Noriyuki Aoi

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

BackgroundLipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis.MethodsTo overcome the problems with lipoinjection, the authors developed a novel strategy known as cell-assisted lipotransfer (CAL). In CAL, autologous adipose-derived 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.ResultsFinal 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.ConclusionsThe 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.

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 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 &mgr;m 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).

Progenitor-Enriched Adipose Tissue Transplantation as Rescue for Breast Implant Complications

Abstract:  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.

Functional Implications of CD34 Expression in Human Adipose-Derived Stem/Progenitor Cells

CD34 is frequently used as a marker of adipose-derived stem/stromal/progenitor cells (ASCs). However, CD34 expression in human ASCs (hASCs) decreases over time in culture, and the implications of this remain unclear. In this study, we sorted shortly-cultured hASCs into CD34+ and CD34- fractions and compared their biological functions. Results indicated that CD34+ hASCs were more proliferative and had a greater ability to form colonies. In contrast, CD34- cells showed a greater ability for differentiation into adipogenic and osteogenic lineages. Both CD34+ and CD34- cells showed similar abilities in migration and capillary formation in response to growth factors. Expression levels of endothelial progenitor markers (Flk-1, FLT1, and Tie-2) were higher in CD34+ cells, whereas those of pericyte markers (CD146, NG2, and alpha-smooth muscle actin) were higher in CD34- cells. Both CD34+ and CD34- cells showed similar expression levels of vascular endothelial growth factor and hepatocyte growth factor, although hypoxia affected the expression levels. Together these results suggest that CD34 expression in hASCs may correlate with replicative capacity, differentiation potentials, expression profiles of angiogenesis-related genes, and immaturity or stemness of the cells. Loss of CD34 expression may be related to the physiological process of commitment and/or differentiation from immature status into specific lineages such as adipose, bone, or smooth muscle.

Characterization of Structure and Cellular Components of Aspirated and Excised Adipose Tissue

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.

Adipose tissue remodeling under ischemia: death of adipocytes and activation of stem/progenitor cells.

Background: 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. Methods: 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. Results: 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 1&agr; 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. Conclusions: 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.

Preserved Proliferative Capacity and Multipotency of Human Adipose-Derived Stem Cells after Long-Term Cryopreservation

Background: Human adipose-derived stem (stromal) cells are promising as a regenerative therapy tool for defective tissues of mesenchymal lineage, including fat, bone, and cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation could be an indispensable fundamental technology, as has occurred in other fields involving cell-based therapies using hematopoietic stem cells and umbilical cord blood cells. Methods: The authors examined the proliferative capacity and multipotency of human adipose-derived stem cells isolated from lipoaspirates of 18 patients in total before and after a 6-month cryopreservation following their defined protocol. Proliferative capacity was quantified by measuring doubling time in cell culture, and multipotency was examined with differentiation assays for chondrogenic, osteogenic, and adipogenic lineages. In addition, expression profiles of cell surface markers were determined by flow cytometry and compared between fresh and cryopreserved adipose-derived stem cells. Results: Cryopreserved adipose-derived stem cells fully retained the potential for differentiation into adipocytes, osteoblasts, and chondrocytes and for proliferative capacity. Flow cytometric analyses revealed that surface marker expression profiles remained constant before and after storage. Conclusions: Adipose-derived stem cells can be cryopreserved at least for up to 6 months under the present protocol without any loss of proliferative or differentiation potential. These results ensure the availability of autologous banked adipose-derived stem cells for clinical applications in the future.

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. STEM CELLS 2009;27:238–249

Ultrasound assessment of deep tissue injury in pressure ulcers: possible prediction of pressure ulcer progression.

Background: The concept of deep tissue injury under intact skin helps us understand the pathogenesis of pressure ulcers, but the best method for detecting and evaluating deep tissue injury remains to be established. Methods: Intermediate-frequency (10-MHz) ultrasonography was performed to evaluate deep tissue injury. The authors analyzed 12 patients (nine male patients and three female patients aged 16 to 92 years) who showed deep tissue injury–related abnormal findings on ultrasonography at the first examination and were followed up until the pressure ulcer reached a final stage. Results: The stage of ulcer worsened in six of 12 cases compared with baseline, and healed in the remaining six patients. The authors recognized four types of abnormal signs unique to deep tissue damage in ultrasonography: unclear layered structure, hypoechoic lesion, discontinuous fascia, and heterogeneous hypoechoic area. Unclear layered structure, hypoechoic lesion, discontinuous fascia, and heterogeneous hypoechoic area were detected at the first examination in 12, 10, seven, and five patients, respectively. Unclear layered structure and hypoechoic lesion were more commonly seen in pressure ulcers in deep tissue injury than the other features, but the follow-up study suggested that discontinuous fascia and heterogeneous hypoechoic area are more reliable predictors of future progression of pressure ulcers. Conclusions: The use of intermediate-frequency ultrasound reliably identified deep tissue injury and was believed to contribute to prevention and treatment of pressure-related ulcers. The results suggest that specific ultrasonographic characteristics may predict which pressure ulcers will progress.