Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells.

BACKGROUND: Lipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis. METHODS: To overcome the problems with lipoinjection, the authors developed a novel strategy known as cellassisted lipotransfer (CAL). In CAL, autologous adiposederived stem (stromal) cells (ASCs) are used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is freshly isolated from half of the aspirated fat and recombined with the other half. This process converts relatively ASC-poor aspirated fat to ASC-rich fat. This report presents the findings for 40 patients who underwent CAL for cosmetic breast augmentation. RESULTS: Final breast volume showed augmentation by 100 to 200 ml after a mean fat amount of 270 ml was injected. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Cyst formation or microcalcification was detected in four patients. Almost all the patients were satisfied with the soft and natural-appearing augmentation. CONCLUSIONS: The preliminary results suggest that CAL is effective and safe for soft tissue augmentation and superior to conventional lipoinjection. Additional study is necessary to evaluate the efficacy of this technique further.

Possibility of Using Standard Plates for Orbital Fracture.

OBJECTIVES: Absorbable plates are sometimes grafted for treating orbital fractures. These plates cannot be readily processed to fit the shape of the fracture site, particularly when the fracture encompasses a broad area from the medial toward the inferior wall. Preparing the plates in a standard shape beforehand will be useful. Thus, in this study, the authors measured the orbital wall distance in healthy orbits to determine the mean orbital size with the ultimate goal of developing and clinically applying a standard plate for orbital fracture. METHODS: Measurements were performed for the left eye orbit on computed tomography images using a three-dimensional medical image processing workstation. The authors measured the orbital wall distances and angle of healthy orbits in 40 males and 40 females to determine the mean size of the orbit. RESULTS: In healthy orbits, no significant difference was noticeable in the angle between medial wall and inferior wall between males and females. The medial, inferior, and medial + inferior wall distances were markedly longer in males than in females (P < 0.05). DISCUSSIONS: The orbital shapes had the same pattern in males and females. The standard plate would be adaptable to all cases if it were produced with the medial wall + inferior wall distance greater than the maximum value in males and trimmed to fit the orbit form of the patient. CONCLUSIONS: The results would be the basis of creating a standard plate and using it after appropriate adjustments.

Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column.

BACKGROUND AIMS: Adipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells. METHODS: We applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells. RESULTS: The column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples. CONCLUSIONS: Our results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.

Therapeutic potential of fibroblast growth factor-2 for hypertrophic scars: upregulation of MMP-1 and HGF expression.

Although hypertrophic scars (HTSs) and keloids are challenging problems, their pathogenesis is not well understood, making therapy difficult. We showed that matrix metalloproteinase (MMP)-1 expression was downregulated in HTS compared with normal skin from the same patients, whereas type 1 and 3 collagen and transforming growth factor-ß (TGF-ß) were upregulated. These differences, however, were not seen in cultured fibroblasts, suggesting the involvement of microenvironmental factors in the pathogenesis of HTS. Fibroblast growth factor-2 (FGF-2) highly upregulated the expression of MMP-1 and hepatocyte growth factor (HGF) in both HTS-derived and control fibroblasts; the upregulation was reversed by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors. An animal study using human HTS tissue implanted into nude mice indicated that controlled-release FGF-2 resulted in significantly less weight and decreased hydroxyproline content in HTS. Degradation of collagen fibers in FGF-2-treated HTS was also confirmed histologically. Western blotting showed that FGF-2-treated HTS expressed significantly higher MMP-1 protein than control. Decreased MMP-1 expression may be an important transcriptional change in HTS, and its reversal as well as upregulation of HGF by FGF-2 could be a new therapeutic approach for HTS.

Adipose injury-associated factors mitigate hypoxia in ischemic tissues through activation of adipose-derived stem/progenitor/stromal cells and induction of angiogenesis.

