Background: Clinical outcomes following fat grafting are variable and technique dependent, and it is unknown how the graft is revascularized. The authors recently observed that living and dead adipocytes can be differentiated not with hematoxylin and eosin staining but with immunohistochemistry for perilipin. Methods: The viability of cellular components (adipocytes, adipose stem/stromal/progenitor cells, vascular endothelial cells, and hematopoietic cells) in human adipose tissue was evaluated using (1) stored lipoaspirates, (2) cultured cells, and (3) organ-cultured adipose tissue. In addition, the groin fat pad (150 to 200 mg) in mice was transplanted under the scalp, and the graft was stained at 0, 1, 2, 3, 5, 7, or 14 days. Results: In vitro studies revealed that adipocytes are most susceptible to death under ischemic conditions, although adipose-derived stromal cells can remain viable for 3 days. The in vivo study indicated that most adipocytes in the graft began to die on day 1, and only some of the adipocytes located within 300 μm of the tissue edge survived. The number of proliferating cells increased from day 3, and an increase in viable adipocyte area was detected from day 7, suggesting that repair/regeneration of the dead tissue had begun. Conclusions: The authors show convincing evidence of very dynamic remodeling of adipose tissue after nonvascularized grafting. The authors observed three zones from the periphery to the center of the graft: the surviving area (adipocytes survived), the regenerating area (adipocytes died, adipose-derived stromal cells survived, and dead adipocytes were replaced with new ones), and the necrotic area (both adipocytes and adipose-derived stromal cells died).

Injective transfer of autologous aspirated fat is a popular option for soft tissue augmentation, but several issues require attention, including unpredictability and a low survival rate due to partial necrosis. In this study, histologic features and yield of adipose-derived stromal (stem) cells (ASCs) were compared between human aspirated fat and excised whole fat. Aspirated fat contained fewer large vascular structures, and ASC yield was lower in aspirated fat. Aspirated fat was transplanted subcutaneously into severe combined immunodeficiency mice with (cell-assisted lipotransfer; CAL) or without (non-CAL) vascular stromal fractions containing ASCs isolated from adipose tissue. The CAL fat survived better (35% larger on average) than non-CAL fat, and microvasculature was detected more prominently in CAL fat, especially in the outer layers. DiI-labeled vascular stromal fraction cells were found between adipocytes and in the connective tissue in CAL fat, and some of these cells were immunopositive for von Willebrand factor, suggesting differentiation into vascular endothelial cells. Another experiment that used vascular stromal fractions taken from green fluorescent protein rats also suggested that ASCs differentiated into vascular endothelial cells and contributed to neoangiogenesis in the acute phase of transplantation. These findings may partly explain why transplanted aspirated fat does not survive well and suggest clinical potential of the CAL method for soft tissue augmentation.

Background: Adipose tissue is an easily accessible tissue for use as a soft-tissue filler and source of adult multipotent cells, called adipose-derived stem/stromal/progenitor cells. However, many aspects of its anatomy remain unclear because of the fragile structure of adipocytes and adipose tissue. Methods: Aspirated (n = 15) or intact (n = 9) subcutaneous adipose tissue samples were evaluated by (1) whole-mount histology with triple-fluorescence staining for three-dimensional visualization of living adipose tissue, (2) glycerol-3-phosphate dehydrogenase assay, (3) multicolor flow cytometry (CD34, CD31, and CD45), and (4) adherent cell culture of stromal vascular fraction cells for viable adipose-derived stromal cell yield. Results: Whole-mount histology revealed the presence of a capillary network running alongside adipocytes, which was partly disrupted in aspirated adipose tissues. Aspirated adipose tissue also lacked large vasculature structures and showed significantly larger numbers of small lipid droplets (ruptured adipocytes) (p = 0.00016) and dead cells (p = 0.0038) compared with excised adipose tissue. Adipocyte number was less than 20 percent of total cellularity, and vasculature-associated cells, including endothelial cells and adipose-derived stromal cells, constituted over one-half of total cells in both intact and aspirated adipose tissue. Glycerol-3-phosphate dehydrogenase assay suggested that greater than 30 percent and 5 percent of adipocytes were ruptured in aspirated and excised adipose tissue, respectively (p = 0.032). Multicolor flow cytometric analysis indicated a much higher percentage of blood-derived cells (CD45⁺) contaminated in aspirated adipose tissue (p = 0.0038), and adipose-derived stromal cell yield in aspirated adipose tissue was approximately one-half that in excised tissue (p = 0.011). Conclusion: The authors’ results indicate the differential structure and cellular composition of the two tissues, and significant tissue damage and progenitor yield deficiency in aspirated adipose tissue.

