percent). A comparison of all individual groups are displayed in Figure 6 and showed a significantly increased retention rate in the 10 × 106 adipose-derived stem/stromal cells/ml group compared to that in the nonenriched control group [14.4 percent (95 percent CI, 9.6 to 21.6 percent) versus 9.2 percent (95 percent CI, 6.9 to 12.1 percent); adjusted p = 0.022]. No other significant differences were observed between the cell-enriched groups (p > 0.66), and no difference in graft retention rates was observed between the pooled ex vivo–expanded adipose-derived stem/stromal cells and stromal vascular fraction (p = 0.49), as shown in Figure 7.
Effect of Cell Enrichment on Fat Graft Morphology
The histologic examinations are summarized in Figure 8, and examples of microscopic images are shown in Figure 9. All fat grafts showed a significantly better resemblance to normal fat tissue in the periphery than in the center, where necrosis was the most predominant of the four histologic parameters assessed (p < 0.009). However, statistically significant differences in fat graft morphology were not observed between any of the groups when the periphery and the center were analyzed and compared separately (p > 0.14). Overall fat graft morphology, as defined by the combined histology scores from both the periphery and the center of the fat grafts, did not show significant differences between any of the groups (p > 0.17), indicating that cell enrichment had no effect on fat graft morphology in this porcine model.
In the present study, we compared graft retention between cell enrichment and nonenriched control in a porcine model for the first time. When pooling all cell-enriched concentrations, we observed a significant overall increase in the relative graft retention of 41 percent (95 percent CI, 11 to 79 percent) compared with controls (p =0.0045), supporting the concept of cell enrichment. No significant difference in fat graft retention was observed between the individual treatment groups, and surprisingly, the highest concentration of adipose-derived stem/stromal cells did not result in the highest graft retention. Moreover, no significant difference in graft retention was observed between ex vivo–expanded adipose-derived stem/stromal cells and stromal vascular fraction (p = 0.49).
Thus, we were not able to show increasing graft retention rates by increasing the concentration of adipose-derived stem/stromal cells, as proposed in a previous study.1 However, enrichment with 10 × 106 adipose-derived stem/stromal cells per milliliter resulted in the highest fat graft retention rate, possibly indicating that higher concentrations of enriched cells (20 × 106 adipose-derived stem/stromal cells per milliliter) are redundant to optimize graft retention further. Interestingly, the only previous study investigating different concentrations of pure ex vivo–expanded adipose-derived stem/stromal cells for enrichment in a murine model also found that 10 × 106 adipose-derived stem/stromal cells per milliliter produced the highest graft retention rate.16 Enrichment with various concentrations of stromal vascular fraction, in contrast, has been investigated in several previous studies, revealing a trend toward “less is more,” meaning that low numbers of added cells provided the best effects on graft retention.9,10,17–19 A possible explanation for this finding is that the addition of cells above a certain threshold level produces a large group of competitors for the scarce resources in the nutrient-poor, hypoxic, newly placed fat graft.10,20 A maximum adipose-derived stem/stromal cell–to-adipocyte ratio is therefore plausible, and a study by Mojallal et al. has shown that the effect of adipose-derived stem/stromal cells on graft retention was superior when adipose-derived stem/stromal cells were co-transplanted with adipocytes compared with the grafting of pure adipose-derived stem/stromal cells.21 This finding supports the hypothesis that an upper limit of added cells exists because of their need for a “scaffold.”
In stromal vascular fraction enrichment, the majority of added cells are not adipose-derived stem/stromal cells, and the mechanism of action is probably mediated primarily by other trophic factors or cells in the heterogenic cell population. Therefore, the optimal stromal vascular fraction cell concentration necessary for fat graft enhancement may differ from the concentration required for adipose-derived stem/stromal cell enrichment.9,10,19
Our observation of a nonsignificant difference in graft retention between adipose-derived stem/stromal cells (pooled) and stromal vascular fraction differs from the results reported in a study by Moseley et al., which is the only previous study to directly compare the effects of stromal vascular fraction and adipose-derived stem/stromal cells on graft retention.22 They used a murine model and observed a significant increase in graft retention using stromal vascular fraction compared with adipose-derived stem/stromal cells in small-volume fat grafts (0.1 ml).
Only two clinical trials using culture-expanded adipose-derived stem/stromal cells for enrichment have been conducted previously.7,23 In a study by Koh et al.23 using structural fat grafting, an increase in relative retention of 49 percent was observed when comparing grafts enriched with 10 × 106 adipose-derived stem/stromal cells per milliliter to the control. These results are comparable to the 41 percent overall relative retention reported in our study. Our relative graft retention rates are also comparable to the rates reported in several previous studies in small animals.16,24,25 However, a study by Kølle et al.7 reported a notably higher increase in the relative retention rate of nearly 400 percent using bolus fat grafting. Even though we used the same experimental design with 30-ml bolus fat grafts, we were not able to replicate these results in our porcine model.