Based on the analysis of exudates from injured adipose tissue, we prepared a mixture containing the injury-associated growth factors at the same proportion as the exudates, named adipose injury cocktail (AIC). We hypothesized that AIC induces a series of regenerating and angiogenic processes without actual wounding. The purpose of this study is to elucidate the therapeutic potentials of AIC. AIC preferentially activated adipose-derived stem/progenitor/stromal cells (ASCs) to proliferate, migrate, and form networks compared with vascular endothelial cells, whereas vascular endothelial growth factor did not induce mitogenesis or chemotaxis in human ASCs. Each component growth factor of AIC was differently responsible for the ASC activation. AIC-treated ASCs tended to differentiate into adipocytes or vessel-constituting cells rather than into other cell types. In ischemic adipose tissues of mice, induced by either a surgical intervention or diabetes, AIC administration enhanced proliferation, especially of CD31(-)/CD34(+) ASCs, and mitigated tissue hypoxia by increasing capillary density and reducing fibrogenesis. These results suggest that AIC may have therapeutic potentials for various ischemic/hypoxic conditions by inducing adipose remodeling and neovascularization through activation of ASCs and other cells. Treatment with AIC has many advantages over cell-based therapies regarding morbidity, cost, and physical risks and may be used as an alternative therapy for improving tissue oxygen.

Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug.

BACKGROUND: The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells. METHODS: Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression. RESULTS: Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3. CONCLUSIONS: Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism.

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs.

Progenitor-enriched adipose tissue transplantation as rescue for breast implant complications.

Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor-supplemented adipose tissue (cell-assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 +/- 50 mL (Right; mean +/- SD) and 143 +/- 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants.

TGF-beta is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis.

Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming growth factor (TGF)-beta(2) to be highly expressed in cultured hDPCs. Keratinocyte conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(2) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D(3) analogue was found to promote TGF-beta(2) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(2) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(2) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs.

Differential expression of stem-cell-associated markers in human hair follicle epithelial cells.

Several putative biomarkers have been suggested for identifying murine follicular stem cells; however, human hair follicles have a different pattern of biomarker expression, and follicular stem cell isolation methods have not been established. To isolate a stem cell population applicable to clinical settings, we conducted a comprehensive survey of the expression of stem-cell-associated (K15, CD200, CD34, and CD271) and other biomarkers (K1, K14, CD29, and CD49f) in immunohistological sections of the human epidermis and follicular outer root sheath (ORS). We also examined freshly isolated and cultured epidermal or follicular cells with single- and multicolor flow cytometry or immunocytochemistry. After sorting cells by CD200, CD34, and forward scatter (FSC) values (cell size), colony-forming assays were performed. We found that biomarkers were differentially expressed in the epidermis and ORS. Basal bulge cells were mainly K15+CD200+CD34(-)CD271(-), and suprabasal cells were K15(-)CD200+CD34(-)CD271(-). We categorized follicular cells into nine subpopulations according to biomarker expression profiles. The CD200+CD34(-) bulge cells had much higher colony-forming abilities than the CD34+ population, and were divided into two subpopulations: a CD200+CD34(-)FSC(high) (K15-rich, basal) and a CD200+CD34(-)FSC(low) (K15-poor, suprabasal) population. The former formed fewer but larger-sized colonies than the latter. Follicular epithelial cell cultivation resulted in loss of K15, CD200, CD34, and CD271 expression, but maintenance of K14, CD29, and CD49f expression. We found that the bulge contained two populations with different localizations, cell sizes, and colony-forming abilities. We showed that K15, CD200, CD34, and CD271 were useful biomarkers for characterizing freshly isolated human follicular epithelial cells in diverse stages of differentiation.

Evaluation of animal models for the hair-inducing capacity of cultured human dermal papilla cells.