Following various types of plastic surgery, such as adipose grafting and flap elevation, adipose tissue undergoes ischemia, leading to hypoxia and nutrient depletion. However, few studies have examined ischemic and/or hypoxic changes in adipose tissue. The authors established surgically induced ischemia models by severing blood vessels supplying the inguinal fat pads in mice. The partial pressure of oxygen in adipose tissue was measured with an oxygen monitor, and ischemic changes were analyzed by whole-mount staining, immunohistochemistry, flow cytometry, and Western blotting. The authors also examined cell survival under a hypoxic condition in vitro. Models for three degrees (mild, intermediate, and severe) of ischemia showed approximately 75, 55, and 20 percent of the partial pressure of oxygen level in normal adipose tissue (50.5±1.3 mm Hg), respectively. Adipose tissue atrophy with substantial fibrosis on day 28 was seen, depending on the severity of ischemia. Intermediate and severe ischemia induced elevated expression of hypoxia-inducible factor 1α and fibroblast growth factor 2 on day 1 and degenerative changes (i.e., apoptosis, necrosis, and macrophage infiltration and phagocytosis) in adipose tissue. Dead cells included adipocytes, vascular endothelial cells, and blood-derived cells, but not adipose-derived stem/progenitor cells. Subsequent to degenerative changes, regenerative changes were seen, including angiogenesis, adipogenesis, and proliferation of cells (adipose-derived stem/progenitor cells, vascular endothelial cells, and blood cells). The authors found that, in vitro, the experimentally differentiated adipocytes underwent apoptosis and/or necrosis under severe hypoxia, but adipose-derived stem/progenitor cells remained viable. Severe ischemia/hypoxia induces degenerative changes in adipose tissue and subsequent adaptive tissue remodeling. Adipocytes die easily under ischemic conditions, whereas adipose-derived stem/progenitor cells are activated and contribute to adipose tissue repair.

Lipoinjection is a promising treatment, but its efficacy in recontouring facial lipoatrophy remains to be established. The objective was to evaluate the efficacy and adverse effects of lipoinjection and supplementation of adipose-derived stem/stromal cells (ASCs) to adipose grafts. To overcome drawbacks of autologous lipoinjection, we have developed a novel strategy called cell-assisted lipotransfer (CAL). In CAL, stromal vascular fraction containing ASCs was freshly isolated from half of an aspirated fat sample and attached to the other half of aspirated fat sample with the fat acting as a scaffold. This process converts relatively ASC-poor aspirated fat into ASC-rich fat. We performed conventional lipoinjection (non-CAL; n=3) or CAL (n=3) on six patients with facial lipoatrophy due to lupus profundus or Parry-Romberg syndrome. All patients obtained improvement in facial contour, but the CAL group had a better clinical improvement score than did the non-CAL patients, although the difference did not reach statistical significance (p=.11). Adipose necrosis was found in one non-CAL case who took perioperative oral corticosteroids. Our results suggest that CAL is both effective and safe and potentially superior to conventional lipoinjection for facial recontouring. The authors have indicated no significant interest with commercial supporters.

Many features of adipose tissue-specific stem/progenitor cells, such as physiological function and localization, have recently been examined. Adipose-tissue turnover is very slow and its perivascular progenitor cells differentiate into adipocytes in the next generation. The progenitor cells play important roles in physiological turnover, hyperplasia and atrophy of adipose tissue, as well as in incidental remodeling, such as postinjury repair. Adipose tissue has been used as an autologous filler for soft tissue defects, despite unpredictable clinical results and a low rate of graft survival, which may be due to the relative deficiency of progenitor cells in graft materials. A novel transplantation strategy, termed cell-assisted lipotransfer, involves the enrichment of adipose progenitor cells in grafts; preliminary results suggest this approach to be safe and effective.