Despite reporting promising increases in relative retentions, the absolute volume retentions in the present study were very limited in all groups, as shown in Table 1. Limited improvement in absolute terms would arguably make it questionable whether the procedures and cost required for cell enrichment are justified in clinical practice or should be reserved for selected special cases (e.g., patients at high risk for low-volume retention, large-volume fat grafts, limited donor tissue availability). Nonetheless, the limited absolute retentions observed in the present study were expected because of the use of bolus fat grafts as opposed to structural fat grafting, which is the gold standard for fat grafting in clinical practice.26–28 However, bolus injections in an experimental setting have the advantages of a more precise volume determination on magnetic resonance imaging and easy histologic examination. Bolus injections constitute a “less than ideal scenario” because of maximum hypoxia and ischemia in the large central area of the fat graft, which thereby tests the ability of cell enrichment to improve graft retention under suboptimal conditions.7,29 Future studies will possibly determine whether the relative improvements shown here can be reproduced using structural fat grafting and thereby further translated into clinical practice.
A certain amount of central necrosis was also expected because of the use of bolus injections.30,31 The histopathologic examinations did not differ significantly between the groups, which is in contrast to the results of most previous studies where the enriched grafts more closely resembled normal fat tissue.7,9,10,16,18,25 The stromal vascular fraction–enriched grafts in this study had a tendency of greater histologic deterioration in the periphery compared with the control group. In contrast, the grafts enriched with 10 × 106 adipose-derived stem/stromal cells per milliliter produced a slightly better histology score. These differences, however, did not reach statistical significance (Fig. 7).
Patient-to-patient variation in native adipose-derived stem/stromal cells occurs in autologous fat grafting and, subsequently, fat graft retention.32 To eliminate this variation and strengthen the study design, the graft placement was randomized, and each pig served as its own control. Despite the use of this robust design, the study was limited by fair similarity between values within each group, as presented in the spaghetti plot shown in Figure 10.33 This vast heterogeneity was unexpected and caused by an unknown source, despite the use of every effort to align the procedures. The heterogeneity may also have been partially responsible for our ability to determine the efficacy of cell enrichment at only one of the concentrations used.
Ex vivo–expanded adipose-derived stem/stromal cells are not legally approved for clinical use in some countries, which is why the use of stromal vascular fraction is more appealing. Because vast numbers of added cells might result in limitations or adverse effects, the use of lower concentrations or using stromal vascular fraction is of clinical interest. In the present study, we did not observe significant differences in fat graft retention rates among the various adipose-derived stem/stromal cell concentrations used or between adipose-derived stem/stromal cells and stromal vascular fraction. These results could indicate that the use of lower concentrations of adipose-derived stem/stromal cells or the use of stromal vascular fraction would potentially result in comparable increases in fat graft retention. Use of stromal vascular fraction has the advantage over ex vivo–expanded adipose-derived stem/stromal cells that it is widely legally approved, and the large laboratory setup and cost required for the expansion of adipose-derived stem/stromal cells in large numbers is obsolete, leading to increased feasibility of cell enrichment.
A major challenge in fat grafting is to provide good outcomes in large-volume fat grafting, where ischemia is more pronounced. Graft retention also differs among fat grafts of various sizes,34 and the relationship between the number of added cells needed for maximum retention and the fat graft volume may not be linear. Cell enrichment might play a future role in large-volume fat grafting, and especially if the relative increased volume retention presented in this study could be translated to a comparable or better increase using structural fat grafting. However, additional studies are needed to further elucidate this relationship and ultimately determine whether cell enrichment is superior over two consecutive sessions of conventional fat grafting.
Cell enrichment significantly increased the overall relative graft retention rate by 41 percent (13.0 percent versus 9.2 percent; p = 0.0045) in this porcine model. No significant difference in graft retention rates was observed among the various adipose-derived stem/stromal cell concentrations used or between the adipose-derived stem/stromal cell and stromal vascular fraction-enriched grafts and likewise for the histologic morphology. The results could thus indicate that adipose-derived stem/stromal cells and stromal vascular fraction are comparable alternatives for cell enrichment, but because of limited statistical power, we were unable to characterize any possible dose-response relationship in detail. The results should also be interpreted with caution because of the substantial variation within each group and overall limited absolute volume retention. Cell enrichment in this porcine model, however, was effective and feasible, and the model could be used for future studies of cell-enriched fat grafting with clinically relevant volumes.
The work has been funded by the Danish Cancer Scociety (Kræftens Bekæmpelse) and The Research Foundation of Rigshospitalet (Rigshospitalets Forskningspuljer). The authors thank the staff of the Department of Experimental Medicine, Panum Institute, University of Copenhagen, for professional and dedicated assistance with the minipigs during the study period.
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