BACKGROUND: The dermal papilla (DP) interacts with epithelial cells for folliculogenesis. For translational research on cell therapies for hair regrowth with cultured human DP cells (hDPCs), a model to evaluate the capacity of hDPCs to induce hair formation is inevitable. METHODS: Chamber models were constructed by transplanting 4 different combinations of mouse or human epithelial and mesenchymal cells into a silicone chamber implanted onto the back of nude mice. In parallel, 3 types of sandwich constructs were created by inserting hDPCs or human DP tissue between the epidermis and dermis of isolated rat footpad skin or human facial skin, and subcutaneously transplanting the constructs into the back of nude mice. Four to six weeks later, skin sections of each model were histologically examined. RESULTS: Folliculoneogenesis was detected in both chamber and sandwich models, although the induction rate and maturity of the hair follicles varied among cell combination subgroups in each model. The difference in hair induction rate was not statistically significant between 2 representative chamber and sandwich subgroups using cultured hDPCs. The sandwich model, however, required fewer hDPCs, did not require human keratinocytes, and exhibited a higher rate of successful sample collection. CONCLUSIONS: Although there is no significant difference in hair induction rate, the sandwich model using cultured hDPCs and the rat sole skin is more feasible than the chamber model using human cultured keratinocytes and hDPCs as a tool to evaluate the hair-inducing capacity of cultured hDPCs.

Adipose tissue remodeling in lipedema: adipocyte death and concurrent regeneration.

Lipedema is a disease with unknown etiology presenting as bilateral and symmetric enlargement of the lower extremities due to subcutaneous deposition of the adipose tissue. Here we describe the histopathological features of the lipedema tissue and nonaffected adipose tissue obtained from a typical patient with severe lipedema. Immunohistochemical analyses indicated degenerative and regenerative changes of the lipedema tissue, characterized by crown-like structures (necrotizing adipocytes surrounded by infiltrating CD68+ macrophages; a feature commonly seen in obese adipose tissue) and proliferation of adipose-derived stem/progenitor/stromal cells (Ki67+CD34+ cells), respectively. These findings suggested increased adipogenesis in the lipedema tissue, which may further lead to hypoxia similar to that seen in obesity, resulting in adipocyte necrosis and macrophage recruitment. The confinement to the lower extremities and the difference from systemic obesity warrants further elucidation in future studies.

Cell-assisted lipotransfer for facial lipoatrophy: efficacy of clinical use of adipose-derived stem cells.

BACKGROUND: Lipoinjection is a promising treatment, but its efficacy in recontouring facial lipoatrophy remains to be established. OBJECTIVE: The objective was to evaluate the efficacy and adverse effects of lipoinjection and supplementation of adipose-derived stem/stromal cells (ASCs) to adipose grafts. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Evaluation of the In Vivo Kinetics and Biostimulatory Effects of Subcutaneously Injected Hyaluronic Acid Filler.

BACKGROUND: Because subcutaneously injected hyaluronic acid filler is absorbed over 6 months to 1 year after the treatment of facial wrinkles, frequent retreatment may be required. However, persistent long-term effects are often clinically observed when hyaluronic acid filler is injected as a bolus for facial augmentation. Therefore, the authors investigated, over time, the changes in volume and histologic features of subcutaneous bolus injections of hyaluronic acid. METHODS: Hyaluronic acid filler was subcutaneously injected as a bolus into the dorsum of 6-week-old rats. At several time points (immediately after injection and 4, 8, 16, 32, and 64 weeks thereafter), magnetic resonance imaging was introduced to observe morphologic changes and to measure volume. Histologic examination of sectioned tissues was also performed. RESULTS: The average volume increased for up to 4 weeks after injection and then gradually decreased, with 74.8 percent of the injected volume remaining after 64 weeks, with no statistical difference compared to the initial volume. Histologic analysis revealed that lattice structures were created by fibroblasts and collagen fibers, and blood vessels and adipocytes were also generated in the filler. CONCLUSIONS: Although subcutaneous bolus injections of hyaluronic acid filler exhibited flattening, the total volume was maintained even after 64 weeks. Histologically, hyaluronic acid filler acted as a scaffold for autogenous tissue replacement by means of fibroblast migration and proliferation, collagen induction, and angiogenesis, followed by proliferation of adipocytes. This study demonstrates that the total volume is maintained long-term by replacing part of the injected hyaluronic acid filler with autologous tissues.

The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.

BACKGROUND: Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases. METHODS: Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing. RESULTS: Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively. CONCLUSIONS: Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.

Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.

Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3+ cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues.

Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis.

The heterogeneous stromal vascular fraction (SVF), containing adipose-derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell-processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side-effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell-based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice-level cell processing area.

The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes.

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).